Biocontrol Science and Technology - Nuclear Sciences and ...

Biocontrol Science and Technology, 

Vol. 19, S1, 2009, 3 22 

REVIEW ARTICLE 

Improving the cost-effectiveness, trade and safety of biological control 

for agricultural insect pests using nuclear techniques 

Jorge Hendrichs a *, Kenneth Bloem b , Gernot Hoch c , James E. Carpenter d , 

Patrick Greany e , and Alan S. Robinson a 

a Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, International 

Atomic Energy Agency, Wagramerstrasse 5, A-1400 Vienna, Austria; b Center for Plant Health 

Science & Technology (CPHIST), USDA-APHIS-PPQ, 1730 Varsity Drive, Suite 400, 

Raleigh, NC 27606, USA; c Department of Forest and Soil Sciences, BOKU University of 

Natural Resources and Applied Life Sciences, Vienna Hasenauerstrasse 38, A-1190 Vienna, 

Austria; d USDA-ARS Crop Protection and Management Research Unit, Tifton, GA 31793, 

USA; e 2770 Pine Ridge Road, Tallahassee, FL 32308, USA 

If appropriately applied, biological control offers one of the most promising, 

environmentally sound, and sustainable control tactics for arthropod pests and 

weeds for application as part of an integrated pest management (IPM) approach. 

Public support for biological control as one of the preferred methods of managing 

non-indigenous and indigenous pests is increasing in many countries. An FAO/ 

IAEA Coordinated Research Project (CRP) addressed constraints related to 

costly production systems for biological control agents, and the presence of 

accompanying pest organisms during their shipment. These constraints can be 

alleviated using nuclear techniques such as ionizing radiation or X-rays to reduce 

production and handling costs (e.g., by expanding the period of host suitability, 

increasing shelf life, avoiding unnecessary sorting steps before shipment, etc.), and 

to eliminate the risk of shipping fertile host or prey pest individuals or other 

hitchhiking pests. These nuclear techniques can also help to reduce the risks 

associated with the introduction of exotic biological control agents, which can 

become pests of non-target organisms if not carefully screened under semi-natural 

or natural conditions. Radiation is also a very useful tool to study host-parasitoid 

physiological interactions, such as host immune responses, by suppressing 

defensive reactions of natural or factitious hosts. Applied at a very low-dose, 

radiation may be used to stimulate reproduction of some entomophagous insects. 

Additionally, radiation can be applied to semi- or completely sterilize hosts or 

prey for deployment in the field to increase the initial survival and build-up of 

natural or released biological control agents in advance of seasonal pest 

population build-up. Finally, the work carried out under this CRP has 

demonstrated the feasibility of integrating augmentative and sterile insect releases 

in area-wide IPM programmes, and to utilise by-products from insect massrearing 

facilities in augmentative biological control programmes. This special 

issue provides an overview of the research results of the CRP. 

Keywords: biological control; radiation; biocontrol agents; weeds; irradiated host; 

prey; insects; nematodes; parasitoids; sterile insects; sterile insect technique; 

inherited sterility; F1 sterility 

*Corresponding author. Email: j.hendrichs@iaea.org 

First Published Online 18 June 2009 

ISSN 0958-3157 print/ISSN 1360-0478 online 

# 2009 Taylor & Francis 

DOI: 10.1080/09583150902985620 

http://www.informaworld.com

4 J. Hendrichs et al. 

Introduction 

Many pests, including arthropods and weeds, adversely affect agricultural production, 

and pre- and post-harvest losses of the order of 30 40% are common 

(Yudelman, Ratta, and Nygaard 1998). Management of these pests still relies heavily 

on the use of pesticides with their associated limitations. For these reasons there 

is pressure to develop improved methods of pest control, with an emphasis on 

biologically and ecologically based tactics that can be applied as part of an area-wide 

integrated pest management (AW-IPM) approach (Vreysen, Robinson, and Hendrichs 

2007). If appropriately applied, biological control offers one of the most 

promising, environmentally sound, and sustainable tools for control of arthropod 

pests and weeds (van Lenteren, Bale, Bigler, Hokkanen, and Loomans 2006; van 

Driesche, Hoddle, and Center 2008). Public support for biological control as one of 

the preferred methods of managing non-indigenous and indigenous pests is increasing 

in many countries. There appear to be significant opportunities for increasing the use 

and cost-effectiveness of the application of classical and augmentative biological 

control through nuclear techniques for the production, shipping and release of 

biological control agents. 

Nuclear techniques are already applied in certain areas of entomology (Bakri, 

Heather, Hendrichs, and Ferris 2005a) and include the use of radiation sources 

for (1) studying sperm precedence, parasitoid host interaction studies, etc., (2) 

post-harvest disinfestation for quarantine or phytosanitary security in support of 

agricultural international trade (IDIDAS 2004), and (3) insect sterilization as part of 

the application of the Sterile Insect Technique (SIT) (Dyck, Hendrichs, and 

Robinson 2005), where exposure to carefully selected irradiation doses of gamma 

or X-rays maximizes the induction of dominant lethal mutations in germ cells of pest 

insects, while minimizing other physiological changes (Bakri, Mehta, and Lance 

2005b). Nuclear techniques in a wider sense also include the use of stable isotopes to 

study insect biology, behaviour, and physiology (IAEA 2009), although their use in 

biological control will not be considered here. 

In classical biological control, non-indigenous biological control agents, usually 

selected from the suite of parasitoids, predators and diseases that co-evolved with the 

pest, are introduced into the target area. One of the key concerns in this approach is 

the host specificity and host range of the introduced biological control agents. There 

are numerous examples of situations where introduced biological control agents have 

‘jumped hosts’ (Follet and Duan 2000; Lockwood, Purcell, and Howarth 2001; 

Wajnberg, Scott and Quimby 2001), prompting some serious criticisms of this 

control method (Hamilton 2000; Louda, Pemberton, Johnson, and Follett 2003). In 

view of the growing awareness and concern, countries and their respective national 

plant protection organizations are increasingly implementing stringent environmental 

risk assessment methods in order to screen potential biological control agents 

before release (van Lenteren et al. 2006). In cases where doubts remain about very 

promising natural enemies of weeds or insect pests, the release of such biological 

control agents that have been radiation-sterilized would enable a more definite 

and safe assessment of host specificity under natural conditions without any risk of 

permanent establishment. 

Inundative biological control involves the use of indigenous or non-indigenous 

biological control agents against indigenous or non-indigenous pests. These control

Biocontrol Science and Technology 5 

agents generally do not establish permanently, often because of adverse seasonal ecoclimatic 

conditions in the area of introduction, and are mass-reared and released 

in very large numbers, often several times each season. Though the commercial 

biological control agent industry is growing, it still only represents a niche market 

with less than 3% of pest control sales (Cornell 2007). Regulatory, technical 

and other constraints on biological control have kept the market share relatively 

small. Challenges facing augmentative biological control include the high cost of 

production of biological control agents, adequate quality control and assurance, 

trade barriers and regulations that complicate shipping. 

Lack of enabling regulations probably has been amongst the most important 

barriers to the wider implementation of biological control globally. Appropriate 

regulations are needed that facilitate the importation and use of natural enemies. In 

many countries, ‘gatekeeper’ regulations place barriers in the way of efficient 

introduction of agents. However, the Secretariat of the International Plant Protection 

Convention, at the Food and Agriculture Organization of the United Nations (FAO) 

has developed and recently revised the International Standard for Phytosanitary 

Measures on ‘Guidelines for the export, shipment, import, and release of biological 

control agents and other beneficial organisms’ (ISPM No. 3) (FAO 2005), which 

should help to solve some of these problems and therefore increase cross-boundary 

trade in biological control agents. 

There are several ways in which nuclear techniques can improve the efficiency of 

augmentative biological control and the Joint FAO/IAEA Programme of Nuclear 

Techniques in Food and Agriculture initiated a 6-year Coordinated Research Project 

(CRP) entitled ‘Evaluating the Use of Nuclear Techniques for the Colonization and 

Production of Natural Enemies of Agricultural Insect Pests’ (Greany and Carpenter 

1999) to address some of these aspects. For example, the cost of production may be 

decreased by simplifying the rearing process, increasing host suitability and shelf life, 

improving diets and dealing with disease and contamination. Possible trade barriers 

related to shipment of biological control agents include the accidental inclusion of 

fertile pest individuals or other hitchhikers, or the deliberate inclusion of live fertile 

food (the prey or pest insect for entomophagous agents) in the shipments, problems 

which can be overcome by irradiation. Properly timed distribution of the appropriate 

stage of a biological control agent is another crucial component for success. 

Providing irradiated hosts/prey in the field at the time of introduction would provide 

greater flexibility in timing the release and allow for development of the biological 

control agent population so that the most effective stage is present for optimum pest 

suppression. 

This introductory paper presents an overview of the research carried out under 

this CRP. Some of the results are described in research papers collected in this special 

issue, while some others are already published elsewhere. 

The FAO/IAEA Coordinated Research Project (CRP) 

This international research network consisted of 18 research teams from 15 

countries. The general objective of the CRP was to assess potential applications 

of nuclear techniques to increase the cost-effectiveness, trade and safety in the use of 

biological control agents of agricultural insect pests. It focused on the six major areas


listed in Table 1. The main research results in these major areas are described below, 

and a summary of the main findings is presented in Table 2. 

Improving rearing 

Suppressing host immune reactions 

Host immune reactions may reduce rearing efficiency of biological control agents or 

prevent the use of factitious or non-habitual hosts that are easier or more economical 

to mass-rear. Exposure to radiation has been shown to suppress host immune system 

responses (Vey and Causse 1979) and it can also make older instars of irradiated 

larvae suitable for parasitoid development and thus increase rearing efficiency and 

parasitoid quality. Irradiated haemolymph of the greater wax moth Galleria 

mellonella (L.) showed severely reduced capability to encapsulate artificially 

introduced Sephadex beads (both in vivo and in vitro) probably due to radiationdamage 

to haemocytes. G. mellonella larvae irradiated with 65 Gy were found to be 

suitable for parasitization by Venturia canescens (Gravenhorst), thus facilitating the 

use of G. mellonella as a potential factitious host for the rearing of this biological 

control agent (Genchev, Milcheva-Dimitrova, and Kozhuharova 2007). 

The use of prey or hosts that are easier or more economical to mass-rear was also 

facilitated for the green lacewing predator Chrysoperla carnea (Stephens), where 

irradiated Sitotroga cerealella (Olivier) eggs, provided as a prey substitute, increased 

larval survival, fecundity and the proportion of female predators (Hamed, Nadeem, 

and Riaz 2009). Rearing of the parasitoid Psyttalia concolor (Szépligeti) on a 

factitious host, i.e., irradiated Mediterranean fruit fly Ceratitis capitata (Wiedemann) 

larvae, allowed mass-releases of this parasitoid against the olive fruit fly 

Bactrocera oleae (Gmelin) (Hepdurgun, Turanli, and Zümreog˘lu 2009a). 

Behavioural and physiological interactions between hosts and parasitoids are 

complex, often difficult to study, and not well understood in terms of improving 

rearing efficiency. Certain physiological processes in the host (e.g., defence 

mechanisms, hormone metabolism) can be selectively modified by radiation, thereby 

facilitating the study of particular host parasitoid interactions. Radiation can 

likewise be used to modify or terminate certain parasitoid processes that affect 

host physiology and behaviour, e.g., by sterilizing the wasps or parasitoid eggs (Bai, 

Chen, Cheng, Fu, and He 2003). Irradiation of Glyptapanteles liparidis (Bouché) 

wasps caused temporary sterilization and a reduction in oviposition and reduced the 

total number of eggs laid per female, but did not reduce longevity (Tillinger, Hoch, 

and Schopf 2004). These wasps were used to study the action of the parasitoid’s 

polydnavirus and venom in gypsy moth Lymantria dispar (L.) larvae. During 

oviposition the irradiated wasps injected non-viable eggs, together with polydnavirus 

and venom in normal amounts and with minimal traumatic impact on the host 

(Hoch, Marktl, and Schopf 2009a). This ‘pseudoparasitization’ of L. dispar larvae by 

irradiated female wasps caused delayed larval development, morphological abnormalities 

and high mortality during pupation or in the pupal stage. The immune 

response (haemocytic encapsulation and haemolymph melanization) of the host 

larva was also suppressed by pseudoparasitization (Tillinger et al. 2004; Hoch et al. 

2009a), as was juvenile hormone esterase activity (Schafellner, Marktl, and Schopf 

2007). Moreover, pseudoparasitization of L. dispar host larvae by irradiated

Table 1. Potential applications of nuclear techniques assessed to increase the cost-effectiveness, 

trade and safety in the use of biological control agents focused on six major areas. 

Areas of assessment Description of specific potential nuclear applications 

1. Improving rearing (a) Understanding host-parasitoid physiological 

interactions to be able to modulate defensive reactions of 

hosts/prey; 

(b) expanding the time window suitable for host 

parasitisation; 

(c) allowing for increased storage and stockpiling of hosts 

or prey; 

(d) using very low-dose radiation to affect the sex ratio or 

to stimulate reproduction by entomophagous insects; 

(e) improving rearing media (either natural hosts/prey or 

artificial diets) through irradiation to reduce the microbial 

load and also to allow terminal sterilization after 

packaging; and 

(f) utilising by-products of mass rearing 

facilities producing sterile insects for simultaneous 

production of biological control agents. 

2. Facilitating handling, shipment, 

trade and release 

3. Supplementing hosts in the field 

for survival or early build-up 

of biological control agents 

4. Integrating SIT or F1 sterility 

and biological control 

5. Reproductively inactivated hosts 

as sentinels in the field 


(a) Eliminating the cost and logistics of holding and 

separating of parasitoids and non-parasitized pest adults 

before being able to ship to customers; 

(b) avoiding the emergence of pest adults from nonparasitized 

immature stages; 

(c) ameliorating concerns relating to the incidental 

presence in commercial shipments of fertile individuals of 

other hitchhiking pests; and 

(d) provisioning of sterilized natural prey to be used as 

food during commercial shipments of predators. 

(a) Provisioning of sub-sterile or sterile hosts or prey in 

the field as supplemental food to increase the initial 

survival of inoculatively released biological control 

agents; and 

(b) provisioning of sub-sterile or sterile hosts or prey in 

the field as supplemental food to increase the early buildup 

of native biological control agents in advance of pest 

population build-up. 

(a) Integrating natural enemies with the Sterile Insect 

Technique (SIT) or inherited sterility results in synergistic 

action, particularly with biological control agents reproducing/feeding 

on sterile or substerile offspring; and 

(b) applying the SIT against biocontrol agents that have 

become pest insects. 

(a) Exploring for, and collection of, new biological control 

agents; and 

(b) monitoring of populations of parasitoids, predators 

and microorganisms.


Table 1. (Continued). 

Areas of assessment Description of specific potential nuclear applications 

6. Screening classical biological 

control agents under field 

conditions 

Facilitating the importation of exotic species for classical 

biological control, through sterilisation (particularly 

insect herbivores of weeds), where host specificity doubts 

remain, so that they can be released and assessed in the 

field without the risk of establishing breeding populations. 

G. liparidis increased spore production of several species of entomopathogenic 

microsporidia co-infecting the hosts probably due to immune suppression as well as 

modified nutritional physiology (Hoch, Solter, and Schopf 2009b). 

Expanding the time window when a host is suitable for parasitization 

Normal host development limits the time window when a host is suitable for 

parasitization and it is known that radiation can delay normal insect development 

and thus may extend the time window for host parasitization or modify the internal 

host environment to the benefit of the biological control agent. This was assessed 

for parasitoids of the Mediterranean flour moth Ephestia kuehniella (Zeller), the 

house fly Musca domestica (L.), the Indian meal moth Plodia interpunctella 

(Hübner) and S. cerealella (Fatima, Ahmad, Memon, Bux, and Ahmad 2009; 

Hamed et al. 2009; Zapater, Andiarena, Pérez-Camargo and Bartoloni 2009). Also, 

in sugarcane stemborer Chilo infuscatellus (Snellen) larvae, a dose of 60 80 Gy 

allowed the normally unsuitable fourth and fifth instar larvae to be successfully 

parasitised by Cotesia flavipes Cameron (Fatima et al. 2009). Furthermore, 

irradiation of carambola fruit fly Bactrocera carambolae Drew & Hancock eggs 

with 30 50 Gy extended the larval period available for parasitization by Psyttalia 

incisi (Sylvestri) and Fopius vandenboschi (Fullaway) (Kuswadi, Himawan, Indarwatmi, 

and Nasution 2003); and irradiation of third instar Anastrepha spp. larvae 

with a dose of 45 Gy extended the parasitization period and increased the quantity 

and quality of the parasitoid Diachasmimorpha longicaudata (Ashmead) developing 

in these hosts (Cancino, Ruíz, López, and Sivinski 2009b). Exposing irradiated hosts 

sequentially to larval and pupal parasitoids was another way that was explored to 

expand the suitability window for parasitization in order to maximize the use of 

hosts in the mass production of fruit fly parasitoids (Cancino, Ruíz, Hendrichs, and 

Bloem 2009a). 

Allowing for storage and stockpiling of hosts or prey 

The limited shelf life of hosts and prey for biological control agents restricts their use 

during mass-production. For certain insect species, radiation can be used to arrest 

development and thus allow for storage and stockpiling of hosts or prey. Studies on 

the Mediterranean flour moth E. kuehniella, M. domestica, S. cerealella, and the 

cotton leafworm or tobacco cutworm Spodoptera litura (F.) showed that irradiation 

caused a prolongation in the development of host stages suitable for parasitization, 

thus facilitating the use of these hosts under mass-rearing conditions (Celmer-Warda

Table 2. Listing of some of the studies of nuclear applications conducted in conjunction with 

the FAO/IAEA Coordinated Research Project to improve the cost-effectiveness, trade and 

safety of biological control of agricultural insect pests using nuclear techniques. 

Constraints 

addressed Pest species 

Suppressing host 

immune reactions 

Expanding the 

period of host 

suitability 

Extending storage 

and stockpiling time 

for hosts or prey 


Biological control 

agent References 

Galleria mellonella (L.) Venturia canescens 

(Gravenhorst) 

Genchev et al. (2007) 

Lymantria dispar (L.) Glyptapanteles 

liparidis (Bouché) 

Hoch et al. (2009a) 

Lymantria dispar (L.) Microsporidia Hoch et al. (2009b) 

Ephestia kuehniella Venturia canescens Celmer-Warda (2004) 

(Zeller) 

(Gravenhorst) 

Chilo infuscatellus Cotesia flavipes Fatima et al. (2009) 

(Snellen) 

Cameron 

Sitotroga cerealella Chrysoperla carnea Hamed et al. (2009) 

(Olivier) 

(Stephens) 

Anastrepha spp. Various parasitoids Cancino et al. (2009a,b) 

Plodia interpunctella Venturia canescens Celmer-Warda (2004) 

(Hübner) 

(Gravenhorst) 

Sitotroga cerealella Trichogramma Fatima et al. (2009) 

(Olivier) 

chilonis Ishii 

Musca domestica (L.) Spalangia endius 

Walker 

Zapater et al. (2009) 

Bactrocera carambolae Fopius vandenboschi Kuswadi et al. (2003) 

Drew & Hancock (Fullaway) Psyttalia 

incisi (Sylvestri) 

Exorista sorbillans Nysolynx thymus Hasan et al. (2009) 

(Wiedemann) (Girault) 

Spodoptera litura (F.) Steinernema glaseri 

(Steiner) 

Seth et al. (2009) 

Ephestia kuehniella Trichogramma Tunçbilek et al. (2009b) 

(Zeller) 

evanescens (Westwood) 

Sitotroga cerealella Trichogramma Tunçbilek et al. (2009b) 

(Olivier) 



Walker 


Phthorimaea 

operculella (Zeller) 

Trichograma spp. Saour (2009)



Constraints 




Stimulation effects 

of low dose radiation 

Ephestia kuehniella Trichogramma Genchev et al. (2008) 

(Zeller) 


Helicoverpa armigera Trichogramma chilonis Wang et al. (2009) 

(Hübner) 

Ishii 

Utilisation of 

by-products from 

insect mass rearing 

facilities 

Ceratitis capitata Diachasmimorpha Viscarret et al. (2006) 

(Wiedemann) longicaudata (Ashmed) 

Ceratitis capitata Orius laevigatus Steinberg and Cayol 

(Wiedemann) (Fieber) 

(2009) 

Ceratitis capitata Spalangia cameroni Steinberg and Cayol 

(Wiedemann) Perkins 

(2009) 

Anastrepha spp. Various parasitoids Cancino et al. 

(2009b,c,d) 

Avoiding unnecessary 

handling and sorting 

steps before shipment 


(Steiner) 

Seth and Barik (2009) 

Bactrocera oleae Psyttalia concolor Hepdurgun et al. 

(Gmelin) 

(Szépligeti) 

(2009a) 


Walker 


Ephestia kuehniella Trichogramma Tunçbilek et al. (2009a) 

(Zeller) 


Sitotroga cerealella Trichogramma Tunçbilek et al. (2009a) 

(Olivier) 


Anastrepha spp. Diachasmimorpha 

longicaudata (Ashmed) 

Cancino et al. (2009b) 

Ceratitis capitata Diachasmimorpha Cancino et al. (2009b) 

(Wiedemann) tryoni (Cameron) 

Anastrepha ludens Fopius arisanus Cancino et al. (2009c) 

(Loew) 

(Sonan) 

Anastrepha ludens Various pupal Cancino et al. (2009d) 

(Loew) 

parasitoids 

Avoiding the 

shipment and 

release of fertile 

pest individuals 


Walker 


Tetranychus urticae Phytoseiulus persimilis Baptiste et al. (2003) 

Koch 

Athias-Henriot


Constraints 


Bactrocera oleae 

(Gmelin) 

Shipping sterilized 

hosts or prey in the 

absence of biological 

control agents 




Anastrepha spp. Various parasitoids Cancino et al. (2009d) 

Psyttalia concolor 

(Szépligeti) 

Hepdurgun et al. 

(2009b) 

Musca domestica (L.) Zapater et al. (2009) 

Ceratitis capitata 

(Wiedemann) 

Steinberg and Cayol 

(2009) 

Synergising 

biological control 

agents and 

F1 sterility 


(Hübner) 

Ishii 

Phthorimaea Trichogramma Saour (2009) 

operculella (Zeller) principium (Sugonyaev 

& Sorokina) 

Lymantria dispar (L.) Various Novotny and Zubrik 

(2003); Zubrik and 

Novotny (2009) 


(Steiner) 

Seth et al. (2009) 

Thaumatotibia Trichogrammatoidea Carpenter et al. (2004) 

( Chryptophlebia) cryptophlebiae 

leucotreta (Meyrick) Nagaraja 

Liriomyza bryoniae Diglyphus isaea Steinberg and Cayol 

(Kaltenbach) (Walker) 

(2009) 

Exorista sorbillans Nysolynx thymus Hasan et al. (2009) 

(Wiedemann) (Girault) 

Using SIT against 


agents that have 

become a pest 

Exorista sorbillans 

(Wiedemann) 

Hasan et al. (2009) 

Cactoblastis cactorum 

(Berg) 

Hight et al. (2005) 

Building up natural 

enemies in advance 

of pest populations 

Lymantria dispar (L.) Various Zubrik and Novotny 

(2009) 


(Hübner) 

Ishii



Constraints 


Monitoring natural 

enemies in the field 

Screening classical 


agents in the field 

Chilo infuscatellus 

(Snellen) 



Trichogramma chilonis 

Ishii 

Fatima et al. (2009) 

Lymantria dispar (L.) Various Novotny and Zubrik 

(2003); Zubrik and 

Novotny (2009) 

Ephestia kuehniella Venturia canescens Celmer-Warda (2004); 

(Zeller) 

(Gravenhorst) Celmer (2006) 

Plodia interpunctella Venturia canescens Celmer-Warda (2004) 

(Hübner) 

(Gravenhorst) 

Ceratitis capitata Diachasmimorpha Jordao-Paranhos 

(Wiedemann) 

Helicoverpa armigera 

(Hübner) 

longicaudata (Ashmed) 


Ishii 

Episimus unguiculus 

Clarke 

Cactoblastis cactorum 

(Berg) 

et al. (2003) 

Wang et al. (2009) 

Moeri (2007); Moeri 

et al. (2009) 

Tate et al. (2007, 2009) 

2004; Seth, Barik, and Chauhan 2009; Zapater et al. 2009). Host eggs of E. kuehniella 

irradiated at 200 Gy could be stored at 48C for up to 30 days without any quantitative 

or qualitative loss in the production of Trichogramma evanescens (Westwood) and for 

up to 60 days with only a minor decrease in quality (Tunçbilek, Canpolat, and Sumer 

2009b). Parasitoids in diapause could be stored inside irradiated host eggs for a period 

of 50 days without adverse effect on emergence, and irradiation of eggs did not affect 

acceptance by parasitoids (Tunçbilek et al. 2009b). The parasitoid C. flavipes 

irradiated at 20 Gy could be stored as pupae for 2 months at 108C without apparent 

loss of quality (Fatima et al. 2009). 

Utilisation of by-products from insect mass-rearing facilities 

Insect mass-rearing facilities often have excess production of particular insect life 

stages, and they also generate batches of sub-standard insects that are normally 

discarded. In addition, they create significant amounts of by-products during the 

production process (large biomass of discarded adults, larvae and pupae from 

the various colonies and quality control testing). Instead of having to invest in their 

disposal, these by-products may be processed (Nakashima, Hirose, and Kinjo 1996) 

or irradiated, if still alive, to support the production of biological control agents. 

Examples include the use of excess eggs, as well as the use of remnant larvae and 

pupae to produce egg, larval and pupal parasitoids, respectively. It was shown that


irradiated excess eggs of C. capitata and the Mexican fruit fly Anastrepha ludens 

(Loew) produced in mass-rearing facilities could be used to produce egg parasitoids, 

and that the discarded larvae and pupae could be used to produce larval and pupal 

parasitoids (Cancino, Ruíz, Pérez, and Harris 2009c; Cancino, Ruíz, Sivinski, 

Gálvez, and Aluja 2009d). Another example is the commercial use of C. capitata 

eggs and pupae, respectively, for the production of the minute pirate bug Orius 

laevigatus (Fieber), a highly effective predator of Western flower thrips, Frankliniella 

occidentalis Pergande (Steinberg and Cayol 2009), and the parasitoid Spalangia 

cameroni Perkins (Hymenoptera: Pteromalidae), a parasitoid of M. domestica and 

other Diptera (Geden 2007). The effective use of these by-products from insect 

rearing facilities can greatly increase the efficiency and the economics of the rearing 

process. 

Mass-rearing of C. capitata genetic sexing strains, which are now used in a 

majority of Mediterranean fruit fly production facilities (Cáceres et al. 2004), allows 

separating sexes at the larval or pupal stages with the possibility to employ a 

majority of males for sterile insect releases, while any excess females not returning to 

the colony can be used to produce larval and pupal parasitoids (Viscarret, La Rossa, 

Segura, Ovruski, and Cladera 2006). 

Reproductive stimulation by use of very low dose radiation 

The controversial phenomenon known as ‘radiation hormesis’ (Luckey 1991) refers 

to the use of very low dose radiation to stimulate biological processes. Two of the 

studies related to this CRP (Genchev, Balevski, Obretenchev, and Obretencheva 

2008; Wang, Lu, Liu and Li 2009) report noting a stimulation of reproduction and 

parasitization parameters in the parasitoids Habrobracon hebetor (Say), Trichogramma 

chilonis Ishii and V. canescens after exposure to very low doses of radiation, 

an intriguing discovery warranting further investigation. 

Facilitating handling, shipment, trade and release 

Avoiding unnecessary handling and sorting steps before shipment 

The continued development and emergence of non-parasitised fertile hosts, as well as 

of unused prey (pest) insects during mass-production of biological control agents 

often requires additional handling steps. These procedures, involving separation of 

significant proportions of non-parasitised hosts (or unused prey individuals) from 

the biological control agents, decrease efficiency in large scale mass-rearing. During 

parasitoid mass-production, radiation was used to reproductively sterilize prey, 

hosts, and factitious hosts such as E. kuehniella, S. cerealella, P. interpunctella, 

C. infuscatellus, and C. capitata, thereby inhibiting their further development and 

preventing the need for costly separation procedures. This also removed the delays 

inherent to the traditional production processes, which are related to waiting for the 

emergence from pupae of fertile unused pest individuals (Cancino et al. 2009b; 

Fatima et al. 2009; Tunçbilek, Canpolat, and Ayvaz 2009a). In the case of fruit flies 

such as the West Indian fruit fly Anastrepha obliqua (Macquart), the sapote fruit fly 

Anastrepha serpentina (Wiedemann), and A. ludens, irradiation of larvae is used 

routinely in the mass-production of tens of millions of parasitoids of these pest fruit


flies (Cancino et al. 2009b). Irradiation of A. ludens eggs and the pupae of M. 

domestica and A. ludens avoided having to wait for adult emergence as well as 

unnecessary handling during the mass-rearing of egg and pupal parasitoids for these 

pests (Cancino et al. 2009c,d; Zapater et al. 2009). 

Avoiding the shipment of fertile pest individuals 

There exists a real or perceived risk that shipping biological control agents with 

hosts/prey material could lead to the introduction of non-native, pesticide resistant 

or new strains of pest insects into new areas or countries and this may exacerbate the 

ever-stricter quarantine regulations required to obtain permits for their shipment. 

Procedures are required to guarantee that customers receive pest-free shipments. 

Research on the use of eggs of the spider mite Tetranychus urticae Koch to provision 

shipments of several species of predatory mites has confirmed that radiation at a 

dose of 280 Gy or less, depending on the age of the host eggs, can be used to 

eliminate the risk of introducing fertile mites, or other hitchhiking arthropods. At 

the same time, the provisioning of living eggs allows the inclusion of nutritional 

supplements in the form of prey material to maintain predator quality during 

shipment (Baptiste, Bloem, Reitz, and Mizell 2003). 

Irradiation of house fly pupae was shown to be very beneficial for the 

commercial shipment of house fly pupal parasitoids, allowing early shipment of 

recently parasitised pupae while ensuring that clean shipments were not contaminated 

with unparasitised pupae that would emerge later with the customers (Zapater 

et al. 2009). In another example, fruit fly parasitoids have been sent from Mexico to 

South America after being reared on irradiated A. ludens, which is a quarantine pest 

in this region (J. Cancino, personal communication). Furthermore, the feasibility of 

inoculative and augmentative releases of entomopathogenic nematodes within 

sterilized hosts was proposed to establish a safe mode of transport and dispersion 

without concern for the inadvertent release of uninfected fertile hosts. Laboratory 

studies demonstrated that hosts irradiated at 40 Gy were suitable for nematode 

development (although nematode parasitisation efficacy was better in non-irradiated 

host larvae) and could thus be used for safe, inoculative releases of these biocontrol 

agents (Seth and Barik 2009). 

Shipping sterilized hosts or prey in the absence of biological control agents 

Needs and opportunities exist for some commercial biological control companies to 

ship mass produced sterile hosts/prey in the absence of natural enemies for 

redistribution, both within and between countries, for use as host/prey at smaller 

rearing facilities. This alternative can be implemented in order to gain efficiencies in the 

production of biological control agents or to standardize the use of strains of host/prey 

material to ensure product quality (Steinberg and Cayol 2009; Zapater et al. 2009). 

Supplementing hosts in the field for survival or early build-up of biological control 

agents 

Many insect pests show cyclical population outbreaks that temporarily escape 

natural control. By increasing the number of host insects prior to the pest outbreak,


the population density of the biological control agents can be significantly increased. 

Radiation can be used to produce sterile host insects or host insects generating sterile 

F1 individuals to be released as hosts for the biological control agents without 

increasing the risk that the released host insects will become pests themselves. 

Irradiated eggs, as well as sterile F 1 eggs and larvae resulting from irradiated 

parents of the gypsy moth, L. dispar, were distributed in a natural forest and found 

to be acceptable and suitable as hosts for a number of parasitoid species. Most 

importantly, the parasitoids did not differentiate, under these natural conditions, 

between sterile F1 larvae and untreated larvae (Novotny and Zubrik 2003; Zubrik 

and Novotny 2009). Similar results were obtained with irradiated cotton bollworm 

Helicoverpa armigera (Hübner) and diamondback moth Plutella xylostella (L.) 

adults released in field crops during critical periods where their sterile eggs served as 

hosts for feral egg parasitoids resulting in parasitoid population increases (Wang 

et al. 2009). In crops with low damage thresholds, the early season use of trap crops 

in rows or in the surroundings may be required in some situations to minimize any 

potential damage by such semi-sterile Lepidoptera hosts. 

In sugarcane fields, the provision of supplemental hosts (irradiated sterile host 

eggs) to T. chilonis early in the season allowed populations of parasitoids to build-up 

and enhanced their survival during critical periods thereafter. This approach is 

currently providing effective management of several species of sugarcane borers in a 

40,000-ha area of sugarcane in Pakistan (Fatima et al. 2009). 

Integrating SIT or F1 sterility and biological control 

The release of sterile or semi-sterile insects together with biological control agents has 

been known to have synergistic effects for population suppression when applied 

simultaneously (Knipling 1992; Wong, Ramadan, Herr and McInnis 1992; Bloem, 

Bloem, and Knight 1998). This synergy results from the sterile insects impacting on 

the adult stage, while the biological control agents target mostly the immature stages, 

including reproducing on the F1 offspring in inherited sterility releases. Laboratory 

and field trials with H. armigera, the corn earworm Helicoverpa zea (Boddie), 

L. dispar, the potato tuber moth Phthorimaea operculella (Zeller), P. xylostella, 

S. litura, and the beet armyworm Spodoptera exigua (Hübner) indicated that sterile 

progeny from semi-sterile moths were acceptable as hosts for egg and larval 

parasitoids (Novotny and Zubrik 2003; Saour 2009; Wang et al. 2009; Zubrik and 

Novotny 2009). 

Experiments under large laboratory cage conditions showed that F1 sterility 

and releases of Trichogramma principium (Sugonyaev & Sorokina) are effective in 

suppressing Ph. operculella. Properly timed releases of T. principium together with 

moths irradiated at 250 Gy produced the greatest reduction in P. operculella F3 

progeny, demonstrating the synergistic effects of combining F1 sterility with egg 

parasitoids (Saour 2009). 

Eggs of the false codling moth Thaumatotibia ( Chryptophlebia) leucotreta 

(Meyrick) treated with 150 200 Gy were acceptable and suitable for development of 

the egg parasitoid Trichogrammatoidea cryptophlebiae Nagaraja under laboratory 

conditions. Field-cage evaluations in citrus orchards in South Africa revealed 

that releases of irradiated (150 and 200 Gy) moths combined with releases of 

T. cryptophlebiae provided synergistic suppression of false codling moth populations


(Carpenter, Bloem, and Hofmeyr 2004). These findings have encouraged the 

establishment of a private company by the South African citrus export industry in 

the Western Cape Province for the area-wide suppression of false codling moth, a 

major polyphagous pest that has developed resistance against many insecticides. 

The compatibility of the application of entomopathogenic nematodes with F 1 

sterility for population suppression of S. litura was demonstrated in laboratory 

experiments. Various feasible modes of integration of these two bio-rational strategies 

have been proposed (Seth et al. 2009). 

Another system under development for integrating augmentative parasitoid 

releases with the SIT is the release of Diglyphus isaea (Walker), the parasitoid of 

celery miner fly Liriomyza bryoniae (Kaltenbach), a serious pest of vegetables and 

ornamentals, together with sterile males of L. bryoniae for application in greenhouses 

(Kaspi and Parella 2008; Steinberg and Cayol 2009). 

Developing the SIT against biocontrol agents that have become pest insects 

themselves is another application linking nuclear techniques with biological control 

agents. One case is the cactus moth Cactoblastis cactorum (Berg), a textbook example 

of very effective classical biological control of introduced cactus Opuntia spp., which 

has invaded the south-eastern USA, and where its westward expansion is being 

contained by the integrated application of SIT (Hight, Carpenter, Bloem, and Bloem 

2005; Bloem et al. 2007; Tate, Carpenter, and Bloem 2007) to protect native Opuntiabased 

ecosystems in the south-western USA and Mexico. 

The Uzi-fly Exorista sorbillans (Wiedemann) is an endoparasitoid of the silkworm 

Bombyx mori (L.) and as such is a pest due to its negative impact on silk production. 

Radiation studies have been carried out to assess if the SIT can contribute to an 

integrated control of this pest. In addition, Nesolynx thymus (Girault), a hyperparasitoid 

of the Uzi-fly, has been identified and rearing studies have been carried out 

on irradiated Uzi-fly pupae to assess if combined releases of sterile insects and 

parasitoids are feasible (Hasan, Uddin, Khan, and Reza 2009). 

Mass releases of the olive fruit fly parasitoid P. concolor, reared on irradiated 

Mediterranean fruit fly larvae, were integrated with mass trapping for an environmentally 

friendly suppression method of olive fly populations (Hepdurgun, 

Turanli, and Zümreog˘lu 2009b). 

Reproductively inactivating hosts as sentinels in the field 

The exploration for, and collection of new exotic biological control agents and the 

monitoring of field populations of native biological control agents are sometimes 

complicated by the fact that hosts are rare or difficult to locate. Reproductively 

sterilized host insects may be placed in the field in strategic locations as sentinels to 

aid in these efforts (Jordao-Paranhos, Walder, and Papadopoulos 2003). 

Several approaches were evaluated including the use of (1) irradiated eggs of 

S. cerealella, a factitious host of Trichogramma, to monitor effects of seasonal 

environmental conditions on the establishment of released Trichogramma in 

sugarcane fields (Fatima et al. 2009), (2) sterile F1 larvae from irradiated L. dispar 

for monitoring the density and type of parasitoids and pathogens in forests (Novotny 

and Zubrik 2003; Zubrik and Novotny 2009), (3) reproductively inactivated larvae 

(400 and 600 Gy) of E. kuehniella and P. interpunctella to monitor the density of 

V. canescens and Habrobracon hebetor (Say) in warehouses and mills (Celmer-Warda


2004; Celmer 2006), and (4) sterilized M. domestica pupae in traps to monitor wild 

populations of pteromalid parasitoids in the field and under conditions of livestock 

production (Zapater, personal communication). 

Screening classical biological control agents under field conditions 

Classical biological control has resulted in many significant successes, but also many 

cases of direct and indirect non-target impacts have been documented (Howarth 

1991; Thomas and Willis 1998; Henneman and Memott 2001). Also, inundative 

biological control can result in environmental problems (van Lenteren et al. 2003), 

which has fostered growing concerns about the need to preserve biodiversity and 

natural ecosystems. Therefore, the importation of exotic biological control agents, 

particularly insect herbivores of invasive plants, is becoming increasingly difficult 

due to concerns over the possibility that imported species may shift hosts and 

become pests of crops or protected species. In some cases, despite careful selection 

(Briese 2006; van Lenteren et al. 2003; van Lenteren et al. 2006) and extensive prerelease 

studies under quarantine conditions, the release of promising biological 

control agents is ultimately rejected because of remaining doubts about their host 

specificity. 

In such situations, exotic biological control agents may be sterilized using 

radiation so that they can be released and studied under actual field conditions 

without the risk of establishing permanent breeding populations in space and 

time. The use of sterilized individuals allows further assessment and confirmation 

of oviposition behaviour and host (acceptability) associations. Also, the use of F1 

sterile larvae of exotic herbivores being considered for introduction and release 

against plant pests would allow field-testing of larval feeding preferences and the 

ability of these larvae to develop and survive on related weeds, crops and other native 

plants that are of concern (Greany and Carpenter 1999; Carpenter, Bloem, and 

Bloem 2001). 

A model system that includes Opuntia spp. and C. cactorum has been developed 

to study the host range of an exotic herbivore. Radiation biology studies revealed 

that the optimum dose at which females are sterilized and males remain partially 

fertile and produce sterile progeny is 200 Gy (Carpenter et al. 2001). Whole plant 

and single cladode host preference tests demonstrated that C. cactorum females 

mated with males irradiated at 200 Gy exhibited normal oviposition preferences and 

can be used safely under field conditions to predict the host range, as well as to study 

possible interactions with natural enemies (Hight et al. 2005; Tate, Hight, and 

Carpenter 2009). Another system under evaluation involves the exotic herbivore 

Episimus unguiculus Clarke (E. utilis Zimmerman), which is currently in quarantine 

in Florida, for the eventual biological control of the Brazilian pepper tree Schinus 

terebinthifolius Raddi (Moeri 2007; Moeri, Cuda, Overholt, Bloem, and Carpenter 

2009). 

Conclusion 

The results generated by this CRP have confirmed that nuclear techniques have a 

significant role to play in facilitating the use and increasing the cost-effectiveness and 

safety of biological control agents (Table 2). Nevertheless, a major constraint faced


by practitioners and producers of biological control agents who would like to adopt 

some of these technologies is access to radiation sources as these sources are only 

available to the larger commercial producers of biological control agents, some 

research institutions, or programmes involved with the production of sterile insects 

for release. Radiation sources represent a major financial investment for any 

company and bring with them considerable logistic and regulatory constraints. 

The final paper in this issue provides a review of the smaller radiation sources 

available as well as a comparison of some key parameters (Mehta 2009). In the 

future, in view of the increasing difficulty of transporting radioactive materials 

(IAEA 2008), non-isotopic sources such as those emitting X-rays may become the 

equipment of choice. 

Acknowledgements 

We would like express our appreciation to Jean Pierre Cayol and Marc Vreysen for useful 

suggestions and comments to improve this manuscript, and also to thank all the participants 

of the CRP for their enthusiastic and productive participation in this coordinated research 

network. 

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Vol. 19, S1, 2009, 23 34 

Gamma radiation-induced pseudoparasitization as a tool to study 

interactions between host insects and parasitoids in the system 

Lymantria dispar (Lep., Lymantriidae) Glyptapanteles liparidis 

(Hym., Braconidae) 

Gernot Hoch*, Robert C. Marktl, and Axel Schopf 

Department of Forest and Soil Sciences/Institute of Forest Entomology, Forest Pathology 

and Forest Protection, BOKU-University of Natural Resources and Applied Life Sciences, 

Vienna, Austria 

Larvae of the koinobiont endoparasitoid Glyptapanteles liparidis (Hym., Braconidae) 

need to suppress the immune responses of parasitized Lymantria dispar 

host larvae while maintaining them at high nutritional quality. We used the 

method of g-radiation-induced pseudoparasitization to study the effects of the 

parasitoid’s polydnavirus and venom in these processes. To achieve pseudoparasitization, 

G. liparidis females were irradiated in a cobalt-60 irradiator; such 

wasps injected during oviposition nonviable eggs along with polydnavirus and 

venom into the host. Glyptapanteles liparidis eggs or larvae were implanted into 

unparasitized or pseudoparasitized L. dispar larvae together with or without the 

parasitoid’s teratocytes. Eggs or larvae of G. liparidis implanted into unparasitized 

hosts were readily encapsulated by the host hemocytes. The further 

development of the hosts was not impaired. Implantation into pseudoparasitized 

hosts prevented encapsulation; complete endoparasitic development, however, 

was only possible when also teratocytes were implanted along with parasitoids 

into the L. dispar larva. These parasitoids required longer to emerge from the host 

compared to natural parasitization, but they were able to complete metamorphosis 

into imagines. Analysis of trehalose levels in the host hemolymph and 

glycogen in host tissue revealed that G. liparidis polydnavirus/venom is 

responsible for an alteration of carbohydrate metabolism in L. dispar that is 

probably beneficial for the developing parasitoid. 

Keywords: parasitoids; host defense; polydnavirus; parasitoid nutrition; 

g-radiation; Glyptapanteles liparidis; Lymantria dispar 

Introduction 

Interactions between insect parasitoids and their hosts have been shown to be a 

complex struggle between the two counterparts. Fine mechanisms have evolved in 

parasitic insects, particularly in endoparasitic koinobionts, to keep the balance 

between succeeding over the host’s defense reactions and maintaining the host at high 

quality for the developing parasitoid. Braconid endoparasitoids use parasitoid 

associated factors, such as symbiotic polydnaviruses (PDV), venom, and teratocytes, 

to manipulate physiological functions of the host. During oviposition, particles of 

PDV that replicate in the calyx region of the wasp’s ovary are injected into the 

*Corresponding author. Email: gernot.hoch@boku.ac.at 

First Published Online 16 October 2008 



DOI: 10.1080/09583150802434059 


24 G. Hoch et al. 

hemocoel of the host together with venom. One of the main functions of PDV, in 

many cases supported by venom, is the suppression of the host immune responses to 

prevent damage to the endoparasitoid (Lavine and Beckage 1995; Strand and Pech 

1995; Shelby and Webb 1999; Beckage and Gelman 2004). PDV are injected as virions 

which infect cells in the lepidopteran host, most importantly hemocytes, where 

specific viral genes are expressed (Kroemer and Webb 2004). The cellular immune 

response is directly impaired by causing failure of hemocyte spreading behavior or 

induction of apapotosis of hemocytes (Beckage and Gelman 2004). Moreover, PDV 

have been shown to manipulate host development by interfering with its hormone 

metabolism (Beckage 1997; Cusson, Laforge, Miller, Cloutier, and Stoltz 2000; 

Beckage and Gelman 2004; Schafellner, Marktl, and Schopf 2007). Teratocytes are a 

third factor that support the parasitoids. These cells are released from the serosal 

membrane of the parasitoid egg. They live individually in the host hemocoel and grow 

to large size; teratocytes have been shown to aid nutrition of the parasitoid, exert 

immunological and antimicrobial functions, and play a role in altering the host’s 

endocrine system (Dahlman 1990; Dahlman and Vinson 1993; Nakamatsu, Fujii, and 

Tanaka 2002; Bell, Kirkbride-Smith, Marris, and Edwards 2004). 

The gregarious larvae of Glyptapanteles liparidis (Bouché) (Hym., Braconidae) 

are endoparasitic koinobionts that maintain their host larva in suitable physiological 

condition until completion of the larval development and emergence from the host. 

The wasp parasitizes young to mid-stage larvae; depending on host size at time of 

oviposition, females lay five to 50 eggs into the host’s hemocoel. After 2 3 weeks of 

endoparasitic development through two instars, the larvae emerge from the host 

while molting to the third instar, spin a cocoon and pupate (Schopf and Steinberger 

1996; Schopf and Hoch 1997). Parasitism by G. liparidis leads to alterations in host 

development as well as in levels of certain nutrients, such as trehalose and fatty acids 

(Schopf and Steinberger 1996; Hoch and Schopf 2001; Hoch, Schafellner, Henn, and 

Schopf 2002). We explored a method to study the effect of PDV plus venom in the 

system G. liparidis L. dispar by sterilizing female wasps with g-radiation from a 

cobalt-60 source (Tillinger, Hoch, and Schopf 2004). Such irradiation is normally 

used in sterile insect (SIT) programs (Bakri, Mehta, and Lance 2005; Klassen and 

Curtis 2005). Our method followed the idea of Soller and Lanzrein (1996) who 

employed X-rays to sterilize female Chelonus inanitus to study effects of polydnavirus 

and venom. In reference to Jones (1985) who reported on parasitized larvae that 

showed symptoms typical for parasitization but did not contain obvious or living 

parasitoids at that time, we use the term ‘pseudoparasitization’ for this procedure. 

One advantage of this method in comparison to injection of purified PDV and 

venom is that it reflects the natural situation of parasitism as far as amount of 

injected parasitoid associated substances and minimal impact on the host larva 

during the process of injection are concerned. A disadvantage is that PDV and 

venom can only be studied together. Radiation-induced pseudoparasitization by G. 

liparidis was shown to affect both, host development resulting in prolonged 

development and frequently incomplete metamorphosis and immune competence 

(Tillinger et al. 2004). 

In this study, we used the technique of g-radiation-induced pseudoparasitization 

to study interactions in the host parasitoid system, L. dispar G. liparidis and 

complemented it with implantation experiments. The goal was to develop a 

technique that can be used as a basic tool for our future research. We established


methods to harvest G. liparidis at different developmental stages as well as 

teratocytes and to subsequently implant them into new host larvae. PDV and 

venom were administered via pseudoparasitization. The experiments demonstrate 

that implanted parasitoids require both PDV/venom and teratocytes to successfully 

complete their development. We report the effects of the various implantation 

treatments on host and parasitoid development. In the second part of the study, we 

applied radiation-induced pseudoparasitization to test whether PDV and venom of 

G. liparidis are responsible for observed alterations in carbohydrate metabolism of 

parasitized L. dispar larvae. 

Materials and methods 

Insects 

Lymantria dispar larvae were obtained from egg masses provided by the USDA/ 

APHIS Otis Methods Development Center at Cape Cod, MA, USA. Larvae were 

reared on high wheat germ diet (Bell, Owens, Shapiro, and Tardiff 1981) in groups in 

250-mL plastic cups at 20918C and 16 h L:8 h D photoperiod. Larvae that had 

received parasitoid implants were reared individually in 90-mm diameter Petri dishes 

under temperature and light regime as above and checked daily. Glyptapanteles 

liparidis were obtained from our laboratory colony, originating from parasitized L. 

dispar collected in oak forests in Burgenland, Austria. Adult wasps were reared on 

water and honey at 15/108C and 16 h L:8 h D photoperiod. To achieve controlled 

parasitization, host larvae were offered to wasps with a pair of forceps until the sting 

occurred. This procedure results in approximately 95% successful parasitism and 

avoids superparasitism. 

Irradiation of parasitoid wasps 

Glyptapanteles liparidis females were g-irradiated based on the method outlined in 

Tillinger et al. (2004). Six-day-old female wasps with at least one oviposition 

experience were placed in ventilated 10 10 15-cm plastic boxes and irradiated 

with 96 Gy (at a dose rate of 25.6 Gy/min) in a Gammacell 220 cobalt-60 irradiator 

(Atomic Energy of Canada Ltd.) at the FAO/IAEA Agriculture and Biotechnology 

Laboratories, Seibersdorf, Austria. Wasps were used for the experiments within 48 h 

post irradiation for a maximum of four ovipositions in order to prevent recovery of 

egg viability. 

Implantation of G. liparidis into L. dispar larvae 

To study the effects of parasitoid associated factors on the development of G. 

liparidis and the L. dispar host larvae, we implanted different parasitoid stages into 

unparasitized or pseudoparasitized L. dispar larvae. Development of both, host and 

parasitoids was monitored. G. liparidis were harvested either from normally 

parasitized hosts (larvae hatched in vivo) or derived from parasitoid eggs that 

hatched in vitro. To harvest in vivo larvae, a normally parasitized L. dispar larva 

(donor host) was dissected at a certain stage post parasitization. Parasitoid larvae 

were washed three times in 1 mL TC100 insect medium (Sigma-Aldrich) to remove


teratocytes and adhering host hemolymph and immediately implanted into an 

unparasitized or pseudoparasitized gypsy moth larva. Both, recipient and donor 

larvae were surface sterilized with 70% ethanol. To harvest larvae that hatched in 

vitro and their released teratocytes, five parasitoid eggs were dissected out of 

regularly parasitized L. dispar larvae and incubated in 10 mL TC100 medium in well 

plates (96 well microtiter plates with V-bottom, Roth GmbH) at 278C in darkness. 

The parasitoid larvae and teratocytes were retrieved from the wells together with the 

culture medium 24 h after hatch and implanted into the unparasitized or 

pseudoparasitized host larva with micro capillary pipettes after thoroughly washing 

them in culture medium. To perform the implantations, recipient host larvae were 

anesthetized with CO 2. A small incision was cut with ocular scissors at the base of an 

abdominal leg and the G. liparidis larvae were implanted through this opening with a 

micro capillary pipette. Recipient larvae were placed individually on filter paper in 

Petri dishes until bleeding ceased. Then they were supplied with wheat germ diet and 

reared as described above. 

Implantation of G. liparidis eggs or larvae into unparasitized recipient hosts: G. 

liparidis eggs or newly hatched larvae were harvested from donor hosts 4 or 5 days 

post-parasitization (dpp), respectively. Either five parasitoid eggs or five parasitoid 

larvae were implanted into L. dispar larvae on day 1 in the fifth instar as described 

above. One group of larvae received only 10 mL of TC100 insect medium; controls 

remained untreated. 

Implantation of G. liparidis larvae plus teratocytes into pseudoparasitized hosts: 

L. dispar larvae were pseudoparasitized in premolt to the fourth instar (indicated by 

slipped head capsule). One group received five newly hatched parasitoid larvae 

together with their teratocytes on day 3 of the host’s fourth instar. The other group 

of L. dispar larvae received five newly hatched G. liparidis larvae plus teratocytes on 

day 1 of the host’s fifth instar. In the first case, we harvested parasitoid larvae in vivo. 

For the second implantation we switched to harvesting in vitro (see above), which 

appeared to be more feasible because it offers controlled conditions. 

Implantation of G. liparidis larvae without teratocytes into pseudoparasitized 

recipient hosts: L. dispar larvae were pseudoparasitized in premolt to fourth instar. 

Glyptapanteles liparidis larvae that had been harvested in vivo were implanted on day 

3 in fourth instar. The following treatments were tested: 10 larvae received one 

second instar G. liparidis larva (harvested from the donor host 11 dpp), 10 larvae 

were injected with 10 mL TC100 medium, controls were pseudoparasitized but did 

not receive any implants or injections. After rearing for 8 or 9 days, the recipient 

larvae were dissected and the status of the implanted parasitoids was evaluated 

under the dissecting microscope. Harvesting larvae without teratocytes for implantations 

was only possible when the parasitoids had molted into second instar. 

Younger parasitoids had always significant numbers of teratocytes attached that 

could not be completely removed by several washings in insect medium. 

Effects of pseudoparasitization on host carbohydrate metabolism 

Lymantria dispar larvae were pseudoparasitized in premolt to the third instar. 

Unparasitized larvae served as controls. Larvae were reared in groups in 250-mL 

plastic cups and checked daily. On days 5, 7, 9 and 11 post-pseudoparasitization, 

samples for nutrient analysis were taken from larvae that were of the same age in the


respective instar. After cutting one proleg of a L. dispar larva, 20 mL of hemolymph 

were collected with a micro capillary pipette and transferred into a reaction tube 

containing 180 mL ice-cold 50% (v/v) aqueous methanol. After collecting the 

hemolymph sample, larvae were immediately dissected on parafilm (American 

National Can) in a wax dish on ice. The guts were removed and the carcass rinsed 

with ice-cold distilled water. The washed L. dispar carcasses were lyophilized and 

weighed. Hemolymph samples were prepared and analyzed for trehalose as described 

in Hoch et al. (2002). Briefly, after adding 30 mL 0.1% pentaerythrit (Sigma) as an 

internal standard, the samples were centrifuged to eliminate cellular debris. The 

supernatant was washed in chloroform. The methanolic phase was dried under 

vacuum and resuspended in Milli-Q ultrapure water (Millipore). Sugars were 

separated by HPLC (Hewlett-Packard 1050 with Biorad Aminex HPX-97P column) 

followed by refractive index detection (Hewlett-Packard 1047A). The lyophilized 

carcasses were prepared for glycogen analysis by homogenizing and washing with 

ethanol and water to eliminate low molecular weight carbohydrates. Glycogen was 

cleaved into glucose by incubating the sample with heat-stable a-amylase (500 U/mL) 

from Bacillus licheniformis (Sigma). After centrifugation, aliquots of the supernatant 

were incubated with amyloglucosidase (20 U/mL) from Aspergillus niger (Boehringer 

Mannheim) (Hoch et al. 2002). These samples were then analyzed for glucose 

contents by HPLC as described above. 

Statistical analyses 

Statistical analyses were carried out with SPSS 12.0.1 for Windows (SPSS Inc., 

2003). Data were analyzed for normal distribution with Kolmogorov Smirnov Ztest. 

Homogeneity of variances was tested with Levene’s test. Means of normally 

distributed data were compared by one-way ANOVA, and post-hoc analyzed by 

Scheffe test or Tamhane’s T2. Comparisons of two means of normally distributed 

data were carried out by Student’s t-test. 

Results 

Development of G. liparidis implanted into L. dispar larvae/effects on host larvae 

When G. liparidis were implanted into unparasitized L. dispar larvae, parasitoids 

were unable to develop. Dissections of recipient hosts revealed that all implanted 

parasitoids, both egg and larval stages, had been encapsulated by host hemocytes 

(Figure 1). Moreover, implantation of parasitoids did not alter the development of 

the host. Only a short but statistically significant prolongation of the fifth instar 

occurred. The same prolongation occurred also in larvae into which only TC100 

medium was injected. Duration of the pupal stage did not differ among treatments 

(Table 1). 

Pseudoparasitization of hosts prevented lethal encapsulation of implanted G. 

liparidis larvae by host hemocytes. However, no parasitoids were able to emerge from 

the recipient hosts when they had been implanted without teratocytes. Hosts that had 

received second instar parasitoids were dissected 9 days post-implantation (equaling 

a total age of the parasitoid of 20 days). All implanted parasitoids were full grown, 

normally developed second instars and alive at the time of dissection but showed 

partially melanized surfaces indicating host immune response, although at a reduced


Figure 1. Newly hatched G. liparidis larvae implanted into unparasitized L. dispar larvae 

were encapsulated by a thick layer of host hemocytes (arrows). 

level. Moreover, these larvae were in very close contact with lobes of the host’s fat 

body that could hardly be removed from the larvae upon dissection (Figure 2). 

Successful development into adults was only possible when G. liparidis larvae 

were implanted together with teratocytes into pseudoparasitized hosts. Ninety-four 

percent of G. liparidis implanted into fourth instar hosts and 85% of parasitoid 

larvae implanted into fifth instar hosts completed development; this was not 

significantly different from natural parasitism in the respective instars. However, 

the duration of endoparasitic development of implanted parasitoids as well as 

total development to adults was significantly longer than in natural parasitization 

(Table 2). 

Effects of pseudoparasitization on host carbohydrate levels 

Pseudoparasitization by G. liparidis led to alterations in hemolymph and tissue 

carbohydrate levels in L. dispar larvae. Trehalose concentrations in the hemolymph 

showed a steady increase within the fourth instar. This increase was steeper in 

pseudoparasitized larvae; consequently, trehalose concentrations were significantly 

elevated 9 and 11 days post-pseudoparasitization (Figure 3). In a similar way, 

glycogen content of the larvae increased during the fourth instar after a pronounced 

Table 1. Duration of fifth instar and pupal stage of unparasitized male L. dispar that received 

implants of five G. liparidis eggs (harvested 4 dpp), five G. liparidis larvae (harvested 5 dpp), or 

10 mL of TC100 medium and of untreated controls. 

Controls TC100 5 eggs 5 larvae 

Days in fifth instar 12.091.6 a 13.691.3 b 12.591.2 ab 13.491.7 b 

Days in pupa 16.490.8 a 16.891.3 a 16.490.8 a 15.990.9 a 

N 10 9 14 11 

Means9SD in a row followed by different letters are significantly different at PB0.05 (one-way ANOVA, 

post-hoc test: Scheffe).


Figure 2. G. liparidis larvae implanted into pseudoparasitized L. dispar hosts without 

teratocytes were able to develop but showed melanization on their surface and were tightly 

surrounded by lobes of the host fat body (total age of parasitoids at dissection was 20 days). 

decline during the molting process. Glycogen content per mg dry host tissue was 

significantly higher 9 and 11 days post-pseudoparasitization compared to 

unparasitized larvae (Figure 3). Pseudoparasitized larvae showed also modified 

development, manifested in significantly higher dry mass on day 11 postpseudoparasitization 

than unparasitized controls (22.791.4 vs. 16.691.3 mg). 

Discussion 

Implantations of G. liparidis were only successful when host larvae were treated with 

PDV/venom. Without immune suppression by these associated factors, the host 

responded with immediate encapsulation of the parasitoid by hemocytes, sometimes 

accompanied by melanization. The host immune system coped with the implanted 

parasitoids; no effect other than a short prolongation of the host instar following the 

Table 2. Duration of endoparasitic and total development (in days) and developmental 

success of G. liparidis after regular parasitization or implantation of five parasitoid larvae 

together with teratocytes into pseudoparasitized L. dispar larvae. 

d2 in L3 1 

Regular parasitization by 

G. liparidis 

d3 in L4 1 

d1 in L5 1 

Implantation of 

parasitoids 

d3 in L4 1 

d1 in L5 1 

Endoparasitic development [d] 14.890.8 b 12.290.6 a 16.391.4 c 18.391.3 d 18.190.7 d 

Total development [d] 21.290.8 b 18.090.6 a 23.191.5 c 25.490.9 d 25.191.1 d 

No. of hatched wasps per host 33.49 13.4 37.3914.4 34.1914.1 4.090.8 4.490.6 

% Successful development 2 

96.9 a 95.1 ab 94.6 ab 84.6 b 93.9 a 

n 35 33 34 11 14 

Means9SD within a row followed by different letters are significantly different (PB0.05; one-way 

ANOVA, post-hoc tests: Scheffe or Tamhane’s T2). 1 Developmental stage of host at treatment: e.g. d2 in 

L3 second day in third instar. 2 Data were arcsin-transformed before statistical analysis. The table shows 

values before transformation.


* * * * 

Figure 3. Trehalose concentration in hemolymph and glycogen content of tissue (n 10 12) 

of L. dispar larvae 5, 7, 9, and 11 days post-pseudoparasitization by G. liparidis. Arrows 

indicate the molt of the host larvae to fourth instar. Values from pseudoparasitized (gray 

boxes) and unparasitized larvae (white boxes) within a day marked with an asterisk are 

significantly different (PB0.01; t-test). 

implantation was noticed. This prolongation was apparently due to the trauma of 

the treatment rather than energetic cost of encapsulation. Parasitoids were killed 

before they could have caused any alterations of host development due to 

interference with the host endocrine system (Schafellner, Marktl, Nussbaumer, and 

Schopf 2004). Also, teratocytes are unable to escape the immune response in L. 

dispar; when injected into unparasitized larvae they are totally cleared from the 

hemolymph (Schafellner et al. 2007). 

When G. liparidis were implanted into pseudoparasitized hosts without teratocytes, 

the parasitoid larvae were able to continue development. PDV/venom 

prevented the fatal immune response. Previous work had already shown that 

pseudoparasitized L. dispar larvae show reduced encapsulation of implanted 

artificial objects and reduced hemolymph melanization (Tillinger et al. 2004). 

However, as is suggested by our findings from the dissections, G. liparidis larvae 

implanted without teratocytes still suffered some attack from the host immune 

system and were apparently unable to complete their development. Pseudoparasitization 

alone did not permanently reduce hemolymph melanization. This is 

demonstrated by substantial melanin deposition on G. liparidis larvae implanted 

without teratocytes. Also C. kariyai implanted into hosts without teratocytes showed 

melanized surfaces 4 days post-implantation (Nakamatsu et al. 2002). Teratocytes of 

other parasitoid species were shown to exert immune suppressive functions such as 

reduction of host phenoloxidase activity (Bell et al. 2004) or of cellular immune 

response (Andrew, Basio, and Kim 2006). Moreover, it was interesting to observe 

that implanted G. liparidis larvae were always closely attached to the fat body 

pieces of which would remain attached to the larvae upon dissection while they are


normally floating freely in the hemolymph. This is in agreement with the finding that 

larvae of the braconid endoparasitoid Cotesia kariyai consume the fat body of their 

hosts with the help of teratocytes (Nakamatsu et al. 2002). We also noticed that the 

fat body of L. dispar normally parasitized by G. liparidis was always clearly reduced 

in volume compared to unparasitized larvae (personal observations). Generally, 

important involvement of teratocytes in nutrition of parasitoids is known from other 

systems (Dahlman 1990; Beckage and Gelman 2004). These cells can synthesize and 

release a fatty acid binding protein believed to aid fatty acid nutrition of the 

parasitoid (Falabella et al. 2005), proteins that inhibit protein synthesis in the host 

(Rana, Dahlman, and Webb 2002), or interfere with host development (Dahlman 

1990; Bell et al. 2004). The nutritional deficiency and lack of a hormonal cue may 

have prevented emergence of the developed parasitoid larvae in our case. 

Only the complete re-assemblage of all components, i.e. G. liparidis larvae, 

teratocytes, and PDV/venom allowed successful development in our study. The 

successful implantation of G. liparidis demonstrates that administration of PDV/ 

venom by pseudoparasitization functions as during the act of regular parasitization; 

it conditions the host such that implanted parasitoids can develop successfully. 

Teratocytes were shown to be necessary to allow successful completion of 

endoparasitic development and emergence from the host. 

Pseudoparasitized insects showed significantly elevated levels of trehalose in the 

hemolymph and glycogen in the tissue. This demonstrates that PDV/venom assists 

the developing parasitoid by altering the nutritional situation in the host, which 

seems to be an important feature in addition to the suppression of the immune 

system. These nutritional alterations are in accordance with previous findings from 

normally parasitized L. dispar. There, trehalose titers are elevated in the early stage 

of parasitism but are significantly reduced towards the end of the endoparasitic 

phase. In contrast, glycogen content is maintained at the same levels as in 

unparasitized larvae (Schopf and Nussbaumer 1996; Hoch et al. 2002). On the 

other hand, fatty acid levels in the hemolymph as well as total lipids of normally 

parasitized L. dispar are significantly reduced (Bischof and Ortel 1996; Hoch et al. 

2002). This indicates some action of the parasitoid to redirect the host’s energy 

metabolism for the parasitoid’s advantage. Alterations of the host’s metabolism 

towards increased trehalose titers in the hemolymph have been shown in several host 

parasitoid systems and were interpreted as beneficial for the hemolymph feeding 

parasitoids (Thompson, Lee, and Beckage 1990; Vinson 1990; Thompson and 

Dahlman 1999). We were able to demonstrate that PDV/venom of G. liparidis are 

involved in such alterations of host metabolism. Thereby, PDV/venom could assist 

the developing parasitoids by having higher levels of nutrients easily available in the 

hemolymph. The finding that treatment of Pseudaletia separata larvae with C. 

kariyai PDV/venom led to increased approximate digestibility but decreased 

efficiency of conversion of digested food by the host larva (Nakamatsu, Gyotoku, 

and Tanaka 2001) further supports this assumption of an important supportive 

function of parasitoid associated factors in parasitoid nutrition. 

Overall, our study demonstrates the use of g-radiation-induced pseudoparasitization 

as a tool to investigate interactions between host insects and their parasitoids. 

PDV and venom administered to L. dispar host larvae by pseudoparasitization 

suppressed the host immune response and altered its metabolism such that implanted 

G. liparidis larvae can develop successfully. This tool will be highly useful in future


studies on the delicate physiological interaction between this parasitoid and its host. 

We were able to use a g-radiation source at the FAO/IAEA laboratories for our 

study, where it is used for research and development in sterile insect technique (SIT) 

programs. Sterilization of insects is normally done with either cobalt-60 and 

caesium-137 irradiators. Both must meet the very high standards for nuclear safety 

and must be licensed by national atomic energy authorities (Bakri et al. 2005). 

Moreover, transportation of nuclear material is becoming increasingly problematic. 

Therefore, alternatives such as electron beams or X-rays are being explored also for 

SIT programs (Robinson and Hendrichs 2005; Mehta, in press). Such X-ray sources 

that can be used for insect sterilization as well as pseudoparasitization may 

eventually become more accessible to researchers than g-radiation sources. 


We are grateful to Dr Alan Robinson of the FAO/IAEA Agriculture and Biotechnology 

Laboratory, Seibersdorf, Austria, for irradiation of the parasitoid wasps. We thank Dr D.L. 

Dahlman, University of Kentucky, Lexington for introducing R.C.M. to in vitro techniques 

with teratocytes. Gypsy moth eggs were kindly provided by USDA/APHIS Otis Methods 

Development Center at Cape Cod, MA, USA. We thank Ms Andrea Stradner and Mr Petr 

Zabransky for their technical assistance. Funding was provided from grant P13603-BIO by the 

Austrian Science Fund (FWF) to A.S. This work is part of the FAO/IAEA coordinated 

research project CRP D4.30.02 (project coordinator: Dr Jorge Hendrichs). 

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Vol. 19, S1, 2009, 35 42 

Treatment of Lymantria dispar (Lepidoptera, Lymantriidae) host larvae 

with polydnavirus/venom of a braconid parasitoid increases spore 

production of entomopathogenic microsporidia 

Gernot Hoch a *, Leellen F. Solter b , and Axel Schopf a 

a Department of Forest and Soil Sciences, BOKU University of Natural Resources and Applied 

Life Sciences, Vienna, Austria; b Center for Economic Entomology, Illinois Natural History 

Survey, Champaign, IL, USA 

Female Glyptapanteles liparidis (Hym., Braconidae) were irradiated in a caesium- 

137 irradiator; these wasps oviposit nonviable eggs along with polydnavirus and 

venom into the host ( pseudoparasitization). When Lymantria dispar larvae were 

infected with microsporidian species for which they are permissive or semipermissive 

hosts, spore production was higher in pseudoparasitized than in 

unparasitized larvae. 

Keywords: microsporidia; Nosema portugal; Vairimorpha necatrix; host suitability; 

polydnavirus; immune suppression 

Introduction 

When Lymantria dispar larvae are parasitized by the endoparasitoid Glyptapanteles 

liparidis (Hym., Braconidae) and infected with the microsporidium Vairimorpha 

disparis they die more quickly than unparasitized, infected hosts. Moreover, 

reproduction of the microsporidium, measured in numbers of mature spores, is 

higher in the parasitized hosts (Hoch, Schopf, and Maddox 2000). In a follow-up 

study we showed that treatment of the hosts with the parasitoid’s polydnavirus 

(PDV) and venom also induced higher spore yield of V. disparis (Hoch and Schopf 

2001). We hypothesized that a reduced immune response in PDV/venom treated 

hosts could be responsible for this effect. Like other braconids and ichneumonids, G. 

liparidis possesses a symbiotic PDV that occurs in the calyx region of its ovary (Krell 

1991). PDV is injected together with venom into the host hemocoel during 

oviposition. In several other host-parasitoid systems, these substances were shown 

to play a crucial role in preventing encapsulation of the parasitoid egg by the host 

immune system (e.g. Edson, Vinson, Stoltz, and Summers 1981; Lavine and Beckage 

1995; Strand and Pech 1995; Shelby and Webb 1999) and to manipulate host 

development by interfering with hormonal metabolism (reviewed in Beckage 1997). 

Glyptapanteles liparidis PDV, e.g. is responsible for suppressed juvenile hormone 

esterase activity in parasitized L. dispar larvae (Schafellner, Marktl, and Schopf 

2007). To study the effects of PDV and venom of G. liparidis we modified a method 

described by Soller and Lanzrein (1996) and used gamma radiation to sterilize eggs 

inside the ovaries of wasps; these wasps then insert nonviable eggs into the host 

*Corresponding author. Email: gernot.hoch@boku.ac.at 




DOI: 10.1080/09583150802364868 



hemocoel together with active PDV and venom ( pseudoparasitization) (Tillinger, 

Hoch, and Schopf 2004). Compared to injection of purified PDV and venom, 

pseudoparasitization has the advantage that a physiological dose is administered 

during oviposition and that the host is not affected by the application procedure 

itself. Pseudoparasitization by irradiated G. liparidis altered host development and 

interfered with larval-pupal molt as well as metamorphosis. Moreover, it suppressed 

the immune response of L. dispar larvae; encapsulation of implanted artificial 

objects was reduced and hemolymph melanization occurred at lower level (Tillinger 

et al. 2004). 

The responses of the insect immune system to microsporidian infections are not 

well known. A typical microsporidian life cycle in a lepidopteran host larva begins 

with the invasion of the midgut tissues after oral ingestion of infective spores. The 

pathogen then spreads to target tissues, e.g. other midgut cells, fat body or silk 

glands, where a secondary developmental cycle leads to production of persistent 

environmental spores (Maddox et al. 1999). In the laboratory, L. dispar is a semipermissive 

host for a variety of entomopathogenic microsporidia isolated from other 

lepidopteran hosts. These infections, however, are often atypical and result in very 

low spore production (Solter and Maddox 1998). Hemocytic nodulation was 

regularly observed in one microsporidian species for which L. dispar was barely 

permissive. The most intense melanization of hemolymph, however, occurred as a 

consequence of heavy infections by microsporidia for which L. dispar is permissive 

and was, therefore not interpreted as sign of successful immune response (Hoch, 

Solter, and Schopf 2004). 

In the present study, we used pseudoparasitization by irradiated G. liparidis to 

test whether polydnavirus/venom-induced changes in host physiology enhance 

reproduction of microsporidia for which L. dispar larvae are permissive or semipermissive 

hosts. Therefore, we measured microsporidian spore load in infected 

hosts. In addition, a qualitative evaluation was done by observing the progress and 

quality of infection under the light microscope. 


Lymantria dispar egg masses were provided by the USDA/APHIS Otis Method 

Development Center, Cape Cod, MA. Larvae were reared on high wheat germ diet 

(Bell, Owens, Shapiro, and Tardiff 1981) at 24918C, 55 60% relative humidity, and 

16 h L:8 h D photoperiod. G. liparidis were obtained from the laboratory colony at 

BOKU University, Vienna. Adult wasps were reared on water and honey and L. 

dispar larvae were used as hosts. 

All microsporidian isolates were available from the microsporidia germplasm 

collection at the Illinois Natural History Survey and were used in previous studies. 

We used the following microsporidia isolated from L. dispar: (1) Nosema portugal 

and (2) Nosema sp., hereafter referred to as N. sp. [Schweinfurth]. These species were 

propagated in L. dispar larvae; spores were harvested and processed as described in 

Hoch et al. (2004). The following microsporidia, originally isolated from other 

lepidopteran hosts were also used in the experiments: (1) Vairimorpha necatrix, 

originally isolated from Pseudaletia unipuncta; (2) Vairimorpha sp. from Hyphantria 

cunea, hereafter referred to as V. sp. [H. cunea]; (3) Nosema sp. from Malacosoma 

americanum, hereafter referred to as N. sp. [M. americanum]. Details on these species


as well as production of inoculum are given in Hoch et al. (2004). All microsporidia 

were stored in liquid nitrogen (Maddox and Solter 1996) until used in the 

experiments, but no longer than 2 months. 

To achieve pseudoparasitization, wasps were temporarily sterilized by irradiation 

with 50 Gy at a dose rate of 7.6 Gy/min in the caesium-137 irradiator at the 

Department of Molecular and Integrative Physiology, University of Illinois. All 

female wasps were approximately 1-week-old, mated, and had previous oviposition 

experience. They were put in ventilated 30-mL plastic cups for irradiation. The day 

after irradiation, the wasps were allowed to sting up to four L. dispar larvae in 

premolt to third instar as described in Tillinger et al. (2004). 

On the second day in the third instar, larvae were inoculated with microsporidia 

at dosages of 1 10 4 spores per larva (except N. sp. [M. americanum], for which the 

dosage was 1 10 5 ). Exact dosages were administered via 4-mm 3 diet cubes that were 

contaminated with 1 mL of spore suspension (Hoch et al. 2000, 2004). The larvae 

were then reared in groups of three to four in 30-mL plastic cups. Both, L. dispar 

larvae pseudoparasitized with G. liparidis and unparasitized larvae were infected with 

each microsporidian species from the same batch out of liquid nitrogen storage. 

Spore production in infected L. dispar was quantified following the method 

described in Hoch et al. (2000). Larvae were weighed and frozen 18 dpi unless 

otherwise indicated. Dead larvae were decapitated and homogenized in water in a 

tissue grinder. The homogenate was cleaned by filtration through cheese cloth and 

centrifugation in tap water. The spore pellet was re-suspended in water by filling vials 

to a total volume of 20 mL. Spores were counted in a Neubauer hemacytometer; four 

counts were carried out per sample. Infections with N. sp. [Schweinfurth], which 

reproduces mainly in the silk glands of L. dispar larvae, were additionally assessed by 

measuring the percentage of infested silk gland area on fresh preparations. The silk 

glands were dissected out of unparasitized and pseudoparasitized larvae 12 and 16 

dpi (10 larvae per day and treatment) and photographed under a dissecting 

microscope. After coloring infested areas that can easily be recognized by their 

opaque color (as opposed to the clear uninfested areas), the digital images were 

analyzed using Lucia 4.21 software (Laboratory Imaging, Ltd.). The percentage of 

infested area of the projection of the silk gland was calculated. Besides these 

quantitative methods, infection levels in unparasitized and pseudoparasitized larvae 

were compared qualitatively by examination of fresh preparations of infected tissues 

under phase contrast microscopy. 

Statistical analysis was done using SPSS 12.0.1 for Windows (SPSS, Inc.). Data 

were tested for normal distribution with Kolmogorov Smirnov Z-test. Means of 

normally distributed data were compared between unparasitized and pseudoparasitized 

larvae by independent samples t-test. Data lacking normal distribution were 

compared by Mann Whitney U-tests. Percent values were arcsin transformed before 

analysis. Spore production of N. portugal in unparasitized and pseudoparasitized 

hosts at different points in time was analyzed by two-way ANOVA using the GLM 

procedure of SPSS. The relationship between fresh mass and spore load of larvae 

was analyzed by computing Pearson’s correlation coefficient or Spearman’s r when 

data were not normally distributed. Relative frequencies were compared with x 2 cross 

table analysis.


Table 1. Number of microsporidian spores produced per host, fresh mass of the infected 

host, and correlation between number of spores and fresh mass in unparasitized (np) and 

pseudoparasitized (psp) L. dispar larvae measured 18 days post-infection. 

N. portugal 6. 9 10 8 93.5 10 7 

N. sp. 

[Schweinfurth] 

V. sp. 

[H. cunea] 

No of spores per host$ Fresh mass (mg)$ Correlation % 

np psp np psp np psp 

P 0.036 

4.5 10 7 95.6 10 6 

P 0.007 

1.3 10 8 92.2 10 7 

P 0.000 

V. necatrix 1.5 10 8 93.7 10 7 

P 0.045 

8.2 10 8 95.0 10 7 

7.7 10 7 99.9 10 6 

3.9 10 8 96.0 10 7 

2.6 10 8 95.2 10 7 

593931 

P 0.624 

328926 

P 0.001 

478939 

P 0.166 

337933 

P 0.293 

613928 0.466** 0.699** 

464928 0.627** 0.674** 

561944 0.154 0.356* 

383928 0.268 0.132 

$Means9SE, n 34 35. Probability values refer to independent samples t-test (except spores/host in V. 

necatrix. 

Mann Whitney U-test) comparing unparasitized and pseudoparasitized hosts within microsporidian 

species. 

%Pearson’s correlation coefficient (except V. necatrix: Spearman’s r); asterisks denote significant 

correlations at the 0.05 and 0.01 level, respectively. 

Results and discussion 

The total spore production after 18 days of infection was generally higher in 

pseudoparasitized L. dispar larvae than in unparasitized hosts (Table 1). We counted 

significantly more spores in pseudoparasitized larvae infected with the L. dispar 

microsporidia N. portugal and N. sp. [Schweinfurth] as well as with microsporidia 

from other Lepidoptera, V. necatrix and V. sp. [H. cunea], for which L. dispar has 

been shown to be a semi-permissive host. The spore loads of the two L. dispar 

microsporidia, N. portugal and N. sp. [Schweinfurth], in the host were positively 

correlated with the fresh mass of the larva, both with or without pseudoparasitization, 

indicating optimal utilization of the host tissue by the microsporidium. 

Nevertheless, pseudoparasitization led to a further increase in total spore production. 

For N. sp. [Schweinfurth] that reproduces mainly in the silk glands the higher 

spore load appears to be due to increased larval weight of pseudoparasitized hosts. 

There were no significant differences between pseudoparasitized and unparasitized 

larvae in spore load per mg fresh mass (t-test: P 0.287). It is known from previous 

studies that PDV/venom of G. liparidis alters host growth and development, resulting 

in bigger larvae and heavier pupae (Tillinger et al. 2004). Analysis of the 

photographs of infected silk glands revealed no significant difference in the 

percentage of infected area in unparasitized (12 dpi: 70.594.6%; 16 dpi: 79.49 

2.2%) and pseudoparasitized larvae (12 dpi: 72.993.5%; 16 dpi: 71.294.5%; t-test: 

P 0.740 and P 0.146, respectively). For N. portugal, which develops in the fat 

body as well as in the silk glands, the situation was different; pseudoparasitization 

also led to higher spore load per unit fresh weight. The increase in spore production 

was analyzed in more detail for N. portugal on days 10, 12, and 18 post-infection. A 

two-way ANOVA with date of count and parasitization as factors and fresh mass as


Figure 1. Number of Nosema portugal spores (logarithmic scale) produced in unparasitized 

and pseudoparasitized L. dispar larvae quantified 10, 12 and 18 days post-infection. Two-way 

ANOVA was performed with parasitization treatment and day of count as factors and fresh 

mass (FM) of the infected larva as covariate. 

covariate showed a significantly higher amount of spores produced in pseudoparasitized 

larvae together with a significant increase in spore load over time (Figure 1). 

Gypsy moth is a semi-permissive host for V. necatrix; infection of the fat body is 

light and variable, and infection of silk glands remains localized (Hoch et al. 2004). 

There was no correlation between spore load in the host and fresh mass (Table 1) 

indicating that the microsporidium was not able to utilize the semi-permissive host 

optimally. The lack of correlation was due to a high percentage of larvae that showed 

only low infestation levels; 60% of the unparasitized and 43% of pseudoparasitized 

larvae had a spore load of less than 5 10 7 spores. Spore counts at an earlier date (13 

dpi) did not reveal any significant difference (P 0.319) but pseudoparasitization led 

to a slight but significant increase in total spore load compared to unparasitized 

hosts 18 dpi (Table 1). The V. sp. [H. cunea] was likewise promoted by 

pseudoparasitization of the L. dispar larvae, which are also semi-permissive hosts 

for this species (Hoch et al. 2004); total spore load was significantly higher in 

pseudoparasitized larvae than in unparasitized L. dispar (Table 1). While unparasitized 

hosts showed no relationship between fresh mass and spore load there was a 

moderate but significant correlation in pseudoparasitized larvae infected with V. sp.


[H. cunea]. Apparently, the microsporidium was able to better utilize the tissue of the 

pseudoparasitized hosts. We could not quantify reproduction of N. sp. [M. 

americanum], the third microsporidium, for which L. dispar is only a semi-permissive 

host. Although it was fed to larvae at 10 times higher dosages than the other 

microsporidia, infectivity of this species was clearly below 100%. Moreover, only few 

and frequently atypically shaped spores were produced in L. dispar larvae. Of 

unparasitized larvae, 52.2% (n 46) were diagnosed positive when dissected between 

17 and 25 dpi. For pseudoparasitized larvae the percentage increased to 70.2% (n 

47). This difference was not statistically significant (x 2 

3.188, P 0.05). Based on 

our microscopic observations, the level of the infections did not differ in the two host 

types. We found very few spores and only in infected silk glands of both host types. 

Also, atypically shaped spores (Solter and Maddox 1998) occurred regularly in both 

unparasitized and pseudoparasitized hosts. Microscopy likewise revealed no 

qualitative differences of infections with V. necatrix and V. sp. [H. cunea] in the 

semi-permissive L. dispar larvae with or without pseudoparasitization. Progression 

of infection and infection of the respective tissues was not affected. In pseudoparasitized 

hosts even the strongest infections remained localized. Thus, the improvement 

in host quality through pseudoparasitization seems to occur mostly on a 

quantitative level; a host classified as semi-permissive did not become permissive 

after pseudoparasitization. This appears to be different from baculovirus infections 

in lepidopteran larvae. Refractory hosts, such as Helicoverpa zea and Manduca sexta 

react to a systemic infection with Autographa californica multienveloped nucleopolyhedrovirus 

by encapsulation of viral foci in the tracheae and thereby block a 

subsequent spread of the infection. In hosts that were immunosuppressed either by 

chemicals or parasitization by hymenopteran parasitoids, virus infection caused 

higher mortality and was more widespread than in infected, immune competent 

hosts (Washburn, Kirkpatrick, and Volkman 1996; Washburn, Haas-Stapleton, Tan, 

Beckage, and Volkman 2000). Infection with microsporidia following oral inoculation 

differs also from injection of microorganisms into the hemocoel of larvae, in 

which case even normally non-pathogenic yeasts were shown to cause lethal 

infections in PDV-treated insects because the hemolymph was not cleared of 

infection (Stoltz and Guzo 1986). 

Pseudoparasitization of L. dispar suppressed hemolymph melanization after 

infection with the virulent microsporidium N. portugal. Melanization levels were 

significantly lower in pseudoparasitized, infected larvae than in unparasitized, 

infected larvae 7 dpi; levels further decreased to levels in unparasitized, uninfected 

controls 9 dpi (unpublished data). But overall, our findings suggest that it is not the 

level of host defense reaction alone that determines host suitability for microsporidia. 

The gradual increase in suitability of semi-permissive hosts and of permissive 

hosts may also be caused by better nutritional quality of pseudoparasitized larvae. 

Microsporidia are obligate intracellular parasites and are thus completely dependent 

on resources provided by the host cell. We showed that infection of L. dispar larvae 

by V. disparis leads to total depletion of carbohydrates from the host as well as 

significantly reduced levels of lipids (Hoch, Schafellner, Henn, and Schopf 2002). A 

redirection of the host’s metabolism for the parasitoid’s advantage by its PDV/venom 

could, likewise, be beneficial for a developing microsporidian infection. Such 

alterations of the host’s metabolism by several parasitic wasps have been demonstrated 

(e.g. reviewed in Nakamatsu, Gyotoku, and Tanaka 2001), and we showed


that PDV/venom of G. liparidis leads to increased trehalose titers in the hemolymph 

and increased glycogen levels in the tissue of L. dispar larvae (Hoch et al., 

submitted). Hence, such changes in host metabolism may well be profitable for a 

developing microsporidium by supplying higher amounts of nutrients or energy for 

the parasite. 


We are grateful to Dr H.S. Ducoff, Department of Molecular and Integrative Physiology, 

University of Illinois, for irradiation of G. liparidis and dosimetry. Mr J. Tanner, USDA/ 

APHIS Otis Method Development Center, kindly provided the L. dispar used in this study. 

This research was funded by the Austrian Science Fund FWF (Erwin Schrödinger Fellowship 

J2027 to G.H.), the Illinois Natural History Survey, and the Office of Research/Illinois 

Agricultural Experiment Station Project No. 65-344_S301. The work is part of the FAO/IAEA 

coordinated research project CRP D4.30.02 (project coordinator: Dr Jorge Hendrichs). 

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Hormones and Behavior, ed. N.E. Beckage, New York: Chapman and Hall, pp. 3 36. 

Bell, R.A., Owens, C.D., Shapiro, M., and Tardiff, J.R. (1981), ‘Mass Rearing and Virus 

Production’, inThe Gypsy Moth: Research toward Integrated Pest Management, eds. C.C. 

Doane and M.L. McManus, Washington, DC: USDA (US Dept. Agric. For. Serv. Tech. 

Bull. 1584), pp. 599 600. 

Edson, K.M., Vinson, S.B., Stoltz, D.B., and Summers, M.D. (1981), ‘Virus in a Parasitoid 

Wasp: Suppression of the Cellular Immune Response in the Parasitoid’s Host’, Science, 211, 

582 583. 

Hoch, G., and Schopf, A. (2001), ‘Effects of Glyptapanteles liparidis (Hym.: Braconidae) 

Parasitism, Polydnavirus, and Venom on Development of Microsporidia Infected and 

Uninfected Lymantria dispar (Lep.: Lymantriidae) Larvae’, Journal of Invertebrate 

Pathology, 77, 37 43. 

Hoch, G., Schopf, A., and Maddox, J.V. (2000), ‘Interactions between an Entomopathogenic 

Microsporidium and the Endoparasitoid Glyptapanteles liparidis within their Host, the 

Gypsy Moth Larva’, Journal of Invertebrate Pathology, 75, 59 68. 

Hoch, G., Schafellner, C., Henn, M.W., and Schopf, A. (2002), ‘Alterations in Carbohydrate 

and Fatty Acid Levels of Lymantria dispar Larvae Caused by a Microsporidian Infection 

and Potential Adverse Effects on a Co-Occurring Endoparasitoid, Glyptapanteles liparidis’, 

Archives of Insect Biochemistry and Physiology, 50, 109 120. 

Hoch, G., Solter, L.F., and Schopf, A. (2004), ‘Hemolymph Melanization and Alterations in 

Hemocyte Numbers in Lymantria dispar Larvae Following Infections with Different 

Entomopathogenic Microsporidia’, Entomologia Experimentalis et Applicata, 113, 77 86. 

Krell, P.J. (1991), ‘Polydnaviridae’, inAtlas of Invertebrate Viruses , eds. J.R. Adams and J.R. 

Bonami, Boca Raton, FL: CRC Press, pp. 321 338. 

Lavine, M.D., and Beckage, N.E. (1995), ‘Polydnaviruses: Potent Mediators of Host Insect 

Immune Dysfunction’, Parasitology Today, 11, 368 378. 

Maddox, J.V., and Solter, L.F. (1996), ‘Long-Term Storage of Viable Microsporidian Spores in 

Liquid Nitrogen’, Journal of Eukaryotic Microbiology, 43, 221 225. 

Maddox, J.V., Baker, M.D., Jeffords, M.R., Kuras, M., Linde, A., Solter, L.F., McManus, 

M.L., Vavra, J., and Vossbrinck, C.R. (1999), ‘Nosema portugal, n.sp., Isolated from Gypsy 

Moths (Lymantria dispar L.) Collected in Portugal’, Journal of Invertebrate Pathology, 73, 

1 14. 

Nakamatsu, Y., Gyotoku, Y., and Tanaka, T. (2001), ‘The Endoparasitoid Cotesia kariyai (Ck) 

Regulates the Growth and Metabolic Efficiency of Pseudaletia separata Larvae by Venom 

and Ck Polydnavirus’, Journal of Insect Physiology, 47, 573 584.





868. 

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Immunity’, Journal of Insect Physiology, 45, 507 514. 

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Spodoptora littoralis (Noctuidae)’, Journal of Insect Physiology, 42, 471 481. 

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Indicator of Ecological Host Specificity’, Journal of Invertebrate Pathology, 71, 207 216. 

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Host Relationships’, Annual Review of Entomology, 40, 31 56. 




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Viruses’, Nature, 383, 767. 

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Vol. 19, S1, 2009, 43 48 

Use of gamma radiation for improving the mass production of 

Trichogramma chilonis and Chrysoperla carnea 

Muhammad Hamed*, Sajid Nadeem, and Asia Riaz 

Nuclear Institute for Agriculture & Biology (NIAB), Faisalabad, Pakistan 

Gamma radiation studies were conducted to improve the existing mass rearing 

capabilities for an egg parasitoid, Trichogramma chilonis Ishii and a predator, 

Chrysoperla carnea Stephens as part of a program to achieve an area-wide control 

of cotton and sugarcane pests. The suitability of host (Sitotroga cerealella Olivier) 

eggs for parasitization by T. chilonis was restored and prolonged from 3 to 7 days 

pre-hatching with the application of gamma radiation in the range of 5 55 Gy. 

The findings indicated that all treatment doses were effective but varied 

significantly in parasitization of eggs. This effect was similar during the first 2 

days ranging from 78 to 94% parasitization, but it decreased drastically at lower 

doses (5 and 15 Gy) in succeeding days. High doses were better than low doses. 

For the highest treatment dose (55 Gy), successful parasitization declined only 

gradually, resulting in 45% parasitization even after 7 days. This finding would 

allow for reliable supply of viable host eggs to small insectaries in remote areas. 

Studies on C. carnea showed that feeding of irradiated prey eggs increased larval 

survival, fecundity and female sex ratio. Larval survival improved by 89% over 

the control when C. carnea was fed eggs irradiated with a dose of 45 Gy. The 

effects of radiation-treated prey eggs on survival and fecundity of C. carnea 

persisted in successive generations, but were considerably less in the F2 than in the 

F1 and P generations. At 45 Gy, fecundity was highest with 444 eggs/female in the 

parent, 397 in F1, and 311 in F2 generations, whereas it decreased significantly at 

lower doses and in untreated eggs. 

Keywords: gamma radiation; Trichogramma chilonis Ishii; Chrysoperla carnea 

Stephens; Sitotroga cerealella Olivier; viability; fecundity; sex ratio; generations 

Introduction 

The egg parasitoid, Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) 

and the predator, Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) are 

prevalent and widely used in Pakistan in biological control (Cheema, Muzaffar, and 

Ghani 1980; Mohyuddin et al. 1997). Rearing of these parasitoids and predators has 

been done mostly on eggs of the Angoumois grain moth, Sitotroga cerealella Olivier, 

because this host is easy and inexpensive to produce (Marston and Ertle 1973; Mark, 

Jenings, Welty, and Southard 1983). An essential aspect improving the economics of 

the mass production and area-wide release of beneficial insects is to use some means 

to extend the shelf-life of host eggs to ensure their continuous supply. 

It is known that suitability of host eggs for parasitization by Trichogramma species 

is decreased significantly with the increase in their age (Reznik and Umarova 1985; 

*Corresponding author. Email: hamrazapak@yahoo.com 




DOI: 10.1080/09583150802433846 


44 M. Hamed et al. 

Somchoudhury and Dutt 1989; Ruberson and Kring 1993; Takada, Kawamura, and 

Tanaka 2000). Therefore, a sufficient number of fresh eggs should be preserved to use 

as hosts for production of parasitoids. A variety of options may be used for egg 

preservation, including gamma radiation, cold storage, liquid nitrogen storage, etc. 

(Smith 1996). The early hatching of fresh host eggs during transportation to small 

insectaries at outstations highlighted the need for a critical evaluation of the effect of 

gamma radiation on the hatching of hosts and their acceptance and suitability for 

parasitization. The other objective of using radiation was to improve the production 

capacity of the predator Chrysoperla carnea under mass rearing conditions. 


The egg parasitoid T. chilonis, the predator C. carnea, and their factitious host S. 

cerealella were obtained from stock cultures maintained separately in small insectaries 

for running mass rearing laboratories. Laboratory conditions were set at 27918C, 

6595% RH and 14 L: 10 D h period. To determine the percent T. chilonis 

parasitization, freshly laid host eggs (ageB12 h) were glued on cards, each with 50 

eggs. Immediately thereafter, these eggs were given gamma radiation doses of 5, 15, 

25, 35, 45 or 55 Gy through a cobalt-60 gamma irradiator with a dose rate of 872 Gy/ 

h (Brower and Tilton 1973; Diop, Marchioni, Doudou, and Hasselman 1973). Three 

replicates were made for each dose according to methods by Zhengdong, Ping, Zulin, 

and Jianqing (1993) and Brower, Tilton, and Cogburn (1971). The host eggs of each 

dose were exposed, starting at the same day, to two male and two female newly 

emerged parasitoids in glass jars (11 7 cm) with honey streaks inside and covered on 

top with muslin cloth. This procedure was continued daily for all treatments up to 7 

days with 1 day older eggs each day. After 24-h exposure to T. chilonis, the parasitized 

eggs on the cards were taken out of jars and placed in Petri dishes for incubation. 

After complete development of parasitoids, the host eggs were examined under a 

binocular microscope to determine percent parasitization. 

For experiments on C. carnea, 1-day-old untreated or irradiated (5, 15, 25, 35 and 

45 Gy) S. cerealella host eggs were fed to the larvae in parent, F1 and F2 generations 

(0.1 g eggs/day per larva), each in a separate glass vial (7.7 1.3 cm) till pupation. 

Five replicates were made for each radiation treatment with 25 larvae per replicate. 

Observations were taken on percent survival of larvae, fecundity and sex ratio. Data 

were subjected to least significant difference, and one- and a three-way factorial 

analysis of variance (ANOVA) with Duncan’s multiple range tests. The associated 

statistics are presented in the legends of the appropriate figures. 

