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In crustacean aquaculture, size dimorphism between males and females is the main key factor determining the advantage of monosex aquaculture over that of mixed populations. This factor is particularly relevant for the freshwater prawn, Macrobrachium rosenbergii, for which intensification of cultures is complicated by a complex social structure in which large dominant males are territorial and inhibit the growth performance of smaller males and females. It has therefore been suggested that all-female mono-culture could be the practice of choice, since females are less aggressive and less territorial and are believed to exhibit a relatively homogenous growth pattern. Here we report the first large-scale comparative field study of all-female and mixed populations under extensive and intensive stocking conditions in earthen ponds. The study was facilitated by application of our novel biotechnology based on a single injection of suspended hypertrophied androgenic gland cells. Under both our intensive and extensive conditions, the all-female cultures showed better performance than the mixed cultures in most key aquaculture parameters including survival rate and yield per hectare. Also, the intensively stocked all-female ponds showed better feed conversion ratio than mixed ponds. Furthermore, while the mean size of the animals did not differ significantly between the two treatments, the all-female populations exhibited significantly higher size uniformity. Our study suggests that for M. rosenbergii, female monosex aquaculture is a sustainable method to yield a homogenous crop.

The cultivation of monosex populations is common in animal husbandry. However, preselecting the desired gender remains a major biotechnological and ethical challenge. To achieve an efficient biotechnology for all-female aquaculture in the economically important prawn (Macrobrachium rosenbergii), we achieved – for the first time – WW males using androgenic gland cells transplantation which caused full sex-reversal of WW females to functional males. Crossing the WW males with WW females yielded all-female progeny lacking the Z chromosome. We now have the ability to manipulate – by non-genomic means – all possible genotype combinations (ZZ, WZ and WW) to retain either male or female phenotypes and hence to produce monosex populations of either gender. This calls for a study of the genomic basis underlying this striking sexual plasticity, questioning the content of the W and Z chromosomes. Here, we report on the sequencing of a high-quality genome exhibiting distinguishable paternal and maternal sequences. This assembly covers ~ 87.5% of the genome and yielded a remarkable N50 value of ~ 20 × 106 bp. Genomic sex markers were used to initiate the identification and validation of parts of the W and Z chromosomes for the first time in arthropods.

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.

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.

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.

Sexual differentiation in male crustaceans is known to be controlled by the androgenic gland (AG), possibly through a peptide hormone. Recently, three freshwater and one marine crustacean decapod genes encoding AG-specific insulin-like peptides were characterized. We report here the molecular cloning of the complete sequence encoding an AG-specific insulin-like peptide (Pm-IAG) in the commercially important marine Penaeid prawn, Penaeus monodon. The deduced precursor sequence consists of a signal peptide, B chain, C peptide and an A chain. It exhibits the same structural organization as that of previously identified crustacean insulin-like androgenic gland specific peptides (IAGs). The positions of cysteine residues of the putative A and B chains, which govern the folding of the mature peptide via the formation of disulfide bridges, are highly conserved among the prawn and other crustaceans, while the rest of the amino acids show low sequence similarity. Gene expression analysis of Pm-IAG in several tissues, including the closely juxtaposed sperm duct and muscle, confirmed that it is specifically expressed in the AG. The findings suggest that with an appropriate intervention, sexual differentiation could be manipulated and thus might be instrumental for the establishment of monosex culture in this bimodally growing shrimp.

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.

Early malacological literature suggests that the outbreak of schistosomiasis, a parasitic disease transmitted by aquatic snails, in the Senegal River basin occurred due to ecological changes resulting from the construction of the Diama dam. The common treatment, the drug praziquantel, does not protect from the high risk of re-infection due to human contact with infested water on a daily basis. The construction of the dam interfered with the life cycle of the prawn Macrobrachium vollenhovenii by blocking its access to breeding grounds in the estuary. These prawns were demonstrated to be potential biological control agents, being effective predators of Schistosoma-susceptible snails. Here, we propose a responsible restocking strategy using all-male prawn populations which could provide sustainable disease control. Male prawns reach a larger size and have a lower tendency to migrate than females. We, therefore, expect that periodic restocking of all-male juveniles will decrease the prevalence of schistosomiasis and increase villagers' welfare. In this interdisciplinary study, we examined current prawn abundance along the river basin, complemented with a retrospective questionnaire completed by local fishermen. We revealed the current absence of prawns upriver and thus demonstrated the need for restocking. Since male prawns are suggested to be preferable for bio-control, we laid the molecular foundation for production of all-male M. vollenhovenii through a complete sequencing of the insulin-like androgenic gland-encoding gene (IAG), which is responsible for sexual differentiation in crustaceans. We also conducted bioinformatics and immunohistochemistry analyses to demonstrate the similarity of this sequence to the IAG of another Macrobrachium species in which neo-females are produced and their progeny are 100% males. At least 100 million people at risk of schistosomiasis are residents of areas that experienced water management manipulations. Our suggested non-breeding sustainable model of control—if proven successful—could prevent re-infections and thus prove useful throughout the world., Author Summary Schistosomiasis is a chronic parasitic disease that infects millions of people, especially in Africa. Schistosomes are transmitted by direct contact with water sources infested by freshwater snails, which are intermediate hosts for the parasite. The cure in humans is a drug, praziquantel, that kills the mature parasites inside the human body. The main problem with controlling the parasite by drug treatment is the high re-infection rate, since individuals are in contact with infected water on a daily basis. To efficiently combat the disease, an integrated management program is needed that includes control of infection in the intermediate host snails. We suggest the use of non-migrating, all-male populations of freshwater prawns that efficiently prey on these snails. Here, we describe the case of the Senegal River basin as an example of human actions (dam construction) that resulted in severe ecosystem changes, including exclusion of the native river prawns and expansion of snails hosting schistosomiasis. We have conducted an interdisciplinary study that documents the reduction of prawn abundance in the Senegal River and lays the molecular foundation for technology to produce all-male prawn populations to be used as part of an integrated disease control program, including both periodic stocking of juvenile prawns and chemotherapy.

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 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 ER-like localization pattern. Moreover, co-expressing 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 androgenic gland 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. On this note, Cq-MAG's 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 androgenic gland hormone.

In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish.

In Crustacea, an early evolutionary group (;50000 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 IAGencoding 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 insulinlike 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. gene expression, insulin-like androgenic gland hormone, invertebrates, sex differentiation, sexual manipulation biotechnology

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.