38th Annual Meeting of the Society for Invertebrate Pathology

August 7-11, 2005  Anchorage, Alaska, U.S.A

Analysis of midgut proteinases from Bacillus thuringiensis susceptible and resistant Heliothis virescens (Lepidoptera: Noctuidae)

Lohitash Karumbaiah1,Brenda Oppert3,Juan.L.Jurat-Fuentes1, Michael.J.Adang1,2
Departments of Entomology1, Biochemistry and Molecular Biology2, University of Georgia, Athens, GA-30602. USDA ARS Grain Marketing and Production Research Center3, Manhattan, KS-66502.

Heliothis virescens is a major lepidopteran pest of cotton in the United States and the target insect of Bacillus thuringeinsis (Bt) transgenic cotton. We conducted an analysis of gut proteases from Bt susceptible and resistant H.virescens strains, including the susceptible strain, YDK, and three resistant strains, YHD2-B, CXC, and KCBhyb. Casein zymogram analysis of YDK and YHD2-B gut extracts did not reveal significant differences. However, two unique bands of caseinolytic activity were observed in CXC and KCBhyb. Kinetic microplate assays with a trypsin substrate demonstrated that proteinases in YDK gut extract had more alkaline pH optima compared to YHD2-B, CXC and KCBhyb. In assays with a chymotrypsin substrate, enzymes in YDK extracts had lower alkaline pH optima in contrast to those in YHD2-B gut extract. Enzymes from KCBhyb gut extracts had the highest activity of all strains in alkaline buffers, particularly at pH 10.6, similar to the physiological pH of the lepidopteran midgut. Temporal Cry1Ac protoxin activation indicated that YHD2-B gut extract processed protoxin at a slower rate than that of YDK. Because gut proteinases are a critical component of Bt toxin mode of action, these differences may contribute to decreased toxicity in the Bt-resistant strains.

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