Identification of Candidate Male-Reproduction-Related Genes from the Testis and Androgenic Gland of Macrobrachium nipponense, Regulated by PDHE1, through Transcriptome Profiling Analysis.

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.

CRISPR/Cas9-mediated gene mutation of EcIAG leads to sex reversal in the male ridgetail white prawn Exopalaemon carinicauda.

In the culture of crustaceans, most species show sexual dimorphism. Monosex culture is an effective approach to achieve high yield and economic value, especially for decapods of high value. Previous studies have developed some sex control strategies such as manual segregation, manipulation of male androgenic gland and knockdown of the male sexual differentiation switch gene encoding insulin-like androgenic gland hormone (IAG) in decapods. However, these methods could not generate hereditable changes. Genetic manipulation to achieve sex reversal individuals is absent up to now. In the present study, the gene encoding IAG (EcIAG) was identified in the ridgetail white prawn Exopalaemon carinicauda. Sequence analysis showed that EcIAG encoded conserved amino acid structure like IAGs in other decapod species. CRISPR/Cas9-mediated genome editing technology was used to knock out EcIAG. Two sgRNAs targeting the second exon of EcIAG were designed and microinjected into the prawn zygotes or the embryos at the first cleavage with commercial Cas9 protein. EcIAG in three genetic males was knocked out in both chromosome sets, which successfully generated sex reversal and phenotypic female characters. The results suggest that CRISPR/Cas9-mediated genome editing technology is an effective way to develop sex manipulation technology and contribute to monosex aquaculture in crustaceans.

Monosex Populations of the Giant Freshwater Prawn Macrobrachium rosenbergii-From a Pre-Molecular Start to the Next Generation Era.

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.

Isolation of an Insulin-Like Receptor Involved in the Testicular Development of the Mud Crab Scylla paramamosain.

Insulin-like androgenic gland hormone (IAG) is a key regulator of male sexual differentiation in crustaceans that plays important roles in secondary sexual characteristics and testicular development. As a hormone, IAG interacts with its membrane receptor to initiate downstream signal pathways to exert its biological functions. In this study, we isolated a full-length cDNA of an insulin-like receptor (Sp-IR) from the mud crab Scylla paramamosain. Sequence analysis revealed that this receptor consists of a Fu domain, two L domains, three FN-III domains, a transmembrane domain, and a tyrosine kinase domain, classifying it as a member of the tyrosine kinase insulin-like receptors family. Our results also suggested that Sp-IR was highly expressed in the testis and AG in males. Its expression in the testis peaked in stage I but significantly decreased in stages II and III (p < 0.01). Next, both short- and long-term RNA interference (RNAi) experiments were performed on males in stage I to explore Sp-IR function in mud crabs. The results showed that Sp-vasa and Sp-Dsx expression levels in the testis were significantly down-regulated after the specific knockdown of Sp-IR by RNAi. Additionally, the long-term knockdown of Sp-IR led to a considerable decrease in the volume of seminiferous tubules, accompanied by large vacuoles and a reduced production of secondary spermatocytes and spermatids. In conclusion, our results indicated that Sp-IR is involved in testicular development and plays a crucial role in transitioning from primary to secondary spermatocytes. This study provided a molecular basis for the subsequent analysis of the mechanism on male sexual differentiation in Brachyuran crabs.

IAG Regulates the Expression of Cytoskeletal Protein-Encoding Genes in Shrimp Testis.

Insulin-like androgenic gland hormone (IAG) is the master regulator of sexual differentiation and testis development in male crustaceans. However, the molecular mechanism on how IAG functions during testis development is still largely unknown. Here, the transcriptional changes were analyzed in the testes of shrimp after LvIAG knockdown in Litopenaeus vannamei. Differential expression analysis identified 111 differentially expressed genes (DEGs), including 48 upregulated DEGs and 63 downregulated DEGs, in testes of shrimp after LvIAG knockdown. Gene ontology (GO) analysis showed that these DEGs were apparently enriched in cytoskeleton-related GO items. Gene function analysis showed that genes enriched in these GO items mainly encoded actin, myosin, and heat shock protein. Interestingly, these genes were all downregulated in testis after LvIAG knockdown, which was confirmed by qRT-PCR detection. Furthermore, injection of LvIAG protein that was recombinantly expressed in insect cells upregulated the expression levels of these genes. The present study revealed that shrimp IAG might function in testis development through regulating the expression of cytoskeletal protein-encoding genes, which would provide new insights into understanding the functional mechanisms of IAG on male sexual development of crustaceans.

