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Abstract:This paper includes four sections. 1. Significant changes in Chinese modern fisheries were explicitly expressed by three data sets (i.e. total fisheries production, per capita production of aquatic products, and aquaculture as a percentage of total production) over the past half century. Such changes document the substantial contributions of Chinese “aquaculture-primary” fisheries development mode, which not only make greatly contribution to ensuring food security, nutrition security and ecological security, but also overturn the development modes of global fisheries oriented or dominated by capture fisheries since 1840. The concept of “aquaculture-primary” has become the main direction of modern fisheries development. 2. The driving forces that stimulate the rapid and sustainable development of Chinese “aquaculture-primary” fisheries were elaborated, including advanced mental preparation, solid technological support, correct development decisions, and clear development concepts. These four aspects will remain to be important for future development. 3. The unique composition of aquaculture species and their considerable carbon sink functions constitute the major pivots for stable and sustainable development of Chinese “aquaculture-primary” fisheries. These characteristics remain essential foundation for supporting stable and sustainable development of Chinese modern fisheries oriented. 4. For the future, green and high-quality development will be a necessary strategy for sustainable development of Chinese modern fisheries. The top priorities are to build an environment-friendly aquaculture and implement an aquaculture carrying capacity planning and management system, and build a stock conservation-based capture fisheries and strengthen the catch quota management system and conservation measures. Therefore, the basic and applied research on sustainable fisheries goods needs to be strengthened for green and high-quality development of fisheries.

Abstract:Both population and economic development are driving the rapid adoption of aquaculture development worldwide. The unique attributes of the intensive factory recirculating aquaculture system, including high density, low pollution, and high efficiency, have made this system an important avenue for aquaculture transformation and improvement, and this fits the current concept of green development in aquaculture. Flow patterns are an important environmental factor in the recirculating water aquaculture systems, with this parameter directly affecting the growth and welfare of fish within the system. However, it is also worth noting that the presence and movement of the fish also affect the construction of the system flow pattern. This study presents a comprehensive analysis of the effects of flow field conditions in recirculating aquaculture systems on different fish species, including the effects of flow rate on the growth and development, physiological indicators, and survival rates of commonly cultured fish, as well as the effects of other hydrodynamic conditions, such as circulating water and circulating water exchange rates, on fish behavior. The effects of fish and their locomotor behavior on hydrodynamic conditions and performance in culture ponds are also discussed, including the effects on turbulence intensity, flow-field flow patterns, and water mixing in these systems. The methods used to study the effect of fish movement on the flow field characteristics of any system can be broadly categorized into real measurement methods and numerical studies, where the real measurement methods include the visualization of research objects using the particle image velocimetry (PIV), acoustic Doppler velocimetry (ADV), and rhodamine water tracer fluorescence method. In contrast, numerical studies primarily rely on the application of the computational fluid dynamics (CFD) method. The advantages and disadvantages of both approaches are evaluated in this study. Our evaluations include a discussion of the common problems with the current system for constructing the circulating water aquaculture systems in industry, such as neglecting the flow field construction, lack of cross-disciplinary research, poor regularity, and repeatability of research, and suggest that a systematic experimental program should be established in combination with refined numerical simulation methods to facilitate the development of more accurate complex models. The aim of this study was to clarify the design of hydrodynamic conditions in these aquaculture systems in order to promote the development of better circulating water aquaculture systems for the industry in the hope of improving both the fish and water balance.

Abstract:The Eastern Central Atlantic Ocean is located on the west coast of the African continent and the East coast of the Atlantic Ocean. The United Nations Food and Agriculture Organization (FAO) has classified the operational area into 34 fishing areas. Under the influence of the canary Current (cold current), equatorial countercurrent (warm current), and ocean trade winds, an upwelling fishing ground has been formed in the broad continental shelf area of the Eastern Central Atlantic Ocean, which is abundant in fishery resources and is a globally important fishing area. Studies have shown that the Eastern Central Atlantic Ocean has been overfished by the European Union and other countries, which damaged the fishery resources and ecosystem in this fishing area. Therefore, analyzing the composition of catches of different countries and regions in this area can objectively evaluate the situation of fishing and utilization of fishery resources in different countries and regions, and provide a strong basis for rational development and scientific management of fishery resources in this fishing area. Using the catch data of the Eastern Central Atlantic Ocean provided by FAO from 1950 to 2019, the dominance analysis was used to analyze the composition and contribution of catches and catch species in the Eastern Central Atlantic, as well as in the coastal and non-coastal countries and regions in different decades. Previous works have illustrated that the total catch in the region has been increasing since 1950. The top ten identifiable species of fish in the region during the mentioned period were Sardina pilchardus, Jack and horse mackerels nei, Sardinellas nei, Sardinella aurita, Scomber colias, Ethmalosa fimbriata, Katsuwonus pelamis, Engraulis encrasicolus, Thunnus albacares, and Sardinella maderensis accounting for 63.61% of the total catch in this fishing area. Statistics of catches by age revealed that five species including Sardina pilchardus, Sardinellas nei, Sardinella aurita, Ethmalosa fimbriata, Thunnus albacares ranked in the top ten in each decade, and Sardina pilchardus, and Ethmalosa fimbriata catch increased in each decade, and Sardina pilchardus ranked first in each decade. From 1950 to 2019, the total catch of the top ten coastal countries and regions reached 10974.80×104 t, accounting for 94.95% of the total catch of the coastal countries and regions, representing the catch level of the coastal countries and regions. The sum of the total catches of the top ten non-coastal countries and regions from 1950 to 2019 reached 6440.01×104 t accounting for 80.40% of the total catch of the non-coastal countries and regions in this fishing area, representing the catch level of non-coastal countries and regions. Dominant analysis showed that four species of Sardina pilchardus, Ethmalosa fimbriata, Sardinella aurita, and Scomber colias were dominant in this fishing area. With an average combined contribution of 52.06% in each decade, Morocco, Senegal, Ghana, Cameroon, and Nigeria contributed the maximum to the fishing, among which Morocco and Senegal contributed more than 10% in each decade. The European Union, Russia (Soviet Union), and Japan were the main contributing countries among non-coastal countries and regions with an average total contribution of 82.71% in each decade and the average total contribution of the European Union was 60% in each decade. The cumulative catch of the Chinese mainland in this fishing area was merely 55.71×104 t, which was less than 18% of Japan´s contribution. According to this study, the fishery resources in the Eastern Central Atlantic Ocean have been overfished or overexploited to varying degrees threatening the survival of major catch species such as Sardina pilchardus, cephalopods. In this fishing area, the European Union, Japan, and other non-coastal countries and regions had captured a sizable catch with high economic value (such as tuna). Therefore, the monitoring and management of overfishing and illegal fishing must be strengthened to accelerate the monitoring and conservation of major catch species such as sardines and cephalopods. Coastal countries and regions should formulate reasonable fishery management measures, reject unreasonable fishery assistance and fishery agreements, scientifically evaluate fishery development systems such as bottom trawling permits, and reduce the damage to demersal marine fish resources and social impacts. Non-coastal countries and regions should keep the intensity of fishing appropriately to help resource assessment and conservation of overfished fishery resources in this fishing area. Coastal countries and regions should strengthen regional cooperation, break the current situation of unbalanced development between regions, and promote the sustainable development of fishery resources in the Eastern Central Atlantic Ocean. Based on the dominance analysis, this study comprehensively analyzed the fishery resources in the Eastern Central Atlantic Ocean from the perspective of catch composition and difference in catch size of countries and regions, objectively reflected the importance of different catch species, as well as the contribution and utilization of different fishing countries and regions to the fishery resources, which provided scientific support for the sustainable utilization of fishery resources in this fishing area. It also provided a new opinion for the analysis of the utilization of fishery resources in various fishing areas around the world.

