Author
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Shapiro, Jeffrey |
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Ferkovich, Stephen |
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Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 4/29/2002 Publication Date: 10/1/2002 Citation: Shapiro, J.P., Ferkovich, S.M. 2002. Yolk protein immunoassays (YP-ELISA) to assess diet and reproductive quality of mass-reared Orius insidiosus (Hemiptera: Anthocoridae). Journal of Economic Entomology. 95:927-935. Interpretive Summary: Insect predators are commercially produced for mass release to suppress pest populations in field and greenhouse. To mass-produce beneficial insects effectively, the producer must be able to practically assess the quality of the insects being produced. One useful measure of quality is the reproductive rate of the beneficial insect, but it is often difficult or laborious to measure rates of egg production by females, especially with very small insects, and also may not be possible prior to shipment. USDA- ARS scientists at the Center for Medical, Agricultural and Veterinary Entomology, Gainesville, FL, are developing an immunological method, a sort of quantitative early pregnancy test for insects, to predict the reproductive success of insects. Using eggs of the minute pirate bug, monoclonal antibodies were produced and used in a test to measure very small quantities of yolk protein in the bugs. When fed on a range of diets, ,the bugs responded by producing large quantities of yolk protein on nutritious diets and lesser quantities on less nutritious diets. The amount of yolk protein produced correlated well with the number of eggs laid over a period of 10 days. After eggs were laid, their content of yolk protein decreased as they developed prior to hatching. If this and similar tests can be developed and made commercially available as a service, insectaries can use them to rapidly certify the quality of adult insects that they produce, and to keep close watch on the production process. Technical Abstract: A yolk protein enzyme-linked immunosorbent assay (YP-ELISA) was developed for the predator Orius insidiosus. The YP-ELISA is intended to assess reproductive response to dietary and other rearing conditions, and to assist in quality control and diet development for mass rearing. Hybridomas and monoclonal antibodies were produced against homogenates of eggs dissected from females. Hybridomas were selected for secretion of IgG that reacted with extracts of both females and their eggs, and that did not react with male extracts. Each cloned hybridoma produced a monoclonal antibody that specifically reacted on western blots against one of the two major yolk polypeptides, apoVn-I (180,000 molecular weight) or apoVn-II (40,000). Yolk protein ELISAs were developed with these antibodies to assess yolk protein content of female O. insidiosus as a measure of reproductive fitness and as a potential predictor of fecundity. Protocols for an indirect antigen ELISA and double antibody sandwich ELISA were developed to assess yolk protein contents of eggs and total contents in whole body homogenates. ELISA standards consisted of homogenates of eggs collected 0-24 h following oviposition. As determined with the sandwich ELISA, yolk protein contents of eggs declined with age prior to hatch, with a half-life of 32-34 h. Results were similar whether the detecting antibody-enzyme conjugate was anti-apoVn-I or anti-apoVn-II. Optimal conditions and sampling parameters were developed for the sandwich ELISA, which demonstrated minimal nonspecific interference in whole-insect extracts. In an initial application of the YP-ELISA, oviposition rates over a 10-day period were compared with yolk protein contents at the end of that period, dependent on diets of differing nutritional composition and quality. High and low yolk protein contents correlated with oviposition rates on respective diets, though oviposition. |
