Abstract:At present, for aquatic animals, successive selection is mainly used in the breeding of fish and shrimp species. However, there are some relevant research studies on bivalves, and many attempts have been made and have obtained positive and encouraging results. Specifically, in oyster, all selection experiments on growth characteristics were successful in altering the selected trait in the direction of selection. For other bivalves, positive or successful responses to selection have also been observed in catarina scallop , Japanese scallop , hard clam P. maxima), and other species. The Kumamoto oyster is an important wild oyster resource in Southeast Asia, including China, Japan, and Korea. They not only occur naturally in China but also live in abundance over a wide geographical distribution, ranging from Jiangsu to Guangxi, including Hainan Island. Traditionally, this oyster was a wild fishery resource; farmers captured these oysters from the reef or stones in the inter-tidal zone. Although the artificial breeding of this oyster was successfully conducted, little relative information for genetic improvement was found. A selective breeding program for improving growth rate in Kumamoto oyster was initiated in China in 2012 after the breakthrough of large-scale artificial breeding. To compare differences in growth between two strains, mass selection for the first and second generation selection between two strains was performed from 2012 to 2014. As expected, a relatively higher response to selection and realized heritability was obtained for two populations after the first two generations. To investigate whether the response to selection after one generation of selection among different strains was sustained, we examine responses to selection, realized heritability, and genetic gain for growth traits of cultchless Kumamoto oyster taken from the third generation in southern China. The selection effect, strain origin, and their interaction were evaluated using multi-ridge and non-ridge strains by selecting the largest 10% of the oysters from the second generation parents for the selected groups, while an equal number of the parents were randomly chosen as the control groups before the removal of parents for truncation selection. The results show that the realized heritability of growth traits of multi-ridge strains was higher than those of non-ridge strains with a high genetic improvement level, while the non-ridge strains showed moderate heritability at a realistic level with a certain degree of genetic improvement potential. During grow-out for the adult stage, shell height and total weight of response to selection, and realized heritability and genetic gain were 0.70, 0.76; 0.40, 0.43; and 7.02%, 12.29% for multi-ridge strains; and these indices were 0.36, 0.33; 0.20, 0.19; and 3.74%, 5.72% for non-ridge strains, respectively. Growth traits were mainly affected by strain origins; a second factor was selection effects, and no interaction was found during the whole process. Hence, the selection response of the two strains continually decreased with increasing generations. This study provides an excellent base for new variety culture of Kumamoto oysters.

Abstract:is an important representative species of the Pleuronectiformes. It has a fast growth rate, most parts of its body are edible, and the meat is tender and delicious; all these features make a good aquaculture candidate. In order to explore the relationship between morphometric traits and body weight in , full length (FL), body length (BL), body height (BH), head length (HL), head height (HH), caudal peduncle length (CL), caudal peduncle height (CH), snout length (SL), and body weight (BW) of at 11, 14, and 17 months of age were measured. Correlation and path analysis were conducted, and the effects of each morphometric trait on body weight were separated into direct and indirect effects. Regression analyses were conducted with morphological traits as independent variables and body weight as the dependent variable at each growth stage. The results showed that (1) different morphometric traits had significant path coefficients at each growth stage; at the 11 month stage, the morphometric traits that significantly affected body weight were BH and BL, at the 14 month stage, the significant morphometric traits were FL, BH, and HL, at the 17 month stage, the significant traits were FL and BH. (2) The regression equations at 11, 14, and 17 months of age were =-362.484+13.145, and , respectively. (3) Besides the morphometric traits included in this study, body thickness is another potentially important trait that can affect body weight. This study demonstrated the relationship between morphometric traits and body weight. The results provide valuable information and theoretical guidance for breeding programs.

