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Abstract:Neurokinin B (NKB) is a hypothalamic neuropeptide containing 10 amino acids and is an essential member of the tachykinin family. Based on studies in mammals, Kiss, NKB, and dynorphin A are located in the common neurons, named KNDy neurons, and NKB and Kiss can stimulate the GnRH pulses. In humans, the mutations of NKB and its receptor NK3R can lead to hypogonadotropic hypogonadism and infertility. NKB is also involved in many other physiological activities in mammals and has received increasing attention. NKB was first purified from porcine spinal cord extracts and named neurokinin B or neuromedin K. Subsequently, NKB has been identified in various species, and its encoding gene was named tac2 in ruminants and rodents, and tac3 in other mammals, birds, reptiles, amphibians, and fish. In teleosts, the NKB system was first identified in zebrafish by three laboratories in 2012; it was also found in grass carp, goldfish, Nile tilapia, European eel, orange-spotted grouper, and half-smooth tongue. As teleosts have experienced the third round of genome duplication (3R), the bony fish have two forms of tac3 genes, namely tac3a and tac3b. However, only tac3a is present in highly evolved fish species, such as Nile tilapia and orange-spotted grouper, whereas the tac3b gene has been lost. tac3 in fish and amphibians can encode two mature peptides, NKB and an NKB-related peptide (NKBRP), whereas there is only NKB in mammals, birds, and reptiles. NKB and NKBRP sequences are highly conserved, and sequence analysis showed that NKB and NKBRP share an identical -FXGLM motif at the C-terminus, and X is a hydrophobic or aromatic amino acid residue, which plays an important role in binding to homologous receptors. NKB exerts biological effects by activating the endogenous receptor NK3R. The NK3R receptor belongs to the G protein-coupled receptor family and has a typical seven-transmembrane structure. NK3R is encoded by the tacr3 gene. There are two tacr3 subtypes in teleosts: tacr3a and tacr3b. There was an additional gene, called tacr3a2, in the zebrafish and grass carp, and it was produced by local genome duplication of tacr3a1. The tissue distribution shows that tac3 and tacr3 are widely expressed in the central nervous system and peripheral tissues, indicating that they have essential physiological functions. The specific expression patterns vary depending on the species. They are mainly expressed in the brain, with high expression levels in the pituitary, intestine, and gonads. NKB may participate in regulating reproduction and feeding in teleosts. Currently, studies in teleosts mainly focus on reproductive and feeding regulation. Due to the existence of multiple forms of tac3 and tacr3 genes in teleosts, the action of the NKB system on reproduction control is more complex. Using different experiment methods, such as intraperitoneal injection, intramuscular injection, and incubation of pituitary cells or pituitaries, NKB affected the expression of gnrh, kiss, lhβ, and fshβ and the secretion of LH, FSH, and E2 in fish. The physiological effects varied depending on the gonadal development stages, species, sex, treatment methods, treatment time, and dose. In addition, NKB can act as an anorectic peptide to inhibit food intake and promote gastrointestinal motility. It could also affect the expression of growth-related genes. In summary, as a neurotransmitter or neuromodulator in the central nervous system and a major member of the brain-gut peptides, the NKB system plays an important role in teleosts. Tachykinins activate receptors by coupling to Gαs and Gαq proteins, transducing its signals via PKA and PKC pathways. NKB and NKBRP could activate the PKA/PKC pathway via cognate receptors in some teleosts. This could be verified because different pathway inhibitors could block the effects of NKB and NKBRP. Upon binding to NK3R, NKB induces the secretion of related neuropeptides and the expression of related genes through the AC/cAMP/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II cascades. The C-terminal motif of NKB is crucial to binding receptors; once it is changed, NKB cannot activate the downstream signaling of NK3R, indicating that the integrity of the NKB/NK3R system is essential for the normal functioning of NKB. There are many deficiencies in the research regarding the function of NKB in teleosts. The specific effects and mechanisms of regulating reproductive endocrinology remain unclear, and the functional and signaling interactions between NKB and other neuroendocrine factors such as Kiss, GnIH, and GnRH require further study. In addition, the physiological functions of NKB in fish are mainly focused on reproductive regulation, with less attention given to feeding regulation and other physiological effects. In conclusion, this review provides a summary of the research progress on the NKB system in teleosts, including the identification, tissue distribution, physiological functions, and signaling mechanisms of NKB and its receptors, to enhance the understanding of the NKB system in fish and provide a reference for future research.

Abstract:Epigenetics refers to heritable changes that do not affect DNA sequences. Compared to genetic changes, epigenetic changes affect gene expression and protein products in cells, and these changes are reversible and dependent on the environment. There are three major types of epigenetic changes: DNA methylation, histone post-translational modifications (PTMs) increase the functional diversity of the proteome through the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of whole proteins), and non-coding Ribonucleic Acid.. This study focused on post-translational histone modifications. There are five main histone types: H1/H5, H2A, H2B, H3, and H4. Genes encoding histones do not contain introns and are among the most conserved proteins in eukaryotes. Histones are basic structural proteins comprising eukaryotic chromosomes. Generally, two molecules, H2A, H2B, H3, and H4 form a histone octamer that combines with DNA to form a structural unit called a nucleosome. This nucleosome appears every 200 bp and is connected by H1 histones to form chromatin. Histone modification refers to the addition of functional groups to histone tails, most commonly lysines. This process regulates gene expression by altering chromatin structure through condensation and depolymerization. Additionally, histone modification creates binding sites for various proteins. Histone modifications reported in animals include methylation, acetylation, phosphorylation, ubiquitination, SUMOylation (which is a small ubiquitin-related modifier involved in post-translational modification of proteins), ADP-ribosylation (which is a small ubiquitin-related modifier involved in post-translational modification of proteins), and short-chain lysine acylation. Many studies have shown that chromatin remodeling is a key step in spermatogenesis, involving the transformation of histones to protamines. Briefly, protamine replacement requires (Ⅰ) histone PTMs to promote the opening of histone-based chromatin structures, especially histone hyperacetylation and incorporation into histone variants; (Ⅱ) binding of bromine domain proteins to acetyl residues and remodeling of chromatin; (Ⅲ) formation and repair of DNA strand breaks in chromatin remodeling; and (Ⅳ) incorporation of protamine. Herein, we focused on Process (Ⅰ). In bisexual reproduction, sperm, as a paternal information carrier, is a key factor in a species continuation. Spermatogenesis includes various stages, including spermatogonia, primary and secondary spermatocytes, round sperms, and mature sperms. During round sperm transformation into mature sperm, chromatin remodeling occurs and cell morphology undergoes dramatic changes, in which histone PTMs and variants are essential. Histone PTMs patterns affect gene expression over a wide range, such as methylation, which is mainly related to gene expression activation or inhibition; acetylation, which activates transcriptional activity and participates in histone deposition and DNA repair; phosphorylation, which promotes post-transcriptional modification or participates in DNA double-strand break repair; and ubiquitin, which regulates various protein substrates in different cellular pathways. Histone variants have special functions in regulating chromosome structure. For example, histone H1 variants inhibit transcription during differentiation, histone H2A and H2B variants play a unique role in sperm chromatin packaging, H3.3 is the most important variant of H3, which is expressed in all stages of the cell cycle and participates in chromosome formation outside the S phase, Histone H4 is one of the slowest evolving proteins, and no histone variant has ever been found. Focusing on post-translational histone modifications, this study reviews the latest progress in methylation, acetylation, phosphorylation, and ubiquitination. Subsequently, the histone variants and their functions in chromatin remodeling are summarized. Finally, using Cynoglossus semilaevis as an example, this study briefly introduces the implications of these studies on spermatogenesis in aquatic animals. Elucidating the effect of PTMs on spermatogenesis will aid in exploring the regulatory mechanism of specific sperm (W-type) absence, which expands the fundamental theory of reproductive biology and provides novel solutions to monosex fry cultivation in aquaculture.

Abstract:Water temperature is one of the most important ecological environmental factors affecting fish survival and growth. When the water temperature is higher than the optimal temperature for fish survival, fish homeostasis may be disrupted. The heart plays a crucial role in maintaining vertebrate homeostasis. In this study, TMT and LC-MS/MS were performed to analyze differentially expressed protein (DEP) profiles in the hearts of turbot under high-temperature stress to explore the key proteins and metabolic pathways related to the high-temperature tolerance traits of turbot. Ninety turbot (mean weight 23.01±3.63 g, mean length 10.34±0.76 cm) were used in experiments. Three temperature points for heat stress experiments were designed: control=(14.0±0.5)℃, HT1=(20.0±0.5)℃, and HT2=(28.0±0.5)℃. Three parallel groups were established for each temperature group, with 10 turbots placed in each group. Fold Change≥1.2 or < 0.83, P < 0.05 was used as a criterion to screen for DEPs in turbot. Through mass spectrometry analysis of heart tissues from turbot, 85,731 spectra were identified, with 35,434 peptides, of which 31,704 were unique, and 4,765 proteins were identified. The HT1/control group in the heart of turbot had a total of 87 differentially expressed proteins, including 77 upregulated and 10 down-regulated proteins; the HT2/control group in the heart of turbot had 171 DEPs, including 149 upregulated and 22 down-regulated proteins; the HT2/HT1 group in the heart of turbot had a total of 188 DEPs, including 130 upregulated and 58 downregulated proteins. Through bioinformatics analysis, the DEPs between the 28℃ group and the 14℃ group, as well as the 28℃ group and the 20℃ group, were mainly enriched in KEGG pathways related to fatty acid metabolism, inflammation, and immune defense. Multiple proteins in the heat shock protein family (HSP90B, HSP70, etc.) were significantly upregulated in the 28℃ group compared to the 14℃ group. The protein interaction network revealed that HSP90B exhibited the highest correlation. A new discovery is that the expression levels of fibrinogen-related proteins (FGA, FGB, and FGG) increased, and their role in the thermal response requires further research. These results indicate that extremely high temperatures may cause fatty acid metabolic disorders in turbot, leading to inflammatory reactions. The upregulation of heat shock protein family proteins may have a positive effect on alleviating stress symptoms under heat stress. This study identified DEPs, such as HSP90B, HSPA5, and HYOU1, which were significantly upregulated under heat stress, as candidate proteins for high-temperature tolerance breeding in turbot and provided a theoretical basis for further elucidating the molecular mechanism of heat stress in turbot.

