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Abstract:DNA methylation is an important epigenetic regulatory mechanism in organisms that regulates genome stability through chromosome and protein structures without altering gene sequences. DNA methylation has been applied in the fields of medicine, agriculture, forestry and animal husbandry, and has attracted great attention in the field of fish genetics and fish breeding. Methyl groups are transferred to cytosine residues by specific DNA methyltransferases in fish DNA molecules, such as DNMT3, and the existing DNA methylation cell patterns are maintained by the methylation maintenance enzyme, DNMT1. Finally, the methyl group is removed by the oxidation of ten-eleven translocation dioxygenase (Tet1/2/3). DNMT2 catalyzes the transfer of methyl groups from the cofactor S-adenosylmethionine (SAM) to carbon 5 of the cytosine residues of the cytoplasmic tRNA—SAM is also converted to S-adenosine homocysteine. These DNA methylated transferases are widely present in many cells and tissues and play an important role in fish. DNA methyltransferase catalyzes the transfer of methyl groups from SAM to biomolecules (DNA, RNA, proteins, and small molecules) in vivo. There are many species of fish DNA methyltransferases, including two homologous DNMT1 enzymes (DNMT1a and DNMT1b), one DNMT2 enzyme, and eight homologous DNA methyltransferase 3. The naming of DNA methyltransferase 3 homologous genes is complicated; however, they are all parologous genes of DNMT3a and DNMT3b. Demethylation refers to the demethylase-mediated removal of methyl groups from DNA, which plays a key role in gene expression regulation, cell differentiation, embryonic development, and disease occurrence and development. Demethylation refers to the removal of methyl groups from DNA by demethylases. DNA demethylation patterns can be divided into passive and active DNA demethylation patterns. In passive demethylation, methylated DNA undergoes demethylation in successive replication cycles by inactivation or nuclear rejection of DNA methylation transferases that maintain methylation patterns, as well as ubiquitin-like proteins containing PHD and RING finger domains1 (uhrf1). In active DNA demethylation, methylcytosine is first oxidized by TET1/2/3 and then excised by thymine DNA glycosylase. During this process, DNA 5-methylcytosine is oxidized to 5-hydroxymethylcytosine. These oxidation products act as intermediates in DNA demethylation and are replaced by unmodified cytosines to achieve demethylation. The biological function of DNA methylation in fish is similar to that observed in other organisms, such as mammals, and is involved in gene regulation and cell development. DNA methylation occurs in three C environments: CG, CHG, and CHH (where H is any basic group other than G). DNA methylation occurs primarily at the CG site and allows fish to precisely regulate gene expression and adapt to different environmental factors. Differential methylation—cytosine-phosphate-guanine sites—is involved in apoptosis, epigenetic regulation, autophagy, collagen metabolism, cell membrane function, and homeobox protein generation through gene expression regulation. DNA methylation leads to changes in DNA conformation and stability, and the manner in which DNA interacts with RNA (or proteins) to control gene expression. It can interact with its binding proteins to inhibit gene expression in fish. DNA methylation affects genome expression regulation by activating or inhibiting transcription at the transcriptional level. Methylation near the transcriptional initiation site blocks initiation, but in the gene body it does not block and may even stimulate transcriptional elongation. It plays an important role in fish biological functions—gene expression regulation, embryonic development, reproductive development, muscle growth, body color, disease, and evolution. It can also provide insights into how genes are regulated during development and how these patterns are passed on to future generations, contributing to the understanding of epigenetics. Fish are often used as model organisms in endocrine disruption studies because of their high sensitivity to environmental factors. Environmental factors—temperature, heavy metals, starvation stress, nutritional feed, and hormones—affect the regulation of DNA methylation in fish, affecting their growth, development, and overall health. Recently, DNA methylation has attracted increasing attention as an important epigenetic regulator. The pattern and biological function of DNA methylation in fish, as well as its relationship with important environmental factors, have been gradually recognized; however, knowledge of its depth and breadth are insufficient. For example: (1) because of the wide variety of fish species, their DNA methylation characteristics still need researching; (2) there are still many important epigenetic relationships between DNA methylation, and more genes associated with it need to be explored to improve the application efficiency of fish breeding; (3) the specific mechanisms of some important variations and DNA methylation levels are still unclear; (4) the genetic mechanism of DNA methylation levels in different generations is still unclear; and (5) the interaction of core regulators of DNA methylation and the regulation and differentiation mechanisms are not clear. Further studies of these scientific issues will reveal the mechanism of fish methylation regulation of growth and development and the environmental factor response mechanisms, enrich the theoretical system of fish epigenetics, and provide a theoretical basis for the application of genetic breeding.

Abstract:The abundant and widely distributed resources of small pelagic fish in marine ecosystems constitute significant targets for commercial fisheries. The Japanese anchovy (Engraulis japonicus) is a prominent representative of small pelagic fish and is considered one of the most commercially valuable species in the northwestern Pacific region. This species is crucial in the Yellow Sea marine ecosystem, serving as a vital food source for various marine predators and contributing to the area's overall biological diversity. However, the sustainability of anchovy populations is increasingly threatened by factors such as overfishing and climate change, which have led to notable inter-annual fluctuations in the catch quantities. Consequently, focusing on effectively recruitment of anchovy populations is essential for maintaining ecological balance, particularly during their early growth stages, which are critical for survival and long-term population stability. This study investigated the marine environment of the Yellow Sea and the complex internal mechanisms that affect the early growth of anchovies. By analyzing samples collected from 2016 to 2018, we used otolith microstructural analysis, a method that allows for precise tracing of hatching dates and the evaluation of growth patterns over time. Furthermore, we employed mixed-effects models to explore the relationships between intrinsic factors, specifically age and maternal effects, and the early growth of anchovies. The distance measured from the otolith core to the annulus, representing complete absorption of the yolk sac, served as an indicator of growth status during the yolk sac stage, providing a quantifiable measure of the maternal effect on the early growth of anchovy larvae. To assess the influence of environmental factors on early growth, we applied a combination of gradient forest models and generalized additive models (GAM). The selected environmental factors included sea surface temperature (SST), mixed layer depth (MLD), northward sea water velocity (V), eastward sea water velocity (U), sea surface height (SSH), sea surface salinity (SSS), and chlorophyll-a concentration (CHL). This analysis identified the three most significant environmental factors influencing early growth. By classifying anchovy larvae into two distinct age groups (˂ 15 days and 15–40 days old), we used sliding windows to examine the lagged effects of environmental factors on different age stages. A relative time window was integrated into our analysis, and to mitigate the impact of random occurrences among candidate environmental factors, we conducted 500 randomization trials. The environmental factors that showed lagged effects were incorporated as fixed external effects into the best fixed internal effects model for further analysis, which aimed to predict their impact on early growth. Our study indicated a close relationship between larval growth and intrinsic factors. Specifically, the daily increment of anchovy otoliths was positively correlated with age, revealing a gradual flattening of growth trends as age increased. This supports the "bigger is better" hypothesis, suggesting that larger individuals tend to have lower mortality rates, enhanced predation capabilities, and improved predator evasion. Moreover, the results showed a linear positive correlation with maternal effects, underscoring the notable influence that mothers have during the initial growth phase of anchovies. The "maternal effect" hypothesis posits that, compared to first-time spawners under average conditions, repeat spawners and physiologically superior females produce larger eggs, which provide more nutrition and energy during the early growth stages, thereby promoting growth. Despite quantifying the influence of intrinsic factors on early growth, the underlying mechanisms behind these effects remain unclear and warrant further exploration. The study identified SST, MLD, and SSS as the three most critical environmental factors affecting early growth. However, their relative importance varied by month, indicating different regulatory mechanisms for anchovies hatching in the same area during different times of the year. This study found that the lag effect of environmental factors on otolith growth was approximately one week. Among the three environmental factors analyzed, SST was the only factor exhibiting a lag effect, which varied with the age of the anchovy. For larval fish younger than 15 days, SST had a one-day lag effect on growth; for those younger than 40 days, the lag effect extended to two days. The relatively short lag time of SST on early growth in this study may be attributed to a previous study where anchovies reached an age of 90 days, whereas the maximum age in this study was 40 days. The high sensitivity of early larval fish to environmental changes resulted in a slightly longer lag effect of SST on 40-day-old anchovies compared to those that are 15 days old. This indicates that as the age of the anchovy increases, its physiological state may prolong the response time to environmental changes. Further predictive analysis suggests that the early growth of anchovies increases with increasing sea surface temperatures. Additionally, within the same temperature range, otolith increment widths for anchovy larvae aged 5–40 days were greater than those for larvae younger than 15 days. However, as indicated by the Generalized Additive Model (GAM) analysis, the relationship between SST and growth often becomes complex due to the synergistic influence of various factors. Therefore, future research should adopt an energy balance perspective to comprehensively elucidate the growth mechanisms of anchovies. This study integrates several models in the investigation of early growth in anchovies, providing a scientific basis for early growth and laying a foundation for the conservation and scientific management of anchovy resources in the Yellow Sea from the perspective of internal and external factors affecting early life history stages.

