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Macrobrachium rosenbergii is an economically important crustacean worldwide. Based on the important role of the androgenic gland (AG) in sex determination in crustaceans, this study comparatively analyzed the transcriptomes of AG and ovary tissues using Illumina sequencing technology. A total of 91,810 unigenes were generated from the six libraries after splicing and assembly, and 28,516 (31.1%) were annotated by BLASTx comparison with Nr and other databases. A total of 20,793 differentially expressed genes including 105 potentially related to sex determination and sex differentiation were identified in both tissues, of which 69 were upregulated in the ovary, 19 were upregulated in AG, 12 were ovary-specific, and 5 were AG-specific. This study screened out differentially expressed genes related to sex determination and differentiation by comparing the transcriptome expression differences between the ovary and AG of M. rosenbergii, accumulating important data for further studies on the mechanisms of sex determination and differentiation in M. rosenbergii. [ABSTRACT FROM AUTHOR]

The previous publication identified that pyruvate dehydrogenase E1 (PDHE1) positively regulated the process of male reproduction in M. nipponense through affecting the expressions of insulin-like androgenic gland hormone. The present study aimed to identify the potential male-reproduction-related genes that were regulated by PDHE1 through performing the transcriptome profiling analysis in the testis and androgenic gland after the knockdown of the expressions of PDHE1 by the injection of dsPDHE1. Both RNA-Seq and qPCR analysis identified the significant decreases in PDHE1 expressions in the testis and androgenic gland in dsPDHE1-injected prawns compared to those in dsGFP-injected prawns, indicating the efficiency of dsPDHE1 in the present study. Transcriptome profiling analysis identified 56 and 127 differentially expressed genes (DEGs) in the testis and androgenic gland, respectively. KEGG analysis revealed that the energy-metabolism-related pathways represented the main enriched metabolic pathways of DEGs in both the testis and androgenic gland, including pyruvate metabolism, the Citrate cycle (TCA cycle), Glycolysis/Gluconeogenesis, and the Glucagon signaling pathway. Thus, it is predicted that these metabolic pathways and the DEGs from these metabolic pathways regulated by PDHE1 may be involved in the regulation of male reproduction in M. nipponense. Furthermore, four genes were found to be differentially expressed in both the testis and androgenic gland, of which ribosomal protein S3 was down-regulated and uncharacterized protein LOC113829596 was up-regulated in both the testis and androgenic gland in dsPDHE1-injected prawns. The present study provided valuable evidence for the establishment of an artificial technique to regulate the process of male reproduction in M. nipponense. [ABSTRACT FROM AUTHOR]

Sexual manipulation in the giant freshwater prawn Macrobrachium rosenbergii has proven successful in generating monosex (both all-male and all-female) populations for aquaculture using a crustacean-specific endocrine gland, the androgenic gland (AG), which serves as a key masculinizing factor by producing and secreting an insulin-like AG hormone (IAG). Here, we provide a summary of the advancements from the discovery of the AG and IAG in decapods through to the development of monosex populations in M. rosenbergii. We discuss the broader sexual development pathway, which is highly divergent across decapods, and provide our future perspective on the utility of novel genetic and genomic tools in promoting refined approaches towards monosex biotechnology. Finally, the future potential benefits of deploying monosex prawn populations for environmental management are discussed. [ABSTRACT FROM AUTHOR]

Many decapod crustaceans with sexual dimorphism in body size are economically significant species for fisheries and aquaculture, making monosex culture a desirable practice to increase farmers' yield and profits. It is well established that the male‐specific androgenic gland (AG) mediates masculinization in decapods by producing and secreting the insulin‐like AG hormone (IAG). However, IAG is not a sex‐determining factor; hence, iag silencing in decapods does not always lead to successful sex change. Since the establishment of monosex populations in the giant freshwater prawn Macrobrachium rosenbergii, the same AG/iag manipulation approach has been attempted in economically significant penaeid shrimps, crabs, crayfish and spiny lobsters, to no avail. There are many factors at play with species‐specific intricacies which require close examination when addressing monosex production. This review provides a refined roadmap to successful sexual manipulation in decapod crustaceans, highlighting the key caveats to be considered and critical gaps in knowledge such as the timing of iag expression compared to the development of sexual characteristics, the relationship between iag and a master regulator, as well as silencing capacity. Lastly, this review examines what the future might hold for monosex aquaculture in decapods, taking into consideration novel technologies such as gene editing. [ABSTRACT FROM AUTHOR]

