Abstract:The Chinese sturgeon (Acipenser sinensis) is one of the key aquatic animals under first-degree protection in China, but its germplasm resources, such as spermatogonial stem cells, are not well protected by cryoprotection technology. In this study, we investigated the effects of thawing temperature, antioxidants, and antifreeze proteins on the cryopreservation of spermatogonial stem cells of Chinese sturgeon based on the formulation of the spermatogonial stem cell cryopreservation solution used for the American paddlefish (Polyodon spathula). The aim was to establish a highly efficient method for ultra-low-temperature cryopreservation of spermatogonial stem cells of Chinese sturgeon. The best cryopreservation effect was achieved at a thawing temperature of 25 ℃ among the temperatures tested (20, 25, 30, and 40 ℃). The number of cells after thawing was (3.86±0.51)×10⁵ , and the cell viability reached (96.36±0.53)%. The effects of different concentrations (500 mg/L, 1000 mg/L, and 1500 mg/L) of glutathione, ascorbic acid, and α-tocopherol on the cryopreservation of spermatogonial stem cells of Chinese sturgeon were examined. The results showed that the experimental group with the addition of 1000 mg/L α-tocopherol to the cryopreservation solution had the highest number of cells (7.64±0.34)×10⁵ after thawing, which was significantly higher than that of the other groups with antioxidants added, and the cell viability reached (92.82±0.72)%. The effects of different concentrations (0.1, 1.0, and 10 μg/mL) of two types of antifreeze proteins (AFPI and AFPIII) on the cryopreservation of spermatogonial stem cells of Chinese sturgeon were examined. The experimental group with 1.0 μg/mL AFPI had the best defrosting effect, and the number of cells obtained after defrosting was (6.85±0.19)×10⁵ , which was significantly higher than the number in the other experimental groups, and the cell viability was (86.89±0.73)%. In summary, in the present study, we obtained the optimal thawing temperature for the cryopreservation of spermatogonial stem cells of Chinese sturgeon, evaluated the effects of three types of antioxidants and antifreeze proteins on the effect of cryopreservation, and established a highly efficient cryopreservation technology for spermatogonial stem cells of Chinese sturgeon. These data provide technical support for the cryopreservation and restoration of the Chinese sturgeon’s germplasm resources.

Abstract:NLRP3, a classic pattern recognition receptor, combines with ASC and pro-caspase 1 to form the NLRP3 inflammasome, which is responsible for the activation of pyroptosis in teleost fish. To further investigate the role of NLRP3 protein in largemouth bass (Micropterus salmoides), this study amplified an Msnlrp3 segment using largemouth bass cDNA as the template and constructed the pET-32a-MsNLRP3 prokaryotic recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) cells, and a soluble fusion protein was expressed after induction with 0.8 mmol/L isopropyl β-D-1-thiogalactopyranoside overnight at 16 ℃. The obtained recombinant protein was purified using affinity chromatography, and then identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analysis to confirm the successful construction of the prokaryotic expression system for MsNLRP3. Subsequently, the purified MsNLRP3 protein was used to immunize Japanese long-eared rabbits and Balb/C mice to obtain polyclonal antibodies, respectively. The titers and specificity of the obtained antibodies were determined using enzyme-linked immunosorbent assay (ELISA) and western blotting. Results showed that the obtained antisera, after immunization, could specifically recognize the recombinant and endogenous NLRP3 protein in largemouth bass tissues, as indicated by a single, clear target band consistent with the expected molecular weight. Meanwhile, the titers of rabbit and mouse antisera were 1∶10240 and 1∶1024, respectively. To test the application potential, a Flavobacterium columnare infection model via immersion was constructed in largemouth bass, in which the gills exhibited significant histopathological changes. Western blotting analysis with the polyclonal antibody obtained from this study indicated that NLRP3 protein levels were significantly increased in gills after bacterial infection. Therefore, the polyclonal antibody against MsNLRP3 prepared in this study provides an important material basis for the functional research of NLRP3 protein in largemouth bass.

