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Abstract:Biodiversity assessment in fisheries plays a crucial role in conserving marine ecosystem diversity. Accurate survey data serve as the foundation for ensuring the precision of quantitative analysis and the effectiveness of resource conservation measures. However, conducting fishery resource surveys in the ocean is costly and constrained by on-site conditions. Therefore, meticulous sampling design is key to maximizing data quality and survey efficiency. While increasing the number of sampling stations can enhance result accuracy, it escalates survey costs and may adversely affect the marine environment and ecosystems. Hence, sampling design must balance sample size and precision to meet management objectives and budget constraints. Optimizing fishery resource survey sampling station design is widely recognized as an effective approach to enhance survey precision, and numerous studies have been conducted on this topic both domestically and internationally. Recent studies have indicated that stratified sampling offers high precision, making it a preferred choice for optimizing fishery resource survey site selection. However, most research primarily focuses on optimizing either stratification or sample allocation and pays less attention to methods that simultaneously optimize both stratification and sample allocation. This study aims to provide valuable insights into the simultaneous optimization of stratification and sample allocation for biodiversity-focused fisheries surveys. In this study, we take Maoming as a case study and employ the R package "SamplingStrata" to optimize the sampling design through stratified sampling under a multivariate scenario. This package, based on a genetic algorithm, can determine the optimal stratification, sample size, and sample allocation to meet precision constraints in the presence of multiple stratification variables and multiple target variables. When using this package, it is essential to clearly define the stratification and target variables while predefining the precision requirements for the target variables. To maximize the efficiency of the samples, the selection of stratification variables should be based on their correlation with the target variables. Choosing stratification variables that are best correlated with the target variables can enhance the representativeness of the samples. Additionally, the precision requirements are expressed using the coefficient of variation (CV) for each target variable. The CV value reflects the magnitude of the sample estimate variance relative to its mean for each target variable. In this study, we use environmental data (dissolved oxygen, water temperature, pH, and salinity) and water depth as stratification variables and define the Shannon–Wiener diversity index (H′), Margalef richness index (D), Pielou evenness index (J′), and species number as target variables. A maximum CV value is set for the target variables at 0.2, 0.15, 0.1, 0.05, 0.04, 0.03, 0.02, and 0.01. Subsequently, samples are selected from the optimized stratification design, and the relative errors are calculated. The results show that biodiversity in the coastal waters of Maoming exhibits seasonal differences, with diversity indices being higher in autumn than in spring. Additionally, the number of required sampling stations increases as the maximum CV decreases. However, when the maximum CV decreases below 0.05, the number of sampling stations increases significantly with each reduction of 0.01 CV, requiring one or more additional stations. At CV = 0.05, when stratified by environmental factors, spring conditions require eight sampling stations. In contrast, autumn and non-seasonal conditions require 7 and 11 sampling stations, respectively. When stratified by water depth, spring requires 12 sampling stations, whereas autumn and non-seasonal conditions require 14 and 21 sampling stations, respectively. According to relative error analysis, when stratified by environmental factors and water depth, the mean relative errors for H′, D, and J′ are 2.38, 2.02, 2.85, and 0.43, 3.14, and 1.74, respectively, with stratification by water depth resulting in smaller mean relative errors for all indices except J′. Through stratified sampling and appropriate sample allocation, reducing the number of required sampling stations while maintaining data accuracy, thus minimizing adverse impacts on marine ecosystems, is possible. In the coastal waters of Maoming, setting the maximum CV equal to 0.05 for the data precision requirement and using depth as the stratified variable are shown to be suitable considerations. The number of sampling stations for spring, autumn, and non-season-specific surveys is 12, 14, and 21, respectively. This study optimizes fisheries resource survey station selection in the coastal waters of Maoming, offering an effective sampling station optimization method for fisheries resource surveys and providing guidance for future fisheries resource survey station design.

Abstract:The warm-temperate coastal waters of Qingdao, including Aoshan Bay, Jiaozhou Bay and Lingshan Bay, serve as crucial spawning and feeding grounds for diverse fishery organisms and as important fishing grounds. These areas have experienced significant ecological shifts due to escalated human activities, marine pollution, and increased fishing pressure, leading to changes in fishery resources towards miniaturization and domination by low economic value species. To understand the characteristics of the structural changes of the fishery resources in the coastal waters of Qingdao, the present study analyzed the composition, community structure, ecosystem stability, and biodiversity of fishery resources within the coastal waters of Qingdao, based on the data from the fishery surveys conducted in the summer (July-August) and winter (November-December) of 2022. The keystone species were identified using a food web topology, which is crucial for regulating marine ecosystems. A total of 92 and 86 fishery species were collected in the summer and winter with a notable replacement rate of 42.5% respectively. Specifically, Metapenaeopsis dalei is the predominant species in both seasons. The study also highlighted the seasonal dominance of Enedrias fangi and Hexagrammos otakii in summer and Loligo spp., Trachypenaeus curvirostris, Charybdis bimaculate and Syngnathus acus in winter. A discernible shift was observed in the structure of fishery resources, with the increasing dominance of invertebrates and a trend toward miniaturization and devaluation across fish, crustaceans, and cephalopods. In terms of biodiversity indices, the Shannon-Weaver (H') and Pielou evenness (J') indices were significantly higher in summer, whereas the Margalef richness index (D) was higher in winter, indicating normal ecosystem diversity and demonstrating stable seasonal horizontal structures and consistent community compositions. Notable ecological improvement was observed over the past decade, benefiting from the implementation of measures to restore fishery resources. The food web topology for the ecosystem, developed based on the feeding relationships of the local feeding relationships, incorporated 26–29 major species (S) and 116–165 feeding relationships, indicating substantial seasonal variations. The node density (Dd) in summer was greater (0.22>0.18), whereas other indices i.e., average path length (1.17<1.27), interspecific connectance (C) (0.17<0.20) and clustering coefficient (Cl) (0.18<0.25) were lower than those in winter. Based on a comprehensive analysis of the topological index ranking (D, Dout, Din, BC, CC, IC, TI1, TI3, TI5, K, Kb, Kt, F, and DF), Lophius litulon and Metapenaeopsis dalei were identified as key species, with the former being a primary predator and the latter an important prey species. Overall, the food web of the coastal waters of Qingdao exhibited relatively high connectivity and complexity, presenting strong stability and resilience against disturbances and thereby ensuring the provision of vital ecosystem services. Furthermore the role of keystone species must be fully considered in the subsequent conservation of fishery resources and adaptive management to achieve precise management. The findings of this study provide scientific data and knowledge for understanding the structure of the marine food web and biodiversity conservation in this area.

Abstract:Green tide is a harmful ecological phenomenon caused by the explosive proliferation or high aggregation of certain large green algae (such as Ulva prolifera) in seawater under specific environmental conditions, leading to discoloration of the water. The green tide formed by a large number of floating algae can not be effectively disposed of in a short period of time, which will lead to the accumulation and decay of green algae, thereby affecting the coastal ecological landscape. The outbreak of U. prolifera has been one of the main causes of green tide occurrences in the Yellow Sea since 2008. U. prolifera is an algal plant belonging to the family Ulvaceae, which has the characteristics of high nutrient absorption efficiency, fast growth rate and strong reproductive ability, and is more competitive than other species. The explosive proliferation of a large number of U. prolifera during a short period of time has a serious impact on marine environmental ecology, aquaculture, tourism and public health and safety. The study mainly investigated the environmental physicochemical parameters, cultivable microorganisms, dominant culture bacteria, and virulence and resistance genes of dominant bacteria in water and algae samples collected before and after the outbreak of U. prolifera in the main coastal areas of Qingdao were investigated. Environmental physicochemical factors, including the coliform and fecal coliform contents, chemical oxygen demand (COD), biochemical oxygen demand (BOD5), ammonia nitrogen (NH3-N) levels, nitrite, active phosphate, inorganic nitrogen, cyanide, sulfide, arsenic, mercury, copper, zinc, selenium, total chromium, nickel, cadmium, and lead ion levels, in the seawater were assessed. At the same time, the levels of organic carbon, sulfides, coliforms, fecal coliforms, and mercury, arsenic, cadmium, lead, zinc, copper, and chromium ions in the algae were measured. Research on cultivable microorganisms included an assessment of the abundance and composition of cultivable bacteria and Vibrio species in seawater and algae. The distribution of common virulence genes (VAC, vhh, chxA, tlh, and flaA) and resistance genes (strA, strB, QnrVC, and sul2) in the dominant Vibrio bacteria was examined to evaluate the potential virulence risk of the dominant Vibrio bacteria. Comparative analysis results showed that after the outbreak of U. prolifera, the COD, NH3-N, inorganic nitrogen, arsenic ion, and active phosphate levels in the coastal waters of Qingdao increased significantly (P<0.05), and the levels of dissolved oxygen and cadmium, lead, chromium, copper, and other metal ions decreased significantly (P<0.05). The organic carbon content in the algal samples from the aggregated areas of U. prolifera was lower than that from non-aggregated areas. In contrast, the content of coliforms, fecal coliforms, sulfide, mercury, cadmium, zinc, and copper ions significantly increased compared to that in open areas (P<0.05). Moreover, the proportion of Vibrio bacteria increased from 7% to 45%, representing an increase of 38%. The bacterial identification results based on 16S rDNA showed that before the outbreak of U. prolifera, there were 79 dominant bacterial and 62 Vibrio strains in the coastal waters and algae bodies of Qingdao, belonging to 22 species. The main Vibrio species were V. gigantis, V. tasmaniensis, and V. atlanticus. After the outbreak of U. prolifera, 79 dominant bacterial and 65 Vibrio strains were isolated, belonging to 24 species, with the main Vibrio species being V. fortis, V. alginolyticus, and V. owensii. The distribution of virulence genes (VAC, vhh, chxA, tlh, and flaA) and resistance genes (strA, strB, QnrVC, and sul2) in dominant Vibrio bacteria isolated from water bodies and algae before and after the outbreak of U. prolifera was tested. The results showed that only two virulence genes, tlh and flaA, were detected before the outbreak of U. prolifera, and no resistance genes were detected. Five virulence genes and four resistance genes were identified after the outbreak of U. prolifera. Correlation analysis between the number of cultivable bacteria and the environmental physicochemical factors in the water and algae of Qingdao coastal waters before and after the outbreak of U. prolifera showed that the abundance of cultivable bacteria and Vibrio species in the water and algae was correlated with environmental factors such as temperature, pH, NH3-N, nitrite, inorganic nitrogen, active phosphate, total chromium, and nickel. The results revealed the impact of the outbreak of U. prolifera on the bacterial community structure and environmental ecology in the coastal environment of Qingdao. At the same time, the harmful microbial species caused by the outbreak of U. prolifera and the risk of virulence genes and drug resistance genes carried by the outbreak of U. prolifera were evaluated. The relevant research results provide a theoretical basis for the prevention and control of green tide, marine ecological protection, healthy aquaculture in the coastal waters of the Yellow Sea and Bohai Sea.