Results and discussion 

Trichogramma studies 

The percent parasitization by T. chilonis of gamma irradiated host eggs varied 

significantly (for day 1, F 8.0, P 0.0007; day 2, F 5.721, P 0.0034; day 3, 

F 119.311, P 0.00; day 4, F 35.161, P 0.00; day 5, F 41.740, P 0.00; day 

6, F 68.346, P 0.00; day 7, F 94.275, P 0.00 and df 6 for all treatments) 

among radiation doses and age at which host eggs were exposed for parasitization 

(Table 1). The untreated (control) eggs were most heavily parasitized on the first


Table 1. Comparison between means (9SE) for daily parasitization (%) by T. chilonis on 

un-irradiated (control) and irradiated host (S. cerealella) eggs of increasing age in successive 

24-hour periods up to the seventh day against radiation doses. 

Day of parasitization 

Radiation 

Doses (Gy) 1 2 3 4 5 6 7 

Control 94.790.66 a 86.791.76 ab 78.091.15 a 0.00 0.00 0.00 0.00 

5 92.091.15 a 84.790.66 b 33.391.76 d 30.093.05 c 28.091.15 d 25.390.66 d 16.091.15 d 

15 94.091.15 a 90.792.90 a 44.093.05 c 42.093.05 b 40.093.05 c 42.091.15 bc 18.091.15 d 

25 92.092.31 a 88.091.15 ab 80.091.15 a 60.092.31 a 52.091.15 b 45.391.76 b 34.791.76 c 

35 92.791.57 a 87.391.76 ab 70.091.15 b 65.392.90 a 50.791.76 b 40.092.00 c 40.091.76 b 

45 92.790.66 a 86.091.15 ab 69.391.33 b 60.091.15 a 54.091.15 ab 56.091.15 a 44.791.76 a 

55 82.092.00 b 78.091.76 c 76.091.15a 66.791.76 a 58.091.03 a 56.091.15 a 44.790.66 a 

Means in the same column sharing the same letter are statistically non-significant (df 6, PB0.05) 

according to Duncan’s multiple range test. 

day followed by the second and third day. Thereafter, as usual, they started to hatch 

on the fourth day. Irradiated eggs proved somewhat suitable as hosts through the 

seventh day after irradiation in a dose-dependent fashion, whereas none of the 

untreated eggs were suitable after day 3. 

This finding, that irradiation extends host eggs’ suitability for parasitization and 

that the viability of host eggs for parasitization gradually decreases over time, agrees 

partly with Brower (1982), who found that irradiated host eggs of the Indian meal 

moth, Plodia interpunctella (Hubner) were preferred by T. pretiosum over eggs from 

irradiated females. Our data completely agree with the findings of Zhengdong et al. 

(1993) that the preservation time of Antheraea pernyi (Guérin-Méneville) eggs for 

parasitization with Trichogramma species was remarkably extended as a result of 

irradiation. There is a negative correlation between the age of the eggs and their 

sensitivity to radiation treatment (Chand and Sehgal 1978). Seal and Tilton (1986) 

observed that radio-sensitivity of eggs of hide beetles decreased with increasing 

embryonic development. The reasons of the minimum 33 and 44% parasitism of eggs 

at 5 and 15 Gy in our results might be the incomplete arrest of the embryo 

development at day 3 and onward, whereas the increases in doses and the resulting 

decreases in embryo development encouraged parasitism. 

Chrysoperla studies 

The assessment of the effects of feeding the predator C. carnea on control or 

irradiated prey eggs in terms of larval survival (Figure 1) indicate that the percent 

survival to the adult stage of larvae fed treated eggs increased significantly (P 0.05) 

in parent, F1 and F2 generations (LSD 2.676). Even though the overall interactions 

between radiation doses and generation means were non-significant, with untreated 

eggs, the survival was only 56%, whereas feeding of irradiated eggs significantly 

enhanced larval survival. The subsequent effects of feeding on a diet of control or 

irradiated prey eggs on the fecundity of C. carnea for P, F 92293.53, P 0.00; for 

F1, F 43896.59, P 0.00; for F2, F 16041.68, P 0.00 and df 5 for all 

treatments varied significantly (PB0.05) among radiation doses and generations 

(Table 2). Fecundity increased in the parent generation with increased radiation


Figure 1. Effect of feeding on control or irradiated prey (S. cerealella) eggs on percent 

survival of C. carnea larvae in the parental and two successive generations. 

doses to prey eggs, whereas it decreased at 5 Gy. There was a successive decrease in 

fecundity in F1 and F2 than the parent generation. However, it was comparatively 

high in F 1 at 25 Gy and onward and in F 2 at 45 Gy. The resulting C. carnea male to 

female sex ratio (Figure 2) was non-significant in successive generations, whereas it 

Table 2. Comparison among means (9SE) for the effect of larval feeding on irradiated prey 

(S. cerealella) eggs on fecundity of C. carnea in successive generations against radiation 

treatments. 

Generations 

Radiation doses (Gy) Parent F1 F2 

Control 271.890.63 e 273.290.98 d 273.691.30 b 

5 216.090.43 f 192.590.54 f 209.290.66 d 

15 332.090.89 d 222.890.70 e 178.090.67 f 

25 400.290.43 b 336.991.33 c 196.890.93 e 

35 349.690.09 c 371.190.66 b 240.791.03 c 

45 443.690.81 a 396.690.84 a 311.290.49 a 

Means in the same column sharing similar letters are non-significant (df=5, PB0.05) according to 

Duncan’s multiple range test.

Male to female ratio 

5.0 

4.5 

4.0 

3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 


LSD value for: 

Generation means = Non-significant 

Radiation doses = 0.9797 

Interaction = Non-significant 

P F1 F2 

0 5 15 25 35 45 

Radiation doses 

Figure 2. Effect of larval feeding on irradiated prey (S. cerealella) eggs on sex ratio of C. 

carnea in successive generations. 

varied significantly (LSD 0.9797) among radiation doses. The overall mean values 

for male to female ratios were significantly higher with 3:1 at 5 Gy followed by 2:1 at 

15, 2:1 at 25 Gy and 1:1 with the control. At the highest doses (35 and 45 Gy), the sex 

ratio decreased even further in all generations. The overall mean values for male to 

female ratio for 35 and 45 Gy treated eggs were 0.9:1 and 0.3:1, respectively. In the 

present studies, radiation doses, fecundity and female to male ratio were directly 

correlated to each other. 

There are few studies on insects reared for several generations on irradiated diets 

and irradiated prey with respect to predator. Ye and Cheng (1986) reared Chrysopa 

sinica on UV-irradiated eggs of Corcyra cephalonica and reported that all stages of the 

predator developed normally for three successive generations. Brower et al. (1971) 

conducted research on the effects of irradiated diets on Indian meal moth on several 

generations and found statistically non-significant but biologically significant and 

increasing effects on progeny, fecundity and sex ratio in successive generations. In 

similar studies on the rearing of three stored product insects on irradiated wheat, 

fecundities were slightly but consistently higher than that of the control (Hodges and 

Guyer 1958). 

Conclusions 

Gamma radiation prolonged the viability of host eggs for T. chilonis parasitization up 

to 7 days. High doses proved better to prolong egg viability than low doses. Treatment 

of 55 Gy resulted in similar levels of parasitization as the control during the first 3 

days, and gave 45% parasitization on the seventh day. Supply of irradiated host eggs 

enabled us to run insectaries in remote areas with low rearing cost and fulfill the 

requirement of area-wide releases of T. chilonis. Feeding of irradiated prey eggs (45-Gy 

dose) to C. carnea increased significantly the percent larval survival (89%), fecundity


(444 eggs/female) and female to male ratio (1:0.5) in the parental generation. The 

effect on some of these parameters was less pronounced in the F2 generation. 

References 

Brower, J.H. (1982), ‘Parasitization of Irradiated Eggs and Eggs from Irradiated Adults of the 

Indian Meal Moth (Lepidoptera: Pyralidae) by Trichogramma pretiosum (Hymenoptera: 

Trichogrammatidae)’, Journal of Economic Entomology, 75, 939 944. 

Brower, J.H., and Tilton, E.W. (1973), ‘Comparative Gamma Radiation Sensitivity of 

Tribolium madens (Charpentier) and T. castaneum (Herbst)’, Journal of Stored Product 

Research, 9,93 100. 

Brower, J.H., Tilton, E.W., and Cogburn, R.R. (1971), ‘Effects of Irradiated Diets on 

Production of Progeny by Successive Generations of the Indian Meal Moth, Plodia 

interpunctella (Hubner)’, Radiation Research, 48, 283 290. 

Chand, A.T., and Sehgal, S.S. (1978), ‘Sensitivity of Gamma Radiation of Corcyra cephalonica 

Eggs Related to Their Age’, Entomon, 3,7 9. 

Cheema, M. A., Muzaffar, N., and Ghani, M.A. (1980), ‘Investigation on Phenology, 

Distribution, Host Range and Evaluation of Predators of Pectinophora gossypiella 

(Sunders) in Pakistan’, The Pakistan Cotton, 24, 140 176. 

Diop, Y.M., Marchioni, E., Doudou, B.A., and Hasselman, C. (1973), ‘Radiation Disinfestation 

of Cowpea Seeds Contaminated by Callosobruchus maculates’, Journal of Food 

Processing and Preservation, 21, 69 81. 

Hodges, R., and Guyer, G. (1958), ‘The Effects of an Irradiated Wheat Diet on the Confused 

Flour Beetle, Granary Weevil, and the Angoumois Grain Moth’, Journal of Economic 


Mark, W.H., Jenings, D.T., Welty, E., and Southard, S.G. (1983), ‘Progeny Production of 

Trichogramma minutum, Utilizing Eggs of Choristoneura fumiferana and Sitotroga 

cerealella’, Canadian Entomologist, 115, 1245 1252. 

Marston, N., and Ertle, L.R. (1973), ‘Host Influences on the Bionomics of Trichogramma 

minutum’, Annals of the Entomological Society of America, 66, 1155 1162. 

Mohyuddin, A.I., Gilani, G., Khan, A.G., Hamza, A., Ahmad, I., and Mahmood, Z. (1997), 

‘Integrated Pest Management of Major Cotton Pests by Conservation, Redistribution and 

Augmentation of Natural Enemies’, Pakistan Journal of Zoology, 29, 293 298. 

Reznik, S. Ya., and Umarova, T. Ya. (1985), ‘The Reaction of Females of Trichogramma 

cacoeciae (Hymenoptera, Trichogrammatidae) to the Duration of Development of the Eggs 

of the Host’, Zoologicheskii Zhurnal, 64, 709 714. 

Ruberson, J.R., and Kring, T.J. (1993), ‘Parasitism of Developing Eggs by Trichogramma 

pretiosum (Hymenoptera: Trichogrammatidae): Host Age Preference and Suitability’, 

Biological Control, 3,3946. 

Seal, D.R., and Tilton, E.W. (1986), ‘Effect of Gamma Radiation on the Metamorphic Stages 

of Dermestes maculates DeGeer (Coleoptera: Dermestidae)’, International Journal of 

Radiation and Applied Instruments [A], 37, 531 535. 

Smith, S.M. (1996), ‘Biological Control with Trichogramma: Advances, Successes, and 

Potential of Their Use’, Annual Review of Entomology, 41, 375 406. 

Somchoudhury, A.K., and Dutt, N. (1989), ‘Influence of Hosts and Hostages on the 

Bionomics of Trichogramma perkinsi Girault and Trichogramma australicum Girault’, 

Indian Journal of Entomology, 50, 374 379. 

Takada, Y., Kawamura, S., and Tanaka, T. (2000), ‘Biological characteristics: Growth and 

Development of the Egg Parasitoid Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae) 

on the Cabbage Armyworm Mamestra brassicae (Lepidoptera: Noctuidae)’, 

Applied Entomolohy and Zoology, 35, 369 379. 

Ye, Z.C., and Cheng, D.F. (1986), ‘Rearing the Green Lacewing, Chrysopa sinica on 

Ultraviolet-Irradiated Eggs of the Rice Moth’, Chinese Journal of Biological Control, 2, 

133 134. 

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650.


Vol. 19, S1, 2009, 49 79 

Colonization and domestication of seven species of native 

New World hymenopterous larval-prepupal and pupal fruit fly 

(Diptera: Tephritidae) parasitoids 

Martín Aluja a *, John Sivinski b , Sergio Ovruski c , Larissa Guillén a , 

Maurilio López a , Jorge Cancino d , Armando Torres-Anaya a , 

Guadalupe Gallegos-Chan a and Lía Ruíz d 

a Instituto de Ecología, A.C., Xalapa, Veracruz, México; b Center for Medical, Agricultural 

and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA; c Planta Piloto de Procesos 

Industriales Microbiológicos y Biotecnología (PROIMI), División Control Biológico de Plagas, 

San Miguel de Tucumán, Argentina; d Subdirección de Desarrollo de Métodos, Campaña 

Nacional Contra Moscas de la Fruta, Tapachula, Chiapas, México 

We describe the techniques used to colonize and domesticate seven native New 

World species of hymenopterous parasitoids that attack flies within the genus 

Anastrepha (Diptera: Tephritidae). All parasitoid species successfully developed 

on artificially reared Mexican fruit fly, Anastrepha ludens (Loew) larvae or pupae. 

The parasitoid species colonized were the following: Doryctobracon areolatus 

(Szépligeti), Doryctobracon crawfordi (Viereck), Opius hirtus (Fischer), Utetes 

anastrephae (Viereck) (all Braconidae, Opiinae), Aganaspis pelleranoi (Bréthes) 

and Odontosema anastrephae Borgmeier (both Figitidae, Eucoilinae) (all larvalpupal 

parasitoids), and the pupal parasitoid Coptera haywardi (Ogloblin) 

(Diapriidae, Diapriinae). We provide detailed descriptions of the different rearing 

techniques used throughout the domestication process to help researchers 

elsewhere to colonize local parasitoids. We also describe handling procedures 

such as number of hosts in parasitization units and compare optimal host and 

female age, differences in parasitism rate, developmental time, life expectancy and 

variation in sex ratios in each parasitoid species over various generations. In the 

case of D. crawfordi and C. haywardi we also provide partial information on massrearing 

techniques such as cage type, parasitization unit, larval irradiation dose 

and adult handling. 

Keywords: hymenoptera; Braconidae; Figitidae; Diapriidae; Tephritidae; 

Anastrepha; biological control; parasitoids; rearing 

Introduction 

Historically the release of exotic (i.e. non-native) parasitoid species to deal with fruit 

fly pests has been the norm (Wharton 1989; Aluja 1994; Purcell 1998; Ovruski, 

Aluja, Sivinski, and Wharton 2000). In comparison, native parasitoids of indigenous 

pestiferous species have received little attention except for systematic studies and 

surveys of parasitoids of flies in the economically important genera Anastrepha (e.g. 

Wharton, Gilstrap, Rhode, Fischel, and Hart 1981; Aluja et al. 1990, 2003; Katiyar, 

Camacho, Geraud, and Matheus 1995; López, Aluja, and Sivinski 1999; Canal and 

*Corresponding author. Email: martin.aluja@inecol.edu.mx 




DOI: 10.1080/09583150802377373 


50 M. Aluja et al. 

Zucchi 2000; Ovruski, Schliserman, and Aluja 2004), Bactrocera (e.g. Wharton and 

Gilstrap 1983) and Rhagoletis (e.g. Wharton and Marsh 1978; AliNiazee 1985; 

Hoffmeister 1990; Gut and Brunner 1994; Feder 1995). The unstated perception has 

perhaps been that the long-standing co-existence of native parasitoids with flies that 

have remained pests was evidence that they were unable to exert economically 

significant levels of control. However, recent interest in the augmentative release of 

parasitoids (e.g. Sivinski et al. 1996; Purcell 1998; Montoya et al. 2000), with the 

possibility of strategically increasing the mortality inflicted by native species 

(Sivinski, Aluja, and López 1997; López et al. 1999; Sivinski, Piñero, and Aluja 

2000), has given new impetus to studies of their colonization and mass rearing. 

Around 205 species of the Neotropical genus Anastrepha have been described to 

date (Norrbom 2004). In Mexico, 37 species have been reported to date (Hernández- 

Ortíz and Aluja 1993; Hernández-Ortíz 1998, 2004; Hernández-Ortíz, Manrique- 

Sade, Delfín-González, and Novelo-Rincón 2002) and the larvae and/or pupae of 

these species are hosts for a diversity of parasitoids (Aluja et al. 1990, 2003; 

Hernández-Ortíz et al. 1994; López et al. 1999). Species such as Diachasmimorpha 

longicaudata (Ashmead), Psyttalia incisi (Silvestri), P. concolor (Szépligeti), Fopius 

arisanus (Sonan), F. vandenboschi (Fullaway), Aceratoneuromyia indica (Silvestri) and 

Pachycrepoideus vindemiae (Rondani) were introduced into Mexico as biological 

control agents, beginning in 1954 in an attempt to curb populations of the Mexican 

fruit fly, Anastrepha ludens (Loew) (Jiménez-Jiménez 1961; Wharton 1989; Ovruski 

et al. 2000). With similar intentions, non-native parasitoids were released in El 

Salvador, Nicaragua, Costa Rica, Panamá, Colombia, Perú, Brazil and Argentina 

(Wharton, Gilstrap, Rhode, Fischel, and Hart 1981; Ovruski et al. 2000). However, 

despite the large numbers of individuals introduced, few parasitoid species have 

successfully established (Ovruski et al. 2000). The shortcomings of this handful of 

exotic species has turned attention to the many native parasitoid candidates for 

augmentative release. Their diversity suggests that suitable species would be available 

for programs faced with an assortment of pests occurring in a variety of 

environments (Sivinski et al. 1997; Aluja, López, and Sivinski 1998; Sivinski and 

Aliya 2003). 

To facilitate native parasitoid colonization efforts in other parts of the world, we 

describe the colonization and domestication of the following seven native Anastrepha 

parasitoids found in Mexico and various other countries in Latin America (in some 

cases the US) (updates on exact distribution can be found in Ovruski et al. 2000; 

Ovruski, Wharton, Schliserman, and Aluja 2005): Doryctobracon areolatus (Szépligeti), 

Doryctobracon crawfordi (Viereck), Opius hirtus (Fischer), Utetes anastrephae 

(Viereck) (all Braconidae, Opiinae), Aganaspis pelleranoi (Brèthes) and 

Odontosema anastrephae Borgmeier (both Figitidae, Eucoilinae) (all larval-prepupal 

parasitoids), and the pupal parasitoid Coptera haywardi (Ogloblin) (Diapriidae, 

Diapriinae). Recent findings on the biology, ecology, and behavior of the latter 

parasitoid species have been reported by Sivinski (1991), Sivinski et al. (1996, 1997, 

2000), Sivinski, Aluja, Holler, and Eitam (1998a), Sivinski, Vulinec, Menezes, and 

Aluja (1998b), Sivinski, Aluja, and Holler (1999), Sivinski, Vulinec, and Aluja 

(2001), Aluja et al. (1998), Aluja et al. (2003), López et al. (1999), Guillén, Aluja, 

Equihua, and Sivinski (2002), Eitam, Holler, Sivinski, and Aluja (2003), Eitam, 

Sivinski, Holler, and Aluja (2004), Ovruski and Aluja (2002), Ovruski et al. (2004), 

Ovruski et al. (2005) and Guimarães and Zucchi (2004).


The most common and widely distributed Anastrepha native parasitoid species in 

the Neotropics and subtropics is D. areolatus (Ovruski et al. 2000). It is a larvalprepupal 

braconid parasitoid and broadly distributed from Mexico to Argentina 

(Wharton and Marsh 1978). When introduced into Florida in 1969, it became one of 

the most common parasitoids of A. suspensa (Loew) (Sivinski et al. 1998a; Eitam et 

al. 2004). In Mexico, D. areolatus and U. anastrephae are among the most numerous 

native species parasitizing larvae of A. obliqua (Macquart), a fruit fly that is an 

economically important pest of mango (Mangifera indica L.) and tropical plum 

(Spondias purpurea L.) (Aluja et al. 1996). Utetes anastrephae is also a larval-prepupal 

braconid parasitoid, but in comparison to D. areolatus, has the shortest ovipositor of 

any of the braconids sampled (Sivinski et al. 2001; Sivinski and Aluja 2003). This 

parasitoid species occurs naturally from Florida to Argentina (Ovruski et al. 2000). 

Doryctobracon crawfordi is a larval-prepupal opiine parasitoid commonly 

associated with A. ludens (Plummer and McPhail 1941; López et al. 1999). Reported 

for the first time by L. de la Barrera (see Herrera 1905), this species apparently 

prefers more temperate climates (Aluja et al. 1998) and higher altitudes (Sivinski et 

al. 2000). Opius hirtus is another larval-prepupal parasitoid that commonly attacks 

the relatively rare A. cordata Aldrich in Tabernaemontana alba Mill. (Apocynaceae) 

(Hernández-Ortíz et al. 1994). It has also been reported attacking A. obliqua in 

Tapirira mexicana Marchand and Spondias mombin L (both Anacardiaceae) 

(Hernández-Ortíz et al. 1994; Sivinski et al. 2000), A. alveata Stone in Ximenia 

americana L. (Olacaceae) (López et al. 1999), Toxotrypana curvicauda Gerstaecker 

and Ceratitis capitata (Wiedemann) (Wharton 1983). Aganaspis pelleranoi and O. 

anastrephae are two figitid larval-prepupal parasitoids that gain access to A. striata 

Schiner and A. fraterculus (Wiedemann) in guavas through wounds or holes in the 

fruit (Ovruski 1994; Sivinski et al. 1997; Ovruski et al. 2004). A. pelleranoi is more 

widely distributed and has a broader host range than O. anastrephae (Wharton, 

Ovruski, and Gilstrap 1998). 

One native, pupal endoparasitoid that has potential for fruit fly biological control 

is C. haywardi (Baeza-Larios, Sivinski, Holler, and Aluja 2002a; Guillén et al. 2002). 

It was originally discovered in Argentina attacking A. fraterculus and A. schultzi 

Blanchard pupae (Loiácono 1981). In 1994, C. haywardi was found in Veracruz, 

Mexico, attacking A. ludens pupae (López et al. 1999). More recently, this diapriine 

species was recovered from A. striata and A. serpentina (Wiedemann) pupae in 

Venezuela (García and Montilla 2001) and from A. fraterculus and A. sororcula 

Zucchi pupae in Brazil (Aguiar-Menezes, Menezes, and Loiácono 2003). Unlike 

many other pupal parasitoids of Diptera, it has a relatively restricted host range and 

is known only to parasitize Tephritidae (Sivinski et al. 1998b). 


Source of insects 

In every case with the exception of C. haywardi, we obtained parasitoids by 

harvesting mature fruit from the tree or retrieving fallen fruit from the ground and 

transporting it to our laboratories in Xalapa, Veracruz, where they were processed 

following the methods described in Aluja et al. (1998), López et al. (1999) and 

Sivinski et al. (2000). In the case of C. haywardi, specimens stemmed from pupae that


Table 1. Location and host plant from which the individuals stemmed that were used to 

establish the first successful colonies. 

Locality Host plant Fruit fly host Parasitoid species 

Llano Grande 1 and Tejería 2 , 

Municipality of Teocelo, 

State of Veracruz, Mexico 

La Mancha 3 , Santiago 

Tuxtla 4 and San Andrés 

Tuxtla 5 , State of Veracruz, 

Mexico 

Vicinity of Tapachula 6 , 

State of Chiapas, Mexico 

Playa Escondida 7 and 

Sontecomapan 8 , Los 

Tuxtlas, State of Veracruz, 

Mexico 

Spondias mombin L. 

(Anacardiaceae) 

Psidium guajaba L. 

(Myrtaceae) 

Citrus sinensis L. 

(Rutaceae) 

P. guajaba L. 

(Myrtaceae) 

Anastrepha 

obliqua 

Doryctobracon areolatus 

Utetes anastrephae 

A. obliqua 

pupae 

Coptera haywardi 

A. fraterculus D. crawfordi, Aganaspis 

and/or 

pelleranoi, Odontosema 

A. striata 

anastrephae 

A. ludens D. crawfordi 

A. fraterculus 

and/or 

A. striata 

O. anastrephae 

Ximenia americana L. 

(Olacaceae) 

A. alveata O. anastrephae 

Tabernaemantana A. cordata 

Opius hirtus 

alba Mill. 

(Apocynaceae) 

larvae 

1 (19822’08’’ N, 96851’57’’ W), 2 (19822’07’’ N, 96854’59’’ W), 3 (19835’23’’ N, 96822’49’’ W), 4 (18828’31’’ N, 

95818’40’’ W), 5 (18826’42’’ N, 95811’53’’ W), 6 (14854’21’’ N, 92815’33’’ W), 7 (18836’47’’ N, 95803’45’’ 

W), 8 (18825’07’’ N, 95812’48’’ W). 

were collected underneath fruit naturally infested in the field or from lab reared 

pupae artificially exposed to parasitization in the field (details in López et al. 1999). 

Details on fruit fly (fruit) and parasitoid host (fruit fly larvae) species and the 

geographical location where the specimens for founding the colonies were collected 

are provided in Table 1. 

Laboratory conditions 

During the initial phases of the colonization and domestication processes, we 

maintained parasitoid colonies at the Fruit Fly and Parasitoid Laboratory of the 

Instituto de Ecología, A.C., Xalapa, México, at 25918C, 7095% RH, and a 

photoperiod of 12:12 h. Over time (i.e. several years of observations), much insight 

into the particular idiosyncrasies of each species was gained, and as a result, we 

moved established colonies of D. crawfordi and C. haywardi into a laboratory 

maintained at a lower temperature (23928C). As previously noted, both species are 

common in areas above 800 m, with lower year-round temperatures. All the other 

species, typically found in warmer climates, were maintained in laboratories at 259 

18C. A separate laboratory, kept at 27918C, 7095% RH, 12:12 h photoperiod) was 

used to rear A. ludens adults, while larvae and pupae were kept at 30918C, 7595% 

RH in an additional room without light (i.e., full darkness). This species was used as 

a host for all the parasitoid species. Yet another laboratory was used to mass-rear D. 

crawfordi in Metapa de Domínguez, Chiapas (24928C, 70910% RH, 12:12 h 

photoperiod).

Rearing of A. ludens larvae as parasitoid hosts 

Our A. ludens strain was originally provided by the Comité Estatal de Sanidad 

Vegetal (DGSV-SAGARPA) in Xalapa, Veracruz, where it had been kept for over 

200 generations. We placed 200 mL of A. ludens pupae in 30 30 60-cm Plexiglas 

cages. Between 2,500 and 3,000 adults emerged 1 2 days later and were fed ad libitum 

with a mixture of hydrolyzed protein (Greif Bros. Corporation, Delaware, OH) and 

locally available refined sugar (no particular brand). Water was provided ad libitum 

by using 300-mL plastic bottles with a cotton wick. After 8 days, flies were provided 

with an artificial oviposition medium placed inside the cage, which originally 

consisted of a 10-cm dome-like, hollow, dark green hemisphere made of green 

cheesecloth (dyed with commercial fabric dye (Mariposa † , Colorantes Importados, 

S.A. de C.V., México D.F., Mexico) and paraffin (McPhail and Guiza 1956). This 

oviposition device was later replaced by a 12-cm diameter Petri-type plastic dish 

covered with green linen cloth and filled with transparent silicon or ‘fuseleron’ 

(Devcon † , Junta Flex, ITW Poly Mex SA de CV, Mexico). The plastic dish was 

placed upside down on top of the fly-holding cage so that females could insert their 

aculeus through the cloth and lay eggs into the ‘fuseleron’. Once flies reached 8 days 

of age, eggs were collected daily over an 8-day period and washed in a solution of 2 g 

of sodium benzoate (Baker, J.T. Baker S.A. de C.V., Xalostoc, Edo. de México) 

dissolved in 1 L of purified water. After washing, eggs were placed on pieces of filter 

paper (Whatman No. 1, Whatman Int., Ltd., Maidstone, England) in Petri dishes, 

incubated for 4 days and then placed (2 mL per unit) in a 11 26 32-cm plastic 

washbowl containing an artificial diet (ingredients in Appendix 1). Once the desired 

larval stage was reached (2nd and 3rd stage depending on parasitoid species), 

exposure to parasitoids was carried out according to the technique used for each 

particular species (details follow). 

In the particular cases of the D. crawfordi and C. haywardi strains sent from 

Xalapa, Veracruz to the Laboratorios de Desarrollo de Métodos, Campaña 

Nacional Contra Moscas de la Fruta in Metapa de Domínguez, Chiapas, Mexico 

for mass-rearing purposes, parasitoids were exposed to irradiated A. ludens larvae 

(pupae in case of C. haywardi) produced locally (Domínguez, Hernández, and 

Castellanos 2002). For D. crawfordi we used larvae irradiated at 40 Gy and in the 

case of C. haywardi, irradiation dose for pupae was 30 Gy (Cancino, Ruiz, Sivinski, 

Gálvez, and Aluja 2008). Since irradiated larvae support parasitoid development but 

do not mature into fertile flies, removal of unattacked hosts from the colony is 

greatly simplified (Sivinski and Smittle 1990). Larvae (32,000) and pupae (25,000) 

were placed in 1-L containers and irradiated, in an atmosphere containing oxygen, 

using a Gammacell irradiator with a cobalt-60 source (Cancino et al. in press) 

located at the Medfly mass rearing facility in Metapa de Domínguez, Chiapas. 

Cages for holding parasitoids 


Various sizes of Plexiglas cages, covered with fiberglass and aluminum screen, were 

used to house parasitoids. Screen mesh size and cage size depended on the size of the 

parasitoid species kept inside (details in Table 2). In the case of Plexiglas cages, one 

side of each cage was covered with plastic wrap (Kleen Pack † ; Kimberly Clark de 

México S.A. de C.V.) held in place by three strips of masking tape (Shurtape † ,

Table 2. Summary of rearing procedures and handling conditions used during the domestication and colonization of seven native Anastrepha 

parasitoid species (all parasitoid colonies were maintained at 25918C, 7095% RH, 12:12 h photoperiod) (see Figures 1 4 for further details on rearing 

cages and parasitization devices such as FF, SD and M-PD). 

Species 1 

Doryctobracon 

crawfordi 

No. of parasitoids 

per rearing cage 

Rearing 

Plexiglas cage 

size Female Male 

30 30 30 1,2 

D. areolatus 25 25 25 1 

Utetes anastrephae 25 25 25 1 

Opius hirtus 30 30 60 1 

Aganaspis 

pelleranoi 

Odontosema 

anastrephae 

30 30 30 1 

30 30 30 1 

Host stage 

attacked 

Host age 

(days) 

Type of parasitization 

devices (and No. 

hosts per unit) 

30 15 Larva 8 Fruit filled with guava 

FF (50) Sandwich- 

type oviposition 

Host 

exposure 

periods (h) 

No. of exposed hosts 

per parasitoid female 

and per hour 

36 0.05 larvae 

device one SD1 


(250) Sandwich type 

oviposition 

device two 

(250) 

SD2 

7 19 larvae 

30 15 Larva 8 FF (50) 36 0.05 larvae 

SD1 (250) 36 0.23 larvae 

40 20 Larva 7 8 FF (50) 48 0.03 lavae 

Modified Petri dish 

M-PD (250) 


SD2 (250) 7 0.91 larvae 

40 20 Larva 8 FF (50) 36 0.04 larvae 

SD1 (250) 24 0.26 larvae 

SD2 (250) 7 0.91 larvae 

30 15 Larva 9 Uncovered Petri dish 

UP (250) 


UP (250) 7 1.19 larvae 

30 15 Larva 9 UP (250) 24 0.35 larvae 

54 M. Aluja et al.

Table 2 (Continued) 

Species 1 

Coptera haywardi 30 30 30 1 

No. of parasitoids 

per rearing cage 

Rearing 

Plexiglas cage 

size Female Male 

Host stage 

attacked 

Host age 

(days) 

Type of parasitization 

devices (and No. 

hosts per unit) 

30 15 Pupa 1 2 Covered pupae 

(500) 

CP 

125 125 Pupa 2 Naked pupae 

(800) 

NP 

Host 

exposure 

periods (h) 

No. of exposed hosts 

per parasitoid female 

and per hour 

168 0.10 pupae 

72 0.09 pupae 

1 2 

The fiberglass screen that covered the cage frame had a 0.3-mm mesh size. When D. crawfordi was mass-reared (details in text) we used an aluminum cage frame covered 

with a metallic screen (1-mm mesh size). 

Biocontrol Science and Technology 55


0.14 M 

0.24 M 

Shurtape Technologies, Inc., Hickory, NC). A 150-mL container holding one or two 

orange, mango or guava (depending on availability) branches with five to eight leaves 

each, was placed in every cage to provide resting sites and adequate conditions for 

mating activities. In the case of C. haywardi, 10 10-cm pieces of black paper were 

used to form small (5 8 cm) resting shelters that were placed on cage floors (1 2 per 

cage). In each clean, sealed cage, we placed a predetermined number of newly 

emerged males and females from a given parasitoid species (details in Table 2). 

For mass-rearing purposes (case of D. crawfordi), we used a 40 30 30-cm cage 

with an aluminum frame, covered with a metallic mesh (1 mm) known as the 

‘Metapa’ cage (Figure 1). In the cage front, there are two 15 1.5-cm openings that 

project inside of the cage by means of two 17 11.5-cm hollow aluminum squares 

(width of 2 cm) covered with the same 1-mm metallic mesh used to cover all cage 

walls. Inside the hollow squares, we slid the oviposition units, which consisted of 

empty compact disk cases (10 5 1 cm, length width depth) in which the top 

had been replaced by organdy cloth held tightly to the frame. Between the disk case 

bottom and the cloth cover, we placed 2,000 third instar A. ludens larvae mixed with 

some of the diet the larvae had been reared in (Figure 1). Each cage contained 1,500 

parasitoids (sex ratio close to 1:1) that were allowed to parasitize larvae over a period 

of 4 h daily over 10 days. After this, they were replaced with a new cohort. 

Feeding, and handling of adults 

0.30 M 

0.40 M 

0.30 M 

Adults were fed with diluted honey (70% honey, 30% water) (Miel Carlota † ; Herdez 

S.A. de C.V., Cuernavaca, Morelos, Mexico). Pieces of cotton (Zuum † ; Universal 

Productora S.A. de C.V., México D.F.) saturated with this liquid diet were placed in 

Petri dishes (10 cm in diameter) and offered to the parasitoids ad libitum (see 

Bautista, Harris, and Vargas 2001). Food was changed on a weekly basis. Water was 

0.25 M 

0.055M 

0.11M 

0.165 M 

Figure 1. ‘Metapa‘ cage used in initial D. crawfordi mass-rearing efforts. ‘Cassette-type’ 

oviposition units (compact disk cases) filled with larvae (2000 third instar larvae mixed with a 

small amount of rearing diet) were slid into cage openings in walls. Each cage contained 1,500 

parasitoids.


also administered on a piece of cotton and was changed two times per week. At the 

same time that food and water were changed, dead parasitoids were removed from 

the cages to avoid problems with fungi, bacteria, mites, and other insect pathogens. 

To keep parasitoids from escaping the cages while maneuvering objects within them, 

we temporarily shut the lights in the laboratory and used a 22-W lamp to attract the 

parasitoids towards the light. 

Diagnostic features for quick recognition of the sexes 

To facilitate quick recognition of the sexes, the following diagnostic features were 

used. In the case of braconid species, differences among the sexes were obvious 

because the female, besides being larger than the male, has an exerted ovipositor that 

is clearly visible (Sivinski et al. 2001; Sivinski and Aluja 2003). In the case of figitids, 

the most obvious character for identifying the sexes is the size and shape of the 

antenna, since the ovipositor is not apparent in females. Male antennae are filiform 

and 1.6 1.8 times longer than female antennae which are moniliform (Ovruski and 

Aluja 2002). In the case of C. haywardi, sex can also be distinguished by clearly 

different antennal lengths. Female and male antennae measure, respectively (mean9 

SE), 1.790.1 mm (N 20) and 3.090.2 mm (N 20). 

General conditions for the reproduction, management and care of parasitoids 

Once field-collected larvae had pupated and adult parasitoids emerged, the 

domestication phase ensued. It initially consisted of adapting adults of each species 

to the artificial housing and rearing conditions associated with the laboratory. The 

first step was to identify and manipulate environmental conditions, such as 

temperature, required by each species. In addition, preliminary observations of 

mating and oviposition behaviors were conducted to determine which species 

parasitized larvae and which attacked pupae and what circumstances enhanced 

mating. To confirm that C. haywardi exclusively parasitized pupae (and not late third 

larval instars), females were offered two guavas containing 50, third instar A. ludens 

larvae. These fruit were removed before the larvae had pupated. At the same time, 

parasitoids were exposed to pupae (0 2 days old) for 7 days (168 h). 

Description of oviposition units utilized to colonize each species of parasitoid 

We tried to fabricate the cheapest and most natural oviposition devices to entice 

females to accept the artificial laboratory conditions (details in Figures 2 4). In what 

follows we describe the oviposition devices that worked best for us after several failed 

attempts. 

Oviposition substrates for larval-prepupal parasitoids 

Fruit filled with larvae (FF). Our objective was to simulate a naturally infested fruit 

that would be attractive to wild parasitoids, particularly in the initial stages of the 

domestication process. Commercial guava (Psidium guajava) was chosen as the 

preferred parasitization unit because: (a) almost all species of larval-prepupal 

parasitoids described in this work were found parasitizing fruit fly larvae in guavas in


Figure 2. Description of the ‘fruit filled with larvae (FF)’ oviposition substrate used during 

the initial colonization stages of larval-prepupal parasitoids. (1 3) Cutting of fruit, with 

proximal quarter functioning as ‘lid’ and rest as ‘base’. (4 6) Removal of pulp to create cavity 

(hollow ‘base’). (7 8) Filling of hollowed ‘base’ with larvae mixed with diet. (9) Joining of 

‘base’ and ‘lid’ with aid of 1.5 10-cm parafilm strip (‘belt’). (10) Pricking of holes into of 

fruit. (11 12) Paper clip inserted into parafilm ‘belt’ to hang fruit from cage roof. (13) Fruit 

hanging from cage roof. (14) Parasitoids ovipositing in FF unit.


Figure 3. Modified Petri dish (M-PD) oviposition unit used to rear U. anastrephae, the 

parasitioid species with the shortest ovipositor (left). For comparative purposes (i.e. 

distinguish differences in thickness of oviposition unit), a ‘sandwich-type oviposition device’ 

is also shown (right). 

the field (López et al. 1999) and (b) because guava can be obtained year round in 

local markets and supermarkets at a reasonable price. Guavas were cut open 

transversally along the peduncle, about one-quarter down the length of the fruit as 

measured from the proximal end (Figure 2). The proximal quarter sections 

functioned as ‘lids’ for the filled fruits and the remainder of the fruit served as 

‘bases’ for filling. Mesocarp and endocarp (pulp) were extracted in the bases to


Figure 4. Preparation of the ‘sandwich-type oviposition devices (SD)’. (A) Exposure of 

naked larvae without fruit skin. (B) One-mm (thickness) guava epicarp (skin) pieces placed on 

top of chiffon cloth covering larvae placed to entice female parasitoids to land on oviposition 

unit and parasitize larvae. 

create cavities that could be filled with larvae and diet. Guavas had to be mature 

(yellow and soft, but not watery) and emit the characteristic odor associated with 

this fruit (i.e. not sealed with wax). However, if wax residues were encountered, they 

were removed by gently washing the fruit with diluted soap. The optimal size for 

guavas was 45 55 g and 4 5 cm in diameter. Larger fruit typically yielded smaller


numbers of parasitoids because females were unable to reach larvae feeding deep 

within the fruit (Sivinski 1991). The short ovipositor of U. anastrephae (Sivinski et al. 

2001) restricts females to parasitizing larvae in small fruit such as Spondias mombin 

(López et al. 1999). As a consequence, we were forced to use small (25 30 g and 3 4 

cm in diameter) larvae-filled guavas to colonize this species. We filled each fruit with 

ca. 50, laboratory-reared, second or third instar A. ludens larvae, and hung three or 

four guavas per rearing cage (Figure 2). Larval stage was associated to parasitoid 

species as described in Table 2. Once guava ‘bases’ were filled with larvae, they were 

covered with their corresponding ‘lids’ and the different parts tightly joined with 

1.5 10-cm strips of parafilm (Parafilm ‘belts’) (Parafilm † Laboratory Film, 

American National Can Tm, Chicago, IL). Four to five holes were pricked into 

the fruit with a 1-mm metal needle to allow for aeration. Plastic paper clips were 

inserted into the Parafilm ‘belts’ to hang fruit from the cage ceilings where parasitoid 

density was usually highest. A variant of this technique was used in the case of O. 

anastrephae and A. pelleranoi, whose females prefer to enter into fruit interiors to 

search for fruit fly larvae (Ovruski 1994; Sivinski et al. 1997). For these species a 2mm 

orifice was left in the upper portion of each guava (between the ‘lid’ and the 

‘base’) to serve as an entrance for female parasitoids. Because adults of these two 

figitid species prefer to forage on the ground (Ovruski et al. 2004), fruit were not 

hung, but rather placed on cage floors. 

Modified Petri dish (M-PD). This technique was only used in the case of U. anastrephae, 

which as noted before, has the shortest ovipositor of the species we were 

attempting to colonize. The oviposition unit consisted of 10-cm diameter Petri dishes, 

which we made shallower by scraping down ca. 50% of the walls (height was lowered 

from 0.9 to 0.4 cm) (Figure 3). We placed A. ludens larvae mixed with the diet on 

which they had been reared on the lowered Petri dish ‘bottom plate’ and tightly 

covered it with a stretched-out piece of Parafilm (original size was 5 5 cm). We chose 

to use Parafilm, because we had observed that the organdy cloth, which worked well 

in the case of other species, apparently did not provide the necessary mechanical 

aculeus stimulation that U. anastrephae females needed before parasitizing larvae. 

Sandwich-type oviposition devices (SD). Once the parasitoids had reproduced for 

several generations using the ‘fruit filled with larvae’ technique (FF), the next step in 

the colonization process was to develop an artificial oviposition substrate for 

parasitoid females that was inexpensive and easy to handle. For this reason, we 

began to adapt adult parasitoids to ‘sandwich-type devices’ (SD) which were similar 

to the Petri dish methodology employed for mass rearing exotic opine parasitoids 

such as D. longicaudata and D. tryoni (Cameron) in Hawaii (Wong and Ramadan 

1992). We used two kinds of SD devices (Figure 4). 

Sandwich-type oviposition device one (SD1). This parasitization unit was suitable 

during the initial rearing stages of D. crawfordi, D. areolatus, and O. hirtus (Figure 2). 

It consisted of a 11.5 1.6-cm (diameter height) plastic ‘dish’ with a bottom made 

of a 15 15-cm piece of chiffon cloth. On the cloth surface we placed ca. 250 A. 

ludens larvae mixed with the diet on which they had been reared. The age of the 

larvae depended on the species of parasitoid being reared (details in Table 2). The 

dish containing larvae and diet was covered with another 15 15-cm piece of chiffon 

cloth that was tied to the base by a 11.7 0.8-cm (diameter height) plastic ‘ring’


put in place by pushing against the base (i.e. pressure exerted with index fingers). 

After the ‘sandwich’ was built, we completely covered the chiffon cloth top with a 

layer of guava epicarp (skin) ca. 1 mm in thickness. The thin skin pieces were 

obtained by finely slicing the guava epicarp with a razorblade or sharp knife. The 

ultimate goal was to entice females to oviposit by mechanical and olfactory 

stimulation with the fragrant guava epicarp. 

Sandwich-type oviposition device two (SD2). The parasitization unit was the same as 

described under SD1, but in this case larvae were exposed in naked form (i.e. not 

mixed with diet). Furthermore, we did not place a layer of guava epicarp but instead 

soaked the chiffon cloth with liquid guava pulp. This method turned out suitable to 

entice wild D. crawfordi, D. areolatus, O. hirtus, and U. anastrephae females to 

oviposit. 

Uncovered Petri dish (UP). We discovered that the females of the figitids A. pelleranoi 

and O. anastrephae were suffering severe ovipositor damage while attempting to 

parasitize larvae in the oviposition units covered with chiffon cloth. Furthermore, 

because females of these species like to enter fruit in search of the larvae feeding 

inside, we used an uncovered unit. We used the bottom part of a Petri dish half filled 

with diet mixed with larvae. At the same time, half a guava was added to the artificial 

diet with larvae. The fruit, including seeds, was macerated into pieces and thoroughly 

mixed with the diet. In general, endocarp and mesocarp were utilized because the 

fruit’s fragrance appeared to attract females and stimulate oviposition behavior. 

Oviposition substrate for pupal parasitoid 

Initial exposure of A. ludens pupae to C. haywardi was done in 500-mL plastic 

containers containing a ca. 10-cm layer of moistened soil (50 70% water content) 

and some leaf litter. Soil was brought from the original collection locality of Tejería, 

Veracruz (López et al. 1999), and was predominantly clay (Guillén et al. 2002). 

Approximately 500 recently formed pupae (1 2 days from pupation) were placed in 

the plastic container and mixed with the soil (referred to as CP method, i.e. covered 

pupae, in the text). Then, a mature guava placed on a galvanized wire screen was 

inserted into the container to lure parasitoids to the pupae underneath. The wire 

screen measured 10 10 cm with 1 1-cm mesh openings. Pupae were exposed to 

parasitism over a period of 7 days (Table 2). The guava was only inserted into the 

oviposition unit during the first three generations, after which time the parasitoids 

seemed to respond well to A. ludens pupae alone. After the 21st generation, soil and 

leaf litter were eliminated and only ‘naked’ pupae (referred to as NP-method, i.e. 

naked pupae, in the text) were exposed during 3 days on a 11.5 1.6-cm (diameter 

height) plastic dish (Table 2). 

Maintenance of parasitized larvae and pupae 

In the case of M-PD (modified Petri dish), SD1 (sandwich-type oviposition device one 

(larvae mixed with diet)), and UP (uncovered Petri dish) exposures, larvae were 

cleaned of diet and guava residues by placing them in a fine mesh plastic colander and 

rinsing them under running tap water. Once clean, larvae were placed in 500-mL 

plastic containers with 2.5 cm 3 of moistened vermiculite where they formed puparia.


All containers were labeled, protected with a top made of chiffon cloth, and 

maintained under laboratory conditions (25918C, 7095% RH) until fly or parasitoid 

adults emerged. In the case of FF (fruit filled with larvae) exposures, fruit was placed 

in 200-mL plastic vials, which in turn were placed inside 500-mL plastic containers 

with 2.5 cm 3 of moistened vermiculite. This was done to allow larvae to exit the fruit, a 

process that many times caused the fruit to disintegrate, spilling larvae and diet onto 

the floor of the 200-mL vial. On day 4, any diet or fruit residues were rinsed from 

pupae and larvae as described above and transferred to a 500-mL plastic container 

with moistened vermiculite, where they remained until adult emergence. The double 

container technique allowed us to avoid fungal and bacterial contamination that 

usually ensues if the vermiculite is mixed with fruit and diet residues. 

Handling of emerged parasitoids and flies 

Once parasitoids and flies had emerged, they were transferred to a clean, empty 

Plexiglas cage and provided with food and water. The size of the cage and the 

number of males and females per cage depended on the species (see Table 2). Daily 

inspection of containers with pupae was critical to make sure that emerging adults 

did not escape or suffer stress because of lack of food and water. Length of pupal 

period and associated timing of parasitoid emergence was species-specific and may 

occur before, after, or in synchrony with host emergence. In the case of parasitoids 

that emerge before their host (i.e. U. anastrephae), there was no need to separate 

adult parasitoids from adult flies since unemerged A. ludens pupae were simply 

removed and discarded once the adult parasitoids had emerged. In the case of 

parasitoid species whose emergence is more synchronous with host emergence (i.e. O. 

hirtus, D. crawfordi, and D. areolatus), we were forced to separate adult parasitoids 

from adult flies. This was done utilizing a standard aspirator. Adult parasitoids that 

were very sensitive to ‘rough’ handling (i.e. aspirator) like D. areolatus, or that were 

destined for behavioral studies, were separated using 10-mL glass vials into which 

insects walked. When parasitoids had a more prolonged pupation interval than their 

hosts (i.e. A. pelleranoi, O. anastrephae, and C. haywardi), the emerged adult flies and 

empty puparia were separated to leave only parasitized pupae. Separation of flies and 

empty puparia was critical to avoid fungal growth and to significantly lower the risk 

of contamination by mites. 

Determination of percent parasitism, sex ratio, and pupal viability per generation 

To measure percent parasitism, two 10-mL samples of parasitized A. ludens larvae 

(approximately 220 larvae) were processed per generation. The first sample was 

taken when parasitoid females reached, 4 and the second one when they reached 

10 days of age. In the case of C. haywardi, instead of larvae, two random samples of 

100 pupae were processed. The handling procedure for these larvae and pupae was 

the same as that described earlier. Once parasitoid adults had emerged, number and 

sex were recorded. Relative percent parasitism was estimated by dividing the total 

number of parasitoids that emerged by the total number of larvae exposed in the 

parasitization unit as we were not interested in an exact determination of the ‘killing 

power’ of each parasitoid species at this juncture (i.e. a certain proportion of larvae/ 

pupae were parasitized and killed and therefore ended up not yielding an adult


parasitoid). Pupal viability was determined as the total number of pupae that yielded 

flies and parasitoids divided by the total number of unemerged and emerged pupae. 

Demographic studies. Doryctobracon areolatus, D. crawfordi, and O. hirtus 

Adults used in these tests stemmed from colonies that were 14 generations old. 

Utetes anastrephae, A. pelleranoi and O. anastrephae had been reared over nine 

generations, and C. haywardi over 24 generations. For these studies, braconid larvalprepupal 

species were only reared using A. ludens larvae in the FF (fruit filled with 

larvae) method, while figitid larval-pupal species were reared using A. ludens in the 

UP (uncovered Petri dish) method (Table 2). In all cases, 30 host larvae were exposed 

daily to 15 parasitoid pairs (i.e. 15 females and 15 males totaling 30 individuals per 

cage) for 24 h during their entire adult lifespan in Plexiglas rearing cages containing 

water and honey (details on size in Table 2). After exposure to parasitoid attack, host 

larvae were placed in plastic trays (500 mL) and provided with fresh larval diet. 

Three days after, formed pupae were separated from diet and transferred to other 

500-mL trays with 150 mL of moistened vermiculite. All trays were taken into a room 

at 25918C, 7095% RH, and full darkness, where they remained until fly and 

parasitoid adults emerged. After all died (no food or water was provided), they were 

counted and sexed. In the case of the pupal parasitoid C. haywardi, 30 pairs (30 

females and 30 males totaling 60 individuals per cage) were exposed daily to 20 twoday-old 

A. ludens pupae in 5 1.5 (diameter height) plastic Petri dishes covered 

with 1 cm of vermiculite. Cages in this case, were 10 10 10 cm in size, with glass 

walls and aluminum frame. Each study (i.e. one per species) was replicated five times. 

Life table parameters (lx, fraction of the original cohort surviving to age x; px, period survival; qx, period mortality; dx, fraction of the original cohort dying at age 

x; ex, expectation of life; Mx, average number of male and female offspring produced 

by female at age x; mx, female offspring per female at age x; Carey 1993, 1995) were 

calculated from daily mortality records and offspring data for cohorts of all larvalprepupal 

and pupal parasitoids. These values were used to determine reproductive 

parameters such as gross fecundity rate (GFR in text), net fecundity rate (NFR in 

text), cohort lifespans, and offspring sex ratios (as female proportions) and 

population parameters such as Ro (net reproductive rate), r (intrinsic rate of 

increase), l (finite rate of increase), and T (mean generation time) (Carey 1993; 

Vargas et al. 2002). These demographic parameters helped us to estimate the relative 

population growth vigor of the first colonized cohorts. 

Experiments to determine optimal pupal age to rear C. haywardi 

We conducted two types of experiments with mated, 7-day-old females: no choice 

and choice tests. In each case we tested six treatments, each corresponding to an age 

class of the host (A. ludens pupae). Pupal age classes (days) tested were: 0 2, 3 5, 6 8, 

9 11, 12 14, and 15 17. We used 30 pupae per age class, 10 per age included in every 

age class (i.e. in age class 0 2, there were 10 pupae each of ages 0 (B24 h or prepupa), 

1 and 2 days). In the no choice experiment, we released 15 C. haywardi females 

together with 30 pupae of a determined age class in a 500-mL plastic container that 

was halfway filled with sterilized clayey soil (pupae were superficially buried). 

Exposure period was 24 h and the experiment was replicated five times for every age

Table 3. Parasitization rates (mean percent parasitism), proportion of emerged females, and 

pupal viability in all seven native Anastrepha fruit fly parasitoids as the domestication and 

colonization process proceeded. 

Parasitoid species Rearing method 

D. crawfordi a 

U. anastrephae b 

O. hirtus a 

D. areolatus a 

A. pelleranoi a 


% Parasitism e 

(Mean9SEM) 

% Emerged females 

(Mean9SEM) 

% Pupal viability e 

(Mean9SEM) 

FF 38.792.9 54.592.8 55.992.9 

SD1 20.891.4 47.992.5 58.893.3 

SD2 37.992.1 44.792.6 43.692.3 

FF 26.194.3 58.792.9 72.892.6 

M-PD 20.494.1 45.493.3 50.494.9 

SD2 25.293.9 50.693.7 39.794.4 

FF 24.792.1 55.692.7 61.093.6 

SD1 16.591.0 45.492.2 55.591.4 

SD2 13.791.3 56.893.9 64.792.2 

FF 24.391.6 60.191.9 56.192.8 

SD1 11.191.2 58.592.6 54.491.6 

UP-24h 26.491.8 58.392.7 50.092.4 

UP-7h 35.692.9 46.892.9 65.694.3 

O. anastrephae c UP-24h (bisexual) 30.592.6 61.193.2 52.994.1 

UP-24h (unisexual) 24.491.2 100.0 56.291.8 

C. haywardi d 

CP 3.890.3 57.191.3 8.490.3 

NP 4.590.3 48.292.8 8.390.4 

a Data from first 14 generations; b data from first nine generations; c data from first 20 generations, d data 

from first 12 generations. FF (fruit filled with larvae), M-PD (modified Petri dish), SD1 (sandwich-type 

oviposition device one [larvae mixed with diet]), (sandwich-type oviposition device two [naked larvae]), UP 

(uncovered Petri dish filled with larvae mixed with diet and fruit pulp; 7 and 24 h refer to exposure period), 

CP (pupae covered with soil), NP (pupae exposed naked [without soil cover]). 

class. In all cases (each replicate) we used a new cohort of females (i.e. no repeated 

measures on same cohort). In the multiple choice experiment, we released 90 females 

together with 180 pupae encompassing all age classes (30 pupae per age class) in a 

15 10-cm (diameter height) plastic container that was also half-filled with 

sterilized clayey soil. To distinguish pupae of every age class, they were individually 

marked with a dot of acrylic paint (six colors used) (Colores Acrílicos Indelebles 

Politec, Distribuidora Rodin, Mexico). Exposure period in this case was 36 h and we 

replicated each experiment five times. The pupae were handled as already described 

before until all parasitoids emerged and were counted. 


Owing to the fact that colonization efforts where not simultaneous and that we 

typically only had access to a small number of individuals of any given species at any 

particular time, we could not run any formal statistical analyses comparing the 

performance of the various rearing methods. Nevertheless, overall trends can be 

ascertained by visually comparing data summarized in Tables 3 and 5. In the case of 

the experiment to determine optimal A. ludens pupal age to rear C. haywardi, we ran a 

one-way ANOVA comparing percent parasitism, sex ratio and proportion of


unemerged puparia (sometimes the host is killed due to single or multiple parasitoid 

stings). Post-hoc mean comparisons were done by means of a Tukey honest significant 

difference test (HSD) at an a of 0.05. Proportions were arcsine square root 

transformed prior to analysis, but untransformed means are presented in the text. 

Results 

Colonization and adult handling conditions 

A summary of parasitoid rearing and handling procedures is provided in Table 2. In 

what follows, we report the most relevant results of the colonization efforts on a per 

species basis to facilitate domestication and colonization efforts in other parts of the 

world. We place emphasis on sex ratios, percent parasitism and mean proportion of 

pupae yielding a parasitoid given that these parameters greatly influence the success 

rate of the domestication/colonization process early on. 

Doryctobracon crawfordi. The domestication process of this species was initiated in 

October 1994, using the FF (fruit filled with larvae) method over 10 generations. 

Then gradually, between the 10th and 15th generations, we exposed the parasitoids 

to the SD1 (sandwich-type oviposition device using larvae mixed with diet) method. 

The length of the larval exposure period was the same in both cases (Table 2). The 

sex ratio for both FF and SD1 parasitoids varied throughout the colonization 

process. For example, for FF parasitoids, the smallest proportion of females 

occurred in the first four generations (0.4 0.9:1). From generation 5 to 42 and 

with only one exception (generation six, 0.7:1), the sex ratio consistently favored 

females (1.1 7.0:1). Similarly, the lowest proportion of SD1 females was observed in 

the first eight generations (0.3 0.9:1), whereas the highest appeared after generation 

9 (1.1 2.6:1; generations 9 14). Starting with generation 14, the SD1 technique was 

replaced by method SD2 (sandwich-type oviposition device using naked larvae). The 

sex ratio in the SD2 strain varied sharply from generation to generation over the 44 

generations recorded (most likely due to variations in host quality). The lowest 

proportions of SD2 females were 0.2:1, whereas the highest proportions were 6:1 

(mean values in Table 3). Percent parasitism levels during the first 14 generations 

using the three rearing methods varied between 15.5 62.3% (FF), 9.1 41.8% (SD1) 

and 20.0 56.8% (SD2) (mean values in Table 3). 

Doryctobracon areolatus. This parasitoid species presented various challenges during 

the early stages of the domestication/colonization process. Among the most difficult 

ones to overcome was a propensity to enter what appeared to be a reproductive 

diapause from late November until almost March (coldest time of the year), despite 

the fact that we controlled temperature and lighting conditions inside the laboratory. 

As a result, from 1993 to 1997 we were only able to keep temporary colonies (all 

eventually died out) by using plums (Spondias purpurea and S. mombin) and mangos 

(Mangifera indica) naturally infested by A. obliqua (collected in the field) and 

parasitized by D. areolatus. Later, in July of 1997, we were able to successfully 

establish two D. areolatus colonies using artificially reared A. ludens larvae as a host, 

taking advantage of an unusually high parasitism rate in A. obliqua developing in the 

above mentioned fruit species. One of the colonies was maintained employing the SD1 

(sandwich-type oviposition device using larvae mixed with diet) method while the


other colony was maintained using the FF (fruit filled with larvae) technique (see 

Table 2 for details). As was the case with D. crawfordi, sex ratios in both successfully 

colonized strains tended to be initially male-skewed. However, in subsequent 

generations the proportion of males and females was gradually equalized or favored 

females. The lowest proportion of FF females was observed in first and second 

generations (0.7 0.8:1) and thereafter (up to generation 69) it reached a maximum of 

3.5:1. Overall, sex ratios of SD1 parasitoids were female skewed but intergenerational 

variation was greater than that observed in FF parasitoids (Table 3). Parasitism rates 

during generations 1 69 (68 in the case of the SD1 method) using the FF method 

varied from 8.2 to 36.4% between first and 69th generation, whereas employing the 

SD1 technique varied from 1.4 to 25.9% between first and 68th generation (Table 3). 

Opius hirtus. Domestication of the first strain of this species was initiated in October 

1994, through FF (fruit filled with larvae) exposures. However, the colony was lost in 

generation 6 (March 1995). We believe that failure hinged principally on the fact that 

females were probably not mating because of saturation of the environment with sexual 

pheromones (avery strong fruit-like bouquet was perceived near the cage). We therefore 

doubled cage size and introduced citrus brancheswith ample foliage as resting sites (tips 

of branches were inserted into 60-mL glass vials covered with cotton to prevent the 

parasitoids from drowning). After the original failure, a new colonization attempt was 

initiated in January 1996 with a few (B20) parasitoids obtained from a rare Anastrepha 

species (A. cordata) collected in the few remaining patches of tropical evergreen 

rainforest in Southern Veracruz, Mexico. Due to the difficulties involved in finding 

parasitoids in nature and considering our initial failure, we maintained three strains 

along the domestication/colonization process. Initially, we used the FF technique and 

then (generation six), started a new line using the SD1 (sandwich-type oviposition 

device using larvae mixed with diet) method (Table 2). Three generations later, we 

started a third line, by switching to the SD2 (sandwich-type oviposition device using 

naked larvae) method. In the latter case, we reduced the exposure period 5-fold with 

respect to the other rearing methods. Because in nature O. hirtus females are faced with 

very low host densities, we wanted to reduce the risk of larvae being marked with a 

marking pheromone that would have caused females to quickly leave the ‘resource 

patch’. Sex ratios in the FF strain tended to be initially (generations 1 7) male-skewed 

(0.4 0.9:1), but in subsequent generations (8 37), favored females (1.3 5.6:1). In 

general, sex ratios of SD1 parasitoids were more male-skewed than FF parasitoids. In 

the case of the SD2, sex ratios were highly variable over time (0.2 5.3:1 over 115 

generations). Mean parasitism was highest under the FF rearing method (Figure 2). 

Parasitization rates varied between 9.5 59.1, 7.2 26.4, and 6.8 31.4% in the FF, SD1, 

and SD2 lines, respectively. Pupal viability was highest in the FFand SD2 lines (Table 3). 

Utetes anastrephae. This parasitoid presented a particularly difficult challenge because 

of its extremely short ovipositor and the fact that it is usually reared from only very 

small fruit in nature (e.g. S. mombin, Tapirira mexicana;López et al. 1999; Sivinski et 

al. 2000, but see Eitam et al. 2004 for exceptions to the rule). The first unsuccessful 

colonization attempt was made in October 1996 using the FF rearing method (after 

fourth generation no adults emerged). In September 1999, another attempt was 

made using ca. 800 female parasitoids collected from A. obliqua larvae infesting S. 

mombin. The original colony was divided into FF (fruit filled with larvae) and M-PD 

(modified Petri dish) strains. After four generations, we initiated a third strain (SD2


(sandwich-type oviposition device using naked larvae)) with M-PD material. 

Exposure periods in the M-PD and SD2 strains were reduced 2 7-fold with respect 

to the FF strain to avoid superparasitism caused by easier access to larvae (Table 2). 

Sex ratios in the FF strain were slightly male-skewed in the first two generations 

(0.8 0.9:1), but then remained relatively stable over the next 11 generations, with a 

consistent tendency for more females to emerge than males (1.1 6.5:1). In contrast, 

sex ratios in the M-PD and SD2 strains were highly variable between generations. 

The lowest proportion of females fluctuated between 0.3 and 0.9:1 in both M-PD 

and SD2 strains, while the greatest proportions fluctuated between 1.1 2.3:1 and 

1.0 7.0:1 in the M-PD (first 11 generations) and SD2 (first nine generations) (mean 

9 SE values in Table 3). Parasitization rates varied between 6.8 51.8, 1.8 56.4, and 

3.6 60% in the FF, M-PD, and SD2 rearing methods, respectively (mean 9 SE 

values in Table 3). Finally, we found that pupal viability in insects stemming from FF 

lines was higher than those stemming from M-PD and SD2 lines (Table 3). 

Aganaspis pelleranoi. A colony of this figitid parasitoid was initiated in September of 

1994, using adults obtained from field-infested P. guajava. At first, parasitoids were 

reared with the variant of the FF (fruit filled with larvae) technique described in 

Section 2, but few individuals were obtained per generation. Therefore, beginning 

with the fifth generation, this technique was replaced by the UP (uncovered Petri 

dish) rearing method, allowing us to reduce exposure periods 3-fold (Table 2). In 

general, and with few exceptions (e.g. generation one), sex ratio in UP-24h (24 h 

refers to the exposure period in hours) parasitoids favored females over the first 14 

generations. In the case of the UP-7h strain, sex ratios were highly variable, ranging 

between 0.2 and 8:1 (78 generations considered). Parasitization rates varied between 

20.0 68.2 and 11.8 43.6% in the UP-7h and UP-24 h lines, respectively. Also, pupal 

viability in UP-7h lines was higher than in UP-24h lines (Table 3). 

Odontosema anastrephae. The first unsuccessful attempt at colonization was started 

in November of 1995. For the first two generations, we employed the variant FF 

(fruit filled with larvae) method, but extremely low yields forced us to switch to the 

UP (uncovered Petri dish) technique using 36-h exposure periods. However, 

extremely low oviposition activity by females and an extremely male-biased sexratio 

(as low as 0.2:1), lead to the demise of the colony after eight generations. After 

a 3-year search for sufficient wild material, we were finally able to start a new colony 

between September and November 1998, using the UP rearing technique. A second 

O. anastrephae colony was started in February 2000, with wild material stemming 

from guavas. Interestingly, starting with generation 11 (December 2000) essentially 

only females emerged (such a pattern has remained steady over more than 75 

generations). On occasion, one or two males emerged (sex ratio of 1: 0.008), but 

when such was the case, we immediately removed them given our interest in 

maintaining a theliotokous line. In both lines, exposure period was gradually reduced 

to 6 h (details in Table 2). In the case of the bisexual O. anastrephae colony, sex ratios 

varied greatly between generations, fluctuating between 0.1:1 (first four generations) 

and 1.1 8.0:1 in the remaining generations (29 generations considered). Parasitism 

rates varied between 7.7 79.5 and 12.0 37.8%, in the unisexual and bisexual 

colonies, respectively. Pupal viability in the bisexual and unisexual O. anastrephae 

colonies was similar (Table 3).

Coptera haywardi. This endoparasitic pupal parasitoid was first colonized in November 

of 1994 by means of the CP-method (covered pupae (artificially buried in soil)). 

Starting with the 21st generation, we replaced this rearing technique with the NPmethod 

(naked pupae (soil removed)), which is still currently used because of its 

practicality. Sex ratio in CP-line favored females in all generations (1.1 2: 1), except 

one (generation 15, 0.7:1; data stem from generations 4 to 20). In the case of the NPline, 

sex ratios varied more, fluctuating between 0.5 and 2.5:1 (34 generations 

considered). Parasitism rates varied initially between 2.2 6.0 and 2.8 7.4% in the CP 

and NP lines (first 12 generations obtained using each rearing method). Currently 

(generations 35 42 in NP method), parasitism rates have reached 21.491.1% (range 

11.3 27.1%, n 16), and sex ratios fluctuate between 0.4 and 2.5:1. Data on mean 

parasitism rates, mean proportion of emerged females and pupal viability for the first 

12 generations are shown in Table 3. 

Results of the experiments to determine optimal pupal age are summarized in Table 

4. Under choice conditions, parasitism in pupal age classes 0 2, 3 5, and 6 8 was 

significantly higher than in age classes 9 11, 12 14, and 15 17 (one-way ANOVA, 

F5,24 78.43, PB0.0001). Similar results were obtained in the no-choice experiment 

(one-way ANOVA, F 5,24 31.46, PB0.0001). Mean parasitism in the optimal pupal 

age class varied between 60 and 70% in the no-choice experiment and between 36 and 

55% in the choice one (further details in Table 4). With respect to sex ratios, in both 

choice and no-choice experiments, mean proportion of females was similar in 

parasitoids emerging from pupae within the first 5 age classes (i.e. 0 2, 3 5, 6 8, 9 

11, 12 14) but different when compared to the sixth age class (15 17 days) (one-way 

ANOVA, F 5,24 3.50, P 0.0161 and F 5,24 6.26, P 0.0008 for the choice and nochoice 

conditions, respectively) (Table 4). There were no statistically significant 

differences among age classes with respect to the proportion of unviable (i.e. 

unemerged) pupae in the choice experiment (F 5,24 0.99, P 0.4425). The situation 

Table 4. Percent parasitism, sex ratio (proportion of females) and proportion of uneclosed 

pupae in the experiments designed to determine the optimal host age (Anastrepha ludens 

pupae) for Coptera haywardi. Experiments conducted under choice (pupae of varying ages 

offered simultaneously to ovipositing females) and no-choice conditions (females offered pupae 

of only one age class). 

Choice experiment (mean9SEM) No-choice experiment (mean9SEM) 

Age 

class of 

A. ludens 

pupae % Parasitism % Females 


% Unemerged 

pupae % Parasitism % Females 

% Unemerged 

pupae 

0 2 55.392.3a 52.794.1a 37.395.8a 60.795.0a 45.197.0a 30.795.9ab 

3 5 46.093.9ab 52.297.2a 44.795.9a 68.095.1a 55.2912.8a 17.394.6a 

6 8 36.795.5b 32.395.9a 57.396.4a 60.998.0a 55.796.2a 28.798.1ab 

9 11 20.092.9c 42.2917.4a 58.099.6a 43.3910.7a 76.192.2a 41.395.5ab 

12 14 3.391.1d 20.0920.1a 66.798.8a 12.792.7b 48.3920.5a 52.095.7b 

15 17 0.090.0d 0.090.0b 52.7916.1a 0.090.0b 0.090.0b 51.792.2b 

Means within a column followed by the same letter are not significantly different (Tukey HSD test, P 

0.05).


changed in the case of the no-choice experiment, since significant differences were 

detected (F5,24 5.91, P 0.0011) (Table 4). 

Demographic parameters 

Reproductive and population parameters for larval-prepupal and pupal parasitoid 

species are summarized in Table 5. Highest GFR, NFR, Ro, r,andlwere recorded in 

the diaprid C. haywardi and in the braconid D. crawfordi. Mean generation time (T) 

was longest in the case of C. haywardi and A. pelleranoi, while it was short and 

similar in D. areolatus, D. crawfordi, and O. hirtus. Mean life spans in all larvalprepupal 

parasitoid species were quite short (B15 days). In contrast, in the pupal 

parasitoid C. haywardi lifespan was almost twice as long (Table 5). Survivorship 

curves for all species are shown in Figure 5. 

Discussion 

Our multiyear effort aimed at domesticating and colonizing various native fruit fly 

parasitoids resulted in many practical lessons that will hopefully facilitate similar 

efforts elsewhere in the world. Clearly, there were a number of major hurdles to 

overcome before successful establishment of stable colonies was achieved: (1) 

availability of large enough numbers of wild parasitoids to start a colony in the 

cases of rare species like O. hirtus. (2) Availability of a stable supply of high quality 

larval or pupal hosts. (3) Finding a fruit species that is available year round and that 

emits volatiles attractive to as many parasitoid species as possible and that can 

therefore be used to entice females to lay eggs under highly artificial laboratory 

conditions (e.g. guava in our case). (4) Building oviposition units that expose 

sufficient larvae to the attack of females with varying ovipositor sizes. (5) 

Overcoming the initially highly male-biased sex ratio, presumably due to lack of 

mating that in many cases led to the demise of the incipient colony. (6) Overcoming 

apparent pheromone saturation in the small rearing cages that can lead females to 

not mate or do so reluctantly. (7) Finding ideal environmental conditions to suit the 

idiosyncrasies of each species. (8) Cost considerations as the domestication and 

colonization processes are labor and material intensive and therefore end up being 

expensive. 

As noted by Vargas et al. (2002), knowledge on parasitoid demographic 

parameters is critical when trying to select candidate species for fruit fly biological 

control. Our data here, added to the wealth of knowledge already accumulated on 

the basic biology and ecology of native Anastrepha parasitoids (e.g. Sivinski et al. 

1997, 2000; Aluja et al. 1998, 2003; Eitam et al. 2003, 2004) highlights the potential 

that species such as D. crawfordi, O. hirtus and C. haywardi have for augmentative 

release programs in regions with variable climatic and host density conditions. 

Furthermore, in many Latin American countries, in addition to dealing with 

pestiferous Anastrepha species, the presence of C. capitata is often the main concern 

for growers. Two of the species colonized here (i.e. A. pelleranoi and C. haywardi) are 

the only native parasitoids shown so far to be able to attack this important 

agricultural pest (Ovruski et al. 2004, 2005). 

Being able to choose among many parasitoid species opens up the possibility to 

release the one best adapted to the particular climatic and ecological conditions of a

Table 5. Basic demographic parameters for seven native Anastrepha larval-prepupal and pupal parasitoids successfully colonized. 

Demographic 

Parameter (Mean9SEM) D. areolatus 1 

Parasitoid species 

D. crawfordi 1 U. anastrephae 2 O. hirtus 1 

A. pelleranoi 2 O. anastrephae 2 C. haywardi 3 

Offspring sex ratio (female 

proportion) 

58.5896.45 50.4392.98 49.4192.58 60.2593.84 55.0293.79 30.9094.97 56.4192.47 

Cohort lifespan (days) 9.8290.41 11.0990.11 10.5091.37 11.2393.02 7.9491.11 5.3490.35 28.0491.87 

GFR (gross fecundity rate) 

(offspring/female) 

6.6191.75 29.1598.30 2.8790.40 6.2792.04 13.5791.84 13.2692.20 85.1396.63 

NFR (net fecundity rate) 

(offspring/female) 

2.1990.41 10.6891.40 2.6490.34 2.1490.37 5.1790.74 5.5490.81 63.7090.76 

Ro (net reproductive rate) (female 

offspring/generation) 

1.3990.16 5.3690.66 1.3490.20 1.2790.13 2.8490.53 1.4490.21 35.2490.78 

r (intrinsic rate of increase) (per 

female per day) 

0.0390.01 0.2490.04 0.0790.04 0.0390.01 0.1390.03 0.0990.03 0.2590.01 

l (finite rate of increase) (per day) 1.0490.01 1.2790.05 1.0890.04 1.0390.01 1.1590.03 1.0990.03 1.2890.01 

T (mean generation time) (days) 8.6590.87 7.6991.44 3.0890.39 8.4690.68 7.4990.45 4.0790.73 14.3790.39 

1 Individuals stemmed from colonies that were 14 ( 1 ), 9 ( 2 ) and 24 ( 3 ) generations old, respectively. 



Survivorship 

1 

0.9 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

C. haywardi 

D. areolatus 

U. anastrephae 

D. crawfordi 

O. hirtus 

A. pelleranoi 


0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 

Age (days) 

Figure 5. Survivorship (lx) curves for Doryctobracon areolatus, D. crawfordi, Opius hirtus, 

Utetes anastrephae, Aganaspis pelleranoi, Odontosema anastrephae and Coptera haywardi. 

fruit growing region (e.g. temperature, rainfall, host density, larval host) which can 

greatly influence the efficacy of the control agent released or strategy implemented 

(Ovruski et al. 2000; Sivinski et al. 2000). In Mexico, a good example of the latter is 

represented by the native D. crawfordi and the recently introduced (1954 1955, 

quoted in Jiménez-Jiménez 1956) exotic species D. longicaudata which, given the 

short time of their interaction (B50 years), have not been able to partition the niche 

in which they forage in nature (Miranda 2002). Both have long ovipositors (Sivinski 

et al. 2001) and thus are able to attack third instar A. ludens larvae in large fruit such 

as Citrus sinensis, C. paradisi and M. indica in perturbed environments (López et al. 

1999) where they exhibit similar distributions in tree canopies (Sivinski et al. 1997). 

Of the two species, D. longicaudata has already been successfully released 

augmentatively to reduce populations of A. ludens and A. obliqua in mango 

plantations in warm, lowland areas of the Soconusco region in Chiapas, Mexico 

(Montoya et al. 2000). Interestingly, here we found that D. crawfordi was not only the 

species exhibiting the highest r values of all larval-prepupal parasitoids studied 

(Table 5), but its intrinsic rate of population increase was twice as high as the one 

reported for D. longicaudata reared on Bactrocera dorsalis (Hendel) under laboratory 

conditions (Vargas et al. 2002). As documented by Sivinski et al. (2000), D. 

crawfordi, in contrast to D. longicaudata, prefers more humid, temperate environments 

and does not enter diapause (which is the case with D. longicaudata; Aluja et 

al. 1998). According to Miranda (2002), each species should be released singly in 

different environments owing to the fact that they compete for the same resource. A 

particularly interesting potential release site for D. crawfordi is in areas where the 

native A. ludens host (Casimiroa greggii [S. Watts]) is abundant (e.g. canyons and 

mountain slopes in Tamaulipas and Nuevo León, Mexico), allowing fly populations 

to increase and cause damage to commercial citrus groves planted nearby. D. 

crawfordi is indigenous to those areas (González-Hernández and Tejada 1979), 

rendering augmentative releases of this native species instead of the exotic D. 

longicaudata, more environmentally friendly (Simberloff and Stiling 1996). 

Despite the fact that D. areolatus was one of the native species with one of the 

lowest r values, it nevertheless exhibits certain ecological advantages over D.


crawfordi. For example, it is the most widely distributed native fruit fly parasitoid in 

the Neotropics (i.e. Florida to Argentina) and exhibits a close association with A. 

obliqua in native fruit species within the Anacardiaceae (Ovruski et al. 2000). As is the 

case with the exotic D. longicaudata, D. areolatus also prefers warm and drier 

environments at lower altitudes (Sivinski et al. 2000; but see below). Based on the fact 

that Vargas et al. (2002) reported a 4-fold higher intrinsic rate of increase in D. 

longicaudata when compared to what we found here for D. areolatus, the logical 

inference would be that the exotic species is a better candidate for augmentative 

releases. But recent evidence gathered in Florida where both species coexist (Eitam et 

al. 2004), indicates that at least in that part of the world, the distribution of D. 

longicaudata was negatively related to variance in monthly temperatures (it was most 

abundant in southern Florida along the Atlantic and Gulf coasts). These authors also 

reported that D. longicaudata may depend on a constant supply of hosts. In contrast, 

D. areolatus, a species that is able to diapause over extended periods (11 months; D. 

longicaudata did so only over a 7-month period) (Aluja et al. 1998), was the dominant 

species in most interior locations (Eitam et al. 2004). Based on the findings of Eitam 

et al. (2004), in Florida D. areolatus is apparently a superior searcher, while D. 

longicaudata a superior intrinsic competitor. So, as was the case with the previous 

example (D. crawfordi/D. longicaudata), augmentative releases of D. longicaudata 

need to be tailored to local conditions and are not warranted in every location. 

Opius hirtus exhibited similar r and fecundity values as D. areolatus, but together 

with D. crawfordi, was one of the larval-prepupal species that lived longest. Of all the 

braconid species that we successfully colonized, it is the least common and most 

specialized parasitoid (Sivinski et al. 2000; Aluja et al. 2003). Recently, García- 

Medel, Sivinski, Díaz-Fleischer, Ramírez-Romero, and Aluja (2008) showed that it is 

very effective at parasitizing hosts at very low densities and that it is able to coexist 

with other species such as D. longicaudata. As indicated by LaSalle (1993), many 

times rare parasitoid species exert a significant regulatory effect on pests. All the 

above renders O. hirtus an interesting candidate for more wide scale tests. 

The fourth species of native braconid parasitoid that we were able to colonize 

was U. anastrephae. In nature, this species is specialized at attacking A. obliqua and 

A. fraterculus in small fruit within the Anacardiaceae (e.g. Spondias spp.) and 

Myrtaceae (e.g. Psidium spp., Eugenia spp., Myrcianthes spp.), respectively (Sivinski 

et al. 1997; López et al. 1999; Ovruski et al. 2004). The detailed studies by Sivinski et 

al. (1997) discovered an apparent partitioning of the niche in S. mombin trees, with 

U. anastrephae being most abundant in interior parts of the canopy preferentially 

infesting smaller fruit, while D. areolatus was most abundant in exterior parts of the 

canopy and infested larger fruit. Program managers would have to ascertain if any of 

these characteristics are of interest when deciding about new potential candidates for 

augmentative releases. 

Of the two figitid species we were able to successfully colonize, A. pelleranoi offers 

various interesting attributes. On the one hand, and in contrast to the braconid 

species, it preferentially forages on the ground where it attacks larvae in fallen fruit 

(Sivinski et al. 1997; Ovruski et al. 2004). It does so in a wide range of hosts that 

varies greatly with respect to physical and chemical characteristics (Wharton et al. 

1998; Ovruski et al. 2000). On the other hand, it is one of the few native parasitoid 

species in the New World that is able to attack C. capitata (Baeza-Larios et al. 2002a; 

Ovruski et al. 2004, 2005).


Coptera haywardi (the only pupal parasitoid the colonization of which we describe 

here), was the species exhibiting the longest survival and highest fecundity, and 

exhibited r values similar to those found in D. crawfordi. We also show here that it can 

attack pupae of highly contrasting age classes (i.e. 0 2 to 9 11 days of age). 

Furthermore, C. haywardi produced high rates of pupal mortality (85 92%). Similar 

observations were previously reported by Sivinski et al. (1998b) with A. suspensa 

(Loew) and Guillén et al. (2002) with A. ludens pupae. Considering all the above, and 

the fact that C. haywardi is an endoparasitoid that only attacks tephritid flies (Sivinski 

et al. 1998b), among them C. capitata and several species within Anastrepha, it 

represents an ideal candidate to substitute generalist, cosmopolitan species such as 

P. vindemiae, Spalangia endius Walter and S. cameroni Perkins, which are known 

primarily as parasitoids of synantropic flies (e.g. in poultry sheds) (Morgan 1986). 

We conclude that, given the relatively fast adaptation of these organisms to 

laboratory conditions, it is feasible to mass rear most of them. As a matter of fact, in 

the case of D. crawfordi, A. pelleranoi, and C. haywardi, successful attempts at massrearing 

have already taken place in the fruit fly and parasitoid mass-rearing facilities 

of the Medfly Program in Metapa de Domínguez, Chiapas, Mexico and, in the case of 

C. haywardi, the La Aurora rearing facility in Guatemala City, Guatemala (see Baeza- 

Larios, Sivinski, Holler, and Aluja 2002b). Furthermore, as reported by Cancino et al. 

(2008), with the exception of A. pelleranoi and O. anastrephae, native parasitoids can 

be successfully reared using irradiated larvae or pupae. As discussed above, 

demographic parameters from well-established colonies such as ours might guide 

mass-rearing and control programs. They indicate, all other things being equal, which 

parasitoids might increase at the greater rate and thus are cheaper to produce. 

Furthermore, the effectiveness of the parasitoid species successfully colonized here 

should not be limited to Mexico, but rather they should be amenable to introduction, 

augmentation and conservation in many other tropical areas (e.g. Costa Rica, 

Colombia, Venezuela, Brazil, Bolivia, Argentina) where fruit flies such as A. 

fraterculus, A. obliqua, A. ludens, A. serpentina, A. striata, and A. sororcula are 

important pests. Gates et al. (2002) have highlighted three important benefits of the 

use of native parasitoids in biological control: (1) avoidance of costly and prolonged 

trips abroad in search of candidate species, (2) avoidance of cumbersome importation 

and quarantine protocols, and (3) avoidance of potential non-target effects on local 

fauna (also see Simberloff and Stiling 1996). Importantly, and given the massive rate 

of deforestation prevalent in Latin America, on top of searching for native species as 

potential fruit fly biological agents, we also need to foster the conservation of natural 

habitats, to enhance local parasitoid reservoirs and prevent the local extinction of rare 

species such as O. hirtus (Aluja 1996, 1999; Aluja et al. 2003). 


We thank two anonymous reviewers and the editor for helping us produce a more polished final 

product. We also wish to acknowledge Juan Rull, Jaime Piñero, Diana Pérez-Staples, Astrid 

Eben, Francisco Díaz-Fleischer and Ricardo Ramírez (all Instituto de Ecología, A.C.) and 

Patrick Dohm for revisions and suggestions on improving the manuscript. We thank Drs Robert 

Wharton (Texas A&M University) for identifying all the braconid and figitid parasitoids 

described here and Lubomir Masner (Canadian Bureau of Land Resources), for identifying 

C. haywardi. Thanks are due also to Jorge A. Müller and his collaborators (Comité Estatal de 

Sanidad Vegetal, Xalapa, Veracruz, México) for donating the A. ludens laboratory strain we 

used to rear all parasitoids. We appreciate the important technical support of Isabel Jácome,


Emmanuel Herrera, Cesar Ruíz, Gloria Lagunes, Guadalupe Trujillo, Cecilia Martínez, 

Alejandro Vázquez, Graciano Blas, Alberto J. Mata, and Braulio Córdoba (all Instituto de 

Ecología, A.C.). We especially thank Faustino Cabrera Cid and family (Tejería), Othón 

Hernández (Llano Grande) and Pablo Ventura (Los Tuxtlas) for allowing us to work in their 

orchards in order to collect parasitoids. Thanks are due to Nicoletta Righini and Alberto 

Anzures (both INECOL) for formatting and final preparation of this manuscript, and to Darío 

García-Medel for preparing Figures 2 4. This work was principally financed with resources 

from the Mexican Campaña Nacional Contra Moscas de la Fruta (Secretaría de Agricultura, 

Ganadería, Desarrollo Rural y Pesca Instituto Interamericano de Cooperación para la 

Agricultura [SAGARPA-IICA]) and the US Department of Agriculture, Agricultural Research 

Service (USDA-ARS Agreement No. 58-6615-3-025). Complementary resources were 

provided by the USDA Office of International Cooperation and Development (OICD Project 

No. 198-23), the Mexican Comisión Nacional para el Conocimiento y Uso de la Biodiversidad 

(Project No. H-296), and the Consejo Nacional de Ciencia y Tecnología Sistema Regional del 

Golfo de México (Project 96-01-003-V). Sergio Ovruski acknowledges the CONICET- 

Argentina for support during his research stays in México during 2003 and 2004. MA also 

acknowledges support from CONACyT through a Sabbatical Year Fellowship (Ref. 79449) and 

thanks Benno Graf and Jörg Samietz (Forschungsanstalt Agroscope Changins-Wädenswil 

ACW), for providing ideal working conditions to finish writing this paper. 

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N.C. Leppla, Boulder, CO: Westview Press, pp. 405 426. 

Appendix1. Composition of the artificial diet used to rear A. ludens larvae under laboratory 

conditions. 

Amount Ingredients 

100 g Dried Yeast (Type B-Torula), Lake States, Rhinelander, WI, USA 

100 g Natural wheat germ, Nutrisa SA de CV, México DF 

100 g Refined sugar 

150 g Sugar cane bagass (from local sugar refinery) OR Corn cob fractions, Mt. Pulaski, 

Products, Inc. 

8 g Sodium benzoate, Baker (J.T. Baker SA de CV, Xalostoc, Edo. de México) 

2 mL Hydrochloric acid, Baker (J.T. Baker SA de CV, Xalostoc, Edo. de México) 

2 u Viterra Plus capsules, Pfizer (Pfizer SA de CV, Toluca, Edo. de México) 

750 mL Distilled water 

These amounts are recommended for seeding of 2 mL of eggs and the production of 2,500 to 3,000 larvae.


Vol. 19, S1, 2009, 81 94 

The suitability of Anastrepha spp. and Ceratitis capitata larvae as 

hosts of Diachasmimorpha longicaudata and Diachasmimorpha tryoni: 

Effects of host age and radiation dose and implications for quality 

control in mass rearing 

Jorge Cancino a *, Lia Ruíz a , Patricia López a , and John Sivinski b 

a Desarrollo de Métodos, Campaña Nacional Contra Moscas de la Fruta, Tapachula, Chiapas, 

México; b Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 

Gainesville, FL, USA 

The emergence of parasitoids from irradiated tephritid host larvae of different 

species and ages was evaluated. Parasitoid and fly longevity and fecundity resulting 

from each treatment were also assessed. Doses of 5, 10, 20, 30, 40, 50, 80, 100 and 

150 Gy were applied to samples (100 larvae) of 6-, 7-, 8- and 9-day-old Anastrepha 

spp. larvae (A. ludens (Loew), A. obliqua (Mcquart) and A. serpentina (Wiedemann)) 

and 6- and 7-day-old Ceratitis capitata (Wiedemann) larvae. Anastrepha 

larvae were exposed to Diachasmimorpha longicaudata (Ashmead), and C. capitata 

larvae to D. tryoni (Cameron). Following larval exposures of 20 Gy, fly emergence 

was totally suppressed in all larval ages of A. ludens and A. serpentina, while in A. 

obliqua and C. capitata, total suppression was achieved at 30 Gy. In all species, fly 

emergence decreased with increasing radiation dosages from 5 to 20 Gy. Emerged 

fly fertility and longevity also decreased as the radiation increased. On the other 

hand, parasitoids did not suffer decreases in longevity or fecundity as host 

radiation dose increased. Larval age at the time of irradiation did not influence 

emergence, longevity and fecundity of either flies or parasitoids. When the 

irradiated cohort size was raised to one liter of larvae (about 32,000 Anastrepha 

or 50,000 C. capitata larvae) a dose of 40 Gy in A. ludens, A. serpentina and A. 

obliqua totally suppressed fly emergence but permitted D. longicaudata emergence, 

while for C. capitata larvae, it was necessary to increase the dose to 60 Gy. Quality 

control tests under mass rearing conditions were applied to D. longicaudata reared 

using irradiated A. ludens larvae. There was no statistical difference between 

parasitoids derived from irradiated or non-irradiated host for most parameters. 

Only percent pupation after 72 h differed, along with the consequent differences 

between the percent emergence and pupal weight. The conclusions drawn from this 

study lead to a greater flexibility in the use of irradiated hosts in the mass rearing of 

the fruit fly parasitoids D. longicaudata and D. tryoni. 

Keywords: Diachasmimorpha longicaudata; Diachasmimorpha tryoni; irradiated 

host; fruit fly parasitoids; mass-rearing parasitoids; quality control; Anastrepha; 

Ceratitis 

Introduction 

Radiation is an important and novel technique in the mass rearing of natural 

enemies. Radiation has led to advances such as host storage, emergence suppression, 

*Corresponding author. Email: jcancino@ecosur.mx 



DOI: 10.1080/09583150802379577 


82 J. Cancino et al. 

and ease of host manipulation (Morgan, Smittle, and Patterson 1986; Sivinski and 

Smittle 1990; Roth, Fincher, and Summerlin 1991). The mass rearing of fruit fly 

parasitoids has taken an important step forward with the use of radiation. Host 

irradiation has permitted the suppression of emergence from unparasitized hosts 

without affecting the fecundity or longevity of the adult parasitoids that emerge 

(Sivinski and Smittle 1990; Cancino, Ruíz, Gómez, and Toledo 2002). The earliest 

experiments applied radiation to Bactrocera dorsalis (Hendel) pupae that had been 

previously parasitized by Diachasmimorpha longicaudata (Ashmead) in order to 

obtain parasitoids and sterile flies at the same time. Unfortunately, sterility was 

found in both species (Ramadan and Wong 1989). Sivinski and Smittle (1990) 

reported the emergence of D. longicaudata parasitoids and the suppression of 

Anastrepha suspensa (Loew) emergence when host larvae were irradiated before 

exposure to parasitoids. As a result, irradiation was incorporated into mass-rearing 

procedures for various species of fruit fly parasitoids. Some adjustments and 

applications were proposed by Cancino et al. (2002) using Anastrepha ludens (Loew) 

as host to D. longicaudata that further simplified the management of large quantities 

of parasitoids. Host irradiation in the mass rearing of fruit fly parasitoids is currently 

found useful in many laboratories (Brazil, Florida, Guatemala, Mexico and Peru) 

(Sivinski et al. 1996; Baeza, Sivinski, Holler, and Aluja 2002; Cancino et al. 2002). 

However, some effects of radiation on different species remain to be analyzed. 

There is little known about the effects of the radiation on larval hosts of different 

ages. This information is vital because the age of a host larva is a determinant of host 

size and weight, percent parasitism, and emergence of adult parasitoids (Wong and 

Ramadan 1992; Wong 1993). Other aspects, such as the optimal dose for massrearing 

in the different host species and the effects of dose on parasitoid quality 

control parameters must also be assessed. 

In this study, larvae of three species of the genus Anastrepha: A. ludens, (Loew) A. 

obliqua (Mcquart) and A. serpentina (Wiedemann) were evaluated at different ages as 

hosts to D. longicaudata. In the same fashion, different larval ages of Ceratitis 

capitata (Wiedemann) were compared as hosts for Diachasmimorpha tryoni 

(Cameron). Various radiation doses were then evaluated in each host species with 

their respective parasitoids. The effects of host irradiation on parameters of quality 

control in D. longicaudata were also determined. All these appraisals will ultimately 

improve the use of radiation in the mass rearing of larval fruit fly parasitoids. 


Parasitoids and flies evaluated were taken from their respective colonies maintained 

in Moscamed-Moscafrut Program ubicated in Metapa de Domínguez, Chiapas, 

Mexico. Adult parasitoids of D. longicaudata and D. tryoni were obtained from the 

Moscafrut Plant and the Biological Control Department. These strains have been 

maintained under laboratory conditions for over 300 generations. Larvae of A. 

ludens, A. obliqua and A. serpentina were obtained from the laboratory colonies of 

the Rearing and Colonization Department. Ceratitis capitata larvae were taken from 

the strain kept under mass rearing conditions. 

The irradiation of larvae was performed in a Gammacell 220 with a Co 60 source 

of g radiation. The doses were applied with a range of 2.5 3.0 Gy/min in free oxygen. 

Exposure times were determined with Fricke dosimetry (IAEA 2001).


Irradiating host Anastrepha and C. capitata larvae at different ages and radiation 

effects on emergence of parasitoids 

Anastrepha ludens, A. obliqua and A. serpentina larvae aged 6, 7, 8 and 9 days old and 

C. capitata larvae aged 6 and 7 days old were subjected to irradiation doses of: 5, 10, 

20, 30, 40, 50, 80, 100 and 150 Gy. The irradiated larvae of Anastrepha were then 

exposed to D. longicaudata and C. capitata larvae to D. tryoni as described below. 

Non-irradiated larvae were used as the control. 

A sample of 100 larvae per species, age and dose were exposed to 30à:15ß 

parasitoids for 2 h. Five- to 10-day-old parasitoids were put into a ‘Hawaii-type’ 

screen cage (27 27 27 cm) (Wong and Ramadan 1992). The larval exposure 

unit consisted of a Petri dish cover containing larvae and diet, and then covered 

with a piece of organza cloth held in place with a circular plastic top. Following 

exposure a cylindrical plastic container (8 4 cm) with vermiculite was placed 

into each treatment and held at 268C and 60 80% R.H. The data taken 

consisted of the number of emerged adults (parasitoids and flies), larval, and 

pupae weight. 

Fecundity and longevity in adults derived from irradiated hosts 

Samples of 10à:5ß adults from each treatment were evaluated for longevity and 

fecundity. For each D. longicaudata treatment, 50 A. ludens larvae were exposed to 

parasitoids that had emerged from A. ludens, A. obliqua and A. serpentina larvae. 

Fifty C. capitata larvae were exposed to D. tryoni parasitoids emerged from C. 

capitata larvae. The larval exposure and holding were as described above. Following 

adult emergence, daily fecundity was calculated as the number of offspring eclosed 

daily per female (offspring/female/day). Fecundity of females from age 5 to 15 days 

was evaluated. Dead parasitoids found in each cage were removed and counted daily 

for 15 days. 

The fertility of the emerged flies was also evaluated with samples of 10à:10ß. 

When females reached 12 days of age, an oviposition substrate was provided per cage 

for 2 h daily. The oviposition unit was a green agar ball (2 cm diameter) covered with 

parafilm paper (Boller 1968). The eggs oviposited in the unit were collected with a 

scalpel. A sample of 100 of the collected eggs was then immediately incubated inside 

a Petri dish with a piece of filter paper saturated with water. After 5 days, the eggs 

were observed with a stereoscope to count the number hatched. Fertility was 

calculated as the number of hatched eggs/female/day. 

Longevity, fecundity and fertility of adults emerged from irradiated larvae were 

compared with the control treatments of parasitoids and flies emerged from nonirradiated 

larvae. 