Abstract:Fish eggs, larvae, and juveniles are important food sources for fish populations. Correct identification of fish eggs, larvae, and juveniles can let us understand which species are spawning where and when, their hatching and nursery grounds, and their possible migration routes during their early life history. At present, fish larval and juvenile identification mostly relies on morphological characters. But since different species often share similar characters and species change greatly during their early development stages, identification of fish larvae and juveniles is very hard, most of which can only be identified to the family or genus level, except for a few fish juveniles that can be identified to species. Moreover, because traditional morphological methods rely too much on personal experience and observation methods, it is easy to misidentify fish larvae and juveniles and difficult to identify to the genus or species level. In view of the limitations of traditional morphological methods, DNA barcode techniques could be a rapid tool to survey many uncertain species, species composition, and cryptic species and to distinguish morphologically similar species. To study the applicability of this technique to identify fish larvae and juveniles, samples were collected in Fujian coastal areas from July 17 to 20, 2015, and 80 samples were selected for DNA barcoding analysis. A total of 73 valid cytochrome C oxidase subunit I (CO I) gene sequences of fish larvae and juveniles in Fujian coastal areas were obtained. Five species were identified to the level of genus, two were identified to the level of family, and another 26 species were identified to the level of species. The results showed that the average Kimura-2-parameter (K2P) distances within species, genus, family, and order were 0.0023, 0.1797, 0.1937, and 0.2420, respectively. The interspecific genetic distance was much larger than the intraspecific distance, and K2P genetic distance values increased with taxonomic level. All sequences formed species units in a neighbor-joining phylogenetic tree, indicating that the DNA barcode can be used to identify these 33 species. Consequently, CO I barcoding is one of the useful tools to identify fish larvae and juveniles. However, this test highlights the limitations of DNA barcoding. The neighbor-joining tree cannot analyze cluster relations clearly above the taxonomic level of family, which corresponds to the slower-increasing trend of genetic distance values with the higher taxa. This means that CO I barcoding is only suitable for phylogenetic analysis of lower taxa, and is not applicable for species identification of higher taxa. Four sequences having high similarity (greater than 98%) with many species in the database were identified only to genus ( sp.). That indicates that some fish species are similar not only in shape but also in CO I sequences, and sequence identification cannot be done using only CO I barcoding. We should strengthen the research of multi-gene barcode technology in order to overcome the deficiency of the single CO I gene sequence. Another sequence, limited by the number of sequences that can be used in the DNA database, with a maximum similarity between 85%-86%, was identified to the level of family. It is suggested to further improve the sequence quantity and quality of the DNA barcode database, so as to let it be more useful for species identification.

Abstract:SOX9 is a member of the SOXE group in the SOX family, which plays an important role in sex determination and differentiation of vertebrates. is specifically expressed in the testes of mammals, birds, and reptiles, and is expressed in both the testes and ovaries in amphibians and fishes. To measure the expression of in the gonads of invertebrates, we cloned a 2835 bp full-length cDNA of the scallop ) which contains a 1413 bp open reading frame (ORF) encoding a 470-amino acid sequence with the HMG-box of the SOX family and the highly conserved region of the SOXE group, although there is no Pro-Gln-Ser-rich region at the C-terminus of vertebrate SOXE proteins. -SOX9 polyclonal antibodies were produced based on the full-length cDNA sequence. ISH and immunohistochemistry detection showed that SOX9 proteins were located in all germ cells of testes and ovaries at different developmental stages. The expression patterns between mRNA and SOX9 protein were similar. In the testes, the intensity of positive signals was highest in the spermatocytes and lowest in the spermatozoa. In the ovaries, the intensity of positive signals gradually decreased across the oogonia, oocytes, and mature oocytes. The expression characteristics of gonads were different from that most of vertebrates, which show variability in expression between sex, suggesting its function of gonad development and gametogenesis might be different from vertebrates.

Abstract:The hybrid offspring from black porgy () have been successfully farmed in an attempt to develop new marine aquaculture species with optimal target traits. To analyze the molecular mechanism of different growth traits between parent fish (As) and hybrids (PA), the full-length cDNA sequences of ) from each were cloned and identified. The expression profiles of genes in six different tissues of two-year-old and 30 d larvae of F1 generation hybrids and pure individuals were evaluated and compared using real-time quantitative PCR. The gene is 1241 bp, both including an open reading frame (ORF) of 450 bp and encoding a peptide of 149 amino acids with an estimated molecular mass of 16.84 kD and a predicted isoelectric point (pI) of 4.09. Sequence alignment, structure comparison, and bioinformatics analyses showed that family. Functional domain analysis indicated that the protein has four EF-h and calcium-binding domains, with sixteen Ca²⁺ binding sites. are non-secretory and non-transmembrane proteins, and likely have their functional location in the cytoplasm. Quantification of expressed mRNA showed comparatively high levels in the brain and gonadal tissues of two-year-old fish and in larval fish. in gill, muscle, and gonadal tissue, as well as in larvae, but not significantly different in the brain, liver, or kidney tissues ( transcripts in the hybrid larvae, and the highest enrichment of in growth and development. This study provides a basis for further research into gene function and differing genetic traits between the parent and hybrid offspring of genus crosses of Sparidae.