The Australian red claw crayfish Cherax quadricarinatus, an emerging species within the freshwater aquaculture trade, is not only an ideal species for commercial production due to its high fecundity, fast growth, and physiological robustness but also notoriously invasive. Investigating the reproductive axis of this species has been of great interest to farmers, geneticists, and conservationists alike for many decades; however, aside from the characterisation of the key masculinising insulin-like androgenic gland hormone (IAG) produced by the male-specific androgenic gland (AG), little remains known about this system and the downstream signalling cascade involved. This investigation used RNA interference to silence IAG in adult intersex C. quadricarinatus (Cq-IAG), known to be functionally male but genotypically female, successfully inducing sexual redifferentiation in all individuals. To investigate the downstream effects of Cq-IAG knockdown, a comprehensive transcriptomic library was constructed, comprised of three tissues within the male reproductive axis. Several factors known to be involved in the IAG signal transduction pathway, including a receptor, binding factor, and additional insulin-like peptide, were found to not be differentially expressed in response to Cq-IAG silencing, suggesting that the phenotypic changes observed may have occurred through post-transcriptional modifications. Many downstream factors displayed differential expression on a transcriptomic level, most notably related to stress, cell repair, apoptosis, and cell proliferation. These results suggest that IAG is required for sperm maturation, with necrosis of arrested tissue occurring in its absence. These results and the construction of a transcriptomic library for this species will inform future research involving reproductive pathways as well as biotechnological developments in this commercially and ecologically significant species. [ABSTRACT FROM AUTHOR]

Many researchers have focused on male sexual differentiation and development of the Macrobrachium nipponense in recent years. In this review, we highlight the status of knowledge about male sexual differentiation and development in this species by summarizing the chromosome analyses, major regulatory factors, identification of male reproduction‐related genes and gene functional analyses. Fifty‐two chromosomes have been identified in the testis of M. nipponense, which led to the identification of sex‐related chromosomes in this prawn. Studies have shown that hormones secreted by the eyestalk, androgenic gland, and testis as well as 17‐estradiol and environmental factors regulate the process of male sexual development in M. nipponense. The sex differentiation and development sensitive period was identified as post‐larva 10 (PL10) to PL22; therefore, this time span is critical in the identification of male reproduction‐related genes from M. nipponense. In this study, 80 male sex‐related homologous genes were identified based on functional analysis in other species, and another 45 genes were considered to be strong male reproduction‐related candidates based on their expression changes through performing multi‐omics profiling analyses. Quantitative polymerase chain reaction analysis revealed 25 genes that were involved in the process of male sexual development in M. nipponense, and RNA interference showed that knockdown of the expression levels of SDHB, PDHE1, NFkBa, HSDL1 and ferritin delayed testis development. Use of Crispr‐Cas9 technology and analysis of noncoding RNA should be further investigated, which will be effective in establishing an artificial technique to regulate the process of male sexual development in M. nipponense. [ABSTRACT FROM AUTHOR]

Since females grow faster in penaeid shrimp, all-female aquaculture was proposed. Environmental conditions in the Pacific white shrimp did not found to affect genetic sex determination (ZZ/ZW system). The androgenic gland (AG)-secreting insulin-like androgenic gland hormone (IAG) is a key controlling factor in crustacean male differentiation. However, functional sex reversal (neo-male) in penaeid shrimp has not yet been achieved by manipulating the IAG-sexual switch. Therefore, understanding the molecular mechanisms of gonadal differentiation may help build appropriate tools to generate neo-male (ZW) for all-female breeding. This study describes the potential role of the novel penaeid-specific testicular zinc finger protein (pTZFP) in the gonads of Pacific white shrimp. First, pTZFP transcripts show a male-bias expression pattern in undifferentiated gonads, which is then exclusively expressed in the testis and absent or slightly expressed in the ovary and other tissues. Besides, the knockdown of pTZFP in undifferentiated males results in smaller testes but no sex reversal. Immunohistochemical (IHC) staining of proliferating cell nuclear antigen (PCNA) further confirmed that the smaller testes in pTZFP-deficient males are due to the lower proliferating activity of spermatogonia. These data reveal that pTZFP may be involved in testicular development but have fewer effects on gonadal differentiation. Moreover, testicular pTZFP transcription levels were not reduced with estradiol-17β (E2) administration or AG excision. Therefore, our data suggest that pTZFP may regulate testicular development through downstream genes regulating spermatogonia proliferation. Moreover, our data provide an appropriate molecular marker for identifying the sex of undifferentiated gonads., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Molecules related to the insulin signaling pathway are widely conserved in the animal kingdom and are responsible for the regulation of various biological functions including growth, metabolism, and reproduction. Information on insulin-like peptides (ILPs) is, however, limited to insulin-like androgenic factor (IAG) in crustaceans. To date, in the kuruma prawn Marsupenaeus japonicus, only Maj-IAG, which is thought to be a crustacean androgen, and ovarian-specific Maj-ILP1 are the only members of the insulin family that have been reported. In this study, we identified two additional ILPs, Maj-ILP2 and Maj-gonadulin (Maj-GON), from the transcriptome data of M. japonicus. Maj-ILP2 gene expression was observed in the brain ganglia of both sexes and testis, while Maj-GON gene expression was limited to the testis. A more detailed tissue expression analysis of three male-dominant ILPs showed clear site-specific gene expression patterns in the gonads: Maj-ILP2 in the testis, Maj-GON in the vas deferens, and Maj-IAG in the androgenic gland attaching to the seminal vesicle. The results of this study show that, in addition to Maj-IAG, a crustacean androgen, there are two more ILP molecular species in the male gonad of M. japonicus, and suggest that they may serve as signaling molecules involved in the regulation of some male-specific functions. [ABSTRACT FROM AUTHOR]