Abstract:Dalian boasts deep-water coastline and fishery resources and is rich in offshore biological resources. Regarding carbon sink fisheries, as the city with the longest coastline in China, Dalian has blue-carbon resources such as marine shellfish and macroalgae and is uniquely positioned to develop the natural conditions required for a blue-carbon economy. The seawater aquaculture industry in Dalian has formed a diversified aquaculture system with diversified modes and species. The aquaculture of scallops, Undaria pinnatifida, and mussels holds the leading position in China, and the production scale and output of scallops and U. pinnatifida account for more than 95% of the total output from the country. Dalian has become an outstanding representative of China's sea ranch construction. By the end of 2020, Dalian had 22 state-level sea ranch demonstration zones, which is the largest number of sea ranches in the country at the municipal level. Dalian's green ecological aquaculture industry is an important offshore sink-boosting resource, with the industrial foundation and capacity necessary for the development of a blue-carbon economy. In 2022, Dalian's mariculture area was 4.57×105 hm2 its mariculture production was 2.08 million tons, and the total economic output value of its fishery industry was 76.60 billion yuan, which makes Dalian's mariculture production the highest in the country. However, no report assesses the carbon sink capacity of marine shellfish and macroalgae in Dalian. Therefore, this study selected the main aquaculture shellfish species (scallop, oyster, clam, blood clam, mussel, Saccharina japonica, and U. pinnatifida) in Dalian in 2022 as the research object and calculated carbon sink capacity. The aim of this analysis was to provide a scientific basis for the contribution to and capacity of shellfish aquaculture in Dalian and provide a theoretical foundation and technological support for the development of carbon-sink fisheries in Liaoning Province. The results show that in 2022, the total carbon sink capacity of marine shellfish and macroalgae in Dalian reached 826,900 t, of which the net carbon sink represented 538,400 t and the removed carbon represented 261,500 t, which is equivalent to a reduction in carbon dioxide emissions of 1,974,300 t and has a value of carbon aggregation equal to 79,447,100 yuan. Shellfish aquaculture production in Dalian accounted for approximately 64.48% of the province's total aquaculture production, with a carbon sink contribution rate of 55.58%; macroalgae production accounted for 94.75% of the province's total, with a carbon sink contribution rate as high as 94.68%. Among algae and shellfish in Dalian, kelp and clams had the highest carbon sinks, representing 66.12% and 31.37%, respectively, of the entire carbon sinks of algae and shellfish in Liaoning Province. Mussels, as a fishery shellfish with high carbon content, represented only 0.72% of the total carbon sink in Dalian due to factors such as small farming areas and low production. Additionally, it is recommended to strengthen the aquaculture of algae and mussels with high carbon sink contribution rates and to promote the integrated multi-trophic aquaculture (IMTA) of shellfish and macroalgae to optimize the structure of the aquaculture industry (aquaculture species, spatial layout) in Dalian while realizing the effective expansion of carbon sinks of marine organisms, changing the pattern of carbon circulation in the aquaculture industry, and promoting the healthy development of aquaculture in Dalian.

Abstract:China is the largest aquaculture country in the world, with mariculture production accounting for more than 50% of the total production in the world. In 2021, the production of shellfish in China increased to 1.546 × 107 tons, accounting for approximately 70% of the mariculture production. Filter-feeding bivalves such as oysters and clams are the main species of mariculture in China. In addition to the important economic values, filter-feeding bivalves influence ecosystem nutrient cycling through feeding, metabolism, and biodeposition and play roles in increasing the water transparency, preventing harmful algal blooms, controlling eutrophication, and promoting carbon storage. The physiological activities of filter-feeding bivalves, especially ingestion and metabolism, form the link between planktonic and benthic ecosystems, and their physiological indicators are the basic parameters for evaluating the energy budget and carrying capacity. However, although researchers have conducted a series of studies on the physiological activities of filter-feeding bivalves, some limitations in monitoring and the subsequent data processing remain. Therefore, it is urgent to improve the measurement of the physiological activities of filter-feeding bivalves, including the accuracy of data collection and the rigorousness of data processing, to ensure the accuracy of the experimental results. Mudflats are located in the interaction zone between the land and sea and are important areas for the habitat, growth, and reproduction of several macrobenthic organisms. As the dominant species of macrobenthic communities, mudflat-buried shellfish play a crucial role in the material and energy flows of a mudflat ecosystem. However, recently, with the expansion of shellfish aquaculture, the mudflat environment has been deteriorating accompanied by a series of ecological problems, such as high mortality and slow growth rates and alteration in the structure of phytoplankton community, which has led to significant losses to the shellfish aquaculture industry. Therefore, the ability of the ecosystem to support shellfish production must be evaluated, and its carrying capacity must be estimated. Generally, numerical methods for estimating the carrying capacity of shellfish based on food limiting indicators include physical-biological ecosystem modelling, trophodynamic modeling, and energy balance modeling. Current methodologies for estimating shellfish carrying capacity are divided into two main categories: dynamic and static modellings. Compared with the dynamic estimation model, static estimation methods are based on the environment of the target area and the key physiological parameters of shellfish and have been widely applied in some aquaculture areas such as Sanggou Bay, Jiaozhou Bay, and Zhangzidao in China. Here, to explore the energy budget and carrying capacity of the surf clam, Mactra veneriformis, and the estuarine clam, Potamocorbula laevis, in Geligang, Liaodong Bay, a portable particle counter and a continuous oxygen monitoring system were used in combination with flow-through chambers to detect the feeding and metabolic parameters of M. veneriformis and P. laevis. Furthermore, the carrying capacity of two mudflat-buried bivalves in Liaodong Bay was estimated based on the organic carbon supply-demand balance model. The results indicated that 1) the clearance rates of M. veneriformis and P. laevis were (4.87±0.85) L/(h·g) and (6.46±2.25) L/(h·g), respectively, and the oxygen consumption rates were (0.94±0.45) mg/(h·g) and (0.22±0.14) mg/(h·g), respectively. The energy absorption of M. veneriformis and P. laevis ranged from 748.97 to 1 333.52 J/(h·g), and 931.55 to 1 647.08 J/(h·g), respectively. 2) Using organic carbon supply-demand balance model combined with the primary productivity and shellfish clearance rate, we found that the carrying capacity of M. veneriformis in Geligang, Liaodong Bay, was 57, 47, and 34 ind./m2 for age-1 (total wet weight 6.7 g), age-2 (total wet weight 9.3 g) and age-3 (total wet weight 14.6 g), respectively; and the carrying capacity of P. laevis was 346, 143, and 99 ind./m2 for age-1 (total wet weight 0.14 g), age-2 (total wet weight 0.69 g), and age-3 (total wet weight 1.25 g), respectively. These results provide basic data for the rational exploitation and utilization of shellfish resources and the conservation of biodiversity in a mudflat ecosystem.

Abstract:Marine ranching is a fishery model that can improve the ecological environment of sea areas, increase and conserve fishery resources, and realize the sustainable utilization of fishery resources. It is widely used worldwide. In Shandong Province, marine ranching is divided into the following types: Reef casting, game fishing, bottom seeding, pastoral, and equipment, based on the production mode. Among them, pastoral marine ranching refers to marine ranching dominated by three-dimensional ecological aspects such as raft culture facilities. Raft culture facilities are a culture framework that must be constructed for the culture of bivalves such as oysters, scallops, and mussels in China’s coastal waters. These facilities include six parts: a wooden pile, pursed rope, float, suspended cage, suspended rope, and floating rope. In the process of bivalve culture, the construction of a raft culture facility provides a good adhesion substrate for other organisms in the ocean, and a large number of fouling organisms will inhabit and survive on the culture facility. The activity of these fouling organisms will soften the raft culture facility and shorten the service life of the raft culture facility. Furthermore, the fouling organisms can plug the mesh, leading to the death of the farmed shellfish due to lack of oxygen, causing economic losses. However, relevant studies have also shown that the presence of fouling organisms can accelerate the rate of nutrient salt exchange between suspended organisms and aquaculture waters, which can promote the growth of phytoplankton, improve primary productivity, provide more bait for bivalve culture, and increase the fertility of bivalve culture, which is beneficial to bivalve culture. There are differences in the relationship between the fouling organisms' species number and biomass and environmental factors across different raft aquaculture areas. Therefore, basic research must be conducted on fouling organisms in raft culture areas to provide reference for healthy bivalve culture and further provide basic data for exploring the impact of fouling organisms on the ecological environment in raft culture facilities. To explore the seasonal changes of fouling organisms in raft culture facilities of pastoral marine ranching and their relationship with environmental factors, a 4-quarter survey was performed in the pastoral marine ranching area of Rushan Huaxin Foodstuffs Co., Ltd., from July 2022 to April 2023. A total of four fixed stations were set up in the raft culture area of oysters for each survey. The fouling organisms were investigated at each station. An area of 10 cm × 10 cm in the float, a length of 30 cm of the floating rope, and an area of 20 cm × 20 cm in the suspended cage were sampled. The collected samples were bottled and numbered; then, the sample was fixed and stored in 5% formaldehyde solution, and returned to the laboratory for species identification and counting. The species, biomass, diversity, dominant species, and community structure of the fouling organisms at three different sampling sites were analyzed, and the relationship between environmental factors and dominant species was determined by redundancy analysis (RDA) and Spearman analysis. The results showed that 35 species of fouling organisms were found in eight categories, including 13 species of Crustacea, 8 species of Mollusca, 4 species of Polychaeta, 3 species of Chordate, 2 species of Cnidaria and Phaeophyta, and 1 species each of Rhodophyta, Chlorophycophyta, and Echinodermata. There were significant seasonal variations in the community structure of fouling organisms. Furthermore, there were significant differences in the community structures of fouling organisms in suspended cages, floating ropes, and floats, and the biomass varied in the ranges of 31.48~1 645.82 g/m2, 23.22~580.76 g/m2, and 5.76~30.12 g/m2, respectively. The maximum biomass of fouling organisms appeared in summer. The richness index, evenness index, and diversity index of the fouling organisms ranged from 1.38 to 3.61, 0.33 to 0.92, and 0.88 to 2.58, respectively. The lowest richness index was observed on the summer float, the lowest evenness index and diversity index were observed on the summer suspended cage, and the three indexes reached the highest values on the spring suspended cage. The dominant species of fouling organisms belonged to the phyla Crustacea and Mollusca. Mytilus edulis was the dominant species on the floating ropes and suspended cages, and Caprella aino was the dominant species on the floats. The dominant species of fouling organisms in the float showed obvious seasonal changes. The dominant species on the float in summer was Perinereis aibuhitensis, and that in autumn was Crassostrea gigas. Gracilaria vermiculophylla was the dominant species on the float in winter and spring. The RDA results of dominant species and environmental factors showed that transparency, temperature, silicate levels, and chlorophyll were the main environmental factors affecting the community structure of fouling organisms, and the correlation between different species and environmental factors was also different. In this study, the seasonal variation of fouling organisms and their relationship with environmental factors were investigated in the Rushan oyster raft culture facility, and our results can provide reference for healthy oyster culture in this area.