Radiation dose and host emergence under mass rearing conditions 

In these experiments, the radiation doses that suppress emergence of unparasitized 

flies were analyzed. Again, larvae of A. ludens, A. obliqua and A. serpentina (8 days 

old) were exposed to D. longicaudata and C. capitata larvae (7 days old) were 

exposed to D. tryoni. ForA. ludens and A. serpentina larvae, doses of 20, 30, 40 and 

50 Gy were used. In A. obliqua and C. capitata larvae, 30, 40, 50 and 60 Gy were


applied. In each treatment, 1 L of larvae (approximately 32,000 Anastrepha spp. 

larvae and 50,000 C. capitata larvae) were irradiated. A sample of 200 larvae from 

each liter was then exposed to parasitism. Exposure periods lasted 2 h and the 

subsequent procedures were similar to those used in mass rearing (Cancino 2000). 

After exposure, larvae were removed from their diet and placed in trays (8 4 cm) 

with vermiculite. Before adult emergence, 14 days following pupation, three samples 

of 100 pupae per treatment were taken and put into a cylindrical plastic container 

(8 4 cm). The number of emerged parasitoids and flies was counted. 

Quality parameters in the mass rearing of D. longicaudata with irradiated 

A. ludens larvae 

Ten lots of mass produced D. longicaudata from irradiated and non-irradiated larvae 

were evaluated by applying various quality control tests. The parameters were: 

Percent mortality and pupation of the host at 72 h. Three samples of 100 larvae per lot 

were taken 72 h after exposure to parasitoids. In each sample, the numbers of pupae, 

live and dead larvae were counted and the data were used to calculate the percent 

pupation and mortality. 

Emergence and sex-ratio. Three samples of 100 pupae were taken per lot and put into 

cylindrical plastic containers (8 4 cm). Following emergence, the numbers of 

parasitoids and flies and their sex-ratio were obtained. 

Percentage of adults capable of flight. Three samples of 100 pupae per lot were 

individually introduced into black PVC tubes (10 10 cm). The inside walls were 

covered with talcum powder. Each tube was put into a cage (0.5 1 1 m) fitted with 

a light source. Following emergence, the parasitoids walking inside the tube were 

considered ‘non-fliers’ and those outside the tube ‘fliers’. 

Longevity with and without food. 30à:15ß newly emerged adult parasitoids per lot 

were held in screen cage and were either provided with honey and water or deprived 

of both. Daily mortality inside the cages was registered from day one. The test was 

carried out until the 15th day when honey and water were present and only until the 

7th day when no food or water were provided. 

Fecundity. One sample per lot of 30à:15ß was placed into a screen cage. When the 

females were 5 days old and continuing until they were 15 days old, daily host 

exposures were carried out with a Petri dish-type oviposition unit containing 50 A. 

ludens larvae. Following exposure, larvae were maintained in a container with 

vermiculite for 15 days at 268C and6080% R.H. The number of offspring per day 

was related to the number of live females. 

Olfatometric measurements. Thirty groups of three parasitoid females per lot were 

introduced into a ‘Y’ glass tube olfactometer with arms 21 cm long and 3 cm in 

diameter, and separated by an angle of 858. Air flow (400 ml/min) was provided by 

a pressurized tank. The air flow in each arm bore volatiles from one of two sources: 

mango fruit infested with A. ludens larvae and uninfested mango. A positive 

response was defined as a walk of at least 10 cm into an arm within 5 min. 

Onset of oviposition activity. Samples of 10 female (5 days old) parasitoids per lot 

were introduced into a screen cage with an oviposition unit containing 50 A. ludens


larvae. Over three consecutive days, for a period of 4 h, the number of females posing 

and ovipositing on the artificial unit was observed and recorded hourly. 

Longevity and fecundity under field conditions. A sample of 100à:50ß parasitoids per 

lot were placed into a cage (1 1 0.5 m). Over 15 days (from the first to the 15th 

day of age), the daily mortality of both females and males was recorded, and for a 

period of 10 days (from the 5th to the 15th day) a fresh oviposition unit with 50 A. 

ludens larvae was placed inside daily. A leafy branch was added to the cage to 

simulate environmental conditions. This test was carried out at 25 308C and 70 

90% H.R. The fecundity data were calculated from offspring emergence per day 

and its relationship to the number of live females. Longevity data were obtained 

from the proportion of live parasitoids per day. 

Data analysis 

Twenty replicates were performed for the evaluations of the effects of radiation on 

different aged larvae and their subsequent suitability as hosts. The data were 

obtained by applying a bifactorial design (factors: age of larvae and doses) and 

analyzed by two-way ANOVA. Data with zero value were compared using 

Bonferroni’s test. Fecundity of the progeny was also analyzed with two-way ANOVA 

and Tukey’s multiple range test was used to distinguish means. In the comparison of 

various radiation doses, 10 replicates were carried out and the results were analyzed 

using ANOVA and Tukey’s multiple range test. The means for the quality control 

parameters were obtained from 10 lots of D. longicaudata, and a Student’s t-test was 

applied to these parameters for statistical analysis. Prior to statistical analysis, data 

were checked for ANOVA assumptions and transformed, if needed, by log(x 1), 

arcsine and Box cox transformation. 

Results 

Irradiating host Anastrepha and C. capitata larvae at different ages and radiation 

effects on the emergence of parasitoids 

The percentage emergence and sex-ratio of adult D. longicaudata and D. tryoni 

parasitoids emerged respectively from Anastrepha and C. capitata larvae are shown 

in Table 1. The parasitoid emergence in A. ludens and C. capitata decreased with the 

age of larvae (two-way ANOVA A. ludens: df 3, F 26.98, PB0.000; C. capitata: 

df 1, F 49.42, PB0.000). This inverse relationship was not observed in the other 

species. However, there were significant differences between particular ages in A. 

obliqua and A. serpentina (two-way ANOVA A. obliqua: df 3, F 4.56, P 0.004; 

A. serpentina: df 3, F 2.70, P 0.045). Parasitoid sex ratios were not different 

statistically in A. obliqua and C. capitata (two-way ANOVA A. obliqua: df 3, F 

0.40, P 0.755; C. capitata: df 1, F 3.30, P 0.070). In A. ludens and A. 

serpentine, there were significant differences (two-way ANOVA A. ludens: df 3, F 

6.22, P 0.000; A. serpentina: df 3, F 13.09, PB0.000), but these had no clear 

relationship with larval age. There were no interactions between irradiation doses 

and larval age in any host species.


Table 1. Mean (9 S.E.) of emergence and sex-ratio of parasitoids reared on Anastrepha spp. 

and C. capitata irradiated larvae at different ages. 

Age of larva (days) n Parasitoid emergence (%) Sex-ratio (female/males) 

A. ludens 

6 158 67.5191.10 a 2.1790.12 ab 

7 168 60.9091.07 b 2.4490.11 a 

8 158 57.3991.11 bc 1.7690.11 b 

9 

A. obliqua 

175 57.0991.05 c 2.0390.11 ab 

6 93 38.3391.06 a 1.5190.07 a 

7 100 36.2891.01 ab 1.4290.07 a 

8 99 32.7291.02 b 1.4890.07 a 

9 

A. serpentina 

96 36.9191.04 a 1.4190.06 a 

6 102 50.1091.29 ab 5.1290.07 c 

7 118 48.9291.20 ab 6.0790.64 bc 

8 111 48.4491.25 b 7.9790.70 a 

9 

C. capitata 

88 52.6691.41 a 7.2790.76 ab 

6 206 36.3990.79 a 7.0990.41 a 

7 183 28.3390.84 b 5.9790.42 a 

Means followed by different letters into the same column indicate a significant difference. Data was 

analyzed through two-way ANOVA followed by Tuckey Multiple Range test (8 0.05) 

Irradiating host Anastrepha and C. capitata larvae at different ages and radiation’s 

effects on the emergence of flies 

Only in C. capitata was the fly emergence between ages statistically different. Averages 

of 13.1790.99 and 8.6891.05 flies emerged from 6- and 7-day-old larvae, respectively 

(df 1, F 6.31, P 0.013). The fly emergence from irradiated larvae of Anastrepha 

spp. were not different between ages (A. ludens: df 3, F 1.69, P 0.169; A. 

serpentina: df 3, F 0.29, P 0.833; A. obliqua: df 3, F 2.07, P 0.107). In 

Table 2. Mean (9 S.E.) of fly emergence from unparasitized hosts of different species of 

Anastrepha and C. capitata, exposed to various radiation doses. 

Irradiation A. ludens A. obliqua A. serpentina C. capitata 

Dose (Gy) Fly emergence 

0 6.8890.72 a 25.4592.04 a 3.2191.03 a 16.2491.55 a 

5 7.590.72 a 25.6992.07 a 4.6191.17 a 21.2591.55 a 

10 1.2390.72 b 8.492.04 b 0.1491.19 b 18.3291.61 a 

20 0 c 0.5392.04 c 0 c 0.1291.50 b 

30 0 c 0 d 0 c 0.1091.54 b 

40 to 150 0 c 0 d 0 c 0 c 

Means followed by different letters into the same column indicate a significant difference. Data was 

analized through a two-way ANOVA followed by Tuckey Multiple Range test (8 0.05).


general, Anastrepha fly emergence began to be reduced at 10 Gy. In all species, 

emergence at 10 Gy was significantly less than at 5 Gy. Fewer flies emerged at each 

progressively higher dose (A. ludens: df 2, F 57.67, PB0.000; A. obliqua: df 3, 

F 80.93, PB0.000; A. serpentina: df 2, F 18.04, PB0.000). At 30 Gy, fly 

emergence was totally suppressed in the three species of Anastrepha. This complete 

suppression was obtained above 40 Gy in C. capitata. There were statistical differences 

between doses in C. capitata (df 4, F 220.39, P 0.000) (Table 2). 

Longevity, fecundity and fertility in adults derived from irradiated hosts 

Neither the longevity (Table 3) nor fecundity of parasitoids (Figure 1 and 2) were 

affected consistently by host irradiation dose, although there were some significant 

differences amoung host ages. Fertility in flies emerged from unparasitized irradiated 

larvae was very sensitive to dosage (Table 4). In Anastrepha, fertility decreased 

significantly at 5 Gy (A. ludens: df 7, F 5.95, PB0.0001; A. serpentina: df 7, 78, 

F 4.37, P 0.0004; A. obliqua:df 7, 78, F 22.56; PB0.0001). A few flies emerged 

at 10 Gy but they showed abnormalities in their body (wings, legs, antennae, etc.) and it 

was not possible to evaluate their fertility. A clear reduction of fertility was obtained in 

A. ludens and A. obliqua; this was variable with A. serpentina because the oviposition 

unit (green ball of agar) was not an efficient substrate for this species. The larvae of C. 

capitata were more resistant to radiation at 10 Gy than were those of Anastrepha spp. 

But beyond this level, fertility was notably reduced (C. capitata:df 5, 61, F 13.68, 

PB0.0001). For practical reasons, the number of eggs oviposited by the offspring was 

Table 3. Results of statistical analysis of longevity of D. longicaudata and D. tryoni adults 

emerged from different aged larvae of Anastrepha spp. and C. capitata exposed to different 

irradiation doses. 

Age of larva (days) d.f. x 2 

A. ludens 

6 9 4.513 0.875 

7 9 23.33 0.005 

8 9 8.52 0.482 

9 

A. obliqua 

9 19.97 0.018 

6 9 13.74 0.132 

7 9 10.04 0.348 

8 9 29.82 0.000 

9 

A. serpentina 

9 49.37 B0.0001 

6 9 23.78 0.005 

7 9 25.53 0.002 

8 9 35.79 B0.000 

9 

C. capitata 

9 33.25 0.000 

6 9 19.58 0.021 

7 9 16.49 0.057 

P


Longevity (%) 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

A. ludens 

0 5 10 20 30 40 50 80 100 150 

A. obliqua 

A. serpentina 

C. capitata 

larvae age (days): 

6 7 8 9 

0 5 10 20 30 40 50 80 100 150 

0 5 10 20 30 40 50 80 100 150 

0 5 10 20 30 40 50 80 100 150 

Doses (Gy) 

Figure 1. Percent adults surviving to the 15th day of Diachasmimorpha longicaudata and 

D. tryoni emerged from different aged larvae of Anastrepha spp. and Ceratitis capitata exposed 

to different irradiation doses. 

not recorded. Nonetheless, we observed that fewer fly eggs were oviposited by adults 

which had been subjected to higher doses of radiation. 

Adjusting dose to avoid host emergence under mass rearing conditions 

The emergence of adult flies from 1 L of Anastrepha spp. and C. capitata larvae was 

totally suppressed at higher doses (Table 5) (A. ludens: df 2,95, F 529.22, PB 

0.0001; A. obliqua: df 1, 23, F 359.47, PB0.0001; A. serpentina: df 1,21, F 

452.55, PB0.0001; C. capitata: df 2,79, F 219.53, PB0.0001). In Anastrepha 

larvae, doses above 30 Gy were necessary to suppress fly emergence and complete 

suppression of C. capitata was obtained at 50 Gy. Again, there were no negative sideeffects 

of higher doses on parasitoid emergence or sex ratio.

Offspring/female/day 

20 A. ludens 

larvae age (days): 6 7 8 9 

15 

10 

5 

0 

20 

15 

10 

5 

0 

20 

15 

10 

5 

0 

10 

5 

0 

A. obliqua 

A. serpentina 

C. capitata 


0 5 10 20 30 40 50 80 100 150 

0 5 10 20 30 40 50 80 100 150 

0 5 10 20 30 40 50 80 100 150 

0 5 10 20 30 40 50 80 100 150 

Doses (Gy) 

Figure 2. Means (9SE) of fecundity (offspring/female/day) of Diachasmimorpha longicaudata 

and D. tryoni emerged from different aged larvae of Anastrepha spp. and Ceratitis 

capitata exposed to different irradiation doses. 

Quality parameters in the mass rearing of D. longicaudata parasitoids with 

irradiated hosts 

Only the percentage of host pupation at 72 h, pupal weight, and the percentage of D. 

longicaudata parasitoid emergence were statistically different between the parasitoids 

that developed in 45 Gy irradiated versus non-irradiated A. ludens larvae (percentage 

of host pupation at 72 h: t 2.35, P 0.03; pupae weight: t 2.72, P 0.008, and the 

percentage of parasitoid emergence: t 2.48, P 0.015). No significant difference 

was found for the other parameters evaluated (Table 6). 

Discussion 

These results indicated broad tolerances in the use of irradiation for the rearing of 

fruit fly parasitoids. There were no effects of radiation dosage on host-suitability nor


Table 4. Means (9 S.E.) of fertility (percent egg hatch) of flies that emerged from irradiated 

larvae. 

Dose 

(Gy) 

Percent egg hatch 

Host 

age 

(days) A. ludens A. obliqua A. serpentina C. capitata 

0 6 85.2793.07 ab 73.3195.97 ab 5.5893.63 b 73.0499.71 a 

7 89.2792.66 a 78.7592.24 ab 34.8696.46 a 90.43 91.52 a 

8 83.2793.31 ab 85.5792.51 a 9.1496.05 b 

9 80.692.92 ab 92.6291.20 a 7.1593.05 ab 

5 6 58.895.71 c 42.83910.26 c 090 b 74.7699.26 a 

7 75.4693.71 abc 12.6395.86 d 11.6394 ab 82.8295.72 a 

8 77.693.75 abc 51.7596.80 bc 090 b 

9 6895.95 bc 28.9497.27 cd 7.1093. ab 

10 6 11.8594.55 b 

7 28.61912.21b 

Means followed by different letters within each column are significantly different. Data was analyzed 

through a ANOVA followed by Tuckey Multiple Range test (8 0.05). 

did the age of the various larvae substantially interact with dosage. The major 

difference was found between Anastrepha species and C. capitata, i.e. a higher dose 

was required to suppress adult-host emergence in the latter, and this may be related 

with the larval size. During these evaluations, the mean larval weights for all ages of 

A. ludens and A. serpentina were above 21 mg. In A. obliqua, the minimum weight 

was 17.4 mg, while in C. capitata, weight never exceeded 13 mg. The effects of 

radiation appear related to the size of the receiving body (Balock, Burditt, and 

Christenson 1963; Bustos, Enkerlin, Toledo, Reyes, and Casimiro 1992). Fertility of 

emerged flies was affected at a lower dose, 5 Gy, in the larger Anastrepha spp. 

Fertility was maintained in the smaller C. capitata up to 10 Gy. Similar results have 

been published in diverse evaluations of fruit fly larvae irradiated during postharvest 

treatments (Arthur and Wiendl 1996; Hallman and Worley 1999; Toledo, 

Bustos, and Liedo 2001). The studies performed by Bustos et al. (1992) provided 

important support for the irradiation of larval hosts prior to exposition to D. 

longicaudata parasitoids. 

Parasitoids which emerged from irradiated host larvae demonstrated adequate 

levels of longevity and fecundity. The results obtained in these evaluations 

demonstrate that the use of an irradiated host does not have any negative 

repercussions on parasitoid development. The irradiation affects fly pupal development 

and it is independent of parasitoid physiology (Nation, Smittle, Milne, and 

Dykistra 1995). The availability of irradiated larvae in Anastrepha spp. and C. 

capitata could be extended to other fruit fly larval parasitoids particularly larvalprepupal 

parasitoids of the braconid subfamily Opinae (Cancino, Ruíz, Sivinski, 

Gálvez, and Aluja, 2009). 

The radiation doses that were effective for suppression of fly emergence in small 

lots were not effective, however, when large lots of mass reared larvae were exposed. 

The radiation doses used for 1 L of larvae (about 32,000 larvae in Anastrepha spp. 

and 50,000 in C. capitata) had to be raised to 40 Gy for A. serpentina, A. ludens and 

A. obliqua, and 60 Gy were necessary for C. capitata. In addition to species/size

Table 5. Means (9 S.E.) of fly and parasitoid emergence and sex-ratio of D. longicaudata and 

D. tryoni reared on Anastrepha spp. and C. capitata from 1 L of larvae irradiated at different 

doses. 

Irradiation 

Dose (Gy) 


Fly emergence 

(%) 

Parasitoid emergence 

(%) 

Sex-ratio 

(female/males) 

A. ludens 

0 31.1391.37 a 65.4791.39 a 1.1390.03 a 

20 5.3990.46 b 67.6491.33 a 1.1690.02 a 

30 0.6690.21 c 67.8291.32 a 1.2090.01 a 

40 0 d 68.3691.51 a 1.1690.01 a 

50 

A. obliqua 

0 d 68.5991.18 a 1.1790.02 a 

0 30.9393.71 a 20.9391.77 a 1.1290.12 a 

30 0.3690.28 b 20.1892.23 a 1.5590.31 a 

40 0 b 22.3392.70 a 1.0390.31 a 

50 0 b 20.2091.67 a 1.8190.35 a 

60 

A. serpentina 

0 b 21.6293.03 a 2.1890.61 a 

0 19.0894.15 a 34.5093.66 a 1.9490.29 a 

20 0.0890.08 b 41.5893.68 a 1.6890.26 a 

30 0 b 41.7593.57 a 1.4790.09 a 

40 0 b 40.0093.91 a 1.4090.12 a 

50 

C. capitata 

0 b 43.1793.88 a 1.6190.11 a 

0 23.1591.62 a 27.5291.61 a 1.6690.20 a 

30 5.2391.21 b 25.8791.73 a 1.9690.27 a 

40 1.6090.30 c 25.4391.69 a 2.2190.33 a 

50 0 d 26.9392.07 a 2.4090.38 a 

60 0 d 24.3191.93 a 2.5890.36 a 

Means followed by different letters in the same column indicate statistical difference. Data was analyzed 

through a ANOVA followed by Tuckey Multiple Range test (8 0.05). 

affects, other factors, such as the physical condition of the larvae after they have been 

taken off their diet, the number and volume of larvae, and the status of the irradiator, 

influence the required radiation dose. Prior evaluations to determine the optimal 

doses for large quantities of larvae have been carried out. For example, in Mexico, in 

the mass rearing of D. longicaudata at the Moscafrut Plant in Metapa de 

Domínguez, Chiapas, 45 Gy are routinely applied to 10 million A. ludens larvae 

daily with the objective of suppressing adult fly emergence (Cancino et al. 2002). In 

the mass production of D. tryoni using C. capitata larvae as hosts, 70 Gy were 

necessary to avoid the adult emergence of the non-parasitized flies. 

Several mass release field studies in Mexico have reported a high efficiency of 

parasitoids mass reared in irradiated hosts (Montoya et al. 2000). The longevity and 

fecundity data obtained from parasitoids emerged from irradiated larvae in this 

study confirm that these attributes do not suffer any reduction. Moreover, the 

analysis of quality control parameters of parasitoids reared with irradiated and nonirradiated 

larvae did not show any significant difference.


Table 6. Means (9 S.E.) of quality control parameters of D. longicaudata reared on 

irradiated to 45 Gy and non-irradiated larvae of A. ludens. 

Parameter Irradiated host Non irradiated host 

QUALITY OF THE PROCESS 

Host mortality at 72 h (%) 1.1790.20 a 1.2590.22 a 

Host pupation at 72 h (%) 97.0390.28 b 98.3190.24 a 

Pupae weight (mg) 

QUALITY OF THE PRODUCT 

11.6190.12 b 12.0690.12 a 

Emergence of flies from non-exposed larvae (%) 0 b 90.1691.15 a 

Emergence of flies from exposed larvae (%) 0 b 4.4490.60 a 

Parasitoid emergence (%) 65.9491.63 a 59.6991.91 b 

Sex-ratio of parasitoids (female/male) 3.9590.30 a 3.8690.25 a 

Parasitoid fliers (%) 88.3291.18 a 88.7990.90 a 

Longevity and fecundity with food at 20 th day (percent of alive adults) 

Females 69.7095.97 a 66.6795.02 a 

Males 66.6793.68 a 60.0094.87 a 

Offspring/female/day 4.2690.13 a 4.3190.12 a 

Longevity without food at 7 th day (percent of alive adults) 

Female 21.5298.18 a 36.3799.65 a 

Male 

Behavior test 

15.0495.98 a 21.6297.07 a 

Female with positive response to infested mango 

Search and oviposition activity of female 

61.1197.35 a 51.3996.24 a 

Female posing 40.0092.67 a 39.3392.91 a 

Female ovipositing 

Evaluation under field conditions 

20.0092.30 a 20.6792.75 a 

Longevity at female at 20 th day (percent of alive 53.6797.82 a 5096.18 a 

females) 

Longevity of male at 20 th day (percent of alive male) 4596.13 a 3797.35 a 

Offspring/female/day 4.6190.38 a 4.4490.25 a 

Means followed by different letters by row implicate significant differences. Data was analyzed through a 

ANOVA followed by Tuckey Multiple Range test (8 0.05). 

Only the percentage of pupation at 72 h was higher in the non-irradiated larval 

groups. This suggests that irradiated larvae were slower in their transformation to 

pupae, perhaps due to damage of some component(s), structures, glands, hormones, 

etc., that are crucial to the pupation process. This effect was mentioned by Zdérek 

(1985), who included irradiation as an important abiotic factor in affecting the 

pupation time. 

Fortunately, this delay in the pupation of irradiated larvae is not a problem in the 

mass rearing process of these parasitoids. The proper temperature and the artificial 

vermiculite medium promote a high percentage of pupation 72 h after the exposure. 

In general, the use of irradiation in larval hosts of fruit fly parasitoids does not cause 

negative effects to the mass rearing process. The use of irradiation in the mass 

rearing process of fruit fly parasitoids is clearly positive and even indispensable to the 

application of augmentative parasitoid releases as part of a fruit fly integrated pest 

management program.



We thank Paula Hipólito and Floriberto López for their technical help. We would also like to 

thank Yeudiel Gómez and the irradiation staff at the Medfly Plant. We appreciate the 

comments given by Francisco Díaz-Fleisher and Pablo Montoya in the first draft of this 

manuscript. Parasitoids and flies were kindly given by Flor de Ma. Moreno, Julio Domínguez, 

Eduardo Solis and Emilio Hernández. This work was carried out thanks to the support 

received under contract No. 10848 with the IAEA in cooperation with the Medfly Program 

SAGARPA. 

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Vol. 19, S1, 2009, 95 109 

RESEARCH ARTICLE 

Evaluation of sequential exposure of irradiated hosts to maximize 

the mass rearing of fruit fly parasitoids 

J. Cancino a *, L. Ruíz a , J. Hendrichs b , and K. Bloem c 

a Desarrollo de Métodos, Campaña Nacional contra Moscas de la Fruta, Central Poniente 14, 

30700 Tapachula, Chiapas, México; b Joint FAO/IAEA Programme, PO Box 100, A-1400 

Vienna, Austria; c Center for Plant Health Science & Technology (CPHST), 

USDA-APHIS-PPQ, 1730 Varsity Drive, Suite 300, Raleigh, NC 27606, USA 

A series of evaluations were carried out to assess the feasibility of sequentially 

exposing tephritid hosts to a primary and a secondary parasitoid to ascertain if 

the larvae not parasitized by the primary parasitoid could be attacked by the 

secondary parasitoid in the pupal stage, thus optimizing the mass production of 

two or more species of parasitoids. Larvae or pupae of Anastrepha ludens (Loew) 

were exposed either to no parasitoids, the larval parasitoid Diachasmimorpha 

longicaudata (Ashmead) (primary parasitoid), the pupal parasitoids Coptera 

haywardi (Oglobin), Dirhinus sp., and Eurytoma sivinskii (Gates & Grissell) 

(secondary parasitoids), or sequentially to combinations of both the larval and 

pupal parasitoids. As part of all evaluations, host larvae were either irradiated or 

unirradiated. Typically, host larvae are irradiated under parasitoid mass rearing 

to avoid fly emergence from unparasitized hosts. Results show that a second host 

exposure did not increase parasitoid production mainly due to a high incidence of 

mortality caused by multiparasitism. With the exception of pupae exposed to 

C. haywardi obtained from irradiated larvae previously exposed to D. longicaudata, 

multiparasitism was around 50%. This resulted in a reduction in 

emergence of both parasitoids. To some extent, pupal parasitoids discriminated 

among pupae, preferring to oviposit in pupae that were not superparasitized 

previously by D. longicaudata. Notably, pupae resulting from irradiated larvae 

were not appropriate for the development of C. haywardi. In contrast, in the cases 

of Dirhinus sp. and E. sivinskii, adult parasitoids did emerge from pupae resulting 

from irradiated larvae, although emergence was significantly lower than when 

pupae from unirradiated larvae were used. Our findings offer critical insights for 

fine-tuning the possible use of sequential host exposure to maximize parasitoid 

mass production. 

Keywords: Tephritidae; Hymenoptera; irradiation; parasitoid mass rearing; 

multiparasitism; superparasitism; sequential exposure 

Introduction 

In the mass rearing of parasitoids, it is nearly impossible to approximate 100% 

parasitism. There are a number of factors that prevent complete utilization of hosts, 

including defensive adaptations of the host (Godfray 1994; Blumberg 1997; 

Kraaijeveld, Hutcheson, Limentani, and Godfray 2001), parasitoid-induced mortality 

through host-feeding and superparasitism, and technical difficulties in 




DOI: 10.1080/09583150902764140 



exposing all the members of a host cohort to parasitoids during a limited period of 

developmental time. In the mass rearing of native parasitoids of Anastrepha fruit fly 

(Diptera: Tephritidae) percent parasitism is commonly well below 50%. Only in longcolonized 

species such as Diachasmimorpha longicaudata (Ashmead) that have been 

mass reared for more than 300 generations, are parasitism levels of 80% routinely 

reached (Biological Control Department, National Program against Fruit Flies, 

SAGARPA, Mexico, unpublished rearing records). 

Because not all hosts are attacked, the presence of fertile flies in parasitoid 

cohorts destined for augmentative release represents a problem in operational 

programs. The use of irradiated hosts, which preclude fly development but allow that 

of the parasitoid, has been employed as an efficient measure to obtain pure cohorts 

(Sivinski and Smittle 1990; Cancino, Ruíz, Gómez, and Toledo 2002). But despite the 

fact that using irradiated hosts solves the problem of fertile flies, loss of expensive 

host material remains as one of the major problems in parasitoid mass rearing 

protocols. 

There are two means to gain a benefit from these unparasitized hosts. The first is 

to apply a low radiation dose to allow both the emergence of sterile flies and 

parasitoids of good quality (Greany and Carpenter 2000). The other is to employ a 

second parasitoid that can utilize the unparasitized larvae or pupae during a second 

exposure (Sivinski, Vulinec, Menezes, and Aluja 1998). The first option can have the 

disadvantage that, at suboptimal radiation doses, flies often emerge from irradiated 

larvae with physical deformations in wings, antennae or feet (Bustos, Enkerlin, 

Toledo, Reyes, and Casimiro 1992). Furthermore, such flies exhibit low sexual 

competitiveness (Rull, Brunel, and Mendez 2005). The second option is to expose 

hosts that have not been attacked by a primary parasitoid (e.g., an egg or larval 

prepupal parasitoid) to a secondary parasitoid (i.e., a pupal parasitoid). For this 

approach to work optimally, pupal parasitoids should have the capacity to 

discriminate against already parasitized hosts and so avoid multiparasitism (Menezes 

et al. 1998; Sivinski et al. 1998). 

Based on the above, our aim here was to assess parasitism potential of pupal 

parasitoids (Coptera haywardi [Oglobin], Dirhinus sp., and Eurytoma sivinskii [Gates & 

Grissell] [Hymenoptera: Diapriidae, Chalcididae and Eurytomidae, respectively]) 

when offered pupae that stemmed from Mexican fruit fly (Anastrepha ludens [Loew]) 

larvae previously exposed to a larval prepupal parasitoid (D. longicaudata). We also 

wanted to assess the effect of using irradiated larvae on pupal parasitoid performance. 


Study site and insects 

All experiments were carried out in laboratories belonging to the Biological Control 

Department (Methods Development Unit) of the Moscamed-Moscafrut Program in 

Metapa, Chiapas, Mexico. A. ludens larvae, as well as larval and pupal parasitoids, 

were obtained from colonies maintained under laboratory conditions in the various 

mass rearing facilities of the Moscafrut Plant (details in Cancino 2000; Domínguez, 

Castellanos, Hernández, and Martínez 2000, Cancino, Ruíz, Sivinski, Gálvez, and 

Aluja 2009a).


Irradiation of A. ludens larvae 

Eight-day-old larvae handled as described by Cancino, Ruíz, López, and Sivinski 

(2009b), were irradiated using a Gammacell 220 irradiator (Co 60 source) at a rate of 

2.5 3.0 Gy/min. Larvae were exposed until reaching a radiation dose of 40 Gy in a 

cylindrical plastic container (9 4.5 cm). Radiation times were determined using a 

Fricke dosimetry (IAEA 2001). 

Experimental design 

Our study involved the following treatments: (1) irradiated and unirradiated 

A. ludens larvae exposed to the larval pupal parasitoid D. longicaudata; (2) pupae 

derived from irradiated and unirradiated larvae exposed to one of three pupal 

parasitoids, C. haywardi, E. sivinskii and Dirhinus sp.; and (3) irradiated and 

unirradiated larvae exposed first to the larval pupal parasitoid D. longicaudata and 

then the resulting pupae exposed to one of three pupal parasitoids, C. haywardi, 

E. sivinskii and Dirhinus sp. Our design also included irradiated and unirradiated 

A. ludens larvae not exposed to any parasitoid. In the case of single species 

exposures, unirradiated and irradiated larvae (pupae) were exposed separately to 

cohorts of each of the parasitoid species under study over a 10-day period (cohorts 

of larvae [pupae] replaced daily). We tested three cohorts (each one in a separate 

cage) per treatment simultaneously. In the case of sequential, multiple-species 

exposures, we first separately exposed both types of larvae to the larval prepupal 

parasitoid D. longicaudata (primary parasitoid) and then, once the host larvae had 

pupated, to one of the following pupal parasitoids (secondary parasitoid): 

C. haywardi, E. sivinskii and Dirhinus. As was the case with single-species exposures, 

sequential, multiple-species exposures were replicated three times. We did not test 

sequential exposures within the pupal parasitoid guild. 

Larval and pupal exposure and overall parasitoid handling procedures 

Exposure of irradiated larvae to D. longicaudata followed within an hour after the 

irradiation procedure was completed. To expose irradiated and unirradiated larvae 

to the parasitism of D. longicaudata (primary parasitoid), we placed 200 larvae with 

diet in Petri dishes (0.7 9 cm, depth diam) tightly covered with a circular piece of 

organdy cloth (Wong and Ramadan 1992). The 8-day-old larvae were exposed for 

2 h to 30à and 15ß D. longicaudata adults inside ‘Hawaii type’ cages (27 27 27 

cm wooden frame covered with a 1 mm plastic mesh and with a 10 5 cm elliptical 

opening at the base) (Wong and Ramadan 1992). As noted before, every day, over a 

10-day period, we removed the exposure unit (Petri dish containing 200 larvae) once 

the 2-h exposure period was over, and repeated the procedure the next day using a 

new cohort of larvae of the same age. Given that some parasitoids died along the 

way, every morning we replaced all dead individuals with live ones transferred from 

cages that had been kept under exactly the same experimental conditions (i.e., 

parasitoids were also offered larvae every day for 2 h). That way, we assured that 

each cage had the same number of females over the entire experiment and that the 

parasitoids used to replace dead ones were of the same age and had been exposed to 

the exact same experimental conditions up to that point in time. D. longicaudata


individuals were 5-days-old when first exposed to larvae (ended up being 15-days-old 

when experiment was ended). 

After exposure to parasitism, larvae were returned to plastic washbowls filled 

with clean rearing diet (Domínguez et al. 2000) to which we had added corncob grit 

(Mt. Pulaski Products, Inc TM , Mt. Pulaski, Ill) to facilitate handling. Larvae were 

allowed to feed for 24-h and then removed from the washbowl and washed with tap 

water to remove all diet residues. After this, they were placed in a cylindrical plastic 

container (9 4.5 cm) to which a shallow (1.5 cm) layer of vermiculite had been 

added to stimulate pupation (Cancino 2000). Two days later, from these containers 

randomly chosen cohorts of 200 pupae were removed and placed in a Petri dish 

(1.3 9 cm, depth diam) containing a small amount of lightly moistened vermiculite 

(not covering pupae) and exposed to 50à and 50ß adults of one of the 

following three species of pupal parasitoids: C. haywardi, Dirhinus sp., and 

E. sivinskii. All pupal parasitoids were 8-days-old when first exposed to larvae and 

18-days-old when the experiment ended. Inside the Petri dish we placed a piece of 

cardboard (11 cm 2 ) to simulate leaf litter and to also simulate a dark, subterranean 

environment. Experimental procedure was exactly the same as with D. longicaudata 

(i.e., daily exposures of pupae over a 10-day period with daily replacement of dead 

individuals until the experiment was over). After a 24-h exposure period, we removed 

all pupal parasitoids and maintained the pupae in plastic containers (9 4.5 cm) at 

ca. 268C(928C) and 60 80% relative humidity for an additional 15 35 days to allow 

for life cycle completion (species vary with respect to emergence time). 

Unexposed larvae, unirradiated and irradiated, were handled exactly as described 

above, with the exception that they did not suffer parasitism or otherwise had any 

contact with parasitoids. 

Determination of parasitism levels 

Puparia from each species/treatment combination were randomly divided into lots of 

100 pupae each and placed in plastic containers with a small amount of lightly 

moistened vermiculite. On the fourth day after exposure to the pupal parasitoids 

(secondary exposure), we removed a sample of 10 puparia from each container for 

every replicate in each corresponding treatment. These puparia were used to 

determine if super- or multiparasitism had occurred. First, the puparia were 

observed under a stereomicroscope (Stemi SV6, Carl Zeiss, México D.F., Mexico) 

to determine presence and number of oviposition scars. Then, pupae were dissected 

over the following four consecutive days to assess immature-stage development 

(Ramadan and Beardsley 1992; Kazimírova and Vallo 1999). 

In some containers, no puparia were removed until flies and parasitoids started 

to emerge. Flies invariably emerged before any of the pupal parasitoids (independent 

of species). In the case of D. longicaudata, the majority of adults emerged before the 

flies, but after day three, flies and parasitoids started to emerge simultaneously. 

Adult parasitoid emergence was recorded daily starting on the 15th day following 

host-exposure. Development time of the parasitoid species was variable: 

D. longicaudata and E. sivinskii began to emerge at 15 days after exposure to larvae 

(pupae in the case of E. sivinskii), while in the cases of C. haywardi and Dirhinus sp. it 

took up to 35 days before any adult emerged from the puparia. Based on parasitoid 

and fly emergence we calculated percent parasitism.



Analysis of adult emergence data was carried out using a model of repeated measures 

with a correlation structure of either compound, Huynth Feldt, or unstructured 

symmetry (Brown and Pressat 1999). In all cases, a bi-factorial design was applied, 

using as factors double or simple exposure, and larval host condition (irradiated or 

not). Comparison of means was carried out through orthogonal contrasts. For the 

analysis, SAS systems for Windows release 8.02 TS level 02M0 was used. The levels 

of multiparasitism per parasitoid species with irradiated and unirradiated larvae 

(pupae) were analyzed by one-way ANOVA. Data on scar numbers in relation to the 

number of immature stages inside pupae were submitted to a regression analysis. 

Alpha in all cases was 0.05. 

Results 

Results involving the combination of D. longicaudata and C. haywardi are presented 

in Table 1. In the case of D. longicaudata, there was significant difference in 

parasitoid emergence between the hosts that were exposed to both parasitoids and 

those that were exposed to only one (df 1, 96.812; F 55.05; PB0.0001). The 

emergence of D. longicaudata decreased when pupae stemming from exposed larvae 

were utilized as a host for C. haywardi. In both cases, the emergence of 

D. longicaudata derived from irradiated hosts was significantly higher (df 1, 95.2, 

F 7.22, P 0.0085). Importantly, there was no emergence of C. haywardi adults if 

females had parasitized pupae that stemmed from irradiated larvae. In both 

parasitoid species, percent emergence decreased when the host was exposed twice. 

In C. haywardi, this difference was highly significant (df 1, 17.9, F 102.88, 

PB0.0001). Different factors such as natural mortality, superparasitism, and 

multiple parasitism were responsible for lower parasitoid emergence relative to the 

number of exposed hosts. Similar results were obtained in subsequent evaluations. 

As in the previous case, the emergence of D. longicaudata was significantly lower 

when Dirhinus sp. was used as the second parasitoid (df 1, 33.4, F 50.78, 

PB0.001) (Table 2). Based on the statistical analysis, these treatments were 

independent and there was no interaction; analyzed separately there was no 

significant difference between average D. longicaudata emergence from irradiated 

and unirradiated larvae in both treatments (df 1, 33.5, F 2.24, P 0.1440). In the 

case of Dirhinus sp., emergence was statistically similar for pupae resulting from 

irradiated and unirradiated larvae that had been previously exposed to 

D. longicaudata. However, the emergence of Dirhinus sp. from pupae not exposed 

as larvae to D. longicaudata was significantly different between irradiated and 

unirradiated larvae (df 1, 35.8, F 183.09, PB0.0001). When the pupae exposed 

to Dirhinus sp. had not been previously exposed to D. longicaudata in the larval stage, 

then emergence from unirradiated hosts was much higher. Average Dirhinus sp. 

emergence from pupae exposed and those not exposed previously as larvae to D. 

longicaudata was significantly different (df 1, 35.8, F 6.62, P 0.0144) (Table 2). 

In trials where E. sivinskii was the secondary parasitoid, results were quite similar 

to those obtained with Dirhinus sp. (Table 3). As was the case in the previous 

experiment, there was no interaction between treatments. Accordingly, the mean 

percent emergence of D. longicaudata was not statistically different between

Table 1. Parasitoid and fly emergence (mean percentage9SE) in different treatments with one or two exposures of irradiated or unirradiated A. ludens 

host larvae to D. longicaudata and/or C. haywardi. 

Mean proportion of adult emergence 

First exposure Seconnd exposure Treatments D. longicaudata C. haywardi A. ludens 

D. longicaudata C. haywardi Irradiated larvae 48.4891.53 Aa 0 0 

Unirradiated larvae 43.4492.49 Ab 21.0092.77 a 20.4592.26 a 

D. longicaudata No exposure Irradiated larvae 60.9691.85 Ba 0 0 

Unirradiated larvae 56.8891.54 Bb 0 28.6391.68 b 

No exposure C. haywardi Irradiated larvae 0 0 0 

Unirradiated larvae 0 47.4593.25 b 38.4593.47 c 

No exposure No exposure Irradiated larvae 0 0 0 

Unirradiated larvae 0 0 93.0091.74 d 

Means followed by different letters within columns indicate statistical differences. Parasitoid emergence was analyzed using a model of repeated measures with a compound 

symmetry correlation structure; fly emergence was analyzed with a Huynth Feldt structure. Comparison of means was carried out through orthogonal contrasts (a 0.05). 

100 J. Cancino et al.

Table 2. Parasitoid and fly emergence (average percentage9SE) in different treatments with one or two exposures of irradiated or unirradiated 

A. ludens host larvae to D. longicaudata and/or Dirhinus sp. 


First exposure Second exposure Treatments D. longicaudata Dirhinus sp. A. ludens 

D. longicaudata Dirhinus sp. Irradiated larvae 39.7293.57 Aa 11.3591.44 Aa 0 

Unirradiated larvae 34.9692.39 Aa 11.2591.29 Aa 1.3590.82 a 

D. longicaudata No exposure Irradiated larvae 62.5093.75 Bb 0 0 

Unirradiated larvae 60.9292.80 Bb 0 6.4291.15 b 

No exposure Dirhinus sp. Irradiated larvae 0 1.5390.81 Bb 0 

Unirradiated larvae 0 48.7693.15 Bc 24.3092.98 c 


Unirradiated larvae 0 0 84.5792.38 d 

Averages followed by different letters within columns indicate statistical differences. Parasitoid and fly emergence was analyzed using a model of repeated measures with a 

compound symmetry correlation structure. Comparison of means was carried out through orthogonal contrasts (a 0.05). 


Table 3. Parasitoid and fly emergence (average percentage9SE) in different treatments with one or two exposures of irradiated or unirradiated 

A. ludens host larvae to D. longicaudata and/or E. sivinskii. 


First exposure Second exposure Treatments D. longicaudata E. sivinskii A. ludens 

D. longicaudata E. sivinskii Irradiated larvae 46.8092.23 Aa 5.0490.67 Aa 0 

Unirradiated larvae 44.4092.60 Aa 9.6590.95 Ab 7.8692.06 a 

D. longicaudata No exposure Irradiated larvae 61.4792.69 Ba 0 0 

Unirradiated larvae 59.3392.24 Ba 0 9.4692.00 a 

No exposure E. sivinskii Irradiated larvae 0 2.1490.58 Bc 0 

Unirradiated larvae 0 47.1792.93 Bd 33.7493.85 b 


Unirradiated larvae 0 0 86.9291.43 c 

Averages followed by different letters within columns indicate statistical differences. D. longicaudata emergence was analyzed using a model of repeated measures with a 

compound symmetry correlation structure. E. sivinskii and A. ludens emergence were analyzed with a non-structured model. Comparison of means was carried out 

through orthogonal contrasts (a 0.05). 


irradiated and unirradiated larvae analyzed by treatment (df 1, 36.4, F 0.80, 

P 37.80). But as was the case with the other two pupal parasitoids, emergence of 

D. longicaudata decreased significantly when the host was subsequently exposed to 

parasitism by E. sivinskii (df 1, 30.3; F 78.72, PB0.0001). The emergence of 

E. sivinskii from pupae exposed as larvae to D. longicaudata was significantly lower 

than that obtained from pupae not exposed as larvae (df 1, 39.5, F 74.67, 

PB0.0001) (Table 3). Also, the emergence of E. sivinskii from pupae resulting from 

irradiated and unirradiated larvae was significantly different (df 1, 39.5, F 498.5, 

PB0.0001). The highest values of emergence of E. sivinskii were obtained with 

pupae resulting from unirradiated larvae that were not exposed to D. longicaudata. 

As the decreases in individual and summed parasitoid emergences with double 

exposures may be due to mortality caused by multiparasitism, we performed scar 

counts and dissections. When dissected, over 50% of sequentially exposed puparia 

(unirradiated) revealed multiparasitism (Figure 1). No statistical differences in 

multiparasitism were found in the D. longicaudata/Dirhinus sp. combination between 

irradiated and unirradiated hosts (df 19, F 0.1256, P 0.7272). Similarly, there 

were no statistically significant differences in multiparasitism between irradiated and 

unirradiated hosts exposed to the combination D. longicaudata/E. sivinskii (df 19, 

F 0.80, P 0.3829). However, when C. haywardi was employed as the secondary 

parasitoid, significantly more multiparasitism occurred in unirradiated hosts 

(df 19, F 14.2258, P 0.0014). 

Oviposition scars resulting from the penetration of D. longicaudata’s ovipositor 

through the larval cuticle or by that of a pupal parasitoid which pierces the puparium 

directly, facilitate estimates of superparasitism. The relationship between the number 

of scars and the number of immature stages of D. longicaudata and C. haywardi per 

puparium is shown in Figure 2. Note that this relationship is stronger in 

D. longicaudata (i.e., r 2 values were higher for both irradiated and unirradiated 

larvae). The relationship between the number of scars and parasitoid immature 

stages was also higher in D. longicaudata when the host was subsequently exposed to 

Dirhinus sp. and E. sivinskii (Figures 3 and 4). Furthermore, multiparasitism and 

heterospecific parasitism was more frequent in pupae that were not superparasitized 

% Multiparasitised pupae 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

a 


C. haywardi C. haywardi 

b 

a 

a 

a 

Unirradiated 

Irradiated 

Dirhinus sp. Dirhinus sp. E. sivinskii E. sivinskii 

Figure 1. Average percentage (9SE) of multiparasitized A. ludens pupae after exposure to 

two different fruit fly parasitoids, first to the larval parasitoid D. longicaudata and second to 

either the pupal parasitoids C. haywardi, Dirhinus sp., or E. sivinskii. 

a


a) 

No. of immature stages 

b) 


16 

14 

12 

10 

8 

6 

4 

2 

0 

16 

14 

12 

10 

8 

6 

4 

2 

0 

D. longicaudata 

C. haywardi 

r 2 = 0,2446 D. longicaudata 

r 2 = 0,0073 C. haywardi 

0 5 10 15 20 25 

No. of scars 


r 2 = 0,0004 C. haywardi 

0 5 10 15 20 25 

No. of scars 

Figure 2. Relationship between number of pupal scars and the number of immature stages of 

parasitoids C. haywardi and D. longicaudata. (a) Irradiated larvae, (b) unirradiated larvae. 

by D. longicaudata (Figure 5). Finally, superparasitism by pupal parasitoids was 

higher in Dirhinus sp. and E. sivinskii. 

Discussion 

Our results indicate that the use of a primary (D. longicaudata) and a secondary 

(either C. haywardi, E. sivinskii or Dirhinus sp.) parasitoid in sequential fashion to 

optimize their mass rearing is not a viable proposition, at least under the conditions 

tested and with the parasitoid species combinations used. 

Our results agree with first attempts to assess sequential parasitism in mass 

rearing. Menezes et al. (1998) reported similar results with the sequential exposure of 

irradiated and unirradiated Anastrepha suspensa (Loew) to D. longicaudata and 

C. haywardi. The results were very similar in terms of the percentages of parasitism 

reached by each parasitoid with unirradiated larvae. In addition, development of

a) 


b) 


16 

14 

12 

10 

8 

6 

4 

2 

0 

16 

14 

12 

10 

8 

6 

4 

2 


Dirhinus sp. 



r 2 = 0,0675 Dirhinus sp. 

0 5 10 15 20 25 30 35 40 

No. of scars 


r 2 = 0,0046 Dirhinus sp. 

0 

0 5 10 15 20 

No. of scars 

25 30 35 40 


parasitoids Dirhinus sp. and D. longicaudata. (a) Irradiated larvae, (b) unirradiated larvae. 

C. haywardi in pupae resulting from irradiated larvae was not viable. Even though 

sequential exposure of D. longicaudata with Dirhinus sp. and E. sivinskii did allow 

pupal parasitoid development in pupae resulting from irradiated larvae, the situation 

was quite similar. The main problem detected was multiparasitism, which probably 

increased host mortality. 

The phenomenon of multiparasitism is common in nature (Bautista and Harris 

1997; Cusson et al. 2002; Ribeiro, d’Almeida, and Pinto de Mello 2005), and several 

cases have been reported in fruit flies (Pemberton and Willard 1918; van den Bosch 

and Haramoto 1953; Palacio, Ibrahim, and Ibrahim 1991; Leyva 1982; Bautista and 

Harris 1997). In the present study, superparasitism by C. haywardi (an endoparasitoid) 

was relatively low, more frequent in Dirhinus sp., and most common in 

E. sivinskii. This situation was perhaps caused by the paucity of interspecifically 

recognized markers that allow ovipositing females to discriminate between 

parasitized and unparasitized hosts (Godfray 1994; Cusson et al. 2002). However, 

in our studies, oviposition by pupal parasitoids, while present under all conditions,


a) 


b) 


20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 


E. sivinskii 


r 2 = 0,0095 E. sivinskii 

0 5 10 15 20 25 30 35 

No. of scars 


r 2 = 0,1299 E. sivinskii 

0 

0 5 10 15 20 25 30 35 

No. of scars 


parasitoids E. sivinskii and D. longicaudata. (a) Irradiated larvae, (b) unirradiated larvae. 

was less frequent in pupae that had been previously superparasitized by 

D. longicaudata (puparia with multiple oviposition scars). This may not be due to 

discrimination by ovipositing pupal parasitism. Pupae superparasitized by 

D. longicaudata suffer significant biochemical changes when the number of supernumerary 

immature stages is high (Lawrence 1988a,b). Such changes may partially 

explain lower levels of pupal parasitoid survival. 

Pupae resulting from irradiated larvae were less suitable hosts than those derived 

from unirradiated larvae for all of the pupal parasitoids examined, particularly the 

endoparasitic C. haywardi which in our dissections never developed past the second 

instar. This was perhaps due to biochemical and morphological changes resulting 

from the irradiation dose and timing selected. 

We note that when recently irradiated 4-day-old pupae, rather than irradiated 

larvae that are allowed to pupate, are used as hosts for pupal parasitism, then high 

yields of all three species of pupal parasitoids are achieved (Cancino et al. 2009a).

70 

60 

50 

40 

30 

20 

10 

0 

70 

60 

50 

40 

30 

20 

10 

0 

70 

60 

50 

40 

30 

20 

10 

0 


No Irradiated 

larvae 

No Irradiated 

larvae 

No Irradiated 

larvae 

Irradiated 

larvae 

C. haywardi 

1 >1 1 >1 

Irradiated 

larvae 

Dirhinus sp. 

1 >1 1 >1 

Irradiated 

larvae 

E. sivinskii 

1 >1 1 >1 

Larvae of D. longicaudata by host 

Figure 5. Frequency distribution (shades of grey) of number of immature stages of pupal 

parasitoids (y-axis) in either D. longicaudata single parasitized (1) or D. longicaudata 

superparasitized ( 1) unirradiated or irradiated A. ludens host. 

Morgan, Smittle, and Patterson (1986) similarly found that irradiated pupae were 

suitable hosts for a pteromalid pupal parasitoid of calypterate flies. In the present 

case, presumably the type of host development parasitoids require occurs sometime 

between the last day of larval life and the fourth day of pupation. 

Given that (1) multiparasitism, as tested here, results in fewer summed 

parasitoids, and (2) that larval irradiation inhibits the development of the candidate 

pupal parasitoids, the practicality of sequential rearing with irradiated hosts seems 

questionable. However, there remain some possibilities of application. For example, 

while parasitism by Dirhinus sp. and E. sivinskii in pupae from irradiated larvae is 

relatively low, they might still contribute useful numbers if there were a means of 

separating larvae parasitized by D. longicaudata from those left unattacked. The 

latter could be ‘recycled’ to pupal parasitoids without the concerns raised by 

5 

4 

3 

2 

1


potential multiparasitism. Different pupation times between parasitized and 

unparasitized irradiated larvae (J.C. unpublished data), may make such a scheme 

viable. 


Floriberto López offered outstanding technical support throughout the study. We thank 

Larissa Guillén and Darío García-Medel for support during the final stages of manuscript 

preparation. Martín Aluja contributed significant support and significant ideas to the final 

presentation of this manuscript. Javier Valle-Mora proposed the use of statistical methods for 

the analysis of data. This work is the result of research funded by the International Atomic 

Energy Agency (IAEA) under contract No. 10848 and the Mexican Campaña Nacional contra 

Moscas de la Fruta (SAGARPA-IICA). 

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Vol. 19, S1, 2009, 111 125 


Assessment of infective behaviour and reproductive potential over 

successive generations of entomopathogenic nematodes, Steinernema 

glaseri (Rhabditida: Steinernematidae), reared within radiosterilized 

host larvae, towards Spodoptera litura (Lepidoptera: Noctuidae) 

Rakesh K. Seth* and Tapan K. Barik 

Department of Zoology, University of Delhi, Delhi 110 007, India 

The infective behaviour of entomopathogenic nematodes (EPNs), Steinernema 

glaseri (Steiner), reared within radiosterilized host larvae of the tropical pest, 

Spodoptera litura (Fabricius), was ascertained towards the unirradiated same host 

(S. litura) for successive generations and compared with the infectivity of controls 

(EPNs derived from unirradiated host larvae). A primary goal was to establish a 

safe mode of transport and dispersal of EPNs without concern that uninfected, 

reproductively competent hosts would be inadvertently released. Based on prior 

studies on radiation-mediated effects, two gamma doses (40 and 70 Gy), were 

used for radiosterilization of last instar S. litura larvae. Tests were performed 

using the following parameters: Regimen I (Control) with normal infective 

juveniles (N-IJs) vs. normal (N) hosts; Regimen II with N-IJs vs. Irradiated hosts; 

Regimen III with F1 IJs (harvested from Regimen II) vs. N-hosts; and Regimen 

IV with F2 IJs (harvested from Regimen III) vs. N-hosts. The infective 

performance of F1 IJs was affected more at 70 Gy than at 40 Gy, but the effect 

was not great enough to nullify the infective efficiency of IJs emerged from 

irradiated hosts; thus, these IJs could be effectively utilized in pest biocontrol. 

Furthermore, the infective performance of F2 IJs was almost equivalent to that of 

the controls, especially at 40 Gy. Hosts radiosterilized at 70 Gy could be 

considered safer than those exposed to 40 Gy for inundative release of EPNs as 

biocontrol agents. Hosts radiosterilized at 40 Gy or 70 Gy could be conveniently 

used, with greater efficiency at 40 Gy, for inoculative release of EPNs for longterm 

pest management. 

Keywords: entomogenous nematodes; host-irradiation; pest management; 

biocontrol agents; killing efficiency; Spodoptera litura; Steinernema glaseri 

Introduction 

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), a common cutworm of 

polyphagous nature, is an economically serious pest in the Indian subcontinent 

(Lefroy 1908; Moussa, Zaher, and Kotby 1960; Thobbi 1961; Chari and Patel 1983), 

and it is considered one of the major threats to the present day intensive agriculture 

and changing crop patterns worldwide. Increasing environmental hazards 

from chemical pesticides and development of insecticide resistance in S. litura 

(Ramakrishnan, Saxena, and Dhingra 1984; Armes, Wightman, Jadhav, and Ranga 

Rao, 1997) have prompted the development of ecologically sound alternative 

*Corresponding author. Email: rkseth@del2.vsnl.net.in 

First Published Online 27 May 2009 



DOI: 10.1080/09583150902981603 


112 R.K. Seth and T.K. Barik 

methods to control this pest. Among the eco-friendly tactics being developed for 

management of Spodoptera spp., biological control and other biorational approaches 

are paramount (Narayanan and Gopalakrishnan 1987; Seth and Sehgal 1993; 

Barclay and Judd 1995; Armes et al. 1997). 

One such ecologically compatible approach would be to release entomopathogenic 

nematodes (EPNs) as potential augmentative biocontrol agents to control the 

pest, S. litura. EPNs often have a marked ability to find and kill their hosts rapidly. 

EPNs exhibit favourable characteristics including high reproductive rate, virulence, 

and safety for non-target organisms (Kaya 1986; Jansson 1993; Ehlers and Peters 

1995; Boemare, Laumond, and Mauleon 1996; Ehlers and Hokkanen 1996). The 

potential of nematodes as biocontrol agents and their biology, ecology and 

behaviour have been studied extensively (Poinar 1983; Kaya 1985; Gaugler and 

Kaya 1990; Smart 1995; Grewal, Ehlers, and Shapiro-Ilan 2005). Steinernematids 

and heterorhabditids have been reported to infect a number of species of insects in 

several orders (Poinar 1975; Hom 1994). Third stage ‘dauer’ juveniles (J3) are the 

infective, non-feeding stage. In soil, these infective juveniles (IJs) can locate hosts 

with varying degrees of efficiency. Steinernematid and heterorhabditid nematodes 

are symbiotically associated with specific bacterial species in the genera Xenorhabdus 

(Thomas and Poinar 1979) and Photorhabdus (Boemare, Akhurst, and Mourant 

1993), respectively. Once the nematodes have penetrated the host insect, they release 

their bacteria, whose multiplication produces proteolytic enzymes that kill the host 

within 24 48 h (Poinar 1986). Nematodes spend their free-living stage in soil and 

thus soil insect pests and/or soil inhabiting stages of the foliage feeding insects can be 

the ideal targets. 

The residual effect of nematode treatment was reported to be greater than that of 

standard chemical pesticide (Bari and Kaya 1984). The feasibility of using 

entomopathogenic nematodes for the control of some noctuids was demonstrated 

using Steinernema spp. and Heterorhabditis spp. against Pseudaletia unipuncta 

Haworth (Morris 1985; Morris, Converse, and Harding 1990; Morris and Converse 

1991), S. feltiae (Otio) against P. unipuncta and Spodoptera exigua (Hübner) (Kaya 

1985), and S. carpocapsae (Weiser) against S. exigua (Kaya and Hara 1980; Hara 

and Kaya 1983; Begley 1990), Helicoverpa zea (Boddie), S. littoralis (Boisduval) 

(Glazer, Galper, and Sharon 1991), and Actebia fennica Tauscher (West and Vrain 

1997). Steinernema glaseri (Steiner) has been employed successfully for the management 

of target pest species (Bareth, Bhatnagar, and Sharma 2001). 

Nuclear techniques have been useful in elucidating and promoting the genetic 

and biological characteristics of insects, and radiation has played a significant role in 

insect pest management (North 1975; Seth and Sethi 1996). Nuclear techniques can 

prove useful in biological control in various ways, viz., reproductive sterilization of 

insect hosts, provision of non-reproductive supplemental hosts for biocontrol agents, 

safe transport of parasitoids in irradiated hosts, improvement of the suitability of 

hosts for mass rearing, and sterilization of synthetic media to maintain mass culture 

(Greany and Carpenter 2000). 

To enable biocontrol agents to be released along with their hosts into the 

ecosystem in an environmentally safe manner, factitious insect hosts can be used as 

well as radiosterilized hosts, including those of insect pest species. For this approach 

to be successful, it is necessary to verify the parasitization capacity of parasitoids on


radiosterilized hosts and their ability to remain infective after being reared within the 

radiosterilized hosts. 

Entomopathogenic nematodes may be applied in infected insect cadavers 

(Creighton and Fassuliotis 1985; Jansson, Lecrone, and Gaugler 1993), and in this 

approach, nematode-infected cadavers are disseminated and pest suppression is 

subsequently achieved by the progeny IJs that exit the cadavers. Field application of 

EPNs in infected hosts may be superior to application in aqueous suspension, in 

terms of infectivity, dispersal and survival (Shapiro and Glazer 1996; Shapiro and 

Lewis 1999). EPNs can survive dry or harsh conditions or desiccation for extended 

periods within host cadaver (Brown and Gaugler 1996; Koppenhofer et al. 1997). 

Improved persistence within the host cadavers (Perez, Lewis, and Shapiro-Ilan 2003) 

has been reported as compared to aqueous suspensions wherein EPNs might face 

osmotic stress. However, EPNs carried within infected hosts are compromised by 

limitations of storage and application. To an extent, this constraint can be solved by 

improved formulations (Shapiro-Ilan, Lewis, Behle, and Mcguire 2001). It is 

reported that EPNs have the ability to seek out and quickly kill hosts within 

24 48 h (Gaugler 1981) and EPN entry into hosts can occur within 6 10 h (Lewis, 

Campbell, Griffin, Kaya, and Peters 2006). Therefore, after an adequate time of 

exposure to IJs, the host can be transported immediately to the field, but this may 

pose a serious limitation if some viable, non-parasitized host insects escape 

parasitization. This problem can be overcome by radiosterilization of hosts before 

host exposure to IJs, and transport to the field before the hosts die and turn into 

cadavers. In this manner, the IJs from infected hosts can then directly interact with 

the ecosystem after emergence and seek new hosts (target pests) for progeny 

propagation. 

Steinernema glaseri (Steiner) was chosen as a model entomopathogenic species in 

the present study due to its tropical origin (Grewal, Selvan, and Gaugler 1994) and 

relatively large size (Stuart, Lewis, and Gaugler 1996). S. glaseri, originally 

discovered in larvae of the Japanese beetle (Popillia japonica Newman), has been 

reported to prefer host beetles in the families Chrysomelidae, Curculionidae, 

Elateridae and Scarabeidae, as well as various moth larvae in the families, 

Galleriidae, Noctuidae and Pyralidae (Poinar 1979). Prior studies by Kondo and 

Ishibashi (1986); Kondo and Ishibashi (1987); Kaya and Koppenhofer (1996); and 

Park, Yu, Park, Choo, Bae, and Nam (2001), showed that Spodoptera litura can serve 

as a suitable host for S. glaseri and it has also been reported as one of the preferred 

hosts for S. glaseri in terms of the EPNs sensory response elicited towards insect 

emitted attractants (Bilgrami, Kondo, and Yoshiga 2000). Steinernema glaseri has 

been reported to infect last instar larvae of S. litura, which may be invaded through 

natural openings and the cuticle. This nematode species has been found to be slowly 

but steadily attracted toward hosts, followed by invasion, rapid development and the 

establishment of a high population of EPNs within the host (Kondo and Ishibashii 

1986). EPNs invasion efficiency also has been reported to be better in last instar 

larvae of Spodoptera litura than in the greater wax moth, Galleria mellonella L. 

(Kondo and Ishibashi 1987). Seth and Barik (2007) showed that the infection rate of 

IJs of S. glaseri in S. litura (normal as well as radiosterilized) was quite high (79 

100%) and that this host was suitable for rapid proliferation, growth and 

development of S. glaseri.


Since EPN infectivity is affected by intrinsic (host specific) and extrinsic factors, 

and the host specific chemical cues are involved in host finding by EPNs (Kaya and 

Gaugler 1993), there is a possibility that EPNs might be conditioned in the particular 

host while developing in vivo. Hence, it was presumed that EPNs, due to their broad 

host range, would exhibit better attraction and infectivity toward the same host 

species from which they emerged or most recently encountered rather than showing 

an innate preference for a particular host species. 

Therefore, the present study assessed the infective potential and proliferation 

of S. glaseri, reared within radiosterilized hosts (S. litura), and the infective 

performance was evaluated up to two successive generations on the normal (nonirradiated) 

same host, S. litura. Comparisons were made of the relative suitability of 

hosts exposed to 40 and 70 Gy of radiation and the infectivity of IJs deriving from 

irradiated hosts vs. those reared in non-irradiated hosts. The intent of these studies 

was to develop a protocol that would facilitate inoculative and inundative 

augmentative biological control programmes against S. litura. 


Maintenance of host insects 

Spodoptera litura was mass reared on a semi-synthetic diet (Seth and Sharma 2001) 

at a temperature of 27.0918C, 7595% relative humidity and a regimen of 12 h 

light (06:00 18:00) and 12 h dark in an insectary for the experimental investigations. 

G. mellonella (often an acceptable host for entomogenous nematodes) 

was mass-reared on a semi-synthetic diet (Woodring and Kaya 1988). The culture 

of this moth was maintained at 29 308C. Last instar larvae were used for 

parasitization by EPNs. 

Maintenance of entomopathogenic nematodes 

An isolate of an entomogenous nematode, Steinernema glaseri procured from 

Biosys, USA, was maintained on G. mellonella and S. litura. Optimal environmental 

conditions of 25918C, 7595% relative humidity and a 12 h L:12 h D 

photoperiod regimen were maintained in the rearing facility. Steinernema glaseri 

was selected for the present study due to its known persistent viability and 

virulence in a tropical atmosphere (Grewal et al. 1994), for assessing its potential to 

infect S. litura. EPN survival and viability were persistently monitored and any 

batch with reduced infective response and survival less than 80 85% was not used 

for further bioassays. Viability criterion in the EPN population was taken as 80% 

or more survival with responsiveness, as suggested by Epsky and Capinera (1994). 

The experimental studies of EPNs were conducted using last instar (L6) S. litura 

larvae as hosts, because its positive geotactic behaviour for seeking a pupation site 

would make it most likely to be encountered by soil inhabiting EPNs. At 258C 

the infective juveniles could complete the entire cycle in about 7 9 days, after entry 

into the host.


In vivo rearing of EPNs on factitious host, Galleria mellonella 

Steinernema glaseri was maintained on the factitious insect host, G. mellonella, by the 

method of Woodring and Kaya (1988). Galleria mellonella was chosen as host for 

stock culture of nematodes since it appears to be a universal host to steinernematid 

and heterorhabditid nematodes. Multiplication of the nematodes was done by 

infecting 10 15 surface-sterilized last instar G. mellonella larvae with a known 

number of IJs, dispersed on a Whatman #1 filter paper bed made inside a 9 cm 

diameter inoculation chamber (a pair of sterilized Petri dishes). Surface sterilization 

of the G. mellonella larvae was done with 1% formalin, followed by three washes with 

0.1% formalin and treatment with sterilized distilled water. The infected larvae 

became flaccid at a later stage due to EPN infection. If heavily contaminated by 

microbes other than bacterial associates of the nematodes, the host larvae turned 

black and exhibited a putrid odour. To collect IJs, White Traps (White 1927) were 

prepared by draping Whatman #1 filter paper (9 cm diameter) over a platform (an 

inverted 5 cm diameter Petri dish) kept in a 9 cm diameter Petri dish. About 10 mL 

of 0.1% formalin was added to the large base Petri dish, and the filter paper covering 

the platform was in contact with the formalin solution. Surface sterilization of 

infected hosts with formalin was done as above, and the infected hosts were placed 

on the rim of the inverted Petri dish. After 8 10 days, IJs began to emerge and 

migrated through the filter paper into the formalin solution. These IJs were 

harvested daily until their emergence was reduced considerably or ceased. After 

collecting the dauers (infective juveniles) from the host cadaver in White traps, they 

were rinsed 2 3 times with 0.1% formalin solution. The viable IJs were allowed to 

pass through Whatman #42 filter paper. These IJs were stored in 0.1% formalin 

solution in sterilized distilled water at 25.08C for 3 4 weeks; whereas a proportion of 

the IJs population was stored at 6 88C in the refrigerator and a viable culture 

of EPNs could be revived from this up to 26 weeks, thereafter. IJ viability, to the level 

of 90%, was better retained at 258C up to the first two generations, although the 

freshly harvested IJs were taken for the experimental studies. 

In vivo rearing of EPNs on Spodoptera litura 

Similar procedures (as stated above) were adopted for rearing EPNs on S. litura, but 

since S. litura can be cannibalistic, individual larvae were exposed to EPNs in Petri 

dish chambers (5 cm diameter), rather than by mass exposure when G. mellonella 

larvae were used as hosts. 

Irradiation of insects 

The irradiation facility at the Institute of Nuclear Medicine and Allied Sciences 

(INMAS), Ministry of Defence, Delhi, was used for irradiation of 1-day-old sixth 

instar (L6) S. litura larvae. Sixth instar host larvae were considered as a proper stage 

for irradiation due to their large size (resulting in a large harvest potential of IJs from 

infected hosts), and high water and nutrient content (responsible for maintaining 

viability of EPNs) (Seth and Barik 2007). A 60 Cobalt source, emitting radiation at a


dose rate of 95 110 Gy/min was employed. Radiation doses of 40 and 70 Gy were 

evaluated for the efficacy studies of EPNs developed within irradiated hosts. 

Bioassay for assessing infective behaviour of EPNs of Steinernema glaseri developed 

within radiosterilized hosts 

The infective potential and proliferation of EPNs reared in radiosterilized hosts were 

assessed for two consecutive generations and compared with controls. The doses 

selected for radiosterilization, 40 and 70 Gy, were based upon the earlier work of 

Seth and Barik (2007). Various regimens evaluated for infective behaviour of EPNs 

were as follows: Regimen I (Control): Normal (N) IJs vs. Normal (N) host; Regimen 

II: N-IJs vs. Treated (T) host; Regimen III: F1 IJs vs. N-host, (where F1 IJs were 

derived from Regimen II, hereafter termed as ‘F1 IJs’); Regimen IV: F2 IJs vs. N-host 

(where F2 IJs were derived from Regimen III, hereafter termed as ‘F2 IJs’). Regimens 

II IV were evaluated for both the doses. 

Individual 1 2-day-old L6 larvae of average weight ranging from 520 to 590 mg 

were placed in a Petri dish (50 15 mm) lined with 2 times folded filter paper 

(Whatman #1). The freshly harvested IJs in all experimental regimens were taken 

from the culture maintained on S. litura, and transferred to each Perti dish by 

distributing them evenly onto the filter paper base to expose them to host larvae at 

the rate of 25 IJs released in 1 mL water solution per individual host (as described by 

Koppenhofer, Kaya, Shanmugam, and Wood 1995). Incubation was performed at a 

constant temperature of 258C. 

Parasitization behaviour and development of EPNs 

Host killing efficiency of EPNs was assessed based on the time taken for an insect 

host to become moribund and die. These responses were recorded at 4 6-h intervals. 

Morbidity is an initial behavioural response due to toxins released into the host’s 

haemolymph by symbiotic bacteria derived from IJs. Morbidity of infected larva 

represented a sluggish nature, a reduced response upon being probed, and delayed 

resumption of a normal posture and slight torsion in the body when turned upside 

down. 

For assessment of percent parasitization, a cohort of 45 50 larvae bioassayed (by 

individual exposures) constituted each replicate. After insect death, the parasitized 

larvae were allowed to incubate until the next generation of IJs developed and began 

emerging from the host cadaver. After 7 8 days, IJs were seen wriggling at the outer 

surface of the insect cadaver. At this stage, the cadavers were removed from the 

inoculative Petri dishes and transferred to sterilized ‘harvesting dishes’ (90 mm 

diameter) (also known as White trap dishes). The harvest of IJs was done by using 

White traps. 

The incubation time of EPNs (i.e., the period required from host-exposure by IJs 

to emergence of next generation IJs from host cadaver) was determined. Daily 

emergence of IJs from host cadavers and their total harvest period (duration of 

emergence of IJs from cadavers) were recorded. The harvest potential was 

determined as cumulative yield of IJs per host and IJ yield per mg host weight.


Analysis of data 

The experiments were usually replicated 10 15 times. To assess host morbidity time 

and host mortality time by EPNs, incubation time (of EPNs within host until next 

generation IJs emerged) and total harvest potential of IJs, individual host 

observations were conducted and each replicate represented the mean value of 

observations on a set of five to seven individuals. These characteristics were studied 

in 15 replicates, except in the case of mortality inducing time, percent parasitization, 

and IJ harvest (cumulative as well as per unit fresh host weight), where 10 replicates 

were conducted. 

Data was computed for means, standard error and further analysis of variance 

(ANOVA) using SPSS, version 11.0 (SPSS Statistics, www.spss.com). Percentage data 

were transformed using arcsine âx before ANOVA. Means were separated at the 5% 

significance level by least significant difference (LSD) test (Snedecor and Cochran 

1989). 

Results 

The infective behaviour and proliferation of Steinernema glaseri EPNs, that were 

cultured in irradiated (using 40 or 70 Gy) host larvae of S. litura were ascertained for 

two successive generations on normal host larvae of S. litura, in different regimens as 

described below, to assess its potential for use in augmentative biological control. 

Killing efficiency and parasitization by EPNs 

The time taken for EPNs to cause host morbidity was slightly delayed due to parent 

host irradiation in case of F1 and F2 generation IJs (F 2.89; df 6, 98; P50.05) 

(Table 1). For instance, time to morbidity was recorded as maximum (27.9 h) for 

Regimen III (F1 IJs vs. N-host) at 70 Gy, whereas it was 23.4 h in the controls. 

Further, EPNs caused host mortality in 46 h in the controls (Regimen I) and the 

mortality time was found to be slightly extended by host irradiation (F 6.49; df 6, 

63; PB0.01) (Table 1). Hence, the time taken by EPNs to induce host morbidity and 

host mortality was influenced by host irradiation, with greater impact at 70 Gy (than 

at 40 Gy), although the effect was minimal in the case of host-morbidity. The effect 

of host irradiation on mortality inducing time was greater in Regimen III (F1 IJs vs. 

N-host) than in Regimen IV (F2 IJs vs. N-host) at each of the gamma doses tested. 

Time to mortality of host larvae exhibited by IJs in Regimen IV (F2 IJs vs. Normal 

host) was 64 h at 70 Gy (39% more than the controls), but at 40 Gy it was 53 h 

(statistically not different as compared to controls; PB0.05). 

Host parasitization by EPNs was adversely influenced by host irradiation 

(F 5.15; df 6, 63; PB0.01) as compared to the controls (91.4%) (Table 1). The 

parasitization response of EPNs was not affected on radiosterilized hosts in Regimen 

II (N-IJs vs. T-host). Further, the parasitization response by F1 EPNs (Regimen III) 

was determined to be less than that by F2 EPNs (Regimen IV) at 40 Gy as well as 70 

Gy, but the percentage parasitization by F2 EPNs was almost equivalent to the 

response of normal EPNs on treated hosts at 40 or 70 Gy, although it was slightly 

less than the controls.

Table 1. Infective behaviour and parasitization of the entomopathogenic nematodes, Steinernema glaseri, reared in irradiated Spodoptera litura larvae. 

Host 1 

irradiation 

dose (Gy) Regimen Nature of parasite 2 

Nature of host 

Time required for 

morbidity (h) 

Time required for 

mortality (h) % Parasitization 

0 Gy I Normal IJs (Control) Normal host 

(non-irradiated) 

23.4a91.1 46.1a92.3 91.4a93.8 

40 Gy II Normal IJs Irradiated host (40 Gy) 24.8ab91.2 54.2b92.9 87.1ab93.9 

III F1 IJs from treated host 

(40 Gy) 

Normal Host 25.7ab91.1 59.4bcd93.2 75.1bc93.6 

IV F2 IJs from treated host 

40 Gy) 

Normal Host 24.6ab91.2 53.2ab92.5 88.7a92.9 

70 Gy II Normal IJs Irradiated host (70 Gy) 24.9ab91.7 55.2bc93.5 83.5ab94.1 


(70 Gy) 

Normal Host 27.9b91.3 67.3d93.3 69.7c93.4 


(70 Gy) 

Normal Host 26.6ab91.3 64.1cd93.2 86.9ab94.3 

1 L6 of S. litura irradiated as 0 1-days-old, and exposed at 1 2-days-old, to EPNs at a dose rate of 25 IJs/host for bioassay. 

2 IJs-infective juveniles of entomopathogenic nematode (EPNs), F1 IJs: IJs harvested from EPNs infecting radiosterilized host in Regimen II (Normal-IJs vs. Treated-host), 

F 2 IJs: IJs harvested from F 1 EPNs infecting Normal-host in Regimen III (F 1 IJs vs. Normal-host). 

Percentage data were transformed (arcsine) before ANOVA, but data in table are back transformations. 

Means9SE followed by the same letter in a column are not significantly different at P50.05 level (ANOVA followed by LSD post-test); n 10 (except n 15 in case of 

‘Time required for morbidity’). 

118 R.K. Seth and T.K. Barik

Table 2. Development and reproduction of the entomopathogenic nematode, Steinernema glaseri, reared in irradiated Spodoptera litura larvae. 

Host 1 

irradiation 

dose (Gy) Regimen Nature of parasite 2 

Nature of host 

Harvest (yield) 

Incubation time 3 

(h) IJs per host IJs per mg host Period (days) 

0 Gy I Normal IJs (Control) Normal host 

(non-irradiated) 

183.6a96.6 20119ab9948 34.1a91.6 12.2a90.41 

40 Gy II Normal IJs Irradiated host (40 Gy) 208.2bc98.5 17569bc9879 31.7ab91.5 11.5abc90.56 


(40 Gy) 

Normal Host 201.1abc98.7 16224cd9726 28.6bc91.4 10.9abc90.54 


(40 Gy) 

Normal Host 182.9a98.2 21182a91059 33.9a92.8 11.8ab90.71 

70 Gy II Normal IJs Irradiated host (70 Gy) 221.5c99.0 16278cd9828 27.7bc91.3 10.2bc90.51 


(70 Gy) 

Normal Host 214.1bc99.8 14779d9738 25.8c91.2 09.8c90.34 


(70 Gy) 

Normal Host 196.2ab97.8 17602bc9880 30.8ab91.6 11.2abc90.66 

1 L6 of S. litura irradiated as 0 1-days-old, and exposed as 1 2-days-old to EPNs at a dose rate of 25 IJs/host for bioassay. 

2 IJs-infective juveniles of entomopathogenic nematode (EPNs), F1 IJs: IJs harvested from EPNs infecting radiosterilized host in Regimen II (Normal-IJs vs. Treatedhost), 

F2 IJs: IJs harvested from F1 EPNs infecting Normal -host in Regimen III (F1 IJs vs. Normal -host). 

3 Time required from inoculation of IJs to emergence of next generation IJs from infected host. 

Percentage data were transformed (arcsine) before ANOVA, but data in table are back transformations. Means9SE followed by the same letter in a column are not 

significantly different at P50.05 level (ANOVA followed by LSD post-test); n 10 (except n 15 in case of ‘Incubation period’ and ‘Harvesting period’ of IJs). 



Development of EPNs in vivo and IJ harvest 

Host irradiation had a small prolongation effect on the incubation period of 

infecting EPNs (F 6.81; df 6, 98; PB0.01) (Table 2). The incubation period of 

infecting N-IJs on radiosterilized hosts (Regimen II) was extended more than that 

of F1 IJs and F2 IJs, with more impact at 70 Gy, and the incubation period of 

infecting F1 IJs was more than that exhibited by F2 IJs due to host irradiation (at 40 

Gy as well as 70 Gy). Further, the incubation period of F2 IJs, recorded as 182.9 and 

196.2 h from hosts irradiated at 40 and 70 Gy, respectively, was similar to that in the 

controls (183.6 h). 

The proliferation of EPNs was assessed as cumulative IJ harvest per host and IJ 

harvest per mg fresh weight of host. The cumulative IJ harvest per individual host 

was affected by host irradiation (F 7.77; df 6, 63; PB0.01) (Table 2). The IJ 

harvest per host in the controls (Regimen 1) was ca. 20.1 10 3 IJs and it decreased at 

40 Gy to 17.5 10 3 IJs and 16.2 10 3 IJs in Regimens II and III, representing 12.6 

and 19.3% reductions with respect to controls, respectively. At 70 Gy, it was 

decreased by 19% in Regimen II and by 26.5% in Regimen III. Further, in terms of IJ 

harvest per mg host weight, host irradiation again was found to affect the IJs 

emergence from infected hosts (F 3.94; df 6, 63; PB0.05), but this impact was 

less than that on cumulative IJ harvest per host. 

The IJ yield in Regimen III (F1 IJs vs. N-host) was less than the IJ yield in 

Regimen II (N-IJs vs. T-host) and in the controls, with a significant difference 

(PB0.05) with respect to the latter. The influence of host irradiation on the IJ 

harvest in Regimen III was more apparent at 70 Gy, indicating a dose dependent 

debilitating effect. The IJ harvest in Regimen IV (F2 IJs vs. N-host) was more than 

that in Regimen II (N-IJs vs. T-host) at 40 Gy as well as 70 Gy. IJ harvest from host 

cadavers was found to be affected by 9 12% in Regimen IV (F2 IJs vs. N-host) at 70 

Gy, but the degree of IJ emergence in this Regimen was almost equivalent to the 

controls at 40 Gy. Similarly, the harvest period of developing IJs from the host 

cadaver also was found to be slightly influenced by host irradiation (F 5.59; 

df 6,98; PB0.05) (Table 2). The effect of host irradiation was more apparent in the 

harvest period exhibited by infecting F1 IJs (Regimen III) at 70 Gy as compared to 

the non-irradiated controls. The harvest period exhibited by infecting F2 IJs 

(Regimen IV) was almost equivalent to that of controls (Regimen I) at both the 

doses (40 and 70 Gy). 

Discussion 

The favourable performance of Steinernema glaseri EPNs reared in irradiated hosts 

supported the feasibility of using irradiated Spodoptera litura larvae for mass rearing 

of this entomopathogenic nematode based upon several parameters, including time 

to host morbidity, time until mortality, percent parasitization, incubation time and IJ 

harvest (yield). In the present study, S. litura was considered for host irradiation to 

enable in vivo transport of EPNs, as presumably the EPNs derived from this host 

would be better acclimatized to act as more effective (in terms of parasitization) and 

elicit better orientation towards same host, S. litura, as a pest in the field (Seth and 

Barik 2007).


EPNs mass-reared in irradiated hosts (i.e., F1 progeny of EPNs that infected 

irradiated hosts for mass-rearing), showed little decline in performance as compared 

with those IJs reared in non-irradiated L6 S. litura larvae, and the parasitization 

performance of ensuing generation IJs of F1 EPNs, (i.e., F2 IJs that developed from 

the infection of F 1 IJs) was better than that of F 1 IJs and not drastically affected with 

respect to the controls (N-IJs vs. N-host). It also was noticed that the impact of host 

irradiation on IJ harvest per unit host weight was relatively less distinct as compared 

to cumulative emergence of IJs per host, which indicated that host irradiation did 

not critically affect the host’s nutritional quality for the developing EPNs. 

Use of 70 Gy for host radiosterilization would be preferable to use of 40 Gy for 

inundative releases because reproductive sterilization is more assured, but 40 Gy 

should be acceptable for inoculative releases in view of the parastization performance 

of these EPNs mass-reared in irradiated hosts (at 40 Gy) and its next progeny (i.e., F 1 

and F 2 IJs), where F 2 IJ parasitization performance was similar to that of controls. 

The level of reproductive inhibition of S. litura required for use in inoculative 

releases would allow for an occasional viable S. litura adult to be released. Even so, 

the chances of releasing reproductively competent S. litura adults using only 40 Gy 

would be minimal, as shown by Seth and Barik (2007), who found that 40 Gy 

induced 80 91% sterility and a 28 47% reduction in mating success with respect to 

control, along with reduced adult emergence (53.9%), and a pronounced (61.2%) 

incidence of malformations. However, using 40 Gy for host radiosterilization would 

better preserve the infective potential of EPNs in subsequent generations and this 

would be helpful in inoculative release programs. 

Applications of nematodes via radiosterilized infected insect hosts may have 

great potential for adoption in developing countries (especially tropical) because it is 

simple and requires no special equipment or water for application, and this would be 

cost effective with no need for formulation. Altogether, these findings lend support 

to the use of irradiated S. litura larvae for mass production of Steinernema glaseri to 

enable their distribution in the field without fear that reproductively competent, nonparasitized 

S. litura larvae might be inadvertently released. As biocontrol agents 

derived from these radiosterilized hosts would interact with normal existing 

populations of insect pests prevailing in that ecosystem exposed to other control 

measures, it would be desirable to study the compatibility of these biocontrol agents 

with other control approaches being used. Therefore, such studies on compatibility 

of integrated tactics involving EPNs are in progress. 


Financial assistance by the International Atomic Energy Agency, Vienna is gratefully 

acknowledged for supporting this research work under Research Contract No. IAEA/IND- 

10847/RB in a Coordinated Research Project. Thanks are due to Zubeda and Mahtab Zarin 

for the technical support. 

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Vol. 19, S1, 2009, 127 138 


Suitability of irradiated and cold-stored eggs of Ephestia kuehniella 

(Pyralidae: Lepidoptera) and Sitotroga cerealella (Gelechidae: 

Lepidoptera) for stockpiling the egg-parasitoid Trichogramma 

evanescens (Trichogrammatidae: Hymenoptera) in diapause 

Aydin S. Tunçbilek*, Ulku Canpolat, and Fahriye Sumer 

Department of Biology, Erciyes University, Faculty of Arts & Sciences, 38039 Kayseri, Turkey 

The use of irradiated and cold-stored host eggs could be one option to facilitate 

the mass rearing of egg parasitoids to control lepidopteran pests. The effect on 

Trichogramma evanescens (L.) wasp quality after 3-month storage of host eggs of 

the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) 

and the Angoumois grain moth, Sitotroga cerealella (Olivier) (Lepidoptera: 

Gelechidae), that had previously been irradiated with gamma radiation, was 

investigated. Efficiency of T. evanescens was studied by measuring parasitization, 

adult and female emergence. There was no significant difference in parasitization 

and in adult and female T. evanescens emergence between gamma radiation doses 

and the untreated control for up to 30 days for E. kuehniella eggs and, thereafter 

they decreased drastically as the storage time increased for up to 60 and 30 days 

for E. kuehniella and S. cerealella eggs, respectively. No parasitization was 

observed when the eggs were stored longer and then offered to T. evanescens 

females. Data obtained from diapaused T. evanescens stored at 38C for 20, 70, 100 

and 150 days indicated that pre-storage temperatures affected the induction of 

diapause. It was possible to induce diapause in developmental stages of T. 

evanescens by exposing the immature stages (prior to the pre-pupal stage) inside 

host eggs to 10 and 128C for 30 days. Under these conditions, parasitoids could be 

stored for a period of 50 days without adverse affects on emergence. Emergence 

appeared to decrease with an increase in the duration of storage for a period up to 

150 days for the eggs of E. kuehniella. Parasitoids failed to enter diapause when 

pre-storage conditions were 3 and 78C for host eggs of both E. kuehniella and S. 

cerealella. The long-term storage of parasitoids in diapause improved the mass 

rearing potential for lengthened releases of this species. 

Keywords: Trichogramma evanescens; cold storage; diapause; host egg; gamma 

radiation; Ephestia kuehniella; Sitotroga cerealella 

Introduction 

Moth species of the genus Ephestia, especially the Mediterranean flour moth, 

Ephestia kuehniella Zeller, and Angoumois grain moth, Sitotroga cerealella (Olivier), 

are serious pests in cereal-based food processing facilities, stored maize and other 

cereals in Turkey (Ministry of Agriculture and Rural Affairs 1995a). Typically, 

control of these pests is undertaken by regular treatment of infested areas with a 

pesticide such as malathion, dichlorvos, and methyl bromide (Ministry of Agriculture 

*Corresponding author. Email: tunca@erciyes.edu.tr 



DOI: 10.1080/09583150902985588 


128 A.S. Tunçbilek et al. 

and Rural Affairs 1995b). The disadvantages of using chemicals, together with the 

effect of fumigants on the ozone layer and the development of pest resistance, have all 

contributed to the urgency of the current search for grain protection systems that 

target the pest species. 

Biological control is an often-underutilized component of integrated pest 

management of stored grains (Brower, Smith, Vail, and Flinn 1996; Schöller, Prozell, 

Al-Kirshi, and Reichmuth 1997). Recent legislation in the USA has allowed for 

augmentative releases of beneficial insects in stored products. Parasitoids from the 

genus Trichogramma are of interest for control of pyralid moths in flourmills because 

they attack the egg stage and kill the pest before it reaches the larval stage and starts 

to produce webbing (Hansen and Jensen 2002). Eggs of E. kuehniella and S. 

cerealella are suitable hosts for Trichogramma evanescens (L.) (Hansen and Jensen 

2002; Pitcher, Hoffmann, Gardner, Wright, and Kuhar 2002), a parasitoid that could 

be effectively used for suppression of stored products moths. However, if all fertile 

host eggs that are deployed are not parasitized, the eggs that hatch will serve to 

increase the number of moth larvae present. This would be unacceptable. One way to 

avoid this problem is to kill the moth egg embryos by gamma radiation before they 

are deployed in food storage facilities. Therefore, before placement in food storage 

facilities, the host eggs are irradiated with gamma radiation to prevent development. 

In our previous study (Tunçbilek, Canpolat, and Ayvaz 2009), we showed that the 

irradiated E. kuehniella and S. cerealella eggs presented to female T. evanescens in 

choice experiments were equally acceptable and suitable for parasitoid development. 

The objective of a mass rearing programme is ‘to produce the maximum quantity 

of quality-assured individuals by predetermined dates at a minimal cost’ (King 

1993). An important aspect in mass production and deployment of biological control 

agents is development of storage techniques to provide flexibility and efficiency of 

their use, and to make standardized stocks available for use in research (Ravensberg 

1992; Greenberg, Nordlund, and King 1996; Leopold 1998). Many insects and mites 

are able to overwinter at sub-zero temperatures in a supercooled state (Lee 1991). 

Few studies have been conducted to examine storage temperatures below 08C to 

facilitate mass production and/or release. Short-term storage has been used in the 

range of 3 158C to accumulate organisms for shipment to a consumer, to 

synchronize a specific stage for peak release or to maintain them in a quiescent 

state during shipment to prevent damage or loss. Long-term storage would benefit a 

programme and be cost effective in terms of maintaining the low temperatures and 

conducting the necessary pre-programme treatments, and where it is desired to shut 

down mass-rearing during the off-season (Leopold 1998). 

Currently, cold storage for most species that are used in biocontrol, Sterile Insect 

Technique (SIT), or an Integrated Pest Management (IPM) programme involves 

chilling at temperatures above 08C. The two main cold storage techniques that have 

been used in mass rearing of Trichogramma spp. are either with and/or without 

previous diapause induction (Greenberg et al. 1996). Immature stages of several 

Trichogramma species can enter diapause or become quiescent within host eggs, 

allowing them to tolerate long periods of subfreezing temperatures (Smith 1996). 

Such an approach has been attempted for a number of Trichogramma species by 

exposing the parasitoids to a range of cold storage conditions (Laing and Corrigan 

1995; García, Wajnberg, Pizzol, and Oliveira 2002; Pitcher et al. 2002). To date, very 

little data are available on the diapause requirements of T. evanescens and on storage


techniques for irradiated host eggs. Therefore the objectives of this study were (1) to 

evaluate the effects of gamma radiation and storage temperature on two types of 

host eggs and on their parasitization, and (2) to determine the role of these factors 

on the induction of diapause in T. evanescens, as a way of facilitating extended 

storage. 


Host rearing 

Strains of E. kuehniella and S. cerealella were obtained from the Department of 

Plant Protection, Faculty of Agriculture of Ankara University and Adana Plant 

Protection Research Institute, respectively. E. kuehniella was reared on a mixture 

consisting of one kg wheat flour, 5% yeast and 30 g wheat germs (Marec, Kollarova, 

and Pavelka 1999). S. cerealella was reared on wheat grain. Throughout the rearing, 

cultures were kept in a rearing room at 27918C and7095% RH and under a light 

regime of 14 h light followed by 10 h darkness (14 h L:10 h D). To obtain eggs for the 

tests, large numbers of 1 2-day-old adults of E. kuehniella and S. cerealella were 

collected from stock cultures and placed in plastic jars with screen bottoms. Eggs 

that fell through the screen were collected the following days and sifted to remove 

insect parts and frass, and placed in a Petri dish. The eggs removed daily were 

exposed to parasitoids in glass tubes for 24 h. 

Rearing of Trichogramma evanescens 

The T. evanescens strain used in this experiment was obtained from Adana Plant 

Protection Research Institute. It originated from Ostrinia nubilalis (Lep: Pyralidae) 

eggs collected in southern Turkey in 1999. In the laboratory, T. evanescens was massreared 

on E. kuehniella eggs for several generations. Throughout the rearing, cultures 

were kept in the rearing room at 24918C and 7095% RH, and under a light regime 

of 14 h L:10 h D. Parasitoid cultures were started from a single female on 

E. kuehniella eggs and maintained in glass rearing vials (2 7.5 cm). 

Preparation of egg cards 

For each experiment, a series of egg selection tests were performed with ‘egg cards’. 

These were prepared using certain numbers of moth eggs as described by Brower 

(1982). Strips of lightweight cardboard (2 2.5 or 2.5 4 cm) were glued with gum 

Arabica, and the gum was allowed to dry for at least 1 h. Eggs were then counted and 

equal numbers (150910) were sprinkled on these cards. When exposed to 

parasitoids during the experiments, the parasitoid to host ratio was high in these 

tests to ensure that most acceptable eggs would be parasitized. 

Storage experiments 

Large numbers of 1-day-old E. kuehniella and S. cerealella eggs were placed in glass 

Petri dishes and irradiated in a calibrated 60 Co irradiator (Therathronics 780C) with 

a source strength of ca. 3811 Ci and at a dose rate of ca. 1 Gy/min. The dose rate was


verified with Fricke dosimetry, the best known chemical radiation dosimeter, which 

relies on oxidation of ferrous ions into ferric ions in an irradiated ferrous sulphate 

solution (Andreo, Seuntjens, and Podgorsak 2005). The eggs were exposed to gamma 

radiation doses of 0, 50, 100, 150 or 200 Gy to prevent development. After 

irradiation exposure, these eggs were placed at 48C and 7095% RH in the dark for a 

period of 30, 60 or 90 days. Following each period of storage at 48C, eggs were 

transferred to normal room temperature. 

Eggs glued to lightweight cardboard cards as described above were placed in 

tubes along with a single female T. evanescens. A single female per tube was obtained 

by capturing a 24-h-old female from a group of females scattered on a piece of white 

paper: the glass tube was placed over a medium size female and the female was 

allowed to walk up the vial towards the light (replicated 10 times). All females had 

no previous contact with host eggs, were fed with honey and allowed to mate in the 

tubes. The lid of the tube was covered tightly with plastic hardware cloth to prevent 

the wasp from leaving the tube. 

After 24 h, the wasps were discarded from the tubes and eggs incubated at 

controlled conditions. The parasitization was assessed by counting the number of 

black eggs after 5 days of development. Emergence was determined by counting 

emergence holes on black eggs. Longevity of wasps was measured every 24 h (from 

the time of emergence until death) by keeping the adults individually in a 1 5cm 

glass tube with food. The sex ratio was determined by examining fully developed 

dead adults under a microscope. Parasitization, adult emergence, and sex ratio were 

scored. Females that died during the experiment were excluded from evaluation. 

Diapause experiments 

Our studies on diapause concentrated on immature stages of T. evanescens, especially 

pre-pupal and pupal stages. To induce diapause in immature stages of the parasitoid, 

less than 24-h-old eggs were presented to the wasps on the egg cards after irradiation 

(200 Gy). Each egg card (150910 eggs) was exposed to five mated T. evanescens 

females (B24-h-old) inside a glass rearing tube (18 180 mm) with a drop of honey 

solution to provide parasitoids with a carbohydrate source. After 24 h exposure to 

parasitoids at 2490.58C, 7095% RH and a photoperiod of 14 h L:10 h D, egg cards 

were assigned to each of the four different pre-storage temperatures (3, 7, 10 and 

128C) for 30 days. After pre-storage, these egg cards were placed in the dark at 38C 

(García et al. 2002) for a period of 20, 70, 100 or 150 days for storage. Finally, eggs 

were incubated at normal room temperature in the rearing room mentioned above. 

