Abstract:The grass carp (Ctenopharyngodon idella) represents an economically important fish species in freshwater aquaculture within China. However, the urgency to develop superior varieties has become paramount due to significant germplasm degeneration in grass carp. Germline stem cell transplantation emerges as a promising and potent technique for reducing the sexual maturity cycle in fish. Consequently, our objective was to shorten the generation interval for obtain functional gametes of grass carp by employing barbel chub (Squaliobarbus curriculus) as a surrogate host. The accurate identification of germ cells between donor and recipient species is a critical step in the successful establishment of germline stem cell transplantation. However, the method of identification of germ cells in grass carp and barbel chub is not clear. In the present study, the full-length cDNA of reproduction-related gene nanos2 were cloned from grass carp and barbel chub, which are 649 and 636 base pairs, encoding 145 and 144 amino acids, respectively. Sequence analysis revealed that the grass carp Nanos 2 (CiNanos2) amino acid sequence exhibits a high degree of sequence identity to that of barbel chub (91.67%), and to that of zebrafish (Danio rerio) (65.49%). Phylogenetic tree analysis showed that grass carp is clustered together with barbel chub, indicating the closest genetic relationship between them. The expression of nanos2 transcripts in grass carp and barbel chub was predominantly observed in the gonads, with significantly higher levels detected in the testis compared to the ovary, suggesting that nanos2 might plays a crucial role in the development of gonad. Species-specific and common primers were designed based on the alignment of nanos2 sequences between grass carp and barbel chub for PCR analysis. The results demonstrated that the grass carp-specific primers had exclusively target product (179 bp) in the gonad of grass carp, while the barbel chub-specific primers had only expected product (265 bp) in the gonad of barbel chub gonads. Additionally, the common primers were able to amplify indiscriminately in the gonads of both grass carp and barbel chub, producing a target product of 251 bp, implying that the germ cells of grass carp and barbel chub could be efficiently distinguished via species-specific primers by PCR. Our study laid the foundation for further investigating the mechanism of the nanos2 gene in gonad development of grass carp and barbel chub. Meanwhile, it provided an effective method for monitoring the chimerism and development of grass carp germ cells in the gonads of barbel chub.

Abstract:Heat hardening in Mytilus galloprovincialis is a survival strategy for enhancing thermal tolerance in response to extreme low tides during consecutive low-tide periods in spring. Transcriptome sequencing was used to analyze the gill tissues of mussels in the heat-hardening (H) and control (N) groups to uncover the mechanisms of thermal tolerance enhancement under heat hardening. The results showed that, compared to the control group, the heat-hardening group had 651 differentially expressed genes, including 159 significantly upregulated and 492 significantly downregulated genes. Gene ontology (GO) analysis revealed significant enrichment of differentially expressed genes in biological processes such as metabolism, substance transport, and signal transduction. Gene set enrichment analysis (GSEA) of significantly enriched items in the GO analysis revealed the upregulation of biological processes related to macromolecule metabolism, nucleic acid metabolism, and metabolism of compounds containing nuclear bases, which are associated with macromolecule metabolism, substance metabolism, and energy metabolism. Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that differentially expressed genes were significantly enriched in signaling pathways related to metabolic regulation, such as Rap1, apoptosis-multiple species, and MAPK. These findings indicate that thermal hardening in Mytilus galloprovincialis may enhance tissue functional capacity by activating various macromolecular metabolic processes while also strengthening the ability of gill tissues to actively remove damaged cells, thus enabling early physiological regulation and adaptive responses to subsequent high-temperature stress, leading to better functional performance. This study explored the biological processes and metabolic pathways associated with thermal hardening in Mytilus galloprovincialis, providing a foundation for investigating the molecular mechanisms underlying heat-hardening in this species.