Abstract:To explore the molecular mechanism of dwarf fish formation in the cultured population of Cyprinus carpio rubrofuscus and the functions of vascular endothelial growth factor (VEGF) in fish growth, the features of growth trait, feed intake, and feed utilization of dwarf fish were detected and analyzed. Vegf121 cDNA was amplified and sequenced in this study. The expression levels of vegf121 were analyzed by quantitative real time PCR (qRT-PCR) in 11 tissues of normal fish, and in the heart, pituitary, brain, muscle, and hypothalamus of dwarf fish. The results showed that under the culture condition in ponds, the body length distribution of the typical five-month-old C. carpio rubrofuscus population with dwarf individuals showed two obvious independent normal distribution curves. Furthermore, the dwarf individuals formed a small population that distinguished it from the normal and fast-growing fish in terms of body size. The results of the feeding experiment showed that the daily feed intake (DFI) and the weight gain rate (WGR) of fish in the dwarf group were significantly lower than those in the normal group, whereas the feed conversion rate (FCR) showed no significant difference between the two groups. Meanwhile, vegf121 was found to be differentially expressed in C. carpio rubrofuscus. The partial cDNA sequence of vegf121 gene was 1,235 bp with an open reading frame of 432 bp encoding 144 amino acids. According to the predicted amino acid sequence, the protein belongs to the VEGF121 isoform and has a relatively high sequence identity to the VEGF121 proteins of 11 known fish with the lowest identity of 79.86% aligned to Denticeps clupeoides. However, the identities to VEGF121 isoforms to those of Homo sapiens and Mus musculus were lower, at only 51.02% and 52.74%, respectively. The low homology and different spatial and temporal distribution of VEGF121 isoforms between animals and fishes may be related to breathing regulators and the differences in vascularization patterns in the respiratory system. The qRT-PCR results of the expression levels of vegf121 mRNA detected in the tissues of the normal growth fish showed that the relative expression levels were the highest in the muscle, followed by those in the heart, gill, brain, stomach, pituitary, eye, liver, kidney and spleen, and the lowest in the hypothalamus. This suggested that vegf121 has different regulatory functions in the development of blood vessels in different tissues of C. carpio rubrofuscus. Comparing the expression of vegf121 in the five tissues between the dwarf and normal-growth fish group revealed that the expression levels of vegf121 mRNA in the hypothalamus of the dwarf fish group were significantly higher than those of the normal fish group. The hypothalamus, as a high-level nerve center regulating endocrine and metabolic activities in humans and fish, may undergo some pathological changes under the action of certain environmental or genetic factors. The higher expression of vegf121 in the hypothalamus may have disrupted hormone secretion in C. carpio rubrofuscus and subsequently destroyed the metabolic balance in the body, resulting in a decrease in the appetite and feeding ability of fish. However, there was no significant change in the expression of vegf121 mRNA in two other neural tissues, the brain and pituitary, between the two groups. While the expression levels of vegf121 in the both muscle and heart were slightly lower than those in the normal-growth group. The abnormally higher expression of vegf121 in the hypothalamus indicated angiogenesis or lesions in the hypothalamus, which may have affected the feed intake regulation pathway signals in the fish body. This study provides a scientific basis to further explore the mechanism leading to dwarfism in C. carpio rubrofuscus. Genetic studies on the reasons leading to dwarf individuals in cultured economic fish populations can help increase the yield and productivity in aquaculture.

Abstract:Conger myriaster has a high price on the international market and occupies an important position in aquatic product export in China, and it is widely distributed in the Yellow Sea, the Bohai Sea, the East China Sea, the coastal waters of Japan from southern Hokkaido to northern Okinawa, and the sea near the Korean Peninsula. The experimental aquaculture techniques of C. myriaster include pond culture, industrialized open-flowing water culture, and industrialized recirculating aquaculture. No report has been published on the morphological characteristics or the effects of various morphological traits on the body weight and net body weight of C. myriaster in an industrialized open-flowing water culture. In this study, C. myriaster was collected under this industrialized culture mode and assessed using traditional measurement methods, statistical analysis, and other means to systematically measure and analyze morphological traits. A total of 90 C. myriaster were collected equally from three aquaculture companies along the coast of the Yellow Sea. The body weight (BW), net body weight (NW), total length (TL), body length (BL), body height (BH), soma width (SW), head length (HL), head height (HH), head width (HW), proboscis length (PL), proboscis width (PW), eye diameter (ED), vertical eye diameter (VED), and distance between eyes (DE) were measured individually using an electronic balance, a ruler, and a Vernier caliper. We analyzed the relationships between morphological traits and constructed a truss network diagram of morphological traits. The effects of the morphological traits on BW and NW were studied by correlation analysis, path analysis, multiple regression analysis, and curve fitting. The correlation analysis showed that the correlations of BW with TL, BL, BH, SW, HH, and HW were extremely significant (P<0.01), with correlation coefficient values of 0.818, 0.829, 0.611, 0.697, 0.667, and 0.642, respectively. The correlations of NW with TL, BL, BH, SW, HH, HW, and VED were extremely significant (P<0.01), with correlation coefficients of 0.848, 0.857, 0.574, 0.591, 0.617, 0.564, and 0.519, respectively. Path analysis revealed that the path coefficients between BW and three morphological traits (namely BL, BH, and SW) were larger than those for other morphological traits, at 0.631, 0.204, and 0.374, respectively. The path coefficients between NW and these three morphological traits (BL, BH, and SW) were larger than those for other morphological traits, at 0.703, 0.213, and 0.239, respectively. Through determination coefficient analysis, it was observed that the determination coefficient for BL to BW was the largest, with a value of 0.398. The combination of SW and BL had the largest determination coefficient for BW, with a value of 0.162. This indicated that BL was a core morphological trait to determine the BW, and the SW was an important morphological trait to determine the BW. The determination coefficient of BL to NW was the largest at 0.494. The combination of SW and TL had the largest coefficient of determination for NW, with a value of 0.116. This showed that the BL was the core morphological trait to determine the NW, and SW was an important morphological trait to determine the NW. A stepwise regression method was used to establish a regression equation in which the three morphological traits were independent variables and the BW or NW was the dependent variable. The multiple regression equation of the important morphological traits for BW was BW = –642.699 + 1.086 × BL + 3.874 × BH + 7.917 × SW (P<0.01). The multiple regression equation of the important morphological traits for NW was NW = −526.995 + 1.033 × BL + 3.438 × BH + 4.317 × SW (P<0.01). Further curve fitting was performed for each morphological trait with BW and NW in the equation. The best fitting models of BL, BH, and SW for BW were the logarithmic function, logarithmic function, and power function, respectively, and the model equations were BW = −3,542.357 + 608.061lnBL (R2 = 0.929), BW = 14.313BH0.941 (R2 = 0.908), and BW = −1,028.49 + 416.452lnSW (R2 = 0.920), respectively. The best-fitting models of BL, BH, and SW for NW were the logarithmic, linear, and logarithmic functions, respectively. The model equations were NW = −2,983.881 + 516.411lnBL (R2 = 0.930), NW = −22.252 + 11.392BH (R2 = 0.914), and NW = −774.583 + 329.017lnSW (R2 = 0.921), respectively. In summary, BL was the core morphological trait affecting the BW and NW of C. myriaster in an industrialized culture model. BH and SW were important morphological traits affecting the BW and NW of C. myriaster in the industrialized culture model. These results provide a theoretical basis for the future identification of germplasm characteristics, genetic breeding, and healthy aquaculture of C. myriaster.

