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Abstract:The aquatic ecosystem is essential for human survival and development. Food web studies on aquatic ecosystems describe many key processes, such as material cycling and energy flow processes. The results are of great significance for protecting the diversity, stability, and functionality of biological and ecological systems. However, the extreme complexity of aquatic ecosystem food webs has puzzled ecological researchers. The development of stable isotope technology has contributed significantly to our understanding of intricate feeding relationships and structure of food webs. The carbon and nitrogen-stable isotope compositions of animal tissues are closely related to their food sources, reflecting the comprehensive characteristics of all food isotopic compositions assimilated over a period of time by animal tissues. The carbon isotope composition of animals can indicate their food source, while the nitrogen-stable isotope composition can reflect the trophic structure. However, the animal diet is not composed of a single food source, and the complexity and unpredictability of food sources complicate the interpretation of carbon and nitrogen-stable isotope data regarding the contribution of food sources. In recent years, the rapid development of stable isotope mixing models (SIMMs) has provided a powerful means to solve the complexity of aquatic food webs, particularly with regard to determining the contribution of food sources and comparing consumer niches. In this study, we reviewed the historical evolution of stable isotope models, the characteristics of the most prominent models, and factors to consider when utilizing such models. This study summarized the application prospects and limitations of stable isotope mixing models in the research field of aquatic ecosystem food webs in order to provide a reference and scientific basis for subsequent research. The development of stable isotope models can roughly be divided into three stages: the early initial stage (1976–2001), which mainly involved the development of linear models; the supplementary stage (2001–2008), during which numerous uncertain factors were added to the model analysis as a result of the advancement of computer technology; the Bayesian mixed model stage (2008–current). The mixed model stage is based on mass conservation, and the Bayesian framework greatly improves the accuracy and credibility of model analysis. Simmr, MixSIAR, and SIBER have become the primary isotope models used today. Stable isotope technology has a long history of development, and the emergence of various stable isotope models has expanded the application of isotope technology in the research field of food webs. These models have powerful analytical capabilities and provide researchers with a powerful means for analyzing the structure of the food web. However, owing to the limitations of isotope analysis and models, researchers should pay more attention to the relevant prerequisites and practical ecological significance when selecting models to analyze isotope data. Researchers should also devise reasonable field investigation and experimental treatment plans based on their own experimental conditions and economic capacity in order to avoid the error analysis and misuse of isotope models and reduce the uncertainty of model analysis results as much as possible. Based on the previous research progress, the authors share their experience and understanding of the application of isotope mixing models. The purpose of this study was to assist fellow researchers in quickly understanding the development process and matters requiring the attention of isotope models, as well as to provide fundamental data and theoretical support for related research.

Abstract:At present, the world is still experiencing a climate warming trend, which has severe implications for Earth’s sustainable development. Increasing carbon sinks mitigates climate change and improves national and social development through carbon trade. As the largest carbon pool on the planet, the ocean serves as a major carbon sink. The fishery carbon sink is an important part of the ocean carbon sink, which can increase the capacity of aquatic fishery ecosystems to absorb atmospheric CO2. Marine ranching is a typical example of a carbon sink fishery and an effective model for ensuring the sustainability of the carbon sink effect of aquatic fishery ecosystems. Despite the fact that marine ranching plays an important role in enhancing the carbon capacity of fishery carbon sinks, there have been few studies on marine ranching carbon storage. Reef fish species are the target population for marine ranching conservation and enhancement and are the most typical biological community of an artificial reef ecosystem, with substantial ecological and economic benefits. Reef fish species have an important influence on the carbon cycling, deposition, and removal processes in the ocean. It is one of the key carbon storage components of the marine ranching carbon sink. However, there are currently only a few studies on the carbon storage capacity of reef fish. Therefore, estimating and predicting the carbon storage of reef fish are useful for assessing the carbon sink potential and formulating a stock enhancement strategy for reef fish based on the carbon sink target. Based on this, we conducted research on five species of reef fish. On the one hand, we determined the carbon content of reef fish and used the standing biomass of reef fish harvested by cage nets from Shique Beach marine ranching in April 2017, January 2018, May 2019, and December 2020 to estimate the carbon capacity of reef fish in the artificial reef area and contrast areas in spring 2017, winter 2018, spring 2019, and winter 2020. On the other hand, we used the Grey-Markov model, a prediction model that we initially employed in a fishery study, to predict the carbon potential of reef fish in the artificial reef area in spring 2021, winter 2022, spring 2023, and winter 2024. The results showed that the mean carbon contents of dry reef fish samples were 42.95%~50.19% and that the mean carbon contents of fresh reef fish samples were 11.05%~13.25%. The standing biomass carbon storage of reef fish decreased on a regular basis in reef areas, whereas it fluctuated in contrast areas. The standing biomass carbon storage values of reef fish in the reef area in spring 2017, winter 2018, spring 2019, and winter 2020 were 293.46 t, 104.49 t, 119.40 t, and 48.48 t, respectively. This equates to approximately 0.73 × 104~4.40 × 104 USD carbon sink economic value. The standing biomass carbon storage values of reef fish in contrast areas in spring 2017, winter 2018, spring 2019, and winter 2020 were 21.64 t, 59.07 t, 6.73 t, and 0 t, respectively. Reef fish in reef areas had much higher standing biomass carbon storage values than those in contrast areas. The average relative error of the validation data of the Grey-Markov model was 8%, which was 12% better than the prediction accuracy of the GM(1,1) model; therefore, we were able to fully demonstrate the superiority of the Grey-Markov model for accurate short-term prediction of the carbon storage of reef fish. The standing biomass carbon storage values of reef fish in reef areas in spring 2021, winter 2022, spring 2023, and winter 2024 were 64.84 t, 49.84 t, 25.28 t, and 19.43 t, showing a decreasing trend. In summary, reef fish species have a strong carbon storage capacity and a high ecological value, which give them an important role in fishery carbon sinks. However, the carbon storage capacity of reef fish is expected to decline in the future, which may be related to their overexploitation. Reef fish species are the primary targets of traditional fisheries. Therefore, we can take the following measures: First, we should consider the carbon storage effect of reef fish; Second, marine ranching operators can strengthen their investment in the construction of artificial reef areas and conduct active stock enhancement of reef fish, which can increase reef fish resources and subsequently reef fish carbon storage. Third, we can strengthen the environmental protection and management of marine ranching. The findings of this study not only provide a basis for assessing the carbon storage potential of reef fish but also serve as a scientific reference for establishing a strategy to develop reef fish resources in marine ranching based on fishery carbon sinks.

Abstract:With the rapid development of aquaculture in China in recent decades, the country now faces environmental problems, such as high energy consumption and severe environmental pollution. The Yangtze River Basin is the most important area for freshwater aquaculture production in China, accounting for more than half of the total production. The environmental issues induced by aquaculture have received widespread consideration in this area. However, the freshwater farming-induced environmental issues in the Yangtze River Basin were rarely evaluated in prior research. Life cycle assessment (LCA) is a valid tool for assessing environmental impacts and resources and is widely deployed in the industrial sector. It can quantify the impact of a product or service on different environmental indicators at various stages of the production process, thereby aiding in the identification of the best measures to reduce environmental impacts. The LCA is more effective than traditional environmental impact assessment methods; thus, it has been gradually applied in other fields, such as agriculture and aquaculture, in recent years. In aquaculture, LCA has been used to evaluate the environmental impact of various farming models or species in a laboratory setting. However, little attention has been paid to large-scale systems, such as the Yangtze River Basin. The two most important farming models in China are the pond farming model and the integrated rice-fish farming model. Among them, the integrated rice-fish farming model is regarded as environmentally friendly and an important step towards realizing the sustainable development of aquaculture in terms of the efficient utilization of water and land resources. Therefore, in this study, the environmental effects of pond farming and integrated rice-fish farming models were investigated in the Yangtze River Basin using the LCA method. A total of 20 monitoring points were established in the Yangtze River Basin, including 5 for integrated rice-fish farming and 15 for pond culture. In the integrated rice-fish farming model, the life cycle consists of two stages: Feed supply and breeding. In pond culture, the life cycle consists of three stages: Feed supply, power supply, and breeding. We analyzed the values of energy use (EU), global warming potential (GWP), acidification potential (AP), eutrophication potential (EP), and water use (WU) in the pond farming and integrated rice-fish farming models using the LCA method, assessed the contribution rate of each phase for environmental indices, and compared the environmental impact of the two farming models. In order to establish the inputs and outputs of an aquaculture system, experimental measurements and surveys were conducted to collect data. The collected data mainly included feed consumption, electricity consumption, water consumption, total phosphorus and total nitrogen emissions, and greenhouse gas (CH4 and N2O) emissions. The data were analyzed by the LCA software eBalance. In this study, the weight gain of 1 ton of aquaculture products served as the functional unit, and the results were standardized and evaluated in order to compare the environmental impacts of various impact indicators. The results showed that the standardized and weighted evaluation values of WU, EP, GWP, AP, and EU in the integrated rice-fish farming model were 11.650, 0.770, 0.141, 0.096, and 0.003, respectively, and the total environmental impact indicator was 12.660. In the pond farming model, the values of WU, EP, GWP, AP, and EU were 31.453, 1.187, 0.210, 0.174, and 0.007, respectively, and the total environmental impact indicator was 33.031. The environmental impacts of integrated rice-fish farming were lower than those of pond farming. The contribution rate analysis of the three stages of the life cycle to environmental impact showed that the contribution rates of EU, GWP, and AP in the feed supply stage were higher than those in the power supply and breeding stages. EP was primarily associated with the feed supply stage and pollutant discharge stages of the breeding process, and WU was principally concentrated on the breeding stage. Overall, the LCA results showed that the integrated rice-fish farming model had a more environmentally friendly effect than the pond farming model, which indicates that the Yangtze River Basin has considerable development potential. WU was the most influential environmental impact indicator and the main restriction factor in aquaculture; it was mainly associated with the breeding stage. Therefore, moderately increasing stocking density was an effective strategy for reducing water resource consumption and improving water resource utilization rates. In addition to WU, the main environmental impact index of the pond culture model and integrated rice-fish farming models comprised EP, followed by GWP, AP, and EU. The contribution rate analyses of the two models showed that the aquaculture-induced environmental impacts could be mitigated by improving feed production technology, establishing accurate feeding technologies, applying advanced effluent water treatment technologies, and appropriately increasing stocking density in the Yangtze River Basin. In summary, in this study, we compared the environmental impacts between the pond culture and integrated rice-fish farming models in the Yangtze River Basin using the LCA method. In addition, we analyzed the differences between environmental impacts at different production stages. These data served as a reference for the sustainable development and optimization of aquaculture in the Yangtze River Basin.

