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Abstract: From 2005 to 2015, Ostrinia nubilalis were collected in the most important maize‐growing areas in Europe where MON 810 was cultivated. The susceptibility of these O. nubilalis collections to the Cry1Ab protein was determined using overlay bioassays and compared to that of reference (control) strains. Larvae that died or did not moult after 7 days were used to calculate a moulting inhibition concentration (MIC). Two different batches of Cry1Ab protein were used over the course of this study. Between 2005 and 2015, 145 collections of O. nubilalis from 14 areas were analysed. The Cry1Ab susceptibility of populations from different geographic regions differed only slightly across years. The greatest variability in the MIC50 for field samples collected from 2005 to 2011 and tested with batch 1 was 6.6‐fold in 2006. For field‐collected O. nubilalis, the difference between MIC50 values of the most susceptible and most tolerant samples was 13.1‐fold for this period. For samples collected in 2012–2015 and tested with batch 2, the greatest variability was 4.1‐fold in 2014. A diagnostic concentration (MIC99) was calculated for batch 1 (48 ng/cm2) using the results from all the collections in 2005–2012. Bridging experiments indicated that the diagnostic concentration for batch 2 was 28 ng/cm2. From 2006 onwards, no O. nubilalis reached the 2nd larval stage when the diagnostic concentration of either batch of Cry1Ab was used. Only one insect collected from Romania in 2012 and two insects collected as reference strain from Spain in 2015 survived exposure to a dosage of 20 ng/cm2, and none of these larvae survived on MON 810 maize. Our results indicate that there has been no significant change in susceptibility to Cry1Ab in European populations of O. nubilalis over the period 2005–2015. [ABSTRACT FROM AUTHOR]

A laboratory study was carried out to assess the potential prey-mediated effects of Cry3Bb1-expressing Bt maize on the fitness and predatory ability of Atheta coriaria Kraatz (Coleoptera: Staphylinidae), using Tetranychus urticae Koch (Acari: Tetranychidae) as prey. The concentration of Cry3Bb1 toxin through the trophic chain significantly decreased from Bt maize (21.7 μg g(-1) FW) to mites (5.6 μg g(-1) FW) and then to A. coriaria adults (1.4 μg g(-1) FW), but not from mites to A. coriaria L1-L3 larvae (4.1-4.6 μg g(-1) FW). Interestingly, the toxin levels detected in A. coriaria larvae represent more than 20% of the concentration found in Bt maize, and the toxin was detected up to 48 h after exposure. To our knowledge, this is the highest level of exposure ever reported in a predatory beetle to the Cry3Bb1 protein. When A. coriaria larvae were reared on Bt-fed mites, Bt-free mites or rearing food, no significant differences among treatments were observed in development, morphological measurements of sclerotized structures and body weight. Moreover, no negative effects on reproductive parameters were reported in adults feeding on Bt-fed prey after 30 days of treatment, and survival was not affected after 60 days of exposure. Similarly, predatory ability and prey consumption of A. coriaria larvae and adults were not affected by exposure to the toxin. All together, these results indicate a lack of adverse effects on A. coriaria, a species commonly used as a biological control agent. The use of A. coriaria as a surrogate species for risk assessment of GM crops that express insecticidal proteins is discussed.

The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the optimisation of the use of protease inhibitors for pest control, and on the identification of endoprotease-mediated resistance to Bacillus thuringiensis Cry-toxins is discussed.