Rivka Manor

Temporal Silencing of an Androgenic Gland-Specific Insulin-Like Gene Affecting Phenotypical Gender Differences and Spermatogenesis

Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis.

The eyestalk-androgenic gland-testis endocrine axis in the crayfish Cherax quadricarinatus

In decapod crustaceans, a number of neurohormones regulating a variety of physiological processes, including reproduction, are to be found in the X-organ-sinus gland complex of the eyestalk. Bilateral eyestalk ablation was thus performed in mature males of the Australian red claw crayfish Cherax quadricarinatus with the aim of studying the role of eyestalk-borne hormones on spermatogenic activity in the testis and on the androgenic gland (AG). The latter gland controls the differentiation and functioning of male sexual characteristics in crustaceans. Eyestalk ablation caused hypertrophy of the AG, as indicated by an increase in gland weight (3.9 +/- 0.44 mg vs < 0.1mg in intact males) and by overexpression of AG polypeptides. In the testes of eyestalk-ablated males, empty spermatogenic lobules were common, while lobules containing primary spermatocytes were infrequent. These findings were reflected in decreased amounts of DNA in these testes and a consequent increase in the relative weights of the sperm ducts. Since it was found that eyestalk ablation affected both the AG and the reproductive system, in vitro experiments were conducted to study the direct effects of the sinus gland on the AG and testes and of the AG on the testes. Sinus gland extracts inhibited by 30% the incorporation of radiolabeled amino acids into AG polypeptides and almost totally inhibited the secretion of radiolabeled AG polypeptides into the culture medium. However, sinus gland extracts had no significant effects on testicular tissue. On the other hand, AG extracts affected the in vitro phosphorylation of a testicular polypeptide (of 28 kDa), in a time- and dose-dependent manner, suggesting a direct effect of AG-borne hormones on the testes. The above findings, together with the evidence for direct inhibition by the sinus gland on the AG, suggest an endocrine axis-like relationship between the sinus gland, the AG, and the male reproductive system in decapod crustaceans.

Cloning of an insulin-like androgenic gland factor (IAG) from the blue crab, Callinectes sapidus: Implications for eyestalk regulation of IAG expression

In malacostracan crustaceans, sex differentiation is uniquely regulated by a hormone secreted by the male-specific androgenic gland (AG). An isopod AG hormone was the first to be structurally elucidated and was found to belong to the insulin superfamily of proteins. Recently, it has been found that the AGs of several decapod crustaceans express insulin-like androgenic gland factors (IAGs), whose function is believed to be similar to that of the isopod AG hormone. Here we report the isolation from the blue crab Callinectes sapidus of the full-length cDNA encoding a candidate insulin-like AG hormone, termed Cas-IAG. The predicted protein Cas-IAG was encoded as a precursor consisting of a signal peptide, the B chain, the C peptide, and the A chain in that order. While the AG was the main source of Cas-IAG expression, as found in other decapod species, the hepatopancreas of male Callinectes sapidus crabs displayed minor Cas-IAG expression. Eyestalk ablation confirmed the presence of a possible endocrine axis between the eyestalk ganglia and the AG, implying that Cas-IAG expression is negatively regulated by (a) substance(s) present in the eyestalk ganglia.

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.

Androgenic gland implantation promotes growth and inhibits vitellogenesis in Cherax quadricarinatus females held in individual compartments

Summary Androgenic glands (AGs) were implanted into young female red claw crayfish, Cherax quadricarinatus, with the aim of investigating the role played by the AG in the balance between growth and reproduction under conditions of minimal social interaction (individual compartments). The growth rate of the females with AG implants was significantly higher than that of the control non-implanted females (0.11 ± 0.03 g/day vs. 0.08 ± 0.02 g/day). This difference was attributed to the larger molt increments and slightly shorter molt intervals of the females with implants vs. the control females. At the end of the experiment (538 days), the mean weight of the implanted females was significantly higher than that of the control females (64.58 ± 18.24 g vs. 51.07 ± 12.71 g, respectively), a lead of 26.4% for the implanted females that started 91 days after implantation and became significant at 153 days after implantation. By that time, 55.5% of the implanted females had developed typical male secondary characters, such as the red patch on the propodus. The shift of energy from female reproduction to growth was further demonstrated by the level of expression of the vitellogenin gene in the hepatopancreas: gene expression was high in control females but lower by several orders of magnitude in the AG-implanted females, as shown by real time RT-PCR relative quantification. Confirmation of these findings was provided by an ELISA test, which showed that the level of vitellogenic cross-reactive protein in the hemolymph of AG-implanted females was significantly lower than that in intact females. The significant growth promotion in AG-implanted females was clearly not due to social interaction. It may be attributed to a direct growth factor—like the effect of androgens in vertebrates—in combination with an indirect effect, through the shift of energetic investment from reproduction and vitellogenesis to growth. Since the AG implant had a more marked effect on molt increment than on molt interval, it seems likely that the AG acts as a growth promoter rather than as a molt enhancer.

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.