Abstract:Procambarus clarkii is an economically important aquaculture species in China, and pathogens, especially Vibrio parahaemolyticus, have caused significant economic losses in large-scale farming. However, the mechanism of the immune system response to pathogen invasion remains unclear. To delve into the molecular mechanisms behind the immune regulation of the Procambarus clarkii, this study analyzed the transcriptome data of its hemocytes that had been co-cultured with 3-hydroxybutyrate. By analyzing the transcriptome data of hemocytes from P. clarkii co-cultured with 3-hydroxybutyrate, significant differential expression of cytoskeleton genes was observed, suggesting a potential association with immune regulation in P. clarkii. This study annotated and classified cytoskeleton genes and validated their impact on cellular immune regulation. The annotation results showed that out of five cytoskeleton genes, four belonged to the kinesin family (KIF). All contained KISc domains, and they encoded amino acids primarily composed of glutamate and leucine. The secondary protein structure was mainly composed of α-helices. The genes encoding KIFs were generally similar across species, except for variations in the number, type of conserved motifs, and gene structure among kinesin-like-protein genes in different species. Quantitative reverse transcription-polymerase chain reaction results showed that the expression levels of KIFs were higher in the hemopoietic tissues and gills of P. clarkii than in tissues such as eyestalks and muscles (P＜0.05). RNA interference results demonstrated that the optimal knockdown effect was achieved with a dose of 0.10 μg/g body weight of dsKIF11, reducing the expression level of KIF11 to 8.01% of its normal level (P＜0.05). Moreover, compared to the control group, after the knockdown of the KIF11 gene, the phagocytic rates of hemocytes towards green-fluorescence-labeled V. parahaemolyticus and red fluorescent microspheres decreased by 36.26% (P＜0.01) and 41.94% (P＜0.01), respectively. The abovementioned results indicate that KIFs exhibit high conservation and are involved in the phagocytic activity of hemocytes. This finding provides a basis for further investigation of the function and mechanism of KIFs in innate immunity.

Abstract:Due to excessive human disturbance, the germplasm resources of Quasipaa spinosa are experiencing a decline, and the gradual reduction in genetic diversity has become an urgent issue that needs to be addressed. The objective of this study was to identify the germplasm of different geographical populations, comprehensively evaluate their characteristics, and identify excellent breeding populations. This would provide important reference materials for subsequent population selection and the optimization of germplasm resources. The study employed methods such as single-factor analysis of variance, principal component analysis, cluster analysis, and discriminant analysis to conduct morphological analyses of 120 cultured individuals from four geographical populations (Guixi YJFGX, Yifeng LHSYF, Yudu LHSYD, and Taining LHSTN). Genetic differences among Quasipaa spinosa populations were also analyzed based on simplified genome sequencing results. The findings revealed variations in the morphological characteristics among the four geographical populations. Particularly notable were the differences between LHSYD and the other three populations; however, there were similarities in morphological traits between LHSTN and YJFGX, without significant differentiation. Four geographical populations exhibit a relatively common sexual dimorphism in body limb length and other related indicators. Based on morphological characteristics, the four populations can be roughly divided into three major branches: the LHSTN population and the YJFGX population each form a branch, while the LHSYF population and the LHSYD population comprise another branch. The observed heterozygosity (H_o) ranged from 0.035 to 0.038, lower than the expected heterozygosity (H_e) ranging from 0.103 to 0.129. Moreover, the average inbreeding coefficient ranged from 0.197 to 0.287, indicating a high level of inbreeding within these populations. A relatively high degree of genetic differentiation was observed between LHSTN and LHSYF (F_st=0.303), LHSYD (F_st=0.277), and YJFGX (F_st=0.276) populations; however, only moderate genetic differentiation existed among LHSYF, LHSYD, and YJFGX populations. In summary, (1) there is morphological variation among the four studied populations, but not at the subspecies level and (2) the overall genetic diversity of the population was relatively low, indicating a high degree of genetic differentiation and independent genetic information. This characteristic makes them suitable as parental sources for crossbreeding, thereby enhancing the genetic diversity of the local population and optimizing germplasm resources.