Abstract:Sciaenidae species are economically important in China and contribute to the coastal fisheries industry. They are also key species in many aquatic ecosystems, occupying important food chain and ecosystem positions. However, resources of Sciaenidae species such as Pseudosciaena crocea and Collichthys lucidus have recently declined significantly owing to the combined effects of overfishing, adverse climate change, and marine environmental pollution. Studies on the ecological niche and interspecific relationships of Sciaenidae species are key to understanding their species and population dynamics. Such studies can reveal the ecological positions occupied by Sciaenidae species, their resource utilization, and their interactions, which can serve as a basis for formulating corresponding conservation and management measures. Currently, studies on Sciaenidae species in the offshore waters of southern Zhejiang Province mainly focus on the biological characteristics, age, growth, and feeding of individual fish species; however, a fundamental gap exists in studies on the interspecific relationships of sciaenid fishes. This hinders the conservation and management of these economically important fishery resources. To comprehensively investigate the ecological niche characteristics and interspecific interactions of Sciaenidae species in the offshore waters of southern Zhejiang Province, this study used data from four quarterly fishery resource surveys conducted from May 2020 to January 2021. Ecological niche measurements, cluster analysis, chi-square test, association coefficient, and redundancy analysis were employed to examine the temporal and spatial ecological niche characteristics and interspecific associations of Sciaenidae species in the area. The survey identified nine species of Sciaenidae species, with temporal niche widths ranging from 0.17 to 1.08. Pseudosciaena polyactis had the widest temporal niche width (1.08), followed by C. lucidus at 1.02, whereas Pennahia macrocephalus had the narrowest at 0.17. Spatial niche widths ranged from 1.40 to 2.50, with Atrobucca nibe exhibiting the widest spatial niche width, followed by P. polyactis (2.24), and C. lucidus had the smallest. The spatiotemporal niche widths varied from 0.32 to 2.42, with the P. polyactis exhibiting the largest combined niche width (2.42), which was significantly greater than the other eight species, followed by the Penahia argentata at 1.65. P. macrocephalus had the smallest at 0.32, which was significantly less than the other eight species. These results indicated that P. polyactis had a larger niche width across all three dimensions and was competitively advantageous. In contrast, P. macrocephalus and A. nibe were more affected by the temporal dimension, particularly P. macrocephalus, which was at a competitive disadvantage. Cluster analysis categorized Sciaenidae species into narrow, medium, and wide niche groups based on temporal, spatial, and spatiotemporal. Overall, the niche overlap of this area was low. Regarding time, 30.56% of species pairs had temporal niche overlap above 0.600, 25.00% between 0.300 and 0.600, and 44.44% below 0.300. The highest overlap values were between P. macrocephalus and A. nibe and between Pennahia argentata and Johnius distinctus, both at 0.998, whereas the overlap between the P. macrocephalus and the Miichthys miiuy was the lowest, at only 0.007. In terms of space, most species pairs had low overlap values, with 72.22% below 0.300, 22.22% between 0.300 and 0.600, and only M. miiuy and A. nibe (0.659), as well as P. macrocephalus and P. argentata (0.616), had higher spatial niche overlap values above 0.600. Regarding spatiotemporal, all species pairs had low overlap values, with no pair exceeding 0.600, only 13.89% between 0.300 and 0.600, and the remaining 86.11% below 0.300, indicating that species can coexist through differentiation strategies. The overall association among Sciaenidae species in the area showed a significant positive correlation. The chi-square tests revealed an extremely significant positive correlation only between P. argentata and A. nibe, whereas other species pairs showed non-significant levels of association. Association coefficients indicated that more than half of the species pairs tended to be independent, with only 11.11% showing a high degree of positive association and another 25% exhibiting negative associations. The percentage of co-occurrence indicated that only 8.33% of species pairs had the strongest positive association, whereas the rest (91.67%) had moderate or lower levels of positive association, suggesting that most species pairs distribute independently of each other. This indicates that although a strong overall association exists among Sciaenidae species in the area, implying a certain level of interdependence, the association between species pairs gradually decreases, trending towards an independent distribution pattern. Redundancy analysis results suggest that surface water temperature and salinity are the main environmental factors that affect the distribution of sciaenid fishes in the area, with salinity having the greatest impact.

Abstract:The mariculture industry has rapidly developed in recent decades owing to population growth and the increasing demand for seafood. Phytoplankton is an important indicator for assessing the carrying capacity of bivalve mariculture as well as a limiting factor for large-scale and high-density bivalve cultivation. Phytoplankton can be categorized by size into picophytoplankton (<2 μm), nanophytoplankton (2–20 μm), and microphytoplankton (20–200 μm). Considering that the retention rate of picophytoplankton by filter-feeding bivalves is very low, using the total amount of phytoplankton available for aquaculture capacity assessment would result in an overestimation. Therefore, understanding the particle size composition and spatiotemporal distribution characteristics of phytoplankton in the target sea area can improve the accuracy of assessing bivalve culture capacity and provide scientific guidance for marine bivalve aquaculture. Blue mussel Mytilus edulis and triploid oyster Crassostrea gigas are the most common bivalve species in Haizhou Bay, a typical bivalve mariculture area in China. This study aimed to understand the size-fractionated phytoplankton and its environmental influencing factors, including water temperature, salinity, dissolved oxygen, pH, total nitrogen, total phosphorus, phosphate, silicate, nitrate, nitrite, ammonia nitrogen, dissolved inorganic nitrogen, N/P, N/Si and Si/P, both in bivalve mariculture areas (Area 1 and 2) and non-mariculture areas (channel and reference areas). Phytoplankton biomass was investigated in March, July, September, October, and December 2023 by measuring Chl-a of size-fractionated phytoplankton. Two-way variance and redundancy analyses were used to analyze the effects of environmental factors on size-fractionated phytoplankton. (1) The annual variation range of total Chl-a concentration in the investigated area was 0.86–18.49 µg/L, and the seasonal difference was significant (P < 0.05). The annual ranges of pico Chl-a, nano Chl-a, and micro Chl-a concentrations were 0–0.90, 0.13–6.12, and 0.35–15.30 µg/L, respectively, with significant seasonal differences (P<0.01). However, no significant differences were observed in the concentrations of Chl-a in March (1.98±0.61 µg/L) and December (3.69±1.55 µg/L). In July and October, the average concentrations of Chl-a in Area 1 were (9.80±2.04) µg/L and (12.34±6.27) µg/L, respectively, which was much higher than those in other areas (P<0.05). This may be due to the higher nutrient concentration in the coastal waters. In September, the Chl-a concentration in Area 2 (1.47–1.94 µg/L) was significantly lower than in the non-bivalve mariculture areas (P<0.01). Simultaneously, the nitrate and nitrite concentrations in Area 2 were significantly higher than in the other areas. This period marked the rapid growth of bivalves and was presumed to be caused by bivalve feeding and excretion. (2) Phytoplankton communities exhibit notable spatiotemporal variation. In March, the phytoplankton communities of Area 1 and the channel area were dominated by microphytoplankton, whereas Area 2 and the reference area were dominated by nanophytoplankton. In July, nanophytoplankton dominated in Area 1 and the channel area, whereas microphytoplankton dominated in Area 2 and the reference area. In September, microphytoplankton dominated Area 1, Area 2, and channel area, whereas nanophytoplankton dominated the reference area. The proportion of picophytoplankton in the reference area was significantly higher than that in other areas (P<0.05). In October, the contribution rate of microphytoplankton increased gradually in all areas, and the value added in bivalve mariculture areas was significantly higher than that in the non-bivalve mariculture areas (P<0.05). In December, no significant difference was observed in the contribution rate of particle size in different areas, but the contribution rate of microphytoplankton continued to increase across different areas, with an average value of 89.01%. The contribution rate of picophytoplankton was mainly affected by seasonal factors (P<0.001), whereas the contribution rate of nanophytoplankton and microphytoplankton was significantly affected by seasonal and seasonal and regional interactions (P<0.05). (3) Seasonal and regional differences exist in the response of the particle size structure of phytoplankton to environmental factors in the survey areas. The redundancy analysis showed that the first two axes explained 79.31%, 86.94%, 88.35%, and 99.09% of the species variation in Area 1, Area 2, channel area, and reference area, respectively. Seasonal and regional differences influenced the response of the particle size structure of phytoplankton to environmental factors in the four areas. A significant negative correlation was observed between nanophytoplankton and N/Si in Area 1. In Area 2, picophytoplankton was significantly negatively correlated with total nitrogen, and nanophytoplankton was significantly positively correlated with N/P. In the channel area, the phytoplankton of three sizes were significantly negatively correlated with ammonia nitrogen. Microphytoplankton was positively correlated with DIN in the reference area (P<0.05). Consequently, nutrient salts mainly regulated the Chl-a concentration of phytoplankton in Area 1, whereas the effects of nutrient salts and cultured bivalve influenced the Chl-a concentration of phytoplankton in Area 2. For the entire survey area, seasonal changes in environmental conditions are the main cause of phytoplankton particle size structure variation. In addition, the presence of seasonal and regional interactions in nanophytoplankton and microphytoplankton suggests that bivalve farming may also affect the size-fractionated phytoplankton.

Abstract:“Hao Gen” is a common name for a small fish inhabiting the tidal flat of Jiaozhou Bay of the Yellow Sea. It is a target catch of a traditional subsistence fishery in Qingdao (Shandong, China), focusing on a single fish species, and is a famous specialty seafood of Jiaozhou Bay. However, the taxonomic classification and scientific name of “Hao Gen” have long been unclear. Recently, the catch of “Hao Gen” has continuously declined, indicating that its resources may be under threat. In this study, taxonomic identification using morphological methods and DNA barcoding technology was conducted to clarify the species identification and distribution information of “Hao Gen”. Ten “Hao Gen” fish samples were collected from a traditional fishing area of the tidal flat in the northern part of Jiaozhou Bay in April 2023. In the morphological study, seven countable traits (spine and ray numbers of dorsal fin and anal fin, pectoral fin ray numbers, longitudinal scales, rows of scales, scales before dorsal fin, and vertebrae numbers) and eight measurable characteristics (body length, body depth, head length, snout length, eye diameter, interorbital space, and caudal peduncle length and height) were selected and used for the quantitative analysis of samples. By comparing with historical literature records, the species was preliminarily identified as Acanthogobius elongatus (Fang, 1942). We examined the DNA barcode fragment with 542 bp of the mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) gene of all samples. Six haplotypes were obtained, and all sequences were submitted to the GenBank database (Accession numbers: PQ407566–PQ407571). BLAST searches were performed to identify similar sequences in the GenBank. The comparative analysis and species identification also included homologous sequences from closely related species. Phylogenetic relationships among eight species of the family Gobiidae were reconstructed by MEGA Ⅹ using the maximum likelihood (ML) method based on the best-selected model Jukes-Cantor. The net genetic distance was calculated based on the Kimura 2-parameter (K2P) model. The results indicated that all DNA barcode sequences of the “Hao Gen” samples showed a high degree of similarity to the homologous sequences of the species A. elongatus, and the genetic distance between them was at the intraspecific level. In the phylogenetic tree, all the “Hao Gen” samples clustered with DNA barcode sequences of A. elongatus, published in GenBank. The results of morphological and DNA barcoding analysis were consistent, indicating that the “Hao Gen” samples could be identified as A. elongatus. According to the literature, the species of A. elongatus is distributed along the coast from Liaoning to Zhejiang in China, as well as the west coast of the Korean Peninsula, and it often inhabits the brackish waters of river estuaries. The species is classified as vulnerable and included in the “China Red List of Species”. In this study, the effective identification of “Hao Gen” fish and the first record of A. elongatus in Jiaozhou Bay address the long-standing issue of limited scientific understanding of the subsistence fishery catch species. These findings enhance the knowledge of fish diversity in Jiaozhou Bay and provide a scientific basis for the conservation of fishery resources and improving fisheries management in the area.