Abstract:The genus Thymallus, belonging to the Salmoniformes, Salmonidae and Thymallinae, is distributed in three main areas in China, namely, the Irtysh River Basin, the Heilongjiang River Basin and the Yalu River Basin. Research has shown that the diversity of extant Thymallus in China is much higher than that in the vast European and North American regions, but the taxonomic results of some species are still controversial. Among them, those whose taxonomic status has been clarified and whose nomenclature is not controversial are T. arcticus, T. grubii and T. tugarinae, and the one that is still controversial is T. yaluensis. Named by Mori in 1928, T. yaluensis is mainly distributed in the Yalu River Basin and is geographically isolated from the T. arcticus, T. grubii and T. tugarinae. In 2007, a study on the comparison of countable traits of Thymallus found that the countable traits of the T. yaluensis were in the middle between the T. grubii and T. tugarinae, and the validity of the taxonomic status of T. yaluensis has been formally questioned ever since; in 2008, a study using mitochondrial DNA control region sequences to study the evolutionary relationships of fishes in the genus Thymallus pointed out that the T. yaluensis is synonymous with the T. grubii. In 2016, researchers used mitochondrial genome data to confirm once again that there was no difference between the T. yaluensis and the T. grubii. However, this conclusion does not seem to be accepted by the academic community, and in recent years, there are still some scholars who regard the T. yaluensis as an independent species. Morphological methods are the most commonly used methods in species identification, which can be categorized into traditional morphological method and framework measurement method. The traditional morphological method mainly analyzes the horizontal and vertical traits of the fish body, and it is mostly limited to the head and tail traits, which can't measure the whole surface of the fish body comprehensively. The framework measurement method, however, is based on a certain number of anatomical coordinate points and measures the entire fish body in multiple directions, which can provide a more comprehensive response to the morphological differences of the fish body. So far, the studies on the interspecific differences of Thymallus in China have mostly focused on the molecular biology level, while the morphological differences have been less studied, only in T. grubii and T. tugarinae. The rapid development of molecular marking technology provides an important auxiliary means for species classification, but the apparent morphological differences are still recognized as the most intuitive basis for species classification. In order to clarify the morphological differences of Thymallus in China, this study comprehensively used traditional morphological methods and framework measurement method, and took the T. grubii, T. arcticus, T. yaluensis and T. tugarinae as the research objects, Statistical analysis was conducted on their 10 segmentation characteristics and 32 standardized metric features. The results showed that: (1) there was no significant difference (P>0.05) between all the subsection characteristics of T. yaluensis and T. grubii, and there were significant differences (P<0.05) in more than five segmentation characteristics between T. grubii, T. arcticus and T. tugarinae. (2) The coefficient of variation analysis showed that except for caudal peduncle length/body length, the differences in morphological characters between T. yaluensis and T. grubii were at the level of differences between geographic populations within species. (3) The principal component analysis screened out eight principal components with eigenvalues greater than 1, and the cumulative contribution rate reached 81.170%, among which the first and second principal components with the highest contribution rate mainly explained the differences in trunk and head between Thymallus, respectively. The construction of scatter plots of the first and second principal components revealed that the individual crossover between T. yaluensis and T. grubii had a high degree and could not be distinguished individually, while T. tugarinae and T. grubii show a lower level of overlap and partial distinction. Only T. arcticus can be distinctly separated. (3) Discriminative analysis was used to screen out 14 standardized measurement features to establish the Fisher linear discriminant function, and the discriminative rate of individuals being correctly assigned to their respective groups was as high as 97.30%. (4) Cluster analysis showed that compared with T. grubii, T. yaluensis had the highest degree of similarity with it, followed by T. tugarinae, and T. arcticus had the lowest degree of similarity with T. grubii. The comprehensive analysis showed that: (1) T. yaluensis should not be designated as a valid species and its scientific name should be revised to T. grubii. (2) Morphological differences among T. grubii, T. arcticus and T. tugarinae are mainly reflected in their segmentation characteristics and the measurement characteristics of trunk and head. The results of this study will provide a reference for the study of species classification and evolutionary relationships of the genus Thymallus.

Abstract:The black rockfish, Sebastes schlegelii, is the main marine fish species produced via offshore cage culture in China. Moreover, it is essential to select a convenient, safe, and harmless anesthetic that can effectively reduce the physiological stress response of juveniles during land–sea relay transport. MS-222 has been widely used in fish and other aquatic creatures during handling and transportation because of its secure induction and rapid recovery. In this study, the anesthetic effect of MS-222 (tricaine methanesulfonate; anesthetic effect: 20–130 mg/L, interval of 10 mg/L, total of 12 concentration gradients; simulated transportation: 30–60 mg/L, interval of 10 mg/L) on three specifications of juvenile black rockfish (W10, W40, and W80; average body weight of (10.11±2.13) g, (42.38±5.19) g, and (80.79±6.65) g, respectively) was investigated. The times to deep sedation (A2) and anesthesia (A4) under different concentrations of MS-222 and the optimal sedation and anesthetic concentrations of MS-222 were determined. The results showed that in water with a temperature of (14.5±0.5) ℃, pH of 7.85, salinity of 30 and dissolved oxygen concentration above 7.5 mg/L, the time required for W10, W40, and W80 juveniles to reach the A2 and A4 stages decreased with an increase in anesthetic concentration and increased with an increase in body weight at the same anesthetic concentration. Quadratic regression analysis was performed to analyze the relationship between the A2 and A4 stage effect times and the concentrations of MS-222. The equations describing the relationship between the A2 stage effect time (y) and anesthetic concentration (x) in W10, W40, and W80 groups were y = 0.210 7x2  21.207x + 602.6 (R2 = 0.95), y = 0.202 1x2  21.501x + 642.6 (R2 = 0.98) and y = 0.089 3x2  14.153x + 606 (R2 = 0.91), respectively. Those for A4 stage effect time and the concentration of MS-222 were y = 0.043 1x2  10.755x + 813.57 (R2 = 0.97), y = 0.062 1x2  16.221x + 1 205.6 (R2 = 0.97) and y = 0.059 6x2  15.954x + 1 229.9 (R2 = 0.92), respectively. Therefore, the optimal sedative and anesthetic concentrations of MS-222 for juvenile W10, W40, and W80 black rockfish were 27.38, 29.94, and 40.39 mg/L (A2), and 95.32, 107.32, and 116.58 mg/L (A4), respectively, according to the optimal effect time of 180 s. No significant difference was observed between the respiratory rates of juvenile fish in the A2 stage and those in the control group; however, the respiratory rates of juvenile fish in the A4 stage gradually decreased with an increase in anesthetic concentration and were significantly lower than those of the control group (P < 0.05). The results of simulated transport over 8 h demonstrated that the survival rate of all fish remained at 100% within a concentration range of 30–50 mg/L for MS-222. However, when the concentration reached 60 mg/L, the survival rate decreased to approximately 60% for the W10 group and 80% for the W40 group, though it was still maintained at 100% for the W80 group. Therefore, suggested transport concentrations for juvenile W10, W40, and W80 black rockfish are in the ranges of approximately 27.38–50.00, 29.94–50.00, and 40.39–60.00 mg/L, respectively. These findings provide valuable guidance for the safe transportation of juvenile black rockfish during stock enhancement and offshore cage land-sea transportation. Moreover, the optimal anesthetic concentration can serve as a reference point for biological experiments involving measurement, labeling, or sample collection with juveniles of varying sizes.

Abstract:Heat shock proteins (HSPs), also known as heat stress proteins, can be divided into five main families, including HSP100, HSP90, HSP70, HSP60, and small heat shock proteins (sHSPs), according to their molecular weight. Further studies have shown that HSPs represent a class of highly conserved proteins that widely exist in the subcellular compartments of prokaryotic and eukaryotic cells. They have many functions, such as protein folding, repair, and immunity, and play important roles in organism growth and development. For example, Dmhsp70-A and Dmhsp70-B showed similar trends in the development of asexual embryos in Daphnia magna. However, they exhibited different trends in the development of sexual embryos despite high expression in both embryo types. Penaeus japonicus is one of the most important commercial shrimp species in China, Japan, and Southeast Asian countries. Relevant studies have shown that hsp genes widely exist in P. japonicus and play important roles in growth and development. For example, hsp10 and hsp60 were expressed in all tissues of P. japonicus, and the differential expression of hsp60 was identified in the early development stages. However, previous studies were based on single genes, and more systematic studies based on the role of the hsp gene family in the growth and development of P. japonicus have not been published. Our laboratory has completed the genome sequence of P. japonicus, which provides basic data for analyzing the molecular mechanism of hsp in the growth and development of P. japonicus. To further study the characteristics of the hsp gene family in P. japonicus and the expression characteristics of hsp genes during different development stages, the hsp gene family of P. japonicus was identified and analyzed using bioinformatics methods, and variation in its expression during the development of P. japonicus was measured using reverse transcription and quantitative real-time (RT-qPCR). Fifteen members of the hsp gene family were identified from the whole genome of P. japonicus via bioinformatics analysis, and their gene structures, motif compositions, chromosome localizations, and phylogenetic characteristics were analyzed. The expression of hsp genes in maternal-to-zygotic cells, blastocysts, gastrulae, limb buds, intramembranous nauplii, stage 1 nauplii, stage 1 zoeae, stage 1 myses, and stage 1 post-larval juveniles of P. japonicus was measured using qPCR. A total of 15 hsp genes were identified, including 1 hsp10, 2 hsp20, 2 hsp40, 1 hsp60, 6 hsp70, and 3 hsp90 genes. The physicochemical properties of seven proteins were stable, with instability coefficients less than 40. Subcellular localization results showed that most of the hsp gene family members were located in the cytoplasm, as seven hsp genes were localized to the cytoplasm, two to the nucleus, two to the extracellular region, two to the endoplasmic reticulum, and two to the mitochondria. Isoelectric point (pI) is related to the number and proportion of acidic and basic amino acids contained in a protein. Most HSPs exhibited a pI <7, suggesting they were acidic proteins. The 15 hsp genes were located on 11 chromosomes. Phylogenetic tree analysis showed that most of the HSP family members were first clustered with HSP family members from species such as Penaeus monodon, Litopenaeus vannamei, and Fenneropenaeus chinensis and then with HSP family members of other species, which was consistent with the traditional taxonomic status. A few HSP family members were clustered first with HSP family members from Eriocheir sinensis, D. melanogaster, and other insects, and then with HSP family members from other species. Motif analysis showed that the hsp gene family was highly conserved; however, the functions of different families were quite different. Structural domain analysis showed that the HSP family included cpn10-, metazoan ACD-, DnaJ bact superfamily-, GroEL-, PTZ00009 superfamily-, dnaK-, HSP70 superfamily-, HSP90 superfamily-, PRK14083 superfamily-, and HATpase_HSP90-like domains. Each gene family contained corresponding domains and was important in molecular chaperone processes. hsp genes were expressed in different developmental stages of P. japonicus and played important roles in growth and development. The trend of Pjhsp10-19.517 was similar to that of Pjhsp60-19.518, which was highly expressed during embryonic development and significantly downregulated during larval development. This is consistent with the conclusion of previous studies that HSP60 and HSP10 are molecular chaperones for each other to ensure the correct folding of the target protein. The hsp genes such as Pjhsp40-15.349, Pjhsp70-39.287, Pjhsp70-1.298, and Pjhsp90 were highly expressed in the embryonic period; Pjhsp20, Pjhsp70-3.662, Pjhsp70-15.369, Pjhsp70-32.916, and Pjhsp90-12.759 were highly expressed during the larval period. The hsp genes expressed at high levels during the embryonic period satisfy the high protein requirements of embryos by playing the role of molecular chaperone and regulating the transcription of vitellogenin to meet the embryonic need for vitellin. The hsp genes expressed at high levels during the larval period protect the body from environmental pressure, pathogens, and xenobiotics through a synergistic immune effect. The authors believe that hsp genes play important roles in the growth and development of P. japonicus, and the specific pathways and mechanisms should be further studied. This study provides basic data for further investigations of the role of hsp genes in the growth and development of P. japonicus.