Abstract:Marine stock enhancement is not only commonly reported to amplify the target species production but also induces some potential threats or effects at multiple ecological levels in the receiving ecosystem. Studies on the interaction between released and naturally occurring organisms are necessary to avoid or minimize the adverse impacts of invasive species from stock enhancement in the receiving system. Chinese shrimp Fenneropenaeus chinensis, are an important stock enhancement species, and in recent years approximately 10 billion have been released annually into the coastal waters of China. Owing to fishing pressure and the ecological complexity of the open sea, the interaction between the released shrimp and native species may be weak and undetectable. Studies on the ecological impacts of such intensive releasing activities are limited. Therefore, in this study, an in-situ mesocosm experiment was carried out to determine if the released shrimp altered the relative composition and niche characteristics of fish, the results of which could serve as a reference to the local risk management of the species stock enhancement. The mesocosm was located in the shrimp-releasing channel of Weihe, Laizhou Bay, from April to September 2019. A cross design of four treatments with three replicates was conducted in the mesocosm. The habitats were homogenized across the replicates by mixing substrates and filtering seawater with a 0.28 cm sieve. The number of individual shrimp larvae released into net enclosures in each of the four treatments were: A) 2 400, B) 1 800, C) 1 200, and O) 0. The treatments were designed to represent the release of 1 billion, 750 million, 500 million, and no shrimp larvae (none-releasing) in the coastal waters of Laizhou Bay, respectively. The released shrimp larvae had an average body length of (1.63±0.18) cm and an average body width of (0.38±0.12) cm. At the end of the experimental period in September, shrimp and fishes were recaptured and their Levin’s niche breadth and overlap indices were examined in each enclosure. Results indicated that within the enclosures of the four treatments, the recapture rates of shrimp ranged from 8.16% to 12.69% and had an average value of 9.87%. The number of recaptured shrimp increased with the number released, and had the highest value in enclosure A. However, the total biomass, average individual mass, and recapture rate of shrimp did not show a similar pattern among enclosures. The recapture rate of shrimp was significantly different among the treatments and had the highest value in enclosure C. The fish species composition was similar across all four treatment enclosures and included six species with three different feeding habits: Omnivores (Konosirus punctatus and Liza haematocheila), zooplanktivores (Thryssa mystax and Sardinella zunasi), and fish/shrimp predators (carnivores) (Acanthogobius sp. and Sparus microcephalus czerskii). Of the fish species, Acanthogobius sp. dominated in all enclosures, while S. m. czerskii occurred in only a few. There was no significant difference in the number and biomass of total fishes and carnivorous fishes among enclosures. There were more omnivorous fishes in the released enclosures A–C, while the number of zooplanktivorous fishes decreased when shrimp were released and had the highest number and biomass in the non-released enclosure O. Moreover, fishes with different feeding habits had variable niche breadths among the four treatment enclosures. Most fish species had a wider niche breadth in enclosures with released shrimp (enclosure A–C) than in enclosure O. Both the fish´s niche overlap indices and the species pairs with high niche overlap (>0.75) decreased in the order non-released enclosure O > released enclosure B >enclosure C >enclosure A. The carnivorous fish Acanthogobius sp. had more pairs with niche overlap indices (>0.75) in the non-released enclosure O than in the other three treatments. A similarity analysis and multidimensional plot indicated that enclosures A and O could be differentiated by the relatively long similarity distance and little overlap area. The results of the experiment suggest that the shrimp recapture efficiency did not increase when shrimp were released. In the waters of the study area, released shrimp could have a positive effect on the total output of both shrimp and fish, especially for omnivorous and carnivorous fishes, as the shrimp could share predation pressure and provide food for them. In addition, the shrimp release may affect the relative composition and niche characteristics of fish by increasing their niche breadth and reducing the overlap between fishes with different feeding habits, and the impacts may be more detectable as the shrimp number increases.

Abstract:The known universal primers for environmental DNA (eDNA) metabarcoding of fish are mainly located in the mitochondrial ribosome gene regions. Not only are the reference sequences insufficient but also some relative fish species could not be identified using these 12S and 16S primers. In this study, the freshwater fishes of Hainan Island were selected to meet six investigation targets. (1) Six conserved regions were selected through the cytochrome c oxidase subunit I (COⅠ) sequences of 8 orders, 26 families, 101 genera, and 150 species; (2) Based on the base variation, species identification, eDNA degradation, and read length requirements of high-throughput sequencing, 26 primers were designed from four flanking conserved regions; however, six primers did not meet the Premier score requirements. (3) The first-round PCR results of 72 freshwater fish species on Hainan Island showed that 11 primers had high universality. Among them, there were 5 primers with more than 70 species successfully amplified and brighter PCR bands than marker. The second-round of PCR results showed that the amplification success rate of 3×2 (forward and reverse, respectively) primer combinations generated by the five primers was 100%, and the optimal primer combinations after PCR band length and brightness screening were "HN-A-F4, HN-D-R3" (hereinafter referred to as HN-COⅠ). (4) High-throughput sequencing results of 30 water samples showed that the total number of clean reads, fish sequences, operational taxonomic units (OTUs), and fish OTUs generated by HN-COⅠ were 0.77, 1.22, 0.85, and 1.32 times those of MiFish-U, respectively. (5) The proportion of fish OTUs annotated to species, genus, and family was 81.40%, 11.63%, and 6.98% for HN-COⅠ and 81.54%, 4.62%, and 13.85% for MiFish-U, respectively. The non-metric multidimensional scaling (NMDS) clustering pattern of "primer + water sample" formed two distinct groups with an obvious boundary (stress=0.15). The minimum and average values of pairwise genetic distances of species amplicons within genera of HN-COⅠ were 1.23 and 1.57 times those of MiFish-U, respectively. (6) The correlation coefficient of the linear regression equation of common carp biomass and sequence copies of the six indoor density groups was low. HN-COⅠ and MiFish-U could not accurately reflect the biomass of common carp. In this study, the advantages and disadvantages of HN-COⅠ were screened and compared with those of MiFish-U. The results showed that HN-COⅠ primers have a high targeting ability for freshwater fishes on Hainan Island. HN-COⅠ are advantageous in preventing eDNA contamination from non-target organisms, such as microorganisms and mammals, and is also conducive to the detection and accurate identification of freshwater fishes.

Abstract:In this study, three genes associated with the metabolism of Pampus argenteus, namely dehydropeptidase (dp), carboxypeptidase A (cpa), and sulfotransferase (sult), were cloned and evaluated using transcriptomics analysis, and their functions in the digestion and absorption of jellyfish were discussed. First, full-length cDNA sequences for dp1, cpa2l, and sult2 were cloned using RACE and confirmed using sequencing. The total length of the dp1 was 2 522 bp, with this transcript including an open reading frame (ORF) of 1 272 bp, a signal peptide composed of 23 amino acids, and an amide hydrolase superfamily domain. The cpa2l transcript was 1 421 bp long and encoded a 1 260 bp ORF. This protein consisted of a signal peptide of 16 amino acids and was characterized by a typical M14 metal carboxypeptidase family domain. sult2 was 1 834 bp in length, producing an ORF of 714 bp, which included a typical sulfatase domain. In addition, homology evaluations of these three P. argenteus genes revealed that they were most similar to the bluefin tuna (Thunnus maccoyii), and phylogenetic tree analysis showed that dp1, cpa2l, and sult2 were all closely related to their corresponding genes from T. maccoyii. RT-qPCR was then used to evaluate the expression levels of these three genes in different tissues and the changes in expression patterns in response to jellyfish ingestion. These experiments revealed that dp1 expression was naturally the highest in the liver (P<0.05) and significantly increased in the brain, gills (P<0.01), midgut, and kidney (P<0.05), but significantly decreased in the muscle (P<0.05) when compared to that in the non-feeding control. cpa2l expression was highest in the middle intestine in the non-jellyfish feeding group and the kidney in the jellyfish feeding group (P<0.05) and was shown to be significantly increased in the liver (P<0.01) but significantly decreased in the midgut and muscle (P<0.01) when compared to that in the non-feeding group. sult2 expression was highest in the liver (P<0.05) and significantly increased in the midgut, brain, gill, liver, and kidney in the feeding group compared to that in the non-feeding group (P<0.05). Taken together, these results suggest that dp1, cpa2l, and sult2 play important roles in nutrient digestion, absorption, and metabolism in P. argenteus, especially in response to jellyfish feeding. In addition, the results of the tissue expression patterns for each of these genes following jellyfish ingestion suggest that dp1 is primarily involved in the regulation of digestion, absorption, and nutrient deposition; cpa2l is involved in the regulation of nutrient metabolism in the liver; and sult2 may play an important role in the overall process of digestion, absorption, and metabolism.

Abstract:Takifugu rubripes are warm temperate fish, suggesting that the reduced seawater temperatures in winter are likely to have a substantial impact on their survival. Considering this, there is likely to be some industrial value in breeding extremely low-temperature tolerant varieties of this fish. Here, we evaluate the expression changes in six major QTL candidate genes (tacc2, fsip1, exoc4, arhgap44a, pde10a, and unc5b) in response to reduced temperature in an effort to understand cold tolerance in T. rubripes. The expression changes of these six genes in the liver, heart, and kidney were detected using real-time quantitative PCR. This study used three groups of 8-month-old fish, all from the same family established by our research group, exposed to three different temperature gradients, where 8 ℃ and 13 ℃ acted as the minimum in the low temperature groups and 18 ℃ acted as the minimum in the control group. Our results showed that all six genes were expressed at different levels across each of these three tissues at different temperatures. The relative expression of pde10a first increased and then decreased in all three tissues, whereas the relative expression of tacc2 and exoc4 were distinctly different in the liver, kidney, and heart at 8 ℃. In this case, these transcripts first decreased and then stabilized in the liver, increased and then stabilized in the kidneys, and increased and then decreased in the heart. The relative expression of unc5b was low in the liver and heart, but high in the kidney following a second week of low-temperature growth, whereas arhgap44a expression was slightly upregulated in the liver and stable in the kidney and heart. fsip1 expression demonstrated a downward trend in the liver but seemed to first increase and then decrease in the heart and kidney. Taken together, these results demonstrate that all six of these genes are differentially expressed in different tissues of T. rubripes, with these differences exhibiting dynamic changes with respect to tissue origin and temperature. In addition, this data clearly revealed a positive correlation between cold stress and the expression of these QTL candidate genes. Thus, we can conclude that these six QTL candidate genes may play a substantial role in the low temperature adaptation of T. rubripes. This is significant because although low temperature is known to be an important factor limiting the development of the industrial utility of T. rubripes, there are still relatively few reports describing their cold stress response. This study provides a theoretical basis for the study of signaling pathways related to the low temperature tolerance response of T. rubripes and the development of low temperature tolerant varieties.

