R. Jeff Whitworth

Effect of Corn Hybrids Expressing the Coleopteran-Specific Cry3Bb1 Protein for Corn Rootworm Control on Aboveground Insect Predators

Field and laboratory studies were conducted to determine the effect of transgenic Bacillus thuringiensis (Bt) corn, Zea mays L. (YieldGard Rootworm), expressing the Cry3Bb1 protein on aboveground nontarget insect predators (minute pirate bug, ladybird beetles, and carabids). Visual counts of adult and immature Orius insidiosus (Say), Coleomegilla maculata (DeGeer), Hippodamia convergens Gurin-Meneville, and Scymnus spp. occurring in Bt corn and its non-Bt isoline were made at Manhattan, KS, in 2002 and at Manhattan and Scandia, KS, in 2003. No significant differences were found between the Bt corn and non-Bt isoline plots in the abundance (number per plant) of O. insidiosus, C. maculata, H. convergens, and Scymnus spp. Field predation on Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) egg masses was also observed during the silking stage of corn at Manhattan and Scandia in 2003. No significant differences were observed among treatments in predation rate for predators with chewing versus sucking mouthparts. Two laboratory studies determined the effect of Cry3Bb1 protein expressed in Bt corn pollen on C. maculata and carabids. The larvae of C. maculata were reared on Bt pollen, non-Bt pollen, or greenbugs, Schizaphis graminum (Rondani). The duration of larval and pupal stages, developmental time from egg hatch to adult emergence, percentage of survival, and elytra length were compared among treatments. There were no significant differences in developmental time of larvae fed pollen or greenbugs during their first two instars. However, significantly prolonged development of the third (1 d) and fourth instars (2 d) was observed for larvae fed greenbugs only. Total time for larval development was significantly longer for larvae that fed on greenbugs versus larvae fed on pollen. No significant differences were observed among treatments in the percentage of larvae that pupated or pupal stage duration. Larvae that fed on greenbugs had higher pupal and adult weights compared with pollen-fed larvae. However, pupal and adult weights did not vary between the Bt and non-Bt pollen treatments. No significant differences occurred in longevity and elytra length of beetles among all treatments. Two carabid species, Harpalus caliginosus F. and Harpalus pensylvanicus DeGeer, were reared on moistened dog food sprinkled with Bt or non-Bt corn pollen. No significant differences in mortality of H. caliginosus and H. pensylvanicus were detected among any of the treatments. There was no significant effect of Bt pollen on fecundity and egg viability of H. caliginosus. Our studies showed that YieldGard Rootworm had no effect on the selected coleopteran predators; therefore, this Bt corn hybrid could be used in an integrated pest management system.

Hessian Fly-Associated Bacteria: Transmission, Essentiality, and Composition

Plant-feeding insects have been recently found to use microbes to manipulate host plant physiology and morphology. Gall midges are one of the largest groups of insects that manipulate host plants extensively. Hessian fly (HF, Mayetiola destructor) is an important pest of wheat and a model system for studying gall midges. To examine the role of bacteria in parasitism, a systematic analysis of bacteria associated with HF was performed for the first time. Diverse bacteria were found in different developmental HF stages. Fluorescent in situ hybridization detected a bacteriocyte-like structure in developing eggs. Bacterial DNA was also detected in eggs by PCR using primers targeted to different bacterial groups. These results indicated that HF hosted different types of bacteria that were maternally transmitted to the next generation. Eliminating bacteria from the insect with antibiotics resulted in high mortality of HF larvae, indicating that symbiotic bacteria are essential for the insect to survive on wheat seedlings. A preliminary survey identified various types of bacteria associated with different HF stages, including the genera Enterobacter, Pantoea, Stenotrophomonas, Pseudomonas, Bacillus, Ochrobactrum, Acinetobacter, Alcaligenes, Nitrosomonas, Arcanobacterium, Microbacterium, Paenibacillus, and Klebsiella. Similar bacteria were also found specifically in HF-infested susceptible wheat, suggesting that HF larvae had either transmitted bacteria into plant tissue or brought secondary infection of bacteria to the wheat host. The bacteria associated with wheat seedlings may play an essential role in the wheat-HF interaction.

