A Sexual Shift Induced by Silencing of a Single Insulin-Like Gene in Crayfish: Ovarian Upregulation and Testicular Degeneration

Ohad Rosen,Simy Weil,Assaf Linial,Eliahu D Aflalo,Tomer Ventura,Amir Sagi,Rivka Manor,Ohad Gafni,Laszlo Orban

doi:10.1371/journal.pone.0015281

Abstract

In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins.

Highlights

Understanding sexual shifts and differentiation in both vertebrates and invertebrates is of major interest, given our changing environment
While previous studies have demonstrated the influence of the androgenic gland (AG) on a wide array of characteristics related to sexual development and growth of C. quadricarinatus [10,12], only recently a specific AG factor accounting for the male phenotype in this species has been suggested with the identification of Cq-IAG, the first insulin-like AG specific gene to be revealed in decapods [15]
Screening of an AG-derived cDNA library revealed that Cq-IAG comprises about 26% of the ESTs in the library, possibly hinting at the importance of this gene in AG function

Summary

Introduction

Understanding sexual shifts and differentiation in both vertebrates and invertebrates is of major interest, given our changing environment. In vertebrates, such as fish, the regulation of such processes can be attributed to genetics and to the effects of hormones and/or environmental factors [1,2]. Too, diverse factors are involved in the regulation of sexual differentiation. It is believed that female sexual development and secondary characteristics are exhibited only in the absence of the AG, thereby establishing femaleness as a matter of default, negatively regulated by the AG [7]. In the Australian red-claw crayfish, Cherax quadricarinatus, the implantation of AGs into immature females led to the replacement of female characteristics with male traits, as well as to the cessation of vitellogenesis [10,11,12]

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Conclusion