Abstract:The construction of dams impedes energy exchange and material circulation in rivers, and the operation of hydropower stations negatively impacts the function of river ecosystems. Countries across the globe have implemented various fish protection countermeasures and conducted many associated hydraulic and ecological studies to mitigate the impact of hydropower development on fish survival, maintain the abundance and diversity of fish populations, and restore riverine and lacustrine fish migration routes and habitats. Remarkably, wild fish generally migrate in groups, whereas most contemporary studies on fish passage facilities focus on individual fish. Hence, the behavioral characteristics of fish schools are worth investigating. Given that schooling behavior is a pervasive feature of fish communities and plays an essential role in dealing with potential risks, improving self-adaptation, expanding resilience, and enhancing population sustainability, this study concentrated on investigating its effect on upstream fish migration and decoding the internal mechanism. We conducted an experiment targeting grass carp (Ctenopharyngodon idella) and silver carp (Hypophthalmichthys molitrix), two economically important freshwater fish species in China, on a non-uniform flow ground with low turbulence. The experiment quantified the impact of fish schooling on their ability to overcome flow barriers by examining the ascending sustainability and swimming performance of five-fish groups and one-fish groups at three flow velocity levels (0.25–0.50 m/s, 0.30–0.60 m/s, and 0.35–0.70 m/s) utilizing a novel index system. The new index system employed nondimensionalized ascending sustainability, first-attempt endurance, and first-attempt ascending energy consumption to indicate the fish's persistent ascending ability, ascending efficiency, and ascending energy cost, respectively. Finally, the ascending trajectory of fish was investigated simultaneously to determine the distribution of trajectory and the distribution of the hydrodynamic force factor at the trajectory points. It could be concluded that (1) the influence of schooling behavior on ascending fish behavior was related to the ascending sustainability of individual fish. Schooling behavior significantly increased the ascending sustainability while producing no specific impact on the ascending efficiency at low-level individual ascending sustainability. Inversely, schooling behavior significantly decreased the ascending efficiency while having no particular effect on high-level individual ascending sustainability. Moreover, the contribution of schooling behavior to the ascending sustainability of grass carp varied with the flow velocity, as the ascending sustainability of the five-fish groups was significantly higher (P = 0.030) and lower (P = 0.048) than the one-fish group at the velocity levels of 0.30–0.60 m/s and 0.35–0.70 m/s, respectively. In contrast, schooling behavior holistically improved the upward swimming of silver carp, significantly increasing the ascending sustainability of the five-fish groups at velocity levels of 0.30–0.60 m/s (P = 0.004) and 0.35–0.70 m/s (P < 0.001). (2) The endurance of the first attempt in the juvenile grass carp group was significantly higher than that of the single fish at the velocity levels of 0.25–0.50 m/s (P < 0.001) and 0.35–0.70 m/s (P = 0.005), while the endurance of the first attempt in the juvenile silver carp group was significantly higher than that of the single fish at the velocity levels of 0.25–0.50 m/s (P < 0.001) and 0.30–0.60 m/s (P = 0.005). The endurance of the first attempt in juvenile silver carp decreased significantly in schools (P < 0.001) solely at the velocity level of 0.35–0.70 m/s. In addition, the flow velocity generally increased the first-attempt endurance and cumulative energy consumption of individual and grouped fish. However, since the burst-coast swimming mode forced on juvenile silver carp in high-velocity conditions significantly improved ascending efficiency, the endurance of the first attempt initially increased and then decreased with the flow velocity. (3) Schooling behavior enabled grass carp to swim with less energy and significantly lowered their energy cost at the velocity level of 0.25–0.50 m/s (P < 0.001), whereas it augmented the accumulated energy consumption of five-fish groups in silver carp and significantly increased their energy cost at the velocity level of 0.25–0.50 m/s (P = 0.050). (4) Collectively, juvenile silver carp could find an ideal ascending trajectory more rapidly than juvenile grass carp. The ascending trajectory of grass carp tended to concentrate at first and then disperse with increasing velocity, whereas the trajectory of silver carp tended to concentrate with increasing velocity. In brief, schooling is an unsubstituted behavior in the upstream migration of fish migrants, motivating and inhibiting the fish's upstream movement performance with its primary effect on locomotion in terms of energy consumption, visual response, and the ability to overcome flow barriers. The findings could improve the design of fish protection measures and provide specific recommendations for the operation of fish passage facilities. For example, when silver carp migrate through a fishway, the additional light source can adequately promote schooling behavior and improve their capacity to overcome flow barriers. When grass carp migrate through a fish passage, the additional light source in the rest pond of the fishway can effectively promote schooling behavior, thereby preventing the return of fish owing to a lack of ascending sustainability. Considering that the hydrodynamic environment of natural waters and the size of fish groups are highly complex and variable, the schooling behavior characteristics of fish groups in different water flow environments should be investigated in future research to enrich the database for the planning and implementation of fish protection engineering measures.

Abstract:Sebastes schlegelii is a cold temperate ovoviviparous fish found near the seabed layer and is widely distributed in the Yellow Sea and Bohai Sea of China, as well as in the coasts of South Korea and Japan. It is characterized by its tender meat, delicious taste, rich nutrition, and high medicinal value, thus it is an economically important marine fish species in the northern coastal areas of China. In recent years, deep-water cage cultures have rapidly developed in China. Among the economically important fish species suitable for cage culture in northern China, S. schlegelii is the most important local breed with excellent quality and characteristics. However, with the expansion of the scale of the aquaculture industry, aquaculture diseases including those caused by Vibrio harveyi, Vibrio anguillarum, and Photobacterium damselae subsp. damselae, occur frequently. The authors isolated and identified a dominant pathogenic bacterium, the P5W of V. harveyi, from the lesions of diseased S. schlegelii and studied the response of S. schlegelii to V. harveyi infection. Multitissue transcriptome analysis screened the immune differential gene protein kinase B. Protein kinase B, also known as Akt protein, is a serine or threonine protein kinase, which mainly conducts signal transduction by phosphorylating other proteins. It plays an important role in various biological processes such as cell metabolism, transcriptional regulation, cell cycle regulation, immune defense, and embryonic development. Many studies have shown that during viral infection of cells, the Akt pathway is activated, thereby inhibiting viral proliferation. To clarify the sequence information, evolutionary characteristics, and biological functions of the Akt gene in S. schlegelii and to understand the immune response mechanism of S. schlegelii to bacterial invasion, the full-length cDNA of the SsAkt gene was obtained using molecular biology, and the sequence characteristics, tissue expression rules of the gene, and the response rules to Micrococcus luteus and V. anguillarum were studied. The results showed that the length of the open reading frame was 1 440 bp. The predicted relative molecular mass of the SsAkt protein was 55.80 kDa and the isoelectric point was 5.64. Sequence analysis revealed that the SsAkt protein contains three conserved domains: the PH domain (residues 6–109), serine/threonine protein kinase catalytic (S_TKc) domain (residues 148–405), and serine/threonine protein kinase (S_TK_X) domain (residues 406–475). The conserved amino acid site appears in the center of the S_TKc domain and at the end of the S_TK_ X domain: Thr305 and Ser472. Homologous alignment showed that the amino acid sequence of this gene was highly conserved among different species. Phylogenetic tree analysis showed that SsAkt is closely related to Akt in vertebrates, such as Sebastes umbrosus, Lates calcarifer, Larimichthys crocea, and Cyprinus carpio. The relative expression of SsAkt in different tissues of S. schlegelii was detected by real-time fluorescence quantitative PCR. The results showed that SsAkt was expressed in all tested healthy tissues of S. schlegelii, which indicated the relative expression of the SsAkt gene in S. schlegelii. This expression has distinct tissue specificity. The expression of SsAkt in the kidney is the highest, and in the blood and brain, it is also higher than that in other tissues. Therefore, it is speculated that higher expression of SsAkt in the kidney and brain is closely related to immune defense, neurodevelopment, and other processes. To study the role of SsAkt in the immune response of S. schlegelii, we designed a bacterial challenge experiment. The results showed that the transcription level of SsAkt changed significantly, and the relative expression of SsAkt in the three tissues increased after stimulation with M. luteus and V. anguillarum compared with the control group (PBS). After infection with M. luteus, its expression only increased sharply in the kidney, whereas its expression increased slowly in the blood and liver. In the experimental group infected with V. anguillarum, the expression increased significantly in the liver and kidney, and the two relative expression peaks in the kidney occurred at 12 h and 72 h after infection, respectively. The results revealed that the SsAkt gene may play an important role in the immune defense of S. schlegelii, which can further enrich the sequence information and evolutionary data of the Akt gene in marine fish and provide reference materials for an in-depth study of the biological function of this gene. In follow-up experiments, we will explore the specific mechanism of SsAkt in the innate immune defense of S. schlegelii to provide theoretical guidance for the study of the immune signaling pathway of S. schlegelii and to lay a theoretical foundation for revealing the role of SsAkt in other important biological functions in growth and development.

Abstract:α-Tocopherol transfer protein (α-TTP) is a protein that exhibits a marked ligand specificity that selectively recognizes α-tocopherol (vitamin E) and plays a key role in regulating concentrations of vitamin E. The primary function of α-TTP is maintaining adequate vitamin E levels. However, the available data are insufficient to comprehensively understand the mechanisms by which α-TTP regulates vitamin E supplementation. Related studies have been mainly focused on humans, mice, and rats as the primary subjects; however, little is currently known about fish. The pituitary gland plays an important role in the growth and reproduction of fish. It synthesizes growth hormone and gonadotropin after receiving signals from the brain and releases the hormones into the blood through the axons. It is then transported to the target cells (tissues or organs) through the blood to perform the physiological function of hormones. As the pituitary tissue is very small, being nearly half the size of a grain of rice, it is difficult to detect the nutrient contents (such as vitamin E, which has been verified to play an important role in the growth and reproduction of fish) in the tissue. This limits the evaluation of the effect of nutrients on the pituitary gland. Therefore, this experiment was conducted to investigate the effects of vitamin E concentrations on the expression of the α-TTP gene in the pituitary tissue of a half-smooth tongue sole (Cynoglossus semilaevis). DL-alpha-tocopherol acetate (vitamin E, Sigma) was added to the basic diet, which was formulated with fish meal, casein, and wheat meal as protein sources, as well as phospholipids, fish oil, and soybean oil as lipid sources. The finalized crude protein was 56.0%, and the crude lipid was 14.1% at concentrations of 0, 200, 400, 800, and 1 600 mg/kg to feed half-smooth tongue sole (464.0±2.6 g) for 60 d. The fish were maintained in polyethylene tanks (diameter: 150 cm, height: 60 cm) with flowing filtered seawater at a rate of 50 L/min, and during the feeding trial, the water temperature ranged from 24 to 27 ℃, salinity from 30 to 31, and pH from 7.8 to 8.1. Dissolved oxygen was greater than 5.65 mg/L. In addition, the pituitary glands of similar fish were collected, minced into small pieces (about 1 mm3), and digested with 0.25% trypsin-EDTA (Gibco) for approximately 15 min at 25 ℃. Then, the digested glands were filtered through 70 μm nylon mesh (BD Falcon), filtrates were collected and centrifuged at 100 ×g for 10 min at 4 ℃, and cell deposits were resuspended in 1 mL L-15 medium containing 5% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Subsequently, the pre-dissolved vitamin E in ethanol solutions of 0, 18, and 54 μmol/L were added to the L-15 medium for the primary pituitary cell culture of 3 d. The ethanol content in the cell medium (0.1% v/v) was tested to ensure that it was not harmful to the cells, and its purpose was to dissolve the fat-soluble vitamin E in the culture medium. Then, the α-TTP gene was cloned using rapid amplification of cDNA ends PCR (RACE-PCR) technology, and the relative gene expression levels were analyzed via quantitative real-time PCR (qRT-PCR) in the tissues and the primary pituitary cells. The results showed that the total length of the α-TTP gene was 3 964 bp, encoding a total of 293 amino acids. The evolutionary gene tree showed that the α-TTP gene from C. semilaevis had a further genetic relationship with other fish. The α-TTP gene was expressed in all 11 tissues of the half-smooth tongue sole, with the highest expression in the spleen, followed by the kidney, and the lowest expression in the stomach. The feeding experiment indicated that the expression of the α-TTP gene showed an initial increase and then decreased with the increase in dietary vitamin E content. The α-TTP gene expression of 400 mg/kg in this group was significantly higher than that in other groups (P<0.05). As vitamin E concentrations in the cell culture medium increased, the relative expression of the α-TTP gene increased significantly (P<0.05), and no declining trend was observed. These findings suggested that vitamin E was involved in the expression of the pituitary α-TTP gene, whose expression level was affected by the vitamin E dose. Since the pituitary α-TTP gene expression could be a potential evaluation indicator to demonstrate the effect of vitamin E on fish growth and reproduction, the association should be further investigated and verified in subsequent related studies. In conclusion, vitamin E supplemented in the diet at the appropriate concentration of 400 mg/kg can significantly increase the expression of the α-TTP gene in half-smooth tongue soles. This study offers novel insights into the underlying mechanisms of α-TTP in mediating the effect of vitamin E on fish growth and reproduction.