Abstract:This study aimed to enhance our understanding of the changes in the intestinal microbiota of juvenile largemouth bass (Micropterus salmoides) resulting from changes in dietary habits. The intestinal contents of 13-, 19-, 24-, and 46-day-old largemouth bass were continuously collected, representing the feeding live-bait period (LP), the first conversion diet period (CP1), the second conversion diet period (CP2), and the combination feed period (FP). 16S rRNA amplicon sequencing was used to investigate species diversity and differences in relative species abundance in the intestinal microbiota of juvenile largemouth bass. A Goods coverage value of 1 was obtained for the intestinal microbiota across all periods, suggesting that the sequencing results reflected the microorganisms present in the intestinal tract. The conversion of bait significantly affected the gut microbial diversity of the larvae and juveniles. Significant changes in species type were observed in the intestinal microbiota during CP1 and CP2, with the species composition being simpler. During LP and FP, the intestinal microbiota exhibited high evenness in species composition. The highest biodiversity and structural complexity in the intestinal microbiota were observed during FP, with 16 microbial communities having average relative abundances greater than 1% at the genus level. This number was only eight species for LP, and two or fewer species for both CP1 and CP2. Significantly higher average relative abundance values for Pseudomonas were observed in CP1 and CP2 (93.27% and 97.33%, respectively). Furthermore, significant differences in the relative abundance of dominant bacterial species were identified at different periods, indicating that diet conversion had a notable impact on gut-related functional microorganisms. This may be the intestinal response of largemouth bass to the absorption and utilization of feed, which has complex ingredients. This study provides preliminary data for the study of fish domestication and diet conversion and offers new insights into improving or optimizing largemouth bass feeding technology from the perspective of gut microbiota regulation.

Abstract:In this study, we aimed to explore the effects of low-temperature dormancy on survival rate, apparent traits, immune function, antioxidant capacity, and muscle quality of Procambarus clarkii, providing a reference for off-season listing of P. clarkii. Experimental animals included P. clarkii with a body weight of (17.98±0.83) g and a body length of (6.55±0.07) cm. Initially, P. clarkii were cooled from 25 ℃ at a rate of 5 ℃/h to determine the critical dormancy and death temperatures. Subsequently, cooling and heating rates were set at 2 ℃/h, 3 ℃/h, 4 ℃/h, and 5 ℃/h, with different dormancy temperatures applied to P. clarkii. Optimal dormancy conditions were determined based on different cooling rates, dormancy temperatures, and survival rates of P. clarkii. Changes in apparent traits, serum immune and antioxidant indicators, and muscle quality of P. clarkii under optimal dormancy conditions were then assessed. The critical dormancy temperature for P. clarkii was 7 ℃, and the critical death temperature was 0 ℃. The highest survival rates were observed at a cooling rate of 2 ℃/h, 5 ℃ dormancy for 60 days, and a warming rate of 2 ℃/h, with survival rates of (97.50±1.12)%, (60.50±2.02)%, and (98.33±1.05)%, respectively. Therefore, the optimal dormancy conditions were a cooling rate of 2 ℃/h, 5 ℃ dormancy, and a warming rate of 2 ℃/h. No significant differences in body weight, body length, condition factor (CF), hepatopancreas weight, or hepatopancreas index (HSI) were observed after 60 days of dormancy compared with 0 days under optimal conditions. Similarly, no significant differences in ACP activity, SOD activity, and MDA content were observed in the serum of P. clarkii after 15, 30, 45, and 60 days of dormancy (P＞0.05). AKP activity at 30 days of dormancy was significantly higher than that at other time points (P＜0.05), while AKP activity at 60 days was significantly lower (P＜0.05). After 60 days of dormancy, muscle hardness and recovery of P. clarkii were significantly reduced (P＜0.05), and cohesion was significantly increased (P＜0.05); changes in muscle umami amino acids alanine, aspartic acid, glutamic acid, and glycine were not significant (P＞0.05). In conclusion, the cooling rate of 2 ℃/h, dormancy at 5 ℃, and heating rate of 2 ℃/h were optimal low-temperature dormancy survival conditions. After 60 days of dormancy under these optimal conditions, the apparent traits of P. clarkii did not change significantly. However, with prolonged dormancy, immune and antioxidant regulatory functions weakened, and survival rates decreased. Although low-temperature dormancy caused some changes in the physical and chemical properties of P. clarkii muscle, muscle flavor did not change significantly.

