Abstract:To investigate the molecular characteristics, interaction, and nutritional regulation of CDK2 in grass carp (Ctenopharyngodon idella), we obtained the grass carp cdk2 sequence by cloning; its open reading frame is 897 bp, encoding 298 amino acids. It exhibited a high degree of sequence and structure conservation in the analyzed species. The results of codon bias and cluster analyses based on CDK2 suggested that the grass carp has the closest relationship with zebrafish. Compared with clustering based on cdk2 RSCU, a phylogenetic tree analysis based on CDK2 sequence was more consistent with the conventional species classification. Tissue expression analysis showed the highest cdk2 mRNA levels in blood (P＜0.05), liver, and spleen; molecular docking and yeast two-hybrid assays were employed to analyze the interaction between grass carp CDK2 and its downstream phosphorylated substrate protein CDX2. The results showed that grass carp CDK2 interacted with CDK2. Furthermore, the regulation of different inclusion levels of free amino acid CAA (free amino acids: lys and glu), dipeptide KE (lys-glu, KE), tripeptide GHK (gly-his-glu, GHK) on cdk2 expression were examined, and the results revealed that compared with the control and other treatment groups, cdk2 expression showed the highest expression in the 0.5% GHK group; cdk2 expression of the CAA groups was higher than those of the KE dipeptide group which showed similar amino acids as the CAA group. In summary, cdk2 expression in grass carp exhibits tissue specificity; CDK2 interacted with CDX2; and its expression was regulated by the inclusion level of amino acids and oligopeptides. This study provides new clues for studying the molecular characteristics of grass carp cdk2 exploring the regulatory mechanism of intestinal function in other teleost fish, and provides a theoretical basis for further study on the molecular regulating mechanism of cdk2 on transporting and absorption of amino acids and oligopeptides.

Abstract:Eriocheir sinensis is an important economic crab in China; it has a typical migratory habit, including an annual reproductive migration from November to May and for mating and spawning at the confluence of salty and fresh water at the mouth of the Yangtze River. When the broodstock crab spawns, the fertilized egg will commence a new life cycle. At this time, temperature is one of the key environmental factors regulating the development of the embryo, which is particularly sensitive to ambient temperature during its development, and even small temperature differences may cause stagnation of embryonic development. Therefore, it is important to investigate the effect of temperature on embryonic development of E. sinensis. In order to explore the molecular mechanism of temperature-regulated embryonic development in E. sinensis, three experimental groups were set up, namely, constant temperature of 9 ℃, constant temperature of 15 ℃, and gradually increasing temperature from 9 ℃ to 15 ℃. We utilized transcriptomics to screen genes associated with embryonic development and to identify the signaling pathways involved in the temperature-regulated embryonic development. The results showed that temperature significantly affected embryonic development process and effective cumulative temperature. At a constant temperature of 9 ℃, the embryos were stagnant at the egg cleavage stage, and their effective cumulative temperature was 1961.95 h·℃. In the experimental group, with a gradual temperature increase from 9 ℃ to 15 ℃, the embryos were able to develop to the prothallus larval stage, and embryo development time and effective cumulative temperature were reduced by 144 h and 1303.77 h·℃, respectively, compared with that under a constant temperature of 15 ℃. Temperature changes significantly regulated the expression patterns of genes related to embryo development. A total of 4767 differentially expressed genes were identified under a temperature increase from 9 ℃ to 15 ℃, including 1800 up-regulated genes and 2967 down-regulated genes. The gap gene (LOC126998238) related to embryonic development was significantly down-regulated, and the functional pathway was mainly enriched in endoplasmic reticulum protein targeting. At a constant temperature of 15 ℃, a total of 2061 differentially expressed genes were identified, including 1326 up-regulated genes and 735 down-regulated genes. Further analysis revealed that the maternal effect gene (LOC126984051) was significantly up-regulated in embryos, and the functional pathways were mainly enriched in small molecule metabolism. The results showed that embryo development accelerated with increasing temperature within a certain range and that variable temperature accelerated embryo development more than constant temperature. Variable temperature significantly increased the expression of genes related to embryonic development and embryonic energy metabolism, and it accelerated embryonic development. The results of this study can provide a theoretical reference for the interpretation of the regulatory mechanism of temperature on the embryonic development of E. sinensis.

