The transforming growth factor-beta (TGF-beta) signaling pathway is a central regulator of skeletal muscle growth in vertebrates, but the function of Acvr1b (activin receptor type 1B) remains poorly characterized in teleosts. Here, we used CRISPR/Cas9 to disrupt Acvr1b in grass carp (Ctenopharyngodon idella) and evaluated growth performance, muscle histology, and myogenic gene expression. Cas9 protein and sgRNAs targeting exons 2 and 3 were co-injected into fertilized eggs, and edited juveniles were identified by T7E1 assay and Sanger sequencing. Acvr1b-edited fish showed significantly improved growth relative to wild-type controls, including a 25.8% increase in body weight and a 7.39% increase in body length. qRT-PCR revealed reduced Acvr1b expression in all examined tissues. Consistent with enhanced muscle growth, mstn was downregulated, myog was upregulated, and myod was unchanged in edited fish. Histological analysis showed increased muscle fiber size and density, indicating that both hypertrophy and hyperplasia contributed to the growth advantage. Together, these findings identify Acvr1b as a promising target for genetic improvement in grass carp aquaculture.

Cyclic AMP response element-binding protein 1 (CREB1), a conserved bZIP transcription factor, is crucial for animal reproduction. Therefore, this study investigated its role in gonadal development of the freshwater snail Bellamya purificata using qRT-PCR, ISH, RNAi, and DNA methylation analysis. Results revealed that the creb1 CDS (867 bp, 288 aa) features a highly conserved BRLZ domain. Significantly, tissue expression showed markedly higher creb1 transcript levels in testes versus ovaries (p < 0.01). ISH localized enriched mRNA in ovarian oocytes/follicular walls and testicular spermatogonia/sperm. Functionally, efficient RNAi (max. 71.98% silencing) caused acute downregulation of sex genes: female β-catenin, cyp17a1 and sox9 expression fell 27%-34%, while male expression dropped 58%-77%. Critically, prolonged interference induced testicular defects, namely reduced mature sperm and increased spermatogonia, implicating Bpcreb1 in spermatogenesis—potentially via tssk and sox9 regulation. Furthermore, epigenetic analysis found consistently low Bpcreb1 promoter methylation (<5%) in both sexes, yet RNAi did not alter this hypomethylation, hinting at non-methylation regulatory mechanisms. In summary, creb1 demonstrably regulates gonadal development in B. purificata, with a pivotal role in spermatogenesis. Consequently, these findings provide a theoretical foundation for studying its gonadal development and monosex breeding strategies.

The reproductive toxicity of microcystin-LR (MC-LR) threatens declining freshwater mussels, many of which have a doubly uniparental inheritance (DUI) system that may confer sex-specific vulnerability. However, whether this toxicity is driven by ferroptosis and mediated by the DUI system remains unknown. Therefore, using the mussel Hyriopsis cumingii, we investigated whether ferroptosis mediates MC-LR-induced gonadal toxicity and sex-specific responses under DUI. Male and female mussels were exposed to environmentally relevant concentrations of MC-LR (0, 10, 40, and 70 mu g/L) for up to 4 d. Gonadal tissues were subsequently analyzed for oxidative stress markers, mitochondrial function, ferroptosis-related gene expression, and ultrastructural changes. MC-LR exposure induced a classic time- and concentration-dependent oxidative stress response, characterized by the collapse of antioxidant defenses (SOD, CAT, GSH) and the accumulation of ROS and MDA. This was accompanied by mitochondrial dysfunction, evidenced by a sharp decline in mitochondrial membrane potential (Delta Psi m) and ultrastructural damage. Intracellular Fe2+ levels increased, the expression of the key antiferroptotic gene GPX4 was suppressed, while pro-ferroptotic genes ACSL4 and PTGS2 were significantly upregulated. Principal component analysis revealed distinct sex-specific clustering, demonstrating that male gonads were strongly associated with markers of oxidative damage and ferroptosis, whereas female gonads maintained a stronger antioxidant capacity. These results demonstrate that ferroptosis underlies MC-LR reproductive toxicity, with sex-specific vulnerability linked to the DUI system, providing insights for ecological risk assessment and conservation of freshwater bivalves.