Transcriptomic Changes Following Induced De-Masculinisation of Australian Red Claw Crayfish Cherax quadricarinatus.

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.

RNA interference analysis of potential functions of cyclin A in the reproductive development of male oriental river prawns (Macrobrachium nipponense).

Cyclin A (CycA) plays essential roles in regulating multiple steps of the cell cycle, and it affects gonad development in mammals and invertebrates. Previous RNA interference (RNAi) analysis revealed that knocking-down the expression of CycA in female oriental river prawns (Macrobrachium nipponense) inhibited ovarian development. CycA was also predicted to have regulatory roles in reproductive development of male M. nipponense based on significant changes of Mn-CycA expression after eyestalk ablation. The goal of this study was to investigate the potential functions of CycA in the reproductive development of male M. nipponense using RNAi and histological observations. Quantitative real-time PCR analysis revealed that both single-side and double-side eyestalk ablation stimulated the expressions of Mn-CycA, and the expression was higher in prawns with double-side eyestalk ablation (p < 0.05). Mn-CycA expression was significantly higher in the testis and androgenic gland during the reproductive season than during the non-reproductive season (p < 0.05). In the RNAi analysis, Mn-CycA expression significantly decreased after prawns were injected with dsCycA, and the expression of insulin-like androgenic gland hormone (Mn-IAG) also decreased as Mn-CycA expression decreased. This result indicated that CycA positively regulated the expression of IAG in M. nipponense. Histological observations revealed that the number of sperm decreased dramatically to <5% of the total cells in the testis of the dsCycA-treated group compared to that of control group on day 14, indicating that knockdown of Mn-CycA expression inhibited testis development by affecting the expression of Mn-IAG in M. nipponense. These results highlighted the functions of CycA in male reproductive development of M. nipponense, which can be applied to future studies of male reproduction in other crustacean species.

RNA Interference Analysis of the Functions of Cyclin B in Male Reproductive Development of the Oriental River Prawn (Macrobrachium nipponense).

Cyclin B (CycB) plays essential roles in cell proliferation and promotes gonad development in many crustaceans. The goal of this study was to investigate the regulatory roles of this gene in the reproductive development of male oriental river prawns (Macrobrachium nipponense). A phylo-genetic tree analysis revealed that the protein sequence of Mn-CycB was most closely related to those of freshwater prawns, whereas the evolutionary distance from crabs was much longer. A quantitative PCR analysis showed that the expression of Mn-CycB was highest in the gonad of both male and female prawns compared to that in other tissues (p < 0.05), indicating that this gene may play essential roles in the regulation of both testis and ovary development in M. nipponense. In males, Mn-CycB expression in the testis and androgenic gland was higher during the reproductive season than during the non-reproductive season (p < 0.05), implying that CycB plays essential roles in the reproductive development of male M. nipponense. An RNA interference analysis revealed that the Mn-insulin-like androgenic gland hormone expression decreased as the Mn-CycB expression decreased, and that few sperm were detected 14 days after the dsCycB treatment, indicating that CycB positively affects testis development in M. nipponense. The results of this study highlight the functions of CycB in M. nipponense, and they can be applied to studies of male reproductive development in other crustacean species.

New insights for the regulatory feedback loop between type 1 crustacean female sex hormone (CFSH-1) and insulin-like androgenic gland hormone (IAG) in the Chinese mitten crab (Eriocheir sinensis).