Hermaphrodite systems offer unique opportunities to study sexual differentiation, due to their high degree of sexual plasticity and to the fact that, unlike gonochoristic systems, the process is not confined to an early developmental stage. In protandric shrimp species, such as Hippolyte inermis and Pandalus platyceros, male differentiation is followed by transformation to femaleness during adulthood. The mechanisms controlling sexual differentiation have not been fully elucidated in crustaceans, but a key role has been attributed to the insulin-like hormone (IAG) produced by the androgenic gland (AG), a crustacean masculine endocrine organ. To uncover further transcriptomic toolkit elements affecting the sexual differentiation of H. inermis, we constructed eye and whole body RNA libraries of four representative stages during its protandric life cycle (immature, male, young female and mature female). The body libraries contained transcripts related to the reproductive system, among others, while the eye libraries contained transcripts related to the X-organ-sinus gland, a central endocrine complex that regulates crustacean reproduction. Binary pattern analysis, performed to mine for genes expressed differentially between the different life stages, yielded 19,605 and 6,175 transcripts with a specific expression pattern in the eye and body, respectively. Prominent sexually biased transcriptomic patterns were recorded for the IAG and vitellogenin genes, representing, respectively, a key factor within the masculine IAG-switch, and a precursor of the yolk protein, typical of feminine reproductive states. These patterns enabled the discovery of novel putative protein-coding transcripts exhibiting sexually biased expression in the H. inermis body and eye transcriptomes of males and females. Homologs to the above novel genes have been found in other decapod crustaceans, and a comparative study, using previously constructed transcriptomic libraries of another protandric shrimp, P. platyceros, showed similar sexually biased results, supporting the notion that such genes, mined from the H. inermis transcriptome, may be universal factors related to reproduction and sexual differentiation and their control in other crustaceans. This study thus demonstrates the potential of transcriptomic studies in protandric species to uncover unexplored layers of the complex crustacean sex-differentiation puzzle.

A large number of decapod crustaceans with sexual dimorphism in body size, most of which, are important economic animals. Besides, some of them such as the red swamp crayfish Procambarus clarkii possess strong invasive ability and would damage ecosystem environment. Thereby, monosex culture of decapods would contribute to not only increasing yield and profits, but also reducing the risks of biological invasion. Insulin‐like androgenic gland hormone was reported to be a key regulator in sex differentiation. All‐male populations in Macrobrachium rosenbergii were successfully produced through insulin‐like androgenic gland hormone manipulation. In addition to sex differentiation, insulin‐like androgenic gland hormone was reported to be involved in male secondary sex characteristics maintenance, growth, metabolism, morphology change, ovary development and other functions. Considering its important application value for decapods, insulin‐like androgenic gland hormone has attracted much attention in recent years. This hormone was reported to have the structure and function similar to androgenic gland hormone of isopods. In this study, the origin of name of androgenic gland hormone and insulin‐like androgenic gland hormone was investigated, and the recent research on structure, function, application and molecular regulation mechanism of insulin‐like androgenic gland hormone of decapods was reviewed. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