Abstract:Ras homologue enriched in the brain (Rheb) is an important positive regulator on the upstream of the mammalian mechanistic target of rapamycin (mTOR) signaling pathway. The cDNA sequence encoding Rheb in Eriocheir sinensis (EsRheb) was cloned using RT-PCR in order to investigate the role of this gene in the regulation of growth, development, and nutrient metabolism of Eriocheir sinensis via the mTOR signaling pathway. The sequence and structural characteristics of EsRheb were analyzed using bioinformatics. The tissue distribution of EsRheb mRNA and the effects of animal starvation and eyestalk ablation on EsRheb transcription were detected using qRT-PCR. The results showed that EsRheb cDNA encoded 182 amino acids; its amino acid sequence, conserved domain, and functional sites were highly similar to the Rheb of a wide range of species; and its three-dimensional structure was highly consistent with that of human Rheb. EsRheb mRNA expression was high in the hepatopancreas, stomach, myocardium, skeletal muscles, and Y organ, but was much lower in the eye stalk. Starvation treatment of animals significantly decreased the transcriptional expression of EsRheb in the skeletal muscle but had no significant effect on its expression in the hepatopancreas. The removal of the eye stalk significantly increased the transcriptional expression of EsRheb in the Y organs and skeletal muscles. This study provides a basis for elucidating the function of EsRheb in regulating the mTOR-mediated growth and metabolism of E. sinensis.

Abstract:In the present study, the hypoxia tolerance and alternation of hematological, physiological, and biochemical parameters, gill histology, and hepatic antioxidant capacity subjected to hypoxia stress were investigated in black scraper Thamnaconus modestus. The closed static water method was used to analyze the dissolved oxygen (DO) levels at critical oxygen tension (P_crit) and loss of equilibrium (LOE) in black scraper. Subsequently, changes in hematological, physiological, and biochemical parameters, gill histology, and hepatic antioxidant enzyme activity were detected at P_crit, LOE, reoxygenation 3 hours (R3) and 6 hours (R6). The results showed that the DO concentrations of the black scraper [(101.23±14.28) g] at P_crit and LOE were (4.0±0.2) mg/L and (2.2±0.2) mg/L, respectively. Hypoxia significantly increased glucose and cortisol levels. White and red blood cell numbers also increased with the highest value observed at LOE (P＜0.05). The hemoglobin concentration and hematocrit showed an upward trend at LOE, but the difference was not significant (P＞0.05). Meanwhile, hypoxia significantly increased the length, width, basal epithelial thickness, and perimeter of the secondary lamellae, and decreased interlamellar distance and the proportion of available gas exchange (P＜0.05). Hypoxia stress also induced branchial clubbing, fusion, and matrix hyperplasia of the secondary lamellae. Hepatic superoxide dismutase, catalase, glutathione peroxidase activity, and malondialdehyde levels significantly increased under hypoxia (P＜0.05), peaking at LOE. Reoxygenation treatment for 6 hours alleviated the stress mentioned above effects. These results indicated that hypoxia stress lead to significantly alterations in the black scraper's hematological, physiological and biochemical profiles, gill morphology, and hepatic antioxidant capacity, which are substantially alleviated after 6 hours re-oxygenation. These findings provide valuable insights into the physiological mechanisms underlying hypoxia tolerance and support the development of efficient and healthy breeding practices for black scraper.

