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The insulin-like androgenic gland hormone (IAG) secreted from the androgenic gland (AG) is a unique endocrine controller (the IAG-switch) of crustacean sexual differentiation. However, while previous studies of the IAG-switch focused mainly on sexual differentiation at early developmental stages of gonochoristic species, this mechanism is yet to be deciphered during naturally occurring sexual shifts in hermaphrodite species. The Northern spot shrimp, Pandalus platyceros, is a protandric hermaphrodite species, native to the North Pacific Ocean. We collected four stages of spot shrimp in Southeast Alaska including: juveniles, adult males, transitionals, and adult females. The AG was dissected from each stage and characterized histologically. Additionally, the IAG mRNA was sequenced. The function of the IAG-switch during the life history of this protandric species was demonstrated through monitoring IAG gene expression. Transcript levels were highest at the juvenile stage, then decreased significantly in mature males and became negligible in the transitional and female stages. Moreover, manipulating the IAG-switch via IAG loss of function in males through RNAi, induced the expected masculine to feminine sexual transformation that naturally occurs in this species. This included reduction in IAG transcript levels in males, elevation of vitellogenin gene expression in hepatopancreas and transformation of the gonad from an ovotestis containing both ovarian and testicular tissue to a true ovary with vitellogenic oocytes. Furthermore, a transcriptomic library from tissues associated with the endocrine axis upstream and downstream the IAG-switch yielded 1,801, 1,707, 1,946, and 182 differentially expressed genes between males, transitionals, and females in the AG, eyestalk, gonad, and hepatopancreas, respectively. Among these genes, the transcriptional pattern of six of them (all of them in the AG), between males, transitionals and females, had similar or inverted transcriptional pattern to that of IAG in P. platyceros. Five of these putatively IAG-switch associated genes are downregulated and one is upregulated throughout the P. platyceros shift from maleness to femaleness. Homolog protein sequences for the above novel genes were found in 17 other decapod species suggesting that they might represent conserved factors associated with the IAG-switch and involved in universal crustacean sexual differentiation mechanisms.

The androgenic gland (AG) – a unique crustacean endocrine organ that secretes factors such as the insulin-like androgenic gland (IAG) hormone – is a key player in crustacean sex differentiation processes. IAG expression induces masculinization, while the absence of the AG or a deficiency in IAG expression results in feminization. Therefore, by virtue of its universal role as a master regulator of crustacean sexual development, the IAG hormone may be regarded as the sexual 'IAG-switch.' The switch functions within an endocrine axis governed by neuropeptides secreted from the eyestalks, and interacts downstream with specific insulin receptors at its target organs. In recent years, IAG hormones have been found – and sequenced – in dozens of decapod crustacean species, including crabs, prawns, crayfish and shrimps, bearing different types of reproductive strategies—from gonochorism, through hermaphroditism and intersexuality, to parthenogenesis. The IAG-switch has thus been the focus of efforts to manipulate sex developmental processes in crustaceans. Most sex manipulations were performed using AG ablation or knock-down of the IAG gene in males in order to sex reverse them into 'neo-females,' or using AG implantation/injecting AG extracts or cells into females to produce 'neo-males.' These manipulations have highlighted the striking crustacean sexual plasticity in different species and have permitted the manifestation of either maleness or femaleness without altering the genotype of the animals. Furthermore, these sex manipulations have not only facilitated fundamental studies of crustacean sexual mechanisms, but have also enabled the development of the first IAG-switch-based monosex population biotechnologies, primarily for aquaculture but also for pest control. Here, we review the crustacean IAG-switch, a unique crustacean endocrine mechanism, from the early discoveries of the AG and the IAG hormone to recent IAG-switch-based manipulations. Moreover, we discuss this unique early pancrustacean insulin-based sexual differentiation control mechanism in contrast to the extensively studied mechanisms in vertebrates, which are based on sex steroids.

Recently, the crustacean female sex hormone (CFSH), which is considered a female-specific hormone, has been shown to play a crucial role in female phenotypes in crustaceans. In this study, two transcripts (Sp-CFSH1 and Sp-CFSH2) encoding the same CFSH precursor were cloned from the mud crab Scylla paramamosain. Homology and phylogenetic analysis showed that CFSHs were homologous to interleukin-17 and highly conserved among brachyuran crabs. PCR analysis revealed that Sp-CFSH was expressed exclusively in the eyestalk ganglion of both prepubertal males and females, and surprisingly, the abundance of Sp-CFSH transcripts detected in the males were not significantly different from that of the females (P > 0.05). In addition, mRNA in situ hybridization showed that Sp-CFSH was localized in the X-organ of the male eyestalk ganglion. During the development of the androgenic gland (AG), the level of Sp-IAG mRNA in AG remained at low levels from stages I to II (early stage) but had a significant increase at stage III (mature stage). In contrast, the level of Sp-CFSH transcripts in the eyestalk ganglion was high in the early stage but extremely low in the mature stage. To investigate the potential function of CFSH in male S. paramamosain, the recombinant protein (∼20 kDa) was expressed in Escherichia coli and was subsequently added to AG explants in vitro. It was demonstrated that recombinant Sp-CFSH protein significantly reduced the expression of Sp-IAG in the AG explants at a concentration of 10−6 M (P < 0.05). In conclusion, our study provides the first piece of evidence that shows CFSH from the eyestalk ganglion acts as a negative regulator inhibiting the development of AG in crustaceans.