In crustaceans, the male sexual differentiation and maintenance are specially regulated by androgenic gland (AG). However, little is known about the genes involved in the regulation process. RNA-Seq was performed on AG with ejaculatory duct (AG_ED) and ejaculatory duct (ED) as control in Eriocheir sinensis, one of the most important economic and fishery crabs with typically sex dimorphism. A total of 925 unigenes were identified as differentially expressed genes (DEGs) and the expression of nine genes randomly selected was confirmed by qRT-PCR. 667 unigenes were up-regulated in AG_ED, being supposed to be AG preferential genes. Among them, the full length of insulin-like androgenic gland factor (IAG) cDNA named as Es-IAG was obtained as a logo gene of AG, which together with the genes insulin-like receptor (INR), and single insulin binding domain protein (SIBD), might constitute the sex regulation pathway. Several sex related genes were identified, and their function will have to be investigated. Also, the identification of juvenile hormone epoxide hydrolase 1 (JHEH1), ecdysteroid 22-hydroxylase (DIB) and ecdysone receptor (ECR) preliminarily clarified the molecular regulation mechanism of eyestalk-AG-testis axis, which plays important roles in molting and reproduction. The results will enhance our understanding for the molecular basis of the AG involved in male sex regulation in crabs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The androgenic gland produces hormones that play crucial roles in driving male sexual development. In this study, we investigated the regulatory proteins and pathways affecting male sexual development of M . nipponense , by analyzing the proteomes of their androgenic glands during their reproductive and nonreproductive seasons using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 13 sex-related protein homologs out of 999 total proteins were identified in the proteome of M . nipponense based on comparisons with the published literature. A total of 32 proteins were differentially expressed in the androgenic gland between the reproductive and nonreproductive seasons of M . nipponense . However, only 10 differentially expressed proteins (DEPs) were annotated in the Nr, COG, GO, and KEGG databases. Other non-annotated DEPs may also play vital roles in the male sexual development of M . nipponense . The qPCR analysis indicated that the mRNA expression patterns of the 10 annotated DEPs were consistent with that determined by iTRAQ analysis. Additional qPCR analysis of the 10 DEPs in testes, ovaries, and androgenic glands showed that Gem, Ferritin, and Rev3 were highly expressed in the androgenic gland, implying importance in male sexual development, given the important role of the androgenic gland in male differentiation and development in many crustacean species. Our study provided valuable information about DEPs in androgenic glands between reproductive and nonreproductive seasons for M . nipponense and identified their potential roles in male sexual development, which will improve our understanding of the development process in both M . nipponense and other crustaceans. [ABSTRACT FROM AUTHOR]

The androgenic gland (AG) is an important endocrine gland for male reproductive function in crustaceans. In the present study, we investigated the histological characteristics of the androgenic gland of peppermint shrimp, Lysmata wurdemanni. The peppermint shrimp matures as male first, then some individuals may become euhermaphrodite after several moltings (transitional phase). Euhermaphrodite-phase shrimp acts as male at intermolts. However, it can be fertilized as a female immediately after molting. Considering the male reproductive function acts in its lifespan except for at larval stages, and female reproductive system starts to develop at transitional phase, we hypothesized that AG activity might be reduced to allow and promote vitellogenesis onset in early transitional phase and the following euhermaphrodite phase. So AG cell structure might be different in three phases in L. wurdemanni. The results showed that AG exists in the male in transitional and euhermaphrodite phases. The gland cell clusters surrounding the ejaculatory ducts locate at the roots of the fifth pereopods. The nucleus diameters are similar in the three phases while the nucleus- to-cell ratio is the lowest in euhermaphrodite phase. Our results indicated that for the individuals that will become euhermaphrodite, the cellular structure of AG changes since transitional phase. Male reproductive function which is still available in euhermaphrodite-phase shrimp should be due to the existence of the gland. [ABSTRACT FROM AUTHOR]

Crustacean aquaculture production has developed rapidly in recent years because there has been an increase in crustacean market demands in different regions of the world. The growth rate of the most cultured crustacean species depends on genders. Therefore, monosex crustacean aquaculture, all male or all female, is more suitable for achieving higher yields, with the ecological benefits of reducing the risk of cannibalism. In addition, feminization is also appropriate for increasing individual number in populations because males are able to copulate with more females. For this reason, sexual manipulations are important in crustacean reproduction units. This review focuses on the sex differentiation mechanism and the use of different strategies for feminization of economically important crustaceans. In this review, feminization strategies are divided into two main methods: direct feminization and indirect feminization (masculinization). Direct feminization includes (1) ablation of androgenic gland (AG), (2) silencing and suppression AG and androgenic gland hormones (AGHs), (3) hormone therapy (i.e., 17β-estradiol), and (4) polyploidy induction. This review also emphasizes how feminization is carried out for crustaceans belonging to the different sex determination systems (ZW or XY) by using indirect method. The intension of controlling sexual differentiation for crustaceans will continue to increase in the next a few years, either for basic research or for its utilization to crustacean aquaculture; hence, this review will be profitable for crustacean farmers and researches. [ABSTRACT FROM AUTHOR]

