Pierce-sucking insects, such as plant bugs or stink bugs, cause damage in cotton fields worldwide. A novel genetically engineered (GE) cotton (Gossypium hirsutum) is protected against hemipteran pests and thrips by producing the modified Bacillus thuringiensis (Bt) mCry51Aa2 protein. Herbivores that consume insect-protected GE crops, and their natural enemies, can be exposed to plant-produced insecticidal proteins. We investigated tritrophic interactions to evaluate the potential impact of the

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... ovel Bt cotton on a non-target herbivore, the spider mite Tetranychus urticae, and on a generalist predator, the pirate bug Orius majusculus. Pirate bugs belong to the same insect order as the target pests and might thus be adversely affected by mCry51Aa2. Enzyme-linked immunosorbent assays showed that levels of mCry51Aa2 in T. urticae were one order of magnitude, and those in O. majusculus were three orders of magnitude lower than in Bt cotton leaves. O. majusculus nymphs fed with spider mites collected from Bt cotton had lower survival, increased developmental time, and reduced fecundity compared to nymphs fed spider mites from non-Bt near-isogenic cotton. Because Bt cotton did not affect the survival and growth of the spider mites, we conclude that indirect prey-quality mediated effects of the Bt cotton on the predatory bugs are unlikely and that O. majusculus are directly affected by the Bt protein. Follow-up studies are indicated to assess whether the effects observed under worst-case conditions are mitigated under more realistic exposure conditions where alternative prey with low Bt protein levels is available.