Proteolytic activity of gut bacteria isolated from the velvet bean caterpillar Anticarsia gemmatalis

The development of proteinase inhibitors as potential insect control agents has been constrained by insect adaptation to these compounds. The velvet bean caterpillar (Anticarsia gemmatalis) is a key soybean pest species that is well-adapted to proteinase inhibitors, particularly serine-proteinase inhibitors, which are abundant in the caterpillar host. The expression of diverse proteolytic enzymes by gut symbionts may allow the velvet bean caterpillar to circumvent proteinase inhibitors produced by the host plant. In this study, we characterized the proteolytic activity of the four nonpathogenic species of gut bacteria isolated from the velvet bean caterpillar-Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii and Staphylococcus xylosus. Two proteinase substrates, N-alpha-benzoyl-l-Arg-p-nitroanilide (l-BApNA) and N-alpha-p-tosyl-l-Arg methyl ester (l-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-alpha-tosyl-l-lysine chloromethyl ketone (TLCK)] as well as CaCl2, pH and temperature profiles were used to characterize the expressed proteolytic activity of these bacterial strains in vitro. Kinetic parameters for proteolytic activity were also estimated. The results of these experiments indicated that serine- and cysteine-proteinase activities were expressed by all four gut bacteria symbionts of the velvet bean caterpillar. The cysteine- and serine-proteinase activities of these gut symbionts were distinct and different from that of gut proteinases of the caterpillar itself. This finding provides support for the potential involvement of gut symbionts in the mitigation of the negative effects of serine-proteinase inhibitors in the velvet bean caterpillar.