41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

The Western Corn Root Worm (Diabrotica v. virgifera) first time occurred in Germany in July 2007. Four different maize cultivars including Diabrotica-resistant MON88017, were assessed in respect to its effects on saprophagous Diptera and predators out of Carabidae and Staphylinidae. The methodological approach comprised a hierarchic order of different ecological scale levels (agro-ecosystem, population, organisms). Abundance and species composition of both Diptera and their predators were recorded in the field. Most saprophagous Diptera belong to Sciaridae (fungus gnats), of which the predominant Lycoriella castanescens was used for feeding trials. It was tested whether mortality, pupation, hatching rates, duration of larval development and pupation were affected by uptake of Cry3Bb1-contminated plant tissues. Species of Carabidae and Staphylinidae were fed with Sciaridae-larvae reared on Bt- and non-Bt-maize-litter respectively. In a similar way Diabrotica-larvae were offered as prey. Toxin analyses of saprophagous Diptera and predators reared with Bt-plant parts or feeding on Bt-contaminated prey contained Bt-toxin up to 1.6% (decomposers) and 14.0% (predators) of the toxin level recorded in the source material. Predators collected from Bt-maize fields stated these findings. Thus, Bt-toxin is transferred into the food chain. Predators feeding on prey containing Cry3Bb1-toxin showed a significant delay in accepting the prey in comparison to prey free of Bt-toxin, but this didn't result in higher mortality or less longevity. However, predators which were fed with Sciaridae-larvae containing Bt-toxin produced significantly less offspring than those feeding on prey reared with non-Bt-maize litter. Thus, an uptake of Cry3Bb1-toxin by carnivorous beetles doesn't lead to a higher mortality, but results in subtle effects like lower fertility of the females.

The Western corn rootworm (WCR) is one of the economical most important pests in corn. For its control, genes encoding Bacillus thuringiensis toxins (e.g. Cry3Bb1, Cry3A, Cry34Ab1/Cry35Ab1) were introduced into corn. The cultivation of transgenic corn expressing the respective B.t.-toxins may result in the development of resistant pest populations. In general, the resistance of insects to B.t.-toxins can be located at any step of the toxic pathway. However, in other B.t.-toxin-pest-systems, the resistance mechanisms are mainly proteinase- or receptor-mediated. To establish reference systems for the identification of resistance mechanisms in potential available resistant individuals, studies on proteinase activities and binding analysis were carried out with midgut fluid and midgut epithelium of WCR 3rd instar larvae. Studies on the identification and quantification of proteinase activities in the midgut fluid were conducted using photometrical tests with specific chromogenic substrates - mainly peptidyl-p-nitroanilid (pNA) - and specific inhibitors. As a result, the digestive serine endopeptidases trypsin, chymotrypsin, and elastase were identified. Besides, high digestive activities were observed for the serine endopeptidases cathepsin G, plasmin, and thrombin. Due to the acid midgut fluid, in Chrysomelidae cysteine endopeptidases were expected. Accordingly, high activities of cathepsin L, papain, cathepsin B, and cathepsin H were observed in the midgut fluid of WCR (pH 5.75). Besides, the metallo endopeptidase saccharolysin as well as the exopeptidases aminopeptidase and an omegapeptidase - acylaminoacylpeptidase - were identified. For aspartic endopeptidases no specific pNA substrates were available. Using the general proteinase substrate azocasein, the activity of the aspartic endopeptidase pepsin was demonstrated. Furthermore, with midgut epithelium binding analysis were carried out to study binding site competition of B.t.-toxins Cry3Bb1 vs. Cry34Ab1/Cry35Ab1. From the midgut epithelium brush border membrane vesicles (BBMV´s) were prepared. To examine the toxin binding, biotin labeled B.t.-corn-toxins, and the ligand-blot technique as well as streptavidin-horseradish-peroxidase-conjugat and the ECL system were used.