Abstract:Turbot (Scophthalmus maximus) is one of the most economically important fish in the northern coastal areas of China. Recently, low salinity stress is an important factor influencing the development of the turbot industry. Lately, the industry has faced challenges posed by low salinity stress, a key factor influencing turbot development. Salinity fluctuations represent a crucial environmental stressor in aquaculture, impacting osmotic pressure and inducing abnormal energy metabolism. Our preliminary research had identified that low salinity stress contributes to lipid metabolism disorders in turbot. This study aims to investigate the impact of varying lipid concentration levels on the lipid metabolism disorders induced by low salinity stress. To achieve this, feeding experiments were conducted with different lipid concentration levels under low salinity stress conditions, determining the optimal lipid requirement for turbot to cope with reduced salinity. Three salinity gradients (10, 20, and 30) were employed, with four lipid concentration gradients (8%, 12%, 16%, and 20%) for each salinity group. The results revealed that at a lipid concentration of 16%, no significant difference in the growth performance of juvenile turbot was observed between the salinities of 20 and 30. However, the growth performance under a salinity of 10 was lower than that under salinities of 20 and 30. Notably, under a salinity of 10, the growth performance of juvenile turbot increased with higher lipid concentrations, reaching its peak at 20%, surpassing values under the salinities of 20 and 30. Regarding the expression of genes related to lipid metabolism, under the salinities of 10 and 20, the expression levels of genes related to lipid synthesis, lxrα, cyp7a1, and srebp-1, showed a trend of first increasing and then decreasing with increasing lipid concentration, particularly under the salinity of 10. The expression level of the 12% lipid group was higher than that of other groups and the expression level of the 20% lipid group was significantly lower than that of other groups (P<0.05). In addition, under a salinity of 10, the expression levels of acc and fas also showed a similar situation. They were both suppressed in the 20% lipid group, and the expression levels were higher in the 12% lipid group. The expression level of the gene apoa-IV related to lipid absorption in the 20% lipid group under a salinity of 10 was markedly higher than that in other groups under the same salinity conditions (P<0.05) and it showed a change with salt under the condition of 20% lipid concentration. The increasing and decreasing trends were contrary to the trends in other lipid groups. The aforementioned results show that after low-salt stress affects lipid metabolism, feeds with different lipid concentrations can alleviate the adverse effects of the stress from the perspectives of lipid synthesis and lipid absorption, and this mitigation effect is more reflected at the level of lipid absorption, thereby improving growth performance under low salt stress. The results reveal the adaptability of fish to low salt from the perspective of salinity affecting lipid metabolism, enrich the concentration of fish stress physiology, and provide theoretical and technical support for the breeding of low-salt tolerant turbot varieties.

Abstract:The investigation of fish bioenergetics primarily relies on using an energy budget equation to examine how the allocation of energy and nutrients in their diet impacts their growth rate and reproductive capabilities. By accurately forecasting the distribution of feeding intake energy towards growth, fecal excretion, and metabolism within the overall energetic framework of a fish, and considering diverse physiological factors or ecological influences that affect each aspect of this energetic balance sheet, scientific discoveries derived from these studies can provide valuable insights for making informed decisions regarding optimal dietary choices to enhance fish well-being. Additionally, it aids in refining rearing management strategies leading to improved feed utilization efficiencies, ultimately resulting in more sustainable aquaculture practices aimed at minimizing environmental pollution caused by excessive feeding activities. Temperature affects various physiological processes and growth potential, thereby, influencing feed requirements and utilization efficiency, which ultimately determine the allocation of feeding intake energy among growth, fecal, excretory, and metabolic energies. Extensive studies have been conducted to explore how temperature influences fish growth rates along with their overall energy budgets. The effects of temperature on the growth and energy budgets in triploid turbot, Scophthalmus maximus, were investigated in this study by establishing a series of five temperature gradients (13 ℃, 16 ℃, 19 ℃, 22 ℃, and 25 ℃). Measurements of the growth and energy budget allocations were conducted on large-sized triploid turbot juveniles with mean body weight of (120.24±17.20) g at the aforementioned temperatures for 49 d. The results indicate that under conditions of salinity 28.6, pH 7.8, dissolved oxygen content above 7.8 mg/L, light intensity of 300 lx, and a light period of 16 L∶8 D ratio; the feeding rates (FR) and weight gain rates (WG) of triploid juveniles initially increased with increasing temperature before declining, with the peak FR of (1.02±0.06)% and (0.95±0.04)% observed at 19 ℃ and 22 ℃, respectively, whereas the highest WG was recorded at 19 ℃ (62.17±3.10)% (P<0.05). The relationship between specific growth rate (SGR) or feed conversion efficiency (FCE) and temperature, calculated based on different parameters including wet weight (SGRw and FCEw), dry weight (SGRd and FCEd), protein content (SGRp and FCEp), and energy content (SGRe and FCEe), was observed to follow a quadratic regression equation. The highest recorded temperature for the SGR values was observed at 19 ℃. At 25 ℃, the group exhibited significantly reduced levels of SGRs compared to that of other experimental groups, except for the data recorded at 13 ℃ (P<0.05), where 13 ℃ and 25 ℃ showed relatively low SGR values. Additionally, no significant differences were observed in SGR values between 16 ℃ and 22 ℃. The maximum FCE values were also attained at 19 ℃ (P<0.05). In contrast, the FCE values recorded at 13 ℃ and 25 ℃ exhibited considerable reductions when compared to those obtained from other experimental groups (P<0.05). Regression analysis revealed that the optimal temperatures for SGRw, SGRd, SGRp, and SGRe were determined as 18.4 ℃, 18.7 ℃, 18.9 ℃, and 18.5 ℃, respectively, whereas the temperatures for achieving maximum FCEw, FCEd, FCEp, and FCEe were 18.1 ℃, 18.6 ℃, 18.9 ℃, and 18.2 ℃, respectively. Additionally, the rates of nitrogenous excretion and fecal production exhibited an initial decrease followed by an increase with increasing temperature, reaching their highest levels at 25 ℃. Dry matter, protein, and energy apparent digestibility coefficients at 25 ℃ were significantly lower than those observed at other temperatures. The feeding intake energy and growth energy proportion demonstrated an inverted "U" shape with increasing temperature, reaching its highest value at 19 ℃. The allocation of energy in triploid juveniles was primarily dominated by growth and metabolism energies, whereas the proportion of metabolism energy to feeding intake energy displayed a "U" shape trend with increasing temperature. The proportion of growth energy to feeding intake energy ranged from 17.86% to 34.58%, whereas the proportion of fecal energy varied from 4.12% to 5.57%. Furthermore, the proportion of excretion energy to feeding intake energy ranged from 6.61% to 9.19%, and metabolism energy accounted for 54.69% to 67.38% of feeding intake energy. The energy budget equation of triploid turbot juvenile at 19 ℃ was 100.00 C (feeding intake energy) = 34.58 G (growth energy) +4.12 F (fecal energy) +6.61 U (excretion energy) +54.69 R (metabolism energy) or 100.00 A (assimilated energy) = 38.74 G+61.26 R. The triploid turbot juveniles exhibited an energy allocation pattern characterized by high growth efficiency and low metabolic consumption. Consequently, the optimal rearing temperature for triploid turbot juveniles with an average weight of 120 g ranged from 18.1–18.9 ℃. These findings offer valuable insights into optimizing aquaculture conditions, improving feed efficiency, and controlling water environment pollution in the context of triploid turbot farming.

Abstract:The waters adjacent to the Changdao Islands are a key gateway for fish recruitment and 0-year-old juveniles migrating between the Yellow and Bohai seas, and there is a lack of local observations of early life history characteristics of fish in this area. To investigate monthly variations in the abundance, community structure, and functional diversity of early life resources of fish in waters adjacent to the Changdao Islands, monthly surveys were conducted from March to December, 2021. This study examined the spatiotemporal patterns of the early life resource abundance of fish in waters adjacent to the Changdao Islands using data on fish eggs, larvae, and the environment, and analyzed monthly variations in community structure and functional diversity. Spatial interpolation, Garrison centroid, and similarity percentage analysis methods were employed, along with functional trait indices. A total of 36 early life fish resource species comprising 21 separate species of fish eggs and larvae were collected from waters adjacent to the Changdao Islands between March and December 2021. The total quantity of fish eggs was 71,695, and that of larvae was 1,161. The main spawning period was from May to August. The main spawning species were Engraulis japonicus, Nibea albiflora, Saurida elongata, Acanthopagrus schlegelii, Sillago japonica, Pennahia argentata, Callionymus beniteguri, and Cynoglossus joyneri. The dense area of fish eggs was situated in the waters adjacent to the Miaodao Strait, extending from the eastern to southwestern waters of the study area. The main distribution period of fish larvae was from May to July and from November to December. The main larval species were E. japonicus, Hypoatherina valenciennei, Hexagrammos otakii, and Ammodytes personatus. The dense area of fish larvae was situated in the eastern, southwestern, and northwestern waters of the study area. The richness, evenness, and dispersion indices of early life resources community structure and functional diversity of fish were highest during the spring-summer season (May to July). In the northeast and southwest regions of the study area, there were abundant early life fish resource species with high functional richness and divergence, and moderate evenness. Local fish communities have strong resistance to disturbance, and the recruitment of fish transported from this area can occupy a large ecological niche in spawning grounds, thereby promoting the ecological resilience of early life fish resource communities. In the summer-autumn (August to September) and autumn-winter (October to December) seasons, as the spawning season of migratory fish comes to an end, there was a significant decline in the number of early life fish resource species. This led to a reduction in the ecological niche space and a decrease in the functional diversity of early life fish resource communities in the waters adjacent to the Changdao Islands. In conclusion, the abundance, community structure, and function of fish eggs and larvae in the waters adjacent to the Changdao Islands exhibited significant spatiotemporal heterogeneity. The migration of reproducing fish populations also appeared to have a profound effect on the early life fish resource communities in the waters adjacent to the Changdao Islands. Community structure is mainly influenced by the migration of spawning herring, and the dominance of warm-water species, such as Cynoglossus joyneri and Sillaginidae species, increases in the summer. This indicates that the succession pattern of early life fish resource communities in the waters adjacent to the Changdao Islands shares common characteristics with the spawning grounds in the central-northern Yellow and Bohai seas. In addition, compared with seawater spawning grounds with intact coastlines, large water depth gradients, and relatively stable currents, the dense areas of early life fish resources in the waters adjacent to the Changdao Islands during the warming period in the spring-summer season are relatively scattered. The stability of the community is greatly affected by the spawning and migration of fish, and there is a significant difference in the spawning intensity of recruitment groups. As an area adjacent to island groups in the Yellow and Bohai seas and as a migratory channel for fish, the early life fish resources in this region have distinct features. The abundance and diversity of early life fish resources in this area may reflect the characteristics of local communities, as well as the stability of new communities formed by recruitment groups transported from this area to spawning grounds. Therefore, mastering the annual succession pattern of early life fish resource communities in waters adjacent to the Changdao Islands is conducive to an overall understanding of the early life history of fish in the central-northern Yellow and Bohai seas, and facilitates a deeper understanding of the fish population recruitment process in adjacent waters of temperate island ecosystems. Research on the early life history of fish in the waters adjacent to the Changdao Islands should not be limited to a traditional spawning site perspective. The intermediary centrality of this area in the fish migration network of the Yellow and Bohai seas is also worth highlighting.