The Cyp11a gene is essential for steroid hormone synthesis, which is closely associated with gonadal development. To explore the role of Cyp11a in the gonadal development of Hyriopsis cumingii, we cloned the 2951 bp H. cumingii Cyp11a gene (Hccyp11a), which includes a 2376 bp open reading frame (ORF) encoding 791 amino acids. Phylogenetic analysis revealed that HcCYP11A is closely related to other molluskan homologs. Tissue distribution analysis showed that Hccyp11a was predominantly expressed in the gonad, with significantly higher expression in the ovary compared to the testis (p < 0.01). In juveniles, Hccyp11a expression peaked at 5 months of age. In the gonads of 12-, 24-, and 36-month-old individuals, Hccyp11a consistently exhibited higher expression in females. During ovarian development, Hccyp11a expression was highest at the ovulation stage. In situ hybridization localized Hccyp11a mRNA to the follicular wall and oocytes. RNA interference of female Hccyp11a significantly reduced the expression of Hsd3b and Srd5a1 genes, estradiol (E2) and methyltestosterone (MT) levels. Female H. cumingii were exposed to low (40 ng/L) and high (200 ng/L) concentrations of 17α-methyltestosterone (17α-MT) and 17β-estradiol (17β-E2) for 24 d. Low concentrations of 17α-MT and high concentrations of both 17α-MT and 17β-E2 significantly increased (p < 0.05) Hccyp11a expression. These findings suggest that Hccyp11a is involved in sex hormone synthesis and may regulate gonadal development in H. cumingii through its effects on steroidogenic gene expression and hormone levels.

Star3 is located in sex hormone synthesis signaling pathway associated with gonadal development. In this study, star3 was first cloned from the gonad of Hyriopsis cumingii. The cDNA of star3 in H. cumingii was 2072 bp in length, concluding an open reading frame (ORF) of 1419 bp, encoding 472 amino acids with a conserved START structural domain. Tissue distribution analysis showed star3 highest expressed mainly in gonad, which had higher transcription levels in testis than that in ovary. Significant hybridization signals were detected on the follicle wall cell of male gonad. Gonad expression patterns revealed star3 highest expressed at two months old during juvenile stages. After RNA interference with star3, expression levels of cyp17a and 17 beta-hsd11 was repressed. Furthermore, contents of pregnenolone, testosterone, and estradiol in gonad were reduced significantly. These results suggest star3 may be involved in sex hormone synthesis and play an important role in early gonadal development of H. cumingii.

Nanoplastics pollution has emerged as a global issue due to its widespread potential toxicity. This study delved in to toxic effects of nanoplastics on juvenile P. clarkii and molecular mechanisms from perspectives of growth, biochemical, histopathological analysis and transcriptome level for the first time. The findings of this study indicated that nanoplastics of different concentrations have varying influence mechanisms on juvenile P. clarkii. Nanoplastics have inhibitory effects on growth of juvenile P. clarkii, can induce oxidative stress. The biochemical analysis and transcriptome results indicated that 10mg/L nanoplastics can activate the antioxidant defense system and non-specific immune system in juvenile P. clarkii, and affect energy metabolism and oxidative phosphorylation. While 20mg/L and 40mg/L have a destructive influence on the immune function in juvenile P. clarkii, leading to lipid peroxidation and oxidative damage, and induce apoptosis, can affect ion transport and osmotic pressure regulation. The findings of this study can offer foundational data for delving further into impacts of nanoplastics on crustaceans and toxicity mechanism.

Matrix proteins play important roles in shell formation by regulating the assembly of organic matrix and minerals. Here, we obtained a new matrix protein (hicraqin) from Hyriopsis cumingii. The amino acid sequence of hicraqin contains multiple aggregated (Gly)n (n > 2) residues, a feature unique to silk-like matrix proteins. In situ hybridization studies and tissue expression patterns demonstrated that hicraqin may be a prismatic layer matrix protein. In vitro experiments were performed using the peptide N-hicraqin (the N-terminal free sequence of hicraqin). In the in vitro crystallization of calcium carbonate, crystals resembling dumbbell, spindle, and lotus aragonite crystals were observed under scanning electron microscopy and confirmed as calcite by Raman spectroscopy. In the in vitro crystallization system of calcium carbonate with the addition of magnesium ions, aragonite plates were generated with 50 μg/mL of the peptide N-hicraqin. The fluorescent labeling analysis indicated that N-hicraqin was involved in the crystallization process. The crystallization rate experiment showed that the peptide N-hicraqin plays a role in promoting crystallization. Following the silencing of the hicraqin gene by RNA interference, its expression was reduced by about 61 %. There was incomplete formation of the organic framework outside the prismatic layer. Overall, the present study showed that N-hicraqin participates in the crystallization process and acts as a framework protein that influences the formation of the organic framework of the prismatic layer.

