"Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors" (2000).
AbstractProteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiolactivators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or l-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM l-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN 2 and Z-Phe-Ala-CHN 2 , suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.
The parasitic wasp Pediobius foveolatus (Crawford), was successfully reared in the laboratory on EpiJachna varivestis Mulsant. The feasibility of maintaining overwintering stocks of this parasite was demonstrated. The optimum parasite-host ratio used in terms of adult parasites produced/host larvae was 0.4: 1. Reproductive capacity of adult !j! diminished with age, and those held for longer than 90 days at 13°C failed to successfully reproduce. The optimum age for reproduction was less than 12 days. The parasitic eulophid wasp Pediobius foveolatus (Crawford) has been effectively utilized in the suppression of the Mexican bean beetle, Epilachna varivestis Mulsant, on soybeans in Maryland (Stevens et at. 1975). The parasite is unable to overwinter and must be released annually. This necessitates maintenance of overwintering stocks and subsequent increase to sufficient numbers for inoculative releases every spring. This paper deals with laboratory rearing of the Mexican bean beetle and Pediobius foveolatus, emphasizing the effects of parasite age and parasite-host ratios on production of the parasite. Materials and Methods Rearing of Mexican Bean Beetle Campbell and Brett (1966), Kogan (1972), and others have conducted host plant selection and nutritional studies with the Mexican bean beetle in the laboratory. Kogan (1971) reported rearing 3rd and 4th instars on artificial media, but continuous rearing of large numbers of Mexican bean beetles has not been reported. Since a satisfactory artificial diet is not available, large numbers of all stages of this insect were reared on lima beans, Phaseolus lunatus L., under controlled conditions in the laboratory. Plant Material.-Soil medium, premixed in a cement mixer, consisted of the following materials:b u (17.62 liters) each of sphagnum and Michigan peat moss, 1 bu (35.24 liters) vermiculite, 150 g ground limestone, and 150 g 5-10-5 granular fertilizer. 'Henderson' bush lima beans, 5 seeds/ pot, were planted in ll-cm clay pots containing the soil medium. All plant material was grown in the greenhouse at 24-27°C under l5-h light. Rearing and Storage.-Insect colonies were established with adult beetles, 2-3 wk old, collected from soybeans on the Eastern Shore of Maryland in late 1 Coleoptera: Coccinellidae. • Hymenoptera: Eulophidae. • Scientific Article No. 2108, Contribution No. 5065 of the MD Agric. Exp. Stn. This research was sponsored under USDA-ARS Coop. Agreement No. 12-14-1001-41 between the Beneficial InsecĨ
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