Abstract:The Pacific white shrimp or white leg shrimp, Penaeus vannamei, is native to the tropical coastal regions of Central and South America. It is one of the three major shrimp species with high production worldwide. It was brought from Hawaii, USA, in 1988 and introduced into China. It has been widely promoted and cultivated due to its adaptability to the environment, fast growth rate, and suitability for high-density industrial farming. As of 2022, China's total production of P. vannamei reached 2.09 million tons, accounting for >90% of the country's total shrimp farming production. It has become one of the pillar industries in China's aquaculture sector. With the continuous expansion of aquaculture, diseases are becoming increasingly severe. Among them, acute hepatopancreatic necrosis disease (AHPND) hinders the development of the global shrimp farming industry and causes catastrophic economic losses. After infection, most shrimp show hepatopancreatic enlargement, which appears pale white or light yellow. In the later stages of infection, some diseased shrimp may exhibit atrophy of the hepatopancreas, appearing reddish-brown and blackened, with a hardened texture. Within 20–30 d after infection, death can occur, with a mortality rate of up to 90%. As of 2021, the economic losses caused by this disease to the major P. vannamei farming areas (China, Malaysia, Thailand, Vietnam, and Mexico) have exceeded 43 billion US dollars. Cultivating novel P. vannamei germplasm with AHPND resistance is an effective way to solve AHPND. Recently, domestic researchers have established independent shrimp breeding systems and cultivated 12 novel P. vannamei strains, such as "Hai Xing Nong No.2", "Ren Hai No.1", and "Zheng Jin Yang No.1". These varieties mainly target traits such as growth, resistance to white spot syndrome virus, farming survival rate, and temperature, salinity, and ammonia nitrogen tolerance. However, no reports of novel varieties specifically targeting AHPND resistance are available. Growth traits are essential economic traits in the genetic breeding of aquatic animals. Among the 12 novel P. vannamei varieties in China, 11 have excellent traits related to growth. Evaluating the genetic relationship between growth and AHPND resistance in the core breeding population of P. vannamei is important for fully utilizing existing high-quality germplasm and breeding novel varieties with composite traits and AHPND resistance. Genetic parameters reflect the genetic variation of target traits in a breeding population and are important references for breeding decisions. Due to the complex genetic background of existing P. vannamei germplasm resources in China, genetic parameters are influenced considerably by the tested population, infection methods, and other factors. Before breeding, stable infection tests must be conducted on the base population and obtain accurate parameters. This study used two independently bred P. vannamei strains as research objects to test their growth and AHPND resistance. Among them, 40 families were selected for high resistance (20 individuals per family, total 800 experimental individuals), and 30 families were selected for fast growth (32 individuals per family, total 960 experimental individuals). The test was conducted at P70–P90 by feeding with VpAHPND quantitative oral infection. Feeding with toxic bait was stopped after 50% of the experimental individuals died. The genetic heritability and correlation of the weight, individual survival time after VpAHPND infection, and half-lethal survival rate of the two strains were evaluated using individual animal and male and female threshold models. The results showed that the weight heritability estimate of the highly resistant strain was 0.599±0.120, indicating a high level of genetic heritability. The heritability estimates of survival time and half-lethal survival rate were 0.240±0.072 and 0.173±0.051, respectively, indicating a moderate to low level of genetic heritability, and all significantly different from 0 (P<0.01). The weight heritability estimate of the fast-growth strain was 0.266±0.082, indicating a low level of genetic heritability. The heritability estimates of survival time and half-lethal survival rate were 0.374± 0.096 and 0.257±0.048, respectively, indicating a moderate level of genetic heritability, and all significantly different from 0 (P<0.01). The genetic correlation between weight and AHPND resistance traits (survival time and half-lethal survival rate) in both strains showed a low positive correlation (0.061–0.235), and no significant difference was observed from 0 (P>0.05). The genetic correlation between survival time and half-lethal survival rate in both strains was highly positively correlated, with estimates of 0.997±0.129 and 0.967±0.044, respectively. The results indicate that growth and AHPND resistance can be included as selection indicators in the comprehensive selection index for breeding and seed production in the actual breeding process. Under limited conditions, the infection program can be simplified, and the half-lethal survival rate can be used as the AHPND resistance indicator for the breeding population. This study provides a primary reference for utilizing existing germplasm resources to develop high-quality P. vannamei strains for growth and AHPND resistance breeding.

Abstract:This study aimed to investigate the effects of physical mutagenesis on the development and adaptive traits of early-stage shrimp larvae and to explore the feasibility of increasing genetic diversity and utilizing genetic variation in breeding populations through mutation technology. Atmospheric and room-temperature plasma (ARTP) mutagenesis technology was used to perform batch mutagenesis on fertilized eggs (gastrula stage) of four different families (A, B, C, and D) of Penaeus vannamei at the same power (360 W) and different times (2, 4, 8, and 12 min). Hatching rates, limb bud stage hatching deformity rates, survival rates, metamorphosis rates, and indicators of juvenile shrimp hypoxia tolerance and ammonia nitrogen stress were analyzed. The experimental results showed that all groups except for the 2 min mutation group showed hatching deformity rates significantly higher than those of the control group for the A and D families (P<0.05). The deformity rate of each treatment group showed no significant differences from that of the control group in Family B. The malformation rate for Family C in the 8 min mutation treatment group was significantly higher than that in the control group. However, there was no significant difference between the 2 and 4 min treatment groups. In all four families, the hatching rate of fertilized eggs decreased with an increase in mutation time, and when the mutation time was greater than or equal to 4 min, the hatching rates of all four families were less than 15%. Compared with that of the control group, all treatment groups of the families except for the 2 min mutation group from Family A showed a significant decrease in hatching rate (P<0.05). The hatching rate of the 2 min group for families A, B, C, and D was significantly higher than that of the 4, 8, and 12 min mutation groups, and the hatching rate of the 4 min mutation group from Family B was significantly higher than that of the 8 min mutation group (P<0.05). The hatching and limb bud stage hatching deformity rates were moderately and highly correlated with mutagenic time, respectively. Compared with the control group, the survival rates of families A, B, C, and D in the same treatment group at the same stage showed no significant differences. The survival rate of the 8 min mutation group from Family A was significantly reduced (P<0.05), that of the 4 and 8 min mutation groups from Family C was significantly increased, and that of the 2 and 4 min mutation groups from Family D was significantly reduced relative to the control value, whereas that of the 8 min group from Family D was significantly increased. Considering Family A as an example, metamorphosis rates in each stage were analyzed. The metamorphosis rates of the control group and the 2 min mutation group showed a significant decreasing trend during the three stages of larval development (P<0.05); however, the metamorphosis rate in the M–P stage was significantly lower than that in the N–Z and Z–M stages. The metamorphosis rate in the Z–M stage of the 4 min and 12 min groups was significantly lower than that during the N–Z and M–P stages, whereas the metamorphosis rate during the N–Z stage of the 8 min group was significantly lower than that of the Z–M stage. After low-dose (0 and 2 min) mutagenesis of fertilized eggs of P. vannamei, the peak period of death mainly occurred in the late stage of larval development. The peak period of larval death after relatively high doses of mutagenesis (4, 8, and 12 min) mainly occurred in the middle or early stages of larval development. The peak period of death varied among different treatment groups. The results caused by radiation treatment showed directionality, and this trend of peak mortality was similar between all families. Hypoxia tolerance and ammonia-nitrogen stress testing in Family A of juvenile shrimp revealed that almost all shrimp in the 4, 8, and 12 min groups died after 5.5 h of hypoxia stress. In contrast, the control group died after 9 h of stress, and the 2 min group died after 10 h of stress. The results indicate that low doses of radiation (2 min) may improve hypoxia tolerance to some extent. The survival rates of the 2 min and 8 min groups were highest on the fourth and seventh days of the ammonia nitrogen stress experiment, respectively. Mutation had a certain impact on the ability of larvae in Family A to resist ammonia-nitrogen stress. The experimental results suggest that ARTP induced the mutation of fertilized P. vannamei eggs with non-directional changes in adaptive traits. These findings suggest the feasibility of novel methods for creating new germplasms and preparing functional research materials for P. vannamei and provide basic data and a scientific basis for the mutagenic breeding of P. vannamei.