Abstract:Before culturing adult Chinese mitten crabs (Eriocheir sinensis), aquaculture ponds are sun-dried to enhance the bottom environment for the next cultivation stage. However, during the sunning stage, the sediment in these ponds may release significant amounts of CO₂ into the atmosphere. We focused on E. sinensis aquaculture ponds in Suqian, Jiangsu Province, using soil carbon flux measurement instruments to monitor and analyze CO₂ emission fluxes over two and a half months, from mid-December 2023 to early March 2024. Continuous monitoring was conducted both day and night to determine CO₂ emission fluxes throughout the sunning stage. CO₂ emission fluxes were measured three times a day and night on December 19, 2023, January 24, 2024, and February 28, 2024. Each monitoring session began at 8:00 AM and continued every three hours. Additional measurements were taken on December 14, 2023, December 24, 2023, January 4, 2024, January 14, 2024, January 24, 2024, February 3, 2024, February 13, 2024, February 23, 2024, and March 4, 2024, between 8:00–10:00 AM. Simultaneous meteorological and soil indicators were measured to analyze environmental factors affecting CO₂ emission fluxes. Results showed that the average daily CO₂ emission fluxes from the pond bottom after one week, one month, and two months of sunning were (1214±633) mg/(m²·d), (1055±422) mg/(m²·d), and (407±93) mg/(m²·d), respectively. Emission fluxes peaked in the afternoon and were lowest in the early morning. A diurnal pattern was observed, with higher emission fluxes during the day and lower at night. As sunning time extended, the day-night difference in emission fluxes gradually decreased, though a significant difference persisted. The range of CO₂ emission fluxes at the sediment-atmosphere interface during the sunning stage was 351– 2331 mg/(m²·d), with an average of (1040±647) mg/(m²·d). The CO₂ emission fluxes showed a trend of initial increase followed by a decrease, indicating that the entire sunning stage was a source of CO₂ emissions. Stepwise regression analysis identified soil temperature and soil porosity as the primary environmental factors influencing CO₂ emission fluxes. A significant positive correlation was found between soil temperature and CO₂ emission fluxes, while soil porosity was negatively correlated due to the effects of soil moisture and thermal conditions. Cumulative CO₂ emissions increased gradually, although the growth rate slowed over time, with total cumulative CO₂ emission fluxes during the sunning stage calculated at 89.3 g/m². In this study, we reveal the changes in CO₂ emission fluxes during the sunning stage of E. sinensis aquaculture ponds, highlighting these ponds as significant CO₂ emission sources during this period. There is substantial potential for CO₂ reduction in the E. sinensis aquaculture industry. The data provided by this study support efforts to reduce greenhouse gas emissions and develop corresponding emission reduction strategies in E. sinensis aquaculture ponds.

Abstract:Channa maculata, an economically significant freshwater fish in China, has been subject to a limited number of population studies. To explore the genetic diversity of naturally distributed populations of C. maculata, single nucleotide polymorphism (SNP) markers were developed and genetically analyzed in seven populations (Yangchun, Guangdong; Huazhou, Guangdong; Nanning, Guangxi; Hezhou, Guangxi; Jianghua, Hunan; Shaowu, Fujian; Taipei, Taiwan) using the simplified genome sequencing technology of SLAF. The findings revealed the detection of 2502887 SNP loci, with an average sequencing depth of 20.19×, and average Q30 and GC contents of 95.31% and 41.16%, respectively. Population genetic structure analysis revealed individual clustering of all seven populations, each with a distinct genetic structure. The polymorphic information content of the seven C. maculata populations was 0.2672–0.2872, the expected heterozygosity (H_e) was 0.3304–0.3580, and the observed heterozygosity (H_o) was 0.2145–0.3393. The populations had a medium level of genetic diversity. The genetic differentiation index (F_st) was 0.15300–0.78658 and the gene flow (N_m) was 0.24118–0.62035, showing a medium-high genetic differentiation status among populations. Phylogenetic analysis revealed that the Hezhou population was most closely related to the Jianghua population. Given their close geographical proximity in Guangxi and Hunan, the Hezhou and Huazhou populations can be considered part of the same population unit. The Taibei group exhibited the smallest differences in H_e and H_o, indicating a high degree of genetic purity. Conversely, both the Hezhou and Huazhou groups displayed lower H_o than H_e and substantial differences. Furthermore, the seven populations exhibited high genetic differentiation from each other, with the Taipei group showing extreme differentiation, likely due to the strait’s isolation effect. This study elucidated the genetic background and relationships among wild C. maculata populations across various regions, and serves as a valuable reference for the strategic utilization of spotted snakehead germplasm resources and genetic breeding endeavors. Particular emphasis should be placed on introducing new lineages in future breeding efforts to enhance genetic diversity within C. maculata populations and facilitate the rational exploitation of C. maculata germplasm resources.