Abstract:Black rockfish (Sebastes schlegelii) inhabit the coastal regions of the northwestern Pacific Ocean, including the East China Sea, Yellow Sea, and the coastal areas of Korea and Japan. It is highly favored by consumers because of its delicate flavor and high nutritional content. Black rockfish is among the most economically cultivated marine species in northern China. The juveniles were raised in offshore net cages. The optimum temperature for black rockfish growth ranges from 18 ℃ to 24 ℃. In summer, the seawater temperature sometimes exceeds 28 ℃ in black rockfish farms. As global warming intensifies, long-term exposure to high temperatures in summer will affect the growth and survival of black rockfish cultured in net cages, indicating that the effects of chronic heat stress on black rockfish merit further research. However, the molecular mechanisms underlying the responses of black rockfish to chronic heat stress remain largely unknown. Understanding these mechanisms will improve fish welfare and farm production. Marker genes to monitor heat stress are required to identify heat-resistant fish. In this study, we conducted RNA-seq analysis to characterize the genes and pathways involved in chronic thermal stress responses in the gills, liver, and intestines of black rockfish. Healthy black rockfishes were cultured at a normal temperature (18 ℃) and a high temperature (27 ℃). For the chronic heat stress treatment, water temperature was increased from 18 ℃ to 27 ℃ at a constant rate of 1 ℃ per day, and maintained for 10 days. The gill, liver, and intestinal tissues were used as experimental materials in both the heat stress and normal groups. Total RNA was extracted, and 18 mRNA libraries were constructed and sequenced using the Illumina HiSeq-4000 technology platform. Differentially expressed genes were analyzed using edgeR. Bioinformatic analysis was performed on the GO and KEGG functions of the differentially expressed genes, and the key differentially expressed genes were further validated using RT-qPCR. In total, 306 annotated differentially expressed genes (DEGs) were identified in the gill, of which 96 and 210 were up-and downregulated, respectively. In total, 806 annotated differentially expressed genes (DEGs) were identified in the liver, of which 382 were upregulated and 424 were downregulated. A total of 343 annotated differentially expressed genes (DEGs) were identified in the intestine, among which 162 and 181 were up-and downregulated, respectively. A Venn diagram showed that 12 DEGs were shared among three tissues. And 40, 53, and 49 DEGs were shared between liver and gill, liver and intestine, gill and intestine, respectively. Furthermore, GO functional enrichment analysis revealed that the DEGs were mainly enriched in proteolysis, extracellular region, structural molecule activity, receptor regulator activity, and receptor-ligand activity in the gill; in lipid metabolic process, cellular amino acid metabolic process, cytoplasm, and oxidoreductase activity in the liver; and in oxidation-reduction process, extracellular region, and cofactor binding in the intestine. Among the up-regulated genes under heat stress included the heat shock proteins 90α, period circadian protein homolog 2, serpin H1, and the down-regulated genes included period circadian protein homolog 1. Ten DEGs were subjected to reverse transcription quantitative PCR (RT-qPCR) for relative quantification to assess the differences in gene expression between the normal and high temperature groups. The expression trends observed in the RT-qPCR analysis were consistent with those identified in the RNAseq data, which confirmed the reliability of the transcriptomic sequencing results. These results provide abundant data for further studies on the molecular mechanisms of the chronic heat stress response in Sebastes schlegelii.