Abstract:The ridgetail white shrimp (Exopalaemon carinicauda), belonging to the Palaemonidae family of crustaceans, is an economically important small-to-medium-sized shrimp in China that is naturally distributed on the coasts of the Yellow and Bohai Seas. Due to its rapid growth and high tolerance for environmental stress, the scale at which E. carinicauda is cultured has expanded in recent years. The reproductive performance of broodstock is crucial to the yield and quality of larvae, as the egg production of E. carinicauda is much lower than that of other economically important shrimps. Meanwhile, E. carinicauda seedling production mainly relies on wild shrimp or breeding parents, resulting in low fertility and poor seed quality, such that the cultivation of E. carinicauda is often hindered by a slow growth rate and poor disease resistance, seriously limiting the development of the E. carinicauda farming industry. Optimizing growth and reproductive traits is vital for the animal aquaculture industry, as these traits are crucial for developing high-quality seedlings and are directly related to yield. Cultivating new varieties with high fertility and fast growth rates can effectively improve the aquaculture of E. carinicauda. In this study, we estimated the genetic parameters of growth and reproduction traits in E. carinicauda. We constructed 40 full-sibling lines of E. carinicauda and 25 half-sibling lines by pairing one male with two females. Thirty shrimps were randomly selected from each line at 50 d and 80 d of age, and the body length, total abdominal length, and body weight were measured to calculate the genetic parameters. The number of eggs, body length, body weight, and incubation time of the first spawning were measured to ensure that the culture conditions for each line were consistent. The main baits used during incubation were folded brachyuran rotifers (Brachinonus plicatilis) and brine shrimp (Artemia sinica). The heritability, phenotypic correlations, and genetic correlations of body weight, body length, total abdominal length traits, time of first spawning, absolute egg count, egg count per unit of body length, and egg count per unit of body weight in E. carinicauda were estimated at the ages of 50 and 80 d using a general linear model. The results showed that the heritability estimate values for body length, total abdominal length, and body weight at 50 d were approximately 0.27–0.69, 0.31–0.44, and 0.20–0.65, respectively, and those for body length, total abdominal length, and body weight at 80 d were approximately 0.39–0.54, 0.31–0.47, and 0.33–0.71, respectively. The heritability estimate values for the number of eggs, the number of eggs per unit of body length, the number of eggs per unit of body weight, and the time of the first spawning were approximately 0.45–0.52, 0.30–0.49, 0.43–0.48, and 0.33–0.77, respectively. The heritability estimate values for all three growth-related traits and four reproduction traits almost reached the level of medium-high heritability. The heritability estimate values for growth and reproduction traits based on full siblings reached a highly significant level according to t-tests. Therefore, the unbiased estimates of narrow-sense heritability for growth and reproduction traits were those estimated by the full-sibling variance component. The phenotypic and genetic correlations for growth and reproduction traits were approximately 0–0.756 9 and –0.006 5–0.951 4, respectively. The genetic correlation coefficients of body weight with body length and egg count were 0.951 4 and 0.205 8, respectively. The phenotypic correlation coefficients of body weight with body length and egg count were 0.742 0 and 0.212 4, respectively. However, the genetic and phenotypic correlations between body length and egg count were poor. Therefore, we suggest that body length and egg count be set as two different targets in breeding programs. The results indicate that the heritability estimate values for body length, total abdominal length, and body weight at 50 and 80 d and for reproduction traits including egg count, egg count per unit length or weight, and time of first spawning almost reached the level of medium-high heritability. The growth and reproductive traits of E. carinicauda can be selected through individual or population-phenotypic selection. The above results provide important basic data to support the breeding of E. carinicauda and have considerable application value for future breeding programs.

Abstract:Exopalaemon carinicauda has the highest production in the Yellow Sea and Bohai Sea of China. Compared with those of other shrimp species, E. carinicauda has a short reproductive cycle, fast growth, and adaptability. The pond mono-culture and mixed culture mode is more common in small economic shrimp and can be used as an excellent biological material for crustacean biology research. Dissolved oxygen, an important environmental factor for aquatic animal survival, affects the growth, behavior, reproduction, immunity, and metabolism of aquatic animals and is highly susceptible to hypoxia due to high temperatures, flushing, water pollution, the expansion of the scale of aquaculture, high-density aquaculture, high temperatures, and climate change. Recently, studies have been conducted on the effects of hypoxia on aquatic animals, mainly focusing on immune response, energy consumption, respiration, and antioxidant character. Gill and hepatopancreas tissue are major organs in crustaceans and increasing evidence reveal that gut bacterial community are involved in host immune defense, nutrient absorption, and antioxidant processes. Therefore, investigating the effects of hypoxic stress on the tissue structure and gut bacterial community of E. carinicauda will help to elucidate the mechanism of the response of E. carinicauda to hypoxic environments, which will be useful for its healthy aquaculture. In this study, we collected gill, hepatopancreas, and gut tissues from experimental and control groups after 24 h of hypoxic stress (2.5±0.2) mg/L. The gills and hepatopancreas were sectioned and observed and the changes in gut bacterial community before and after hypoxic stress were analyzed using 16S rRNA gene sequencing. The experimental results showed that gill and hepatopancreas tissues underwent different degrees of damage after hypoxic stress; the gut bacterial community changed, some pillar and epithelial cells of gill tissues were disordered, the number of chloride cells was significantly reduced, chloride cells were changed from irregularly flattened to rounded, the secondary lamellae was aggravated, and the gill tissues changed their morphology and structure to alleviate the hypoxic stress. The number of storage cells in hepatopancreas tissues did not change significantly compared with that of the control group, the lumen contracted significantly, the morphology and structure of the entire hepatic tubule contracted, the volume of transport vacuole increased significantly and even ruptured, and the number of secretory cells decreased significantly. Although shrimp gut bacteria are numerous and diverse the vast majority of gut bacteria are reported to be concentrated in a few dominant bacterial phyla, such as Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. The richness and diversity of gut bacterial community of individuals in the experimental group changed significantly after hypoxic stress and Proteobacteria and Firmicutes were the dominant bacterial phylum in the guts of the control group, accounting for 81.42% and 11.18% of the total amount, respectively. The amounts of Bacteroidota and Actinobacteriota were significantly higher (P<0.05), and that of Proteobacteria were significantly lower (P<0.05) in the experimental group. At the family level, the numbers of Prevotellaceae and Lachnospiraceae were significantly higher (P<0.05) in the experimental group than those in the control group. Rhodobacteraceae bacteria had a relative high abundance in the gut of healthy E. carinicauda and their amounts were significantly reduced after hypoxic stress. In addition, the numbers of some potential pathogenic bacteria were significantly higher (P<0.05) in the experimental group. At present, multiple studies are being conducted on aquatic animals under hypoxic stress; however, the effects of hypoxic stress on the tissue structure and intestinal flora of E. carinicauda remains unexplored. In this study, we observed the damage of tissue structure and simultaneously analyzed the changes in intestinal flora of E. carinicauda under hypoxic stress, to deeply study the physiological response to stress under hypoxic conditions of E. carinicauda and provide basic scientific research on the actual production and cultivation of a novel species of E. carinicauda that is resistant to hypoxic conditions. This will provide basic scientific research information for the actual production and cultivation of novel hypoxia-tolerant varieties of E. carinicauda.

Abstract:Eriocheir sinensis, commonly known as the Chinese mitten crab, is widely distributed in China from the Liaohe River in the north to the Pearl River in the south and in the shallow waters of the estuary into the sea, among which the population in the Yangtze River system has the largest yield and the most delicious taste, and is favored by domestic consumers. It is an important breeding species in the Chinese fishing industry. Since artificial breeding began in the 1980s, the production of Chinese mitten crabs has increased, and this species has become one of the most economically important crabs in the world and the most economically important freshwater crab in China. From the perspective of aquaculture areas, the Yangtze River Basin region accounts for over 80% of the total output in China, with Jiangsu and Anhui provinces ranking first and third, respectively. Therefore, the culture of Chinese mitten crabs in Jiangsu and Anhui plays a pivotal role in the country. After more than 30 years of development, China has established ecological and large-scale breeding systems for juvenile crabs. Therefore, artificial seedlings dominate the market but are mainly distributed in coastal areas such as Jiangsu. However, to reduce production costs, some breeding farms blindly introduce Chinese mitten crabs to one another, and do not perform germplasm detection during breeding, causing germplasm resources to be random and mixed, which directly affects the quality of juvenile crabs and results in the risk of germplasm decline. To evaluate the genetic background of cultured Chinese mitten crab populations in Jiangsu and Anhui in odd-number years, and to provide a scientific basis for subsequent genetic improvement and breeding of new varieties, 10 pairs of highly polymorphic microsatellite markers (simple sequence repeats) were used to analyze the genetic diversity and genetic structure of six cultured populations with 177 individuals in total. Each population contained 28–30 adult individuals with an approximate sex ratio. The leg muscle of the sampled crabs was extracted, and genomic DNA was extracted according to the instructions of the genome extraction kit (TianGen DP304). The microsatellite primers were modified using fluorescent labels (FAM and HEX) prior to PCR, and the PCR products were subjected to capillary electrophoresis. Genemarker 2.2 software was used to interpret typing results. The number of alleles (Na), effective number of alleles (Ne), expected heterozygosity (He), observed heterozygosity (Ho), inbreeding coefficient (Fis), and genetic distance (Dn) were calculated using Popgene 1.32 software, and Hardy-Weinberg equilibrium analysis was performed using the Markov chain method. PIC_CALC 0.6 software calculates polymorphism information content (PIC). Analysis of molecular variance (AMOVA) and the coefficient of genetic differentiation (Fst) were performed using Arlequin 3.5 software. A UPGMA tree was constructed based on inter-population genetic distance using MEGA 4.0 software. According to the frequency of alleles, mutation-drift equilibrium was detected using Bottleneck software. Genetic structure analysis was performed using the Structure 2.3.4 software. The results showed that the genetic diversity levels of the six cultured populations of E. sinensis were high and similar (Na=16.0–18.4, Ne=10.1–12.4, Ho=0.759–0.836, He=0.897–0.916, PIC=0.870–0.892). Genetic distance among populations (0.015–0.277) and the coefficient of genetic differentiation (Fst: 0.001–0.011) were low, and genetic variation among populations accounted for only 0.47% of the total variation in AMOVA. These analyses consistently indicated no significant genetic differentiation among the populations. The phylogenetic tree showed that the six cultured populations had a common ancestor and that the Gaochun population had further relationships with the other populations. Owing to its high genetic diversity, the Gaochun population can be used as a base population for mass selection. Genetic structure analysis showed that the genetic composition of each cultured population was diverse, and the proportions were similar. In this study, the inbreeding coefficient (Fis) of 10 loci in six populations had 43 positive values and 17 negative values, and there were both positive and negative values in each population, indicating that there was not only inbreeding to a certain extent but also a small amount of distant breeding in the six cultured populations of Chinese mitten crabs. The results of genetic structure analysis showed that the number of optimal genetic cluster groups was K=3. However, the chaotic genetic structures among the three groups failed to gather into a relatively independent group. Furthermore, each population had a genetic composition similar to those of the three genetic lineage sources. Considering the high level of genetic diversity in these populations, it is possible that the parental population sources are more complex. This indicated that the cultured population of Chinese mitten crabs had a large number of exogenous genetic hybrids. In conclusion, the genetic diversity of cultured Chinese mitten crab populations in the Jiangsu and Anhui areas is still high, which has potential development and utilization value, but there may be germplasm confounding. Therefore, it is necessary to conduct further studies on the breeding population during the subsequent breeding of improved varieties, such as using molecular markers with high sensitivity that can be used to identify different water systems to analyze the sources of mixed germplasm, and conducting germplasm detection before breeding to purify the germplasm and make reasonable and sustainable use of its germplasm resources.