Abstract:Premelanosome protein (pmela) is a key gene in the melanin synthesis pathway, which has an important impact on the skin color of animals. This study was conducted to explore the role of pmela in rainbow trout (Oncorhynchus mykiss) skin color variation. First, the full-length cDNA sequence of pmela was obtained using the rapid amplification of cDNA ends (RACE) technique, and bioinformatic analysis of the encoded protein was performed. Second, quantitative real-time PCR (qRT-PCR) was performed to detect the relative expression of pmela in different developmental stages [from the fertilized-stage to 3 months post-hatching (3 M)] and tissues of wild-type rainbow trout (WT), yellow mutant rainbow trout (YM), and their F1 generation. The results showed that the total length of pmela was 3 476 bp, including a 2 532 bp open reading frame encoding 843 amino acids. Bioinformatic analysis revealed that Pmela was a hydrophobic protein and had a PKD functional domain. Homology alignment showed that the similarity of the Pmela amino acid sequences of rainbow trout and sockeye salmon (Oncorhynchus nerka) was 97.75%. Phylogenetic analysis indicated that rainbow trout was most closely related to sockeye salmon and most distantly to humans (Homo sapiens) and mice (Mus musculus). qRT-PCR results showed that pmela was expressed in all developmental stages of WT and YM, and the expression level of the gene from the fertilized-stage to the 16-cell stage was higher in WT than in YM, whereas the opposite expression pattern was identified after hatching. Moreover, there were significant differences in the expression of pmela at the same stages between WT and YM, including at the fertilized-stage, 4-cell, 16-cell, gastrula, neurula, somites, heartbeating, 1 day post-hatching (dph), 3 dph, 5 dph, 7 dph, 10 dph, 1 M and 2 M. In various tissues, pmela expression was significantly higher in the dorsal skin and eyes of WT and YM than in other tissues. In the hybrid F1 generation, the expression pattern of pmela was similar to those of both parents, and there was a significant difference in expression at the same stages between the F1 generation and the parents in most of the same stages and tissues. The above results indicate that the expression level of pmela is correlated with the skin color of rainbow trout and may be involved in the formation of its skin color, which provides basic data for further studying the role of pmela in the variation of rainbow trout skin color.

Abstract:Variation in fish temperature is determined by changes in the external aquatic environment, and temperature serves as a crucial regulatory factor for the behavior, physiology, and molecular processes of fish. A rapid decline in water temperature can elicit a strong stress response in fish, potentially inhibiting growth and even leading to mortality. Low temperature stress significantly impacts various life processes of organisms, including growth and development, energy metabolism, and reproductive development. In recent years, there has been increasing research on the effects of low-temperature stress on organisms, with a greater focus at the molecular level. Low temperatures induce a decrease in cellular activity and even growth arrest, resulting in apoptosis. Apoptosis is a form of programmed cell death ubiquitous in the biological world, and it plays a vital role in maintaining the normal physiological function of an organism. Once apoptosis occurs, it is irreversible and cannot be stopped. Temperature regulation is crucial for fish, and current knowledge of the role of leptin in fish primarily focuses on the regulation of feeding, lipid energy metabolism, and reproduction, with limited reports on its role in temperature regulation. Leptin, a protein hormone produced by adipocytes, has long been recognized as a product of the obese gene, which regulates organismal metabolism, neuroendocrine function, and other physiological processes. In animals, it exerts diverse physiological functions related to cell growth, proliferation, apoptosis, and metabolism. The homology of the leptin gene in fish is relatively low compared to that in mammals, and multiple leptin proteins encoded by multiple leptin genes can be produced due to genomic duplication events. Regarding the role of leptin in temperature regulation, more research has been conducted on mammals, mainly related to the regulation of energy metabolism and the influence on temperature adaptation evolution. There has been limited research on the regulatory mechanism of leptin in fish during cold stress. Dissostichus mawsoni belongs to the group of Antarctic vertebrates and has lived in the cold and isolated environment of the Southern Ocean at a temperature of –1.9 ℃ for ~30 million years. It has adapted to the extremely low temperatures of the surrounding Antarctic waters and is an excellent material and living fossil for studying temperature adaptation mechanisms in extreme environments. Compared with other fish species at the same depth, it has a large amount of lipid deposition in its subcutaneous and muscle tissues, mainly triglycerides (TGs). Predictions based on 3D structural models suggest that the partial absence of the lepB structure in D. mawsoni leads to only three α-helices, and lepA has four α-helices and a short and twisted E-helix as well as several irregular turns, forming a hollow barrel-like structure, which differs from the protein structure of all other known leptins. Moreover, previous studies have found a close correlation between lepA and temperature evolution, leading to speculation that Antarctic fish lepB may play an essential role in low-temperature adaptation. In this study, a eukaryotic expression vector of the Antarctic toothfish lepb gene was constructed and transfected into ZFL cells to establish a model of overexpression of the D. mawsoni lepb (DMLB) gene in the ZFL cells. After conducting a cell culture temperature experiment, 10 ℃ was selected as the significant difference temperature. Following 2 weeks of cold stimulation, the DMLB experimental group cells remained viable, whereas the control group cells died. The results indicated that the overexpression of the DMLB gene provided strong resistance to low-temperature conditions. By detecting changes in cell proliferation, apoptosis, reactive oxygen species (ROS) level, and ATP content under low-temperature stress, it was discovered that DMLB maintained cell growth and proliferation, reduced ROS production, and inhibited cell apoptosis. DMLB effectively maintained ATP levels in cells under low-temperature stress, which helps maintain the mitochondrial status and reduce the effects of apoptosis and necrosis caused by low-temperature stress, thereby protecting cells from cold stress. Additionally, the results of Oil red O staining and TG detection suggested that the DMLB gene may slow down the depletion of cell lipids under low-temperature stress, perhaps by lowering lipid metabolism to preserve lipids to cope with low-temperature damage. Consequently, the DMLB gene may be functional in the cold resistance of D. mawsoni by protecting cells from damage at low temperatures, but its specific molecular regulatory mechanism needs to be further explored. In this study, 10 ℃ was used as the key temperature, providing a basis for understanding the regulation of energy homeostasis by the lepb gene in D. mawsoni under low-temperature stress. This study explored the role of the lepB gene in the adaptation of D. mawsoni to extremely low-temperature environments and provided fundamental data for further study of the evolution of similar species in the Antarctic region. Furthermore, this study enriched the basic data related to low-temperature tolerance and provided reference materials for further investigation of the low-temperature tolerance mechanism of leptin in Notothenioids, as well as laid a foundation for further study of the mechanism of leptin in temperature regulation.

Abstract:Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used molecular techniques, often implemented to allow for the detection and quantitation of gene expression because of its high sensitivity, specificity, and reproducibility. However, reliable and comparable relative quantitative results using qRT-PCR require the application of appropriate reference genes in order to eliminate non-biological variations caused by initial RNA templates, efficiency of cDNA synthesis, and laboratory procedures. However, previous studies have reported that the stability of many reference genes may vary across species, tissue types, cell lines, developmental stages, and experimental treatments, yielding inaccurate or incorrect gene expression results. Therefore, the selection and validation of stable reference genes for different tissues from a specific species are especially important for obtaining accurate target gene expression results. The striped jack, Pseudocaranx dentex, belonging to the order Perciformes and family Carangidae, is a pelagic migratory fish with high nutritional value. This fish has already received extensive attention in global aquaculture production and is regarded as a candidate species for far-reaching marine aquaculture in China. Given this, there are currently a large number of fairly extensive molecular biology and genetics studies of P. dentex underway, which in turn have increased the demand for quantitative gene expression analysis by qRT-PCR in these animals. However, few studies have evaluated the reference genes for this species. Thus, the objective of this study was to identify suitable reference genes in different tissues of P. dentex, in an effort to provide the necessary tools to support subsequent gene expression pattern analysis. We evaluated nine commonly used reference genes, including beta actin (β-actin), ribosomal protein L13 (RPL13), elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase (HPRT), peptidylprolyl isomerase A (PPIA), beta 2-microglobulin (β2M), beta tubulin (TUB), and serine/threonine-protein phosphatase 2A catalytic subunit (PP2A) using qRT-PCR analysis across various P. dentex tissues. These evaluations included the study of their expression stability across ten tissues, including the brain, gill, heart, intestine, kidney, liver, spleen, stomach, slow-twitch muscle, and fast-twitch muscle, from three adult individuals of P. dentex using four independent methods, namely BestKeeper, NormFinder, geNorm, and RefFinder. These results were then validated in the qRT-PCR profiling of the myoblast determination protein 1 (myod1) gene in both muscle (slow-twitch and fast-twitch muscles) and non-muscle tissues (kidney and gills) using the various recommended reference genes or their combinations. Expression analysis showed that RPL13 was the most highly expressed gene in these samples, followed by EF-1α, β2M, β-actin, PPIA, HPRT, TUB, and PP2A, whereas GAPDH was the most weakly expressed across all ten P. dentex tissues. In addition, all nine candidate reference genes exhibited relatively inconsistent variations in Ct value across various tissues, with the BestKeeper stability assay, which uses standard deviation (SD) and coefficient of variation (CV) to score the candidates, suggesting that these genes could be ordered from most stable to least stable as follows: EF-1α > RPL13 > PP2A > PPIA >HPRT > β2M > TUB > GAPDH > β-actin. NormFinder suggested that the ranking was best described as follows: RPL13 > EF-1α> HPRT > TUB > PP2A > PPIA > β2M > GAPDH > β-actin. Evaluations using geNorm, which is based on the idea that the lower the expression stability value (M), the better the stability of gene expression, suggested that the expression stability of these genes is best described as follows: RPL13 = EF-1α > β-actin > GAPDH > PP2A > TUB > PPIA > HPRT > β2M. Finally, RefFinder analysis showed that the comprehensive stability ranking of each gene was RPL13 > EF-1α > PP2A > HPRT > PPIA > TUB > β2M > β-actin > GAPDH. In addition, all of the paired coefficients of variation, Vn/(n+1), reference genes had little effect on the V value. The combination of the two best reference genes is a valid normalization strategy, and the results can be used to correct the expression levels of various target genes. Given this, we can conclude that RPL13 and EF-1α are the two best reference genes for adult tissues. These outcomes were then validated in a myod1 expression assay in the slow-twitch muscle, fast-twitch muscle, kidney, and gills. These experiments revealed no significant differences in relative outcomes when using RPL13, EF-1α, and their combination as reference genes, whereas significant differences were identified when using the three least stable genes β2M, β-actin and GAPDH. This result confirmed that stability evaluation using the four methods was both necessary and effective. Based on our evaluations, we recommend that RPL13, EF-1α, and their combination are the ideal reference gene combinations for all previously evaluated adult tissue types in P. dentex, as verified by four individual algorithms. The results of this study provide the basis for improved standardization of qRT-PCR and transcriptomic evaluations in this species, which in turn should support more accurate evaluation of functional genes and provide technical support for the comprehensive and systematic molecular evaluation of more mature P. dentex samples.