Genetic Structure of Tribolium castaneum (Coleoptera: Tenebrionidae) Populations in Mills

ABSTRACT The red flour beetle, Tribolium castaneum (Herbst), is primarily found associated with human structures such as wheat and rice mills. Such structures are predicted to be spatially isolated resource patches with frequent population bottlenecks that should influence their genetic structure. Genetic diversity and differentiation among nine populations of T. castaneum collected from wheat and rice mills (ranging from <1–5,700 km apart) were investigated using eight polymorphic loci (microsatellites and other insertion-deletion polymorphisms, each with 3–14 alleles). Seventy-two locus-by-population combinations were evaluated, of which 31 deviated significantly from Hardy—Weinberg equilibrium, all because of a deficiency of heterozygotes. AMOVA analysis indicated significant differences among populations, with 8.3% of the variation in allele frequency resulting from comparisons among populations, and commodity type and geographic region not significant factors. Although there were significant differences in genetic differentiation among populations (FST values = 0.018–0.149), genetic distance was not significantly correlated with geographic distance. Correct assignment to the source population was successful for only 56% of individuals collected. Further analyses confirmed the occurrence of recent genetic bottlenecks in five out of nine populations. These results provide evidence that populations of T. castaneum collected from mills show spatial genetic structure, but the poor ability to assign individuals to source populations and lack of isolation by distance suggest greater levels of gene flow than predicted originally.

Pheromone Trapping to Determine Hessian Fly (Diptera: Cecidomyiidae) Activity in Kansas

Abstract The Hessian fly (HF), Mayetiola destructor (Say), has historically been a significant pest of wheat throughout the Great Plains, including Kansas. However, it has been many decades since the flies’ activity has been monitored in the field. This paper presents research on the activity of the HF throughout the year in Kansas, i.e., examining when the fly is active and how moisture events may play a role. Results of a newer technology, pheromone trapping, in four counties in Kansas shows that HF males are active in the fall, at least 6 wk later than the historical fly-free dates established nearly 100 yr ago. Therefore, the ‘Hessian Fly-Free Date’ is not as valid as previously thought and might be better referred to as the ‘Best Pest Management Planting Date’. Using pheromones for fall and spring trapping also indicated that HF is more active throughout the spring than previously thought, with almost continuous fly emergence and numerous emergence peaks in both spring and fall. Pheromone traps were also used to determine if fly emergence peaked after moisture events as previously thought. Fly emergence could not be positively correlated with any moisture event.

Massive Shift in Gene Expression during Transitions between Developmental Stages of the Gall Midge, Mayetiola Destructor.

Mayetiola destructor is a destructive pest of wheat and has six developmental stages. Molecular mechanisms controlling the transition between developmental stages remain unknown. Here we analyzed genes that were expressed differentially between two successive developmental stages, including larvae at 1, 3, 5, and 7 days, pupae, and adults. A total of 17,344 genes were expressed during one or more of these studied stages. Among the expressed genes, 38–68% were differently expressed between two successive stages, with roughly equal percentages of up- and down-regulated genes. Analysis of the functions of the differentially expressed genes revealed that each developmental stage had some unique types of expressed genes that are characteristic of the physiology at that stage. This is the first genome-wide analysis of genes differentially expressed in different stages in a gall midge. The large dataset of up- and down-regulated genes in each stage of the insect shall be very useful for future research to elucidate mechanisms regulating insect development and other biological processes.