Abstract:Branched chain amino acids (BCAAs) are neutral amino acids containing branched aliphatic chains on the α-carbon, including leucine (Leu), isoleucine, and valine (Val), accounting for 18%–20% of the total amino acids in animal and plant proteins. BCAAs are essential amino acids for animal growth; however, they cannot be synthesized in animals and can only be obtained from the diet. BCAAs participate in the metabolism of protein, fat, and carbohydrates; promote intestinal development and intestinal amino acid transport; and improve the immune capacity of the body. However, the imbalance of BCAAs in diets leads to poor growth and metabolic disorders in animals. BCAAs have similar chemical structures and catabolic pathways and compete for the same amino acid transporters when passing through cell membranes, resulting in antagonism. Sea cucumbers (Apostichopus japonicus) have rich nutritional value and are an important seafood source in northern China. In recent decades, the study of amino acid nutritional requirements of aquatic animals mainly focused on individual amino acid requirements, and few studies focused on the interaction between amino acids with strong correlations, particularly BCAAs. At present, the interaction of BCAAs has been studied in species such as Chinook salmon (Oncorhynchus tshawytscha), Rainbow trout (Oncorhynchus mykiss), Channel catfish (Ictalurus punctatus), Tilapia GIFT (Oreochromis niloticus), and Tiger puffer (Takifugu rubripes); however, the results have been inconsistent. The purpose of this experiment was to study the interaction effect of Leu and Val on the growth, body wall composition, and digestive ability of sea cucumbers. In this experiment, white fishmeal, algae powder, and wheat flour were used as the primary protein sources, and fish oil and soybean lecithin were used as the main lipid sources to design a basic diet with crude protein and lipid contents of 18.10% and 2.80%, respectively. In a two-way experimental design, 0%, 1.06%, 2.34%, and 3.40% coated Leu and 0%, 1.74%, 2.50%, and 3.48% coated Val were added to the basic feed. Sixteen groups of isonitrogen and isolipid diets were prepared with four Leu levels (actual content: 1.00%, 1.50%, 2.10%, and 2.60%) and four Val levels (actual content: 0.65%, 1.40%, 1.70%, and 2.20%). A total of 960 healthy sea cucumbers with an initial average weight of 16.80±0.18 g were selected and randomly assigned to 48 cylindrical circulating buckets. They were divided into 16 experimental groups, with three replicates in each group and 20 sea cucumbers in each replicate. The feeding period was 60 d. Bait was fed once a day at a fixed time (16:00). The water was changed every 2 d, and a siphon was used to withdraw the residual bait and feces from the bottom of the bucket. The amount of water changed was 50% of the water level in the bucket. During the breeding period, the water temperature was 14–17 ℃, pH was 7.4–8.2, and dissolved oxygen was at least 6 mg/L. A low-light environment was maintained indoors. The results showed that the Leu and Val contents and their interaction significantly affected the weight gain (WG) and specific growth rate of sea cucumbers. When the Val content was 1.4%, the crude lipid contents of the body wall increased with increasing dietary Leu contents, and there was a synergistic effect between Leu and Val; the L1.0V1.4 group was significantly lower than the other groups. The interaction between Leu and Val significantly affected the Val, Met, Tyr, and Phe contents in the body wall of sea cucumbers. When the Leu content was 2.1%, the intestinal lipase and amylase activities first increased and then decreased with increasing dietary Val contents; the interaction between Leu and Val showed a synergistic and then antagonistic effect. The amylase activity reached a maximum value in the L2.6V1.4 group and was significantly higher than that in other groups. In conclusion, Leu and Val contents and their interaction significantly affected the growth performance, body wall amino acid composition, and digestive ability of sea cucumbers. Taking WG as the evaluation index, the ratios of Leu and Val in sea cucumber feed were 1.90 (Leu content was 2.6%, Val content was 1.4%).

Abstract:The shallow-sea net-cage culture of sea cucumber (Apostichopus japonicus) has been developing rapidly in recent years, and the culture mode of feeding formula feed has been accepted by farmers. However, the optimal culture density under the feeding mode is still unknown. To evaluate the influence of culture density on the shallow sea net-cage culture of sea cucumber under a feeding mode, this study examined the growth, digestive enzymes, and gut bacterial community structure of sea cucumber at different culture densities. The culture density was set to 10 kg/cage, 20 kg/cage and 30 kg/cage, marked as F10, F20 and F30, respectively. Sea cucumber seedlings with an initial body weight of (75.11±2.99) g were randomly assigned into 9 cages (3 replicates of each density level) according to the density settings. The net cage is cuboidal and its length, width and height were 4 m, 4 m, and 3 m, respectively. After a 42-day experimental period, the total weight of sea cucumber in each cage was weighted and 20~30 individuals in each cage were randomly sampled for individual weight and body wall weight. Then, intestines from 3 individuals were sampled and preserved at –80 ℃ freezer. Digestive enzyme (Trypsin, Amylase, and Lipase) activities were determined spectrophotometrically using enzyme activity assay kits (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s instructions. Gut microbial DNA was extracted, and the V3～V4 region of the prokaryotic ribosomal RNA gene (16S rDNA) was amplified and sequenced on an Illumina Novaseq platform. Sequence data were then analyzed on the platform BMKCloud (www.biocloud.net). The results showed that as culture density increased, the weight gain rate of sea cucumbers in net-cage culture significantly decreased, and both the F20 and F30 groups had significantly lower weight gain rates compared to the F10 group (P<0.05). In addition, increasing culture density resulted in a significant increase in net production and body wall production rates, although none of the differences were significant (P<0.05). The feed conversion ratio of the F10 group was the lowest and not significantly different from that of the F20 group, but significantly lower than that of the F30 group (P<0.05). The enzyme activities of all three digestive enzymes showed a decreasing trend with the culture density increase. The digestive enzyme activity of F30 was significantly lower than that of F10 and F20 (P<0.05). The similarity of the bacterial community structure between the F10 group and F20 group was relatively high. The ACE index of F10 group was significantly lower than that of the F20 group (P<0.05), while the Chao1 index, Shannon's index and Simpson's index showed no significant difference from that of the F20 group (P>0.05). All the microbial diversity indices of the F30 group were significantly lower than those of the other density groups (P<0.05). The relative abundance of dominant organisms varied with culture density. The relative abundance of Firmicutes and Actinobacteria tended to decrease with increasing density, while the opposite was true for Bacteroidota and Proteobacteria. The relative abundance of Actinobacteria in group F10 was significantly higher than that of group F30 (P<0.05). The relative abundance of Lachnospiraceae, Bifidobacteria and Streptococcus was significantly higher in group F10 than in groups F20 and F30 (P<0.05), and the relative abundance of Oscillospiraceae and Rikenellaceae was significantly higher in the F20 group than in the F10 and F30 groups (P<0.05). LEfSe analysis showed that F10, F20, and F30 were mainly significantly enriched in Streptococcus, Ruminococcus UCG_005 and Alkanindiges, respectively. Pearson's correlation analysis showed that three gut bacteria genera were significantly positively correlated with digestive enzyme activities, including Streptococcus, unclassified_ Muribaculaceae and UCG_005. According to the benefit estimation, there was little difference in culture benefit between the F20 and F30 groups, both of which were significantly higher than the F10 group. The results indicate that higher culture densities can improve the net-cage culture production of sea cucumber, thus increasing the overall benefit. However, too high culture density could negatively affect the growth, digestive enzymes, and the gut microbial balance of the sea cucumber. Therefore, to improve the overall benefit, it is better to increase the culture density to 20 kg/cage in the current small net-cage feeding mode. These results provide references for the scientific culture of sea cucumbers in shallow-sea net-cage systems, leading to improvements in both quality and culture efficiency.