Abstract:Liparis tanakae is a dominant species of the Yellow Sea ecosystem, and its biological characteristics—such as high seasonal variations in population structure, rapid growth, relatively short lifespan, and pronounced response to environmental changes—substantially contribute to population maintenance mechanisms. To date, relevant ecological studies of L. tanakae have mainly focused on seasonal and interannual changes in its spatial distribution, feeding ecology, genetic classification, and osteological and myological characteristics. However, research on its reproductive ecology remains insufficient, limiting our scientific understanding of the mechanisms underlying its dominance. As a pivotal life history phase, reproduction is essential for the survival and persistence of fish populations. Furthermore, the reproductive capabilities of species form the basis for broader ecological adaptations and success within their environments. To investigate the biological reproductive characteristics of L. tanakae, histological studies of the ovaries at various developmental stages were conducted using paraffin sections and hematoxylin and eosin staining. Samples were obtained from fishery resource surveys conducted in June and August 2022 and October 2023 in the Yellow Sea as well as during the winter (January and February 2022, December 2023, and January 2024). In total, 71 specimens were collected, 50 of which were used for ovary sample sectioning. The morphology and diameter distribution characteristics of oocytes at different developmental stages were described in detail, as well as the spawning type. The results showed that oocyte development could be divided into six phases. In phase Ⅰ, the oocyte is just differentiated from the oogonium, with a small cell volume and relatively large nucleus; the oocyte in phase Ⅱ enlarges, and a follicle membrane appears around the cells; in phase Ⅲ, yolk vacuoles start to appear in the cytoplasm of the cell and yolk begins to accumulate; zona radiate appears between the cytoplasm and follicle membranes, and the follicle membrane becomes two-layered; in phase Ⅳ, yolk begins to fill up the yolk vacuoles, gradually forming yolk platelets; in phase Ⅴ, yolk merges into a single large yolk ball, and the nucleus dissolves; in phase Ⅵ the oocytes degenerate, the yolk is gradually absorbed to form a cavity, and some oocytes are irregular in shape. Ovary development can also be divided into six stages, and there are obvious differences in oocyte composition at different ovarian development stages. In stage Ⅱ, the ovary is mainly composed of oocytes in phase Ⅰ and Ⅱ with the percentages of 34.92% and 65.08%, respectively. In stage Ⅲ, the ovary is mainly composed of oocytes in phase Ⅱ (49.47%) and Ⅲ (34.73%). In stage Ⅳ, the percentage of oocytes in phase Ⅲ account for 51.55%, whereas that of oocytes in phase Ⅳ increase to 32.99%. In stage Ⅴ，the ovary is mainly composed of oocytes in phase Ⅲ (34.62%) and Ⅳ (32.28%). In stage Ⅵ, the percentages of oocytes in different phases are relatively uniform, with some degraded oocytes and empty follicles. The egg diameter in stages Ⅳ and Ⅴ exhibited a unimodal distribution with the dominant groups between 0.90–1.00 and 1.70–1.80 mm, respectively. The egg diameter in stage Ⅵ showed a bimodal distribution, with dominant groups between 0.50–0.70 and 1.10–1.20 mm, and high oocyte proportions in the small-growth and degraded phases were observed. Compared to previous results (1985–1986 and 2011–2012), our study (2022–2024) revealed a notable increase in both the distribution range of egg diameters and the proportion of large egg diameter groups. Hatchability was positively correlated with the egg diameter, and an increase in the egg diameter of L. tanakae improved the hatching rate. This may be an adaptive response of L. tanakae to the multiple pressures from a rapidly changing external environment, caused by climate change and fishing activities. In addition, other economically important fish species in the Yellow Sea ecosystem, such as Larimichthys polyactis and, Gadus macrocephalus have declined in the past few decades. This has decreased the inter-specific competition pressure faced by L. tanakae to some extent, and the relatively abundant prey environment might have contributed positively to the egg diameter increase of L. tanakae. The results indicate that L. tanakae ovaries exhibited considerable developmental potential and sustainability, and we consider L. tanakae to be a non-synchronized multiple batch spawner. Our study reveals the developmental characteristics of L. tanakae ovaries and provides a theoretical reference for further enrichment of the reproductive ecology of this species.

Abstract:Recently, the rapid development of the Chinese coastal economy and the expansion of aquaculture have led to an increased reliance on plastic materials within marine aquaculture systems. These materials, such as impermeable membranes, fishing nets, buoys, cages, and ropes, are extensively used for their beneficial properties. They are lightweight, durable, and cost-effective. However, the large-scale production and extensive use of plastics in aquaculture have increased concerns about their potential environmental impacts, particularly regarding the pollution of the marine environment and the associated risks to ecosystems. The primary concern is the plastic additives that are incorporated into these materials. These additives, including organophosphate esters (OPEs), are often physically mixed into the polymer matrices rather than chemically bonded. This physical integration increases their susceptibility to release into the surrounding environment through various processes, such as volatilization, leaching, abrasion, and dissolution, during the product’s lifecycle. Despite these risks, the mechanisms and patterns of additive release, as well as the exposure risks presented to marine organisms, remain poorly understood. This study focused explicitly on OPEs, a common group of plastic additives, to investigate their presence and behavior in plastic impermeable membranes used in marine aquaculture ponds. The study aimed to (1) identify the types and concentrations of OPEs present in the plastic impermeable membranes collected from marine aquaculture ponds, (2) simulate the dynamic dissolution of these OPEs in artificial seawater, and (3) evaluate the bioaccumulation potential of these OPEs in aquatic organisms, using Artemia as a model species. We collected plastic impermeable membranes from wild marine aquaculture ponds and analyzed their OPE content using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Our analysis revealed the presence of seven different types of OPEs, which included three aliphatic OPEs (TnBP and TiBP), one aromatic OPE (TCP), and three chlorinated OPEs (TCEP, TDCPP, and TCPP). TCPP was the most abundant, with a concentration of (395.15±48.05) ng/g, accounting for 63.12% of the total OPEs detected in the membranes. TnBP followed, exhibiting concentration of (52.96±5.25) ng/g. In contrast, TCP had the lowest concentration, contributing only 0.33% to the total OPEs. To understand the dissolution behavior of OPEs in these membranes, we conducted a 240-h laboratory simulation. The impermeable membranes were submerged in artificial seawater with a salinity of 30, and water samples were collected every 48 h for analysis. The OPE concentrations in these water samples were determined using solid-phase extraction (SPE) followed by HPLC-MS/MS. At the end of the experiment, the total amount of OPEs dissolved from the membranes reached 12.24 ng/g. Chlorinated OPEs exhibited the highest dissolution rates, with TCEP showing a dissolution rate of 40.6%. The general dissolution order was chlorinated OPEs > aromatic OPEs > aliphatic OPEs. Notably, the dissolution rate was highest during the first 48 h of the experiment and then gradually decreased over time. By approximately day 6, the dissolution rate reached a near-equilibrium state, likely due to the depletion of OPEs from cracks and pores on the membrane surface. At this stage, the membranes had likely reached a dynamic equilibrium between the release and adsorption of OPEs. In addition to the dissolution experiments, we assessed the bioaccumulation potential of OPEs in Artemia, a species commonly used in marine studies. Plastic impermeable membranes were added to artificial seawater at concentrations that mimicked those typically found in marine aquaculture ponds. After exposure, Artemia and the surrounding water samples were collected, and the OPE concentrations were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Five OPEs—TCP, TDCPP, TnBP, TCPP, and TiBP—were detected in Artemia. We evaluated the degree of bioaccumulation using the bioaccumulation factor (BAF). The results indicated that TCP and TDCPP had significant bioaccumulation potential, with logBAF values greater than 3.7. TnBP exhibited a moderate bioaccumulation effect, with a logBAF between 3.3 and 3.7. However, TCPP and TiBP showed no significant bioaccumulation potential, as their logBAF values were below 3.3. In conclusion, our study identified six distinct OPEs in the plastic impermeable membranes used in marine aquaculture, with chlorinated OPEs being the most prevalent. The results of the dissolution experiments revealed that OPEs are initially released rapidly from surface cracks and pores of the membranes, followed by a slower release as equilibrium is reached. Furthermore, the bioaccumulation experiments demonstrated that certain OPEs, particularly TCP and TDCPP, present bioaccumulation risks to marine organisms such as Artemia. These findings underscore the importance of managing plastic additives in marine aquaculture systems to mitigate the potential environmental impacts of plastic waste and its associated contaminants. The study provides valuable insights that can inform future strategies for reducing the ecological risks of plastic additives in marine environments.

