Abstract:This paper classifies and compares different types of mariculture and equipment involved in deeper offshore aquaculture according to China’s national conditions, and reviews the opportunities, challenges and development strategies facing nearshore and offshore aquaculture, especially deeper offshore aquaculture. Mariculture can be divided into coastal aquaculture, off-the-coast aquaculture, offshore aquaculture, and deeper offshore aquaculture, and the first two can be collectively referred to as nearshore aquaculture. Deeper offshore aquaculture or equipped offshore aquaculture refers to offshore aquaculture that employs steel framework and automatic feeding system, that withstands or can avoid strong typhoons. The equipment of deeper offshore aquaculture includes offshore pens, composite steel cages (including semi-submersible and fully submersible ones), vessel-shaped cages (powered or unpowered) and sea-based aquaculture tanks (including aquaculture vessels and closed aquaculture tanks). The development from coastal to off-the-coast and further offshore mariculture has been an academic consensus and an inherent demand of the aquaculture industry in China. Stocking appropriate species and quantity of juveniles according to the ecological carrying capacity and social carrying capacity of the aquaculture waters, carrying out multi-trophic integrated aquaculture, and implementing industrial integration development are efficient ways to improve the production and economic benefits of off-the-coast and offshore aquaculture. Considering the coexistence of nearshore aquaculture, offshore aquaculture and deeper offshore aquaculture at present, deeper offshore aquaculture can only be profitable by cultivating higher-quality species that are difficult to cultivate in ponds, coastal waters and off-the-coast waters, or species that can gain additional income through deeper offshore aquaculture. Fish-oriented intelligentization based on knowledge of fish behavior and physiology is the current shortcoming of intelligent deeper offshore aquaculture, which can only be solved through in-depth cooperation among experts in aquaculture, fish biology, engineering and information technology. The development of deeper offshore aquaculture is in urgent need of a large-scale innovative operation model that integrates aquaculture, food processing, logistics, wind power generation, tourism etc., to achieve a triple win for environmental protection, economy and people’s wellbeing.

Abstract:Sex determination and differentiation are basic events of development and key processes of sexual reproduction. The mechanism of sex determination and differentiation is both baffling and intriguing. Mollusca is the second largest animal phylum, harboring diverse sexual systems including hermaphroditism, gonochorism, protandry and sex reversal. Therefore, molluscs are an ideal animal group to study the mechanisms of sex determination and differentiation. Although efforts towards understanding sex determination and differentiation of molluscs can be traced back to the 1940s, the mechanisms of sex determination and differentiation of molluscs remain enigmatic. Solving the riddle of sex determination and differentiation and identifying key genes would help us better understand the biology of molluscs and provide insights into sexual evolution in the animal kingdom. Here we review the research progress and the prospects of sex determination and sex related genes such as Dmrt, Foxl2, Sox, Wnt4, Dax1, β-catenin, Fem-1, Vasa, Nanos and GnRH in molluscs. We also propose several goals about mechanistic underpinning of sex determination in molluscs which require further study.

Abstract:The ubiquitin-proteasome system is an important protein modification pathway, involved in many biological processes. Ubiquitin-conjugating enzyme E2 is the key enzyme in regulating protein ubiquitination. In order to explore the function of ubiquitin-conjugating enzyme E2 in female gonad development of Cynoglossus semilaevis, the Open Reading Frame (ORF) of Cs-UBE2D4 gene was cloned in this study. Its expression pattern was measured in various tissues of male and female adults, and at different developmental stages of gonads as well. Then, we constructed Cs-UBE2D4 promoter reporter vector (pGL3-Cs-UBE2D4) and detected its activity. The ORF of Cs-UBE2D4 gene was 360 bp, encoding 119 amino acids with a conserved ubiquitin-conjugating enzyme E2 domain. qPCR results showed that Cs-UBE2D4 was widely expressed in all tissues of females, especially in ovary and liver, but it was hardly detected in males. The gene expression increased from 90 days post-hatcing (dph), peaked at 6 months post-hatcing (mph), and decreased afterwards. Its expression level was lowest at 1.5 years post-hatching (yph) and increased at 3 yph. During this period, its expression was 1.97-fold higher at 6 mph than that at 90 dph. Dual luciferase assay showed strong activity of the promoter of Cs-UBE2D4 gene, indicating its participation in transcription. This study provides a theoretical basis for future research on Cs-UBE2D4 gene in ovarian development and gender-related mechanisms of Cynoglossus semilaevis.

