The Regulatory Relationships Between the Gonad-Inhibiting Hormone and Insulin-Like Androgenic Gland Hormone-Binding Protein Genes in the Eyestalk-Androgenic Gland-Testis Axis of Macrobrachium rosenbergii

Gonad-inhibiting hormone (GIH) belongs to a family of neuropeptides that are released from the eyestalks of male crustaceans and plays key roles in gonadal maturity, reproduction, and molting. However, the detailed mechanisms underlying the effects of GIH on sexual regulation have yet to be elucidated. In the present study, we aimed to demonstrate how GIH mediate the activity of the androgenic gland (AG) to affect sexual regulation. To do this, we cloned and characterized a GIH sequence from Macrobrachium rosenbergii (MrGIH). The open reading frame (ORF) of MrGIH was 360 bp and codes for a polypeptide of 119 amino acids and a putative protein of 13.56 KDa. Tissue analysis showed that MrGIH is widely expressed in a range of tissues but particularly, the eyestalk, intestine, and nerve cord. Following the dsRNA silencing of MrGIH for 24 h, the expression levels of MrGIH were down-regulated in both the eyestalk and AG when compared with the negative control, but significantly increased the expression of Macrobrachium rosenbergii insulin-like androgenic gland hormone-binding protein (MrIAGBP) in AG, thus suggesting that MrGIH is an inhibitory factor for MrIAGBP. In addition, we found that eyestalk removal on certain days led to increased expression levels of MrIAGBP expression. The expression levels of MrIAGBP peaked at 2 d in the AG after unilateral and bilateral eyestalk ablation, exhibiting a 7.27- and 6.03-fold increase, respectively. Afterward, the expression of GIH protein levels were down-regulated and IAGBP protein levels were up-regulated after GIH silencing using immunohistochemistry method, combined with the increase of IAGBP protein levels after eyestalk ablation, confirming that MrGIH is an inhibitory factor that can moderately regulate AG development and IAGBP expression. Collectively, our findings enriched the mechanisms that control the sexual regulation pathway of male M. rosenbergii, and provided significant information for further explorations of the mechanism underlying sex regulation in other decapod crustaceans.

Lentiviral-Transduced Ectopic Expression of Androgenic Hormone in a Crustacean Hematopoietic Primary Cell Culture

Novel monosex biotechnologies in crustacean aquaculture involve the induction of sex reversal through manipulations of the androgenic gland (AG), and its most prominent factor, the insulin-like androgenic gland (IAG) hormone, during early developmental stages. In the giant freshwater prawn Macrobrachium rosenbergii, all-female populations are desirable for aquaculture, since the females can be stocked at higher densities and exhibit more uniform growth than males. Recently, a novel biotechnology for all-female aquaculture was developed based on injection into post-larvae of suspended primary AG cells from mature M. rosenbergii males. However, this biotechnology depends on the availability of appropriate male donors and it requires delicate surgical procedures on the small endocrine AG to produce appropriate quantities of cells for the sex manipulation. We therefore established a new platform for the production of M. rosenbergii IAG (Mr-IAG) in hematopoietic cells. The method rests on the induction of ectopic Mr-IAG expression under the white spot syndrome virus (WSSV) immediate-early 1 (IE1) promoter via a novel lentiviral vector. Our results demonstrate that M. rosenbergii primary cells infected with the Mr-IAG lentiviruses are capable of transcription, translation, and secretion of Mr-IAG in culture. Our new platform, which produces easy-to-harvest cells in abundance, could replace the AG cells used in the first step of the above-mentioned biotechnology for all-female aquaculture and, importantly, pave the way for producing monosex populations in other edible crustacean aquaculture species. In addition, a lentiviral system for crustacean cells provides a useful tool for basic and applied research in crustacean species.

Identification of candidate genes from androgenic gland in Macrobrachium nipponense regulated by eyestalk ablation

The eyestalk of crustaceans, such as Macrobrachium nipponense, contains many neurosecretory hormones affecting the process of reproduction, molting, metabolism of glucose, and other functions. In this study, important metabolic pathways and candidate genes involved in male sexual development were selected from M. nipponense. The methodology involved performing long-read and next generation transcriptome sequencing of genes from the androgenic gland after eyestalk ablation. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation of both the single-side (SS) and double-side (DS) eyestalk, compared with the control group (CG). The long-read transcriptome generated 49,840 non-redundant transcripts. A total of 1319, 2092 and 4351 differentially expressed genes (DEGs) were identified between CG versus SS, SS versus DS and CG versus DS, respectively. These data indicated that ablation of the double-sided eyestalk played stronger regulatory roles than the single-side ablation on male sexual development in M. nipponense. This was consistent with the qPCR analysis. Cell Cycle, Cellular Senescence, Oxidative Phosphorylation, Glycolysis/Gluconeogenesis and Steroid Hormone Biosynthesis were the primary enriched metabolic pathways in all three comparisons, and the important genes from these metabolic pathways were also selected. qPCR permitted secondary confirmation of ten DEGs identified through RNA-seq. RNAi-mediated silencing analyses of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has a positive regulatory effect on testes development. This study provides valuable insight into male sexual development in M. nipponense, including metabolic pathways and genes, paving the way for advanced studies on male sexual development in this species and in other crustaceans.

