Mechanisms of resistance to the Cry1Ab Bacillus thuringiensis toxin in the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae)

Insect resistance to Bacillus thuringiensis toxins expressed in crops such as corn is perhaps the biggest concern among the public and scientific community. This thesis characterizes Bt resistance among European corn borer (ECB), Ostrinia nubilalis, (Hübner) populations selected with Cry1Ab protoxin by establishing cross-resistance patterns for other B. thuringiensis toxins, and the possible mechanisms of resistance evolved in these colonies. Toxicological studies showed that three different colonies of O. nubilalis selected with Cry1Ab protoxin incorporated into artificial diet developed significant levels of resistance (2.0- up to 10-fold). However, a higher level of resistance to the Cry1Ab toxin was observed among the colonies (up to 39-fold). Selection with Cry1Ab protoxin caused decreased susceptibility to Cry1Ac (up to 51.0-fold) and Cry1F (up to 5.0-fold) to which colonies were not previously exposed. Cross-resistance patterns suggested the possible role of gut proteases in the resistance. Assays of luminal gut proteases using trypsin-like, chymotrypsin-like, and elastase-like substrates as well as Cry1Ab protoxin and toxin proteolysis provided no indication of proteolysic-mediated resistance as major mechanism of resistance in these colonies. Activation of the 130-kD Cry1Ab protoxin occurred rapidly in all colonies tested with no apparent differences among colonies. Alternatively, immunoblotting analysis showed that reduced binding to BBMV receptors is likely the major mechanism of resistance in the selected colonies. Real-time kinetic analysis using an optical biosensor showed significantly (P < 0.05) reduced affinity of Cry1Ab to BBMV from Europe-R [60.9 ± 6.45 (nM)] and RSTT-R [94.0 ± 10.65 (nM)] selected colonies compared to Europe-S [34.5 ± 2.85 (nM)]. Cadherin-like protein was identified as major Cry1Ab-binding receptor and reduced Cry1Ab binding to this protein was observed in one of the selected colonies suggesting its association with resistance. Expression analysis of an O. nubilalis cadherin gene showed no reduction in transcripts from resistant colonies compared with susceptible suggesting that reduced number of receptors is not associated with resistance. The major conclusion of this work is that receptor-toxin binding alteration appears to be the major mechanism of resistance to B. thuringiensis Cry1Ab in these O. nubilalis colonies.