Abstract:Smoothened (SMO) is a seven-transmembrane protein related to the G protein-coupled receptor and plays a central role as transducer in the Hedgehog (Hh) signaling pathway, which has been demonstrated to play an important role in the differentiation and development of gonadal cells in mammals. However, the role of Smo signaling in fish testis development remains unknown by now. To elucidate the role of Smo signaling in Oreochromis niloticus testis, the O. niloticus smo (Onsmo) cDNA was cloned and characterized. Its expression profile in different tissues was investigated by RT-PCR and in testis by fluorescence in situ hybridization (FISH). After treatment with Smo specific agonist SAG and inhibitor cyclopamine, the proliferation and apoptosis of germ cells and somatic cells in an ex vivo testicular organ culture system were evaluated by EdU incorporation and TUNEL staining, respectively. The full open reading frame of Onsmo is 2 478 bp in length, it encodes a putative 825 amino acids protein, including seven transmembrane domains. The identity of amino acid sequence with human SMO is up to 77%. Onsmo was expressed in multiple examined tissues including testis. According to FISH results, Onsmo in testis was expressed in spermatogonia, spermatocytes, Sertoli cells (SC) and Leydig cells (LC). In the testicular organ culture system, our experimental data show that SAG significantly promoted the proliferation of spermatogonia, whilst cyclopamine significantly promoted the apoptosis of testicular SC and LC. Taken together, our study suggests that Smo signaling plays an important role in the proliferation of spermatogonia and the survival of testicular somatic cells in O. niloticus. To our best knowledge, this study firstly reports the role of Smo signaling in fish testicular cells. This study not only enriches our understanding of the role of Smo signaling in fish testicular cells, but also lays an important foundation for further elucidation of its mechanism.

Abstract:The kuruma shrimp (Marsupenaeus japonicus) includes two morphologically similar varieties, which are distributed mostly allopatrically but coexist in the northern South China Sea. In order to further analyze the phylogenetic relationship between the two phenotypic varieties of M. japonicus, the gene structure, genetic distance and codon preference of their mitochondrial whole genomes. The results showed that there were differences in gene overlap region, gene spacing and codon preference between the two genomes. The similarity of the nucleotide sequences of the mitochondrial protein-encoding genes was between 91.10% and 95%, and the similarity of amino acid sequences was between 96.15% and 100%. The sequence homology of A+T rich regions was only 82.60% and the divergence was 15.40%, indicating a high degree of genetic differentiation. The nucleotide sequence divergence of protein-coding genes between the two varieties was higher than that between Fenneropenaeus and Orconectes, while the divergence of amino acid sequence was smaller than that between the latter. The study found that the interspecific genetic distance based on cytb gene was more than 10 times the intraspecific genetic distance. The interspecific genetic distance based on cox1 gene was 14.6 times and 5.2 times of the intraspecific genetic distance for variety Ⅰ and variety Ⅱ, respectively. The Ka/Ks values of nd1, nd4 and nd5 genes were greater than 1, indicating positive selection. Phylogenetic trees were constructed based on the protein coding sequence of 20 Penaeus species belonging to 9 genera, and the result showed that each genus could be effectively differentiated. In the phylogenetic tree, the two varieties of M. japonicus were clustered first, and then clustered with Melicertus latisulcatus. In view of these differences between the two Marsupenaeus species, we believe that it is essential and urgent to establish a genetic database for each and reevaluate their suitable ecological conditions in order to improve species-specific culturing techniques.

Abstract:Various diseases caused by metabolic abnormalities have caused great losses to the breeding industry of Eriocheir sinensis. Therefore, the study of the metabolic system of E. sinensis is very important for its healthy growth and development. However, there is still a lack of systematic analysis and research on the metabolic process of E. sinensis. , and this study aims to construct a systematic tool for the study on metabolic process of E. sinensis. Firstly, the proteins with unknown molecular functions were annotated based on the protein-protein interaction network (PIN) by annotating adjacent nodes. Secondly, based on the global network, the metabolic PIN of E. sinensis was constructed by extracting metabolism related proteins and their interaction relationships from the global network according to the GO annotation. The GO terms were organized in a hierarchical structure, among which, the highest level of metabolic system is annotated as GO: 0008152 (metabolic process). All the proteins that can be back traced to GO: 0008152 through the hierarchical structure were considered as metabolism related proteins. Then, the metabolism related proteins and their interaction relationships were extracted from the global network, which formed the metabolic PIN. Subsequently, the proteins in the network were annotated according to their molecular function and cellular component. Furthermore, the pathway distribution of the proteins in the metabolic PIN was analyzed according to the KEGG annotation of unigenes. The molecular functions of 932 proteins were determined, accounting for 97% of all unknown functional proteins in the network. The extracted metabolic PIN contains 2 045 proteins and 15 927 interactions. According to the molecular function and cellular component annotations, the functions and subcellular locations of the proteins in the network were further analyzed. 94.2% (1 926/2 045) of the proteins in the network had subcellular location information, and most of them were distributed in membranous organelles. 96.1% of the proteins (1 966/2 045) have molecular function information, and most of them have catalytic and binding activities. Analysis on the distribution of proteins in 40 subsystems showed that more proteins were involved in translation and amino acid metabolism, and some of them were involved in immune and transport processes. In this study, the metabolic PIN of E. sinensis was constructed for the first time, and the molecular function, subcellular location and pathway of the proteins were analyzed, which provided data basis for further study of metabolic activities and metabolism related proteins in E. sinensis. The results of this study can provide important data for the study of the function and location of metabolic proteins in E. sinensis.

