Androgenic Gland Research Articles

Ocean acidification influences plant-animal interactions: The effect of Cocconeis scutellum parva on the sex reversal of Hippolyte inermis.

Ocean acidification (O.A.) influences the ecology of oceans and it may impact plant-animal interactions at various levels. Seagrass meadows located at acidified vents in the Bay of Naples (Italy) are considered an open window to forecast the effects of global-changes on aquatic communities. Epiphytic diatoms of the genus Cocconeis are abundant in seagrass meadows, including acidified environments, where they play key ecological roles. A still-unknown apoptogenic compound produced by Cocconeis triggers the suicide of the androgenic gland of Hippolyte inermis Leach 1816, a protandric hermaphroditic shrimp distributed in P. oceanica meadows located both at normal pH and in acidified vents. Feeding on Cocconeis sp. was proven important for the stability of the shrimp’s natural populations. Since O.A. affects the physiology of diatoms, we investigated if, in future scenarios of O.A., Cocconeis scutellum parva will still produce an effect on shrimp’s physiology. Cell densities of Cocconeis scutellum parva cultivated in custom-designed photobioreactors at two pH conditions (pH 7.7 and 8.2) were compared. In addition, we determined the effects of the ingestion of diatoms on the process of sex reversal of H. inermis and we calculated the % female on the total of mature individuals-1 (F/mat). We observed significant differences in cell densities of C. scutellum parva at the two pH conditions. In fact, the highest cell densities (148,808 ±13,935 cells. mm-2) was obtained at day 13 (pH 7.7) and it is higher than the highest cell densities (38,066 (±4,166) cells. mm-2, day 13) produced at pH 8.2. Diatoms cultured at acidified conditions changed their metabolism. In fact, diatoms grown in acidified conditions produced in H. inermis a proportion of females (F/mat 36.3 ±5.9%) significantly lower than diatoms produced at normal pH (68.5 ±2.8), and it was not significantly different from that elicited by negative controls (31.7 ±5.6%).

Screening for Dmrt genes from embryo to mature Macrobrachium rosenbergii prawns

The doublesex and mab-3 related transcription factor (Dmrt) gene family is known to be related to the sexual regulators doublesex of arthropods and mab-3 of annelids and to hold highly conserved functions in sexual determination and differentiation across phyla. Here, we report a study of the Dmrt gene family in the freshwater prawn Macrobrachium rosenbergii, a crustacean whose sexual differentiation has been widely researched. A wide transcriptomic screen, from the embryo to the adult M. rosenbergii, identified five novel Dmrt genes (MroDmrts) and confirmed two known MroDmrts. The seven MroDmrts encode proteins of 275–855 amino acids; each protein contained at least one conserved DNA-binding DM domain, which is typical of Dmrt proteins, and five proteins contained 1–4 transactivation domains (TADs). Importantly, in the embryonic, larval and post-larval stages, MroDmrt genes exhibited time-dependent expression patterns rather than sex-specific expression. In-silico screening of the expression of the MroDmrt genes in adult males revealed the enrichment of MroiDmrt1b and MroiDmrt1c in the androgenic gland (AG) as compared to the eyestalks. In vivo silencing of the androgenic gland insulin-like (IAG) encoding gene significantly decreased the expression of the above two Dmrt genes, while not affecting the expression of control genes, thereby suggesting the possible role of these two genes in the IAG-switch and in sex-differentiation processes.

A Simple Genome Walking Strategy to Isolate Unknown Genomic Regions Using Long Primer and RAPD Primer.

BackgroundGenome walking is a DNA-cloning methodology that is used to isolate unknown genomic regions adjacent to known sequences. However, the existing genome-walking methods have their own limitations.ObjectivesOur aim was to provide a simple and efficient genome-walking technology.Material and MethodsIn this paper, we developed a novel PCR strategy (termed SLRA PCR) that uses a single long primer (SLP), a set of gene specific primers (GSP), and a random amplified polymorphic DNA (RAPD) primer for genome walking. SLRA PCR consists of two processes: the first amplification using SLP, and three successive rounds of nested PCR amplified by GSP and RAPD primer. The novelty of the approach lies in the use of long primers (SLP and GSP) and same annealing and extension temperature 68℃ in combination. This method offers higher amplification efficiency, superior versatility, and greater simplicity compared with conventional randomly primed PCR methods for genome walking.ResultsThe promoter regions and the first introns of the insulin-like androgenic gland hormone (IAG) gene and the hemocyanin gene of Macrobrachium nipponense were cloned using SLRA PCR, respectively.ConclusionsThis genome walking strategy can be applied to a wide range of genomes.

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense.

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.