Abstract:The waters of the Changdao Archipelago are located at the confluence of the Yellow and Bohai seas, which have high species diversity and spatial and temporal heterogeneity. This area is an important route for migratory fish and macroinvertebrates to enter the Bohai Sea for spawning or to leave the Bohai Sea southward for overwintering. In recent decades, with increasing human activities, fishing pressure, and ocean pollution, the ecosystems of the Yellow and Bohai seas have become fragile with many fishery species evolving towards miniaturization and precociousness. Important fishery resources are unable to fulfill the minimum requirements during the fishing season, and the support function of the Yellow and Bohai seas is diminishing. Research on fishery species in the waters of the Changdao Archipelago is less focused on their composition, community structure, and fishery biological health evaluation. To understand the species composition and diversity of demersal fishery species in the waters of the Changdao Archipelago and to identify keystone species that play important regulatory roles in the biological community, we analyzed the biological diversity and identified the dominant species based on the Changdao Archipelago fishery resource survey data from March to December 2021. Keystone species were determined by food web topology and social network analysis, and the fishery biological health status was determined based on the 2021 survey data. A total of 38 species of fish (mainly warm-water, demersal, and low-value fish) and 50 species of invertebrates (mainly small crustaceans) were caught in the area. There were obvious seasonal variations in the composition of the dominant species, with invertebrates, such as Alpheus japonicus and Oratosquilla oratoria, dominating in the spring, Chaeturichthys stigmatias and O. oratoria in the summer and autumn, and only A. japonicus and C. stigmatias in the winter. The species Margalef richness index (R) was highest in the summer, and the Shannon-Wiener diversity index (H´) and Pielou evenness index (J´) were highest in the autumn, in the waters of the Changdao Archipelago. To further investigate the food web topology of the bottom fishery community in the waters of the Changdao Archipelago, and to identify keystone species, seasonal changes in the topology of the study area were analyzed. Based on field survey data, the main species (IRI > 100) were constructed. The area contained 13–23 main species (S) and 32–113 feeding relationships (L) in four seasons. The node density (Dd) ranged from 0.18–0.41, link density from 2.46–5.65, connectance (C) from 0.18–0.28, clustering coefficient (Cl) from 0.334–0.401, and weighted clustering coefficient (W-Cl) from 0.276–0.373, consistent with the interspecific feeding relationships in communities under natural conditions. There were significant seasonal variations in each topological index. Based on the topological structure index, we analyzed the robustness of keystone species at the bottom of the food web in the waters of the Changdao Archipelago. Using different removal methods, we found that these three species have differences in maintaining the robustness of the food web. To study the importance of keystone species, species were removed in ascending and descending order according to the node degree (D). Under the descending order of removal, the stability of the food web showed a downward trend, showing a “staged” change of first increasing and then decreasing. This change may be due to the reconstruction of the food web caused by the loss of some species. Even if new feeding relationships are created among other species, the number of corresponding feeding relationships decreased exponentially, while feeding relationships with ascending removal patterns showed an inverted U-shaped trend with a significantly slower rate of decline. The most important species in the food web were removed first in descending order, which had the greatest effect on the stability of the food web and the number of feeding relationships. Among them are species that play important roles in the robustness of the food web—C. stigmatias, Pennahia argentata, A. japonicus, and O. oratoria are both dominant and keystone species. The keystone species in the waters of the Changdao Archipelago are diverse and small, as most of the bottom organisms in the area are omnivores with diverse feeding relationships and include many migratory species in addition to native species, which leads to the diversity of keystone species—C. stigmatias, P. argentata, A. japonicus and O. oratoria are both dominant and keystone species. The keystone species are characterized by diversification and miniaturism. When keystone species were removed from the food web, the linkage robustness and number of feeding relationships decreased significantly, indicating that keystone species play important roles in maintaining the stability of the food web. In winter, keystone predator species change from O. oratoria and P. argentat to C. stigmatias, and A. japonicus changes from the keystone prey species to the keystone intermediate species. Niche replacement of keystone species leads to the simplification of the bottom of the food web and, to some extent, increases the vulnerability of the bottom community structure in the waters of the Changdao Archipelago. Therefore, in future studies, dynamic monitoring and protection of keystone species should be strengthened to maintain their robustness function in the food web, to maximize the protection of natural resources in the waters of the Changdao Archipelago.

Abstract:Integrated multi-trophic aquaculture has attracted widespread attention and is considered an effective way to purify the water environment and improve the sustainability of aquaculture. However, the biodeposition produced by large-scale, high-density raft-type shellfish and seaweed aquaculture activities has been shown to be the main reason for the enrichment of organic matter in sediments. Organic matter enrichment changes the biogeochemistry of sediment, which is the main reason for hypoxia in the benthic environment. Especially in the summer, water column stratification in terms of water and salinity can lead to hypoxia in the benthic environment. Besides, the hypoxic conditions in sediment promote anaerobic metabolism and sulfate reduction, which increase the dissolved sulfide concentration in the interstitial water. It was reported that dissolved oxygen (DO) in the sediment is depleted within a range of a few millimeters to a few centimeters in surface sediments. The diffusion boundary layer (DBL), which generally varies between a tenth of a millimeter and a few millimeters above the sediment, greatly influences substance exchange across the sediment-water interface. Current studies on the environmental effect of aquaculture mainly focus on the influence of aquaculture activities on the environment, and the influence mechanism of aquaculture activities on the sediment microenvironment remains unknown. Therefore, it is necessary to study the influence of aquaculture activities on the sediment microenvironment and to explore the influence mechanism of aquaculture activities on the distribution of physical and chemical factors in the sediment microenvironment. In this study, microscale variations in the distribution and consumption of oxygen at the sediment-water interface under high temperature conditions was investigated in the summer of 2022 in Sanggou Bay, a typical integrated multi-trophic aquaculture area in northern China. A high-resolution microelectrode system was used to investigate the profile distributions of DO, hydrogen sulfide (H2S), and pH in sediments with a spatial resolution of 1 mm collected from the shellfish monoculture area (SF), shellfish and seaweed polyculture area (SF-SW), seaweed monoculture area (SW), and offshore area (OF). The thickness of the DBL of different aquaculture areas was determined according to the DO distribution at the sediment-water interface and the inflection point of the profile distribution. Sediment cores were sliced at a thickness of 1 cm and the profile distribution of sediment organic carbon, the median grain size, and other factors in the surface sediment in different aquaculture areas were measured. The diffusive oxygen flux at the water-sediment interface was calculated from the gradient of the DO concentration in the DBL, and the sediment oxygen consumption rate in different aquaculture areas was calculated from the DO profile distribution in the sediment. The results showed that the mean thickness of the DBL in the SF, SF-SW, SW, and OF areas were (1.5±0.3), (1.0±0.3), (2.0±0.8), and (1.3±0.2) mm, respectively; there was no significant difference between different aquaculture areas. The mean sediment oxygen penetration depth (OPD) was (12.49±1.59), (12.17±0.09), (15.49±0.79), and (14.87±1.27) mm, respectively. The sediment OPDs in the SF-SW and SF areas were significantly lower than those in the SW and OF areas. The maximum concentrations of H2S were (5.73±0.04), (4.80±0.08), (3.30±0.19), and (3.97±0.38) μmol/L, respectively. The mean diffusive oxygen flux in the SF, SF-SW, SW, and OF areas were (24.10±1.89), (49.53±10.24), (26.69±13.13), and (24.79±7.95) mmol/(m2·d), respectively. The diffusive oxygen flux in the shellfish and kelp polyculture area was significantly higher than that in other areas. The diffusive oxygen flux at the sediment-water interface was affected by the DO concentration of the overlying water and the sediment oxygen consumption rate. The diffusive oxygen flux in the SF area was influenced by the thickness of the DBL, the DO concentration of the overlying water, and the DO concentration at the sediment-water interface. The main factor influencing the diffusive oxygen flux in the SW and SF-SW areas was the DO concentration of the overlying water. In conclusion, the biodeposition produced by aquaculture activities significantly affects material cycling in surface sediments, which promotes sediment oxygen consumption and the production of hydrogen sulfide. The influence of biodeposition produced by shellfish farming on the sediment chemical characteristics and microprofiles may be stronger than that of kelp debris. The DO concentration of the water environment is the main reason affecting organic matter mineralization in the sediments of aquaculture areas. This study also provides scientific and technological support for an in-depth understanding of the influence of aquaculture on the sediment microenvironment and the benthic carbon cycle.