Abstract:Brachymystax tsinlingensis Li (1966) is sensitive to light and susceptible to death by strong light during long-term artificial breeding, especially during early developmental stages. The development of visual organs and the characteristics of retinal development were systematically observed using histological methods to analyze the physiological causes of this phenomenon and improve the efficiency of large-scale artificial breeding. The results showed that the embryo entered the organ differentiation stage, and the optic cup was formed 177 hours post-fertilization (HPF). At 213 HPF, a primitive retina and crystal sac were formed, and the cuboidal epithelium constituted the cornea. The crystal sac had developed into the lens, the cells of the neural layer of the retina had increased notably, and the staining of the pigment cell layer deepened at 357 HPF. At 452 HPF, the sclera appeared, and the retina differentiated into four layers (pigment epithelium, outer nuclear layer, inner nuclear layer, and optic ganglion cell layer). After 508 h, the fertilized egg broke the membrane and hatched larvae. The visual organs of hatchlings were relatively well-developed, and the lens is fully differentiated and has the same structure as that of the adult; the retina has been differentiated into 8 layers, and the pigment granules in the pigment layer are clearly visible; the sclera consists of elastic fibers and cartilage; the iris is in the form of a single-layer ring; and the choroid is present. At 5 days after hatching (DAH), a small number of cone cells appeared, and the visual cell layer was pure cone cell structure. At 10 DAH, the retina differentiated into a complete 10-layer structure, ON/SC was 1.27＞1, a few optic rods began to appear in the visual cell layer, and the inner nuclear layer was differentiated into three clear layers with the initial visual imaging system. At 12 DAH, ON/G was 1.07 ＞ 1, the retinal network was less convergent, and the number of nuclei in the outer nuclear layer was comparable to the number of ganglion cells, which was sufficient to receive signals from the visual cells. At 16 DAH, the mean value of the retinal bright-adapted pigmentation index (PI) was 0.75, whereas the mean value of the dark-adapted PI was significantly reduced to 0.55 (P ＜ 0.05). At 21 DAH, the number of optic rod cells increased, and the cornea differentiated into a complete 5-layer structure; at 41 DAH, the iris differentiated, the choroid was perfected, and all parts of the visual organs were fully developed. Therefore, the retinal structure and visual characteristics of B. tsinlingensis show strong light sensitivity, and vision plays a major role in its behavior and feeding activities. Reducing environmental light appropriately during early breeding of B. tsinlingensis could reduce the stress response and improve the survival rate of seedlings. The results of this study provide a reference for the proliferation, conservation, and utilization of B. tsinlingensis resources.

Abstract:This study used artificially bred Brachymystax tsinlingensis Li (1966) larvae [body weight: (0.06± 0.02) g, body length: (1.85±0.11) cm] as experimental subjects to explore the swimming ability and behavioral characteristics of B. tsinlingensis larvae and scientifically solve the problems of water velocity environment selection, during processes such as seedling cultivation and artificial release. The induced velocity, critical swimming speed, and burst swimming speed were measured using the increasing velocity method at (11.4±0.17) ℃, and the sustained swimming ability and durable swimming ability were measured using the fixed velocity method. The results showed that the induced velocity, critical swimming speed, and burst swimming speed were (0.03±0.01) m/s, (0.14±0.03) m/s, and (0.22±0.03) m/s, respectively. Relative induced velocity, relative critical swimming speed and relative burst swimming speed were (1.62±0.29) BL/s, (7.55±1.61) BL/s, and (11.48±1.79) BL/s, respectively. The maximum sustained swimming speed and the maximum durable swimming speed were 0.13 m/s and 0.25 m/s, respectively. This was similar to the mean critical swimming speed and mean burst swimming speed. Continuous and endurance experiments showed that the swimming duration of B. tsinlingensis larvae was negatively correlated with flow rate (lgT=−24.48X+7.09, R² =0.79, P＜0.01), tail beat frequency was positively and linearly correlated with flow rate (TFB=0.70U+5.54, R² =0.89, P＜0.01), and stride length was linearly and positively correlated with flow rate (SL=0.09U+0.11, R² =0.99, P＜0.01). Therefore, artificial breeding and release were performed at a flow rate of 0.04–0.14 m/s. The results of this study contribute to improving the survival rate of artificial cultivation and release effectiveness of B. tsinlingensis and provide a reference for the selection of resource conservation and utilization environments.

