Sexual differentiation and maintenance of masculinity in crustaceans has been suggested as being regulated by a single androgenic gland (AG) insulin-like peptide (IAG). However, downstream elements involved in the signaling cascade remain unknown. Here we identified and characterized a gene encoding an insulin-like receptor in the prawn Macrobrachium rosenbergii (Mr-IR), the first such gene detected in a decapod crustacean. In mining for IRs and other insulin signaling-related genes, we constructed a comprehensive M. rosenbergii transcriptomic library from multiple sources. In parallel we sequenced the complete Mr-IR cDNA, confirmed in the wide transcriptomic library. Mr-IR expression was detected in most tissues in both males and females, including the AG and gonads. To study Mr-IR function, we performed long-term RNA interference (RNAi) silencing in young male prawns. Although having no effect on growth, Mr-IR silencing advanced the appearance of a male-specific secondary trait. The most noted effects of Mr-IR silencing were hypertrophy of the AG and the associated increased production of Mr-IAG, with an unusual abundance of immature sperm cells being seen in the distal sperm duct. A ligand blot assay using de novo recombinant Mr-IAG confirmed the existence of a ligand-receptor interaction. Whereas these results suggest a role for Mr-IR in the regulation of the AG, we did not see any sexual shift after silencing of Mr-IR, as occurred when the ligand-encoding Mr-IAG gene was silenced. This suggests that sexual differentiation in crustaceans involve more than a single Mr-IAG receptor, emphasizing the complexity of sexual differentiation and maintenance.

Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into "neo-males" bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.

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Male sexual differentiation in crustaceans is controlled by the androgenic gland (AG), a unique male endocrine organ that, in decapods, is located at the base of the 5th pereiopod. In these animals, the insulin-like androgenic gland hormone (IAG) is the major factor secreted from the AG to induce masculinization and maintain male characteristics. It has, however, recently been proposed that this hormone also plays a role in growth and ovarian development in females. In this study, we tested such a possibility by searching for the IAG gene in the marbled crayfish, a parthenogenetic animal that reproduces asexually to form an all-female genetic clone. Based on the phylogenetic relationship between the marbled crayfish and Procambarus fallax, a gonochoristic species of the same North American Cambaridae family, we searched for the IAG gene in the marbled crayfish and then fully sequenced it. The open reading frame of the gene was found to be completely identical in the two species, and their introns shared over 94% identity. It was also found that, in addition to its expression at the base of the 5th pereiopod and in the testes of male P. fallax crayfish, IAG was expressed in the muscle tissue of P. fallax males and females and even of the parthenogenetic marbled crayfish. These findings provide new insight into possible functions of IAG, in addition to its role as a masculinization-inducing factor, and also constitute the basis for a discussion of the evolutionary relationship between the above two species.