The objective of this study is to explore the effects of light intensity (600 1800 lx) and photoperiod (4 24 h) on the growth performance and ovarian development of red swamp crayfish (Procambarus clarkii). The feeding rate (FR), weight gain rate (WGR), specific growth rate (SGR), condition factor (CF), hepatosomatic index (HSI), gonadosomatic index (GSI), and mature oocyte ratio in the ovary were measured. The results of the study showed that the first-order effect of photoperiod and light intensity has significant impacts on FR, WGR, SGR, CF, HSI, GSI, and mature oocyte proportion of P. clarkii; the second-order effect of photoperiod and light intensity has significant impacts on the FR, SGR, and CF of P. clarkii; and the interaction effect of photoperiod and light intensity has significant impacts on WGR, SGR, CF, HSI, GSI, and mature oocyte proportion of P. clarkii. Within the range of conditions in this study, as the photoperiod extended, the FR, WGR, SGR, CF, HSI, and GSI gradually increased. As the light intensity increased, the FR, WGR, SGR, and CF first decreased and then increased, while HSI and GSI showed a gradually increasing trend. The results indicated that the change of light intensity (enhanced or weakened) and the extension of photoperiod in the range of conditions in this study could promote the growth and ovarian development of P. clarkii. The photoperiod and light intensity have important effects on the growth and ovarian development of P. clarkii. The results in this study provided a theoretical basis for biological research of P. clarkii and basic data for artificial reproduction and intensive culture of P. clarkii.

China leads the world in freshwater pearl production, an industry in which the triangle sail mussel (Sinohyriopsis cumingii) plays a pivotal role. In this paper, we report a high-quality chromosome-level genome assembly of S. cumingii with a size of 2.90 Gb—the largest yet reported among bivalves—and 89.92% anchorage onto 19 linkage groups. The assembled genome has 37,696 protein-coding genes and 50.86% repeat elements. A comparative genomic analysis revealed expansions of 752 gene families, mostly associated with biomineralization, and 237 genes under strong positive selection. Notably, the fibrillin gene family exhibited gene family expansion and positive selection simultaneously, and it also exhibited multiple high expressions after mantle implantation by transcriptome analysis. Furthermore, RNA silencing and an in vitro calcium carbonate crystallization assay highlighted the pivotal role played by one fibrillin gene in calcium carbonate deposition and aragonite transformation. This study provides a valuable genomic resource and offers new insights into the mechanism of pearl biomineralization.

Procambarus clarkii (Girard, 1852) has important economic value in China and internationally. In this research, the comparative transcriptome analysis was used to reveal molecular mechanisms of influences of photoperiod and light intensity on ovarian development in P. clarkii for the first time. Some genes (such as laminin, collagen, integrin beta, catenin) and pathways (including TGF-beta signaling pathway, focal adhesion, ECM-receptor interaction) associated with ovarian development and oocyte maturation were significantly upregulated. Some genes related to circadian clock (such as CLK, PER) were identified in this research. The results indicated that when light intensity or photoperiod increased, P. clarkii could up-regulate the expression levels of the laminin and collagen, thereby synthesizing related proteins, promoting meiosis of the oocytes, thus increasing the number of oocytes in the ovary. At the same time, P. clarkii could up-regulate the expression levels of integrin beta, integrin alpha 6, and diacylglycerol to synthesize related proteins, thereby promoting the formation of proteins and fats such as triglycerides, these proteins and fats can provide material basis for maturation and development of oocytes, resulting in oocyte maturation and ovarian development. P. clarkii could synthesize related proteins by upregulating expression levels of genes (such as catenin), these proteins or hormones can adhere to other actins (such as integrins), thereby stabilizing the morphology of the oocytes and ensuring normal development. Meantime, the increase in light intensity or photoperiod could cause release GSH and VTG, resulting in oocytes development and maturation. The data in this research can reveal molecular mechanisms of impacts of photoperiod and light intensity on oocyte maturation and ovarian development in P. clarkii, can offer crucial genomic data for studying developmental mechanisms of ovary and oocyte in crustacean.