Abstract:The Japanese grenadier anchovy Coilia nasus Temminck & Schlegel, 1846 belongs to the family Engraulidae of the order Clupeiformes. This species is widely distributed in China, Japan, and Korea and in Northwest Pacific Ocean. In China, C. nasus is found in the Bohai Sea, the Yellow Sea, the East China Sea, and the middle and lower reaches and the estuaries of some main rivers, such as the Liao River, the Yellow River, and the Yangtze River. The life history of C. nasus is diverse and it is found to have three main ecological types (i.e., anadromous migratory, freshwater resident, and freshwater landlocked), indicating its strong adaptability to different habitats. Because of its commercial importance in China, C. nasus has been heavily exploited for years. Due to the significant decline of the wild stocks and the degeneration of freshwater spawning grounds in its distribution region, C. nasus was listed in the IUCN Red List of Threatened Species as “Endangered” category in 2018. Currently, the exact distribution boundary of C. nasus to the southern limit of China is unclear. Based on the fishery surveys in the southern Zhejiang Province for the past 15 years, the adjacent waters of Ou River estuary in Wenzhou are the known southernmost distribution area for C. nasus. The distribution of C. nasus in Fujian waters, both freshwater and seawater was documented from 1960—2008; however, the sampled individuals were very rare. It is impossible to understand the habitat history and life history pattern of C. nasus using traditional fishery survey methods. In this study, three C. nasus individuals were collected by trawl net, set net, and gill net in Fuzhou and Ningde waters in 2017—2019 during various fishery surveys along the coastline of Fujian. To reconstruct the life history of C. nasus in Fujian waters, the X-ray electron probe micro-analysis (EPMA) approach was used to analyze otolith (sagitta) microchemistry patterns of the fish for the first time. Otolith microchemistry is an effective tool for revealing the anadromous pattern and the spatial and temporal distribution of fishes. The otolith Sr/Ca ratios are powerful indicators for identifying freshwater, brackish water, and seawater habitats throughout the life history of fish. The results of this study showed that the Sr/Ca ratios of the otolith core areas of all three C. nasus individuals were less than 3, and the core areas exhibited a blue pattern in the X-ray intensity maps of Sr content. Both results indicated that the three individuals originated from freshwater and hatched in freshwater habitat. Among the three individuals, the individual collected in Fuzhou waters was more dependent on freshwater at its early life stage and did not enter brackish water until it was almost 1-year-old. Combined with the sampling site and the adjacent water of Min River, it is deduced that it might have originated from the pure freshwater habitat of Min River. Two individuals collected in Ningde waters were less dependent on freshwater habitat at their early life stage. Combined with the sampling sites, i.e., the outer water of Sansha Bay, it is deduced that the two individuals from Ningde might have originated from the pure freshwater habitat of Jiao Brook in Ningde, Ou River in Wenzhou, or Min River in Fuzhou. According to the fluctuations of the Sr/Ca ratio from the core to the edge of the otoliths and X-ray intensity maps of Sr content through the otolith core, all three C. nasus individuals were confirmed to be typically anadromous ecological types. The habitat history of the only one C. nasus individual in Fuzhou was a freshwater-brackish type; its hatching and early life stage were also associated with freshwater habitat, and then it entered the brackish water habitat and traveled back and forth between brackish water and freshwater habitats to grow out until it was captured. The habitat history of the other two C. nasus individuals in Ningde was freshwater-brackish-seawater type; their hatching and early life stage were associated with freshwater habitat, and then they entered high salinity habitat and traveled back and forth between brackish water and seawater habitats for growth until captured. Moreover, we found that the higher salinity in the capture waters of the three C. nasus individuals did not correspond to the Sr/Ca ratio at the edge of the otoliths, indicating a certain time delay in otolith responding to the salinity of their most present living habitats. This study not only revealed the diversity of C. nasus life history patterns and habitat histories but also confirmed the current distribution of anadromous C. nasus in Fujian waters. Since February 2019, the Yangtze River, the most important habitat for C. nasus and many endangered fishes and mammals has initiated a new management measure, i.e., a 10-year fishing ban plan. However, there is still a lack of sufficient understanding of the distribution, the locations of spawning grounds and the population size of C. nasus in Fujian waters. Further studies on the key habitats of C. nasus such as spawning ground and ecological type are needed to formulate effective management measures for its natural resource conservation in Fujian waters.

Abstract:Coilia nasus is a species of anadromous migratory fish, which predominantly lives in the sea. During the breeding season, the reproductive population migrates from the sea to rivers to reproduce and clusters to form a fishing season. The Yangtze Estuary is an important migration channel for this species. In recent years, due to the influence of upstream dam construction, water conservancy projects along the Yangtze River, environmental pollution and overfishing, the populations of this species in the Yangtze River are on the verge of extinction. In order to better protect C. nasus in the Yangtze River, a large number of studies on the conservation and management of its resources, migration habits, genetic structure, and the effects of resource recovery on this species have been conducted. As for the nutritional composition of C. nasus, studies have been conducted on the different sources, ecotypes, breeding methods, and stages of gonad development. These studies have mainly focused on the muscle nutrition of C. nasus from the Yangtze River; however, none have investigated the nutritional composition of different tissues of the reproductive populations from the Yangtze Estuary. This study is the first to determine the nutritional status of the reproductive population that migrates to the Yangtze Estuary with mature ovaries, and to compare and analyze the distribution characteristics of total lipids and fatty acids in different tissues, and provides reference information for ascertaining the nutritional status and reproductive performance of this species in the Yangtze Estuary. In this study, the contents of moisture, total lipid, and fatty acid composition in muscle, liver, and ovary tissues of adult female C. nasus with gonad development stage IV were measured and analyzed using standard GB methods. The results indicated that: The moisture content declined successively in muscle, liver, and ovary tissues, with measurements of (77.91±1.61)%, (75.01±1.33)%, and (46.77±5.58)%, respectively. The total lipid content of dry matter increased successively in muscle, liver, and ovary tissues, with measurements of (16.21±1.09)%, (21.94±1.23)%, and (55.21±1.35)%, respectively. The saturated fatty acids (SFA) content was (29.89±0.81)%, (37.60±3.06)%, and (18.33±1.61)% in the muscle, liver, and ovaries, respectively, with the highest in the liver and the lowest in the ovaries. There were significant differences in SFA contents among the three tissues (P<0.05). The content of C18:1n9c was highest in mono-unsaturated fatty acids (MUFA), with values of (40.88±0.19)%, (35.06±1.84)%, and (42.85±2.14)% in muscle, liver, and ovary tissues, respectively. The content of C18:1n9c in liver was significantly lower than that in the other two tissues (P<0.05). In polyunsaturated fatty acids (PUFA), the content of docosahexaenoic acid (DHA) was the highest, followed by that of eicosapentaenoic acid (EPA). The total content of DHA and EPA in muscle, liver, and ovary tissues was (12.42±0.49)%, (12.41±2.32)%, and (16.20±1.49)% respectively, with the highest content in the ovary. The contents of PUFA, n3-PUFA and DHA increased successively in the muscle, liver, and ovary tissues, and were significantly higher in ovary tissue than in the muscle and liver (P<0.05), but showed no significant difference between muscle and liver (P>0.05). The ratio of UFA/SFA in muscle, liver, and ovary tissues was 2.35, 1.67, and 4.49, respectively, and the ratio of n3-PUFA/n6-PUFA in the same three tissues was 4.94, 3.87, and 5.13, respectively. These ratios were highest in the ovary, followed by the muscle, and were the lowest in the liver. In summary, the total lipid, PUFA, EPA, DHA, and n3-PUFA contents and the ratios of UFA/SFA and n3-PUFA/n6-PUFA were highest in ovary tissues, and the distribution characteristics of the corresponding composition in different tissues was closely related to the reproductive habits of this species during gonad development stage IV. The accumulation of rich lipids and reasonable essential fatty acid nutrition in the ovary can improve the reproductive performance of C. nasus in this stage and provide sufficient nutrition and energy sources for the development of eggs and larvae in the subsequent stage. In this study, it was found that the lipid and polyunsaturated fatty acid nutrition of C. nasus adults with ovarian development to stage IV selectively accumulated in the ovary, which is an adaptation to the reproductive activities of this species at this stage. The results provide basic data for determining the differences in nutritional demands of C. nasus adults at different ovarian development stages and the mechanisms for the synthesis and transformation of essential fatty acids. Additionally, this study lays a foundation for further exploration of the relationship between ovarian nutritional status and egg quality. Relevant data can enrich the reproductive biology knowledge of this species and provide theoretical guidance for protecting the reproductive population and the maintenance of C. nasus spawning grounds in the waters adjacent to the Yangtze Estuary.