Abstract:Garra mirofronits is classified under the order Cypriniformes, family Cyprinidae, subfamily Labeoninae, and genus Garra. This species is endemic to the Lancang River and is exclusively found in the middle and lower reaches of the river within China. Its distribution is confined to an altitude range of 500‒700 meters above sea level, and its wild populations have declined. G. mirofronits exhibits a cylindrical body shape with a distinctive frontal groove anterior to the nostrils, which forms two frontal processes. Each process terminates in a prominent conical keratinous projection. This species prefers habitats characterized by swift currents and rocky substrates, where it adheres to rocks using its adhesive discs and primarily feeds on filamentous algae. In recent years, the progressive development of the Lancang River, including the construction and operation of hydropower stations, has altered the river's hydrological conditions, thereby impacting the migratory, reproductive, and feeding behaviors of fish. Notably, the decrease in flow velocity and the reduction in the length of flowing water have severely compromised the hatching success of drifting eggs, including eggs of G. mirofronits. Consequently, the wild populations of G. mirofronits have experienced a significant decline, necessitating urgent conservation measures. This study aims to investigate the temporal characteristics of the early development of G. mirofronits, enrich research data on its reproductive and developmental biology, and offer technical support for artificial breeding and large-scale seedling production of this species. Sexually mature individuals of G.mirofronits were collected from the wild and used to obtain fertilized eggs through artificial induction of ovulation and dry insemination. Then, the early development process of the fertilized eggs was observed and recorded in detail. Three artificial breeding experiments were carried out in total. On average, the ovulation induction rate was 100%, the fertilization rate was (89.93±0.72)%, and the hatching rate was (87.83±1.00)%. The fertilized eggs were light gray, with a thin and transparent egg membrane, non-viscous, and a diameter of (1.12±0.04) mm. After water absorption and expansion, the egg membrane diameter reached (3.37±0.09) mm, with an expansion coefficient of 300.89%. At water temperatures between 18‒21 ℃, hatching occurred after 24‒26 hours, with an accumulated hatching temperature of 496.04 ℃·h. The early development process went through nine stages including fertilized egg, blastoderm stage, cleavage stage, blastula stage, gastrula stage, neurula stage, organogenesis stage, heartbeating stage, and hatching stage. The total length of newly hatched larvae was (4.50±0.07) mm, with 32 myotomes. The period of the yolk sac larvae lasted for 5 days, with the specific growth rate of the total length at 9.22%/d. The larval stage lasted for a total of 16 days, with the specific growth rate of the total length at 7.02%/d. The temporal characteristics of the early development of G.mirofronits follow the basic laws of osteichthyes development and are in line with the general characteristics of the embryonic development of drifting eggs. It is thus a typical fish species that produces drifting eggs.

Abstract:In order to elucidate the effect of nucleus insertion stimulation on hemolymphocytes and their function in pearl mineralization of the freshwater pearl mussel Hyriopsis cumingii, the present study explored the morphology and classification of hemolymphocytes, including four types by means of light and electron microscopy based on cellular complexity, cell size, and nucleo-cytoplasmic ratio. Giemsa staining and flow cytometry analysis showed that the granularity complexity of hemolymphocytes increased significantly, cell diameter became larger after nucleus insertion, the proportion of granulocytes increased significantly (P＜0.05), and the proportion of hyalinocyte decreased significantly (P＜0.05). The results of histological observation showed that a large number of hemolymphocytes gathered near the insertion site and granulocytes were recruited. Ca²⁺ content in hemolymphocytes and serum continued to significantly increase 2, 5, and 10 d after insertion (P＜0.05) and then significantly decreased 20 and 50 d after insertion (P＜0.05), which revealed that the number composition, distribution, and Ca²⁺ level of hemolymphocytes all respond to nucleus insertion. In vitro calcium carbonate crystallization experiments showed that hemolymphocytes and serum were able to influence the morphology of calcite but did not change the crystal shape. In summary, hemolymphocytes respond rapidly to the stimulation of nucleus insertion and can regulate biomineralization. This study lays a theoretical foundation for further research on the regulatory role of hemolymphocytes in shellfish biomineralization.

