pathogenic effect of metarhizium anisopliae on the larval growth and ...

Pak. Entomol. Vol. 31, No.2, 2009PATHOGENIC EFFECT OF METARHIZIUM ANISOPLIAE ON THE LARVALGROWTH AND DEVELOPMENT OF OCINARA VARIANS WALKER(LEPIDOPTERA: BOMBYCIDAE)Abid Hussain, Lin Ruan, Mingyi Tian and Yurong HeDepartment <strong>of</strong> Entomology, South China Agricultural University, Guangzhou, 510640, ChinaABSTRACTOcinara varians Walker is considered as an important pest <strong>of</strong> Ficus microcarpa and other ornamental plants.The present study explained the <strong>pathogenic</strong> <strong>effect</strong> <strong>of</strong> Metarhizium <strong>anisopliae</strong> on the growth (body weight),mortality, development and various growth indices on newly moulted (III-V) instar larvae at a concentration <strong>of</strong>1 × 10 7 spores/ml. Our laboratory experiments showed that there was no difference in the susceptibility <strong>of</strong> thedifferent larval stages tested in terms <strong>of</strong> <strong>effect</strong>iveness on O. varians. These fungal spores retarded the larvalgrowth (35%-76%), which resulted in a prolonged developmental time <strong>of</strong> the infected larvae. Food consumption(21-27%), relative consumption rate (31-48%), efficiency <strong>of</strong> conversion <strong>of</strong> ingested food (18-69%), efficiency<strong>of</strong> conversion <strong>of</strong> digested food (26-70%), and relative growth rate (42-84%) values declined significantly, butconcurrently a significant increase in approximate digestibility (3-36%) was observed. These results suggest thatM. <strong>anisopliae</strong> may be an alternative for synthetic pesticides in order to protect ornamental plants against O.varians.Keywords: Biological control, consumption, food utilization, growth, larval instars, Ocinara variansINTRODUCTIONThe lepidopteran, Ocinara varians Walker(Bombycoidea: Lepidoptera) is an important pestresponsible for the defoliation <strong>of</strong> Ficus microcarpa(Ou et al., 2006) and F. religiosa (Rajavel andShanthi, 2007). The early instar larvae damage leaves,twigs and tender tips <strong>of</strong> the plant and make the leavestransparent. The late instars completely defoliate thetrees and degrade the aesthetic value <strong>of</strong> the gardens.Until now, the control <strong>of</strong> O. varians larvae reliesmainly on the use <strong>of</strong> synthetic insecticides. But theuse <strong>of</strong> chemicals around homes, gardens andrecreational parks possesses direct danger to humansand environment. These problems promoted a searchfor the alternative methods <strong>of</strong> plant protection. As aresult <strong>of</strong> that, interest in bio-pesticides is growingbecause they are capable <strong>of</strong> attacking alldevelopmental stages <strong>of</strong> insects (Ferron, 1978),environmentally safe and have pest selectivity (Kingand Coleman, 1989).Current information on the use <strong>of</strong> Metarhizium<strong>anisopliae</strong> on insects indicates that these insectparasites attach their spores into the epidermis <strong>of</strong> theinsects. Then they pierce the epidermis, aftergerminating infiltrates the insect. Thisentomo<strong>pathogenic</strong> fungus begins to suck thehaemolymph <strong>of</strong> the insect once it enters the insect,grows and proliferates inside the body <strong>of</strong> the host,interacts with the insect’s defense mechanisms andfinally sporulation occurs on the cadaver <strong>of</strong> the host.Then the infected insect soon dies by the loss <strong>of</strong>water and after a few days its body hardens. Theinteractions that occur between the fungus and theinsects are exceedingly complex and are dependentupon specific host-pathogen interactions (Hajek andSt Leger, 1994). During this period, the insects maycontinue feeding, adding to crop damage before theirdeath (Hajek, 1989; Fargues et al., 1994). There arefew reports about the use <strong>of</strong> biocontrol agents againstO. varians. The previous laboratory investigationsinvolve the use <strong>of</strong> Trichogramma wasps havingparasitizing capacity (Ou et al., 2006) and OvNuclear polyhedrosis virus (Ou et al., 2007) againstO. varians.Considering the situation, the present study was setup at the Department <strong>of</strong> Entomology, South ChinaAgricultural, University, Guangzhou to investigatethe influence <strong>of</strong> M. <strong>anisopliae</strong> on the mortality,growth and food utilization indices <strong>of</strong> O. variansthird, fourth and fifth instar larvae in the laboratory.The overall objective is to determine the potential <strong>of</strong>116