Abstract:To study the effect of Miaoling Mountain as a geographical barrier on the genetic structure and diversity of Siniperca scherzeri, the genetic diversity of seven wild S. scherzeri populations sampled from the Liujiang, Yuanjiang, and Wujiang River systems on both sides of Miaoling Mountain was examined by sequencing the mitochondrial DNA D-loop and analyzing their genetic variation and population dynamics. The results showed that out of 365 sequences of S. scherzeri, 57 polymorphic loci and 56 haplotypes were identified. The high-frequency haplotype hap3 was an ancestral haplotype shared by the three river systems. S. scherzeri in the Miaoling Mountain area can be divided into the northern (Yuanjiang and Wujiang Rivers) and the southern (Liujiang River) geographical populations. The southern region exhibited low genetic variability and a lack of genetic diversity, while the northern region displayed a high degree of variation and abundant genetic diversity. Significant genetic differentiation and barriers to genetic communication existed between the two geographical populations. Populations of S. scherzeri in Miaoling Mountain experienced demographic expansion in the early and middle of the MedioPleistocene period. The findings of this study indicated that the southern region had a small, closed population, and the original population structure of the ancestors was maintained due to an inbreeding depression effect, whereas the northern region had a large population with an ideal habitat; therefore, it had great potential for genetic evolution. It is inferred that after the population expansion of Siniperca chuatsi in the Middle Pleistocene interglacial period, it developed and evolved along different directions due to the barrier of Miaoling Mountain and differences in habitat, thus shaping the formation of two geographic populations with obvious genetic differentiation between the northern and southern regions. The southern population faces a survival crisis, as it can be seen as an evolutionarily significant unit. It is suitable for establishing a germplasm resource reserve of S. scherzeri in the upper Yuanjiang River system. The effects of the geographical barrier on the genetic structure and diversity of wild S. scherzeri populations in the Miaoling Mountain area were revealed at the molecular level, enriching the genetic background data of S. scherzeri in China and providing a theoretical basis for germplasm conservation and exploitation of S. scherzeri.

Abstract:Illex argentinus is a short-lived cephalopods species, and its resource abundance were sensitive to environment variation. Mesoscale eddies are the main dynamics in its habitat sea area, which cause the variation of environment. In order to investigate the characteristics of eddies in Patagonia sea area and the relationship between I. argentinus resources and mesoscale eddies in the sea area, The angular momentum eddy detection and tracking algorithm (AMEDA) was utilized to identify mesoscale eddies in Patagonia from 2013 to 2019, and analyze the temporal and spatial characteristics of the eddies in this region. Based on the fishery data of Argentine shortfin squid Illex argentinus and eddy data, the characteristics of the suitable environment within a twofold radius (±2R) of the anticyclonic and cyclonic eddies were determined, and the relationship between mesoscale eddies and fishery resources was revealed. During the study period, there were 2262 eddies with a complete life cycle, and they were mainly distributed in the Falkland current basin and its vicinity. The number of cyclonic eddies was slightly less than the number of anticyclonic eddies, but the lifespan, radius, velocity, and abundance of fishery resources were higher for cyclonic eddies than for anticyclonic eddies. The abundance of fishery resources was higher at the edge of the twofold radius of the eddies than in the inner area of the eddies, and the high abundance values significantly appeared on the west side of the cyclonic eddies and the northwest and southeast sides of the anticyclonic eddies. The distribution patterns of sea surface temperature, seawater temperature at a depth of 200 m, and chlorophyll-a concentration were consistent with the fishery resource abundance in the area of the twofold radius of the anticyclonic and cyclonic eddies in the fishing ground. Suitable environmental conditions occurred more frequently within gas vortices predominantly along the edges of the cyclonic eddy. These results provide crucial scientific insights into the role of marine mesoscale eddies in ecosystems, while elucidating their impact on fishery resources.