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Abstract:Based on fishery-independent survey data from 2016 to 2020 in the offshore waters south of Zhejiang, the relationship between fish resource density and environmental factors was explored using a generalized additive model, and the spatial and temporal distribution of fish resource density was predicted using the optimal models and environmental data of 2020. The results showed that the optimal models had deviances of 47.9%, 68.0%, 56.6% and 45.6% in spring, summer, autumn, and winter, respectively. The average slope of the cross-validation regression line was 0.74–1.02, and the model had good fitting and prediction abilities. Water temperature, salinity, and chlorophyll were significant factors that affected fish resource density in the offshore waters south of Zhejiang, and they had different influencing mechanisms in different seasons. In general, in summer and autumn, water temperature had a significant impact on fish resource density (P<0.01). And there is a negative correlation between water temperature and fish resource density in autumn (r= –0.225, P<0.05). In autumn, fish density increased with an increase in salinity, and in winter, fish density first increased and then decreased, reaching the maximum value at a salinity of 31.5. In spring, summer and autumn, chlorophyll was significantly correlated with fish resource density (P<0.05). Overall, the results showed that the fish resource density in autumn and winter was relatively lower than that in spring and summer in 2020. In spring, the fish resources in Wentai fishing ground were significantly greater than those in Yushan fishing ground. In summer, fish resources in Wentai and Yushan fishing grounds were relatively concentrated, primarily distributed at 27.8°~28.4°N, 121.7°~122.9°E and at 28.9°N, 122°E, respectively.

Abstract:Interleukin 12 (IL-12), a key molecular switch in the immune response, is a pleiotropic cytokine composed of the p35a and p40c subunits. It has been proven that IL-12 can be used as a vaccine adjuvant to enhance the immune response level of fish, and is used in the development of vaccines against pathogen infection. In this study, we cloned the coding regions of the p35a subunit and p40c subunit of IL-12 in Chinese tongue sole (Cynoglossus semilaevis), with a length of 651 bp and 984 bp, respectively. The phylogenetic analysis showed that the p35a and p40c of C. semilaevis were clustered with the corresponding genes in other fishes. The sequence of amino acid homology analysis showed that p35a and p40c of C. semilaevis had the highest similarity with Takifugu rubripes (52.04%) and Gasterosteus aculeatus (48.67%), respectively. The tissue expression analysis showed that p35a was highly expressed in the gill, brain, heart, and ovary, and p40c was highly expressed in the liver, spleen, gill, and heart. We further analyzed the expression patterns of p35a, p40c, and their related genes (ifn-γ and il-10) after infection with Vibrio harveyi. The expression of p35a increased significantly at 48 h (P<0.05), and then gradually decreased in the spleen, and its expression increased significantly at 72 h in the liver and intestine. The expression of p40c increased significantly at 6 h in spleen and liver, and at 48 h in kidney. Its expression also began to increase at 6 h and peaked at 48 h in the intestine. The expression of ifn-γ increased significantly before the upregulated expression of p35a in the liver and intestine, and later than p35a in the spleen. The expression of il-10 was opposite to that of p40c in the liver, spleen, kidney, and intestine. The expression of the immune related cytokines (tnf-α, tnf-β, ifn-γ, and il-10) in the lymphocytes of C. semilaevis was detected after the overexpression of the p35a and p40c genes. The results showed that p35a and p40c could significantly increase the expression of tnf-α under lipopolysaccharide stimulation. The results indicate that the p35a and p40c subunits of IL-12 respond to stimulation by V. harveyi in C. semilaevis, and then participate in the immune response by inducing the expression of the tnf-α gene, which provides a theoretical basis for the development of IL-12 as an adjuvant for the C. semilaevis vaccine against V. harveyi.

Abstract:The innate immune system is the main defense against viral infections in teleost fish. As an important part of the innate immune system, the TRIM protein family participates in the regulation of the immune network during virus infection. Among such proteins, TRIM25 has been reported to play an important role in the immune response of many types of fish. In this study, 16 copies of the trim25 gene in common carp (Cyprinus carpio) were compared with those of other species by phylogenetic and syntenic analyses. Functional domain structures for 16 copies of the TRIM25 protein were predicted, and the expression and cis-regulatory network in tissues for each gene copy were compared. Sequence alignment and phylogenetic analysis showed that the TRIM25 protein structure of C. carpio was highly similar to that of Sinocyclocheilus grahami and Danio rerio, but distinct from that of other species beyond Cyprinidae. The results of gene synteny indicated that the upstream and downstream genes of trim25 were relatively conserved in the evolution of different species. Structural analysis of TRIM25 showed that six of the sixteen copies in C. carpio had complete functional domains; five of these copies were highly expressed in liver and brain tissue. In the expression quantitative trait loci (eQTL) regulatory network for trim25, 5 and 17 SNPs were determined to regulate trim25 expression in liver and brain tissue, respectively. In this study, the sequence differences of multiple copies of trim25 in common carp were compared, and the evolutionary relationship and synteny of trim25 were identified. The diversity of the structure and tissue expression of trim25 genes in the common carp were revealed, and the SNP sites that may regulate trim25 gene expression were identified, providing a reference for future research on trim25 related regulation and disease resistance in the C. carpio.

