Impact of Spodoptera frugiperda neonate pretreatment conditions on Vip3Aa19 insecticidal protein activity and laboratory bioassay variation.

BACKGROUND: Variation in response to insecticidal proteins is common upon repetition of insect bioassays. Understanding this variation is a prerequisite to detecting biologically important differences. We tracked neonate Spodoptera frugiperda (J.E. Smith) susceptibility to Vip3Aa19 over 17 generations using standardized bioassay methods. Five larval pretreatment conditions and one bioassay condition were tested to determine whether susceptibility was affected. These included: storage time; prefeeding; storage at reduced temperature; storage at reduced humidity; colony introgression of field-collected individuals. Extremes of photoperiod during the bioassay itself were also examined. RESULTS: LC50 values for two strains of S. frugiperda varied 6.6-fold or 8.8-fold over 17 generations. Storage time and humidity had no impact on Vip3Aa19 susceptibility, whereas prefeeding significantly reduced subsequent mortality (by 27%). Storage at reduced temperature increased mortality for one colony (from 45.6 to 73.0%) but not for the other. Introgression of field-collected individuals affected susceptibility at the first generation but not for subsequent generations. A 24 h bioassay photophase significantly reduced susceptibility (by 26%) for both colonies. CONCLUSION: Certain pretreatment and bioassay conditions were identified that can affect S. frugiperda Vip3Aa19 susceptibility, but innate larval heterogeneity was also present. Our observations should help to increase the consistency of insecticidal protein bioassay results.

Fitness costs of Cry1Ab resistance in a field-derived strain of Ostrinia nubilalis (Lepidoptera: Crambidae).

The study of fitness costs associated with resistance to toxins from Bacillus thuringiensis Berliner (Bt) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) is important for understanding resistance evolution and for evaluating resistance management practices that prevent or mitigate resistance to transgenic corn, Zea mays L. Resistant individuals identified from a field collection in Kandiyohi, MN, were used to generate a Cry1Ab-resistant strain. We used susceptible and resistant strains with similar genetic background to establish crosses and estimate dominance of fitness costs by measuring fitness components and population parameters determined by fertility life tables. Spermatophore volume and mating frequency also were compared to identify potential effects of resistance on fertility. Inheritance of fitness costs in O. nubilalis varied from recessive to incompletely recessive among the parameters evaluated. Selection for resistance to Cry1Ab significantly reduced the fitness of O. nubilalis. Resistant insects exhibited reduced pupal weight and increased developmental time compared with susceptible and F1 larvae derived from reciprocal crosses of resistant and susceptible parents. In addition, it was observed that resistant insects exhibited a higher proportion of unsuccessful matings and lower fertility than the susceptible strain. Despite the differences observed in resistant insects, our results did not indicate strong evidence of fitness costs in the F1 progeny.

On-plant survival and inheritance of resistance to Cry1Ab toxin from Bacillus thuringiensis in a field-derived strain of European corn borer, Ostrinia nubilalis.

BACKGROUND: The high dose plus refuge is one of the major components of the resistance management plan mandated for transgenic corn expressing Cry toxins from Bacillus thuringiensis Berliner (Bt) that targets the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae). This strategy was based on assumptions such as functional recessive inheritance, which has not been previously tested for O. nubilalis. The authors used a field-derived resistant strain of O. nubilalis to define the nature of resistance to Cry1Ab toxin by examining the inheritance and on-plant survival of susceptible and resistant insects and their F(1) progeny. RESULTS: The resistant strain exhibited > 800-fold resistance to Cry1Ab. Resistance was primarily autosomal and controlled by more than one locus or multiple alleles at one locus. The degree of dominance D calculated on the basis of LC(50) values was - 0.45(h' = 0.27), indicating that resistance was incompletely recessive. No survivors were found on vegetative-stage Bt corn, although both resistant larvae and their F(1) progeny were able to survive on reproductive corn 15 days after infestation. CONCLUSIONS: A field derived O. nubilalis strain exhibited high levels of resistance to Cry1Ab and survived on transgenic corn by feeding on tissues with low Cry1Ab expression. The Cry1Ab resistance was primarily autosomal, incompletely recessive and polygenic. Tissue and on-plant survival data indicated that dominance varies depending on plant stage.

Increased survival of western corn rootworm on transgenic corn within three generations of on-plant greenhouse selection.

To delay evolution of insect resistance to transgenic crops producing Bacillus thuringiensis (Bt) toxins, nearby "refuges" of host plants not producing Bt toxins are required in many regions. Such refuges are expected to be most effective in slowing resistance when the toxin concentration in Bt crops is high enough to kill all or nearly all insects heterozygous for resistance. However, Bt corn, Zea mays, introduced recently does not meet this "high-dose" criterion for control of western corn rootworm (WCR), Diabrotica virgifera virgifera. A greenhouse method of rearing WCR on transgenic corn expressing the Cry3Bb1 protein was used in which approximately 25% of previously unexposed larvae survived relative to isoline survival (compared to 1-4% in the field). After three generations of full larval rearing on Bt corn (Constant-exposure colony), WCR larval survival was equivalent on Bt corn and isoline corn in greenhouse trials, and the LC(50) was 22-fold greater for the Constant-exposure colony than for the Control colony in diet bioassays with Cry3Bb1 protein on artificial diet. After six generations of greenhouse selection, the ratio of larval recovery on Bt corn to isoline corn in the field was 11.7-fold greater for the Constant-exposure colony than the Control colony. Removal from selection for six generations did not decrease survival on Bt corn in the greenhouse. The results suggest that rapid response to selection is possible in the absence of mating with unexposed beetles, emphasizing the importance of effective refuges for resistance management.

Comparison and validation of methods to quantify Cry1Ab toxin from Bacillus thuringiensis for standardization of insect bioassays.

Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC(50) values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.