Abstract:This study aims to explore the genetic characteristics of the translocon-associated protein alpha subunit (TRAPα) of Litopenaeus vannamei and its role in resistance to white spot syndrome virus (WSSV). The ORF (open reading frame) sequence of TRAPα from L. vannamei was obtained through PCR and Sanger sequencing techniques. The gene was named Lv-trapα and bioinformatics analysis was conducted. Real-time PCR was used to analyze the expression level of the Lv-trapα gene in the hepatopancreas, gills, muscle, and eyestalk of healthy L. vannamei and those infected with WSSV at different time points. Meanwhile, bisulfite sequencing PCR (BSP) was used to detect the methylation level of the upstream DNA sequence of the Lv-trapα gene in the hepatopancreas of healthy L. vannamei and those infected with WSSV after 96 h. The results showed that the ORF of Lv-trapα was 873 bp in length, encoding 290 amino acids. The predicted relative molecular mass was 32466.4 and theoretical isoelectric point was 4.45. Multiple sequence alignment with various species including Penaeus chinensis, Procambarus clarkii, and Portunus trituberculatus revealed that conservation of the TRAPα protein was relatively high. Through PCR and Sanger sequencing techniques, eight single nucleotide polymorphism (SNP) sites were found in the Lv-trapα DNA sequence, among which one SNP site was located in the exon region and belonged to a missense mutation, while the other seven SNP sites were located in intron regions. Real-time PCR showed that the Lv-trapα gene was expressed in the hepatopancreas, gills, muscle, and eyestalk of L vannamei, and its expression was significantly up-regulated (P＜0.05) after infection with WSSV. Notably, 96 h post infection with WSSV, the expression level of Lv-trapα in the hepatopancreas of L. vannamei with different internal viral loads showed significant differences. The expression level of Lv-trapα in the HPC group was significantly higher than that in the LPC group (P＜0.05), suggesting that the expression level of Lv-trapα was positively correlated with the replication level of WSSV. Results of bisulfite sequencing technology showed that the methylation level of an upstream CpG site (located at position 360336-360337 in the NCBI database NW_020872863.1) of the Lv-trapα gene was negatively correlated with the expression level of Lv-trapα, and the methylation level of this CpG site was also negatively correlated with the WSSV viral load in L. vannamei. This study provides a theoretical reference for in-depth research on the molecular mechanisms of L. vannamei resistance to WSSV and molecular-assisted breeding for disease-resistance.

Abstract:The Forkhead box (Fox) protein family is a family of transcription factors with wing-like helical structured DNA-binding region and is widely distributed in biological groups ranging from yeast to mammals. The Fox protein family is divided into 19 subfamilies, A to S, among which the forkhead box O (FoxO) subfamily is the most intensively studied. Forkhead box O4 (FoxO4) is a member of the FoxO subfamily, which includes FoxO1, FoxO3, and FoxO6 in mammals. These members share structural and functional similarities as well as regulations. In certain fish species, seven highly homologous members of the FoxO subfamily exist: FoxO1a, FoxO1b, FoxO3a, FoxO3b, FoxO4, FoxO6a, and Fox06b. The involvement of FoxO in the regulation of various physiological functions, including the cell cycle, apoptosis, DNA damage repair, oxidative stress, cell differentiation, and glucose metabolism, has been demonstrated in multiple studies. Furthermore, its activity is modulated through diverse mechanisms such as phosphorylation, acetylation, and ubiquitination. To explore the molecular characteristics of foxo4 in grass carp (Ctenopharyngodon idella) and its relationship with the nutritional regulation of dietary proteins, we obtained the grass carp foxo4 sequence through homologous cloning. Its open reading frame is 1875 bp, encoding 624 amino acids, and it consists of three functional domains: FH, Pfam (FOXO_KIX_bdg), and Pfam (FOXO_TAD). Amino acid sequence alignment analysis showed that the foxo4 gene in C. idella is highly homologous to that in Danio rerio, Homo sapiens, Mus musculus, and Electrophorus electricus. Analysis of codon usage bias revealed that foxo4 exhibited a strong preference for CUG and AGC codons across all examined species, whereas C. idella and Pimephales promelas shared certain similarities in their codon preferences, suggesting a close evolutionary relationship between them. Phylogenetic tree analysis showed that the foxo4 gene of grass carp has the highest homology to that of Pimephales promelas. Tissue expression analysis of foxo4 revealed that the mRNA expression level of foxo4 was most significant in grass carp muscle (P＜0.05), followed by the heart, liver, intestine, brain, spleen, and kidney. In addition, circadian rhythm analysis showed that the expression of foxo4 was the highest at 18:00 and lowest at 24:00. This study also explored the effects of dietary protein on the expression of foxo4. The results of different protein source experiments showed that after 14, 21, and 35 days of the feeding trial, the expression of the foxo4 gene in the intestinal tissue of grass carp was significantly upregulated in both the rapeseed meal and fish meal groups compared with that in the soybean meal group (P＜0.05). These findings suggest a close association between the expression of foxo4 gene in grass carp and dietary protein sources. A feeding trial with different levels of l-alanyl-l-glutamine dipeptide (Ala-Gln) showed a gradual decrease in the expression of foxo4 in the intestines of grass carp as the ratio of Ala-Gln in the feed increased. The control group showed the highest expression level, whereas the 1.5% addition group displayed the lowest expression level. In summary, the expression of foxo4 in grass carp exhibits tissue specificity and is regulated by dietary protein sources and dipeptide levels. This study establishes a foundation for revealing the molecular characteristics of foxo4 in fish and its protein nutritional response and provides basic data for subsequent studies on the molecular mechanism of foxo4 gene regulation in fish protein metabolism in teleosts.