Abstract:Sierra Leone is located on the west coast of Africa, and owing to its unique geographical location and climate, it has relatively suitable hydrological conditions on the continental shelf for providing ideal habitats and breeding grounds for many fish species. The fisheries play a crucial role in Sierra Leone's economy. Sphyraena guachancho is a critical economic fish and a significant protein source for coastal livestock due to its wide distribution, excellent meat quality, low price, and easy capture. This study focused on the basic biological characteristics of S. guachancho, providing a reference for further in-depth research and the rational development of fishery resources. The relationship between body length and weight is an essential aspect of fisheries biology research, and obtaining the accurate and appropriate body length and weight relationship is crucial for fisheries management. It can provide information on the size and body shape of a fish, allow the estimation of the average weight of fish of a certain length in a population, indicate the health status of a fish, allow comparison of the growth status of the same fish species in different regions, and provide vital information for fish breeding research. The sex ratio provides information on the composition of males and females in a group and indicates the dominant gender. This is the basic information necessary for understanding fish reproduction and conducting a population assessment. A model for predicting the quantity and quality of food required by fish based on feed levels and satiety provides an ecological basis for rationally utilizing aquatic feed resources, improving aquaculture efficiency, and investigating fish populations. In this study, 2,135 S. guachancho were sampled from six scientific expeditions (September, October, December, January, April, and May) from 2019 to 2021, and their basic biological characteristics, such as body length and weight composition, sex ratio, gonadal maturity, body length at initial sexual maturity, food intake level, and plumpness were studied. The results showed that the body length range of the S. guachancho was 50–845 mm with a single peak distribution. That for the advantage group was 175–345 mm, accounting for 74.52%. The weight range was 0.9–4,000.8 g, and that for the advantage group was 25–225 g, accounting for 71.33%. The relationship between the body length and weight was W=3.339×10-5L2.758, indicating negative allometry growth. The male-to-female ratio was 0.56:1, which is significantly different from 1:1. The maturation of the gonads was mainly in stages Ⅱ and Ⅲ, with a relatively small proportion of individuals in stages Ⅴ and Ⅵ. The body length of male and female individuals at initial sexual maturity was (269.982±2.340) mm and (259.846±3.397) mm, respectively. The feeding level was mainly level 0, and as the body length increased, the proportion of fish with an empty stomach gradually increased. The plumpness range was between 0.59 and 1.25, with an average value of 0.86±0.02. There were certain differences in fertility levels between different months. As the body length increased, the plumpness showed a trend of first increasing and then decreasing. The statistical analysis results showed significant differences in the body length and mass distribution between male and female populations of S. guachancho (P<0.01). The body length and mass distribution in different months differed significantly (P<0.01). The body length and body mass distribution in different seasons also had highly significant differences (P<0.01). The body length significantly affected gonadal maturity (P<0.01). Below a certain body length, there was no significant linear correlation between the month and gonadal maturity (P>0.05). The body length significantly affected the feeding intensity of S. guachancho (P<0.01), whereas the month had no significant impact on this (P>0.05). The maturity of the gonads significantly affected the level of food intake (P<0.01). The body length significantly affected the level of fullness (P<0.05), whereas the month had no significant impact on this (P>0.05).

Abstract:Stress is a nonspecific response that occurs when the body is stimulated by internal and external stimuli. When the stress levels exceed the body´s tolerance threshold, physiological and biochemical changes occur that affect homeostasis within the internal environment. Takifugu fasciatus is an important cultured fish species in China, and its demand in the aquatic market has increased in recent years. Stress is extremely harmful to T. fasciatus farming, and cortisol is considered an essential marker for determining the stress response of fish, as it changes significantly within the organism under stress. The intestine is a contact medium between fish and the external environment and can respond to physiological stress levels. Although the study of fish intestinal responses to stress is a global research focus, the role of the T. fasciatus intestinal tract in response to stress remains unclear. In this study, intestinal epithelial cells of T. fasciatus were cultured using four different methods: Trypsin (0.25%) with DMEM, trypsin (0.25%) with RPMI 1640, typeⅠcollagenase (1 mg/mL) in DMEM, and typeⅠcollagenase (1 mg/mL) in RPMI 1640 to establish an in vitro culture method for primary intestinal epithelial cells of T. fasciatus. The effects of different concentrations of cortisol on oxidative stress, apoptosis, and lipid metabolism were investigated. The study methodology involved disinfecting of T. fasciatus with 75% alcohol and subsequently anesthetizing with MS-222. Furthermore, the intestines were removed and cleansed using PBS containing 100 IU/mL penicillin and 100 μg/mL streptomycin. The intestines were then sectioned into 1.5 mL centrifuge tubes with digestive solutions and digested at 28 ℃ for 30 min. Cells were subsequently centrifuged at 1 000 r/min for 5 min at 4 ℃, and resuspended in a new complete medium (DMEM containing 20% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin). The proliferation rate of intestinal epithelial cells under the four culture methods was determined within 5 d using the CCK-8 method, and cell growth was observed under a microscope after 24 h of culture. Cortisol solutions at varying concentrations were diluted with complete medium, and the cell viability of T. fasciatus was measured using the CCK-8 method after cortisol treatment (0, 100, 1 000, 2 000, 3 000, and 5 000 nmol/L) for 24 h. The morphological structure of intracellular mitochondria was observed through transmission electron microscopy after cortisol treatment (0, 10, 100, and 1 000 nmol/L) for 24 h. After cortisol treatment (0, 10, 100, and 1 000 nmol/L) for 3, 6, 12, and 24 h, the expression patterns of oxidative stress-related genes, apoptosis-related genes, and lipid metabolism-related genes in intestinal epithelial cells were measured by real-time fluorescence-based quantitative PCR (qRT-PCR). Cell apoptosis was measured using the Annexin V-FITC/PI method, and the contents of triglycerides, total cholesterol, and free fatty acids were measured using kits from Nanjing Jiancheng Co. The results showed that the intestinal epithelial cells cultured with trypsin (0.25%) digestion and DMEM had the highest cell proliferation rate among the four culture methods, with predominantly fibroblast-like cell morphology and the best apposition. At a cortisol concentration of 1 000 nmol/L, the intestinal epithelial cell viability was 0.8 times that of the control group, with no significant difference between the 1 000 nmol/L cortisol-treated group and the 2 000 nmol/L cortisol-treated group. However, the cell viability significantly decreased when the cortisol concentration exceeded 2 000 nmol/L (P<0.05). Thus, 1 000 nmol/L or lower was selected for cortisol treatment. Transmission electron microscopy revealed that the mitochondrial structure of intestinal epithelial cells was altered in all the cortisol-treated groups, with an increase in the number of mitochondria. The expression of oxidative stress-related genes (sod, cat, and gsh-px) and apoptotic index significantly increased with increasing cortisol treatment concentration and time. Pro-apoptotic genes (caspase-3, caspase-7, caspase-9, bax and p53) increased significantly, while anti-apoptotic gene bcl-2 expression decreased significantly with the increase of cortisol treatment concentration (P<0.05). The expression of lipid synthesis-related genes (g6pd, 6gpd, pparγ, fas and acc) decreased significantly and lipolysis-related genes (hsl, pparα, lpl and cpt-1) increased significantly with increasing cortisol treatment concentration (P<0.05). Triglyceride and total cholesterol contents decreased significantly, whereas the free fatty acids content increased significantly with increasing cortisol treatment concentration and time (P<0.05). These results indicate that trypsin (0.25%) digestion with DMEM is optimal for culture of primary intestinal epithelial cells of T. fasciatus. Cortisol promotes oxidative stress, apoptosis, and lipid decomposition but suppresses lipid synthesis in intestinal epithelial cells of T. fasciatus. This study established the optimal isolation and culture method for primary intestinal epithelial cells of T. fasciatus and investigated the mechanism of the intestinal response to cortisol stress in T. fasciatus, thus providing a theoretical basis for the subsequent anti-stress culture of fish.

Abstract:Megalobrama amblycephala, which belongs to the genus Megalobrama of the subfamily Culterinae in the family Cyprinidae, is an important freshwater cultured species in China because of its low culture cost and high growth rate. Culter alburnus, belonging to the subfamily Culter, is another slender carnivorous species, whose meat is more delicate and delicious than that of M. amblycephala; however, the scales of C. alburnus are small and thin and are easily injured and fall off, and these fish are slow-growing and expensive to feed. Although both the fish are quite different in terms of diet, growth, and stress resistance, they are highly complementary to each other. Crossbreeding can combine the desired characteristics of both parents; however, this advantage is usually limited to the F1 generation. Theoretically, because the gonads are almost undeveloped during triploid growth and development, F1 heterosis can be maintained to avoid germplasm decline caused by continuous reproduction, and energy can be saved to accelerate growth and improve muscle quality. This will greatly expand the breeding space of new hybrid varieties and ensure high economic, social, and ecological value. Therefore, the triploids of M. amblycephala ♀ × C. alburnus ♂ have superior growth and economic value. To understand the growth and morphological characteristics of the triploids of M. amblycephala ♀ × C. alburnus ♂, the triploid population of M. amblycephala ♀ × C. alburnu ♂ was induced by hydrostatic pressure, and successfully induced triploid (hybrid-3n) and uninduced diploid (hybrid-2n) populations were obtained. At the same time, two self-breeding populations of Megalobrama (MA) and Culter (CA) were established. The growth performance and morphological characteristics of the four kinds of fish were compared and analyzed in growth contrast experiments. The results showed that during the 210-day growth cycle, the absolute weight gain (0.88±0.11 g/day) of hybrid-3n was 8.64% higher than that of hybrid-2n, 20.55% higher than that of MA, and 120.00% higher than that of CA. In terms of countable traits, the hybrid index of hybrid-3n and hybrid-2n was 41.05 and 36.07, respectively. In terms of measurable traits, the hybrid index of hybrid-3n and hybrid-2n was 36.73 and 57.57, respectively. Cluster analysis showed that hybrid-3n was first grouped with hybrid-2n, then grouped with maternal MA, and finally grouped with paternal CA. The results showed that hybrid-3n and hybrid-2n were closer to their mothers in quantifiable traits and frame structure, showing a maternal effect. Discriminant analysis showed that the discriminant accuracy of hybrid-3n was 90%, and the comprehensive discriminant rate of the four populations was 95%. A scatterplot of the discriminant analysis showed that the distribution centers of hybrid-3n and hybrid-2n were located between the parents and closer to the mothers. Hybrid-3n and hybrid-2n occupied partially overlapping areas, which indicates that the two are similar in proportion and frame structure, making it difficult to distinguish between them. These results are basically consistent with those obtained by cluster analysis. Most of the 11 biological traits used to construct the discriminant function were related to the longitudinal axis of the body, especially the ratio of the height of the body to the trunk, which is similar to the results obtained by principal component analysis. During principal component analysis, four principal components with a cumulative contribution rate of 75.10% were obtained, which mainly reflect morphological variations of body height and trunk length. In this study, the experimental fish were cultured in still water ponds with abundant bait. The morphological differences may be attributed to the adaptation of the fish to this ecological environment. Comprehensive analysis showed that: Hybrid-3n has a fast growth rate and has the basic conditions for promotion and application in production; The body size of hybrid-3n and hybrid-2n is between the parents, and both are slightly biased towards the mother, which mainly reflect the morphological variations of body height and trunk length; In this study, three multivariate analyses were used to effectively reflect the morphological differences among the four populations of hybrid-3n, hybrid-2n, MA, and CA from different perspectives, which has made them irreplaceable. In conclusion, this study confirmed the superior breeding potential of triploids of M. amblycephala ♀ × C. alburnu ♂, and provided basic data for the morphological comparison of hybrid offspring of M. amblycephala and C. alburnu, which is significant for the identification and protection of fish resources, and is expected to provide a theoretical basis for the establishment of new strains of M. amblycephala and C. alburnu. The results are of great significance for population identification and ploidy breeding of hybrid offspring of M. amblycephala and C. alburnu.