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain. This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens (ChqIAG), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 50 and 30 rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 50 untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 30 UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain, which are clustered together phylogenetically. [ABSTRACT FROM AUTHOR]

Indirect evidence indicates that penaeids may have a W/Z sex heritability mechanism with the androgenic gland (AG) mediating masculine differentiation. The present study evaluated the immune response against grafts and the expression of primary and secondary male sexual characters in Litopenaeus vannamei females implanted with terminal ampoules and associated AGs. Experiments included grafting females at PL 34–48 . Grafting at PL 37 generated few cases of melanization exhibiting dark coloration covering parts or the whole implant while most implants showed white coloration, without any trace of melanization. Histology of melanized implants showed necrotic tissue, encapsulated by black depositions. These melanized capsules were externally surrounded by normal haemocytes and flat haemocytes in the middle. An experiment evaluating possible absorption of the grafts based on recovery, gross coloration, size of implants, and histology showed that Complete terminal ampoule-AG implants, Control-vas deferens implants and Abdominal muscle implants, recovered at day 7 and 15, exhibited gradual and statistically significant decrease in size, which was more evident for muscle implants. Grafts with normal appearance from this experiment remained viable during 7 and 15 days post-implantation; these tissues showed a normal cellular structure with neither signs of melanization nor haemocyte infiltration. Appendices masculinae did not develop in any implanted females or controls. Concerning petasma development, two females implanted with AG showed a male phenotype in their first endopodites, characterized by a straight shape at the distal region, a middle protuberance and absence of setae along the middle curved edge of the structure. Regenerated and intact endopodites of Control-proximal terminal ampoule females showed the typical female phenotype with a slender shape with a curved distal edge, without a middle protuberance, and with setae along the middle curved edge of endopodites. Implanted females showed no evidence of masculinization or abnormal development of oocytes. In the light of the present study the effects of AG transplantation on sex in penaeids should be further evaluated in younger PL stages. Statement of relevance This contribution presents new information on sex reversal technology on Litopenaeus vannamei based on androgenic gland implants . The findings are novel for the family Penaeidae concerning the immunological response to tissue grafting and the plasticity for sex reversal in this world aquaculture species. [ABSTRACT FROM AUTHOR]

The recent introduction into aquaculture of RNA interference (RNAi) for producing the preferred all-male monosex cultures, which give improved yields, has raised awareness of the need to investigate the consequences of such novel biotechnological manipulations. Here, we present meta-analysis style study on data from observations of three consecutive cultured all-male (ZZ) generations of the giant freshwater prawn Macrobrachium rosenbergii (De Man). Each consecutive generation comprised the progeny of RNAi-manipulated sex-reversed males. The manipulation was achieved through the administration of dsRNA encoding the insulin-like androgenic hormone into males (which transformed them into ‘neofemales,’ ZZ) of the previous all-male generation. Each generation was cultured in a separate earthen pond for a short (~ 4–5 months) growout period. At harvest of each of the generations, the typical M. rosenbergii population structure comprising three male morphotypes was obtained. An anatomical examination of the male reproductive system of a representative specimen of the third all-male generation showed normal reproductive outputs, even though the prawns had been grown without the presence of females (WZ) for three generations. At the molecular level, expression of vital male-specific genes in the third generation of all-male M. rosenbergii culture was demonstrated. Thus, the present study showing the lack of any overtly apparent long-term consequences of the RNAi-based biotechnology provides support for the responsible use of temporal RNAi in aquaculture. Finally, the absence of the W chromosome for three generations raises questions with regard to its role and content with respect to crustacean sexual determination and differentiation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This research was implemented to study the effects of androgenic gland ablation in the white shrimp Litopenaeus vannamei to explore sex reversal potential as an alternative technology for monosex female mariculture based on the sex dimorphic growth of this species. The surgical procedure was applied to male postlarvae ( PL) at different ages, after external sex differentiation, as well as in sex undifferentiated PL. Andrectomized males regenerated normal appendices including pereiopods and pleopods; however, body growth and relative size of regenerated petasmas and appendices masculinae were statistically inferior ( P ˂ 0.05) to control males. Spermatogenesis in andrectomized males was active, but a phenomenon of degradation of spermatids and reproductive tissues was detected. No sex reversal was accomplished regardless of PL age from sex undifferentiated stages ( PL38) to sex differentiated stages (˃ PL55). The complete regeneration of sexual characters in andrectomized L. vannamei (Dendrobranchiata) is different from previous reports from Decapoda. [ABSTRACT FROM AUTHOR]

