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-α-benzoyl-L-Arg-p-nitroanilide (L-BApNA) and N-α-p-tosyl-L-Arg methyl ester (L-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-α-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.

Purification of active trypsin in the digestive process of insects is essential for the development of potent protease inhibitors (PIs) as an emerging pest control technology and research into insect adaptations to dietary PIs. An important aspect is the presence of proteolytic microorganisms, which contribute to host nutrition. Here, we purified trypsins produced by bacteria Bacillus cereus, Enterococcus mundtii, Enterococcus gallinarum, and Staphylococcus xylosus isolated from the midgut of Anticarsia gemmatalis. The trypsins had a molecular mass of approximately 25 kDa. The enzymes showed increased activity at 40°C, and they were active at pH values 7.5-10. Aprotinin, bis-benzamidine, and soybean Kunitz inhibitor (SKTI) significantly inhibited trypsin activity. The l-1-tosyl-amido-2-phenylethylchloromethyl ketone (TPCK), pepstatin A, E-64, ethylenediamine tetraacetic acid, and calcium ions did not affect the enzyme activity at the concentrations tested. We infer the purified trypsins do not require calcium ions, by which they differ from the trypsins of other microorganisms and the soluble and insoluble trypsins characterized from A. gemmatalis. These data suggest the existence of different isoforms of trypsin in the velvetbean caterpillar midguts.