Abstract:Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used molecular techniques, which allows the detection and quantification of gene expression due to its high sensitivity, specificity, and reproducibility. To increase the reliability of the experimental results, internal reference genes are used to normalize the data. However, previous studies have shown that the stability of internal reference genes is influenced by experimental conditions and interspecies variations, and no universal internal reference genes have been identified for all species. Therefore, suitable internal reference genes must be identified for each species. The Hong Kong catfish (Clarias fuscus) has strong adaptability, high nutritional value, and tender flesh, and it was first introduced to large-scale aquaculture in the Guangdong and Guangxi provinces in the early 1970s. Currently, it is a key aquaculture species in the Guangdong-Guangxi region and one of the freshwater economic fish cultured in southern China. Thus, fairly extensive molecular biology and genetics studies of C. fuscus have been conducted, which in turn has increased the demand for quantitative gene expression analysis by qRT-PCR in these animals. Consequently, there is a growing demand for quantitative gene expression analysis by qRT-PCR in C. fuscus for various molecular biology and genetics studies. However, few studies have evaluated the internal reference genes for this species. Therefore, we aimed to identify suitable internal reference genes in different tissues and different stages of gonadal development of males and females to provide the necessary tools to support subsequent gene expression pattern analysis. In this study, we examined the expression of 13 internal reference genes (actb1, actb2, ef1a, eef1b2, rpl13a, rpl13, ccdc124, cfl1, nm23, eif3g, rap1a, ikbkg, and ywhab) in different stages of gonadal development and different tissues of adult fish of C. fuscus. We used four software tools, namely BestKeeper, GeNorm, NormFinder, and RefFinder, to evaluate the expression stability of the internal reference genes. BestKeeper software evaluates the stability of internal reference genes by calculating the standard deviation (SD) and coefficient of variation (CV). The results showed that in different tissues of adult fish, the stability ranking of these genes was as follows: actb1＞actb2 =ef1a＞rpl13＞ywhab＞eef1b2＞rpl13a＞nm23＞eif3g＞ccdc124＞rap1a＞cfl1＞ikbkg. In different stages of male and female gonadal development, the stability ranking of these genes was as follows: actb2＞rpl13＞ef1a＞rpl13a＞actb1＞ywhab＞cfl1＞rap1a＞eef1b2＞eif3g＞ccdc124＞ikbkg＞nm23. NormFinder suggested that the best stability ranking was as follows: actb2=rpl13＞actb1＞rpl13a＞ywhab＞nm23＞rap1a＞cfl1＞eif3g＞ef1a＞ccdc124＞ikbkg＞eef1b2 in different tissues of adult fish and actb2=actb1＞rpl13＞ef1a＞rpl13a＞cfl1＞ywhab＞rap1a＞nm23＞eif3g＞ccdc124＞ikbkg＞eef1b2 in different stages of male and female gonadal development. The geNorm screens for the most stable internal reference genes by comparing the stability value (M) of each internal reference gene. It indicated that the final stability ranking in different tissues was actb2=rpl13＞rpl13a＞actb1＞ef1a＞ccdc124＞ywhab＞rap1a＞eif3g＞nm23＞eef1b2＞ikbkg＞cfl1, and actb1=rpl13＞actb2＞ef1a＞rpl13a＞cfl1＞ywhab＞ccdc124＞rap1a＞eif3g＞nm23＞eef1b2＞ikbkg in different stages of male and female gonadal development. Finally, RefFinder analysis integrated the results of the other three software tools and showed that the comprehensive stability ranking of each gene in different tissues was actb2＞rpl13＞cfl1＞rap1a＞ef1a＞rpl13a＞ikbkg＞eif3g＞nm23＞actb1＞eef1b2＞ywhab＞ccdc124. In different stages of male and female gonadal development, the comprehensive stability ranking was actb2＞rpl13＞actb1＞cfl1＞rap1a＞ef1a＞rpl13a＞ikbkg＞eif3g＞nm23＞eef1b2＞ywhab＞ccdc124. Given this, we can conclude that actb2 is the best internal reference gene for C. fuscus. To validate the accuracy of the results, we selected actb2 and rpl13 as internal reference genes and compared their expression levels with those of four genes (zp3, atp2b3, slc13a5 and parp8) from transcriptome RNA-seq data. The fold changes in the expression levels were closer to the transcriptome data when using actb2 than when using rpl13, indicating that actb2 is more stable than rpl13. This study demonstrated that actb2 showed good stability among the 13 internal reference genes and could be used as an internal reference gene for different stages of gonadal development and different tissues of adult fish in C. fuscus. The results can provide technical support for the subsequent research on the functional gene expression patterns of C. fuscus and are expected to be applicable to other catfish species.

Abstract:Penaeus vannamei, also known as white foot shrimp, is globally one of the three high-yield shrimp farming varieties. The wild species is found along the Pacific coast of South America. Since 1988, China has introduced P. vannamei. Due to its strong environmental adaptability, high feed conversion rate, fast growth rate, and high tolerance to ammonia nitrogen and nitrite, it has been widely promoted in aquaculture. By 2021, the aquaculture output of P. vannamei in China reached 1.977 million tons, accounting for approximately 37% of the world’s total production, and has extremely high economic value. Growth traits are among the most important economic factors in the production of P. vannamei. With the intensive development of shrimp farming and degradation of germplasm, the white spot syndrome virus (WSSV) is a serious disease faced by the global shrimp industry. The infection can cause symptoms such as reduced food intake, enlarged liver and pancreas, pale red body color, and white spots on the head and chest armor in shrimp, resulting in widespread death. At present, China has cultivated 12 new varieties of P. vannamei, which to some extent alleviates its dependence on imports for high-quality P. vannamei. However, the excellent traits of domestic shrimp species are singular, and cultivating new varieties of P. vannamei with fast growth and strong disease resistance is an urgent demand in the market. Genetic parameter evaluation is the fundamental work of selection breeding. Heritability reflects the genetic variation in traits in the breeding population, which is of great significance in the development of a selection index, prediction of selection response, comparison of selection methods, selection breeding planning, and other breeding processes. There are different degrees of genetic correlation among various quantitative traits of shrimp, and genetic correlation coefficients are important for selecting target traits. The estimation of genetic correlation can be used to develop a comprehensive selection index and breeding program of multiple traits, which can improve the selection efficiency and breed better varieties with multiple traits. The higher the genetic correlation between traits, the better the effect of indirect selection. The estimation of genetic parameters is greatly affected by the test population, breeding management, analysis methods, and other factors. To ensure the accuracy of multi-trait composite breeding for growth and WSSV resistance, accurate evaluation of growth and WSSV resistance needs to be carried out for specific breeding populations. There are two commonly used target traits for measuring WSSV resistance in P. vannamei: individual survival time after infection and half-lethal survival rate (SS50) of families. In practice, measuring the half-lethal survival rate of families is more convenient. However, the correlation between these two traits has not yet been reported. To promote the growth and WSSV resistance of P. vannamei, 59 families (1,770 individuals) were tested for WSSV infection. We recorded the survival time and individual body length of shrimp after infection and analyzed the mean, standard deviation, maximum and minimum values, and coefficient of variation of half-lethal survival rate and test traits for each line. The heritability and genetic correlation coefficients of growth, survival time, and half-lethal survival rate were calculated. Variance components and genetic parameters for growth and survival traits were estimated using a two-trait animal model and a sire-dam threshold model. The genetic parameters of body length were corrected by including age as a covariate. The genetic parameters of WSSV survival time were corrected by including body length as a covariate. The estimated heritability of body length was medium (0.17±0.05), and the estimated heritabilities of survival time and half-lethal survival rate were medium (0.18±0.05 and 0.14±0.05). Further, the estimated heritabilities of the three traits were significantly different from zero (P<0.01). The genetic correlation between body length and survival time and that of body length and half-lethal survival rate were low (0.15±0.20 and 0.25±0.22). There were no significant differences between the genetic correlations and zero (P>0.05). The genetic correlation between survival time and half-lethal survival rate was high (0.96±0.03). There was no significant difference between the genetic correlation and one (P>0.05). The results showed that a comprehensive selection index of growth and WSSV resistance of P. vannamei can be established to carry out multi-trait composite breeding. In this breeding population, the growth and WSSV resistance of P. vannamei can be combined with multiple traits according to the breeding requirements, and the comprehensive selection index can be formulated by assigning values. In addition, to optimize the breeding of each generation, the half-lethal survival rate of WSSV can be used as an indicator of WSSV resistance. This study provides basic data and theoretical support for breeding varieties of P. vannamei with excellent growth and WSSV resistance.