Abstract:The effects of salinity acclimation on the morphological characteristics and gonadal development of 5-year-old female Japanese eels (Anguilla japonica) were investigated. A 150-day salinity acclimation experiment was conducted to analyze the morphological features and gonadal development of female eels under different salinity conditions. The results showed that, as salinity increased from 0 to 35 over a period of 90 d (with 30 d of acclimation at salinities 15, 25, and 35, respectively), the pectoral circumference (PC), pectoral fin index (PI), gonadosomatic index (GSI), and liver index (LI) of female eels significantly increased (P＜0.05), while the digestive tract index (DTI) and eye diameter index (EI) showed no significant changes with increasing salinity (P＞0.05). Ovarian histological observations revealed a significant increase in oocyte diameter (OD) with increasing salinity (P＜0.05), although the oocytes remained in the oil droplet stage. After continued acclimation to salinity 35, no significant differences were observed in the aforementioned seven indices at 90, 120, and 150 d (P＞0.05). To investigate the effects of flowing water on female eel gonadal development, a flowing water group was acclimated to varying salinities. The results showed that the trends of changes in the seven indicators in the flowing water group of female Japanese eels during the salinity acclimation process were consistent with those of the still water group. Except for OD, the other six indicators in the flowing water group at the same acclimation salinity level showed no significant differences compared with the still water group (P＞0.05). The OD in the flowing water group was significantly larger than that in the still water group at the same salinity (P＜0.05). After 30 d of acclimation at salinity 35, oocytes in the flowing water group entered the primary yolk globule stage, with an oocyte diameter of (161.97±7.46) μm, which was significantly larger than that of the still water group at (140.46±9.36) μm. Determination of gonadotropin levels in the serum of female eels in the still water and flowing water groups revealed that during the entire salinity acclimation period, no significant changes were present in luteinizing hormone (LH) levels at various salinities for both still water and flowing water groups (P＞0.05). The LH levels of female eels in the flowing water group were not significantly different from those in the still water group at the same salinity level (P＞0.05). Salinity acclimation had a significant impact on follicle-stimulating hormone (FSH) levels in both the still-and flow-water groups. After 30 d of acclimation to a salinity of 35, the FSH level in the flowing water group reached (39.66±1.78) U/L, which was significantly higher than that in the still water group (36.97±1.52) U/L (P＜0.05). In summary, salinity is a crucial triggering factor for the gonadal development of female Japanese eels, and the stimulation of flowing water accelerates the early development of oocytes, promoting the synthesis of follicle-stimulating hormones. These findings provide fundamental data and reference information for reproductive biology research on Japanese eels.

