A Newly Established ELISA Showing the Effect of the Androgenic Gland on Secondary-Vitellogenic-Specific Protein in the Hemolymph of the Crayfish Cherax quadricarinatus

Abstract

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to monitor the onset of secondary vitellogenesis in Cherax quadricarinatus females and in intersex individuals (having both male and female reproductive systems) after removal of the androgenic gland (AG). As a prerequisite for the assay, the 106-kDa polypeptide was separated from newly laid C. quadricarinatus eggs by SDS–PAGE, and anti-106-kDa antibody was raised in rabbit. The specificity of the anti-106-kDa polypeptide for proteins specific for the hemolymph of secondary-vitellogenic females was confirmed by double immunodiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity between 16 and 500 ng (r=0.953) and was sensitive for amounts as low as 8 ng. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, respectively. Only traces of egg HDL equivalents were detected in the hemolymph of male and primary-vitellogenic females (11 to 110 μg/ml), confirming the specificity of the assay, whereas high levels of such a protein (8–35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. Removal of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in females subjected to AG transplant (79.3 μg/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the expression of a vitellogenic-specific protein in female and intersex C. quadricarinatus, confirming the central role of this gland in tuning sexual plasticity in this species.