Background Oriental river prawn Macrobrachium nipponense is an economically important aquaculture species in China, Japan, and Vietnam. In commercial prawn farming, feed cost constitutes about 50 to 65% of the actual variable cost. Improving feed conversion efficiency in prawn culture will not only increase economic benefit, but also save food and protect the environment. The common indicators used for feed conversion efficiency include feed conversion ratio (FCR), feed efficiency ratio (FER), and residual feed intake (RFI). Among these, RFI is much more suitable than FCR and FER during the genetic improvement of feed conversion efficiency for aquaculture species. Results In this study, the transcriptome and metabolome of hepatopancreas and muscle of M. nipponense from high RFI low RFI groups, which identified after culture for 75 days, were characterized using combined transcriptomic and metabolomic analysis. A total of 4540 differentially expressed genes (DEGs) in hepatopancreas, and 3894 DEGs in muscle were identified, respectively. The DEGs in hepatopancreas were mainly enriched in KEGG pathways including the metabolism of xenobiotics by cytochrome P450 (down-regulated), fat digestion and absorption (down-regulated) and aminoacyl-tRNA biosynthesis (up-regulated), etc. The DEGs in muscle were mainly enriched in KEGG pathways including the protein digestion and absorption (down-regulated), glycolysis/gluconeogenesis (down-regulated), and glutathione metabolism (up-regulated), etc. At the transcriptome level, the RFI of M. nipponense was mainly controlled in biological pathways such as the high immune expression and the reduction of nutrients absorption capacity. A total of 445 and 247 differently expressed metabolites (DEMs) were identified in the hepatopancreas and muscle, respectively. At the metabolome level, the RFI of M. nipponense was affected considerably by amino acid and lipid metabolism. Conclusions M. nipponense from higher and lower RFI groups have various physiological and metabolic capability processes. The down-regulated genes, such as carboxypeptidase A1, 6-phosphofructokinase, long-chain-acyl-CoA dehydrogenase, et. al., in digestion and absorption of nutrients, and the up-regulated metabolites, such as aspirin, lysine, et. al., in response to immunity could be potential candidate factors contributed to RFI variation for M. nipponense . Overall, these results would provide new insights into the molecular mechanism of feed conversion efficiency and assist in selective breeding to improve feed conversion efficiency in M. nipponense .

In bivalves, the heterogeneity of mitochondrial DNA and its unique mode of transmission have been the focus of attention, which is called doubly uniparental inheritance (DUI). Prohibitin-2 (phb2) is a mitochondrial inner membrane protein that is a key mitophagy receptor for parental mitochondrial removal. Hyriopsis cumingii is a freshwater bivalve in China, the full-length cDNA of H. cumingii phb2 (named Hcphb2) is 2917 bp and encodes a total of 300 amino acids, a highly conserved sequence. Hcphb2 was highly expressed in the ovary. In the gonadal tissues of 5- to 8-month-old female mussels, the expression level of Hcphb2 continued to significantly increase. After Hcphb2 siRNA interference in 6-month-old female mussels, the expression of M-COII, a marker gene on M-type mitochondria, showed a considerable increase (p < 0.05). In contrast, the expression of autophagosome formation and maturation-related genes, atg4b, atg5, atg12, and atg16l, in the ATG family genes was significantly decreased (p < 0.01). Subcellular localization showed that Hcphb2 appeared in spermatogonia, spermatocyte, spermatid, and sperm, and its location changes synchronize with the behavior of M-type mitochondria location changes in DUI species. And it was found that miR-184 negatively regulated Hcphb2. The above results suggest that the mitochondrial autophagy receptor gene Hcphb2 may be associated with the degradation of M-type mitochondria in the freshwater mussel. This process requires multiple genes to participate, of which Hcphb2 and autophagy genes are only some of those that may play a role.