Abstract:The amount and species diversity of fish and other biological resources in island and reef waters are significantly higher than those in the surrounding waters, which is closely related to seaweed beds. As a typical offshore aquatic plant near islands and reefs, more than 90% of its energy enters the food chain in the form of detritus, providing an extremely important energy basis for the conservation and production of biological resources in island and reef waters. In order to explore the decomposition process of macroalgae, the composition and sedimentation characteristics of algal debris, and explore the contribution of macroalgae detritus to the potential scale of ecological functions of the seaweed beds, detritus of Ulva pertusa and Grateloupia livida from the seaweed beds of Gouqi Island was collected by on-site bag hanging and laboratory hydrostatic sedimentation process, and then analyzed in its composition, particle size and chemical characteristics. Results of the present study showed that the detritus density of U. pertusa and G. livida decreased gradually during the decomposition process, and there were no significant differences among different decomposition stages (P>0.05). The content of organic C and organic N in the detritus of G. livida had no significant effect on the change in density (P>0.05), while the content of organic C in the detritus of U. pertusa had no significant effect on its density (P>0.05), whereas the content of organic N had a significant effect on this index (P<0.05). U. pertusa fragmental δ¹³C and δ¹⁵N had a significant effect on its density (P<0.05), and the density of G. livida detritus was only affected by δ¹³C (P<0.05). In the decomposition process, the lignin and cellulose contents of the fragments of the two seaweeds showed a downward trend, and the contents of the two components of the fragments of U. pertusa were higher than those of C. ligulate. The density of both algal detritus was significantly affected by lignin content (P<0.05), while the cellulose content had no significant effect on their density (P>0.05). Particle size is one of the critical factors for the settling speed of the two species of seaweed detritus. The settling speeds of large detritus (>0.830 mm), medium detritus (0.380-0.830 mm) and small detritus (0.180-0.380 mm) of G. livida were 0.016-0.023, 0.008-0.015 and 0.004-0.005 m/s, respectively, and those of U. pertusa were 0.011-0.014, 0.005-0.006 and 0-0.003 m/s, respectively. This study provides useful scientific evidence for studies on the detritus of macroalgae as well as for studies on the ecological radiation range of seaweed beds.

Abstract:In order to explore the molecular regulation mechanism of growth traits of Larimichthys crocea, in this experiment, 182 individuals with fast growth rate [average body weight (527.1 ± 83.6) g] and 230 individuals with slow growth rate [average body weight (238.4 ± 52.3) g] were selected from L. crocea cultured in the same cage, a total of 412 individuals were studied. The muscle tissues were subject to total RNA extraction and sequenced by transcriptome, and the differential expression of genes between the two groups was analyzed. According to Padj < 0.05 and abs[log₂(FoldChange)] > 1, 227 differentially expressed genes were screened, including 125 up-regulated genes and 102 down-regulated genes. The annotation rates of differentially expressed genes in NCBI-nr and Uniprot (Swiss-Prot) databases were 99.12 % and 81.94 %, respectively. Based on the functional annotation results of differentially expressed genes, candidate functional genes that may be related to muscle growth, such as gdf9, ckm, tnni2 and des, were screened. GO and KEGG analysis predicted some information related to muscle growth, such as actin filament organization, actin cytoskeleton organization, actin filament-based process, microtubule organizing center and developmental growth in GO term, and cytokines and growth factors, fatty acid biosynthesis, transforming growth factor-beta signaling pathway (TGF-β signaling pathway), insulin signaling pathway and regulation of actin cytoskeleton in KEGG pathway. WGCNA analysis showed that there was a strong correlation between purple module and body weight traits, and its core genes myoz1, tpm1 and tnni2 may be related to muscle growth. These results provide a reference for further verification of the functions of related genes and the analysis of the molecular regulation mechanism of growth traits of L.crocea.