Abstract:Penaeus chinensis is one of the most economically important species in northern China. Strong market demand requires the expansion of aquaculture production capacity of P. chinensis. Saline-alkali water covers approximately 46 million hectares in China alone, primarily distributed in the northeast, northwest, and coastal areas. Low salinity, high carbonate alkalinity, high pH, and complex ionic composition are characteristics of such waters, which cause stress to aquatic animals by interfering with physiological homeostasis. High carbonate alkalinity can directly damage the gill tissue of crustaceans. The inhabitation of ion-exchange can result in alkalosis. The understanding of response mechanisms to saline-alkali stress in P. chinensis will contribute to the sustainable development of the shrimp industry. ATP-binding cassette (ABC) transporters are one of the most prominent families of transmembrane proteins. ABC transporters can transport molecules, such as inorganic ions, sugars, amino acids, lipids, peptides, specialized metabolites, and xenobiotic agents, across membranes by binding and hydrolyzing ATP (adenosine triphosphate). Members of the ABCG subfamily consist of a single ABC cassette in the amino terminal followed by six putative transmembrane domains, and thus, are referred to as half-sized ABC transporters. Members of this family play an important role in the efflux transport of cholesterol. The ABCG subfamily participates in signal transduction, antiviral defense, and antigen presentation through hormone transport and lipid metabolism, thus helping plants adapt to changing environments. Several members of this gene family show different expression patterns in P. chinensis when exposed to saline-alkali stress. Therefore, we aimed to explore the ABC functions in resistance to alkalosis in P. chinensis. In this study, ABCG5 (GenBank accession number: OQ318160) was identified in P. chinensis and named PcABCG5. The full length of PcABCG5 was 1,923 base pairs, encoding 640 amino acids. The estimated molecular mass was 71.39 kDa, and the theoretical isoelectric point was 9.12. Subcellular localization prediction showed that PcABCG5 was located in the endoplasmic reticulum. The PcABCG5 protein contained an NBD (nucleotide-binding domains) and a TMD domain (transmembrane domain) and had no signal peptide. Homology and phylogenetic analysis showed that PcABCG5 was highly conserved and that mature PcABCG5 shared 98.91% and 97.97% similarity with ABCG5 sequences from Penaeus monodon and Penaeus japonicus, respectively. PcABCG5 expression profiles were assessed by qPCR. PcABCG5 mRNA was detected in the eyestalk, gills, heart, muscle, intestine, hepatopancreas, stomach, and hemolymph. Our results showed that saline-alkali stress induced significant upregulation of PcABCG5 and that the expression of PcABCG5 was highest in the gills. This may be owing to the location of the gills between the external and internal environments. Osmotic pressure regulation by ABCG transporters in the gills is the primary mechanism by which P. chinensis copes with saline-alkali stress. To determine the function of PcABCG5, dsRNA against PcABCG5 was successfully injected and PcABCG5 was downregulated by 82.9% (P<0.05). At 48 h after RNAi, we observed a 20% increase in mortality of PcABCG5 mRNA knockdown shrimp, which verified that PcABCG5 participated in the response and improved the survivability of P. chinensis under acute saline-alkali stress. Co-expressed ABCG5 and ABCG8 formed heteromeric dimers that participated in lipid transport. They also affected membrane permeability by regulating the asymmetric distribution of membrane lipids. It was also evident that saline-alkali stress induced ion and osmotic stress. We speculated that PcABCG5 may participate in the maintenance of osmotic homeostasis by hydrolyzing ATP to release energy and enhance the transport function. In conclusion, the full length cDNA of PcABCG5 was cloned in P. chinensis. PcABCG5 was upregulated under saline-alkali stress. Moreover, RNAi resulted in increased mortality of PcABCG5-silenced shrimp under saline-alkali stress. Therefore, it can be concluded that PcABCG5 is involved in the response to saline-alkali stress in P. chinenesis. Our findings provide information for further understanding the genetic basis of saline-alkali tolerance and exploring the molecular breeding of P. chinensis.

Abstract:Pacific white shrimp Penaeus vannamei, as one of the most productive and economically valuable shrimp species in the world, have many favorable characteristics, such as a short growth cycle, strong adaptability and disease resistance, and a low nutritional demand. They are omnivorous animals whose diet mainly consists of animal-derived food. Conventional feed used in their culture contains fishmeal as the main protein source. Feed costs account for 40%~60% of their production costs. Therefore, breeding P. vannamei strains without demand for fishmeal would be an effective way to address the issues of fishmeal shortage and high feed costs. Improving utilization efficiency for diets without fishmeal is key to breeding such P. vannamei strains. In this study, genetic parameters for feed efficiency traits (feed efficiency ratio, feed intake, and weight gain) and genotype by environment interaction were evaluated using two diets, one without fishmeal (38% protein) and one with 25% fishmeal (42% protein). Thirty families of P. vannamei were selected for a 40-day feeding experiment. A total of 900 shrimp (30 animals per family) were selected. Fifteen animals per family were fed a zero-fishmeal diet, and the other 15 animals were fed a high fishmeal diet. The feed intake and body weight of all animals were measured for calculating the feed efficiency ratio and weight gain. The variance components for these traits were estimated using a univariate model and an average information restricted maximum likelihood algorithm, and genetic (phenotypic) correlations between traits were estimated using bivariate analysis. The results showed that shrimp fed a high fishmeal diet exhibited a significantly higher mean weight gain (62.00%) and feed efficiency ratio (124.00%) than those fed a zero-fishmeal diet, which had a mean weight gain of 23.50% and feed efficiency ratio of 49.40%. There was a small difference in the mean feed intake between shrimp fed a high fishmeal diet (7.09 g) and those fed a zero-fishmeal diet (7.28 g). The heritability estimates for feed intake of shrimp fed zero- and high fishmeal diets were 0.699 ±0.155 and 0.556±0.139, respectively. For weight gain, the heritability estimates of shrimp fed zero- and high fishmeal diets were 0.458±0.140 and 0.667±0.146, respectively, which were high. However, the differences between diets and between traits were not statistically significant (P>0.05). The heritability estimates for feed efficiency ratio of shrimp fed zero- and high fishmeal diets were 0.186 ±0.098 and 0.341±0.110, respectively, which were low to medium. The differences in feed efficiency ratio between diets was not statistically significant (P>0.05). All the above heritability estimates were overestimated, to some extent, because common environmental effects were not estimated. Therefore, there was not much genetic variation in feed efficiency ratio in shrimp fed a zero-fishmeal diet, suggesting limited potential in selection breeding of this trait. Regardless of diet, genetic correlations among feed intake, weight gain, and feed efficiency ratio were moderate to high (0.580±0.188 to 0.945±0.040). Genetic correlations between diets were 0.580± 0.171 and 0.676±0.155 for feed intake and weight gain, respectively, indicating that these two traits had moderate genotype by diet interactions. The genetic correlation between diets was only 0.299±0.304 for feed efficiency ratio, which indicates a serious genotype by diet interaction. According to the above results, use of a zero-fishmeal diet is not good for the culture and breeding of P. vannamei. In the future, fishmeal content in the diet may be appropriately increased, and genetic parameters and potential feed efficiency traits should be evaluated in low fishmeal diets.

Abstract:The Pacific white shrimp Penaeus vannamei is one of the most productive shrimp species in China and the world. Domestic demand for P. vannamei has increased annually because of its fast growth, easy breeding, strong resistance to stress and disease, and high meat yield. The primary challenge in the development of the shrimp industry is seed rearing. The demand for broodstocks of P. vannamei in China is as high as 1,000,000 pairs per year, and the total weight of shrimp seedlings exceeds 1.3 trillion. China needs to import 300,000 pairs of P. vannamei every year. The cost of each pair of P. vannamei is approximately 200 US$, thus equating to hundreds of millions of US$ in total. During the production of P. vannamei, the spawning frequency varies greatly among female shrimp. Cultivating high-fecundity P. vannamei through genetic improvement is an effective way to reduce the cost of seedling farms and improve economic benefits. To further study the molecular mechanisms of fecundity differences in female P. vannamei, the ovarian histology and expression of four candidate genes in populations with high- and low-fecundity were analyzed in this study. Before the experiment, the 7-month-old parent shrimp of P. vannamei were fed a commercial parent shrimp fortified diet, a maturation promoting diet, and squid for nutritional enhancement. After one month of promoting maturity, the unilateral eyestalk of female shrimp was cut using forceps, and next-generation family construction began after 15–20 days of recovery. The spawning frequency of 439 female shrimp in the 30-day production cycle was calculated and used to indicate fecundity. Those with a spawning frequency of 0 were included in the low-fecundity group, while those with a spawning frequency of >3 were included in the high-fecundity group. The ovarian development of P. vannamei was divided into four stages (proliferation, small growth, large growth, and maturation) according to the color, size, and shape of the ovaries. In the high-fecundity group, the ovaries exhibited development up to stageⅣ, indicating their attainment of maturity. In the low-fecundity group, ovarian development was limited to stageⅢ, with progression to stageⅣ proving difficult. Ovarian tissues from the same part of female shrimp of stageⅠ–Ⅳ in the high-fecundity group and stageⅠ–Ⅲ in the low-fecundity group were taken and immediately immersed in tissue fixative for paraffin sectioning. The research team previously identified candidate genes related to fecundity in genomic regions undergoing selective sweep in isolated populations. These included thioredoxin 2 (TRX2), partitioning-defective 3 (PARD3), phospholipase Cβ4 (PLCβ4), and arginine-glutamate acid dipeptide repeats (RERE), which play important roles in oocyte maturation, cell proliferation, and early development in other species. qRT-PCR was used to analyze and compare their expression in the ovary and eyestalk tissues of high- and low-fecundity females at different stages of ovarian development. The results of paraffin section showed that the ovarian development of the low-fecundity group was slower than that of the high-fecundity group. In the high-fecundity group, stageⅠ ovaries primarily consisted of oogonia and oocytes during the early phase of yolk formation, with the emergence of small spherical yolk granules. StageⅠ ovaries in the low-fecundity group primarily consisted of oogonia, with no observable yolk granule formation. StageⅡ ovaries in the high-fecundity group were mainly composed of oocytes with vitelline formation, accompanied by a notable augmentation in both the size and number of yolk granules. StageⅡ ovaries in the low-fecundity group primarily comprised oocytes in the early phase of vitelline formation with yolk granules beginning to form. StageⅢ ovaries in the high-fecundity group consisted mainly of oocytes during the late phase of yolk formation. Many spherical yolk granules were formed in the cytoplasm, and cortical rods began to appear, although the lengths of the cortical rods were short. StageⅢ ovaries in the low-fecundity group were composed of oocytes during yolk formation. The cytoplasm was full of spherical yolk granules in the absence of cortical rods. In the high-fecundity group, stage Ⅳ ovaries were filled with mature oocytes, large yolk granules filled the entire cytoplasm, and thick cortical rods were formed in the oocytes. qRT-PCR showed that the four genes maintained high expression levels in the mature ovaries of the high-fecundity group. Their expression levels in the ovaries of the high-fecundity group were higher than those in the ovaries of the low-fecundity group in the small and large growth stages. TRX2, PLCβ4, and RERE expression levels were significantly different between the groups (P<0.05). In eyestalk tissues at different stages, TRX2, PARD3, PLCβ4, and RERE expression levels in the low-fecundity group were higher than those in the high-fecundity group. The results suggest that these four genes may play important roles in the ovarian development of P. vannamei. The expression levels of all four genes in the high-fecundity group were lower than those in the low-fecundity group in eyestalk tissues. However, in ovarian tissues, except for the PARD3 gene in stageⅠ, which was expressed at lower levels in the high-fecundity group than in the low-fecundity group, the expression levels of all four genes in the other stages were higher in the high-fecundity group than those in the low-fecundity group, which is opposite to the expression trend in eyestalk tissues. Therefore, we speculate that these genes also promote cell proliferation, development, and target gene expression in the eyestalks of the low-fecundity group. Their high expression in the eyestalks of the low-fecundity group may enhance the secretion of ovarian inhibitory factors, thereby inhibiting ovarian development. Further research is needed to reveal their functional mechanisms. The above results provide an important reference for the in-depth study of the molecular mechanisms of differences in the fecundity of female P. vannamei.