Abstract:Mystus macropterus has delicious taste, rich nutrients and fast growth rate. It is a high-quality breed requiring further development. To investigate the biological characteristics of M. macropterus sperm and its adaptability to the external environment, the pH, osmotic pressure, sperm density, and sperm motility parameters of semen were determined, and the structure of the sperm was observed. The effects of environmental factors, such as pH, glucose, and ions (including NaCl, KCl, and CaCl₂), on sperm motility were also investigated. Based on the experimental data, the sperm of M. macropterus possessed a head, neck, and tail, and lacked acrosomes and had lateral fins. The mean sperm density of M. macropterus was 2.50×10⁹ , the pH of semen was stable between 7.0 and 7.2, and the osmotic pressure was stable at (634.16±6.66) kPa. Na⁺ content was the highest in the seminal plasma, followed by K⁺ , Mg²⁺, Ca²⁺, Fe³⁺, and Zn²⁺ and no Cu²⁺ was detected. The total amount of hydrolyzed amino acids in seminal plasma was approximately 169239.21 μmol/L. The largest proportion was composed of leucine, whereas the methionine content was relatively low. When sperm was activated by deionized water, the percentage of motile sperm, fast movement time, and life time were (48.61±14.85)%, (34.00±4.00) s, and (396.50±9.50) s, respectively. The straight line velocity, curvilinear velocity, and average path velocity were (11.50±6.26), (24.21± 8.68), and (14.00±5.99) μm/s, respectively. Sperm activity was most powerful in a slightly alkaline water environment with a pH of 7.0 to 9.0, with the optimal pH being 8.0. Simultaneously, when the concentrations of NaCl, KCl, and CaCl₂ were 1, 5, and 6 g/L, respectively, the percentage of motile sperm reached a maximum. Sperm motility was the greatest when glucose levels were maintained at 7 g/L. In conclusion, the sperm of M. macropterus had the best living environment in a pH range of 7.0 to 9.0. At the same time, appropriate concentrations of NaCl, KCl, CaCl₂, and glucose could also effectively improve the sperm motility of M. macropterus. In this study, we discuss the biological characteristics of spermatozoa and changes in their motility under the influence of different environmental factors. However, further research is required to establish a more effective breeding strategy and improve the artificial breeding efficiency of M. macropterus.

Abstract:Oxygen is necessary for fish survival, and low oxygen levels affect the growth, reproduction, and survival of fish. miR-462 and miR-731 are located in the same gene cluster (miR-462-731), which is unique to teleost fish; the expression of this gene cluster is significantly upregulated in hypoxic environments. Mitochondria are the centers of energy metabolism and use oxygen for aerobic respiration to power the body. The function of miR-462-731 is speculated to be related to the mitochondria. To investigate whether the miR-462-731 cluster could regulate mitochondrial function in grass carp (Ctenopharyngodon idella), we analyzed the effects of miR-462 and miR-731 overexpression on the biological function of mitochondria in grass carp liver cells. The mRNA expression levels of key enzyme genes in the tricarboxylic acid cycle were detected by qRT-PCR after the overexpression of miR-462 and miR-731, and the expression of mdh, ogdh and cs significantly decreased. Simultaneously, the ATP content was significantly reduced. The JC-1 probe was used to detect the mitochondrial membrane potential of cells after transfection. The content of reactive oxygen species (ROS) in the cells increased significantly, whereas the total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity decreased significantly; however, the content of malondialdehyde (MDA) increased. Electron microscopy revealed that the overexpression of miR-462 and miR-731 led to mitochondrial structural damage. In conclusion, this study found that overexpression of miR-462-731 could reduce cellular energy metabolism and the antioxidant capacity of grass carp liver, destroy mitochondrial structure, and affect mitochondrial function. This study further improved the functional study of miR-462-731 and provided a theoretical basis for the hypoxic regulation mechanism and hypoxic tolerance of grass carp and other bony fishes.