Transcriptome Analysis Reveals the Endocrine Regulation on the Expression of IAG in Litopenaeus vannamei

The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation and spermatogenesis in crustaceans. The expression of IAG is usually negatively regulated by neuropeptide hormones through the “eyestalk-AG” endocrine axis. However, the underlying mechanism is still largely unknown. In the present study, we performed a comparative transcriptome analysis on the androgenic gland (AG), the main secretory organ of IAG, of L. vannamei before and after unilateral eyestalk ablation. A total of 67 differentially expressed genes (DEGs) were identified, including some putative genes involved in sexual development. Interestingly, several genes related to molting and endocrine processes were found differentially expressed between the two treatments. Further investigation on the expression profiles of these genes and Lv-IAG showed that their expression patterns were closely correlated throughout the molting cycle. In addition, injection of 20-hydroxyecdysone (20E) could inhibit the expression of Lv-IAG at different molting stages in a dosage-dependent manner. The data indicated that ecdysteroids played important roles in regulating the Lv-IAG expression and might bridge the endocrine axis between eyestalk and AG. The present study provided new insights into understanding the regulation of sexual development in male crustacean.

The Regulatory Mechanism of Sexual Development in Decapod Crustaceans

Crustacean culture has been developing rapidly in various parts of the world. Therefore, it is important to understand their reproductive biology. Insulin-like androgenic gland hormone (IAG) secreted from the androgenic gland (AG) is widely accepted as a key regulator of sexual differentiation in male crustaceans. However, recently several sex-related genes (i.e., CFSH, DEAD-box family, Tra-2, Sxl, Dsx, Fem-1, Sox gene family, Foxl2, and Dmrt gene family) have been identified via transcriptomic analysis in crustaceans, indicating that sexual differentiation in crustaceans is more complicated than previously expected. It has been found that several non-coding RNAs (i.e., miRNAs, lncRNAs, and piRNAs) and IAG receptors may be involved in the sexual development of decapods. Identification and study of the regulation mechanism of sex-related genes, non-coding RNAs, and IAG receptors will provide valuable information regarding sexual development in decapods. In this review, the roles of hormonal and genetic factors in both males and females are discussed. In males, crustacean female sex hormone (CFSH), Sxl, Dmrt gene family, Dsx, Sox gene family, GEM, Fem-1, l-GnRH-III, and corazonin play important roles in IAG regulation in the “eyestalk-IAG-testis” endocrine axis. Unlike males, the regulation mechanism and interaction of sexual genes are relatively unknown in females. However, CFSH, IAG, Fem-1, FAMeT, Slo, UCHLs, Erk2, Cdc2, EGFR, Vg, VgR, and VIH seem to play crucial roles during ovarian development. This study summarizes the available information in the field, highlights gaps, and lays the foundations for further studies and a better understanding of the regulatory mechanism of sexual development in decapods.

RNAi Reveals Role of Insulin-Like Androgenic Gland Hormone 2 (IAG2) in Sexual Differentiation and Growth in Hermaphrodite Shrimp

Insulin-like androgenic gland hormone (IAG) is the most widely known hormone that regulates sexual differentiation in crustaceans. Previously, a transcriptome study described two transcripts of IAGs (Lvit-IAG1 and Lvit-IAG2) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Herein, we explored the function of Lvit-IAG2 via RNA interference (RNAi) experiments, and then compared the functional differences between the two IAGs. We demonstrated that Lvit-IAG2 positively regulated the expression of crustacean hyperglycemic hormone (Lvit-CHH) in eyestalk ganglion but exhibited no significant effect on the expression of gonad-inhibiting hormone (Lvit-GIHs) and crustacean female sex hormone (Lvit-CFSHs). Besides, Lvit-IAG2 gene knockdown caused degeneration in appendices masculinae (AM) and suppressed germ cells at the secondary spermatocyte stage. Moreover, silencing the Lvit-IAG2 gene impeded ovarian development, including smaller previtellogenic oocytes, and lower expression of vitellogenin (Lvit-Vg) gene in hepatopancreas and vitellogenin receptor (Lvit-VgR) gene in the ovarian region. Notably, silencing the Lvit-IAG2 gene impeded individual growth of the species. Collectively, findings from this study demonstrate that Lvit-IAG2 and Lvit-IAG1 coordinatively function to modulate sexual differentiation in L. vittata; meanwhile, Lvit-IAG2 stimulates the growth of the PSH species.

Identification of Candidate Genes of Male Sexual Development From Androgenic Gland in Macrobrachium Nipponense Through Performing Long-reads and Next Generation Transcriptome Sequencing After Eyestalk Ablation

The eyestalk of crustacean species contained many neurosecretory structures, affected the process of reproduction, molting, metabolism of glucose and other function in crustaceans. In this study, we aimed to selected important metabolic pathways and candidate genes involved in the male sexual development through performing the long-reads and next generation transcriptome sequencing of androgenic gland after the ablation of eyestalk from Macrobrachium nipponense. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation both of the single-side (SS) and double-side (DS) eyestalk, compared with that of control group (CG). The long-reads transcriptome generated 49,480 non-redundant transcripts. A total of 1,319, 2,092 and 4,351 differentially expressed genes (DEGs) were identified between CG vs SS, SS vs DS and CG vs DS, respectively, indicating the ablation of double-side eyestalk has more important regulatory roles than that of single-side ablation on male sexual development, which was consistent with that of qPCR analysis. Cell cycle, Cellular senescence, Oxidative phosphorylation, Glycolysis/Gluconeogenesis and Steroid hormone biosynthesis were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 GEDs were as the same as that of RNA-seq. The qPCR and RNAi analysis of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has the positive regulatory effected on testis development. This study provided valuable evidences on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustacean as well.