Abstract:Ctenophyngodon idella is one of the most important aquaculture species in the world, and suffered from local adaptation during the process of introduction and aquaculture. However, most studies of C. idella local adaptation were based on genetic variation, and the effect of epimutation was still widely unknown. In order to explore how DNA methylation affects the domestication and environmental adaptation of the C. idella populations in Asia. A whole-genome bisulfite sequencing was performed between 5 farmed populations and the wild background population, followed by further differential methylation analysis and enrichment analysis. A total of 308.15 Gb sequencing data were generated with a sequencing depth of 31× and a mapping rate of 62.97 % in average, where 76 422 differentially methylated loci (DML), 3 737 differentially methylated regions (DMR), and 1 950 differentially methylated genes (DMG) were identified. Gene Ontology enrichment analysis showed that the DMGs were significantly enriched in development process, such as angiogenesis, neural crest cell migration, and cranial skeletal system development. Annotation with the KEGG pathway database exhibited these genes were mainly involved in adhesion junction, Notch signaling pathway, and Wnt signaling pathway. Among the DMG, some might be functional in the developmental process of neural, immunity, bone, and muscle tissues, like sema3d, sema5bb and nrg2a. This study enhances the understanding of the epigenetic mechanism of domestication and environmental adaption during the process of aquaculture and provides valuable data for the conservation and utilization of the C. idella germplasm resources.

Abstract:Fenneropenaeus chinensis is one of the most commercially important cultured shrimps in China. It belongs to the family Penaeidae of Crustacea. F. chinensis exhibits a significant sexual dimorphism, the color of adult female shrimps tends to be blue, while the males tend to yellow, and the females were noticeably bigger in body size than males. However, no sex-determining region has been reported. A closely related species, Pacific white shrimp, Litopenaeus vannamei, which also belongs to the Penaeus, has been completely sequenced, and an assembled genome of F. chinensis was published recently, which accelerated the progress of genome research on shrimp culture. Sex-differentiation study has been carried out only in recent years, and this study aspires to screening out the potential sex-linked regions on the genome of F. chinensis. A total of 11 male and 10 female F. chinensis shrimps at 4-months-old were picked randomly, and the DNA samples of these 21 shrimps were extracted from muscle and sequenced using the BGISEQ platform individually. 104.5 Gb clean data of female and 115.4 Gb clean data of male were obtained, and the reference genome of F. chinensis (assembly ASM1692082v1) and L. vannamei (assembly ASM378908v1) were both used. The mapped result was used to evaluate the genetic differentiation between two sexes across the genome, and fixation index (F_st) was calculated across the genome. According to the results using reference genome of F. chinensis, there were many disorderly peaks of F_st on the genome, and the F_st value showed an overall increasing trend on LG5. According to the results using reference genome of L. vannamei, there were 2 most significant peaks located on scaffold 2550 (Accession: NW_020870010.1) and scaffold 3683 (Accession: NW_020871268.1), respectively. Many variants were found showing genetic differentiation between the two sexes. The 2 peaks of the F_st value on 2 scaffolds indicate that the corresponding regions on the genome of F. chinensis may be responsible for sex differentiation. RNA-seq data of gonad and muscle of female and male F. chinensis shrimps at 5-months-old were aligned to the reference genome of L. vannamei to obtain the expression information of genes on these two scaffolds. On scaffold 2550, two genes expressed differentially in muscle, while three genes expressed differentially in gonad. On scaffold 3683, no DEG was found in muscle. Four DEGs were filtered out in gonad, all of which had higher expression levels in males. Coincidentally, they all located on the range of F_st peak. Two DEGs encoded peripheral-type benzodiazepine receptor-associated protein 1, which is a key factor in steroid hormone biosynthesis. The peripheral-type benzodiazepine receptor was detected in many peripheral tissues but showed uniquely high densities in the adrenal cortex and in Leydig cells of the testes. Therefore, it is speculated that these male-biased expression genes were involved in the sex-specific physiological mechanism of male shrimps and participated in the sex differentiation of F. chinensis. In this study, we preliminarily located the sex-linked regions in genome of F. chinensis and filtered candidate genes related to the sex differentiation. The study provides a basis for further research of sexual dimorphism in F. chinensis.