Abstract:Cyprinus carpio var. Quanzhounensis, native to Quanzhou County, Guilin City, Guangxi, has a dark brown body color, translucent gill cover, and abdominal skin, and is an important farmed species in the local integrated rice-fishery industry. A comparative study on the skin of Cyprinus carpio var. Quanzhounensis revealed a lack of reflective guanine crystals on the body surface and a significantly higher melanin content than that of C. carpio var. Jian, which was tentatively considered the direct cause of variations in body color of this group. Iridocytes are a pigment cell species that contain regularly arranged guanine crystals, which are the key material basis for the metallic luster of the fish body surface. In species such as medaka (Oryzias latipes) and zebrafish (Danio rerio), the absence of guanine crystals is considered a manifestation of the absence of iridocyte differentiation and therefore is ideal for studying the mechanism of pigment cell differentiation. The pnp4a, Gbx2, sox10, tfec genes and other iridocyte-related genes have been mined using mutant materials, and we have uncovered the differentiation mechanism that regulates the formation of iridescent cells. C. carpio var. color has abundant genetic variation in body color and is a good material for studying the mechanism of body color determination in fish. The roles of ASIP and MC1R in the aggregation and distribution of melanin and formation of black spots in C. carpio var. color were verified. The guanine crystalline deletion trait of C. carpio var. Quanzhounensis may be loaded with regulatory mechanism diversity and mutation loci related to guanine crystal formation or iridescent cell differentiation. In addition, as a rice-fish culture species, the living environment of C. carpio var. Quanzhounensis harvestmen differ significantly from pond and net-pen culture species, requiring high resistance to disease, adversity, and transport. It is unclear whether the absence of guanine crystals in their skin leads to changes in their basal physiological state, and in-depth studies are beneficial for accurate assessment of culture performance. To reveal the structural basis and transcriptomic characteristics of guanine crystalline deficiency traits in the skin of C. carpio var. Quanzhounensis in this study, we selected the subscale tissues with the most significant differences in guanine crystalline distribution as the control material, and transmission electron microscopy was used to observe the tissue structure and full-length transcriptome sequencing to understand the structural and transcriptomic characteristics of guanine crystalline deficiency in the skin of C. carpio var. Quanzhounensis. The results of this study provide information for the analysis of body color traits, identification of economic traits, and utilization of germplasm resources. Transmission electron microscopy of the subscale tissue sections revealed two significant differences in the histological structure of C. carpio var. Quanzhounensis, and C. carpio var. Jian. First, guanine crystals were absent in C. carpio var. Quanzhounensis, whereas guanine crystals were widely present in the tissues of C. carpio var. Jian and cascading cavities were observed in the sections after guanine crystals were dislodged. Second, the number and density of melanin particles in the tissues of C. carpio var. Quanzhounensis harvestmen were significantly higher than those of C. carpio var. Jian, showing smaller, darker, and more numerous particles, which is consistent with the darker color and lack of silvery reflective material on the body surface of C. carpio var. Quanzhounensis harvestmen. The transcriptome characteristics were analyzed using Oxford Nanopore (ONT) sequencing technology, and 2.88~3.26 Gb of high-quality data were obtained for each sample. The number of full-length sequences after filtering ribosomal RNA for all sample data was 2 203 826~2 412 500, and the proportion of full-length sequences for each sample was 87.06%~88.57%. The comparison rate was 90.35%~92.46%. Variable splicing events in the transcripts were counted; 3 075 variable splicing events and 57 624 variable polyadenylation events were detected; and 15 615 new coding region sequences and 771 long-stranded non-coding RNAs were predicted. The number of exon jumps and intron retention in variable splicing events differed significantly (P<0.01) between species, and the number of transcripts with five polyadenylation sites differed significantly (P<0.01) between species, indicating that variable splicing and polyadenylation are involved in regulatory processes related to trait formation. A total of 15 615 open reading frames (ORFs), including 9 890 complete ORFs, were predicted in this study. A total of 841 differentially expressed transcripts were screened in this study; 183 transcripts were upregulated and 658 transcripts were downregulated in C. carpio var. Quanzhounensis compared to C. carpio var. Jian (JH), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment in extracellular matrix-receptor interactions and adherent spots. KEGG pathway analysis showed that it was enriched for extracellular gaps, transcription factor complexes, integrin complexes, and other terms. The most significantly enriched KEGG pathway and GO terms were closely associated with the extracellular matrix, and it is speculated that these transcriptional changes may lead to changes in the composition, density, and conformation of the skin extracellular matrix.

Abstract:Mannose receptor C-type 1 (MRC1) is a member of the C-type lectin superfamily that encodes the mannose receptor. This pattern recognition receptor plays a key role in the innate immune response. The role of the MRC1 gene in the immune response of mammals has been extensively investigated but less so in fish. The emergence of high-density intensive culture in recent years has increased the frequency with which diseases caused by pathogenic microorganisms have occurred. Aeromonas hydrophila is one of the most prevalent pathogenic bacteria. In this study, 11 copies of the MRC1 gene were identified in Cyprinus carpio for the first time. Domain prediction, collinearity analysis, multiple sequence alignment, and phylogenetic analysis were conducted. The results showed that the MRC1 gene has been highly conserved during evolution. We found that the MRC1 gene exhibited varying degrees of the multicopy phenomenon in different species, ranging from 2 copies in most fish to 11 copies in C. carpio. Simultaneously, the expression level of each copy in the brain, muscle, liver, and spleen tissues of healthy carp was examined and compared. It was discovered that immune-related tissue expression was relatively higher in the spleen than that in other tissues. Further comparative analysis of expression in the spleen at 4 h, 12 h, and 24 h after infection with A. hydrophila revealed that the expression characteristics of different copies were distinct, with the expression of HHLG13g0734 being significantly up-regulated after 4 h of infection, HHLG13g0734 being significantly up-regulated after 24 h of infection, and HHLG3g0497 being significantly down-regulated throughout the infection. It indicated that only a portion of the MRC1 gene of C. carpio retained immune-related functions and participated in the immune response. Our findings contribute to a better understanding of the immune function of the MRC1 gene during the process of defending against A. hydrophila infection and serve as a fundamental data reference for the molecular-assisted breeding of new disease-resistant strains.

Abstract:The rare and endemic largemouth bronze gudgeon (Coreius guichenoti) inhabits the middle and upper reaches of the Yangtze River. It is a typical migratory river fish found in the vicinity of rapids. Over the years, the construction of water conservancy facilities and overfishing by humans have harmed the ecological habitat of this species, resulting in a considerable population decline. Thus, the largemouth bronze gudgeon has emerged as an important conservation objective and artificial stocking target for protecting and restoring the ecological environment in the Yangtze River Basin. Artificial stocking is one of the most effective methods for restoring the population of rare and endangered fish, and it is widely used domestically and abroad. Culturing a healthy and sufficient number of fry is critical to stocking success. In the production of largemouth bronze gudgeon, the formulation of an effective feeding strategy plays an important role. A scientific and reasonable feeding rate and frequency serve as guidelines for improving the production and quality of cultured fry, which directly impact the survival rate, growth performance, and feed consumption of fish. In order to determine the appropriate feeding strategy for improving fry breeding efficiency, an 8-week feeding trial in a 5 × 2 two-factorial design was conducted to investigate the effects of feeding levels (1%, 2%, 3%, 4%, and 5%) and feeding frequency (2 and 3 times/d) on growth, whole-body composition, digestive enzyme activity, and liver structure of juvenile largemouth bronze gudgeons with an initial weight of (4.91±0.11) g. The results were as follows: After 8 weeks of culture, the survival status of largemouth bronze gudgeon in each treatment group was good. Only two fish mortalities were observed, one in each of the two feeding frequency groups at the 5% feeding level. There was no mortality in the other groups. Feeding level, feeding frequency, and the interaction of the two items had significant effects on the weight gain rate (WGR), specific growth rate (SGR), and feed efficiency (FE) of individuals, whereas only the feeding frequency had significant effects on the condition factor (CF) and viscerosomatic index (VSI). WGR and SGR significantly increased with the feeding level. Meanwhile, the FE increased with increasing feeding levels and then stabilized at the 3% feeding level. Fish fed 3 times/d had higher WGR, SGR, and FE than fish fed 2 times/d when feeding levels did not exceed 3%. As feeding frequency increased, the CF and VSI of individuals tended to decrease. Both whole-body moisture content and crude fat content were significantly affected by the feeding level. The increase in feeding level decreased the whole-body moisture content while increasing the whole-body crude fat content. Digestive enzymes in the foregut of individuals were not significantly affected by feeding level, feeding frequency, or their interactions; however, the feeding level had significant effects on hepatic trypsin (TPS) and lipase (LPS) activities. Increasing feeding levels considerably reduced the TPS activity when the feeding levels were higher than 3%, whereas the LPS activity initially increased and then decreased with increasing feeding levels at a feeding frequency of 3 times/d. Hepatic HE staining revealed that the hepatocytes of fish fed 2 times/d continued to increase in size and progressively swelled and vacuolated, exhibiting indications of a fatty liver when the feeding level reached 3% and increased further. Hepatocytes of fish fed 3 times/d were surrounded by a large number of round and ovoid lipid droplets at the 3% and 4% feeding levels, but there were no discernible changes in the structure of the cells. When the feeding level reached 5%, hepatocytes began to enlarge and swell in groups at a feeding frequency of 3 times/d, but to a lesser extent than the groups fed 2 times/d. In conclusion, under the conditions of this experiment, a comprehensive assessment of growth performance, feed utilization efficiency, and hepatic health revealed that feeding juvenile largemouth bronze gudgeons three times daily at a level of 3% body weight was the optimal feeding strategy. The objective of this study was to determine the optimal feeding strategy for the fry culturing process. The findings provide theoretical guidelines for the artificial reproduction of largemouth bronze gudgeon and contribute to the recovery of its native population. Additionally, as a species that inhabits torrential habitats, the largemouth bronze gudgeon possesses distinct digestive and physiological properties. This study lays the foundation and groundwork for future research on the nutritional requirements, feed production, and artificial breeding of these species.