Abstract:Yadong trout (Salmo trutta fario) is an important and characteristic aquaculture fish species in Yadong, Xizang, China. Farmed Yadong trout has been produced on a large scale. Usually, two different-colored eggs (yellow and orange) are produced during Yadong trout farming and show differential reproductive performance. There has been very limited information on the nutrient composition of eggs with different colors, and relevant results have only been reported for rainbow trout. The present study aimed to analyze the nutrient compositions in different-colored Yadong trout eggs to elucidate the factors responsible for the different reproductive performances of the two egg types. The experimental samples were collected from Yadong Industry Park, where the broodstocks were reared with artificial compound feeds. Each egg type was analyzed in 3 replicates. Yellow eggs were produced by 2–3 year-old broodstocks, and orange eggs were produced from 3–5 year-old broodstocks. After spawning, the eggs were cryopreserved and transported to the laboratory by plane. The samples were lyophilized and used for the determination of proximate composition, fatty acids, amino acids, and other reproduction-related nutrients. The crude protein and crude lipid contents were analyzed using the Kjeldahl and Soxhlet extraction methods, respectively. The fatty acid composition (expressed as % total fatty acids) and amino acid composition (expressed as % dry matter) were assayed using gas chromatography and an automatic amino acid analyzer, respectively. The protein carbonyl and malondialdehyde concentration were assayed using commercial kits supplied by Nanjing Jiancheng Bioengineering Institute. The α-vitamin E and astaxanthin were assayed using national standard methods, and the α-vitamin E was measured from wet samples. The statistics of proximate composition, fatty acids, and amino acids data were determined using a t-test for independent samples. The results were expressed as mean ± standard error, and showed that yellow eggs had lower contents of crude protein and ash contents, but higher contents of crude lipid and moisture than orange eggs. There was no significant difference in carbohydrate and energy content between yellow and orange eggs. The saturated fatty acid (SFA), n-3 polyunsaturated fatty acid (n-3 PUFA), and docosahexaenoic acid (DHA) contents of yellow eggs were significantly lower than those of orange eggs, while the n-6 polyunsaturated fatty acids (n-6 PUFA) and EPA contents did not differ significantly between the two egg types. The C16:0 and C20:0 contents of yellow eggs were significantly lower than those of orange eggs, while the contents of monounsaturated fatty acids (MUFA), including C16:1n-7, C18:1n-9, C20:1n-9, and C22:1n-9, of yellow eggs were significantly higher than those of orange eggs. The contents of essential amino acids, non-essential amino acids, and total amino acids were slightly lower in yellow eggs compared to orange eggs. Among the essential amino acids, the contents of valine, methionine, leucine, lysine, and histidine in yellow eggs were significantly lower than those in orange eggs. Among the non-essential amino acids, except for glycine and alanine which were significantly lower in yellow eggs than in orange eggs, the contents of other amino acids did not differ significantly between the two egg types. The contents of protein carbonyl and malondialdehyde in yellow eggs were higher than those in orange eggs, while the contents of α-vitamin E and astaxanthin in yellow eggs were lower than those in orange eggs. In conclusion, orange eggs had higher contents of protein, some essential amino acids, DHA, α-vitamin E, and astaxanthin, but lower contents of MUFA, crude lipid, and peroxidation products (protein carbonyl and malondialdehyde) than yellow eggs. These differences in nutrient composition may contribute to the superior reproductive performance of orange eggs over yellow eggs.

Abstract:Shell morphological traits are important quantitative traits in shellfish. Shell morphology is usually affected by many factors, such as the ecological environment and shellfish genetics. It is considered the results of comprehensive actions of natural selection, micro-evolution, and heredity. Measuring shell morphological traits aids our understanding of the current situation of germplasm resources among shellfish species or intraspecific populations and also provides important breeding traits for shellfish breeding programs. For most shellfish, quality traits (eg. live weight and soft body weight) are considered the important breeding traits, but there are practical issues in using these traits, such as the inability or difficulty in measuring them in vivo. It is well known that shellfish quality traits are closely related to shell morphological traits. However, a simple correlation analysis between quality and morphological traits cannot adequately explain all the intrinsic links between these traits. In this study, Manila clams (Ruditapes philippinarum) were collected from nine geographical locations along the north and south coasts of China. The shell morphology and quality characteristics were measured and analyzed for the nine different populations of clams. Furthermore, the main factors affecting the quality traits were analyzed using cluster analysis to reveal the genetic relationships among the populations. The optimal regression equations were constructed using the morphological traits with the soft weights of these populations. For all populations, shell length, shell height, shell width, and shell thickness were measured using a vernier caliper (with an accuracy of 0.01 mm) and a thickness gauge. After wiping the surface of clams with filter paper, their live weight was measured on an electronic balance (with an accuracy of 0.01 g). A scalpel was used to sample the soft body from the clams. After drying, soft body weights were calculated using the difference between live weight and shell weight. The morphological data, including mean values, standard deviation, and coefficient of variation, were calculated using SPSS 26.0. To eliminate the effects of size differences among individuals, two morphological scale parameters (shell width/shell length and shell height/shell length) were calculated to reflect the morphological characteristics of the clams. In addition, stepwise linear regression analyses were performed to obtain the correlation coefficients, direct path coefficients, correlation indexes, indirect path coefficients, and determine the coefficients using SPSS26.0 software. The correlation coefficient and multiple regression analyses of the traits were evaluated. The multiple regression equations were established for all populations. Cluster analysis was used to assess each trait using the calibrations (to calculate the ratio of each trait to shell length). The heatmap displayed the shortest distance method for the Euclidean distance between the different populations. There were significant differences in the effects of shell length, shell height, shell width, and shell thickness on live weight and soft body weight of clams. Morphological traits were significantly correlated with body weight and soft weight (P<0.05), except for shell thickness. The results of determination coefficient revealed shell width had the greatest impact on live weights of the Putian, Jinzhou, and Chaozhou populations, with determination coefficients of 0.277, 0.232, and 0.307, respectively. The determination coefficients of soft body weights indicated that shell width played very important roles in determining soft body weights of the Putian and Chaozhou populations, with determination coefficients of 0.249 and 0.443, respectively. By calculating the total of determination coefficients, the results are consistent with the correlation index R2 for each trait. For the total determinant effects, the sum of determination coefficients for the morphological traits with soft body weights were greater than 0.850 in most populations. In contrast, the sum of determination coefficients for the morphological traits with soft body weights were less than 0.850 in Laizhou, Zhuanghe, Donggang, and Jinzhou. Based on the path analysis and the determination coefficient analysis, shell width had the greatest impact on live weights and soft body weights in most populations. The maximum values of the two morphological proportion parameters (shell width/shell length and shell height/shell length) were found in the Laizhou population (0.49 and 0.74), while the minimum proportion parameters were detected in the Dalian Donggang population (0.42 and 0.67). Among the shell morphological traits, shell thickness had the largest coefficient of variation (22.74), while shell height had the smallest coefficient of variation (9.47). By testing for statistically significant variation in the partial regression coefficient, the optimal regression equations of the morphological traits for soft body weight were constructed for all the populations. The cluster analysis revealed that different clam populations did not have a typically regional characteristic in terms of the shell morphological traits. There was an irregular north-south alternating clustering phenomenon. According to the present findings, shell thickness and morphological ratio parameters (ratio of shell height to shell length and ratio of shell width to shell length) are important indicators for evaluating the growth potential and nutritional status of clams in aquaculture. The present results provide a scientific basis for morphological discrimination and germplasm resource evaluation for geographical populations of clams and also aid breeding strategies to predict breeding traits for aquaculture breeding programs, including guidance for the selective breeding of clams.