Abstract:An eight week experiment was conducted to investigate the effects of dietary arginine on the growth performance, body composition, serum biochemical parameters, and liver enzyme activities of juvenile Korean rockfish (Sebastes schlegelii) and to determine the dietary arginine requirement. Six isonitrogenous and isolipidic diets were formulated to contain graded dietary arginine (1.5%, 1.0%, 2.0%, 2.5%, 3.5%, and 4.5% dry diet). The crystalline amino acid mixture was supplemented in the test diets to simulate the amino acid profile of the juvenile S. schlegelii muscle protein, except for arginine. The initial average body weight of the juvenile S. schlegelii was (12.03±0.03) g. The results showed that, with increasing dietary arginine, the weight gain rate (WGR), specific growth rate, and protein efficiency ratio significantly increased at first and then decreased; these were significantly higher in the 2.34% and 2.80% arginine diets than the 1.39% arginine diet, but the feed conversion ratio showed the opposite trend. The crude protein content of the muscle was significantly affected by dietary arginine levels and was significantly higher in the 2.34% arginine diet than the other groups. The concentrations of both total essential amino acids and total amino acids first increased and then decreased in the whole fish and muscle tissues. The levels of albumin and nitrogen oxide in the serum significantly increased first and then decreased (P<0.05), whereas the serum blood urea nitrogen content first increased and then plateaued. However, the glutamic pyruvic transaminase in the serum significantly decreased at first and then increased, and was significantly lower in the 2.34% arginine diet than in other groups. The activities of superoxide dismutase, lysozyme, aspartate aminotransferase, total antioxidant enzymes, alkaline phosphatase, total nitric oxide synthase, and inducible nitric oxide synthase in the liver first significantly increased and then decreased, whereas the content of malonaldehyde first significantly decreased and then increased. With WGR as an evaluation index, the optimal arginine requirement of juvenile S. schlegelii was 2.78% (5.56% dietary protein).

Abstract:The purpose of this study was to evaluate the effects of dietary Pennisetum sinese Roxb meal on the growth, antioxidant response, and intestinal health of juvenile grass carp (Ctenopharyngodon idellus). Three isonitrogenous and isoenergetic semi-purified diets containing 0% (control group C), 10%, and 20% P. sinese Roxb meal (P1 and P2) were designed (protein level 36%, fat level 9%) and fed to juvenile grass carp (28.51±0.04) g for 8 weeks. The results showed that the weight gain rate and survival rate of fish in the P1 and P2 groups were significantly higher than those in the C group (P<0.05). Serum malondialdehyde (MDA) levels in the P2 group were significantly lower than those in the C group (P<0.05). The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of the P1 and P2 groups were significantly lower than those of the C group (P<0.05). Fish in the P1 and P2 groups significantly increased their complement of C3 and C4 contents in the serum compared to those in the C group (P<0.05). Meanwhile, these two groups also showed increased hepatic 3-hydroxyacyl-CoA dehydrogenase and cytochrome C oxidase activities (P<0.05). The intestinal microstructure showed that the intestinal villus height and fold depth in the P1 and P2 groups were significantly increased compared with those in the C group, but the thickness of the muscle layer was reduced (P<0.05). In terms of transcripts, the relative gene expression of intestinal pro-inflammatory cytokine tumor necrosis factor alpha (tnf-α) and interleukin 1β (il-1β) were significantly down-regulated in the P1 and P2 groups compared with those in the C group. In conclusion, our data showed that 10% and 20% Pennisetum sinese Roxb meal dietary supplementation could enhance the growth, reduce the oxidative response, and improve the health status of the intestine in juvenile grass carp.

Abstract:Mastacembelus armatus is primarily distributed in the Yangtze River and aquatic systems in the south of China, and is affiliated with Mastacembelus, Mastacembelidae, and Symbranchiformes. It has great economic value and breeding prospects owing to its high meat quality and balanced nutritional content. However, with overfishing and human influences on its habitat, the population of M. armatus has gradually decreased. We characterized the morphology and histology of the M. armatus digestive tract, aiming to investigate the relationship between the morphological and histological features of the digestive tract and its feeding behavior. Anatomy, paraffin sections, HE staining, and stomach content analysis were used to study samples collected from the Dongjiang River and Taojiang River in Ganzhou, Jiangxi Province. The results indicated that the digestive tract was composed of the oropharyngeal cavity, esophagus, stomach, and intestine. The oropharyngeal cavity contained the tongue, pharyngeal bone, and callous pad. The mucosal surface of the esophagus was covered with squamous epithelial cells, and the inner wall was full of longitudinal folds and secondary branches. The muscle layer consisted of striated and smooth muscles. The gastric wall and intestinal wall were composed of the mucosal layer, submucosa, muscle layer, and serosal layer. The mucosal layer was a single layer of the columnar epithelium, and the muscle layer was a smooth muscle that was divided into circular and longitudinal muscles. The “V-shaped” stomach was divided into the cardia, gastric body, and pylorus. The thickness of the muscular layer in each part was significantly different. There were no goblet cells in the mucosa, but many glands were located in the cardia or body of the stomach. The gastric pylorus muscle layer was extremely developed. There were two pyloric caeca between the stomach and intestine. The intestine had a curve and included three parts: The foregut, midgut, and hindgut. The average intestinal coefficient of M. armatus was 0.325±0.050. There were significant differences among the anterior, middle, and posterior intestinal villi. Nine prey items were identified in the digestive tract of M. armatus, including shrimp, aquatic insects, fish, snails, eggs, and plant detritus. The average percentage of the stomach fullness index was 2.86%. The prey diversity H’ was 2.19, and prey dominance D was 0.33. Shrimp were dominant in the stomach contents, accounting for the relative importance index percentage of 97.75%, numerical percentage of 92.29%, and quality percentage of 73.07%. In conclusion, the histological structure of the digestive system of M. armatus is related to its function and feeding habits. This study provides a theoretical basis for wild resource conservation and nutrition research on M. armatus.