Recently, the crustacean female sex hormone (CFSH), which is considered a female-specific hormone, has been shown to play a crucial role in female phenotypes in crustaceans. In this study, two transcripts (Sp-CFSH1 and Sp-CFSH2) encoding the same CFSH precursor were cloned from the mud crab Scylla paramamosain. Homology and phylogenetic analysis showed that CFSHs were homologous to interleukin-17 and highly conserved among brachyuran crabs. PCR analysis revealed that Sp-CFSH was expressed exclusively in the eyestalk ganglion of both prepubertal males and females, and surprisingly, the abundance of Sp-CFSH transcripts detected in the males were not significantly different from that of the females (P > 0.05). In addition, mRNA in situ hybridization showed that Sp-CFSH was localized in the X-organ of the male eyestalk ganglion. During the development of the androgenic gland (AG), the level of Sp-IAG mRNA in AG remained at low levels from stages I to II (early stage) but had a significant increase at stage III (mature stage). In contrast, the level of Sp-CFSH transcripts in the eyestalk ganglion was high in the early stage but extremely low in the mature stage. To investigate the potential function of CFSH in male S. paramamosain, the recombinant protein (∼20 kDa) was expressed in Escherichia coli and was subsequently added to AG explants in vitro. It was demonstrated that recombinant Sp-CFSH protein significantly reduced the expression of Sp-IAG in the AG explants at a concentration of 10−6 M (P < 0.05). In conclusion, our study provides the first piece of evidence that shows CFSH from the eyestalk ganglion acts as a negative regulator inhibiting the development of AG in crustaceans.

This study investigated the seasonal variation in the structure and secretory activity of the androgenic gland (AG) in the freshwater crab: Travancoriana schirnerae. The androgenic gland is an elongate structure, attached to one side on the wall of the ejaculatory duct. Histological studies showed the presence of three cell types, which differ in size, shape of nuclei, and presence or absence of secretory vesicles. Type I cells are small with large nuclei whereas type II cells are large with small nuclei. Type III cells are intermediate in size and exhibited streak-like nuclei and transparent cytoplasm. Seasonal changes were discerned in the morphology, histology and secretory activity of the gland. March-June appeared to be the active season with type II cells containing secretory vesicles. The mode of release of secretion found to be holocrine. The secretory activity almost completed by July-August (the mating season) with vacuolization of type II cells. The gland remained inactive from September-December with abundance of vacuoles, scattered pycnotic nuclei, indistinct cell membranes and total cellular degeneration. January-February was the revival period with type I cell proliferation. The present study revealed that the secretory activity of the gland is in tune with the male reproductive cycle.

Monosex male culture of C. quadricarinatus is proposed for increasing yields because males reach a larger size at harvest than females. An experiment was done to evaluate the effects of androgenic gland extract and temperature on sex reversal and somatic growth in early juvenile females. Females were exposed to the following experimental conditions: C: control food and temperature maintained at 26 ± 1°C; HT (high temperature): control food and temperature maintained at 28. 5 ± 1°C; VHT (very high temperature): control food and temperature maintained at 31 ± 1°C; AG: food with enriched with androgenic gland (1/10 dose for each juvenile per day) and temperature maintained at (26 ± 1)°C. The juveniles were weighed, sexed, and growth increment (GI) and growth rate (GR) were calculated twice a month. The HT and AG groups differed from the C group in GI and GR indicating a greater somatic growth. At the end of the experiment, the HT and AG groups had similar weight but only the HT group had enhanced oocyte diameter, with some vitellogenic oocytes compared to the C group. In the HT group, there was significant sex reversal, as indicated by development of male and intersex male characteristics in different individuals. Both temperature and AG diet have a high potential for culture of this species because of their enhancement of somatic growth; higher temperature increases the proportion of males, a desirable characteristic of cultured populations. Fil: Sánchez, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Lopez, Laura Susana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina