Randall A. Higgins

Survival of Kansas Dipel-Resistant European Corn Borer (Lepidoptera: Crambidae) on Bt and Non-Bt Corn Hybrids

Abstract The Kansas Dipel-resistant and susceptible European corn borer, Ostrinia nubilalis (Hübner), were evaluated in the greenhouse on different Bt transgenic events expressed in corn hybrids. There were important differences in the resistance offered by the different Bt event corn hybrids. Hybrid comparison tests indicate that these Dipel-resistant first-instar European corn borer were not able to survive to adulthood on whorl-stage MON810, Bt11, or 176 Bt event corn plants. Third instars did not survive to adulthood on whorl-stage MON810 or Bt11 event corn plants but a small number of fifth instars were found on whorl-stage DBT418 plants infested with Dipel-resistant larvae. First and third instars of these Dipel-resistant European corn borers caused more leaf-feeding damage and more tunneling on whorl-stage Bt-corn plants than did the Dipel-susceptible European corn borers. However, in the single Bt corn hybrid test, there was no survival of the Dipel-resistant European corn borers on DK580BtX or MAX454 Bt plants 35 to 42 d after they had been infested with first instars. These results demonstrate that the current Kansas selection of Dipel-resistant European corn borer strain cannot establish reproducing populations in the tested Bt corn lines and hybrids.

No Effect of Bacillus thuringiensis Corn and Bacillus thuringiensis on the Predator Orius insidiosus (Hemiptera: Anthocoridae)

Abstract Laboratory feeding studies were conducted to determine the effects on Orius insidiosus nymphs of feeding on 1-d-old European corn borer, Ostrina nubilalis (Hübner), larvae that had ingested a diet containing Bt toxins. A commercial formulation of Bacillus thuringiensis subsp kurstaki (Dipel ES) was incorporated into a meridic diet used to feed European corn borer larvae; they then were offered as food to O. insidiosus nymphs. Immediately after adult eclosion, O. insidiosus sex was determined, body weight and length were measured, and developmental time was calculated. Another feeding study was conducted to determine the effect of Bt corn silk on mortality of immature O. insidiosus. Fresh silks of Bt and non-Bt corn plants were offered to O. insidiosus nymphs until they reached adulthood. Mortality counts were made daily. Finally, visual counts of O. insidiosus were made on Bt and non-Bt corn in fields at three locations in Kansas. The numbers of O. insidiosus nymphs and adults were recorded on 40 plants per location on two sampling dates. No significant differences occurred in developmental time, body weight, or body length of mature O. insidiosus or mortality of immature O. insidiosus when reared on European corn borer larvae that had fed on a diet containing Dipel ES. The nymphs feeding only on Bt or non-Bt corn silk suffered 100% mortality. No significant difference occurred in mortality of immature O. insidiosus when they were fed on Bt or non-Bt silk one day and on corn earworm eggs the next day. Numbers of O. insidiosus adults and nymphs in fields of Bt corn and non-Bt corn did not differ significantly in most cases. Our results suggest that Bt corn does not have a significant effect on the predator O. insidiosus.

Expressed sequence tags from larval gut of the European corn borer (Ostrinia nubilalis): Exploring candidate genes potentially involved in Bacillus thuringiensis toxicity and resistance

BackgroundLepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST) libraries from the guts of lepidopteran larvae. Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxins, and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis), one of the most destructive pests of corn in North America and the western world. Our goals were to establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB.ResultsWe constructed two cDNA libraries from the guts of the fifth-instar larvae of ECB and sequenced a total of 15,000 ESTs from these libraries. A total of 12,519 ESTs (83.4%) appeared to be high quality with an average length of 656 bp. These ESTs represented 2,895 unique sequences, including 1,738 singletons and 1,157 contigs. Among the unique sequences, 62.7% encoded putative proteins that shared significant sequence similarities (E-value ≤ 10-3)with the sequences available in GenBank. Our EST analysis revealed 52 candidate genes that potentially have roles in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 18 chymotrypsin-like proteases, 13 aminopeptidases, 2 alkaline phosphatases and 1 cadherin-like protein. Comparisons of expression profiles of 41 selected candidate genes between Cry1Ab-susceptible and resistant strains of ECB by RT-PCR showed apparently decreased expressions in 2 trypsin-like and 2 chymotrypsin-like protease genes, and 1 aminopeptidase genes in the resistant strain as compared with the susceptible strain. In contrast, the expression of 3 trypsin- like and 3 chymotrypsin-like protease genes, 2 aminopeptidase genes, and 2 alkaline phosphatase genes were increased in the resistant strain. Such differential expressions of the candidate genes may suggest their involvement in Cry1Ab resistance. Indeed, certain trypsin-like and chymotrypsin-like proteases have previously been found to activate or degrade Bt protoxins and toxins, whereas several aminopeptidases, cadherin-like proteins and alkaline phosphatases have been demonstrated to serve as Bt receptor proteins in other insect species.ConclusionWe developed a relatively large EST database consisting of 12,519 high-quality sequences from a total of 15,000 cDNAs from the larval gut of ECB. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Our work provides a foundation for future research to develop an ECB gut-specific DNA microarray which can be used to analyze the global changes of gene expression in response to Bt protoxins/toxins and the genetic difference(s) between Bt- resistant and susceptible strains. Furthermore, we identified 52 candidate genes that may potentially be involved in Bt toxicity and resistance. Differential expressions of 15 out of the 41 selected candidate genes examined by RT-PCR, including 5 genes with apparently decreased expression and 10 with increased expression in Cry1Ab-resistant strain, may help us conclusively identify the candidate genes involved in Bt resistance and provide us with new insights into the mechanism of Cry1Ab resistance in ECB.