Residual Effect of Insecticide Treatment Plus Use of Sticky Traps on Brown Recluse Spiders (Araneae: Sicariidae) on Two Surfaces

Abstract There is a need for safe and consistent management of the brown recluse spider, Loxosceles reclusa Gertsch & Mulaik, a toxic spider found in homes throughout Kansas and the south and central United States. This study examined the efficacy of two general-use insecticides with brown recluse on their labels, .05% bifenthrin with 0.0125% zeta-cypermethrin (Insecticide 1), and 0.03% lambda-cyhalothrin (Insecticide 2) against the brown recluse spider at three times post-treatment, 1, 24, and 96 hr, and on two substrates, carpet and tile. This study also evaluated the efficacy of these insecticides on spiders exposed for three lengths of time, 0.5, 1, and 30 min on each substrate. Results indicate that Insecticide 2 had better overall residual efficacy than Insecticide 1. In addition, in one experiment efficacy was better when the spiders were exposed on tile surfaces. Residual efficacy for Insecticide 2 was greater one and 24 hr after application but decreased at 96 hr. The efficacy of Insecticide 1 was not affected by length of time between application and spider exposure, on either surface. Spider mortality was also significantly impacted by the length of time the spiders were exposed to the treated surface. There was no significant difference in spider mortality with either insecticide at 0.5 or 1 min exposure. However, exposures of 30 min led to significantly greater spider mortality for both insecticides, and on both substrates. 100% mortality occurred when spiders were exposed to either insecticide for 30 min on tile. This study also examined the effect of sticky traps placed in homes and in combination with an insecticide application of 0.03% lambda-cyhalothrin. The sticky traps alone did not reduce the infestation but, in combination with one insecticide treatment, spider trap numbers decreased over a 4 wk period.

Transcriptomic Analyses of Secreted Proteins From the Salivary Glands of Wheat Midge Larvae

Abstract Both the wheat midge (Sitodiplosis mosellana) (Géhin) (Diptera: Cecidomyiidae) and the Hessian fly (Mayetiola destructor) (Say) (Diptera: Cecidomyiidae) belong to a group of insects called gall midges (Diptera: Cecidomyiidae), and both are destructive pests of wheat. From Hessian fly larvae, a large number of genes have been identified to encode secreted salivary gland proteins (SSGPs), which are presumably critical for the insect to feed on and manipulate host plants. For comparison, we conducted an analysis on transcripts encoding SSGPs from the first instar larvae of the wheat midge. In total, 3,500 cDNA clones were sequenced, from which 1,301 high-quality sequences were obtained. Approximately 25% of the cDNAs with high-quality sequences encoded SSGPs. The SSGPs were grouped into 97 groups based on sequence homology. Among the SSGP-encoding transcripts, 206 encoded unique proteins with no sequence similarity to any known protein and 29 encoded proteins similar to known proteins including proteases, serpines, thioesterases, ankyrins, and ferritins. Most (~80%) SSGP-encoding genes appear under strong selection for mutations that generate amino acid changes within the coding region. Identification and characterization of SSGPs in wheat midge larvae provide a foundation for future work to reveal molecular mechanisms behind wheat midge–wheat interactions and the role of these putative effector proteins in insect virulence. Availability of the SSGP transcripts will also facilitate comparative analyses of insect effectors from related species.

An insect nucleoside diphosphate kinase (NDK) functions as an effector protein in wheat - Hessian fly interactions

Like pathogens, galling insects deliver effectors into plant tissues that induce gall formation. The gall midge Mayetiola destructor, also called Hessian fly, can convert a whole wheat seedling into a gall by inducing the formation of nutritive cells at the feeding site, inhibiting wheat growth, and reprogramming metabolic pathways of the attacked plants. Here we demonstrated the identification of a secreted Hessian fly protein, the nucleoside diphosphate kinase (NDK), in infested wheat plants through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blots. In association with the NDK presence, enzymatic activity of NDK increased significantly in wheat tissues at the feeding site. In addition, there was a sudden increase in ATP abundance at the feeding site of infested susceptible wheat seedlings 24 h following Hessian fly larval infestation. Even though a direct link between cause and effect remains to be established, our data points to the direction that Hessian fly larvae injected NDK into wheat tissues as an effector protein, which plays a role in manipulating host plants and converting the plants into galls.