Abstract:The Ras-related proteins in brain (Rab) subfamily is the largest group of small molecule guanine nucleotide-binding regulatory proteins (GTP-binding proteins). These proteins are widely distributed across various eukaryotes and consist of approximately 200 amino acids, producing a molecular weight of approximately 20~25 kDa. Rab is known to be an important regulator in the transport of membrane vesicles and is embedded in almost all membrane-related proteins. Rab is primarily regulated through GTP binding and hydrolysis within the organelles and is known to perform several important functions across various stages of vesicle transport. Current research on Rab proteins produced by aquatic animals has revealed that the Rab gene plays an important role in immune response. The Pearl oyster, Pinctada fucata martensii, is the most common species used in the cultivation of seawater pearls, making them economically important. These oysters are primarily distributed along the coastal areas of Guangdong, Guangxi, and Hainan provinces in China. However, they display weak tolerance to low temperatures, which severely limits their cultivation area and the overall production of seawater pearls. Given the desire to expand their cultivation northward, we designed this study to help create low-temperature resistant P. f. martensii breeding lines F3 (R). We used genome resequencing technology to compare and analyze the R and Beibu Gulf wildtype populations (W), and identified a group of candidate genes, including the Rab gene, that were strongly positively selected during the breeding process. In addition, the full-length sequence of Rab7 of P. f. martensii (Pm-Rab7) was cloned using RACE technology, and the expression levels of Pm-Rab7 in adductor muscle, gill, gonad, hepatopancreas, foot, and mantle, and their expression patterns under temperature stress (17 ℃ in the low temperature group, 22 ℃ in the control group, and 32 ℃ in the high temperature group) were detected using bioinformatics analysis. Furthermore, single nucleotide polymorphisms (SNPs) in the exon regions of Pm-Rab7 in the R and W were screened, and this information was used to determine the genetic polymorphism, haplotype, and frequency rates for each mutation. Sequence analysis also revealed that the Pm-Rab7 gene displayed a full length of 1 153 bp, with the 5' and 3' UTR adding 30 bp and 505 bp, respectively. This gene was also shown to encode a single open reading frame (ORF) of 618 bp and 205 amino acids, producing a theoretical protein of 23.04 kDa with the isoelectric point at 5.40. This was later confirmed using the cloned Pm-Rab7 gene, which produced 5 conserved amino acid sequences, including a RAB conserved domain. Similarity evaluations revealed a high degree of overlap with C. gigas (92.79%), whereas phylogenetic tree construction revealed that the Rab7 gene produced a tree with three branches, one each for protostomes, echinoderms, and vertebrates. Closer inspection of the protostomes branch revealed that Pm-Rab7 first clusters with other mollusks, and then with arthropods to produce a large clade, suggesting that this protein is relatively well conserved during the evolutionary process. Pm-Rab7 was also shown to be expressed in all the tested tissues, with the most Pm-Rab7 expression recorded in the gonad and gill (P<0.05). Time-course results from the gill tissues of temperature stressed samples revealed that Pm-Rab7 expression first increased and then decreased in response to low temperature (17 ℃) exposure, with all time points showing a significant increase in Pm-Rab7 expression when compared to that in the 22 ℃ control from 6 h to 3 d (P<0.05). In addition, we noted an expression peak at 1 d. The expression of Pm-Rab7 was generally stable in response to growth at 32 ℃ (high temperature group), but was also significantly increased when compared to that in the control group following 12 h of exposure (P<0.05). This suggests that Pm-Rab7 is most likely linked to the low temperature response process in these shellfish. We also identified a total of seven SNPs in the exon region of Pm-Rab7, three (g.112712470, g.112712503, and g.11271477) of which demonstrated significant differences in occurrence rate between the R and W populations (P<0.05). Genetic evaluations of all seven SNPs revealed that three could be classified as low polymorphism loci (PIC<0.25) and four could be classified as moderate polymorphism loci (0.25

Abstract:Paralytic shellfish toxins (PSTs) are some of the most harmful algal neurotoxins in the world. They easily accumulate in bivalve shellfish and are transmitted through the food chain, causing symptoms such as nausea and vomiting, muscle paralysis, dyspnea and even asphyxiation in consumers, leading to food poisoning in humans. Therefore, a widely accepted limit standard of 800 μg STXeq/kg has been established as a safe limit for PSTs. PSTs are produced by some microalgae, among which Alexandrium tamarense is one of the predominant toxic algae found along the coast of China. It was found that the detection rate and over-standard rate of Chlamys farreri in bivalve shellfish sold in China are relatively high. PSTs are mainly stored in visceral masses and are characterized by their fast accumulation and slow metabolism. PSTs are neurotoxins that exert their toxic effects by blocking sodium channels and inhibiting nerve conduction. Studies have shown that PSTs can cause stress responses in bivalves, including production of a large amount of reactive oxygen species (ROS), antioxidant stress (including enzymatic and non-enzymatic defense), imbalance of intracellular redox homeostasis, and cell damage (i.e., lipid peroxidation). As one of the main products of lipid peroxidation, the content of malondialdehyde (MDA) can directly reflect tissue and cell membrane damage caused by PSTs. In addition, superoxide dismutase (SOD) and peroxidase (POD) are often used as indicators to evaluate the level of antioxidation, and glutathione peroxidase (GSH-Px) plays a key role in antioxidant defense. The changes in lipid peroxidation and antioxidant enzymes are commonly used in existing studies to reflect the injury and degree of stress in organisms. Some studies have also shown that PSTs can cause tissue damage and induce abnormal gene expression in C. farreri, but research on the changes of gene expression and regulatory mechanism of PST-induced tissue damage in C. farreri is still lacking. This information is important for establishing and perfecting food safety risk assessment technology. In this study, C. farreri was exposed to a strain of A. tamarense (AT5-3). We measured the toxin accumulation, oxidative stress kinase activity, and its transcriptional regulation in the visceral mass of C. farreri in control and experimental groups. Further, the ultrastructure of the visceral mass in the control group and the experimental group was observed to explore the initial stress response mechanism of C. farreri exposed to PSTs. The 2-year-old scallop C. farreri was selected as the experimental animal. AT5-3 was cultured in L1 medium at temperatures of (20±1) ℃, light intensity of 54 μ/Em2·s, and a light-dark ratio of 12 h:12 h in the laboratory, and Chlorella vulgaris was cultured simultaneously. The experimental group was fed with algal solution in the exponential growth period and the cell density was 4×104–4.2×104 cells/mL. The control group was fed with the same amount of C. vulgaris. The experiment lasted 20 days, of which the first six (days 0–6) were the exposure stage and the remaining 14 days (days 7–20) were the metabolic stage. During the exposure stage, C. farreri were fed regularly with the AT5-3 strain in exponential growth period twice a day with a feeding dose of 8×106 cells/items/day. The same amount of C. vulgaris was fed in the metabolic stage. The results of toxin accumulation showed that paralytic shellfish toxins could accumulate rapidly in the visceral mass of C. farreri, but the metabolic rate was slow. The toxin content was highest on day 6 of the experiment, and the maximum accumulated content was approximately 15 times higher than the limit standard. The toxin residue reached its highest level (62.4%) on 20th day of the experiment. The results of enzyme activity tests showed that the stress due to MDA, GSH-Px, and POD in the visceral mass was significantly increase (P<0.05), and the SOD activity was significantly inhibited (P<0.05) after a brief increase. The results also showed that PSTs could induce lipid peroxidation in C. farreri, and POD, SOD, and GSH-Px were significantly stressed to eliminate the adverse effects of PSTs. The pathological changes in the visceral mass were observed under a transmission electron microscope and included vacuolation, chromatin aggregation, and nucleoplasmic pyknosis. Tissue damage worsened as the exposure time increased and although the toxin content in C. farreri decreased after the exposure period, the tissue damage was further aggravated. The results of transcriptome analysis showed that 933 differentially expressed genes (DEGs) were screened from the visceral mass of C. farreri after PST exposure. The results of KEGG and GO annotations showed that DEGs were mainly annotated in amino acid metabolism, energy metabolism, and other metabolic processes. Weighted gene co-expression network analysis showed that apoptosis and the glutathione metabolic detoxification pathway were significantly up-regulated, mapping ALOX5, AfGST-σ11, caspase-8 and Bax4 key transcription factors. In the experimental group, the expression of ALOX5 and AfGST-σ11 increased significantly when the accumulation rate was highest (P<0.05). The expression of caspase-8 and Bax4 was highest when toxin accumulation was high, which was significantly higher than that in the control group (P<0.05). In summary, PSTs can cause lipid peroxidation stress and cell damage to C. farreri. In addition to antioxidant stress, C. farreri can activate characteristic apoptosis and resist PSTs toxicity by glutathione detoxification metabolism. However, this effect is limited and the persistent damage caused by high residual PSTs cannot be eliminated. This study provides a basis for further study on the potential toxicity of PSTs to the scallop C. farreri and its immunomolecular mechanisms.

Abstract:Human norovirus (HuNoV) is a non-enveloped, single-stranded, positive-stranded RNA virus belonging to the family Caliciviridae. HuNoVs are important pathogenic microorganisms responsible for causing food safety incidents. Numerous species of shellfish are characterized by their abundant nutritional value, excellent healthcare function, and a high economic value. As a filter-feeding animal, shellfish filter the seawater at a rate of 4–20 L per hour and ingest microalgae to meet their physiological needs. Additionally, shellfish are able to continuously accumulate viruses from seawater into their bodies, resulting in a concentration ten or even thousand times higher in their tissues than in the environment. In recent years, more attention has been paid to HuNoV contamination in shellfish in China, and related monitoring studies have been conducted in different areas. However, most of these studies focused on a certain region in a certain period of time, and some species, which are not conducive to a comprehensive understanding of the overall prevalence of HuNoVs in shellfish in China. Meta-analysis refers to the methods which focus on contrasting and combining results from different studies for identifying patterns among the study results or other interesting relationships that may come to light in the context of multiple studies. This study aimed to collect cross-sectional data on the studies conducted on shellfish contamination in China. The search terms used were Norwalk virus, norovirus, shellfish, bivalves, oysters, mussels, clams, cockles, and scallops in all the databases (CNKI, VIP, CBM, WanFang Data, PubMed, Web of Science, Embase, and Cochrane Library). A total of 600 studies on HuNoV contamination of shellfish were initially included in the data. First, 303 duplicate studies were removed, leaving 297 studies remaining. One meta-analysis, 11 reviews, and 16 meeting abstracts were excluded from the first analysis. In the second analysis, abstracts of the remaining 269 studies were read by two independent reviewers, further excluding 221 studies in which experimental designs did not meet the inclusion criteria. After carefully reading the full text of the 48 studies, 11 of them were excluded. Ultimately, 37 studies were included in the final analysis. The total sample size in these 37 studies was 17 162 among which the maximum number of samples in a single study was 2 955, and the minimum was 52, and the total number of HuNoV-contaminated samples was 1 970. The meta-analysis was conducted using Stata 14.0, and the effect size was defined as the prevalence of HuNoVs (percentage). Moreover, the heterogeneity of the studies was examined using Q test (P-value) and I2. The pooled prevalence of HuNoVs in shellfish was found to be 15% with an I2 value of 97.22%, which indicated a strong heterogeneity among the 37 studies. We then grouped them based on genogroups, area, season, and species using a random effect model. As a result, GⅠ and GⅡ were found as the two most prevalent genogroups. As revealed by the results, the prevalence of contamination of GⅠ alone, contamination of GⅡ alone, and the combined contamination of GⅠ and GⅡwas found to be 3%, 10%, and 1%, respectively; both Beijing and Guangdong contributed the most with seven studies, followed by Zhejiang with five studies. In addition, there were four studies conducted each in Jiangsu and Shandong, and Fujian contributed with three studies. Two studies were conducted in Gansu, Guangxi, Hebei, Liaoning, and Shanghai. Only one study has been conducted in Hainan. The geographical location exhibited a significant impact on the prevalence of HuNoVs (P<0.01), and its prevalence in South China (Guangdong, Guangxi, Hainan), North China (Beijing, Hebei), and East China (Fujian, Jiangsu, Shandong, Shanghai, and Zhejiang) reached 19%, 17%, and 11%, respectively, while those in Northeast China (Liaoning) and Northwest China (Gansu), which were not coastal areas, were 4% and 9%, respectively. HuNoVs contamination in shellfish was found to be significantly correlated with the season. At low temperatures, the virus is more persistent, and shellfish metabolism may be inhibited. Our results showed that the prevalence of HuNoVs was the highest in winter (25%), only 10% in summer, and 16% and 12% in spring and autumn, respectively. HuNoVs may contaminate a wide variety of species during the pre-harvest or post-harvest stages. Oysters, clams, and mussels have been recognized as the most common seafood on the table and therefore these are the most investigated. The results of this meta-analysis revealed that the prevalence of HuNoVs in oysters, mussels, and clams was 16%, 10%, and 9%, respectively. The possible reasons why oysters showed the highest prevalence among all shellfish are as follows. One, the oyster farming area is located in shallow bays, which are easily contaminated by domestic sewage. Second, different oyster tissues contain HuNoV receptors (human histo-blood group antigens (HBGAs)-like carbohydrates) and protein-ligands (oyster heat shock protein 70), which can specifically bind to HuNoVs. In summary, HuNoV contamination in shellfish is common in China, and the region, season, and species exhibit significant effects on the prevalence of HuNoVs. The results of this study are beneficial for gaining insights into the HuNoV contamination in shellfish, demonstrating the importance of continuous HuNoV monitoring. Future studies should establish some effective control measures to ensure the sound growth of the shellfish industry in China.