Pak. Entomol. Vol. 31, No.2, 2009The relative consumption rate (RCR) is a measure <strong>of</strong>the relative rate at which nutrients enter the digestivetract, reflecting the insect’s physiological andbehavioral response to the food provided. Theinfected insects remained significantly lower (31-48%) compared with the uninfected controlcounterparts in all the larval instars. Along with this,the RCR generally declined over the course <strong>of</strong> larvaldevelopment (III-V instar) for both infected anduninfected larvae (Table 1).The approximate digestibility (AD) <strong>of</strong> the O. varianslarvae infected with fungal spore suspension hadsignificantly higher AD values (3-36%) whencompared with uninfected larvae at all the studiedlarval growth stages. Additionally, there was adecreasing trend in the AD values over the course <strong>of</strong>larval development for both infected and uninfectedlarvae.Like RCR, the RGR <strong>of</strong> the infected larvae wassignificantly reduced (42-84%) compared with theuninfected healthy insects <strong>of</strong> all the growth stages.The RGR <strong>of</strong> the infected and uninfected larvaedeclined with the age <strong>of</strong> the larvae (3 rd to 5 th ).Generally, the late instars had lower RGR valuescompared with the 3 rd instar larvae.The response <strong>of</strong> O. varians infected larvae towardsECI (index that expresses the efficiency <strong>of</strong>conversion <strong>of</strong> ingested food to insect biomass) andECD (efficiency <strong>of</strong> conversion <strong>of</strong> assimilated food toinsect biomass production) was quite similar. Boththe food utilization indices (ECI and ECD values)declined in infected larvae and remained significantlyat lower level than uninfected. This decreasing trendwas observed in all the studied larval instars(Table 1).DISCUSSIONIn this study, the results clearly demonstrated that M.<strong>anisopliae</strong> spores parasitized O. varians during themost destructive stage <strong>of</strong> the life cycle <strong>of</strong> O. varians(III-V larval stages), thus indicating potential as abiological control agent against O. varians.The immersion <strong>of</strong> O. varians larvae in sporesuspension <strong>of</strong> M. <strong>anisopliae</strong> exhibited retardedgrowth with significantly lower body weight andconsumption (compared with control). The differencein growth (body weigh) and consumption wasobserved at all the studied instars. As a result <strong>of</strong> that,the infected larvae took longer time to complete theirinstars. The lower body weights are consistent withthe results reported by other researchers; Cossentineand Lewis (1987) and Grundler et al. (1987) whodescribed decline in the body weight <strong>of</strong> Agrotisipsilon (Hufnagel) infected with V. necatrix. Thesignificant reduction in consumption is attributed tothe production <strong>of</strong> toxic substances by theentomo<strong>pathogenic</strong> fungi inside the host that lead tomechanical disruption <strong>of</strong> the insect structuralintegrity by hyphal growth (Tefera and Pringle, 2003).The destructive <strong>effect</strong>s <strong>of</strong> pathogen proteases oninsect cuticle have also been reported by St. Leger etal., (1991). Previous investigations are in line withour findings which have shown significant reductionsin feeding after treatment with entomo<strong>pathogenic</strong>fungal spore suspensions (Moore et al., 1992;Fargues et al., 1994; Seyoum et al., 1994; Thomas etal., 1997; Tefera and Pringle, 2003; Hussain et al.,2009).In our study, the developmental time among theinfected larvae in comparison with control wasevidently higher. The same results were obtained byHenn and Solter (2000), by the application <strong>of</strong>microsporidium Vairimorpha sp. on the diet <strong>of</strong>Lymantria dispar. In this case the feeing period waslonger and the infected larvae died without pupationcompared with control counterparts. While anothertendency <strong>of</strong> infected V instar larvae with M.<strong>anisopliae</strong> (strain 2049), had already been observedin which the infected larvae <strong>of</strong> Ocinara varianspupate (Hussain et al., 2009).Along with extended developmental time, most <strong>of</strong> theinfected larvae died without pupation. Previousstudies have explained that these differences areprobably due to the fungal cells which completelydestroy the fat body cell and thus destroying thefunction <strong>of</strong> organ. With the destruction <strong>of</strong> fat bodies,the larva loses its ability to synthesize nutrients andits capacity to store nutrients. Consequently, the larvadoes not pupate and disrupted the life cycle (Mitchelland Cali, 1994).The entomo<strong>pathogenic</strong> hyphomycetes are capable <strong>of</strong>attacking all developmental stages <strong>of</strong> host (Ferron,1978) but the differences in susceptibility does existamong different developmental stages <strong>of</strong> the samehost (Inglis et al., 2001). These differences in119