Abstract:Saline-alkaline water resources are widely distributed in China. However, their imbalanced ionic composition, high pH, and elevated carbonate alkalinity significantly impair normal growth and reproduction of fish. Although previous studies have explored the effects of saline-alkaline stress on the growth performance of aquatic organisms, these investigations have largely focused on low-concentration environments, leaving the impacts of high-concentration conditions on growth performance and associated metabolic changes insufficiently understood. This study aimed to elucidate the mechanisms by which carbonate-alkaline environments affect the growth performance and physiological metabolism of crucian carp (Carassius auratus). Experimental fish were randomly assigned to three groups: a freshwater control group (Con), a 20 mmol/L NaHCO3 exposure group (T), and a 40 mmol/L NaHCO3 exposure group (F), subjected to carbonate-alkaline exposure for 56 days. Through the application of growth indices, biochemical assays, and non-targeted metabolomics analysis using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) coupled with multivariate techniques such as principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and pathway enrichment tools like MetaboAnalyst 5.0 and KEGG database, the study systematically examined the effects of carbonate-alkaline exposure on the growth, oxidative defense, and metabolomics profile of crucian carp. The results revealed that carbonate-alkaline exposure significantly suppressed growth indices, including weight gain rate, specific growth rate, and feed conversion efficiency. Moreover, with increasing carbonate-alkaline concentrations, antioxidant enzyme activities in the liver—such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)—and blood biochemical parameters, including blood ammonia, blood urea nitrogen (BUN), total cholesterol (TC), and triglycerides (TG), underwent notable alterations. Pathway enrichment analysis identified 17 statistically significant differential metabolites (DEMs) in the Con vs. T group, enriched in 27 metabolic pathways, including unsaturated fatty acid biosynthesis, purine metabolism, glycerophospholipid metabolism, and glutathione metabolism. In contrast, the Con vs. F group identified 100 DEMs enriched in 35 pathways, encompassing additional processes such as arginine biosynthesis, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, arachidonic acid metabolism, and sphingolipid metabolism. The findings indicate that carbonate-alkaline exposure imposes a substantial energetic burden on crucian carp, undermining the energy availability for growth and thereby significantly inhibiting growth rates. This condition induces oxidative stress by disrupting the balance between reactive oxygen species (ROS) and the oxidative defense system, leading to severe hepatic tissue damage. As carbonate-alkaline concentration increases, critical metabolic pathways associated with growth, immune defense, and fatty acid metabolism—such as arginine biosynthesis, unsaturated fatty acid biosynthesis, purine metabolism, glycerophospholipid metabolism, sphingolipid metabolism, and glutathione metabolism—experience pronounced disruption. This exacerbates hepatic damage, compromises immune defense, destabilizes cellular membrane integrity and function, and suppresses cellular proliferation, ultimately impairing normal growth.By integrating growth performance, biochemical analysis, and metabolomics, this study provides a comprehensive understanding of the adverse effects of carbonate-alkaline environments on the growth and hepatic metabolism of crucian carp. The findings offer critical insights for optimizing aquaculture practices involving freshwater bony fish in saline-alkaline waters, contributing to the sustainable utilization of saline-alkaline water resources with significant ecological, economic, and societal benefits.

Abstract:Fishing pressure and environmental factors can not only affect the abundance and distribution of fish, but also affect the age-and length-distributions. Length-based indicators of fish community are useful tools for fishery management. Based on the fishery resource survey data collected in the Haizhou Bay and its adjacent waters in spring and autumn from 2013 to 2022, seven length-based indicators including median length (L₅₀), mean length (mL), 10th and 90th-percentile of the length distribution (L₁₀, L₉₀), mean length of the 10% largest fish (L_max), large fish index (LFI) and size-diversity (SD) were analyzed to examine the dynamics of fish community structure in the Haizhou Bay as well as its response to the fishing pressure and environmental factors. The results showed that the robustness of L10, mL and L90 was better, and they were suitable for assessing the status of fish community in the Haizhou Bay. Random forest model results suggested that length-distributions of fish community in the Haizhou Bay were affected by a combination of fishing pressure and environmental factors. In terms of interannual variation, L₁₀, L₅₀ and mL showed a upward trend, while L₉₀, Lmax and SD fluctuated, and LFI showed a declining trend, reflecting the recovery of small and medium-sized fish resources in the Haizhou Bay. However large-sized fish are still affected by fishing pressure, and more efforts should be made to protect and manage the fishery resources in Haizhou Bay. This study explored the potential of using body length-based indicators for setting fishery management objectives, which will help to gain a deeper understanding of the ecological status of fish community dynamics in the Haizhou Bay, and provide technical support for the protection and restoration of the Haizhou Bay fish community.