Twenty-five egg cards (150 parasitized host eggs in each) were randomly assigned 

to each period of exposure at the respective pre-storage temperatures. Subsequently, 

from each period of storage at 38C, 5 egg cards (i.e., replicates) were transferred to 

the rearing room until adult emergence. This was carried out at steeply increasing 

deacclimation temperatures (7, 10, 12 and 208C) in different environmental 

chambers, holding the egg cards for 24 h progressively in each of these chambers 

(Table 1). Females that died during the experiment were excluded from the 

experiment. Parasitization, total adult emergence, and female emergence were 

recorded.

Table 1. Experimental design for inducing diapause in T. evanescens regarding pre-storage, 

storage and deacclimation temperatures and durations. 

Pre-storage 

temperature 

(8C) for 30 days 


Storage 

temperature and time 

(8C) (days) 

Deacclimation 

temperature (8C) 

(24 h each) 

Rearing 

temperature 

(8C) 

3 20 

7 3 70 7 10 12 20 24.5 

10 100 

12 150 

Statistical analysis 

Data collected from storage and diapause experiments were analyzed using a twofactor 

analysis of variance, with storage time and dose of radiation for the storage 

experiment, and pre-storage temperature and storage time for the diapause 

experiments as sources of variation (ANOVA) (SPSS 1999). All data were 

transformed to square root before statistical analysis was performed. When 

significant differences occurred, Tukey-HSD was used for separation of means. 

Results 

Storage experiments 

We used the number of parasitized eggs as an estimation of fecundity because actual 

oviposition was difficult to measure. Typically, only one parasitoid emerges from 

each E. kuehniella or S. cerealella egg. There was no significant difference between 

control and stored eggs for up to 30 days for parasitization, adult emergence, and 

female emergence of T. evanescens (Figure 1 and Table 2) for E. kuehniella eggs. 

Nevertheless, there was a significant difference for adult emergence and female 

emergence (87 and 84%, respectively) of the parasitoid stored for 60 days 

(F 1358.165, df 3, 180, PB0.001; F 159.178, df 3, 180, PB0.001, respectively). 

On the other hand, data from S. cerealella eggs stored for 30 days were 

significantly lower when compared with the equivalent data obtained from stored 

eggs of E. kuehniella. The parasitization and emergence of the wasp from stored eggs 

of S. cerealella was only 63 and 54% after 30 days, drastically lower than the control 

(Figure 1 and Table 3) (F 977.540, df 2, 135, PB0.001; F 606.598, df 2, 135, 

PB0.001). No wasp emergence was recorded after 90 and 60 days of storage for 

E. kuehniella and S. cerealella eggs, respectively. 

When the complete results were evaluated, while parasitization, adult emergence 

and female emergence of T. evanescens were not significantly affected by irradiation 

doses for host eggs of E. kuehniella (Figure 1), the same parameters for S. cerealella 

were significantly decreased by irradiation (F 3.925, df 4, 135, P 0.005; 

F 3.832, df 4, 135, P 0.006). The difference in female emergence was not 

significant (Tables 2 and 3). 

On the other hand, parasitization, adult emergence and female emergence of 

T. evanescens were not influenced by gamma radiation for both host eggs, but they 

were influenced by storage time. Mean parasitization and adult emergence decreased


(a) 

40 

Mean 

(b) 

Mean 

(c) 

Mean 

35 

30 

25 

20 

15 

10 

5 

0 

40 

35 

30 

25 

20 

15 

10 

5 

0 

40 

30 

20 

10 

0 

E. 

Parasitized egg 

E. kuehniella S. cerealella 

0 Gy 

50 Gy 

100 Gy 

150 Gy 

200 Gy 

0 30 60 90 0 30 60 90 

Storage (Day) 

kuehniella 

Adult emergence 

cerealella 

0 Gy 

50 Gy 

100 Gy 

150 Gy 

200 Gy 

0 30 60 90 0 30 60 90 

Storage (Day) 

E. 

kuehniella 

Female emergence 

0 30 60 90 0 30 60 90 

Storage (Day) 

significantly with the length of storage time. There was interaction between gamma 

radiation doses and storage time for eggs of E. kuehniella and S. cerealella (Tables 2 

and 3). Parasitization, adult emergence and female emergence of T. evanescens reared 

on the irradiated eggs of E. kuehniella were significantly higher than on similarly 

treated eggs of S. cerealella. 

Diapause experiments 

Data obtained from diapaused T. evanescens in relation to pre-storage and storage 

temperatures for host eggs of E. kuehniella and S. cerealella are presented in Figure 2. 

Results indicated that pre-storage temperatures affected the induction of diapause. 

The parasitization of T. evanescens was significantly higher in the eggs of E. kuehniella 

S. 

S. 

cerealella 

0 Gy 

50 Gy 

100 Gy 

150 Gy 

200 Gy 

Figure 1. Mean numbers of percent parasitization, adult emergence and female emergence 

(9SD) of T. evanescens reared on eggs of E. kuehniella and S. cerealella after storage at 48C 

for 0, 30, 60 or 90 days. The eggs were irradiated at 0, 50, 100, 150 or 200 Gy before storage.

Table 2. Two-way ANOVA results comparing the effects of each storage time and irradiation 

dose treatments for parasitization, adult emergence and female emergence of T. evanescens 

from eggs of E. kuehniella. 

Treatments Parameters N df 

Mean square 

(treatment) 

Mean 

square 

(error) F Significance 

Storage time Parasitization 200 3,180 209.51 0.11 1983.46 B0.001 

Adult 

emergence 

200 3,180 187.42 0.14 1358.17 B0.001 

à emergence 200 3,180 140.46 0.88 159.18 B0.001 

Radiation dose Parasitization 200 4,180 0.19 0.11 1.81 0.129 

Adult 

emergence 

200 4,180 0.22 0.14 1.60 0.176 

à emergence 200 4,180 0.51 0.88 0.58 0.678 

Storage time Parasitization 12,180 0.24 0.11 2.23 0.012 

Radiation dose Adult 

emergence 

12,180 0.31 0.14 2.27 0.010 

à emergence 12,180 1.37 0.88 1.55 0.109 

pre-stored at 10 and 128C and eggs of S. cerealella pre-stored at 128C than at 7 and 

108C, respectively. These values were drastically reduced in the eggs stored at 38C as 

pre-storage temperature. Our results showed that it was possible to induce diapause in 

developmental stages of T. evanescens by exposing the immature stages (prior to the 

pre-pupal stage) to 10 and 128C for 30 days. 

The two-way ANOVA analysis showed that parasitization, adult emergence and 

female emergence of T. evanescens were significantly affected by the duration of 

Table 3. Two-way ANOVA results comparing the effects of each storage time and irradiation 

dose treatments for parasitization, adult emergence and female emergence of T. evanescens 

from eggs of S. cerealella. 



Mean square 

(treatment) 

Mean 

square 



Adult 

emergence 

150 2,135 132.71 0.22 606.60 B0.001 

à emergence 150 2,135 97.30 0.43 228.23 B0.001 

Radiation dose Parasitization 150 4,135 0.82 0.21 3.93 0.005 

Adult 

emergence 

150 4,135 0.84 0.22 3.83 0.006 

à emergence 150 4,135 0.30 0.43 0.71 0.588 

Storage time Parasitization 8,135 0.64 0.21 3.05 0.003 

Radiation dose Adult 

emergence 

8,135 0.74 0.22 3.36 0.002 

à emergence 8,135 1.79 0.43 4.20 B0.001


(a) 

Mean 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

(b) 

100 

Mean 

(c) 

Mean 

80 

60 

40 

20 

0 

80 

70 

60 

50 

40 

30 

20 

10 

0 

E. 

kuehniella 

Parasitized egg 

S. 

cerealella 

3ºC 

7ºC 

10ºC 

12ºC 

Pre 20 70 100 150 Pre 20 70 100 150 

Storage (Day) 



3ºC 

7ºC 

10ºC 

12ºC 

Pre 20 70 100 150 30 20 70 100 150 

Storage (Day) 



3ºC 

7ºC 

10ºC 

12ºC 

Pre 20 70 100 150 Pre 20 70 100 150 

Storage (Day) 

Figure 2. Mean numbers of percent parasitization, adult emergence and female emergence 

(9SD) of T. evanescens reared on irradiated eggs of E. kuehniella and S. cerealella after 30 

days of exposure to: 3, 7, 10 or 128C, followed by storage at 38C for 0, 20, 70, 100 or 150 days. 

storage at 38C for both host eggs (F 4.453, df 4, 80, P 0.003; F 61.016, df 4, 

80, PB 0.001; F 54.738, df 4, 80, PB0.001 for E. kuehniella; F 38.219, df 4, 

80, PB0.001; F 31.317, df 4, 80, PB0.001; F 31.899, df 4, 80, PB0.001 for 

S. cerealella) (Tables 4 and 5). Interestingly, parasitization for both host eggs was 

significantly higher in each period of storage compared to the control regardless of 

irradiaton doses, pre-storage and storage duration. Wasp emergence from E. 

kuehniella eggs was high and remained the same up to day 20 of storage after 30 

days of pre-storage. This was higher than that of S. cerealella eggs. Thereafter, it 

drastically decreased in the eggs stored for 70 days and continued decreasing to 150 

days when it reached its lowest level for E. kuehniella eggs.

Table 4. Two-way ANOVA results inducing diapause effect of each storage time and 

temperature treatments for parasitization, adult emergence and female emergence of 

T. evanescens from eggs of E. kuehniella. 


Mean square 

(treatment) 

Mean 

square 


Storage time Parasitization 150 4,80 9.94 2.23 4.45 0.003 

Adult 

emergence 

150 4,80 71.91 1.18 61.02 B0.001 

à emergence 150 4,80 60.67 1.11 54.74 B0.001 

Temperature Parasitization 150 3,80 385.49 2.23 172.67 B0.001 

Adult 

emergence 

150 3,80 69.28 1.18 58.79 B0.001 

à emergence 150 3,80 58.28 1.11 52.58 B0.001 

Storage time Parasitization 112,80 21.51 2.23 9.64 B0.001 

Temperature Adult 

emergence 

112,80 3.28 1.18 2.78 0.003 

à emergence 112,80 2.94 1.11 2.65 0.005 

The two-way ANOVA showed that parasitization, adult emergence and female 

emergence of T. evanescens were significantly affected by the pre-storage temperature 

for both host eggs (F 172.674, df 3, 80, PB0.001; F 58.790, df 3, 80, PB 

0.001; F 52.582, df 3, 80, PB0.001 for E. kuehniella; F 156.292, df 3, 80, 

PB0.001; F 22.429, df 3, 80, PB0.001; F 18.179, df 3, 80, PB0.001 for S. 

cerealella) (Tables 4 and 5). These values at the pre-storage temperatures of 3 and 

78C decreased considerably and the parasitoids failed to enter diapause for both host 

Table 5. Two-way ANOVA results inducing diapause effect of each storage time and 

temperature treatments for parasitization, adult emergence and female emergence of 

T. evanescens from eggs of S. cerealella. 



Mean square 

(treatment) 

Mean 

square 



Adult 

emergence 

100 4,80 9.81 0.31 31.32 B0.001 

à emergence 100 4,80 5.56 0.17 31.90 B0.001 

Temperature Parasitization 100 3,80 215.49 1.38 156.29 B0.001 

Adult 

emergence 

100 3,80 7.03 0.31 22.43 B0.001 

à emergence 100 3,80 3.17 0.17 18.18 B0.001 

Storage time Parasitization 12,80 15.41 1.38 11.18 B0.001 

Temperature Adult 

emergence 

12,80 2.25 0.31 7.17 B0.001 

à emergence 12,80 1.21 0.17 6.96 B0.001


eggs. In contrast, the development of parasitoids that were held at 10 and 128C for 30 

days was arrested in pre-pupal stage, indicating that wasps entered diapause, 

tolerating storage at 38C (Figure 2). 

Discussion 

In this study, the host eggs of both E. kuehniella and S. cerealella were irradiated with 

gamma radiation and then stored at 48C for up to 90 days. When T. evanescens 

parasitoids were reared on E. kuehniella, there were no significant differences in 

terms of parasitization and adult emergence between control and stored eggs for up 

to 30 days, but there was a difference in the number of adult emergence and female 

emergence after 60 days. The adult and female emergence from the treatments for 30 

and 60 days was still comparable to our stock culture (87 and 84%, for adult and 

female emergence, respectively) and quality control parameters of IOBC (2002). 

These values obtained from S. cerealella eggs were less than for eggs of E. kuehniella. 

A maximum storage of 4 weeks is reported for irradiated Ephestia eggs held at 

28C and 90% RH (Bigler 1994). Parasitized S. cerealella eggs stored at 9 and 128C 

allowed comparatively good emergence of T. ostrinia after storage of 4 weeks 

(Pitcher et al. 2002). These results are similar to those of Iacob and Iacob (1972) who 

found that 9 128C was an acceptable range to store T. evanescens. They also reported 

that storage for more than 6 weeks caused emergence to decline to levels that would 

not be commercially acceptable. There are also several reports of reductions in fitness 

traits of insects, including Trichogramma, after cold storage (Frei and Bigler 1993; 

Laing and Corrigan 1995). Contrary to these findings, our results showed that adult 

and female emergence were still acceptable after 60 days. These were much higher 

than the findings of Bigler (1994) and Pitcher et al. (2002). Therefore, the irradiated 

eggs, especially E. kuehniella eggs, can be stored more effectively for the parasitoids 

to be used after the low production season. The access production of the eggs of 

E. kuehniella and S. cerealella can be stored at 48C after irradiation at a dose of 200 

Gy without any loss in number and quality of subsequent parasitoid production. 

Data obtained from diapaused T. evanescens indicated that pre-storage temperatures 

affected the induction of diapause. It was possible to induce diapause in 

immature stages of T. evanescens by exposing them within their hosts to 10 and 128C 

for 30 days. Under these conditions, the parasitoid could be stored at 38C for 

a period of 50 days, without adverse effects on emergence. The emergence of 

T. evanescens appeared to decrease with longer duration of storage of E. kuehniella 

eggs towards 150 days. While diapause induction resulted in an increase in 

parasitization after exposure to storage at the low temperature, emergence of the 

wasp decreased after 70 days of diapause induction. This may be interpereted in two 

ways: one likely explanation is that parasitized eggs may also have developed to the 

pupal stage in cold storage, but could not to reach the adult stage. The other possible 

explanation is that parasitized eggs cannot be identified soon after parasitization 

(before the pre-pupal stage), and that is why it could be not determined how many 

eggs were actually parasitized by the female wasp at the time of the first day of 

storage. 

Among insects, including Trichogramma, diapause generally occurs in a specific 

stage (Zaslavki and Uvarova 1990). Thus, as indicated by Leopold (1998), there is a 

need to determine the most cold tolerant stage of parasitoids before cold storage.


García et al. (2002) reported that a mimimum conditioning period (30 days) and a 

precise temperature regime (108C) during a particular immature stage (prior to prepupal 

stage) are necessary to induce diapause in the pre-pupae of T. cordubensis 

(García et al. 2002). 

Knowledge from the parasitoid emergence and fitness portions of the experiment 

leads us to conclude that host eggs stored at a low temperature and cold-stored T. 

evanescens in diapause within irradiated host eggs facilitate the mass rearing process 

by allowing stockpiling of parasitoids for future releases without any loss in 

parasitoid production. Further research on the effects of fluctuating temperatures for 

inducing diapause is needed to improve the storage of T. evanescens. 


The authors would like to thank the International Atomic Energy Agency, Vienna, Austria for 

the support through Research Contract No: IAEA/TUR-10782. We thank Dr. Gernot Hoch 

for comments on earlier drafts of the manuscript and Mrs S. Öztemiz for supplying the 

Trichogramma evanescens used in the experiments, and the Department of Radiation 

Oncology for allowing the use of the Co 60 irradiator. 

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Vol. 19, S1, 2009, 139 155 


Interaction of entomopathogenic nematodes, Steinernema glaseri 

(Rhabditida: Steinernematidae), cultured in irradiated hosts, with 

‘F 1 sterility’: Towards management of a tropical pest, 

Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae) 

Rakesh K. Seth*, Tapan K. Barik, and Sonal Chauhan 

Department of Zoology, University of Delhi, Delhi 110 007, India 

Efficacy of the entomopathogenic nematode (EPN), Steinernema glaseri, (Steiner) 

cultured in radio-sterilized host, was studied vis-à-vis radiation-induced F1 sterility 

on a tropical lepidopteran pest, Spodoptera litura (Fabr.). To ensure safe transport 

of S. glaseri EPNs in vivo, host radio-sterilization was done; and the parasitising 

performance of S. glaseri infective juveniles (IJs), cultured in irradiated last instar 

S. litura larvae (with either 40 or 70 Gy of gamma rays) was evaluated on F1 sterile 

insects (progeny of male moths treated with 100 Gy, 130 Gy). S. glaseri EPNs 

cultured in radio-sterilized larvae at 40 Gy, had better infective potential than those 

cultured in sterilized host larvae at 70 Gy. F1 sterile larval hosts (progeny from 100 

or 130 Gy treated parents) were equally acceptable to the EPNs cultured in radiosterilized 

hosts, although the nematode harvest was reduced on F1 sterile hosts at 

130 Gy. Infectivity of IJs derived from F1 sterile host was almost similar on F1 sterile 

larvae and normal larvae of S. litura, although their parasitisation efficacy on 

the F1 sterile hosts was relatively less than the controls. The IJs performance was 

little influenced by irradiation of IJs’ parent host and current host’s irradiation 

history individually, but both the parameters together did not have any further 

negative interaction on the performance of IJs. The present results indicate that 

S. glaseri harvested from F1 sterile larval hosts (progeny from 100 or 130 Gy treated 

parents) retained a reasonably high degree of infectivity on normal and F1 sterile 

S. litura hosts (61 83% of controls). Two highly promising operational modes of 

integrating S. glaseri EPNs with ‘F1 sterility’ to suppress S. litura populations 

(initial releases of EPNs to strongly suppress the density of the pest followed by use 

of F1 sterility vs. simultaneous use of both techniques) are discussed. 

Keywords: Spodoptera litura; cutworm; gamma irradiation; entomopathogenic 

nematodes; Steinernema glaseri; parasitoid; population suppression; inherited 

sterility; F1 sterility 

Introduction 

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is a common defoliator of a 

polyphagous nature (recorded on more than 120 host plants), and it has attained an 

economically serious status in the Indian subcontinent (Lefroy 1908; Moussa, Zaher, 

and Kotby 1960; Chari and Patel 1983; Higuchi, Yamamoto, and Suzuki 1994). 

Increasing environmental hazards from the use of chemical pesticides and development 

of insecticide-resistance in this pest (Ramakrishnan, Saxena, and Dhingra 

*Corresponding author. Email: rkseth@del2.vsnl.net.in 

First Published Online 21 April 2009 



DOI: 10.1080/09583150902814515 


140 R.K. Seth et al. 

1984; Armes, Wightman, Jadhav, and Ranga Rao 1997) have encouraged entomologists 

to seek environmentally sound alternative measures to control this pest. One 

such ecologically compatible pest control strategy is biological control. Increased 

efforts in recent years have been focused on biological control using entomopathogenic 

nematodes in the families, Heterorhabditidae and Steinernematidae. Entomopathogenic 

nematodes (EPNs) of these two families kill their hosts due to the action 

of their endo-symbiotic bacteria; and they have a broad host range, can be massproduced 

using conventional fermentation technology, and are exempted from 

registration requirements in several countries (Kaya and Gaugler 1993). Therefore, 

they are commercially available for insect control in nurseries, greenhouses, and 

turfgrass around the world (Grewal and Georgis 1998). The EPNs mainly attack soil 

insects in the families Chrysomelidae and Curculionidae. They are also reported to 

parasitize some insects in the order Lepidoptera (Poinar 1979). Successful application 

of Steinernema carpocapsae (Rhabditida: Steinernematidae) against the beet 

armyworm, Spodoptera exigua was attained in a commercial nursery in Florida 

(Kaya and Hara 1980; Begley 1990). The residual effect of the nematode treatment 

lasted longer than that of standard chemical pesticides (Bari and Kaya 1984). The 

biocontrol potential of S. carpocapsae against the cutworm, S. litura has been 

suggested (Narayanan and Gopalakrishnan 1987; Choo, Kaya, and Reed 1989; 

Sezhian, Sivakumar, and Venugopal 1996). 

The feasibility of using induced ‘F1 sterility’ as a genetic control method has been 

studied for several species of Lepidoptera (Knipling 1970). Since high doses of 

gamma irradiation (200 350 Gy) are required to induce complete sterility in 

Lepidoptera (North and Holt 1971) and exposure to high radiation levels also 

adversely affects male mating behaviour and competence, one approach to reduce the 

negative effects of radio-resistance in Lepidoptera has been the use of inherited or F1 

sterility (North 1975; Carpenter, Bloem, and Marec 2005). In view that F1 sterile 

progeny are produced in the field, the release of partially sterile insects offers greater 

suppressive potential than the release of fully sterile insects (LaChance 1985) and is 

more compatible with other pest control mechanisms or strategies (Carpenter 1993). 

Knipling (1970) explored the theoretical basis for the application of F1 sterility for 

control of lepidopteran pests by using mathematical models, which was further 

supported by later studies (Carpenter 1993; Anisimov 1998). Many studies have 

shown that F1 sterility can be effectively combined with other biological control 

tactics such as pheromone disruption (Bloem, Bloem, Carpenter, and Calkins 2001), 

entomopathogens (Hamm and Carpenter 1997), host plant resistance (Carpenter 

and Wiseman 1992a,b) and natural enemies (Carpenter, Hidrayani, and Sheehan 

1996; Greany and Carpenter 1999). As a result of these studies, F1 sterility is 

regarded as the best genetic method for combined use with other suppressive 

measures against Lepidoptera (Carpenter and Bartlett 1999). 

F1 sterility, as a parabiological (genetic) control measure, has been proposed for the 

suppression of S. litura (Seth and Sehgal 1993; Seth and Sharma 2001), instead of 

using the Sterile Insect Technique (SIT) wherein the high (100%) sterilizing dose 

impairs the mating competitiveness of the moths. A range of 100 130 Gy was 

suggested to be employed in order to implement F1 sterility for the suppression of S. 

litura populations, because under the influence of this range of doses of ionising


radiation, the mating competitiveness of S. liturawas not drastically impaired unlike in 

case of the higher and fully sterilizing radiation doses. Therefore, two doses, viz. 100 

and 130 Gy were selected in the present study to ascertain the interaction of EPNs with 

host insects derived from irradiated male parents. 

Further, it has been suggested that the EPNs carried within the host (i.e., in vivo) 

retain better viability than in aqueous releases. For instance, the dispersal abilities of 

EPNs such as Heterorhabditis bacteriophora Poinar (HP88 strain) and S. carpocapsae, 

were reported to be significantly greater when nematodes were applied in 

cadavers than when they were applied in aqueous suspension. This difference in 

migration ability was presumed to be due to physiological or behavioural differences 

between nematodes exiting hosts and those kept in aqueous suspension; and there 

could also be a difference in fitness and behaviour of nematodes carried in vivo as 

compared to nematodes used in aqueous application (Shapiro and Glazer 1996; 

Shapiro and Lewis 1999). Since entomopathogenic nematodes applied in infected 

hosts (in vivo) may have dispersal advantages and increased efficacy in biological 

control, it was desirable to understand the efficacy of EPNs carried within their host 

in relation to other compatible control measures. 

Steinernema glaseri (Steiner) was selected as model entomopathogenic species in 

the present study due to its tropical origin and persistent viability in tropical 

conditions (Grewal, Selvan, and Gaugler 1994) and relatively large size (Stuart, 

Lewis, and Gaugler 1996). Further, S. glaseri has been reported to parasitize S. 

litura (Kondo and Ishibashi 1986; Kaya and Koppenhofer 1996), and S. litura has 

been reported as one of the preferred hosts for S. glaseri in terms of sensory 

response elicited towards insect emitted attractants (Bilgrami, Kondo, and Yoshiga 

2000). 

A fraction of the individuals of the pest species that are used with the intention 

that they carry EPNs may escape parasitisation and add to pest population in the 

ecosystem. Therefore, the radio-sterilization of the insect-hosts was considered 

necessary to have a risk-free mode of carrying EPNs in vivo, so as to ensure safe 

transport of EPNs. The present study for assessing the parasitising performance of 

EPNs transported in vivo was designed to ensure the transport of viable EPNs within 

irradiated host, and to study the interaction of S. glaseri EPNs, carried within radiosterilized 

S. litura hosts with F1 sterility (substerilizing radiation induced genetic 

control method). An attempt was made to evaluate (i) the parasitising performance 

of S. glaseri EPNs against normal and F1 sterile S. litura hosts, (ii) the infective 

potential of S. glaseri EPNs cultured in radio-sterilized hosts against normal and F 1 

sterile S. litura hosts, and (iii) the parasitising performance of S. glaseri EPNs 

harvested from F1 sterile S. litura hosts. 


Maintenance of insect hosts 

Maintenance of S. litura as a potential host of entomopathogenic nematodes 

Mass rearing of S. litura was conducted on semi-synthetic diet (Seth and Sharma 

2001) at 2791 o C, 7595% relative humidity and 12 h L:12 h D regimen in an 

insectary for experimental investigations.


Maintenance of Galleria mellonella as a factitious host of S. glaseri EPNs 

The greater wax moth, Galleria mellonella (L.) was reared on a semi-synthetic diet 

consisting of 350 mL honey, 350 mL glycerin, 400 g corn starch, 200 g wheat flour, 

200 g wheat bran, 200 g milk powder, and 50 g yeast granules. A mixture of honey 

and glycerin was prepared. Then the dry constituents were mixed and added to 

honey-glycerin mix. This amount of diet could accommodate about 100 larvae. The 

culture of this moth was maintained at 3091 o C (as described by Woodring and 

Kaya 1988). 

Maintenance of S. glaseri entomopathogenic nematodes 

A core culture of an entomogenous nematode species, S. glaseri, procured from 

Biosys, USA, was maintained on the factitious insect-host, the greater wax moth, G. 

mellonella, larvae at optimum environmental conditions, viz., 2590.5 o C, 7595% 

relative humidity, by the method of Woodring and Kaya (1988). G. mellonella was 

chosen to serve as host for the stock culture of nematodes as it was found to be quite 

susceptible to infection and to be an acceptable host. S. glaseri dauers (infective 

juveniles) stored in 0.1% formalin solution in sterilized distilled water at 6 88C 

maintained proper viability for 2 4 weeks. These EPNs were allowed to acclimatize 

at ambient temperature (25 o C) for 24 h before being applied to host larvae for 

ongoing in vivo rearing or for experimental studies. 

Irradiation of insects 

In the irradiation facility of the Institute of Nuclear Medicine and Allied Sciences 

(INMAS), Ministry of Defence, Delhi, S. litura was irradiated with a Cobalt-60 

source at the dose rate of 80 95 Gy/min for the present investigations. A dose range 

of 40 70 Gy was used for radio-sterilization of last instar larvae of S. litura. A 

sublethal dose range of 100 130 Gy was used to irradiate male moths (0 1-day-old) 

to produce partially sterilized males that could be released to mate with normal 

females and produce F1 sterile progeny. These F1 sterile progeny derived from substerile 

male parents were used for the present experimental study. 

Bioassay of S. glaseri EPNs 

A bioassay requiring detailed observations was designed to assess the various 

features of the nematode’s infective behaviour and reproduction. 

Inoculation 

Individual 1 2-day-old sixth instar S. litura larvae (L6) were each placed into a Petri 

dish (50 15 mm) lined with 2-fold filter paper (Whatman #1). Freshly harvested (up 

to 2 weeks old) infective juveniles (IJs) in all experimental regimens were transferred 

into each Petri dish by distributing them evenly onto the filter paper base in order to 

inoculate at the rate of 25 IJs per individual host. Incubation was performed at 25 o C 

and 7595% relative humidity. Each assay for ascertaining IJs infective behaviour 

was conducted in an individual mode with respect to host (comprising of single L6


larva inoculated by 25 IJs per assay) due to the cannibalistic behaviour of host 

larvae. 

Parasitisation performance and harvesting of nematodes 

The time profile for the induction of morbidity and mortality in the exposed S. litura 

larvae was recorded at 4 6 h intervals. Morbidity is an initial behavioural response to 

haemolymph septicemia caused by toxins released by symbiotic bacteria of EPNs, 

followed by the host’s resultant mortality. Morbidity criteria of infected larva 

included their minimal response to a probe, sluggish nature, and delayed resumption 

of the normal posture with slight torsion in the body when turned upside down. 

After host death, the parasitised (host) larvae were incubated until the next 

generation of IJs were developed. After 7 8 days, IJs were seen wriggling at the 

outer surface of the insect cadaver. The incubation times of EPNs (i.e., from 

inoculation to emergence of next generation IJs from host cadaver) were recorded. 

The harvest of IJs was done using the White Trap method (White 1927). For this, the 

cadavers having proliferating IJs inside were removed from the inoculative Petri 

dishes and transferred to sterilized harvest dishes (90 mm diameter). The daily 

profiles of emergence of IJs out of host cadavers and their total emergence (harvest) 

period were recorded. Their harvest potential (IJs’ yield) was determined in terms of 

cumulative number of IJs harvested over the total harvest period per host, and the 

number of IJs harvested per mg fresh weight of host. IJs when dead appeared to have 

lost weight, because they tended to float as compared to surviving nematodes. Dead 

IJs remained completely straight. The viability criterion in the S. glaseri EPN 

population was taken as 80% or more survival with responsiveness, as suggested by 

Epsky and Capinera (1994). 

For assessment of host morbidity timing and its mortality timing by EPNs, 

incubation time of EPNs (within host) and harvest potential of IJs, individual host 

observations were conducted and each replicate represented the mean value of 

observations on a set of five to seven individual hosts; whereas for the assessment of 

percent parasitisation, a cohort of 25 host larvae (evaluated by individual exposures) 

constituted each replicate. 

Interaction of S. glaseri EPNs with radiation induced F1 sterility in S. litura 

Bioefficacy of EPNs on F1 sterile insect hosts 

Firstly, the bioefficacy of S. glaseri EPNs was assayed as indicated above against F1 

sterile S. litura larvae derived from matings in which their male parent had been 

treated with sub-sterilizing gamma doses (100 or 130 Gy). Bio-efficacy of S. glaseri 

was assessed by recording the times needed to induce morbidity and mortality, the 

incubation time, the percent parasitisation, development and proliferation (as 

computed by the harvest potential) of EPNs. 

Bioefficacy of S. glaseri IJs cultured in radio-sterilized insect hosts 

The bioefficacy of S. glaseri IJs cultured in radio-sterilized insect hosts was evaluated 

in terms of times taken to morbidity and mortality of host, parasitising efficacy,


incubation period and IJs’ harvest potential against normal and against F1 sterile S. 

litura hosts. Two doses, viz., 40 and 70 Gy, were selected for radio-sterilization of S. 

litura sixth instar larvae in view of viability and EPNs’ parasitising performance on 

radio-sterilized hosts (Seth and Barik 2007). 

Bioefficacy of S. glaseri IJs cultured in F1 sterile insect larvae 

Lastly, the parasitising performance and harvest potential of IJs (indicating 

reproductive potential) of the entomogenous nematode cultured in parasitised F1 sterile insects (progeny of irradiated male parents), was ascertained against normal 

and against F1 sterile S. litura hosts. 

Data analysis 

All the characteristics related to host killing efficiency and parasitization performance 

of S. glaseri were studied in 10 12 replicates. The data were computed for 

means, standard error and further analysis of variance (ANOVA, SPSS, 11.0). Twoway 

ANOVA was used to test the interaction of irradiation history of IJs’ parent host 

(either as radio-sterilized host or F1 sterile larval progeny of irradiated male parent) 

and current host’s irradiation background on the performance of IJs. Percentage 

data were transformed using arcsine âx before ANOVA. Means were separated at 

the 5% significance level by least significant difference (LSD) test (Snedecor and 

Cochran 1989). 

Results 

Bioefficacy of EPNs on F1 sterile insects 

The bioefficacy of normal EPNs (i.e., cultured in un-irradiated insect host) was 

ascertained against F1 sterile S. litura larvae produced in matings of untreated 

females with substerilized male moths, irradiated with 100 and 130 Gy, in order to 

understand the degree of acceptability and suitability of F1 progeny as potential 

hosts to entomophilous nematodes (Table 1, Figure 1). ANOVA performed on data 

pertaining to bioefficacy of EPNs on F1 sterile insects indicated that there was a 

significant influence of induced sterility of the host (F1 sterile insects) on the 

parasitisation efficacy of EPNs, but not on the mortality induction process. The 

onset of morbidity and mortality induced by normal IJs (i.e., IJs derived from untreated 

host) was not different in F1 sterile hosts and un-irradiated controls (i.e., 

untreated host) (P 0.05). The incubation time taken by IJs on F1 sterile hosts was 

slightly prolonged, being significantly longer in F1 hosts derived from 130 Gy treated 

male parent (PB0.05). Parasitisation response was reduced in a dose dependent 

manner on F1 sterile hosts, with a significant impact on F1 larvae derived from 

fathers that had been treated with 130 Gy (PB0.05). The IJs’ harvest was found to 

be moderately reduced on F1 hosts, but the effect was significant on F1 hosts at 130 

Gy (PB0.05). The number of IJs harvested from F1 sterile host was reduced by 

about 8 11% at 100 Gy and 17 20% at 130 Gy with respect to controls. The harvest 

period was also slightly affected by the radiation dose applied to the host in the 

previous generation (PB0.05).

Table 1. Infective performance of entomopathogenic nematodes (EPNs), Steinernema glaseri, 

on F 1 sterile Spodoptera litura larvae (progeny of matings of untreated females and males 

irradiated with sub-sterilizing gamma doses of 100 or 130 Gy). 

Nature of 

EPN 

Normal IJs 

(Control) 

Nature of 

host 

F 1 host from 

unirradiated 

male parent 

(Control) 

Normal IJs F1 host from 

100 Gy 

irradiated 

male parent 

Normal IJs F1 host from 

130 Gy 

irradiated 

male parent 


Time 

required 

for 

morbidity 

(h) 

22.8a 

91.1 

23.8a 

91.2 

23.1a 

90.9 

Time 

required 

for 

mortality 

(h) 

48.6a 

91.8 

50.9a 

92.2 

49.3a 

91.5 

Incubation 

time (h) 

177.4a 

94.9 

182.9ab 

95.2 

192.8b 

94.1 

Harvest (yield) of IJs 

IJs per 

host 

20855a 

91022 

18426ab 

9894 

16667b 

91006 

IJs per 

mg body 

wt 

34.9a 

91.2 

31.9ab 

91.1 

28.9b 

91.7 

Period 

(days) 

11.9a 

90.4 

11.2a 

90.4 

9.6b 

90.5 

Sixth instar host larvae (1 2-day-old) were bioassayed, IJs, infective juveniles of EPNs, Means9SE 

followed by same letter in a column are not significantly different at P50.05 level (ANOVA followed by 

LSD post-test); n 12. 

Bioefficacy of EPNs cultured in radio-sterilized host larvae 

The infective performance of EPNs cultured in radio-sterilized host was studied 

against normal and F1 sterile host-insects to ensure the viability and virulence of 

EPNs transported in radio-sterilized host (Table 2, Figure 2). There was no 

significant interaction between irradiation of the IJs’parent-host (in which the IJs 

had been produced) and the nature of the current host, i.e., F1 sterile larvae (i.e., 

having radiation dose applied to its male parent) on parasitisation performance of 

EPNs (P 0.05), except in case of the time to mortality (F 8.9;df 4, 99; PB0.01) 

and the harvest period (F 5.04; df 4, 99; PB0.01) as indicated by two-way 

ANOVA. Further, as per one-way analysis of variance, both the factors, viz., 

irradiation of IJs’ parent host and nature (quality) of current F1 sterile host (i.e., the 

degree of sterility induced by irradiated male parent), individually had a significant, 

though not drastic, impact on time to mortality, percent parasitisation, incubation 

period and the number of IJs harvested from host cadaver (PB0.05). Infection was 

induced a little later by IJs that had been cultured in irradiated hosts than cultured in 

the untreated control, i.e., IJs emerging from normal hosts. Time taken for inducing 

morbidity was slightly more in case of IJs harvested from 70 Gy irradiated hosts 

than by IJs from 40 Gy irradiated hosts. The time to mortality induced by IJs that 

had been cultured in radio-sterilized host was slightly delayed on both normal and 

F1 sterile hosts, as compared to that induced by control IJs (PB0.05), although the 

IJs from 40 Gy irradiated hosts killed host faster than the IJs from 70 Gy irradiated 

hosts. 

The incubation time taken by IJs from radio-sterilized hosts was prolonged on 

normal as well as F1 hosts. The effect was a little more apparent when the IJs that


Figure 1. Parasitisation efficacy of entomopathogenic nematodes (EPNs), Steinernema 

glaseri, against normal (N) and F 1 sterile Spodoptera litura larvae. The latter were the 

progeny of matings of untreated S. litura females with males irradiated with sub-sterilizing 

doses (100 or 130 Gy) of gamma radiation. Sixth instar host larvae (1 2-day-old L6) were 

bioassayed to ascertain the parasitisation by EPNs (25 IJs/host larva). Means9SE (of the 

bars) denoted by the same letter are not significantly different at PB0.05 level (calculated 

using ANOVA followed by LSD post-test); percentage data were arcsine transformed before 

ANOVA, but data in the figure are back transformations; n 12 (a cohort of 25 host larvae, 

evaluated by individual exposures, constituted each replicate). 

had been cultured in 70 Gy irradiated hosts pursued parasitisation, and also it was 

more apparent when parasitisation occurred towards F1 hosts at 130 Gy (PB0.05). 

Bioassays showed that the parasitisation efficacy (Figure 2) of IJs derived from 

radio-sterilized hosts was impaired (PB0.05) by 14 16% at 40 Gy and by 18 21% at 

70 Gy with respect to controls. F 1 sterile hosts from 100 Gy or 130 Gy treated male 

parents appeared to be almost equally acceptable to IJs from irradiated hosts. The 

EPNs’ harvest was moderately but significantly reduced when IJs that had been 

cultured in larvae irradiated with 40 Gy infected normal hosts (PB0.05). The 

harvest of EPNs was further reduced in host larvae irradiated with 70 Gy (PB0.05). 

Moreover, EPNs’ harvest was further negatively affected when IJs that had been 

cultured in irradiated hosts infected F1 sterile hosts. This resulted in the evident 

reduction of the harvest in the case of F1 sterile hosts at 130 Gy. Similarly the harvest 

period was shortened, the effect being more apparent on F 1 hosts at 130 Gy (PB 

0.05). On the other hand, the harvest period was not significantly shortened when IJs 

from 40 Gy irradiated hosts parasitised normal or F1 host insects (P 0.05). 

Bioefficacy of EPNs cultured in F1 sterile host larvae 

The infective performance of IJs cultured in parasitised F1 sterile S. litura larvae was 

ascertained on normal (unirradiated) hosts and on F1 sterile host-progeny of 

irradiated male parents, so as to understand the persistence of infective viability of 

IJs harvested from F1 sterile hosts (Table 3, Figure 3). It is worth noting that no 

evident interaction was noticed between the irradiation background of IJ’s parent

Table 2. Infective performance of entomopathogenic nematodes (EPNs, Steinernema glaseri, 

cultured in radio-sterilized Spodoptera litura larvae (40 Gy, 70 Gy) and applied to normal and 

F1 sterile S. litura larvae (progeny of matings of untreated females and males irradiated with 

sub-sterilizing gamma doses of 100 or 130 Gy). 

Nature of 

EPN 

Normal IJs 

(Control) 

IJs from 40 

Gy treated 

host 

IJs from 40 

Gy treated 

host 

IJs from 40 

Gy treated 

host 

IJs from 70 

Gy treated 

host 

IJs from 70 

Gy treated 

host 

IJs from 70 

Gy treated 

host 

Nature of 

host 

Normal host 

(Control) 

Time 

required 

for 

morbidity 

(h) 

22.8a 

91.2 

Normal host 23.9ab 

91.6 

F1 host from 

100 Gy 

irradiated 

male parent 

F1 host from 

130 Gy 

irradiated 

male parent 

24.2ab 

91.2 

24.4ab 

91.1 

Normal host 26.7b 

91.2 

F1 host from 

100 Gy 

irradiated 

male parent 

F 1 host from 

130 Gy 

irradiated 

male parent 


24.4ab 

90.9 

26.2b 

90.8 

Time 

required 

for 

mortality 

(h) 

47.5a 

91.7 

68.7c 

93.4 

54.7b 

91.1 

54.3ab 

92.6 

69.3c 

92.6 

55.7b 

93.6 

58.1b 

92.9 

Incubation 

time (h) 

179.3a 

94.2 

199.2bc 

97.4 

188.9abc 

97.1 

195.9bc 

95.9 

214.1c 

97.8 

194.5abc 

96.2 

201.5bc 

99.1 


IJs per 

host 

21010a 

9911 

16986bc 

9889 

17459b 

9981 

15270bc 

9839 

15940bc 

9797 

16222bc 

9785 

14413c 

9653 

IJs per 

mg body 

wt 

35.1a 

92.2 

30.2abc 

91.5 

31.1ab 

91.6 

28.7bc 

91.4 

27.0bc 

91.3 

29.1bc 

91.5 

26.4c 

91.3 

Period 

(days) 

11.8a 

90.5 

10.1bc 

90.5 

11.1ab 

90.5 

10.1bc 

90.4 

9.1c 

90.4 

10.1bc 

90.5 

9.7bc 

90.5 

Note: Sixth instar host larvae (1 2-day-old) were bioassayed, IJs, infective juveniles of EPNs, Means9SE 

followed by same letter in a column are not significantly different at P50.05 level (two-way ANOVA 

followed by LSD post-test); n 12. 

host, i.e., F1 sterile larvae (in which the IJs used had been cultured) and the nature 

(quality) of the current host (i.e., F1 sterile host) on parasitisation behaviour of EPNs 

(P 0.05) except in case of time to mortality (F 2.94; df 4, 81; PB0.05), as 

reflected by two-way ANOVA. However, one-way analysis of variance reflected an 

evident impact (but not drastic) of these factors individually, on time to mortality, 

percent parasitisation, incubation time and the numbers of nematodes harvested 

from host larva (PB0.05). Induction of morbidity and mortality in normal host 

larvae caused by IJs cultured in F 1 sterile host larvae (progeny of matings of 

untreated females with males irradiated with 100/130 Gy) occurred at a similar time 

as in the controls (P 0.05), although their infective response was slightly delayed on 

F 1 sterile host larvae (PB0.05). Incubation time taken by IJs that had been cultured



glaseri, cultured in radio-sterilized Spodoptera litura larvae (40 or 70 Gy) and applied to 

normal (N) and F1 sterile S. litura larvae (progeny of matings of untreated females and males 

irradiated with sub-sterilizing doses (100 or 130 Gy) of gamma radiation). The parasitisation 

bioassay was conducted by applying 25 infective nematode juveniles to each sixth instar S. 

litura larvae (1 2-day-old L6). Means9SE (of the bars) denoted by the same letter are not 

significantly different at PB0.05 level (calculated using ANOVA followed by LSD post-test); 

percentage data were arcsine transformed before ANOVA, but data in figure are back 

transformations; n 12 (a cohort of 25 host larvae, evaluated by individual exposures, 

constituted each replicate). 

in F1 sterile hosts was not significantly affected on normal host larvae with respect to 

the control, whereas it was slightly prolonged on F1 sterile host larvae. The effect was 

especially apparent when IJs that had been cultured in F 1 sterile host larvae infected 

F 1 sterile host larvae at 130 Gy (PB0.05). 

Parasitisation efficacy (Figure 3) was reduced by 18 38% when IJs, which had 

been cultured in F1 sterile host larvae, were allowed to infect normal and F1 sterile 

insects, as compared to the controls (90.5% efficacy). IJs emerged out of F1 sterile 

host did not show any differential infective response towards F1 sterile host as 

compared to normal host larvae (P 0.05), except the significantly reduced 

parasitisation response by IJs from F 1 sterile hosts (progeny of 130 Gy treated 

male) towards F1 sterile host larvae at 130 Gy (55.4%; PB0.05). The IJs’ harvest was 

reduced by 16 25% when IJs that had been cultured in F1 sterile hosts (progeny of 

100 Gy treated male) parasitised F1 sterile hosts, with respect to controls. Further, a 

harvest reduction of 18 31% was recorded in case of infection by IJs that had been 

cultured in F 1 sterile hosts at 130 Gy. The reduction in harvest potential of IJs 

depended upon the gamma dose administered to male parent of F1 insects (as hosts). 

The IJs’ harvest potential exercised by the infection of IJs that had been cultured in 

F1 sterile hosts was reasonably high on normal insect-hosts and nearly similar to

Table 3. Infective performance of entomopathogenic nematodes (EPNs), Steinernema glaseri, 

cultured in F 1 sterile Spodoptera litura larvae and applied to normal and F 1 sterile S.litura 

larvae (progeny of matings of untreated females and males irradiated with sub-sterilizing 

gamma doses of 100 or 130 Gy). 

Nature of 

EPN 

Normal IJs 

(Control) 

IJs from F1 

host (derived 

from 100 Gy 

treated male) 

IJs from F1 

host (derived 

from 100 Gy 

treated male) 

IJs from F 1 

host (derived 

from 100 Gy 

treated male) 

IJs from F 1 

host (derived 

from 130 Gy 

treated male) 

IJs from F1 

host (derived 

from 130 Gy 

treated male) 

IJs from F1 

host (derived 

from 130 Gy 

treated male) 

Nature of 

host 

Normal host 

(Control) 

Time 

required 

for 

morbidity 

(h) 

22.9a 

91.2 

Normal host 23.6ab 

90.9 

F1 host from 

100 Gy 

irradiated 

male parent 

F 1 host from 

130 Gy 

irradiated 

male parent 

25.8bc 

91.1 

24.3abc 

91.9 

Normal host 25.2abc 

91.2 

F1 host from 

100 Gy 

irradiated 

male parent 

F1 host from 

130 Gy 

irradiated 

male parent 


25.9c 

91.3 

27.4c 

91.5 

Time 

required 

for 

mortality 

(h) 

47.5a 

91.6 

46.7a 

92.2 

52.9ab 

92.3 

54.8bc 

92.7 

52.4ab 

91.6 

62.2cd 

93.1 

65.4d 

92.5 

Incubation 

time (h) 

180.1a 

95.9 

185.2a 

94.6 

188.5a 

96.9 

217.7b 

910.4 

191.3a 

96.9 

215.9b 

99.5 

226.2b 

910.1 


IJs per 

host 

21202a 

91055 

19139b 

9845 

17724b 

9886 

17774bc 

9890 

18197b 

9711 

17343b 

9893 

15412c 

9691 

IJs per 

mg 

body wt 

36.7a 

91.8 

33.1ab 

91.2 

30.7bcd 

91.2 

27.6cd 

91.3 

31.3b 

91.1 

25.4d 

91.2 

26.6d 

91.3 

Period 

(days) 

11.18a 

90.6 

10.6ab 

90.5 

9.2bc 

90.4 

9.1bc 

90.4 

10.1ab 

90.3 

9.7ab 

90.4 

8.9c 

90.2 

Note: Sixth instar host larvae (1 2-day-old) were bioassayed, IJs, infective juveniles of EPNs, Means9SE 

followed by same letter in a column are not significantly different at P50.05 level (two-way ANOVA 

followed by LSD post-test); n 10. 

control. The harvest period of IJs was also found to be slightly affected, the effect 

being especially apparent on F1 hosts derived from 130 Gy treated male (PB0.05). 

Discussion 

The bio-infective performance of normal IJs, in terms of inducing morbidity and 

mortality, was similar towards F1 sterile host (derived from irradiated male parent) 

and control S. litura larvae, whereas the parasitisation and the harvest of EPNs were 

diminished on F1 hosts at 130 Gy.



glaseri, cultured in F 1 sterile Spodoptera litura larvae and applied to normal (N) and F 1 

sterile S. litura larvae (progeny of matings of untreated females and males irradiated with substerilizing 

doses (100 or 130 Gy) of gamma radiation). The parasitisation bioassay was 

conducted by applying 25 infective nematode juveniles to each sixth instar S. litura larvae (1 

2-day-old L6). Means9SE (of the bars) denoted by the same letter are not significantly 

different at PB0.05 level (calculated using ANOVA followed by LSD post-test); percentage 

data were arcsine transformed before ANOVA, but data in figure are back transformations; 

n 10 (a cohort of 25 host larvae, evaluated by individual exposures, constituted each 

replicate). 

The parasitising performance of IJs cultured in 40 Gy treated hosts was better 

and their viability was greater than that of IJs cultured in 70 Gy treated host larvae. 

Although 70 Gy was ascertained to be complete (reliable) sterilizing dose for L6, the 

overall sterilizing impact 40 Gy (that induced 80 91% reproductive suppression in 

L6 in different irradiated crosses) was apparently much more (almost complete) if 

coupled with 28 47% reduction in mating success (with respect to control) plus 

reduced adult emergence (53.9%) with pronounced degree of malformation (61.2%) 

at this dose. Hence, even 40 Gy dose could also be considered as safe for radiosterilization 

of L6, in order to ensure an almost risk free mode of transport of EPNs 

(by avoiding potential pest release) (Seth and Barik 2007, Seth unpublished). 

The bio-infective performance of IJs cultured in radio-sterilized hosts exhibited a 

reasonable degree of parasitisation potency, with better performance from IJs 

derived 40 Gy irradiated host than that from 70 Gy irradiated hosts towards F1 

sterile host progeny of irradiated male parents. Time profile of onset of morbidity 

caused by these IJs (from irradiated host) was more or less similar in F1 sterile insects 

and in normal hosts. That indicated that release of the toxins by endosymbiotic 

bacteria must have occurred in a similar fashion in F1 insects as in the controls. 

Further, it was interesting to note that F1 hosts probably possessed less immunity or


offered less resistance towards the infecting IJs cultured in irradiated hosts; hence 

mortality induced by these IJs occurred more quickly in F1 sterile insects than in 

normal hosts. The F1 progeny from the 100 Gy treatment were found to be more 

acceptable and suitable hosts (than F1 larvae from 130 Gy treated male parent) for S. 

glaseri EPNs transported in the radio-sterilized host. 

The bio-infective performance of IJs harvested from F1 sterile host towards 

normal and F 1 sterile insects, however, indicated a great degree of persistent efficacy 

of these EPNs (emerging out of parasitised F 1 sterile host) that would interact with 

the insect population in the ecosystem. The relatively reduced (though not drastic) 

performance of IJs, which had been cultured in F 1 sterile insects (especially at 130 

Gy) might be attributed to probably lower host quality in terms of nutrition and 

secondary chemicals, which could be correlated with affected nutritional efficiencies 

and energy budget of S. litura as reported by Seth and Sehgal (1993). Further, it was 

interesting to note that the IJs cultured in the F1 sterile host (derived from treated 

males at 100 Gy, 130 Gy) could also retain their infective potential up to 61.2 82.5%, 

with 70 91% harvest potential of IJs (with respect to controls). This reflects that 

these IJs, if released in inoculative mode, in conjunction with F1 sterility, could be 

used quite effectively in the field for several generations keeping in view the 

acceptability and suitability of F1 sterile insects as potential host coupled with 

tremendous recycling capacity of these EPNs within F1 insects. 

Inherited sterility induced by irradiated male parent moths has been proposed 

for the reproductive suppression of lepidopteran pests. Combination with certain 

ecologically safe strategies like biological control may further improve the control of 

lepidopteran pests using F 1 sterility; hence, the probability of integrating S. glaseri 

EPNs with this genetic control measure was investigated in the present study on S. 

litura. It is difficult to find complementary control strategies for synergistic use in 

conjunction with sterile moth release programmes. Gouge, Lee, Bartlett, and 

Henneberry (1998) suggested that S. carpocapsae may be an ideal entomopathogenic 

nematode to be used in conjunction with inherited sterility for the management of 

the pink bollworm, Pectinophora gossypiella, asS. carpocapsae would more likely 

infect the mobile native pink bollworm larvae than the sedentary F1 larvae from 

irradiated parents. S. carpocapsae basically is a passive ambusher, but in our present 

experiments, we have used the species S. glaseri, which is a cruise forager, and highly 

mobile and responsive to long-range host volatiles. S. glaseri is best adapted to 

parasitize hosts possessing low mobility and residing within the soil profile, as 

suggested by Gaugler, Campbell, Selvan, and Lewis (1992). 

We have attempted to study the feasibility of integrating the use of EPNs with F1 

sterility and have focused on investigations regarding the interaction with F 1 sterile 

insects of normal EPNs, and EPNs that had been cultured in radio-sterilized insects. 

F 1 insects, derived from 100 Gy irradiated male parent, were acceptable and suitable 

hosts for EPNs, almost with the same degree as untreated insects (control), whereas 

F1 insects (from 130 Gy treated parents) were relatively less acceptable hosts for 

parasitisation by S. glaseri EPNs. These F1 larvae (progeny of 130 Gy treated male) 

were slightly sluggish as well. Probably the host volatiles and nutritional quality of 

these treated larvae were adversely affected; and this could be one of the causes of 

the reduced infectivity potential of S. glaseri. Therefore, simultaneous release of 

EPNs (in inundative mode) with F1 insects from 100 Gy treated parents might be 

good proposition unlike with F1 insects from 130 Gy treated parents. Moreover, due


to reduced mating competitiveness and reduced sperm transfer by F1 progeny insects 

(from 130 Gy male parent), 130 Gy has been proposed as relatively less preferred 

than 100 Gy dose to be employed in F1 sterility technique (Seth and Sharma 2001). 

Hence, release of EPNs along with F1 sterile insects might limit or influence the 

effectiveness of F 1 sterility for pest suppression, depending upon the gamma dose (to 

be used in F 1 sterility) and the timing of EPNs release. Further, since the 

compatibility of two control measures was confirmed, and the pest population 

suppression was feasible by both techniques, a management strategy could be 

devised. In this situation, simultaneous application of both tactics, due to 

acceptability and suitability of F1 insects as host for EPNs reared in radio-sterilized 

host, may show an additive effect, because these two methods are not antagonistic to 

each other. Provided timing and logistics are taken into consideration, synergy may 

be achieved in response to inoculative release of EPNs along with F 1 sterility. 

As per review of our investigations, the use of ‘genetic pest control method’ (F 1 

sterility technique) in conjunction with EPNs could be a feasible strategic 

component in IPM of S. litura, in which operational modality might be either (i) 

‘sequential’, i.e., EPNs application preceding the use of F1 sterility so as to reduce 

the load of release of sub-sterilized moths, or (ii) ‘simultaneous’ for some initial 

specific phase, because F1 insects (at 100 Gy) would be equally acceptable as normal 

insects, and pest suppression would be operating against different stages of the life 

cycle, i.e., against larvae and pupae (through EPNs) and against adults (via F 1 

sterility). The intermittent (inundative) releases of EPNs could also be effectively 

pursued, alternately with F1 sterility, so as to keep the pest population below the 

economic threshold. Further, in a situation where F1 sterility has been successful in 

suppression of pest population, the inoculative releases of EPNs could be considered 

for biological pest management, especially as a quarantine measure along with 

release of partially sterile insects, in view of bio-infective potential of IJs, cultured in 

F1 sterile insects, observed in the present study. 

Cautious field simulated studies are warranted to judge the operational approach 

with respect to pest density and timing, so as to optimally integrate the use of EPNs 

with F1 sterility. The inundative and inoculative releases of EPNs might be possible 

and effective in view of acceptability and suitability of normal and irradiated S. litura 

and their F1 sterile progeny as hosts of EPNs, according to the present investigation, 

where the pest was found to be responsive towards both control tactics. 


Financial assistance by the International Atomic Energy Agency, Vienna is gratefully 

acknowledged for supporting this research work under Research Contract No. IAEA/IND- 

10847/R0/RB as part of a Coordinated Research Project. Thanks are due to the technical 

support provided by Mr Manas K. Dhal. 

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Vol. 19, S1, 2009, 157 165 


Parasitism rate and sex ratio of Psyttalia ( Opius) concolor 

(Hymenoptera: Braconidae) reared on irradiated Ceratitis 

capitata larvae (Diptera: Tephritidae) 

Bahriye Hepdurgun*, Tevfik Turanli, and Aydin Zümreog˘lu 

Plant Protection Research Institute, Gençlik caddesi No. 6, 35040 Bornova, I˚zmir, Turkey 

Tests were conducted to evaluate use of irradiated Mediterranean fruit fly 

(medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), larvae as 

factitious hosts for mass-rearing the parasitoid Psyttalia concolor (Szepligeti) 

(Hymenoptera: Braconidae). In order to prevent the release of the alternative host 

adults, eclosion from unparasitized larvae must be prevented. Exposure of medfly 

larvae to 40, 50 or 60 Gy of 60 Co gamma radiation showed that 60 Gy was the 

most effective dose in inhibiting adult medfly eclosion. The most suitable 

host:parasitoid ratio was found to be three larvae per parasitoid regardless of 

exposure time. When three larvae per parasitoid were exposed for 3 h, there was 

no significant difference in parasitism rates using irradiated vs. unirradiated host 

larvae (16.4 vs. 18%, respectively). Irradiation of host larvae also had no 

significant effect on the sex ratio of resulting parasitoids. Implementation of 

this practice will improve the efficiency of mass production and release of this 

biocontrol agent. 

Keywords: gamma radiation; Ceratitis capitata; Bactrocera oleae; Psyttalia 

concolor; mass rearing 

Introduction 

The olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tepritidae), is one of the most 

important pests in olive orchards in Turkey. Infested fruits fall and the level of oil 

acidity may increase by 10 12% in damaged fruits. Damage levels of 30 50% are not 

uncommon (Hepdurgun, Koçlu, Zümreog˘lu, and Turanli 2004) and can reach 90 

100% damage in epidemic years (Aysu 1957), especially in early maturing pulpy and 

oily olive fruit cultivars. 

Aerial bait-spraying (bait attractant insecticide), organized by the government 

(Plant Protection Research Institute, PPRI, of Bornova, Turkey), has been used to 

achieve area-wide control over 15 million trees in coastal areas of the Aegean Region 

since 1980 (Pala, Zümreog˘lu, Fidan, and Altin 1997). Although bait-spraying is less 

harmful to beneficial organisms and the environment than traditional cover spraying 

(insecticide alone), non-target effects still occur. Accordingly, alternate strategies that 

further minimize detrimental treatment side-effects, including mass-rearing and 

release of parasitoids, continue to gain in importance in the control of B. oleae. 

To date, a substantial amount of B. oleae biological control research has been 

carried out throughout the Mediterranean basin (Bjelis, Pelicaric, and Masten 

*Corresponding author. Email: hepdurgun@hotmail.com 

First Published Online 6 July 2009 



DOI: 10.1080/09583150903090479 


158 B. Hepdurgun et al. 

2003). Exploration for B. oleae natural enemies led to discovery of a larval pupal 

endoparasitoid in Tunisia by Marchal (1910), described as Opius concolor 

(Hymenoptera: Braconidae) by Szepligeti. This species was placed in the subgenus 

Psyttalia by Fischer (1987) and subsequently elevated to generic rank by Wharton 

(1987). Most of the countries that suffer from B. oleae damage have conducted 

studies on the rearing of P. concolor for use in biological control of the pest 

(Biliotti and Delanoue 1959; Jannone and Binaghi 1959; Monastero 1959; 

Monastero and Genduso 1962; Fenili and Pegazzano 1965; Brnetic 1973; Avilla 

and Albajes 1983; Raspi and Loni 1994). Additional studies on the host 

parasitoid relationship were conducted by Avilla and Albajes (1984). It was 

found by Delanoue in 1958 (Arambourg 1983) that C. capitata could serve as a 

factitious host for rearing P. concolor. Because techniques for mass rearing C. 

capitata are advanced, it was chosen as a preferred laboratory host for parasitoid 

mass production. 

Nuclear techniques can play an important role in augmentative biological 

control, for example by facilitating mass rearing (Greany and Carpenter 2000). In 

recent years, irradiating host larvae before exposure to parasitoids has been an 

important technique in the mass rearing of fruit fly parasitoids to prevent the 

emergence of adult flies, thus eliminating concerns about releasing or needing to 

separate host material when conducting parasitoid releases (Sivinski and Smittle 

1990). Large scale rearing of the braconid parasitoid Diachasmimorpha longicaudata 

(Ashmead) has been undertaken in the USA and Mexico using irradiated Anastrepha 

suspensa (Loew) (Sivinski et al. 1996) and Anastrepha ludens (Cancino, Ruiz, Gomez, 

and Toledo 2002), respectively as hosts, and the technique has been examined in 

numerous other locations and with a variety of parasitoid species (e.g., this volume). 

The studies described below were conducted using C. capitata to mass rear 

P. concolor as a means to develop an environmentally friendly approach for areawide 

control of B. oleae. The radiation dose needed to completely prevent adult 

eclosion of C. capitata was determined and optimal parasitization exposure time and 

host:parasitoid ratios were established. The parasitism of irradiated vs. unirradiated 

mature medfly larvae was compared, and the affect of radiation on parasitoid sex 

ratio determined. 


Stock cultures of C. capitata and P. concolor were kept at 2591 o C, 6595% relative 

humidity and a photoperiod 16 h L:8 h D. C. capitata larvae were reared according 

to the method described by Zümreog˘lu (1979), using an artificial medium. For the 

mass rearing of P. concolor, rearing procedures outlined in the parasitoid rearing 

manuals from Hawaii and Guatemala were used (Anonymous 1997, 1998). Mature 

third instar medfly larvae to be used as hosts in the rearing of P. concolor were 

collected in trays of water placed at the bottom of cabinets after they exited the 

rearing trays. The mass reared P. concolor strain in Turkey was originally set-up 

with wasps derived from the National Agricultural Research Foundation (NA- 

GREF) of Greece, which had origins from collections in Greece and Italy (Karam 

et al. 2008).


Irradiation dose 

Irradiation tests were performed to determine the dose of gamma radiation 

applied to medfly larvae required to prevent adult emergence. Groups of ca. 2500 

mature larvae in water in Petri dishes were irradiated at 40, 50 or 60 Gy; ca. 2500 

larvae from the same batch were left untreated as controls. Irradiation was 

performed using a type C-146, 7000 C: Cobalt-60 Theratron irradiator, delivering 

107.33 cGy/min. 

Percent emergence for the irradiated (40, 50 and 60 Gy) and non-irradiated 

control larvae was determined by placing each group of 2500 larvae in a wooden 

box containing fine sand and kept in a pupation room at 2091 o C and 7095% RH. 

Pupae were sifted 2 days before the expected emergence date and four separate 

groups of 400 pupae were randomly selected from each treatment and placed in 

946 mL polystrene containers. Cube sugar and moist sponges were placed on the 

screen tops of the containers to provide food and water for the emerged flies. Adult 

emergence from the pupae was checked every other day for 10 days and the 

numbers of deformed pupae, half-emerged flies and fully emerged flies were 

recorded. 

Determination of optimal host:parasitoid ratio and exposure time 

Tests were conducted to determine the optimal host:parasitoid ratio and exposure 

duration. For these tests, the following host larvae (L):à parasitoid (P) ratios were 

evaluated: 1:1, 2:1 and 3:1 and these ratios were tested using 1-, 2-, and 3-h exposure 

periods. Thus, for each series of tests, nine cages were set-up (3 L:à P ratios 3 

exposure times). The tests were carried out under laboratory conditions (2591 o C, 

6595 RH) using 30 40 30 cm aluminum cages (frame and bottom) covered with 

fine mesh organdy cloth. Each cage was populated with 50 à and 50 ß parasitoids 

that had emerged on the same day a given test series was initiated. Medfly larvae 

were exposed to the parasitoids using ‘sting units’, which were prepared using two 

interlocking PVC rings approximately 11 cm in diameter, with unirradiated third 

instar medfly larvae sandwiched between two pieces of fine mesh organdy cloth held 

in place by the rings. Each sting unit contained either 50, 100 or 150 larvae 

depending on the L:P ratio being tested (1:1, 2:1 or 3:1, respectively) and was placed 

in a cage for 1 3 h. This procedure was followed using the same cages of parasitoids 

for 5 consecutive days. Means for each treatment over the 5 consecutive days were 

considered as one replicate. The entire procedure was replicated on five separate 

occasions. 

Larvae taken from the sting units from each cage after parasitoid exposure were 

transferred to labeled jars (12 cm height, 15 cm diameter) containing fine sawdust. 

The jars were ventilated using organdy cloth and kept at 2591 o C, 6595 RHfor adult emergence. Following completion of adult emergence and death of the 

individuals, all parasitoids produced in each jar were counted as male or female. 

The 5-day means of each parameter were subjected to SAS statistical analysis and 

pooled. The five replicates over time were also subjected to SAS statistical analysis 

(randomized complete block design) and the means were ranked according to the 

LSD test.


Parasitoid development on irradiated and unirradiated medfly larvae 

For this experiment, two cages each were provisioned with 500 pairs of newly 

emerged parasitoids. Sting units were then prepared containing either ca. 750 

unirradiated or 750 irradiated (60 Gy) mature medfly larvae. Two of the same type 

sting units were introduced into each cage (3L:1P) for a period of 3 h. This procedure 

was followed twice-a-day for 5 consecutive days. The larvae were taken from each 

cage/sting unit at the end of each parasitization period and were transferred to 

labeled plastic trays containing fine sawdust. Pupae were sifted 6 days after pupation, 

left for 1 day in pupal trays for air circulation, and then placed in Petri dishes labeled 

according to their parasitism dates and times and whether or not they were from 

irradiated or unirradiated larvae. 

From each day’s collection of pupae, 50 pupae were randomly selected from each 

of the AM and PM exposures (100 total) for dissection to determine ‘expected’ rates 

of parasitism and another 100 pupae similarly selected were set-up in emergence 

grids to determined ‘observed’ rates of parasitism. The remaining pupae from each 

day were pooled (AM & PM exposures) and placed in separate emergence cages, five 

(one for each day) with pupae from irradiated larvae and five with pupae from 

unirradiated larvae. Upon emergence, 100 à and 100 ß were randomly selected from 

each emergence cage and placed in individual parasitation cages. A sting unit was 

then placed in each cage for 3 h that contained 5 mL larvae (ca. 300), giving a ratio 

of 3L:1P. This process was repeated once-a-day for 10 consecutive days. Pupae were 

collected as previously described. Parasitism efficiency comparing F1 adults reared 

from irradiated and unirradiated larvae was determined by placing 100 pupae from 

each trial into eclosion grids. Emerged F2 individuals were counted and sexed to 

determine percent successful parasitism and sex ratio. 

Results 

Our experiments showed that no medfly adults emerged when mature third instar 

medfly larvae were irradiated at 60 Gy using a 60 Co gamma radiation source. The 

percentages of adult emergence from pupae whose larvae were irradiated at the doses 

of 50 and 40 Gy were 0.56 and 9.18%, respectively. Percent adult emergence from 

pupae of unirradiated larvae averaged 94.75% (Table 1). 

Tests to determine the optimal host larvae (L) to female parasitoid (P) ratio and 

duration of larval exposure to parasitoids indicated that the highest numbers of 

parasitoids were recovered from treatments using the highest numbers of larvae 

regardless of the exposure time. Percent parasitism for the 3L:1P ratios using 150 

larvae to 50 à parasitoids were 40.32, 43.96 and 42.84% for 1-, 2- and 3-h exposures, 

respectively (Table 2). Percent parasitism using 2L:1P and 1L:1P and a 3-h exposure 

were 23.60 and 7.48%, respectively. 

The rate at which irradiated (60 Gy) larvae were parasitized was not significantly 

different from that of unirradiated larvae. In these experiments, the mean parasitism 

rate of irradiated larvae based on pupal dissections was 18.4 vs. 19.2% for 

unirradiated larvae. The mean parasitism rates based on parasitoid emergence 

from pupae of irradiated and unirradiated larvae were 16.4 and 18.0%, respectively 

(Table 3). The use of irradiated vs. unirradiated host larvae also did not affect the sex 

ratio of emerging F1 parasitoids. In both cases the proportion of females was higher

Table 1. Comparative effect of various irradiation doses applied to third instar medfly larvae 

on adult eclosion. 

Treatment Replicate 1 

Deformed 

pupae (%) 

Half-emerged 

pupae (%) 

Adult 

eclosion (%) 

40 Gy 1 84.50 8.75 6.75 

2 82.25 9.00 8.75 

3 74.00 13.50 12.25 

4 80.50 10.50 9.00 

Mean 80.31 10.43 9.18 

50 Gy 1 99.50 0.25 0.25 

2 99.50 0.50 0.00 

3 98.00 0.00 2.00 

4 99.75 0.25 0.00 

Mean 99.18 0.25 0.56 

60 Gy 1 0 0 0 

2 0 0 0 

3 0 0 0 

4 0 0 0 

Mean 0 0 0 

Non-Irrad. Control 1 2.75 2.00 95.25 

2 3.00 1.25 95.75 

3 5.50 2.00 92.50 

4 2.50 2.00 95.50 

Mean 3.43 1.81 94.75 

1 400 pupae were set-up per replicate. 

than males 1.0:2.9 male:female using irradiated larvae and 1.0:2.1 male:female 

using unirradiated larvae. 

The use of irradiated larvae as hosts did not affect the quality of the F 1 adults 

produced based on their parasitism rates over a 10-day period, as none of the day- 

Table 2. Percent parasitism by Psyttalia concolor at various unirradiated host:parasitoid 

ratios and exposure times. 

No. host 

larvae (L) 


Treatment 

No. à 

parasitoids (P) 

L:P 

ratio 

Exposure 

time (h) 

% Parasitism 

(Mean9SE) 1 

50 50 1:1 1 13.88 de 92.43 

100 50 2:1 1 27.52 c 97.87 

150 50 3:1 1 40.32 ab 98.59 

50 50 1:1 2 12.20 e 93.86 

100 50 2:1 2 30.92 bc 97.50 

150 50 3:1 2 43.96 a 99.73 

50 50 1:1 3 7.48 e 93.28 

100 50 2:1 3 23.60 cd 95.64 

150 50 3:1 3 42.84 a 99.53 

1 Means followed by different letters are significantly different (LSD test, P 0.05).


Table 3. Parasitism rates by Psyttalia concolor and sex ratio of emerging F1 progeny using 

irradiated (60 Gy) and unirradiated medfly larvae. 

Expected 1 

parasitism rate (%) 

Observed 2 

parasitism rate (%) 

Sex ratio of 

emerging adults (M:F) 

Exposure day Irrad. Unirrad. Irrad. Unirrad. Irrad. Unirrad. 

1 10 16 6 8 1:5.0 1:1.7 

2 10 22 7 18 1:1.3 1:1.0 

3 44 22 25 23 1:4.0 1:1.6 

4 12 16 28 24 1:2.5 1:1.4 

5 16 20 16 17 1:1.7 1:4.7 

Mean 18.4 19.2 16.4 18 1:2.9 1:2.1 

1 Expected percent parasitism was determined by pupal dissections. 

2 Observed percent parasitism was determined by adult eclosion. 

wise comparisons between adults from irradiated vs. unirradiated larvae were 

significantly different (Table 4). The sex ratios of the F2 adults produced in these 

trials were also similar. There was a marked decline in total percent parasitism over 

the 10-day period by F1 parasitoids obtained using both irradiated and unirradiated 

medfly larvae, decreasing from around 60% on day 1 to around 16% on day 10. 

Discussion 

Our results showed that irradiated medfly larvae could be used to mass rear the olive 

fruit fly parasitoid P. concolor without having to worry about separating or releasing 

adult medflies. Our results also indicated that the quality of the parasitoids produced 

using irradiated and unirrradiated medfly larvae was not significantly different. The 

Table 4. Percent parasitism by F1 P. concolor adults produced from either irradiated 

(60 Gy) or unirradiated medfly larvae. 

Exposure day 

F 1 adults from 

irrad. larvae 

Parasitism rate 1 (%) Sex ratio of emerging F2 adults 

(M:F) 


unirrad. larvae 


irrad. larvae 


unirrad. larvae 

1 60.0 60.6 1:2.7 1:3.7 

2 49.6 47.0 1:3.0 1:3.3 

3 42.2 40.0 1:2.0 1:2.9 

4 32.4 27.8 1:2.4 1:3.2 

5 28.4 28.6 1:3.1 1:5.5 

6 20.2 21.4 1:3.4 1:5.7 

7 13.2 13.0 1:6.3 1:2.1 

8 16.8 18.4 1:3.0 1:3.2 

9 22.8 11.8 1:2.5 1:4.9 

10 16.2 16.0 1:2.4 1:6.3 

Mean 30.18 28.46 1:3.1 1:4.1 

1 No day-wise comparisons were significantly different after transformation using a chi-square analysis.


complete inhibition of adult eclosion from mature medfly larvae irradiated with 

60 Gy 60 Co gamma radiation, as was found in the studies presented here, is in 

keeping with the results of Delrio (1994). Likewise, Cancino, Ruiz, López, and 

Sivinski (2009) found that a dose of 60 Gy was needed to completely prevent the 

eclosion of adult medflies when these larvae were utilized in the rearing of the 

parasitoids D. longicaudata and D. tryoni (Cameron). Based on these studies, this 

technique was successfully used to conduct a field trial to control olive fruit fly 

populations in Turkey through a combination of mass trapping and mass releases of 

the parasitoid P. concolor reared on irradiated medfly larvae (Hepdurgun, Turanli, 

and Zümreog˘lu 2009). As noted by Greany and Carpenter (2000), this is a more 

useful application of gamma radiation than that of Ramadan and Wong (1989), who 

exposed pupae of the Oriental fruit fly Bactrocera dorsalis (Hendel) to gamma 

radiation after having already exposed the larvae to parasitization by D. longicaudata. 

This resulted in sterility of the adult parasitoids. 

In the present studies, parasitism rates were relatively low (generally below 50%), 

with the highest parasitism rates for P. concolor being achieved using three larvae per 

adult female. In hindsight, the low parasitism rates may have been due to a lack of 

sufficient host larvae resulting in superparasitism. Lawrence, Greany, Nation, and 

Baranowski (1978) found that for D. longicaudata, when 15 or more A. suspensa 

larvae were provided per female, the females discriminated between parasitized and 

non-parasitized hosts and oviposited preferentially in non-parasitized ones, which 

resulted in 70% parasitoid progeny survival. However, when there were only six 

host larvae per female, superparasitism occurred and parasitoid progeny survival 

was B30%. Ashley and Chambers (1979) also found that the production of progeny 

by D. longicaudata was affected by host availability, as well as parasitoid density, age, 

and previous ovipositional experience. In their experiments, maximum rearing 

efficiency was achieved at a host larva to parasitoid ratio of 4:1. Future studies may 

find further increases in rearing efficiency can be achieved for P. concolor using 

irradiated medfly larvae if higher larvae to parasitoid ratios than 3:1 are used. 


The authors thank Dr. Jorge Hendrichs, Project Coordinator and Head of the Insect Pest 

Control Section, Joint FAO/IAEA, for his valuable support and interest during this project 

(no. 10783/TUR). Thanks are also due to Felipe Jeronimo and Gustavo Baeza for their kind 

assistance during the training of the author in rearing techniques at the Moscamed 

Programme facilities in Guatemala. 

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Vol. 19, S1, 2009, 167 177 

Irradiation of Anastrepha ludens (Diptera: Tephritidae) eggs for the 

rearing of the fruit fly parasitoids, Fopius arisanus and 

Diachasmimorpha longicaudata (Hymenoptera: Braconidae) 

Jorge Cancino a *, Lia Ruíz a , Jorge Pérez a , and Ernest Harris b 


Mexico; b US Pacific Basin Agricultural Research Center, USDA-ARS, Honolulu, HI, USA 

Irradiated eggs of Anastrepha ludens were evaluated as hosts of two fruit-fly 

parasitoids for mass rearing. Three different ages of A. ludens eggs (24-, 48- and 

72-h-old) were analyzed for hatchability after being subjected to radiation doses 

of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 Gy. No significant 

reduction in hatchability occurred with the 72-h-old eggs at any of the radiation 

dose levels and no adult emergence occurred at radiation doses greater than 25 

Gy. Seventy two-h-old eggs irradiated above 25 Gy were found to be the best age 

and dose for fruit fly egg hosts to be used in mass rearing the egg parasitoid 

Fopius arisanus. It was demonstrated that larvae hatching from the irradiated A. 

ludens eggs can also be used as hosts for Diachasmimorpha longicaudata. 

Parasitoid emergence of both species was not statistically different from the 

control group (parasitoids emerged from non-irradiated host). The fecundity of 

parasitoids emerged from irradiated hosts also was similar to that obtained with 

parasitoids reared with non-irradiated hosts. There were some statistical 

differences between the curves for longevity. However, these were not clearly 

correlated with radiation dose. The results of this study will aid in the design of 

improved methods for mass rearing and release of fruit-fly parasitoids. 

Keywords: egg irradiation; irradiated host; parasitoid mass-rearing 

Introduction 

Irradiated larvae have proven to be a significant resource for the mass rearing of fruit 

fly parasitoids (Sivinski and Smittle 1990; Cancino, Ruíz, Gómez, and Toledo 

2002a). Irradiated hosts that are not parasitized do not emerge, so that it is possible 

to work only with the parasitoids. Many activities such as host exposure, packing, 

and mass releases would be extremely difficult to carry out without the use of 

irradiation. Nowadays, in the mass rearing of parasitoids, the use of irradiated hosts 

is imperative. However, there may be opportunities to increase the efficiency of the 

irradiation procedures. Currently Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: 

Braconidae) is reared in irradiated larval hosts. These are irradiated 

before exposure to the parasitoids. Many facilities that raise D. longicaudata use 

irradiated hosts, for example, in Piracicaba, Brasil, La Molina, Lima, Peru; La 

Aurora, Guatemala; Florida, USA; and the Moscafrut Plant in Mexico (Sivinski 




DOI: 10.1080/09583150802439827 



et al. 1996; Rodríguez, Quenta, and Molina 1997; Matrangolo, Nascimento, 

Carvalho, Melo, and De Jesús 1998; Menezes et al. 1998; Cancino et al. 2002a). 

If fruit fly eggs could be irradiated as opposed to late instar larvae, then smaller 

volumes would need to be exposed to radiation, decreasing the time required to 

handle host materials. At the same time, it would be easier to collect eggs from 

‘oviposition cages’ than to remove larvae from diet, again resulting in increased 

savings of time and space. To this end, we examined the ability of the widely used 

larval-prepupal parasitoid D. longicaudata to develop in larvae derived from 

irradiated eggs. We compared both the survival of the parasitoid and the propensity 

of the host to complete its development when eggs are exposed to a variety of 

radiation doses. At the same time, we determined the ability of another parasitoid, 

Fopius arisanus (Sonan), an egg-prepupal braconid species, to develop in irradiated 

eggs. This species has been developed on Anastrepha eggs (Lawrence, Harris, and 

Bautista 2000; Zenil et al. 2004), although nowadays there are no strong results in its 

mass-rearing with Anastrepha. This could be a reason to believe that F. arisanus is 

not a good option as a natural enemy for Anastrepha fly species. However, recently in 

Mexico, mass-rearing of F. arisanus has been established successfully on Anastrepha 

ludens (Loew) eggs (Cancino and Ortíz 2002). This could offer opportunities to 

propose new ideas for the use of F. arisanus against Anastrepha spp. 

The literature provides little guidance on parasitoid host egg irradiation. The 

range of doses reported in this paper was in part derived from previous studies of fly 

mortality following exposure to radiation (Rigney 1989). Post-harvest radiation 

treatments have been explored in Anastrepha spp. (Bustos, Enkerlin, Toledo, Reyes, 

and Casimiro 1992), but none of the studies investigated the effects that radiation 

might have on parasitoids. 


A. ludens eggs used in the evaluations were provided by the Moscafrut Plant located 

in Metapa de Domínguez, Chiapas, Mexico. The colony was maintained in 

accordance with the procedures described by Domínguez, Castellanos, Hernández, 

and Martínez (2000). The F. arisanus parasitoids that were used had developed from 

host A. ludens eggs. Parasitoids from the 10 20th generation were used during the 

development of the tests. The colony of the parasitoid D. longicaudata had been 

maintained for approximately 300 generations under mass-rearing conditions when 

the tests were initiated. 

A Gammacell 220 Co-60 irradiator with a dose rate of 2.5 3.0 Gy/min was used. 

The eggs were irradiated with an oxygen-free substitute for air (IAEA 1982) in a 

plastic container (5 3 cm, high wide) with 1 mL of eggs suspended in 3 mL of 

water. The times of exposure to the different doses were determined prior by Fricke’s 

dosimetry according to the Manual High-Dose Dosimetry in Industrial Processing 

(Zavala, Fierro, Schwarz, Orozco, and Guerra 1985; IAEA 2001; FAO/IAEA/ 

USADA 2003). The evaluations were performed as follows. 

Irradiation of A. ludens eggs at different ages 

The first step was to evaluate the viability of A. ludens eggs using different ages and 

different doses of radiation. Eggs incubated for 24, 48 and 72 h were irradiated at


2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 Gy. The hatching percentage was taken from a 

sample of 100 eggs placed on a piece of black paper held in a closed Petri dish 

containing a piece of wet filter paper. With the aid of a dissecting microscope, the 

number of eggs hatched was recorded every 24 h and was used to calculate the 

maximum percentage of eggs hatched for each age and dose of radiation. This test 

was replicated 13 times. 

Effects of irradiation on 72-h-old A. ludens eggs 

The next step was to analyze the data from the previous step in order to select the 

best age for the egg hatching percentage component of the study. The best age was 72 

h. Twelve different doses of radiation were applied to 72-h-old eggs of A. ludens. 

Doses of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 Gy were applied. We 

increased the doses applied to 72-h-old eggs to assure no emergence of flies. The 72h-old 

eggs were not affected by these high doses. The eggs were incubated at 268Cfor 

24 h. The eggs hatched in the diet medium (larval diet mix of torula yeast, corn flour, 

corn cob fractions, sugar, citric acid, nipagin, sodium benzoate and water) and the 

larvae remained there for 9 days. 

After 9 days, the larvae were separated from the artificial diet medium by 

washing. The volume of larvae obtained for each dose was recorded. Samples 

containing 100 larvae were taken for each treatment. Each sample was placed in a 

cylindrical plastic container (9 4.5 cm) packed with fine vermiculite (SUNGRO 

Horticulture † ), held at 268C, and allowed to develop until reaching the pupal stage. 

One day before emergence (on day 14 of the 15 days needed to complete pupal 

development), the samples of pupae were weighed for each treatment. The pupae 

were returned to the container for adult emergence. 

After emergence, the number of adult flies was counted for each treatment. With 

a sample of 10 à:10 ß flies within a 10-cm 3 glass cage, their longevity and fertility 

were recorded. Adult longevity was determined by monitoring mortality daily. When 

the females were 12 days old, their eggs were collected daily as they were laid into a 

green agar ball (3 cm diameter) covered with a piece of parafilm. Fertility was 

estimated using the percentage of hatched eggs. Percent hatch was obtained by 

counting the eclosed eggs on a piece of wet, black paper on the bottom of a Petri 

dish. Nine replications for longevity and fertility were done. 

Exposure of irradiated eggs to F. arisanus 

For the 12 irradiated treatments, 72-h-old samples (about 1 mL of eggs) of A. 

ludens eggs were irradiated at 2.5, 5. 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 

30 Gy. One treatment consisting of eggs that were not treated with radiation was 

used as a control. The 13 treatments were exposed in a ‘papaya unit’ (Harris and 

Okamoto 1991) which consisted of a piece of papaya (4.5 4.5 2.5 cm) with six 

holes carved into its surface. About 0.5 mL (approximately 12,000 eggs) of eggs per 

treatment were distributed in the holes. These units were introduced into a ‘Hawaiitype’ 

cage (27 27 27 cm) (Wong and Ramadan 1992) containing 30 à:15 ß of F. 

arisanus parasitoids and held for 10 h. Afterwards, the parasitized eggs were seeded 

onto the larval artificial diet for development. The mature larvae were then


separated from the diet and kept in trays of vermiculite to allow pupation. 

Fourteen days after pupation, samples of 100 pupae were placed into containers 

(9 4.5 cm) and the number and percentage of parasitoids emerging at each 

treatment dose was recorded. In order to confirm the longevity and fecundity of 

parasitoids emerged from each dose, a sample of 30 à:15 ß emerged parasitoids 

was taken per treatment and placed inside a ‘Hawaii-type’ cage. Daily mortality 

was recorded starting from day 1. The fecundity was recorded by monitoring the 

daily exposition of eggs from 5- to 10-day-old parasitoids. The eggs were treated as 

mentioned above. The numbers of offspring emerged were compared with the 

number of live females per day. This test was replicated 10 times. 

Exposure of larvae hatched from irradiated eggs exposed to D. longicaudata 

Twelve treatments were prepared consisting of 200 fruit fly larvae each, developed 

from 72-h-old eggs exposed to one of 12 different doses of radiation. The doses were 

the same as those applied previously. These larvae were put into an oviposition unit 

(plastic Petri dish covered with a fine mesh) containing artificial diet medium. The 

unit was placed inside a ‘Hawaii-type’ cage (27 27 27 cm) containing 30 à:15 ß 

D. longicaudata parasitoids for a period of 2 h. 

After exposure, the larvae were kept in cylindrical plastic containers (9 4.5 cm) 

with fine vermiculite at 268C in order to stimulate pupation. The flies and parasitoids 

that emerged from each treatment were counted to obtain the percent emergence. Ten 

replicates were carried out. The adult parasitoids that emerged were sampled, and a 

group of 30 à:15 ß per treatment were placed into ‘Hawaii-type’ cages. These samples 

were evaluated to confirm the longevity and fecundity of parasitoids emerged from 

each dose. Mortality was recorded daily until the 20th day. 

When D. longicaudata females were 5 days old, an oviposition unit with 200 A. 

ludens (8-day-old) larvae was exposed to these parasitoids for 2 h. Larval exposure to 

parasitoids was performed during the following 10 days. The exposed larvae were 

kept in a container with fine vermiculite for 14 days. The number of adults that 

emerged each day was related to the respective number of living females in order to 

obtain the number of offspring per female per day. 

Data analysis 

The means for percentage hatched eggs, larval yield, and adult emergence (parasitoids 

and flies) were analyzed using ANOVA and Tukey’s multiple range test (a 0.05). A 

Long-rank test (Francis, Green, and Payne 1993) was applied to the longevity data. 

These data were analyzed with the JMP software (SAS Institute 2002). 

Results 

Irradiation of A. ludens eggs at different ages 

Figure 1 shows the percentage of eggs hatching at different radiation doses. There 

were clear differences in the percent hatching in 24- and 48-h-old eggs with 

increasing doses of radiation (24 h: F 30.18, df 8,108, PB0.001; 48 h: F 

255.23, df 8,108, PB0.001). The 72-h-old eggs did not suffer the effects of

% 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

a a a 

24 48 72 

a a a a ab 

a a a a a 

ab 

bc 

c cd 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Doses (Gy) 

irradiation, with percent egg hatch ranging from 72 to 82% (n 13, F 0.993, a 

0.05). The following evaluations were performed using 72-h-old eggs. 

Effects of irradiation on 72-h-old A. ludens eggs 

The averages of hatching percent, larval yield, pupal weight and fly emergence are 

shown in Table 1. The hatching percent and volume of larvae yielded were not 

significantly different for the doses of radiation applied (hatching percent: F 0.558, 

df 12,117, P 0.871; volume of larvae: F 0.571, df 12, 102, P 0.860). 

d cd 

d 

cd 

d 

e 

d 

e d e 

Figure 1. Percent hatching in 24-, 48- and 72-h-old Anastrepha ludens eggs subjected to 

different doses of radiation. 

Table 1. Means (9SE) of percent hatching, larval yield, pupal weight and fly emergence 

from 72-h-old Anastrepha ludens eggs irradiated at different doses. 

Doses 

(Gy) 

Hatching 

percentage (%) 


Percentage (%) flies emerged 

Larval yield 

(mL) Weight of pupa (mg) Females Males 

0 88.8 9 2.0 a 105.3 9 12.15 a 19 9 0.05 ab 41.1 91.44 a 38.6 9 1.7 ab 

2.5 88.9 9 2.1 a 90.2 9 14.49 a 20.2 9 0.04 a 40.1 91.22 a 42.6 9 1.2 a 

5.0 86.2 9 1.8 a 105.5 9 12.65 a 19.8 9 0.04 ab 38.3 91.44 ab 39.6 9 1.3 ab 

7.5 85.8 9 1.8 a 94.6 9 12.16 a 19.5 9 0.05 ab 36.3 91.59 abc 35.5 9 1.8 b 

10.0 86.4 9 2.1 a 88.9 9 11.62 a 18.8 9 0.03 ab 34.99 0.85 bc 37.5 9 1.6 ab 

12.5 86.4 9 2.1 a 92 9 11.41 a 18.1 9 0.04 abc 31.6 9 0.93 cd 29.1 9 1.8 c 

15.0 85 9 2.0 a 90.7 9 10.29 a 18.3 9 0.06 abc 26.6 9 1.31 d 27.6 9 1.7 c 

17.5 87.4 9 1.8 a 95.7 9 10.37 a 16.9 9 0.06 bcd 17.1 9 1.79 e 10.0 9 0.9 d 

20.0 89 9 1.5 a 82.6 9 15.48 a 15.6 9 0.07 cd 5.0 9 0.66 f 4.4 9 0.6 de 

22.5 85.3 9 1.4 a 77.4 9 9.13 a 14.8 9 0.07 de 1.4 9 0.29 f 1.0 9 0.2 e 

25.0 86.1 9 1.8 a 89.3 9 8.46 a 11.8 9 0.07 f 0.2 9 0.07 f 0.0 9 0.0 e 

27.5 87.6 9 1.5 a 82.7 9 11.87 a 12.6 99 0.07 ef 0.0 9 0. 00 f 0.0 9 0.0 e 

30.0 87.6 9 1.6 a 76.3 9 10.48 a 10.4 9 0.09 f 0.0 9 0.00 f 0.0 9 0.0 e 

*Means with different letters in each column indicate statistical differences. ANOVA, Tukey’s test (a 

0.05).


Percent of eggs 

hatched 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

12 13 14 

15 16 17 

18 19 20 

21 adult age (d) 

0 2.5 5 7.5 10 12.5 15 17.5 

Doses (Gy) 

Figure 2. Daily decrease in fertility of Anastrepha ludens flies emerged from 72-h-old eggs 

irradiated at different doses. 

However, there were significant differences among the average pupal weights (F 

29.24, df 12, 385, PB0.0001) at different doses. The decrease in pupal weight 

progressed as the radiation dose increased. Adult emergence suffered with increasing 

radiation as well. At 27.5 Gy, flies did not emerge, even though the larvae had 

satisfactorily achieved pupation. 

The flies that did emerge after receiving different radiation doses showed 

decreasing fertility with increasing dose. In Figure 2, the fertility curves, representing 

the percentage of hatched eggs, show that the flies’ fertility began to decrease at 7.5 

Gy. For example, at 10 Gy the hatching average was 54.7%, which was significantly 

lower than the 81.1% hatching rate obtained in the control group. Above 15 Gy the 

flies that emerged were sterile. 

Exposure of irradiated eggs to F. arisanus 

In general, there was a high variability in the emergence data obtained. The only 

common thread was that parasitoid emergence occurred in every sample. Although 

there were significant differences in the parasitoid emergence averages at different 

doses, there was no relationship between the decrease of emergence and higher doses. 

Similar variability was obtained in the sex-ratio data of parasitoids (female 

emergence: F 3.340, df 12, 117, P 0.0003) (Table 2). 

The longevity presented as averages of live parasitoids at 20 days and fecundity in 

emerged parasitoids are shown in Table 3. With the exception of the 17.5, 22.5 and 

27.5 Gy doses, the percentage of living females at day 20 was upwards of 40% or 

more. In males, the longevity obtained at 2.5, 15 and 25 Gy was below 15%. For the 

remainder of the group, longevity was higher. The long-rank analysis reported 

statistical difference between the longevity results (females: x 2 

44.27, df 12, PB 

0.0001; males: x 2 

30.80, df 12, P 0.0021). However, it did not show a tendency 

to reduce the longevity at higher radiation levels. The fecundity data were lower than 

one offspring/female/day at all radiation levels except 17.5 Gy, which had a rate of 

1.2 offspring/female/day.


Table 2. Means (9SE) for emergence and sex-ratio of Fopius arisanus parasitoids using 

irradiated 72-h-old eggs as host. 

No. of parasitoids emerged 

Doses (Gy) Females Males Sex-ratio (female:male) 

0 1.5 9 0.7 c 1.4 9 0.68 ef 1.1:1 

2.5 5.8 9 1.8 abc 2.8 9 0.90 bcd 2.1:1 

5 6 9 2.00 ab 2.9 9 0.92 bcd 2.1:1 

7.5 5.2 9 2.42 abc 2.6 9 1.16 bcde 2.0:1 

10 2.8 9 0.88 abc 2.4 9 0.98 bcde 1.2:1 

12.5 6.5 9 2.11 a 4.7 9 1.95 a 1.4:1 

15 4.4 9 1.24 abc 3.2 9 1.2 bc 1.4:1 

17.5 3.19 0.3 abc 2.1 9 0.3 cde 2.0:1 

20 4.1 9 2.68 abc 2.4 9 1.44 bcde 1.7:1 

22.5 2 9 1.12 bc 1.9 9 0.88 de 1.1:1 

25 1.6 9 0.86 bc 0.4 9 0.22 f 4.0:1 

27.5 4.8 9 2.63 abc 0.3 9 1.6 f 1.6:1 

30 5.0 9 2.91 abc 3.4 9 1.72 b 1.6:1 

*Means followed by different letters in the columns are significantly different. ANOVA, Tukey?s test (a 

0.05%). 

Exposure of larvae hatched from irradiated eggs to D. longicaudata 

The average larval yield per treatment was statistically similar (F 0.819, df 12, 39, 

P 0.630). There were no differences in percentages of male parasitoid emergence 

(males: F 0.971, df 12, 117, P 0.480). However, for female emergence, there 

were minor statistical differences (females: F 0.739, df 12, 117, P 0.710) (Table 

4). The emergence of flies from unparasitized larvae decreased as the dose of 

radiation increased. Above 10 Gy, fly emergence dropped considerably. Moreover, 

Table 3. Longevity and fecundity of Fopius arisanus parasitoids emerged from 72-h-old eggs 

treated with different radiation doses. 

Live parasitoids at the 20th day (%) 

Doses (Gy) Females Males Offspring /female/day 

0 60 53 0.45 

2.5 70 13.3 0.22 

5 43 20 0.20 

7.5 40 40 0.12 

10 63 47 0.34 

12.5 43 27 0.44 

15 43 13.3 0.09 

17.5 27 27 1.21 

20 50 20 0.95 

22.5 27 27 0.99 

25 67 13 0.74 

27.5 27 6.7 0.31 

30 60 73 0.50


Table 4. Mean (9SE) volume of larval yield and percentage of Diachasmimorpha longicaudata 

parasitoids and flies emerged from larvae developed from 72-h-old eggs irradiated at 

different doses. 

