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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.)

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 .

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.

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.

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., (Copyright © 2020 Levy and Sagi.)

The effect of glyphosate, both pure and formulated (Roundup Ultramax®), was evaluated on males of the estuarine crab Neohelice granulata, by means of both in vivo and in vitro assays. The in vivo assays comprised the exposure for 30 d to 1 mg/L of the herbicide, until finally assessing weight gain, levels of energy reserves, sperm number per spermatophore, proportion of abnormal spermatophores, and sperm viability. At the end of this assay, significant (p < 0.05) decrease in weight gain and muscle protein levels was detected by effect of both pure and formulated glyphosate. In spermatophores from the vas deferens, a significant (p < 0.05) decrease of the sperm count was observed by effect of Roundup, while a significant incidence (p < 0.05) of abnormal spermatophores was observed either with glyphosate or with Roundup treatment. No changes were seen in the spermatophore area or in vas deferens secretions. Since no sperm mortality was induced by the formulated herbicide, we propose a probable inhibiting effect on spermatogenesis might explain the observed sperm count decrease. In this sense, an in vitro assay was designed by incubating testes and vasa deferentia with Roundup, in order to corroborate the possible interference of glyphosate with the secretion of the androgenic gland hormone that controls the spermatogenesis, in the presence or absence of the androgenic gland. Although the herbicide per se was able to reduce the sperm count to some extent, the increase in the number of spermatozoa/spermatophore produced by the co-incubation with the androgenic gland was completely reverted by the addition of Roundup (1 mg/L of glyphosate a.e.), suggesting that an inhibition on the secretion and/or transduction of the androgenic gland hormone could be taking place., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

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.

The androgenic gland in malacostracan crustacean species produces and secretes androgenic gland hormone, which is responsible for male sexual differentiation, such as the induction and development of male sexual traits, and in turn the suppression of female sexual traits. Marmorkrebs, Procambarus fallax forma virginalis, which was identified as the first parthenogenetic species in decapod crustaceans, produces only female offspring. In this study, in order to reveal whether the Marmorkrebs crayfish is sensitive to androgenic gland hormone, we transplanted an androgenic gland from a related congener, P. clarkii, to P. fallax f. virginalis. In androgenic gland-implanted specimens, partial masculinization was confirmed: the masculinization of several external sexual characteristics (i.e., thickening of the first and second pleopods; formation of reverse spines on the third and fourth pereopods) was detected, whereas that of internal sexual characteristics (e.g., the formation of ovotestes and male gonoducts) was not. Our results imply that P. fallaxf. virginalis still has sensitivity to the androgenic gland hormone and, at least partly, the hormone should be able to induce male characteristics, even in parthenogenetic Marmorkrebs.

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., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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 © 2014 Elsevier B.V. All rights reserved.)

Neurotransmitters and neurohormones are agents that control gonad maturation in decapod crustaceans. Of these, serotonin (5-HT) and dopamine (DA) are neurotransmitters with known antagonist roles in female reproduction, whilst gonadotropin-releasing hormones (GnRHs) and corazonin (Crz) are neurohormones that exercise both positive and negative controls in some invertebrates. However, the effects of these agents on the androgenic gland (AG), which controls testicular maturation and male sex development in decapods, via insulin-like androgenic gland hormone (IAG), are unknown. Therefore, we set out to assay the effects of 5-HT, DA, l-GnRH-III, oct-GnRH and Crz, on the AG of small male Macrobrachium rosenbergii (Mr), using histological studies, a BrdU proliferative cell assay, immunofluorescence of Mr-IAG, and ELISA of Mr-IAG. The results showed stimulatory effects by 5-HT and l-GnRH-III through significant increases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). In contrast, DA and Crz caused inhibitory effects on the AG through significant decreases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). Moreover, the prawns treated with Crz died before day 16 of the experimental period. We propose that 5-HT and certain GnRHs can be now used to stimulate reproduction in male M. rosenbergii, as they induce increases in AG and testicular size, IAG production, and spermatogenesis. The mechanisms by which these occur are part of our on-going research., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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

Male Procambarus clarkii were matched by size and largest claw length and observed interacting in pair matches before and after removal of the androgenic gland or a sham operation. Although results were not significant, trends suggested that males showed less aggression after the removal of the androgenic gland. Average bout duration did not decrease, but mean intensity of interaction decreased. This exercise was part of a student lab for non-majors. Students were positive about the lab, indicating that they learned about quantifying behavior, about hormonal involvement in aggression, and that the lab made them want to do more science.