Abstract:In recent years, the number of small greenhouse farms in China has expanded rapidly. Within the aquaculture process of these small greenhouses, microorganisms play a key role in decomposing residual bait and feces in the water column. However, the lack of nutrient-degrading microbial taxa in the water column, combined with excessive baiting by farmers, often leads to the accumulation of inorganic nitrogen. This accumulation negatively affects the yield and economic benefits of cultured species. The present study was conducted to investigate the differences in water quality, as well as the structure and function of particle-associated bacteria (PAB) and non-mobile free-living bacteria (NFB) in small greenhouse farms. This investigation focuses on the turbidity of the water column caused by the increasing concentration of suspended particles during the aquaculture process, in order to reveal the factors influencing the culture yield. Differences in the structure and potential function of the two bacteria were analyzed by high-throughput sequencing of 16S rRNA. Additionally, real-time fluorescence quantitative PCR was used to detect the abundances of ammonia-oxidizing bacteria amoA and nitrite-oxidizing bacteria nxrB, both of which are associated with nitrogen transformation in suspended particles. The results of this study showed that the concentration of suspended particles was significantly higher in high-yield small greenhouses compared to low-yield small greenhouses (P＜0.001). Additionally, the concentration of suspended particles in the water column was closely related to shrimp production. The Chao1 and Shannon indices of PAB were found to be higher than those of NFB in the aquaculture water column of small greenhouses, indicating greater diversity and abundance of PAB. Notably, Flavobacteriaceae were significantly enriched in PAB, indicating that they were the dominant functional microorganisms in PAB. The abundance of biosynthesis, material, and energy metabolism functional pathways in PAB was significantly higher than that in NFB (P＜0.05). Among these ways, nitrification-denitrification was notably upregulated in PAB, indicating that PAB is more capable of material synthesis and nutrient metabolism. Real-time fluorescence quantitative PCR results revealed that the abundances of major functional genes of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were higher in the PAB of high-yield small greenhouses than in those of low-yield small greenhouses, indicating that nitrifying bacteria were more mature in the PAB of high-yield small greenhouses. This study demonstrated that PAB play an essential role in the removal of nitrogenous pollutants from aquaculture water in small greenhouse farms, which further affects shrimp production by regulating water quality. The results of this study provide an important theoretical basis for water quality control and efficient shrimp culture in small greenhouse farms.

Abstract:We quantitatively analyzed the changing patterns of phytoplankton after being treated by purification units in this study with three groups of recirculating pond aquaculture systems—a composite pond recirculating aquaculture system (A), a paddy field-fish pond recirculating aquaculture system (B), and a combined wetland-pond recirculating aquaculture system (C)—to explore the influence law of aquaculture water purification on phytoplankton community structure and diversity. The removal efficiency of different pollutants varied among the three systems, with system A having the highest removal efficiency for potassium permanganate index, total nitrogen, and suspended solids, whereas system C had the lowest efficiency for these contaminants. Compared to the inflow water, the Chl a concentrations in the effluent water were decreased by 52.08%, 67.61%, and 27.06% in systems A, B, and C, respectively. During the study period, 138 species from seven phyla of phytoplankton were identified, which were dominated by species from Chlorophyta, Cyanophyta, and Bacillariophyta. The number of phytoplankton species varied between the different systems; however, the dominant species and their dominance were not significantly different. Compared to the inflow water, the density and biomass of different phytoplankton species in the purification units were substantially decreased in systems A and B, and the algal removal rates exhibited an increasing trend along the cross-sections of the purification unit. Phytoplankton density fluctuated greatly in the purification unit of system C. Overall, phytoplankton species from different phyla were similarly affected by the purification units. The diversity of phytoplankton in the purification units was increased compared to the inflow water in the three systems, with the H′ and J index values increasing significantly in systems A and B (P＜0.05). Pearson correlation and redundancy analyses indicated that the densities of phytoplankton were significantly positively correlated with factors such as total phosphorus, total nitrogen, and potassium permanganate index in the water (P＜0.05), but there were differences in the factors influencing phytoplankton among different systems. This study revealed that aquacultural water purification significantly affects the phytoplankton community. Optimizing the purification unit environment may be useful for improving the purification efficiency of pollutants and phytoplankton.

