Yolk Protein Immunoassays (YP-ELISA) to Assess Diet and Reproductive Quality of Mass-Reared Orius insidiosus (Heteroptera: Anthocoridae)

Abstract

A yolk protein enzyme-linked immunosorbent assay (YP-ELISA) was developed for the predator Orius insidiosus (Say). 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 before 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-d 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 showed more graded response to diets than did yolk protein. Improvements in sampling methods are discussed.