To clarify the hormone control on sex determination and differentiation, we studied the Chinese mitten crab, Eriocheir sinensis (Henri Milne Edwards, 1854), a species with importantly economic and ecological significance. The crustacean female sex hormone (CFSH) and the insulin-like androgenic gland hormone (IAG) have been found to be related to the sex determination and/or differentiation. CFSH-1 of E. sinensis (EsCFSH-1) encoded a 227 amino-acid protein including a signal peptide, a CFSH-precursor-related peptide, and a mature CFSH peptide. Normally, EsCFSH-1 was highly expressed in the eyestalk ganglion of adult female crabs, while the expression was declined in the intersex crabs (genetic females). The intersex crabs had the androgenic glands, and the expression level of EsIAG was close to that of male crabs. During the embryogenesis and larval development, the changes of EsCFSH-1 and EsIAG genes expression in male and female individuals were shown after the zoea IV stage. Next, we confirmed the existence of the regulatory feedback loop between EsCFSH-1 and EsIAG by RNA interference experiment. The feminization function of EsCFSH-1 was further verified by examining the morphological change of external reproductive organs after EsCFSH-1 knockdown. The findings of this study reveal that the regulatory interplay between CFSH and IAG might play a pivotal role in the process of sex determination and/or differentiation in decapod crustaceans.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5'-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Chemical synthesis and functional evaluation of the crayfish insulin-like androgenic gland factor.

Insulin-like androgenic gland factor (IAG) from the marbled crayfish Procambarus virginalis is an insulin-like heterodimeric peptide composed of A and B chains and has an Asn-linked glycan at the B chain. IAG is considered to be a male sex hormone inducing the sex differentiation to male in decapod crustacean, although there is no report on the function of IAG peptide in vivo. In order to characterize P. virginalis IAG, we chemically synthesized it and evaluated its biological function in vivo. A and B chains were prepared by the ordinary solid-phase peptide synthesis, and three disulfide bonds were formed regioselectively by dimethyl sulfoxide oxidation, pyridylsulfenyl-directed thiolysis and iodine oxidation reactions. An IAG disulfide isomer was also prepared by the same manner. Circular dichroism spectral analysis revealed that the disulfide bond arrangement affected the peptide conformation, which was similar to the other insulin-family peptides analyzed so far. On the other hand, the glycan moiety attached at the B chain had no effect on the peptide secondary structure. Injection of the synthetic IAG and its disulfide isomer to female crayfish did not induce male characteristics on the external morphology, but both peptides suppressed the oocyte maturation in vivo. These results suggest that IAG has a pivotal role on the suppression of female secondary sex characteristics.

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation.

Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

Insulin-like androgenic gland hormone from the shrimp Fenneropenaeus merguiensis: Expression, gene organization and transcript variants.

Male sex differentiation in the crustacean is best known to be controlled by the insulin-like androgenic gland hormone (IAG). In this report, the cDNA and gene of the shrimp Fenneropenaeus merguiensis FmIAG were studied and characterized. FmIAG gene shares a high sequence identity in the coding region as well as the promoter region with that of F. chinensis. FmIAG gene is most likely consists of 5 exons and 4 introns. The cDNA reported here is the most abundant transcript that retained cryptic intron 4. The use of different splicing acceptor sites in exon 2 can produce a long-form FmIAG transcript variant with 6 additional amino acids inserted. Splicing of cryptic intron 4 would produce a transcript variant with a different C-terminal end. Therefore 4 different FmIAG transcripts can be produced from the FmIAG gene. During the molt cycle, the expression level of FmIAG was low in the early intermolt, increase steadily towards the late premolt and decreased rapidly in the early postmolt. In addition to the androgenic gland, FmIAG is also expressed in the hepatopancreas and ovary of adult females. Unilateral eyestalk ablation caused a significant increase in FmIAG transcript suggesting that the eyestalk consists of inhibiting factor(s) that suppressesFmIAGexpression. To explore the function of FmIAG in males, injection of FmIAG dsRNA knock-down the expression of FmIAG and up-regulated the expression of the vitellogenin gene in the testis and hepatopancreas. Interestingly a CHH-like gene identified in the androgenic gland was down-regulated. CHH-like gene knock-down resulted in altered expression of FmIAG in males suggesting that the CHH-like may be involved in FmIAG's regulation. RT-PCR with specific primers to the different transcript variant were used to determine if there is an association of different sizes of male and the type of IAG transcript. Results indicated that a high percentage of the large male shrimp expressed the long-form of FmIAG. The results suggested that FmIAG may be useful as a size marker for male shrimp aquaculture. In summary, the results of this study have expanded our knowledge of shrimp insulin-like androgenic gland hormone in male sex development and its potential role as a marker gene for growth regulation in shrimp.