Abstract:Insulin-like peptide (ILP) is a member of the insulin superfamily with evolutionary conservation and is one of the most important factors affecting animal life activities. In this study, a full-length of ILP1 gene in Pacific white shrimp, Litopenaeus vannamei, was cloned, and the mRNA length consisted of 812 bp with an open reading frame (ORF) of 543 bp, encoding 180 amino acids. Sequence analysis showed that the predicted molecular weight of LvILP1 protein was 20.81 kDa, and the theoretical isoelectric point was 9.45 and the instability coefficient was 96.20. There was a signal peptide, no transmembrane structure. It was deduced that it was located outside the cell and is an alkaline unstable secreted protein. Structure prediction found that LvILP1 protein had the conserved IlGF domain of the insulin superfamily, which was composed of the N-terminal signal peptide, B-chain, C-peptide and A-chain, as well as six conserved cysteine sites and two cleavage sites. Phylogenetic analysis found that LvILP1 was most closely related to ILP7 in Penaeus monodon, and clustered with ILP1 of Crustaceans to form a branch, and then clustered with ILP7 of Invertebrate, Relaxin, Insulin and Insulin-like growth factor (IGF) of vertebrate; ILP7 of Invertebrate was evolutionarily closest to outgroup sea anemone ILP1, suggesting that it may be more similar to the ancestor gene of the insulin superfamily. Transcription factor prediction found that the possible transcription factors of LvILP1 are Forkhead box protein O3 (FoxO3), Glucocorticoid receptor (GR), CAAT region/enhancer binding protein (C/EBP) and Signal transduction and transcription activator protein (STAT); The protein interaction analysis found that LvILP1 interacted with Insulin receptor (IR) on the cell membrane, nerve signaling molecules (VGLUT1, SYT 1_3), Glycoprotein hormone beta 5 (GPHB5), Bursicon alpha, etc. By analyzing the biological function analysis of these transcription factors and interacting proteins, it is speculated that LvILP1 may play an important role in regulating shrimp growth and development, response to hormonal stimulation, nervous system homeostasis, carbohydrate homeostasis, postmolt tissue remodeling and reproductive development. The analysis showed that LvILP1 was expressed in the early developmental stage of shrimp, and expressed in all tissues of adults, but the expression level in the eye stalk was the highest. This study provides important information for in-depth understanding of the structure, evolution, function and expression of ILP in shrimp, as well as clues for molecular breeding and healthy farming of shrimp.

Abstract:Aquaculture in large water bodies has become an important culture mode of Litopenaeus vannamei in coastal waters. Fractionated chlorophyll-a (Chl-a) and environmental factors of the large water ponds with high salinity (54, n=3) and the control ponds (32, n=3) were investigated from May to July 2020 to explore the variations in Chl-a, phytoplankton particle size, and the response to environmental factors during the aquaculture season. Pearson correlation analysis was performed to analyze the relationship between the environmental factors and the size-fractionated Chl-a concentration. Partial redundancy analysis (RDA) was applied to assess the effects of environmental factors (including silicate, active phosphate, ammonia salt, nitrite, nitrate, water temperature, salinity, dissolved organic nitrogen, and dissolved organophosphorus) on total Chl-a, Chl-a of micro phytoplankton (micro Chl-a), Chl-a of nano phytoplankton (nano Chl-a), and Chl-a of pico phytoplankton (pico Chl-a). The following results were obtained: 1) Diurnal variation of Chl-a: Total Chl-a of the high-salinity group showed no significant diurnal variation (P>0.05). Total Chl-a of the control group showed significant diurnal change in May and June (P<0.05). The highest value of total Chl-a in May occurred at 15:00, while the highest value of that in June was at 08:00. For size-fractionated Chl-a, pico Chl-a in the high-salinity group showed significant diurnal variation in July (P<0.05), with the highest value appearing at 12:00. Micro Chl-a in the control group showed significant diurnal changes in May, June, and July (P<0.05), and nano Chl-a in the control group showed significant diurnal changes in June (P<0.05). 2) Monthly changes of Chl-a: The lowest and highest values of total Chl-a occurred in June and July, respectively. Total Chl-a in July was significantly higher than that in May and June (P<0.05). For size-fractionated Chl-a, the pico Chl-a and nano Chl-a of the high-salinity group in July were significantly higher than those in May and June (P<0.05) and showed no significant difference between May and June (P>0.05). Pico Chl-a, nano Chl-a, and micro Chl-a of the control group in July were significantly higher than those in May and June (P<0.05) and showed no significant difference between May and June (P>0.05). 3) Contribution of size-fractionated phytoplankton in high-salinity and control groups: The contribution of micro Chl-a, nano Chl-a, and pico Chl-a to total Chl-a in the high-salinity group were (15.64±0.16)%, (73.81±0.13)%, and (10.55±0.06)%, respectively. Nano Chl-a was dominant in May, June, and July. The contribution of pico Chl-a increased from 6.43% in May to 16.81% in July, and exceeded that of micro Chl-a. The contributions of micro Chl-a, nano Chl-a, and pico Chl-a to total Chl-a in the control group were (52.29±0.10)%, (41.82±0.10)%, and (5.59±0.01)%, respectively. Micro Chl-a concentration had a major advantage in May and June, accounting for 59.64% and 57.49%, respectively. Nano Chl-a concentration accounted for 35.46% and 36.90%, respectively. By July, nano Chl-a had a major advantage, contributing to 53.09%. 4) Pearson correlation analysis showed no significant correlation between the diurnal variation of Chl-a and the environmental factors of the high-salinity group in May and June (P<0.05). Yet, the concentrations of nano Chl-a and total Chl-a were negatively correlated with the concentration of nitrate in July (P<0.05). The concentrations of micro Chl-a and total Chl-a were positively correlated with those of silicate (P<0.05). For the control group, Pearson correlation analysis showed a significant positive correlation between nano Chl-a and water temperature (P<0.05). Total Chl-a and phosphate were negatively correlated in May (P<0.05). There was a significant negative correlation between pico Chl-a and nitrate in July (P<0.05). 5) For the high-salinity group, RDA revealed a significant positive correlation between Chl-a and water temperature, and the contribution of nano Chl-a increased with the increase in temperature. Total Chl-a was positively correlated with silicate and negatively correlated with phosphate, dissolved organic nitrogen, and dissolved organophosphorus in the high-salinity group. For the control group, RDA showed that total Chl-a was positively correlated with dissolved organic nitrogen and negatively correlated with silicate and nitrite. In general, Chl-a in high-salinity ponds has a small diurnal variation, and the phytoplankton particle size gradually decreased with cultivation, which may be caused by the increasing temperature and high organic nitrogen concentration.

Abstract:Crustacyanin (CRCN), a family of lipocalin proteins specific to crustaceans, was primarily found in the exoskeleton of crustaceans. By binding with astaxanthin to form the astaxanthin-binding protein complex, it resets the proton at the end of astaxanthin and modifies the acidic site in astaxanthin, thereby regulating crustacean shell color. In addition to regulating shell colors, crustacyanin is involved in the transport of small molecule lipids such as steroid hormones and pheromones and has important biological functions in moult growth, gonadal and nervous system development, resistance to heavy metal lipid metabolism, and hypoxic stress. It was first discovered that the pigment in the blue shell of lobster could be extracted with ammonium chloride via a complex combination of organic bases and lipid pigments. It was later proposed that the pigment was a combination of astaxanthin and multimeric protein complex known as α-CRCN. This complex is composed of an octomer of dimeric β-CRCN subunits, with this dimer formed by two types of CRCN A and C in association with two astaxanthin molecules. Natural crustacyanins are all α-CRCN, comprising a total of 16 molecules. Procambarus clarkii, a member of the order Crustacea (Decapoda, Crayfish), is native to North America and is an important freshwater crustacean in China. It had been established that P. clarkii is susceptible to low oxygen stress during the culture process. The low oxygen environment inhibits the metabolic rate of P. clarkii, leading to increased susceptibility to pathogens; therefore, the ability of P. clarkii to tolerate and physiologically regulate stress due to other environmental factors was also affected, which can lead to irreversible damage and even death in severe cases. To understand the role of crustacyanin-like genes in gonadal development and hypoxic-reoxygenation stress in P. clarkii, a cDNA sequence of the PcCRCN-L gene was isolated from the hepatopancreas of this organism. The structural characteristics and evolutionary patterns of the PcCRCN-L gene were analyzed, and the expression characteristics of the PcCRCN-L gene in different tissues and gonad development stages were investigated. The expression response pattern of PcCRCN-L under hypoxic-reoxygenation stress was investigated. The DNA sequence of the PcCRCN-L gene was 6 130 bp long and located on chromosome 12 of the P. clarkii genome. The cDNA sequence was 2 700 bp, and its open reading frame (ORF) length was 1 587 bp. It contained five exons and four introns, encoded 528 amino acid residues, had a theoretical isoelectric point of 5.71 and a relative molecular weight of 55 613.55, and was a hydrophilic protein. The intron/exon splicing pattern was in accordance with the GT-AG rule. The PcCRCN-L protein had a complete lipocalin domain, which included the typical sequences G-X-W of conserved region I (SCR1), T-D-Y of conserved region II (SCR2), and arginine R of conserved region III (SCR3). The results of multiple sequence alignments and phylogenetic analyses showed that PcCRCN-L, as well as the crustacyanin A and crustacyanin C subgroups, were separately clustered into a branch. The expression characteristics of the PcCRCN-L gene in different tissues showed that PcCRCN-L was expressed in all the tissues; however, the highest expression level was found in the hepatopancreas. The expression trend of the PcCRCN-L gene in the ovary and the hepatopancreas was similar in different stages of gonadal development. The expression level of the PcCRCN-L gene was significantly decreased in stages Ⅰ~Ⅱof ovarian development (P<0.05), but no significant difference was found in stages Ⅱ~Ⅴ (P>0.05). The expression of the PcCRCN-L gene was significantly decreased under hypoxia stress for 1 h (P<0.05), but there was no significant difference between hypoxia stress for 1 h and hypoxia stress for 6 h. After reoxygenation for 1 h, the expression of the PcCRCN-L gene was significantly up-regulated (P<0.05). Compared with reoxygenation for 1 h, the expression of the PcCRCN-L gene was significantly increased at 12 h (P<0.05). The results showed that PcCRCN-L should be closely involved in the regulation of gonadal development and hypoxia-reoxygenation stress in P. clarkii.

