Abstract
Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.
Highlights
IntroductionSeveral studies reported a decrease in the susceptibility of different lepidopteran species, including H. armigera, to crystal proteins (Cry) toxins as the larval development increases [5,6,7,8,9,10,11]
Two hypothesis could explain these differences: 1) The gut surface proteins bound by Cry1Ac change during the course of development resulting in differences in toxicity, and 2) Gut proteases increase in late instars resulting in degradation of the crystal proteins (Cry) toxin
We determined the toxicity of Cry1Ac toxin to each larval instar of H. armigera
Summary
Several studies reported a decrease in the susceptibility of different lepidopteran species, including H. armigera, to Cry toxins as the larval development increases [5,6,7,8,9,10,11]. We evaluated the toxicity of Cry1Ac against different larval instars of H. armigera and found significant differences in the susceptibility of the first instars versus late instar larvae. Two hypothesis could explain these differences: 1) The gut surface proteins bound by Cry1Ac change during the course of development resulting in differences in toxicity, and 2) Gut proteases increase in late instars resulting in degradation of the Cry toxin. By pull-down assays, the Cry1Ac binding proteins from early and late instar larvae-BBMV that may be candidates for the differences observed in toxin susceptibility among these larval stages
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