Evidence of Evolving Carbaryl Resistance in Western Corn Rootworm (Coleoptera: Chrysomelidae) in Areawide-Managed Cornfields in North Central Kansas

Abstract Susceptibility of adult populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte, to carbaryl was determined by a survey in 1996 before the implementation of an areawide management program near Scandia in north central Kansas. Subsequently, the susceptibility of western corn rootworm adults to carbaryl has been monitored throughout the program from 1997 to 2000 in both control and managed areas. In 1996, adults were highly susceptible to carbaryl with a mean LC50 value of 0.64 μg/vial. This value was comparable to those for adults collected from other regions within Kansas. However, adult susceptibility to carbaryl decreased rapidly within the managed area, where the cucurbitacin-carbaryl-based bait SLAM has been used as the primary tool to control adults in this project since 1997. In 1999, adults collected from the managed area were 9- and 20-fold less susceptible to carbaryl at the LC50 and LC90 levels, respectively, than those evaluated in 1996. In contrast, adults collected from the control area were only 2- and 3-fold less susceptible to carbaryl at the LC50 and LC90 levels, respectively, than adults evaluated in 1996. Although field adult populations of western corn rootworm were relatively low in 2000, evaluations showed trends similar to those in 1999 regarding their carbaryl susceptibility in the managed and control areas. These results provide evidence that western corn rootworm has been evolving carbaryl resistance rapidly in response to the use of SLAM in areawide-managed cornfields near Scandia.

Modeling the Development of Resistance by Stalk-Boring Lepidoptera (Crambidae) in Areas with Irrigated Transgenic Corn

Abstract The population dynamics and population genetics of two bivoltine species of corn borers are modeled in a hypothetical region of irrigated transgenic and nontransgenic corn. European corn borer, Ostrinia nubilalis (Hübner), adults were assumed to disperse throughout the landscape for both mating and oviposition. Southwestern corn borer, Diatraea grandiosella Dyar, adults were assumed to have very localized dispersal behaviors. Resistance developed quickly in both species when the allele for resistance to the transgenic toxin was dominant. When the allele for resistance was not dominant and few or none of the heterozygous larvae survived the toxin, the behaviors of adult insects determined the speed of resistance development. With block refuges of 10–40% the European corn borer developed resistance within 15–38 yr, but the southwestern corn borer never developed resistance within the 100 yr simulated. A row-strip refuge configuration did not change the time for resistance to develop in the European corn borer; however, row-strip refuges cannot be recommended for the southwestern corn borer. Uncertainty about adult behaviors in irrigated corn led us to examine the potential impact of behavior on resistance development. Adult behaviors influenced resistance development more than refuge size. For instance, if the first flight of moths exhibit random mating and uniform oviposition throughout the landscape and the second flight exhibits localized mating and oviposition, resistance developed at least five times faster in the southwestern corn borer population and three times slower in the European corn borer population compared with our standard assumptions. We discuss the implications of adult behavior, refuge configuration, refuge placement within the landscape and year-to-year relocation on resistance management plans.

Susceptibility of dipel-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae) to individual Bacillus thuringiensis protoxins.

