John P. Purcell

Examination of midgut luminal proteinase activities in six economically important insects

A biochemical survey of insect midgut fluid proteinase activities was conducted to determine which proteinase inhibitors could play roles in insect control strategies. In this study, midgut juices from six insects of economic importance have been isolated and their proteolytic activities classified. Three lepidopterans (black cutworm, corn earworm, and tobacco budworm) and the boll weevil have maximal midgut fluid proteolytic activity in vitro at pH 10–11. Soybean trypsin inhibitor inhibits lepidopteran and boll weevil activities by 63–72%, indicating that their major proteinases are “trypsin-like” serine proteinases. Varying levels of inhibition were seen against the midgut fluid proteinase activities whereas bovine trypsin was inhibited 95–98% by each of four different trypsin inhibitors. Southern corn rootworm and Colorado potato beetle have maximal midgut fluid proteolytic activity in vitro at pH 6–7. E-64 inhibits southern corn rootworm (69%) and Colorado potato beetle (100%) midgut fluid proteinase activity, demonstrating that their major activities are provided by cysteine proteinases. Feeding studies found no mortality nor stunting associated with feeding high levels of trypsin inhibitors to boll weevil or tobacco budworm larvae despite the fact that these proteins were very good inhibitors 60–78% inhibition) of midgut proteolytic activity in vitro.

Cholesterol oxidase: an oöstatic and larvicidal agent active against the cotton boll weevil, Anthonomus grandis

The enzyme cholesterol oxidase (E.C. 1.1.3.6), purified from Streptomyces culture filtrate was previously found to have oral insecticidal activity on neonate larvae of the boll weevil (Anthonomus grandis grandis Boheman) from a laboratory population. In the present study, second instar larvae were also controlled by the enzyme at diet concentrations similar to those which control neonates (12 day LC50 = 2.4 μg.ml−1 in diet). Larvae from field‐collected adults were similarly susceptible to cholesterol oxidase in the diet. When ingested by adult females during the mating/pre‐oviposition period, cholesterol oxidase greatly reduced subsequent oviposition (83% reduction in eggs laid as compared to controls) and larval survival (97% reduction from controls). Dissection of treated adult females revealed poorly developed ovaries and few developing oöcytes. These studies were conducted to further evaluate the utility of cholesterol oxidase in a program to establish boll weevil‐resistant transgenic cotton.

Studies on the mode of action of cholesterol oxidase on insect midgut membranes

Cholesterol oxidase (EC 1.1.3.6.) is an insecticidal protein known to have potent activity against the boll weevil, milder activity against a number of lepidopteran species, and virtually no activity against other insects. Several factors that could explain its species-dependent differential activity were examined. We compared cholesterol concentrations and rates of cholesterol oxidation in the midgut membranes from larvae of boll weevil (Anthonomus grandis grandis Boheman), southern corn rootworm (Diabrotica undecimpunctata howardi Barber), tobacco budworm (Heliothis virescens Fabricius), and yellow mealworm (Tenebrio molitor Linnaeus). Results showed that cholesterol concentration alone could not account for the differences in insecticidal activity and that midgut brush-border membranes of all species tested were generally susceptible to oxidation by cholesterol oxidase in vitro. We also demonstrated that cholesterol oxidase stability in the midgut environment was similar for the species tested and thus could not account for the differential activity. However, comparison of the pH of the insect midgut fluids with the pH optimum of cholesterol oxidase indicated that the lower sensitivity of lepidopteran larvae to the enzyme may be partially due to the alkaline nature of their midgut environments. In some species, oxidation caused significant changes in the activities of brush-border membrane alkaline phosphatase, and these changes did correlate with the susceptibility of the insect to cholesterol oxidase. Arch. Insect Biochem. Physiol. 34:429–442, 1997.