The "IAG-Switch"-A Key Controlling Element in Decapod Crustacean Sex Differentiation.

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.

Molecular Characterization of the Insulin-Like Androgenic Gland Hormone in the Swimming Crab, Portunus trituberculatus, and Its Involvement in the Insulin Signaling System.

The insulin-like androgenic gland hormone (IAG) is mainly produced in the androgenic gland (AG) of the male crustaceans and is a crucial regulator in male sexual differentiation. In the current study, the full-length cDNA of IAG in the swimming crab, Portunus trituberculatus (Pt-IAG), was cloned and characterized. Similar to other reported IAGs, the deduced amino acid sequence of Pt-IAG consists of signal peptide, B chain, C peptide, and A chain, containing six conserved cysteines that form two interchain disulfide bonds and one intra-B chain disulfide bond. Tissue distribution analysis suggested that the Pt-IAG cDNA was highly expressed in the AG and was slightly expressed in several other tissues. A short-term silencing of PtIAG with double-stranded RNA was found to reduce the transcript levels of insulin receptor (Pt-IR) and insulin-like growth factor-binding protein (Pt-IGFBP), suggesting the Pt-IAG might perform its biological function through the insulin family-based signaling system. Bilateral eyestalk ablation (ESA) induced the expression of Pt-IAG in the AG at 4 and 7 days after surgery, while the transcript levels of Pt-IR in the AG and testis and Pt-IGFBP in the muscle, testis, and thoracalia ganglia were significantly decreased from 1 day after surgery. The results suggested that the Pt-IR and Pt-IGFBP might also be the targets of eyestalk neuropeptides and responded to the ESA independent of IAG regulation.

Insight into the Regulatory Relationships between the Insulin-Like Androgenic Gland Hormone Gene and the Insulin-Like Androgenic Gland Hormone-binding Protein Gene in Giant Freshwater Prawns (Macrobrachium rosenbergii).

Giant freshwater prawns (Macrobrachium rosenbergii) are commonly found throughout the world. The size of the male giant freshwater prawn is much larger than that of the female. Therefore, understanding the molecular mechanism that underlies the sexual differentiation of M. rosenbergii is of both commercial and scientific importance. Insulin-like androgenic gland hormone (IAG) plays a key role in the differentiation of sex in M. rosenbergii. Although IAG has been investigated, the regulatory relationship between IAG and its binding protein partner, the insulin-like androgenic gland hormone-binding protein (IAGBP), has not been studied in M. rosenbergii. Here, we cloned and characterized the IAGBP from M. rosenbergii (Mr-IAGBP) for the very first time. Transcriptomic analysis showed that Mr-IAGBP mRNA was detected in a wide array of tissues with the highest expression found in the androgenic gland. The importance of IAG in male development was further demonstrated by an increase in IAG transcripts during the development of the androgenic gland and Mr-IAG was only highly transcribed in the androgenic gland of M. rosenbergii. Interestingly, we found that the Mr-IAG gene expression started during the 20th-day larva after hatching stage (LH20), followed (20th-day post-larval stage, PL20) by a gradual elevation of Mr-IAGBP levels. The levels of both genes peaked at the adult stage. The relationship between Mr-IAGBP and Mr-IAG was further analyzed using RNA interference. The injection of Mr-IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of Mr-IAG, while the amount of Mr-IAGBP mRNA and the translation of IAGBP protein was significantly reduced by the injection of Mr-IAG dsRNA. These results revealed that IAGBP is involved in IAG signaling. Furthermore, our data supports the hypothesis that (IAG and IAGBP)-IAG receptor signaling schemes exist in M. rosenbergii. Our results will provide important information for the further study of determining the sex of M. rosenbergii.

siRNA-Mediated MrIAG Silencing Induces Sex Reversal in Macrobrachium rosenbergii.

