Ohad Rosen

From the discovery of the crustacean androgenic gland to the insulin-like hormone in six decades

Over the past six decades, a unique crustacean endocrine organ, the androgenic gland (AG), has occupied the minds of groups researching Crustacea the world over. Unlike male sexual differentiation and maintenance of sexual characteristics in other arthropods, in crustaceans these processes are regulated by the unique male AG. Crustaceans present a particular case in which the gametogenic organ (testis) is clearly separated from the organ regulating sex differentiation (the AG), enabling endocrine manipulations. The AG was first discovered in a decapod species and later investigated in detail not only in decapods but also in amphipods and isopods. The key role of the AG in regulating sex differentiation was subsequently validated in a number of representative species of a wide array of Malacostraca. It was in an isopod species that the AG hormone was first discovered. Later, orthologous genes were found in isopods and decapods, with all these genes sharing the key features of the insulin-like superfamily of peptides. This review unfolds the story of the AG and AG-specific insulin-like factors (IAGs) from a historical perspective, highlighting the main achievements in the field and giving a glimpse of future challenges to be addressed.

A sexual shift induced by silencing of a single insulin-like gene in crayfish: ovarian upregulation and testicular degeneration.

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.

Timing Sexual Differentiation: Full Functional Sex Reversal Achieved Through Silencing of a Single Insulin-Like Gene in the Prawn, Macrobrachium rosenbergii

ABSTRACT In Crustacea, an early evolutionary group (∼50 000 species) inhabiting most ecological niches, sex differentiation is regulated by a male-specific androgenic gland (AG). The identification of AG-specific insulin-like factors (IAGs) and genomic sex markers offers an opportunity for a deeper understanding of the sexual differentiation mechanism in crustaceans and other arthropods. Here, we report, to our knowledge, the first full and functional sex reversal of male freshwater prawns (Macrobrachium rosenbergii) through the silencing of a single IAG-encoding gene. These “neofemales” produced all-male progeny, as proven by sex-specific genomic markers. This finding offers an insight regarding the biology and evolution of sex differentiation regulation, with a novel perspective for the evolution of insulin-like peptides. Our results demonstrate how temporal intervention with a key regulating gene induces a determinative, extreme phenotypic shift. Our results also carry tremendous ecological and commercial implications. Invasive and pest crustacean species represent genuine concerns worldwide without an apparent solution. Such efforts might, therefore, benefit from sexual manipulations, as has been successfully realized with other arthropods. Commercially, such manipulation would be significant in sexually dimorphic cultured species, allowing the use of nonbreeding, monosex populations while dramatically increasing yield and possibly minimizing the invasion of exotic cultured species into the environment.

Expression of an Androgenic Gland-Specific Insulin-Like Peptide during the Course of Prawn Sexual and Morphotypic Differentiation

The crustacean male-specific androgenic gland (AG) regulates sexual differentiation. In the prawn Macrobrachium rosenbergii, silencing an AG-specific insulin-like encoding transcript (Mr-IAG) inhibited the development of male sexual characters, suggesting that Mr-IAG is a key androgenic hormone. We used recombinant pro-Mr-IAG peptide to generate antibodies that recognized the peptide in AG cells and extracts, as verified by mass spectrometry. We revealed the temporal expression pattern of Mr-IAG and studied its relevance to the timetable of sex differentiation processes in juveniles and after puberty. Mr-IAG was expressed from as early as 20 days after metamorphosis, prior to the appearance of external male sexual characters. Mr-IAG expression was lower in the less reproductively active orange-clawed males than in both the dominant blue-clawed males and the actively sneak mating small males. These results suggest a role for Mr-IAG both in the timing of male sexual differentiation and in regulating reproductive strategies.

A crayfish insulin-like-binding protein: Another piece in the androgenic gland insulin-like hormone puzzle is revealed

Background: In Crustacea, a male-specific insulin-like androgenic hormone governs sexual differentiation. Results: A crayfish insulin-like growth factor-binding protein (Cq-IGFBP) was expressed and found to bind the androgenic hormone. Conclusion: Cq-IGFBP is part of the scheme modulating the crustacean androgenic hormone. Significance: Cq-IGFBP is the first IGFBP found that interacts with an insulin-like gender-specific ligand linking early evolution of insulin pathways with gender. Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.

A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture

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.

An androgenic gland membrane-anchored gene associated with the crustacean insulin-like androgenic gland hormone

SUMMARY Crustacean male sexual differentiation is governed by the androgenic gland (AG) and specifically by the secreted insulin-like AG hormone (IAG), thus far identified in several decapod species including the Australian red claw crayfish Cherax quadricarinatus (termed Cq-IAG). While a few insulin-like AG genes have been identified in crustaceans, other AG-specific genes have not been documented until now. In the present study, we describe the recent identification of a non-IAG AG-specific transcript obtained from the C. quadricarinatus AG cDNA library. This transcript, termed C. quadricarinatus membrane-anchored AG-specific factor (Cq-MAG), was fully sequenced and found to encode a putative product of 189 amino acids including a signal anchoring peptide. Expression of a recombinant GFP fusion protein lacking the signal anchor encoding sequence dramatically affected recombinant protein localization pattern. While the expression of the deleterious fusion protein was observed throughout most of the cell, the native GFP::Cq-MAG fusion protein was observed mainly surrounding the periphery of the nucleus, demonstrating an endoplasmic reticulum (ER)-like localization pattern. Moreover, co-expression of the wild-type Cq-MAG (fused to GFP) and the Cq-IAG hormone revealed that these peptides indeed co-localize. This study is the first to report a protein specifically associated with the insulin-like AG hormone in addition to the finding of another AG-specific transcript in crustaceans. Previous knowledge suggests that insulin/insulin-like factor secretion involves tissue-specific transcripts and membrane-anchored proteins. In this regard, Cq-MAGs tissue specificity, anchoring properties and intracellular co-localization with Cq-IAG suggest that it may play a role in the processing and secretion of this insulin-like AG hormone.

The gene encoding the insulin-like androgenic gland hormone in an all-female parthenogenetic crayfish

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.