Abstract:This study investigated the effects of UV irradiation on sperm motility and duration of sperm activity and determined the optimal UV irradiation dosage to genetically inactivate sperm, while retaining adequate motility to fertilize eggs. Milt from several males was diluted 1:30 with Ringer's solution and UV irradiated at an intensity of 2200 μW/(cm²·s), and used to fertilize eggs. The fertilized eggs were divided into a cold-shock group (3℃ for 10 min at 2 min 30 s after fertilization) and a control group without cold-shock. Early embryo survival rate and larvae hatching rate were measured and compared. The results showed that:1) motility of sperm was negatively correlated with UV treatment time, and the rapid changes in sperm showed a typical Hertwig effect; 2) early embryo survival rate induced by sperm in the control groups was negatively correlated with the UV exposure time, while the larvae hatching rate showed a typical Hertwig effect; 3) early embryo survival rate and larvae hatching rate induced by sperm in the cold-shock group showed a typical Hertwig effect with the increase in UV irradiation time, reaching peaks at 2 min 20 s and 1 min 30 s, respectively (the peak values of the survival and hatching rates of were (38.3±4.3)% and (66.5±5.1)%, respectively; the peak values of the survival and hatching rates of were (43.3±3.3)% and (67.7±6.3)%, respectively). The results of this study indicated that the optimal UV dosages for eggs were 308 mJ/cm² and 198 mJ/cm², respectively. These results provide information for the improvement of gynogenetic technology for .

Abstract:This study determined the effects of different sources of dietary lipid on survival rate, weight gain, molting, digestive enzyme activity, and fatty acid composition in male juvenile Chinese mitten crab, . Five diets were formulated to contain different amounts of fish oil, soybean oil, and linseed oil. The control feed (Group F1) was formulated with 6% fish oil, whereas the other 4 experimental diets were formulated with 6% soybean oil (Group F2), 6% linseed oil (Group F3), 3% fish oil and 3% soybean oil (Group F4), and 3% fish oil and 3% linseed oil (Group F5). Juvenile crabs (with initial body weight 2.15 g±0.10 g) were randomly allocated to one of the diet groups. Twelve juvenile crabs per diet group were individually stocked in plastic tanks. After 112 d, weight gain and specific growth rate of Group F5 was significantly higher than that of the other groups (<0.05). No significant difference was observed in molting interval or hepatosomatic index. Analysis of digestive enzyme profiles from the hepatopancreas revealed positive correlations between enzyme activity and lipid sources:tryptase activity in Group F1 was significantly higher than that in the other groups (<0.05); pepsin activity in Group F3 was significantly higher than that in the other groups (<0.05); lipase activity in Groups F1, F2, and F4 was significantly higher than that in the other two groups (<0.05). Different diets had no significant effects on amylase activity. Fatty acid composition of the hepatopancreas and muscles followed the fatty acid composition of the diet. Eicosapentaenoic acid (C_20:5n3) and docosahexaenoic acid (C_22:6n3) in Group F1 was significantly higher than that in the other groups () in Group F2 was significantly higher than that in the other groups () in Group F3 was significantly higher than that in the other groups (<0.05). These results suggested that replacing 50% fish oil with soybean or linseed oil had a positive effect on growth, but a negative effect on survival rate in juvenile Chinese mitten crab, . Both soybean oil and linseed oil had a significant effect on tryptase activity, pepsin activity, lipase activity, and fatty acid of the hepatopancreas and muscles in juvenile Chinese mitten crab, .

Abstract:A 90-day feeding trial was conducted to investigate the effects of fish oil substitution with microalgae meals on growth performance, tissue proximate composition, and biochemical indices in juvenile starry fiounder, (initial body weight 7.35 g±0.03 g). Four groups of isonitrogenous and isolipidic diets were formulated using different primary lipid sources:fish oil (FO), meal (NO), and the mixture of these two algae meals (MO), with the remaining part filled with corn oil. Results showed that growth performance was not significantly different between FO and SO, whereas specific growth rate (SGR), protein efficiency ratio (PER), and viscerosomatic index (VSI) of NO and MO were significantly lower than FO (<0.05). Feed efficiency (FE) of MO and NO was the opposite. The body crude protein content of MO was significantly higher than that in the other groups (<0.05), while body crude lipid content of FO and MO were significantly higher than that in the other groups (<0.05). The muscle ash content of FO was significantly lower than that in the other groups (<0.05). Fatty acid composition of muscle and liver were directly influenced by dietary fatty acid composition ( and DHA in muscle were positively correlated with that of the diets (<0.05), and the percentages of C_14:0 and C_16:1n-7 were significantly positively correlated with that of the diets (18:2n-6, n-6 PUFA, and DHA:EPA in liver were also positively correlated with that of the diets (16:1n-7 showed a significantly positive correlation (<0.01). Compared with FO, levels of serum alanine aminotransferase (ALT), triglyceride (TG), cholesterol (CHO), and alkaline phosphatase (ALP) of NO and MO were significantly decreased (<0.05). Additionally, levels of serum aspartate aminotransferase (AST) and glucose (GLU) of NO were significantly lower than those in other groups ( < 0.05). In summary, there were no negative effects on growth and physiology when fish oil was replaced with P. stellatus to a certain extent.