Abstract:Since the 1980s, China's aquaculture industry has experienced rapid development and sustained growth in aquaculture production, not only providing large quantities of high-quality protein for humans but also becoming an important supplement to ensure global food security. The salinity adaptation range of Penaeus vannamei is extensive; therefore, it can be cultured in both saline and freshwater environments. China has 690 million acres of saline water resources, offering considerable potential for the aquaculture of P. vannamei. The ongoing aquaculture boom incurs environmental costs, while providing protein to the fast-growing human population. Carbon emissions are generated during aquaculture; thus, assessing the carbon footprint of seafood aquaculture is essential for establishing targeted emission reduction and carbon sink enhancement strategies, vital for achieving the "Carbon Peak and Carbon Neutrality" goals. The carbon footprint is defined as the total amount of greenhouse gases emitted, either directly or indirectly, by an individual, organization, event, or product throughout its life cycle and is typically expressed in terms of CO2 equivalents (CO2e). This metric provides a comprehensive and intuitive understanding of the environmental impacts of human activities and facilitates the development of targeted emission reduction and carbon sink enhancement strategies. The most commonly employed method for carbon footprint accounting is the Life Cycle Assessment (LCA), which consists of four key parts: (1) target and scope definition, (2) inventory analysis, (3) impact assessment, and (4) interpretation of results. Here, the carbon footprint of P. vannamei pond culture was assessed using the "gate-to-gate" LCA methodology, based on primary data from on-site monitoring of culture ponds and aquaculture enterprises in Shandong Province. The carbon footprint of producing 1 kg of P. vannamei was 5.60 kgCO2e, with total carbon emissions of 7.69 kgCO2e. These emissions primarily stem from the use of chicken manure, lime, and feed, whereas the net carbon uptake by the pond ecosystem—attributed to the abundance of various algal species—is 2.09 kgCO2e. Notably, carbon emissions from the aquaculture process alone reached 7.42 kgCO2e, constituting 96.5% of total emissions, while pond construction and decommissioning emissions were only 0.27 kgCO2e, accounting for 3.5%. Among the sources of carbon emissions, chicken manure used for fertilization represented the largest share, contributing 5.62 kgCO2e and accounting for 73.0% of the total emissions during the shrimp culture phase. This was followed by compounded feeds, contributing 0.87 kgCO2e and representing 11.3% of the total emissions, while quicklime accounted for 10.7% of the total emissions at 0.82 kgCO2e. Additionally, diesel fuel and materials such as electricity and polyethylene contribute ~5% of the total carbon emissions. The predominant farming methods of P. vannamei in China include factory farming, semi-intensive ponds, small-shed cultures, and pond aquaculture. Existing research analysis indicates that the carbon footprint of 1 kg of P. vannamei farmed year-on-year with different farming methods are in the following order: large surface culture (5.60 kgCO2e) < small shed culture (18.25 kgCO2e) < semi-intensive ponds (52.3 kgCO2e) < factory farming (198 kgCO2e). Therefore, it is necessary to explore a win-win aquaculture model for economic and ecological benefits from a multidimensional perspective of green, sustainable, and efficient aquaculture. Under equivalent intake conditions, substituting seafood products with P. vannamei with low-carbon emissions can effectively reduce overall carbon emissions. Several recommendations have been proposed to mitigate emissions from P. vannamei aquaculture in ponds with large water surfaces. First, the excessive use of chicken manure as fertilizer—as indicated by the pond monitoring results—should be moderated to optimize application rates. Currently, farming enterprises often apply chicken manure without adequate scientific guidance, necessitating further research to refine fertilization practices. Second, problems such as less-refined culture techniques and lower bait utilization exist during large water surface cultures. Therefore, it is necessary to improve shrimp survival, reduce carbon emissions through refined pond culture management, and optimize feeding strategies and bait structures. Moreover, the recapture rate of shrimp in large surface cultures is currently <30%, which can be improved by adding filter-fed shellfish to shrimp ponds. This approach would not only reduce carbon emissions associated with organic pollutants but also enhance the efficiency of material cycling in aquatic ecosystems and improve water quality, ultimately benefiting shrimp survival rates. Finally, establishing and refining P. vannamei carbon footprint labeling can guide the green consumption market and promote sustainable development of green industries. Consequently, scientific fertilization, precision feeding, and algae-shellfish integrated aquaculture are critical for achieving emission reduction and enhancing carbon sinks in large surface culture systems of P. vannamei. This study supports carbon accounting for P. vannamei pond aquaculture.

Abstract:In the face of growing concerns over global climate change and carbon emissions, the study of marine carbon sinks has garnered significant attention. Shellfish aquaculture offers a promising pathway for carbon sequestration in marine ecosystems. Therefore, the determination of shell carbon content and the study of shell carbon degradation are critical approaches to understanding the carbon sink potential of shellfish farming. Filter-feeding shellfish not only achieve long-term carbon sequestration through shell calcification but also significantly impact the marine carbon cycle through their physiological activities and post-mortem degradation processes. Filter-feeding bivalves, as a crucial group in coastal aquaculture ecosystems, play a substantial role in carbon sequestration by filtering particulate organic carbon, such as phytoplankton and organic debris, during their growth processes. Additionally, they incorporate inorganic carbon into their shells, indirectly contributing to the enhancement of carbon sink functionality. The carbon content of bivalve shells in marine aquaculture accounts for 63.44%–92.64% of their total biomass carbon. The degradation of organic matter and the rate of organic carbon breakdown within the shells are also key factors determining the potential for long-term carbon sequestration in the shells. However, little is known about the carbon content in different bivalve species and various parts of their shells. Thus, precise measurements of shell carbon content and insights into the kinetics of organic carbon degradation are crucial for comprehending the carbon sequestration capacity of bivalves and evaluating the overall carbon sink potential of bivalve aquaculture. This study aims to accurately quantify the carbon content of shells and understand the degradation rates of shell carbon, providing fundamental data for deeper investigation into the role of shellfish in marine carbon sequestration. The following six species of filter-feeding bivalves were collected from Sanggou Bay in September 2022: Mytilus edulis, Chlamys farreri, Patinopecten yessoensis, Crassostrea gigas, Ruditapes philippinarum, and Scapharca subcrenata. An elemental analyzer was employed to measure the total carbon content and organic carbon content in different parts of the shells (edge, umbo, and entire shell). The total and organic carbon degradation rates were determined through indoor natural degradation experiments. The results indicated that the total and organic carbon contents varied among the different species. The total carbon content in the six species ranged from 11.31% to 13.37%, whereas the organic carbon content ranged from 0.53% to 2.17%. The total and organic carbon contents in M. edulis were significantly higher than those in the five other species (P<0.05), with no significant differences in total carbon content among the five remaining species (P>0.05). S. subcrenata exhibited significantly higher organic carbon content than the four other species, except for M. edulis (P<0.05). The total and organic carbon contents at the edge of M. edulis were significantly higher than those at the umbo (P<0.05), whereas the organic carbon content at the edge of S. subcrenata was also significantly higher than that at the umbo (P<0.05). No significant differences in total and organic carbon contents were found between the edge and the umbo in the four remaining species (P>0.05). The degradation rates of total and organic carbon in the shells varied by species, with higher organic carbon content leading to a faster total carbon degradation rate. The total carbon degradation rates (k445) of the six types of shells ranged from 0.021 9 a–1 to 0.080 3 a–1, with an average of (0.0511 ± 0.022 1) a–1. S. subcrenata had the highest total carbon degradation rate (k445 = 0.080 3 a–1), whereas R.phlippinarum had the lowest (k445 = 0.021 9 a–1). The degradation rates of S. subcrenata and M. edulis were significantly higher than those of the four other species (P<0.05). No significant differences in degradation rates were found among C. farreri, P. yessoensis, and C. gigas (P>0.05), whereas R.phlippinarum had a significantly lower degradation rate than the five other species (P<0.05). The organic carbon degradation rates (k445) ranged from 0.032 9 a–1 to 0.609 6 a–1. S. subcrenata had the highest organic carbon degradation rate (k445 = 0.609 6 a–1), whereas C. gigas had the lowest (k445 = 0.032 9 a–1). The degradation rate of S. subcrenata was significantly higher than those of the five other species (P<0.05), with no significant differences between C. farreri and R. phlippinarum (P>0.05) or among M. edulis, P. yessoensis, and C. gigas (P>0.05). Except for S. subcrenata, where the organic carbon degradation rate was significantly higher than the total carbon degradation rate (P<0.05), no significant differences were observed between total carbon and organic carbon degradation rates in the other species (P>0.05). In conclusion, R. philippinarum and C. gigas, as widely distributed representative species in coastal aquaculture systems, exhibit significant carbon sequestration potential. Therefore, further research and optimization of the cultivation models for these bivalves hold considerable theoretical and practical significance for enhancing marine carbon sequestration capacity and mitigating global climate change.