Abstract:Litopenaeus vannamei, also known as the Pacific white shrimp or white foot shrimp, is a member of the Crustacea, Decapoda, Palaemonidae, and Litopenaeus families. This species is mainly distributed on the Mexican and Pacific coasts of South America. Owing to its excellent immune characteristics, L. vannamei is currently one of the most important economic shrimp species in China. In the aquaculture process and under natural environmental conditions, low dissolved oxygen (DO) condition or even hypoxia frequently occurs. Under high-density culture operations, acute hypoxia is one of the major factors affecting the survival rate of shrimp and the quality of aquaculture water, causing not only a large number of shrimp deaths in the short term but also water decay and fermentation, increased turbidity, and other phenomena. The genetic improvement of L. vannamei, as well as the screening and breeding of exceptionally tolerant species under low DO conditions, are crucial to the sustainable development of the shrimp industry. Global selective breeding efforts based on quantitative genetics to improve genetic progress in L. vannamei have been extensive, and the main targeted traits were growth, survival, disease resistance, and stress tolerance. Few studies have been conducted on the evaluation of genetic parameters and screening of strains for hypoxic tolerance traits in L. vannamei; in particular, the genetic parameters and cytological characteristics of key tissues of L. vannamei under short-term highly lethal DO levels (0.3–0.5 mg/L) have not been reported. In this study, we used two strains of L. vannamei, GK (a strain with high disease resistance) and K (a strain with fast growth characteristics), each with a total of 6 560 shrimp from 41 families, to count survival traits at highly lethal DO levels, analyze differences in hypoxia tolerance traits between strains and within families of the same strain, and evaluate genetic parameters. Individual gill, muscle, and hepatopancreas tissues from families with significantly different tolerance levels were histologically investigated and compared at the cellular level. The results showed that there was a significant difference (P<0.05) in semi-lethal survival (SS50) between families within both the GK and K strains when overall semi-lethality was reached under hypoxic environmental conditions of 0.3 mg/L, with SS50 values of 49.30% for GK and 42.52% for K. The coefficients of variation for survival times of families within each strain were 60% and 45% for GK and K, respectively. Using the threshold trait animal model, the genetic parameters were estimated to be between 0.345±0.031 and 0.378±0.029 using the survival status of the individual at semi-lethal levels as the observed value (1 for survival and 0 for death), and the heritability was between 0.219±0.031 and 0.237±0.029 following transformation on a continuously varying scale of observed values, indicating a moderate level of heritability. Moreover, the gill, muscle, and hepatopancreas tissues of the hypoxia-tolerant and hypoxia-sensitive families in the GK strain exhibited varying degrees of damage and different degrees of variations in the physiological characteristics of resistance among different families. This may be related to the resistance tolerance strength of different GK families. In contrast to the gill, muscle, and hepatopancreas tissues of the control shrimp, the equivalent tissues of shrimp from tolerant and sensitive families showed different degrees of changes following stress. The gill lumen of the filaments of the experimental group from sensitive families increased, the number of blood cells increased, the structure of the epithelial layer was gradually destroyed or even disintegrated, the vacuolation of the hepatopancreas was severe, the lumen of the ducts became irregularly deformed, and the muscle tissue muscle bundle interval widened with a certain degree of deformation. These results suggested that there was abundant genetic variation in the tolerance traits of L. vannamei at highly lethal DO levels and that this species was amendable to selective breeding practices. This study provides a reference and basis for the selection and breeding of hypoxia-tolerant traits in L. vannamei.

Abstract:Eriocheir sinensis is an important fishery resource in the Yangtze River basin, which has brought about huge socio-economic benefits. The estuarine spawning grounds of E. sinensis, however, has experienced negative effects, such as habitat alteration and degradation of the water environment, in the mid- to late 1980s as a result of the construction of water conservation projects, land reclamation along the estuary, and pollution input. In addition, the overexploitation of E. sinensis resources has contributed to a sharp decline in its resources. To develop rational and scientific habitat management plans, the estuarine spawning habitats of E. sinensis have received attention in recent years. Currently, the distribution pattern and habitat characteristics of E. sinensis spawning grounds in the Yangtze River estuary have been explored from the perspective of resource distribution; however, habitat quality has not been further investigated. Studies on habitat quality can more effectively support the development of fishery habitat management strategies by serving as a tool to objectively assess the functional status of aquatic habitats. The habitat quality of the Yangtze River estuary has a substantial impact on the resources of E. sinensis as it is the only spawning ground for this species in the Yangtze River basin and is also the largest in China. Therefore, the behavior of E. sinensis during reproduction and spawning, which reflects the habitat quality of the spawning grounds, served as the foundation for this study. The evaluation of the habitat quality of E. sinensis spawning grounds was based on the parameters of reproductive performance and quantitative distribution. The habitat quality index (HQI) of E. sinensis spawning grounds was built by screening evaluation indices, weight analysis of evaluation indices, and construction of evaluation criteria. To provide the necessary scientific tools for examining E. sinensis spawning grounds in the Yangtze River estuary and to establish a solid scientific foundation for managing estuarine spawning grounds for this species. The spawning grounds of the Yangtze River estuary were monitored using egg-holding crabs of E. sinensis, and sampling was performed using gillnets positioned at certain locations. Key environmental parameters influencing the spawning activity of E. sinensis, such as water depth, turbidity, dissolved oxygen (DO) in bottom water, salinity (SAL), and temperature (T) were gathered during the sampling process. Through anatomical measurements of collected egg-holding crabs, physiological characteristics including carapace width, absolute fecundity, relative fecundity, condition factor, hepatopancreas index, and reproductive effort were gathered and collected. The characteristics of egg-holding crab population distribution and reproductive success were key factors considered when choosing the evaluation indices. The catch per unit effort of egg-holding crabs is a quantitative distribution characteristic, and the reproductive performance characteristics primarily rely on correlation analysis to determine the traits that can objectively respond to the reproductive performance of egg-holding crab, which are RF and HSI. Subsequently, a hierarchical evaluation system is created based on the characteristics of the evaluation indices. The response relationship between the environmental elements and each evaluation index serves as the foundation for the weight analysis of the evaluation indices. The environmental factors were analyzed separately with each evaluation index for redundancy analysis (RDA), and the results of the RDA were used to understand the degree of influence of each evaluation index on environmental characteristics. It is also used as the basis for the weight value of the evaluation indices, and the weight value of each evaluation index is determined by combining the hierarchical analysis method. Finally, a multi-index comprehensive scoring technique was used to determine the HQI of the E. sinensis spawning grounds, and guidelines for evaluating habitat quality were developed. The findings revealed a strong response relationship between egg-holding crab reproductive performance characteristic indicators and environmental factors (91.9%, F=9.0, P=0.042), which has significant application value when assessing the habitat quality of their spawning grounds. The evaluation revealed that the survey sites´ HQIs ranged from 0.33 to 0.84, and 47% of the survey sites assigned a "medium" or higher rating to the habitat quality level. Overall, the northern bank of Hengsha had a greater habitat quality than the deepwater canal. The HQI developed in this study has obvious advantages in quantitatively assessing the habitat quality of E. sinensis spawning grounds because it has a greater interpretation rate of environmental elements (94.9%, F=12.0, P=0.038) than quantitative distribution characteristics. The maximum percentage of HQI was explained by DO, SAL, and T when each environmental factor was used as an independent explanatory variable, accounting for 74.0% (F=22.7, P=0.004), 44.0% (F=6.3, P=0.044), and 33.0% (F=3.9, P=0.076), respectively. DO, SAL, and T were the key environmental factors influencing the habitat quality of E. sinensis spawning grounds. In this study, the Yangtze River estuary was chosen as the spawning ground for E. sinensis, and the quantitative distribution and reproductive performance characteristics of this species were employed as the foundation for evaluating habitat quality. The spawning grounds of the Yangtze River estuary for E. sinensis were further investigated in terms of habitat function using the HQI, which was built based on the characteristics of the evaluation indices. This study identified the major habitat factors that influence the habitat quality of these spawning grounds.

Abstract:Bivalves are affected by various stressors, such as global warming and microplastics, in the marine environment. Microplastics are one of the most concerning pollutants worldwide, and high seawater temperatures caused by global warming influence the survival of marine organisms. However, little is known about the combined effects of elevated temperature and microplastics (MPs) on marine organisms, and most studies conducted in recent years have investigated the two factors, respectively. Thus, it is necessary to investigate the combined effects of elevated temperature and MP exposure on marine life. The Pacific oyster Crassostrea gigas is a widely distributed marine mollusk, and has very important economic value. The aim of the current study was to explore the toxic effects of elevated temperature and microplastic co-exposure on the hemocyte function, immune-related gene expression, and energy metabolism of C. gigas. In the current study, oysters were exposed to three levels of microplastics (no microplastics, 6 μm microplastics: SPS-MPs, and 50~60 μm microplastics: LPS-MPs) and two temperature levels (20 ℃ and 25 ℃) for 21 days, and the phagocytosis rate and reactive oxygen species (ROS) content of hemocytes, glycogen content in digestive glands, and immune-related gene expression in digestive glands and gills were examined on the 21st day. 2',7'-Dichlorodihydrofluorescein diacetate and fluorescent microspheres were used to measure the ROS content and phagocytosis ratein hemocytes of C. gigas by flow cytometry, respectively. The glycogen content was measured using detection kits. Total RNA was isolated by TRIzol reagent, and the concentration was measured by Nanodrop. M-MLV Reverse Transcriptase was used for cDNA synthesis. The expressions of immune-related genes [inhibitor of NF-κB (IκB), p53, and heat shock protein 90 (HSP90)] were examined by quantitative real-time PCR in the digestive glands and gills of oysters from each treatment group. Two-way ANOVA was used to analyze the interactive effects of elevated temperature and microplastics on tested parameters of oysters using SPSS software. The results showed that exposure to SPS-MPs could elevate ROS content and reduce phagocytosis in hemocytes, but no significant interaction was found between elevated temperature and microplastic effects on ROS content and phagocytosis rate in hemocytes (P>0.05). The 25 ℃+LPS-MPs exposure significantly decreased phagocytosis in hemocytes compared with single LPS-MPs and elevated temperature exposures (P<0.05). Single SPS-MPs exposure significantly decreased phagocytosis in hemocytes compared with single LPS-MPs exposure (P<0.05). In digestive glands, there was a significant interaction between elevated temperature and microplastics in glycogen content (P<0.05), and the combined exposure could increase the glycogen content compared with other treatments. In digestive glands, the 25 ℃+LPS-MPs exposure significantly increased glycogen content compared with single elevated temperature and single LPS-MPs exposure (P<0.05). In digestive glands and gills, there was a significant interaction between elevated temperature and microplastics in the expressions of HSP90, IκB, and p53 genes (P<0.05). The 25 ℃+SPS-MPs exposure significantly upregulated the expression of HSP90, IκB, and p53 genes in the digestive glands of oysters compared with single SPS-MPs and single elevated temperature exposures (P<0.05). The 25 ℃+SPS-MPs exposure significantly downregulated the expression of the HSP90 gene in the gills of oysters compared with single SPS-MPs exposure (P<0.05). Single elevated temperature and single microplastics exposure significantly upregulated the expression of the IκB gene compared with the control in gills (P<0.05). The combined exposure of elevated temperature and microplastics showed a significant antagonistic effect on the expression of the p53 gene in gills. Microplastics exposure downregulated p53 gene expression compared with the control at 20 ℃, while it upregulated p53 gene expression compared with single elevated temperature at 25 ℃. These results indicated that the p53 gene plays an important role in regulating the immune response in both digestive glands and gills. The interaction between elevated temperature and microplastics on the mRNA expression of HSP90 and IκB genes in digestive glands of C. gigas was size-dependent: A synergistic effect was found between SPS-MPs and elevated temperature, and an antagonistic effect was found between LPS-MPs and elevated temperature. A significant antagonistic effect was found between elevated temperature and microplastics on the mRNA expression of the IκB gene in gills, and the regulation pattern was different from that in the digestive glands, indicating that the regulation effect of the IκB gene was tissue-specific. In conclusion, the combined exposure of elevated temperature and microplastics can increase the glycogen content in the digestive glands of C. gigas, induce an immune response in digestive glands and gills, and trigger the oxidative stress response in hemocytes. Microplastics can cause stronger oxidative stress in hemocytes than elevated temperature. Moreover, a significant interactive effect was found between elevated temperature and microplastics on glycogen content in digestive glands and the expression of immune-related genes (HSP90, p53, and IκB) in digestive glands and gills. The results of this study provide valuable information for evaluating the toxic effects of microplastics on marine organisms under a global warming background.