Doses 

(Gy) 

Parasitoids emergence (%) Fly emergence (%) 

Volume of larval 

yield (mL) Females Males Females Males 

0 125910.41 a 30.793.32 ab 16.593.92 a 10.692.55 a 9.893.32 abc 

2.5 101.2917.60 a 30.994.15 ab 1994.15 a 8.391.77 abc 10.792.35 abc 

5 117.5917.97 a 28.793.03 ab 11.991.45 a 7.991.50 abc 12.392.98 ab 

7.5 13598.66 a 30.293.37 ab 15.492.64 a 7.591.59 abc 10.492.93 abc 

10 107.5916.52 a 31.492.85 ab 16.592.85 a 9.291.75 a 12.992.89 a 

12.5 105921.02 a 34.493.84 ab 17.991.98 a 5.691.31 bcd 7.591.61 abcd 

15 115911.90 a 30.594.90 ab 17.993.53 a 5.591.03 cde 792.07 bcde 

17.5 115911.90 a 2592.61 b 15.193.74 a 3.190.71 def 5.191.12 def 

20 102.5910.31 a 35.294.51 ab 12.490.73 a 1.990.84 ef 3.391.33 ef 

22.5 118.7917.12 a 37.694.19 a 19.494.20 a 1.390.66 f 1.690.86 ef 

25 137.5911.09 a 36.593.94 a 12.791.48 a 0.090.00 f 0.090.00 f 

27.5 123.7912.81 a 32.694.61 ab 11.390.89 a 0.090.00 f 0.090.00 f 

30 97.594.79 a 32.994.90 ab 18.792.86 a 0.090.00 f 0.090.00 f 

*Means followed by different letters in the columns are significantly different. ANOVA, Tukey’s test (a 

0.005). 

there were significant differences between the averages for emergence suppression in 

males and females. At 25 Gy, total suppression of fly emergence was obtained, while 

the emergence of parasitoids maintained similar values in comparison with the other 

doses. The longevity and fecundity of the emerged parasitoids did not appear to be 

affected by radiation dose (Table 5). These data were widely variable. A range of 

three to six male offspring/female/day was obtained without any relationship with 

levels of radiation. Statistic differences were found in the longevity results (females: 

x 2 

51.96, df 12, PB0.0001; males: x 2 

36.68, df 12, P 0.0003), but they 

were not related to the increase in radiation dose. 

Discussion 

The irradiation of host eggs is an important new development in the mass rearing of 

fruit fly parasitoids. Previously, radiation had been successfully applied to larvae in 

the mass rearing of fruit fly parasitoids of larvae (Sivinski and Smittle 1990; Cancino 

et al. 2002a), but the irradiation of eggs improves the mass-rearing of larval 

parasitoids and makes the rearing of pure egg-parasitoid cohorts possible. 

The present study was inspired by Rigney (1989) who found that if a fruit fly egg 

is irradiated, the adult will not emerge. In general, insect eggs are one of the most 

sensitive stages to irradiation. Radiation effects are inversely proportional to the 

degree of differentiation. The rate of cell division in insect eggs is high, although 

during a brief period just before molting, cell division slows down. This could 

explain why the 72-h-old eggs were less sensitive to radiation. Previous research also 

suggests that the static stages are less sensitive to radiation (IAEA 1977). After 72 h 

of development, the A. ludens eggs could be considered ready to start hatching. This


Table 5. Mean longevity and fecundity of Diachasmimorpha longicaudata emerged from 

larvae developed from 72-h-old irradiated eggs at different doses. 

Live parasitoids at 20th day (%) 

Doses (Gy) Females Males Offspring/female/day 

0 83 87 3 

2.5 55 50 4.8 

5 70 83 3 

7.5 58 70 4.9 

10 68 63 3.7 

12.5 50 57 6.1 

15 57 80 6.1 

17.5 53 87 4.5 

20 52 50 5.2 

22.5 73 67 3.8 

25 78 80 3.6 

27.5 65 50 4.1 

30 47 43 6 

is an advantage for F. arisanus which is a parasitoid of older eggs and young first 

instar larvae of Tephritidae (Bautista and Harris 1996). 

In the case of F. arisanus, this is a particularly crucial improvement since it makes 

it possible to obtain pure cohorts of adult parasitoids for mass-release without any 

contamination by fertile flies. When C. capitata eggs are used as hosts, parasitoid 

emergence occurs at almost the same time as adult fly emergence making separation 

at this point particularly difficult. When A. ludens eggs are used as hosts, flies emerge 

earlier but the labor to separate the insects and sanitation problems caused by 

starved host adults still makes egg radiation an attractive alternative (Zenil et al. 

2004). 

The mass rearing of D. longicaudata may also benefit from using irradiated eggs 

as host. Egg irradiation would require the exposure of less volume of host material 

and eggs are easier to gather and prepare for oviposition than larvae that must be 

removed from the diet. About 22,000 A. ludens eggs can be contained in a volume of 

1 mL, while millions can fit into a litre. The irradiation of this volume of eggs 

requires a smaller and simpler irradiating device than is used for other biological 

control operations. 

Again, cages, oviposition units, and procedures for developing immature stages 

can be designed taking into consideration the emergence of only parasitoids. These 

advantages have already been implemented in the design of mass rearing procedures 

for parasitoids reared on irradiated host larvae (Sivinski and Smittle 1990; Cancino, 

Villalobos, and De la Torre 2002b). The irradiation of biological material at mass 

rearing facilities already using an irradiator is easily performed. This point is of 

considerable importance in many cases, since many parasitoid mass rearing facilities 

have been built away from fruit fly mass rearing facilities. For example, when the 

larvae are to be subjected to radiation, they are typically crowded into a small 

container where metabolic heat can collect and cause a rise in the temperature inside 

the container. Depending on the length of time, this increased temperature can lead


to a reduction in longevity and high mortality of parasitoid hosts 72 h after 

oviposition. In mass-rearing conditions, a mortality of hosts (dead larvae) greater 

than 10% brings about a significant reduction in the parasitoid emergence (Cancino 

et al. 2002b). When host eggs are irradiated, the risks of increasing host mortality 

due to such packaging procedures are easier to manage. 

Another significant benefit of using host egg irradiation in the mass rearing of 

fruit fly parasitoids will be evident in the packing and releasing components of the 

program. Suppressing fly emergence (from unparasitized hosts) gives new freedom in 

designing broader methodologies for packing and mass releasing. Recently, several 

new packing methods have been developed thanks to the use of irradiated larval 

hosts in the mass rearing of D. longicaudata. However, in the case of the packing and 

release of F. arisanus, little work has been done. Some methods have been previously 

designed for the packing of F. arisanus using Bactrocera dorsalis (Hendel) eggs, but 

since their development takes different lengths of time, separating emerging flies 

from emerging parasitoids causes fewer problems (Bautista, Harris, and Vargas 

2001). 


We thank Edgar de León for his technical help. We are also grateful to M.C. Yeudiel Gómez 

and his team for their help in the irradiation of eggs. This work was supported by the IAEA in 

cooperation with the Medfly Program SAGARPA under contract No. 10848. 

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Vol. 19, S1, 2009, 179 191 


Effects of gamma radiation on suitability of stored cereal pest eggs and 

the reproductive capability of the egg parasitoid Trichogramma 

evanescens (Trichogrammatidae: Hymenoptera) 

A.S. Tunçbilek*, U. Canpolat, and A. Ayvaz 

Department of Biology, Erciyes University, Faculty of Arts & Sciences, 38039 Kayseri, Turkey 

A study was conducted to determine parasitization suitability and preference of 

irradiated and untreated eggs of the Mediterranean flour moth (MFM), Ephestia 

kuehniella Zeller, and the Angoumois grain moth (AGM), Sitotroga cerealella 

(Olivier) for mass-production and augmentative releases of T. evanescens Westwood 

in cereal storage and processing facilities. Eggs of both species irradiated 

with 200 Gy could be effectively used for propagation of T. evanescens in the 

sterilized host eggs. The irradiated host eggs of E. kuehniella were markedly 

preferred over eggs of S. cerealella. A dose of 200 and 150 Gy prevented adult 

emergence of E. kuehniella and S. cerealella, respectively. Treatment of immature 

stages of the parasitoid inside the host eggs or treatment of adults of T. evanescens 

with low-level doses (ranging between 0 and 140 Gy) resulted in significant 

reduction in the number of parasitized eggs, adult emergence and progeny 

production (F1) as radiation dose increased. Our study showed that MFM and 

AGM eggs killed by gamma radiation could be used for the rearing and release of 

T. evanescens into commodity storages without any risk of increasing the pest 

population. 

Keywords: parasitization; Trichogramma evanescens; Ephestia kuehniella; 

Sitotroga cerealella; biological control; gamma radiation; irradiated host eggs; 

stored cereal pests management 

Introduction 

Moth species of the genus Ephestia, especially the Mediterranean flour moth 

(MFM), Ephestia kuehniella Zeller, as well as the Angoumois grain moth (AGM), 

Sitotroga cerealella (Olivier) are serious pests in cereal-based food processing 

facilities, stored maize and other cereals (Anonymous 1995a). They have three to 

four overlapping generations per year in Turkey (Yildirim, Özbek, and Aslan 2001). 

Typically, control of these pests is undertaken by regular treatment of infested 

facility areas with pesticides such as malathion, dichlorvos and methyl bromide 

(Anonymous 1995b). Use of chemicals is very costly, as it requires the shut-down of 

the food processing factory and the interruption of the production process. 

Therefore, research is needed to find improved methods for control. Egg parasitoids 

are promising biological control agents because they eliminate the pest before it 

causes damage and they can be economically produced (Knipling 1992; Nurindah 

and Gordh 1999). 

*Corresponding author. Email: tunca@erciyes.edu.tr 




DOI: 10.1080/09583150902790269 



Recent investigations suggested the use of inundative releases of parasitoid wasps 

of the genus Trichogramma in cereal stores. Trichogramma is often reared on eggs of 

the stored product pests, and therefore, releasing large numbers of Trichogramma 

into a warehouse might greatly suppress the pest population (Brower 1983). The use 

of Trichogramma requires repeated (inundative) releases at regular time intervals in 

order to ensure a long-term effect (Gwinner, Harnisch, and Mück 1996). 

Trichogramma turkestanica Meyer has been considered as a potential candidate for 

control of pyralid moths in stored grain and grain processing (Hansen and Jensen 

2002). The first step in the evaluation of a proposed biological control agent is 

definition of the complete group of species on which the agent can survive and 

develop under controlled conditions (McEvoy 1996; Onstad and McManus 1996). If 

the agent is not monophagous when tested in a no choice situation, it is useful to 

assess host preference through choice tests with more than one host species present 

(Field and Darby 1991; Blossey, Schroeder, Hight, and Malecki 1994; Silva and 

Stouthamer 1999). Choice tests indicate if the target host is preferred over other 

physiologically acceptable hosts or if all acceptable hosts are equally suitable. 

Considerable technological advances, including the use of radiation, have been 

made in mass rearing of parasitoids and natural enemies. It has been reported that 

parasitization by egg parasitoids was increased in the eggs of irradiated lepidopteran 

pests (Marston and Ertle 1969; Mannion, Carpenter, and Gross 1995). Marston and 

Ertle (1969) tested the acceptability of irradiated moth eggs to Trichogramma 

minitum Rile, and reported that irradiated eggs were as suitable as non-irradiated 

eggs for parasitoid development. Indian meal moth Plodia interpunctella Hubner 

eggs killed by irradiation were used for rearing and release of Trichogramma 

pretiosum Riley into commodity storage facilities (Brower 1982). Irradiation can also 

be used to reproductively sterilize hosts to prevent the emergence of non-parasitized 

hosts or to prolong the development of host stages suitable for parasitization, thus 

facilitating the use of these hosts under mass rearing conditions. There appear to be 

significant opportunities for increased use of classical and augmentative biological 

control through nuclear techniques for production, shipping, and release of 

biological control agents. Moreover, Tillinger, Hoch, and Schopf (2004) showed 

that gamma radiation can be used as a tool to study interactions between the 

braconid endoparasitoid associated factors and its host larva. On the other hand, 

when the field performance of laboratory-reared parasitoids is a concern, very low 

doses of radiation may be useful in improving field performance of laboratory-reared 

parasitoids which may stimulate some physiological and behavioral processes in 

parasitoids. 

The aim of this study was to evaluate the effect of gamma radiation on the eggs 

of E. kuehniella and S. cerealella on their suitability as hosts of T. evanescens. The 

study also evaluated the effect of low dose gamma radiation on T. evanescens 

reproductive capability to increase progeny production of the parasitoid. 


Host rearing 

E. kuehniella and S. cerealella were obtained from the Department of Plant 

Protection, Faculty of Agriculture of Ankara University and Adana Plant Protection


Research Institute, respectively. E. kuehniella was reared in a mixture of 1 kg wheat 

flour, 5% yeast and 30 g wheat germ (Marec, Kollarova, and Pavelka 1999). S. 

cerealella were reared on wheat grain. Throughout the rearing, cultures were kept in a 

rearing room at 27918C and 7095% RH and under a light regime of 14 h L:10 h D. 

To obtain eggs, large numbers of 1 2-day-old adults of E. kuehniella and S. cerealella 

were collected from stock cultures and placed in plastic jars with screen bottoms. Eggs 

that fell through the screen were collected the following days and sifted to remove 

insect parts and frass, and placed in Petri dishes. Eggs were removed daily and exposed 

to parasitoids in glass tubes for 24 h. 

Rearing of Trichogramma evanescens 

T. evanescens used in this experiment were initially obtained from Adana Plant 

Protection Research Institute. They were collected from Ostrinia nubilalis Hubner 

(Lep: Pyralidae) eggs in southern Turkey in 1999. In the laboratory, T. evanescens 

were mass-reared on E. kuehniella eggs for several generations. Throughout the 

rearing, cultures were kept in the rearing room at 24918C and 7095% RH, under a 

light regime of 14 h L:10 h D. Parasitoid cultures were started from a single female 

on E. kuehniella eggs and maintained in glass rearing vials (2 7.5 cm). 

Sterility tests 

Large numbers of 1-day-old MFM and AMG eggs were placed in glass Petri dishes 

and irradiated in a calibrated 60 Co irradiator (Therathronics 780C) with a source 

strength of ca 3811 Ci and a dose rate of ca. 1 Gy/min; dose rate was verified with 

Fricke dosimetry. The eggs were exposed to doses of 0, 50, 100, 150, 200, 250, 300, 

350, 400, 450, 500 and 550 Gy, using 120 eggs at each dose (three replicates of 40). 

Immediately after treatment, the eggs were transferred to 300 mL jars each 

containing 100 g of medium and incubated in the conditioned chamber. An 

unirradiated control population was started similarly. The number of eggs hatched 

and adult emergence was noted. Data were analyzed using analysis of variance 

(ANOVA) and probit analysis was used to estimate SD50 and SD99 values (SPSS 

1999). 

In the parasitization test, 1-day-old eggs obtained from irradiated E. kuehniella 

were glued on pieces of white cardboard (2 2.5 cm) and placed in individual tubes 

along with young female adult T. evanescens (five replicates of 50). All females were 

fed with honey, mated and had no previous contact with host eggs. A single female 

per tube was obtained by capturing a 24-h-old female from a group of females 

scattered on a white piece of paper. A single tube was placed over an adult female of 

medium size and the female was allowed to walk up the vial towards the light. Data 

collected on the longevity of T. evanescens adults emerged from irradiated host eggs 

were compared by Kruskall Wallis test. 

No choice and choice experiments irradiated host eggs 

In the no choice experiment, 1-day-old MFM and AMG eggs were placed in glass 

tubes and irradiated with the doses mentioned above, using approximately 250 eggs 

at each dose level (five replicates of 50). The parasitoid to host ratio was high in these


tests to ensure that most acceptable eggs would be parasitized (i.e., a female wasp 

may lay about 35 40 eggs in each day). The egg cards were prepared using a number 

of moth eggs as described by Brower (1982). Equal number of eggs were counted and 

sprinkled on strips of lightweight cardboard (2 2.5 or 2.5 4 cm). The cards were 

glued with gum arabica, and the gum was allowed to dry for at least 1 h. Eggs were 

placed in tubes along with young female adult T. evanescens. All females had no 

previous contact with host eggs, were fed with honey and mated. Single females per 

tube were obtained by capturing a 24-h-old female from a group of females scattered 

on a white piece of paper. A single tube was placed over an adult female of medium 

size with open end and the female was allowed to walk up the vial towards the light. 

Newly emerged Trichogramma that climbed from emergence glass tubes into an 

experimental tube were used, ensuring that more than 90% were female, since 

females are more phototactic than males (McDougall and Mills 1997). The glass 

tube was covered tightly with plastic hardware cloth to prevent the wasp from 

escaping. After 24 h at 248C, 70% RH and a photoperiod of 14 h L:10 h D, the wasps 

were removed from the tube cards, and the parasitized eggs were incubated in a 

controlled environment. Each morning, the tubes were checked for the number of 

live and dead T. evanescens. To determine whether parasitoid eggs hatched, we 

counted the eggs that had turned black after 4 7 days of incubation at 248C. The 

numbers of parasitized eggs, adult and female emergence were recorded as 

parameters. Data were analyzed using analysis of polynomial regression analysis, 

with dose of radiation (independent variable), the number parasitized eggs, adult 

emergence, and the number of females (dependent variables) as sources of variation 

(SPSS 1999). 

In the choice experiment, the procedures were the same as above with the 

following exceptions. Five categories of radiation doses ranging from 0 to 200 Gy 

were defined and the experiment was replicated 10 times (50 vs. 50 eggs of each host 

type in each replication). The following combinations were tested: 0 vs. 50, 50 vs. 0, 

50 vs. 50, 150 vs. 150, and 200 vs. 200 Gy, E. kuehniella egg versus S. cerealella egg, 

respectively. In this test, 1-day-old irradiated and unirradiated eggs (50 eggs from 

each) were glued on pieces of white cardboard (2 2.5 cm) 5 mm apart and arranged 

crosswise. 

This test was performed to determine the relative attractiveness of irradiated E. 

kuehniella and S. cerealella eggs for T. evanescens. The eggs of irradiated and 

unirradiated E. kuehniella and S. cerealella were offered simultaneously to single 

female of T. evanescens in glass tubes. A single female per tube was obtained by 

capturing a 24-h-old female as mentioned above. After 24 h, the wasps were 

discarded and the cards were incubated under controlled conditions. Mean 

parasitization of the corresponding E. kuehniella eggs vs. S. cerealella eggs was 

compared by independent samples t-test (SPSS 1999). 

Irradiation of parasitoids 

Irradiated T. evanescens immature stage experiments 

Parasitized host eggs (1.5-h- and 5-day-old, stored at room temperature) were 

exposed to five different doses ranging from 0 to 20 Gy, using about 250 eggs in each 

dose (five replicates of 50). Data were analyzed by polynomial regression analysis,


with dose of radiation (independent variable), the number parasitized eggs, and the 

number of adult emergence (dependent variables) as sources of variation (SPSS 

1999). 

Irradiated T. evanescens adult experiments 

One-day-old adult T. evanescens were exposed to five different doses ranging from 0 

to 140 Gy. After irradiation, untreated eggs of MFM were offered to single 

irradiated females, using about 250 host eggs in each dose (five replicates of 50). The 

numbers of parasitized eggs and adult emergence were recorded as parameters. Data 

were analyzed by polynomial regression analysis, with dose of radiation (independent 

variable), number parasitized eggs, and number of adult emergence (dependent 

variables) as sources of variation (SPSS 1999). Data from longevity of irradiated T. 

evanescens adults were compared by Kruskall Wallis test. 

Irradiated T. evanescens adult and host egg experiments 

Host eggs were irradiated with 0, 100, 140 and 200 Gy and parasitized by irradiated 

T. evanescens that were exposed to doses from 0 to 4 Gy, using about 500 eggs in each 

dose (10 replicates of 50). Data were analyzed using a two-factor analysis of variance 

(ANOVA), with dose of radiation applied vs. host eggs and dose applied vs. adults 

parasitoids as factors and the number of parasitized eggs, adult emergence and 

female emergence as parasitization efficiency dependent variables (PROC ANOVA & 

PROC GLM) (SPSS 1999). All data were transformed to square root before 

statistical analysis; when significant differences occurred Tukey-HSD was applied as 

a means of separation. Back transformed data are presented in regression equations. 

Results 

Sterility test irradiated MFM and AGM eggs 

There was a significant reduction in adult emergence with increasing radiation doses, 

F 146.383; df 4; PB0.001 for E. kuehniella and F 160.968, df 3; PB0.001 for 

S. cerealella. A dose of 200 and 150 Gy prevented adult emergence of E. kuehniella 

and S. cerealella, respectively. Male-to-female ratios in the adult stage were skewed in 

favor of male moths with increasing doses (from 0.60:1 to 1.25:1; ß:à). Using probit 

analysis, estimates were obtained of the doses required for 50 and 99% egg sterility. 

The SD50 values were 110.9 and 37.1 Gy for E. kuehniella and S. cerealella, 

respectively, and the SD99 were 210.3 and 188.5 Gy for E. kuehniella and S. 

cerealella, respectively (x 2 

27.262; df 2; PB0.001; x 2 

5.977; df 2; P 0.050). 

At least 75% of the Trichogramma females that emerged from irradiated eggs 

survived until the 8th day of adult life when given access to honey and an unlimited 

supply of E. kuehniella eggs (Figure 1). The mean female longevity was 4.6193.98, 

5.7293.92, 6.593.72, 6.5393.66 and 5.5393.90 days for 0, 50, 100, 150 and 200 Gy, 

respectively. Longevity of T. evanescens adults from irradiated 1-day-old E. 

kuehniella eggs was not significantly influenced by increasing doses and was similar 

to that of the untreated control hosts (Figure 1). The Kruskall Wallis test showed 

that there was no statistically significant difference among the medians at the 95%


Survival 

11 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

confidence level (x 2 

males). 

0 Gy 50 Gy 100 Gy 150 Gy 200 Gy 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Longevity (Day) 

1 2 3 4 5 6 7 8 9 

Figure 1. Longevity of female and male adults of T. evanescens originating from irradiated 1day-old 

E. kuehniella eggs. The host eggs were treated with 0, 50, 100, 150 and 200 Gy. 

14, df 13, P 0.374 for females; x 2 

8, df 7, P 0.333 for 

No choice and choice experiments irradiated host eggs 

The T. evanescens parasitization curves in the no choice experiments are shown in 

Figure 2. The number of 1-day-old E. kuehniella eggs parasitized by T. evanescens 

was not affected by the dose of radiation given to host eggs (Y 22.58 0.009X 

0.000027X 2 , R 2 

0.020; P 0.59). Similarly, adult parasitoid emergence as well as 

female parasitoid emergence were not significantly affected by irradiation (Y 

21.62 0.00009X 0.000015X 2 , R 2 

0.027; P 0.49 and Y 16.59 0.0048X 

0.000024X 2 ; R 2 

0.02; P 0.57, respectively). 

A different trend was observed for irradiated 1-day-old host eggs of S. cerealella 

eggs (Figure 2). Here, the number of parasitized eggs was significantly affected by the 

dose of radiation given to host eggs (Y 27.21 0.0173X 0.0000689X 2 , R 2 

0.20; 

P 0.003). Likewise, adult and female parasitoid emergence were significantly 

affected by irradiation (Y 21.78 0.01X 0.000063X 2 ; R 2 

0.29; PB0.0001; Y 

19.06 0.006X 0.000034X 2 ; R 2 

0.24; P 0.0009, respectively). 

For E. kuehniella, irradiating eggs had no significant effect on their acceptability 

by T. evanescens. However, the acceptability of irradiated eggs of S. cerealella 

declined significantly as the dose of radiation increased. 

In choice experiments, when E. kuehniella and S. cerealella eggs had been 

exposed to different combinations of radiation (0 vs. 50, 50 vs. 0, 50 vs. 50, 150 vs. 

150 and, 200 vs. 200 Gy for E. kuehniella eggs versus S. cerealella eggs, respectively), 

and then T. evanescens females were given a choice between E. kuehniella and S. 

cerealella eggs, there was no preference (tB0.001, P 1.000). 

Irradiation of parasitoids 

Irradiated T. evanescens immature stages experiments 

When the host eggs were irradiated after parasitization (1.5 h or 5 days post 

parasitization) by T. evanescens, the survival of parasitoids and adult emergence 

decreased with increasing doses (Figure 3). Survival of parasitoids differed 

significantly among doses when parasitized host eggs were treated with gamma

50 

40 

30 

20 

10 

0 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

50 

40 

30 

20 

10 

0 


Number of parasitized eggs 

E. kuehniella 

S. cerealella 

0 50 100 150 200 250 300 350 400 450 500 550 

Dose (Gy) 

Number of adults emerged 

E. kuehniella 

S. cerealella 

0 50 100 150 200 250 300 350 400 450 500 550 

Dose (Gy) 

Number of females emerged 

E. kuehniella 

S. cerealella 

0 50 100 150 200 250 300 350 400 450 500 550 

Dose (Gy) 

Figure 2. Effect of radiation dose administered to 1-day-old E. kuehniella and S. cerealella 

eggs on parasitization, adult emergence and female emergence of T. evanescens. 

radiation after 1.5 h (Y 0.11669X 2 

5.2091X 38.743; R 2 

0.9734; PB0.0001), 

but this difference disappeared when parasitized eggs were treated with gamma 

radiation after 5 days (Y 0.0029X 2 0.2349X 36.063; R 2 

0.0351; P 0.6749). 

Adult parasitoid emergence decreased significantly with increasing dose (Y 

0.1731X 2 5.0589X 33.937; R 2 

0.9194; PB0.0001 and Y 0.056X 2 

0.168X 

27.2; R 2 

0.6498; PB0.0001 for irradiation at 1.5 h and 5 days post parasitization, 

respectively). Female parasitoid emergence also decreased significantly with dose 

(Y 0.1623X 2 

4.6137X 29.274; R 2 

0.9082; PB0.0001 and Y 0.0657X 2


50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

80 

70 

60 

50 

40 

30 

20 

10 

Irradiated T. evanescens (1.5 h post parasitization) 

Parasitization 



0 5 10 15 20 

Dose (Gy) 

Irradiated T. evanescens (5 d post parasitization) 




0 

0 5 10 

Dose (Gy) 

15 20 

Figure 3. Effect of radiation dose administered to the endoparasitic developmental stages of 

T. evanescens within host eggs on parasitization, adult emergence and female emergence of T. 

evanescens. Regression equations are given in the text. 

0.4463.6137X 18.514; R 2 

0.6204; PB0.0001 for irradiation at 1.5 h and 5 days 

post parasitization, respectively). 

Irradiated T. evanescens adult experiments 

The mean number of parasitized eggs, as well as adult emergence of parasitoids, 

obtained from irradiated T. evanescens adults decreased with increasing dose of 

radiation. There were no parasitized eggs at doses of 60 Gy and above (Figure 4). 

There were significant relationships between irradiation doses and number of 

parasitized eggs (Y 0.016X 2 

1.3227X 23.37; R 2 

0.7594; PB0.0001), adult 

parasitoid emergence (Y 0.0126X 2 

1.0112X 16.641; R 2 

0.7239; PB0.0001), 

and female parasitoid emergence (Y 0.0102X2 0.8134X 13.395; R 2 

0.7287; PB 

0.0001). In the F1 generation egg production, (Y 0.0053X 2 

1.0799X 42.881; 

R 2 

08896; PB0.0001), adult parasitoid emergence from F1 generation (Y 

0.0124X 2 

1.382X 37.712; R 2 

0.8465; PB0.0001) and female parasitoid emergence 

(Y 0.011X 2 

1.2168X 31.772; R 2 

0.7977; PB0.0001) also decreased with 

increasing dose and, there was no progeny production at doses of 40 Gy and above. 

T. evanescens are more sensitive to gamma radiation than host eggs. All of the

35 

30 

25 

20 

15 

10 

5 

0 

50 

40 

30 

20 

10 

T. evanescens adults that were irradiated with 0, 1, 2 and 3 Gy when 1-day-old 

survived until the 5th day when given access to honey; at 4 Gy this value was 80%. 

Longevity of T. evanescens adults irradiated with lower doses was not 

significantly influenced by increasing doses and was similar to that of the adults 

from untreated control hosts (Figure 5). The Kruskall Wallis test shows that there is 

no statistically significant difference among the medians at the 95% confidence level 

(x 2 

14, df 13, P 0.374). However, mortality rates of adult wasps irradiated with 

Survival (%) 

0 10 20 30 40 50 60 

Dose (Gy) 

100 

80 

60 

40 

20 

0 


Irradiated T. evanescens adults 

F1 generation from irradiated T. evanescens 







0 

0 10 20 30 

Dose (Gy) 

40 50 60 

Figure 4. Effect of radiation dose administered to the adult of T. evanescens on 

parasitization, adult emergence and female emergence of T. evanescens. Regression equations 

are given in the text. 

0 Gy 

1 Gy 

2 Gy 

3 Gy 

4 Gy 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Longevity (Day) 

Figure 5. Longevity of T. evanescens adults when irradiated with low level gamma radiation 

of 0, 1, 2, 3, or 4 Gy.


Mean number 

40 

35 

30 

25 

20 

15 

10 

5 

0 

Parasitizes egg 

Progeny production 

0 1 2 3 0 1 2 3 

Dose (Gy) 

2 Gy were a little higher than those from control host eggs, but this differences was 

not statistically significant (P 0.005). 

Irradiated T. evanescens adult and host egg experiments 

When 1-day-old T. evanescens adults were irradiated, the two-way ANOVA analysis 

showed that the mean number of parasitized host eggs and adult emergence (F1) of 

T. evanescens were significantly reduced (F 56.535; df 3; PB0.001; F 52.407; 

df 3; PB0.001, respectively). On the other hand, when E. kuehniella eggs were 

irradiated, the mean number of parasitized eggs was not affected, but progeny 

production of T. evanescens was significantly decreased by irradiation of host egg 

(F 5.511; df 3; PB0.001) (Figure 6). 

Discussion 

We conducted a series of experiments to determine the acceptability and suitability of 

irradiated and unirradiated eggs of both E. kuehniella and S. cerealella to 

parasitization by T. evanescens under choice and no choice experimental design. In 

practice, unless all of the fertile host eggs were parasitized, which is unlikely, the eggs 

that hatched would increase the number of pest moth larvae present. This would be 

unacceptable, and one way to avoid this problem is to kill or sterilize the moth eggs 

before they are exposed to the T. evanescens adults (Brower 1982; Takada, 

Kawamura, and Tanaka 2000). The present results with both host species confirmed 

earlier work (Ayvaz and Tunçbilek 2006), which showed that doses of 200 Gy and 

above prevented adult emergence from irradiated E. kuehniella eggs. Similarly, 

studies on sterilization of E. kuehniella adults by gamma radiation showed that egg 

hatch was significantly reduced at 150 Gy (Marec et al. 1999). 

Irradiating eggs of the two species had no significant effect on their acceptability 

as hosts by T. evanescens. The test was carried out with 24-h-old females to evaluate 

0 Gy 

100 Gy 

140 Gy 

200 Gy 

Figure 6. Effect of radiation dose administered to T. evanescens and to 1-day-old E. 

kuehniella host eggs on parasitization (number of parasitized eggs) and progeny production 

(F 1)ofT. evanescens adults.


the performance of the wasps in respect of host acceptance, fecundity, emergence and 

longevity. These are the most important traits for the performance of mass-reared 

Trichogramma parasitoids (Mansfield and Mills 2004) and host suitability was 

shown to be strongly correlated with host acceptance (Pak and Van Lenteren 1984; 

Wackers, De Groot, Noldus, and Hassan 1987). 

Based on the regression analysis of non choice experiments, the irradiated host 

eggs of E. kuehniella were equally acceptable as control eggs. The choice experiments 

showed that the irradiated MFM and AGM eggs presented to female T. evanescens 

were also equally acceptable for oviposition and suitable for parasitoid development. 

The present results indicate that the tendency of females to attack irradiated host 

eggs was similar to that of unirradiated eggs. Thus, host fertility did not appear to 

play a role in host preference of T. evanescens. These findings are in agreement with 

Brower’s results (1983) on irradiated and unirradiated host eggs. Similar findings 

were made by Lewis and Young (1972) in Trichogramma spp. and by Makee (2006) in 

Trichogramma cacoeciae Marchal. Roriz, Oliveira, and García (2006) found that 

when host preference was analyzed by offering eggs of two host species simultaneously 

to a single female of T. cordubensis Vargas & Cabello, the mean number of 

parasitized eggs differed significantly, and the host species with heavier eggs were the 

most parasitized. 

Our results on egg hatching after irradiation corresponded with those of 

Cogburn, Tilton, and Brower (1973) on the eggs of almond moth, Cadra cautella 

Walker. Thus, eggs irradiated at 200 Gy could be effectively used for propagation of 

T. evanescens in the sterilized host and could be used in warehouses and mills, 

without any risk of increasing the pest population. In addition, irradiated moth eggs 

could be used for laboratory mass rearing programmes in order to avoid any 

problems posed by hatching host eggs. 

Data obtained from both irradiated immature and adult stages of T. evanescens 

experiments indicated that there was no stimulatory effect of irradiation on 

T. evanescens, rather it caused significant adverse effects on parasitoid competence 

with increasing doses. Tillinger et al. (2004) found that irradiation had no 

negative effect on the lifespan of Glyptapanteles liparidis Bouche (Braconidae), 

but when female wasps were irradiated with 48 Gy, oviposition was significantly 

reduced and only 10% of these eggs were viable. This might be due to the 

vulnerability of sensitive stages of the developing parasitoid within the host at the 

time of irradiation. Mechanisms underlying any stimulatory response induced by 

low dose irradiation are still unclear, but several hypotheses have been considered 

(Yamaoka, Edamatsu, Itoh, and Mori 1994; Cai, Satoh, Tohyama, and Cherian 

1999). 

Earlier studies (Brower 1982; Prozell and Schöller 1998; Schöller and Hassan 

2001) support the assumption that the inundative release of Trichogramma egg 

parasitoids may be practicable for the control of stored product moths. Comparing 

the results obtained for irradiated eggs from different host species, we found that the 

acceptability and suitability of irradiated eggs for parasitoid production is a strong 

argument for the development of biological control strategies for control of MFM 

and AGM under commodity storage conditions.



We acknowledge the support of the project by FAO/IAEA to enable us to carry out this work 

under Research Contract No: IAEA/TUR-10782 as part of a Coordinated Research Project 

and The Unit of Scientific Research Projects, Erciyes University (EÜBAP 02-012-09). We 

thank Dr G. Hoch and Dr Canhilal for comments on early drafts of the manuscript, Mrs S. 

Öztemiz for supplying the T. evanescens used in the experiments, and Department of Radiation 

Oncology for using 60 Co irradiator. 

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Vol. 19, S1, 2009, 193 209 

Rearing of five hymenopterous larval-prepupal (Braconidae, Figitidae) 

and three pupal (Diapriidae, Chalcidoidea, Eurytomidae) native 

parasitoids of the genus Anastrepha (Diptera: Tephritidae) on 

irradiated A. ludens larvae and pupae 

Jorge Cancino a , Lía Ruíz a , John Sivinski b , Fredy O. Gálvez a , and 

Martín Aluja c * 


México; b Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 

Gainesville, FL, USA; c Instituto de Ecologia, Xalapa, Veracruz, México 

The aim of this study was to ascertain if eight species of native larval-prepupal 

and pupal Anastrepha (Diptera: Tephritidae) parasitoids which have been recently 

domesticated and colonized (Aluja et al. in press) could be reared on irradiated 

larvae and pupae, and if such was the case, determine the optimal irradiation dose 

so that only adult parasitoids (not flies) would emerge. The species considered 

were: Doryctobracon crawfordi, Utetes anastrephae, Opius hirtus (all larvalprepupal 

braconids), Aganaspis pelleranoi, Odontosema anastrephae (both larval-prepupal 

figitids), Coptera haywardi, Eurytoma sivinskii and Dirhinus sp. 

(diapriid, eurytomid and chalcidoid pupal parasitoids). Eight-day-old A. ludens 

larvae or 3-day-old A. ludens pupae were irradiated with 0, 5, 10, 15, 20, 25, 30, 

35, 40, 50, 60 and 70 Gy under free oxygen and then subjected to parasitoid 

attack. Emergence of the unparasitized host was completely halted at 20 25 Gy 

but such was not the case with the three braconid parasitoids that emerged even if 

subjected to doses as high as 70 Gy. In the case of the figitids, the emergence of 

the host and the parasitoids was completely halted at 20 and 25 Gy, respectively. 

Some parasitoid emergence was recorded at 5 15 Gy but at this irradiation dose, 

fly adults also emerged rendering the fly/parasitoid separation procedures 

impractical. Finally, in the case of the pupal parasitoids, A. ludens adults emerged 

from unparasitized pupae irradiated at 15 Gy. Beyond this dose, only parasitoids 

emerged. With the exception of the figitid larval-prepupal parasitoids, irradiation 

did not negatively affect adult longevity or fecundity. Our results show that 

parasitoid mass rearing with irradiated hosts is technically feasible. 

Keywords: fruit fly parasitoids; mass rearing; host irradiation; Tephritidae; 

Braconidae; Figitidae; Diapriidae; Eurytomidae; Chalcidoidae 

Introduction 

In the New World, some species of fruit flies in the genus Anastrepha (Diptera: 

Tephritidae) (e.g. A. grandis [Macquart], A. fraterculus [Wiedemann], A. obliqua 

[Macquart], A. ludens [Loew], A. serpentina [Wiedemann], A. suspensa [Loew]) 

represent important agricultural pests that also significantly hinder fruit exports 

(Aluja 1994). With increasing public resistance to widespread insecticide use (Clark, 

*Corresponding author. Email: martin.aluja@inecol.edu.mx 




DOI: 10.1080/09583150802377423 



Steck, and Weems 1996), regional efforts are underway attempting to combine the 

use of the sterile insect technique (SIT) and augmentative releases of parasitoids. For 

example, in Mexican mango and citrus growing regions (e.g. Nayarit, Sinaloa, 

Nuevo León), sterile A. obliqua and A. ludens adults are being released in 

conjunction with the exotic parasitoid, Diachasmimorpha longicaudata (Ashmead) 

(Anonymous 2003). Despite the fact that this parasitoid has been proven effective at 

significantly lowering A. suspensa and A. ludens populations when repeatedly 

released in large numbers (Sivinski et al. 1996; Montoya et al. 2000) and that it is 

easily and cheaply mass-reared (Montoya and Cancino 2004), there has been a recent 

upsurge in interest at determining the potential of native parasitoids which had been 

so far neglected in fruit fly biological control programs. Native parasitoids, given 

their long-term evolutionary interaction with their host, could prove quite effective 

at lowering fly populations under certain circumstances (e.g. Sivinski, Aluja, and 

López 1997; Eitam, Sivinski, Holler, and Aluja 2004). For example, in fruit growing 

regions, officially declared as low fruit fly prevalence areas, a native parasitoid may 

be better suited at detecting and parasitizing the few larvae present. Furthermore, 

some authors have proposed that releasing large numbers of exotic parasitoids may 

be detrimental to native, non-target insects (e.g. Williamson 1996). In this sense, 

native parasitoids may represent a more environmentally friendly alternative. 

There are three fundamental prerequisites to the use of native parasitoids in 

Anastrepha biological control programs. The first is to obtain basic knowledge of 

their natural history, ecology and behavior, and significant progress in this field has 

been made over the past 10 years (Sivinski et al. 1997; Aluja, López, and Sivinski 

1998; Sivinski, Aluja, and Holler 1999; Sivinski, Vulinec, and Aluja 2001; Guillén, 

Aluja, Equihua, and Sivinski 2002; Ovruski and Aluja 2002; Aluja et al. 2003; Eitam 

et al. 2004; Guimarães and Zucchi 2004; Ovruski, Schliserman, and Aluja 2004; 

Ovruski, Wharton, Schliserman, and Aluja 2005). The second one is related to their 

domestication and colonization. Recently, Eitam et al. (2004) described some rearing 

techniques useful in the initial stages of the colonization of D. areolatus in Florida. 

Related to the work being reported here, we have successfully domesticated and 

colonized D. areolatus, D. crawfordi, U. anastrephae, O. hirtus (all larval-prepupal 

braconids), A. pelleranoi (Brèthes), O. anastrephae (both larval-prepupal figitids), 

Coptera haywardi (Oglobin), E. sivinskii and Dirhinus sp. at the Instituto de Ecologia, 

A.C. in Xalapa, Veracruz, Mexico (Aluja et al. in press). Thirdly, on top of having 

access to an established colony, parasitoids need to be mass-reared. Two efforts stand 

out in this respect. A fairly recent effort by Menezes et al. (1998) aimed at rearing the 

native pupal parasitoid C. haywardi in irradiated A. suspensa and Ceratitis capitata 

(Wiedemann) larvae. The other, is a yet unpublished but successful effort, directed at 

mass-rearing D. crawfordi in Mexico (L.R., unpublished data). 

Our aim here was to ascertain if eight of the species of native larval-prepupal and 

pupal Anastrepha (Diptera: Tephritidae) parasitoids recently domesticated and 

colonized at the Instituto de Ecologia, A.C., in Xalapa, Veracruz, Mexico (Aluja 

et al. 2008) could be reared on irradiated larvae and pupae and if such was the case, 

to determine the optimal irradiation dose. Our approach was based on the 

pioneering effort by Sivinski and Smittle (1990), who successfully tested the idea 

of mass rearing the exotic parasitoid D. longicaudata on irradiated A. suspensa 

larvae. We also wanted to develop a technique that would facilitate the use of excess 

mass reared larvae that sometimes are left over in mass rearing facilities with the idea


of finding an irradiation dose that would allow healthy adult parasitoids but not flies 

to emerge, as the latter would greatly facilitate handling procedures and reduce costs 

of production. 


Study site 

All experiments were carried out under controlled environmental conditions in 

facilities and laboratories belonging to the Subdirección de Desarrollo de Métodos 

and the Programas MoscaMed/MoscaFrut, Campaña Nacional Contra Moscas de 

la Fruta in Metapa de Domínguez, Chiapas, México. Mean temperature, relative 

humidity and illumination regime were as follows: 24928C, 60 80% RH, and 

12:12 h. Fly rearing and irradiation procedures took place in separate buildings. 

Insects 

All parasitoids were reared on A. ludens larvae stemming from a laboratory strain 

that had been kept for over 300 generations (Domínguez, Castellanos, Hernández, 

and Martínez 2000). Doryctobracon crawfordi, U. anastrephae, O. hirtus, A. 

pelleranoi, O. anastrephae, C. haywardi, and E. sivinskii colonies were obtained 

from the Instituto de Ecología, A.C. in Xalapa, Veracruz, Mexico and reared for 

over 25 generations in our laboratories in Metapa de Domínguez, Chiapas before 

being used for this study. Dirhinus sp. was discovered during a parasitoid survey in 

the Soconusco region (near the city of Tapachula, Chiapas) and subsequently 

domesticated and colonized in our laboratories. 

Irradiation procedures 

Eight-day-old larvae and 3-day-old pupae of A. ludens were exposed to 0, 5, 10, 15, 

20, 25, 30, 35, 40, 50, 60 and 70 Gy, respectively. Experiments were replicated 30 

(braconids), 50 (figitids), 25 (C. haywardi), 35 (E. sivinskii) and 20 (Dirhinus sp.) 

times (replication level determined on the basis of result variability (e.g. high in the 

case of the two figitids, low in the case of Dirhinus sp.)). We used a Gammacell 220 

irradiator (g radiation with a Co 60 source), applying a dose ranging between 2.5 and 

3.0 Gy/min under free oxygen. Exposure times were determined by Fricke’s 

dosimetry (IAEA 1977). Before being exposed to radiation, larvae were removed 

from their rearing medium (artificial diet in a plastic washbowl) and rinsed with tap 

water until all diet residues had been washed away. In the case of pupae, we removed 

excess vermiculite (pupation medium) with the aid of a sieve. 

Exposure of A. ludens larvae to parasitoids 

The method used to expose irradiated larvae or pupae to parasitism was tailored to 

the idiosyncrasies of the parasitoids. In the case of the braconids, 100 A. ludens 

larvae mixed with diet (same diet used for rearing them) were placed in a Petri dish 

that was covered with organza cloth kept in place with a rubber band. The 

parasitization unit was then placed in a Hawaii-type holding cage (27 27 27-cm


wooden structure cage covered with 0.5-mm caliber mesh) (Wong, Ramadan, Herr, 

and McInnis 1992) into which 60 (30à and 30ß) 5 10-day-old parasitoids had been 

released. Exposure periods were 4, 6 and 8 h for D. crawfordi, O. hirtus and U. 

anastrephae, respectively. Given that not all species are equally adapted to the 

artificial rearing conditions, varying exposure times are required to, on the one hand 

avoid superparasitism (case of D. crawfordi) and on the other, secure minimally 

acceptable rates of parasitism (case of U. anastrephae). In the case of the two figitids 

that preferentially parasitize larvae in fallen fruit where they seek them out by 

penetrating the fruit, we did not cover the Petri dish to allow the female’s direct 

access to the larvae. In this case, 100 larvae were exposed to 100 adults (50à:50ß) 

inside a 30 30 30-cm Plexiglass cage. Exposure periods were 4 and 6 h for A. 

pelleranoi and O. anastrephae, respectively. Finally, in the case of the three pupal 

parasitoids, 100 pupae were mixed with vermiculite after irradiation and placed in a 

Petri dish with a paper ‘roof’ to secure a darkened environment for the foraging 

females. In all cases (i.e. all three species), we released 100 parasitoids (50à:50ß) and 

allowed them to parasitize pupae over a 24-h period. 

Parasitoid developmental times and emergence 

After exposure to parasitoid attack, larvae were again rinsed with tap water (to 

remove all diet residues) and placed in 4 8-cm plastic containers with moistened 

vermiculite as a pupation medium. Exposed pupae were also handled as described 

for larvae but were not rinsed with water. After 15 days had elapsed, the vermiculite 

was removed to facilitate emergence of fly and parasitoid adults, which varied among 

parasitoid species. After all insects had emerged, we counted the number of females 

and males, and transferred the insects into cages as described in what follows. 

Determination of parasitoid longevity and fecundity 

After emergence, parasitoid adults were sorted out by species and irradiation 

treatment, and transferred to holding cages to determine their longevity and 

fecundity on a per treatment and replicate basis (three per species). Type of cage, 

parasitization unit and exposure period also varied according to species (details in 

section 2.4). Cohort size in each cage was 10à: 5ß in all cases (i.e. all species). 

Survival was measured over a 30-day period from the moment of emergence. 

Fecundity was measured over a 10-day period starting at age 5 days by offering 

females a parasitization unit that contained non-irradiated larvae and that was 

replaced daily after the exposure period was covered. Exposed larvae and pupae were 

then handled as described in Parasitoid developmental times and emergence. 

Parasitoids had ad libitum access to water and honey throughout the test period. 


Mean number of flies and parasitoids that emerged, sex ratio, and number of 

offspring per female per day (i.e. fecundity; OFD), were subjected to a one-way 

ANOVA (each variable analyzed independently). Quadratic trends in OFD data were 

also ascertained but given extremely low r 2 values (B0.05), results are not reported. 

To compare means, we used Bonferroni’s test (Snedecor and Cochran 1980). OFD


values were obtained by dividing the number of offspring by the number of live 

mothers per day. The proportion of living parasitoids per day (i.e. longevity) was 

analyzed by means of a log-rank test (Francis, Green, and Payne 1993). 

Results 

Emergence patterns of irradiated and non-irradiated hosts 

Developmental times (egg to adult) varied sharply among parasitoid species: 15 days 

for U. anastrephae, O. hirtus, E. sivinskii, 20daysforD. crawfordi, A. pelleranoi, O. 

anastrephae and Dirhinus sp., and 30 days for C. haywardi. Furthermore, we found 

that development of irradiated A. ludens larvae or pupae not subjected to parasitism 

by any of the eight parasitoid species under study here was completely halted at 25 

Gy (Table 1). 

In the case of the braconid parasitoids and their host (exposed to parasitism), 

highly significant differences were found when comparing the effect of irradiation 

on emergence patterns of the host (A. ludens) but not the parasitoid (A. ludens, 

F11 19.31, P 0.0001, D. crawfordi, F11 0.6543, P 0.781; A. ludens, F11 20.58, 

P 0.0001, U. anastrephae, F11 0.7321, P 0.7075; A. ludens, F11 15.74, P 

0.0001, O. hirtus, F11 1.7138, P 0.0708). Complete suppression of adult 

emergence for irradiated A. ludens larvae exposed to unsuccessful parasitism was 

achieved at doses of 20 Gy (Table 2). In the case of the parasitoids, over 30% 

emergence was recorded at doses as high as 70 Gy (Table 2). With respect to sex 

ratio, there were no statistically significant differences among any of the three 

parasitoid species under study (D. crawfordi, F11 0.999, P 0.447; U. anastrephae, 

F11 0.394, P 0.957; O. hirtus, F11 0.6154, P 0.815). Despite the latter, sex 

ratio was consistently skewed towards females. 

The two figitid species were much more susceptible to irradiation. As shown in 

Table 3, emergence was completely halted at 25 Gy, with a highly significant drop 

apparent at 20 Gy. At lower doses, even though emergence was observed in both 

Table 1. Mean proportion (9SE) of A. ludens adults emerging from unparasitized larvae and 

pupae that were subjected to irradiation. 

Dose (Gy) Larva Pupa 

0 82.5892.12 a 88.3792.52 a 

5 81.5192.60 a 85.3092.46 a 

10 38.5194.73b 2.8095.97b 

15 5.9593.12c 0.3090.02c 

20 0.1390.09c 0.1190.02c 

25 0c 0c 

30 0c 0c 

35 0c 0c 

40 0c 0c 

50 0c 0c 

60 0c 0c 

70 0c 0c 

Means within columns followed by the same letter are not significantly different (one-way ANOVA, 

followed by Bonferroni’s test).

Table 2. Mean number (9SE) of flies and parasitoids and sex-ratio of three species of Opiinae parasitoids that emerged from irradiated fruit fly larvae 

that were subjected to parasitism (values are means9SE). 


D. crawfordi U. anastrephae O. hirtus 

Mean number emerged Sex-ratio Mean number emerged Sex-ratio Mean number emerged Sex-ratio 

Dose (Gy) Flies Parasitoids (à: ß) Flies Parasitoids (à: ß) Flies Parasitoids (à: ß) 

0 20.3791.91a 32.2293.77a 2.7090.44a 31.0792.86a 40.3093.23a 1.2890.13a 36.3193.37a 36.8794.38a 1.0090.16a 

5 10.6492.22b 27.6692.51a 2.1390.26a 27.9293.24a 42.9693.39a 1.0490.11a 29.5493.29a 42.0090.91a 1.0390.08a 

10 3.7591.20b 36.6192.90a 2.2390.45a 8.4292.07b 41.9694.03a 1.4190.42a 14.2092.03b 27.7391.64a 1.2290.16a 

15 0.0790.07c 35.6893.02a 2.0190.17a 0.4690.26b 33.5792.97a 1.5190.47a 0.3690.30c 31.7691.70a 1.0790.12a 

20 0 c 36.6894.04a 1.6390.13a 0c 38.5193.00a 1.1390.15a 0 32.6091.77a 1.2790.16a 

25 0 c 35.1392.66a 1.8590.15a 0c 35.6892.93a 1.1790.12a 0 33.3291.61a 1.1290.09a 

30 0 c 31.2592.70a 2.1990.66a 0c 35.2792.89a 1.1390.17a 0 32.1691.61a 1.3190.12a 

35 0 c 34.8292.97a 2.1790.23a 0c 38.8493.24a 1.1990.13a 0 32.4491.45a 1.0890.10a 

40 0 c 35.3993.06a 2.9490.55a 0c 37.2893.87a 1.0190.14a 0 32.7291.50a 1.1790.13a 

50 0 c 34.5093.19a 2.7190.60a 0c 39.6493.60a 1.3390.23a 0 30.4891.36a 1.0290.10a 

60 0 c 33.8693.21a 1.9990.15a 0c 40.5093.42a 1.3190.12a 0 31.2491.59a 1.0690.13a 

70 0 c 34.8793.09a 1.9290.29a 0c 40.8493.06a 1.2590.17a 0 30.1391.73a 1.0690.12a 

Means within columns followed by the same letter are not significantly different (one-way ANOVA, followed by Bonferroni’s test). 


Table 3. Mean number (9SE) of flies and parasitoids and sex-ratio of two species of Figitidae parasitoids that emerged from irradiated fruit fly larvae 



A. pelleranoi O. anastrephae 

Mean number emerged Sex-ratio Mean number emerged 

Dose (Gy) Flies Parasitoids (à: ß) Flies Parasitoids Mean no. a females Mean no. b males 

0 7.2491.19a 22.6892.08a 2.6790.65a 32.1192.08a 35.2391.84a 35.0891.85a 0.1490.10a 

5 8.8491.29a 23.0291.84a 2.3690.27ab 16.6791.81b 32.0091.87a 31.9391.87a 0.0790.04a 

10 3.0990.57b 17.8691.87a 2.8290.49a 7.5491.41c 15.4291.73b 15.4291.73b 0a 

15 1.6191.19b 5.1591.45b 1.0690.28bc 0.6590.31d 2.8291.10c 2.0490.69c 0.0290.02a 

20 0 c 0.3490.15c 0.1090.05c 0 d 0.0290.02d 0.0290.02c 0a 

25 0c 0d 0c 0d 0d 0c 0a 

30 0c 0d 0c 0d 0d 0c 0a 

35 0c 0d 0c 0d 0d 0c 0a 

40 0c 0d 0c 0d 0d 0c 0a 

50 0c 0d 0c 0d 0d 0c 0a 

60 0c 0d 0c 0d 0d 0c 0a 

70 0c 0d 0c 0d 0d 0c 0a 

a,b Instead of sex-ratio, we provide actual emergence values for each sex to highlight fact that almost all emerged adults were females (apparently because we are dealing 

with a thelytokous strain). Means within columns followed by a common letter are not significantly different (one-way ANOVA, followed by Bonferroni’s test). 


Table 4. Mean number (9SE) of flies and parasitoids and sex-ratio of three species of fruit fly pupal parasitoids that emerged from irradiated pupae 



C. haywardi E. sivinskii Dirhinus sp. 

Mean number emerged Sex-ratio Mean number emerged Sex-ratio Mean number emerged Sex-ratio 

Doses (Gy) Flies Parasitoids (à: ß) Flies Parasitoids (à: ß) Flies Parasitoids (à: ß) 

0 22.2191.22a 37.7892.07a 1.3590.10a 61.9092.46a 24.8891.38a 1.1490.07a 49.3292.74a 31.0593.62a 0.9290.11a 

5 18.3292.00a 38.7091.39a 1.2690.06a 63.1092.46a 20.2791.54a 1.2990.14a 46.2492.88a 27.0593.46a 0.9490.12a 

10 8.9591.30b 38.5291.47a 1.3290.09a 14.5092.42b 20.0891.89a 1.6990.37a 17.0592.86b 28.8093.41a 0.8790.10a 

15 2.4091.57c 36.0797.30ab 1.7490.13a 0.9490.47c 22.4891.55a 1.1490.08a 0.2090.2c 32.6293.48a 0.8890.10a 

20 0d 31.2591.99abc 1.7890.22a 0d 22.1991.74a 1.1090.08a 0c 29.7392.43a 1.0690.12a 

25 0d 35.7291.50ab 1.4290.07a 0d 23.0092.20a 1.1890.15a 0c 32.4492.65a 0.9890.09a 

30 0d 30.9091.4abcd 2.9290.56b 0d 24.6492.02a 1.0190.07a 0c 33.5292.91a 1.0590.10a 

35 0d 34.0291.73ab 2.0790.15ab 0d 24.6991.88a 1.2890.19a 0c 33.3792.82a 1.5290.34a 

40 0d 34.1991.47ab 1.799 0.19a 0d 24.2492.23a 1.1490.08a 0c 36.2092.85a 1.1290.10a 

50 0d 29.3191.99bcd 2.269 0.2ab 0d 25.7492.22a 1.1990.13a 0c 31.3593.20a 1.1190.09a 

60 0d 23.7392.04cd 1.899 0.1ab 0d 23.1891.97a 1.1490.10a 0c 31.4593.70a 0.9690.14a 

70 0d 23.2591.18d 2.209 0.2ab 0d 24.3891.44a 1.1690.11a 0c 23.3592.78a 1.4690.28 

Means within columns followed by the same letter are not significantly different (one-way ANOVA, followed by Bonferroni’s test). 


species, a highly significant effect of irradiation was also detected, particularly in the 

case of O. anastrephae (A. ludens, F11 8.91, P 0.0003, A. pelleranoi, F11 20.89, 

P 0.0001; A. ludens, F11 47.15, P 0.0001, O. anastrephae, F11 33.72, P 

0.0001). Sex ratios were highly skewed towards females in both species, with 

statistically significant differences detected when adult emergence was recorded (B 

25 Gy) (A. pelleranoi, F 11 8.26, P 0.001; O. anastrephae, F 11 134.64, P 

0.0001). 

Finally, in the case of the pupal parasitoids, emergence was observed at doses as 

high as 70 Gy, while the host exposed to unsuccessful parasitism (in this case irradiated 

in the pupal stage) totally ceased emerging at doses of 20 Gy (Table 4). The effect of 

irradiation dose was highly significant with respect to emergence of the host and in the 

case of C. haywardi (A. ludens, F11 19.97, P 0.0001, C. haywardi, F11 9.44, P 

0.0001; A. ludens, F 11 128.4, P 0.0001, E. sivinskii, F 11 0.9568, P 0.48; A. ludens, 

F 11 38.89, P 0.0001, Dirhinus sp., F 11 1.148, P 0.324). With respect to sex 

ratios, significant differences were also only detected in the case of C. haywardi (C. 

haywardi, F11 4.11, P 0.0001; E. sivinskii, F11 0.439, P 0.937; Dirhinus sp., 

F11 0.849, P 0.590) (details in Table 4). 

Fecundity and longevity of parasitoid offspring 

Mean fecundity of the braconid species studied was significantly affected by 

irradiation (D. crawfordi: F11 3.51, P 0.0003, U. anastrephae: F11 2.32, P 

0.013, O. hirtus: F11 3.99, PB0.0001) (Figure 1). With respect to longevity, there 

was no statistically significant effect of irradiation in any of the three species (D. 


3 

2.5 

2 

1.5 

1 

0.5 

0 

ab 

ab 

a 

b 

ab 

a 

ab 


ab 

a 

ab 

ab 

a 

b 

ab 

a 

ab 

a 

a 

b 

ab 

a 

ab 

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a 

b 

b 

a 

D. crawfordi 

O. hirtus 


0 5 10 15 20 25 30 35 40 50 60 70 

Irradiation dose (Gy) 

Figure 1. Fecundity of D. crawfordi, O. hirtus and U. anastrephae (Braconidae: Opiinae) 

stemming from larvae irradiated at varying gamma radiation doses. The larvae offered to the 

adult parasitoids were not irradiated. 

ab 

b 

a 

ab 

ab 

a 

a 

ab 

a


Proportion alive 

1 

0.8 

0.6 

0.4 

0.2 

0 

1 

0.8 

0.6 

0.4 

0.2 

0 

1 

0.8 

0.6 

0.4 

0.2 

0 

D. crawfordi 

0 3 6 9 12 15 18 21 24 27 30 

Age (day) 


0 3 6 9 12 15 18 21 24 27 30 

Age (day) 

O. hirtus 

0 3 6 9 12 15 18 21 24 27 30 

Age (day) 

0 Gy 5 Gy 10 Gy 15 Gy 20 Gy 25 Gy 

30 Gy 35 Gy 40 Gy 50 Gy 60 Gy 70 Gy 

Figure 2. Longevity of D. crawfordi, O. hirtus and U. anastrephae (Braconidae: Opiinae) 

stemming from larvae irradiated at varying gamma radiation doses. 

crawfordi, x 2 11 9.23, P 0.60, U. anastrephae, x 2 11 45.97, P 0.001, O. hirtus, x 2 11 

16.32, P 0.129). Of the latter, D. crawfordi lived the longest (Figure 2). 

In the case of A. pelleranoi and O. anastrephae, no statistically significant 

influence of irradiation on fecundity was detected in the few cases where adequate


0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 


0 5 10 15 20 25 30 35 40 50 60 70 


A. pelleranoi 


Figure 3. Fecundity of A. pelleranoi and O. anastrephae (Figitidae: Eucoilinae) stemming 

from larvae irradiated at varying gamma radiation doses. The larvae offered to the adult 

parasitoids were not irradiated. 

emergence was observed (up to 15 Gy) (A. pelleranoi, F11 0.726, P 0.542; O. 

anastrephae, F11 0.142, P 0.934; details in Figure 3). With respect to longevity, 

and particularly in the case of A. pelleranoi, irradiation had a marginally significant 

effect (A. pelleranoi, x 2 3 7.54, P 0.056, O. anastrephae, x 2 3 0.272, P 0.965) 

(Figure 4). 

As for pupal parasitoids, fecundity was only influenced by irradiation in the case 

of C. haywardi and E. sivinskii (C. haywardi, F 11 5.595, P 0.0001; E. sivinskii, 

F 11 3.824, P 0.0001; Dirhinus sp., F 11 0.26, P 0.99). Remarkably, offspring 

was produced even at doses as high as 70 Gy (Figure 5). With respect to longevity, no 

statistically significant differences were detected when comparing the different 

irradiation doses in all three species (C. haywardi, x 2 11 4.58, P 0.949; E. sivinskii, 

x 2 11 11.74, P 0.383; Dirhinus sp., x 2 11 12.84, P 0.303). As can be seen in Figure 

6, large numbers of adults were still alive after 30 days. 

Discussion 

Several points of basic physiological and applied significance emerged from our 

study: (1) irradiating larvae or pupae to mass rear native Anastrepha larval-prepupal 

and pupal parasitoids appears technically feasible in all but two of the species under 

study here. With the exception of A. pelleranoi and O. anastrephae (both figitids), 

host emergence was completely halted at doses that did not negatively affect 

parasitoid emergence, fecundity or survival. This capacity to develop in irradiated 

hosts is paralleled in certain Old World species such as D. longicaudata (Sivinski and 

Smittle 1990; Cancino, Ruiz, Gómez, and Toledo 2002). (2) Sex ratios were 

consistently (albeit not significantly) female biased, and did not vary when compared



1 

0.8 

0.6 

0.4 

0.2 

0 

1 

0.8 

0.6 

0.4 

0.2 

0 

0 Gy 5 Gy 10 Gy 15 Gy 

A. pelleranoi 

0 3 6 9 12 15 18 21 24 27 30 

Age (day) 


0 3 6 9 12 15 18 21 24 27 30 

Age (day) 

Figure 4. Longevity of A. pelleranoi and O. anastrephae (Figitidae: Eucoilinae) stemming 

from larvae irradiated at varying gamma radiation doses. 

to the control. The latter adds significantly to the practical benefit of irradiation on 

native parasitoid mass rearing. (3) All three species of pupal parasitoids developed 

on irradiated hosts, although C. haywardi seemed the most sensitive to host 

irradiation, perhaps due to its unusual endoparasitic feeding habits and possible 

damage to host organs and physiology. (4) While C. haywardi is unable to develop in 

pupae resulting from irradiated larvae (Menezes et al. 1998), it was found to develop 

in irradiated pupae, suggesting some necessary early pupal development in the host. 

(5) Finally, it appears that A. ludens is highly susceptible to irradiation, as is A. 

obliqua (Toledo, Rull, Oropeza, Hernández, and Liedo 2004), highlighting the urgent 

need to reexamine currently used irradiation doses that seem unnecessarily high.


1 

0.8 

0.6 

0.4 

0.2 

0 

1 

0.8 

0.6 

0.4 

0.2 

0 

1 

0.8 

0.6 

0.4 

0.2 

0 


C. haywardi 

0 3 6 9 12 15 18 21 24 27 30 

Age (day) 

E. sivinskii 

0 3 6 9 12 15 18 21 24 27 30 

Age (day) 

Dirhinus sp. 

0 3 6 9 12 15 18 21 24 27 30 

Age (day) 

0 Gy 5 Gy 10 Gy 15 Gy 20 Gy 25 Gy 

30 Gy 35 Gy 40 Gy 50 Gy 60 Gy 70 Gy 

Figure 5. Longevity of Dirhinus sp., C. haywardi and E. sivinskii (Chalcidoidea, Diapriidae 

and Eurytomidae, respectively) stemming from pupae irradiated at varying gamma radiation 

doses. The pupae offered to the adult parasitoids were not irradiated. 

The opiine braconids contribute a number of important fruit fly biological 

control agents (Wharton and Marsh 1978; Wharton and Gilstrap 1983; Ovruski, 

Aluja, Sivinski, and Wharton 2000). Our present experiments found that, like the 

Old World species D. longicaudata and D. kraussii (Fullaway), the New World species 

D. crawfordi, U. anastrephae and O. hirtus develop as well or better in irradiated host-



3 

2.5 

2 

1.5 

1 

0.5 

0 

a 

a 

a 

b 

b ab 

bab 

b a 

larvae (see Sivinski and Smittle 1990). However, this capacity is not universal in the 

subfamily. Attempts to rear Psyttalia spp. on irradiated hosts have been unsuccessful 

(E. Harris, unpublished data). It is possible that irradiation prevents some important 

developmental process in the host that subsequently prevents parasitoid development. 

For example, Thomas and Hallman (2000) documented that irradiating late 

third instar A. ludens larvae at 20 Gy (gamma radiation), retarded protein 

metabolism and arrested development at the transition from cryptocepahlic to 

phanerocephalic pupa. Evidence of required host development for maturation of the 

endoparasitic pupal parasitoid C. haywardi can be obtained by comparing the 

capacity of the insect to develop in pupae derived from irradiated larvae and pupae. 

In the first instance, C. haywardi is unable to develop (Sivinski et al. 1999), while 

development is completed if radiation is applied after pupation (our data here). 

In addition to retarding host development, irradiation might damage vital 

structures in the host required by the immature parasitoid. For example, radiation 

damages the nervous and endocrine systems of Anastepha suspensa (Loew) larvae 

(Nation, Smittle, Milne, and Dykstra 1995). None of the two figitid parasitoids 

emerged at doses above 20 Gy. These species have a longer developmental period, 

20 25 days, than braconids, and this relatively slow development could be a 

disadvantage when irradiated hosts eventually begin to decompose. In addition, 

apparently larvae start as endoparasitoids but move outside the host with increasing 

size. Given that parasitoid larvae may need to use the empty spaces between the host 

and the puparium (Ovruski 1994), unsatisfactory formation of the pupae might 

b 

a 

a 

b 

a 

b 

b 

b 

Dirhinus sp. 

C. haywardi 

Eurytomidae 

b 

a 

a 

b 

0 5 10 15 20 25 30 35 40 50 60 70 


Figure 6. Fecundity of Dirhinus sp., C. haywardi and E. sivinskii (Chalcidoidea, Diapriidae 

and Eurytomidae, respectively) stemming from pupae irradiated at varying gamma radiation 

doses. 

a 

b


result from irradiation. However, damage to the host need not be detrimental to the 

developing parasitoid. Increasing levels of irradiation could possibly suppress 

the immune system of the host and inhibit its ability of for example, encapsulate 

the parasitoid developing inside. 

In conclusion, the results obtained here represent a significant step forward in the 

use of native parasitoids in fruit fly biological control. Although their augmentative 

release has to date not been formally tested, the use of irradiated hosts may provide 

various advantages in other activities. For example, tests to determine movement 

ability with artificial traps and studies of foraging behavior using irradiated hosts 

may be carried out under field conditions without the risk of releasing pests. 


We thank two anonymous reviewers and the editor for helping us produce a more polished 

final product. Francisco Díaz-Fleischer (Subdirección de Desarrollo de Métodos, Campaña 

Nacional Contra Moscas de la Fruta [CNCMF]), Mariano Ordano, Juan Rull, Ricardo 

Ramírez and Larissa Guillén (all Instituto de Ecología, A.C. [INECOL]) also made many 

useful comments on an earlier draft. Javier Valle Mora (El Colegio de la Frontera Sur) and 

Francisco Díaz-Fleischer (CNCMF) made important suggestions on data analyses. We 

appreciate the technical support provided by Edelfo Pérez, Mario Pineda, Javier Robledo, 

Floriberto Pérez, Velizario Ribera and Marbel Monjaraz (all Subdirección de Desarrollo de 

Métodos, CNCMF). We are also grateful to Yeudiel Gómez and his staff (Programa 

MoscaMed) for having irradiated all the larvae and pupae used in this study. Thanks are due 

to Nicoletta Righini and Alberto Anzures (both INECOL) for formatting and final 

preparation of this manuscript. This work was financed by the International Atomic Energy 

Agency (IAEA) through contract No. 10848, the Mexican Campaña Nacional Contra Moscas 

de la Fruta (Secretaría de Agricultura, Ganadería, Desarrollo Rural y Pesca Instituto 

Interamericano de Cooperación para la Agricultura [SAGARPA-IICA]) and the Instituto de 

Ecología, A.C. (INECOL). MA also acknowledges support from CONACyT through a 

Sabbatical Year Fellowship (Ref. 79449) and thanks Benno Graf and Jörg Samietz 

(Forschungsanstalt Agroscope Changins-Wädenswil ACW), for providing ideal working 

conditions to finish writing this paper. 

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Vol. 19, S1, 2009, 211 224 


Control of the olive fruit fly, Bactrocera oleae, (Diptera: Tephritidae) 

through mass trapping and mass releases of the parasitoid 

Psyttalia concolor (Hymenoptera: Braconidae) reared on 

irradiated Mediterranean fruit fly 

Bahriye Hepdurgun*, Tevfik Turanli, and Aydin Zümreog˘lu 

Plant Protection Research Institute Gençlik cad. No. 6, 35040 Bornova, I˙zmir, Turkey 

Field studies were performed from 2002 to 2004 on Gökçeada Island, Turkey, to 

determine the effectiveness of releases of the larval pupal parasitoid Psyttalia 

concolor Szepligeti against the olive fruit fly, Bactrocera oleae (Gmelin), alone and 

in combination with mass trapping, using EcoTraps † . For this, the parasitoid was 

reared on a factitious host, irradiated larvae of Ceratitis capitata (Wiedemann). 

Preparatory to making open-field augmentative releases, initial parasitoid releases 

were conducted throughout the 2001 season using confinement cages over 

branches bearing naturally infested olives into which parasitoids were introduced, 

using 1 ß:à pair per 3 fruit oviposition punctures. Percent reduction in fly 

emergence due to parasitization in these cages was 26.9, 27.6, 18.0, and 24.7% from 

the first to the fourth olive fruit fly generations during the season, respectively. In 

2002, open-field experiments were conducted in an experimental area (EA-1) 

containing 2500 olive trees. In this area, augmentative parasitoid releases and 

mass-trapping (MT) were combined, using 2000 EcoTraps. Following the first fruit 

oviposition punctures, parasitoids were released throughout the season, using ca. 

26 40 parasitoids per tree per occasion. Damage of olives was reduced from an 

average of 87.6% in the control areas to only 18.1% in areas receiving mass trapping 

plus parasitoids. In 2003, the experiments were conducted in two areas. In EA-1 

( EA-1 in 2002), only parasitoid releases were made throughout the season, using 

ca. 27 34 parasitoids per tree per occasion. In EA-2, which contained 2000 olive 

trees, parasitoid releases were combined with 2000 EcoTraps and the parasitoids 

were released using ca. 20 30 parasitoids per tree per occasion throughout the 

season after occurrence of the first fruit oviposition punctures. Overall fruit 

damage rates of 10.6 and 10.1% were recorded in EA-1 (parasitoids only) and 

EA-2 (parasitoids MT), respectively. Damage in the control area was 35.5%. In 

2004, only parasitoid releases were conducted in EA-1. However, that year releases 

were begun early (in May) to attack the spring olive fly generation. Early season 

releases were made at ca. seven parasitoids per tree and late releases involved ca. 

35 parasitoids per tree. Overall damage throughout the season was 12.2% in EA-1 

vs. 37.9% in the control area. Our studies suggest that parasitoid releases are not 

enhanced by use of EcoTraps at the times and rates they were deployed. Despite the 

positive effects of both mass trapping and parasitoid releases, the reduction of 

damage by these means alone was not adequate to meet the requisite economic 

threshold of one to six larvae per fruit for table- and oil-varieties, respectively. 

Keywords: Bactrocera oleae; Ceratitis capitata; Psyttalia concolor; EcoTraps; pest 

management; gamma radiation 

*Corresponding author. Email: hepdurgun@hotmail.com 



DOI: 10.1080/09583150903056926 



Introduction 

The olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tepritidae), is the major 

pest of olives in Turkey, as it is in other Mediterranean countries (Haniotakis 2005 

and references therein). In the Mediterranean region, it has been one of the most 

devastating olive pests for more than 2000 years. Infestation of olive fruit by the 

larvae causes premature fruit drop and reduces fruit quality for both table olives and 

for olive oil production (Michelakis and Neuenschwander 1983). Many studies and 

experiments have been carried out to suppress the pest (Haniotakis 2005). Among 

the methods used, chemical control measures are most widely applied, both as 

general cover sprays and as aerial and ground bait sprays. However, because of the 

detrimental effects of these chemicals on the environment and beneficial insects, an 

increasing effort is being made to develop biorational control strategies, including 

more biotechnical approaches. For this purpose, the sex pheromone of the olive fruit 

fly has been synthesized and used with traps developed to help monitor and control 

the pest (Haniotakis, Mazomenos, and Tumlinson 1977; Mazomenos, Haniotakis, 

Ioannou, Spanakis, and Kozirakis, 1983; Montiel 1986; Broumas and Haniotakis 

1987; Cristofaro, Cristofaro, Tenaglia, Fenio, and Tronci 2007). In recent years, in 

order to protect the beneficial fauna in the ecosystem, mass trapping has become an 

important management tool (Haniotakis, Kozyrakis, and Fitsakis 1991; Montiel 

and Jones 2002; Ragoussis 2002; Mazomenos, Haniotakis, Ioannou, Spanakis, 

and Kozirakis 2002; Lentini, Delrio, and Foxi 2003; Tedeschini, Isufi, Uka, Bacaj, 

and Pfeffer 2003; Rizzi, Petacchi, and Guidotti 2005; Caleca, Rizzo, Battaglia, and 

Piccionello 2007; Iannotta, Pellegrino, Perri, Perri, and de Rose 2007). 

Biological controls using parasitoids are also being developed. In the Mediterranean 

and sub-Saharan Africa, the olive fruit fly is attacked by a number of parasitoid 

species including a braconid wasp, Psyttalia concolor Szepligeti, which was 

introduced into Italy from Tunisia in 1914 (Clausen 1978). This larval pupal 

parasitoid was later introduced into France, Greece and other Mediterranean 

countries. Numerous studies have been conducted to develop improved rearing 

techniques for P. concolor to facilitate its use against B. oleae (Biliotti and Delanoue 

1959; Jannone and Binaghi 1959; Monastero 1959; Monastero and Genduso 1962; 

Fenili and Pegazzano 1971; Bmetic 1973; Kapatos, Fletcher, Pappas, and Laudeho 

1977; Liaropoulos, Louskas, Canard, and Laudeho 1977; Kapatos and Fletcher 

1984; Jimenez, Castillo, and Lorite 1990). This species is believed to be relatively 

ineffective as a classical biological control agent in Europe. One reason for its poor 

performance may be a lack of synchronization between the life cycles of the 

parasitoid and fly (Clausen 1978). However, because of increasing concerns for the 

environment and maintaining a more natural balance of plants and animals in 

managed ecosystems, P. concolor is still routinely used in the Mediterranean region 

for inoculative (Delrio, Lentini, and Satta 2003, 2005) and augmentative releases 

against the olive fly (Kimani-Njogu, Trostle, Wharton, Woolley, and Raspi 2001). 

After the first discovery of olive fruit fly in California in 1998, rearing of P. concolor 

was also initiated in Guatemala using the Mediterranean fruit fly (medfly) as a host 

and shipments of the adult parasitoids were made to California (Yokoyama, Rendon, 

and Sivinski 2008 and references therein). Continuing efforts are being made in 

California to use P. cf. concolor from Kenya in inoculative releases against B. oleae, 

importing parasitoid adults reared in Guatemala on C. capitata larvae (Yokoyama


et al. 2008). Efforts also are being made in Europe to use augmentative releases of P. 

concolor alone and in combination with other control tactics such as mass trapping 

(Liaropoulos, Mavraganis, Broumas, and Ragoussis 2005). 

To facilitate augmentative releases, rearing of P. concolor on C. capitata was 

achieved by rearing P. concolor for multiple generations on this factitious host 

to achieve a medfly-adapted strain (Loni and Canale 2005). Detailed studies on host 

suitability of C. capitata for P. concolor were conducted by Mohamed, Overholt, 

Lux, Wharton, and Eltoum (2007). Hepdurgun, Turanli, and Zümreog˘lu (2009) 

demonstrated that P. concolor could be successfully reared on irradiated C. capitata 

larvae, which would allow field release of parasitized host puparia without fear of 

release of viable C. capitata adults from unparasitized pupae. 

In Turkey, efforts are now underway to test combinations of tactics and to apply 

control measures on an area wide basis in an attempt to find more effective 

and environmentally safe olive fly control strategies. In this study, we examined 

augmentative releases of P. concolor alone and in combination with mass trapping 

using EcoTraps. 


Experimental area 

Field experiments were conducted on Gökçeada Island (Imbroz), which is ca. 30 km 

from continental Turkey and 300 km 2 in size. It has ca. 120 000 productive olive 

trees. Although the olive oil variety ‘Megaritiki’ is dominant, the table olive variety 

‘Gemlik’ also is present. Treatment orchards were located in the Zeytinli zone of 

the island, while the untreated control orchard was located ca. 2 km distant in the 

Baraj zone. Tree height in all of the orchards was 7 10 m and trees were planted at 

an average density of 150 trees/ha. No olive fly control measures other than the 

experimental treatments were applied in the orchards from 2001 to 2004. 

Parasitoid rearing 

The parasitoid P. concolor was acquired from the National Agricultural Research 

Foundation (NAGREF), Greece, and was adapted to continuous rearing on medflies 

using procedures described in USDA manuals (Anonymous 1997, 1998). Further 

details on parasitoid rearing using irradiated C. capitata larvae are given in 

Hepdurgun et al. (2009). 

Field-cage studies on the efficacy of Psyttalia concolor 

As a first step in preparing for eventual augmentative open-field releases of 

P. concolor to control olive fly, the efficacy of the parasitoid was initially investigated 

in cage tests, using confinement cages over fruit-bearing branches. The cages served 

to exclude wild olive flies and to confine introduced parasitoids. For this purpose, 

80 mesh organdy cloth tree-branch cages 51 cm in height and 28 cm in diameter were 

used. All 80 cages were installed over fruit-bearing branches prior to observing the 

first oviposition punctures, on June 28, 2001, but 20 were not closed initially, so that 

they allowed access for oviposition by wild olive fly females.


For the first generation, these 20 cages were closed after samples revealed that 

they contained sufficient punctured fruit as of July 11, 2001. Eleven days after 

observing the first punctures, mated individuals of P. concolor were released into 

10 of these cages. The other 10 cages were maintained as controls. The number of 

parasitoids released was determined according to the number of punctures on 

the fruits inside the cages. The release ratio was one ß:à pair of parasitoids per 

3 punctures (potentially representing 3 host larvae). Small plastic tubes containing 

wet wicks and a honey water solution were placed into the branch-cages to provide 

food and water for the enclosed parasitoids. 

For the second and subsequent generations, 20 additional cages out of the total 

of 80 cages were opened to allow access for oviposition by the time the number of 

olive fly adults of second and subsequent generations were present, as evidenced by 

trap captures. The cages were closed again after 7 9 days, when enough olive fruit fly 

punctures were seen on the fruits. Parasitoids were released into 10 cages 10 12 days 

later. The other 10 cages were left as controls. Parasitoids were released into the cages 

right away in second generation tests because we noticed that larval development 

had begun. This process was also followed for the third and fourth olive fly 

generations, except that for the fourth generation, the test was replicated only 7 times 

(7 release and 7 control cages) due to inadvertent damage to some of the cages. Thus, 

for each olive fruit fly generation, 10 experimental and 10 control replicates/cages 

were employed (except that only 7 cages were used for the fourth generation). 

Assessment of parasitoid efficacy in cages 

For each generation, the cages were opened 11 20 days after parasitoid introduction 

and punctured fruits were collected and placed into labeled culture boxes with a 

pupation medium (sand) for any larvae completing development. Upon adult 

eclosion, olive fruit flies were counted along with unemerged puparia so that the 

percent successful development of B. oleae could be determined and the influence of 

parasitoid activity assessed. 

Field tests 

Mass-trapping applications against Bactrocera oleae 

For mass trapping, EcoTraps † (Vioryl, S.A. Athens, Greece) were used. These traps 

were made of a 15 20 cm green paper envelope with an internal plastic lining for water 

and air proofing. Each trap contained 70 g ammonium bicarbonate salt, a powerful 

food attractant for both sexes, and on its surface, 15 mg a.i. of deltamethrin especially 

formulated for protection of the active ingredient from natural UV light. A pheromone 

dispenser contained 80 mg of synthetic racemic 1,7-Dioxaspiro (5-5) undecane. In 

2002, the experiment was conducted in Experimental Area (EA) 1, which had 2500 

olive trees, and 2000 EcoTraps were distributed on June 26th at a density of ca. one trap 

per tree. In 2003, 2000 EcoTraps were hung on 2000 olive trees in EA-2 (across the road 

from EA-1) on 29th July. EcoTraps were placed approx. 2 m high and in the middle the 

canopy of the olive trees, in the shade, without coming in contact with leaves or 

branches. EcoTraps were hung in the trees before the emergence of the first generation 

of olive flies and before the olive fruits became susceptible to infestation. Traps were

applied once during the season. This application was intended to decrease the B. oleae 

population before releasing P. concolor. 

Parasitoid releases 

Parasitoid releases were combined with mass-trapping in EA-1 in 2002 and in EA-2 

in 2003, respectively, as shown in Table 1. In 2003 and 2004, in EA-1, only parasitoid 

releases were made; i.e., without mass-trapping. 

Table 1. Number of Psyttalia concolor released in experimental areas throughout study. 

Year Area 

2002 Experimental Area 

1 (combination of 

mass-trapping and 

parasitoid releases) 


1 (parasitoid 

releases only) 

Releasing No. of parasitoids released 

Experimental Area 

2 (combination of 

mass-trapping and 

parasitoid releases) 


1 (parasitoid 

releases only) 


Date 

(dd/mo) à ß Total 

Average no. 

parasitoids 

released/tree 

05.09 37,84 28,571 66,455 26.6 

18.09 52,940 50,933 103,873 41.5 

02.10 45,308 20,385 65,693 26.3 

24.10 37,961 28,896 66,857 26.7 

Total 174,093 128,785 302,878 121.1 

23.09 36,023 32,718 68,741 27.5 

07.10 39,371 22,168 61,539 24.6 

21.10 45,287 38,827 84,114 33.6 

04.11 45,144 39,868 85,012 34.0 

Total 140,825 113,581 254,406 101.7 

23.09 22,050 19,380 41,430 20.7 

07.10 23,335 19,882 43,217 21.6 

21.10 33,336 28,336 61,672 30.8 

04.11 30,423 22,998 53,421 26.7 

Total 109,144 90,596 199,740 99.9 

26.05 10,681 7,478 18,159 7.3 

23.06 11,054 8,820 19,874 7.9 

29.07 15,301 9,910 25,211 10.1 

18.08 32,677 26,151 58,828 23.5 

07.09 62,345 56,541 118,886 47.6 

22.09 48,550 42,326 90,876 36.4 

07.10 44,827 41,499 86,326 34.5 

20.10 49,814 38,556 88,370 35.3 

Total 275,249 231,281 506,530 202.6


Parasitoid releases were initiated after the number of olive fly trap catches 

increased commensurate with discovery of fruit punctures in 2002 and 2003. 

However, releases were begun in May 2004 against the overwintering individuals 

and spring generation. Laboratory-reared P. concolor individuals were released into 

the groves after they had mated and matured. Before releases, the adult eclosion rate 

and sex ratio were determined by using eclosion grids. Thus, the numbers of 

parasitoids released in 2002 2004 were estimates (Table 1). 

For assessment, before every release and at harvest, a total of 1000 randomly 

selected fruit were collected from the trees and the number of infested fruits was 

determined by checking for oviposition punctures. This was done in each area, core 

(orchard centre), buffer (15 rows beyond core), neighboring (10 rows beyond buffer) 

and control (2 km distant). Additionally, 500 fruits were collected from the ground. 

Infested fruits were held under laboratory conditions. After 15 20 days, infested 

fruits were checked and the number of eclosed olive flies and P. concolor were 

recorded. In addition, McPhail and EcoTraps baited with pheromone were checked 

to observe the population trend of B. oleae in experimental areas and in the control. 

Mass-trapping and parasitoid releases were evaluated together where these two 

techniques were combined. 

Population trend of Bactrocera oleae 

The population trend of B. oleae was observed by use of McPhail traps containing 

DAP 2% and yellow sticky traps with pheromone. Six traps of each were distributed 

as pairs in the experimental and control areas. 

The traps were checked weekly and the McPhail trap solutions were renewed after 

3 weeks, along with the pheromone capsules (Vioryl, S.A. Athens, Greece). 

Results 

Branch cage trials (2001) 

When parasitoid release cage and control cage results are compared, the reduction in 

olive fly survival as a result of parasitoid activity in the cages was 26.8, 27.6, 18.0 and 

24.7%, respectively, for the four generations (Table 2) The highest parasitization rate 

occurred in the third and forth generations, when adult emergence of B. oleae also 

was highest. 

Open field trials 

In 2002, the average fruit damage throughout EA-1, which received parasitoids plus 

mass trapping, was only 14.1% in the core area, 22.1% in the buffer area, and 44.3% 

in the neighboring area (18.1% overall) vs. 87.6% in the control (Table 3). In 2003, 

the damage in EA-1, which received only parasitoids, averaged 10.4% (core), 10.9% 

(buffer) and 18.9% (neighboring). Damage in EA-2, which received parasitoids plus 

mass trapping, averaged 10.1% (core), 10.2% (buffer), and 18.9% in the same 

neighboring area vs. 35.5% damage in the control area (Table 4). The overall fruit 

damage rates for all regions of EA-1 and EA-2 were 10.6 and 10.1%, respectively.

Table 2. Overall results of the branch cage studies. 

Generation 

no. Treatment 


Mean no. 

punctured fruit 

per cage 

B. oleae adult eclosion 

from recovered 

puparia (%) 

Reduction in B. oleae 

survival associated with 

parasitoid activity (%) 

I Control 11.0 54.3 26.8 

Parasitoids 

released 

12.1 27.4 

II Control 14.3 37.7 27.6 

Parasitoids 

released 

14.8 10.1 

III Control 16.3 66.3 18.0 

Parasitoids 

released 

17.3 48.3 

IV Control 17.4 86.5 24.7 

Parasitoids 

released 

18.7 61.8 

In 2004, since the parasitoid releases were made early (in May instead of 

September or October as in 2002 and 2003) because trapping results indicated the fly 

population was peaking early, beginning in August, as compared to September in 

earlier years. Fruit counting was initiated on August 18, when the first fruit 

punctures were seen. According to the assessment made at harvest, the average 

damage in the core, buffer and neighboring areas was 11.7, 12.7 and 18.3% (12.2% 

overall) vs. 37.9% in the control (Table 5). 

Discussion 

The preliminary field cage studies conducted in 2001 clearly established that 

P. concolor reared on irradiated C. capitata larvae (Hepdurgun et al. 2009) were 

able to find and successfully attack B. oleae under our conditions. Consequently, we 

initiated larger-scale trials involving releases of this parasitoid alone and in 

combination with EcoTraps during 2002 2003. 

In 2002, when mass trapping and P. concolor releases were combined in EA-1, it 

was found that by using a combination of parasitoids and traps, damage was reduced 

to a marked degree, culminating in only 14.1% damage in the core area at harvest vs. 

87.6% damage in the untreated control area. In 2003, comparisons between use of 

only parasitoids vs. parasitoids plus mass trapping showed similar outcomes, with 

only about 10% damage in the core areas of each experimental area vs. 35.5% 

damage in the control area. This indicated that the use of EcoTraps might not be cost 

effective to use along with parasitoid releases. Tests performed in 2004 using only 

parasitoids again indicated that the parasitoid release strategy (without traps) could 

substantially reduce damage, in this case to around 10% vs. about 38% in the control 

area, reaffirming the earlier study. It is generally accepted that almost all alternate 

pest management methods, including mass trapping, depend upon a large experimental 

site (of at least 1000 trees) for success to be achieved. 

Mass-trapping experiments carried out in Greece (Mazomenos et al. 2002) 

showed that high population densities decreased the success of this technique.

Table 3. Effectiveness of combining Psyttalia concolor releases and mass-trapping (MT). 

Parcel Damage (%) 

Experimental Area 1 (Parasitoids MT) 

Core Buffer Neighboring Control 

Date Tree Ground Mean Tree Ground Mean Tree Ground Mean Tree Ground Mean 

2002 05.09 0.5 1.3 2.0 3.4 

18.09 1.1 1.2 1.1 2.1 2.0 2.1 5.0 4.9 4.9 8.0 10.9 9.5 

02.10 1.2 1.5 1.4 2.9 3.3 3.1 6.1 5.1 5.6 12.3 18.7 15.5 

24.10 11.3 14.4 12.9 14.8 25.3 20.0 20.1 26.5 23.3 36.2 44.5 40.4 

Harvest 

20.11 

12.4 15.8 14.1 16.1 28.2 22.1 39.9 48.7 44.3 81.0 94.2 87.6 

218 B. Hepdurgun et al.

Table 4. Efficacy of releasing Psyttalia concolor alone and in combination with EcoTraps (MT). 

DAMAGE (%) 

Experimental Area 1 Parasitoids only Experimental Area 2 (Parasitoids MT) 

Parcel Core Buffer Neighboring Area Core Buffer Control 

Date Tree Ground Mean Tree Ground Mean Tree Ground Mean Tree Ground Mean Tree Ground Mean Tree Ground Mean 

2003 23.09 1.2 1.4 1.3 0.4 0.4 1.8 

07.10 1.5 1.9 1.7 2.0 1.8 1.9 2.3 1.4 1.9 0.7 1.0 0.9 0.7 1.0 0.9 3.8 3.0 3.4 

21.10 4.3 2.2 3.2 5.1 3.2 4.1 6.2 4.6 5.4 1.2 5.0 3.1 1.4 3.8 2.6 7.2 7.4 7.3 

04.11 9.1 10.3 9.7 9.8 8.9 9.3 13.6 10.9 12.3 8.7 10.8 9.7 9.2 10.2 9.7 22.7 28.4 25.6 

Harvest 

19.11 

9.5 11.2 10.4 10.6 11.2 10.9 18.2 19.6 18.9 9.2 10.9 10.1 9.6 10.8 10.2 31.5 39.6 35.5 


Table 5. Efficacy of releasing Psyttalia concolor alone. 

Experimental Area 1 (parasitoid release area) 

Damage (%) 

Parcel Core Buffer Neighboring Control 

Date Tree Ground Mean Tree Ground Mean Tree Ground Mean Tree Ground Mean 

2004 18.08 0.1 0.1 0.1 0.1 0 

07.09 0.8 0.8 1.4 1.1 1.6 1.4 1.8 1.9 1.9 2.2 2.0 2.1 

23.09 1.4 1.2 1.3 1.9 1.7 1.8 2.2 2.6 2.4 5.6 8.2 6.9 

07.10 2.0 2.8 2.4 2.2 3.1 2.7 3.2 5.4 4.3 8.8 11.4 10.1 

20.10 5.2 6.4 5.3 5.8 6.9 6.4 12.3 10.8 11.6 28.0 32.1 30.1 

Harvest 

03.11 

10.1 13.2 11.7 10.6 14.8 12.7 17.8 18.6 18.3 36.4 39.3 37.9 

220 B. Hepdurgun et al.


Accordingly, it was stated that in such circumstances, additional control methods 

would be needed to achieve an acceptable result (Mazomenos et al. 2002). Similarly, 

Liaropoulos, Mavraganis, Broumas, and Ragoussis (2002) performed an EcoTrap 

trial in an olive orchard containing 150 trees, using 1 trap per tree and released a 

total of 17,000 P. concolor pupae (113,333/tree) on two different dates in October 

2002. Despite this, damage rates were 78.4 and 68.1% in control and experimental 

areas, respectively because of the high population density. Because the olive fly 

population density generally is low from early May to late August in our test area, 

the parasitoids released within this period suppressed the olive fly population fairly 

effectively, but still did not achieve the local economic threshold of about 1% for 

table varieties or 6 8% for oil varieties. 

Conclusions drawn from our experiments may be outlined as follows: in 

Gökçeada Island, since the growers do not apply any control measures due to the 

ecological/organic farming techniques undertaken, olive fly populations typically 

exceed the economic threshold, which is considered to be only 1% damage for 

table variety olives and 6 8% for oil production olives. Although the overall results 

obtained were encouraging in experimental areas where either parasitoids and mass 

trapping were used together, or only parasitoid releases were employed, damage rates 

below the generally accepted economic threshold level were not achieved. This 

illustrates the importance of an area-wide application strategy in the control of pests 

like the olive fruit fly, which has high dispersal capabilities and a high reproduction 

potential. Additional biotechnical tools may need to be used to achieve adequate 

control. By way of example, in Hawaii, the related species Psyttalia fletcheri 

(Silvestri) was used along with sterile flies to suppress the melon fly, Bactrocera 

curcurbitae (Coquillet) (McInnis et al. 2004; Vargas et al. 2004). More recently, 

inclusion of sanitation (Klungness et al. 2005), reduced-risk protein bait sprays, and 

male lure treatments also have been integrated into effective IPM systems for fruit 

flies in Hawaii (Vargas et al. 2001; Prokopy et al. 2003; Mau, Jang, and Vargas 2007; 

Vargas, Mau, Jang, Faust, and Wong 2008). GF-120 NF Naturalyte Fruit Fly Bait TM 

(Peck and McQuate 2000; Vargas et al. 2001; Prokopy et al. 2003), and male lures 

have been used on organically certified farms in Hawaii (Vargas et al. 2008). To 

achieve the desired degree of economic control of the olive fly, it may be possible to 

utilize additional tools such as those used synergistically in Hawaii to complement 

use of parasitoids and traps. 


The authors would like to thank the International Atomic Energy Agency, Vienna, Austria for 

their support of the project that is number 10783/TUR and the Vioryl firm (Athens, Greece) to 

support the EcoTraps during the study. 

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Vol. 19, S1, 2009, 225 234 


Effect of early oviposition experience on host acceptance in 

Trichogramma (Hymenoptera: Trichogrammatidae) and application 

of F 1 sterility and T. principium to suppress the potato tuber moth 

(Lepidoptera: Gelechiidae) 

George Saour* 

Atomic Energy Commission (AECS), Department of Biotechnology, P.O. Box 6091, 

Damascus, Syria 

Laboratory experiments with Trichogramma principium Sugonyaev and Sorokina 

females offered potato tuber moth Phthorimaea operculella (Zeller) eggs demonstrated 

that wasps’ rates of oviposition were highest the first day and decreased 

gradually thereafter. In addition, when T. principium females were sequentially 

offered eggs from 250 Gy irradiated parents or obtained from non-irradiated 

moths, the probability of host acceptance was not influenced by treatment of host 

eggs. In a concurrent laboratory study, a large cage test with combinant releases 

of T. principium and 250 Gy irradiated moths produced the greatest reduction in 

potato tuber moth F3-emerged progeny. Reductions obtained with irradiated 

moths alone, single release of irradiated moths with T. principium, and one or 

three releases of parasitoids were significantly higher than those in the control. 

From a pest management perspective, T. principium releases would synergistically 

complement the effects of F1 sterility against potato tuber moth infestation. 

Keywords: Phthorimaea operculella; Trichogramma; F1sterility; pest management; 

nuclear techniques; gamma radiation 

Introduction 

The cultivated potato Solanum tuberosum L. is one of the world’s major food crops. 

