title: Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae).
abstract: Insects with altered proteinases can avoid intoxication by Bacillus thuringiensis (Bt) toxins. Therefore, proteinase activities from gut extracts of Bt-susceptible (YDK) and -resistant (YHD2-B, CXC and KCBhyb) Heliothis virescens strains were compared. The overall pH of gut extracts from YDK and CXC were statistically similar (9.56 and 9.62, respectively), while the pH of extracts from KCBhyb and YHD2-B were significantly more alkaline (9.81 and 10.0, respectively). Gut extracts from YHD2-B and CXC larvae processed Cry1Ac and Cry2Aa protoxin slower than extracts from YDK larvae, suggesting that differences in proteolysis contribute to resistance in these strains. Casein zymogram analysis of gut extracts revealed both qualitative and quantitative differences in caseinolytic activities among all strains, but the overall caseinolytic activity of YHD2-B gut extract was lower. Kinetic microplate assays with a trypsin substrate (l-BApNA) demonstrated that proteinases in YDK gut extract had increased alkaline pH optima compared to resistant strains YHD2-B, CXC and KCBhyb. Gut extracts from YHD2-B had reduced trypsin-like activity, and activity blots indicated that YHD2-B had lost a trypsin-like proteinase activity. In assays with a chymotrypsin substrate (SAAPFpNA), enzymes from all Bt-resistant strains had increased pH optima, especially those from KCBhyb. Activity blots indicated that CXC had lost a chymotrypsin-like proteinase activity. Because serine proteinases are a critical component of Bt toxin mode of action, these differences may contribute to decreased toxicity in the Bt-resistant strains.
authors.affiliation: Department of Entomology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Lohitash
authors.initials: L
authors.lastname: Karumbaiah
authors.forename: Brenda
authors.initials: B
authors.lastname: Oppert
authors.forename: Juan L
authors.initials: JL
authors.lastname: Jurat-Fuentes
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Adang
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000235:genetics / Q000494:pharmacology*; D004064:Digestive System / Q000201:enzymology*; D004731:Endotoxins / Q000235:genetics / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000494:pharmacology*; D007305:Insecticide Resistance / Q000187:drug effects / Q000502:physiology*; D007306:Insecticides / Q000494:pharmacology*; D007915:Lepidoptera / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D013045:Species Specificity
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010447:Peptide Hydrolases