Abstract:Artificial reefs are increasingly used in marine ecosystems to support biodiversity and provide refuge for fish species. To maximize the effectiveness of artificial reefs, understanding the mechanisms by which fish locate and aggregate around them is essential. Sensory cues are critical in guiding fish navigation and aggregation behaviors. Vision and the lateral line system are particularly important for detecting environmental features and predators among the primary sensory modalities. However, the specific contributions of these two sensory systems and their potential interactions in guiding fish toward artificial reefs remain poorly understood. This study investigated the effects of impairing the visual and lateral line systems, both independently and in combination, on the aggregation behavior of Sebastes schlegelii, a species that relies heavily on these sensory modalities for habitat selection. This study aimed to assess whether visual and lateral line system impairments affect the fish's ability to aggregate around artificial reefs. We designed six experimental groups to explore the impacts of various impairments on aggregation behavior. The groups were as follows: (1) Normal group with no sensory impairments; (2) Visual impairment group with visual cues blocked using physical methods to destroy; (3) Mild lateral line impairment group with the lateral line system partially disrupted using a gentle treatment; (4) Visual + mild lateral line impairment group with combined visual and mild lateral line impairments; (5) Severe lateral line impairment group, with the lateral line system extensively disrupted; and (6) Visual + severe lateral line impairment group, with severe impairments to both sensory systems. Aggregation behavior was measured by observing the number of fish within a defined proximity to an artificial reef over a 12-h period, which allowed us to examine immediate and sustained responses. Our findings demonstrated that fish in the normal group exhibited the highest levels of aggregation, confirming that visual and lateral line cues are essential for guiding fish towards artificial reefs. This supports the hypothesis that these sensory systems are crucial in habitat selection and aggregation. Fish in the visual and mild lateral line impairment groups showed significantly reduced aggregation compared with the normal group, suggesting that visual and lateral line cues are critical for efficient navigation and habitat selection. Notably, fish in the severe and visual + severe lateral line impairment groups exhibited even lower levels of aggregation, indicating that more severe impairments to either or both sensory systems result in further decreases in aggregation behavior. Notably, this study found no significant interaction between the two sensory impairments. Although impairing the visual and lateral line systems (either mildly or severely) resulted in a considerable reduction in aggregation compared to impairing either system alone, the effects were additive rather than synergistic. This means that impairing the two sensory systems did not result in a compounded or exaggerated loss of aggregation behavior. Instead, the loss of aggregation behavior owing to sensory impairments occurred independently for each system. For example, fish with only visual impairments showed reduced aggregation, and fish with only lateral line impairments showed a similar reduction. The combined impairments led to a further decrease in aggregation, which was not notably greater than the expected sum of the individual impairments. These results indicate that visual and lateral line systems are crucial for fish aggregation behavior; however, impairments to these systems do not synergistically amplify the overall effect. In particular, impairing both sensory systems reduces aggregation behavior more than impairing either system alone, but the lack of an interaction effect implies that the two systems do not jointly influence aggregation in a compounded manner. This finding has implications for understanding how fish navigate toward artificial reefs and could guide the design of future reef structures. For example, artificial reefs that target the enhancement of one sensory modality (such as visual cues) may remain effective in supporting fish aggregation, as the loss of one sensory system does not appear to severely compromise aggregation behavior. However, notably, these conclusions are based on the behavior of S. schlegelii in the context of this specific study. The lack of interaction between sensory systems in this species may not apply universally to all fish species. Some species may rely more heavily on one sensory modality over another, or they may exhibit different types of sensory integration. Therefore, further studies are needed to explore the sensory preferences and behavior of other fish species in relation to artificial reefs. This study also highlights the broader ecological implications of artificial reef design. Understanding the role of sensory systems in fish aggregation can inform strategies to optimize the placement and features of artificial reefs, making them more effective in supporting marine biodiversity. In particular, this study underscores the importance of considering the sensory ecology of target species when designing artificial habitats and provides valuable insights into how sensory impairments affect fish behavior and ecological interactions.

Abstract:The Chinese spotted sea bass (Lateolabrax maculatus) inhabits the coastal regions of China, Japan, and Korea and ranges from the southern border of Vietnam to the western coast of the Korean Peninsula. This species holds considerable economic significance in China and is highly valued for its nutritional content and taste. Temperature is a crucial environmental factor influencing the biological functions of aquatic animals. Global warming and the widespread practice of intensive aquaculture have rendered high-temperature stress a major challenge for L. maculatus farming. Heat shock proteins (HSPs) are essential in cellular responses to thermal stress. Among them, HSPA5 (also known as GRP78 or Bip) is a member of the HSP70 family that plays an essential role in mitigating endoplasmic reticulum stress induced by various stressors, including temperature fluctuations, oxidative stress, and nutrient deprivation. In this study, we elucidated the molecular response mechanism of L. maculatus to high-temperature stress and focused on the role of HSPA5 in liver tissue. We cloned the hspa5 gene from the L. maculatus, performed amino acid sequence and evolutionary analyses, and investigated its expression and localization in liver tissues to clarify its role in heat stress response. To further investigate the properties and functions of HSPA5, we cloned the open reading frame (ORF) of hspa5 using the L. maculatus reference genome and conducted bioinformatic analyses. The ORF of hspa5 was 1 965 bp in length and encoded a 654-amino-acid protein. The first 1–16 amino acids formed a signal peptide. The protein was predicted to possess a molecular mass of 72.23 kDa, an isoelectric point of 4.95, and an instability coefficient of 31.01. It was characterized as a stable, hydrophilic protein without transmembrane structural domains. Sequence comparison and conserved domain analysis revealed that L. maculatus HSPA5 exhibited higher homology with bony fish than with mammals or birds and possessed a conserved domain characteristic of the HSP70 superfamily. Structural modeling indicated that the secondary structure of HSPA5 was predominantly composed of α-helices, accompanied by some irregular coiling. To investigate the response mechanism of hspa5 in L. maculatus under high-temperature stress, we analyzed its expression in liver tissue using real-time quantitative polymerase chain reaction (qPCR) under a 30 ℃ heat stress condition. The fish were acclimated in an environment with temperatures ranging from 15 to 20 ℃, salinity of 28–30, pH levels of 7.5–8.0, dissolved oxygen >5.0 mg/L, and ammonia nitrogen levels <1.0 mg/L for 2 weeks. The expression of hspa5 mRNA was measured at 0, 6, 12, 24, and 72 h after heat stress exposure. The results showed a gradual increase in hspa5 expression over time, peaking at 12 h, followed by a decline. This indicated the crucial role of hspa5 in the hepatic response to high-temperature stress in L. maculatus. To determine the cellular localization of hspa5 in response to heat stress, we performed in situ hybridization and hematoxylin-eosin (HE) staining of liver tissue sections. In situ hybridization results demonstrated that hspa5 mRNA was predominantly localized in the cytoplasm of hepatocytes and confirmed the active involvement of HSPA5 in the hepatic response to thermal stress. HE staining revealed characteristic liver tissue morphology, including distinct features (e.g., the central vein and hepatocytes), under both normal and high-temperature conditions. These findings suggest that hepatocytes are likely the primary cell type that responds to high-temperature stress and serves a protective function. Under heat stress, hepatocytes may enhance their cellular tolerance to heat injury by upregulating hspa5 expression. This increased expression may aid in alleviating liver damage induced by high-temperature stress and promote liver tissue repair. In summary, this study elucidated the expression pattern of the hspa5 gene in L. maculatus liver tissue under high-temperature stress using real-time qPCR, HE staining, and in situ hybridization. The findings enhance our understanding of the functional role of hspa5 in heat stress responses and offer a theoretical basis for the management, selection, and breeding of high-temperature-tolerant L. maculatus strains.

Abstract:Lateolabrax maculatus belongs to the order Perciformes and is distributed in the coastal waters and estuaries of China, Japan, and the Korean Peninsula. Its muscle protein contains many high-quality amino acids necessary for the human body, which have extremely high edible value. Lateolabrax maculatus has the advantages rapid growth under wide temperature and salt ranges and is suitable for various culture modes, such as cages, ponds, and factories. It is an economically important marine fish occurring in China. Light is a key environmental factor that affects the behavior and physiological and biochemical indices of fish, and optimizing the growth environment of fish by controlling different light color conditions can markedly improve aquaculture efficiency. However, it is not completely clear what kind of light color L. maculatus adapts to, and the influence of different light colors on its growth, feeding, distribution, and metabolism. To explore these effects, blue, green, yellow, and indoor natural lights were used to clarify the relationship between light color and L. maculatus growth, determining the optimal light color for its culture, and providing a theoretical basis for the optimization of artificial culture technology and environmental regulation. Four light color groups—blue, green, yellow, and natural light—were used. Two circular light strips were fixed around the bottom of each experimental pool to provide light sources. The distance between the light strips was 15 cm, and the light intensity was measured 5 cm above the water center. The light intensity was ~300 lx, and the light period was 12 h light: 12 h dark. During the experiment, the compound feed was given twice daily at 08:00 and 18:00, and the single feeding amount was 1.5%–2 % of the total weight of the sea bass in the pond. After feeding for 1 h, residual bait and feces were removed. Video monitoring equipment (Hikvision camera, Smart265) was placed above the experimental pool, and the time from the first experimental fish to the end of feeding was recorded. Feeding time was measured every five days. The distribution of L. maculatus in the experimental pond was recorded 30 min before and after feeding. The number of nodes per minute was recorded, and 60 images were captured for each process. All images of L. maculatus were manually marked according to the division area—black dots represented the location—and compared with the video to ensure the location accuracy. The fish were cultured for 45 days. The results showed that the weight growth and specific growth rates of juvenile L. maculatus under blue light were (45.70±2.20) and (0.90±0.08) (%/d), respectively and were significantly higher than those under yellow and natural lights. The feed coefficient of L. maculatus under blue light was the lowest. The relative expression levels of insulin growth factor (igf-1 and igf-2) and growth hormone receptor 1 (ghr-1) genes in the liver of L. maculatus under blue light were higher than those under natural light. The phototaxis distribution of the fish differed for different light colors, with positive and negative phototaxis for blue yellow light, respectively. Metabolomic analysis showed that the fish under blue light were significantly upregulated by metabolites such as L-isoleucine and lysophosphatidylethanolamine (LPE, 18:2/0:0). This affected nine pathways—including amino acid and glycerol phospholipid metabolism—thereby affecting amino acid and phospholipid synthesis. Under green light, fumaric acid, L- tyrosine, and other metabolites were significantly downregulated. This affected five pathways—including phenylalanine metabolism and oxidative phosphorylation—and protein synthesis and hormone secretion in sea bass. No significant enrichment was observed under yellow light. In conclusion, the relative expression levels of igf-1, igf-2 and ghr-1 in L. maculatus could be improved by blue light illumination, and L-isoleucine and LPE (18:2/0:0) contents could be significantly increased under blue light. This affects amino acid and glycerophospholipid metabolism, and other metabolic pathways, thus increasing the speed of substance synthesis in L. maculatus and significantly improving its growth. Combined with the fact that the distribution behavior of L. maculatus in culture ponds tends toward blue light, this shows that L. maculatus is suitable for culturing under blue light. These results provide a theoretical basis for the selection of light color and the formulation of a culture strategy for L. maculatus.