Potatoes are widely grown over many latitudes and elevations over a wide range of 

agro-ecological zones (FAO 2008). The potato tuber moth (PTM), Phthorimaea 

operculella (Zeller), has been reported as a pest of major economic importance in 

almost all the potato producing areas in the tropics and subtropics (Sporleder, 

Kroschel, Gutierrez Quispe, and Lagnaoui 2004). Systemic organophosphates, plant 

products or insect growth regulators (IGRs) are widely used to control the PTM in 

both field and unrefrigerated, rural storage (Das 1995; Edomwande, Schoeman, 

Brits, and Van Der Merwe 2000; Symington 2003). 

Releases of a biological control agent such as Trichogramma spp. (an oophagous 

parasitoid) have been used successfully to control various lepidopteran pests and 

will likely continue to be a primary component of lepidopterous management 

programmes (Smith 1996; Saour 2004a). Nevertheless, Trichogramma spp. releases 

may be more effective over a wide range of conditions if they are integrated with 

compatible control methods (i.e., sterile insect technique). Other control options, 

*Email: gsaour@aec.org.sy 



DOI: 10.1080/09583150802522838 


226 G. Saour 

however, are limited by the extreme sensitivity of adult Trichogramma spp. to 

various environmental factors (Fournier and Boivin 2000) or pesticide drift 

(Hassan, Hafes, Degrande, and Herai 1998; Hewa-Kapuge, McDougall, and 

Hoffmann 2003). Accordingly, a previous study in our laboratory demonstrated 

that a single release of T. principium along with irradiated moths over potatoes in 

small Plexiglas † 

boxes was more effective in reducing PTM F1-emerged progeny 

than using T. principium or F1 sterility (or inherited sterility) employed separately 

(Saour 2004b). It is well documented that the success of combining Trichogramma 

spp. and the sterile insect technique to suppress lepidopteran pests can be 

influenced by several biotic and abiotic factors (Bloem, Bloem, and Knight 1998; 

Cossentine and Jensen 2000; Carpenter, Bloem, and Hofmeys 2004). For PTM, 

data have been published on the acceptability and suitability of host eggs from 

irradiated parental crosses for three generalist Trichogramma species (Saour 2004b). 

However, data on wasp oviposition rates, in relation to age, early ovipositional 

experience (the ability to learn to discriminate between host eggs of different 

qualities), and treatment synergistic effects on subsequent generations are a 

necessary prerequisite to achieve efficient control of PTM through combined 

releases of egg parasitoids and sterile moths. 

In the current study, females of Trichogramma principium Sugonyaev & Sorokina 

(arrhenotokous species) were provided with normal or F1 sterile PTM eggs arising 

from 250 Gy irradiated parents to study the effects of repeated exposure of T. 

principium to host eggs on oviposition rates and the early ovipositional experience. 

Furthermore, the effects of single or repeated releases of T. principium combined with 

250 Gy irradiated and non-irradiated moths on PTM F3-emerged progeny in large 

cages under laboratory conditions were determined. 


Potato tuber moth 

Moths used in the experiments were obtained from a laboratory stock culture (45th 

generation), which was renewed each year with a field collection of PTM individuals. 

Larvae were reared at a constant temperature of 25928C with 7095% relative 

humidity (RH) and a photoperiod of 12:12 h (L:D) on wax-coated potato slices as 

delineated by Saour and Makee (1997). 

Trichogramma source 

The T. principium colony used in the experiment originated from the Biological 

Control Laboratory at the Aleppo University, Syria. T. principium was cultured on 

eggs of the Mediterranean flour moth, Ephestia kuehniella Zeller. Rearing of E. 

kuehniella was performed in a controlled climate chamber at 25918C, 7095% RH 

and photoperiod of 16:8 h (L:D). Eggs were obtained by placing adult E. kuehniella 

in a transparent Plexiglas † 

cage (50 25 25 cm) with wire mesh covering the 

bottom side. Eggs fell through the wire mesh into a container and were stored at 5 

28C until use. Further details of the rearing method are outlined in Daumal, Voegele, 

and Brun (1975).


Experimental procedures 

Individuals of T. principium were captured by releasing them onto a large sheet of 

white paper and then inverting 9 1 cm glass tubes over dispersing individuals. 

When the captured specimen moved upward off the paper, the tube was quickly 

turned upright and plugged with cotton wads that contained small droplets of pure 

honey on the inner walls as food medium. Male and female T. principium were then 

separated by sex by examining antennal characteristics under a binocular microscope 

at 15 magnification (Kyowa Optical, Japan). The parasitoids were set aside for 

testing. For subsequent tests, two males also were transferred to each test tube to 

ensure female mating. 

Newly emerged adult moths were irradiated at a dose of 250 Gy in order to 

obtain completely sterile females and partially sterile males (Makee and Saour 2004). 

Irradiated males and females were singly paired in 350 ml transparent plastic boxes 

provided with an oviposition support (filter paper) and source of food (10% sucrose 

solution) and held at a constant temperature of 23918C with 7095% RH, and a 

photoperiod of 12:12 h (L:D). Females and males were kept together until death. 

Eggs were removed daily, counted, and left until used. 

A 60 Co source (Gamma irradiator, model Issledova, Techsnabexport Co. Ltd, 

Moscow, Russia) was used to provide the gamma radiation throughout this study 

at a dose rate of 42 Gy/min95%. The absorbed dose was measured using an 

alcoholic chlorobenzene dosimeter. 

Female oviposition rate 

T. principium parasitization activity, i.e., mean number of daily parasitized eggs per 

female, was evaluated by isolating newly emerged females in glass tubes (9 1 cm) 

and offered 40 50 PTM eggs on a filter paper ( 24 h old) each day until parasitoid 

death. Females were fed honey droplets. The exposed eggs were removed daily and 

placed in a glass vial (4 2 cm). Within 72 h after exposure, eggs were evaluated for 

parasitism (parasitized eggs turned black). Daily parasitism and the percentage of 

cumulative parasitism were determined. Each female that failed to oviposit or 

parasitized fewer than 6 host eggs throughout the entire experimental period was 

discarded. Generally, a T. principium female should parasitize more than 15 PTM 

eggs after 24 h of exposure (Saour 2004a). Experiments were held at 25918C witha 

photoperiod of 16:8 h (L:D) and consisted of four replicates, each consisting of 15 

T. principium females. 

Female early ovipositional experience 

The experiment consisted of two treatments. In the first treatment, 1-day-old 

inexperienced single T. principium females were offered sequentially for 24 h 

unlimited numbers ( 50) of 24-h-old eggs obtained either from 250 Gy irradiated 

parents (less preferred host) or from non-irradiated moths (preferred host). In the 

second treatment, T. principium females were either exposed to 10 preferred eggs or 

10 less preferred, irradiated hosts for 6 h, then afterwards they were allowed 

sequentially to oviposit on 50 preferred or less preferred hosts for 24 h. Viable 

and non-viable PTM eggs appeared visually identical within 24 h of oviposition.

228 G. Saour 

The mean number of parasitized eggs was recorded. In each treatment, females 

werekeptat25918C with a photoperiod of 16:8 h (L:D). All tests were repeated 

so that there were a total of three replications, with 30 females per replicate for 

each test. 

Effect of multiple releases of T. principium and sterile moths on PTM F3-emerged progeny 

Irradiated (250 Gy) and non-irradiated moths with or without T. principium 

females were released in large polypropylene mesh cages (1 1 1.2 m) over 12 

kg of intact potatoes placed on a thin layer of sand. Seven experimental 

treatments involving combinations of normal and irradiated moths were assigned 

to the cages (Table 1). The cages were maintained at a constant temperature of 

25928C and a photoperiod of 16:8 h (L:D). Two days after the release of moths, 

Trichogramma females were introduced into the experimental cages. The releases of 

T. principium and irradiated moths for the subsequent generations were performed 

2 days after the emergence of F1 or F2 moths. Fourteen cages that represent two 

replicates were maintained. The experiment was conducted twice to give a total of 

four replicates in time. The number of F3-emerged adults (expressed as moths per 

non-irradiated P1 female) and the fresh weight of potatoes were recorded for each 

replicate. The corrected losses of potato fresh weight during the experimental 

period were calculated according to Kogan (1986) using the formula: C Ie Fe 

(I0/F0). For this, C corrected loss of potato fresh weight, Ie treatment initial 

weight, I0 control initial weight, Fe treatment final weight, and F0 control 

final weight. 

Table 1. Releases of 250 Gy irradiated and non-irradiated potato tuber moth adults with or 

without Trichogramma principiumfor 2 generations througout the experiment. 

Treatment Initial generation (P1) 1st generation 2nd generation 

1 Uninfested potatoes. 

2 15 pairs of non-irradiated moths. 

3 15 pairs of 250 Gy irradiated 

moths 3 pairs of non-irradiated 

moths. 

4 15 pairs of non-irradiated moths 

60 T. principium females. 



moths 60 


6 15 pairs of non-irradiated moths 




moths 60 


60 T. principium females. 60 T. principium 

females. 

15 pairs of 250 Gy 

irradiated moths 60 

T. principium females.


Analysis of variance (ANOVA) at the 5% level (P]0.05) was carried out to 

evaluate the differences in the means of parasitized eggs for female early 

ovipositional experience, number of F3 emerged moths, and corrected losses in 

tuber fresh weight. Significant ANOVAs were followed by Fisher’s protected least 

significant differences method (PLSD) at a 0.05. Data met the assumptions of 

normality before the analysis. All analyses were performed using StatView 

Statistical Software (version 4.02, Abacus Concepts 1994). 

Results 

Female oviposition rate 

Wasps’oviposition rates were highest the first day and decreased gradually thereafter, 

but females continued to parasitize low numbers of PTM eggs each day until their 

death. However, it appears that T. principium females concentrated their activity 

during the first 5 days after their emergence, with a cumulative parasitization rate of 

75% (Figure 1). 

Eggs parasitized/female day 

25 

20 

15 

10 

5 

0 


Daily parasitism 

Accumulative Parasitism 

1 2 3 4 5 6 7 8 9 10 

Days 

Figure 1. Mean 9 SD daily parasitized host eggs per female wasp and percentage 

accumulative parasitism of Trichogramma principium reared on potao tuber moth eggs at 

258C and 16:8 h (L:D) photoperiod. 

100 

80 

60 

40 

20 

0 

Accumulative parasitism (%)

230 G. Saour 

Table 2. Mean number (9SD) of potato tuber moth parasitized eggs when Trichogramma 

principium single female was sequentially offered two different types of eggs resulting either 

from 250 Gy irradiated parents or obtained from non-irradiated moths at 258C and 16: 8 h 

(L:D) photoperiod. 

Exposure duration (h) No. of exposed eggs Eggs type sequence Parasitized eggs 

1st period 2nd 

period 


period 


period 1st period 2nd period 

24 24 50 50 Irradiated Normal* 15.995.0Aa 13.796.3Aa 

Normal Irradiated 19.295.4Ab 11.594.2Ba 

Irradiated Irradiated 15.395.2Aa 11.796.0Aa 

Normal Normal 19.596.7Ab 12.995.4Ba 

6 24 10 50 Irradiated Normal* 7.694.3Aa 14.992.1Bac 

Normal Irradiated 8.692.2Aa 11.894.1Bb 

Irradiated Irradiated 6.994.1Aa 10.494.0Bb 

Normal Normal 8.492.3Aa 15.193.0Bc 

*Eggs from non-irradiated parents. Means in row for each exposure duration followed by the same 

uppercase letter are not significantly different (P50.05, Fisher LSD); means in column for each exposure 

duration followed by the same lowercase letter are not significantly different (P50.05, Fisher LSD). Mean 

of three replicates, 30 T. principium females per replicate. 

Female early ovipositional experience 

When T. principium females were exposed for 24 h to eggs obtained from 250 Gy 

irradiated or normal parents in the first exposure period, they parasitized similar 

numbers of eggs arising either from irradiated or non-irradiated parents in the 

second exposure period. The observed differences in the mean numbers of 

parasitized eggs in the second exposure were not significant (P 0.11). In contrast, 

parasitoids that foraged for only 6 h in eggs from either irradiated or non-irradiated 

moths during the first exposure period significantly (P 0.05) parasitized more eggs 

arising from non-irradiated moths during the second exposure period, i.e., they 

appeared to gain experience and preferred normal eggs during the second period. 

The mean number of parasitized eggs was not influenced by the irradiated parental 

crosses after 6 h of exposure and the differences were not significant (P 0.52). As 

expected, T. principium females significantly preferred eggs from non-irradiated 

parents compared to eggs from 250Gy irradiated moths during 24 h of exposure 

period (P 0.01) (Table 2). 

Effect of multiple releases of T. principium and sterile moths on PTM F3-emerged progeny 

In general, the mean number (97.8) of PTM F3-emerged progeny in the control 

treatment differed significantly versus all other treatments (F 121.4; df 5, 18) (Table 

3). However, the multiple releases of parasitoids reduced by 78% the number of F3emerged 

moths compared to the single release of T. principium. A low emergence of 

moths per non-irradiated P1 female (0.2) was obtained after two releases of T. 

principium and 250 Gy irradiated and non-irradiated moths at a 5:1 ratio. The mean 

number of F3 adults that were obtained after a single release of T. principium and 

irradiated moths (4.1) was not significantly different (P 0.42) than that from two

Table 3. Mean total number (9SD) of potato tuber moth F3-emerged progeny and mean 

losses in tubers fresh weight resulted from one or multiple releases of 250 Gy irradiated and 

non-irradiated moths with or without Trichogramma principium over 12 kg of intact tubers 

placed inside large polypropylene mesh cages. The introduction of T. principium was done 2 

days after the initial moths release or the emergence of F 1 and F 2 adults, at 258C and 16: 8 h (L: 

D) photoperiod. 

Treatments 


No. of F3-emerged moths 

per-non-irradiated 

P 1 female 

Corrected losses in tubers 

fresh weight (g/non-irradiated 

P 1 female) 

1- Uninfested potatoes. 

2- 15 pairs of non-irradiated moths. 97.898.6a 431.0939.8a 

3- 15 pairs of 250 Gy irradiated moths 

of non-irradiated moths. 

3 pairs 28.7912.8b 225.8956.9b 

4- 15 pairs of non-irradiated moths 


60 

29.993.2b 109.3930.2c 

5-15 pairs of 250 Gy irradiated moths 3 pairs 

4.193.0c 58.7922.8cd 

of non-irradiated moths 

females. 

60 T. principium 

6- 15 pairs of non-irradiated moths 


3 releases of 6.691.9c 49.3921.8cd 

7- 15 pairs of 250 Gy irradiated moths 3 pairs 0.290.1c 17.599.6d 

of non-irradiated moths 

T. principium). 

females (2 releases 60 

Means within each column followed by the same letter are not significantly different (PB0.05, Fisher 

LSD). Mean of four replicates. 

releases of T. principium and irradiated moths. Significant losses in potato fresh 

weight per non-irradiated P 1 female occurred among the treatments (F 180; df 5, 

18); however, the corrected loss in tuber weight was determined by the intensity of 

infestation and relative effects of the different treatments (Table 3). 

Discussion 

Trichogramma species are considered to be pro-ovigenic, or partially synovigenic 

(Pak and Oatman 1982; Volkoff and Daumal 1994). In this study, we found that T. 

principium females laid a significant number of their eggs shortly after emergence, 

but it also seemed that the parasitization was distributed over 10 days. The 

propensity for T. principium females to lay eggs soon after emergence and the 

observation that females continued oviposition up to their death are in agreement 

with the results reported for T. principium offered Sitotroga cerealella Olivier 

(Lepidoptera: Gelechiidae) eggs (Reznik, Voinovich, and Umarova 2001) or with 

other trichogrammatids (Leatemia, Laing, and Corrigan 1995; Consoli and Parra 

1996). 

Several studies have assessed Trichogramma spp. early oviposition experience and 

its consequences on host acceptance (Reznik, Umarova, and Voinovich 1997; Keasar, 

Ney-Nifle, Mangel, and Swezey 2001). High- and low-quality hosts were indispensable 

to carry out such an experiment. Usually, fresh hosts are considered to be 

good (in the sense that Trichogramma spp. favors them) and older hosts to be bad. In 

our experiment, two different types of PTM eggs were obtained from the crosses

232 G. Saour 

between 250 Gy irradiated or non-irradiated parents. The results of the present study 

showed that T. principium females tend to maintain parasitization even when 

sequentially exposed to different types of 1-day-old host eggs. This finding 

corroborates results that T. principium parasitization behaviour was stable when 

young (preferred) and old (less preferred) grain moth eggs S. cerealella were offered 

in sequence (Reznik and Umarova 1991; Reznik et al. 1997). 

Theoretically, under release conditions involving irradiated moths and parasitoids, 

Trichogramma spp. females may randomly contact fertile and sterile eggs. 

However, under field or storage conditions, higher parasitization is expected in sterile 

eggs resulting from irradiated parents than in wild eggs. This may be due to the fact 

that fertile eggs continue to develop and are therefore acceptable for Trichogramma 

spp. oviposition for a shorter period. For this reason, using sterile host eggs could 

enhance natural T. principium population build-up. 

F1 sterility (also known as inherited-partial sterility) and Trichogramma spp. have 

been developed and separately field-tested against lepidopterous pests. However, 

combinations of parasitoid and sterile insect releases have been evaluated for 

suppression of Ceratitis capitata (Wiedemann) and Bactrocera cucurbitae (Coquillett) 

(Diptera: Tephritidae) populations in Hawaii (Wong, Ramadan, Herr, and 

McInnis 1992; Vargas et al. 2004). Data reported by Mannion, Carpenter, and Gross 

(1994, 1995) suggest that the use of F1 sterility and the tachinid parasitoid Archytas 

marmoratus (Townsend) (Diptera: Tachinidae), are compatible strategies for managing 

early-season population of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae). 

Accordingly, Bloem et al. (1998), Cossentine and Jensen (2000) and Saour (2004b) 

demonstrated that the combined use of egg parasitoids and F 1 sterility was very 

efficient as a control method against the codling moth Cydia pomonella (L.) and the 

PTM, respectively. 

Based on our cage studies, the data substantiate this potential and suggest that a 

single release of irradiated and non-irradiated moths at a 5:1 over-flooding ratio 

(treatment no. 3), or T. principium females and non-irradiated PTM pairs at 4:1 ratio 

would have detrimental effects on PTM subsequent generations (reductions of 70.7 

and 69.4%, respectively). The combination of T. principium and 250 Gy irradiated 

moths produced the greatest numerical reduction in PTM F3 emergence from tubers, 

particularly when two properly timed releases (2 3 days after moth release or 

emergence) were performed. Moreover, the larval mining of the tubers reduced the 

weight and quality of the potatoes, but when T. principium and irradiated moths were 

released, the potatoes did not suffer feeding damage. Tuber weight loss was only 13.6 

and 4.1%, respectively, when T. principium and irradiated moths were used together 

on a single or repetitive basis (treatments 5 and 7, Table 1). 

The data presented provide positive evidence regarding the synergistic effect 

resulting from combinations between egg parasitoids and sterile moth releases. 

Nonetheless, the greatest likelihood for crosses occurring in the field under area-wide 

releases of sterile moths would be irradiated males irradiated females and normal 

males normal females. Of course, because F1 eggs from the irradiated males wild 

females possess the potential of inherited sterility (based upon radiation-induced 

deleterious effects passed on to F1 generation), it would be advantageous if T. 

principium females seek out and preferentially parasitize fertile eggs produced by 

feral moths rather than the F1 eggs present in the treated areas, which are crucial to 

sustain the F1 population for sterility promotion and subsequent population


collapse. However, our findings should be further tested in order to determine 

whether PTM sterile moths and T. principium releases will be more cost-effective in 

unrefrigerated storage potatoes. 


I thank Dr. I. Othman (General Director) and Dr. N. Sharabi for their help and support. 

Technical and financial assistances were provided, in part, by contract no. 10781 of the 

International Atomic Energy Agency, Joint FAO/IAEA Division of Nuclear Techniques in 

Food and Agriculture, Vienna, Austria. 

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Vol. 19, S1, 2009, 235 242 


Evaluating the use of nuclear techniques for colonization and production 

of Trichogramma chilonis in combination with releasing irradiated moths 

for control of cotton bollworm, Helicoverpa armigera 

Endong Wang a , Daguang Lu c *, Xiaohui Liu b , and Yongjun Li b 

a China Agricultural University, Beijing 100193, China; b Institute of Plant Protection, 

Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, China; c Department 

of International Cooperation, Chinese Academy of Agricultural Sciences (CAAS), 

Beijing 100081, China 

Gamma radiation was tested as a means of increasing production of the egg 

parasitoid Trichogramma chilonis Ishii by improving the suitability of host eggs 

and by stimulating reproduction of the parasitoid females. For manipulation of 

the host eggs’ suitability, radiation was used to either (a) produce developmentally-inactivated 

(DI) eggs incapable of hatching, or (b) to produce F1 sterile host 

eggs. For treatment of the parasitoid females with the intent of stimulating 

reproduction, parasitoid pupae were exposed to very low dose radiation (250 

mGray). For tests on host suitability using radiation-induced DI host eggs, newlylaid 

(B8 h old) host eggs (Helicoverpa armigera Hubner) were exposed to 300 Gy 

of 60 Co gamma radiation. For tests of F1 sterile host eggs, H. armigera moths 

were mated with individuals exposed to 250 Gy as pupae. Tests were performed 

with eggs resulting from all possibilities of normal (N) and sterile (S) à ß 

matings. Both types of DI host eggs (irradiated or sterile), along with untreated 

host eggs (controls), were exposed to T. chilonis females, using the following host 

egg-to-parasitoid ratios: 1:10, 1:30, 1:60 and 1:90. Developmentally-inactivated 

host eggs exposed to 300 Gy did not differ in suitability from normal host eggs at 

a 1:10 parasitoid host ratio, but were significantly more suitable at the higher 

host parasitoid ratios. F1 sterile eggs were not significantly different in suitability 

from normal eggs at a 1:10 host parasitoid ratio but were marginally better at the 

higher host parasitoid ratios. In tests performed using T. chilonis females exposed 

to low-dose radiation (250 mGy), no effects were observed when cohorts of 5 T. 

chilonis females were provided with only 50 host eggs, but when more hosts were 

provided (ratios of 1:30, 1:60 and 1:90), significantly higher rates of parasitization 

were noted for the parasitoids exposed to low-dose radiation. This effect prevailed 

using both normal host eggs and DI host eggs exposed to 300 Gy. The stimulatory 

effect also was noted when F1 sterile host eggs were provided to the irradiated T. 

chilonis females. These results suggest that release of T. chilonis irradiated with 

250 mGy may complement release of irradiated H. armigera moths, which 

produce sterile F1 eggs that can serve as supplemental hosts in the field and 

thereby enhance the pest management system. 

Keywords: Trichogramma; Helicoverpa; biological control; pest management; 

parasitoids; irradiation; low dose irradiation; radiation hormesis 

*Corresponding author. Email: daguang_lu@caas.net.cn 




DOI: 10.1080/09583150902790293 


236 E. Wang et al. 

Introduction 

Releases of insect natural enemies along with irradiated, sterile pest insect eggs 

were evaluated to achieve improved control of the cotton bollworm, Helicoverpa 

armigera Hubner. The irradiated, sterile pest eggs were intended to serve as 

innocuous supplemental hosts for use as a field insectary or to sustain parasitoid 

populations at times of low host populations. Studies by Saour (2004) on the 

potato tuber moth, Phthorimaea operculella (Zeller) showed that release of 

irradiated moths along with three Trichogramma spp. were complementary and 

provided an integrated control approach by using inherited sterility in conjunction 

with these Trichogramma spp. for P. operculella suppression. One way in which 

radiation might be helpful for egg parasitoids such as Trichogramma spp. would be 

to inhibit host egg development beyond a point of optimal suitability. Tunçbilek 

and Ayvaz (2003) and Pak (1986) cite a number of studies in which host age was 

shown to be important for acceptance by egg parasitoids. In studies on the 

influence of host age on parasitism by Trichogramma evanescens Westwood, 

Tunçbilek and Ayvaz (2003) found that newly-laid Ephestia kuhniella Zeller eggs 

were preferred as compared with older eggs. Tunçbilek, Canpolat, and Ayvaz 

(2009) found that eggs of E. kuehniella and Sitotroga cerealella (Olivier) irradiated 

with 200 Gy gamma radiation and exposed to T. evanescens for 24 h were suitable 

for parasitoid development. 

Harwalkar, Rananavare, and Rahaikar (1987) found that Trichogramma 

brasiliense could be successfully reared on radiation-sterilized potato tubermoth 

(Phthorimaea operculella Zeller) eggs and that even after rearing 10 generations of 

the parasitoid on such eggs, no adverse effects were evident. Brower (1982) also 

found that successful parasitization by T. pretiosum Riley on Plodia interpunctella 

(Hübner) eggs could be increased by exposing the eggs at 350 Gy (but not to 500 or 

1000 Gy). He also found that eggs from P. interpunctella adults irradiated at 150 Gy, 

as might be used to achieve F1 sterility, were successfully parasitized at the same rate 

as control eggs. Thus, it appeared that if the eggs are to be used strictly for 

parasitoid production, the better option would be to irradiate the eggs directly 

rather than to irradiate the adult moths, but eggs from moths exposed to up to 150 

Gy to achieve inherited sterility would still be suitable for parasitoids in the field. 

Cossentine and Jensen (2000) also reported that sterile eggs from released, irradiated 

Cydia pomonella (L.) were successfully parasitized by Trichogramma platneri 

Nagarkatti, and these eggs could be used to sustain a population of T. platneri at 

times of low C. pomonella density. These papers indicated that either irradiated 

insect eggs or eggs from irradiated moths may be at least equally acceptable for 

parasitization by Trichogramma spp. as compared with normal eggs. Liu and Chen 

(1983) reported that embryos of eggs from 300 Gy irradiated male and normal 

female adults did not finish development, and were developmentally suspended with 

abundant yolk at this stage egg, resulting in Trichogramma spp. finding irradiated 

eggs suitable for parasitization beyond the point of acceptability for non-irradiated 

eggs. 

Another topic of interest is the possible use of low-dose radiation to stimulate 

reproduction by parasitoids. The phenomenon known as radiation hormesis (Luckey 

1991) refers to the stimulatory effect of very low-dose radiation on biological 

processes. It often refers to accelerated growth of plants due to low-dose radiation, but


it also has been found to influence biological processes in insects. For example, studies 

by Yusifov, Kuzin, Agaev, and Alieva (1990) showed that low doses of ionizing 

radiation (100 4000 times exceeding natural background radiation, or about 2 Gy) 

stimulated embryogenesis in the silkworm, Bombyx mori L. Stimulatory effects on 

growth of B. mori larvae also were found by Abdel-Salam and Mahmoud (1995) in 

response to low levels of gamma radiation (0.01 1 Gy, with the greatest effects at 

1 Gy). 

The goals of the present study were to assess the influence of irradiation of H. 

armigera eggs and eggs resulting from irradiated pupae on their acceptability and 

suitability for parasitization by Trichogramma chilonis (Ishii). We also sought to 

evaluate the influence of very low dose radiation of the parasitoids themselves on 

their parasitization potential in normal H. armigera eggs and in eggs resulting from 

moths irradiated as pupae. 

Methods and materials 

Colonies 

The strain of T. chilonis used in these tests was obtained from the Institute of Plant 

Protection, Beijing Academy of Agricultural Sciences, where it was reared for many 

generations on eggs of Antheraea pernyi Guer. The strain of H. armigera maintained 

in our laboratory was collected from a cotton field in Gaoyang county, Hebei 

province in July, 2002. Tests were performed at 28928C, 6595% RH with a 14 h 

L:10 h D photoperiod regimen. 

Test methods 

Testing irradiated Helicoverpa armigera eggs and F1 sterile eggs for suitability for 


Newly-laid (B8 h old) eggs oviposited by normal mated H. armigera moths were 

collected on napkin paper (‘egg-paper’) and irradiated at a dose of 300 Gy, using a 

60 

Co gamma radiation source located at the Institute for Application of Atomic 

Energy, CAAS, producing a dose rate of 3.14 Gy/min. These ‘egg-papers’ were 

placed in glass tubes and 5 T. chilonis female adults were transferred into these tubes 

and held together for 24 h at a T. chilonis female-to-host egg ratio of 1:10, 1:30, 1:60 

or 1:90 (viz. 5:50, 5:150, 5:300 or 5:450 for no. of T. chilonis to irradiated host eggs, 

respectively), using irradiated host eggs. Control (unirradiated, newly-laid) eggs also 

were exposed in the same fashion and at the same parasitoid-to-host egg ratios. The 

experiment was repeated 5 times. 

For tests involving exposure of F1 eggs from irradiated pupae, the day before 

expected emergence, H. armigera pupae were irradiated using 250 Gy. After 

emergence, treated females (Sà); treated males (Sß); normal, untreated females 

(Nà); and normal, untreated males (Nß) were confined in mating and oviposition 

cages in the following combinations: Nà Nß, Nà Sß, Sà Nß, and Sà Sß. 

The ‘egg-papers’ were like those used for normal H. armigera moths described above 

but only F1 eggs from these crosses were used. The exposure ratios for T. chilonis 

females to H. armigera eggs were the same as above for 1:10, 1:30, 1:60 and 1:90. The 

experiment was repeated 5 times.


Low-dose irradiation of Trichogramma chilonis adults 

For tests on the influence of low dose radiation on T. chilonis reproductive potential, 

H. armigera eggs parasitized by T. chilonis were irradiated when the parasitoids were 

in the pupal stage. We used a dose of only 250 mGy, with a dose rate of only 38.7 

mGy per min to minimize potential damage to the parasitoids. After emergence, 

irradiated T. chilonis females and normal T. chilonis were given an opportunity to 

parasitize normal H. armigera eggs and host eggs exposed to 300 Gy, using the same 

type of oviposition chamber as above. The ratios of T. chilonis females to H. armigera 

eggs employed also were 1:10, 1:30, 1:60 and 1:90. The experiment was repeated 5 

times. 


The data on the parasitization rates of T. chilonis on H. armigera eggs were analyzed 

using an independent sample t-test. The remaining data were analyzed using one-way 

ANOVA followed by a Duncan’s test; these data were checked by homogeneity of 

variance test analysis. All analyses were carried out using SPSS (SPSS Inc. 2004). 

Rejection level was set when P 0.05. 

Results 

The influence of irradiation of Helicoverpa armigera eggs and F1 sterile eggs on 

parasitizing potential of Trichogramma chilonis parasitoids 

The parasitization rate by T. chilonis on 300 Gy irradiated H. armigera eggs was 

significantly higher than that on normal eggs at the following ratios of T. chilonis 

female adults to H. armigera eggs: 1:30, 1:60 and 1:90, but not at a ratio of 1:10 

(Table 1). This suggests that it may be possible to increase the opportunity of T. 

chilonis parasitoids by using host eggs previously exposed to 300 Gy irradiation 

when ratios of at least 30 host eggs per parasitoid female are used. 

When normal and irradiated H. armigera males and females were crossed and the 

resulting eggs were exposed to T. chilonis females, no significant differences in 

parasitization rates were noted among the crosses when only a 1:10 ratio of 

parasitoid to host eggs were tested (F 0.370, df 3, P 0.776) (Table 2). However, 

as the egg-to-parasitoid ratio increased, differences in parasitization rates were 

observed among the crosses. Notably, the parasitization rate at ratios of 1:30 (F 

10.177, df 3, P 0.001) and 1:60 (F 9.346, df 3, P 0.001) was highest for eggs 

Table 1. The parasitization rate (%) of Trichogramma chilonis on normal and 300 Gy 

irradiated Helicoverpa armigera eggs. 

No. T. chilonis female adults: H. armigera eggs 

Type of H. armigera eggs 1:10 1:30 1:60 1:90 

Control 51.697.3a 18.091.6a 9.590.8a 6.190.3a 

300 Gy 52.894.2a 43.294.6b 31.591.8b 22.890.7b 

Note: Means (9SE) followed by different letters in the same column are significantly different (t-test with 

significance level 0.05).


Table 2. The parasitization rate (%) of Trichogramma chilonis on normal Helicoverpa 

armigera eggs and eggs resulting from 250 Gy irradiated H. armigera adults mated with 

normal adults. 

No. T. chilonis female adults/H.armigera eggs 

Cross 1:10 1:30 1:60 1:90 

Nà Nß 55.694.7a 17.691.3a 8.790.4a 6.290.4a 

Nà Sß 53.693.4a 24.391.2b 21.593.5b 10.990.4c 

Sà Nß 49.694.4a 19.390.2a 13.390.4a 12.690.7d 

Sà Sß 52.094.15a 18.790.37a 12.690.3a 9.290.4b 

Note: Means (9SE) followed by different letters in the same column are significantly different (Multiple 

range tests: Duncan test with significance level 0.05). 

from Nà Sß relative to the other treatments, but the ratio of 1:90 (F 31.107, df 

3, P 0.000) was highest for eggs from Sà Nß relative to the other treatments. 

Influence of low dose radiation of Trichogramma chilonis on their reproductive 

potential in normal and F1 sterile Helicoverpa armigera eggs 

The parasitization rate for 250 mGy-irradiated T. chilonis females on 300 Gy 

irradiated H. armigera eggs was significantly greater than that on normal eggs for all 

ratios except 1:10 and 1:60 (Table 3). As in the tests shown in Table 1, this also 

suggests that it may be possible to increase the opportunity of T. chilonis parasitoids 

by using 300 Gy irradiated H. armigera eggs when ratios of T. chilonis female adults 

to H. armigera eggs of 1:30 or 1:90 are used. 

Irradiation of both the parasitoids (with 250 mGy) and their hosts (with 300 Gy) 

positively influenced the parasitization rate at all host parasitoid ratios (Table 4), 

suggesting that low dose radiation may be stimulating the reproductive potential of T. 

chilonis females while they are in the pupal stage. Tests involving parasitoids exposed 

to low dose radiation showed that they also produced significantly higher parasitization 

rates than from control host eggs when provided with F1 sterile eggs resulting 

from Nà Sß (Table 5), just as in the previous tests with normal, non-irradiated T. 

chilonis females (Table 2). This occurred at all host parasitoid ratios above 1:10. 

When parasitoids exposed to low dose radiation were provided with normal host 

eggs vs. eggs from F1 sterile moths, especially from the Nà Sß cross, they generally 

exhibited a higher parasitization rate than the normal, non-irradiated parasitoids 

Table 3. The parasitization rate (%) of 250 mGy low dose irradiated Trichogramma chilonis 

on normal and 300 Gy irradiated Helicoverpa armigera eggs. 

No. T. chilonis female adults/H. armigera eggs 

Type of H. armigera eggs 1:10 1:30 1:60 1:90 

Control 72.094.3a 55.293.9a 47.795.6a 27.291.2a 

300Gy 69.693.2a 78.391.8b 57.295.2a 39.991.4b 

Note: Means (9SE) followed by different letters in the same column are significantly different (t-test with 

significance level 0.05).



on normal and 300 Gy irradiated Helicoverpa armigera eggs. 


Type of H. 

armigera eggs Type of T. chilonis 1:10 1:30 1:60 1:90 

Control Control 51.697.3a 18.091.6a 9.590.8a 6.190.3a 

250 mGy 72.094.2b 55.293.9b 47.795.6b 27.291.0b 

300 Gy Control 52.894.2a 43.294.6a 31.591.8a 22.790.3a 

250 mGy 69.693.2b 78.391.8b 57.295.2b 39.991.4b 

Note: Means (9SE) followed by different letters in the same column for a given host egg type are 

significantly different (t-test with significance level 0.05). 

(Table 6). This was most evident at the host parasitoid ratio of 1:30 and 1:90 for all 

crosses. 

Discussion 

Control of H. armigera by augmentative releases of T. chilonis combined with release 

of 250 Gy irradiated moths to produce sterile F1 progeny might be a promising 

strategy to cope with this pest, which has been a serious problem in recent years in 

China. This methodology must prove cost-effective as well as sustainable and 

environmentally-friendly. The studies we report indicate that irradiated H. armigera 

eggs can be used successfully as hosts for T. chilonis in the insectary, and eggs from 

F1 sterile moths may indeed prove useful as supplemental hosts to maintain and 

increase the feral population of T. chilonis. These findings are in keeping with the 

uses of nuclear techniques in biological control proposed by Wang and Wang (2003). 

We found that irradiation can be useful in three ways: (1) by decreasing loss of 

suitability of H. armigera eggs as hosts for T. chilonis beyond the first day after 

oviposition by exposing them to 300 Gy of radiation to inhibit embryonic 

development; (2) by producing F1 sterile eggs from H. armigera moths exposed to 


on normal Helicoverpa armigera eggs and eggs resulting from 250 Gy irradiated H. armigera 

adults mated with normal adults. 


Cross 1:10 1:30 1:60 1:90 

Nà Nß 50.895.1a 21.691.0a 19.591.6a 16.490.4ab 

Nà Sß 47.293.5a 41.793.2b 24.691.8b 17.890.6b 

Sà Nß 40.493.3a 41.394.9b 22.190.8ab 15.890.8a 

Sà Sß 44.092.5a 36.593.3b 20.190.0a 15.290.4a 

F 1.424 7.601 2.951 3.876 

df 3 3 3 3 

P 0.273 0.002 0.064 0.029 

Note: Means (9SE) followed by different letters in the same column are significantly different (Multiple 

range tests: Duncan test with significance level 0.05).


Table 6. The parasitization rate (%) of normal and 250 mGy low dose irradiated 

Trichogramma chilonis on eggs resulting from 250 Gy irradiated Helicoverpa armigera adults 

mated with normal adults. 


Cross Type of T. chilonis 1:10 1:30 1:60 1:90 

Nà Nß Control 49.293.2a 17.691.3a 8.790.4a 6.290.4a 

250 mGy 50.895.1a 21.691.0b 19.591.6b 16.490.4b 

Nà Sß Control 48.093.2a 24.391.2a 21.593.47a 10.990.4a 

250 mGy 47.293.5a 41.793.2b 24.691.8a 17.890.6b 

Sà Nß Control 41.694.7a 19.390.2a 13.390.4a 12.690.7a 

250 mGy 40.493.3a 41.394.9b 22.190.8b 15.890.8b 

Sà Sß Control 46.094.2a 18.790.4a 12.690.3a 9.290.4a 

250 mGy 44.092.5a 36.593.3b 20.191.0b 15.1290.4b 

Note: Means (9SE) followed by different letters in the same column for a given cross are significantly 

different (t-test with significance level 0.05). 

250 Gy radiation, and by showing that their eggs are highly suitable for oviposition 

by T. chilonis females; and (3) by stimulating the reproductive potential of T. chilonis 

females exposed to a very low dose (250 mGy) radiation while they are in the pupal 

stage. 

In this study, only the parasitization rates of T. chilonis on H. armigera eggs were 

analyzed. There is a critical need to evaluate the fitness of parasitoids emerging from 

irradiated eggs as well as the sex allocation of offspring from irradiated female 

parasitoids. There also is a critical need to field test these findings. These results 

support the possibility that radiation can be an important tool in developing an 

improved rearing and release capability and thereby an improved pest management 

system for use of Trichogramma chilonis and F1 steriles against Helicoverpa armigera. 


Thanks are due to Dr Patrick Greany and Mr Wang Huesong for their help and support. 

Technical and financial assistances were provided, in part, by contract no. 10778 of the 

International Atomic Energy Agency, Joint FAO/IAEA Division of Nuclear Techniques in 

Food and Agriculture, Vienna, Austria. 

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Vol. 19, S1, 2009, 243 259 


Effects of host density, host age, temperature and gamma irradiation on 

the mass production of Nesolynx thymus (Hymenoptera: Eulophidae), an 

endoparasitoid of Uzi fly, Exorista sorbillans (Diptera: Tachinidae) 

Md Mahbub Hasan*, Md Rayhan Uddin, Md Ataur Rahman Khan, and 

Aminuzzaman Md Saleh Reza 

Department of Zoology, Rajshahi University, Rajshahi 6205, Bangladesh 

The Uzifly, Exorista sorbillans Weidemann, is an endoparasitoid of the the 

silkworm moth, Bombyx mori L., and can impact commercial sericulture. Effects 

of Uzifly age, density, temperature and gamma radiation ( 60 Co) on the massproduction 

of the hyperparasitoid Nysolynx thymus (Girault) were investigated. 

There was a direct relationship between progeny production and an increase in 

number of host puparia presented, and a significant decline in the number of 

progeny with increased host-age. Maximum progeny production was obtained by 

maintaining a 1:5 to 1:8 parasitoid-to-host ratio for all the age groups. Two to 4day-old 

host puparia were most suitable for obtaining the maximum progeny 

production of N. thymus for all the host-densities. The intrinsic rate of increase 

(r m day 1 ) increased with the increased host density for all the host-age groups. 

The values of net reproductive rate (R0) and gross reproductive rate (GRR) 

increased for all the density levels and host age groups. On the other hand, the 

values for the doubling time (D) and finite capacity of increase (l) gradually 

decreased with increased host density for all the host age groups. Host density and 

host-age significantly influenced the sex-ratio of progeny in N. thymus. Higher 

proportions of females were observed at higher host density levels and for 

younger host age groups. The progeny production and sex ratio of the parasitoid 

varied significantly with temperature. The maximum mean number of progeny 

was recorded at 258C, while the minimum was at 308C. The trend of progeny 

production at different temperatures was on the order 25 20 308C. The highest 

values for the net reproductive rate (R0) and GRR for the progeny production 

were recorded at 258C compared to 20 and 308C. Both the values for doubling 

time of capacity (D) and finite capacity (l) increased with the trend of 25, 30 and 

208C. The highest value for the intrinsic rate of increase (rm day 1 ) of progeny 

production was recorded at 258C, while the lowest value was at 208C. The sexratios 

were always female-biased at all the temperatures. Temperature had a 

significant effect on the longevity of adult N. thymus. The longevity of the adults 

decreased with an increase of temperature for both sexes. The highest rate of 

parasitism was observed at 208C followed by 25 and 308C. More than 95% 

parasitism was observed at all temperatures. Gamma irradiation significantly 

increased the progeny production of N. thymus when reared either on early or late 

irradiated host puparia, particularly in the parental generation, but irradiated 

early host pupae were more suitable for mass production of N. thymus than the 

irradiated late pupae. The sex ratio of parasitoids developing from gamma 

irradiated host pupae varied significantly. Higher proportions of females were 

observed for all the dose and host-age groups. 

*Corresponding author. Email: mmhbgd@yahoo.com 




DOI: 10.1080/09583150902790319 


244 M.M. Hasan et al. 

Keywords: host density; host age; temperature, gamma irradiation; mass 

production; parasitoid; Nesolynx thymus; Exorista sorbillans; Bombyx mori 

Introduction 

The number of parasitoids inoculated into a system, the synchrony of the parasitoid 

and its host, and the generation time of the parasitoid relative to that of the host are 

key criteria affecting the likely success of the parasitoid as a classical biological 

control agent (Barlow, Goldson, and McHeill 1994). Nesolynx thymus (Girault) is a 

gregarious pupal parasitoid of the Uzifly, Exorista sorbillans Weidemann, a tachinid 

endoparasitoid of silkworm, Bombyx mori, and is globally known as Uzi. The Uzifly 

causes economic injury to the cocoon crop in silkworm cultivating areas of India, 

except those above 400 m above mean sea level (AMSL) in the foothills of the 

Himalayas (Darjeeling). Among the several hymenopteran parasitoids of the Uzifly, 

N. thymus was found to have the best characteristics as a potential control agent 

(Kumar, Kishore, Jayaprakas, and Sengupta 1991). 

The physiological interactions between parasitoids and their hosts are not only 

complex but each association appears to be unique (Vinson and Iwantsch 1980). 

However, for optimal mass-production of any natural enemy, it is essential to 

standardize the host’s age. Hymenopteran parasitoids are known to increase progeny 

production in response to rising host density (Legner 1967). The number of hosts 

parasitized per unit of time depends upon on the ability of the individual parasitoids 

to locate and parasitize a varying number of hosts. A number of parasitoids respond 

by increasing the number of hosts that each individual destroys (functional response) 

or respond to increased host density by increasing their own numbers (numerical 

response) (Solomon 1949). 

Fecundity, the total number of laid eggs, and fertility, the number of viable 

progeny, are variable features of an insect, influenced by a plethora of intrinsic and 

extrinsic factors (Panagiotis, Eliopoulos, and Stathas 2005). The evaluation of a 

natural enemy as a biological control agent requires a thorough study of the main 

effects and possible interactions of such factors on these characteristics (Jervis and 

Copland 1996). In the case of endoparasitoids, however, fecundity is relatively 

difficult to measure, because one or more eggs are laid inside the bodies of multiple 

hosts. Moreover, fertility is a more reliable criterion for evaluation, representing the 

net number of progeny after elimination of individuals that fail to complete 

development (Jervis and Copland 1996). Thus, it is pragmatic to study fertility 

rather than fecundity in endoparasitoids. 

Various developmental processes in insects are influenced by physical factors 

such as temperature (Beck 1980, 1983; Saunders 1982; Denlinger 1985; Ratte 1985). 

Progeny production and adult longevity are two of the major factors influencing the 

abundance of an insect and its population dynamics. Both have been studied in 

relation to temperature for many species of insects (Andrewartha and Birch 1954; 

Syme 1975, 1977; Harrison, King, and Ouzts 1985; Tingle and Copland 1988). 

Nuclear techniques, such as the use of ionizing radiation, could play a prime role 

in augmentative biological control, especially in facilitating the mass rearing of 

insects (Greany and Carpenter 2000). Several potential uses of nuclear techniques 

have been identified by researchers (Ramadan and Wong 1989; Sivinski and Smittle 

1990; Greany and Carpenter 2000), including: (1) improvements in rearing media,


(2) provision of sterilized natural prey to be used as food during shipment, to 

ameliorate concerns relating to the incidental presence of ‘hitchhiking’ pests, (3) 

provision of supplemental food or hosts in the field, to increase the initial survival 

and buildup of released natural enemies, and (4) reproductive sterilization of weedfeeding 

insects that are candidates for biological control, for use in open field trials. 

The objective of the present study was to determine the effect of host age, density 

and temperature on the mass-production of N. thymus prior to successful 

implementation of biological control. The present study also was designed to 

determine the effects of gamma irradiation on the host for mass-production of 

N. thymus to determine the potential value of nuclear techniques in improved 

biological control. 


Effect of host density and age 

Adults of E. sorbillans and N. thymus were collected from the sericultural area in the 

northern region of Bangladesh. They were maintained as stock cultures in the 

Department of Zoology, Rajshahi University. To determine the effect of host-age and 

density on the progeny production of N. thymus, a single male-female pair of 1-dayold 

parasitoids was provided with a single host puparium of either 1, 2, 3, 4, 5 or 6 

days of age in separate glass vials (4 mL). Similarly, cohorts with 1:1, 1:2, 1:3, 1:4, 

1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 parasitoid to host ratios were maintained separately with 

1 2-day-old mated female parasitoids to observe the effect of host density on the 

progeny production of N. thymus. There were 10 observations for each ratio. The 

adult parasitoids were fed on 50% aqueous honey solution. The gender of progeny 

that emerged from the different ages and densities of parasitized puparia was 

determined. The rates of parasitism also were recorded. 

Effect of temperature on adult parasitoid 

Both the Uzifly host pupae and N. thymus were collected from the stock culture. A 

single pair of 1-day-old parasitoids was provided with ten 2-day-old host pupae in 

glass vials. The adult parasitoids were fed on 50% aqueous honey solution. They 

were kept separately in an incubator set at 20, 25 or 308C to observe the effect of 

temperature on progeny production. Parasitoid progeny were counted and separated 

by sex. The longevity of adult parasitoids and the rate of parasitism also were 

recorded. Each temperature experiment consisted of 20 observations. Multivariate 

statistics were used to determine the significance levels of the parameters considered 

in these experiments. 

Effect of irradiated host 

In this investigation, both the Uzi pupae and adult N. thymus were collected from the 

stock culture. To assess the potential value of nuclear techniques in improving host 

suitability, two cohorts of early (2 4-day-old) and late (6 7-day-old) host puparia 

were selected for irradiation. The host pupae were irradiated with 0 (control), 0.5, 1, 

2, 4 or 8 Gy for early pupae and 0 (control), 10, 30, 50, 70 or 90 Gy for late pupae.


The assessment of radiation doses against the host pupae was selected based on 

earlier work (Hasan and Khan 1998; Jahan, Rahman, Hasan, and Islam 1998). 

Single pairs of 1-day-old parasitoids were introduced into glass vials containing 

irradiated host pupae separately according to dose and pupal age. The adult 

parasitoids were fed on 50% aqueous honey solution. The progeny of parasitoids 

emerging from the irradiated host puparia were counted. There were 10 observations 

for each dose and age. The experiments were carried out for three successive 

generations. All the experiments were carried out at 28 8C and 65% RH. 

Gamma irradiation techniques 

The radiation source was a deep-therapy unit of 60 Co at the Bangladesh Atomic 

Energy Commission, Dhaka. The dose rate was approximately 0.78 Gy/min. 


The intrinsic rate of natural increase (rm) expressed as the number progeny per 

individual per day was calculated employing the formula (Birch 1948): 

Xv 1; 

e 

x 1 

rmx lxmx where, v is the age class, lx and mx are the proportion of surviving females at age x 

and the number of progeny produced per female at the age interval x, respectively. 

With a stable age distribution and under given factors, the intrinsic rate of natural 

increase is a useful comparative statistic of population growth potential (Southwood 

1978). 

In addition, gross reproductive rate, GRR /amx or total number of progeny 

produced per female during its lifetime (Price 1997), net reproductive rate, R0 / 

a y 

x 0lxmx or number of progeny produced per female (Krebs 1994), finite capacity of 

increase, l /erm or number of times the population will multiply itself per unit of 

time (Krebs 1994), mean generation time, T In R0/rm (measured in days) and 

doubling time, D In 2/rm (number of days required for the population to double its 

numbers) were calculated for factors (Southwood 1978). Multivariate statistics were 

used to determine the significance levels of the parameters considered in these 

experiments. All the statistical procedures were carried out using the software 

packages including Minitab (Version 9.0) and Excel (XP Microsoft Office). 

Results 

Effect of host density and age 

All the parameters including host density, age, sex and their possible interactions 

showed significant (F5,5861 1119.69; F9,5861 651.70; F4,5861 504.17; F1,5861 

3200.00, PB0.001) effects on the progeny production of N. thymus for all the 

host-age groups. 

There was a significant and positive relationship between progeny production and 

an increase in number of host puparia (Figure 1), and a significant (PB0.001) decline 

in the number of progeny with an increase in host age. Two to 4-day-old host puparia

Progeny (No.) 

350 

300 

250 

200 

150 

100 

Age-1 Age-2 Age-3 Age-4 Age-5 Age-6 

50 

0 

0 1 2 3 4 5 6 7 8 9 10 

Host density (No.) 

Figure 1. Host-density dependent progeny production in N. thymus reared on different ages 

of host pupae (age in days). 

were found to be the most suitable for obtaining the maximum progeny production of 

N. thymus for all the host-densities and parasitoid sex ratios (Figure 1). Maximum 

progeny production was found to be between 100 and 300 for 4-day-old host puparia 

(Figure 1). Maximum progeny production was obtained by providing 2 4-day-old 

host puparia to all the pairs of parasitoids, while minimum progeny production was 

obtained by providing 5 6-day-old host puparia (Figure 1). 

The intrinsic rate of increase (rm day 1 ) increased with the increased host density 

for all the host-age groups (Figure 2). The results also show that the r m day 1 values 

were relatively higher for the progeny production when the parasitoids were provided 

with 2 3-day-old host puparia (Figure 2). This value was found to be greater for all 

the densities and age groups. The net reproductive rate (R 0) and gross reproductive 

rate (GRR) were found to be greater for all the density levels and host age groups. 

On the other hand, the values for the doubling time (D) and finite capacity of 

increase (l) gradually decreased with increased host density for all the host age 

groups. 

The present findings show that the host density and age significantly (F9,5861 

651.70 and F5,5861 1119.69; PB0.001) influenced the sex-ratio of progeny in 

Intricsic rate (r m day -1 ) 

0.85 

0.8 

0.75 

0.7 

0.65 

0.6 

0.55 


0.5 

Age 1 Age 2 Age 3 Age 4 Age 5 Age 6 

0 1 2 3 4 5 6 7 8 9 10 


Figure 2. Host-density and age-dependent r m day 1 values for the progeny production of 

N. thymus (age in days).


Table 1. Host-age and density effects upon male and female progeny production by 

N. thymus. 

Host 

Density 1 

N. thymus. Higher proportions of females were observed for all the host density levels 

and host-age groups (Table 1). It is interesting to note that the proportion of females 

gradually decreased as the host density increased for more or less all the age groups. 

The results show that the highest proportions of females were recorded on 2-day-old 

hosts while the lowest on 5-day-old hosts (Table 1). It also shows that all the femalebiased 

sex ratios were significantly different from a 1:1 ratio. 

The data presented in Figure 3 indicate that the rate of parasitism clearly 

decreased with the increased host density and age; 100% parasitism occurred when 

the parasitoid was released against 1 3-day-old age Uzi pupae. However, the results 

show that the rate of parasitism declined substantially with 6-day-old host pupae, 

where only 55% parasitism was observed at the host density number of 10. The 

results demonstrated that progeny production of the parasitoid varied significantly 

(F2,114 294.26; PB0.001) with temperature (Figure 4). The maximum number of 

Parasitism (%) 

100 

80 

60 

40 

20 

0 

Host age 

1 

2 

3 

4 

5 

6 

Host age (days) 

1-day-old 2-day-old 3-day-old 4-day-old 5-day-old 6-day-old 

Mean no. 

ß:à 

Mean no. 

ß:à 

Mean no. 

ß:à 

Mean no. 

ß:à 

Mean no. 

ß:à 

1 2 3 4 5 6 7 8 9 10 


Mean no. 

ß:à 

1:1 3.6:79.2 3.9:90.6 4.4:64.6 24.9:77.1 5.8:61.6 2.5:42.0 

1:2 5.4:113.4 5.5:135.1 5.8:93.6 12.0:133.9 7.8:81.9 4.4:53.6 

1:3 8.2:139.3 8.2:165.9 6.9:113.6 19.5:166.1 13.7:97.0 4.8:64.0 

1:4 9.4:168.5 12.4:194.4 9.9:145.8 19.9:167.0 16.9:125.1 5.7:69.7 

1:5 8.9:150.0 16.2:163.5 22.5:164.1 15.4:181.2 22.6:158.2 7.2:72.3 

1:6 10.3:159.1 11.8:162.2 22.9:174.2 22.6:232.9 15.1:167.8 7.4:81.0 

1:7 12.2:162.2 11.1:168.9 19.0:191.7 18.3:243.7 12.6:154.2 6.9:77.1 

1:8 16.7:171.6 14.3:190.1 15.0:206.6 25.0:280.2 14.3:152.5 7.4:82.7 

1:9 14.7:168.7 15.3:192.2 26.7:216.4 28.7:275.1 13.6:120.9 7.0:86.9 

1:10 14.9:184.7 14.9:184.7 16.5:202.9 32.2:283.3 11.3:134.9 9.1:82.3 

1 Ratio parasitoids:host pupae. 

Figure 3. Host-density and age-dependent parasitism (%) for N. thymus (age in days).

Progeny (No.) 

250 

230 

210 

190 

170 

150 

130 

110 

90 

70 

50 

20 25 30 

Temperature (°C) 

Figure 4. Effect of temperature on the progeny production of parasitoid N. thymus (vertical 

bars indicate the standard error). 

progeny (over 200) of N. thymus was recorded at 258C, while the minimum was at 

308C (Figure 4). 

The highest values for the net reproductive rate (R0) and gross reproductive rate 

(GRR) for progeny production were recorded at 258C (Figure 5). The lowest values 

for these parameters were recorded at 208C. Both the values for doubling time of 

capacity (D) and finite capacity (l) increased at 25, 30 and 208C, respectively. The 

highest value for the intrinsic rate of increase (rm day 1 ) of progeny production was 

recorded at 258C, while the lowest value was at 208C (Figure 5). The sex-ratio of 

N. thymus also varied significantly (F1,114 360.00; PB0.001) at different rearing 

temperatures. The significant variation was observed in the temperature sex-ratio 

interaction (F2,114 258.34; PB0.001) (Figure 6). The sex ratios were female-biased 

for all the temperatures (Figure 6, Table 1). The highest percentage of female progeny 

GRR 


250 

230 

210 

190 

170 

150 

130 

110 

90 

70 

50 

GRR rm 

20 25 30 


Figure 5. Effect of temperature on the GRR and r m day 1 values for the progeny production 

of parasitoid N. thymus. 

0.8 

0.79 

0.78 

0.77 

0.76 

0.75 

0.74 

0.73 

Intrinsic rate (r m day _ 1 )


Female (%) 

94.80 

94.60 

94.40 

94.20 

94.00 

93.80 

93.60 

93.40 

20 25 30 


Figure 6. Effect of temperature on the sex ratio of the progeny of parasitoid N. thymus. 

was obtained at 20 and 258C, while it decreased at 308C. The results of the test also 

indicate that the percentages for female-biased sex-ratios were more than 93.8 for all 

the temperatures (Figure 6). Temperature had a significant (PB0.001) effect on the 

longevity of adult N. thymus. The longevity of the adults decreased with increase of 

temperature for both sexes (Figure 7). The maximum longevity of adults was 11 and 

18 days at 208C for males and females, respectively (Figure 7). The highest rate of 

parasitism was observed at 208C followed by 25 and 308C, and the average rate was 

more than 95% at all temperatures tested (Figure 8). 

Effect of gamma irradiation on host 

Gamma irradiation of hosts increased the progeny production of N. thymus reared 

either on early (2 4-day-old) or late (6 7-day-old) host puparia (early age, F1,676 3834.63, late age, F1,676 2399.47; PB0.001) The progeny production of N. thymus 

increased with increased doses of gamma radiation for both the early and late host 

puparia (F 4.09; PB0.001) (Figures 9 and 10). A maximum of 272 progeny was 

produced at 8 Gy for a single pair of parasitoids reared on the early host puparia, 

while a minimum of 199 progeny was produced on the control batch (Figure 9). The 

Period days 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

Male Female 

20 25 30 


Figure 7. Effect of temperature on the longevity of adults of parasitoid N. thymus.

Parasitism (%) 

100 

95 

90 

85 

80 

20 25 30 


Figure 8. Effect of temperature on the parasitism percent of parasitoid N. thymus (line bars 

indicate the standard error). 

present findings also show that the irradiated early host pupae were more suitable for 

mass production of N. thymus than the irradiated late pupae (Figures 9 and 10). 

There was a trend of significantly decreased progeny production in two successive 

generations compared to the control batch for both the host age groups (early age, 

F 27.06, PB0.00001; late age, F 6.02, PB0.01) (Figures 9 and 10). 

The values R 0, GRR and r m day 1 for the progeny production increased with the 

increased gamma irradiation dose levels for both the host-age groups. However, the 

F1 and F2 generations for early host age group did not follow the same trends in 

which the declining trend was observed at the moderate dose levels ranging from 2 to 

4Gy. 

The sex ratios of N. thymus developing either from early- or late-irradiated Uzi 

pupae varied significantly, depending upon dose and developmental stage of the host 

Progeny (No.) 

350 

300 

250 

200 

150 

100 

50 

0 


Parental F 1 F 2 

0 0.5 1 2 4 8 

Doses (Gy) 

Figure 9. Progeny production of N. thymus developing from irradiated early (2 4-day-old) 

host Uzi pupae (vertical bars indicate standard errors).


Progeny (No.) 

250 

200 

150 

100 

50 

0 

Parental F 1 F 2 

0 10 30 50 70 90 

Doses (Gy) 

Figure 10. Progeny production of N. thymus developing from irradiated late (6 7-day-old) 

host Uzi pupae (vertical bars indicate the standard error). 

pupae. The proportions of female progeny appeared to be a bit bimodal, with 

somewhat more females being produced at the lowest and highest irradiation doses 

for all generations (parental, F1 and F2), but with a slightly lower proportion of 

female progeny at the intermediate doses (Figures 11 and 12). The maximum female 

proportion was produced at 8 and 50 Gy dose levels from the early- and late-Uzi 

pupae, respectively. 

Discussion 

The number of hosts parasitized per unit of time depends upon the ability of the 

individual parasitoids to locate and parasitize varying numbers of hosts. The present 

Female:Male ratio 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

Parent F 1 F 2 

0 0.5 1 2 4 8 

Doses (Gy) 

Figure 11. Sex-ratio of N. thymus developing from irradiated early (2 4-day-old) host Uzi 

pupae (vertical bars indicate the standard error).

Female:Male ratio 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 


Parent F 1 F 2 

0 10 30 50 70 90 

Doses (Gy) 

Figure 12. Sex-ratio of N. thymus developing from irradiated late (6 7-day-old) host Uzi 

pupae (vertical bars indicate the standard error). 

findings suggest that maximum progeny production could be obtained by providing 

irradiated 2 4-day-old Uzi host to N. thymus. The results also showed that progeny 

production of N. thymus gradually increased with an increase in host density. These 

results are in agreement with Kishore, Sharma, Sharan, Sinhadeo, and Thangavelu 

(2001), who reported that the progeny production in N. thymus increased with the 

density of host puparia. Jalali, Singh, and Ballal (1987) also observed host densitydependent 

progeny production for Cotesia marginiventris (Cresson). Ulleyet (1949) 

in Gyptus sp., Puri and Sangwan (1973), and Utida (1950) observed the same trend 

while working with Neocatalaccus mamezoophagous and Bracon gelechiae Ashmead, 

respectively. 

The progeny production of the parasitoid was clearly affected by the host age, i.e., 

early stage (2 4-day-old) host puparia were more suitable for mass production of the 

parasitoid compared to late stage puparia. These results corroborate the findings of 

Medeiros, Romalho, Lemos, and Zanuncio (2000) who reported that the reproductive 

potential of Podisus nigrispinus (Dallas), a predator of cotton leafworm, 

decreased with an increase of age of the adults. Similar results were observed by 

Morgan, Smittle, and Patterson (1986), who determined that exposing 2-day-old 

Musca domestica L. pupae to Spalangia endius Walker females at a parasitoid to host 

density of 1:5 produced the greatest number of progeny. The present results also 

correlate well with those reported by Barclay (1986) while establishing a hostparasitoid 

dynamics model. 

Host density and host age significantly influenced the sex-ratio of progeny of 

N. thymus. The results are in agreement with the findings of Meunier and Bernstein 

(2002) that parasitoid sex-ratios change as a function of host and parasitoid densities 

and these changes could influence the dynamics of host-parasitoid systems. These 

studies have implications for augmentative biological control programs based upon 

mass rearing of N. thymus. It would appear to be most efficient from a production 

standpoint to use 2 4-day-old parasitized host pupae for inundative release of the 

parasitoid. The study suggests that, although the functional response limits the


potential for N. thymus to generate density-dependent aggregation of parasitism, it 

may still be a promising candidate for biological control of E. sorbillans as well as 

other dipterans due to its aggregative response to host density. Additional studies are 

needed, however, to investigate the impact of this parasitoid on dipteran pest 

populations in natural conditions. 

It is evident from the results that there was significant (PB0.001) variation in 

progeny production of N. thymus when they were reared at different temperatures. 

Temperature-dependent progeny production in parasitoids has been reported by 

several researchers (Urbaneja, Llacer, Garrido, and Jacas 2001; Roy, Brodeur, and 

Cloutier 2003; Daane, Bentley, and Weber 2004). Hinton (1981) reported that there 

are few examples of insects whose reproductive potential is not significantly affected 

by temperature within the temperature range studied. Ratte (1985) mentioned that 

total egg production in insects reached a maximum at a temperature slightly lower 

than the optimum. Minkenberg (1989, 1990) found that the reproductive potential of 

Dacnusa sibirica Telenga, a parasitoid of Liriomyza spp., decreased from 225 to 48 

eggs with increasing temperatures from 15 to 258C, whereas the fecundity of another 

leaf miner parasitoid, Diglyphus isaea (Walker), did not significantly change within 

the same temperature range (209 293 eggs). The present results also agree with 

Taylor (1981) who reported that the optimum temperature for the development of 

Acyrthosiphon pisum (Harris) was 258C. He added that this temperature is consistent 

with that selected in the thermal gradient by non-parasitized aphids, i.e., 24.98C 

(temperature at DT 0). He also reported that the maximum rate of adult 

development of Aphidius rapae (Curtis) occurred at 278C. A remarkably low number 

of progeny at 158C also was reported by Ahmad (1936) (2 5 progeny/day/female). 

Pawson and Petersen (1990) observed a temperature preference for the oviposition 

behaviour of five species of pteromalid wasps using housefly pupae as the host. They 

reported that temperature had a significant effect on oviposition behaviour of 

Musidifurax raptor Girault and Sanders, Pachycrepoideus vindemmiae (Rondani), 

and Urolepis rufipes (Ashmead). Ryoo, Hong, and Yoo (1991) studied the 

reproductive potential of Lariophagus distinguendus (F.) in relation to temperature. 

Jahan and Islam (1998) measured the effects of different constant temperatures, viz. 

18, 22, 26 and 308C on the development of Metaphycus helvolus (Compere), a 

parasitoid of Coccus hesperidum L. They found that the reproductive potential of the 

parasitoid displayed a linear increase at all the constant temperatures ranging from 

18 to 308C. 

In the present experiment, analyses of rm day 1 , R0 and GRR indicate that 

N. thymus was best adapted to temperatures of 258C (Figure 5). The parasitoid’s 

performance in terms of progeny production was poorest at 208C. The low value of l 

for N. thymus at 258C suggests that this temperature is near the lower limit of positive 

population growth. Results also reveal that the intrinsic rate of increase (rm day 1 ) 

did not vary greatly between 20 and 308C, suggesting that performance may not be 

seriously compromised at these temperatures. Roy et al. (2003) mentioned that the 

intrinsic rate of natural increase is useful to estimate the population growth potential 

of insects and mites, which may help predict the outcome of pest-natural enemy 

interactions. They also added that the developmental rate of spider mite prey 

response to temperature has a major influence on the temperature r m day 1 

relationship. The present results agree with the findings of Ren, Stansly, and Liv


(2002), who suggested that the magnitude of intrinsic rate of natural increase was 

greatly influenced by constant temperatures. 

Temperature had a significant (PB0.001) effect on the longevity of adult 

N. thymus. The longevity of the adults decreased with increase of temperatures for 

both the sexes. These findings are consistent with previous results for a number of 

parasitoids (Urbaneja et al. 2001; Bazzocchi, Lanzoni, Burgio, and Fiacconi 2003). 

There were female-biased sex-ratios at all the temperatures studied. Ren et al. (2002) 

reported similar results while working with the whitefly predator Nephaspis oculatus 

(Blatchley). The female-biased sex-ratios of parasitoids influenced by temperature 

have also been reported by several researchers (Murai 2000; Urbaneja et al. 2001; 

Chabi-Olaye, Fiaboe, and Schulthess 2004). Nevertheless, our findings indicate that 

N. thymus exhibits sufficient environmental plasticity to be a useful biological control 

agent against E. sorbillans as well as other dipteran pests under a wide range of 

temperatures. 

Nuclear techniques could also play an important role in augmentative biological 

control, not only in promoting mass rearing, but in several additional ways. It has 

been reported that ionizing radiation may be used to a great advantage to improve 

conventional in vivo rearing strategies for many parasitoids (Greany and Carpenter 

1999). It has also been mentioned that the approach for prolonging the acceptability 

of house fly pupae would be to expose pupae to gamma irradiation and store them at 

cool temperatures (Morgan et al. 1986). Gamma irradiation of the host enhanced the 

progeny production of the parasitoid in their studies. This is consistent with the 

previous results obtained for tephritid fruit fly parasitoids by Hill (1997), and 

Ramadan and Wong (1989). Sivinski and Smittle (1990) reported that gamma 

radiation inhibited development (maturation) of the Caribbean fruit flies, Anastrepha 

suspensa (Lowe), which are attacked by the parasitoid, Diachasmimorpha 

longicaudata (Ashmead). Ramadan and Wong (1989) exposed pupae of the oriental 

fruit fly, Dacus dorsalis (Hendel) to gamma radiation prior to eclosion of the 

parasitoid, D. longicaudata. Sivinski and Smittle (1990) found that gamma radiation 

from a 137cesium source prevented adult eclosion of non-parasitized Caribbean fruit 

flies, but it did not prevent the larvae from serving as viable hosts for 

D. longicaudata. This allowed the investigators to safely release A. suspensa puparia 

from larvae exposed to parasitoids without fear of releasing fertile flies into the area. 

The results of the present findings are also similar to the earlier studies of Morgan 

et al. (1986). They noted that the development of pupae of Musca domestica could be 

inhibited using gamma radiation (500 Gy) prior to mass culture for the parasitoid 

Spalangia endius Walker. Similar results also were obtained by Roth, Fincher, and 

Summerlin (1991), while working with irradiated horn fly pupae as hosts for 

hymenopteran parasitoids. Irradiated house fly pupae could be held successfully for 

an extended period (about 10 weeks) prior to parasitization. Carpenter, Bloem, and 

Hofmeyr (2004) found that the level of acceptability of eggs laid by irradiated false 

codling moth, Cryptophlebia leucotreta (Meyrick) (as hosts for Trichogrammatidae 

cryptophlebiae Nagaraja) was favourable for combined use of sterile insect 

techniques and augmentative releases of parasitoids. Vargas et al. (2004) mentioned 

that the effects of the sterile melon flies and the parasitoids were directed to the adult 

and larval stages, respectively, and would seem to be compatible from an IPM 

perspective, when multiple strategies are desirable. Irradiation of host larvae prior to 

parasitization is used in mass-rearing programmes in Florida, Mexico and


Guatemala to prevent mixed lots of other parasitoid spp. and fertile flies (Sivinski 

and Smittle 1990; Sivinski, Vulince, Menezes, and Aluja 1998). 

It should be noted that irradiation of hosts is not always helpful in parasitoid 

rearing. Menezes et al. (1998) studied the development of the parasitoid Coptera 

haywardi (Oglobin) in irradiated and unirradiated host pupae of Caribbean fruit fly 

and the Mediterranean fruit fly, Ceratitis capitata (Weidemann) and found that 

irradiated host pupae were not acceptable for this parasitoid, for unknown reasons. 

Another application for ionizing radiation that has promise is to inhibit the 

cellular and/or humoral (biochemical) defense reactions of host insects that might 

otherwise serve as optimal factitious hosts for beneficial insects. This approach was 

tested as a means of inhibiting encapsulation of the parasitoid Microplitis croceipes 

Cresson in a candidate factitious host, Galleria mellonella L. (Ferkovich unpublished, 

cf. Greany and Carpenter 1999). Recent studies by Genchev, Milcheva- 

Dimitrova, and Kozhuharova (2007) also showed that 65 Gy of gamma radiation 

enabled the otherwise marginally suitable factitious host Galleria mellonella L. to be 

used as a highly suitable host for the parasitoid Venturia canescens Grav. Although 

not relevant to pupal parasitoids, it has been reported that gamma radiation inhibits 

the behavioural resistance (e.g., defensive attack) of hosts so that they can be made 

more suitable for attack by parasitoids that may otherwise be injured by their hosts 

(Greany and Carpenter 1999). 

The foregoing discussion leads to the conclusion that ionizing radiation offers a 

reliable means to achieve developmental arrest of insect hosts for use in in vivo 

rearing prior to mass production of the parasitoid N. thymus. These findings will 

be further tested in an area-wide demonstration site at a sericultural farming area in 

the northern region of Bangladesh, where both N. thymus and sterile Uziflies will be 

released. It remains to be determined whether sterile flies and augmentative 

parasitoid releases will be cost-effective and sustainable in area wide IPM systems 

in the northern region of Bangladesh. 


This publication derives from a research project funded by the International Atomic Energy 

Agency, Vienna, under a research contract No. IAEA/BGD-10776. We would like to thank the 

Bangladesh Atomic Energy Agency for irradiation facilities and to Dr M.W. Gates, 

Department of Entomology, Smithsonian Institute, USA, for identifying the parasitoid. 

Thanks are due to Dr J. Hendrichs of FAO/IAEA, Vienna and Dr J.E. Carpenter of ARS- 

USDA, Tifton, GA, USA, for advice on experimental design and Dr Patrick D. Greany of 

PDG Consulting, Tallahassee, FL, USA, for critically reviewing the manuscript. We would 

also like to extend our thanks to the Chairman, Department of Zoology, Rajshahi University 

for providing the laboratory facilities. 

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Vol. 19, S1, 2009, 261 270 

Use of irradiated Musca domestica pupae to optimize mass rearing 

and commercial shipment of the parasitoid Spalangia endius 

(Hymenoptera: Pteromalidae) 

Miguel C. Zapater a *, Carlos E. Andiarena a , Gladys Perez Camargo a , and 

Norberto Bartoloni b 

a Cátedra de Genética, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 

4453 (1417), Buenos Aires, Argentina; b Cátedra de Métodos Cuantitativos Aplicados, 

Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453 (1417), 

Buenos Aires, Argentina 

This paper examines the potential for using irradiated Musca domestica pupae as 

suitable hosts of the parasitoid Spalangia endius for its use in biological control 

programs. Prior to being exposed to parasitoids, M. domestica pupae were gamma 

irradiated at 500 Gy and maintained for up to 2 months in anoxia at 68C. The 

parasitization percentage, estimated by parasitoid emergence, decreased 25% after 

26.5 days, 50% after 53.2 days, and 58% after 60 days. This was compared to a 

control group of S. endius parasitoids reared on cold-stored non-irradiated pupae 

whose emergence percentage decreased by 25% after 7.7 days, 50% after 15.5 days, 

and 72% after 22 days. Fecundity and adult longevity of parasitoids emerging 

from irradiated pupae were evaluated as indicators of fitness. There were no 

significant differences in fitness between parasitoids raised on irradiated, coldstored 

pupae and the standard, live pupae presently being used in biocontrol 

programs. If this procedure is implemented for the mass rearing process of S. 

endius, it could allow the production of surplus stocks of pupae, improved 

efficiency, reduced rearing costs, and allow commercial shipments of nonparasitized 

host pupae. 

Keywords: gamma radiation; Musca domestica; Spalangia endius; mass-rearing; 

parasitism; host stockpiling; pupal storage 

Introduction 

The house fly, Musca domestica L. (Diptera: Muscidae), is an important pest 

worldwide and breeds in places where food, warmth, and moisture are present. This 

situation is common in places with intensive animal production, around manure and 

produce composting areas, and near dumps and industrial landfill sites. Urbanization 

close to these facilities has resulted in a gradual lowering of the tolerance threshold 

for nuisance flies. Unfortunately, the close proximity of humans and animals to these 

facilities also often makes it difficult to apply chemicals for fly control. In addition, 

because of frequent applications, which are necessary for effective control, insecticide 

resistance has become a serious problem (Meyer, Georghiou, and Hawley 1987; Scott, 

Roush, and Rutz 1989). Contamination and intoxication are also frequent problems 

on many farms. As a result, biological control using pupal parasitoids within the 

*Corresponding author. Email: mmzapater@arnet.com.ar 



DOI: 10.1080/09583150802439819 


262 M.C. Zapater et al. 

framework of an integrated management system can provide an economical and 

efficient alternative (Zapater, Martínez-Rey, and Mazzoli 1994). 

Spalangia spp. (Hymenoptera: Pteromalidae) are pupal parasitoids of certain fly 

species distributed around the world (Boucek 1963). Natural levels of parasitism due 

to these wasps are generally low. Meyer, Mullens, Cyr, and Stokes (1990) reported 

that 4% and 6.2% of stable flies and house flies, respectively, were being parasitized 

by Spalangia spp. in California dairies. Natural levels of parasitism of house fly 

pupae in installations of caged chickens have been reported at 0.5% (Rutz and Axtell 

1981) and 7.6% (Rueda and Axtell 1985). Parasitism levels of stable flies in feedlots 

are only around 0.1% (Smith, Hall, and Thomas 1987). However, inundative releases 

of Spalangia endius Walker against M. domestica have been shown to increase 

parasitism levels to 80 90% with considerable control; in some instances parasitism 

rates even reached 100% (Morgan, Weidhass, and Patterson 1981). In caged-layer 

poultry houses, Zapater (1997) demonstrated that weekly releases of four S. endius 

per chicken combined with adequate manure management reduced fly populations 

13.2 times compared to those facilities without parasitoid releases. Once the use of 

house fly parasitoids was considered to be innocuous to the environment, seven 

species were released on Easter Island, which has a fragile ecosystem (Ripa 1980, 

1986). Spalangia endius and other muscid fly parasitoids are presently commercialized 

in North America (Hunter 1994), Colombia (Vergara-Ruíz 1996), and 

Argentina (Zapater et al. 1994). Techniques for mass rearing S. endius have been 

reported by Morgan and Patterson (1978), Morgan, LaBrecque, and Patterson 

(1978) and Morgan (1981). 

One of the most important issues for a commercial insectary is the ability to 

guarantee customers regular (e.g. weekly) shipments of parasitoids. In order to deal 

with normal variations in daily pupal production and occasional urgent or increased 

demands for parasitoids, more host pupae are generally produced than are needed. 

Because the optimal age of house fly pupae for parasitism by S. endius is 24 72-h-old, it 

is not possible to stockpile host pupae for any length of time or purchase nonparasitized 

pupae from another insectary. The short period of time that host pupae are 

suitable for parasitism further complicates insectary operations where different species/ 

strains of parasitoids are being reared in different facilities to avoid contamination 

problems. Another issue for commercial insectaries is that not all of the pupae that are 

exposed to parasitoids are parasitized. Fortunately, adult flies emerge from nonparasitized 

pupae before the parasitoids begin to emerge. Therefore, to insure that only 

parasitized host material is shipped and released, exposed pupae must be held for an 

additional 6 days to allow adult flies and empty puparia to be eliminated. 

A potential strategy to address the above mentioned problems is to try to prolong 

the suitability of house fly pupae for parasitism, and prevent the emergence of adult 

flies from non-parasitized pupae using a combination of gamma radiation, anoxia, 

and cold. Morgan, Smittle, and Patterson (1986) showed that no house fly adults 

emerged from pupae irradiated (gamma radiation) at a dose of 500 Gy, and that 

pupae irradiated at this dose were still good hosts for S. endius. They were also able 

to extend the suitability of pupae for parasitism to 8 weeks by storing them at 4.48C 

and adequate humidity. In this paper, we extend this research and further describe 

the effect on S. endius fitness of irradiating house fly pupae and placing them in cold 

storage in anoxia for up to 2 months before using them as host material under mass 

rearing conditions.



Insect strains 

Musca domestica strain 

A 20-year-old house fly laboratory stock originating from the Institute for Pesticide 

Research, Wageningen, The Nertherlands, was provided to the University of Buenos 

Aires in 1999. In August 2000, the colony was invigorated by out-crossing virgin 

laboratory females with wild males collected from a poultry house in Mendoza, 

Argentina. The resulting offspring from these crosses has been used as the parental 

stock in all succeeding experiments. A colony of around 30,000 adults is permanently 

maintained. The average number of pupae in 10 mL is 325924. 

Spalangia endius strain 

A colony of S. endius was established in March 2000 with wild insects collected from 

three different areas in Argentina: Mi Granja, Córdoba province; La Plata, Buenos 

Aires province; and Pergamino, Buenos Aires province. Parasitoids were collected 

from poultry facilities by the placement and retrieval of mesh bags containing 

laboratory-reared house fly pupae. Pupae were removed from the bags and held in 

Plexiglas cages for fly emergence. After adult flies and empty puparia had been 

eliminated, the remaining pupae were held until parasitoids emerged. Parasitoids are 

presently maintained on house fly pupae under mass rearing conditions at a weekly 

production level of about 100,000 adults. 

Irradiation 

Irradiation of the house fly pupae was conducted at ‘IONICS‘, Ingenieros 2475, El 

Talar, Buenos Aires, Argentina, a commercial irradiation facility, using a Cobalt 60 

irradiator with an activity level of (1942.5 10 13 Bq (525,000 Ci). Because of the 

high dose rate, special procedures adjusting the exposure distance were developed by 

the staff at ‘IONICS‘ to ensure that an effective dose of only 500 Gy was delivered at 

a dose rate of 20 Gy/min. The dose was calculated such that no fly emergence was 

observed from material irradiated at 500 Gy, while increasing fly emergence was 

detected at doses of 400 and 300 Gy similar to that reported by Morgan et al. (1986). 

Experiment I: storage potential of irradiated pupae 

Two hundred mL of 48-h-old (912 h) M. domestica pupae were placed in each one of 

54 plastic bags. The bags were hermetically sealed, which caused anoxia to develop 

due to respiration of the pupae, and then irradiated with 500 Gy of gamma radiation. 

The bags of pupae were placed in a cooler before and after transportation to the 

irradiation facility. Later, the bags were maintained permanently at 690.58C ina 

refrigerator. Every day for the first eight days (days 0 7) and every third day for days 

9 63 (total of 27 sampling days), one sample of 50 pupae was extracted from each of 

two bags and the bags discarded. The pupae were placed in small plastic Petri dishes 

(2 0.5 cm, diameter high) and introduced into the parasitization cage containing 

25 30 pairs of parasitoids. The cage was 40 60 40 cm (long wide high), made 

of Plexiglas with a fine mesh screening on two sides for ventilation. An approximate


ratio of one female parasitoid for every four pupae was maintained in the cage and 

parasitoids were replaced every 2 days. The parasitization cage was kept in an 

environmental chamber maintained at 25918C, 14 h L:10 h D, and 70 85% RH. 

After 2 days, the pupae were removed and placed in small plastic tubes (1 3 cm, 

diameter long) covered with mesh to allow for parasitoid emergence. The tubes with 

parasitized pupae were maintained as above. A total of nine repetitions were 

conducted. In addition, two bags of non-irradiated pupae were prepared and exposed 

to parasitoids to compare the percentage of non-irradiated pupae parasitized on day 

0 with that of the irradiated pupae. Percent parasitism was calculated as the number 

of emerged parasitoids, divided by the number of exposed pupae, 100. 

Experiment II: storage potential of non-irradiated pupae 

Similar to Experiment I, plastic bags were prepared consisting of 200 mL of 48-h-old 

(912 h) M. domestica pupae, but in this case they were not irradiated. The bags were 

again maintained at 690.58C in a refrigerator until needed. Every day during 22 

days beginning on day 0 (23 treatments), one sample of 50 pupae was extracted from 

each of two bags and placed into Petri dishes and the bags discarded. The Petri 

dishes were then introduced into similar parasitization cages as in Experiment I and 

exposed to S. endius for 2 days. As before, parasitized pupae were placed in small 

plastic tubes to allow parasitoid emergence for determination of the percent 

parasitism. Fourteen repetitions were performed consisting of 46 bags of pupae 

prepared for each repetition. 

In addition to the samples taken each day to assess the suitability of the pupae for 

parasitism, a second sample of 50 pupae was taken from each bag (one sample from 

each of two bags for days 0 22) to assess fly emergence. Each sample was placed in a 

separate Petri dish. The Petri dishes were then placed within a Plexiglas box 10 10 cm 

and 6 cm high that had a 3 cm hole in the top covered with fine mesh for ventilation 

and maintained at 25918C, 14 h L:10 h D, and 70 85% RH. After 30 days, the 

numbers of fully emerged flies, half emerged flies, and dead pupae were counted. 

Experiment III: fitness of parasitoids reared on irradiated pupae held in cold storage 

in anoxia 

Two simple tests were carried out to evaluate the fecundity and longevity of 

parasitoid females using house fly pupae that had been irradiated and then held in 

cold storage and anoxia for 1, 20 and 40 days as in Experiment I. Pupae from the 

normal colony production that were 48 h old (912 h) and had not been subjected to 

irradiation and cold storage were used for the control treatment. 

Fecundity 

Fecundity was evaluated by placing 400 pupae from each of the four treatments into 

separate 3 cm Petri dishes and then keeping the dishes in the parasitization cage 

containing approximately one female parasitoid for every four pupae. The dishes were 

removed after 48 h and placed in separate 10 10 6 cm Plexiglas boxes kept in an 

environmental chamber 25918C, 14 h L:10 h D, 70 85% RH to allow parasitoid 

emergence. Newly emerged parasitoids (B24 h old) were removed and placed in


additional 10 10 6 cm boxes with excess 24-h-old pupae to allow the parasitoids 

to host feed and mate for 48 h. Ten females from each of the four treatments were then 

selected and placed in individual 1 3 cm plastic tubes containing 25 normal colony 

pupae (48-h-old). The pupae were removed and 25 new pupae added every 24 h for 

4 days. Exposed pupae were held separately in plastic tubes in the environmental 

chamber to allow parasitoid emergence and to determine the number of progeny 

produced per female per day. A total of three replicates were performed. 

Longevity 

Longevity was calculated by selecting 15 newly emerged females from each treatment 

and placing them in individual 1 3 cm tubes containing five normal colony pupae 

(24-h-old). The host pupae were removed every 4 days and replaced with new pupae 

until the female died. Dead insects were counted daily and recorded. Three 

repetitions were done for each treatment. 


Data were analyzed through linear regression analysis (Neter, Kutner, Nachtsheim, 

and Wasserman 1996). Least square estimates were obtained for the relationship 

among response variables and predictive variables, and a measure of how much 

variance in the response variable was explained by the independent variables (R 2 ). In 

Experiment II, we also employed a curvilinear regression (exponential). In the 

analysis of Experiment III, we employed a non-parametric procedure: the Kruskal 

Wallis test ANOVA by ranks (Conover 1980). 

Results 

Experiment I: storage potential of irradiated pupae 

The effect of storing house fly pupae in anoxia in the cold for increasing lengths of 

time on percent parasitism by S. endius is presented in Figure 1. Results indicated that 

there was a decrease in the parasitism rate as the length of pupal storage time 

Figure 1. Mean percent parasitism by Spalangia endius on irradiated Musca domestica pupae 

that had been held in cold storage (690.58C) and anoxia for up to 63 days. N 9 replications.


Figure 2. Mean percent parasitism by Spalangia endius on non-irradiated Musca domestica 

pupae that had been held in cold storage (690.58C) and anoxia for up to 22 days. N 14 

replications. 

increased following the formula Y 53.93 0.50X. The R 2 value for this equation was 

0.51. In accordance with the equation, the rate of parasitism percentage decreased 

25% after 26.5 days, 50% after 53.2 days and 58% near the end of the experiment 60 

days later. This experiment was carried out under simulated mass rearing conditions, 

which probably accounts for the resulting variation in parasitism rates. 

Experiment II: storage potential of non-irradiated pupae 

In Experiment II, using non-irradiated pupae, the rate of parasitism (based on S. 

endius emergence) decreased much more rapidly than in irradiated pupae (Figure 2). 

The initial parasitism values were similar for both experiments, but the parasitism 

rate began decreasing much more rapidly with time, showing a linear regression 

between the average % parasitism and the number of days pupae were stored by: Y 

54.35 1.76X. This function had an R 2 

0.54. In accordance with the regression 

formula, rates of parasitism decreased 25% after 7.7 days, 50% after 15.5 days and 

72% at the end of the experiment at 22 days. 

In the second part of Experiment II, emergence (full emergence and half 

emergence) of adult flies from non-irradiated pupae remained high following 2 3 

days of storage, but decreased rapidly after the following curvilinear equation Y 1/ 

0.01 e 7.03 0.42X (Figure 3). A 25% reduction in adult fly emergence was observed 

after 4 days, 50% after 6.2 days, 75% after 12.5 days and a 99% reduction in fly 

emergence was seen after 16.5 days of pupal storage. 

The correlation between S. endius emergence and fly emergence was calculated 

and resulted in a coefficient of linear correlation, r 0.91. The high correlation 

suggests that S. endius prefers to parasitize live or recently dead fly pupae. 

Experiment III: fitness of parasitoids reared on irradiated pupae held in cold 

storage and anoxia 

Fecundity 

The results of the fecundity experiment comparing the number of progeny produced 

per female reared from normal (control) pupae versus females reared from irradiated


Figure 3. Mean percent adult emergence ( pupae that either fully or partially emerged as 

adults) of non-irradiated Musca domestica pupae that had been held in cold storage and 

anoxia for up to 22 days. N 14 replications. 

pupae held in cold storage for 1, 20 or 40 days are presented in Figure 4. A Kruskal 

Wallis ANOVA test for ranks was employed and no significant differences in 

fecundity were found among females from the different treatments (df 3, N 179, 

H 1.2792, P 0.1692). Females produced an average of 12 14 offspring on day 3, 

which decreased to 5 6onday6. 

Longevity 

The longevity of adult female parasitoids emerging from normal (control) pupae and 

pupae that were stored in anoxia and cold for 1, 20 or 40 days was monitored for 16 

days and cumulative daily averages plotted in Figure 5. A Kruskal Wallis ANOVA 

test for ranks was applied and no significant differences among the four treatments 

Figure 4. Mean daily adult progeny production (fecundity) of Spalangia endius females 

emerged from Musca domestica pupae that had been irradiated and refrigerated under anoxia 

( conserved) for 1, 20, and 40 days and from non-irradiated/non-refrigerated (control) 

pupae. Newly emerged females were allowed to host feed and mate for 2 days and then were 

exposed to 25 fresh pupae each day for 4 days. N 3 replications.


Figure 5. Mean cumulative percent mortality of Spalangia endius females emerged from 

Musca domestica pupae that had been irradiated and refrigerated under anoxia ( conserved) 

for 1, 20 and 40 days and from non-irradiated/non-refrigerated (control) pupae. Each female 

was provided five normal pupae that were replaced every 4 days until the female died. N 3 

replications. 

were discovered (df 3, N 179, H 0.7899, PB0.8519). Fifty percent of the 

females had died by day 8 and 100% by day 16. 

Discussion 

Our tests confirmed that 500 Gy irradiation of house fly pupae prevents adult 

emergence. Results from our experiments also indicated that the combined treatment 

of irradiation, anoxia and refrigeration can extend the suitability of house fly pupae 

for parasitism by S. endius to 30 days or more. For example, when irradiated pupae 

were used, the rate of parasitism based on progeny production decreased by 50% 

from about 60 to 30% after 53.2 days of pupal storage; when non-irradiated pupae 

were used, 50% fewer parasitoids were produced after only 15.5 days. And although 

the percentage of parasitized host pupae that produced viable adult parasiotids 

gradually declined with increased storage time, the parasitoids that were produced in 

this manner from pupae stored up to 40 days were of good quality and lived as long 

and produced as many offspring as parasitoids reared from normal pupae. Thus, 

from a commercial standpoint, it should be possible to guarantee customers a 

specified number of quality adult parasitoids by appropriately adjusting the number 

of parasitized pupae that are sent depending on how long the pupae had been stored. 

The use of irradiated host material for mass rearing parasitoids has a number of 

advantages. First, in the case of S. endius rearing, developing house fly pupae 

produce a significant amount of biological heat. As a result, pupae must be well 

spread out on trays when they are exposed to the parasitoids. However, if the pupae 

are irradiated, development is stopped and more pupae could be placed per unit 

area. Similar concerns about the negative impact of metabolic heat, once parasitized 

pupae have been packaged for shipment, would also be minimized. Second, because 

not all of the exposed pupae are parasitized, current protocols require that the pupae 

must be held for four days to eliminate any flies that emerge before they can be 

shipped to customers. This costs both time and space. If irradiated pupae were used, 

parasitized pupae could be shipped sooner, which would free-up holding space and


create more flexibility in the system for orders to be prepared, shipments to arrive, 

and parasitoids to be delivered to the field. The use of irradiated host material has 

already become standard practice in the mass rearing of fruit fly parasitoids in 

support of sterile insect release programs (Sivinski and Smittle 1990; Cancino, Ruíz, 

Gómez, and Toledo 2002; see also articles by Cancino and Hepdurgun, this issue). 

The ability to store/stockpile pupae for a period of time has a number of 

additional advantages. It would allow facility managers to better plan for the exact 

number of parasitoids needed and avoid the tendency to overproduce both host 

pupae and parasitoids. With proper management, a rotating stockpile of host pupae 

could be maintained such that when parasitoid demand was low, excess host material 

produced during that time could be put into storage. If parasitoid demand increased 

unexpectedly or an opportunity arose to develop new clients, pupae could be brought 

out of storage to quickly meet the need. The ability to store pupae could also reduce 

work, for example, during weekends and holidays. 

Contamination of parasitoid strains can be a significant problem when rearing 

multiple species. If colony host pupae are parasitized by an unintended species prior 

to collection of the pupae for exposure to the intended parasitoid species (in this case 

S. endius), it is easy to contaminate stocks. However, if the host pupae are collected 

and then irradiated before exposure to a particular parasitoid, the window of 

opportunity for contamination by a competing parasitoid species is greatly reduced 

and much easier to manage. The use of gamma irradiation would also make it 

possible for insectaries to trade/sell irradiated host pupae amongst themselves 

instead of parasitized pupae, which occasionally occurs when demand exceeds 

production or problems with a colony develop. This would allow them to continue to 

provide their customers with the same strain they normally provide, rather than 

having to supplement an order with a strain from another facility. 

The large commercial irradiator used in this study had a greater degree of dose 

error at low doses than most traditional Gammacell irradiators. Morgan et al. (1986) 

showed that a dose of 500 Gy was optimal for preventing fly emergence with 3-dayold 

pupae. However, for commercial mass-rearing, non-optimal doses between 250 

and 750 Gy could probably be occasionally tolerated. Unfortunately, the primary 

limitation to the wide scale application of this technology is the availability of an 

irradiation source. Alternatives to irradiation, such as X-ray machines or linear 

accelerators, that would be more accessible, at more reasonable prices, are needed. 


Special thanks to Ionics S.A. for the pupal irradiation and technical advice. The authors would 

also like to thank K. Bloem for helpful edits to earlier drafts of this manuscript. This work was 

funded by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture 

Section, Vienna, Austria, under research contract No 10844/RO. 

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Vol. 19, S1, 2009, 271 275 


Synergism between biological control and sterile insect technique: Can 

commercial mass production of biocontrol agents and sterile insects be 

integrated within the same industrial entity? 

Shimon Steinberg* and Jean-Pierre Cayol 

Bio-Fly Sde Eliyahu, Bet Shean Valley 10810, Israel 

The integration of commercial facilities for mass production of beneficial 

arthropods (Bio-Bee) and sterile insects (Bio-Fly) within the same industrial 

entity in Israel has proven successful. The synergism between the two companies 

has resulted in the integration of nuclear techniques and the use of biocontrol 

agents in area-wide integrated pest management programmes. 

Keywords: SIT; biological control; private sector; host irradiation; Ceratitis 

capitata; mass rearing 

Introduction 

In the last few decades, Israel has been a pioneer and a leader in the development and 

use of biological control techniques for the environment-friendly management of 

insect pests in agriculture. For the past 25 years, Bio-Bee Sde Eliyahu Ltd. has been 

mass producing and using arthropod natural enemies for biological pest control. 

Predatory mites, pirate bugs, parasitic wasps and ladybeetles are used in commercial/ 

augmentative biological control to combat spider mites, whiteflies, thrips, aphids, 

leafminers and mealybugs in protected as well as open field crops. 

Responding to a demand from the plant protection organisations and the fruit 

industry in Israel, the Hashemite Kingdom of Jordan and the Palestinian Territories, 

Bio-Fly, a daughter company of Bio-Bee, was established in 2004 to mass rear the 

Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) for 

Sterile Insect Technique (SIT) purposes (Bassi, Steinberg, and Cayol 2007). 