Abstract:Salinity is a limiting factor affecting gonadal development, mating, oviposition, growth and breeding of Eriocheir sinensis. In the research on the reproductive performance and embryo quality of E. sinensis, the existing reports mainly focus on the effects of different populations, parental specifications and bait composition,while the effects of salinity on the reproductive characteristics and embryo quality of E. sinensis have not been examined. In order to find out the effects of salinity on the reproductive characteristics and embryo quality of E. sinensis parents, the reproductive and oviposition experiments of parents under different salinities (3, 6, 9, 12, 15, 18, 21) were set up. The reproductive characteristics of parents, such as egg production, reproductive index and fecundity, and the egg diameter, weight, biochemical composition and fatty acid composition of embryos were analyzed. The results showed the following. ① The lowest salinity for spawning was 6, at the range of salinity 6-21, the egg production, reproductive index and fecundity of E. sinensis parents increased first and then decreased. When the salinity was 18, the egg production, reproductive index and fecundity of the parents were the highest, and there were significant differences with other groups (P<0.05). ② The embryo diameter was the largest at salinity 6, with an average of (391.13±12.27) μm, which was significantly higher than that in other groups (P<0.05). When the salinity was 12, the wet weight and dry weight of single embryo were the highest, which were (50.64±2.80) μg and (14.85±0.31) μg respectively, and there were significant differences with other groups (P<0.05). ③ With the increase of salinity, the contents of crude protein, total lipid and moisture in the embryo of E.sinensis increased at first and then decreased. When the salinity was 15, the contents of crude protein, total lipid and ash in embryos reached the highest, with an average of (20.64%±3.42%), (8.65%±1.44%) and (1.30%±0.22%) respectively. There was no significant difference in moisturecontent under different salinities (P>0.05). With the increase of salinity, the contents of cholesterol and phospholipid in embryos increased first and then decreased, while the contents of triglyceride did not change significantly. ④ A total of 19 kinds of fatty acids were detected in the embryos of E. sinensis, including 7 kinds of saturated fatty acids, 4 kinds of monounsaturated fatty acids and 8 kinds of polyunsaturated fatty acids. At salinity 6, the contents of C20:4n6 (ARA), C20:5n3 (EPA) and C22:6n3 (DHA) in embryos were the highest, averaging at (0.0098%± 0.0011%), (0.4300%±0.0044%) and (0.0652%±0.0008%), respectively, which were significantly higher than other groups (P<0.05). At salinity 21, the total contents of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) were the highest, and there were significant differences with other groups (P<0.05). Salinity had a certain effect on the quality of embryos, reproductive characteristics of E. sinensis were the best at salinity 18. This experiment explored the relationship between salinity and the reproductive characteristics and embryo quality of E. sinensis, in order to further provide basic dataand reference for the breeding of E. sinensis, and provide theoretical basis and scientific support for seeding production.

Abstract:In order to explore the growth characteristics and sexual differences of Portunus trituberculatus cultured in ponds, this study investigated the growth parameters of pond cultured P. trituberculatus at 1 to 6 months of age, and Logistic model was used to fit eight morphometric traits of P. trituberculatus, including body weight (BW), body length (BL), body height (BH), full carapace width (FCW), carapace width (CW), meropodit length of the claw (MLC), fixed finger length of claw (FFLC) and meropodit length of the first peraeopod (MLFP). The results showed that the growth was different between male and female of P. trituberculatus. The male grew faster at the early stage, while the female grew faster at the late stage of culture, and the mixed sex analysis lied in between. The R² values of all models of the morphometric traits were above 0.990 except MLC and MLFP in female and MLFP in mixed sex. The carrying capacity of BW of female and male were 290.27 g and 195.91 g, respectively, the fast growth interval of female and male were 2.74-5.10 months of age and 2.33-4.14 months of age, respectively, and the inflection points of female and male were 3.92 months of age and 3.24 months of age, respectively. In summary, the growth process of P. trituberculatus conformed to the "slow-fast-slow" pattern. Males entered the fast growth interval earlier than females, but had less duration of fast growth interval. This study provides the characteristics of different growth parameters of P. trituberculatus and the dominant stages for male and female of each parameter under the conditions of mixed sex culture, so as to provide reference data for the fine and efficient culture of this species.