Abstract:Since 2010, iron prawn syndrome (IPS), characterized by sexual precocity and slow growth, has seriously affected the development of Macrobrachium rosenbergii aquaculture. To investigate the molecular mechanism of IPS, we performed transcriptome sequencing of M. rosenbergii with and without IPS using next-generation sequencing technology. This resulted in a total of 56.42 G of high-quality data being obtained. A total of 221 901 unigenes were obtained, and the length range was 201~30 985 bp. The average length was 1572 bp. The length of N50 was 2867 bp, and the length of N90 was 646 bp. Sequence alignment and functional annotation of the unigenes were conducted in the Nr, Nt, Swissprot, KEGG, KOG, GO, and PFAM databases. A total of 103 570 unigenes were annotated, and the GO database had the most annotated unigenes. The differential expression analysis revealed that 2003 genes, including 1209 upregulated genes and 794 downregulated genes, were differentially expressed in the eyestalks of M. rosenbergii with IPS. Consequently, 516 genes were mapped into 242 KEGG pathways, and the number of differentially expressed genes in translation, signal transduction, and the immune system was the largest. Many reproduction-related pathways were identified, including prolactin, estrogen, insulin, gonadotropin-releasing hormone (GnRH), glucagon, oxytocin, glutamatergic synapses, serotonergic synapses, and other metabolic processes related to reproductive regulation. In addition, some differentially expressed genes that have been proven to play an essential role in the immune response were upregulated in the eyestalks of M. rosenbergii with IPS, such as vascular endothelial growth factor receptor 1, serine proteinase inhibitor 6, C-type lectin, arylsulfatase B, prophenoloxidase-activating enzyme 2a, cathepsin B, cathepsin L, and crustin 4. At the same time, several immune-related pathways, such as lysosome, phagosome, antigen processing and presentation, apoptosis, endocytosis, and many others, were annotated. These results support the conclusion from the recent study that a pathogen is associated with IPS in M. rosenbergii. This study provides a foundation for understanding the causes and molecular mechanisms of IPS in M. rosenbergii.

Abstract:Enterocytozoon hepatopenaei (EHP) is a highly infectious intracellular parasite that primarily parasitizes the hepatopancreas, intestine, and muscle of shrimp. It can reproduce by consuming ATP from the host cells, resulting in growth retardation or even growth cessation of the host and increasing individual differences within a population. In recent years, we discovered EHP infection in the breeding process of Exopalaemon carinicauda culture, which has caused losses to the E. carinicauda culture industry. Intestinal microorganisms, which play a very important role in the growth and development of shrimp, can regulate nutritional metabolism, resist pathogen infection, and also have an important impact on the host immune function. Therefore, it is helpful to clarify the pathogenesis of EHP by exploring the differences and functions of the intestinal microflora between healthy and diseased shrimp. To screen potential probiotics for inhibiting or slowing down the spread of EHP, this study analyzed the intestinal microflora structure of shrimp based on 16s rRNA gene sequencing, and further explored the effect of EHP infection on the intestinal microflora of E. carinicauda. The results showed that the intestinal microflora of infected shrimp was significantly different from that of healthy individuals, and the structural diversity of the intestinal microflora was significantly lower than that of the healthy shrimp. Proteobacteria, including Desulfovibrionaceae, Vibrionaceae, unidentified Cyanobacteria, Mycoplasmataceae, and unidentified Alphaproteobacteria were the dominant bacteria in diseased shrimp, whereas Firmicutes including Lactobacillaceae, Bifidobacteria, Bacillaceae, and Chitinophagaceae were dominant in healthy shrimp. Infection with EHP significantly increased the potential pathogenic bacteria level in the intestines of the infected shrimp (P<0.05), and increased their susceptibility to other diseases. In addition, through the Tax4Fun function prediction, we found that the primary function of the intestinal microflora in infected shrimp was metabolism to resist EHP infection, whereas the intestinal microflora of healthy shrimp was primarily involved in individual growth and environmental information processing to ensure growth and survival.

