Vol.12_No.2 - Pesticide Alternatives Lab - Michigan State University

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2The Cry1Ac protein was produced according tothe method in Albert et al. (8) from an E. coli straincontaining the hyper-expressivity recombinant plasmidvector pKK223, kindly provided by Daniel R. Zeigler,Ohio State University, USA. The toxin was purifiedfrom over expressing cells by sonication and extensivewashing with sodium bromide. Proteins werequantified according to Lowry et al. (9) and the toxinwas quantified by SDS-PAGE densitometry beforepreparing dilutions (ranging from 10 to 20000 fold) indistilled water (10). Forty percent of the proteinextracted the recombinant E. coli cultures were foundto comprise Cry1Ac toxin. LC50 values weredetermined for the toxin.Laboratory strains of H. armigera were establishedfrom those collected in cotton fields during thecropping season of 2001-02 from major cotton growingregions of the south Indian cotton ecosystem: Nagpurand Nanded (Maharastra); Guntur, Madhira, andNalgonda (Andhra Pradesh); Dharwad, Raichur, andMysore (Karnataka); Coimbatore, Madurai, andKovilpatti (Tamil Nadu). These 11 sampling locationsrepresent the cotton growing ecosystems of south India(Fig. 1). An insecticide susceptible H. armigeraobtained from ICRISAT, Patancheru, Hyderabad wasused as a baseline susceptible strain for comparison.Larvae were reared on a chickpea-based semi-syntheticdiet (11), individually in 32-well multicavity trays untilpupation. Moths were kept in glass jars at 270C ±10Cand 70% RH and fed with a 10% honey solution. Alayer of muslin cloth was placed on the inner surface ofthe jars for oviposition.Laboratory cultures were established for eachpopulation from 500-650 moths and reared to gethomogenous F1 populations before conductingbioassays. Bioassays were carried out in 32-wellmulticavity culture trays. Six-day-old juvenile larvae(ca: 30-40 mg) were tested, one per well, on cottonleaves dipped in different concentrations of the toxin.In all, 30 larvae in three replicates were tested for eachtreatment. Mortality was recorded daily for six days.All assays were repeated three times and pooled datawere subjected to statistical analysis. Assays wereperformed in the laboratory at conditions at 270C ±10C and 70% RH. Median lethal concentrations(LC50) presented in Table 1 were derived from logdose probit calculations (12) using the MLP 0.38statistical package (13).Cry1Ac protein was found to be toxic to allgeographic populations tested (Table 1). Theinsecticide susceptible ICRISAT laboratory strain wasthe most susceptible. Compared with the others,geographic populations from Nagpur, Nanded, Guntur,Nalgonda, Madhira, and Raichur were found tolerant tothe toxin. Mortality of different populations ispresented in Table 1. LC50 values for Cry1Ac rangedfrom 0.147 to 1.095 µg/ml. The fiducial limits (atP=0.95) of the probit assay data indicated that therewas a good deal of variability in response of differentpopulations to Cry1Ac. The Kovilpatti (Tamil Nadu;extreme southern most part of south Indian cottonecosystem) population was found to be as susceptibleas the laboratory strain for Cry1Ac. The Coimbatoreand Dharwad populations were similar to each other ata resistance factor (RF) of 1.5. Geographic populationsof Guntur and Nanded recorded the highest RF of 8.03and 8.42, respectively. The LC50 values of the testpopulations could be considered as the baselinesusceptibility LC50 values for these individualpopulations and could be used for monitoringresistance in the future.For resistance management programs to beeffective, monitoring, surveillance, and earlydetection of resistant phenotypes in the fieldpopulations are important pre-requisites in orderto initiate timely remedial measures and toevaluate the effectiveness of resistancemanagement strategies. Traditionally, log doseprobit assays and recently diagnostic dose assays,have been routinely used to monitor developmentof resistance to insecticides (3, 14, 15).Diagnostic or discriminatory dose assays arenormally employed to identify individuals in apopulation resistant to the toxin (16), whereas logdose probit assays are useful to assess the level ofresistance of a population as a ration over areference strain or a population, usually asusceptible check. Therefore, for monitoringresistance built up in a population, diagnosticdose assays and log dose probit assays are themost appropriate (10,16,17). The results of thepresent analysis, showing significant differencesin susceptibility to Cry1Ac toxin among11