Abstract:Accurately calculating catch per unit effort (CPUE) is a prerequisite for predicting fishery resource abundance and distribution. In tuna longline fisheries, the statistics of catch and effort are often influenced by spatial scale, and different sources of catch and effort data can result in biased CPUE estimates, which thereby potentially affect the forecast precision of fishing grounds. This study utilized fishery data obtained from longline vessels operating in waters near Micronesia, derived from both fishing logbooks and the VMS (vessel monitoring system), to calculate two types of CPUE for yellowfin tuna (Thunnus albacares). Using a BiLSTM (Bi-directional long-short-term memory) model, both types of CPUE were employed as inputs, alongside spatio-temporal factors and ocean remote sensing data, to develop a predictive model for yellowfin tuna fishing grounds. By SHAP (SHapley Additive exPlanations) analysis, key variables influencing the prediction of yellowfin tuna fishing grounds were identified. The spatio-temporal distribution characteristics of fishing grounds were determined based on the geographical distribution of different CPUE values. This study also evaluated the impact of different CPUE inputs on the accuracy of fishing ground prediction. The results showed that the BiLSTM model effectively predicts yellowfin tuna fishing grounds, with strong performance within the study area. Moreover, the model using CPUE calculated from VMS data demonstrated higher accuracy compared to the model built by using logbook data. Key variables influencing yellowfin tuna fishing ground predictions included chlorophyll-a concentration, dissolved oxygen concentration at 200 m, temperature at 300 m, temperature at 200 m, and longitude. Fishing grounds derived from fishing logbook were dispersed across the north and south, whereas those derived from VMS data were mainly concentrated in the southern region. Based on the vertical profile of temperature and dissolved oxygen, it was inferred that the fishing grounds identified by VMS data are more accurate. In the waters of Micronesia, yellowfin tuna fishing grounds were more densely distributed in the first and second quarters. This study demonstrates that VMS-based CPUE can be calculated for longline fisheries and effectively used for fishing ground prediction. Based on this study, it is recommended to record catch locations individually using the VMS method, when investigating tuna fishing grounds in longline fisheries at smaller spatial scales. This allows for grid-based CPUE calculations, thereby improving the accuracy of fishing ground prediction.

Abstract:Hydrological connectivity is one of the key characteristics that reflect the health and function of estuarine wetland ecosystems, and it has a significant impact on the distribution of estuarine fish communities. This study analyzed the dynamic hydrological connectivity of tidal creeks in the Chongming Dongtan area of the Yangtze River Estuary under different tidal states and its influence on fish community distribution, aiming to provide a scientific basis for the ecological restoration, biodiversity conservation, and water resource management of estuarine wetlands. Twelve hydrological connectivity indicators related to hydrological dynamics and landscape dimensions were selected to explore the relationships and underlying mechanisms between hydrological connectivity patterns and fish distribution patterns. The results showed that factors such as tidal range, the duration of hydrological connection, the duration of flooded retention, the number of nodes, the number of river chains, and the node connection rate β are key hydrological connectivity factors. Canonical correlation analysis (CCA) indicated that the sampling regions within the tidal creek system could be divided into five distinct hydrological connectivity areas, i.e., the high connectivity area during spring tides, the low connectivity area during spring tides, the high connectivity area during neap tides, the low connectivity area during neap tides, and the long-term inundation area. Among them, the high connectivity areas during spring tides, the high connectivity areas during neap tides, and the long-term inundation area are the main habitats of dominant fish species. Dominant fish species exhibited different preferences for different hydrologically connected areas. For example, Xenocypris macrolepis and Pelteobagrus nitidus tended to inhabit the high hydrological connectivity areas during spring tides. Lateolabrax maculatus, Mugilidae, and Gobiidae exhibited a preference for high hydrological connectivity areas during neap tides. Juvenile fish such as Coilia ectenes and Protosalanx chinensis preferred to inhabit areas at the mouths of tidal creeks or near the shore. These findings demonstrate that estuarine tidal creek fish actively exercise selection and exhibit preferences for different hydrologically connected habitats under the influence of tides, which are related to their swimming capabilities, body sizes, and feeding behaviors. This study provides a scientific basis for the ecological restoration of estuarine wetlands and the conservation of fish biodiversity. It also helps to formulate strategies for regulating hydrological connectivity in wetland water resource management, thereby promoting the sustainable development of such ecosystems.