The Eastern spiny lobster, Sagmariasus verreauxi, is commercially important in fisheries, with growing aquaculture potential, driving an interest to better understand male sexual differentiation. Amongst the Decapoda, the androgenic gland (AG) and the insulin-like androgenic gland hormone (IAG) have a well-defined function in male sexual differentiation. However, IAG is not a sex determinant and therefore must be considered as part of a broader, integrated pathway. This work uses a transcriptomic, multi-tissue approach to provide an integrated description of male-biased expression as mediated through the AG. Transcriptomic analyses demonstrate that IAG expression is stage- and eyestalk-regulated (low in immature, high in mature and 6-times higher in hypertrophied glands), with IAG being the predominant AG-specific factor. The low expression of this key factor in immature males suggests the involvement of other tissues in male sexual differentiation. Across tissues, the gonad (87.8%) and antennal gland (73.5%) show the highest malebiased differential expression of transcripts and also express 4 sex-determination regulators, known as Dmrts, with broader expression of Sv-Sxl and Sv-TRA-2. In order to better understand male sexual differentiation, tissues other than the AG must also be considered. This research highlights the gonad and antennal gland as showing significant male-biased expression patterns, including the Sv-Dmrts. [ABSTRACT FROM AUTHOR]

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain. This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens (ChqIAG), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 5′ and 3′ rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 5′ untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 3′ UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain, which are clustered together phylogenetically.

In this study we describe, for the first time in spiny lobsters, the androgenic gland and its putative hormone. The androgenic gland in crustaceans is the key regulator of crustacean masculinity. The transcript encoding the insulin-like androgenic gland specific factor has recently been identified and characterized in a number of decapod crustacean species including commercially important crabs, crayfish, prawns and shrimps. This insulin-like factor has proven to be the androgenic gland masculinizing hormone, and is absent in females. While the androgenic gland and its putative hormone have been identified in all other commercially valuable groups, none had been identified in lobsters. We identified and characterized the androgenic glands of two spiny lobster species ( Sagmariasus verreauxi and Jasus edwardsii ) and conducted a transcriptomic analysis of the S. verreauxi androgenic gland. Bioinformatics analysis led to the discovery and characterization of the insulin-like androgenic gland specific factors in both species studied. Changes in androgenic gland cell size and quantity between sub-adult and sexually mature males were evident. The transcriptomic database established for the S. verreauxi androgenic gland might enable to elucidate the mechanisms through which the insulin-like factor is secreted, transported to the target cells and how it triggers the physiological effects of sexual differentiation towards maleness and maintenance of the male gonad. [ABSTRACT FROM AUTHOR]

Quantitative analysis of hypertrophy and hyperactivity in tissue of the androgenic gland of eyestalk-ablated male Pacific white shrimp Litopenaeus vannamei was made during the molt cycle by measuring the area of androgenic gland cells located in the subterminal region of the terminal ampoule and measuring the expression of the insulin-like androgenic gland hormone precursor gene ( Lv-IAG ) in the distal vas deferens and terminal ampoule, respectively. Data were compared to intact shrimp at the same molt stages. In eyestalk-ablated shrimp, the lack of negative control from the X-organ–sinus gland complex produced hypertrophy in androgenic gland cells, because the area of the cells folded approximately 1.5 times up to the intermolt stage, when compared to the intact group. It also produced hyperactivity because the relative expression of Lv-IAG was significantly higher than the intact group at all molt stages. After gathering data from the vas deferens and terminal ampoule, the relative expression of Lv-IAG was maintained at higher levels, without a significant difference during the molt stages. However, after each tissue was analyzed independently, a bell-shape curve pattern occurred, according to the molt cycle, as also occurred in intact shrimp. This suggests that in the absence of eyestalks, the brain and nervous system are operating secondarily to synchronize Lv-IAG expression into the molt cycle. The relative expression of Lv-IAG was significantly greater in the terminal ampoule than in the distal vas deferens. In summary, these findings support a reproductive endocrine axis in L . vannamei , as an eyestalk–androgenic gland linkage concomitant with the molt cycle. [ABSTRACT FROM AUTHOR]

Mono-sex culture of giant freshwater prawns (Macrobrachium rosenbergii) gives more benefits than mixed-sex culture because of high market demand for male prawns. Unfortunately, sex differentiation in crustaceans could not obviously be observed in their early larval stage. Therefore, sex segregation or sex reversal techniques need to be developed and timing for sex differentiation in M. rosenbergii during larval development needs to be examined. In this study, three genes related to male reproductive hormone (IAG, MRR and MAL) were isolated and amplified from terminal ampulae (TA) of adult male M. rosenbergii. The male specific transcripts demonstrated that they could be used to monitor sex differentiation time during larval development. Observation of male transcripts expression together with macro and microscopic pictures of phenotypic characters, the appendix masculina (AP) and the gonopore complex (GP), during larval and juvenile development demonstrated that the sex differentiation process of male freshwater prawns began when the larvae were 2 months old or after complete metamorphosis. Moreover, this is the first report on SEM depicting an ultra-valve like structure on gonopore complex surface during sex development of male prawn. [ABSTRACT FROM AUTHOR]