Abstract:The full-length cDNA of ATG5 in Fenneropenaeus chinensis was cloned using rapid amplification of cDNA end (RACE) technology and named FcATG5. The tissue expression of FcATG5 and its expression characteristics under pH and carbonate alkalinity stress were analyzed using quantitative PCR. The function of ATG5 was verified using RNAi. Gene analysis showed that the FcATG5 gene cDNA consisted of 2225 bp with an open reading frame of 810 bp, encoding 269 amino acids, and has a predicted protein molecular weight of 31.103 kDa and a theoretical isoelectric point of 5.59. It is a hydrophobin, contains an autophagy-related protein domain (APG5), has no transmembrane structure, and does not contain signal peptides. The homology and phylogenetic analysis showed that FcATG5 was highly conserved and had the highest homology with Litopenaeus vannamei, reaching 98.14%. The tissue expression analysis showed that FcATG5 was expressed in all tissues of F. chinensis, with the highest expression in muscle, and the lowest expression in blood lymphocyte (P<0.05). The expression level of FcATG5 in the gill tissue was the highest at 96 h after pH stress, 2.67 times higher than that of the control group, and was the lowest at 48 h, which was 1.68 times higher than that of the control group. The expression level of FcATG5 in the gill tissue was the highest at 12 h after the carbonate alkalinity stress, 2.77 times higher than that of the control group, and was the lowest at 96 h, which was 1.30 times higher than that of the control group. The results of the interference experiments showed that under pH and carbonate alkalinity stress, silencing the FcATG5 gene significantly increased the mortality of F. chinensis (P<0.05), indicating that the higher the expression of this gene, the higher the survival rate of F. chinensis. The results of the study showed that the expression of FcATG5 was significantly increased under pH and carbonate stress (P<0.05). It is speculated that autophagy may be involved in the regulation of F. chinensis in response to abiotic stress. The results of this study are an important reference for the study of autophagy in aquatic animals, especially crustaceans, and will help advance the research of Chinese shrimp in saline-alkaline aquaculture systems.

Abstract:To determine the influence of temperature and ammonia on the oxygen consumption rate and ammonia excretion rate of the red claw crayfish (Cherax quadricarinatus), a single factor experimental design and closed flow method were used to analyze the influence of temperature and ammonia concentration on the oxygen consumption and ammonia excretion rates of crayfish of three sizes (S, M, and L representing small, medium, and large crayfish, respectively). The results were as follows. Temperature had significant effects on the oxygen consumption and ammonia excretion rates of crayfish. The oxygen consumption rate increased with the increase in temperature (15℃~35℃), and at 35℃, the oxygen consumption rate reached the maximum, wherein the rate of the S group was 0.777 mg/(g·h), significantly higher than that of the other two size classes. The ammonia excretion rate of the crayfish increased first and then decreased with increasing temperature, and reached a maximum at 30℃; among the size classes, the ammonia excretion rate of the S group was 0.061 mg/(g·h) and was significantly higher than the other two sizes. Temperature had a significant effect on the oxygen/nitrogen ratio of the crayfish. 2 The level of ammonia significantly affected the oxygen consumption and ammonia excretion rates of the crayfish. The oxygen consumption and ammonia excretion rates increased with an increase in the ammonia concentration, and then decreased. The oxygen consumption rate reached a maximum at an ammonia concentration of 8 mg/L, which was 0.663, 0.332, and 0.195 mg/(g·h) in the S, M, and L groups, respectively, whereas the ammonia excretion rate reached a maximum at an ammonia concentration of 12 mg/L, which was 0.123, 0.049, and 0.034 mg/(g·h) in the S, M, and L groups, respectively, and was significantly higher than those of the other groups. Furthermore, the ammonia concentration had a significant effect on the oxygen/nitrogen ratio of the crayfish. The Q10 values for the oxygen consumption and ammonia excretion rates ranged from 1.102~3.361 and 0.346~3.417, and at the minimum values were at 25℃~30℃ and 30℃~35℃, respectively.

Abstract:As the most stable metabolite of nitrofurazone (NFZ), semicarbazide (SEM) can be determined after the administration of NFZ in aquatic animals within a few hours. Because of extensive tissue distribution and a long residual period, SEM is used as the exclusive marker for NFZ. However, SEM is naturally present in the shells of crustaceans, such as crayfish, shrimp, prawns, and soft-shell crabs. To avoid false-positive results induced by SEM alone and to precisely identify the illegal use of NFZ, further investigations on the distribution characteristics of SEM in crustaceans is essential. In the present study, samples of shell, muscle, muscle epidermis, internal organs, and compound eye were collected from Macrobrachium rosenbergii shrimp farms that were not exposed to nitrofural, and SEM was determined in order to investigate the background concentrations in different tissues of the commercial shrimp. Samples were evaluated in accordance with Announcement No.783-1-2006 of the Ministry of Agriculture of the People’s Republic of China. As multiple tissue samples, including compound eye, outer shell, limbs, feet, intestine, gill, muscle epidermis, liver, gonad, and muscle were assessed, the method was suitable for monitoring the background concentration of SEM in different tissues of M. rosenbergii. The average concentration of SEM across all tissues was (12.90±2.47) μg/kg. The average concentrations of SEM in compound eyes, shells, and limbs were (33.29±3.06), (29.00±5.67), and (28.10±7.08) μg/kg, respectively. The lowest concentration of SEM was found in muscle samples, with an average value of (1.83±0.24) μg/kg. The concentration of SEM in commercial M. rosenbergii tissues could be ordered as compound eye > outer shell > limbs and feet > intestine > gill > muscle epidermis > liver > gonad > muscle. Considering that it was difficult to distinguish the source of SEM in M. rosenbergii, the background concentration of SEM should be deducted when determining NFZ residues in this species.