Abstract:In recent years, stock enhancement in Shandong province has been carried out in two ways. One is to release hatchery-reared juvenile . The other is to provide suitable spawning substrates made of salt cedar branches or sweet wormwood branches. The materials for the spawning substrates are cheap, and the substrates are also appropriate for conservation and enhancement. However, the materials can't be reused and are easily damaged on transportation and deployment. Furthermore, successive cuttings of salt cedar and sweet wormwood significantly damage the two terrestrial plant species. Therefore, in order to improve stock enhancement and to further expand the stock enhancement scale, identification of environment-friendly spawning substrates has become an important issue. A study on different types of spawning substrates for oosperm adhesion was conducted in an indoor spawning pool. Additionally, the adhesion of oosperms to the spawning substrates was also examined in the field. The results of the indoor experiment showed that the adhesion of the oosperms on salt cedar branches was the best, followed by that on the cruciform spawning substrates, with that on the sea cucumber frame being the worst. The adhesion of oosperms on the white cruciform spawning substrates was significantly better than on the black ones (<0.05). Attaching salt cedar branches to the cruciform spawning substrates significantly increased oosperm adhesion. In addition, oosperm adhesion on the cruciform spawning substrates, which were placed at the pool bottom, was significantly better than those hung in the upper water (<0.01). In the field experiment, the results showed that oosperm adhesion to the cruciform spawning substrates was good in both 2015 and 2016, with highest oosperm adhesion rates being 82.14% and 56.72%, respectively. Oosperm adhesion was not significantly different between the black and the white cruciform spawning substrates (>0.05). Oosperm adhesion to cruciform spawning substrates that were hung at the sea bottom was significantly better than on those hung near the sea bottom (S. esculenta, resource enhancement and comprehensive protection of the spawning population can be achieved effectively by deploying artificial cruciform and collapsible spawning substrates at the sea bottom in its spawning grounds and in the main enhancement and releasing areas of slack water with depths of 15-20 m.

Abstract:Mud crab (), widely cultured in brackish and seawater ponds along the coast of southern China, has become the most productive seawater crab species because of its strong adaptive abilities and high economic value. Bacteria play an important role in pond aquaculture ecosystems; however, little is known about the bacterial composition and diversity in earthen aquaculture pond microbial communities of . Accordingly, we applied Illumina HiSeq high-throughput sequencing to investigate bacterial community structure and diversity in pond-raised intestinal and its aquacultural environment. The results showed that 234575 effective sequences were detected in three samples, and they could be classified into 2812 operational taxonomic units (OTUs), among which 453, 706, and 2547 OTUs belong to the gut of , the aquaculture water, and the sediment, respectively. 184 OTUs were shared among all samples, 197 OTUs between S. paramamosain intestine and sediment, and 572 OTUs between the aquaculture water and sediment. This indicates that bacterial richness and diversity were lower in intestine than in water and sediment. In total, 48 phyla were identified, among them Proteobacteria (39.96%), Tenericutes (23.09%), Firmicutes (16.58%), CKC4 (6.55%), and Bacteroidetes (4.96%), which constituted the predominant components of the intestine; Proteobacteria (63.02%), Actinobacteria (24.96%), Bacteroidetes (8.41%), Cyanobacteria (1.19%), and Firmicutes (0.83%) were the predominant phyla in the water; and the predominant phyla in the sediment were Proteobacteria (75.23%), Bacteroidetes (5.72%), Actinobacteria (3.83%), Chloroflex I (2.65%), and Acidobacteria (1.80%). Analysis of the 10 most abundant bacterial OTUs in different samples revealed that the most dominant bacteria in intestine and its aquacultural environment were totally different, and there were only four kinds of dominant bacteria that have close sequences in the SILVA database. These results suggest that there exists a close correlation between bacterial species composition in the intestine and its aquacultural environment. Meanwhile, intestinal microflora may be a relatively independent microbial ecosystem and the micro-ecosystem could hardly be influenced by the bacteria in the culture environment.