Abstract:Long non-coding RNA (lncRNAs) are involved in various biological processes, including sex differentiation. However, most studies on lncRNAs are limited to mammals, and a few studies are available on their functions in teleost fish. In this study, the sex-specific gene Novel01784 was identified using 126-day-old gonadal transcriptome data from northern pike (Esox lucius), and the full-length 1328 bp sequence was cloned using RACE. The results of CPC2 and CPAT analyses showed that the gene did not encode proteins. BLAST alignment analysis revealed that it was located on chromosome 9, between the sp2 and Abcc10 genes. Based on its location, it was named lincRNA-Abcc10. Transcriptome analysis was used to analyze the expression profiles of lincRNA-Abcc10 and its adjacent 11 genes in the ovaries of northern pikes at 126, 188, and 320 d. The results showed that lincRNA-Abcc10 was downregulated over time, and Mrpl4 and Mrpl45 were downregulated at all time points, similar to the expression pattern of lincRNA-Abcc10. According to the gene annotations for Mrpl4 and Mrpl45, lincRNA-Abcc10 may be involved in the regulation of ion transport, follicular cell development, and mitochondrial activity. Second, the qRT-PCR results of lincRNA-Abcc10 in eight northern pike tissues at three stages of showed that on day 126, the expression of lincRNA-Abcc10 in the female ovary was significantly higher than that in the testis, whereas in other tissues, it was only slightly expressed in the male muscle. On day 188, the expression of lincRNA-Abcc10 in the ovaries was significantly higher than that in the testes. In other tissues, it was slightly expressed in the liver, kidney, and muscle of female fish and in the head kidney and muscle of male fish. At 320 d, the expression of copyright Abcc10 in the ovaries was significantly higher than that in the testes. Other tissues included the brain, intestines, muscles, and kidneys of male fish. According to the comparative analysis of the relative expression in gonads at different developmental stages, the results showed that with the continuous development of the northern pike, the expression of lincRNA-Abcc10 at each stage decreased significantly compared to the previous period. It has also been suggested that it plays an important role in early ovarian differentiation. Additionally, through the isolation of nuclear RNA from 126-day-old northern pike ovaries, qRT-PCR results showed that lincRNA-Abcc10 was distributed in the cytoplasm and might regulate the translation level of adjacent genes in a cis or trans manner. Finally, a lincRNA-Abcc10 mutation model was established using the CRISPR/Cas9 technology, which laid the foundation for further studies on its gene function. This study provides new insights into the roles of lncRNAs in fish sex differentiation.

Abstract:Glucose-regulated protein 78 kDa (Grp78) alleviates endoplasmic reticulum stress induced by external stimuli and enhances cell viability. We studied the molecular response mechanisms of Gymnocypris przewalskii to salinity stress. In this study, Grp78 was cloned from G. przewalskii and the response patterns of Grp78 and related genes were analyzed after 5‰, 10‰, and 15‰ salinity stress using qPCR. The results showed that the open reading frame length of Grp78 was 1962 bp, encoding 653 amino acids and containing the conserved domain of the HSP70 superfamily. Grp78 was expressed in nine tissues of G. przewalskii, and its expression in the intestinal tract, liver, and heart was significantly higher than that in the other tissues (n=3, P ＜ 0.05). In gills, with the increase in salinity and the extension of stress time, compared with the blank control group, the expressions of Grp78, Hyou1, Prdx1, Nqo1, and UBC were first inhibited and then gradually upregulated, while the expression of DNAJC2, Cu/Zn-SOD, Mn-SOD, and Hmox1 was first upregulated and then gradually downregulated and then upregulated again. In the kidney and liver, the expression of DNAJC2, Cu/Zn-SOD, Mn-SOD, Hmox1, and Nqo1 showed similar patterns of upregulation, downregulation, and upregulation, respectively. These results indicated that Grp78 and its related genes have complex responses to salinity stress and may be involved in adaptation and coping with salinity stress in G. przewalskii. In addition, Grp78 and its related genes—Hyou1, DNAJC2, Hmox1, Nqo1, UBC, Prdx1, Cu/Zn-SOD, and Mn-SOD—participate in the regulation of antioxidant stress in the body and play important roles in reducing salinity damage.