On intersexuality in the crayfish Cherax quadricarinatus: an inducible sexual plasticity model

Summary Sexual differentiation is a plastic process. The plasticity may be manifested during embryo-genesis, when one set of primordial reproductive ducts develops while the other degenerates. In adults, many normal (e.g., sequential hermaphroditism) and abnormal (e.g., endocrine disorders or exposure to endocrine disrupters such as estrogenic pollutants) cases are known in which sexual plasticity may be expressed as various degrees of feminization. In crustaceans, the androgenic gland (AG) regulates the development of male characteristics; its absence results in feminization, often including the onset of vitellogenesis. A unique model of intersexuality was found in the crayfish Cherax quadricarinatus, in which some degree of natural sexual plasticity is observed. Two to 14% of the population are intersex individuals, having both male and female genital openings. Intersex specimens always function as males but may also contain an ovary in a permanently arrested, pre-vitellogenic state. This sexual plasticity model was recently characterized and investigated with respect to the role of the AG and the onset of vitellogenesis. Removal of the AG in intersex individuals induced the reproductive system to shift from a permanently active male state to a female state. This shift included changes in morphology, cessation of spermatogenesis and onset of secondary vitellogenesis manifested by a change in the ovarian protein profile, translocation of protein kinase C (PKC) in the ovary and appearance of secondary vitellogenic high-density lipoprotein (HDL) in the hemolymph. The vitellogenin gene was found to be induced in the hepatopancreas of AG ablated intersex individuals suggesting that the AG represses transcription of this gene in intact intersex individuals. The experimentally inducible sex shift in the crayfish provides a unique and controlled model system for the study of sexual differentiation and plasticity at the physiological and molecular levels. The findings presented here also illustrate the central role of the AG in the regulation of sexual differentiation in sexually plastic as well as gonochoristic crustacean species.

Male-like behavioral patterns and physiological alterations induced by androgenic gland implantation in female crayfish.

SUMMARY The androgenic gland (AG) has been shown to regulate male sexual differentiation and secondary male characteristics in Crustacea. This study presents for the first time in crustaceans evidence for masculinization effects of the AG on reproductive behavior, in addition to morpho-anatomical and physiological effects. AG implantation into immature female red claw crayfish Cherax quadricarinatus inhibited secondary vitellogenesis and development of the ovaries, as well as morphological traits that facilitate maternal egg brooding; it also caused the appearance of secondary male characteristics. However, primary male characteristics and a masculine reproductive system were not developed. In pair encounters, aggression was substantially lower in interactions between AG-implanted and intact females than in interactions within AG-implanted or intact pairs. Moreover, elements of mating behavior, i.e. male courtship displays and false copulations, were exhibited by AG-implanted females in several encounters with intact females. In addition to known morpho-anatomical and physiological effects of the AG in crustaceans, the present study suggests that the AG has novel effects on the neural network that generates social behavior.

Gene silencing in crustaceans: from basic research to biotechnologies.

Gene silencing through RNA interference (RNAi) is gaining momentum for crustaceans, both in basic research and for commercial development. RNAi has proven instrumental in a growing number of crustacean species, revealing the functionality of novel crustacean genes essential among others to development, growth, metabolism and reproduction. Extensive studies have also been done on silencing of viral transcripts in crustaceans, contributing to the understanding of the defense mechanisms of crustaceans and strategies employed by viruses to overcome these. The first practical use of gene silencing in aquaculture industry has been recently achieved, through manipulation of a crustacean insulin-like androgenic gland hormone. This review summarizes the advancements in the use of RNAi in crustaceans, and assesses the advantages of this method, as well as the current hurdles that hinder its large-scale practice.

Intersexuality and behavior in crayfish: The de-masculinization effects of androgenic gland ablation

In crustaceans, male differentiation and primary and secondary characteristics are regulated by the androgenic gland (AG). In gonochoristic crustaceans, the AG is also linked to intersexuality. Whereas the co-occurrence of various male and female characteristics has been demonstrated in intersex crustaceans, little is known regarding sexually dimorphic behavior patterns in such individuals. In the present study, we used an intersex crayfish model to investigate--for the first time in crustaceans--the agonistic and mating behavior of intersex individuals, and to explore the effects of AG ablation on behavior, morphology and physiology. As was the case for their morphological and physiological reproductive traits, intersex individuals--despite being genotypically females--generally resembled males in terms of behavior: they engaged in fighting with males and copulated with receptive females. However, fighting durations of intersex animals were intermediate between those of males and females, and the durations of the copulations were remarkably short. Adult intersex individuals that had been AG ablated at the juvenile stage were unlikely to engage in fighting with males (similar behavior to females) and did not exhibit any mating behavior with receptive females. AG ablation resulted in feminine morphological and physiological shifts in the treated intersex individuals and enabled vitellogenin gene transcription and the onset of secondary vitellogenesis. It thus appears that an as-yet-unknown AG-secreted factor(s) regulating maleness also seems to regulate the organization of male behavior in crustaceans.