Abstract:The eutrophication problem caused by various reasons such as excessive nitrogen level in aquaculture wastewater are the most critical obstacle to the sustainability of aquaculture industry in China at present. Denitrification and anaerobic ammonium oxidation (Anammox) are important nitrogen cycling processes in natural water ecosystems, which are the main nitrogen removal pathways in the sediments. Clams could not only purify aquaculture water column by their water-filtering activities and wriggle behaviors, but also change the material transportation which occurs in the sediment/water interface. In the present study, the slurry incubation experiments using the sediment samples collected at the area with or without Manila clam (Ruditapes philippinarum) in a biological wastewater treatment pond in Sept., Oct., Nov. and Dec., 2020 have been performed to investigate the potential denitrification and anammox activity rate, and the relationship between the denitrification or anammox activity rate and the porewater physicochemical parameters have also been analyzed. The results showed that the anammox was found in the sediment of clam culture area in Oct. and Nov., and the denitrification-anammox coupling were also found. The denitrification activity rate of sediments with clam culture in the four months were higher than that of sediments without clam, and the highest rate (0.005 8 μmol/kg·h) was found in the sample of Sept. There was a significant positive correlation (P<0.01) between the denitrification activity rate and porewater temperature in clam culture area, and between the porewater NH4+ level and anammox activity rate. The anammox has been found in the seawater aquaculture pond. The clam promoted denitrification and anammox processes in sediment/water interface which showed a vital role in nitrogen removal in sediment in the present study. The results will not only enrich the nitrogen cycling theory of aquaculture ecosystem, but also provided insights to establishing new biological wastewater treatment measures.

Abstract:The vasa gene is a member of the DEAD-box family of proteins and plays a key role in the formation of germ cells in eukaryotes. In this study, we cloned the full length (2438 bp) of Octopus sinensis vasa cDNA (Os-vasa) via rapid amplification of cDNA end (RACE) methods. With an open reading frame (ORF) of 2067 bp, encoding 688 amino acids, a 5′UTR of 128 bp, a 3′UTR of 244 bp, and included an A-tail. Based on ExPASy, Signal4.1, TMHMM, and SMART biological analysis, the ORF encoded a putative protein, with a predicted molecular weight of 76 580.53 Da, and the theoretical isoelectric point was 5.89. No signal peptide site was detected, and there was a significant signal in the transmembrane region; therefore, it was presumed to be an intracellular protein, and not a membrane protein. There were two domains, DEXDc and HELICc, and nine conserved motifs of the DEAD-box family, indicating that the cDNA cloned in this study belonged to the family of vasa. Real-time fluorescence quantitative PCR was used to analyze the expression patterns of the Os-vasa gene at different stages of the embryo and larva, in the ovaries at two growth stages, and in specific tissues for males and females. The results showed that the Os-vasa gene was especially expressed in the gonads, and the expression level in the ovary was significantly higher than that in the testis; vasa mRNA was expressed in both immature and mature ovaries, and the transcript level of the immature stage was evidently higher than the mature stage, revealing that the Os-vasa gene might play an important role in the development process and the maintenance of ovarian functions. Os-vasa gene transcripts were detected at whole embryonic developmental stages, were weakly expressed first 10 days, and gradually increased from the 13th day to the highest level on the 19th day. In the larval stages, vasa exhibited the lowest and highest expression on the 8th day post-hatching and the 20th day, respectively. The findings of this study can provide information for the study of primordial germ cell origin and migration and differentiation, and can contribute to the understanding of ovarian development and oogenesis of O. sinensis.

Abstract:Scapharca kagoshimensis is a marine shellfish of great economic value. In recent decades, populations of S. kagoshimensis have declined due to environmental destruction and overfishing. To enhance our understanding of the genetic diversity and population-level genetic structure of S. kagoshimensis, microsatellite loci were detected based on the transcriptome data of S. kagoshimensis using MISA software. A total of 3987 Single Sequence Repeats (SSRs) were identified from 35,555 unigenes and the frequency of their occurrence was 11.21%. The main types of repeats were dinucleotides and trinucleotides, which accounted for 58.06% and 19.04%, respectively. A total of 182 types of repeat motifs were classified in all SSRs, and AC/GT was the most abundant among dinucleotide repeats (45.70%). The repeat numbers of SSRs primarily ranged between five and seven, and the number of SSRs gradually decreased as repeat number increased．Motif length was predominantly between 12 and 29 bp, and the SSR polymorphism level was above moderate. Among the 60 designed primer pairs, 14 pairs proved to be polymorphic microsatellite markers and were amplified in 30 wild individuals sampled from Weifang in Shandong Province. The results showed that the average number of alleles (Na), average observed heterozygosity (Ho), average expected heterozygosity (He), and polymorphism information content (PIC) were 15.4, 0.682, 0.852, and 0.817, respectively. All 14 loci were highly polymorphic (PIC≥0.5). After Bonferroni correction, seven of the 14 loci deviated significantly from the Hardy-Weinberg equilibrium (P<0.05). These results indicate that it is feasible to develop microsatellite markers based on the S. kagoshimensis transcriptome. The polymorphic microsatellite loci obtained in this study will facilitate further studies on population genetic management, genetic mapping, and molecular assisted breeding of S. kagoshimensis.