Abstract:Owing to its high economic and nutritional value, Apostichopus japonicus is an important mariculture species in North China. Because of the rapid development of its aquaculture industry in recent years, the limitations of traditional aquaculture modes such as pond aquaculture, cofferdam aquaculture, and beach aquaculture have become increasingly prominent. Therefore, the high-efficiency and healthy northern suspension cage A. japonicus breeding model, with the best comprehensive benefits and the least management problems, came into being. Microorganisms, as an essential part of the aquaculture pond ecosystem, not only play an important role in the material circulation and energy flow of the ecosystem, but also have great significance in maintaining ecosystem balance. As a representative invertebrate, echinoderms such as A. japonicus have a simple digestive structure. Bacteria account for a large proportion of the gut microbiome of A. japonicus, providing more than 70% of their energy demand. The bacterial community is closely related to the healthy growth of the host and plays an important role in digestion and metabolism, defense against pathogens, and immune function. However, the complex bacterial community in the gut of aquaculture species depends on the culture environment, and there is a close symbiotic relationship between the environmental and gut bacterial community which affects the survival and growth of organisms, disease occurrence, and material circulation. Previous studies have shown that the complex bacterial community in the gut of A. japonicus primarily comes from their habitat and maintains a relatively stable dynamic balance with the external environmental community. In order to improve the growth capacity of A. japonicus and quality of the culture water, it is important to understand the structural characteristics of the bacterial communities of A. japonicus and their culture water to support the development of the A. japonicus aquaculture industry. This information will provide a theoretical reference for the healthy aquaculture of A. japonicus and assist with disease prevention and control. Clarifying the complex relationship between the structure and functional characteristics of bacterial communities and the aquaculture environment, as well as the important role of the bacterial community in growth, will support future research on the bacterial community mechanisms, explore ways to improve the ecological regulation of breeding yield, and promote the healthy development of the A. japonicus culture industry. To date, there have been limited studies on the correlation between the gut bacterial community structure of A. japonicus and its culture environment. At present, most of the existing studies are based on the traditional pure culture or separation and enrichment culture methods, which cannot accurately reflect natural bacterial communities. In recent years, high-throughput sequencing technology has introduced a new way to comprehensively analyze the structural and functional characteristics of bacterial communities by combining several bioinformatics methods. With the continuous development of molecular sequencing technology, 16S rRNA high-throughput sequencing technology has become a valuable tool to study the structure of bacterial communities. It has been widely used to study a variety of ecosystems and bacterial community diversity, providing a novel means to study the species diversity and quantity of bacterial communities, and the structural and functional characteristics of bacterial communities. Most of the relevant existing studies are based on the structure and diversity of the bacterial community of A. japonicus cultured in the south, whereas only a few studies have been conducted on the structure and functional characteristics of bacterial communities of A. japonicus cultured in suspension cages in the north. Therefore, in order to investigate the relationship between the bacterial community structure of A. japonicus and the culture water, this study analyzed their structural and functional characteristics in cage-cultured A. japonicus in North China using high-throughput sequencing technology, and preliminarily discussed the correlation between them. The results showed that the diversity and richness of the A. japonicus gut bacterial community were significantly higher than those of the culture water (P<0.05). The dominant bacteria in the gut of A. japonicus and the culture water were Proteobacteria and Bacteroidetes. There were 13 common core bacteria with a relative abundance greater than 0.1%. In addition, the bacterial communities showed some independence; the specific phyla in the gut belonged to Firmicutes and Chloroflexi, represented by Bacillus, Lactobacillus, Halioglobus, Lutimonas, and Woeseia. Based on an analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database, a total of 300 tertiary metabolic pathways was annotated, among which 146 tertiary metabolic pathways had highly significant differences (P<0.001). The specific metabolic pathways in the gut of A. japonicus were mainly carbohydrate digestion and absorption, protein digestion and absorption, and sphingolipid metabolism. This study showed that the bacterial community in the gut of A. japonicus is similar to that of the culture water, but there were significant differences in the relative community abundance. The results of this study provide a theoretical basis for the healthy cultivation of northern A. japonicus in suspension cages.

Abstract:The germ cell-less gene belongs to the BTB/POZ protein-family, whose structural domain plays an important role in the occurrence of mammalian gonads as it specifically combines with other DNA proteins. In this study, a unigene annotated as germ cell-less (Ajgcl) was screened from the gonadal genome of Apostichopus japonicus (sea cucumber) through homology. The full-length Ajgcl cDNA sequence was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). Subsequently, we analyzed the expression characteristic of germ cell-less in A. japonicus using several methods, including multiple sequence alignment, phylogenetic analysis, and real-time quantitative PCR (RT-qPCR). The results showed that germ cell-less attaches to the BTB/POZ superfamily. The full-length cDNA of Ajgcl was 2 316 bp, which contained a 725 bp 3′UTR, a 139 bp 5′UTR, and a 1 455 bp open reading frame (ORF), encoding a protein of 484 amino acids including a conservative BTB/POZ domain. RT-qPCR analysis revealed that germ cell-less was expressed in the ovary, testis, intestines, stomach, longitudinal muscle, respiratory tree, tube feet, and coelomocyte. Remarkably, the expression level in the ovary was the highest and 2.25 times that in the testis. Moreover, as a maternal factor, germ cell-less was expressed throughout embryonic development. During ovarian development, the expression of germ cell-less in growing stage 2 was slightly elevated, but when the ovum developed into mature stage 3, the expression level was significantly down-regulated. It was not until the end of gametogenesis that its expression level returned to its original level. The expression of germ cell-less did not change significantly throughout the developmental stages of the testis. In addition, the online tool Match was used to predict the transcription factor binding sites in the germ cell-less promoter of A. japonicus. Subsequently, a total of 12 transcription factor binding sites were projected, namely Oct-1, FOXD3, PAX-6, CRP, c-Myb and NF-1. Notably, there were four Oct-1 binding sites between -1890 and -608. The characteristics of germ cell-less gene promoter were obtained, and germ cell-less was shown to be a germ cell marker gene for A. japonicus. This study provides data for future studies related to germ cell development in A. japonicus.