Abstract:The sea urchin Strongylocentrotus intermedius was originally found off the coast from Hokkaido, Japan and Far East Russia and was introduced from Japan to China in 1989. Now, is one of the most commercially important cultured sea urchin species in China. Family selection and individual selection have been used to genetically improve its economic traits. In order to clarify the genetic diversity and genetic structure of different breeding populations of S. intermedius, the genetic diversity and genetic structure of a family selection population (FP), an individual selection population, and a unselected common population (CP) were analyzed using SSR-seq technology and 15 microsatellite loci. The results showed that a total of 112 alleles were detected at 15 microsatellite loci, and the average number of alleles (N_a) observed in FP, IP and CP were 5.077, 5.133 and 6.133, respectively, with mean effective alleles (N_e) of 2.816, 2.873 and 3.638, respectively. The mean observed heterozygosities (H_o) in FP, IP and CP were 0.522, 0.441 and 0.501, respectively, and the mean expected heterozygosities (H_e) were 0.595, 0.599 and 0.667, respectively. The mean polymorphic information contents (PIC) in FP, IP and CP were 0.546, 0.543 and 0.623, respectively. The difference between H_e and H_o in FP (0.073) was lower than that in IP (0.158) or CP (0.166). The mean fixation index (F) in FP (0.115) was also lower than that in IP (0.248) or CP (0.246). The coefficients of genetic differentiation (F_st) among the three populations ranged between 0.018 and 0.176, indicating a low-to-medium degree of genetic differentiation. The analysis of molecular variance (AMOVA) results showed that the genetic variation of the three populations mainly come from individuals. Both principal component analysis (PCoA) and clustering phylogenetic tree showed that the three populations were closely related, and the IP population had the highest degree of genetic differentiation. In summary, our results suggest that the three sea urchin populations have high genetic diversity, and multiple generations of family selection and individual selection did not reduce genetic diversity dramatically. Family selection is more beneficial in maintaining population heterozygosity and controlling inbreeding levels. This research can lay a foundation for the evaluation and development of germplasm resources of the sea urchin S. intermedius.

Abstract:The experiment aims to investigate the effect of adding alpha-lipoic acid (α-LA) to high-fat diets on the growth performance and liver lipid metabolism of largemouth bass (Micropterus salmoides). Three isonitrogenous experimental diets were prepared: a basal diet with 14% crude lipid (D1), the second diet with 14% crude lipid added to α-LA (D2) and the third diet with 16% crude lipid added to α-LA (D3). M. salmoides [(5.01±0.02) g initial weight] juveniles ingested the three diets for 8 weeks. The results showed that the weight gain rate and specific growth rate of M. salmoides in groups D3 and D1 were not significantly different (P>0.05), but significantly higher than in group D2 (P<0.05); the protein efficiency and protein deposition rate of M. salmoides in group D3 were significantly higher than those in groups D1 and D2 (P<0.05). Serum triglycerides levels, glutamic oxaloacetic transaminase, and glutamic alanine transaminase activity were significantly lower in groups D2 and D3 compared to group D1 (P<0.05), while the difference in total cholesterol levels was not significant between the groups (P>0.05). Liver vacuolation and fat deposition were significantly lower in groups D2 and D3 than in group D1 (P<0.05). The hepatic metabolome results showed that the biosynthetic metabolic pathway of unsaturated fatty acids was significantly upregulated in group D3 compared to group D1 (P<0.05). Muscle fatty acids were significantly elevated in group D3 for pentadecanoic acid, heptadecanoic acid, and 21 carbonic acids (P<0.05). The contents of arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) in muscle were significantly higher in groups D2 and D3 (P<0.05). High-lipid diet with α-LA enhanced lipolysis, inhibited lipid synthesis through the biosynthetic metabolic pathway of unsaturated fatty acids, and reduced hepatic lipid accumulation, as well as increased ARA, DHA, and EPA deposition in muscle.

Abstract:In general, disease caused by the hemorrhagic pathogen infections, results in hemolysis, in series the hemoglobin (Hb) is discharged into the tissues. Consecutively, oxidation of Hb while exposed to air provokes serious high oxidative damage levels in the body and inducing a large amount of inflammation to release additional proinflammatory mediators. Promptly, it is critical to clear the highly cytotoxic Hb. Macrophages play a key role not only under stress conditions, but also in sustaining homeostasis by promoting erythropoiesis and hemoglobin (Hb) clearance. However, the complete mechanism of the oxidized Hb in teleost fish and the effects of macrophages on it not yet clear. In this study, head-kidney macrophages and Hb of the C. idella were taken as the research model to examine the interaction between Hb and macrophages. Stimulation of the Hb during in vivo studies led to the visible deposition of Hb in the head and middle of the kidney and elevated the levels of antioxidant-related enzymes in serum, and further promoted the expression levels of inflammatory cytokine, such as TNF-α, IL-1β, and IL-10 in head kidney. Thereafter, the indirect immunofluorescence and Prussian blue staining analyses indicated that the macrophages of the head kidney exhibited intense phagocytic activity to Hb. Analysis of gene expression level by quantitative real-time PCR, macrophage markers, including CD68, CD86, CSF, and MHC-Ⅱ genes were significantly elevated through the stimulation of Hb. Likewise, the expression levels of inflammatory cytokines (TNF-α, IL-1 β, and IL-4), chemokines (CCL20 and CCL4), IFN-α and TLR4 genes were significantly up-regulated in the head kidney macrophages after 12 h of Hb stimulation. In summary, these results indicate that C. idella head kidney macrophages possess the phagocytic ability towards Hb, and in turn, Hb also activates the expression levels of macrophage-related markers and associated cytokines. Taken together, our findings explicitly improve the understanding of Hb macrophage interactions of C. idella and provided a new reference for healthy fish farming.