Abstract:Foxl2, an important member of the Fox (forkhead transcription factor) gene family, plays an important role in ovarian development and sex regulation. To explore the mode of expression regulation in the sex differentiation of the scallop Patinopecten yessoensis, we analyzed the Foxl2 gene sequence characteristics of P. yessoensis using bioinformatics methods, and the expression of Foxl2 in different tissues was detected via semi-quantitative real-time polymerase chain reaction (RT-PCR). Quantitative RT-PCR and in situ hybridization revealed the temporal and spatial expression changes of Foxl2 in the four stages of gonadal development (proliferation, growth, maturation, and emission stage). The results showed that the Foxl2 sequence of P. yessoensis contains the FH domain shared by the Fox gene family, and the multiple sequence comparison analysis showed that P. yessoensis has the highest similarity with Chlamys farreri and Pecten maximus (98% and 96%, respectively). The phylogenetic tree analysis showed that the Foxl2 gene is highly conserved in different species and in the process of evolution. The results of the quantitative analysis and in situ hybridization showed that the positive signal of the in situ hybridization was mainly located in the cytoplasm of the germ cells. A small amount of PyFoxl2 transcripts were detected in the gills, kidneys, hepatopancreas, and testes. The expression level of Foxl2 was the highest in the ovary and peaked in the mature stage of the ovary. In contrast, with the development of the testis and the gradual differentiation of the male gametes, the expression of Foxl2 showed a declining trend, which is consistent with the results of the in situ hybridization. In conclusion, Foxl2 may play a key role in the ovarian development of P. yessoensis. It is a key gene in the regulation of sex differentiation in female P. yessoensis. This study provides a theoretical basis for advancing our understanding of the sex differentiation and gonadal development of P. yessoensis.

Abstract:In recent years, environmental pollution and ecological problems caused by cage aquaculture have become increasingly prominent in shallow seas. The cultivation technique of integrated multi-trophic aquaculture (IMTA) can be applied to minimize the impact of nutrient enrichment from organic fisheries. The IMTA mode was implemented in September to November 2018 in this study, which included fish cage aquaculture, the shellfish Pinctada martensi, and the alga Eucheuma striatum. The feeding physiology indicators of P. martensi and the nutrient uptake rate of E. striatum were measured at different temperatures in the laboratory concurrent with conducting field experiments, and the improvement of the water quality in the cage aquaculture area by IMTA of shellfish and algae were compared and analyzed. The results showed that: (1) the specific growth rate (SGR) of P. martensi in the marine fish cage area was significantly higher than that in control area, and the average shell height of P. martensi in the 0.50~0.62 m water layer in the cage area was significantly higher than the other water layers. (2) Eucheuma striatum had the fastest SGR in the 0.60~0.90 m water layer, and the net wet weight gain of E. striatum was significantly higher than that in the control area. (3) At 24.45℃, the average feeding rate, absorption rate, and absorption efficiency of P. martensi on particulate organic matter were, respectively, (4.13±0.77) mg/h·ind., (1.04±0.24) mg/h·ind., and (25.00±2.51)%, which were significantly higher than those at 20.74℃. Within the experimental temperature range, the absorption rates of nitrogen, phosphorus, and silicon first increased then decreased with increasing temperature, and the maximum values appeared at 30℃. (4) It is estimated that the total amount of filtered particulate organic matter and the total amount of absorbed particulate organic matter by P. martensi in the marine fish cage area during the culture experiment was 778.08 g and 144.47 g, respectively, and the total amount of nitrogen, phosphorus, and silicon removed by E. striatum in the marine fish cage area was 8.55 g, 1.11 g, and 9.18 g, respectively; all the above indicators were higher than those in the control area. Temperature, depth of cultivation, particulate organic matter, and nutrient concentrations were the primary factors affecting the growth and physiological metabolism of P. martensi and E. striatum. This study provides data and theoretical support for the establishment of IMTA technology.