Insulin-like androgenic gland hormone (IAG) produced by androgenic gland (AG) in male crustaceans is regarded as a key regulator of sex differentiation. As a member of the insulin/insulin-like growth factor family, IAG is also likely involved in regulating somatic growth. In this study, a full-length cDNA of IAG (termed Sp-IAG ) was isolated from the mud crab, Scylla paramamosain . Genomic DNA of Sp-IAG was also cloned, analysis of which reveals that Sp-IAG gene is organized in a 4 exon/3 intron manner. RNA in situ hybridization analysis detected positive signals in both type I and type II AG cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that Sp-IAG was expressed not only in AG, but also in many other tissues. Sp-IAG expression levels in ovaries were examined at different stages of ovarian development (stages I to V); it was found that the expression was maintained at low levels during undeveloped stage (stage I) to late vitellogenic stage (stage IV) and then increased significantly at mature stage (stage V), suggesting that Sp-IAG may participate in inhibiting oocyte growth and vitellogenesis. The expression pattern of Sp-IAG during the molting cycle of the first stage crabs (C1) was also determined. Sp-IAG expression level continuously decreased from 0 h C1 (postmolt) crabs to 96 h C1 (premolt) crabs, and then increased significantly in the newly molted second stage crabs (C2, postmolt). The combined results suggested for the first time that IAG is involved in regulating ovarian development and somatic growth in crustaceans. [ABSTRACT FROM AUTHOR]

The location of the androgenic gland in the genital organ in male Pacific white shrimp Litopenaeus vannamei (Boone, 1931) was analyzed by histology and expression of the insulin-like androgenic gland hormone precursor gene (Lv-IAG). The androgenic gland in L. vannamei is a group of three conspicuous semicircular cellular masses interconnected by cord-like androgenic gland cells, in parallel and attached to the distal vas deferens and terminal ampoule, which form a hook-like morphology. Lv-IAG expression occurs mainly at the terminal ampoule, but also at the distal part of the descending medium vas deferens and the narrow distal vas deferens. These specific regions of Lv-IAG expression basically match the anatomical localization of the androgenic gland observed by histology. The complete cDNA nucleotide sequence of Lv-IAG precursor had 87% to Fenneropenaeus chinensis (Osbeck, 1765), 84% identity to Penaeus monodon Fabricius, 1798 (Pm-IAG), and 82% to Marsupenaeus japonicus (Bate, 1888) (Maj-IAG). The deduced encoding sequence from an open reading frame of 507 bp contained a signal peptide, a B chain, a C peptide, and an A chain that correspond to the androgenic gland insulin-like hormone polypeptide in decapods, where six highly conserved cysteine residues of the A and B chains fold the mature peptide through disulfide bridges. The amino-acid sequence showed high identity to P. monodon (80%), F. chinensis (80%), and M. japonicus (72%), whereas the match to other decapod families Palaemonidae, Parastacidae, and Portunidae was low (24-32%). This study extends our knowledge of the molecular structure of insulin-like peptides related to sex differentiation in malacostraca. [ABSTRACT FROM AUTHOR]

Abstract: In crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), controls the differentiation of the primary and secondary male sexual characters. In this study, we amplified 12 new AGH cDNAs in species belonging to five different families of the infra-order Ligiamorpha of terrestrial isopods. Putative essential amino acids for the production of a functional AGH protein exhibit signatures of negative selection and are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motif on the A chains and the eight Cys positions. An insulin-like growth factor motif was also identified in Armadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguish two main clades, corresponding to members of the Armadillidiidae and the Porcellionidae families which are congruent with the narrow specificity of AG heterospecific grafting. An in-depth understanding of the regulation of AGH expression would help deciphering the interaction between Wolbachia, widespread feminizing endosymbiotic bacteria in isopods, and the sex differentiation of their hosts. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] The androgenic gland (AG) controls male reproduction in Macrobrachium rosenbergii. [•] The AG cells produce insulin-like androgenic gland hormone (Mr-IAG). [•] 5-HT and l-GnRH-III have stimulatory effects on the AG cell proliferation and Mr-IAG production. [•] DA and Crz have inhibitory effects on the AG cell proliferation and Mr-IAG production. [ABSTRACT FROM AUTHOR]