Abstract:White spot syndrome virus (WSSV) poses a significant threat to red swamp crayfish (Procambarus clarkii) aquaculture, a major crustacean industry in China. This virus' ability to remain latent in hosts and its complex transmission pathways make prevention and early detection crucial. However, current diagnostic methods, including the WOAH-recommended nested PCR, have limitations in speed, sensitivity, and convenience. This study aimed to develop a rapid, sensitive, and specific one-step conventional PCR assay for WSSV detection in P. clarkii, addressing the need for improved surveillance and early intervention strategies. In our study, specific primers (V1) were designed to target a 196 bp region within the WSSV vp19 gene. The assay's analytical specificity was rigorously evaluated using DNA/cDNA from 10 different pathogens in crayfish (5 viruses and 5 bacteria). To determine analytical sensitivity, serial dilutions of a recombinant plasmid (pMD19T-V1) containing the vp19 target sequence were employed. WOAH recommended TaqMan qPCR was used as the reference method in performance parameters evaluation. The assay’s performances parameters including analytical specificity (ASp), analytical sensitivity (ASe), diagnostic specificity (DSp), diagnostic sensitivity (DSe), and positive rate between the one-step PCR and nested PCR were compared. Results showed that developed one-step PCR assay demonstrated high ASp for WSSV detection, no cross-reactivity was detected against other 9 common pathogen samples.ASe tests revealed impressive detection limits: using the WSSV standard plasmid as a template, the theoretical minimum detection limit was 1.83 copies/μL, while with mixed DNA from field samples, the limit reached 0.82 fg/μL. Notably, in both scenarios, this method outperformed the WOAH-recommended nested PCR in terms of sensitivity. Comparison of diagnostic performance on 317 field samples showed that the one-step PCR achieved a DSp of 97.01% and a DSe of 95.08%, both superior to the WOAH-recommended nested PCR. In conclusion, this study introduces an innovative one-step PCR method for WSSV detection in P. clarkii, based on the vp19 gene, achieving a remarkable detection limit of 1.83 copies/μL while maintaining simplicity and cost-effectiveness. Outperforming the WOAH-recommended nested PCR, this highly sensitive approach excels in identifying latent infections and facilitating epidemiological investigations. Its ability to detect low-level infections enhances its versatility for both routine screening and research, providing a powerful tool for year-round dynamic monitoring and early warning of WSSV. While acknowledging current limitations, this groundbreaking method lays a solid foundation for future advancements in aquatic pathogen detection. By combining accuracy, sensitivity, and practicality, this research represents a significant step forward in WSSV detection technology, with far-reaching implications for the sustainable development of crayfish aquaculture in both scientific and economic contexts.