Abstract Dipel-resistant and -susceptible strains of Ostrinia nubilalis (Hübner) were evaluated for larval mortality and growth inhibition when fed diets containing individual Bacillus thuringiensis protoxins. Resistance ratios for four of the protoxins in Dipel (Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa) were 170-, 205-, 524-, and >640-fold, respectively, considerably higher than the 47-fold resistance to Dipel. The Dipel-resistant strain was 36-fold resistant to Cry1Ba, a protoxin not present in Dipel. Another non-Dipel protoxin, Cry1Ca, did not cause significant mortality for either resistant or susceptible larvae with doses as high as 1.0 mg/ml. In an evaluation of larval growth inhibition, resistance to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ba was significant at concentrations of 0.054 and 0.162 μg/ml. However, growth inhibition with Cry2Aa was not significant at either dose. These data provide information on the spectrum of resistance and cross-resistance to individual Cry protoxins in this strain.

Heritability and stability of resistance to Bacillus thuringiensis in Ostrinia nubilalis (Lepidoptera: Pyralidae)

Realized heritability, h 2 , of resistance in European corn borer, Ostrinia nubilalis (Hubner), to Bacillus thuringiensis Berliner ssp. kurstaki endotoxins was examined in five resistant laboratory colonies. These colonies were reared on a meridic diet that incorporated a commercial formulation of B. thuringiensis , Dipel ES. Resistance in these colonies reached 42–67× by the seventh to twentieth selected generations and then plateaued. The realized heritability of resistance averaged 0.17–0.31 over all selected generations for the five colonies. In the three Iowa colonies, the highest realized heritability, 0.18–0.33, occurred during the second period of selection (seventh to thirteenth selected generations). In the two Kansas colonies, the highest realized heritability, 0.36 and 0.46, occurred during the first period of selection (first to sixth selected generations). In the absence of selection pressure, resistance in the southwest Kansas colony decreased from 62× to 42× after two generations, and remained at about that level for the next five generations.

Larval survival and development of susceptible and resistant Ostrinia nubilalis (Lepidoptera: Pyralidae) on diet containing Bacillus thuringiensis

Abstract  1 Larval survival and development of Dipel‐susceptible and ‐resistant strains of European corn borer, Ostrinia nubilalis (Hübner), were assayed using diets incorporating low doses of a commercial formulation of Bacillus thuringiensis var. kurstaki Berliner (Dipel ES).

Pollen Drift from Bacillus thuringiensis Corn: Efficacy Against European Corn Borer (Lepidoptera: Crambidae) in Adjacent Rows of Non-Bt Corn

Abstract Commercial non-Bacillus thuringiensis (Bt) corn was planted adjacent to Bt corn to determine the effects of Bt corn pollen falling on non-Bt plants for control of European corn borer larvae, Ostrinia nubilalis (Hübner). Field plots in Iowa and Kansas consisted of two center rows of Bt corn with eight rows of adjacent non-Bt corn on each side. In mid-September 1996 and 1997, we counted European corn borer larvae and larval tunnels in the stalk and ear shank. There were no significant differences in European corn borer numbers across non-Bt rows and the slope of the regression line was not significantly different from zero. In a single plot in Iowa, however, fewer tunnels were observed in rows of corn that were closer to Bt corn. This site was isolated from natural infestations and probably does not reflect a typical field situation. Our results suggest that Bt pollen has minimal or no control of European corn borer larvae in adjacent rows of non-Bt corn under natural conditions. Bt pollen drifting onto adjacent non-Bt plants should not increase the risks related to resistance management.

Larval feeding behavior of Dipel‐resistant and susceptible Ostrinia nubilalis on diet containing Bacillus thuringiensis (Dipel ES™)

Diet choice tests were conducted to examine the effects of Bacillus thuringiensis Berliner on larval feeding behavior of Dipel‐resistant and susceptible strains of European corn borer, Ostrinia nubilalis (Hübner). Larvae (first through fourth instars) were presented an untreated standard diet and three diets incorporating different concentrations of a commercial formulation of B. thuringiensis, Dipel ES™. Significantly higher proportions of susceptible and resistant larvae were found on the control diet compared to the proportions recorded on any of the Dipel‐treated diets. Soon after release (0.5 to 3 h), the occurrence of larvae on each of the diets was similar across different instars. The number of first and second instars on the control diet increased steadily over 72 h after release, when 43 to 75% of larvae were found on the control diet. The proportion of third and fourth instars on the control diet also increased initially, with an equilibrium occurring 12 to 24 h later when 30 to 40% of larvae were found on the control diet. Both strains appeared to avoid the Dipel‐treated diets and their responses were similar over different Dipel concentrations. The Dipel‐resistant strain showed higher ability to avoid the treated diets than the susceptible strain.