Abstract:Large yellow croaker (Larimichthys crocea) fish are a major marine aquaculture product in China. They mainly farmed in nearshore floating cages, but the irrational layout of nearshore aquaculture and high aquaculture density seriously affect the flesh quality. Offshore aquaculture—such as deep-water cages and offshore ships—has the advantages of a wide area and good water quality, resulting in improvement in the quality of large yellow croaker cultured in offshore systems. Nevertheless, instances exist in which large yellow croaker raised in nearshore floating cages may be misrepresented as being cultured in offshore systems, likely motivated by the higher market value associated with offshore cultivation. Therefore, it is necessary to investigate the quality characteristics of large yellow croaker cultured in different modes and screen for representative indices that can be used to distinguish between these different modes. Thus, large (500–550 g) and small (250–330 g) fish reared in deep-water cages, floating cages, and offshore ship systems were collected from October to November 2023. The deep-water and floating cages were fixed in the Sandu Bay, Ningde City, Fujian Province. In the offshore system, fish were cultured in Guoxin 1, which shifted between the Ningde and Qingdao Sea areas. After collection, the morphology, texture, basic nutrients, amino acids, fatty acids, taste profile, and volatile compound odor of the flesh were analyzed. The results showed that: (1) the morphology of large yellow croaker reared offshore was better than that nearshore with lower condition factor and higher body length/height and caudal peduncle length/height. The condition factor and body length/height ratio of the larger fish were lower than those of the smaller fish in the same culture mode. (2) The hardness, adhesiveness, and stickiness of the fish from offshore ships and deep-water cages were higher than those from floating cages of the same size. For fish from the same culture mode, the hardness, adhesion, and chewiness of the larger fish were higher than that of the smaller fish. The springiness of the smaller fish cultured in floating cages was significantly lower than that of the larger fish. (3) The crude fat content of the larger fish was lower than that of the smaller fish across different farming modes. For the larger fish cultured in floating cages, the crude fat content was significantly higher than that of fish in the other two culture modes and was lowest in the offshore ship system. Fish cultured in the offshore ship system had lower protein levels than those in the other two culture modes. In the same culture mode, the ash content of the smaller fish was lower than that of the larger fish, but the moisture contents were similar. Nutritional evaluation of amino acids suggested that the limiting amino acid of the smaller fish was isoleucine (ILE), while in the larger fish, it was MET and cysteine (CYS). The monounsaturated fatty acid content of the fish reared in offshore ships and deep-water cages was significantly higher than that of fish reared in floating cages. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels of the fish were significantly higher in deep-water and offshore ship systems than in floating cages. (4) Inosine monophosphate (IMP) contributes more to the flavor of large yellow croaker, and in the same culture mode, the IMP content of the larger fish was significantly higher than that of the smaller fish. Large yellow croaker reared offshore exhibited higher umami and sweet tastes, whereas those reared in deep-water cages showed higher richness. Large yellow croaker cultured in floating cages had higher levels of 2-methylbutyraldehyde, 2-methylpropionaldehyde, isopropylidene acetone, acetone, 2-hexanone, 2-pentanone, ethyl caproate, and methyl acetate than those cultured in the other two modes, indicating a richer volatile flavor. Isovaleraldehyde, alpha-pinene, ethyl acrylate, 2-butanone, and butanal-D contents were higher in the smaller fish than in the larger fish, indicating a more intense fruity flavor. Orthogonal partial least squares discriminant analysis identified methyl trans-9-octadecenoate, 2-butanol-M, butanal-M, umami, astringent aftertaste, richness, methionine, and cysteine as important indicators for distinguishing large yellow croaker reared under different culture modes. In summary, emerging culture modes such as offshore ships and deep-water cages are important ways to improve the quality of large yellow croaker by improving the body shape, texture, and taste.

Abstract:The circadian clock is an intrinsic timing mechanism that has evolved in organisms to adapt to the Earth’s periodic diurnal changes. Cryptochrome and Period genes are pivotal in regulating the circadian system. These form PER/CRY heterodimer complexes, which translocate from the cytoplasm to the nucleus and inhibit the transcription of CLOCK/BMAL1-driven E-box elements, thereby functioning as downstream effector genes. These two genes are also influenced by environmental cues, such as external light, which can remodel the periodic rhythm and ultimately facilitate adaptation to environmental rhythmic changes. The boring giant clam Tridacna crocea is a dominant species within the Tridacnidae family, with notable economic and ecological importance. However, this species is currently classified as a rare and protected animal owing to environmental changes, overfishing and other factors. A defining characteristic of this species is its symbiotic relationship with zooxanthellae, in which light plays a fundamental role in the giant clam-zooxanthellae symbiosis. Additionally, light is a crucial regulatory factor influencing the circadian clock. Consequently, exploring the influence of circadian rhythms on the expression of core clock genes in T. crocea can provide crucial data to support conservation efforts and breeding programs for this species. In this study, the SeqMan software was used to assemble sequences obtained through sequencing. The software SignalP 5.0 Server and SMART 4.0 were used to conduct online analysis and prediction of giant clam Cryptochrome and Period functional domains. The ExPASy Server online analysis software was used to analyze and predict the physicochemical properties of the sequences, DNAMMAN software was used to conduct multiple sequence comparison of the sequencing results, and the BLAST option of NCBI database was used to conduct homology analysis of Cryptochrome and Period sequences. The phylogenetic tree of Cryptochrome and Period was constructed and analyzed using the neighbor-joining method in MEGA 7.0. For homology modeling, I-TASSER was adopted, and Hdock was utilized to investigate the interaction mode between Cryptochrome and Period. The interaction mode of the docking results was analyzed with PyMOL 2.3.0. Furthermore, PCR technology was employed to clone and characterize the coding regions of the Cryptochrome and Period from T. crocea. Subsequently, tissue-specific expression analysis of Cryptochrome and Period was performed, and their expression levels in various tissues were quantified under different photoperiods. The results showed that the coding region of Cryptochrome has a base sequence length of 1 641 bp, encoding 546 amino acids, with a theoretical isoelectric point of 6.08 and a molecular weight of 62.98 kDa; the coding region of Period has a base sequence length of 4 386 bp, encoding 1 461 amino acids, with a theoretical isoelectric point of 6.14 and a molecular weight of 164.99 kDa. The Hdock interaction model showed that these two proteins could form heterodimers with a binding energy of –279.88 kcal/mol. Tissue expression analysis indicated that Cryptochrome and Period were expressed in all seven tissues examined, with relatively high expression levels in the outer mantle, inner mantle, gill and adductor muscle (Cryptochrome showed a high level of expression in the heart). Cryptochrome and Period genes exhibited oscillatory expression patterns that varied with the circadian cycle in the outer mantle, inner and outer mantle, gill, and adductor muscle. Under normal lighting conditions, the expression level of Cryptochrome in the outer mantle, inner and outer mantle, and the adductor muscle all reached the maximum value at 1 h of light treatment, whereas the expression level in the gill reached the maximum value at 5 h of dark treatment, and then showed a decreasing trend with increased light time. When the illumination was delayed for 2 h, the expression level of Cryptochrome in the outer mantle, inner and outer mantle, and gill all reached the maximum value at 1 h illumination. The expression level in the adductor muscle reached the maximum value at 7 h darkness treatment, and then all showed a decreasing trend with increased illumination time. Under normal light conditions, the expression level of Period in the outer mantle, inner and outer mantle, and gill reached the maximum value at 5 h of darkness treatment, whereas the expression levels in the adductor muscle reached the maximum value at 1 h of light treatment, and all showed a trend of first decreasing and then increasing with increased light time. When the illumination was delayed for 2 h, the expression level of Period in outer mantle and adductor muscle reached the maximum value at 7 h of darkness treatment, In contrast, the expression levels in inner and outer mantle and gill reached the maximum value at 1 h of light treatment, and both showed a decreasing trend with an increased illumination time. In conclusion, this study represents the initial successful cloning of two essential circadian clock genes, Cryptochrome and Period, from the species T. crocea. Our preliminary validation of the diurnal rhythmic expression patterns of these genes in key tissues provided valuable insights into the behavioral and physiological rhythms of T. crocea, as well as its mechanisms underlying light adaptation.