Abstract:To explore the mechanisms of astaxanthin alleviating the physiological abnormalities caused by blue body syndrome (BBS) in Litopenaeus vannamei, including body color abnormalities, growth retardation, and health deterioration, this study was conducted using two astaxanthin-supplemented diets: 0 mg/kg (control group, CT) and 100 mg/kg (astaxanthin group, AX). Juvenile shrimp [initial weight: (0.77 ± 0.27) g] were reared for 60 days in a recirculating aquaculture system. A systematic comparison of body color, astaxanthin content in various tissues, tissue structure, growth performance, antioxidant capacity, and immune function was performed between the two groups. The results showed: (1) Shrimp in the CT group exhibited blue body coloration, a typical symptom of BBS, whereas AX shrimp showed normal light brown coloration. During the trial, total astaxanthin content in the hepatopancreas, cuticle, and muscle tissues in CT shrimp decreased by 21.51%, whereas that in AX shrimp increased by 95.17%, with significantly higher astaxanthin levels in the hepatopancreas and cuticle (P＜0.05). At the end of the experiment, the blue-body ratio was 8.67% in the AX group, significantly lower than in the CT group (76.00%). These results suggest that exogenous astaxanthin supplementation can significantly increase astaxanthin deposition, improved body coloration, and reduced BBS incidence in shrimp. (2) Histological analysis revealed severe pathological changes in the CT group, including hepatopancreatic cell atrophy and deformation, loose muscle fiber arrangement, and detachment of the intestinal epithelial cells. In contrast, the AX group showed no tissue abnormalities, indicating that BBS caused structural damage to critical tissues, and astaxanthin supplementation helped maintain normal tissue structures. (3) Compared with CT shrimp, AX individuals showed significantly enhanced activities of digestive enzymes (amylase, lipase, and trypsin) and increased relative expression levels of growth-related genes (igf-1 and igf-2), resulting in improved growth performance (P＜0.05). These findings suggest that astaxanthin mitigates BBS-induced growth retardation by enhancing digestive enzyme activity and promoting the expression of growth-related genes. (4) AX shrimp exhibited significantly higher total antioxidant capacity, superoxide dismutase, and catalase activities, and lower malondialdehyde levels than CT shrimp (P＜0.05), indicating that astaxanthin ameliorated the oxidative imbalance caused by BBS through enhancing antioxidant enzyme activities. (5) Expression of immune-related genes, including crustin, penaeidin 3a, proPO, and relish, was significantly upregulated in AX shrimp compared with that in CT shrimp (P＜0.05), demonstrating that astaxanthin alleviates BBS-induced immunosuppression by promoting immune-related gene expression. In conclusion, nutritional metabolic disorders due to insufficient intake of astaxanthin are a critical cause of BBS in L. vannamei. Astaxanthin is preferentially utilized in shrimp to strengthen their antioxidant defense system, protecting tissue structure and physiological metabolism before being deposited for pigmentation, thus ameliorating the body color abnormalities and physiological dysfunctions associated with BBS.

Abstract:In this study, skin ulcer syndrome (SUS) broke out in three cage culture areas of Apostichopus japonicus in Xiaoleijiang, Wenqi and Weijiangcounty of Fujian Province in November 2023. The study analyzed the pathogenesis, bacterial structure, and correlations between bacterial and environmental factors, based on high-throughput sequencing of the 16S rDNA V3‒V4 variable region of microorganisms from sampled tissues of A. japonicus diagnosed with SUS and surrounding water samples. The bacterial community structure analysis results showed that the detected bacteria belonged to 33 phyla, 72 classes, and 350 families. The top three dominant phyla were Proteobacteria, Bacteroidota, and Actinobacteriota. The dominant bacterial families included Flavobacteriaceae, Rhodobacteraceae, Enterobacteriaceae, and Microbacteriaceae, etc. In the water samples, Proteobacteria and Bacteroidota were the two dominant phyla, and the dominant bacterial families consisted of Flavobacteriaceae, Erythrobacteriaceae, Actinomarinaceae, and unclassified Cyanobacteria. Alpha diversity analysis showed that Chao1 and ACE indices exhibited significant differences between the body wall and water samples of A. japonicus from different regions. PCoA analysis indicated that water samples clustered together, surrounding the body wall samples, suggesting that the water bacterial was associated with the health of sea cucumbers. Correlation analysis of the top 20 microflora at the family level in the diseased tissue and water samples revealed that 100 pairs of 18 microflora had significant correlations, with the correlation coefficients between Trichospirillaceae, Spirillaceae, and Muribaculaceae exceeding 0.95. Correlation analysis between microbial flora and environmental factors indicated that environmental factors BOD/Hg/Zn/Ni were significantly correlated with microbial alpha diversity (P＜0.05) of diseased tissues and water samples. The results above indicated that the diseased tissues had certain association with the flora in the water, and the environmental factors had considerable effects on the structure of the flora and the stability of the microecology. This study provided some scientific basis for the prevention and control of A. japonicus skin ulcer syndrome, while supporting the preservation of culture environment and the optimization of the culture process for sea cucumber.