Abstract:Enteric redmouth disease (ERM) is an emerging problem in aquaculture all over the world. ERM is caused by the gram-negative bacterial pathogen , which can infect salmonids and several other fish taxa and cause clinical signs of hemorrhaging on the body surface and in the intestine. Fish suffering from ERM exhibit exophthalmos, darkening skin, subcutaneous hemorrhage of the mouth and throat, perianal swelling with yellow fluid, and other deleterious outcomes up to and including death. When ERM appears in an aquaculture facility, a large number of fish may be affected over a short period of time. Various antibiotics are available for the treatment of ERM and vaccines can also be used in the treatment and prevention of this disease. Several methods such as ultrastructural examination and LAMP have been developed for ERM detection. However, these assays are generally laborious and time-consuming, and are not sufficiently sensitive. To establish a rapid and quantitative method for the detection of , a pair of specific primers was designed and synthesized based on the virulent gene gene was used as a standard to construct a standard curve. SYBR Green I real-time quantitative PCR assay was established for the detection of by optimizing experimental conditions. The established qPCR method was also applied to the detection of in tissues of artificially-infected rainbow trout. Our results showed that the designed primers had good interspecific specificity. The quantitative linear equation was =0.9958). The detection limit of qPCR method was 57 copies/μL, which is approximately 100-fold greater than conventional PCR. This qPCR method can accurately detect in rainbow trout. These results suggest that our qPCR method has the advantages of specificity, sensitivity, rapidity, and quantification, and can be used for rapid diagnosis of early-stage disease and quantitative detection of .

Abstract:In order to determine the impact of pathogenic Litopenaeus vannamei, shrimps with weights of (2.2±0.24) g were challenged with Vp at a concentration of 5×10⁷ CFU/mL for 36 h. The hepatopancreas tissues from the experimental and control groups were collected at 6 h, 12 h, 24 h, and 36 h after the start of the experiment to evaluate antioxidant enzyme activities and immune and mTOR signal pathway-related gene expression levels. The results showed that after challenge with Vp, SOD activity was significantly higher than that of the control group (<0.05), while CAT, GSH-PX, and GST activities all increased at first and subsequently decreased, reaching the peak value at 12 h, 6 h and 24 h, respectively ( were significantly higher than in the control group ( were up-regulated at first and then decreased after reaching their peaks at 12 h and 24 h, respectively, while the level of the gene was significantly lower than that of the control group during the time 12-36 h (< 0.05). These results showed that functions of the immune system were affected by Vp. Several important genes in mTOR signal pathway were also been quantified by qRT-PCR. The relative expression levels of P<0.05). were both up-regulated at first and then decreased after reaching peak values at 12 h and 6 h, respectively ( was significantly lower than that of the control group after challenge (<0.05). The results indicated that Vp challenge had a noticeable impact on antioxidant enzyme activities and immune and mTOR signal pathway-related gene expression of , with significant destructive effect on the shrimp's immune system.

Abstract:This work was conducted to provide a useful basis for development of a new subunit vaccine against Chinese giant salamander iridovirus (CGSIV). In order to express major capsid protein (MCP) of CGSIV in baculovirus expression system, the gene of MCP was subcloned into baculovirus transfer vector (pFastBac1). The recombinant plasmid pFastBac-MCP was identified by restriction enzyme digestion and gene sequencing, and then transformed into DH10Bac competent cells containing baculovirus shuttle vectors. After identification by blue/white selection, PCR analysis, and gene sequencing, a recombinant bacmid (rBacmid-MCP) was obtained. Thereafter, the recombinant bacmid was transfected into Sf9 cells with Insect GeneJuice^{?/SUP> Transfection Reagent and recombinant baculoviruses were obtained in Sf9 cells, which could be observed by eletron microscopy in the ultra-thin sections of infected Sf9 cells and were named AcNPV-MCP. The recombinant baculovirus infected Sf9 cells with different multiplicities of infection (MOI=2, 5, or 10) showed the highest production of recombinant protein of MCP. The result of SDS-PAGE analysis showed that the production of expressed MCP could be the highest with MOI=10. By immunofluorescence assay, the presence of MCP was observed on the surface of infected Sf9 cells. The recombinant protein of MCP was purified with the prepared immunomagnetic bead with monoclonal antibodies against CGSIV MCP and its bioactivity was verified by western blot assay with the rabbit antisera against CGSIV MCP. The results of SDS-PAGE and western-blotting assays indicated that the recombinant protein was highly purified and had good immunogenicity, which could specifically react with the rabbit antisera. In conclusion, the recombinant protein of CGSIV MCP was successfully expressed in baculovirus expression system and purified with the immunomagnetic bead assay, which laid a solid foundation for the development of a new subunit CGSIV vaccine in the future.}

