Resistance To Bacillus Thuringiensis Toxin Cry2Ab In The Cabbage Looper, Trichoplusia Ni
Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the long-term success of transgenic crops expressing the Bt toxins. However, current understanding of Bt resistance evolved in agricultural situations is very limited. The goal of this dissertation research was to understand the greenhouse-evolved resistance to Bt toxin Cry2Ab in a Trichoplusia ni strain (Glen-BGII) with dual resistance to both Cry1Ac and Cry2Ab. Diet overlay bioassays with Cry2Ab determined that the Glen-BGII strain is highly resistant to Cry2Ab, with a resistance ratio > 1,492-fold. Characterization of the inheritance of Cry2Ab resistance revealed that the resistance is incompletely recessive, monogenic, autosomal, and with no maternal effects. Investigation on the genetic association between the resistance to Cry1Ac and Cry2Ab in the Glen-BGII strain demonstrated that Cry2Ab resistance is not genetically associated with Cry1Ac resistance. The Cry2Ab-resistance trait in the Glen-BGII strain was introgressed into an inbred susceptible laboratory strain (Cornell-SS), and the subsequent introgression strain (Glen-Cry2Ab-BCS8) and the Cornell-SS strain were used for comparative analysis to identify the biochemical changes that might be associated with the resistance trait. Biochemical analysis included assays of caseinolytic activity and zymogram profiles of midgut proteases, activities of serine proteases (trypsin, chymotrypsin, and elastase), activity and zymogram profile of midgut esterases, activity of midgut aminopeptidase, activity and protein abundance of midgut alkaline phosphatase, and melanization activity of hemolymph plasma. No biochemical changes were observed to be associated with the resistance. In addition, an in vitro proteolytic digestion assay showed that Cry2Ab was similarly digested by midgut fluid from the susceptible and resistant larvae. Genetic linkage analysis of Cry2Ab resistance with the genes coding for putative midgut receptors for Cry toxins indicated that Cry2Ab resistance is not genetically linked with the cadherin, aminopeptidase N, or alkaline phosphatase genes. The lack of genetic linkage between Cry2Ab resistance and the ATP-binding cassette transporter ABCC2 gene locus indicated that Cry2Ab resistance is not genetically linked with the ABCC2 gene locus. Comparative quantitative proteomic analysis of larval midgut brush border membrane vesicle proteins identified 1,462 proteins and 951 were quantitatively analyzed between the susceptible Cornell-SS and resistant Glen-Cry2Ab-BCS8 strains. Two midgut BBMV proteins were putatively found significantly different in quantity between the susceptible and resistant larvae, but no significant difference was detected by quantitative real-time RT-PCR analysis at the transcript level of the two genes between the two strains. Further functional analysis of these two candidate proteins is required to confirm and understand their potential involvement in Cry2Ab resistance in T. ni.
Bacillus thuringiensis; Cry2Ab; Insect resistance
Scott, Jeffrey Graham; Soderlund, David Matthew
Ph.D. of Entomology
Doctor of Philosophy
dissertation or thesis