Abstract:Larimichthys crocea, an economically important fish species along the coast of China, has faced severe threats to its resources in recent years due to overfishing and loss of genetic diversity. To characterize the phenotypic traits of the less-studied Naozhou group of L. crocea, a combination of fish morphology, otolith morphology, and otolith chemistry was employed to discriminate the two stocks of L. crocea, the Naozhou and Min-yuedong groups sampled in Zhanjiang and Zhangzhou, respectively. The results of the principal component analysis indicated that the two populations could not be separated by the first two principal components of fish morphology, but could be distinctly separated by otolith morphology. Analysis of variance (ANOVA) indicated significant differences in fish and otolith morphologies between the two stocks (P＜0.05). The deformation diagram of fish morphology showed that Naozhou individuals were generally more elongated, with relatively shorter pectoral fin bases than Min-Yuedong individuals (P＜0.05). A deformation diagram of otolith morphology revealed that the otoliths of the Naozhou stock were more compressed overall, exhibiting a thicker neck in the sulcus and a relatively deeper first posterior dorsal depression than those of the Min-Yuedong stock (P＜0.001). Otolith chemical analysis revealed that the Ba/Ca and Sr/Ca ratios in the otoliths of the Naozhou stock were significantly higher than those in the Min-Yuedong stock, whereas the Mn/Ca ratio was significantly lower (P＜0.0001). There were no significant differences in the Mg/Ca ratios (P＞0.05). The Sr/Ca ratios exhibited the most pronounced differences between the two stocks from the core to the edge of the otolith when compared to the ratios of Ba, Mg, and Mn with Ca, with virtually no overlap at the measurement points. Discriminant analysis results indicated that the discrimination accuracy based on fish morphology (85.2%) was lower than that based on otolith morphology (100%) and chemistry (100%). The morphological differences between the two groups may be related to the differences in water flow and temperature between their habitats, with the Naozhou group inhabiting lower-flow and higher-temperature environments. Variations in otolith chemistry can be partly explained by salinity and temperature. Fish morphology, otolith morphology, and microchemical analyses demonstrated sufficient efficacy in the stock identification of the Naozhou and Min-yuedong groups of L. crocea. These findings provide a scientific basis for stock discrimination and fishery management of L. crocea.

Abstract:Based on the fishery biological survey data of the southwestern continental shelf of the Nansha Islands area in spring 2023, the trophic positions of major fishery organisms and the overlap of trophic niche among different functional groups were investigated by applying the carbon and nitrogen stable isotope techniques, and trophic continuum profiles were constructed. The results showed that the δ¹³C values of the fishery organisms ranged from −20.31‰ to −17.56‰, with a mean value of (−19.07±0.72)‰, δ¹⁵N values ranged from 7.79‰ to 10.80‰, with a mean value of (9.28±0.77)‰. Using zooplankton as the baseline organism, the trophic level of the fishery organisms ranged from 2.95 to 4.29. Compared with other marine areas in the South China Sea, the food chain length of the main fishery organisms in the southwestern continental shelf of the Nansha Islands area was relatively short, the initial food source was relatively single, the width of the niche was small, the degree of redundancy of consumers in the food web was high, and competition for food among species was intense. The results of cluster analysis of δ¹³C and δ¹⁵N values of fishery organisms were classified into four trophic groups, namely, zooplankton feeding group, mixed feeding group, large predator feeding group, and benthic feeding group, and the differences among the trophic groups were significant (P＜0.01). The trophic niches of fishery organisms were analyzed according to the δ−space (95% confidence interval) standard ellipse and structural parameter indexes, and it was found that the SEAc=0.66‰² . NR (δ¹⁵N range) and CR (δ¹³C range) of the mixed feeding group were 1.93 and 1.64, respectively, which indicated that the food resources were widely utilized and the food chain was long in this community. The niche overlap rates of the mixed-feeding group with plankton, large carnivores, and benthic feeding groups were 7.32%, 5.42%, and 0.60%, respectively. Compared to the pelagic zone in the central and western parts of the South China Sea, the overlap rate of trophic groups on the southwest shelf of Nansha was relatively low, indicating that the trophic niches of fishery organisms in different layers of the South China Sea have distinct spatial differentiation characteristics.