The biochemical and molecular characterization of trypsins and chymotrypsins from midguts of Bacillus thuringiensis -susceptible and -resistant Heliothis virescens larvae (Lepidoptera: Noctuidae).
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The onset of Bacillus thuringiensis (Bt) toxin resistance is a major threat to the long term use of Bt toxins for insect control. The Bt toxin mode of action involves a multi-step process, and the proteolytic cleavage of protoxin to toxin by midgut serine proteinases is an important step in this process. While altered toxin binding to midgut receptors is the most widely reported mechanism of resistance, the occurance of other mechanisms of resistance should not be discounted. In this dissertation I investigated the possible role of a proteinase-related mechanism of resistance in Cry1Ac and Cry2Aa selected strains of Heliothis virescens. Enzymes in gut extracts of YHD2-B and CXC processed Cry1Ac and Cry2Aa protoxins slower than enzymes in gut extracts of the susceptible strain, YDK. The reduced rate of protoxin processing correlated with the absence of a ~32 kDa trypsin and ~35 kDa chymotrypsin from the gut extracts of the YHD2-B and CXC strains, respectively. Quantitative and qualitative differences in caseinolytic activities, as well as differences in pH optima of trypsin- and chymotrypsin-like proteinases in gut extracts from the different strains, suggest that these differences may contribute to toxin insensitivity in the Bt resistant strains. To characterize changes in proteinase expression I identified four trypsin-like (HvT) and ten chymotrypsin-like (HvC) transcripts from midguts of YDK and CXC larvae fed on diet alone or diet containing Cry1Ac or Cry2Aa toxins. Phylogenetic analyses, as well as pairwise sequence alignments, indicated that HvT sequences were highly similar while HvC sequences showed considerable sequence diversity. SNP analyses found mostly synonymous nucleotide changes; however, one non-synonymous change observed in the HvT1 loop-1 region, results in an amino acid change that could lead to altered substrate specificity. Gene expression analyses demonstrate that HvT3 expression is knocked out in toxin-fed YDK and CXC. Chymotrypsins were generally up-regulated in CXC; however, HvC6 expression was knocked out in Cry2Aa-fed CXC, indicating a toxin-specific down-regulation. I conclude that a proteinase-related mechanism may be a resistance factor in the Bt resistant strain, CXC, and should also be considered in addition to altered toxin binding in the Bt resistant strain, YHD2-B.