Abstract:The razor clam (Sinonovacula constricta) is an economically important species and one of China´s four traditional mariculture mollusks. The ABC transporter family is one of the oldest membrane protein families, widespread across prokaryotes and eukaryotes. By utilizing the energy released by ATP hydrolysis, ABC transporters function to transfer amino acids, lipids, antibiotics, and many other substances across membranes, thereby participating in various physiological processes such as nutrition uptake, antigen presentation, drug excretion, and lipid homeostasis in living organisms. ABC transporters can be classified as full transporters, which contain two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs), a half-transporter (composed of one NBD and one TMD), and a non-transporter (composed of either two NBDs or two TMDs incapable of transporting). The NBD domain is responsible for binding and hydrolyzing ATP, whereas the TMD domain determines substrate specificity. NBD domain sequences are relatively more conserved. Several investigations of heavy metal pollution were conducted in mollusk culture areas, and it was found that the concentrations of some ions exceeded the limits. Previous studies have reported that the multixenobiotic resistance (MXR) mechanism in bivalves is mediated by ABC transporters from the ABCB, ABCC, and ABCG subfamilies, which are important for the cellular efflux of noxious metallic ions. Obtaining a greater understanding of ABC transporters may contribute to the development of a healthier and more scientific method of mollusk culture. Until now, the identification of the ABC transporter family in mollusks has only been systematically performed in three bivalves: Patinopecten yessoensis, Chlamys farreri, and Crassostrea gigas. The analysis and expression pattern of the ABC transporter family of the razor clam have not yet been reported. To systematically study ABC transporters and facilitate an understanding of the evolution and function of ABC transporters in razor clams and mollusks, depending on the genome and transcriptome data, 52 ABC transporter proteins were identified using the local and NCBI BLASTP, TBLASTN programs, and the ExPASy website. The SMART(simple modular architecture research tool) website and FGENESH+ were used to further predict the domain of ABC transporters. After applying the local BLASTN program to the published genome data of the razor clam, the MapInspect software and the CSDS (gene structure display server) website were used to generate graphical representation of the locations of ABC transporter genes on chromosomes and gene structures, respectively. Six vertebrates (Homo sapiens, Mus musculus, Xenopus laevis, Carassius auratus, Danio rerio, and Petromyzon marinus) and seven invertebrates (Diaphorina citri, Tetranychus urticae, Zeugodacus cucurbitae, Patinopecten yessoensis, Chlamys farreri, Crassostrea gigas, and Sinonovacula constricta) were chosen to compare and discuss the differences in subfamilies among species of various evolutionary status. Using the Mev 4.90 software, based on transcriptome data, the heat map of ABC transporter genes expression levels in eight tissues (gill, foot, adductor muscle, hepatopancreas, mantle, siphon, female gonad, and male gonad) and eight development stages (egg, cell, blastula, gastrula, trochophora, d-shaped larvae, umbo larvae, and spat) of the razor clam was completed. Based on sequence similarity, MEGA X software was used to divide the 52 ABC transporters into eight subfamilies, namely ABCA~ABCH, and phylogenetic trees of ABC transporters from four bivalves were drawn. The total of 52 ABC transporter genes was divided into 7 ABCA, 10 ABCB, 13 ABCC, 3 ABCD, 1 ABCE, 3 ABCF, 14 ABCG, and 1 ABCH. The ORF lengths of these genes ranged in size from 1 588 to 7 224 bp, the number of exons ranged from 8 to 44, and the deduced proteins were between 455 and 4 560 amino acids in length. Based on the composition of the protein domain, 52 ABC transporters could be divided into 24 full transporters, 24 half transporters, and four non-transporters. The ABCA subfamily consisted of 1 ABCA1, 2 ABCA2, 2 ABCA3, 1 ABCA5, and 1 ABCA12, all of which were full transporters. The ABCB subfamily consisted of four complete transporters, namely ABCB1a~ABCB1d, and six half transporters, namely ABCB6~ABCB9 and ABCB10a~ABCB10b. The ABCC subfamily comprised 7 ABCC1, 1 ABCC4, 3 ABCC5, 1 ABCC8, and 1 ABCC10, all of which were full transporters. The ABCD subfamily included ABCD2~ABCD4, all of which were half transporters. Non-transporters were observed in the ABCE and ABCF subfamilies, namely ABCE1 and ABCF1~ABCF3. The ABCG subfamily, consisting of 8 ABCG1, 1 ABCG2, 4 ABCG5, and 1 ABCG8, was the largest subfamily and its members were all half transporters. The ABCH subfamily contained only one member, ABCH1, which was a half-transporter. The comparison of ABC subfamilies between different species revealed that tandem duplication events might have resulted in an increase in the numbers of several ABC transporter genes during molluscan evolution and that some genes with functions related to immunity, such as ABCB1 and ABCC5, had multiple copies, indicating a positive influence on the environmental adaptation of mollusks. The analysis of the expression level of ABC transporter genes in different tissues and developmental stages of razor clam showed that the gill and hepatopancreas have relatively more expressed genes, which may be because of their detoxification function. The expression of ABCA and ABCG subfamily genes increased with razor clam development, and the expression levels of many ABCC and ABCG subfamily genes peaked in the spat stage. In general, several members of the ABCB subfamily, as well as all ABCE and ABCF subfamily genes, remained highly expressed in all eight tissues and all eight development stages. The ABC transporter gene family has only been investigated in three species of mollusks. Systematic identification and expression pattern analysis of ABC transporters in razor clams can promote our understanding of the evolution of ABC transporters in mollusks and provide an essential foundation for functional research on ABC transporters in mollusks, which may contribute to healthier mollusk cultur

Abstract:Mactra veneriformis is a common economic benthic shellfish with high nutritional value and is widely distributed in the Shandong and Liaoning provinces. Geligang is located in the northern part of Liaodong Bay. It is formed by the water impact of Shuangtaizi and Liaohe River. It has an area of approximately 10 000 hm2 with a fertile substrate. It is an important habitat of beach shellfish, such as M. veneriformis. Studies on the influencing factors of wild population quality traits and their correlation with morphological traits are helpful for developing genetic breeding of marine shellfish. To analyze the effects of morphological traits on quality traits of M. veneriformis at different ages in Geligang, the shell length (SL), shell width (SW), shell height (SH), body weight (BW), and soft tissue wet weight (RW) of 1–3 years old M. veneriformis were analyzed using correlation analysis, path analysis, and multiple regression analysis. The M. veneriformis used in the experiment was obtained from the Geliang in Liaodong Bay in September 2022. A total of 80 1-year-old, 104 2-year-old, and 168 3-year-old M. veneriformis were obtained. The results showed that the morphological traits of M. veneriformis at different growth stages had different contributions to quality traits, and there were significant differences (P<0.01). SW had the greatest direct effect on the BW of 1–3 year-old M. veneriformis, but had different effects on the RW; SW had the greatest direct effect on 1-year-old M. veneriformis; and SL had the greatest effect on 2- and 3-year-old M. veneriformis. The direct path coefficient of SH to the BW and RW of 2-year-old M. veneriformis did not reach significant level (P>0.05) and was eliminated. The optimal regression equation of quantitative traits to qualitative traits of 1–3 year-old M. veneriformis was established. The results showed that when BW was the target trait, SW was the main selection trait and SL was the auxiliary selection trait for 1–3 year-old M. veneriformis. When RW was the target trait, SW should be the main selection trait for 1-year-old M. veneriformis, supplemented by SL. The SL should be the main selection trait for both 2- and 3-year-old M. veneriformis, but the SW was the auxiliary selection trait for 2-year-old M. veneriformis, while the SH was for 3-year-old M. veneriformis. In this study, correlation analysis, path analysis, and multiple regression analysis were performed on the morphological traits and quality traits of 1–3 years old M. veneriformis to determine the key morphological traits affecting the quality traits of M. veneriformis at different ages. In this study, the R2 values of the morphological traits of 1- and 2-year-old M. veneriformis (0.898 and 0.859, respectively) were greater than 0.850, indicating that the main factors affecting the body weight of living M. veneriformis were found. However, the R2 value of the morphological traits of 3-year-old M. veneriformis (0.815) was below 0.850, indicating that there may be other influencing traits in addition to the traits analyzed in this study. The reason may be that with the growth of M. veneriformis, the thickness of the shell and the effect on the weight of the living body gradually increase. In this study, the R2 values of morphological traits of 1–3 years old M. veneriformis (0.738, 0.648, and 0.538, respectively) on the wet weight of soft tissue were all below 0.850, indicating that there were other key factors affecting the wet weight of soft tissue. The age of sexual maturity of the M. veneriformis is at 1 year. The development of the gonad in the soft part may be closely related to the size of the wet weight of the soft tissue. The sampling period is mainly in the proliferation to depletion period of the gonad development of M. veneriformis. The gonad development is simultaneously affected by the environment. These results can provide reference for breeding M. veneriformis and the selection of parent shellfish.