C-Mos, a proto-oncogene, regulates oocyte maturation by activating the classical MAPK pathway in cells. To examine the function of C-Mos in Hyriopsis cumingii, C-Mos was identified in this study. The full-length cDNA of C-Mos was 2213 bp, including 144 bp in the 5' UTR, 923 bp in 3' the UTR, and 1146 bp in the open reading frame (ORF) region. During early gonad development, the expression of C-Mos from 4 to 6 months of age in H. cumingii was significantly higher than that in other months, with the highest expression in 6-month-old H. cumingii, suggesting that C-Mos may be involved in early gonadal development in H. cumingii. Clear hybridization signals were found by in situ hybridization in the oocytes, oocyte nucleus and oogonium, and a small number of hybridization signals were found in the follicular wall of the male gonads. In addition, the C-Mos RNA interference (RNAi) assay results showed that the knockdown of C-Mos caused a down-regulation of ERK and P90rsk. In summary, these results indicate that C-Mos has a crucial part to play in gonadal development in H. cumingii.

通过RACE技术、实时荧光定量(qRT-PCR)技术、原位杂交和17β-雌二醇注射来探究17β-HSD11基因在三角帆蚌性腺发育和性激素合成中的表达特性和作用.结果显示:17β-HSD11基因的cDNA全长为1 134 bp,其中,5'UTR40 bp,开放阅读框(ORF)923 bp,3'UTR 171 bp,编码307个氨基酸.17β-HSD11基因在肝胰腺、性腺中表达量较高,且卵巢极显著高于精巢.原位杂交结果显示雌性三角帆蚌卵母细胞、滤泡膜和卵膜上均存在杂交信号.注射不同质量浓度17β-雌二醇之后发现17β-HSD11基因在雌雄性腺中的相对表达量均被抑制,其中:低质量浓度注射后,17β-HSD11基因的表达量在卵巢中下降33.38％,在精巢中下降37.74％;而高质量浓度注射后,17β-HSD11基因的表达量在卵巢中下降57.78％,在精巢中下降61.31％.由此推测17β-HSD11可能与三角帆蚌性腺发育和雌激素合成相关.

To clarify the molecular mechanism of the black and yellow shell coloration, we performed a transcriptome analysis of whole tissue of Corbicula fluminea in Hongze Lake (Jiangsu Province, China). After assembly, 335,247 unigenes were obtained, and 136,804 unigenes were functionally identified using public databases (NR, GO, KEGG, eggnog, and Swissprot). 1567 differentially expressed genes (DEGs) were detected through pairwise comparisons, of which 941 DEGs were up-regulated and 626 were down-regulated in the black-shelled clam. We compared the DEGs between two clams and identified some coloration-related genes. Notably, the black-shelled clam was larger than the yellow-shelled. We speculated that higher digestion and anabolic ability of black-shelled clam might lead to this phenomenon. In contrast, the yellow-shelled clam appeared to be more sensitive to environmental stress. The metabolic energy of the yellow-shelled clam was depleted to maintain or recover from stress, and provide less energy for growth. In summary, our finding provides a theoretical basis for the molecular mechanism of pigmentation and the difference of somatotype in bivalve, as well as promotes the future breeding of more elite varieties.

Shells and pearls are the products of biomineralization of shellfish after ingesting external mineral ions. Bone morphogenetic proteins (BMPs) play a role in a variety of biological function, and the genes that encode them, are considered important shell-forming genes in mollusks and are associated with shell and pearl formation, embryonic development, and other functions, but bone morphogenetic protein 10 (BMP10) is poorly understood in Hyriopsis cumingii. In this study, we cloned Hc-BMP10 and obtained a 2477 bp full-length sequence encoding 460 amino acids with a conserved TGF-β structural domain. During the embryonic developmental stages, the cleavage stage had the highest expression of Hc-BMP10, followed by juvenile clams; the expression in the mantle gradually decreased with increasing mussel age. A strong signal was detected on epidermal cells on the mantle edge by in situ hybridization. In both the shell notching and inserting operations of the pearl fragment assay, we found that the expression of Hc-BMP10 increased after the above treatments. RNA interference assays showed that the silencing of Hc-BMP10 resulted in a change in the morphology of the prismatic layer and nacreous layer, with the prismatic layer less closely aligned and the disordered aragonite flakes in the nacreous layer. These findings indicate that Hc-BMP10 is involved in the growth and development of H. cumingii, as well as the formation of shells and pearls. Therefore, this study provides some reference for selecting superior species for growth and pearl breeding of H. cumingii at a molecular level and further investigation of the molecular mechanism for biomineralization of Hc-BMP10.