Abstract:Viral covert mortality disease (VCMD) in cultured shrimp is caused by covert mortality nodavirus (CMNV). The infected shrimp mainly die successively in the deep-water areas at the bottom of farming ponds, which is difficult to observe; as such, the term "covert mortality disease" was coined by local farmers. The disease is known as running mortality syndrome by Southeast Asian farmers because a small number of diseased shrimp die daily in the infected ponds, and more continue to die until harvest time. The infected shrimp mostly show symptoms such as carapace softening, hepatopancreas color fading, atrophy, empty stomach, slow growth, etc. In the acute infection stage, the infected shrimp show opaque abdominal muscles. The prevalence of VCMD has caused severe economic losses in the shrimp farming industry over the past decade. CMNV can infect many major cultured shrimp species, including Penaeus vannamei, P. japonicus, P. chinensis, Macrobrachium rosenbergii, and Exopalaemon carinicauda. Moreover, the virus infects the co-inhabiting organisms in shrimp ponds, which makes it difficult to remove from shrimp farming areas due to its wide host range among invertebrates and its transport by many vectors. To elucidate the prevalence of CMNV in the main cultured shrimp species in China in recent years, an epidemiological investigation of CMNV, including clinical sample collection from shrimp and other organisms inhabiting the shrimp ponds, was carried out in the main shrimp culture areas of China from 2021 to 2022, and the collected samples were systematically analyzed using molecular biology, histopathology, and transmission electron microscopy approaches. During this period, 1,299 samples were collected from Tianjin, Shandong, Jiangsu, Hainan, Hubei, and Xinjiang and included P. vannamei, M. rosenbergii, P. japonicus, P. chinensis, Procambarus clarkia, and other organisms living in shrimp culture ponds. The collected samples were analyzed using CMNV TaqMan probe real-time quantitative reverse transcription PCR (TaqMan RT-qPCR). The results showed that CMNV could be detected in the main cultured shrimp species, including P. vannamei, M. rosenbergii, and P. japonicus. A percentage of the samples collected from Shandong, Jiangsu, Hainan, Xinjiang, Guangxi, and Tianjin were observed to be CMNV-positive in the TaqMan RT-qPCR assays. In addition to cultured shrimp, CMNV was detected in organisms living in the shrimp ponds, such as Artemia collected in 2021 and Perinereis aibuhitensis collected in 2022. The CMNV-positive rate of samples collected in 2021 was 10.04% (69/687), and that of samples collected in 2022 was 11.44% (70/612). Epidemiological investigation showed that CMNV infection in outdoor pond culture usually caused a certain degree of cumulative death, whereas CMNV infection in indoor culture mainly caused the softening and reduced growth of infected shrimp and did not cause a large number of infected shrimp to die. High CMNV loads were detected in samples of Artemia used as fresh bait for shrimp and in samples of P. aibuhitensis living in shrimp pond soils. Accordingly, a high CMNV infection rate was observed in cultured P. vannamei feeding on CMNV-positive Artemia and in that farmed in ponds containing CMNV-positive P. aibuhitensis. The results indicated that CMNV vectors such as Artemia and P. aibuhitensis may pose a high risk of CMNV transition, threatening the health of the shrimp in the culture system. Recent studies on the pathogenesis of CMNV infection indicated that the virus mainly infected epithelial tissues, nerve tissues, and connective tissues in the hepatopancreas, appendages, mid-gut, gills, eye stalks, and nerves of shrimp. Therefore, histopathological and in situ hybridization analyses were performed on samples identified as CMNV-positive using TaqMan RT-qPCR in this study. Eosinophilic virus inclusion bodies were observed in the hepatopancreas and foremidgut tissues, and pathological lesions were observed in the appendicular nerves of diseased Penaeus shrimp. Moreover, obvious purple hybridization signals from the CMNV RNA probe were seen in tissues with lesions, which further confirmed the positive detection results for CMNV in shrimp samples obtained using TaqMan RT-qPCR in this study. The results of this study showed that, compared with previous years, there was still a high CMNV-positive detection rate in many areas of crustacean culture in 2022, and the epidemic risk of this virus was still high. Multiple CMNV-positive results were obtained in the co-inhabiting and feed organisms in crustacean farming ponds. These results remind fishery authorities and stakeholders in the prawn breeding industry to further strengthen the monitoring and early detection of CMNV to prevent and control the spread of the virus in seedlings, reduce the risk of epidemic hazards, and promote the sustainable and healthy development of the crustacean aquaculture industry.

Abstract:The triangle-sail mussel (Hyriopsis cumingii) is the most important freshwater pearl-bearing mussel in China. Nucleated pearls from the mantle of H. cumingii are emerging pearl cultivation products. Compared to traditional non-nucleated and nucleated pearls from visceral mass, nucleated pearls from the mantle show significant cultivation advantages, such as high pearl quality and short nurturing cycles. These nucleated pearls are widely known as “freshwater Akoya” in the pearl market because they resemble Japanese saltwater Akoya pearls. However, techniques for cultivating nucleated pearls from the mantle of H. cumingii are still immature and lack a targeted quality evaluation system. Therefore, this study was conducted to identify growth traits related to the quality of nucleated pearls from the mantle of H. cumingii. Two-year-old H. cumingii were implanted with 10 nuclei, 5 mm each in size per mussel. These host mussels were cultured for 19 months to form commercial pearls. Correlation and path analyses of the key quality traits of pearls (pearl number/pearl formation percentage, maximum diameter, minimum diameter, roundness, nacre thickness) and the growth traits of host mussels (shell length, shell width, shell height, body weight, shell weight, soft tissue weight) and nuclei (maximum diameter, minimum diameter, roundness) were performed. The results showed that the pearl formation rate varied from 50% to 100%, with 43.33% of mussels exhibiting a pearl formation rate of 70% and only 3.33% of mussels exhibiting a pearl formation rate of 50% or 100%. The average pearl formation rate was (70.00±10.82)%. The variation in pearl size (minimum diameter) was 4.72‒9.19 mm with an average of (6.91±0.67) mm. The variation in pearl roundness (diameter variation) was 0‒45.60% with an average of (6.74±7.78)%. The highest percentage of pearls were round, followed by near-round pearls, and then by positive round pearls at 21.90%. The nacre thickness was thin in 1.43% of pearls and extra thick in 98.57%. Pearl quantity and roundness were not correlated with all quantitative traits of pearls, host mussels, or nuclei (P>0.05). Pearl size and nacre thickness were highly significantly and positively correlated with shell width, body weight, and shell weight (P<0.01) and were significantly and positively correlated with shell height and soft tissue weight of the host mussels (P<0.05). Among pearl quantitative traits, path analysis of pearl size and nacre thickness showed (highly) significant correlations with host mussels and nuclei. Stepwise regression analysis was performed with the pearl minimum diameter (y1) and nacre thickness (y2) as dependent variables and the shell length (x1), shell width (x2), shell height (x3), body weight (x4), shell weight (x5), and soft tissue weight (x6) of host mussels, along with the maximum diameter (x7), minimum diameter (x8), and roundness (x9) of the nuclei, as independent variables. The regression equations were y1 = 3.464 + 0.078x2 − 0.055x9 (R2 = 0.75; P<0.01) and y2 = −1.243 + 0.077x2 − 0.055x9 (R2 = 0.75; P<0.01). The direct effect of shell width on pearl size was the highest (0.69). The direct effect of nucleus roundness on pearl size was −0.23. The indirect effect of shell width on pearl size through nucleus roundness (0.05) was significantly greater than that of nucleus roundness on shell width (−0.02). The direct and indirect effects of shell width and nucleus roundness on nacre thickness were similar to those on pearl size. The direct determinant coefficient of shell width on pearl size (0.50) was significantly higher than that of nucleus roundness (0.08) and that of shell width via nucleus roundness (−0.03). The direct and indirect determinant coefficients of nucleus thickness by shell width and nucleus roundness in pearl-bearing mussels showed the same trend. These findings suggest that the shell width of host mussels is a key trait for controlling pearl size and pearl nacre thickness in nucleated pearls from the mantle of H. cumingii. These results will contribute to the development of China´s pearl industry.