Abstract:Homeobox genes are ubiquitous in eukaryotic genomes and can be transcribed into a homeodomain of approximately 60 amino acids. Hox genes are currently the most studied class of homeobox genes. They are usually clustered on chromosomes and play important regulatory roles in embryonic development, cell differentiation, body pattern determination, and tissue and organ formation of organisms. In this study, the bioinformatics methods were used to identify the Hox gene family based on the genome and transcriptome data of Procambarus clarkii to comprehensively understand the distribution, evolution, and function of the Hox gene family in the genome of P. clarkii. The sequence structure, protein physicochemical properties, motif composition, phylogenetic characteristics, and adaptive evolutionary features were analyzed. The expression of Hox genes in different developmental stages of P. clarkii and different tissues of adult crayfish were studied by fluorescence quantitative PCR to explore the possible biological function of Hox genes and provide a theoretical basis for analyzing the morphological development mechanism of P. clarkii. The results showed that eight Hox genes on the same chromosome were identified in the genome of P. clarkii, namely lab, pb, Dfd, Scr, Antp, Ubx, abd-A, and Abd-B. Hox proteins are composed of a YPWM-containing motif and a highly conserved homeodomain, and the homologous domains of Hox proteins are highly similar among different species. The physicochemical properties analysis of Hox proteins showed that the amino acid sequence length of Hox proteins ranged from 278 to 608, the molecular weight ranged from 31 130.52 to 64 355.33 Da, and the isoelectric point ranged from 6.73 to 9.51. All Hox proteins are hydrophilic, unstable proteins that are located in the nucleus. Motif composition analysis showed that the Hox genes clustered into the same cluster were conservative. Motif1 was located in the homologous domain and existed in all Hox genes. Conserved domain analysis showed that Hox proteins contained the homeodomain, and abd-A protein also contained the Abdominal-A domain. Genomic collinearity analysis between species showed collinearity between multiple chromosomes of P. clarkii and Portunus trituberculatus and the Hox genes (pb, lab, and Dfd) on the LG30 chromosome where the Hox gene cluster of P. clarkii was located, and the Hox gene on chromosome 49 of P. trituberculatus showed collinearity. Phylogenetic analysis showed that different Hox gene family members were clustered into one branch. On the branches of pb, Dfd, Scr, lab, and Antp genes, crayfish, and crabs were first clustered into one branch and then clustered into another with prawns. On the branches of abd-A and Abd-B genes, crabs and prawns were first clustered into one branch and then clustered into another branch with crayfish. On the branch of the Ubx gene, the crayfish first clustered with the prawns and then clustered with the crabs. This indicates that different members of the Hox gene family have different phylogenetic histories in shrimp and crab species. The adaptive evolution analysis showed that the Hox genes of crustaceans were mainly subjected to purification selection, and the evolutionary rates of Abd-B and Dfd genes were significantly different between P. clarkii and other crustaceans (P<0.01). Positive selection sites with posterior probability greater than 0.95 were detected in pb and Ubx genes. The relative expression of Hox genes in different developmental stages of P. clarkii showed that except for abd-A and Dfd genes, the expression levels of other Hox genes were the highest in the zoea stage, and all showed a trend of increasing first and decreasing subsequently. Among them, abd-A, Antp, and Ubx genes were lowly expressed in the gastrula and nauplii stages and then highly expressed. The distribution of Hox genes in different tissues of P. clarkii showed that the relative expression levels of different Hox gene family members vary across various tissues in adult crayfish. In addition to the lab gene, the relative expression of Hox genes in different tissues of female crayfish was generally higher than that in different tissues of male crayfish. In summary, this study used bioinformatics methods to perform genome-wide identification, protein structure analysis, amino acid sequence analysis, physical and chemical properties analysis, subcellular localization prediction, collinearity analysis, phylogenetic analysis, adaptive evolution analysis, and expression analysis of Hox genes in early developmental stages and different tissues of adult crayfish. The results showed that eight Hox genes clustered on the same chromosome were identified in P. clarkii. The amino acid sequences and protein physicochemical properties of different Hox genes differed. However, except for the Abd-B gene, they all had a YPWM-containing motif and a highly conserved homeodomain, and the protein structure was similar. Phylogenetic analysis showed that different members of the Hox gene family had different phylogenetic histories in shrimp and crab species. The adaptive evolution analysis showed that Abd-B, Dfd, pb, and Ubx genes had different evolutionary rates among crustacean species. The expression analysis of Hox genes in the early developmental stages and different tissues of adult crayfish showed that lab and Dfd genes may be involved in the differentiation of the head, chest, and abdomen of P. clarkii in the early stage of embryonic development. The expression differences of abd-A, Ubx, and Antp genes between shrimp, crayfish, and crabs may be the potential factors causing the morphological differences in their tails. This study provides a reference for further functional research and analysis of the morphological development mechanism of P. clarkii.

Abstract:Oysters are keystone species in marine ecosystems and essential for restoring ecological function and maintaining biodiversity maintenance. However, global climate change and increasing human activities have intensified environmental fluctuations in marine habitats, which greatly affect the growth and physiological functions of oysters and other marine organisms. Organisms in these environments have developed adaptive regulatory mechanisms. Crassostrea ariakensis is an economically important bivalve that inhabits estuarine areas along the coast of China and crucial for delivering essential ecological services. However, a lack of research exists on the physiological responses of C. ariakensis to frequent and substantial salinity variations in these estuaries. In this study, we investigated the physiological responses and key gene alterations in C. ariakensis under salinity stress by analyzing changes in respiration, ammonia excretion, ingestion, and clearance rates under various salinity conditions. The findings provide valuable data and insights for a comprehensive understanding of the physiological and metabolic responses of oysters to salinity stress. This study had different salinity gradients (5, 15, 25, 35, and 45), whereas a salinity of 25 served as the control group. Seawater, freshwater, and seawater crystals were used to establish environments with different salinities, and oyster individuals were randomly assigned to different salinity groups, with three individuals per group. Following 4 h of salinity stress treatment, the gills, adductor muscle, mantle tissue, and labial palps of the oysters were individually removed and promptly frozen in liquid nitrogen at −80 ℃ for subsequent analysis. The respiration, ammonia excretion, ingestion, and clearance rates of the oysters were measured using the static water method. Based on the β-actin gene as a reference, the expressions of the CarHsp70, CarHyou1, and CarDANJC2 genes were detected using RT-qPCR. The reaction system (10 μL) comprised the following: 0.2 μL each of upstream and downstream primers, 1 μL of template cDNA, 5 μL of 2× ChamQ SYBR Color qPCR Master Mix, and 3.6 μL of diethyl pyrocarbonate water. The reaction conditions were as follows: pre-denaturation at 95 ℃ for 10 min, denaturation at 95 ℃ for 10 s, and annealing at 60 ℃ for 30 s, for 40 cycles. Each group included six biological replicates, with each biological replicate conducted in triplicate. The results demonstrated that within a salinity range of 5–45, the respiration and ammonia excretion rates of C. ariakensis initially increased and subsequently decreased, with peak values observed at a salinity of 35. Similarly, the ingestion and clearance rates of C. ariakensis exhibited an initial increase, followed by a decrease within the same salinity range, with the maximum clearance rate observed at a salinity of 15 and the minimum value observed at a salinity of 45. In addition, the research revealed substantial effects of salinity stress on gene expression, particularly for CarHsp70. The RT-qPCR results showed that the three genes (CarHsp70, CarHyou1, and CarDANJC2) were expressed in the gills, mantle tissue, adductor muscle, and lip tissue of C. ariakensis, with the highest expression level observed in the adductor muscle. Changes in salinity significantly affected the expression of the three genes in the HSP family of C. ariakensis. Following salinity stress, the expression levels of the three genes in gill tissues exhibited an upward trend. Upon a 10-unit rise in salinity, the expression levels of CarHsp70, CarHyou1, and CarDANJC2 were upregulated by 4.36-, 3.58-, and 2.08-fold, respectively, compared to the control group. Conversely, a 10-unit drop in salinity resulted in adjustments to 3.62-, 2.97-, and 2.05-fold, respectively. Upon a 20-unit rise in salinity, the expression levels of the three genes were 7.13-, 4.68-, and 2.72- higher than those of the control group, respectively. Conversely, with a 20-unit drop in salinity, the expression levels were 5.92-, 6.04-, and 2.54- higher than those of the control group, respectively. This study elucidates the physiological and molecular responses of C. ariakensis to different salinity conditions, including respiration, ammonia excretion, ingestion, and clearance rates, and the expression changes of HSP family genes in response to salinity stress. Within a defined salinity range, the respiratory metabolic activities of shellfish progressively increased with rising salinity; however, beyond a specific threshold, these activities were inhibited. The three genes (CarHsp70, CarHyou1, and CarDANJC2) are essential for physiological functions in different shellfish tissues and may exhibit synergistic effects in response to environmental stress. Their expression patterns provide insights into the mechanisms underlying the adaptation of organisms to environmental changes. This research enhances our scientific understanding of the adaptability of C. ariakensis to salinity variations and offers substantial guidance for the sustainable development of the oyster farming industry and genetic improvement. Additionally, it provides reference material for further investigations into the adaptability of oysters to salinity changes.

Abstract:Ruditapes philippinarum—a marine bivalve mollusc known for its adaptability across a range of temperatures, salinities, and habitats—thrives primarily in tidal flats and shallow marine areas. It is an ideal candidate for high-density cultivation in tidal flats, making it one of the four major cultivated shellfish in China. Genetic exchanges are prevalent among populations in adjacent marine areas, and artificial translocation and cultivation of this species in China have affected the genetic resources of local wild populations, notably altering the genetic structure and ecological balance of these groups. Thus, there is an urgent need for an effective genetic identification method to assess their genetic diversity and germplasm resource status. Efficient extraction of larval DNA is essential for genetic research on shellfish; however, methods for extracting and identifying trace amounts of DNA have not been widely reported. This study addresses these limitations by establishing an efficient method suitable for individual larvae of the marine shellfish, R. philippinarum, and other bivalve mollusks. This method can extract high-quality genomic DNA from larvae as small as a few hundred micrometers, with the aim of providing an accurate and efficient DNA extraction method for species identification and genetic analysis of marine shellfish larvae. Here, D-shaped and umbo larvae were selected for DNA extraction. A rapid and efficient method for extracting genomic DNA from a single larva was developed and successfully applied for genetic identification and haplotype analysis. This method involved the use of physical and chemical means to disrupt the cell structure, and release and collect DNA through thermal denaturation and rapid cooling. A single larva was transferred to a PCR tube containing 10 μL of PCR buffer, followed by heating at 100 ℃ for 5 min and quick transfer to an ice bath. After adding protease K (0.5 μL) for digestion at 55 ℃ for 60 min, the tube was heated again at 100 ℃ for 10 min and then centrifuged at 4 ℃ to collect the DNA. The larval DNA quality was evaluated using a NanoDrop UV-Vis Spectrophotometer, agarose gel electrophoresis, and DNA sequencing. The results demonstrated that PCR products amplified by 16SrRNA and COX1 showed a single clear band of target products, corroborated by their precise sequences. This suggests that the extracted larval DNA can be effectively used for genetic identification and haplotype analysis using these markers. Haplotype analysis revealed 14 COX1 haplotypes with a diversity of 0.937, and 13 haplotypes detected by 16SrRNA with a diversity of 0.705. These findings indicate that the PCR buffer DNA extraction method can yield high-quality genomic DNA from larvae several hundred micrometers in size. This method offers an accurate, efficient, and reliable approach for species identification and genetic analysis of shellfish larvae, with broad potential applications in genetic research and molecular breeding.