Abstract:In this study, the infectious hematopoietic necrosis virus (IHNV) isolate SD-12 glycoprotein (glycoprotein, G) gene was cloned into commercial vector pcDNA3.1 (+) to construct an IHNV G expression vector, known as an infectious hematopoietic necrosis (IHN) nucleic acid vaccine and named pIHNsd-G. Rainbow trout (5±0.5) g were immunized with 2 μg of the vaccine by intramuscular injection at the dorsal fin base. The expression of the gene was detected by real-time PCR in the anterior kidney and the muscles from vaccine-delivered sites of the rainbow trout at 4 days and 7 days after immunization, respectively. At 21 days post immunization, the relative protection rate (RPS) of the vaccine was calculated by challenge with SD-12 at a dose of 100% of the tissue culture-infective dose (TCID₅₀) by intraperitoneal injection. The serum neutralizing antibody titers of immunized rainbow trout were tested at 60 and 150 days post-immunization. Finally, the pIHNsd-G promoter sequence and ampicillin resistance gene sequence were used as target genes and the dynamic distribution of pIHNsd-G in the inoculated part of the rainbow trout was monitored by PCR. The results showed that the gene was significantly up-regulated in the kidney and the muscles at the inoculated site and was significantly higher in the muscle tissue than in the head kidney at the same time point. Challenge of rainbow trout showed that the RPS of pIHNsd-G for rainbow trout was 94.4%. The neutralizing antibody titer revealed that at 60 days post-immunization, neutralizing antibodies were found in all of the serum samples of immunized rainbow trout and the highest titer was 320, while the highest antibody titer at 150 days post-immunization was 80. Since then, it has been shown that an effective IHN nucleic acid vaccine has been successfully obtained. The results of PCR following the injection of pIHNsd-G rainbow trout showed that all pIHNsd-G target fragments were detected in the muscle of the injection site on the first day after immunization; it was not possible to amplify the full-length ampicillin resistance gene from the injection site muscle at 84 days, and all target genes disappeared at 150 days. Based on the successful construction of an IHN nucleic acid vaccine and the systematic validation of its effectiveness, this study carried out dynamic analysis of the vaccine at the inoculation site, which provides basic data for the research and development of IHN nucleic acid vaccines and safety evaluations.

Abstract:Chitosanase is important in carbon and nitrogen recycles that occur extensively in nature, and is useful in the preparation of biofunctional chitooligosaccharides. Chitosanase occurs in a variety of microorganisms, including bacteria and fungi. Transformation by genetic engineering has increased the enzyme activity and enzyme content of recombinant chitosanase. To obtain abundant chitosanases possessing high chitosanolytic activity for large-scale production of chitosan-oligosaccharide, the chitosanase of CH2 was best induced by fructose and not induced with chitosan, unlike other chitosanases. This study investigated recombinant expression of chitosanase from , and characterized the application of the recombinant enzyme for chitosan hydrolysis. Chitosanase may have important industrial applications in the utilization of the enormous chitosan substrates. Mass spectrometry (MS) and thin-layer chromatography (TLC) were used to analyze the enzymatic products. The molecular weight of the recombinant chitosanase was estimated to be 29 kD using SDS-PAGE. The specific activity of crude enzymes was 133.60 U/mg. The specific activity of the purified chitosanase was up to 338.08 U/mg. The optimal pH and temperature of the purified chitosanase was 50℃ and 4.5, respectively. The values with soluble chitosan as a substrate were 5.48 mg/mL and 24.39 (μmol/mg)·min^-1, respectively. Fe²⁺, K⁺, Na⁺, Li⁺, Ca²⁺, and especially Mn²⁺ enhanced the enzyme activity of the recombinant chitosanase. Whereas, Ag⁺, Mg²⁺, Hg²⁺, EDTA, EGTA, and SDS inhibited the enzyme activity, and the other metal ion tested had no effect on enzyme activity. This characteristic of the recombinant chitosanase was better than the chitosanase of CH2. Furthermore, the enzymatic production of chitooligosaccharides from chitosans of various deacetylation degrees ranged mainly from chitobiose to chitopentamer, and the enzymic products contained 2-10 degrees of polymerization of chito-oligosaccharide after the recombinant chitosanase hydrolysis, but no monose. These results indicated that the enzyme was an endo-type chitosanase and a typical acidic metalloproteinase, which might be a good candidate for biotechnological application in producing chitooligosaccharides.