Abstract:Antarctic krill (Euphausia superba) is a vital species in the Antarctic ecosystem, boasting substantial reserves and high nutritional value. It has emerged as a significant commercial fishery resource in the Southern Ocean. However, because of its exceptionally high autolytic enzyme activity, it is susceptible to autolysis and blackening during processing, storage, and transportation, resulting in a decline in the sensory quality and nutritional value of raw materials. Shrimp melanosis is caused by a series of enzymatic reactions mediated by phenoloxidase (PO) within the body. PO facilitates the oxidation of phenolic substances, leading to the formation of highly unstable quinone compounds, which further react to produce melanin. The accumulation of melanin significantly affects the appearance of Antarctic krill, thereby reducing its commercial value. On account of their cost-effectiveness and potent anti-blackening effects, sulfites are commonly used as shrimp anti-black agents. Sodium metabisulfite is one of the compounds whose use is allowed for shrimp preservation under GB 2760-2014 regulations. It not only inhibits PO activity, but also reduces quinone compounds to prevent blackening. However, excessive sulfite use can lead to discomfort and health issues such as gastrointestinal problems, liver disturbances, dyspnea, vomiting, diarrhea, and headaches. Therefore, GB 2760-2014 regulations stipulate that the residual SO2 content should not exceed 0.1 g/kg. Currently, the use of sodium metabisulfite in production is widespread but lacks standardized requirements, with limited guidance available on how to regulate its usage. Therefore, to assess the impact of sodium metabisulfite on blackening control in Antarctic krill and refine the treatment process, experiments were conducted to measure the changes in SO2 residue and PO activity in Antarctic krill subjected to various concentrations of sodium metabisulfite solutions and soaking times. Furthermore, observations and analyses were conducted to monitor the blackening of Antarctic krill during cold storage and frozen storage. The results demonstrated a positive correlation between the SO2 residue in Antarctic krill and the concentration of sodium metabisulfite solution. A concentration of 1 g/L sodium metabisulfite solution resulted in a SO2 residue of 0.065 g/kg in Antarctic krill, which is below the national standard limit of 0.1 g/kg. However, treatment with a 2 g/L solution led to a SO2 residue of 0.166 g/kg in shrimp, exceeding the national standard limit. Treatment with a 3 g/L solution resulted in a SO2 residue of 0.254 g/kg in shrimp, which significantly surpassed the national standard limit. Consequently, we recommended using 1 g/L or 2 g/L sodium metabisulfite solutions to prevent blackening in Antarctic krill while maintaining the SO2 residue below 0.1 g/kg. Under these conditions, the treatment time for a 1 g/L sodium pyrosulfite solution could be extended to 300 s, with a relative PO activity of 21.2%. For a 2.0 g/L sodium pyrosulfite solution, a treatment time of 30 s was suitable, yielding a relative PO activity of 34.5%. During storage, the control group exhibited noticeable blackening after storage at 4 ℃ for 24 h and frozen storage for 6 months, predominantly in the head, chest, abdomen, and tail sections, and slight variations were observed in the blackening process among individuals. Compared with the control group, the sodium metabisulfite treatment group displayed significantly lower blackening scores during storage (P<0.05). Specifically, the 1 g/L sodium metabisulfite solution with 300 s treatment group and the 2 g/L sodium metabisulfite solution with 30 s treatment group showed obvious blackening at 48 h and 72 h of cold storage, respectively, and after 12 months of frozen storage, indicating that higher concentrations of sodium metabisulfite solution during treatment led to a better control effect on the blackening of Antarctic krill during storage. Notably, PO activity is just one of the factors influencing the melanosis process. Although the 1 g/L sodium metabisulfite solution with treatment for 300 s could better inhibit PO activity, the anti-blackening effect of this group was not as pronounced as that of the 2 g/L sodium metabisulfite solution with treatment for 30 s during storage. In summary, soaking in 1 g/L sodium metabisulfite solution for 300 s and 2 g/L sodium metabisulfite solution for 30 s resulted in relative PO enzyme activities of 21.2% and 34.5%, respectively. Simultaneously, the SO2 residues in shrimp were measured at 0.094 g/kg and 0.096 g/kg, respectively, meeting the GB 2760-2014 limit of SO2 residue not exceeding 0.1 g/kg. Compared with the control group, sodium metabisulfite treatment effectively delayed the blackening process of Antarctic krill during cold storage and frozen storage, with the 2 g/L sodium metabisulfite solution soaking for 30 s exhibiting superior anti-blackening effects. This suggests that a high concentration of sodium metabisulfite solution and a short treatment period had a better blackening inhibition effect. These findings offer a theoretical foundation and technical guidance for maintaining the quality of Antarctic krill during storage and transportation.

Abstract:With the development of China's economic level and improving people's living standards, the state's supervision of food health and safety has increased. Consumer demand for many people has gradually changed from being satiated to eating well, and high-quality, environmentally friendly, and healthy aquatic products have become increasingly popular with consumers. However, intensive and high-density aquaculture causes the deterioration of water environmental conditions, the increase of anti-nutrient factors and nutritional imbalance caused by the replacement of animal protein with plant protein in feed, and changes in the growth, immunity, and digestive ability of aquatic animals caused by changes in feed nutrients, traditional processing methods, and transportation stress. The above situations encountered in aquaculture have a common impact on aquatic animals and inevitably cause a reduction in the muscle quality of aquatic animals. Therefore, improving the muscle quality of aquatic animals and the quality of aquatic products is an urgent problem with significant research value and potential. To improve aquatic quality, first, it is necessary to first identify the indicators of muscle quality in aquatic animals to improve aquatic quality. However, there is still a lack of a systematic muscle quality evaluation index system in the current aquaculture research. Therefore, this study reviewed the evaluation indices of muscle quality, including meat content, muscle nutrient composition, muscle fiber structure, muscle texture characteristics, and muscle physicochemical indices, to provide a reference for establishing scientific muscle quality evaluation indices. In addition, factors affecting muscle quality must be considered in studies aimed at improving the muscle quality of aquatic animals. Only by understanding the factors affecting muscle quality in aquatic animals can the key factors regulating muscle quality and their mechanisms be studied more comprehensively and scientifically. This study reviewed the factors affecting the muscle quality of aquatic animals, including feed nutrients, feed additives, and the culturing environment, and found that nutrients are key factors in improving the muscle quality of aquatic animals. Protein is an important nutrient, the main component of muscle, and an important indicator of muscle quality. Amino acids are the basic units of proteins and have a vital impact on muscle quality and flavor. Therefore, it is important to study the effects and mechanisms of action of amino acids on muscle quality in aquatic animals. Therefore, the effects of dietary amino acids on the muscle quality of aquatic animals were reviewed in this study, including the effects of amino acids on muscle nutrients, flavor, texture indices, and physical and chemical properties of aquatic animals. Furthermore, the effects of amino acids on muscle quality, including the promotion of protein synthesis, regulation of fat deposition, enhancement of antioxidant activity, and maintenance of intestinal health, were reviewed. In summary, this study summarized the influence of amino acids on the muscle quality of aquatic animals and its mechanism from the aspects of muscle quality evaluation indices, factors affecting meat quality, and the influence of amino acids on muscle quality, providing a reference for further research on the regulation of muscle quality by amino acids and the improvement of feed nutrients on the meat quality of aquatic animals.

Abstract:Cell transfection is a specialized technique for introducing exogenous molecules, such as RNA and DNA, into cells and has become a prevalent method in gene functionality studies. The most frequently employed cell transfection methods include electroporation, viral infection, and liposome transfection. Liposome transfection is a prevalent technique in molecular biology for studying gene function and regulation. Liposome transfection has the advantages of wide applicability, superior efficiency, and simple operation. However, most liposome transfection reagents on the market are designed based on mammalian cells. Cell membrane composition, temperature adaptation range, and oxygen demand in teleost cells differ substantially from those in mammalian cells. Accordingly, the efficiency of liposome transfection in teleost cells is limited, and obtaining stable transfected cells directly through screening is challenging. Therefore, after the transfection of conventional transient expression vectors into teleost cells via liposomes, most of the plasmid copies tend to vanish due to cell proliferation after the duration of screening. Furthermore, the insignificant number of cells retaining plasmids makes it difficult to correctly integrate plasmids into the genome sequence, which hinders screening for stable cell lines. In this study, we investigated a method of rapidly obtaining stable transfected cells of Japanese flounder by transfecting the Tol2 transposon recombinant vector with liposomes. The CMV-EGFP-pminiTol2 vector was modified from the pminiTol2 plasmid, sustaining the functionality of the Tol2 transposable element to facilitate the highly efficient insertion of target genes into genomic DNA. The CMV-EGFP-pminiTol2 plasmid was highly suitable for stable transformation screening in Japanese flounder cells. This advantageous trait was mainly attributed to the following four aspects: (1) The CMV-EGFP-pminiTol2 plasmid carries autonomous transposon elements that can significantly enhance the integration ability of target genes. (2) The CMV-EGFP-pminiTol2 plasmid carries EGFP, allowing researchers to visualize transfection and screening effects directly via fluorescence microscopy. (3) The CMV-EGFP-pminiTol2 plasmid incorporates a neomycin resistance selection marker, which can be used to quickly and successfully screen transfected cells after G418 screening. (4) The multiple cloning site (MCS) region of the CMV-EGFP-pminiTol2 plasmid is mutated to retain two unique cleavage sites (XhoI and XmaI); plasmids can be linearized through these two sites, allowing the target gene to be inserted using homologous recombinase cloning. CMV-EGFP-pminiTol2 and pCS-TP plasmids were co-transfected into flounder cells via liposome encapsulation, and stable transfected cells were obtained through neomycin (800 μg/mL) screening for 2–3 weeks. After 2 weeks of screening, cells were stably cultured for 2–3 weeks without G418 screening. Finally, the EGFP fluorescence of the cells was observed using an inverted fluorescence microscope to confirm the transfection efficiency of the cells. The results revealed that the CMV-EGFP-pminiTol2 plasmid-transfected experimental group successfully expressed EGFP in all cells after G418 selection. In the pEGFP-C1- or pEGFP-N1-transfected control group, only a minor fraction of cells (≤10%) sustained EGFP expression after G418 screening. The CMV-EGFP- pminiTol2 vector is therefore suitable for the preparation of stable transgenic cells in Japanese flounder, offering numerous advantages, including reduced screening time, stable exogenous gene expression, simplified operations, and relatively low cost. Consequently, researchers can insert their desired target genes into the CMV-EGFP-pminiTol2 vector to construct stable transformed cells for investigations such as functional analysis and assessments of subcellular localization and long-term regulation. This study describes a novel approach to rapidly screening stably transformed flounder cells using the Tol2 transposon system. The results can be applied to molecular biology research in flounder and provide novel insights for constructing and screening stable transformations in teleost cells.