Abstract:Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus is an emerging bacterial disease in cultured shrimp. The disease has caused the huge economic losses to the global shrimp aquaculture industry, with annual losses of over $1 billion. All AHPND related V. parahaemolyticus contain a 70 kb plasmid with genes encoding homologues of the Photorhabdus insect-related (Pir) binary toxin PirAB^vp, which is key virulence factor for shrimp. In order to clarify the genetic diversity of the conjugative plasmid of the V. parahaemolyticus strain that causes AHPND in the shrimp culture environment and understand the prevalence of APPND-associated plasmid, this study was conducted in five coastal provinces in China. A total of 100 sediment samples were collected from the shrimp farms in the five coastal provinces. PCR was then conducted to amplify the pirAB and conserved genes encoding the conjugative transfer protein on the plasmids. V. parahaemolyticus strains were then isolated from the pirAB-positive sediment samples to obtain the plasmid. Sanger sequencing was then conducted to obtain the sequence of the plasmid. Finally comparative sequence analysis was performed to assess the plasmid diversity. The results showed that 39 out of 100 samples contained plasmid conjugative transfer protein fragments. 15 strains of V. parahaemolyticus were isolated from 100 sediment samples, 13 of which contained 1-2 plasmids. The results of plasmid sequencing showed that these plasmids can be divided into 8 types/profiles, of which 7 do not carry pirAB gene, but all contain gene clusters encoding conjugation transfer. According to the isolation of 8 types of plasmids carried by V. parahaemolyticus, 8 strains of V. parahaemolyticus were selected for prawn challenge experiments. It was found that the toxicity of these strains to prawns was significantly different, and the mortality rate of prawns was between 15%-100%. Only pirAB positive strains will produce AHPND symptoms, and the mortality rate of shrimp is 100%. The analysis of the plasmid composition shows that the genetic material exchange between plasmids is frequent, and the genetic composition of most of the plasmids comes from a 183 kb super-large plasmid pVP2HP. In this study, we found that the conjugative plasmids isolated from the sediments of shrimp farms had rich genetic diversity. Most of the plasmids are genetically composed of plasmid pVP2HP, and only pirAB positive strains will produce AHPND symptoms. This study improved understanding of plasmid genetic diversity of V. parahaemolyticus and its relationship with AHPND, and provided theoretical guidance for the prevention and control of AHPND.

Abstract:The research of histones and histone-derived peptides is one of the most important study field of antimicrobial peptides. The genus Mytilus is not only of significant economic importance in aquaculture around the world but also shows strong tolerance to a wide range of environmental factors, including various microbes. Although Mytilus antimicrobial peptides had been studied for more than thirty years, there is no reports for now regarding the immunological effects of histones and their derived peptides in this genus. For understanding the roles of histones and their possible derived peptides in Mytilus immunity, the expression levels of H2A and H2B genes were analyzed by fluorescence quantitative PCR after various microbial stress, including Grand-positive, Grand-negative bacterium, and fungi, respectively. In addition, two peptides, Coruscusin Ⅰ and Coruscusin II, derived from the N-terminal of H2A and H2B, respectively, were synthesized by solid phase chemical synthesis. The special configurations of Coruscusin I and Coruscusin Ⅱ were analyzed by circular dichroism spectrometer, and their antimicrobial activities were studied by growth curve suppression method. The results showed that both H2A and H2B of M. coruscus contain classical histone characteristic domain in their sequences, and the α-helix apparently is the predominant conformation for both H2A and H2B. In addition, both H2A and H2B of M. coruscus presented a high sequence similarity with histones of other bivalves species, indicating the conservation of histones in various species. The results of fluorescence quantitative PCR showed that the genes of histone H2A and H2B of M. coruscus were significantly up-regulated in different tissues under different microbial stresses. H2A gene was significantly up-regulated under Staphylococcus aureus induction in digestive gland and gill tissues, and the highest relative expression level were presented at 4 and 24 h for digestive gland and gill, respectively; in blood cell, the gene of H2A was significantly up-regulated at 8 and 24 h under the induction of Vibrio parahaemolyticus and Candida albicans, respectively. In addition, H2B showed sensitivity to V. parahaemolyticus and C. albicans in both digestive gland and hemocytes, and the expression level of H2B gene was significantly up-regulated at 8 and 24 h after induction. In gill, the H2B gene showed sensitivity to S. aureus induction, and the highest expression level was presented at 24 h. These results indicate a very complex immunological responses of histones in Mytilus, and this observation strongly suggests the existence of different recognition mechanisms or signal transduction pathways in mussels. Moreover, Coruscusin-Ⅰ and Coruscusin-Ⅱwere synthesized successfully with the expected molecular masses. Both Coruscusin-Ⅰ and Coruscusin-Ⅱ showed an increasing helix content in 30% of trifluoroethanol compared to that in pure water and PBS buffer, indicating a conformational change in nonpolar solution for both peptides. In addition, both Coruscusin-Ⅰ and Coruscusin-Ⅱ showed significant antimicrobial activity against tested gram-positive and gram-negative bacteria, as well as fungi. However, Coruscusin-Ⅱ showed stronger antifungal activity than that of Coruscusin-Ⅰ. Further helical structure prediction results indicate that the types and distribution characteristics of basic amino acids in the sequences of the two peptides might be the internal cause of the difference in antimicrobial activity. This study provides a foundation for elucidating the role of histones and their derived peptides in mussel immunity and its mechanism, and also provides a scientific basis for the development of new biological antibiotics derived from mussel histones.