Abstract:The trend towards kelp Saccharina japonica cultivation in offshore areas is becoming increasingly obvious, but there is no relevant research report on the influence of different offshore distances on the agronomic traits of kelp aquaculture. The factors influencing the growth (length, width, thickness, fresh weight, and dry weight) of kelp and the seedling drop rate were explored for different kelp cultivars ("Xunshan No.2", "Xunshan No.3", kelp "205"), offshore sea areas (from far to near, defined as offshore, middle area and inshore), and pitches of twist kelp breeding rope (80.8 mm, 65.6 mm, 61.1 mm, 53.4 mm, 48.3 mm); the mechanical properties and aging degree of the breeding rope were also investigated. The results showed that: for the same kelp cultivar in different sea areas, the length, width, and thickness properties of the blade were different. The cultivars "Xunshan 2" and "Xunshan 3" showed better economic traits in the offshore area. The fresh and dry weights of the different cultivars were higher in the offshore area, followed by the middle area, and were lowest in the inshore area; however, offshore cultivation had different effects on the fresh and dry weight enhancement of the different cultivars. Whether before or after use, the breaking strength of the special breeding rope was significantly higher than that of the traditional breeding rope (P<0.05). After using a year, the strength retention rate of the special breeding rope was 93.8%, whereas that of the traditional breeding rope was only 63.6%. The seedling drop rate of the breeding ropes with a twist pitch of approximately 61.1 mm was the lowest, and the seedling drop rate significantly increased if the pitch of twist of the breeding ropes was too large (80.8 mm) or too small (≤53.4 mm). The molecular chain of the breeding rope used in the offshore was oxidized more severely, and the degree of aging was higher. The above results showed that offshore aquaculture can improve the yield of different kelp cultivars, but different cultivars have different adaptabilities to different offshore distances; and controlling the pitch of twist of the culture rope can effectively reduce the kelp seedling drop rate. For kelp culture under offshore sea conditions, a special rope with aging resistance and a twist pitch of approximately 61.1 mm can prolong the use time and reduce the rate of seedling drop. The results of this study can guide the development of the offshore kelp culture industry.

Abstract:Olpidiopsis disease is one of the main diseases affecting laver cultivation in the sea, often causing large-scale rot and serious economic losses. Although the oomycetic genus Olpidiopsis has been identified as the pathogen, little is known regarding its mechanisms, including its interaction with epiphytic microbial communities. In the present study, diversities, structures, and major interactions of epiphytic microbial communities were analyzed using Neopyropia yezoensis in sea infected by Olpidiopsis. The N. yezoensis used in the experiment had different health statuses, including no infection (PyOlpH), partial infection (PyOlpM), and serious infection (PyOlpS). The results showed that no significant difference was observed for the α diversity indices among microbial communities associated with the three groups of N. yezoensis, but those of PyOlpM were higher than the other two. Shared operational taxonomic units (OTUs) among the three groups of communities only accounted for 22.7% of the total OTUs, in addition to significant community dissimilarity (permutational multivariate analysis of variance, R2=0.405, P<0.05). More microbial taxa were obtained when communities associated with more seriously infected N. yezoensis compared to those associated with PyOlpH. In total, 208 genera belonging to 23 phyla were annotated, and α-Proteobacteria, γ-Proteobacteria, and Firmicutes were dominant in all samples. Sixteen of the top 20 genera were also assigned to the three groups, with their relative abundances increasing or decreasing across the infection status from none to severe. Among them, genera Cupriavidus and Sphingomonas were the most connected bacterial taxa in the co-occurrence network, and negative interactions were determined between the two taxa and their positively interacting bacterial taxa. This study provides primary datasets for clarifying the microbial ecological mechanisms and searching for probiotics for Olpidiopsis disease.

Abstract:DNA methylation is an important epigenetic modification that plays a key role in gene expression regulation. In this study, two groups of sea cucumbers (Apostichopus japonicus) were prepared. One group had skin ulceration syndrome body wall (PT16S) under stress from Vibrio splendidus infection at a concentration of 1×106 CFU/mL (LD50); the other group had a healthy body wall (PT10H). Genomic DNA methylation levels and gene expression differences between the two groups were detected using whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing. The key gene ontology (GO) terms and KEGG terms engaged in the immune response were selected using association analysis. The results showed that the total methylation levels of the A. japonicus genome of PT10H and PT16S were (3.59±0.04)% and (3.87±0.27)%, respectively. The methylation levels of the A. japonicus genome under pathogen challenge significantly increased; mCpG accounted for 83.06% and 81.91% of all the methylated sites in PT16S and PT10H, respectively, indicating that mCpG was the most important methylation form in the sea cucumber. A total of 626 677 differentially methylated regions (DMRs) were screened and annotated into 23 706 functional genes. A total of 496 differentially expressed genes were screened, including 214 up-regulated and 282 down-regulated genes. A total of 180 negatively correlated genes were isolated using association analysis between genomic methylation and transcriptome, of which 60 genes had DMRs located in promoter regions. Based on GO and KEGG enrichment of the 180 negatively correlated genes, key genes such as LRR, Hsp20, and CARD were selected to play a critical role in the immune response. The results would provide primary data for the epigenetic regulatory mechanisms of A. japonicus and provide a theoretical reference for A. japonicus breeding.