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 iCq-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-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 AG hormone. [ABSTRACT FROM AUTHOR]

Abstract: The androgenic gland (AG), a male-specific endocrine organ in crustacean, is responsible for the maintenance of male characteristics and gender differentiation. In this study, an AG-specific gene, the Macrobrachium nipponesne insulin-like androgenic gland factor (MnIAG) was isolated from a transcriptome library of M. nipponesne and its full-length cDNA sequences were obtained by RACE method. The cDNA was 1,547bp in length and encoded a precursor protein of 175 amino acids. The deduced precursor protein consisted of a signal peptide, B chain, C peptide and an A chain, which exhibited the same structural organization as that of previously identified insulin-like androgenic gland in crustacean. The mature peptide of the MnIAG owned two additional conserved cysteine residues, which were also found in the Palaemonidae species reported. Results of the tissue distribution and in situ hybridization showed the MnIAG expressed exclusively in androgenic gland. The quantitative RT-PCR results demonstrated that the MnIAG transcript was present at blastula stage and later developmental stages with low levels, which suggested that the primordial cells of the AG might form at these stages. [Copyright &y& Elsevier]

Abstract: Feminizing parasites enhance their vertical transmission to the host offspring by converting genetic male hosts into phenotypic females. Crustacea are the only invertebrates where sexual differentiation is controlled by a specialised endocrine organ, the androgenic gland, rather than by the gonads. We showed that a feminizing microsporidian Microsporidium sp. inhibits androgenic gland differentiation. We investigated the effect of Microsporidium sp. and a second feminizing microsporidium, Nosema granulosis, on the masculinizing function of the androgenic gland in Gammarus duebeni. Androgenic gland implants had a masculinizing effect on the sexual characteristics and sexual behaviour of recipient female hosts, reflecting the masculinizing function of the androgenic gland. Individuals that had received androgenic glands showed changed morphology in comparison with controls; they were bigger overall, they lost their oostegite marginal setae, developed calceoli and acquired a male-like behaviour. This effect was observed in uninfected females, as well as in females infected with the Microsporidium sp. The masculinizing effect of androgenic gland implants was smaller in N. granulosis infected individuals. N. granulosis and Microsporidium sp. fall into distinct clades of the Microspora. It appears that these divergent parasites both act by inhibiting the development of the androgenic gland. However, they differ in their ability to inhibit the host’s response to the hormone that controls male sexual differentiation. [Copyright &y& Elsevier]

We describe an intersex specimen of theMarmorkrebs, the only obligate parthenogenetic freshwater crayfish with an all-female population. The individual was a fully functional female which possessed male-like first pleopods. Nevertheless, it reproduced successfully and the offspring were normally developed parthenogenetic females, lacking any trace of male traits. The general rarity of aberrant sexual traits in freshwater crayfishes, in particular in Procambarus, is discussed. We suggest that a dysfunction of the sex determining system, which controls the anlagen of the androgenic glands during development, caused the partial male-like phenotype of this Marmorkrebs specimen. The application of this organism for investigations of sex determination and differentiation is recommended. [ABSTRACT FROM AUTHOR]

The current knowledge of the precise extension and location of the androgenic gland (AG) in the genera Litopenaeus and Rimapenaeus is partial. Therefore, we analyzed the complete structure of the AG and cellular cord associated with the reproductive system of Litopenaeus setiferus, L. occidentalis, L. stylirostris,and Rimapenaeus byrdi at the stereoscopic microscopy and histological level. Our observations clearly show that the adult male reproductive system has a cellular cord that runs along the entire vas deferens. This cord is a blood vessel that runs along the ampoule, the descending and the ascending medial vas deferens. Terminal ampoules of the four species investigated have a gland-like tissue associated with the blood vessel that is attached to the external surface near to the chamber containing sperm; this structure was identified as the AG and is formed by a homogeneous mass of cells characterized by a small size (7 µm), a cytoplasm with discrete cell boundaries, and an oval nucleus (4 µm). The blood vessel is surrounded by massive layers of adipose tissue at the distal region of the ascending medial vas deferens, near to the large lumen that contains sperm. This tissue has large cells (25-40 µm), with eosinophilic cytoplasms, and small nuclei (4-5 µm) in L. setiferus; L. stylirostris and L. occidentalis also show a cellular structure at the same location, identified as white adipose tissue. This anatomical description further improves the current knowledge of the general organization of the male reproductive system of Litopenaeus and Rimapenaeus. [ABSTRACT FROM AUTHOR]