Abstract:In October 2020, some Pelodiscus sinensis died in a farm in Hubei Province, China. To determine the pathogen and pathogenic characteristics of the disease, a dominant strain A3 was isolated from the liver of the diseased P. sinensis. Based on physiological and biochemical characteristics and 16S rRNA gene sequence identification, it was identified as Citrobacter sp.. Further identification of the recN gene showed that the recN gene sequence identities between A3 and C. braakii were 95.58%-96.01%, while the identities between A3 and other species of Citrobacter were less than 93.72%, which indicated that A3 belonged to a novel species of Citrobacter. Artificial infection tests confirmed its pathogenicity to P. sinensis. Compared with Aeromonas hydrophila, the course of A3 infection was slower, but the mortality was as high. Drug sensitivity tests showed that strain A3 was sensitive to 10 kinds of antibiotics such as meropenem, and resistant to 9 kinds of antibiotics such as florfenicol. Multilocus sequence typing (MLST) showed that strain A3 belonged to a new sequence type (ST). A phylogenetic tree based on the 7 housekeeping gene sequences of Citrobacter from MLST database was constructed. The results showed that the evolution of Citrobacter spp. had no significant relationship with the source and location of isolates. Moreover, strain A3 has the closest genetic relationship with ST120 strain Citfre2580 isolated from human in Europe. In this study, the pathogen of P. sinensis was isolated and identified, and a more reliable method for the identification of Citrobacter was described, in order to arouse people's attention to the pathogenicity of Citrobacter, and provide some reference for the healthy aquaculture of aquatic products.

Abstract:Fish aggregating devices(FADs) have been used for large-scale aggregation of tuna species for more than 30 years. In order to understand the aggregation characteristics of the fish around FADs，this study explored the day of first arrival day and the dynamic aggregation process of fish after the deployment of FADs based on the echo-sounder buoy data of Chinese tuna purse seine vessels in the Western and Central Pacific Ocean in 2021. The U and H tests were used to analyze the difference of fish first arriving day around FADs, and the generalized additive mixed models (GAMMs) was used to analyze the variation of tuna biomass with the change of days at sea and drifting speed of FADs in the study. The results showed as follows: ① The first arriving day of fish was (8.9±9.0) days, including (3.8±4.2) days for tuna species and (16.0±8.9) days for non-tuna species; ② There was a significant difference on the first arriving day of tuna species around different submerged depth of FADs (P<0.05), but no significant difference was found in non-tuna species (P>0.05); ③ The aggregation biomass of tuna species around FADs varied dynamically with the drifting days of FADs, and reached the peak at around the 25th day after deployment, then gradually decreased; ④ The aggregation biomass of tuna decreased with the increase of drifting speed of FADs; ⑤ The random effect showed that the aggregation biomass of tuna around FADs of 80 m was generally the highest, followed by FADs of 60 m, while the FADs of 90 m was the lowest. This study showed that tuna species usually arrived earlier than non-tuna species after FADs were deployed, and the day of first arrival day of tuna species was related to the underwater length of FADs. FADs with shallower submerged length and slower drifting speed were more likely to attract tuna to aggregate around them. Meanwhile, tuna species gradually aggregated after FADs were deployed, and the biomass reached peak at around one month, which might reflect the optimal fishing time after the deployment of FADs. This study was conducive to further understanding the behavior pattern of floating objects associated schools, and provides scientific basis for the design of efficient and eco-friendly FADs, optimization of setting strategies and formulating of conservation and management measures for floating object associated schools.