Molecular Characterization and Functional Study of Insulin-Like Androgenic Gland Hormone Gene in the Red Swamp Crayfish, Procambarus clarkii

Abstract

The androgenic gland (AG) is a male-specific endocrine organ that controls the primary and secondary sexual characteristics in male crustaceans. More evidence indicates that the insulin-like androgenic gland hormone gene (<i>IAG</i>) is the key male sexual differentiation factor, particularly the application of RNA interference (RNAi) technology on <i>IAG</i>. In this study, the full-length cDNA of <i>IAG</i> (termed <i>PcIAG</i>) was isolated from the red swamp crayfish, <i>Procambarus</i><i>clarkii</i>. Tissue distribution analysis showed that in addition to its expression in the AG of male <i>P. clarkii</i>, <i>PcIAG</i> was widely expressed in female tissues and other male tissues. The PcIAG protein was detected in the reproductive and nervous systems of adult male <i>P. clarkii</i>. Additionally, RNAi results showed that the <i>PcIAG</i> expression could be silenced efficiently, and the male sperm maturation and release possibly present a transient adverse interference at lower doses (0.1 μg/g and 1 μg/g) of PcIAG-dsRNA (<i>PcIAG</i> double-stranded RNA). Dramatically, the expression level of <i>PcIAG</i> increased sharply shortly after the injection of higher doses (5 μg/g and 10 μg/g) of PcIAG-dsRNA, which might accelerate the maturation and release of sperm. Moreover, the expression of <i>PcSxl</i> (<i>P. clarkii Sex-lethal</i>) was detected by Quantitative Real-Time PCR (qPCR) after the injection of PcIAG-dsRNA to explore whether the <i>PcIAG</i> gene regulates the <i>PcSxl</i> gene, and we found that the <i>PcIAG</i> did not directly regulate <i>PcSxl</i> in <i>P. clarkii</i>. The study could help accelerate the progress of <i>PcIAG</i> functional research and provide a useful reference for the single-sex selective breeding of <i>P. clarkii</i>.