The insulin-like androgenic gland (IAG) gene is well known in male crustacean, and it is a key regulator in male sexual differentiation and maintaining the male sexual characteristic. The neo-female can be produced by silencing the MrIAG (Macrobrachium rosenbergii Insulin-like Androgenic Gland) in male Macrobrachium rosenbergii. This is the first time to use siRNA approach to silenced MrIAG in male M. rosenbergii. In the current study, the optimal injection dosage to achieve sex reversal is 0.5 µg/g body weight. After MrIAG silencing, the expression level of Dmrt11e, Dmrt99b, MRPINK, Mrr, Sxl1, and Sxl2 decreased significantly. As their long-term silencing effect of MrIAG, the dsRNA and siRNA approaches produce three and two individual neo-females, respectively. The neo-female has a wider brood chamber, ovipositing setae, and ovigerous setae, which is resembled normal female. After a long-term silencing with siRNA, most of the germ cells were arrested in spermatocytes stage, but the spermatocytes in control can further developed into spermatozoon. The seminiferous tubules are loosely arranged and the spermatocytes are more than spermatozoon in the 0.5 µg/g body weight treatment dose. This current study suggests a new path to obtain neo-females through siRNA silencing.

Molecular characteristics and abundance of insulin-like androgenic gland hormone and effects of RNA interference in Eriocheir sinensis.

Insulin-like androgenic gland hormone (IAG) has an important function in sexual differentiation and somatic growth in crustaceans. In this study, there was cloning of the full-length sequence of IAG from Eriocheir sinensis (Es-IAG). The full-length Es-IAG gene was 1392 base pairs long and encoded a protein of 151 amino acid residues. The precursor peptide included a signal peptide, and the protein was a protein that is secreted from the cell in which it is produced with no transmembrane domain. Amino acid sequence alignment indicated there was the greatest homology between E. sinensis and Chaceon quinquedens (47 %), followed by Callinectes sapidus (44 %). Results from analysis of the relative abundances of Es-IAG mRNA transcript at different developmental stages indicated that Es-IAG may have an important endocrine function in early embryonic development, and that Stages I through Ⅲ may be an important period for sexual differentiation in juvenile E. sinensis. In vivo treatment with siRNA-391 resulted in a 66.7 % lesser relative abundance of the Es-IAG mRNA transcript. Three treatments with siRNA-391 to inhibit Es-IAG production during Stages Ⅲ to Ⅴ period resulted in about 10 % of male crabs being transformed into "neo-females." These results provide the basis for further research into the sexual differentiation mechanism and monosex breeding of E. sinensis.

Co-culture of males with late premolt to early postmolt female giant freshwater prawns, Macrobrachium rosenbergii resulted in greater abundances of insulin-like androgenic gland hormone and gonad maturation in male prawns as a result of olfactory receptors.

Insulin-like androgenic gland hormone (IAG) controls development of primary and secondary male sex-characteristics in decapod crustaceans. In male giant freshwater prawns, Macrobrachium rosenbergii, the IAG concentration correlates with male reproductive status and aggressiveness. When female prawns are co-cultured with males this can result in male size variations while this variation does not occur when males are cultured in monosex conditions. It was hypothesized that pheromone-like factors from female prawns may affect the abundance of IAG mRNA and protein in co-cultured males which would affect the pattern of sexual maturation of these males. In the present study, late premolt to postmolt females co-cultured with males for 7 days had a greater abundance of MrIAG mRNA transcript in all male phenotypes as well as for the gonad-somatic indexes (GSI). The abundance of MrIAG mRNA gradually increased from days 1 to 7 and using Western blot procedures MrIAG protein also increased in a similar pattern. Furthermore, with use of BrdU labeling, there was an increased cell proliferation in the spermatogenic zone of testicular tubules and in the spermatic duct epithelium during the 1 to 7 day co-culture period when there were increases in MrIAG mRNA and protein. In contrast, these effects were negated if short lateral antennules of males were ablated. Thus, results of the present study provide evidence that there might be female-molting factors which function as important regulators of androgenic gland function and gonadal maturation that were perceived by males via their short lateral antennules which are the olfactory organs.

Chemical synthesis of N-glycosylated insulin-like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii.

Crustacean insulin-like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N-linked glycan, was synthesized by the combination of solid-phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N-linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin-like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four-disulfide insulin-like peptides.