Location: Stored Product Insect Research Unit
Title: Analysis of Midgut Proteinases from Bacillus Thuringiensis-Susceptible and -Resistant Heliothis Virescens (Lepidoptera: Noctuidae) Authors
|Karumbaiah, Lohitash - UNIVERSITY OF GEORGIA|
|Jurat-Fuentes, Juan - UNIVERSITY OF GEORGIA|
|Adang, Michael - UNIVERSITY OF GEORGIA|
Submitted to: Comparative Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 17, 2006
Publication Date: January 31, 2007
Repository URL: http://www.elsevier.com/locate/cbpb
Citation: Karumbaiah, L., Oppert, B.S., Jurat-Fuentes, J.L., Adang, M.J. 2007. Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae). Comparative Biochemistry and Physiology 146: 139-146. Interpretive Summary: The bacterium Bacillus thuringiensis (Bt) is used to control insects, and the tobacco budworm, a pest of cotton and other crops, has developed high levels of resistance to the insecticidal toxins produced by Bt. This study investigated whether digestive enzymes from the budworm may be responsible for this resistance. Differences in the types and levels of enzymes in Bt-resistant budworms suggest that, in some cases, enzymes may be involved in the resistance. This information can be used in the design of new toxins to prevent this type of resistance development.
Technical 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) H. 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.