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Insect-resistant transgenic plants have been suggested to have unpredictable effects on the biodiversity of the agro-ecosystem, including potential effects on insect natural enemies, beneficial in control of crop pests. Whilst carnivorous as adults, many of these predators may also consume plant tissues, in particular plant pollen and nectar. Coleoptera are important in terms of agro-ecological research not only because of the large number of species in this order, but also because of their role as biological control agents. Thus any detrimental impact on this group of insects would be highly undesirable. The effects of potato expressing the coleopteran-specific Bacillus thuringiensis δ-endotoxin Cry3A (Bt Cry3A) on the ladybird beetle Harmonia axyridis and the carabid beetle Nebria brevicollis were investigated via the bitrophic interaction of the adult ladybird with potato flowers and the tritrophic interaction of the carabid consuming a non-target potato pest. Immunoassays confirmed accumulation of the transgene product in potato leaves and floral tissues (at levels of up to 0.01% (pollen) and 0.0285% (anthers) of total soluble protein). Despite H. axyridis and N. brevicollis belonging to the targeted insect order, no significant effects upon survival or overall body mass change of either beetle were observed. Furthermore, Bt Cry3A had no detrimental effects on reproductive fitness of either beetle species, either in terms of fecundity or subsequent egg viability. Behavioural analysis revealed no significant impact of Bt Cry3A on beetle activity or locomoter behaviour. Ligand blots indicate that this is due to either the absence of Bt-binding sites in brush border membrane vesicles (BBMV) isolated from Nebria brevicollis, or in the case of Harmonia axyridis, the binding did not functionally lead to behavioural or physical effects.