Characterization of digestive proteases in the weevil Aubeonymus mariaefranciscae and effects of proteinase inhibitors on larval development and survival

The major digestive proteinase activities of a new sugar beet pest, Aubeony mus mariaefranciscae Roudier (Coleoptera: Curculionidae), were characterize d. Both larvae and adults of A. mariaefranciscae were found to use a comple x proteolytic system for protein digestion based on at least trypsin-, chym otrypsin-, elastase-, cathepsin D, leucine aminopeptidase-, carboxypeptidas e A- and carboxypeptidase B-like activities. An azocaseinolytic activity at pH 5.0-7.0 was identified, that was not affected by specific inhibitors an d activators, making its classification in any of the mechanistic classes e stablished not possible. According to this proteolytic profile, several ser ine proteinase inhibitors were tested in vitro and in vivo to establish the ir potential as resistance factors against A. mariaefranciscae. Larvae fed from neonate to pupation on diets containing 0.2% (w/w) soybean Bowman-Birk trypsin-chymotrypsin inhibitor, soybean Kunitz trypsin inhibitor, turkey e gg white trypsin inhibitor, or lima bean trypsin inhibitor endure lower sur vival rates and display significant delays in the developmental time to pup ation and to adult emergence. Interestingly, the most significant levels of mortality (about 90%) occurred with larvae fed on diets containing a combi nation of two or three inhibitors, suggesting a synergistic toxicity.