As an important trace element and the accessory factor of many enzymatic processes, heavy metal copper is essential to aquatic animals. The toxic mechanism of copper on gill function of M. nipponense was clarified for the first time in terms of histopathological analysis, physiology, biochemistry and the expression of important genes. The results obtained by present in present research showed that heavy metal copper could affect normal respiratory and metabolic activities in M. nipponense. Copper stress could cause damage to the mitochondrial membrane of gill cells in M. nipponense, and the activity of mitochondrial respiratory chain complex could be inhibited by copper. Copper could affect normal electron transport and mitochondrial oxidative phosphorylation, resulting in the inhibition of energy production. High concentrations of copper could disrupt intracellular ion balance and induce cytotoxicity. The oxidative stress could be induced by copper, leading to excessive ROS. Copper could reduce the mitochondrial membrane potential, lead to the leakage of apoptotic factors, and induce apoptosis. Copper could damage structure of gill, affect normal respiration of gill. This study provided fundamental data for exploring impacts of copper on gill function in aquatic organisms and potential mechanisms of copper toxicity.

Estrogens and androgens that coexist in the aquatic environment could potentially affect shellfish, however, endocrine disrupting effects of them in shellfish are significant. As an important aquaculture shellfish in China, Hyriopsis cumingii has remarkable economic benefits. In this study, the effects of endocrine disrupting chemicals on the steroid synthase Hc-Cyp17a in the male and female gonads of the H. cumingii were assessed by exposing juvenile mussels to cultured waters containing 17β-Estradiol (E2) and 17α-Methyltestosterone (MT) for 28 days. At the same time, the E2 content in the four stages of gonadal development, the expression changes of Hc-Cyp17a in gonadal development and its localization in the mature gonad were measured to explore the relationship between genes and hormones. The results showed that both E2 and MT at 50 ng/L and 200 ng/L could affect the transcription level of Hc-Cyp17a, which was inhibited initially and promoted in post-development. E2 content was positively correlated with gonadal development stage, which was in mussel. By tracing the expression of Hc-Cyp17a, difference was found during different developmental periods. The expression level in ovary was higher than that in testis during gonadal development of 1/ 2/ 3-year-old mussels and showed an increasing trend with age. Furthermore, the expression levels in 6 tissues of mature individuals were measured and it showed that there was a significant difference between male and female in the gonads (p < 0.01). In situ hybridization, it suggested that Hc-Cyp17a was significantly signaled in the follicular wall and oocyte of female and in the follicular membrane of testis, respectively. These results could play a vital role in assessing and understanding the effects of aquatic environment on the endocrine system of H. cumingii.

我国淡水贝类资源丰富、种类繁多,有记录的淡水贝类目前已经超过470种,广泛分布于我国各地的湖泊、河流和山间湿地等生态系统中.其中我国特有种贝类在各主要分布水系的比例均高于50％,特有种质资源十分丰富.淡水贝类不仅在生态系统中扮演着重要的角色,而且具有巨大的经济价值.淡水珍珠蚌中三角帆蚌和褶纹冠蚌,以及一些食用贝类如中华圆田螺、螺蛳、河蚬等的资源开发比较充分,但是由于淡水贝类分布范围广、栖息环境多样,相对于其他一些淡水贝类,目前研究还仅限于资源调查和物种鉴定层面.整体上,国内淡水贝类的种质资源评估不够系统,保护和开发利用未得到足够重视.本文就我国淡水贝类物种多样性与区域分布、淡水贝类种质资源可持续开发与利用情况、引进淡水贝类种质资源与利用现状及淡水贝类种质资源现行保护措施进行概述,并提出有关淡水贝类种质资源保护和可持续利用的建议,以期促进我国淡水贝类资源保护和开发利用协同发展.