The androgenic gland in male morphotypes of the Amazon River prawn Macrobrachium amazonicum (Heller, 1862)

Sexual differentiation and primary and secondary sexual characteristics in male crustaceans are modulated by hormones produced in the androgenic gland (AG). The AG is also responsible for the determination of morphotypes in caridean shrimps, such as Macrobrachium amazonicum that shows four morphotypes: translucent claw (TC), cinnamon claw (CC), green claw 1 (GC1) and green claw 2 (GC2). Here, we verified the anatomical, histological and ultrastructural characteristics of the AG in different morphotypes of this species with both amphidromous and hololimnetic life cycles. In submissive morphotypes (TC and CC), the AGs are reduced and concentrated in the terminal expansion of the distal portion of vasa deferentia (DVD), the ejaculatory ducts (ED). In dominant morphotypes (GC1 and GC2) these glands lie along the DVD and ED. Two morphological stages (I and II) were recorded for AG cells. In submissive morphotypes stage I cells predominated in the AGs, while in dominant morphotypes stage II cells were more common. AG cells in both stages were positive for proteins, confirming the protein nature of the secreted hormone. Stage I cells have abundant rough endoplasmic reticulum (RER) with numerous parallel cisternae, whereas in stage II cells, the cisternae of RER are highly dilated. Stage II cells do not produce secretory granules, but they undergo hypertrophy and the hormone release to hemolymph probably occurs by holocrine secretion. The AGs in TC, GC1 and GC2 morphotypes increase as the animals grow and are larger in GC1 males. On the other hand, AGs decrease in the CC morphotype as the animal grows. These differences are related to the type of reproductive strategy adopted by each morphotype. In M. amazonicum, the AGs show the same morphological, histochemical and ultrastructural patterns between the different life history populations.

Primary molecular basis of androgenic gland endocrine sex regulation revealed by transcriptome analysis in Eriocheir sinensis

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.

Identification of an insulin‐like androgenic gland hormone transcript highly expressed in male precociousness of the Chinese mitten crab Eriocheir sinensis

Identification of an insulin‐like androgenic gland hormone transcript highly expressed in male precociousness of the Chinese mitten crab <i>Eriocheir sinensis</i>

IGFBP-rP1, a strongly conserved member of the androgenic hormone signalling pathway in Isopoda

The first protein which has been described to interact with the malacostracan Androgenic Gland Hormone (AGH) is a binding protein called IGFBP-rP1. It has been identified and studied in several species of decapods, in which its interaction with the masculinizing hormone and its expression patterns have been established in several ways. However, this protein remains uncharacterised to date in the other malacostracan orders, like Amphipoda and Isopoda, although they were historically the first ones in which the androgenic gland and the corresponding hormone were respectively described. In this article, we identified the IGFBP-rP1 of isopods and established its implication in the pathway of the AGH with a silencing approach in the model species Armadillidium vulgare. We also showed that this gene is expressed in all the tissues of males and females, with a similar pattern in animals infected with Wolbachia, a feminizing endosymbiont of several isopod species. The expression pattern did not differ during the development of uninfected and infected animals either. We finally studied the evolution of the IGFBP-rP1 in 68 isopod species, looking for conserved motifs and evidence of natural selection. Altogether, our results showed that this gene is constitutively expressed and strongly conserved in isopods, in which it likely constitutes a key element of the insulin/IGF signalling pathway. However, we also illustrated that IGFBP-rP1 is not sufficient on its own to explain the different developmental paths taken by the males and the females or feminized genetic males.

Chemical synthesis of the crustacean insulin-like peptide with four disulfide bonds.

Among the insulin-family peptides, two additional cysteine residues other than six conserved cysteines are sometimes found in invertebrate insulin-like peptides (ILPs), although the synthetic method for such four disulfide ILPs has not yet been well established. In this study, we synthesized a crustacean insulin-like androgenic gland factor with four disulfides by the regioselective disulfide bond formation reactions using four orthogonal Cys-protecting groups. Its disulfide isomer could be also synthesized by the same method, indicating that the synthetic strategy developed in this study might be useful for the synthesis of other four disulfide ILPs.

Divergent evolution and clade-specific duplications of the Insulin-like Receptor in malacostracan crustaceans

The Insulin-like Receptors (IRs) are an important protein family, represented by three members in vertebrates, two of which are well-known for their implication in metabolism (Insulin Receptor) and growth (IGF Receptor). In contrast, little is known about these receptors in invertebrates, in which a single gene generally exists except for a part of insects and other occasional species-specific duplications. In this study, we used publicly available sequences as well as de novo assembled transcriptomes to investigate the IR evolution in malacostracan crustaceans, animals in which the Insulin/IGF pathway is known to be implicated in sexual development through the androgenic gland hormone. We described the evolutionary divergence of malacostracan IRs compared to all the other metazoan sequences, including other pancrustaceans. We also demonstrated two well conserved duplications of IRs: one specific to the whole malacostracan class, another one specific to the decapod order. The potential implications for malacostracan biology are discussed.

