Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation.

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.

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.

Molecular cloning, expression profiling of a carboxylesterase gene and its potential role in methyl farnesoate degradation in Eriocheir sinensis (Brachyura, Varunidae)

In crustaceans, methyl farnesoate (MF) is an important sesquiterpenoid to regulate many physiological processes, especially reproduction and ovarian maturation. In this study, a 1919 bp cDNA of carboxylesterases (Es-CXE6) with some conserved motifs of the CXE multifunctional enzyme family was cloned from Eriocheir sinensis. Tissue and stage-specific expression results suggested that Es-CXE6 expression in hepatopancreas was highest and associated with the haemolymph MF titer. In vitro and in vivo experiments showed that Es-CXE6 expression was significantly upregulated by MF treatment in the hepatopancreas but not in the ovary. Furthermore, an eyestalk ablation experiment showed that Es-CXE6 expression was significantly upregulated on days 1 and 3 post eyestalk ablation in the hepatopancreas. Together, these results indicate that Es-CXE6 may degrade MF in the hepatopancreas in E. sinensis. Our results offer a potential approach to maintain the MF titer at appropriate levels, which has potential applications in crab aquaculture.

Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: behavioral evaluation

Eyestalk ablation allows the removal of neurohormones that inhibit early reproduction and decreases the time to complete the molting cycle. The present study evaluated the impact of unilateral eyestalk ablation on the behavior of blue crab breeders Callinectes arcuatus in the short term under laboratory conditions. Immature male and female crabs and individuals in the intermolt stage were collected in Ohuira Bay, Ahome, Sinaloa, Mexico. Three bioassays were performed: males, females, and male-females each with three treatments; control + xylocaine (50 μL, 5%), squash ablation + xylocaine (50 μL, 5%), ablation by string ligation + xylocaine (50 μL, 5%), each with three replicas and three periods of evaluation after the ablation procedure (10 min, 2 h, and 24 h). Kruskal-Wallis and Chi-square (χ2) tests were used to evaluate significances (P < 0.05) in the behavior of C. arcuatus. The sizes of blue crab C. arcuatus were 9.72 ± 1.0 cm of carapace width. The feeding behavior of the male-females bioassay was significant at 2 h (P < 0.05). The results obtained were satisfactory using two techniques, unilateral eyestalk ablation by squash and ligation in which pain and aggressiveness indicators were mitigated. Disorientation and aggressiveness in males were significant at 2 h (P < 0.05), and in females after 10 min, and 2 h after ablation (P < 0.05). In the male-females bioassay, aggressiveness was significant at 2 h (P < 0.05). Courtship and copulation were recorded in those crabs subjected to unilateral eyestalk squash ablation (13 and 8, respectively).

A Novel Imprinted Gene (Sp-Pol) With Sex-Specific SNP Locus and Sex-Biased Expression Pattern Provides Insights Into the Gonad Development of Mud Crab (Scylla paramamosain)

Identification and exploring the role of novel sex-related genes is a crucial step for understanding the regulation mechanism of sexual development. In the present study, we identified a novel sex-related gene (designated as Sp-Pol) upstream of a sex-specific single nucleotide polymorphism (SNP). SNP1888 together with Sp-Pol were mapped on LG32 (which is a sex-related linkage group) of a high-density genetic map. The full-length cDNA of Sp-Pol consists of 1703 bp with an open reading frame (ORF) encoding 204 amino acids, a 344 bp 5′-UTR and a 744 bp 3′-UTR. Phylogenetic analysis showed that Sp-Pol may need to be classified as a new gene family due to the very low sequence identity with other known genes (less than 25% identity). The sex-biased expression pattern of Sp-Pol starts from crablet stage V (C5) with approximately three- to four-fold higher in males than in females. Sp-Pol was expressed at a higher level in gonads compared to other tissues, with the highest expression level in the testis. In testis, a downward trend was observed in the expression level of Sp-Pol from the testis stage I (T1) to testis stage III (T3). After unilateral eyestalk ablation, the expression level of Sp-Pol significantly increased in testis and hepatopancreas in males, while it was downregulated in the hepatopancreas of females. Fluorescence in situ hybridization (FISH) assay revealed that Sp-Pol transcripts were strongly localized in the epithelia of seminiferous tubules of the testis, and in the ovary, it was detected in the oogonium cells. These findings showed that Sp-Pol may play crucial roles in the gonad development of S. paramamosain.

Transcriptome Profiling Analysis of the Testis After Eyestalk Ablation for Selection of the Candidate Genes Involved in the Male Sexual Development in Macrobrachium nipponense

The eyestalk of crustacean species secretes many hormones, affecting the process of reproduction, molting, metabolism of glucose, and other functions in crustaceans. In this study, important metabolic pathways and candidate genes involved in the male sexual development were identified through performing the transcriptome profiling analysis of the testis after the ablation of eyestalk from Macrobrachium nipponense. The histological observations revealed that the testis development became vigorous after eyestalk ablation, indicating that the hormones secreted by the eyestalk have negative effects on the testis development in M. nipponense. Transcriptome profiling analysis revealed that 1,039, 1,226, and 3,682 differentially expressed genes (DEGs) were identified between normal prawns (CG) vs single-side eyestalk ablation prawns (SS), SS vs double-side eyestalk ablation prawns (DS), and CG vs DS, respectively, indicating that the ablation of double-side eyestalk has more significant regulatory roles on male sexual development than that of single-side ablation, which was consistent with the histological observations. Lysosome, Apoptosis, Glycolysis/Gluconeogenesis, and Insulin signaling pathway 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 DEGs from these metabolic pathways were the same as those of RNA-seq. The qPCR, in situ hybridization, and RNA interference analysis of Mn-NFkBα revealed that NFkBα has a positive regulatory effect on testis development. This study provided new insights on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustaceans as well.

A Novel Imprinted Gene (Sp-Pol) with Sex-specific SNP Locus and Sex-biased Expression Pattern Provides Insights into the Gonad Development of Mud Crab (Scylla Paramamosain)

Background: We previously discovered a potential sex-specific single nucleotide polymorphism (SNP) locus named SNP1888 in mud crab (Scylla paramamosain). Methods: In this study, we first verified SNP1888 to be truly a female-specific locus and then we identified a novel imprinted gene (designated as Sp-Pol) at the upstream of SNP1888 (SNP1888 is located at the 3’-UTR of Sp-Pol). Moreover, SNP1888 together with Sp-Pol were mapped on LG32 of a high-density genetic map. Results: Phylogenetic analysis showed that Sp-Pol may need to be classified as a new gene family due to the very low sequence identity with other known genes. Sp-Pol was expressed at a higher level in gonads compared to other tissues and its expression level in the testis was much higher than in the ovary. Coincidentally, mono-allelic expression was observed in the ovary. Moreover, Sp-Pol exhibited sex-biased expression with approximately 3- to 4-fold higher in males than in females at fifth (C5) and sixth (C6) crablet stages. During the zoeae development, Sp-Pol had the highest expression at the zoea I stage. After unilateral eyestalk ablation, the expression level of Sp-Pol significantly increased in testis and hepatopancreas in males, while it was downregulated in the hepatopancreas of females. Fluorescence in situ hybridization (FISH) assay revealed that Sp-Pol transcripts were strongly localized in the epithelia of seminiferous tubules of the testis and in the ovary it was detected in the oogonium cells.Conclusion: These results demonstrated that Sp-Pol may play important roles in the sexual development of S. paramamosain.