Abstract:Azaspiracids (AZAs) are a group of lipophilic polyether compounds first discovered in Ireland and have been implicated in shellfish poisoning incidents. Known toxin-producing genera have rapidly increased and now comprise six species, including A. dexteroporum. Approximately 40 different analogs have been reported to differ in structure and toxicity, of which AZA1, AZA2, and AZA3 are the most important ones based on occurrence and toxicity. AZAs are a variety of nitrogen-containing polyether toxins, which include a unique spiral ring assembly, a heterocyclic amine, and an aliphatic carboxylic acid moiety. The consumption of polluted shellfish can cause human intoxication, with symptoms including nausea, vomiting, diarrhea and stomach cramps, which is similar to diarrhetic shellfish poisoning (DSP). Nowadays, in order to guarantee food security, the developed countries have begun to prioritize shellfish monitoring. A regulatory level of 160 μg AZA1eq/kg in the shellfish tissues is implemented in many countries all over the world, but our country does not have a regulation about this. Amphidomataceae, which is known for the production of AZAs. Twenty two out of 25 strains isolated in China have proven to be AZA-producing strains, and show a large variability in the AZA profile among different strains. This status underlines the high-risk potential of blooms with subsequent shellfish intoxication for the Asian Pacific Ocean. AZA accumulation by bivalve mollusks occurs frequently, while blue mussels were found to accumulate by far the highest concentration. Meanwhile, AZAs have been found in various species of bivalve mollusks such as oysters, mussels, scallops, and clams along Chinese coastline. As AZA is a recently discovered toxin, the bloom dynamics, transfer kinetics, and pathways of AZAs into bivalve mollusks are just beginning to be explored. Accumulation and distribution of AZA toxins in bivalve shellfish could be correlated in time and space to blooms of species. Subsequent accumulation and trophic transfer can intoxicate higher trophic-level consumers and eventually pass into human beings. To investigate the hazard formation process, two transfer routes must be taken into consideration:AZAs could be accumulated by bivalve shellfish or plankton vectors from toxin producing algae. In conclusion, a direct link between AZA accumulation by bivalve shellfish and must be investigated for food safety control and monitoring plans. Liquid chromatography coupled with quadrupole linear ion trap tandem mass spectrometry was established for the evaluation of AZA accumulation, distribution, and biotransformation in scallops. Toxin-producing alga Chlamys farreri. The results showed that AZA2 is the dominant toxin of AZDY06, with toxin production capacity up to (7.05 ±0.52) fg/cell. After 12 h exposure experiments with 5x10⁷ cells AZDY06 ingested by every , AZAs in mussels reached the highest concentration and exceeded EU regulatory limits. The concentration was 165.3 μg AZA1eq/kg, while total accumulation efficiency reached 78.2%. The distribution of AZA toxins showed significant differences between organs, with gill > viscera > pallium > muscle. Four AZA analogs including AZA19, AZA12, AZA6, and AZA23 were found in all organs. AZA19 was the main metabolite with a proportion of 40%, while other metabolites were found more rarely. This study proved that the hazard of (AZDY06) isolated from China is strong and underlined that AZA19 was present at significant levels after only 6 h and until the end of the study. Therefore, this analog needs to be considered as the major metabolite that can affect human health. It is highly recommended to quickly formulate AZA limits, based on this analysis of raw bivalves by LC-MS/MS.

Abstract:Three Gorges Dam is a vital project in the development and harnessing of the Yangtze River. Water-level fluctuation zone of Three Gorges Reservoir, which refers to the area along the Three Gorges Reservoir banks bounded by minimum (145 m) and maximum (175 m) water levels of the Three Gorges Reservoir, has provoked a series of ecological problems, which included soil erosion, reduction in ecosystem types, decrease in biodiversity and so on. However, effect of water level fluctuating on fish of early life history stages in the water-level fluctuation zone was not yet reported. In this study, the research area was between 150 km and 330 km from Three Gorges Dam, where fish eggs and larvae, exposed to water or remained in shallows, were collected. Based on distribution of sampling places, the research area was divided into 8 regions, which were mainstream (down), Meixi River, mainstream (middle), Shilu River, Modaoxi River, Tangxi River, mainstream (up), and Pengxi River. According to sampling time and place, and morphological characters of fish eggs and larvae, 132 samples were preserved, including 1735 fish eggs and 150 larvae. Twenty-four samples were collected in mainstream. But only one sample in respective mainstream (middle) and mainstream (down) included respective one and nine larvae, in which no fish egg was found. In Modaoxi River, a tributary of Three Gorges Reservoir, there were the most samples in this study, and 59 samples were collected including 733 fish eggs and 108 larvae. Due to insufficient morphological diagnostic characters in kraurotic fish eggs and larvae, it is easy to misidentify in the genus or species level. So, this study was employed DNA barcoding technology to identify the samples. DNA barcoding technology could be used as a rapid tool to distinguish morphologically similar species, and survey many uncertain species and species composition. Previous researches have shown that only molecular identification could guarantee identification of fish eggs and larvae to the species level. Through mtCO I sequence alignment, and based on mtCO I sequences similarity values (species:>99%, genus:92%-99%, family:85%-92%), the samples were distinguished to 14 types, of which 10 types were identified to species:Cyprinuscarpio, Carassiusauratus, Hemiculterleucisculus, Rhinogobius nagoyae, Rhinogobius giurinus, Opsariichthysbidens, Acheilognathusmacro-pterus, Pseudorasboraparva, Hypophthalmichthys molitrix, and Rhinogobius, Rhodeus, and Rhodeusocellatus and . If fish DNA barcode database of Yangtze River or Three Gorges Reservoir could be completely and accurately constructed, and new molecular markers could be developed for fishes that can be not distinguished by mtCO I barcode, it would help to further identify fish eggs and larvae in the water-level fluctuation zone and investigate fish resources of the Yangtze River. had the largest proportion in the samples, 50.0% and 24.6% respectively, and widely distributed. The number and species of samples in upstream of mainstream was more than downstream in the research area. It may be not only related to its topography, and also its fish resources. In Modaoxi River, the number and species of samples was the most in this research area, which contained nine fish species. The result showed that it was more affected from the water level fluctuating, at the same time, it also indicated that fish resource were rich in Modaoxi River, which should be got more attention in the future fish resources investigation and protection in Three Gorges Reservoir. This study employed DNA barcoding technology to identify fish eggs and larvae collected in partial water-level fluctuation zone, which would contribute to comprehensively assessing the effect of water level fluctuating on fish resources in water-level fluctuation zone of Three Gorges Reservoir.