Crustacean Female Sex Hormone From the Mud Crab Scylla paramamosain Is Highly Expressed in Prepubertal Males and Inhibits the Development of Androgenic Gland.

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.

Isolation and characterization of partial insulin like androgenic gland hormone gene from distal terminal ampoules of three marine shrimp Penaeus species from the Eastern Pacific

The expansion of the shrimp industry depends on scientific advances related to the understanding of reproductive endocrinology that allows the development of aquaculture programs based on biotechnology. An insulin like-androgenic gland hormone (IAG) is synthesized in the male-specific androgenic gland (AG) and it is responsible for sex differentiation in crustaceans. In this study, RNA was extracted from different tissues; amplified cDNA was obtained by reverse transcription-PCR. Nucleotide sequences were analyzed following a bioinformatic approach of the encoding insulin-like androgenic gland hormone gene. This contribution presents the isolation of the partial cDNA encoding IAG from Penaeus vannamei (Lv-IAG), Penaeus occidentalis (LoIAG) and Penaeus stylirostris (Ls-IAG) from the distal region of terminal ampoules. In addition, the molecular characterization of these sequences is provided and the resulting aminoacid sequences were used to establish the taxonomic positioning of the IAG gene among other Decapoda species. The isolation of these partial sequences provides evidence about the anatomical location of the AG in Penaeus and is the basis for further characterize the complete cDNA of the AG-specific insulin-like encoding transcript for all species of the genus Penaeus from the Pacific coast of America.

De novo transcriptome analysis and differentially expressed genes in the ovary and testis of the Japanese mantis shrimp Oratosquilla oratoria by RNA-Seq

The mantis shrimp Oratosquilla oratoria is a widely distributed, commercially important crustacean species. Although its conservation and the development of successful artificial breeding technologies have recently received considerable attention, there are currently no available data regarding the molecular mechanisms in controlling reproduction. In this study, we performed transcriptome sequencing of the testis, ovary, female and male eyestalks and the androgenic gland of O. oratoria, and compared the expression pattern of transcripts from the testis and ovary libraries to identify genes involved in gonadal development. A total of 147,130,937 clean reads were retrieved after removing the adapters in reads and filtering out low-quality data. All the reads were assembled into 94,990 unigenes (23,133 in testis and ovary) with an average length of 783 base pairs (bp) and N50 of 1502 bp. A search of all-unigenes against COG, GO, KEGG, KOG, Pfam, Swiss-Prot and Nr databases resulted in a total of 19,404 annotated unigenes. Comparison of the sequences in the ovary and testis libraries revealed that 1188 unigenes were up-regulated in the ovary and 2732 were up-regulated in the testis. Twenty ovary-up-regulated and 21 testis-up-regulated unigenes were confirmed by quantitative real-time PCR. Additionally, 13,437 simple sequence repeats (SSRs) and 275,799 putative single nucleotide polymorphisms (SNPs) were identified. The important functional genes and pathways identified here provide a valuable dataset for understanding the molecular mechanisms controlling gonad development in O. oratoria, and the numerous (13,437 SSRs and 275,799 SNPs) molecular markers obtained here will provide fundamental basis for functional genomic and population genetic studies of O. oratoria.

A Putative Insulin-like Androgenic Gland Hormone Receptor Gene Specifically Expressed in Male Chinese Shrimp.

The insulin-like androgenic gland hormone (IAG) is the key regulator in crustacean male sexual differentiation. As a secreted peptide hormone, IAG might perform its biological function through interacting with the membrane receptor. However, the receptor of IAG remains unclear. In the current study, a putative IAG receptor gene (FcIAGR) was identified in Fenneropenaeus chinensis. The deduced amino acid sequence of FcIAGR contained several conserved domains of insulin-like receptor proteins, including two L domains (L1 and L2), a cysteine-rich domain, three fibronectin III domains, a transmembrane domain, and an intracellular tyrosine kinase domain. Tissue distribution and in situ hybridization analysis showed that FcIAGR was predominantly expressed in the androgenic gland and testis in male F. chinensis. Protein colocalization analysis in HEK293 cells showed that FcIAGR could colocalize with both FcIAG1 and FcIAG2, respectively. Yeast two-hybrid assay further confirmed the interactions between FcIAGR and FcIAGs. After a long-term silencing of FcIAGR with double-stranded RNA, most of the germ cells in the testis were arrested at the secondary spermatocytes, whereas those in the control developed into sperm cells. The data indicated that FcIAGR was the receptor of FcIAGs in F. chinensis. The current study provides insight into the mechanism that the insulin-like signaling pathway regulates the male sexual differentiation in Decapoda crustaceans.