Abstract:Spondylus aurantius, a member of the Mollusca phylum, Bivalvia class, Pterioida order, and Spondylidae family, is an important economic marine bivalve species that is widely distributed along the warm-water coastal region in Guangdong and Hainan Province in China, and the Philippines. As an important marine commercial bivalve, wild S. aurantius individuals are often captured by local fishermen for their large adductor muscle, which makes delicious seafood, and for their beautiful shells, which are used for displays. According to the China Fishery Statistical Yearbook for 2022, the annual production of cultured mollusks exceeded 15 million tons, with oysters, scallops, and clams accounting for 78.23% of the total aquaculture yield. In southeast coastal provinces, such as Guangdong, Guangxi, and Hainan, the most common cultured mollusks include Hong Kong oysters (Crassostrea hongkongensis), Suminoe oysters (Crassostrea ariakensis), pearl oysters (Pinctada fucata martensii), and noble scallops (Chlamys nobilis). Generally, Hong Kong and Suminoe oysters cannot tolerate high salinity conditions and are often cultured near estuaries; the P. f. martensii industry is rapidly declining because of high mortality rates and nucleus rejection after transplantation in the process of pearl production; and noble scallop rearing, which is primarily practiced in net cages, and a considerable amount of labor is required to clean attachments. Therefore, there is an urgent need to investigate the cultivation of new bivalve species that can adapt to coastal areas with high temperatures and high salinity. Though S. aurantius is traditionally regarded as an edible mollusk, few studies have reported the artificial breeding of this species. In this study, wild adult S. aurantius with shell length of (12.00±1.25) cm and wet body weight of (100.00±5.04) g were selected as the research subjects to artificially ripen mature individuals, observe and compare the development of the embryo and juveniles at different temperatures (28 ℃ and 32 ℃), and study the effects of different substrates and water depths on the adhesion efficiency of juveniles. Our results were as follows: (1) Under artificial conditions, adult S. aurantius gonads could continue to develop and mature, with testes and ovaries filled with milky spermatozoa and orange eggs, respectively; (2) The method of “dry in the shade + flowing water + high temperature” successfully induced spawning and fertilization of S. aurantius; (3) The diameters of fertilized eggs were (130.1±0.3) µm, and the first unequal cleavage was observed 95 min after fertilization. These eggs then developed into multicellular stages (cleavage occurred at approximately 30-min intervals), blastula (after 4–5 h of fertilization), trochophore (after 10–12 h of fertilization), D-shaped larvae (after 25–28 h of fertilization), umbo larvae (after 6–7 d of fertilization), pediveliger (after 14–15 d of fertilization), and juvenile (after 25–27 h of fertilization); (4) Fertilized eggs could develop into the juvenile stage at both 28 ℃ and 32 ℃; however, the survival rates of fertilized eggs under higher temperatures were lower than of those cultured at 28 ℃, although the speed of S. aurantius embryonic development at 32 ℃ was higher than that at lower temperatures; (5) The density of juvenile S. aurantius in deep water (1–1.5 m depth) was evidently higher than that in shallow water (0–0.5 m depth) in an indoor seedling pond (P<0.05); (6) Furthermore, the densities of juvenile S. aurantius were highest on the surfaces of Pinctada margaritifera shells, followed by S. aurantius shells > oyster shells > concrete reels > black shading nets > green polyethylene mesh sheets under indoor conditions, while the densities and growth rate of juvenile S. aurantius attached itself and oyster shells were better after one month culture in the natural coastal area of Wuzhizhou Island, Sanya. In this study, embryonic development in S. aurantius was observed for the first time. Furthermore, the breeding technology of S. aurantius, including the induction of gonadal maturation and spawning, the incubation of fertilized eggs, and the selection of substrates, was established for the first time under artificial conditions, which could contribute to the future large-scale breeding and cultivation of this species.

Abstract:Cephalopods, which have a rapid growth rate and short life cycle, are regarded as an important marine fishing resource. Sepioteuthis lessoniana is among the most widely distributed species in the Loliginidae family in the Indo-Western Pacific Ocean. Its meat is delicious and nutritious. It is widespread in the East China Sea, South China Sea, and other marine areas in China, and it is considered an important local marine resource. In this study, the ultrastructure of the oviducal gland of S. lessoniana was investigated for the first time via anatomical dissection, tissue sectioning, and electron microscope projection. The external morphology and internal structure of the gland were clearly described, as well as the role of the oviduct gland in the reproductive activities of cephalopods. In this experiment, all the samples were collected from the open ocean, with the samples of wild S. lessoniana coming from marine areas in Fujian Province. The body surfaces of the samples were healthy and undamaged, with an average mantle length of (17.5±6.4) cm and an average body weight of (392.0±76.0) g. The samples were dissected using standard anatomical methods, and the glandular characteristics were recorded. The oviducal gland was dissected with a scalpel for tissue sectioning and preserved for electron microscope projection and observation. The experimental results showed that S. lessoniana had a single oviducal gland, which was located on the right side of its abdominal cavity. The sexually mature gland was milky white overall, with brownish-yellow pigmentation near the inner shell. The oviducal gland was enveloped in a transparent membrane, and regular gaps were visible. The gland consisted of three parts: the proximal oviducal gland, the distal oviducal gland, and the transparent valve. The proximal oviducal gland was infundibular and connected with the hyaline oviduct. The distal oviducal gland was cylindrically tapering, and a single mature egg was observed inside. The transparent valve was at the end of the gland, also known as the valve. The microstructure of the oviducal gland was observed via a microscope. The gland was composed of a glandular wall, lamellar, and muscle tissue. The glandular wall tissue was composed of adventitia, loose connective tissue, and a small amount of muscle tissue, blood vessels, and ducts that were scattered in the connective tissue. The transparent valve was composed of columnar epithelial cells and muscle tissue. A small number of water-droplet goblet cells were dispersed between the epithelial cells, while a large number of cilia were generated on the exterior. The lamellar was attached to the glandular wall tissue and distributed in layers within the gland; it was mainly composed of ciliated columnar epithelium and support cells. When the oviducal gland was at various development stages, the cell types of the lamellar and the size of the intercellular space within the leaflet were different. When the gland was immature, the lobe had a regular shape, with more connective tissue in the center and a single layer of columnar epithelial cells on each side. When the gland was about to mature, the connective tissue increased, the number of columnar epithelial cells decreased, and a large number of mucous acinus were simultaneously generated. After the glands matured and spawned, the amount of connective tissue decreased, the columnar epithelial cells disappeared, the mucous acinus ruptured, and the secretory leaflets were filled with secretory substances. The individual oviduct gland secretory cells of S. lessoniana were large and contained many closely arranged mucus granules, which were round or oval in shape. The cytoplasm contained many organelles, such as the endoplasmic reticulum, mitochondria, and Golgi apparatus. In addition, secretory cells were continuously distributed outside the cell with cilia and numerous secretory granules. The nucleus of the muscle cells in the hyaline valve was irregular, and a substantial number of myofilaments, collagen fibers, and capillaries could be seen around it. The oviducal gland is an important gland in the reproductive system of female cephalopods. The morphological characteristics of oviducal glands, such as the number, shape, size, and presence or absence of pigmentation, are some of the distinguishing characteristics between different cephalopod species. The tissue structure and cell types of the cephalopod oviducal glands changed with the growth of individuals. The changes in oviducal glands in S. lessoniana were similar to those in Loligo forbesi. The secretory lobes successively generated an increasing number of mucous acini and secretory substances as cell types shifted. These secreted substances had multiple functions, such as attracting sperm during the sperm-egg hatching process, expanding the chorionic membrane, forming the vitelline space, and regulating the osmotic pressure between the fertilized egg and the ambient seawater. However, the oviducal glands of the orders Cuttlefish and Liliformes differ from those of the order Occarpus, whose oviducal glands had the function of storing sperm. It is concluded that the oviducal gland primarily plays a secretory function in the reproductive activities of cephalopods and that its secretory material forms the second layer of the egg membrane of fertilized eggs, ensuring the normal hatching of fertilized eggs.

Abstract:China is the most productive country in the world in terms of shellfish farming, with seawater shellfish occupying a dominant position in China´s shellfish farming industry. In the past two decades, China´s marine shellfish culture has been enriched in terms of species and culture methods. Moreover, its scale has expanded, with annual production remaining above 10 million tons and the total economic output exceeding 220 billion yuan. However, the economic losses caused by epidemic diseases in the shellfish aquaculture industry are also increasing annually, exceeding 10 billion yuan in 2021. These diseases have become some of the primary limiting factors for the healthy development of the shellfish aquaculture industry. Epidemiological surveys in recent years have shown that vibriosis is the most prevalent bacterial disease and the leading cause of mass mortality in shellfish farming. Vibrio alginolyticus is one of the most common Vibrio pathogens in shellfish diseases, posing a grave threat to the healthy development of the shellfish farming industry. However, effective methods for preventing and controlling V. alginolyticus are still lacking. The pathogenicity of V. alginolyticus is frequently closely related to its virulence factors and biological characteristics, and it is unclear how the virulence factors and biological characteristics of V. alginolyticus vary according to different sources and regions. Therefore, in this study, we aimed to gain a deeper understanding of the pathogenicity and scientific control of V. alginolyticus by analyzing the genetic variation and distribution patterns of V. alginolyticus in different aquaculture environments and shellfish tissues. The study was conducted to observe the external morphological characteristics and conduct 16S identification of twelve V. alginolyticus isolates collected from four regions: Qingdao, Weifang, Weihai, and Yantai. These were isolated and purified in TCBS selective medium; MLST typing of the strains by four housekeeping genes; the distribution of thirteen Vibrio virulence genes in V. alginolyticus; and the resistance of V. alginolyticus to 10 common antibiotics. The findings showed that all V. alginolyticus colonies were yellow or yellowish in color, round and transparent in shape with a raised center and smooth edges, moist, and difficult to harvest. 16S rRNA sequencing showed a homology of greater than 99% with V. alginolyticus, which was initially verified as V. alginolyticus and was consistent with the initial identification results. The ST typing of the twelve V. algolyticus strains differed from each other. Seven strains (A2, A3, B2, B3, C3, D2, and D3) contained ST types already included in the PubMLST database, with ST types 45, 87, 156, 56, 125, 96, and 57; five strains (A1, B1, C1, C2, and D1) formed new ST types owing to allelic locus changes in housekeeping genes, and four of the new ST types were isolated from shellfish tissue. The Qingdao isolate (A1) has similar ST types 38, 131, 134, 46, and 56 in the database; the Weihai isolate (C1, C2) has similar ST types 111, 322, and 61 in the database; and the Yantai isolate (D1) has similar ST types 268, 275, 341, 344, 351, and 358 in the database. These results suggested that V. alginolyticus in the shellfish culture environment had a high genetic diversity and that V. alginolyticus of shellfish origin might be more easily typed than V. alginolyticus from water sources. The MLST typing phylogenetic tree showed that there were four distinct branches: Group 1, Group 2, Group 3, and Group 4. All V. alginolyticus from shellfish tissues were predominantly observed in Group 1; isolates from Group 2 and Group 3 were mainly from marine environments and had closer evolutionary relationships with V. alginolyticus from the same region of the aquatic environment. The evolutionary relationships between V. alginolyticus from different areas of the aquatic environment and V. alginolyticus from shellfish tissues showed different characteristics. All V. alginolyticus strains carried three virulence genes: tlh, fur, and collagenase; VscB, Ompw, FlaA, and toxS virulence genes were present in most strains; UreB and AspA virulence genes were only present in a few strains; and tdh, trh, toxR, and tcpA virulence genes were not detected in any of the strains. The variety and number of virulence factors carried by V. alginolyticus were influenced by factors such as regional distribution. V. alginolyticus of different origins were characterized by multiple drug resistance interactions, but there were differences in the types of antibiotics to which resistance was developed. All V. alginolyticus species showed high susceptibility to cotrimoxazole and chloramphenicol, while they were resistant to penicillin and ampicillin. The majority of V. alginolyticus developed intermediate or high susceptibility to antibiotics such as butyraminecarbana, gentamicin, erythromycin, norfloxacin, and ciprofloxacin. Among all V. alginolyticus, one strain was resistant to ceftolozoline, five strains were intermediated, and six strains were highly sensitive. In addition, five strains were intermediated, and six were highly sensitive to butyraminecarbana, gentamicin, and erythromycin. Two strains were intermediated, and ten were highly sensitive to norfloxacin. Four strains were intermediated, and eight were highly sensitive to ciprofloxacin. Combining the MLST typing results, drug resistance results, and virulence genes in this study, no significant correlation between the three was found at this time. This study showed that V. alginolyticus in a shellfish culture environment was characterized by complex populations and high genetic diversity. There were large differences in virulence gene carriage and drug resistance among strains from different sources. The study provides a theoretical reference framework for understanding the pathogenicity of V. alginolyticus and assisting in the effective control of V. alginolyticus of shellfish origin by investigating the genetic variation and drug resistance of V. alginolyticus from different sources in various regions.