The use of natural enemies and the SIT both rely on the mass production and 

augmentative release of arthropods in the field to control a given target pest. As the 

establishment of Bio-Fly benefited from the experience of Bio-Bee in mass rearing 

procedures, the expertise and know-how that is continuously gained at Bio-Fly’s 

mass rearing facility now offers unique opportunities to synergise between SIT and 

the biological control industry, along three major lines. 

The use of SIT ‘by-products’ for the production of natural enemies 

One of the limiting factors in mass production of beneficial arthropods such as 

predatory insects and mites as well as parasitoids, is the cost of mass producing 

*Corresponding author. Email: s_stein@bio-bee.com 

First Published Online 26 June 2009 



DOI: 10.1080/09583150902884971 


272 S. Steinberg and J.-P. Cayol 

another arthropod species to be used as prey (for predatory insects and mites) or as a 

host (for parasitoids). Very often, the resulting commercial price of beneficial 

arthropods is restricting the use of commercial/augmentative biocontrol to high 

added-value crops. The cheaper the overall production cost of beneficial arthropods, 

the more likely they can be utilized by the agricultural community on a large scale in 

crops that bear marginal profit. The availability of large numbers of Mediterranean 

fruit fly eggs, larvae and pupae, as ‘by-products’ of Bio-Fly’s SIT activities, has 

provided several opportunities to improve the mass production of predators and 

parasitoids by Bio-Bee. 

Predator mass rearing 

The minute pirate bug Orius laevigatus (Fieber) (Heteroptera: Anthocoridae) is a 

highly effective predator of Western flower thrips Frankliniella occidentalis (pergande) 

in protected vegetables, mainly sweet pepper, and is considered to be of key importance 

in the commercial augmentative biocontrol programmes in protected sweet pepper 

throughout Europe. Frozen eggs of the Mediterranean flour moth Ephestia kuehniella 

Zeller represent the most common diet for mass rearing of O. laevigatus, as well as for 

other Orius species and other predators such as Mirid bugs, ladybeetles and lacewings 

(e.g., Schmidt, Richards, Nadel, and Ferguson 1995; Cocuzza et al. 1997; Yano, 

Watanabe, and Yara 2002). The Ephestia eggs are considered to be the most expensive 

component of all other input materials used for industrial mass rearing of O. 

laevigatus. Hence, cheaper alternative food sources are needed. 

A Mediterranean fruit fly larvae-based diet proved to be as efficient as Ephestia 

eggs or as immature stages of the greenhouse whitefly Trialeurodes vaporariorum 

(Westwood) (Homoptera: Aleurodidae) for the production of the Mirid bug 

Macrolophus caliginosus Wagner (Heteroptera: Miridae) (Nannini, Ruiu, and Floris 

2008; Nannini, Foddi, Murgia, Pisci, and Sanna 2008). A thorough study jointly 

conducted by Bio-Bee/Bio-Fly has shown that specially-treated Mediterranean fruit 

fly developmental stages were comparable to Ephestia eggs with respect to their effect 

on O. laevigatus over-all fitness, i.e., developmental time, fecundity and field 

performance. 

Parasitoid mass rearing 

Spalangia cameroni Perkins (Hymenoptera: Pteromalidae) is a solitary pupal 

parasitoid of flies, especially those living in manure, the house fly Musca domestica 

Linnaeus (Diptera: Muscidae) being one of its most important hosts. As generalist 

parasitoids, Spalangia spp. are known to attack Mediterranean fruit fly pupae 

(Stibick 2004). Furthermore, Geden and Kaufman (2007) have shown that progeny 

production of S. cameroni from house fly pupae killed by gamma radiation was not 

significantly different from production on live hosts. It was therefore decided to 

investigate whether gamma irradiated male pupae of the VIENNA-8 genetic sexing 

strain of the Mediterranean fruit fly (Franz 2005), could serve as an appropriate host 

for mass rearing of S. cameroni. By using irradiated Mediterranean fruit fly male 

pupae, it was confirmed that no adult flies emerged from parasitized or notparasitized 

(yet irradiated) hosts. This allows the commercial production of 

S. cameroni at no risk from an agricultural perspective. As a result, the production


of S. cameroni is taking place on gamma irradiated young male pupae of the 

Mediterranean fruit fly. 

In further R&D efforts, fitness parameters of the S. cameroni colony such as rate 

of parasitism, progeny production, offspring sex ratio and long-term genetic 

deterioration will be compared between different hosts such as Mediterranean fruit 

fly irradiated male pupae, house fly pupae and pupae of the green-bottle fly Lucilia 

caesar Linnaeus (Diptera: Calliphoridae). In case of future fitness decrease (though 

not yet detected) in S. cameroni, which would be caused by rearing on a relatively 

small factitious host, other Pteromalid wasps such as Muscidifurax spp. could be 

evaluated. 

Integration of nuclear techniques into biological control procedures 

Kaspi and Parrella (2003) have shown that release of sterile (gamma-irradiated) 

serpentine leafminer Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) can 

significantly reduce the reproductive capacity of a wild population. Their study 

indicated that sterilization of L. trifolii flies is feasible and that sterile males are of 

high quality and competitive with normal males. In a subsequent study, Kaspi and 

Parrella (2008) demonstrated a synergistic interaction between releases of the 

leafminer larval parasitoid Diglyphus isaea (Walker) (Hymenoptera: Eulophidae), a 

well-known natural enemy of leafminers world-wide, and sterile males of L. trifolii. 

Bio-Bee produces the celery miner fly, Liriomyza bryoniae (Kaltenbach) (Diptera: 

Agromyzidae) and its highly effective parasitoid D. isaea. Hence, a similar evaluation 

of this type of synergistic interaction will be carried out. 

Bio-Bee is a leading producer of predatory mites world-wide. Based upon the 

successes mentioned above and the expertise gained by both teams with regards to 

gamma irradiation, use of sub-sterilizing irradiation doses and traditional selection 

to induce mutations for developing strains of predatory mites which are better 

adapted to specific conditions such as tomato plants that are known to be less 

favourable to natural enemies due to presence of various glandular hairs (or 

trichomes) on the plant is being considered. 

In the long-run, with the support and expertise of Bio-Bee in the production of 

natural enemies, Bio-Fly is considering the production of tephritid fruit fly 

parasitoids using Mediterranean fruit fly as a host and combine augmentative 

releases of parasitoids and sterile males of the Mediterranean fruit fly. Potential 

candidates for biological control of the Mediterranean fruit fly are the following 

parasitic wasps: Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae), 

Fopius arisanus (Sonan) (Hymenoptera: Braconidae), F. ceratitivorus (Wharton) 

(Hymenoptera: Braconidae) and Aganaspis daci (Weld) (Hymenoptera: Eucoilidae). 

These species are currently being tested at the Israel Cohen Biological Control 

Institute of the Citrus Growers Board (Yael Argov, personal communication). 

Synergism in the implementation of area-wide integrated pest management 

programmes 

Traditionally, most of the natural enemies produced by Bio-Bee are being used in 

protected environments such as greenhouses. When applied in open fields, given the 

close biocontrol agent-pest-host-plant relationship, the release strategy for the

274 S. Steinberg and J.-P. Cayol 

natural enemies is traditionally based on a field-by-field approach, i.e., strictly 

limited to the fields planted with the crop(s) to be protected. Through its involvement 

in SIT field operations, Bio-Fly has acquired and integrated its expertise on areawide 

integrated pest management (AW-IPM) (Hendrichs, Kenmore, Robinson, and 

Vreysen 2007) into the ‘culture’ of Bio-Bee Sde Eliyahu Ltd. 

Natural enemies and sterile insects, both being non-chemical alternatives for pest 

control, are, by essence, complementary technologies that involve augmentative 

releases. Following several years of damage caused by the Mediterranean fruit fly 

and following the recent ban of malathion, the major producers of table grapes in 

Israel are planning an AW-IPM programme with an SIT component to control 

the Mediterranean fruit fly. Another major pest of table grapes in Israel is the vine 

mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae). The vine 

mealybug will be controlled biologically by seasonal inoculative releases of the 

lady beetle Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinelidae) and 

parasitic wasp Anagyrus near pseudococci (Hymenoptera: Encyrtidae), as part of an 

overall AW-IPM programme integrating biological control and SIT against the two 

major pests. 

Conclusion 

The experience of Bio-Bee/Bio-Fly illustrates that commercial mass production of 

biocontrol agents and sterile insects can be integrated within the same industrial 

entity, benefiting from their mutual experience. In the early days of the establishment 

of Bio-Fly, the expertise of Bio-Bee in mass rearing arthropods and automated 

control of artificial rearing conditions was essential to the establishment of Bio-Fly 

and critical for its success in the first months. As illustrated above, the integration of 

nuclear techniques within the biological control operations of Bio-Bee has been done 

in a progressive manner, from using sterilised by-products provided by Bio-Fly to 

developing specific uses for nuclear techniques in biological control. This progressive 

approach was important as it resulted in the sustainable adoption (and adaptation) 

of the nuclear techniques to the specific problems faced by the biological control 

company and the successful integration of biological control and SIT within a single 

AW-IPM programme in table grapes. To date, there is no doubt that the contribution 

of nuclear techniques to several critical aspects of the biological control led by Bio- 

Bee is invaluable and is here to stay. 

The fact that two technical and commercial entities, one that mass produces and 

releases arthropod natural enemies for biological pest control and the other that 

mass produces and applies tephritid sterile males on an area-wide basis for SIT 

purposes, exist under the same managerial roof, offers a unique opportunity to 

synergise between the two. Bio-Bee/Bio-Fly provides an ideal environment to turn 

this synergy into a reality. 

References 

Bassi, Y., Steinberg, S., and Cayol, J.P. (2007), ‘Private Sector Investment in Mediterranean 

Fruit Fly Mass-production and SIT Operations The ‘Sheep‘ of the Private Sector among 

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Dyck, J. Hendrichs, and A.S. Robinson, The Netherlands: Springer, AA Dordrecht, 

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Vol. 19, S1, 2009, 277 290 


Enhancing biological control of sugarcane shoot borer, 

Chilo infuscatellus (Lepidoptera: Pyralidae), through use of 

radiation to improve laboratory rearing and field augmentation 

of egg and larval parasitoids 

Bilquis Fatima*, Nazir Ahmad, Raza Muhammad Memon, 

Moula Bux, and Qadeer Ahmad 

Nuclear Institute of Agriculture, Tando Jam-70060, Pakistan 

Feasibility studies were conducted to evaluate the use of gamma radiation to 

improve the production and field performance of the egg parasitoid Trichogramma 

chilonis Ishii and to improve production of the gregarious larval endoparasitoid 

Cotesia flavipes Cameron for biological control of the sugarcane shoot borer, Chilo 

infuscatellus Snellen. Sitotroga cerealella (Olivier) eggs were used as hosts for 

T. chilonis and the suitability of non-irradiated host eggs decreased as the age of the 

eggs increased, with no success in parasitization of eggs older than 4 days of age. 

However, irradiation of host eggs using 20 25 Gray (Gy) decreased the age effect 

and significantly more 2-, 3-, 4- and 6-day-old irradiated eggs were successfully 

parasitized than non-irradiated eggs. Radiation doses of 20 and 25 Gy were most 

effective for economical production of T. chilonis. Irradiation of host eggs did not 

affect the hatch percentage up to a dose of 15 Gy. Hatchability was significantly 

reduced at higher doses, with negligible hatching at 50 Gy. Irradiation also skewed 

the sex ratio of S. cerealella in favor of males at higher doses. Radiation at 60 80 Gy 

improved the suitability of C. infuscatellus larvae for parasitism by C. flavipes, 

allowing normally unsuitable fourth and fifth instar larvae of C. infuscatellus to be 

successfully parasitized. The sex ratio of parasitoids reared on irradiated larvae was 

skewed in favor of females. Irradiation also slowed immature development of 

C. flavipes and the combination of irradiation and low temperature (108C) proved 

effective for prolonged storage of the parasitoids. Pupae of C. flavipes irradiated at 

20 Gy could be stored for 2 months at 108 C without apparent loss of quality and 

deferred emergence by 29 30 days. Provisioning with irradiated supplemental hosts 

in the field increased overall pest suppression. The infestation by C. infuscatellus 

was higher in the control sugarcane block, where it remained above the economic 

threshold level (10% infestation) from April to October. High temperatures and 

low relative humidity during May to July reduced increases of the parasitoid 

populations. These findings were used to facilitate the area-wide application of 

biocontrol agents in a 40,000 hectare area to suppress sugarcane borers to subeconomic 

levels (B10% infestation). Sugarcane borer damage was 5.9% in treated 

blocks vs. 19.2% in untreated control blocks. 

Keywords: radiation; augmentative biological control; sugarcane shoot borer; 

Chilo infuscatellus; Trichogramma chilonis; Cotesia flavipes; parasitoids; rearing; 

area-wide; biological management; endoparasitoid; supplemental hosts 

*Corresponding author: Email: niatjam@hyd.paknet.com.pk 



DOI: 10.1080/09583150902793438 


278 B. Fatima et al. 

Introduction 

Interest has increased in using biological control, which has long been recognized 

as an important tool in suppressing insect pests. Beneficial insects have been 

successfully deployed in a variety of augmentation and conservation strategies 

(Nordlund 1984; Mohyuddin 1991; Ashraf, Fatima, Hussain, and Ahmad 1999). 

For example, the use of Trichogramma wasps as biocontrol agents is a recognized 

alternative to use of insecticides and has been applied successfully for the 

management of many insect pests (Browning and Melton 1987; Hassan, Kohler, 

and Rost 1988; Losey, Fleischer, Calvin, Harkness, and Leahy 1995; Mannion, 

Carpenter, and Gross 1995). Similarly, the larval parasitoid Cotesia flavipes 

Cameron, in conjunction with Trichogramma chilonis Ishii, is used to control the 

Chilo infuscatellus Snellen population in India (Suasa-Ard and Charernsom 1999; 

Saikia and Nath 2002; Tanwar and Ashok 2002). Augmentation usually involves 

periodic releases of beneficial insects and environmental management, such as 

providing food or hosts during times of low host density. However, economical 

production of natural enemies is a prerequisite for augmentative biological control 

programs. 

Considerable technological advances have been made in mass rearing of 

parasitoids and predators for augmentative biological control (Leppla, Bloem, 

and Luck 2002; Cohen 2003). Nuclear techniques may play an important role in 

augmentative biological control, not only in facilitating mass rearing, but also by 

inhibiting reproduction by reproductively sterilizing insect hosts, by provisioning 

non-reproductive supplemental hosts for use in field insectaries, by expediting the 

safe transport of parasitoids in irradiated hosts, by improving host suitability for 

mass rearing, and by microbially sterilizing artificial rearing media (Greany and 

Carpenter 2000). Irradiation has improved the suitability of certain lepidopterous 

hosts for parasitization (Mannion, Carpenter, and Gross 1994; Carpenter, 

Mannion, and Hidrayani 1995; Carpenter 1996). Marston and Ertle (1969), tested 

the acceptability of irradiated moth eggs to Trichogramma minutum Riley, and reported 

that irradiated eggs were as suitable as control eggs for parasitoid development. 

Eggs of the Mediterranean flour moth, Ephestia kuehniella Zeller, killed 

with ultraviolet irradiation were suitable for mass rearing of Trichogramma spp. 

(Voegele, Daumal, Brun, and Onillon 1974). Lewis and Young (1972) reported that 

adult males of Helicoverpa (Heliothis) zea (Boddie) sterilized with 320 Gy of 

gamma irradiation paired with untreated females, produced sterile eggs that were 

as suitable as fertile eggs for attack and development of Trichogramma evanescens 

Westwood. 

The studies reported herein were designed specifically to evaluate the feasibility 

of: (1) using radiation-sterilized host eggs for efficient and economical production 

and prolonged storage and release of the egg parasitoid, T. chilonis, a polyphagous 

parasitoid of many lepidopteran pests, (2) using gamma radiation to improve 

production of the larval parasitoid C. flavipes to enhance control of sugarcane borers 

in the field, and (3) providing irradiated supplemental hosts for use as a field 

insectary to enhance the initial survival and population build-up of T. chilonis to 

achieve more cost-effective management of C. infuscatellus. These findings were used 

to implement an area-wide control effort in a large area (ca. 40,000 ha).



Tests on host egg suitability as affected by radiation 

Eggs of the Angumoiis grain moth, Sitotroga cerealella (OIivier), were obtained from 

moths reared in the laboratory at 25928C in 2.5 L glass jars. For this purpose, large 

numbers of 1 2-day-old adults were collected from stock cultures and placed in 

inverted 1 L plastic jars with screen bottoms. Eggs that fell through the screen 

bottoms were collected by sifting them through a 70 mesh screen. Newly laid 

S. cerealella eggs were irradiated with doses ranging from 5 to 50 Gy with 60 Co 

source at the Nuclear Institute of Agriculture, Tando Jam (Theratronic T-780 with 

an emission rate of 175 cGy/min and GWXJ 80 with an emission rate of 225 cGy/ 

min) and were kept in separate Petri dishes at 100 eggs/dish. The whole experiment 

was replicated four times and the hatch percentage in all the doses was recorded. Egg 

cards were prepared by sprinkling ca. 2000 eggs on cards coated with Stickum † , 

which were then allowed to air dry prior to exposure to parasitoids. The eggs were 

exposed to radiation after 1 h at different doses from 5 to 50 Gy with five Gy 

intervals followed by their exposure to the parasitoid T. chilonis in conical flasks 

using eggs of different ages (1 7 days). Twenty pairs of the parasitoid were exposed 

to each card for 24 h. Egg cards were removed from the flasks after 24 h and rate of 

parasitism on each card was recorded. Parasitization per card was recorded, as 

indicated by darkening of the host eggs. The effect of egg age and radiation dose on 

parasitization was determined by counting the number of parasitoids produced vs. 

number of host eggs exposed. 

Seasonal population fluctuation of T. chilonis as a function of temperature and 

humidity 

Seasonal population fluctuations of T. chilonis were determined in sugarcane fields 

by exposing two fresh egg cards of the factitious host S. cerealella per 0.404 ha, for 

24 h at weekly intervals. The cards were returned to the laboratory and parasitization 

was determined by noting the characteristic darkening. The maximum/minimum 

temperatures and relative humidity were recorded in the field. The experiment 

continued throughout the crop-growing season. 

Providing supplemental irradiated host eggs to parasitoids for use in a field insectary 

Tests were conducted to determine whether provision of irradiated supplemental 

hosts could influence the initial survival and establishment of released parasitoids in 

the field. Three sugarcane blocks were selected at the same site. Releases of T. chilonis 

were made in the first block at monthly intervals and no supplemental hosts 

were provided to the parasitoids in the field. In the second block, fresh eggs of 

S. cerealella irradiated at 25 Gy were attached to sugarcane leaves at the time of 

parasitoid releases. Ten cards (ca. 2000 eggs/card) per 0.404 ha were attached to the 

leaves of the sugarcane crop in conjunction with five cards of parasitoids. All cards 

were spaced at uniform distances. The cards with supplemental irradiated eggs were 

provided twice during the first and second parasitoid releases. The third block was 

kept as a control and no plant protection measures were adopted. To record the 

establishment of T. chilonis parasitoids, fresh S. cerealella egg cards were placed for


24 h at two cards per 0.404 ha in each block at 2-week intervals. The cards were 

brought to the laboratory and the percent parasitism was recorded. The borer 

infestation was recorded at 2-week intervals. 

Parasitization of irradiated C. infuscatellus larvae by C. flavipes 

The impact of irradiation on the larvae of C. infuscatellus was evaluated as a means 

of enhancing parasitism by C. flavipes. Larvae of C. infuscatellus were collected from 

the field and reared on natural diet to make a homogenous stock. Different host 

instars (second, third, fourth and fifth) were irradiated at 20 120 Gy and then 

exposed to C. flavipes. Parasitization was recorded for each instar and compared with 

untreated larvae of each stage of development. The numbers of cocoons recovered 

and the percentage of adult parasitoid emergence were recorded from each instar. 

Use of irradiation and low temperatures for prolonged storage of C. flavipes 

The sugarcane-adapted strain of C. flavipes used for these experiments originated in 

Thailand; a laboratory strain was established at the Nuclear Institute of Agriculture, 

Tando Jam and was subsequently field colonized. Third, fourth and fifth instars (100 

larvae for each replicate) of C. infuscatellus were exposed to the C. flavipes 

parasitoids for 24 h at ambient temperature and humidity (24928C, 5 60% R.H). 

After two and eight days of parasitization, C. flavipes were given different doses of 

gamma rays ranging from 5 to 50 Gy to irradiate the eggs and mature larvae of the 

parasitoids, respectively. Mature cocoons also were given the same treatment. The 

irradiated, parasitized larvae along with diet were kept in the incubators set at 10, 15, 

20 and 258C for prolonged storage. The larvae were examined regularly to record 

emergence of the parasitoids for cocoon formation and pupation. Cocoons 

containing pupae were confined in glass tubes plugged with cotton and kept in the 

same incubator where parasitoid larvae completed their development. Upon 

parasitoid emergence, pupal survival and duration was recorded. 

Evaluation of supplemental irradiated host eggs for use as a field insectary 

This study was conducted at the Nuclear Institute of Agriculture Experimental 

Farm. Three 1.62-ha sugarcane blocks were selected and each block was ca. 100 m 

from the next block Sugarcane variety BL4 (Saccharum sp. hybrid) was used in all 

tests. The first block was treated with cards of the egg parasitoid, T. chilonis, at 

monthly intervals from February to October and with no supplemental hosts. The 

second block was treated with egg parasitoids and supplemental irradiated hosts. For 

this purpose, newly laid S. cerealella eggs irradiated at 25 Gy were glued on paper 

cards with ca. 2000 eggs/card and the cards were attached to sugarcane leaves in 

addition to the parasitoid cards. Ten cards per 0.404 ha were attached to the leaves of 

the sugarcane crop in conjunction with five cards with ca. 2000 parasitized eggs at 

uniform distances. Supplemental irradiated cards were provided twice, with the first 

and second release of the parasitoids. The third block was kept as a control and no 

plant protection measures were adopted. Each month, the establishment of the 

parasitoids was monitored by placing two fresh S. cerealella egg cards per 0.404 ha in 

each block for 24 h. The cards were brought to the laboratory and the parasitism


percentage was recorded at 2-week intervals by examination of the appearance of the 

eggs; darkening connoted parasitization as the parasitized eggs became dark after 

3 days whereas, un-parasitized remained yellow. The borer infestation also was 

recorded at 2-week intervals on an internodal basis (total internodes and number of 

infested internodes were counted). 

Scale-up to area-wide management 

T. chilonis parasitoids that were mass-reared on irradiated S. cerealella eggs were 

released at monthly intervals from February to September over a target area of ca. 

40,000 ha during the 2002 2003 seasons. The infestation of sugarcane stem borers was 

recorded at monthly intervals from the entire treated area. Two sugarcane blocks were 

selected at each of two different 1.62 ha sites. Releases of T. chilonis were made in the 

first block at monthly intervals throughout the growing season, i.e., February to 

September, and the second block was left untreated. To evaluate parasitoid efficacy, 

newly laid sentinel irradiated S. cerealella eggs were placed in the field, recovered, and 

evaluated as above. Feral eggs also were collected from both the blocks at the same time 

and the parasitization percentage was recorded. The sugarcane stem borer infestation 

was also recorded during the same 2-week evaluation intervals. 


Statistical analyses were conducted using Statistix † Version 8.1, Analytical Software, 

Inc., Tallahassee, FL, USA. 

Results 

Part I: rearing improvement studies 

Effect of radiation on host egg hatchability and suitability for parasitization by 

T. chilonis 

Radiation significantly reduced the hatch percentage of the irradiated, non-parasitized 

host eggs on a dose-dependent basis and it was negligible at 50 Gy (Table 1). Emergence 

of adult S. cerealella was the same as controls up to a dosage of 25 Gy, and 0 when 

exposed to 50 Gy. The sex ratio of moths emerging from the irradiated eggs was skewed 

in favor of males at higher doses, which may be attributed to the fact that females are 

more radiosensitive than males and some females may have died in the embryonic stage. 

Parasitoids preferred newly laid eggs and host suitability decreased as host age 

increased (Table 2). Radiation of host eggs decreased the age effect and significantly 

(P50.05) more eggs were parasitized on 2-, 3- and 4-day-old irradiated host eggs as 

compared to the normal eggs. Irradiated eggs were successfully parasitized up to 

6 days old, while control eggs older than 4 days were unsuitable as hosts. 

Parasitization of irradiated C. infuscatellus larvae by C. flavipes 

Fourth and fifth instar non-irradiated host larvae were not successfully parasitized, 

suggesting immunity in the host. However, fourth and fifth instar host larvae were 

successfully parasitized at high rates when treated with at least 60 Gy of radiation


Table 1. Effect of gamma radiation on the eggs of S. cerealella. 

Dose 

(Gy) Hatch% 

Pupae 

recovered% 

Sex ratio (%) 

Adult 

emergence% Male Female 

5 88.693.43A 64.493.64BC 65.495.85AB 57.293.11CDE 42.891.92B 

10 80.096.28B 62.293.67C 64.695.85AB 55.094.30DE 45.092.0AB 

15 81.691.94B 66.693.43AB 63.092.23AB 64.293.56BCDE 35.092.86C 

20 76.497.60BC 51.493.2D 66.292.38A 73.695.98BC 26.492.30D 

25 72.492.88CD 50.492.96D 65.693.13A 72.895.31BCD 27.293.70D 

30 68.094.58D 40.494.03E 59.694.98BC 75.097.1BC 25.093.39D 

35 40.492.96E 27.691.81F 68.895.76A 73.694.03BC 26.492.40D 

40 19.695.07F 12.492.40G 56.895.11CD 100.090.00A 0.090.0E 

45 3.691.14G 2.090.70H 50.095.11D 100.090.00B 0.090.0E 

50 0.690.54G 0.690.89H 0.0090.00E 0.0090.00F 0.090.0E 

Control 90.491.94A 68.493.84A 68.693.20A 53.292.68E 46.894.76A 

Means (9SE) in the same column followed by the same letter are not significantly (PB0.05) different by 

LSD analysis. 

(Table 3). These studies indicated that irradiation reduced immunity in the fourth 

and fifth instars of C. infuscatellus for parasitization by this parasitoid. Parasitization 

varied in the larvae when irradiated at different doses and comparatively higher 

numbers of fourth and fifth instar larvae were successfully parasitized when treated 

with 60 or 80 Gy. More parasitoid cocoons were recovered from the irradiated fourth 

and fifth instars than from non-irradiated third instars, which may be due to the 

greater size of fourth and fifth instars compared to third instars. 

Use of irradiation and low temperatures for prolonged storage of C. flavipes 

The immature development of C. flavipes varied by radiation dose (Table 4) and 

significant variations were recorded in developmental rates at different radiation 

doses. The egg-larval period was significantly longer when parasitoids were 

irradiated at 20 Gy than the other tested doses. The pupal period of the parasitoid 

also varied significantly at different radiation doses, with those receiving 40 Gy 

exhibiting a significantly shorter pupal development period (Table 4). Overall, 20 Gy 

irradiation in conjunction with low temperature (108C) prolonged the storage of the 

parasitoids (Table 4). Studies indicated that the C. flavipes larvae irradiated at 20 Gy 

could be stored for 2 months at 108C without apparent damage to quality, as 

indicated by analysis of F1 adult parasitization rates. In addition, parasitoid cocoons 

irradiated at 20 Gy could be stored at 108C to delay adult emergence by 29 30 days 

with no reduction in parasitoid viability. 

Part II: field studies on area-wide management through augmentation with parasitoids 

Seasonal population fluctuation of T. chilonis in sugarcane fields 

Parasitization was low during the month of January when temperatures were lower 

and thereafter increased and reached its first peak in April (Table 5). However, hot 

weather during May through July suppressed the parasitoids, which did not recover

Table 2. Effect of irradiation on suitability of host eggs for parasitization of T. chilonis. 

Parasitization potential (%) in host eggs at different age (days) 

Dose (Gy) 1 2 3 4 5 6 7 

5 19.291.30B 16.492.40BC 11.292.49CD 6.492.30C 0.690.89C 0.090.0C 0.090.0B 

10 23.292.77A 21.093.39A 17.693.84A 11.092.23AB 0.090.0C 0.090.0C 0.090.0B 

15 20.291.92B 17.093.39BC 12.692.70BC 11.892.16A 0.890.83C 0.090.0C 0.090.0B 

20 23.691.14A 20.092.44AB 15.295.26AB 13.293.70A 9.091.5A 3.892.28A 0.090.0B 

25 24.492.70A 19.893.03AB 13.293.76BC 9.292.58B 4.291.9B 1.491.14B 0.890.83A 

30 19.292.28B 16.492.40BC 13.293.11BC 9.491.14B 4.892.5B 1.890.83B 0.090.0B 

35 17.891.64B 15.692.70C 14.693.04ABC 5.491.81C 0.890.83C 0.090.0C 0.090.0B 

40 15.291.92C 13.493.97CD 8.491.94DE 1.491.67D 0.090.0C 0.090.0C 0.090.0B 

45 13.492.51CD 7.692.28E 5.492.30E 0.290.44D 0.090.0C 0.090.0C 0.090.0B 

50 11.691.34C 7.692.40E 1.491.14F 0.090.0D 0.090.0C 0.090.0C 0.090.0B 

Control 18.891.78B 11.292.38DE 7.292.77E 2.090.70D 0.090.0C 0.090.0C 0.090.0B 

Means (9SE) in the same column followed by the same letter are not significantly (PB0.05) different by LSD analysis. 


Table 3. Parasitization of irradiated Chilo infuscatellus larvae by C. flavipes. 

Dose (Gy) 

Third instar Fourth instar Fifth instar 

Mean no. of 

cocoons/larva Emergence % 

Mean no. of 

cocoons/larva Emergence % 

Mean no. of 

cocoons/larva 

Emergence % 

20 29.492.07C 78.496.65A 0.090.0E 0.090.0D 0.090.0E 0.090.0D 

40 31.892.5BC 74.696.22AB 0.090.0E 0.090.0D 0.090.0E 0.090.0D 

60 38.492.30A 81.493.84A 46.495.45B 79.892.28A 50.893.83B 83.6979.0A 

80 36.494.66AB 78.896.57A 53.894.32A 81.892.94A 55.693.13A 84.292.77A 

100 31.694.98BC 71.493.20B 30.691.51C 70.695.17B 39.294.96C 69.897.49B 

120 15.294.08D 57.892.58C 17.492.30D 58.893.49C 23.294.43D 62.293.83C 

Control 30.894.81C 80.094.47A 0.090.0E 0.090.0D 0.090.0E 0.090.0D 


284 B. Fatima et al.

Table 4. Effect of low temperature (108C) in conjunction with radiation on immature 

development of C. flavipes. 

Dose 

(Gy) 

until the more moderate months of August and September (Table 5). The relative 

humidity remained low from February to June. 

Significance of providing supplemental irradiated hosts to the parasitoids for initial 

survival in the field 

Results from August and later months indicated that efficacy of the parasitoids was 

higher in the block where supplemental irradiated hosts were provided to the 

parasitoids, as evidenced by low infestation rates (Table 6). Temperature also played 

an important role in the establishment of the parasitoids in the field and the greatest 

number of parasitoids was observed in the months of April and September when the 

mean temperature ranged between 25 and 308C. Highest infestation rates were 

observed in the control block, where no plant protection measures were applied. 

Table 5. Effect of temperature on the efficacy of T. chilonis in the sugarcane field. 

Months 

Mean egg/larval 

period (days) 

Normal 

host eggs 


Parasitism% 

Mean pupal 

period (days) 

Irradiated 

host eggs 

Mean over all 

developmental 

period (days) 

Max. 


Mean pupal 

survival (%) 

Min. 


F1 parasitism 

Mean no. of 

cocoons/larva 

10 56.891.92BC 29.291.48A 86.092.23B 74.494.66B 28.291.92A 

20 64.292.16A 31.492.30A 95.691.51A 83.693.97A 29.692.07A 

30 58.491.51B 29.093.16A 87.493.04B 78.295.16AB 22.092.64B 

40 53.494.50C 23.892.28B 78.094.69C 58.694.50C 13.092.44C 

50 00.090.00D 00.090.00C 00.090.00D 00.090.00D 00.090.00D 

Control 55.293.76BC 28.692.70A 84.294.14B 74.896.26B 30.295.26A 


LSD analysis. 

% Relative 

humidity 

January 1.091.0E 1.491.14G 18.0 8.5 65.0 

February 11.694.15D 14.494.21EF 29.8 11.8 69.0 

March 30.297.33B 37.697.73B 37.5 15.3 62.0 

April 39.698.47A 45.694.77A 40.0 22.7 67.0 

May 23.695.27BC 29.696.80CD 40.8 28.1 64.0 

June 21.696.69C 24.694.77D 39.2 27.2 65.0 

July 26.095.95BC 31.293.27BC 36.6 26.5 72.0 

August 38.498.04A 47.695.89A 36.1 25.8 73.0 

September 43.297.08A 48.495.81A 33.0 24.0 72.0 

October 28.095.43BC 36.693.50B 32.1 19.0 71.0 

November 12.694.15D 17.094.94E 26.0 14.1 64.0 

December 6.693.57DE 9.492.30F 26.0 11.0 67.0 


LSD analysis.

Table 6. Effect of supplemental irradiated hosts to enhance the performance of T. chilonis in sugarcane fields. 

Month 

Block-1 released parasitoids9supplemental 

host (%) 

Parasitization % 

Block-2 released parasitoids9non 

supplemental host (%) 

Block-3 untreated control 

(%) 

Mean temp. 

(8C) Parasitization Infestation Parasitization Infestation Parasitization Infestation 

February 20.8 39.094.63C 0.890.83G 10.092.23E 0.090.00E 8.294.4A 0.890.83G 

March 26.4 48.093.46B 4.692.7DE 37.894.2C 1.090.70E 2.090.70CD 7.891.92F 

April 31.3 58.095.83A 6.092.2CDE 48.297.1AB 5.691.14D 1.891.09D 11.692.4E 

May 34.4 40.092.91C 7.291.78BC 34.297.46CD 7.092.91CD 2.091.22CD 14.294.43DE 

June 33.2 37.093.39C 8.892.38B 30.094.35D 8.092.44BCD 3.290.83CD 16.893.96CD 

July 31.5 41.094.69C 12.892.68A 32.094.48CD 10.291.92AB 0.890.83D 19.292.49BC 

August 31.2 57.095.24A 7.090.70BCD 45.095.33B 12.093.00A 2.891.30CD 21.692.70AB 

September 30.0 61.096.28A 3.691.14EF 52.895.76A 8.893.19BC 4.691.67BC 24.894.14A 

October 26.0 55.095.43A 1.290.83FG 00.090.00F 5.891.30D 7.292.58AB 12.293.11E 


286 B. Fatima et al.


Both blocks receiving augmentative parasitoid releases, with or without supplemental 

hosts, generally experienced reduced infestation rates throughout the season 

(Table 6). The infestation by C. infuscatellus in the control treatment was higher and 

remained above the economic threshold level (10%) from April to October. 

Area-wide tests 

The infestation of sugarcane stem borers in the entire 40,000 ha area treated by 

provision of supplemental host eggs was successfully managed by the egg parasitoid, 

T. chilonis, resulting in damage levels below economic threshold level. The infestation 

of sugarcane borers on an internodal basis ranged from 0 to 9.7% with an overall 

mean of 4.8% in the treated areas. The mean infestation of the borers in the 

untreated areas was 21.1%, with a range of 16.4 31% (Table 7). 

The percentage recovery of T. chilonis from feral and sentinel eggs were also higher 

in the treated area as compared to the un-treated fields. Moreover, the parasitoid 

recovery was significantly higher during the months of August and September 

followed by June and July when the environmental conditions were conducive. Pupal 

recovery of the parasitoids was much higher in the fields treated with supplemental, 

irradiated host eggs as compared to the untreated fields (Table 8). 

Discussion 

Eggs of S. cerealella are widely used for the production of T. chilonis (Morrison, 

Stinner, and Ridgway 1976; Mannion et al. 1994) and for population suppression of 

sugarcane borers (Carpenter et al. 1995). However, for release programs, all of the 

eggs of the pest placed in the field should be parasitized or developmentally arrested; 

otherwise the eggs that hatch may serve to increase the population of S. cerealella. 

This problem can be avoided by using sterilized eggs. In our studies, gamma 

radiation was very effective in developmentally arresting host eggs without reducing 

their utility as hosts for parasitoids. Radiation doses of 20 and 25 Gy enhanced 

parasitization and reduced the host egg age effect for parasitization. Lewis and 

Young (1972) also reported the acceptance of irradiated host eggs by polyphagous 

Table 7. Chilo infuscatellus infestation in treated and untreated areas. 

% Borer infestation 

Location Village Treated fields Untreated fields 

1 Moosa khatian 3.8 18.7 

2 Abbri 4.7 20.6 

3 Lakhi keti 4.6 17.5 

4 Quba stop 4.0 25.4 

5 Mehran sugar miils area 6.4 31.0 

6 NIA expt. Farm 0.0 16.4 

7 Tando Soomro 4.3 19.8 

8 Chambar 9.7 22.5 

Average 4.8 21.1


Table 8. Percent recovery of T. chilonis from feral and sentinel host eggs in treated and 

untreated sugarcane field. 

Treated area Untreated 

Month Feral eggs Sentinel eggs Feral eggs Sentinel eggs 

January 00.090.0F 0.490.54H 0.090.0D 0.090.0F 

February 00.090.0F 1.091.0F 0.090.0D 1.091.0EF 

March 13.693.71E 18.496.38F 1.691.14D 3.491.14DE 

April 42.6910.31C 46.696.34C 5.492.40BC 7.491.81ABC 

May 36.494.92C 39.295.63D 4.291.92C 6.493.50BC 

June 41.693.04C 45.693.91C 4.691.51C 6.093.39BCD 

July 41.693.20C 46.295.26C 5.491.14BC 5.691.14CD 

August 52.695.49B 57.496.22B 7.091.58AB 8.692.30AB 

September 59.699.44A 66.695.72A 7.691.34A 9.492.88A 

October 28.096.67D 31.693.78E 6.492.60AB 7.692.96ABC 

November 12.493.43E 21.093.30H 1.090.70D 1.691.51EF 

December 00.490.54F 7.492.30G 0.090.0D 0.090.0F 


LSD analysis. 

egg parasitoids. Brower (1982) found that irradiated eggs of Indian meal moth, 

Plodia interpunctella (Hubner) were preferred over normal, untreated eggs. Thus, the 

weight of evidence from our studies and others indicates that irradiated eggs may be 

used effectively for laboratory mass rearing and field releases with no problems 

posed by the hatching of host eggs. 

Our studies also showed that the provisioning of host eggs in the field may be 

helpful. Parker and Pinnell (1972) reported similar results by provisioning hosts early 

in the season along with Trichogramma parasitoids. The irradiated eggs sustained the 

parasitoid population by providing additional host eggs with little risk of increasing 

pest populations. 

Temperature and relative humidity played a significant role in successful build-up 

of the parasitoids. Fewer parasitoids were observed in the field during May to July 

when the temperature remained high and relative humidity low. The parasitoidreleasing 

interval may need to be adjusted on the basis of prevailing environmental 

conditions for successful application of biological agents against sugarcane borers in 

the field. 

Doses of 60 and 80 Gy on host larvae were most effective to improve the mass 

rearing efficiency of C. flavipes by increasing their suitability past the normal third 

instar, enabling fourth instar host larvae to be used. In addition, more pupal cocoons 

were recovered from fourth instar hosts compared to third instars, which may be due 

to their greater size which might favor a gregarious endoparasitoid like C. flavipes. 

The sex ratio of the parasitoids reared on irradiated larvae also was skewed in favor 

of females. The immature development of C. flavipes was reduced in irradiated host 

larvae, which allowed them to be stored for longer periods before use. Host eggs and 

parasitoid larvae and pupae could be stored for 2 months at 108C after irradiation at 

20 Gy with no apparent loss in host quality or parasitoid reproductive potential. 

Regarding the area-wide control trials using T. chilonis, provision of supplemental 

hosts to enhance the efficacy of the egg parasitoids proved effective for


the control of C. infuscatellus as the parasitization rate was higher and the 

C. infuscatellus infestation was lower in the blocks with supplemental hosts as 

compared to the other blocks. There are many factors which affect the establishment 

of parasitoids in the field. Ashraf, Fatima, and Ahmad (2001) reported that longevity 

of both male and female wasps of T. chilonis in the laboratory was significantly 

higher when reared at 22.5% RH. At low relative humidity, the parasitoids may be 

less active as observed in the field. Calvin, Knaff, Welch, Poston, and Etzinga (1984) 

also recorded significant effects of relative humidity on the developmental stages, 

longevity, fecundity and sex ratio of Trichogramma pretiosum Westwood. Similar 

results were observed in the present studies and the parasitization rate was higher 

during the months of April and August when the humidity was relatively high in the 

field. It has long been recognized that temperature is the single most important factor 

influencing the development of the immature stages and the adult maturation rates of 

the majority of insects. Temperature/development rate relationships could be useful 

in predicting phenological events in the field for ecological and pest management 

purposes, the optimization of mass rearing procedures under constant conditions, 

and construction of computer simulation models of population suppression. The 

present studies revealed that population establishment in the field had a linear 

relationship with the temperature. Establishment was slow during hot months and it 

increased in the months when temperature decreased. 

In conclusion, irradiation of host material proved useful for increasing 

the rearing efficiency of T. chilonis and C. flavipes, in addition to expediting 

the development of practical and cost-effective area-wide augmentative biological 

control programs in the field. 

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Sugarcane’, Insect Science and Application, 12, 19 26. 

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Vol. 19, S1, 2009, 291 301 


Impact of gamma radiation on the developmental characteristics of the 

gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae) preparatory 

to their use as supplemental hosts/prey for natural enemy enhancement 

Milan Zúbrik and Július Novotny´* 

Forest Research Institute, Research Station Banská Sˇ tiavnica, Lesnícka 11, 969 23 Banská 

Sˇ tiavnica, Slovak Republic 

The developmental characteristics of irradiated and non-irradiated gypsy moth, 

Lymantria dispar L. eggs and larvae were compared. Gypsy moth eggs were 

irradiated a few days before hatching at 10, 20, 30, 40, 50, 60, 80, or 110 Gray 

(Gy) and first instar larvae at 50, 80, or 110 Gy of gamma radiation and tested for 

differences in their development by comparison with non-irradiated controls. 

Untreated larvae developed to the adult stage more rapidly than irradiated larvae 

treated either as eggs or as larvae and this was dose-dependent. Larval mortality 

and pupal developmental anomalies were dose-dependent. Pupal morphological 

abnormalities occurred in only 1.6% of controls, but in the 110 Gy group, they 

occurred in 93.2 and 95.1% of individuals treated as eggs or larvae, respectively. 

The tests showed that 50 Gy was optimal for irradiating gypsy moth eggs and 

larvae to achieve F1 sterility and extend larval development without excessive 

mortality. This may facilitate use of these sterile larvae as supplemental hosts in 

augmentative biological control programmes. 

Keywords: gypsy moth; Lymantria dispar; gamma radiation; larvae developmental 

characteristics; augmentative biological control; radiation hormesis 

Introduction 

The gypsy moth (Lymantria dispar L.) is one of the most important forest insect pest 

species in Europe, Asia, and North America. Larvae of this moth defoliate large 

areas of broadleaf stands annually. It was imported into Massachusetts in 1869 for 

possible silk production and it accidentally escaped captivity. As of 1994, the US 

Forest Service spent approximately $11 million annually on gypsy moth control 

(Campbell and Schlarbaum 1994). Currently, APHIS and its state cooperators spend 

nearly $10 million annually to prevent gypsy moths from establishing in the western 

portions of the United States (Vic Mastro, USDA APHIS, personal communication). 

Control of the gypsy moth in the United States was reviewed by Liebhold and 

McManus (1999). 

Parasitoids, predators and pathogens are believed to play substantial roles in the 

dynamics of gypsy moth populations in Europe. Studies were performed on L. dispar 

natural enemies in Slovakia (Novotny´ 1989) and in Austria, the latter being carried 

out by the U.S. Department of Agriculture European Parasitoid Laboratory during 

an outbreak in the 1970s (Fuester, Drea, Gruber, Hozer, and Mercadier 1983). The 

*Corresponding author. Email: novotny@nlcsk.org 



DOI: 10.1080/09583150902812188 


292 M. Zúbrik and J. Novotny´ 

Commonwealth Institute for Biological Control discovered diverse complexes of 

parasitoids, predators and pathogens in Europe (Eichhorn 1996). 

There are several possibilities as to how populations of indigenous natural 

enemies build up (Maksimovič and Sivčev 1984; Mills 1990). These studies indicate 

that increasing the efficacy of parasitoids and predators through direct augmentative 

releases would be problematic. Similarly, pathogens might be useful for 

population suppression and they may reduce the host population density very 

quickly and effectively (Podgwaite, Reardon, Walton, and Witcosky 1992; 

Podgwaite, Dubois, Reardon, and Witcosky 1993; Shapiro, Robertson, Injac, 

Katagirik, and Bell 1984; Shapiro and Dougherty 1985). In recent years, dramatic 

collapses of gypsy moth populations in the United States are due, in large part, to 

the fungus Entomophaga maimaiga, collected in Japan and deliberately released 

near Boston between 1910 and 1911 (Hajek, Humber, and Elkinton 1995; Hajek, 

McManus, and Delalibera 2007). As a more cost-effective alternative to augmentative 

releases of natural enemies, it may instead be possible to release sterile, 

developmentally-compromised gypsy moth eggs and/or larvae produced and 

irradiated in the laboratory. These could serve as supplemental hosts/prey to 

achieve a more rapid build up of the natural enemies to preclude the pest 

population from achieving damaging levels. 

Traditional uses of radiation in pest management focus on use of the Sterilie 

Insect Technique (SIT). Since SIT was first used successfully for pest management 

(Knipling 1955), its effectiveness has been demonstrated for many types of pest 

insects. Strategies by which sterile insect releases might be used to control the gypsy 

moth (Maksimovič 1971a,b; Mastro and Schwalbe 1988; Mastro 1993) normally 

involve introducing sterile adults into the pest’s habitat to flood the fertile 

population, with the goal of reducing the pest population by interfering with 

reproduction. A number of studies have been conducted in which pest insects that 

served as laboratory hosts for parasitoids were irradiated in order to arrest their 

development or to preclude release of fertile females when inadvertently released 

along with the parasitoids (reviewed by Greany and Carpenter 2000). However, use 

of radiation to produce innocuous, sterile pest immatures to supplement natural 

populations for the purpose of building up natural enemy populations has not been 

pursued to date and this is the subject of the present research. Studies were therefore 

conducted on the effects of radiation on gypsy moth immatures preparatory to 

enabling this approach. 

Sterility is normally achieved by irradiation of the pupae or adults with adequate 

doses of gamma radiation. Since mature ovaries are not present in the eggs or 

larvae, the eggs and larvae are not usually irradiated. Despite this, it has been shown 

in research on Nezara viridula L. that fully-developed immatures may be irradiated 

and adult sterility achieved (Dyby and Sailer 1999). The aim of the present research 

was to apply a range of radiation doses to gypsy moth eggs and larvae to evaluate 

effects on their developmental characteristics and those of the F1 generation. This in 

turn should establish the optimal rate/timing needed to produce supplemental hosts/ 

prey for natural enemy build up without fear of releasing reproductively competent 

pests.


Material and methods 

1. Insect and bioassay 

We used a laboratory strain of the gypsy moth (Lymantria dispar L.) supplied by the 

USDA APHIS Methods Development Centre in Otis, MA (USA). Eggs and larvae 

in first instar were used as the starting stage for bioassay throughout this study. 

Irradiation was performed at the Entomology Unit of the FAO/IAEA Laboratories 

in Seibersdorf, Austria, using a 60 Co Gammacell 220 (AE of Canada Ltd) with dose 

rates ranging between 50 and 60 Gy/min. 

Newly-laid egg masses were stored in the refrigerator at approximately 5 78C for 

120 days until hatching was possible. Eggs masses (10 masses per dose) were 

irradiated a few days before hatching, at eight different doses (10, 20, 30, 40, 50, 60, 

80, or 110 Gy). Irradiated egg masses were placed into the Petri dishes until the 

larvae hatched. The number of hatched larvae and unhatched eggs were counted. 

Larvae (100 per dose) hatched from the irradiated eggs were moved into plastic 

cups (5 larvae per cup) containing the artificial diet, reared, and tested for differences 

in their development by comparison with non-irradiated controls. The larvae were 

maintained at 20 268C and 60 70% RH. An artificial wheat germ diet (Bell, Owens, 

Shapiro, and Tardif 1981), which is commonly used in gypsy moth rearing, was used. 

From the third instar, larvae were reared separately. Larvae were checked daily. The 

duration of each larval stage was determined by noting the time of moulting. Dead 

larvae were removed from rearing cups. Pupae were stored in boxes (60 60 100 

cm high) until adults emerged. The condition and health of the pupae were 

determined and they were weighed 2 days after pupation. The number of successfully 

emerged imagos was recorded as well as the number of egg masses and the number of 

eggs that females laid. For a comparison of development, we reared a treatment of 

100 non-irradiated larvae to serve as controls. Three replicates were performed. 

In the second experiment, L1 larvae were subjected to 50, 80, or 110 Gy and 

tested for differences in their development in comparison with non-irradiated 

controls. For the laboratory investigation, 100 larvae from each treatment were used. 

The larvae were maintained the same way as in the first experiment. Three replicates 

were performed. 

2. Statistical analyses 

Analyses of variance (ANOVA) were performed on larval duration, pupal weight 

and number of eggs produced by females to determine the effects of irradiation dose 

(Tukey HSD test for unequal number of replicates) using Statistica (StatSoft, Inc.). 

Significant differences were tested at P B 0.05. Standard errors (SE) of the means 

were calculated. 

Results 

1. Developmental time 

The developmental time of male gypsy moth larvae irradiated as eggs varied between 

35.5 and 44.4 days (Table 1). The control group together with 10- and 20-Gy 

treatments developed significantly faster than the 110 Gy irradiation treatment, 

which had the longest development time. The lower irradiation doses (10 20 Gy)

Table 1. Mean developmental time (9SE) in days for larvae of Lymantria dispar treated as eggs with increasing doses of gamma radiation. 

Treatment (Gy) No. larvae reared L1 L2 L3 L4 L5 L6 Total (days) 

0 300 10.090.1 a 5.290.1 b 

Male larvae 

5.790.2 b 5.390.1 b 9.390.2 a 35.5 

10 300 10.690.2 ab 5.490.1 b 5.590.1 ab 4.790.1 a 9.490.1 a 35.6 

20 300 10.590.2 ab 4.690.1 a 5.690.1 b 5.390.1 b 9.590.1 a 35.5 

30 300 10.990.1 bc 5.290.2 ab 6.790.2 b 5.090.1 ab 9.590.2 a 37.3 

40 300 11.290.1 bc 5.390.2 b 6.490.2 bc 5.290.1 ab 9.790.1 a 37.8 

50 300 10.790.1 abc 5.390.1 ab 4.890.1 a 7.390.3 b 11.490.5 b 39.5 

60 300 11.390.1 c 5.0901 ab 5.990.2 bc 6.390.2 c 11.090.3 b 39.5 

80 300 11.590.1 c 5.290.1 ab 7.090.2 d 6.390.2 c 11.990.6 b 41.9 

110 300 12.790.2 d 5.590.1 ab 6.290.1 bcd 5.690.2 bc 14.490.5 c 44.4 

Female larvae 

0 300 10.190.2 a 5.090.1 bcd 5.490.1 ab 5.290.1 b 5.890.2 ab 10.190.2 a 41.6 

10 300 11.090.2 bcd 5.590.2 cde 5.190.1 ab 4.490.1 a 5.590.2 ab 10.190.2 a 41.6 

20 300 10.990.2 bc 4.490.2 ab 5.590.1 abc 5.490.1 bd 5.590.1 ab 9.890.1 a 41.5 

30 300 10.690.2 ab 4.690.2 abc 5.390.3 abc 4.590.1 ac 5.190.1 a 10.090.2 a 39.9 

40 300 10.990.2 bc 4.290.2 a 5.090.1 a 5.590.1 bd 5.190.1 a 9.890.1 a 40.5 

50 300 10.490.1 ab 5.490.2 cde 4.990.2 a 6.190.2 d 7.190.5 c 10.590.3 ab 44.4 

60 300 11.790.1 cd 5.890.2 e 5.990.3 c 5.390.2 b 5.590.3 ab 11.390.2 bc 45.5 

80 300 11.190.1 bcd 5.190.1 abcde 5.890.3d 5.890.5 b 5.790.3 b 12.190.4 c 45.6 

110 300 11.990.1 b 5.691.1 de 6.690.2 bc 5.390.1 bc 6.390.2 bc 12.390.2 c 48.0 

Larvae were reared in the laboratory on artificial diet. Data for male and female larvae are presented separately. L1 L6 larval instars. Means for male or female groups 

followed by different letters (within columns) are significantly different at P50.05 by Tukey HSD. 

294 M. Zúbrik and J. Novotny´


sometimes stimulated the growth rate (Table 1, L2, L3, L4). This may be an 

expression of a radiation-induced hormetic effect (Luckey 1991). Most of the males 

developed through five instars. In the first and fifth instars, larvae from the control 

treatment developed significantly faster than those of all other treatments in these 

stages (P B 0.01). Female developmental time varied between 41.5 and 48 days 

(Table 1). Most of the females developed through six instars. The control group 

needed the shortest time for development. The treatments differ also in the female 

larval stage. We recognized significant differences between treatments in all female 

larval stages, with a shorter development time in control, 10-, 20-, 30-, and 40-Gy 

treatments, which differ significantly from the 50-, 60-, and 110-Gy treatments. 

In experiments with larvae irradiated in the earliest larval stage (L1), very similar 

results were obtained (Table 2). Male development time varied between 35.6 and 40.5 

days (Table 3). The control group developed significantly faster than the 110-Gy 

group, the irradiation treatment with the longest development time. We also 

determined the statistical differences (PB0.05) between the treatments in all male 

larval stages. The female developmental time varied between 41.6 and 50.2 days 

(Table 2). The control group again developed most rapidly. We observed significant 

differences between treatments in all female larval stages (Table 2). 

2. Health condition 

Some irradiated larvae displayed serious health problems during development. 

Larval mortality appeared to be dose-dependent, reaching 15.7% among larvae 

irradiated as eggs receiving 110 Gy (Figure 1a). Similar results were obtained in 

treatments with larvae irradiated in early stage L1 (Figure 1b). Mortality reached 

22% among larvae irradiated by 110 Gy. In the control group of larvae, mortality 

was only 4%. In both sets, differences were statistically significant (PB0.05). 

Dose-dependent effects of radiation on the health condition of pupae also were 

noted. Pupae developing from irradiated larvae or eggs showed a high incidence of 

morphological abnormalities. The thoracic segments of the pupae were undeveloped 

and the antennae also were damaged in different ways. Mortality of pupae, which 

came from irradiated eggs, was relatively high and reached the highest level, ca. 91%, 

at 110 Gy (Figure 1d). Pupae deriving from irradiated larvae exhibited higher rates 

of mortality, reaching 97.5% at 110 Gy (Figure 1c). Differences were statistically 

significant (PB0.05). 

In the control group, morphological abnormalities reached only 1.6%, but in the 

110 Gy treatment, it was 93% (from irradiated eggs) or 95% (from irradiated larvae), 

respectively. Above-mentioned abnormalities affected the ability of the adults to 

emerge. Abnormalities occurred in all treatment groups and were absent among the 

controls. The emergent adults were not able to mate successfully because of these 

morphological problems, the most common being wing damage and the defects of 

mobility. 

Pupal weights for irradiated males and females deriving from treated eggs and 

larvae were significantly (PB0.05) different as a function of radiation dose, with the 

lowest weights occurring for the highest dose treatments (Table 3).

Table 2. Mean developmental time (9SE) in days for larvae of Lymantria dispar treated as larvae in first instar with increasing doses of gamma 

radiation. Larvae were reared in the laboratory on artificial diet. 

Treatment (Gy) Number of larvae followed L1 L2 L3 L4 L5 L6 Total (days) 

Male larvae 

0 300 10.190.1 a 5.290.1 a 5.790.2 ab 5.390.1 a 9.390.2 a 35.6 

50 300 10.690.2 a 5.290.1 a 5.590.1 ab 6.190.2 b 11.990.4 b 39.3 

80 300 12.990.1 b 6.290.2 b 5.390.1 a 6.690.2 b 9.590.6 a 40.5 

110 300 12.790.3 b 6.490.2 b 6.190.2 c 6.790.2 b 8.690.8 a 40.5 

Female larvae 

0 300 10.190.2 a 5.090.1 a 5.490.1 a 5.290.1 a 5.890.2 a 10.190.2 a 41.6 

50 300 9.890.2 a 4.890.1 a 5.390.2 a 6.090.1 abc 7.890.4 b 10.890.3 ab 44.5 

80 300 11.790.2 b 6.290.2 b 5.590.1 a 6.090.1 b 6.290.3 a 14.690.4 c 50.2 

110 300 11.390.3 b 5.890.2 b 6.290.2 b 6.990.3 c 6.490.4 a 12.690.9 bc 49.2 

Data for male and female larvae are presented separately. L1 L6 larval instars. Means for male or female treatment groups followed by different letters (within columns) 

are significantly different at P 5 0.05 by Tukey HSD. 

296 M. Zúbrik and J. Novotny´

Table 3. Weight of pupae from irradiated and control groups of L. dispar fed on artificial diet 

in laboratory conditions. 

Weight of pupae (g) 

Male Female 

Treatment (parents irradiated as eggs) N (X9SE) N (X9SE) 

Control untreated 160 558.392.5 f 128 1476.8913.5 ef 

10 Gy 176 579.693.2 g 108 1637.2913.4 g 

20 Gy 123 466.096.7 d 158 1417.2917.2 e 

30 Gy 132 473.496.1 e 136 1355.0912.2 d 

40 Gy 112 464.493.0 d 151 1218.6911.2 c 

50 Gy 156 570.397.7 g 106 1512.9916.0 f 

60 Gy 144 459.994.3 c 113 1208.7919.1 c 

80 Gy 121 434.996.1 b 134 1060.1925.0 b 

110 Gy 146 362.993.2 a 107 903.6911.5 a 

Treatment (parents irradiated as larvae) 

Control untreated 160 558.392.5 c 128 1476.8913.5 d 

50 Gy 123 533.994.7 c 12 1379.4916.4 c 

80 Gy 140 416.898.8 b 22 1194.4919.7 b 

110 Gy 127 324.898.2 a 20 1097.5918.3 a 

Means in a treatment group followed by different letters are significantly different at P 5 0.05 by Tukey 

HSD. N, the total number of pupae. 

% mortality 

% unhatched pupae 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 

10 

20 

30 

40 

50 

60 

Dose of radiation (in Gy) 

80 

110 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

(a) (b) 

(c) 

0 

10 

20 

30 

40 

50 

60 



80 

110 

% mortality 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 

0 

10 

10 

20 

30 

40 

50 

60 


20 

30 

40 

50 

60 


Figure 1. Effect of dose of radiation on (a) percent mortality for gypsy moth larvae 

irradiated as eggs, (b) on percent mortality for gypsy moth larvae irradiated in early larval 

stage, (c) on percent uneclosed pupae when eggs of gypsy moth were treated with gamma 

radiation, and (d) on percent uneclosed pupae when larvae of gypsy moth in early stage were 

treated with gamma radiation (9SE). 

% unhatched pupae 

(d) 

80 

80 

110 

110


3. Impact of the irradiation dose on the hatching ability of the eggs and F1 eggs 

Egg hatch was influenced by the irradiation dose. In the control group, the 

proportion of unhatched eggs was 9.5%. In all other groups it was much higher up 

to 70.3% in the 110 Gy treatment (Table 4). 

Impact of the irradiation dose on the hatching ability of the F1 eggs also was 

observed (Table 5). In some cases, adults were not able to lay eggs and no eggs for 

calculation were available (Table 5). Mortality reached 98.6% in 60 Gy treatment 

(eggs from parents irradiated as eggs) and 100% in 80 Gy treatments (eggs from 

parents irradiated as larvae). Differences were statistically significant (PB0.05). 

Discussion 

The findings of the present studies should be useful in developing improved 

capabilities for augmentative biological control of the gypsy moth. After its original 

introduction into the United States in 1869, classical biological control of the gypsy 

moth was applied using natural enemies from Europe (Doane and McManus 1981). 

Rather than relying upon natural build up of naturally-occurring and introduced 

predators and parasitoids, it may be possible to accelerate and increase levels of 

control by use of augmentative biological control approaches against gypsy moth, 

similar to use of Trichogramma spp. in greenhouses and under field conditions (Mills 

1990; Smith 1993; Wallace and Smith 1995). 

Maksimovič and Sivčev (1984) introduced into the gypsy moth population a 

large number of (fertile) eggs with the aim to increase the efficacy of the natural 

parasitoids and they found that the parasitism rates associated with the endoparasitic 

wasps Cotesia melanoscela (Ratzeburg) and Apanteles liparidis Bouche were 

slightly increased. The approach they used, using viable gypsy moth eggs to augment 

the natural host population, is risky as there is a possibility of creating an artificial, 

unexpected outbreak of the gypsy moth in the area. To minimize this likelihood, our 

results suggest it might be possible to safely use irradiated gypsy moth eggs and/or 

larvae to augment the natural population. This is valid mostly at the higher doses of 

irradiation. The higher the irradiation dose used, the lower the risk of causing an 

unexpected artificial outbreak. The probability of irradiated eggs, larvae and pupae 

reaching maturity is inversely related to the irradiation dose (Figure 1a c, 

Table 4. Impact of the irradiation dose on the hatching ability of the eggs after irradiation. 

Treatments N Number of unhatched eggs in % (X9SE) 

Control untreated 30 9.590.3 a 

10 Gy 30 9.690.1 a 

20 Gy 30 11.090.1 b 

30 Gy 30 14.390.1 c 

40 Gy 30 15.590.1 d 

50 Gy 30 21.691.4 e 

60 Gy 30 30.190.6 f 

80 Gy 30 50.891.5 g 

110 Gy 30 70.391.5 h 

Means followed by different letters are significantly different at P 5 0.05 by Tukey HSD. 

N, number of egg masses used for calculations.

Table 5. Impact of the irradiation dose on the hatching ability of the F1 eggs (average number 

of the eggs per egg mass; unhatched eggs in%). 

Treatment (parents 

irradiated as eggs) N 


Average number of eggs per egg 

mass (X9SE) Unhatched eggs (%) 

Control untreated 44 445.5930.4 a 9.890.6 a 

10 Gy 10 434.9938.6 a 16.892.1 b 

20 Gy 10 330.3929.2 a 17.591.6 b 

30 Gy 10 291.4925.8 a 31.492.3 c 

40 Gy 10 325.1931.9 a 57.795.6 c 

50 Gy 10 458.1959.8 a 75.096.8 c 

60 Gy 3 158.7923.1 b 98.691.3 c 

80 Gy 0 No eggs available No eggs available 


Average number of eggs 

(X9SE) (X9SE) 

Control untreated 44 445.5930.4 a 9.890.6 a 

50 Gy 4 212.2918.0 a 91.695.1 b 

80 Gy 3 48.6911.8 a 100.0090.0 b 


Means followed by different letters are significantly different at P 5 0.05 by Tukey HSD. 

respectively). Hosts irradiated as eggs as well as larvae would not be able to create a 

new generation because of somatic damage, immobility or sterility, respectively. 

The gypsy moth embryo develops into a small caterpillar and over-winters in the 

egg (Doane and McManus 1981). In our experiments, eggs were irradiated shortly 

before hatch or as neonate (L1) larvae. Little difference was noted in use of either 

stage (see Figure 1, Tables 1 and 2). However, it is much more practicable to use 

irradiated eggs instead of larvae for field release. 

Extension of larval development occurred as a result of irradiating eggs (Table 1) 

and larvae (Table 2) and developmental was dose-dependent. This could have 

practical implications for augmentation. The parasitoids and predators should have 

more time to discover the treated larvae in the field, and parasitoids also would have 

more time for their own development. The very widespread and abundant parasitoid, 

Cotesia melanoscela Ratz. (Hymenoptera: Braconidae) is aided by slow host 

development because females are not very successful at parasitizing fourth and later 

instars for example (Hoch, Zúbrik, Novotny´, and Schopf 2001). The developmental 

time extension for the F1 generation also was noted by Mastro and Schwalbe (1988). 

However, larvae irradiated at higher doses exhibited high mortality (Figure 1a). A lot 

of larvae receiving high doses were weakened and died too early to allow parasitoids 

to complete their development. Similar problems occurred in production of sterile F1 

gypsy moths (Reardon et al. 1987). 

Partial sterility was noted in the F 1 generation (Table 4). A similar effect was 

observed by Mastro and Schwalbe (1988). If the parents are irradiated (usually 

males), F1 sterility is a well-known consequence of irradiation. It is widely used in 

integrated pest management and SIT (Bloem and Carpenter 2001). Irradiation of 

eggs and larvae generally does not cause outright sterility. Larvae do not have 

developed gonads and they generally are not be injured by radiation. However, our


experiments showed that there are effects of irradiation of the eggs and larvae on 

fertility of resulting adults. There is a question whether this is a result of the injury of 

some somatic cells in the larvae that are responsible for the creating the future 

mating organs or if this is a result of the general effect of the irradiation on the 

condition of the larvae and resulting adults. 

In conclusion, these experiments showed that a dose of 50 Gy would be 

appropriate for irradiation of gypsy moth eggs and larvae intended to be used to 

supplement naturally-occurring parasitoid, predator and pathogen populations and 

allow for an increase in the efficacy of the natural enemy complex in the field without 

a fear that they could reproduce and increase the pest’s threat. 


This study was supported by IAEA (grant No. 10849). We thank Dr. A. Robinson 

(Entomology Unit, Seibersdorf, Austria), MUDr. A. Rakytska (Roosevelt Hospital, B. 

Bystrica, Slovakia) for permission to use an irradiation source, Mr Ilkanic for technical 

assistance and Catherine Kwech for correction of English. 

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Vol. 19, S1, 2009, 303 315 


F 1 sterile insect technique: A novel approach for risk assessment of 

Episimus unguiculus (Lepidoptera: Tortricidae), a candidate biological 

control agent of Schinus terebinthifolius in the continental USA 

Onour E. Moeri a *, James P. Cuda a , William A. Overholt b , Stephanie Bloem c , 

and James E. Carpenter d 

a Department of Entomology and Nematology, University of Florida, Gainesville, FL, USA; 

b Biological Control Research and Containment Laboratory, University of Florida, Fort Pierce, 

FL, USA; c Plant Epidemiology and Risk Analysis Laboratory, USDA-APHIS-PPQ-CPHST, 

Raleigh, NC, USA; d USDA-ARS, Crop Protection and Management Research Unit, 

Tifton, GA, USA 

Federal regulations mandate that researchers in the field of classical weed 

biological control follow the precautionary principle when proposing the release 

of an organism that can affect our environment. However, laboratory risk 

assessment experiments often predict a much broader host range than that which 

occurs in the field. Because open-field tests are prohibited in the area of 

introduction, the application of the F1 sterile insect technique (F1SIT) could be 

used to conduct field testing in the proposed release area in a safe and temporary 

manner. In this study, we determined the minimum dose of radiation required to 

field test the tortricid Episimus unguiculus (Clarke), a candidate for biological 

control of Brazilian peppertree, Schinus terebinthifolius Raddi (Anacardiaceae) in 

Florida. Male and female virgin E. unguiculus adults were treated with increasing 

doses of gamma radiation and either inbred or outcrossed to non-treated 

E. unguiculus adults. Pairs of adults were placed in oviposition cages and allowed 

to mate and oviposit. Data from fecundity and fertility counts were recorded. The 

dose at which treated females were 100% sterile was 200 Gy. The dose at which F1 

females and males were 100% sterile was 225 Gy. As the dose of radiation 

increased, there was an increase in sterility, a decrease in fecundity for both 

treated female crosses, and a higher ratio of F1 males to females. The F1 sterile 

insect technique (F1SIT) could be suitably applied to other areas of pest 

management, including risk assessment of potential lepidopteran biological 

control agents of invasive, exotic weeds. 

Keywords: F1 sterile insect technique; Episimus unguiculus; weed biological 

control; inherited sterility; Brazilian peppertree 

Introduction 

Before a candidate weed biological control agent can be released into the environment, 

the host specificity of the organism must be demonstrated. Host-range testing 

is a process of screening potential biological control agents to minimize the risk 

of damage to non-target plant species. Tests involve several different plant species 

*Corresponding author. Email: oemoeri@gmail.com 




DOI: 10.1080/09583150902741932 


304 O.E. Moeri et al. 

closely and distantly related to the family of the targeted host plant (Wapshere 1974). 

Related and sometimes unrelated plant species that are economically important, 

endangered, or native are of high priority and tested (McEvoy 1996; Schaffner 2001). 

Two phases in host-range testing, no-choice and choice tests are performed in the 

laboratory (Marohasy 1998; Withers, Barton-Browne, and Stanley 1999). No-choice 

tests involve larval development and oviposition tests on a single non-target species. 

Choice tests expose the insect to two or more plants at the same time and typically 

include the host plant (McEvoy 1996; Schaffner 2001). Although these tests are 

designed to predict field host range, caged laboratory tests often overestimate host 

specificity because of unnatural behavior exhibited by some candidate biological 

control agents as a result of being in a caged environment. This type of behavior may 

produce ‘false positives’, or acceptance of plants as hosts that would not normally be 

accepted by the potential biological control agent in nature (Marohasy 1998). 

Open field testing provides a more natural assessment of the ecological host 

range of candidate biological control agents. Typically, these tests are performed in 

the native range of the target weed. However, there are serious limitations to using 

this particular approach; not the least of which is the need to import non-native test 

plant species that would most likely be prohibited from entering the country of origin 

of the biological control agent. Other issues include seasonal availability of the test 

plants or potential biological control agent, and mortality of the agent by specialized 

predators and parasitoids in the wild. 

A new approach for risk assessment that could be adopted for some candidate 

biological control agents is field testing in the area of introduction. Open field testing 

can be done in a safe, temporary manner for potential lepidopteran biological control 

agents by using the F 1 sterile insect technique (Greany and Carpenter 2000). 

Advantages of this approach include the exposure of the biological control agent to the 

actual environmental conditions it would experience if approved for release, prediction 

of true field host range, and ability to reverse releases of the biological control agent 

without permanent establishment if non-target damage is detected (Bax et al. 2001; 

Carpenter, Bloem, and Bloem 2001a). The F1 sterile insect technique (F1SIT) is similar 

to the sterile insect technique (SIT), except it uses a lower dose of radiation providing 

partial sterility and a reduced number of progeny. Lepidoptera are highly radioresistant 

and require a large dose of radiation to ensure sterility (LaChance 1985). With 

the use of F1SIT, however, a lower dose could be applied resulting in a more 

competitive insect due to decreased somatic damage (North 1975). Various studies 

have used this approach to demonstrate control of populations of pest Lepidoptera, 

including the codling moth, Cydia pomonella (L.) (Bloem, Bloem, Carpenter, and 

Calkins 1999a,b), false codling moth Thaumatotibia leucotreta (Meyrick) (Bloem, 

Carpenter, and Hofmeyr 2003), potato tuber moth Phthorimaea operculella (Zeller) 

(Makee and Saour 1997, 2003) and cactus moth Cactoblastis cactorum (Berg) 

(Carpenter et al. 2001a,b; Hight, Carpenter, Bloem, and Bloem 2005; Tate, Carpenter, 

and Bloem 2007). Because application of F 1SIT has been demonstrated in pest 

management programs, this approach has potential for evaluating the risks of releasing 

exotic lepidopteran candidates for weed biological control (Dunn 1978; Cullen 1990; 

Greany and Carpenter 2000; Tate et al. 2007). 

An example where F1SIT could be used for risk assessment purposes is in 

classical biological control of Brazilian peppertree, Schinus terebinthifolius Raddi 

(Anacardiaceae) in the continental USA. Brazilian peppertree is native to Brazil,


Paraguay, and Argentina and was introduced into Florida as an ornamental in 1898 

(Austin 1978; Ewel, Ojima, Karl, and DeBusk 1982). This non-native plant is 

considered one of Florida’s worst invasive terrestrial weeds (Austin 1978; Morton 

1978; Schmitz 1994). Brazilian peppertree is distributed widely throughout central 

and southern Florida and listed by the Florida Exotic Pest Plant Council (FLEPPC) 

as a ‘Category 1’ invasive weed because it is drastically altering native plant 

communities. It also is a major problem in Hawaii (Yoshioka and Markin 1991; 

Hight, Cuda, and Medal 2002), California (Randall 2000) and more recently Texas 

(Gonzalez and Christoffersen 2006). The environmental damage from Brazilian 

peppertree results from its ability to produce dense monospecific stands that shade 

out native plants and its allelopathic effects on competing vegetation (Gogue, Hurst, 

and Bancroft 1974; Morgan and Overholt 2005; Donnelly, Green, and Walters 2008). 

Field surveys have been conducted in Brazil since the mid 1980s to identify 

potential biological control agents of Brazilian peppertree (Bennett, Crestana, 

Habeck, and Berti-Filho 1990; Cuda, Habeck, Hight, Medal, and Pedrosa-Macedo 

2004). One of these was a tortricid leafroller Episimus unguiculus Clarke ( E. utilis 

Zimmerman) (Lepidoptera: Tortricidae). Razowski and Brown (2008) recently 

synonymised E. utilis with its senior synonym E. unguiculus. In 1954, the leafroller 

was released in Hawaii, however, it was not found to severely affect Brazilian 

peppertree (Bennett et al. 1990; Yoshioka and Markin 1991; Habeck, Bennett, and 

Balciunas 1994), which was probably due to biotic interference from introduced 

parasitoids and predators of agricultural pests (Krauss 1963; Martin et al. 2004). 

Although E. unguiculus was not effective in controlling Brazilian peppertree in 

Hawaii, the insect may be less prone to biotic mortality from introduced and native 

parasitoids and predators if it were released in Florida (Martin et al. 2004). 

The biology of E. unguiculus and methodology for its laboratory rearing were 

investigated by Martin et al. (2004). Larvae of E. unguiculus inflict damage by 

feeding on leaflets, which can eventually lead to defoliation and a reduction in plant 

growth. Recently, a simulated herbivory study was conducted that showed growth 

and reproduction of Brazilian peppertree are significantly reduced by sustained 

defoliation (Treadwell and Cuda 2007). 

Replicated no-choice and multiple choice tests with E. unguiculus were completed 

as part of the host specificity testing process, and the results showed this insect 

exhibited a wide host range under confined laboratory conditions (J.P. Cuda, 

unpublished data). At least 12 non-target native and cultivated plant species 

representing eight genera in two plant families were unexpectedly accepted as 

developmental hosts (J.P. Cuda, unpublished data). However, California peppertree 

(Schinus molle L.), which is the most closely related congener of Brazilian peppertree, 

was not attacked. We believe that the broad fundamental host range exhibited by 

E. unguiculus in the laboratory is not indicative of the field host specificity of this 

insect in a natural environment (Sheppard, van Klinken, and Heard 2005). This is 

supported by field surveys conducted in Brazil (J.P. Cuda, personal observation) and 

more recently in Argentina, where McKay et al. (2009) found E. unguiculus 

associated only with Brazilian peppertree. More importantly, there are no reports 

of E. unguiculus attacking non-target plants in Hawaii in spite of the establishment of 

the insect for over 50 years. For cases such as these, where the results of laboratory 

tests lead to unexpected false positives with lepidopterans, F1SIT could be an


additional tool to confirm the field host specificity of the candidate biological 

control agent. 

The objectives of the current study were to determine the minimum dose of 

radiation that would sterilize the F1 generation of E. unguiculus and to verify the 

effects of radiation in E. unguiculus. 


Colony rearing 

Colony rearing procedures were similar to those described by Martin et al. (2004). 

Pairs of adult E. unguiculus moths (24 48 h old) were placed on individual Brazilian 

peppertree plants planted in 3.8 L (1 gal) pots (20 22.5 cm, height diam.). Each 

plant was enclosed in a clear acrylic cylinder (45 15 cm, height diam.) with six 

evenly spaced ventilation holes (6.5 cm diam.). The top of the cylinder was covered 

with a sheer polyester fabric (Jo-Ann Fabrics † #449-1676 white casa organza) and 

all six circular ventilation holes were each covered with a mesh, screen size of 150 

150 mm (Green.tek † Inc., Edgerton, WI). The sheer polyester fabric was fastened to 

the top of the cylinder by a metal ring clamp (14.3 21.6 cm) and further sealed with a 

rubber band to prevent small larvae from escaping. Two additional access holes in 

the cylinder (2.5 cm diam.) were plugged with #5 rubber stoppers. Each cylinder was 

provided with a Gatorade † feeder which consisted of a 15-mL glass vial with a 5-cm 

piece of dental wick soaked in Gatorade † as a nectar source for the adults (Cuda, 

Deloach, and Robbins 1990; Martin et al. 2004). When approximately 90% larval 

defoliation of the plant was observed, bouquets of five stems of Brazilian peppertree 

leaves (1 5 days old, field collected in Ft. Pierce, FL) in water filled plastic vials 

(40 mL) were placed near the top of the plant in each cylinder as needed. 

Plants used for colony rearing were sprayed twice weekly with an organic 

insecticide consisting of 15 mL (1 tbsp) each of isopropyl alcohol (70%), insecticidal 

oil, and Ivory † liquid soap mixed in 3.8 L (one gallon) of water to protect the plants 

from damage by aphids and other soft-bodied pests. The rearing room was 

maintained at 25.894.08C and 40 70% RH as recorded by a Fisher Scientific † 

Thermo-Hygro † digital maximum minimum temperature and relative humidity 

recording instrument. Temperature and relative humidity recorded within the 

cylinder were 24.993.88C and6080% RH, respectively. A photoperiod of 14 h 

L:10 h D was maintained by a programmable timer connected to sets of two 60-cm 

20-W fluorescent bulbs (one standard and one Gro-Lux † ) per shelf of colony plants. 

Colony rearing and experiments with E. unguiculus were conducted at the University 

of Florida, Department of Entomology and Nematology Containment Facility, 

Gainesville, FL. 

Radiation biology study 

The procedures used to study the radiation biology were based on methodologies 

developed for the codling moth, C. pomonella (Bloem et al. 1999a,b), false codling 

moth, T. leucotreta (Bloem et al. 2003), and cactus moth, C. cactorum (Carpenter 

et al. 2001a,b). The E. unguiculus moths were collected from the colony at the fifth 

instar (red larval stage) or the pupal stage and placed individually into separate clear


plastic 30 mL (1 oz) diet cups with a 3.5-cm piece of moistened filter paper added to 

each cup to maintain humidity. Temperature and relative humidity in the experiment 

room were 24.694.58C and 50 80% RH, respectively, with a photoperiod of 14 h 

L:10 h D. The cups were checked each morning at the same time and adults were 

removed upon emergence. Male and female virgin E. unguiculus adults (B24 h old) 

were collected and individually exposed to gamma radiation in plastic snap-cap vials 

(12 mL) within an aluminum-lined cardboard canister (8.8 cm height 7.5 cm diam.). 

Doses of 0, 50, 100, 150, 200, 250, and 300 Gy were administered by using a Cesium- 

137 Gammacell † 1000 irradiator with a dose of 12 13 Gy/min (Florida Accelerator 

Services & Technology (FAST), Florida Department of Agriculture and Consumer 

Services, Division of Plant Industry, Gainesville, FL). The dose rate was determined 

by a dosimetry study using Far West † dosimetry film. Two canisters each containing 

10 of the plastic vials were placed on top of each other within the irradiator. 

Dosimetry film was placed in three different positions within four different vials (one 

from the top and bottom of each canister). Results indicated that the canister 

containing the plastic vials should only be positioned at the bottom of the irradiator 

in order to minimize the variance between the levels of irradiation. 

Five treated (T) male or female moths were placed inside a triangular waxed 

paper oviposition chamber (30 19 12 cm) with an equal number of either treated 

(T) or non-treated (N) adult moths of the opposite gender. Five replicates of three 

different crosses (treatments) (Nà Tß, Tà Nß, and Tà Tß) were completed 

for each dose of radiation. The oviposition chamber was then placed inside a 3.8 L 

(1 gal) plastic sealable freezer bag (Ziploc † ) to maintain relative humidity and 

suspended on a string line to maximize the use of the limited amount of space in the 

containment laboratory. Temperature and relative humidity within the oviposition 

chamber were recorded as 24.193.88C and 60 80% RH, respectively. Each 

oviposition chamber included a 2-cm piece of cotton dental wick soaked in 

Gatorade † as a nectar source and a small leaf disc of Pistacia vera L. (2.4 2.4 

cm). Due to inconsistent oviposition in preliminary experiments with non-treated 

moths, small leaf discs of Brazilian peppertree were substituted with P. vera to 

stimulate oviposition. A phytochemical study of the leaves and bark of Schinus 

terebinthifolius had previously found that its compounds show a greater similarity to 

compounds isolated from Pistacia species than to those isolated from other species 

of Schinus (Campello and Marsaioli 1975). It was later determined that the leaf 

material was not a factor in the preliminary results, yet the P. vera leaf discs were 

used throughout the rest of the experiments for consistency. The moths were allowed 

to mate and lay eggs for two intervals of 5 days to take into account the 7-day 

average lifespan for the adults (Martin et al. 2004). After the first 5-day period, they 

were transferred to a new oviposition chamber. At the end of the 10-day period, the 

females were collected, preserved in ethyl alcohol (80%), and subsequently dissected 

to determine their mating status (presence of spermatophores or inflated bursa 

copulatrix) (Ferro and Akre 1975). The egg sheets were then incubated for a period 

of 7 days at 24.694.58C, 50 80% RH, and a photoperiod of 14 h L:10 h D, which 

corresponded to the developmental time of the egg stage (Martin et al. 2004). The 

total number of eggs laid (fecundity) and the number of eggs that hatched (fertility) 

were then counted for each egg sheet per radiation dose.


Inherited sterility study 

Based on the findings from the radiation biology study, five doses of radiation were 

chosen for evaluation of the cross Nà Tß (non-treated female treated male). 

Offspring of this cross would achieve inherited sterility. Radiation doses included 

125, 150, 175, 200, 225 Gy, and a control (0 Gy). The protocol was the same as 

previously described, with the exception that the F1 egg sheets were each placed on 

a Brazilian peppertree plant in a 3.8-L (1 gal) pot enclosed by a clear acrylic 

cylinder (45 15 cm, height diam.) in order to rear the F1 generation. Average 

temperature and relative humidity recorded within the cylinder were 25.293.68C 

and 70 90% RH, respectively. When the larvae hatched, they were allowed to 

develop on the plant. At the fifth instar (red larval stage) or the pupal stage, the 

insects were collected and each individual was placed in a separate clear plastic diet 

cup 30 mL (1 oz) with a 3.5-cm piece of moistened filter paper to maintain 

humidity. Upon emergence, each F1 female or male was outcrossed with a nontreated 

adult moth of the opposite sex. These F1 crosses were made as single pairs 

(1 female 1 male). The protocol for the single pair crosses was the same as 

previously described for the radiation biology study. Ten crosses of F1 females and 

males were attempted for each dose, but due to virgin females (found not to be 

mated upon dissection) and/or limited emergence of adults, there was a range of 

five to 12 replications for each gender per dose. Temperature and relative humidity 

in the experiment room were 25.694.48C and 40 70% RH, respectively, with a 

photoperiod of 14 h L:10 h D. The temperature and relative humidity recorded 

inside the oviposition chamber were slightly higher, averaging 26.194.98C and 

50 60% RH, respectively. 



In order to determine the effect of radiation dose on fecundity, linear regressions 

using fecundity as the response variable (Y) and radiation dose as the treatment 

variable (X) were performed. A separate model was fitted for each of the treatments. 

Effect of radiation dose on fertility was determined by performing simple linear 

regressions of radiation dose predicting fertility for each of the crosses. In some 

cases, a polynomial model was indicated by scatter plots of the data. Alpha level for 

all of the regressions was P 0.05. Regression analyses were performed using 

S-plus † 7.0 for Windows † (Insightful 2005). 


To determine the effects of radiation on the reproductive biology of F1 offspring of 

irradiated males, linear and nonlinear regressions of radiation dose administered on 

fecundity and fertility of the offspring were performed, fitting polynomial models 

where appropriate. Sex ratio was recorded for F1 males and analyzed using a simple 

regression. Alpha level for each of the factors was P 0.05. Regression analyses were 

performed using S-plus † 7.0 for Windows † (Insightful 2005).


Results 


Effects of the radiation treatments on adults of E. unguiculus were dependent upon the 

dose of radiation and gender irradiated. In irradiated males, no significant changes in 

fecundity of mated females were observed as radiation dose increased (Nà Tß; 

F 3.67; df 1, 31; P 0.05; R 2 

0.11), whereas in irradiated females, significantly 

fewer eggs were laid as dose increased (Tà Nß; y 71.67 0.15x; F 11.62; df 1,32; 

PB0.05; R 2 

0.27; Tà Tß; y 81.77 0.21x; F 16.85; df 1, 31; PB0.05; 

R 2 

0.35) (Figure 1). Fertility for treated females also decreased with increasing 

radiation dose (Tà Nß; y 60.01 0.68x 0.0017x 2 ; F 56.31; df 2, 30; PB0.05; 

R 2 

0.79; Tà Tß; y 63.62 0.76x 0.0020x 2 ; F 53.74; df 2, 30; PB0.05; R 2 

0.78) and the same effect was observed for treated males crossed with non-treated 

females (Nà Tß; y 64.43 0.20x; F 57.35; df 1,31; PB0.05; R 2 

0.65) 

(Figure 2). Additionally, the dose of radiation at which treated females were found 

to be 100% sterile was 200 Gy (residual fertility of 0.1% at 150 Gy), whereas males 

irradiated at 200 Gy still had a residual fertility of 18%. Mating was confirmed in all 

adult female moths used in the experiments as determined by the presence of 

spermatophores or inflated bursa copulatrix (Ferro and Akre 1975). 


With respect to the fecundity for F1 females, there was no significant relationship 

between the dose of radiation administered to the treated male in the parental cross 

and fecundity observed in the F1 generation (F1à Nß; F 0.07; df 1, 42; P 0.05; 

R 2 

0.002; Nà F1ß; F 1.11; df 2, 52; P 0.05; R 2 

0.04) (Figure 3). Percent egg 

Figure 1. Fecundity (mean number of eggs laid) per mated female of Episimus unguiculus 

adults for three crosses (Tà Tß, Tà Nß and Nà Tß) treated with increasing doses of 

gamma radiation.


Figure 2. Fertility (mean percentage of eggs that hatched) of Episimus unguiculus adults for 

three crosses (Tà Tß, Tà Nß and Nà Tß) treated with increasing doses of gamma 

radiation. 

hatch for treatments with the F1 males and females both declined with an increased 

dose of radiation (Nà F1ß; y 55.16 0.60x 0.0016x 2 ; F 40.31; df 2, 52; 

PB0.05; R 2 

0.61; F1à Nß; y 63.85 0.32x; F 58.28; df 1, 43; PB0.05; 

R 2 

0.58) (Figure 4). For both the F1 female and male treatments, 100% sterility 

was achieved at 225 Gy, the minimum dose at which no viable offspring could survive. 

Figure 3. Fecundity (mean number of eggs laid) of F1 crosses (F1à Nß, Nà F1ß) of 

Episimus unguiculus adults when increasing doses of radiation were administered to 

parental males.


Figure 4. Fertility (mean percentage of eggs that hatched) of F 1 crosses (F 1à Nß, Nà 

F1ß) ofEpisimus unguiculus adults as a result of radiation administered to parental males. 

Figure 5. Percentage of F1 Episimus unguiculus adult males as a result of radiation 

administered to Episimus unguiculus parental males. 

In addition, an increasing ratio of F1 males to females was positively correlated with an 

increase in radiation dose, although the relationship was marginally significant 

(y 54.98 0.12x; F 7.52; df 1, 4; P 0.05; R 2 

0.65) (Figure 5). 

Discussion 

F1SIT has been used in several studies to control various pest Lepidoptera. The 

technique provides a safe, environmentally friendly approach to pest management. 

Early studies by Proverbs (1962), who first documented partial sterility in the codling 

moth, found that when males were partially sterilized and mated to wild females, the 

progeny number was reduced, mostly male, and highly sterile. Subsequent studies by 

North (1975) and LaChance (1985) comparing the use of partial sterility with


complete sterility in Lepidoptera determined that a partial sterilizing dose of 

radiation would increase competitiveness, possibly cause a delay in development, and 

lower sperm quality in the F1 generation. 

Recent laboratory and field studies have confirmed these effects in the codling 

moth (Bloem et al. 1999a,b) and false codling moth (Bloem et al. 2003), both members 

of the same family (Tortricidae) as E. unguiculus. Results of this study were similar to 

results found in previous studies. Higher doses of radiation resulted in an increase in 

sterility, a higher ratio of F1 males to females, and a declining trend in fecundity for 

both treated female crosses. Irradiated females of E. unguiculus were found to be 100% 

sterile at 200 Gy, which is similar to the female false codling moth but more 

radioresistant than the female codling moth in which complete sterility was observed at 

100 Gy. When treated males of E. unguiculus were mated with non-treated females, 

sterility of the F 1 generation was similar to that reported for other tortricids. In 

particular, the dose at which E. unguiculus was found to be 100% sterile was 225 Gy, 

whereas the dose for the codling moth was 250 Gy (Bloem et al. 1999a,b), and the range 

of partial sterility for the false codling moth was 150 200 Gy (Bloem et al. 2003). We 

therefore determined that a radiation dose of 225 Gy will provide 100% sterility and 

ensure a reduced number of progeny which are more sterile than their parents and 

mostly male. 

Our results clearly show that F1SIT could be appropriately applied to other areas 

of pest management, including risk assessment of potential lepidopteran biological 

control agents of invasive, exotic weeds. A relevant example is the invasive Brazilian 

peppertree in Florida and E. unguiculus, an established biological control agent of 

Brazilian peppertree in Hawaii where no non-target impacts have been documented. 

Because laboratory host specificity tests showed that the fundamental host range of 

E. unguiculus is broader than expected (J.P. Cuda, unpublished data), the cage 

environment in laboratory screening tests potentially inhibited the normal behavior 

of the insects and enabled them to accept plants that they would not normally 

recognize in nature (Withers et al. 1999). Therefore, field testing in the proposed area 

of introduction would provide more accurate results. In this study, we found that a 

dose of 225 Gy can be applied to E. unguiculus adult male moths and upon mating 

with non-treated female moths; complete sterility in the F1 generation is assured. 

Based on fecundity results of females mated with irradiated male parents, no 

significant relationship was found between treatment dose and fecundity of females, 

therefore suggesting that the number of eggs laid would be similar to non-irradiated 

moths. However, fertility recorded in the irradiated male moth treatments (Nà Tß) 

would be greatly reduced. Normal oviposition behavior as well as larval damage and 

feeding would occur under natural conditions except that the F 1 generation would be 

unable to reproduce. Performance of irradiated E. unguiculus males should be similar 

to non-treated males based on results of previous studies examining the effects of 

radiation on other tortricid moths (Bloem et al. 1999a,b, 2003). An additional safety 

factor of the technique is the fact that most of the F1 progeny will be males, therefore 

limiting the number of matings. 

Using F1SIT in addition to laboratory host-range testing can provide a temporary 

and reversible way to test potential biological control agents in the proposed 

area of release as proposed by Bax et al. (2001) and Greany and Carpenter (2000). 

Further studies will be needed to address the performance of irradiated biological


control agents including oviposition, larval feeding preferences and survival, and 

host-finding behavior. 


We thank Dr Burrell Smittle, Carl Gillis, and Suzanne Fraser at The Florida Accelerator 

Services and Technology Gainesville, FL for their support and assistance in the irradiation of 

the E. unguiculus moths. We thank Judy Gillmore and students in the Weed Biological Control 

Laboratory, Entomology & Nematology Department, Institute of Food and Agricultural 

Sciences, University of Florida, for their support and maintenance of E. unguiculus colonies. 

We also thank Veronica Manrique for supplying Brazilian peppertree plants and Sean 

McCann for help with statistical analysis. Finally, we thank the South Florida Water 

Management District, the Florida Fish and Wildlife Conservation Commission (formerly the 

Department of Environmental Protection), and The University of Florida, IFAS, Center for 

Natural Resources for supporting this research. 

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Vol. 19, S1, 2009, 317 333 


Oviposition preference of Cactoblastis cactorum 

(Lepidoptera: Pyralidae) in caged choice experiments and 

the influence on risk assessment of F 1 sterility 

C.D. Tate a , S.D. Hight b , and J.E. Carpenter c * 

a USDA-APHIS-PPQ-CPHST, CPHST Laboratory, Phoenix, AZ, USA; b USDA-ARS- 

CMAVE at Center for Biological Control, FAMU, Tallahassee, FL, USA; c USDA-ARS 

Crop Protection & Management Research Unit, Tifton, GA, USA 

Releases of lepidopteran biological control agents have successfully controlled 

invasive weed species. However, issues with non-target effects of released exotic 

agents have resulted in stringent pre-release host specificity testing. Use of 

inherited (F1) sterility, a radiation induced genetic condition that can cause 

sterility in the F1 generation, could further assess the risk of non-target effects and 

negative ecological effects under field conditions. This technique may aid in 

approving potentially effective and safe biological control agents for release. The 

unintentional arrival of the cactus moth, Cactoblastis cactorum, into the United 

States provides a unique opportunity to evaluate the potential of F1 sterility. This 

study was conducted to assess host oviposition preferences of C. cactorum females 

mated with irradiated and non-irradiated males for cactus species from seven 

groups based on location, cactus growth characteristics (plant structure), spine 

densities, genera, and economic importance. No significant differences in female 

host preference were observed between females mated with normal or irradiated 

males. Lack of significant differences in oviposition preference suggests that 

inherited (F1) sterility has potential as a risk assessment tool for potential exotic 

biological control agents for invasive weed species. Evaluation of the overall 

analysis of female C. cactorum host preference revealed that significantly different 

numbers of eggsticks were oviposited on cactus species. In whole plant cages, 

significantly more eggsticks were oviposited on Opuntia corallicola than any other 

species, and in cladode cages, significantly more eggsticks were oviposited on 

Opuntia humifusa than all other species except Opuntia pusilla. 

Keywords: cactus moth; inherited sterility; invasive species; risk assessment; weed 


Introduction 

Lepidoptera species have been successful biological control agents of invasive 

weeds. For example, the cactus moth, Cactoblastis cactorum (Berg) (Lepidoptera: 

Pyralidae), has been cited as one of the world’s most successful biological control 

programs against weeds (Dodd 1940; Petty 1948; Moran and Zimmerman 1984). 

Release and establishment of C. cactorum in Australia, South Africa, the Caribbean, 

and many other countries resulted in significant reductions in several invasive and 

native pest Opuntia spp. (Dodd 1940; Simmonds and Bennett 1966; Julien and 

*Corresponding author. Email: jim.carpenter@ars.usda.gov 



This work was authored as part of the Contributor’s official duties as employees of the United States Government and is 

therefore a work of the United States Government. In accordance with 17 U.S.C. 105 no copyright protection is available 

for such works under U.S. law. 

DOI: 10.1080/09583150902814507 


318 C.D. Tate et al. 

Griffith 1998; Pemberton and Cordo 2001). However, the unintentional arrival of C. 

cactorum into the United States (specifically the Florida Keys) through either natural 

dispersal (Zimmerman, Moran, and Hoffmann 2001; Stiling 2002) or human 

facilitated introductions, e.g., Opuntia cactus commerce (Pemberton 1995) or 

human-aided dispersal (Stiling and Moon 2001), has become an example of 

potential risks inherent in biological control agents. 