Pak. Entomol. Vol. 31, No.2, 2009susceptibility <strong>of</strong> caterpillar to pathogens and parasitesis positively correlated to melanism on their cuticleand in the midgut (Wilson et al., 2001). Melaninstrengthens the insect’s cuticle, preventing thepenetration <strong>of</strong> parasites and pathogens. Differentdevelopmental stages <strong>of</strong> insects have different levels<strong>of</strong> melanin in the cuticle (Lee and Wilson, 2006). Asa result <strong>of</strong> that, the different levels <strong>of</strong> mortality wereobserved in our case. Such differences in themortality with respect to developmental stage havebeen reported in several investigations on caterpillarsby the application <strong>of</strong> entomo<strong>pathogenic</strong> fungal sporesuspension (Hafez et al., 1997; Nguyen et al., 2007).The significant differences obtained between indicesfrom uninfected (control) and infected assay <strong>of</strong>caterpillars obviously reflect the <strong>effect</strong>s caused by thespore suspension <strong>of</strong> M. <strong>anisopliae</strong>. Significantlylower RCR, RGR, ECI and ECD, as well as anelevated AD values are a typical sign <strong>of</strong> the activity<strong>of</strong> the conidial suspension <strong>of</strong> M. <strong>anisopliae</strong>. Thelower AD values in the late instars <strong>of</strong> infected larvaecan be explained as in late instars these caterpillarsconsumed food indiscriminately to meet the demandfor energy and nitrogen. Accordingly, a large portion<strong>of</strong> ingested food is lost in the form <strong>of</strong> faeces(Muthukrishnan and Pandian, 1987). The higher ADvalues in early the instars <strong>of</strong> infected larvae can beexplained as in early instars these caterpillarsconsumed only some part <strong>of</strong> leaf which might resultin higher AD at this age. O. varians larvae infectedwith entomo<strong>pathogenic</strong> fungi had significantly higherAD values as compared to the healthy control. Thelarvae immersed with entomo<strong>pathogenic</strong> fungiexhibited significantly lower RCR, RGR, ECD andECI values than uninfected larvae, indicating thatmore food was being metabolized for energy and lesswas being converted to body mass. The reduction inthe nutritional indices (RCR, RGR, ECD and ECI)had also been observed in the previous studies oneastern spruce budworm larvae, Choristoneurafumiferana, where microsporidium (Nosemafumiferanaei) (Thomson) spores at a medium lethalconcentration were used (Bauer and Nordin, 1988).From the present results, it may be concluded that theinoculation <strong>of</strong> M. <strong>anisopliae</strong> spores suspensionsreduced the growth <strong>of</strong> the O. varians larvae whichoriginate not only from simultaneously reducedconsumption but also from the worse efficiencies <strong>of</strong>conversion <strong>of</strong> the ingested (ECI) and digested food(ECD). However, the findings presented here for M.<strong>anisopliae</strong> spores could be developed as biologicalcontrol agent as an alternative to conventionalsynthetic insecticides against O. varians larvae inIMP programme.REFERENCESBauer, L. S. and G. L. Nordin, 1988. Nutritionalphysiology <strong>of</strong> the eastern spruce budworm,Choristoneura fumiferana, infected with Nosemafumiferanae, and interactions with dietarynitrogen. Oecologia, 77: 44-50.Cossentine, J. E. and L. C. Lewis, 1987. Impact <strong>of</strong>Vairimorpha necatrix and Vairimorpha sp.(Microspora: Microsporida) on Bonnetia comta(Diptera: Tachinidae) within Agrotis ipsilon(Lepidoptera: Noctuidae) hosts. J. Invertebr.Pathol., 47(3): 303-309.Fargues, J., J. C. Delmas and R. A. Lebrun, 1994.Leaf consumption by larvae <strong>of</strong> the Coloradopotato beetle (Coleoptera: Chrysomelidae)infected with the entomopathogen, Beauveriabassiana. J. Econ. Entomol., 87: 67-71.Ferron, P., 1978. Biological control <strong>of</strong> insect pests byentomogenous fungi. Ann. Rev. Entomol., 23:409-442Goettel, M. S. and G. D. Inglis, 1997. Fungi:Hyphomycetes. In: Lacey, L. A. (Ed.), Manual <strong>of</strong>techniques in insect pathology. Academic Press,San Diego, CA, pp. 213-249.Grundler, J. A., D. L. Hostetter and A. J. Keaster,1987. Laboratory evaluation <strong>of</strong> Vairimorphanecatrix (Microspora: Microsporida) as a controlagent for the black cutworm (Lepidoptera:Noctuidae). Environ. Entomol., 16: 1228-1230.Hafez, M., F. N. Zaki, A. Moursy and M. Sabbour,1997. Biological <strong>effect</strong>s <strong>of</strong> the entomo<strong>pathogenic</strong>fungus, Beauveria bassiana on the potato tubermoth Phthorimaea operculella (Seller). J. PestSci., 70: 158-159.Hajek, A. E., 1989. Food consumption by Lymantriadispar (Lepidoptera: Lymantriidae) larvaeinfected with Entomophaga maimaiga(Zygomycetes: Entomophthorales). Environ.Entomol., 18: 723-727.120

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