Abstract:Shrimp paste, a traditional aquatic fermented product, is more prevalent in Southeast Asia and the coastal areas of China. The traditional shrimp paste production process is straightforward. Usually, small economic shrimp species, such as grasshopper and sesame shrimp, are used as raw materials. Microorganisms and a series of endogenous enzymes are relied upon to create a condiment with a distinct flavor and aroma via natural fermentation. The quality of shrimp paste is influenced by various factors, including the type of raw shrimp used, the freshness of raw materials, fermentation duration, fermentation temperature, salt addition, and storage time. There are several brands of shrimp paste in the market, but their quality varies significantly. There are no studies on the quality evaluation system for commercially available shrimp paste. In this study, a comprehensive evaluation of shrimp paste samples was conducted to investigate the quality differences among commercially available shrimp pastes. 32 brands of shrimp paste were collected from six provinces, namely Shandong, Guangdong, Hebei, Tianjin, Liaoning, and Jiangsu. The color and histomorphology of these samples were assessed via sensory evaluation, whereas their physicochemical indicators, such as moisture, salt, protein, and amino acid nitrogen, were determined. Principal component analysis was utilized to create a comprehensive ranking of 32 commercially available shrimp pastes. The results showed that the odor score of commercially available shrimp paste had the highest coefficient of variation value at 37.64%, indicating that the odor of different brands of shrimp paste varied significantly. The SC/T 3602-2016 "Shrimp paste" standard states that the moisture content of shrimp paste should be ≤ 60%, ash content should be ≤ 35%, salt content should be ≤ 25%, protein content should be ≥ 10.0 g/100 g, and amino acid nitrogen content should be ≥ 0.6 g/100 g. The minimum variation value for the moisture content of shrimp paste products was 12.17%, but the overall moisture content was high, with seven samples exceeding the standard limit by a significant margin. In the shrimp paste production process, with the extension of the fermentation time, the moisture content gradually reduced, and the water activity decreased. The high moisture content of individual samples could be related to the production process that shortens the fermentation cycle of shrimp paste. A total of 32 samples had ash content ≤ 35%, in accordance with the standard requirements of "shrimp paste". Fifty percent of commercially available shrimp paste samples had salt content values greater than 20%, indicating that most commercially available shrimp pastes were high in salt concentrations. Since a diet high in salt may lead to a series of health problems, such as increased blood pressure, reducing the salt content of traditional shrimp paste is more in line with modern consumer values. The protein content of 28 samples met the requirements, and the amino acid nitrogen content of 31 samples met the requirements. The maximum variation value of amino acid nitrogen content was 33.91%, indicating a large difference in amino acid nitrogen content between samples. The amino acid nitrogen content of 78.13% of the shrimp paste samples was greater than 1.0 g/100 g, and a few shrimp paste samples even exceeded 2.0 g/100 g, which was significantly higher than the amino acid nitrogen content of fish sauce and comparable to that of commercially available oyster sauce. Through principal component analysis, three principal components representing eight quality indicators of 32 commercially available shrimp pastes were extracted based on the principle of principal component eigenvalues greater than 1, and the cumulative variance contribution reached 82.32%, indicating that the majority of the information regarding shrimp paste quality indicators could be covered. Through further analysis, it was determined that moisture, amino acid nitrogen, and odor were important indicators affecting the overall quality of shrimp paste, and a comprehensive evaluation model was established as follows: Fsynthesize= 0.497F1 + 0.318F2 + 0.185F3, in order to investigate the differences in the odor composition of different shrimp pastes in greater detail. The samples were subjected to an e-nose analysis, and an e-nose clustering heat map was developed; the results showed that 32 shrimp sauces were clustered into four categories at a Euclidean distance of 4.03, with the main distinction lying in the W1S, W2S, and W3S sensors, and clustering occurred between the sea silver shrimp sauce and grasshopper shrimp sauce. Gas-ion mobility spectrometry (GC-IMS) analysis of shrimp paste derived from three raw materials (i.e., sea silver shrimp, grasshopper shrimp, and white shrimp) showed the presence of 63 volatile components, mainly different alcohols. The high content of alcohol contained in the flavor substances is consistent with the findings of several previous studies. The results indicated that the quality of commercially available shrimp paste differed significantly, with odor, moisture, and amino acid nitrogen being the most influential factors affecting quality. The results of the study provide a benchmark for the quality assessment and production process enhancement of shrimp paste.

Abstract:Chinese mitten crab (Eriocheir sinensis) is an important freshwater aquaculture species in China, favored by consumers for its unique flavor and high nutritional and economic value. Chinese mitten crabs are sold live and their production areas are mainly located in the eastern part of China. Live crabs need to be transported over various distances to be sold nation- and worldwide. In recent years, with the rapid development of the internet and e-commerce, an increasing number of Chinese mitten crab products have moved online, and e-commerce sales are gradually taking over the dominant position of sales. Since the Chinese mitten crab is able to survive for a longer period of time out of water, e-commerce transportation mainly adopts the method of air-exposure transportation for crabs. In the process of air-exposure transportation, factors such as temperature, humidity, oxygen, and mechanical vibration can affect the survival rate and metabolism of crustaceans. To guarantee the safety and high quality of the crab after transportation, increase the survival rate, and prolong the transportation time, Chinese mitten crabs are transported using styrofoam boxes to reduce vibration, in which ice bags or ice bottles as cooling media are added to reduce the temperature of the environment during express transportation. Conditioned transportation allows them to have a weakened metabolism and be semi-dormant or completely dormant, thus reducing the adverse effects of low-oxygen, stress, and other factors during transportation. However, the transportation time varies due to different geographical locations, and the transportation time of e-commerce sales is generally 24–72 h. The e-commerce storage and distribution mode of Chinese mitten crabs is highly developed, and there is an urgent need to clarify the changes in biochemical characteristics and flavor quality during the live logistics transportation process. In this study, the e-commerce transportation conditions of Chinese mitten crabs were simulated by placing female crabs in sealed styrofoam boxes containing ice packs. The temperature and relative humidity inside the box were monitored in real time during the simulated transportation. The vigor of E. sinensis was counted and evaluated for 24 h, 48 h, and 72 h during the simulated express transportation. The changes in the biochemical characteristics of the meat of E. sinensis were analyzed in terms of lactate, total volatile basic nitrogen (TVB-N) and pH, while the changes in the taste quality of the meat were assessed in terms of free amino acids, taste nucleotides, and sensory evaluation. In addition, correlation analysis was carried out on the vigor, biochemical characteristics, and taste quality of female E. sinensis during the simulated express transportation. The results showed that with the increase of transportation time, the temperature in the box during the simulated express transportation showed a trend of first falling and then rising, and the relative humidity inside the box increased with the increase of the simulated transportation time. The mortality rate of female E. sinensis was 0 and vigorous under the 24 h simulated transportation, and the lactate and TVB-N in meat were not significantly different from the 0 h group, and the bitter amino acid content in meat was the lowest in the 24 h group, confirming the importance of same-day delivery of live E. sinensis. After more than 24 h, the vigor of E. sinensis decreased significantly (P<0.05) and the mortality rate increased at 3.33% after 48 h and 40.00% after 72 h of simulated transportation. The lactate, TVB-N, and pH in meat also increased significantly (P<0.05) after 24 h. With the extension of transportation time, the umami, sweet and bitter free amino acid contents in the meat of female E. sinensis showed a trend of first decreasing and then increasing, with the lowest content of bitter amino acids in the crab meat of the 24 h group being 105.10 mg/100 g. In contrast, after 72 h of simulated transport, the content of 5ʹ-inosine monophosphate increased significantly to 97.47 mg/100 g, and the content of guanosine 5'-monophosphate plummeted to 6.49 mg/100 g. After simulated transportation, the equivalent umami concentration of female E. sinensis meat decreased and tended to decrease and then increase with the extension of transportation time. The sensory results showed that the umami intensity of female E. sinensis meat decreased after the simulated transportation. Correlation analysis of vigor, biochemical characteristics and taste quality showed that the vigor of female E. sinensis was significantly positively correlated with moisture content in meat (R=0.68), negatively correlated with TVB-N (R=–0.66) and highly significantly negatively correlated with IMP (R=–0.76). In conclusion, the mortality rate of female E. sinensis increased, vigor decreased, and biochemical characteristics and taste quality changed significantly as transportation time increased, indicating that the optimal transportation time for E. sinensis under the current e-commerce transportation conditions is 0–24 h. This study could provide a reference for the quality changes of Chinese mitten crab during marketing and transportation, as well as a theoretical basis for subsequent research and practice on the optimization of transportation conditions and quality control.