Cactoblastis cactorum continues to migrate northward and westward since 

arriving in the Florida Keys in 1989 (Hight, Bloem, Bloem, and Carpenter 2003). 

Populations have established as far north as Bull Island, near Charleston, South 

Carolina and currently, as far west as the Mississippi barrier Islands of Petit Bois 

and Horn (Hight and Carpenter 2009). The rate of migration and establishment of 

C. cactorum populations poses a potential threat to Opuntia diversity throughout 

North America and the Caribbean basin, and to wild and cultivated Opuntia spp. in 

the southwestern US and Mexico (Strong and Pemberton 2000; Soberón, Golubov, 

and Sarukhan 2001; Perez-Sandi 2001). 

Presence of C. cactorum in the southeastern US and its dual status, both as a 

beneficial and pest species, provides a unique model system to conduct inherited 

sterility proof-of-concept studies using this non-native insect species as a test subject. 

Inherited sterility (F1 sterility) is a radiation-induced genetic condition that can 

cause sterility in the F1 generation (LaChance 1985). Insects carrying this condition 

are not able to reproduce in the field. This unique genetic feature may be used to 

evaluate, under field conditions, the risks of releasing agents that may cause nontarget 

and negative ecological effects, and to increase the chance of approving 

valuable and safe biological control agents for release (Greany and Carpenter 2000; 

Tate, Carpenter, and Bloem 2007; Moeri, Cuda, Overholt, Bloem, and Carpenter 

2009). 

In a classical weed biological control program, an agent’s host specificity must 

first be defined before it can be considered for release into its new homeland. Over 

the years, practitioners of biological control, government regulators, and the public 

have required increasingly narrower host specificity ranges and insurances that nontarget 

native species will not be harmed before a non-native agent is released into the 

wild. Weed biological control has a rigorous protocol of evaluating host range of 

biological control agents (Wapshere 1974; Marohasy 1998; Briese and Walker 2002; 

Briese 2003). Experiments are designed to evaluate oviposition preference of the 

females and performance of the larvae (McEvoy 1996). Testing regimes involve 

quarantine studies on caged native plants in the region of introduction to identify the 

host range of potential biological control agents. An increasing number of host 

specificity evaluations include open field tests in the agents’ homeland (Briese, 

Zapater, Andorno, and Perez-Camargo 2002). However, both of these types of tests 

have limitations. Quarantine studies are conducted inside cages in a laboratory and 

have been shown to interfere with the normal behavior and biology of the biological 

control agent (Marohasy 1998). Open field studies in the agents’ native range are 

conducted in more realistic settings, but these studies are fraught with problems 

relating to the availability and seasonality of test plants from outside the native range 

of the biological control agent. A potential new approach for evaluating host 

specificity is to conduct open field tests in the region where the target weed has 

become invasive with promising biological control agents that are rendered safe 

through F1 sterility (Greany and Carpenter 2000; Moeri et al. 2009).


Before a non-native insect is tested in an open field setting, the sterility of the 

released individuals must be proven. Carpenter, Bloem, and Bloem (2001a) evaluated 

gamma radiation dose effects on the fecundity and fertility of C. cactorum. From 

these evaluations, a minimum dose at which irradiated females and males were 100% 

sterile when mated to fertile males and females was established and the occurrence of 

F1 sterility in this species was verified. Additionally, Carpenter et al. (2001a) 

suggested doses between 100 and 200 Gray (Gy) would allow for maximum 

production of F1 adults while inducing full sterility in the F1 generation. 

When determining the usefulness of an F 1 sterile open field-testing protocol, 

another important aspect is to ensure that the oviposition preference of females 

mated to irradiated males is the same as females mated to normal (unirradiated) 

males. Unaltered oviposition host preference would suggest that F 1 sterility is 

potentially a risk assessment tool for evaluating the host range of non-native 

biological control agents in a more realistic setting. Open field evaluations of F1 

sterile potential biological control agents in their non-native range would be free of 

risks to non-target species. 

In this study, we evaluated host preference in greenhouse cages of female 

C. cactorum mated with either normal or with irradiated male moths. Cactus 

plants native and introduced to the southeastern US, growth characteristics (plant 

structure), spine densities, non-Opuntia cactus genera, and economic importance 

also are considered and discussed in this study. 


Laboratory reared C. cactorum originating from wild populations collected on the 

causeway connecting the Georgia mainland with Jekyll Island, GA and along the 

Florida Gulf Coast were used in this study. Larvae were reared on Opuntia ficusindica 

(L.) P. Miller cladodes (flattened green stems of Opuntia spp.) in rectangular 

plastic containers (34 24 13 cm) at 268C, 70% relative humidity (RH), and a 

photoperiod of 12 h L:12 h D (Carpenter et al. 2001a). Cocoons were collected 2 3 

days after initiation of pupation. Cocoon silk was removed from pupae with a 5% 

sodium hypochlorite (NaOCL) solution. Pupae were sorted by gender, with male and 

female pupae held separately in 475 mL plastic cups at the above conditions. Just 

before emergence, male and female pupae were placed inside separate screen cages 

(30.5 30.5 30.5 cm) and allowed to emerge at room temperature (23918C) 

(Carpenter, Bloem, and Bloem 2001b). 

Virgin male and female C. cactorum were individually placed in separate 30 mL 

plastic containers (BioServ, Frenchtown, NJ, USA) and held at 68C until quiescent 

(30 min). When the moth flight risk appeared low, cohorts of 10 or 20 male moths 

were placed in separate fabric containers (six containers of each density). A piece of 

paper towel was inside each container to provide a place for moths to rest, and an 

additional piece was placed on top of the container to enclose the moths. Half of the 

containers at each density (three containers or 90 moths) were irradiated at 200 Gy 

in a Cobalt60 gammacell 220 irradiator. Equal numbers of female moths were placed 

in all containers with irradiated and control males. Containers were transported to 

the greenhouse for release into oviposition cages. 

Whole potted cactus plants were evaluated in large cages (1 m 3 ) and excised 

cactus cladodes were evaluated in small cages (30 cm 3 ). Whole plants were used to


evaluate plant structure and growth characteristics as possible factors that influence 

moth oviposition preference. Excised cladodes were used to examine the presence of 

other factors that may influence moth oviposition preference, such as plant surface 

properties and plant volatiles. Choice tests were used to evaluate oviposition 

preference of C. cactorum. Potted plants and cladodes were randomly positioned 

in each cage. A single container with 20 pairs of C. cactorum moths was released in 

each large cage and a single container of 10 pairs of C. cactorum moths was released 

in each small cage. Treatments were randomly assigned to large and small cages, 

resulting in a completely randomized design with three replications. 

Cactoblastis cactorum oviposition preference for five native and one 

introduced Opuntia species found in Florida 

Cactoblastis cactorum oviposition was evaluated in greenhouse cages on six Opuntia 

species commonly found in Florida: O. corallicola (Small) Werdermann, O. ficusindica, 

O. humifusa (Rafinesque) Rafinesque, O. pusilla (Haworth) Haworth, 

O. stricta (Haworth) Haworth, and O. triacantha (Wildenow) Sweet. Irradiated 

(200 Gy) and normal male moths paired with normal females at densities previously 

described were placed in large and small cages for 5 days. 

Female C. cactorum stack their eggs on top of one another so that the group of 

eggs resembles a cactus spine, and the spine mimic is called an eggstick. Eggsticks 

were collected daily from each Opuntia spp. in each test cage, and the number of 

eggsticks and eggs within each eggstick recorded. 

Cactoblastis cactorum oviposition preference for cactus species in six select groupings 

Female C. cactorum moth oviposition preference was evaluated in greenhouse cages 

on cactus species in the Genera Opuntia, Cylindropuntia, and Harrisia. Six groups of 

four cacti were selected to evaluate several different characteristics (Table 1). The 

groups were distinguished as follows: Group 1 comparison of Opuntia spp. with 

similar growth characteristics, i.e., tall upright species; Group 2 comparison of 

Opuntia spp. with differing spine and glochid complements virtually lacking spines 

and glochids to dense glochids or dense spines; Group 3 comparison of different 

species in the Genera Opuntia, Cylindropuntia, andHarrisia; Group 4 comparison 

of documented C. cactorum hosts and non-hosts; Group 5 comparison of cactus 

species present in Florida and species native to the southwestern US and Mexico; 

Group 6 comparison of economically important Opuntia species. 

Irradiated (200 Gy) and normal male moths paired with normal females in 

densities previously described were placed in large and small cages for 3 days. The 

number of days for oviposition was reduced from the previous experiment because 

70% of eggsticks were oviposited in the first 3 days. The number of eggsticks 

collected after 3 days and the number of eggs per eggstick were recorded by host 

plant species. 

Analyses 

Cactoblastis cactorum oviposition preference for six species of Opuntia were ranked 

based on numbers of eggsticks oviposited on each plant (Proc Rank procedure of

Table 1. Groupings of cactus species used in experiments evaluating Cactoblastis cactorum 

oviposition preference for six select groups of four cactus species. 

Group no. and characteristic Test species Categorical trait evaluated 

1 Opuntia spp. with similar 

growth habit 

2 Opuntia spp. with different 

spine/glochid densities 

3 Species in the Genera Opuntia, 

Cylindropuntia, &Harrisia 

4 Documented host and non-host 

species 

O. cochenillifera Tall plant, flat stems, lack spines 

O. corallicola Tall plant, flat stems, dense spines 

O. falcata Tall plant, flat stems, lack spines 

O. stricta Tall plant, flat stems, intermediate 

spine 

O. falcata Lack spines/glochids 

O. microdasys Dense glochids 

O. polyacantha Dense spines 

O. stricta Intermediate spine/glochid density 

C. acanthocarpa Round stems 

C. spinosior Round stems 

H. fragrans Angular stems 

O. stricta Flat stems 

C. spinosior Non-host 

H. fragrans Non-host 

O. streptacantha Host 

O. stricta Host 

5 Species native or not native to FL H. fragrans Native to Florida 

O. dellinii Native to Florida 

C. spinosior Native to southwestern US 

O. streptacantha Native to southwestern US 

6 Economically important 

Opuntia spp. 


O. engelmannii Livestock food & ornamental 

O. ficus-indica Human vegetable & fruit; ornamental 

O. streptacantha Human fruit 

O. stricta Wildlife food 

The categorical trait evaluated for each cactus species under each grouping is identified. 

SAS † , SAS Institute Inc. 1999). Rankings were based on a scale from 1 to 24, with 

the lowest number representing the highest rank the species that received the most 

eggsticks. Rankings included all possible treatment combinations among species, 

radiation, and cage size. Resulting rankings, numbers of eggsticks, and numbers of 

eggs per eggstick were analyzed with ANOVA using Proc Mixed procedure of SAS † 

(SAS Institute Inc. 1999). Cage type, cactus species, and treatment (irradiated or 

normal males) were treated as fixed effects, while replication was treated as a random 

effect. 

Oviposition preferences for six select groups of four cactus species separated 

according to growth habit, spine density, family or genus classification, and 

economic importance also were ranked (1 18) based on numbers of eggsticks


oviposited on each plant using Proc Rank procedure of SAS † (SAS Institute Inc. 

1999). Resulting rankings, proportions of eggsticks, and numbers of eggs per 

eggstick were analyzed with ANOVA using Proc Mixed procedure of SAS † (SAS 

Institute Inc. 1999). Cage type, group, cactus species, and treatment (irradiated or 

normal male) were treated as fixed effects, while replication was treated as a random 

effect. 

Degrees of freedom were adjusted using Satterthwaite approximation method in 

all experiments. Means were separated using Tukey Kramer mean separation 

procedures. Data were log transformed when standard deviations were proportional 

to the mean (heteroscedasticity) and distributions were skewed. 

Results 

No significant differences in oviposition preference were found between the two 

main treatments, C. cactorum females mated to normal males versus C. cactorum 

females mated to irradiated males. Differences were not found for females that mated 

with irradiated or unirradiated males in any of the three measured ovipositional 

parameters (ranking of cactus host species that received eggsticks, proportion of 

eggsticks oviposited per host species, or mean number of eggs per eggstick oviposited 

on cactus species) (Table 2). In addition, no significant interactions were found 

between the main treatment effects and any other fixed effect (cage type or cactus 

species). Because the measurements between the two treatments were not different, 

data were pooled and presented as an analysis of oviposition host specificity for C. 

cactorum. 

Cactoblastis cactorum oviposition preference for five native and one introduced 

Opuntia species found in Florida 

A significant cactus species by cage type interaction effect was observed on species 

ranking (Table 3; F 12.09, df 5, 56.7, PB0.0001). Opuntia corallicola and 

O. stricta mean rankings were significantly higher ranked in the large cage, while 

O. humifusa, O. pusilla, and O. tricantha were significantly higher ranked in the small 

cage. Opuntia corallicola and O. humifusa had significantly higher rankings than all 

other cactus species tested, except O. pusilla. 

Cactus by cage type interaction significantly affected the proportions of eggsticks 

oviposited on cactus species (Table 3; F 29.3, df 5, 60, PB0.0001). Mean 

proportions of eggsticks oviposited on O. corallicola were significantly higher in large 

cages than in small cages; conversely, O. humifusa, O. pusilla, and O. tricantha were 

significantly higher in small cages. Significantly higher proportions of eggsticks were 

oviposited on O. corallicola than any other cactus species (Table 3; F 18.2, df 5, 

60, PB0.0001). 

A significant cactus and cage type interaction effect was observed on mean 

numbers of eggs per eggstick (Table 3; F 2.9, df 5, 49.4, P 0.02). No cactus 

species effect (F 1.74, df 5, 49.2, P 0.14) was observed on mean numbers of 

eggs per eggstick: however cage type (F 3.8, df 1, 51.9, P 0.05) had a significant 

effect on mean numbers of eggs per eggstick.

Table 2. Mean (9SE) values for oviposition parameters of the two main treatments; female 

Cactoblastis cactorum mated to normal males, and females mated to irradiated males. 

Ovipostional 

preference 

comparison 

Six common Opuntia 

spp. in FL 

Opuntia spp. with 

similar growth 

characteristics 

Opuntia spp. with 

different spine & 

glochid compliments 

Species of Opuntia vs. 

Cylindropuntia vs. 

Harrisia 

Documented hosts vs. 

undocumented hosts 

Cactus species in FL 

vs. cactus species in 

Southwest 

Economically 

important Opuntia 

spp. 

Normal 

ß 

Ranking Proportion of 

eggsticks 

Irradiated 

ß 

Normal 

ß 

Irradiated 

ß 

Number of 

eggs/eggstick 

Normal 

ß 

Irradiated 

ß 

10.991.9 11.991.9 0.5190.13 0.4990.13 25.292.1 24.892.1 

8.790.4 9.090.4 0.5090.19 0.5090.19 29.391.7 27.691.7 

9.990.8 10.390.8 0.5090.04 0.5090.04 15.893.2 17.093.1 

9.590.8 10.390.8 0.5090.05 0.5090.5 23.192.5 20.292.4 

9.990.7 9.490.7 0.4990.04 0.5190.04 22.392.2 20.392.4 

8.691.8 8.591.8 0.5090.04 0.5090.04 19.894.2 16.594.7 

9.890.9 11.190.9 0.5490.04 0.4690.04 20.892.2 15.894.1 

No significant differences were found between the two treatments for any of the three parameters at a 

P 0.05. 

Table 3. Mean (9SE) Cactoblastis cactorum oviposition preference ranking, proportion of 

eggsticks per plant, and numbers of eggs per eggstick on five native and one introduced Opuntia 

species found in Florida. 

Cage 

O. corallicola 

(Native) 


O. ficus-indica 

(Introduced) 

Cactus species 

O. humifusa 

(Native) 

O. pusilla 

(Native) 

O. stricta 

(Native) 

O. tricantha 

(Native) 

Rankings 

Small 10.892.6B 10.892.6B 2.392.6A 5.792.6AB 21.392.6C 8.292.6B 

Large 2.692.4A* 13.392.4B 15.592.4B* 15.192.4B* 15.992.4B* 15.192.4B* 

Proportion of eggsticks 

Small 0.1190.04C 0.1390.04B 0.2990.04A 0.269.04AB 0.0190.04D 0.2090.04A 

Large 0.5990.03A* 

Eggs per eggstick 

0.1290.03B 0.0790.03B* 0.079.03B* 0.0790.03B 0.0890.03B* 

Small 18.494.2B 26.394.6AB 27.794.2A 24.6 94.2AB 24.099.8AB 17.994.6B 

Large 34.093.6A* 29.093.6AB 26.494.1B 25.093.9B 27.793.6B 16.893.6C 

Uppercase letters denote significant differences between means within a row and an asterisk (*) denotes 

significant differences between cage means within a column for each variable.


Cactoblastis cactorum oviposition preference for cactus species in six select groupings 

Group 1: comparison of Opuntia cacti with similar growth characteristics 

Cactus species ranking was significantly affected by interaction between cactus 

species and cage type (Table 4; F 18.1, df 3, 48, PB0.0001). Mean rank of 

O. corallicola and O. falcata (Ekman & Werdermann) were significantly higher in 

large cages than in small cages; while O. stricta ranked higher in small cages than in 

large cages. Opuntia corallicola (the endangered south Florida species) in large cages 

was ranked significantly higher than all other tall cactus species evaluated. 

Cactus by cage type interactions (Table 4; F 19.6, df 3, 48, PB0.0001) 

significantly affected proportions of eggsticks oviposited on tall cactus species. 

Significantly higher proportions of eggsticks were oviposited on O. corallicola in 

large cages than in small cages; while significantly more eggsticks were oviposited on 

O. stricta in small cages than large cages. Mean proportions of eggsticks oviposited 

on the endangered species (O. corallicola) were significantly higher than all tall 

cactus species evaluated (Table 4; F 55.5, df 5, 60, PB0.0001). 

A significant interaction between cage type and cactus species was observed on 

numbers of eggs per eggstick on cactus species with similar growth characteristics 

(Table 4; F 33.4, df 3, 47, PB0.0001). Mean numbers of eggs per eggstick on 

O. cochenillifera and O. stricta were significantly higher in small cages than large 

cages. Significantly fewer eggs per eggstick were oviposited on O. cochenillifera and 

O. stricta plants in large cages than all other cactus species in any size cage; however, 

significantly more eggs per eggstick were oviposited on O. cochenillifera than on 

O. stricta plants in large cages. 

Group 2: comparison of Opuntia cacti with differing spine and glochid complements 


species and cage type for this group of Opuntia spp. (Table 5; F 4.1, df 3, 48, 

Table 4. Group 1: comparison of four Opuntia species with similar growth characteristics for 

mean (9SE) Cactoblastis cactorum oviposition preference ranking, proportion of eggsticks per 

plant, and numbers of eggs per eggstick. 


Cage O. cochenillifera O. corallicola O. falcata O. stricta 

Rankings 

Small 12.590.8C 6.990.8A 7.090.8A 9.390.8B 

Large 13.390.8C 1.590.8A* 4.890.8B* 15.490.8D* 


Small 0.1090.04B 0.3590.04A 0.3090.04A 0.2590.04A 

Large 


0.0490.04C 0.5190.04A* 0.2790.04B 0.1390.04C* 

Small 39.293.2A 29.093.0B 38.393.0A 32.193.0B 

Large 13.393.0B* 32.693.0A 36.693.0A 6.493.0C* 




P 0.01). Mean rank of O. polyacantha and O. stricta rankings were significantly 

higher in small cages than in large cages. Opuntia falcata (lacking spines and 

glochids) in large cages was ranked significantly higher than all other test cactus 

species. 

Cactus by cage type interaction (Table 5; F 7.0, df 3, 48, P 0.0006) 

significantly affected proportions of eggsticks oviposited on cactus species in the 

second test group. Mean proportions of eggsticks oviposited on cactus species 

evaluated differed among cage types, with the exception of O. microdasys. 

Significantly higher proportions of eggsticks were oviposited on O. falcata in large 

cages than in small cages. However, significantly higher proportions of eggsticks 

were oviposited on O. polyacantha and O. stricta in small cages than large cages. 

Mean proportions of eggsticks oviposited on the non-spiny O. falcata was 

significantly higher than all cactus species evaluated (Table 5; F 19.7, df 3, 

48, PB0.0001). 

A significant interaction effect between cage type and cactus species (Table 5; F 

4.3, df 3, 37.6, P 0.01) was observed on numbers of eggs per eggstick. Cactus by 

cage type interaction (Table 5; F 10.5, df 3, 37.3, PB0.0001) significantly 

affected numbers of eggs per eggstick. Mean numbers of eggs per eggstick on O. 

polyacantha and O. stricta in small cages was significantly higher than in large cages. 

Significantly higher numbers of eggs per eggstick were observed on non-spiny O. 

falcata in large cages. The two species with the most glochids/spines (O. microdasys 

and O. polyacantha) had significantly lower numbers of eggs per eggstick in small 

cages. Significantly more eggs per eggstick were collected from O. microdasys in large 

cages than from O. polyacantha, and O. stricta in large cages. 

Group 3: comparison of Opuntia, Cylindropuntia, and Harrisia cacti 

Cactus species ranking was significantly affected by cage type (Table 6; F 13.4, 

df 1, 48, P 0.0006) and cactus species (Table 6; F 3.39, df 3, 48, P 0.03). 

Table 5. Group 2: comparison of four Opuntia species with differing spine and glochid 

complements for mean (9SE) Cactoblastis cactorum oviposition preference ranking, 

proportion of eggsticks per plant, and numbers of eggs per eggstick. 


Cage O. falcata O. microdasys O. polyacantha O. stricta 

Rankings 

Small 6.391.5A 11.991.5C 9.691.5BC 8.491.5AB 

Large 3.991.5A 11.091.5B 15.191.5C* 14.891.5BC* 


Small 0.4090.07A 0.1690.07B 0.1890.07B 0.2690.07AB 

Large 0.7890.07A* 0.1690.07B 0.0390.07B* 0.0390.07B* 


Small 24.394.2A 12.796.4B 21.394.6AB 26.694.4A 

Large 30.494.2A 11.494.2B 1.594.2C* 2.594.2C* 




Cactus species rankings, based upon numbers of eggs oviposited, were significantly 

higher in small cages compared to large cages, except for C. spinosior whose ranking 

did not differ between the small and large cage. In addition, H. fragans ranked 

significantly lower overall than all other test species. 

Proportions of eggsticks oviposited was not significantly affected by cage type 

(Table 6; F 9.9, df 1, 45, P 0.19). Harrisia fragans tended to receive the lowest 

proportion of eggsticks in both small and large cages, although not always 

significantly fewer than all other test species. 

Similar to mean proportions of eggsticks, no significant interaction and no 

treatment or cactus species main effects were observed on mean numbers of eggs 

per eggstick oviposited on these cactus species. However, numbers of eggs per 

eggstick oviposited was significantly affected by cage type (Table 6; F 82.8, df 1, 

43, PB0.0001). Significantly more eggs per eggstick were collected in small cages 

than large cages with all four species. 

Group 4: comparison of C. cactorum hosts and non-hosts 


species and cage type (Table 7; F 4.6, df 3, 48, P 0.006). Ranking was 

significantly higher in small cages than in large cages for C. spinosior and O. stricta. 

Mean ranking for H. fragrans and O. steptacantha were not significantly different in 

large and small cages. The non-documented host plant species C. spinosior in small 

cages ranked significantly higher than all other cactus species evaluated, while the 

other non-documented host plant H. fragrans in large cages ranked significantly 

lower than other species. 

Cactus by cage type interaction (Table 7; F 8.4, df 3, 45, P 0.0006) 

significantly affected the proportions of eggsticks oviposited on cactus species. 

Significantly higher proportions of eggsticks were oviposited on O. spinosior in 

Table 6. Group 3: comparison of Opuntia, Cylindropuntia, and Harrisia cacti for mean (9 

SE) Cactoblastis cactorum oviposition preference ranking, proportion of eggsticks per plant, 

and numbers of eggs per eggstick. 


Cage C. acanthocarpa H. fragrans C. spinosior O. stricta 

Rankings 

Small 7.491.7AB 10.591.7B 8.191.7AB 4.991.7A 

Large 12.191.7B* 15.591.7B* 8.091.7A 12.691.7B* 


Small 0.3390.09A 0.1790.09A 0.2490.09A 0.2790.09A 

Large 


0.3290.09AB 0.1790.09B 0.2490.09A 0.2790.09A 

Small 28.695.0A 28.795.4A 34.295.4A 36.094.7A 

Large 6.094.7BC* 1.994.7C* 23.094.7A* 14.694.7AB* 




Table 7. Group 4: comparison of Cactoblastis cactorum hosts and non-hosts for mean (9SE) 

Cactoblastis cactorum oviposition preference ranking, proportion of eggsticks per plant, and 

numbers of eggs per eggstick. 


Cage H. fragrans C. spinosior O. streptacantha O. stricta 

Rankings 

Small 13.591.4C 1.891.4A 11.091.4C 6.691.4B 

Large 13.491.4B 10.491.4A* 10.591.4A 10.491.4A* 


Small 0.0390.07C 0.6490.07A 0.1490.07BC 0.2090.07B 

Large 0.1090.07B 0.3490.07A* 0.2890.07A 0.2890.07A 


Small 45.696.9A 25.594.0B 21.495.1B 30.094.0B 

Large 7.294.0B* 15.494.0A* 14.194.0AB 11.194.0AB* 


significant differences between cage means within a column for each variable. 

small cages than in large cages. No significant difference in mean proportions of 

eggsticks on O. stricta, O. streptacantha and H. fragrans in large and small cages 

was observed. Mean proportions of eggsticks oviposited on a non-documented 

host (C. spinosior) in small cages was significantly higher than all cactus species 

evaluated; while mean proportions of eggsticks on the non-documented host 

H. fragrans was significantly lower than other species in both small and large 

cages, except for O. stricta in small cages. 

No significant interaction was found between cactus and cage type for mean 

number of eggs per eggstick. However, mean number of eggs per eggstick was 

significantly affected by cage type (Table 7; F 60.0, df 1, 40, PB0.0001). 

Significantly more eggs per eggstick were collected from cladodes in small cages 

than from plants in large cages. The highest mean number of eggs per eggstick was 

found on H. fragrans in small cages. 

Group 5: comparison of cactus species, H. fragrans and O. dellinii, represented in 

Florida and species, C. spinosior and O. streptacantha, represented in the 

Southwestern US and Mexico 

Cactus species ranking was significantly affected by cage type (Table 8; F 7.0, 

df 1, 38.3, P 0.01) and cactus species (Table 8; F 9.9, df 3, 36.6, PB0.0001). 

Cactus species rankings were significantly higher in small cages compared to large 

cages. Mean ranking of O. streptacantha was significantly higher than other test 

species, with the exception of C. spinosior. Harrisia fragrans ranked significantly 

lower than all cactus species evaluated. 

Proportions of eggsticks oviposited were significantly affected by cactus species 

(Table 8; F 9.9, df 3, 36.2, PB0.0001). Opuntia streptacantha had significantly


Table 8. Group 5: comparison of species, Harrisia fragrans and Opuntia dellinii, represented 

in Florida and species, Cylindropuntia spinosior and Opuntia streptacantha, represented in the 

southwestern US and Mexico for mean (9SE) Cactoblastis cactorum oviposition preference 

ranking, proportion of eggsticks per plant, and numbers of eggs per eggstick. 


Cage H. fragrans C. spinosior O. streptacantha O. dellinii 

Rankings 

Small 12.692.4C 6.292.4AB 1.992.4A 7.192.4B 

Large 13.992.2B 9.092.2A 7.192.2A* 10.692.2AB 


Small 0.0390.09C 0.3490.09AB 0.4190.09A 0.2290.09B 

Large 0.0290.09C 0.3390.09AB 0.4990.09A 0.1690.09BC 


Small 18.597.6B 28.895.2AB 28.494.8AB 33.495.5A 

Large 2.894.3B* 11.194.3AB* 19.294.3A 6.094.3B* 



higher proportions of eggsticks than all test species. Significantly lower proportions 

of eggsticks were oviposited on H. fragrans than remaining test species. 

Numbers of eggs per eggstick was significantly affected by cage type (Table 8; 

F 86.1, df 1, 31.8, PB0.0001) and cactus species (Table 8; F 5.4, df 3, 30.9, 

P 0.004). Significantly more eggs per eggstick were collected from small cages than 

large cages. Significantly higher numbers of eggs per eggstick were collected from 

O. streptacantha in small and large cages than from H. fragrans. Numbers of eggs per 

eggstick collected from O. dellinii in large cages also was significantly higher than 

from H. fragrans. 

Group 6: comparison of economically important Opuntia species 

No significant cactus species main effect was observed on mean ranking (Table 9; 

F 1.6, df 3, 48, P 0.19). Cactus species ranking tended to be higher for 

O. engelmannii in small cages but lower in large cages than the other test species. 

Cactus species ranking was significantly affected by cage type (Table 9; F 6.6, df 

1, 48, P 0.01); oviposition ranking for O. engelmannii was higher in small cages 

than large cages. 

No significant cactus species main effects were observed on mean proportions of 

eggsticks oviposited on these cactus species (Table 9; F 2.4, df 3, 48, P 0.08). A 

significant interaction between species and cage type was observed (Table 9; F 5.9, 

df 3, 48, P 0.01). The proportion of eggsticks oviposited in small cages was 

significantly higher for O. engelmannii, but significantly lower for O. stricta In 

addition, although O. engelmannii received the highest mean proportion of eggsticks 

in small cages, this same species received significantly fewer eggsticks than 

O. streptacantha or O. stricta in large cages. 

Numbers of eggs per eggstick collected was significantly affected by cage type 

(Table 9; F 185.4, df 1, 36, PB0.0001) and cactus species (Table 9; F 3.5, df 3,


Table 9. Group 6: comparison of economically important Opuntia species for mean (9SE) 

Cactoblastis cactorum oviposition preference ranking, proportion of eggsticks per plant, and 

numbers of eggs per eggstick. 


Cage O. engelmannii O. ficus-indica O. streptacantha O. stricta 

Rankings 

Small 5.591.8C 11.891.8A 7.591.8BC 10.591.8AB 

Large 14.891.8A* 13.491.8AB 9.891.8B 10.491.8B 


Small 0.5390.08A 0.1090.08B 0.2390.08B 0.1490.08B 

Large 0.1490.08B* 0.1990.08AB 0.3490.08A 0.3390.08A* 


Small 18.493.7B 28.894.3A 33.394.1A 28.894.7A 

Large 2.193.4B* 5.793.4AB* 10.493.4A* 9.093.4A* 



35.3, P 0.03). Significantly more eggs per eggstick were collected from cladodes of 

all four species in small cages than from plants in large cages. Mean number of eggs 

per eggstick collected from O. ficus-indica, O. streptacantha, and O. stricta was 

significantly higher than from O. engelmannii. 

Discussion 

Open field tests have been used to clarify host specificity test results obtained from 

more traditional cage tests conducted in quarantine facilities. Many weed biological 

control researchers consider open field tests as the most realistic method to evaluate 

host specificity for potential biological control agents, especially for ovipositing 

females that are no longer behaviorally constrained by caging (Sheppard 1999; Briese 

et al. 2002; Heard, Zonneveld, Segura, and Martinez 2004). However, to date, open 

field tests are conducted in the native range of the potential biological control agent 

and not in the native range of the non-target host plants of concern. Potential host 

plants may be shipped to the native range of the control agent but often these nonnative 

plants cannot be planted outside a containment facility because of concerns 

and prohibitions in introducing non-native species. Even if the non-native test plant 

can be planted in an open field setting, additional problems may arise such as the 

time required producing a healthy, mature test plant, presence of specific microhabitat 

growth conditions, or attack/destruction by generalist natural enemies not 

present in the test plants’ native range. The most realistic test for non-target impacts 

from a potential biological control agent would be conducted in the native area of 

the non-target host plants. A safe alternative for assessing the host range of potential 

Lepidoptera biological control agents is open field-testing in the requested area of 

introduction with the F1 sterility technique (Moeri et al. 2009). 

Carpenter et al. (2001b) discussed using F1 sterility as a technique in elucidating 

potential host range of C. cactorum for key Opuntia species across the southern US. 

As an oligophagous feeder, C. cactorum feeds on a wide range of Opuntia spp. within


the subgenus Platyopuntia in the insects’ native region of South America (Mann 

1969; Zimmermann, McFadyen, and Erb 1979) and the insects’ adventive range in 

the southeastern US (Hight et al. 2002). Host specificity for C. cactorum females 

mated to either irradiated or normal males was evaluated in this study as a proof of 

concept for using the F1 sterility technique. Several arrangements of host plant 

choice tests were conducted to insure a rigorous comparison of the two oviposition 

preference treatments. In all tests, female C. cactorum oviposition preference was not 

significantly different for females mated to irradiated males versus females mated to 

normal males. This study supports the use of F 1 sterility as a tool for assessing the 

safety of exotic Lepidoptera for biological control of invasive weeds. F 1 sterile moths 

could be released in the proposed area of introduction as part of a program to 

evaluate the risk of non-target impacts without the risk of the proposed biological 

control agent becoming established. 

Although female C. cactorum oviposition preference was not significantly 

different when mated with irradiated or normal males, there were significantly 

different oviposition preferences in the choice tests conducted in this study. Cactus 

species significantly affected female C. cactorum oviposition preference. Of the 

six Opuntia spp. from Florida, significantly more eggsticks were oviposited on 

O. corallicola. This species was also found to be the preferred oviposition host by 

Johnson and Stiling (1996) in caged tests with three other Florida native Opuntia 

spp., even though the larval performance was significantly poorer. Opuntia 

corallicola is spinier than other test cacti of similar height (Group 1, Table 2) and, 

for most comparisons, was the preferred host. However, in the evaluation of species 

with and without spines (Group 2, Table 3), the plant without spines (O. falcata) 

received significantly more eggsticks than the species with numerous spines, although 

the test did not include the species O. corallicola. This inconsistency is further born 

out by Mafokoane, Zimmermann, and Hill (2007) who found that C. cactorum 

avoided ovipositing on plants with dense spines, but preferred O. ficus-indica, an 

upright, large stemmed plant, with few spines. Evaluating data only from our study, 

a potential pattern of oviposition preference may be found in that tall species 

(O. corallicola, O. falcata and O. ficus-indica) are preferred over short species, spiny 

(O. polyacantha) or less spiny (O. pusilla), but tall spiny species (O. corallicola) are 

preferred over tall not spiny species (O. cochenillifera). 

Our oviposition test of the three different genera (Group 3) identified variations 

in oviposition preference. Certain taxonomists regard Cylindropuntia as sub-genera 

of Opuntia (Benson 1982) and others regard them as independent genera (Anderson 

2001). Either way, the species are closely related and, in general, numbers of 

eggsticks oviposited on species of Opuntia and Cylindropuntia cacti were similar. 

Oviposition on closely related non-Opuntia cacti in a field situation will likely occur. 

In fact, in our studies evaluating non-hosts and known hosts of C. cactorum (Group 

4, Table 6), females oviposited significantly more eggsticks on C. spinosior cladodes 

in small cages than on the other four cactus species including O. stricta, a known 

host plant. Oviposition on a species in the cactus genera Harrisia received 

significantly fewer eggsticks. The genus Harrisia is in the subfamily Cactoideae 

while the genera Opuntia and Cylindropuntia are in the subfamily Opuntioideae. 

Not only cactus species significantly affected C. cactorum oviposition preference, 

but so did cage type. However, the trend was not consistent. Some species (such as 

O. corallicola) showed a relatively consistent increase in preference measures from


small to large cage. Other species (O. spinosior) showed a decrease in preference 

measures from small to large cage. We did not evaluate the physiological changes in 

cladodes excised from plants and placed in the small cages. Cladodes from some 

cactus species may have changed more than others when they were excised. Cage size 

may also have impacted preference by interfering with female visual cues in concert 

with plant volatiles to identify potential hosts. Removal of the cladode from the 

plant also could have caused biochemical changes in excised cladodes. 

However, in addressing the primary objective of this study, we identified no 

difference in oviposition preference for females mated to irradiated or normal males. 

These data support the use of F1 inherited sterility as a tool for assessing the safety of 

exotic lepidopterans for biological control of invasive weeds. Irradiated males and 

non-irradiated females could be released in areas without the risk of the biological 

control agent becoming established. This technique will allow open field studies that 

can be used to evaluate host specificity under more stringent and realistic testing 

procedures to obtain permission for importation and release of exotic biological 

control agents. 


We thank Susan Drawdy and Robert Caldwell (USDA-ARS, Tifton, GA) for technical 

assistance, and Denny Bruck (USDA-ARS, Corvallis, OR) and David Coyle (University Of 

Wisconsin, Madison) for comments on earlier drafts of this manuscript. Mention of trade 

names or commercial products in this publication is solely for the purpose of providing specific 

information and does not imply recommendation or endorsement by the US Department of 

Agriculture. 

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Vol. 19, S1, 2009, 335 362 

Radiation sources supporting the use of natural enemies 

for biological control of agricultural pests 

Kishor Mehta 

Vienna, Austria 

Augmentative biological control as a component of integrated pest management 

programmes involves the release of natural enemies of the pest, such as parasitoids 

and predators. Several potential uses for nuclear techniques have been identified 

which can benefit such programmes; these benefits include facilitating trade, 

protecting the environment and increasing the overall efficacy of the programmes. 

This may involve sterilising feed material, hosts or even the control insects. 

Radiation is currently the most favoured sterilising agent, although availability and 

cost of radiation sources are considered as limiting the use of radiation in support 

of biological control. This paper reviews various radiation sources that may be 

used for this purpose, including a comparison of several key parameters such as 

cost estimates of these radiation sources that should assist in making a judicious 

selection of a suitable irradiator. 

Keywords: natural enemies; pest management; irradiators; radiation; sterilisation 

Introduction 

Ionising radiation 

Ever since ionising radiation was discovered by Marie Curie more than 100 years 

ago, it has been beneficially used in nearly every activity that touches human life, 

from medicine to communication to hydrology to food and agriculture. 

Electromagnetic radiation spectrum includes radiowaves with very long wavelength 

to the exceedingly penetrating X-rays and gamma rays with very short 

wavelength, all of which travel at the velocity of light ( 3 10 8 1 

ms in vacuum). 

The energy associated with radiation is inversely proportional to the wavelength; the 

longer the wavelength, the less is the energy. The energy associated with X-rays and 

gamma rays is greater than the binding energy of an atomic electron, thus this type of 

radiation can ionise an atom or break the molecular bonds. Radiation with such high 

energy is referred to as ionising radiation. Besides gamma rays and X-rays, ionising 

radiation includes high-energy electrons (generally 80 keV). Ionising radiation 

breaks down molecules, modifying chemical, physical or biological properties of 

the irradiated material. Thus, radiation can cause polymerisation of plastics, kill 

pathogens and microorganisms, and damage DNA molecules, leading to applications 

in industry and food processing, sterilisation of health care products, and 

reproductive sterilisation of insects. 

*Email: mehta@aon.at 




DOI: 10.1080/09583150802417849 


336 K. Mehta 

Applications of ionising radiation in entomology 

There are a number of applications of ionising radiation in entomology (Bakri, 

Heather, Hendrichs, and Ferris 2005a), including disinfestation of commodities for 

quarantine and phytosanitary purposes, and reproductive sterilisation of insects for 

pest management programmes using the Sterile Insect Technique (SIT) (Dyck, 

Hendrichs, and Robinson 2005). Radiation can also be applied in various ways to 

facilitate the use of biological agents for control of arthropod pests and weeds 

(Carpenter 1997, 2000; Greany and Carpenter 2000). These authors cite a number of 

potential advantages of nuclear techniques for biological control: 

avoidance of the emergence of pest insects from non-parasitised hosts, 

allowing earlier transport and facilitating trans-boundary shipment, 

improvements in rearing media (either artificial diets or natural hosts/prey), 

provision of sterilised natural prey to be used as food during predator 

shipment, to ameliorate concerns relating to the incidental presence of hitchhiking 

pests, 

provision of supplemental food or hosts in the field, to increase the initial 

survival and build-up of released natural enemies, and 

reproductive sterilisation of weed-feeding insects that are candidates for 

biological control allowing their risk-free field assessment of host specificity. 

Currently, there are hundreds of producers of natural enemies reared specifically 

for biological control purposes that produce several types of parasitoids and 

predators (BCPC 2004). There are no complete data available regarding such 

information, although sales worldwide are generally estimated at about US$ 100 

million (www.anbp.org; www.ibma.ch; www.amrqc.org). In North America alone, 

there are 25 30 major producers, not including small local producers or collectors 

of garden products such as ladybugs, etc. Very few producers or the pest 

management programmes using biological control agents are currently using 

nuclear techniques; either there is no appreciation for such techniques and the 

subsequent benefit to the pest management programme, or they are not aware of a 

suitable radiation source that is also economical to use, or some organic growers 

using biological control agents may object to the use of radiation. This review 

provides relevant and useful information that may motivate these producers to 

apply this beneficial technology. 

There are three types of ionising radiation used in radiation processing, namely 

gamma rays, X-rays and electrons. All have similar effects on the irradiated materials 

(since they have similar relative biological effectiveness), and in particular on the 

irradiated insects (Bakri, Mehta, and Lance 2005b). In a biological organism 

composed of differentiated and undifferentiated cells, mitotically active cells such as 

stem cells and germ cells are the most radiation-sensitive elements. Thus, radiation 

can make an insect reproductively sterile by damaging the DNA of gonial cells. For 

certain insect life stages, several studies found no significant difference in the lethal 

effects between electrons and gamma rays (Adem, Watters, Uribe-Rendón, and de la 

Piedad 1978; Watters 1979; Dohino, Tanabe, and Hayashi 1994). 

Numerous mutagenic chemicals were tested as alternatives to radiation for 

sterilisation of insects in the 1950s and 1960s (Knipling 1979). Efficacy of irradiated 

and chemosterilised insects for population control was generally similar (Guerra,


Wolfenbarger, Hendricks, Garcia, and Raulston 1972; Flint, Wright, Sallam, and 

Horn 1975; Moursy, Eesa, Cutkomp, and Subramanyam 1988). However, chemosterilants 

are rarely used today. Most are carcinogenic, mutagenic and/or teratogenic, 

leading to environmental and human health issues in such areas as the integrity of 

ecological food chains, waste disposal (e.g. spent insect diet), and worker safety 

(Hayes 1968; Bracken and Dondale 1972; Bartlett and Staten 1996). Insect resistance 

to chemosterilants is an additional concern (Klassen and Matsumura 1966). 

Exposure to ionising radiation is now the principal method for inducing reproductive 

sterility in mass-reared insects. 

Irradiation of insects is a relatively straightforward process with reliable quality 

control procedures (FAO/IAEA/USDA 2003). The key parameter is the absorbed 

dose of radiation; efficacy of the irradiation process is guaranteed as long as the dose 

is correctly delivered (Bakri et al. 2005b). Other advantages of using radiation 

(gamma rays, X-rays and electrons) include (1) insignificant increase in temperature 

during the process, (2) treated insects can be used immediately after processing, (3) 

irradiation does not add residues that could be harmful to human health or the 

environment, and (4) radiation can pass through packaging material, thus allowing 

the insects to be irradiated after packaging. 

Radiation dose 

The absorbed dose, D, is radiation energy absorbed in unit mass of a material, and is 

mathematically expressed as the quotient of do by dm, where do is the mean energy 

imparted to matter of mass dm; thus, D do/dm (ICRU 1998). The unit is J/kg. The 

special name for the unit is gray (Gy); thus, 1 Gy 1 J/kg. The unit of absorbed dose 

used earlier was rad (1 Gy 100 rad). Quite often, ‘absorbed dose’ is simply 

referred to as ‘dose’. 

Radiation technology 

Energy transfer from radiation to matter 

Ionising radiation is classified under two categories: directly ionising radiation, and 

indirectly ionising radiation (Attix and Roesch 1968). Charged particles such as 

electrons belong to the first category since they can transfer energy directly and 

ionise the atoms of the irradiated matter (e.g. tissues, insects). On the other hand, 

uncharged particles like photons transfer energy indirectly by first transferring their 

energy to electrons, which in turn ionise the atoms. The nature of this energy transfer 

from radiation (photons and electrons) to the irradiated matter influences the 

distribution of dose (McLaughlin, Boyd, Chadwick, McDonald, and Miller 1989; 

IAEA 2002a). Dose distribution (or dose variation) in the irradiated matter is one of 

the important parameters for insect irradiation. 

When matter is irradiated with a photon beam, the intensity of the beam, and 

thus the dose imparted to the irradiated matter, decreases exponentially with depth as 

radiation penetrates into the matter. The rate of decrease in the intensity depends on 

the photon energy, composition and density of the irradiated material, and 

irradiation geometry. On the other hand, electrons can be roughly characterised 

by a common pathlength, traced out by most such particles of a given energy in a

338 K. Mehta 

specific medium. This is generally referred to as ‘range’. The range depends on the 

electron energy, composition and density of the irradiated material, and the 

irradiation geometry. Dose initially increases with distance, but eventually decreases 

as electron energy is spent. At the end of their range, the electrons come to rest in the 

material having spent all their kinetic energy. 

Figure 1 shows the variation of dose with distance in water for 5 MeV electrons 

and three types of photons (cobalt-60 gamma rays, and 5 MeV and 160 keV X-rays). 

There is no definite ‘range’ for photons, unlike the case with electrons. 

Radiation energy 

To maintain fitness of the irradiated insects and for the safety of the operating 

personnel, induction of radioactivity in the irradiated materials, such as canisters 

(reusable containers) and insects, must be avoided. This is achieved by restricting the 

energy of the radiation used for treating insects as follows (FAO/IAEA/WHO 1999; 

IAEA 2002b; Codex Alimentarius 2003): 

for photons, the energy should be less than 7.5 MeV, and 

for electrons, the energy should be less than 10 MeV. 

Thus, gamma rays from cobalt-60 (photon energies are 1.173 and 1.332 MeV) and 

caesium-137 (0.662 MeV), electrons generated by accelerators with energy less than 

10 MeV, and X-rays generated from electrons with energy below 7.5 MeV are 

acceptable for irradiation of insects. 

Gamma rays as well as X-rays are photons. However, by convention the photons 

created outside the atomic nucleus are referred to as X-rays, and those created inside 

the nucleus during radioactive decay as gamma rays. 

Relative dose 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

5 MeV electrons 

160 keV X rays 

5 MeV X rays 

Co-60 gamma rays 

0 5 10 15 20 25 

cm of water 

Figure 1. Variation of dose in water with distance for a narrow beam of radiation, for 5 MeV 

electrons, cobalt-60 gamma rays, and X-rays of energy 160 keV and 5 MeV.


Gamma irradiators 

To expose insects to gamma radiation, they are treated in a gamma irradiator, which 

consists of an isotopic radiation source, a mechanism for transporting the insects 

through the radiation field, and an operating system to control the exposure of 

insects to radiation. 

More than 1000 different radioisotopes emit gamma radiation, but only two are 

used for radiation processing, namely cobalt-60 and caesium-137. Both have well 

determined half-lives, emit relatively high-energy gamma rays, and decay into stable 

isotopes. Both radioisotopes are produced in nuclear reactors. Source capsules are 

doubly sealed in stainless steel or zirconium alloy for maximum security, which 

ensures that the radioactive material cannot come into contact with the product to be 

irradiated (Brinston and Norton 1994). Because of the difference in the photon 

energies emitted per disintegration (0.662 MeV for caesium compared to total energy 

of 2.505 MeV for cobalt), caesium sources require about four times more activity than 

cobalt sources to provide the same processing capacity. Since production of caesium- 

137 is quite elaborate and it is water-soluble, presently all large gamma irradiators 

employ cobalt-60 as a radiation source. Cobalt is water-insoluble with a high melting 

point, and thus well suited for use as an intense source of gamma radiation. 

Cobalt-60 is deliberately produced in a nuclear power reactor; its production 

starts with natural cobalt (metal) that is an element with 100% abundance of the 

stable isotope cobalt-59 (Brinston and Norton 1994). On absorption of a neutron, a 

cobalt-59 atom converts into a cobalt-60 atom. The radioisotope cobalt-60 decays 

into a stable nickel isotope by principally emitting one negative beta particle (of 

maximum energy 0.313 MeV) with a half-life of about 5.271 years (Unterweger, 

Hoppes, and Schima 1992). Nickel-60 thus produced is in an excited state, and it 

immediately emits two photons of energy 1.173 and 1.332 MeV in succession to 

reach its ground state (Lide 1990). These two gamma-ray photons are responsible for 

radiation processing in the cobalt-60 gamma irradiators. With decay of every cobalt- 

60 atom, the strength or the activity level of the cobalt source is decreasing, such that 

the decrease amounts to 50% in about 5.271 years. 

The radiation source in a gamma irradiator typically consists of several pencils of 

the radioactive isotope cobalt-60. The only variation in the source output is the 

known reduction in activity (strength) caused by the radioactive decay mentioned 

above, which can have a significant impact on the programme (financial as well as 

scheduling) if not taken into account. Because the activity of a cobalt source decreases 

by about 12% annually, the irradiator operator compensates for this loss of activity by 

incrementally increasing irradiation time to maintain the same radiation dose to the 

insects. Because the irradiation times eventually become impractically long, the 

source needs to be replenished at regular intervals, depending on the operational 

requirements. Source pencils are eventually removed from the irradiator at the end of 

their useful life. Generally they are returned to the supplier for re-use, recycling or 

disposal. In about 50 years, 99.9% of cobalt-60 will decay into non-radioactive nickel. 

Two types of gamma irradiators may be used for exposing insects to radiation: 

self-contained irradiators and panoramic irradiators. Thus, irradiators may be 

divided into two broad types: 

small-scale self-contained irradiators, and 

large-scale panoramic irradiators.

340 K. Mehta 

Figure 2. Self-contained dry-storage gamma irradiator suitable for research and small-scale 

irradiations. This irradiator employs cobalt-60 as the radiation source. In preparation for 

irradiation, a canister is being placed in the irradiation chamber when it is in the loading 

(shielded) position. The control panel is visible at bottom right. 

Small-scale self-contained irradiators 

Self-contained irradiators are specially designed for research and for applications 

that need small doses and relatively small throughputs, such as blood irradiation to 

help prevent transfusion-induced graft-versus-host disease (GVHD) and exposure of 

insects for pest management programmes. Most irradiation of insects is currently 

carried out in such irradiators. Generally, these are dry-storage irradiators; the 

radiation source is predominantly cobalt-60 (Figure 2). There are a few old caesium 

irradiators still being used. These irradiators house the source within a protective 

shield of lead or other material; thus, they can be placed very conveniently in an 

existing laboratory or a room without needing extra shielding (hence, self-contained). 

The advantages of such small irradiators are that they provide a high dose rate and 

good dose uniformity within the irradiated sample, which is essential especially for 

radiation research. These characteristics are achieved by surrounding the sample 

with several radiation source pencils, such that it receives radiation from all 

directions. Such an irradiation arrangement places restriction on the sample size, 

limiting it to typically 1 5 L. However, this volume is quite adequate for research and 

small-throughput applications. 

Considering the amount of material to be treated and the required dose, such 

small irradiators would be most suitable for biological control companies. They are 

easy to install and operate, although their installation and use have to follow 

national licensing procedures. To irradiate, a canister of insects is placed in the 

irradiation chamber while it is in the loading (shielded) position, and the timer is set 

to deliver the pre-selected dose (see Figure 2). On the push of a button located on the 

control panel, the irradiation chamber (along with the insect canister) is automatically 

lowered to the irradiation position, and is returned to the unloading 

(shielded) position at the end of the pre-set irradiation time.


These self-contained irradiators are classified by the IAEA as Category I (dry 

storage) and Category III (wet storage). Applications and the procedures for use for 

these two categories of irradiators are described in a Practical Radiation Safety 

Manual published by the IAEA (1996a). 

Large-scale panoramic irradiators 

For large-volume applications, panoramic irradiators are more suitable, where the 

radiation source consists of either several cobalt-60 rods (pencils) arranged in a 

plane, or a single rod. The source is moved into a large irradiation room for 

treatment of the product (e.g. insects), and when not in use it is returned to a separate 

storage room, which is shielded by either water (wet storage) or concrete (dry 

storage). Since isotopic sources emit gamma radiation in all directions, they may be 

surrounded on all sides by canisters with insects for irradiation to increase the energy 

utilisation efficiency; thus, several insect canisters are typically irradiated simultaneously 

(unlike self-contained irradiators). Many panoramic irradiators are run in a 

continuous operation mode, wherein canisters are carried on a conveyor around a 

central source, which also improves dose uniformity in the product. The speed of the 

conveyor is selected to give the required dose to the product. The source is moved to 

the storage room only when the irradiator is not in use. An example of such an 

irradiator is the unit operated in Mexico by the joint USA/Mexico Moscamed 

programme, which was installed more than 25 years ago. Currently it contains about 

30 kCi of cobalt and processes about 15 000 L of fruit fly pupae per week. Generally, 

this type of gamma irradiator is too large and too expensive for the purpose of insect 

irradiation only, especially if small volumes are involved (see discussion in Total 

treatment costs). 

An alternate method is batch operation, which is more suitable for relatively 

small throughputs. In this mode of operation, several insect canisters (a batch of 

canisters) are placed in the irradiation room around the source position while the 

source is in the storage container (or a separate room below the irradiation room). 

After the irradiation room is vacated and locked, the source is moved into the room 

for the length of time required to deliver the desired absorbed dose. To improve dose 

uniformity, each canister is rotated on its own axis during irradiation. After 

irradiation, the source is returned to the storage container, and the irradiated 

canisters are replaced with a new batch of canisters for the next irradiation. 

An example of a typical batch irradiator, which is commercially available, is 

shown in Figure 3. This irradiator features a ring of product turntables surrounding 

the cobalt-60 source. The radius of the ring is adjustable so that the dose rates, 

irradiation time and product batch volume can be varied to a large extent. The 

configuration can facilitate simultaneous irradiation of products of various densities 

because the products on the turntables do not shield each other at any time. The 

rotation of the turntables ensures good dose uniformity for all product densities. 

When the source is raised from the storage container for irradiation, the biological 

shield of thick concrete and the safety interlock system protect personnel. There are 

a few laboratories (insect facilities) where such batch irradiators are currently being 

used. In 1996, such an irradiator with 12 turntables was commissioned by Servicio 

Nacional de Sanidad Agraria (SENASA), Ministerio de Agricultura, Peru, for the 

purpose of insect sterilisation (see Figure 4). Each turntable takes a large cylinder

342 K. Mehta 

Figure 3. A typical panoramic gamma batch irradiator. This unit, marketed by MDS 

Nordion, has four turntables (50 cm in diameter) that can each support product up to 320 kg. 

The number of turntables and their arrangement can be modified as per customer’s 

requirements. (Figure courtesy of MDS Nordion, Canada.) 

that in turn contains 12 smaller cylinders of 10 cm diameter and 70 cm long. The 

present activity is about 17 kCi of cobalt-60, which yields an average dose rate of 

about 1.7 Gy/min at the insect location. There is a similar irradiator at the ARC 

Infruitec-Nietvoorbij Fruit, Vine & Wine Research Institute in Stellenbosch, South 

Figure 4. A gamma batch irradiator in use at SENASA, Lima, Peru for insect irradiation.


Africa. To improve dose uniformity to the insects, each canister is rotated around its 

own axis, while the entire table (90 cm diameter) on which the eight canisters are 

situated is rotated around the source. This irradiator yields a dose rate of about 

5 Gy/min with cobalt-60 activity of about 6.5 kCi. The volume of each canister is 

about 5 L. 

These panoramic irradiators are classified by the IAEA as Category II (dry 

storage) and Category IV (wet storage). Applications and the procedures for use for 

these two categories of irradiators are described in a Practical Radiation Safety 

Manual published by the IAEA (1996b). 

Electron-beam irradiators 

To expose insects to electrons, they are treated in an electron-beam irradiator, which 

consists of a source of electrons (an electron accelerator that emits a narrow beam of 

electrons), a device to broaden the beam to cover the insect canister, a mechanism for 

transporting the canisters through the electron beam, and an operating system to 

control the exposure. 

An electron accelerator does not involve any radioactive material. It yields a 

narrow and intense electron beam, and thus the dose rate can be up to 1000 times 

greater than that for an isotopic gamma ray source. In an accelerator, electrons are 

introduced into an accelerating structure from an injector, where they are accelerated 

to the designed energy; the energy for the acceleration is derived from a variety of 

sources depending on the type of accelerator (ASTM 2007a). Medium-energy 

(between 300 keV and 5 MeV) electrons are commonly produced by potential-drop 

accelerators, whereas high-energy (more than 5 MeV) electrons are commonly 

produced using microwave-powered accelerators. Electrostatic accelerators can be 

used to accelerate electrons, but such systems have seldom been used for radiation 

processing applications. For all types of accelerators, the beam tube through which 

the electrons travel is under vacuum. Steering and focusing of the electron beam is 

accomplished with electromagnets and/or electrostatic fields. After leaving the 

accelerating structure and prior to reaching the insects, the electron beam is usually 

dispersed to accommodate the canister size. This is typically done by sweeping the 

beam back and forth across the canister using an electromagnet with a varying 

magnetic field (referred to as ‘scanning’), although defocusing elements and 

scattering foils are also used. 

The electron beam reaching the insect canister may be characterised by the 

following parameters: 

electron beam energy (in MeV), which determines the penetration distance in 

the insect canister and thus dictates the useful canister size; 

average beam current (in mA), which determines the dose rate; 

beam power (product of beam energy and beam current, in kW), which 

determines throughput for a selected dose; 

beam (scan) width, which is generally selected to cover the canister size; and 

scan uniformity (uniformity of dose on the canister surface along the scanned 

direction).

344 K. Mehta 

Since the penetration by electrons is relatively shallow, electron energy of less than a 

few MeV will not be suitable for insect irradiation. As seen in Figure 1, 5 MeV 

electrons can penetrate about 3.5 cm of water (density of 1 g cm 3 ), and thus, since 

the density of packaged insects is about half of that of water (Parker, personal 

communication, 2006), the penetration will be about 7 cm (including the canister 

wall facing the electron beam). This defines the size of the canister (the dimension 

along the electron beam) suitable for acceptable dose variation. A larger canister may 

be used if a two-sided irradiation is carried out; however, one-sided irradiation is 

preferable for electron irradiation. Because of the slow decrease in dose with 

distance, this is easily done with photon irradiation; however, because of the sharp 

fall-off of dose in the case of electrons (Figure 1), a two-sided irradiation is much 

more complicated and can be problematic. Basically, the electron beam energy 

dictates the useful size of the canister for irradiation, and the average beam current 

(for a fixed beam energy) determines the throughput, e.g. number of canisters 

irradiated per hour. The dose rate at the location of the insect canister is generally 

much higher than that for a gamma irradiator, and hence the canisters move through 

the electron beam at a relatively high speed. However, unlike gamma irradiation, 

only one canister (in reality, only part of a canister) is irradiated at any instant. While 

in operation, there is a high radiation field in the irradiation room just as in a 

panoramic gamma irradiator; the irradiation room is shielded. However, when the 

accelerator is not operating, there is no radiation in the room. For irradiation, the 

canisters are placed on the conveyor outside the irradiation room, and they enter the 

room through a shielded labyrinth (similar to a panoramic gamma irradiator). The 

conveyor speed is adjusted according to the beam current and the dose requirement. 

Considering the dose required for the treatment of insects (taken to be an average 

of about 100 Gy) and the number of insects irradiated at a facility, the power 

requirement will be less than 1 kW, which is equivalent to about 70 kCi of cobalt-60. 

Currently, there is no commercial treatment of insects using electron-beam 

irradiators, since accelerators with 5 MeV and such low power levels are not 

commercially available. The power levels of commercial accelerators currently 

available are generally 10 200 kW. However, use of electrons for insect irradiation 

will likely increase in the near future. 

In principle, similar to gamma irradiators, there could be two types of electron 

irradiators: one with a shielded room to house the accelerator where the products 

enter the room from outside for irradiation, and another being a self-contained 

irradiator where the shielding is part of the irradiator. Some manufacturers are 

considering producing small self-contained 5 MeV accelerators that would be 

suitable for insect treatment (Brown, personal communication, 2005). One possibility 

is a self-contained irradiator that houses a 5-MeV, 750-W accelerator along with a 

beam-scanning device that spreads the beam over 40 cm. This irradiator could 

deliver a dose of 100 Gy to insects at a throughput of about 6 kg per min. The cost of 

the entire irradiator would be about US$ 1 million. 

X-ray irradiators 

When a beam of electrons strikes a high-atomic number material (called a converter), 

such as tungsten, a fraction of the electron energy is converted into X-rays.


Radiation generated in this manner (by rapid deceleration of electrons) is also known 

as bremsstrahlung (literally ‘braking radiation’ in German). Although their effects 

on irradiated materials (insects in this case) are generally similar to those of gamma 

rays, these types of radiation differ in their energy spectra, angular distributions, and 

dose rates. While gamma radiation from a cobalt-60 source has discrete energies, 

X-rays have a broad energy spectrum with a maximum equal to the energy of the 

incident electrons. Conventionally, the incident electron energy is referred to as the 

energy of the X-ray beam. For example, 5 MeV X-rays are generated by 5 MeV 

electrons, but the average photon energy in this X-ray beam is even less than that of 

cobalt-60 gamma rays. 

X-rays have the benefit of high penetration like that of gamma rays, and also the 

benefit of electrons in that X-rays do not need radioactive material (unlike gamma 

rays) for its generation. 

High-energy X-ray irradiators 

A high-energy X-ray irradiator consists of a source of high-energy electrons, a 

converter to generate X-rays from these electrons, and a mechanism to transport the 

insect canisters through the X-ray beam. Since high-energy X-rays are produced by 

high-energy electrons, an electron accelerator is essential for generating them. 

Various types of accelerators referred to above may be used for this purpose. The 

X-ray conversion efficiency (X-ray power emitted in the forward direction divided by 

the electron power incident on the converter) increases with the electron energy and 

the atomic number of the converter material. The heavy metals, such as tantalum, 

tungsten or gold, are suitable materials because of their high atomic number and 

high melting point. 

In contrast to radiographic and therapeutic X-ray generators, which use smalldiameter 

electron beams to generate well-collimated X-ray beams, radiation 

processing applications require electron beams with large cross-sections and X-ray 

converters with large areas to cover the insect canisters. As mentioned above, 

electron beams may be dispersed by scanning magnets, defocusing magnetic lenses, 

or scattering foils. 

The dose rate associated with high-energy X-rays is considerably smaller than 

that for the incident electron beam. Since high-energy X-ray irradiators need electron 

accelerators, they have the same shielding requirements, and the associated high cost. 

This type of irradiator requires a mechanism for transporting the insect canisters 

through the X-ray beam, such as a conveyor system. Because of the high cost, there 

are currently no high-energy X-ray irradiators in use for insect irradiation. 

Low-energy X-ray irradiators 

A low-energy X-ray irradiator consists of an X-ray tube, and a device to transport 

the insect canister through the X-ray beam. The X-ray tube consists of an electron 

source (generally a heated wire, a filament which emits electrons), and a converter to 

generate X-rays. These electrons are electrostatically accelerated through a small 

potential difference (thus no large and costly accelerators are needed), and thus the 

energy will be in the range of a few hundred keV. These X-ray irradiators require

346 K. Mehta 

much less shielding, and thus self-contained irradiators, just like self-contained 

gamma irradiators (Figure 2), are possible. 

Such self-contained X-ray irradiators have been marketed for the last several 

years for the specific purpose of blood irradiation (which requires a dose of about 

25 Gy), and between 50 and 100 units are operating successfully at hospitals and 

medical institutes in North America. Canister volume is about 1.5 L, and the dose 

rate for this irradiator is about 5 Gy/min, which may be relatively low for insect 

irradiation (requiring dose of about 100 Gy) on a commercial basis. However, the 

original developer has recently introduced a new patented tube design that yields 

much higher dose rates. These can be configured to address the requirements of the 

programme/customer (dose and throughput). The tube configuration can also 

further be optimised to be more efficient, thus reducing the cost. A prototype has 

been used in the USA for seafood research for about 2 years. It used two tubes with a 

rating of 5 kW each, and yielded a dose rate of about 25 Gy/min (Kirk, personal 

communication, 2005). The IAEA has recently purchased a unit with one tube rated 

at 10 kW and surrounded by 5 canisters for their SIT programmes (Figure 5). 

Another one at the design stage uses two tubes with a power rating of 10 kW each, 

and would yield a dose rate of 100 Gy/min. A semi-automatic unit has been recently 

installed in Panama with the dose rate calibrated to 12 Gy/min for insect irradiation, 

and uses four tubes with a total power of 25 kW. This unit is about 1.5 2.8 m and 2 

m in height (Figure 6). It utilises a conveyor and the insects are continuously 

irradiated in flat trays. 

These low-energy X-ray irradiators use tubes that generate X-rays with a maximum 

energy of 160 keV, with an average photon energy in the range of 70 75 keV; 

penetration is thus not very deep (see Figure 1). Hence, the canisters are smaller 

compared to those used for gamma irradiators; generally, flat trays are more suitable. 

Figure 5. This self-contained X-ray irradiator (RS 2400) comes in two units. The unit in the 

foreground houses the X-ray tube and the five canisters which can be loaded/unloaded 

through an opening at the top next to the control panel. The other is the water-cooling unit for 

the X-ray tube.

Technical parameters of four types of irradiators 

For the sterilisation process, the technical parameters of primary interest are canister 

size, dose rate and throughput. These are generally governed by the radiation energy, 

the current/flux (i.e. number of photons or electrons emitted), and the power 

(product of energy and current), respectively. These characteristics for the four types 

of irradiators discussed above are listed in Table 1. 

Selection of radiation source 

General 

To make a meaningful selection of the type of irradiator that is suitable for a specific 

application, such as irradiation of insects, it is essential to review and compare the 

Table 1. Relevant technical parameters of four types of irradiator. 

Irradiator 0 

Parameter ¡ 

Energy (MeV) 

(determines canister 

size) 

Current/flux 

(determines dose 

rate) 

Power (determines 

throughput) 


Figure 6. A self-contained X-ray irradiator. (Courtesy of Rad Source Technologies, Inc., 

USA.) 

Gamma rays 

(Co-60) Electrons 

Fixed Choice at 

purchase 

Activity (Ci) 

Choice at 

purchase 

Decays 

Activity 

energy 

Decays 

Current (mA) 

Variable 

X-rays 

(high-energy) 

Choice 

at purchase 

Current (mA) 

Variable 

Current energy Current 

energy conversion 

efficiency 

X-rays 

(low-energy) 

Presently 

fixed 

Current 

(mA) 

Variable 

Current 

energy 

conversion 

efficiency

348 K. Mehta 

key characteristics of the various available irradiators. The needs of the technology 

and the specific programme can then be compared against these various characteristics; 

this can assist the relevant decision-makers in making an optimum selection. 

Such key characteristics relevant to insect irradiation include: 

dose rate, 

canister size, 

dosimetry, 

cost of equipment and accessories, 

total treatment cost: unit cost, 

technology, 

utility requirements, and 

safety and security. 

Dose rate 

There is some limited data that show that low dose rates reduce the adverse effect of 

radiation on the insects (Bakri et al. 2005b). As regards to the irradiation process, the 

dose-rate value at the location of the insect canister determines how long it will take 

to deliver a required dose, and thus the throughput. If the dose rate is too small, the 

long treatment time will result in a low and, depending on the size of the insect 

facility, uneconomical throughput. On the other hand, if the dose rate is too high, 

the canisters will have to be in the irradiation position for a very short time, which 

will make it difficult to consistently give the correct dose. Thus, there is in essence a 

window (albeit a wide one) of dose rate that is suitable for the process. 

Dose rate depends principally on two factors: current/flux of the source (that is, 

radioactivity for an isotopic source, and current for an electron accelerator, see Table 

1) and the irradiation geometry (including the distance from the source). For 

example, in a self-contained gamma irradiator, the canister is surrounded by the 

source, and thus the dose rate is relatively high as compared to the situation for a 

panoramic irradiator where the gamma rays reach the canister from one direction 

only. In the later case, the disadvantage of low dose rate and thus longer irradiation 

time is mitigated by the fact that several large canisters can be treated simultaneously 

(see Figure 4). 

There are basic differences, however, among gamma rays from a radioisotope 

(like cobalt-60), electrons from an accelerator, and X-rays. These differences are 

partly based on how the radiation is emitted from the source. The fact that the 

gamma radiation is emitted in all directions, and that it penetrates much deeper, 

makes the dose rate for a gamma irradiator much smaller than in the case of an 

electron accelerator (see Table 2). However, this also allows a gamma irradiator to 

process several large boxes simultaneously. Also, the dose rate can be increased for a 

gamma irradiator by adding more activity to the source, which may however require 

more shielding. For an electron accelerator, the dose rate can be reduced to a 

manageable value by selecting a low-powered accelerator. However, as mentioned 

earlier, commercially there are no accelerators available for low power (less than 1 

kW) and high electron energy (more than 5 MeV). It is of course possible to operate 

a high-power accelerator at very low power (by reducing the current), but that would 

be completely uneconomical since such units would cost US$ 1 to 2 million.

Table 2. Radiation field distribution for gamma rays, X-rays and electron sources. 

Gamma rays 

(isotopic source, 

panoramic irradiator) 

Emitted in all 

directions, 

radiation is 

distributed 


Electron beam 

(accelerator) 

Emitted mainly in one 

direction, radiation is 

focused 

X-rays 

(high-energy) 

Emitted mainly in 

one direction, 

radiation is focused 

X-rays 

(low-energy, 

RS-2400) 

Emitted in all 

directions, radiation is 

distributed 

High penetration Low penetration High penetration Low penetration 

Low dose rate High dose rate Moderate dose rate Moderate dose rate 

Long processing time Short processing time Moderate processing Moderate processing 

time 

time 

Several large canisters Single flat canister Single large canister Several canisters 

processed together processed at a time processed at a time processed together 

X-rays interact with matter in a similar manner to gamma rays, so they have high 

penetration. High-energy X-rays generated from high-energy (more than 5 MeV) 

and high-power accelerators will have a reasonable dose rate since the X-ray 

conversion efficiency is only a few percentage points. Low-energy X-ray irradiators 

with enough power will also have an adequate dose rate. 

Canister size 

The absorbed dose that is used to induce the required effect in insects is of prime 

importance to the pest management programmes that use these insects. As dose 

increases, the desirable effect increases; however, insect fitness will decrease. Thus, 

optimisation is necessary in selecting treatment dose to balance the desired effect and 

insect fitness, taking into consideration programme requirements (Parker and Mehta 

2006). 

In reality, because of the unavoidable dose variation within a canister (Figure 1), 

an acceptable range of dose to be given to the insects has to be defined according to 

the specific programme requirements. Most often, programmes or regulatory 

officials specify a minimum dose that all insects must receive to ensure sufficient 

radiation. Due to dose variability, most insects actually receive a dose that is 

somewhat higher than that minimum. Thus, it is essential that dose is fairly uniform 

within a canister; the goal is to expose all insects sufficiently without treating large 

proportions with doses that are high enough to substantially reduce their fitness. 

Irradiator operators can often adjust process parameters to achieve the desirable 

dose uniformity in the canister. 

Canister size is one of those parameters; dose variation within the canister 

depends significantly on the canister size, besides the radiation energy and the type of 

radiation. As seen in Figure 1, photons (gamma rays and X-rays) can travel much 

deeper in the irradiated matter than electrons of similar energy. However, dose is 

decreasing in both the cases with distance, and thus there is a limit on the size of the 

canister that can yield acceptable dose variation. If the size of the canister is larger (in

350 K. Mehta 

the direction of the radiation) compared to the rate of decrease of dose, the dose 

uniformity in the canister would be significantly reduced making the canister 

unsuitable for the irradiation treatment. Thus, the limits on the size of the canister 

depend on the type of irradiator. It is obvious from Figure 1 that large canisters can 

be used with cobalt-60 gamma rays and high-energy X-rays. And of course the 

acceptable size can be substantially increased (generally doubled) by irradiating from 

two or more sides (for example, by rotating the canister). As mentioned earlier, this is 

easily done with photon irradiation; however, two-sided irradiation is much more 

complicated and can be problematic in the case of electron irradiation. First, 

irradiating a flat canister of insects from two sides involves either two accelerators or 

passing the canister under the beam a second time after turning it over. Also, because 

of the sharp fall off of dose, slight variation in the bulk density of the insects from 

canister to canister or variation in the electron beam energy during irradiation can 

cause either under-dosing or over-doing the insects in the centre part of the canister. 

Dosimetry 

As mentioned earlier, dose and distribution (variation) of dose in an insect canister 

are very important parameters, and thus should be determined and controlled for the 

efficacy of the irradiation process. A dosimetry system is generally used to determine 

dose. There are several types of dosimetry systems available commercially that are 

suitable for photons and electrons (ASTM 2007b,c). Some of the systems can be used 

for both types of radiation, while some may be suitable only for photons. Also, a 

majority of the dosimeters that are suitable for gamma rays are also suitable for Xrays 

(Mehta, Kojima, and Sunaga 2003). Thus, careful selection of the dosimetry 

system is necessary depending on the irradiator type. Considering various factors, 

the Gafchromic † dosimetry system has been selected by the IAEA based on the 

specific requirements of insect irradiation (IAEA 2004). 

Cost of equipment and accessories 

Since small self-contained gamma irradiators have been available for a long time, it is 

not surprising that currently a large majority of irradiators used for insect 

sterilisation in pest control programmes with an SIT component are of this type; 

with either cobalt or caesium as a radiation source (cobalt is more common). The 

wide usage of such irradiators also opens up the market, resulting in a reasonable 

cost. The main cost components for such irradiators are the radiation source (initial 

source plus periodic replenishment because of radioactive decay), lead shielding and 

transportation. The construction itself is quite simple. Another possibility is smallsize 

panoramic irradiators, preferably used in batch mode. A few are currently in use 

for insect sterilisation as mentioned earlier. For a relatively large biological control 

programme, these may be affordable. 

Nearly all the commercially available electron accelerators with energy greater 

than a few MeV have high power. High energy also requires heavy shielding. Thus, 

accelerator and shielding both tend to increase cost for electron irradiators. In 

addition, an accelerator needs a conveyor system. For some applications, such as 

polymerisation of cables and sterilisation of medical products, these high-power

accelerators are necessary since the relevant doses are in the region of 20 100 kGy, 

and the quantity of the product irradiated is very large. 

A high-energy X-ray irradiator costs as much as or slightly more than a highenergy 

electron accelerator, due to the added cost of the X-ray converter. On the 

other hand, low-energy X-ray irradiators, which can be self-contained, are reasonably 

priced. As mentioned earlier, there are many units already operating for several 

years for blood irradiation, and more recently, other equipment with higher dose 

rates have been introduced. If these prove to be suitable, the cost of these low-energy 

X-ray irradiators would be reasonable. The first two rows of Table 3 list the capital 

cost and operational cost for four types of irradiator described in the next Section. 

Total treatment costs 

Four types of irradiator have been selected for calculating the treatment cost for a 

unit volume (1 L) of insects since they are currently most suitable for insect 

irradiation. In some cases, specific models have been selected to illustrate some 

realistic specifications; this does not, however, imply any endorsement for them. 

These four irradiators are: 

Table 3. Economic and technical characteristics of four types of irradiators. 

Capital cost 

(equipment 

transport) (US$) 

Operational cost 

(US$/year) 

Operators 

per shift 

Average 

dose rate 

(Gy/min) 

Volume of 

irradiation (L) 

Exchange time 

Tex (min) 

Throughput ’ 

(litres/hour) 

Gammacell 220 

- 20 kCi cobalt 

- self-contained 

178,000 

20,000 

18,300 (cobalt)$ 

( 183,000/10) 

Panoramic 

gamma 

irradiator 

- 60 kCi cobalt 

1,200,000 

(incl. transport) 

42,000 (cobalt) 

( 210,000/5) 

RS 2400 

- low-energy 

X-rays 

-10kW 


- 1 tube 

260,000 

5,000 

X-ray tubes 

electricity 

1 2 1 2 

139 

(average over 

10 years) 

2 (1 canister) 700 # 

1 § 


4.5 # 

(average over 

5 years) 

(several 

canisters) 

12.5 12 

20 

(5 canisters) 

Semi-automatic 

- low-energy X-rays 

-25kW 


- 4 tubes 

400,000 

10,000 

5,000* 

X-ray tubes 

electricity 

16 (4 trays) 

10 3 Continuous with 

10% dead time 

70 1310 110 105 

*This irradiator is supplied with an internal conveyor. However, it would need a short conveyor outside the 

unit at an estimated cost of US$ 5000. $This replenishing estimate is by the supplier of the Gammacell 220. 

However, there are other companies that offer this service at a lesser cost. For example, the estimate of the 

Institute of Isotopes, Hungary, is about 65% of this value; however, this procedure involves transfer of 

cobalt at the insect facility, and would require a water pool, at least 1.5 m deep. # This value is based on the 

irradiation configuration of the Lima, Peru irradiator. ’ The treatment dose is assumed to be 100 Gy. § A 

smaller exchange time will probably entail an additional operator per shift.

352 K. Mehta 

Gammacell 220 manufactured by MDS Nordion, Inc 1 . This is a selfcontained 

gamma irradiator and thus needs no external shielding. The 

assumed loading is 20 kCi of cobalt-60. Only one canister can be irradiated at 

a time (Figure 2). 

Panoramic irradiator operated in a batch mode, similar to the one in use in 

Peru (Figure 4). The assumed loading is 60 kCi of cobalt-60. It requires a 

shielded irradiation room as well as a shielded storage container (or a room) to 

house the source when not in use. Several canisters can be irradiated at a time. 

RS 2400 low-energy X-ray irradiator manufactured by Rad Source Technologies, 

Inc. This is a self-contained unit and thus no external shielding is needed. 

It uses one tube rated at 10 kW. The maximum energy of these X-rays is 

150 keV, with the average energy between 70 and 75 keV. Five canisters can be 

irradiated at a time (Figure 5). 

Semi-automatic low-energy X-ray irradiator manufactured by Rad Source 

Technologies, Inc. This is a self-contained unit and thus no external shielding 

is needed. It uses four tubes with a total power of 25 kW. The X-ray energy is 

similar to that for RS 2400. The insects can be continuously irradiated in four 

trays with a conveyor system (Figure 6). 

Table 3 compares relevant economic data for operating these four irradiators. It also gives 

values of some technical parameters used for these analyses. The rationale for selecting 

these values as well as the assumptions made for the analyses are explained below. 

The capital cost has two components as shown in Table 3; the first is an estimated 

cost of the equipment. The supplier should be contacted for a more current and 

accurate price. The second figure is the cost of transportation from the supplier to 

the buyer, which will vary depending on the destination. For the calculation of the 

unit cost, it is assumed that the total capital cost is amortised over 10 years; thus, 

10% of the capital cost may be viewed as an annual debt-servicing cost. 

The operational cost for the gamma irradiators includes the cost of replenishment 

of the cobalt source. Replenishment is a major procedure and is carried out 

either by the original supplier of the irradiator or some other organisation such as 

the national nuclear regulator. The irradiator will be out of service for a few days 

during this procedure. The radiation source is replenished when the throughput 

decreases to an uneconomical or impractical level for the pest control programme 

(e.g. 50% of the initial value). The initial dose rate is quite high in the case of 

Gammacell 220 (see Table 3), and thus the handling time between two irradiations is 

longer than the irradiation time. This results in a relatively slow decrease in the 

throughput with time (see Equation 1). For example, in the case of a Gammacell 220 

with an initial activity of 20 kCi, even though the activity decreases to 50 and 25% in 

about 5.3 and 10.6 years, respectively, the corresponding throughput decreases only 

to 80 and 56%. Thus, for such a unit it is assumed for the present analysis that cobalt 

would be replenished after 10 years 2 . However, for a panoramic irradiator, the hourly 

1 During the final preparation of this article, Nordion has discontinued this product line. 

2 At that point, however, the insect facility may decide to purchase a new irradiator instead of 

replenishing cobalt in the existing one. It may also decide to keep the old one with a lower dose 

rate. This decision will depend on the insect production rate and other prevailing conditions at 

that time.

throughput decreases to 61% after 5.3 years and to 35% after 10.6 years, and thus it 

is assumed that the source will be replenished after 5 years. The annual operational 

cost is then arrived at by dividing the cost of replenishing cobalt by 10 or 5, as shown 

in Table 3. 

The operational cost for the X-ray irradiators includes the cost for tube 

replacement and the use of electricity for these tubes. The manufacturer guarantees 

2000 h of operation for these tubes. Since the tube is dismountable, should the 

filament fail, it can be replaced without the need for a completely new tube. The 

complete tube is expected to be serviceable for 20,000 h. The replacement cost for 

one X-ray tube for the total 20,000-h cycle is about US$ 45,000. Thus, the average 

cost for a 2000-h period is US$ 4500, which is the value averaged over the entire cycle 

of changes. The cost of electricity can be between US$ 0.1 and 0.3 per kWh. 

The cost of labour is based on an annual salary of US$ 15 000 per person per 

shift (can be adjusted based on the local labour costs), a shift being 8 h a day for 7 

days a week (that is, 56 h/week). It is expected that only one person would be 

necessary for the operation of the Gammacell 220 and for the RS 2400, while two 

persons would be needed for routine operation of the panoramic gamma irradiator 

and also for the semi-automatic X-ray irradiator. 

The values in Table 3 for the dose rate and the irradiation volume are estimates 

and could vary by 10 20%. The value for the dose rate is an average value within the 

canister and is based on the supplier information, except for the panoramic 

irradiator, which is based on the measured values in the Peru irradiator (Figure 

4). For the gamma irradiators, the dose rate shown in Table 3 is the value at the 

midpoint between source replenishments. The exact value of the irradiation volume 

will depend on the acceptable dose variation in the canister. 

For the calculations of the throughput in litres of insect treated in 1 h, it is 

assumed that the goal is to deliver an average dose of 100 Gy. The throughput also 

depends not only on the dose rate and the volume of irradiation, but also on the 

handling time between the irradiation of two canisters or two batches. The estimated 

value of this handling or exchange time (T ex) is also listed in Table 3. The value of the 

hourly throughput for the four irradiators listed in Table 3 is based on the following 

expression: 

Hourly throughput (L=h) 


60 (min=h) irradiation volume (L) 

: (1) 

[dose (Gy)=dose rate (Gy=min)] T ex (min) 

To calculate the unit cost for the treatment, it is assumed that the irradiator is 

operated continuously either for one shift or two shifts. Assuming that the irradiator 

is operating for 50 weeks per year, one shift would be 2800 h/year (56 h/week 50 

weeks/year) and two shifts would mean 5600 h/year (112 h/week 50 weeks/year) of 

operation. 

The unit cost (total cost for irradiation of 1 L of insects) is divided into three 

components, one arising from the capital cost (equipment transport), the second 

from the operational cost, and the last from the labour cost. The unit cost is 

calculated using the following expressions: 

Unit cost (US$=L) capital cost operational cost labour cost (2)

354 K. Mehta 

Capital cost 

10%of capital cost (US$=year) 

throughput (L=h) UR (h=year) 

(3) 

Operational (gamma) 

Operational (X-ray) 

replacement cobalt (US$=year) 


(4) 

[(UR=2000 h) 4500 (US$) tubes] [power (kW) UR ER] 


(2:25 No of tubes) (power 

throughput (L=h) 

ER) 

(5) 

where, UR is the usage rate (number of hours the irradiator is used per year, either 

2800 or 5600), and ER is the cost of electricity (assumed here to be US$ 0.2 per kWh 

of electricity). ‘Power’ is the total power (kW) consumed by the X-ray irradiator, 

which is given in Table 3. 

US$15;000 number of operators number of shifts 

Labour cost : (6) 


It can be seen from these expressions that the capital and the labour components 

of the unit cost for all irradiators, and the operational component for the gamma 

irradiators are inversely proportional to the total quantity of insects treated during 

the year (throughput UR). However, the operational component for the X-ray 

irradiators is constant, i.e. it is independent of the quantity treated. 

Table 4 shows the results of the unit cost analyses for these four irradiators for 

the case when the irradiator is operating for either 2800 h (full 1 shift) or 5600 h (full 

2 shifts) during the year. 

It would be rare, however, that the weekly production of the insect rearing facility 

would exactly match the maximum weekly irradiation capacity of the irradiator 

Table 4. Weekly throughput and unit cost for four types of irradiator when used at maximum 

capacity for one- and two-shift operations. 

Gammacell 220 

(Gamma rays) 

Panoramic 

(Gamma rays) 

RS 2400 

(X-rays) 

Semi-automatic 

(X-rays) 

Weekly throughput 

(L/week) Unit cost (US cents/L) 

1-shift 2-shift 1-shift 2-shift 

3920 7840 27.1* 

(10.1 9.3 7.7) 

73 360 146 720 5.2 

(3.3 1.1 0.8) 

6160 12 320 17.4 

(8.6 3.9 4.9) 

5880 11 760 37.6 

(14.1 13.3 10.2) 

(5.1 

17.5 

4.7 

3.0 

7.7) 

(1.6 0.6 

13.1 

0.8) 

(4.3 3.9 

30.6 

4.9) 

(7.1 13.3 10.2) 

*The three components are, respectively, capital cost, operational cost (replenishment for gamma 

irradiators or tube replacement cost plus electricity use for X-ray irradiators), and the labour cost (either 

one- or two-shift).

when operated for 56 or 112 h per week (as shown in Table 4). Thus, the irradiators 

may not be used for all the 56 or 112 h per week. The unit costs would then be 

different than those shown in Table 4. To calculate the unit cost for this non-optimal 

use of the irradiator, it is convenient to use ‘weekly throughput’ of the rearing facility 

instead of ‘UR’ in the above expressions. These two quantities are related through 

the following expression: 

Weekly throughput (L=week) 


UR (h=year) hourly throughput (L=h) 

: (7) 

50 (week=year) 

The dependence of the total unit cost on the weekly throughput is shown in 

Figure 7; where weekly throughput is the weekly production of the insect rearing 

facility. For the Gammacell 220, at the lowest value of the weekly throughput shown 

(1000 L/week), the total unit cost would be slightly more than US$ 1. This value then 

decreases rapidly (open circles) as the weekly throughput increases and reaches a 

minimum of US$ 0.271 per L for a 1-shift operation at about 3920 L/week (see Table 

4). At this point, the irradiation capacity of the Gammacell is reached for a one-shift 

operation. If the insect rearing facility produces more insects than this, it will need a 

two-shift operation; and the unit cost first increases slightly (filled circles in Figure 7) 

and eventually decreases again till it reaches a minimum value of US$ 0.175 for a 

two-shift operation at 7840 L/week (Table 4). This is the best performance that can 

be achieved with this type of irradiator. If the production rate of the insectary is 

more than 7840 L of insects per week, then either a three-shift operation or two 

irradiators are needed. In the present analysis it is assumed that two irradiators with 

the same characteristics are used for a facility with a higher production rate. A oneshift 

operation for this second unit is shown by open circles (Figure 7), and a twoshift 

operation for the second unit by filled circles. However, the first unit will be 

operating at two shifts all the time. 

As mentioned above, it is assumed that the cost of electricity (for the X-ray 

irradiators) is US$ 0.2/kWh. If the cost is either US$ 0.1 or 0.3 per kWh, the unit 

cost curve for these irradiators will shift down or up by US$ 0.009 or US$ 0.024 for 

RS 2400 and the semi-automatic X-ray irradiator, respectively. 

Figure 7 will assist the insect rearing facility manager to select a suitable type of 

irradiator for the specific needs. Based on the weekly production of the facility, the 

value selected on the x-axis determines the unit cost for the various types of 

irradiator. It is recommended that the value of the production rate should also reflect 

the future requirements, at least over the following few years. The outcome of such 

analyses would to an extent depend on each specific case and the location of the 

irradiation facility. Thus, this analysis should be considered only as a guideline to 

assist with further detailed analysis for a specific case. 

In selecting the most suitable irradiator, unit cost is only one of the parameters. 

The others include available capital for investment, concerns with radioactive 

material, availability of technology, etc. 

Technology 

The technology necessary for gamma irradiators is simple, especially for the selfcontained 

types, and for the panoramic irradiators with batch operation and

356 K. Mehta 

Figure 7. Dependence of unit cost for insect irradiation on weekly throughput (production) 

of the insect rearing facility for two types of cobalt-60 gamma irradiator and two types of lowenergy 

X-ray irradiator. Open circles (k) refer to a one-shift operation of the irradiator, while 

close circles (“) refer to a two-shift operation. The first set of open and close circles refers to 

the first irradiator, while the second set refers to the second irradiator of the same type.


dry-storage. In both cases, no conveyor system is necessary, and the electronics is not 

complex. In most cases, the only moving parts are either the source or a device (for 

example, drawer or shuttle) that moves the canister into and out of the radiation 

field. In some cases, rotating turntables are also used. Such simple technology makes 

these gamma irradiators more reliable, and in the case of malfunction they are easy 

to fix. It also means very little downtime during which the irradiator is not available 

for irradiation. Regular periodic maintenance includes replenishment of the 

radiation source to compensate for the radioactive decay; this procedure is elaborate 

and will require external assistance, generally provided by the supplier of the source. 

Electron accelerators (necessary for electron-beam irradiators as well as for highenergy 

X-ray irradiators) are intrinsically more elaborate, and failure in the system 

requires expertise in the electronics and related technology to repair it. However, 

suppliers are making these systems much more robust and reliable, with a resultant 

decrease in downtime. The conveyor system will require periodic maintenance; 

however, this should not pose a problem. 

A low-energy X-ray irradiator is more robust and much less can malfunction. 

Like the isotopic source in a gamma irradiator, the X-ray tube has a finite life and 

thus needs to be replaced periodically, a task that is relatively easy. Being a selfcontained 

X-ray irradiator, there is no conveyor system to be maintained. 

Utility requirements 

The main utility requirements are an electrical power supply and, in some cases, 

cooling water: 

Gamma irradiators: 

self-shielded units: power for the canister movement (normal line voltage); 

panoramic unit (batch mode): power for the source movement, and 

ventilation of the irradiation room and if applicable, the source storage room. 

Electron irradiators: 

electrical power to operate the accelerator (any interruption would shut down 

the electron beam) and for the canister conveyor system. 

X-rays irradiators: 

high-energy: same as for electron irradiators, as well as cooling for the X-ray 

converter; and 

low-energy: power for the electron injector and for the X-ray tubes, as well as 

cooling for the X-ray converter. 

Safety and security 

All irradiators are designed to keep the radiation exposure and dose to workers ‘as low 

as reasonably achievable’ (ALARA), and within predetermined levels. These dose 

limits are based on the recommendations of several agencies of the United Nations 

(UN), including the International Atomic Energy Agency (IAEA), Food and 

Agriculture Organization of the United Nations (FAO), and World Health Organization 

(WHO) (IAEA 1996c). Appropriate safety methods and procedures have been

358 K. Mehta 

developed for each type of irradiator, and when operated correctly with the appropriate 

safeguards, they are safe and easy to use. Gamma irradiators are usually licensed by 

national atomic energy authorities, which set requirements such as restricting access to 

certain areas and authorised persons, a periodic survey of the radiation field in the 

vicinity where workers could be present, the use of personal radiation dosimeters, and 

the availability of radiation survey meters. On the other hand, electron accelerators or 

X-ray units are generally regulated by occupational safety and health agencies, which 

also require operator training, etc., and may require use of personal dosimeters. All 

these requirements are specifically aimed at protecting the workers from radiation. In 

addition, irradiator design incorporates interlocks that prevent unintentional access to 

areas with high-radiation fields. When the useful life of a gamma source is over, the 

irradiator or the source pencils are usually returned to the supplier for storage, reuse, 

recycling, or disposal. This is now becoming an elaborate and costly procedure. 

Security is an issue because of the use of radioactive material, either cobalt-60 or 

caesium-137. The source material is doubly sealed according to ISO standards and 

there is minimal risk of the radioactive material leaking from the capsule, especially 

in dry storage facilities. The main concern is the physical security of the radioactive 

material against unauthorised use of the material. Thus, in recent times, the 

transportation of radioactive material is getting much more elaborate and restrictive. 

The public is also more concerned nowadays with the eventual disposal of the 

radioactive material. However, in reality, that is a much smaller problem compared 

to the disposal of the used uranium fuel from the nuclear power reactors. Additional 

security is always prudent where a radioactive material is used. These issues are 

coming increasingly to the fore and are introducing difficulties with the transportation 

and operation of isotopic radiation sources. These particular issues do not arise 

with electron accelerators or X-ray irradiators; when the power switch is turned off, 

the radiation disappears along with the related concerns. 

The selection process 

Photons and electrons have similar radiation effects, so the choice of the source for 

treating insects is based on other considerations, such as those mentioned above. The 

power emitted by a cobalt-60 source of 50 kCi is roughly equivalent to that of a 750- 

W electron accelerator. Since caesium-137 emits about one-quarter of the energy per 

disintegration compared to cobalt-60, about 200 kCi of caesium would be needed to 

generate the same power. 

As discussed above, there are advantages and disadvantages of each type of 

irradiator. Thus, it is the responsibility of the facility manager to review the 

requirements of the programme concerned and select the most appropriate 

irradiator. The selection process will require optimisation and some level of 

flexibility. 

To summarise: 

Gamma-ray irradiators (self-contained and batch mode panoramic): 

relatively easy to operate, 

dose rate can be controlled by selecting appropriate activity of the source, 

especially for panoramic irradiator (to match the required throughput), 

high penetration allows the use of larger canisters,

do not need conveyor systems, and 

issues related to associated radioactivity. 

Accelerator based irradiators (electron-beam irradiators and high-energy X-ray 

irradiators): 

maintenance-intensive and relatively less easy to operate, 

need conveyor systems to move the canisters, 

electrons have shorter range, and thus smaller canisters are required, 

high throughput 

5MeV accelerators with low power are not currently commercially available 

(thus, there is not yet much field experience), and 

no issues related to radioactivity. 

Low-energy X-ray irradiators (self-contained): 


relatively easy to operate, 

acceptable dose rate, 

low penetration, and thus smaller canisters are required, 

do not need conveyor systems, 

currently only one supplier who is just coming into the market (thus, there is 

not yet much field experience), 

no issues related to radioactivity, 

less regulatory requirements, and 

low security risk. 

There is an important advantage of having a self-contained unit (either gamma ray 

or X-rays). It can be very easily re-located to another location within the insect 

rearing facility or even moved entirely to a new location. On the other hand, it would 

be very complex and costly to relocate a panoramic facility. 

Conclusions 

This review discusses several relevant characteristics of various radiation sources that 

are commercially available and suitable for pest control programmes that use natural 

enemies. The initial investment can vary from US$ 200,000 to about US$ 1 million. 

The selection would depend on several factors discussed extensively in this review. 

From the price of these irradiators, it is obvious that they may not be attractive to 

producers of garden products. However, they may be attractive to major producers 

of sterile insects or natural enemies. Also, with time it is envisaged that small 

producers of biological control agents will merge into a few major producers, 

especially with the rising international market. With usage, the price of these 

irradiators would also become more reasonable and affordable for some smaller 

producers. This is apparent from the fact that some of the irradiators are just coming 

onto the market. 

It is hoped that this review will make the managers of the pest management 

programmes aware of the available radiation sources and their advantages and 

disadvantages, and help them to select the most appropriate irradiator so as to 

improve their programme at an affordable cost.

360 K. Mehta 


I would like to thank the staff of the Insect Pest Control Section of the IAEA for many 

stimulating discussions, especially W. Enkerlin, J. Hendrichs and M. Vreysen, who also 

provided support for some of the figures in this paper. 

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