ITRAQ-based quantitative proteomic analysis of the androgenic glands of the oriental river prawn, Macrobrachium nipponense, during nonreproductive and reproductive seasons

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.

Identification and characterization of a doublesex gene which regulates the expression of insulin-like androgenic gland hormone in Fenneropenaeus chinensis

The doublesex and its homologue genes are important regulators of sexual differentiation which are conserved among animal kingdom. In the present study, we reported a doublesex gene (designated as FcDsx) identified from the Chinese shrimp F. chinensis. The gene structure, nucleotide and deduced amino acid sequences of FcDsx were characterized. The results showed that the deduced amino acid sequence of FcDsx had the common features of Dsx proteins, including a doublesex/male abnormal 3 (DM) domain, an oligomerization domain and a predicted monopartite nuclear localization signal. The expression patterns of FcDsx in different tissues and developmental stages were detected. FcDsx exhibited a sex-biased expression patterns in different tissues and its expression level increased along with developmental stages. In addition, its regulation on the expression of FcIAG, a gene important for sexual differentiation of male crustacean, was also analyzed. Putative Dsx binding site was identified on the promoter region of FcIAG and knockdown of FcDsx could reduce the expression of FcIAG, which suggested that FcDsx might be the upstream regulator of FcIAG. The present data indicated that FcDsx gene might involve in shrimp sexual differentiation process.

The gene encoding the insulin-like androgenic gland hormone in an all-female parthenogenetic crayfish.

Male sexual differentiation in crustaceans is controlled by the androgenic gland (AG), a unique male endocrine organ that, in decapods, is located at the base of the 5th pereiopod. In these animals, the insulin-like androgenic gland hormone (IAG) is the major factor secreted from the AG to induce masculinization and maintain male characteristics. It has, however, recently been proposed that this hormone also plays a role in growth and ovarian development in females. In this study, we tested such a possibility by searching for the IAG gene in the marbled crayfish, a parthenogenetic animal that reproduces asexually to form an all-female genetic clone. Based on the phylogenetic relationship between the marbled crayfish and Procambarus fallax, a gonochoristic species of the same North American Cambaridae family, we searched for the IAG gene in the marbled crayfish and then fully sequenced it. The open reading frame of the gene was found to be completely identical in the two species, and their introns shared over 94% identity. It was also found that, in addition to its expression at the base of the 5th pereiopod and in the testes of male P. fallax crayfish, IAG was expressed in the muscle tissue of P. fallax males and females and even of the parthenogenetic marbled crayfish. These findings provide new insight into possible functions of IAG, in addition to its role as a masculinization-inducing factor, and also constitute the basis for a discussion of the evolutionary relationship between the above two species.

Histological characterization of peppermint shrimp (Lysmata wurdemanni) androgenic gland

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.

Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway

ABSTRACTRNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn Macrobrachium rosenbergii by knocking down the transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA of the entire Mr-IAG ORF sequence (dsMr-IAG – 518bp). Interestingly, in-vivo knockdown success and dsMr-IAG lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in M. rosenbergii. We discovered the Mr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in our M. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1 gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing Mr-SID1 specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments in-vitro, the case presented here demonstrates length dependency in-vivo, suggesting further complexity in the context of the entire organism.

Integrated analysis of microRNA and mRNA expression profiles during the sex-differentiation sensitive period in oriental river prawn, Macrobrachium nipponense

Male oriental river prawns (Macrobrachium nipponense) grow faster than females, and therefore, reach larger sizes by harvest time. Histological observations have indicated that the sex-differentiation sensitive period (which includes the formation of the androgenic gland, the testis, and the ovary) is from post-larvae (PL) developmental stage for M. nipponense. In this study, we prepared four microRNA (miRNA) and mRNA libraries using samples collected from sex-differentiation sensitive period (PL7 to PL16) to perform RNA-sequencing for identifying sex-related candidate miRNAs, genes, and metabolic pathways. A total of nine intersection miRNAs were identified, of which three were highly expressed in the androgenic gland, and their expression was verified by quantitative Real-Time PCR (qPCR). These three miRNAs and their 11 predicted target genes may be strong candidates for sex-related miRNAs and sex-related genes in M. nipponense. Five vital sex-related metabolic pathways were also identified that may regulate other sex-differentiation and sex-determination mechanisms. Finding of the study provide important insights to enhance our understanding on sex-differentiation and sex-determination mechanisms for M. nipponense.