Terrence A. Spencer

Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen

Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings.

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 LC50 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.

Baseline Susceptibility of Western Corn Rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis Toxin

Abstract Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4–7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.

Baseline Susceptibility of the Corn Earworm (Lepidoptera: Noctuidae) to the Cry1Ab Toxin from Bacillus thuringiensis

Abstract Susceptibility to Cry1Ab toxin from Bacillus thuringiensis (Bt) was determined for 12 field populations of neonate corn earworm, Helicoverpa zea (Boddie), from the United States. Earworm larvae were exposed to artificial diet treated with increasing Bt concentrations, and mortality and growth inhibition were evaluated after 7 d. The range of variation in Bt susceptibility indicated by growth inhibition was very similar to that indicated by mortality. Although interpopulation variation in susceptibility to both proteins was observed, the magnitude of the differences was small (less than or equal to fivefold). These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among corn earworm populations rather than variation caused by prior exposure to selection pressures. Therefore, corn earworms apparently are susceptible to Bt toxins across most of their geographic range.

Comparison and Validation of Methods To Quantify Cry1Ab Toxin from Bacillus thuringiensis for Standardization of Insect Bioassays

ABSTRACT 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 LC50 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.

Validation of a novel resistance monitoring technique for corn rootworm (Coleoptera: Chrysomelidae) and event DAS‐59122‐7 maize

Rootworm control tactics have recently expanded to include transgenic maize, which express insecticidal proteins from Bacillus thuringiensis (Bt) to reduce larval injury and protect yield potential. Exceptional root protection, increased grower efficiency and improved safety have led to rapid adoption of this technology in the USA. As a result, there is a recognized need for resistance management programmes aimed at delaying rootworm resistance. An essential component of resistance management programmes is the development and implementation of effective resistance monitoring techniques. Five test populations of Diabrotica virgifera virgifera (LeConte) were used to evaluate the sensitivity of two techniques used to describe population susceptibility to the Bt proteins expressed in event DAS‐59122‐7 maize: a diet bioassay employing purified proteins applied to artificial diet and a novel technique using sub‐lethal measures of larval development on seedling maize. Test populations included Rochelle‐US, an unselected susceptible colony, three populations composed of 5%, 25% or 50% Rochelle‐S mixed with Rochelle‐US, and the Rochelle‐S selected colony. Rochelle‐S was derived from the same founding population as Rochelle‐US, but selected for survival on DAS‐59122‐7 maize. Selections identified a minor trait conferring increased tolerance, and greenhouse plant efficacy evaluations confirmed that after 10 generations of selection with no random mating, Rochelle‐S caused significantly more root injury to DAS‐59122‐7 than Rochelle‐US. Rochelle‐S present at 5% of the test population resulted in measurable but not significant increase in injury to DAS‐59122‐7 maize. The diet bioassay was relatively insensitive to the susceptibility differences between the Rochelle‐US and Rochelle‐S populations. Neither LC50 nor EC50 estimates produced statistically significant differentiation between test populations with 0%, 5%, 25% or 50% Rochelle‐S. The sub‐lethal assay clearly identified differences between Rochelle‐S and Rochelle‐US and an increased rate of larval development was measurable when the test population contained only 5% of Rochelle‐S.

Fitness costs of Cry1F resistance in fall armyworm, Spodoptera frugiperda

Transgenic corn, Zea mays L., expressing the Bacillus thuringiensis Berliner (Bt) protein Cry1F has been registered for Spodoptera frugiperda (J. E. Smith) control since 2003 in the USA. Unexpected damage to Cry1F corn was reported in 2006 in Puerto Rico, and Cry1F resistance in S. frugiperda from Puerto Rico was documented. The study of fitness costs associated with insect resistance to Bt insecticidal proteins is important for understanding resistance evolution and for evaluating resistance management practices used to mitigate resistance to transgenic corn. Currently, no studies have addressed the fitness costs associated with Cry1F resistance in S. frugiperda. In this study, susceptible and resistant strains with similar genetic background and their reciprocal crosses were used to estimate Cry1F resistance fitness costs. Comparisons between life‐history traits and population growth rates of homozygous susceptible, heterozygous and homozygous resistant S. frugiperda were used to determine whether the resistance is associated with fitness costs. Major fitness costs were not apparent in either heterozygotes or homozygous resistant insects. However, there was a slight indication of hybrid vigour in the heterozygotes. Additionally, two lines in which the frequency of the resistant alleles was fixed at 0.5 were followed for seven generations, after which the frequency of resistant alleles slightly decreased in both lines. The lack of strong fitness costs associated with Cry1F resistance in S. frugiperda indicates that initial allele frequencies may be higher than expected in field populations and will tend to remain stable in field populations in the absence of selection pressure (e.g. Puerto Rico).

Development of European corn borer larvae on Event 176 Bt corn : influence on survival and fitness

European corn borer larvae, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) that have completed development on Event 176 Bt corn hybrids have survived exposure to sublethal doses of the Cry1Ab Bt toxin or are exploiting plant tissues that do not express the toxin. To evaluate the impact of such exposure, diapausing larvae were collected from Event 176 and conventional hybrids and compared for rates of pupation, parasitism, fitness (pupal weight, longevity, and fecundity) and susceptibility to the Cry1Ab toxin. Larvae completing development on Event 176 corn exhibited approximately 10% higher survival rates and correspondingly lower parasitism rates than larvae completing development on conventional hybrids. No significant differences were detected in pupal weight, fecundity, longevity or susceptibility to the Cry1Ab Bt toxin. These results indicate that survival on Event 176 corn are not adversely affect fitness and does not cause increased tolerance to the Cry1Ab toxin in subsequent generations.

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance.

Susceptibility of European and North American populations of the European corn borer to the Cry1F insecticidal protein

Baseline susceptibility to the Cry1F toxin from Bacillus thuringiensis was determined for 11 European Union (EU) and 24 United States (US) populations of European corn borer, Ostrinia nubilalis (Hübner). The response to Cry1F exposure was measured as mortality and growth inhibition for each population. The aim of this study was to characterize inter‐population variation in the susceptibility of EU and US European corn borer (O. nubilalis) neonates to Cry1F protein and additionally, intra‐population variation for the EU European corn borer to help develop effective and sensitive resistance monitoring methods. In this study, neonate European corn borer were exposed to a range of concentration of microbially‐produced Cry1F protein applied to the surface of artificial diet. Measures of mortality and growth inhibition of larvae on surface‐treated diet relative to untreated control diet were taken after 7 days. A comparison between European and American corn borer populations showed that the range of variation in Cry1F toxin susceptibility assessed by mortality and growth inhibition was similar. Differences in Cry1F susceptibility between European corn borer populations across continents and among the various populations within each geography were apparent although similar variation among replicates for the same population were also observed in two EU populations. It is likely that these small differences reflect natural variation in sensitivity among populations of European corn borer as well as methodological differences and are not influenced by geographic location. The importance of establishing baseline susceptibilities to resistance detection is discussed.