Abstract:Paralytic shellfish toxins (PSTs) pose significant ecological and food safety hazards in China and the world. Consequently, there is a global consensus to intensify safety monitoring of these toxins to safeguard consumers. In recent years, the occurrence of harmful algal blooms (known as “red tides”) has increased due to human activities and climate change. The contamination risk posed by PSTs is a pressing concern. This problem poses significant ecological and food safety risks in China and the world, leading to a global consensus on strengthening the safety monitoring of PSTs to ensure consumer safety. Several countries and regions, including the European Union, the United States, and Canada, have established strict regulatory limits (800 μg STXeq/kg) for PSTs and implemented monitoring programs. Currently, the main methods for detecting and monitoring PSTs include enzyme-linked immunosorbent assay, mouse bioassay, high-performance liquid chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, LC-MS/MS has gained international attention due to its high throughput and sensitivity, allowing detailed analysis of toxin profiles in contaminated shellfish. However, the complexity of aquatic food matrices often introduces interference during detection, affecting the accuracy and precision of the results. Using matrix reference materials as quality controls during the analysis can effectively mitigate the errors caused by matrix effects. In recent years, research has been conducted in China on the preparation techniques of matrix reference materials. However, existing techniques have limitations, such as unstable raw materials, limited toxin types, and lack of commercial production. There is still a lack of PST standard materials in China that possess traceability, certification, and physical specimens. To meet the quality control and validation needs for PST detection and analysis, and to enhance the regulation of shellfish quality and safety, the preparation of various PST matrix reference materials has become a top priority. In this study, six PST-producing algae species from four major Chinese coastal areas were compared as the raw material for toxin production: Alexandrium catenella (GY-H25), Gymnodinium catenatum (GY-H65), Alexandrium tamarense (GY-H31 and AT5-3), Alexandrium pacificum (TI0989), and Alexandrium minutum (GY-H46). Through three-step extended culture, all strains achieved mean cell densities ranging from 4.0 × 106 to 4.0 × 107 cells/L. Single cell toxin producing capacity was ordered from highest to lowest as GY-H46, GY-H31, AT5-3, GY-H25, GY-H65, and TI0989. After comparing the toxin production per cell, algal cell density, and toxin composition of the six strains, GY-H46, GY-H31, GY-H25, and GY-H65 were selected for the exposure experiments. These four species of toxic algae (up to 1.0 × 105 cells) were fed to mussels (Mytilus galloprovincialis) for 7 days under controlled conditions. The results demonstrated that the toxin composition of contaminated mussels clustered with those of the causative algae and may separate from one another significantly during the different accumulation phases. GY-H46, GY-H31, and GY-H25 showed consistent toxin profiles with those found in mussels, but varied in the proportions of individual toxin components. GY-H65 exhibited significant differences in toxin composition and proportions in mussels. We also detected a new conversion component (dcSTX) with a maximum proportion of 29.6%. The proportion of dcNEO increased from 1.9% to 13.2% in GY-H65 and mussels. In addition, a high proportion of conversion from C2 to C1 was observed in mussels fed GY-H25 and GY-H65. The mean accumulation rates of toxins from the four toxic algae species in mussels ranged from 39.7% to 83.1%, with the highest toxicity reaching (1,151.0 ± 7.6) μg STXeq/kg in mussels. Individual exposure experiments with the four toxic algae species demonstrated that mussels are capable of accumulating multiple components and high levels of PSTs. Based on the above observations, the feeding ratios of the low- and high-density groups in the mixed exposure experiment were determined as 1∶1∶1 ∶4 and 1∶1.6∶2.4∶8 for GY-H25, GY-H65, GY-H31, and GY-H46, respectively. The total algae feeding amount per mussel per day was 3.5 × 104 cells in the low-density group and 2.6 × 105 cells in the high-density group. When examining the PST content in mussels from the two mixed exposure groups, it was observed that the mean accumulation conversion rates were comparable at 41.4% and 44.6%, which were lower than the theoretical rates of 48.7% and 47.5%, respectively. The overall toxicity of the low- and high-density groups was (535.0±5.6) and (2,636.0±12.4) μg STXeq/kg, respectively. After mixed exposure, both experimental groups of mussels accumulated a total of 12 PST components (NEO, dcSTX, dcNEO, GTX1, GTX4, GTX2, GTX3, GTX5, dcGTX2, dcGTX3, C1, and C2). This study demonstrates that mixed exposure of mussels to four toxin-producing algae species, namely A. catenella, A. tamarense, G. catenatum, and A. minutum, can result in the stable and manageable presence of 12 PST components. This provides technical support for the preparation of matrix reference materials that can be used in industrial regulation and industry services. The development of a preparation technology for matrix reference materials with multiple PST components, which do not exceed international limits, holds promise for water quality monitoring and the assessment of marine pollution risks, and provides reference materials for theoretical studies on PSTs risk formation mechanisms.

Abstract:Paralytic shellfish toxins (PSTs) are a class of neurotoxic marine biotoxins that are widely distributed and cause more than 2,000 poisoning events worldwide each year, with mortality rates of up to 15%. PSTs can accumulate through the food chain and are mainly distributed in marine organisms such as bivalve mollusks. Several countries and regions, including the European Union, China, and the United States, have established strict regulatory limits (400 MU/100 g or 800 μg STXeq/kg) for PSTs and implemented monitoring programs. As reported previously, factors such as filtration rate, selective feeding, and the efficiency of the organism in absorbing toxin-producing algae can significantly affect the accumulation of PSTs in bivalve mollusks. Mytilus unguiculatus is one of three major commercial mussel species in China, with important economic value and social impact. Due to its high nutritional value, it is extensively cultured as an important shellfish species in the Zhoushan archipelago of Zhejiang Province in China. Alexandrium spp. are the main toxin-producing algae in the area. PSTs have been detected in mussels between May and July after harmful algal blooms. Research into the elimination characteristics of PSTs accumulation in M. unguiculatus is urgently needed to establish a monitoring and control program. In this study, 760 mussels were randomly selected and fed A. catenella at different cell densities, with a high-density group (7.00105 cells/d), a low-density group (2.80105 cells/d), and a control group. The experimental period lasted for 30 d, during which the accumulation period approximately represented days 1–7 and the elimination period days 8–30 d. A total of 14 sampling points were set up on days 0.5, 1, 2, 4, 6, and 7 of the accumulation period and days 1, 3, 5, 7, 11, 15, 19, and 23 of the elimination period. Six mussels were randomly collected at each sampling point and dissected into soft tissues, hepatopancreas, and edible tissues. Liquid chromatography-tandem mass spectrometry was used to determine the content of PSTs. During the accumulation period, 5 mL culturing seawater was collected during or 1 h after feeding, and Ruge's solution was added. The filtration rate of the mussels was determined by counting the quantity of A. catenella cells in the water. The results showed that the toxins in M. unguiculatus were not equally distributed. The highest PST content in hepatopancreas tissues was 7,458.2 μg STXeq/kg (high-density group) and 2,555.9 μg STXeq/kg (low-density group). The highest PST content in edible tissues was 108.6 μg STXeq/kg (high-density group). The hepatopancreas was identified as a target organ for toxin accumulation. From day 3 to day 7, the filtration rate of mussels decreased, eventually reaching 30% of its initial value. The filtration rate of M. unguiculatus in the high-density group was not significantly different from that of the low-density group during days 1–5 and was significantly lower during days 5–7. During the elimination phase, the PST elimination rate in mussels was 18.4% (hepatopancreas), 18.1% (soft tissues), and 13.1% (edible tissues). At day 30, the residual content of PSTs in the hepatopancreas of mussels was approximately 1 400 μg STXeq/kg in the high-density group and 600.0 μg STXeq/kg in the low-density group. Changes in the proportion of each PST component were transferred from A. catenella to M. unguiculatus. The proportion of C2 was significantly reduced from 74.1% (A. catenella) to 22.6% (high-density group) and 17.1% (low-density group) (P<0.05); the percentage of C1 increased from 10.6% (A. catenella) to 54.1% (high-density group) and 54.0% (low-density group) (P<0.05). No significant difference was observed in the percentage of GTX5 between A. catenella and mussels in the different density groups (P>0.05). No significant conversion was observed between the PST components in the hepatopancreas of mussels throughout the experiment. Our data indicate that the daily accumulation rate of PSTs in M. unguiculatus was lower than that in other mussels. Moreover, the toxin elimination rate was higher than that of other mussels. A negative correlation was observed between the filtration rate of M. unguiculatus and the PST content of each tissue type. These results show that M. unguiculatus is more sensitive to PSTs than other mussels. During the stage of PST transfer from A. catenella to M. unguiculatus, a high proportion of C2 toxin was converted to C1 toxin. After accumulating in the hepatopancreas, the PST profile exhibited relatively stable performance. In summary, we conclude that, due to higher susceptibility to toxins and lower conversion rates, a lower risk is associated with the consumption of M. unguiculatus than with that of other mussels. Our findings will contribute to improving our understanding of the mechanisms underlying the PST accumulation risk in M. unguiculatus and provide valuable scientific insights for developing prevention and risk management strategies concerning PSTs.

Abstract:Oncorhynchus mykiss is a high-quality, edible fish with high nutritional and economic value. Its tender meat, full elasticity, delicious flavor, and rich nutritional value make it favorite among consumers. However, owing to its high water and fat content and high enzymatic activity, O. mykiss is highly susceptible to spoilage during storage, transportation, and sale. To study the effects of different pretreatment methods on the quality of O. mykiss during storage and slow its decay rate, experiments were conducted with two pretreatment groups: Cold saltwater group (salt content 3%, temperature 4 ℃) and slightly acid electrolyzed water group (pH 6.35, ORP 900 mV, ACC 50 mg/L, room temperature). Different pretreatment times were set for each group: 20 min, 40 min, and 60 min of cold saltwater treatment and 5 min, 10 min, and 15 min of slightly acid electrolyzed water treatment. TVB-N value, total bacterial count, water holding capacity, cooking loss rate, pH value, chromaticity, and sensory evaluation were measured as indicators, and untreated O. mykiss meat was used as a control to compare the changes of the quality of O. mykiss meat during a 9-day storage experiment at 4 ℃. The results showed that the optimal pretreatment times were 60 min for the cold saltwater pretreatment and 10 min for the slightly acid electrolyzed water pretreatment. Both preprocessing methods can effectively maintain the freshness of O. mykiss and delay its spoilage. Compared with the cold saltwater pretreatment, the slightly acid electrolyzed water treatment better inhibited the total bacterial count and TVB-N generation of O. mykiss, maintaining its water-holding capacity and delaying the rate of change of pH, chromaticity, and sensory properties during storage. Cold saltwater pretreatment can extend the refrigerated shelf life of O. mykiss by 1 day, whereas slightly acid electrolyzed water pretreatment can extend the shelf life by 2 days. Overall, pretreatment with slightly acid electrolyzed water for 10 min can effectively improve the preservation quality of O. mykiss stored at 4 ℃. Slightly acid electrolyzed water has enormous potential for applications in the preservation of aquatic products. Its unique sterilization effect and safety make it preferred for aquatic product preservation. Slightly acid electrolyzed water can effectively inhibit the growth of microorganisms in aquatic products, extend shelf life, and maintain the freshness and taste of aquatic products. In the future, with advancements in technology and increasing awareness of food safety among consumers, slightly acid electrolyzed water will play an important role in the preservation of aquatic products, providing strong guarantees for the quality and safety of aquatic products.