Abstract:To study the influence of light trapping technology on the quality of fishes cultured in deep-water cages, light trapping equipment was designed and installed in deep-water cages in Qingdao. Light trapping equipment involves green LED light (wavelength 525~530 nm) installed on the cage to attract live prey in the sea, providing natural food for the cultured fishes inside the cage. In this study, three important fish species were selected: Pseudosciaena crocea, Lateolabrax japonicus, and Pagrosomus major. Since there were no artificial feedings throughout the culture period, the fishes are in a semi-wild growth state. The quality of the cultured fish was determined by measuring the fat, fatty acid, and amino acid content of the specimens in the treatment cages (equipped with light trapping equipment) and traditional cages (with artificial feedings and without light trapping equipment) after a 5-months’ culture period. The results showed that the sensory characteristics of P. crocea, L. japonicus, and P. major in the treatment cages were significantly better than the fishes in traditional cages. The body fat of the three fishes cultured by light trapping technology were significantly lower than those of the control groups (P<0.05). The crude fat contents in the muscles of P. crocea and P. major cultured in treatment cages was 75.24% and 46.81%, which were significant lower than those of traditional cage culture fishes (P<0.01). There was no significant difference in the crude fat content in the muscle of L. japonicus (P>0.05). The docosahexaenoic acid content in the muscles of P. crocea, L. japonicus, and P. major in treatment cages were extremely significant higher than those in fishes cultured in traditional cages (P<0.01). Moreover, the total fatty acid content and palmitic acid content were significantly lower than those in fishes cultured in traditional cages (P<0.01). Under the influence of light trapping equipment, the indices including the contents of taste amino acids, umami amino acids, sweet amino acids, total amino acids, and non-essential amino acids in the muscles of the three fish species in the treatment group were significantly higher than those of the traditional groups (P<0.05). And the contents of flavored amino acids, umami flavored amino acids and sweet flavored amino acids in P. crocea in the treatment groups increased 11.78%, 9.17%, and 19.57% than those in traditional groups. Moreover, the difference was significant. All the results show that although the deep-water net cage fish culture technology with light trapping reduces the growth rate of cultured fish, but it can improve the fish quality and increase output efficiency. The system could be used to promote and apply a green and healthy aquaculture model.

Abstract:To explore the effects of different culture mode on the nutritional quality of loaches, differences in body characteristics, nutritional composition, and quality of Paramisgurnus dabryanus ssp. and Misgurnus anguillicaudatus pond-cultivated and paddy-cultivated were evaluated, and the correlation between body characteristics and nutritional quality were discussed. The results showed that the fatness of P. dabryanus ssp. pond-cultivated (PPOC) (1.00±0.01) was significantly higher than that of M. anguillicaudatus pond-cultivated (MPOC) (0.50±0.01) (P<0.05). The crude protein content of M. anguillicaudatus was significantly higher than that of P. dabryanus ssp. (P<0.05), while the crude fat content of M. anguillicaudatus was significantly lower than that of P. dabryanus ssp. (P<0.05). M. anguillicaudatus paddy-cultivated (MPAC) had the highest crude protein content (21.09±0.57)%. The texture properties of P. dabryanus ssp. were significantly better than those of M. anguillicaudatus (P<0.05), except for cohesiveness. The culture model had a significant effect on the body characteristics of loaches. The fatness of PPOC was significantly higher than that of PPAC (P<0.05), and the fatness of MPOC was significantly lower than that of MPAC (P<0.05). The atherogenic and thrombogenic indices of the two pond-cultivated loaches were significantly lower than those paddy-cultivated (P<0.05). Hardness (PPOC)>hardness(PPAC), and springiness(MPOC)1). The results of principal component analysis showed that there was a close relationship between body characteristics and nutritional quality. Fatness was negatively correlated with the ratio of body length to height (–0.996), positively correlated with TPA characteristics and crude fat content, and negatively correlated with moisture content, ash content, and crude protein content. Based on the indexes of fatness and texture et al, PPOC is the most suitable raw material for loach processing.

Abstract:Lipid changes during the freezing process is the primary reason for the deteriorating quality of aquatic products. To explore the lipid changes in the shrimp Litopenaeus vannamei during frozen storage and the effect of ice coating on lipids, the glazed shrimp were stored at −20℃ for 180 d and sampled regularly for the analysis of the thawing loss rate, cooking loss rate, total fat content, lipid composition, acid value (AV), peroxide value (POV), and thiobarbituric acid reactive substances (TBARS). The results showed that the thawing and cooking loss rates of shrimp increased during frozen storage, which were significantly lower in the ice-coated group than in the control group (P<0.01), indicating that ice coating can improve the water holding capacity of shrimp meat in frozen storage. The total lipid content of the shrimp did not change substantially, but the lipid composition did. The concentrations of phospholipids and triglycerides decreased, free fatty acids increased, and cholesterol was relatively stable. The changes in the lipid oxidation indices of the two groups were similar. The AV and TBARS levels increased continuously. The POV first increased and then decreased until 60 d, and then slowly increased. However, the lipid oxidation indicators of the ice-coated group were lower than those of the control group, indicating that ice coating is helpful for maintaining the lipid stability of shrimp during frozen storage.