Abstract:In the cultivation of shrimp, the use of sandworms as high quality bait often carry pathogenic Vibrio and lead to outbreaks of shrimp disease; therefore, the risk of carrying pathogenic Vibrio is expected to be reduced by sterilizing the aquaculture environment of sandworms. Based on the biological characteristics of the pathogenic Vibrio, this study was conducted to explore the sterilization effects on sediment at different temperatures (65 ℃ for 30 min, 100 ℃ for 30 min, and 121 ℃ for 20 min) and the effects of adding probiotics on the physical and chemical indexes of sediment. The results showed that the immediate sterilization results of each temperature sterilization group were good. The sterilization rate of the total colonies in the sediment was 57.5% for 65 ℃- 30 min, 99.45% for 100 ℃-30 min, and 99.99% for 121 ℃-20 min, respectively. This showed that with an increase in sterilization temperature, the sterilization rate gradually increased, and the sterilization rate for the pathogen Vibrio reached 100%. Analysis of the physicochemical indexes of the sediment showed that with the increase in sampling time, the influence of the sterilization treatment on the nitrogen-phosphorus diffusion flux at the sediment-overlying interface decreased, and the addition of probiotics could reduce the NO₃^– and PO₄^3– concentrations in the water and sediment. There were significant differences in the NO₃^– and PO₄^3– diffusion flux between the probiotic and non-probiotic groups at the same sampling time (P＜0.05). The study showed that all Vibrio bacteria in the sediment could be killed at 65 ℃ for 30 min, and no pathogenic Vibrio colonies were detected during the whole experiment. The addition of probiotics could optimize the sediment colony structure, inhibit the increase in total colony number, and affect the nitrogen-phosphorus diffusion flux at the sediment-overlying interface. These findings provide a reference for the application of sediment sterilization and probiotic preparation in aquaculture.

Abstract:Pearl gentian grouper (Epinephelus fuscoguttatus ♀×E. lanceolatus ♂) was studied to explore the cool acclimation process and evaluate its transport stress response through a series of biochemical indices. The effects of different cooling rates, water temperature, salinity, and fish-water ratios on the survival time of the pearl gentian grouper were determined. Response surface methodology was used to optimize the cool acclimation conditions, and a simulated transportation test was carried out on the pearl gentian grouper based on optimal conditions to determine the water quality indicators, serum biochemical indices, and liver antioxidant indicators at different time points; changes in the liver microstructures of the pearl gentian grouper were observed using an optical microscope. The optimal conditions for the acclimation process of pearl gentian grouper were obtained as follows: cooling rate of 1.2 ℃/h, temperature of 15.7 ℃, salinity of 24‰, and weight ratio of fish to water of 1∶ 5. Under these conditions the fish could survive for (55.6±1.7) h. In the simulated transportation, as the operation time increased, the total ammonia nitrogen concentration, nitrite concentration, and conductivity of the water increased rapidly. Transportation operation induced a gradual increase in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities and a rapid increase in cortisol levels, and their content returned to normal after 48 h. Serum albumin, total protein, triglyceride and total cholesterol contents decreased significantly at 48 h, indicating that transportation stress led to the decomposition of proteins and lipids in the serum, which affected the composition and content of the serum. The activities of liver superoxide dismutase, hydrogen peroxidase, and glutathione peroxidase were elevated during transportation as a pathway to counteract the oxidative stress of the organism. However, with the prolongation of transportation time, the organism gradually depleted and reduced the production of these enzymes, and the enzyme activities showed reduced trends at 48 h. Optical microscopy revealed that adverse effects on the liver tissue of pearl gentian grouper gradually increased. After 24 h, the arrangement of liver cells became chaotic and the overall morphology of the liver was lost. The study showed that the optimized cool temperature acclimation conditions were suitable for the pearl gentian grouper in simulated transportation, and different stages of transportation exhibited varying changes in water quality indicators, serum biochemical indicators, liver antioxidant indicators, and microstructure. These results provide technical support for the preservation and transportation of pearl gentian grouper and other marine fish.

Abstract:十足目甲壳动物性别决定与分化途径有多种方式, 其中多数表现出雌雄二态性模式。本文综述了十足目甲壳动物性别决定和分化分子调控机制的研究进展, 总结了在其中发挥重要作用的眼柄、促雄腺等组织, 以及 IAG 和 dmrt 等关键基因的功能, 探讨通过激素诱导和 RNA 干扰技术等获得十足目甲壳动物单性群体的性别控制方案, 以期为十足目甲壳动物性别控制深入研究和单性苗种养殖提供参考。