Abstract:The sea cucumber industry has developed rapidly over recent year with breeding areas gradually expanding to the southern Fujian region. Using the warm water of the southern sea area during the winter, Fujian Province has exploited Apostichopus japonicus cage culture. With <1% of the breeding area of the nation, Fujian Province produces 18% of the national sea cucumber output, cementing a new paradigm, known as “north sea cucumber and south cultivation,” which has accelerated sea cucumber industry growth substantially. However, with the rapid development of the sea cucumber breeding industry in this area, breeding diseases have become common in recent years, significantly affecting both the survival and weight gain rates associated with breeding. To understand the causes of diseases in this culture mode and explore prevention and control measures, we investigated a sea cucumber enteritis outbreak in the cage cultures of the Dongwuyang Sea, Fujian Province. We analyzed the epidemiological characteristics, pathogenic pathology, environmental microbial groups, and the structure of the intestinal microbiota of sea cucumbers. The intestinal tract of the diseased sea cucumbers were small and contained a large amount of water. The connective tissue and muscle layer were separated, with the connective tissue cells in the sick intestinal tract swollen and partially destroyed, and loosely arranged. Microbial culture data analysis of cultivable bacteria and intestinal and ambient samples from sea cucumbers were conducted. The total quantity and Vibrio quantity in the intestinal samples of the diseased sea cucumbers were significantly greater than those in the intestinal samples of healthy sea cucumbers. High-throughput sequencing of the 16S rDNA variable region V3–V4 identified 25 phylums and 311 families. Enterobacteriaceae, Bacillaceae, and Lachnospiraceae represented the top three bacteria families in the healthy group, with relative abundances of 8.69%, 5.53%, and 5.12%, respectively, while Rickettsiaceae, Vibrionaceae, and Enterobacteriaceae represented the top three prominent bacterial families in the diseased group, with relative abundances of 12.66%, 8.22%, and 5.71%, respectively. Vibriaceae were more prevalent in the diseased group than in the healthy group. The relative abundance of Vibriococcaceae was 34.99% and a mixed diet led to the highest environmental sample abundance. Principal co-ordinates analysis (PCoA) and unweighted pair-group method with arithmetic mean (UPGMA) cluster analyses indicated that the samples of each group were well grouped, and the microbiota types of the mixed diet were similar to that of the intestinal flora. In conclusion, the intestinal tissue structure of the diseased sea cucumber was changed, and a significant amount of Vibrio bacteria in the intestinal tract primarily derived from the mixed diet, which posed a threat to sea cucumber health. Operational taxonomic units (OTUs) with a relative abundance of 0.5% were mainly located in the Lutimonas, Aliivibrio, and Formosa genera. Vibrio was mostly pathogenic, while Formosa was mostly probiotic. Healthy and diseased gut microbiotas differed significantly in terms of high abundance OTU. These results indicate that the intestinal tissue structure of diseased sea cucumber has changed, and that the large amount of Vibrio in the intestine mainly originates from mixed feed. The large microbial community in sea cucumber feed poses a certain threat to their health. The probiotic content of the intestinal tract of healthy sea cucumbers was higher than that of diseased sea cucumbers, and pathogenic bacterial content in diseased sea cucumbers was higher than that of healthy sea cucumbers. Our study findings provide a scientific basis for the development and utilization of potential probiotics and potential pathogenic bacterial screening. Furthermore, our results support sea cucumber bait optimization and offer a theoretical foundation for scientific breeding and disease management.

Abstract:China's marine aquaculture is rich and diverse. As China's marine aquaculture industry continues to expand, the impact of disease problems on this sector has become increasingly prominent, leading to substantial economic losses for the country's aquaculture enterprises. Shellfish is an important source of high-quality protein for human beings and simultaneously plays an important ecological service function in the marine ecosystem. Shellfish aquaculture in China mainly focuses on marine shellfish, accounting for the highest proportion of the total marine aquaculture output in China. Recently, pathogenic infections, including bacteria, fungi, and parasites, have caused notable disease outbreaks in China's mariculture shellfish. For example, oyster herpesvirus and its variant viruses can cause disease infections in many shellfish organisms and are important pathogens that lead to shellfish mortality. Additionally, shellfish farming in China's northern Yellow Sea area is often infected by Vibrio canis and Bacillus luminescens. Regarding the developmental changes of diseases, shellfish diseases in mariculture mainly include pathogenic and non-pathogenic diseases. Pathogenic diseases have harmful manifestations, such as bacteria, parasites, fungi, and pathogenic worms, whereas non-pathogenic diseases are mainly caused by mechanical injuries, drug abuse, and physical or chemical stimuli. Shellfish diseases in mariculture often involve multiple infections and cross-contamination, and non-pathogenic diseases can sometimes develop into pathogenic hazards. Around 2003, "viral covert mortality disease (VCMD)" appeared in the Penaeus vannamei culture area in southern China. VCMD is an emerging infectious disease caused by covert mortality nodavirus (CMNV). The cross-species transmission capacity of CMNV has caused huge economic losses to the aquaculture industry. Movement disorder nodavirus (MDNV) is similar to CMNV, both of which are members of the newly discovered α-nodavirus genus. P. vannamei, infected with MDNV, showed sinking characteristics and decreased swimming performance. To understand whether MDNV has the same cross-species transmission ability as CMNV, we collected wild shellfish samples in the offshore area of the Yellow Sea and the East China Sea and systematically analyzed the possibility of the shellfish carrying and infecting by MDNV. TaqMan real-time fluorescence quantitative PCR (TaqMan RT-qPCR), histopathology, and in situ hybridization were used in this investigation. The results of the TaqMan RT-qPCR assay showed that MDNV was detected in Crossostrea gigas and Azumapecten farreri collected in the offshore area of the Yellow Sea and East China Sea. The positive detection rates of MDNV in C. gigas and A. farreri was 16.67% (1/6) and 33.3% (2/6), respectively. MDNV was not detected in Scapharca broughtonii and Ruditapes philippinarum. Histopathology and in situ hybridization analysis showed many eosinophilic inclusions in the mantle tissue of C. gigas, and nuclear pyknosis occurred. In the gill tissue of C. gigas, epithelial cells were severely damaged, and the appearance of vacuolation and necrosis of karyopyknotic nuclei was observed in the gill. The gill filaments were filled with many eosinophilic inclusions. The epithelial cells of A. farreri chlamys were filled with eosinophilic substances, and the karyopyknotic nuclei of some epithelial cells were also observed. The positive hybridization signals of the purple MDNV RNA probe were observed in the lesion sites of C. gigas and A. farreri. In addition, the MDNV RNA-dependent RNA polymerase nucleic acid sequences were cloned and then submitted to NCBI web multi-sequence alignments using the online program. The multiple sequence alignment results showed that the similarity of MDNV sequence fragments of C. gigas and A. farreri was 99.3%, and the similarity between them and the original CMNV isolates was 83.3%. This study shows that MDNV can cross the species barrier under natural conditions, infect wild shellfish in the offshore area of the Yellow Sea, and cause evident pathological damage to target tissues. It also suggests that attention should focus on avoiding the use of MDNV-infected shellfish seed in aquaculture operations to prevent large-scale transmission or epidemic of MDNV in cultured shellfish.

Abstract:Mud crab reovirus (MCRV) present in Scylla paramamosain larvae poses notable risks to the late stage of mud crab aquaculture. The cultivation of MCRV-free larvae and prevention of virus transmission at its source have become critical issues that must be urgently addressed in mud crab aquaculture. Owing to the low viral load in fertilized eggs and larvae of mud crabs, we attempted to establish a more sensitive detection method for MCRV. Most of the current MCRV detection methods are based on the VP1 and VP6 genes of MCRV, which limit the sensitivity of detection methods owing to the low expression of these two genes. Our previous studies found that detection sensitivity was significantly improved by using VP11 as the target gene because VP11 is the highest expression gene of MCRV in mud crabs. In addition, compared with SYBR Green qRT-PCR, TaqMan-MGB qRT-PCR typically has higher sensitivity in theory. Therefore, in this study, we selected the highly expressed VP11 of MCRV as the target gene, designed specific primers and TaqMan-MGB probes, and established a qRT-PCR detection method suitable for MCRV in mud crab larvae by optimizing the reaction system and procedure, using standard plasmid and RNA as the templates. The final reaction components were as follows: 10 μL Premix Ex Taq (Probe qPCR), 0.4 μL VP11-F (10 μmol/L), 0.4 μL VP11-R (10 μmol/L), 0.4 μL VP11-P (10 μmol/L), 0.4 μL ROX, 2 μL DNA template, and 6.8 μL ddH2O. The final procedure was as follows: Initial denaturation at 95 ℃ for 3 min, followed by 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. The results showed a good linear relationship between the log value (starting quality, Sq) and number of reaction cycles within the range of 1×108 – 1×101 copies/µL of standard plasmids (or dsRNA). Standard plasmids or dsRNA can be accurately quantified with a minimum of 10 copies, and the minimum theoretical detection limit is 2.5 copies/reaction of standard plasmids. The coefficients of variation of this detection method for plasmid and dsRNA samples were 1.4% and 0.98%, respectively, showing good repeatability and stability. When MCRV, MCDV, WSSV, DIV1, EHP, and Vibrio parahaemolyticus nucleic acid samples were used as templates for MCRV detection, no specific amplification was observed except for MCRV samples, indicating that this detection method is highly specific for MCRV. To further validate the practicality and sensitivity of TaqMan-MGB qRT-PCR, 14 batches of mud crab larvae with suspected MCRV infection were detected using this method, and SYBR Green qRT-PCR method was also used to analyze the samples simultaneously. The MCRV detection rate of the former was 78.57%, and the latter was only 57.14%, indicating that TaqMan-MGB qRT-PCR has a higher sensitivity than the SYBR Green method. These findings indicate that the newly established detection method has strong specificity and stability, as well as higher sensitivity. This detection method can be applied to screening MCRV in mud crab larvae at different developmental stages and provide technical support for the production of MCRV-free mud crab seedlings.