Abstract:With the development of the aquaculture industry for high efficiency, energy saving, and environmentally friendly methods, recirculating aquaculture has attracted increasing attention from researchers and practitioners. A recirculating aquaculture system (RAS) consists primarily of an aquaculture module and a water purification module. In the aquaculture module, nitrogen in the feed could be released into the water phase through various routes (i.e., the feedstuff residue and aquaculture animal excretion), whose species can be transformed by microbes. Free ammonia and nitrite have acute toxic effects on aquaculture animals, rendering the efficient removal of ammonia and nitrite essential for the RAS. Compared with physical and chemical methods, biological treatment methods based on microbial nitrification can convert ammonia and nitrite into less toxic nitrate with the advantages of good treatment performance, low operation cost, and little secondary contamination, and have been widely utilized in the purification of multiple wastewaters (e.g., municipal wastewater, industrial wastewater, agricultural wastewater, and ammonia-contaminated groundwater). For the treatment of recirculating water with low and fluctuating nitrogen loads, a biofilm process based on the attached growth of microbes is more suitable than an activated sludge process based on the suspended growth of microbes. To date, a variety of biofilm processes have been developed, among which the fixed-bed biofilm reactor (FBBR) and moving-bed biofilm reactor (MBBR) have been widely investigated for the control of ammonia and nitrite in aquaculture wastewater. However, the relevant studies were mostly conducted in laboratory- and pilot-scale RASs. Thus, there is still a lack of comparative investigations of FBBR and MBBR, which are simultaneously operated in a full-scale RAS. Therefore, a parallel FBBR and MBBR were joined to a full-scale RAS for Macculochella peeli. The FBBR and MBBR were simultaneously and continuously operated for 35 d to investigate variations in their water quality and microbial community structures. The results indicate that the FBBR and MBBR had similar variations in ammonia, nitrite, nitrate, and pH in the effluents. Over the entire operational period, the dissolved inorganic nitrogen (DIN) concentrations gradually increased; the ammonia and nitrite concentrations and their proportions in DIN first increased and then decreased stepwise; and the nitrate concentrations increased gradually, while the variation in the nitrate proportions in DIN was opposite to that of ammonia and nitrite. Both the FBBR and MBBR could transform ammonia and nitrite to nitrate, which resulted in nitrate accumulation and a pH decrease in aquaculture water. During the operation period, the nitrification capacity gradually matured, and ammonia oxidation could occur prior to nitrite oxidation. At 35 d, the concentrations of ammonia, nitrite and nitrate were 0.32 (0.29), 0.27 (0.22) and 29.75 (29.76) mg/L with their proportions in DIN of 1.05% (0.96%), 0.90% (0.72%) and 98.05% (98.32%) in FBBR (MBBR) effluent; pH declined from7.62 (7.59) to 7.25 (7.22) in FBBR (MBBR) effluent. The number of operational taxonomic units (OTU) obtained from the FBBR and MBBR samples were 2,088 and 1,852, respectively, and 1,174 OTUs were shared between FBBR and MBBR. The α indices (Chao1, ACE, Shannon, and Simpson) from the biofilm reactors indicated that FBBR possessed higher richness and diversity of the microbial community than MBBR, which could be attributed to the difference in the internal environment between FBBR and MBBR. In total, 16 phyla, 28 classes, and 149 genera were identified in the FBBR samples, which were slightly fewer than those from the MBBR samples (i.e., 19 phyla, 31 classes, and 155 genera). However, the relative abundance of microbes demonstrated that FBBR and MBBR had similar predominant microbes: Proteobacteria (69.42% in FBBR and 86.92% in MBBR) at the phylum level, γ-Proteobacteria (40.71% in FBBR and 64.36% in MBBR) and α-Proteobacteria (26.58% in FBBR and 21.74% in MBBR) at the class level, and Acinetobacter (27.50% in FBBR and 53.29% in MBBR) at the genus level. Nitrosomonas and Nitrospira constituted the nitrifiers in both FBBR and MBBR, but the relative abundance of nitrifiers was higher in FBBR. Furthermore, the relative abundance of Nitrospira was far higher than that of Nitrosomonas, indicating that complete ammonia oxidation bacteria might exist in FBBR and MBBR. In addition, Vibrio was not found in FBBR and MBBR, but Bdellovibrio was observed. The results of this study can provide technical support for the selection of biological purification technology for RAS, and thus improve the green development of the aquaculture industry. Future studies will focus on: Investigating the effects of water quality conditions and operational parameters on the water treatment performance of FBBR and MBBR; Determining the species of complete ammonia oxidation bacteria and their relative abundances on the filler surface in FBBR and MBBR; Effectively eliminating nitrate in the aquaculture water by introducing microbial denitrification process after FBBR and MBBR to control the total nitrogen in RAS; Establishing a flexible and feasible strategy for controlling the pH in recirculating water by exploring the solid-phase buffers that could serve as the slow-release sources of alkalinity in the biofilm reactor.

Abstract:Seagrasses demonstrate complex ecological functions, and their morphological and physiological indicators are affected by various environmental factors. In this study, we conducted six sampling surveys of a Thalassia hemprichii seagrass bed in Xincun Bay, Lingshui, Hainan in 2019. The morphological and physiological characteristics of seagrasses in January, March, May, July, September, and November of a single year were studied, and the effects of various environmental factors on the morphological and physiological characteristics of T. hemprichii were analyzed. The results demonstrated markedly differences in leaf length and width, rhizome length and thickness, and root length across different months. The highest value [(15.05±6.13) cm] of leaf length occurred in May, and the lowest value [(7.19±2.55) cm] in September. The highest [(11.93±1.68) mm] and the lowest [(8.73±1.96) mm] values for leaf width were recorded in November and September, respectively. The highest value [(5.22± 1.71) mm] for rhizome diameter occurred in November, and the lowest value [(4.06±0.74) mm] in March. The C content of the underground seagrass tissues was significantly different across months, with the highest value (31.23%±0.94%) in November and the lowest value (24.90%±3.48%) in March. Seawater temperature had a significantly positive correlation with leaf width and C content of the underground tissues. Salinity was significantly positively correlated with the leaf and rhizome lengths of T. hemprichii and had a negative relationship with the C content of the underground tissues. There was a significant positive correlation between pH and leaf length, leaf width, and C and N content of the underground tissues. The ammonium level in seawater was significantly positively correlated with the N content of aboveground tissues. Nitrate level of seawater was negatively correlated with the C/N ratio of the aboveground parts. There was a significant positive correlation between the dissolved inorganic nitrogen (DIN) in seawater and the N content of the underground parts. Sediment organic carbon had a negative relationship with leaf length and width, and rhizome growth. The results demonstrated that the growth of T. hemprichii in Xincun Bay was greatly affected by temperature, salinity, and pH. A high nutrient level in Xincun Bay may have a negative impact on T. hemprichii. In order to protect the T. hemprichii meadow, measures should be taken to limit the input of nutrients in Xincun Bay. The results of this study may provide data support and serve as a scientific basis for the restoration of declining seagrass.

Abstract:In this study, the effects of three cultivation methods (hydroponics germination promotion + germination insertion treatment, hydroponic germination promotion + germination post-insertion treatment, and substrate cultivation treatment) on seed germination, seedling establishment, and growth of Zostera marina were studied at 15 ℃, and the cultivation costs and physiological responses of Z. marina seedlings to different cultivation methods were analyzed. The experiment lasted for 42 days. The results revealed that there were no significant differences (P>0.05) in the seed germination rate and seedling establishment rate of Z. marina between treatments of hydroponic germination promotion + germination insertion and hydroponic germination promotion + germination post-insertion. The average of the two cultivation methods reached 54.8% and 46.5%, which were 1.3 and 1.6 times higher than those under the substrate cultivation treatment, respectively (P<0.05). There were no significant differences (P<0.05) in the growth indices of Z. marina seedlings between the two cultivation methods of hydroponic germination promotion + germination insertion and hydroponic germination promotion + hydroponic germination post-insertion, but the average were significantly higher (P<0.05) than those under the substrate cultivation treatment. Specifically, the average of root production rate was 1.6 times higher than that under the substrate cultivation treatment. Principal component analysis revealed that the physiological state of Z. marina seedlings was best under the hydroponics germination promotion + substrate implantation treatment. The contributions of the total nitrogen and soluble sugar contents were greater under the two treatments with an average of 16.6% and 15.0%, respectively, which were 1.2 times greater than those under the substrate cultivation treatment (P<0.05). The analysis of cultivation costs showed that the treatment of germination promotion + germination post-insertion cultivation had the lowest cultivation costs (3.6 Yuan/plant), which was 81.9% of that under the treatment of germination promotion + germination insertion cultivation. The results showed that the hydroponic germination promotion + germination post-insertion treatment is a suitable method for cultivating Z. marina seedlings, mainly by enhancing the formation of nonstructural carbohydrates and increasing the photosynthetic pigment content of seedlings.

Abstract:In this study, two kinds of shark repellents, chemical shark repellent and electric pulse shark-repelling device were selected to test their effects on seawater quality as well as the physiology and survival of fish using simulated ecology experiments. Three marine fish species，Japanese flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegelii), and spotted grouper (Oplegnathus punctatus) were selected as test subjects. The results of water quality indicators showed that the chemical shark repellent could significantly reduce the water transparency and pH (P<0.05), while the electric pulse shark-repelling device had no significant effect. The mortality rate of the fishes under the chemical shark repellent was 100% within 15 min, while it was 0% under both the electric pulse shark-repelling device and the blank control. While investigating the effect of the two repellent methods on the survival and physical activities, the gills and fins of juvenile fish in the chemical shark repellent group were rapidly dyed blue reaching a mortality rate of 100% within 15 min, while the juvenile fish in the electric pulse shark-repelling device group resumed their natural activities rapidly except for exhibiting avoidance behavior when the device was started. The blood indices of the species did not show any significant differences in the chemical shark repellent group because of the acute mortality caused by poisoning. However, in the electric pulse shark-repelling device group, the number of erythrocytes and leukocytes significantly increased in all three fishes. In terms of blood biochemical indices, the chemical shark-repellents caused a significant increase in chloride content, blood glucose, and alkaline phosphatase (AKP) enzyme activity in the juvenile fishes, while the blood albumin content reduced significantly in all three fishes. The use of electric pulse shark-repelling device caused a significant increase in blood glucose and glutamate transaminase activity only in the Japanese flounder. Histopathological results showed that chemical shark repellents caused tissue damage to the gill, liver, spleen, and heart in juvenile fishes. It can be seen that compared to chemical shark repellents, the electric pulse shark-repelling device is a more environmentally friendly way to repel sharks as it has less impact on seawater quality as well as less toxic effects on major marine fishes. These results would provide a scientific basis for the selection of shark repellent methods in ocean exploration activities.