Abstract:is a very important commercial fish distributed widely in the Yangtze estuary. It is a migratory species which grows and matures in the coastal waters and then naturally migrates into the Yangtze estuary for spawning. Otolith growth and development at different stages in larval and juvenile can help to understand their characteristics under natural conditions. An understanding of otolith early growth and development is essential not only for wild population conservation but also for artificial seeding production. In this study, a total of 103 larval and juvenile were collected in the Yangtze estuary, from April to August in 2014. Otolith analysis revealed that the age of specimens ranged from 5 to 48 days. To study the early growth and development of different stages, the body length of 103 individual was measured. Statistics showed that the cubic function best described the increase of body length with age. The increase of body length can be divided into three stages, 5-11 d, 12-30 d, and 31-48 d. The growth rate was significantly different among these stages (<0.05). To study the morphological development, the otolith length and otolith width were measured, and the analysis showed that the increase of otolith length and width increased with body length. After 17 to 19 days, the growth rate increased by about 2-fold. Piecewise regression analysis between otolith length, width, and otolith age was carried out in order to determine the allometric growth pattern, and the inflection point of otolith length and otolith width both occurred at 19-20 days. The growth rate before the knot was slow, while it was rapid after the knot. The otolith morphology of changed significantly at different stages in the early life history. The otolith is circular in the yolk-sac and preflexion stages. Otolith length growth is greater than that of otolith width in the flexion stage, and the otolith becomes oval. After the form becomes stable, the ratio of otolith length and width also remains stable.

Abstract:Body size is a basic biological characteristic in fish populations and can reflect individual physiology as well as changing environment conditions. Slight variabilities in some biological parameters may result in complex ecological effects, and affect food web link intensity in trophic cascades. However, the spatial and temporal heterogeneity of size composition within populations have often been ignored in many studies of fish biology. We use Fang's gunnel () in Haizhou Bay as an example for studying the variability of body size on an annual scale. is a low trophic fish and plays an important role in the food web and ecosystem of the Yellow Sea, with increasing dominance in Haizhou Bay. We collected annual bottom trawl surveys in Haizhou Bay in the spring and fall from 2011 to 2016. We used a range of statistical methods, included variable coefficient, covariance analysis, two-sample -test, and Pearson correlation analysis to study the population size composition, length-weight relationship, and relative fatness of in this area. We analyzed the annual and seasonal variability as well as the spatial distribution of body length and relative body mass. The results showed that had multiple age structure in Haizhou Bay. Their length frequency distributions were multi-modal and skewed, with the majority of captured individuals aged 2-3 years. Statistical analysis indicated that there were remarkable temporal and spatial variations in the average size and the parameters of body length-weight relationship of <0.05). The average body length and relative fatness tended to be higher during autumn surveys than spring surveys. Variation in relative fatness was greater between seasons than among years. In spring, the spatial distribution of body length and relative fatness was larger in the southwest area than the northeast area of the bay. A -test on the body length-weight relationship showed that the allometric growth patterns of were generally positive. Correlation analyses between benthic water temperature and the length-weight relationship showed that temperature had a substantial influence on the relative fatness, body length-weight relationship, and mean body length. The spatiotemporal variability of fish size and other parameters may be attributed to their feeding intensity, maturation, fishing pressure, and environmental and habit variation, and is also likely to reflect changes in the fishery ecosystem. We suggest that the spatiotemporal variability of population size composition should be fully considered in fisheries resource management, as these basic parameters can contribute significantly to fishery ecosystem modelling and management strategy evaluation.

Abstract:Oufei Beach is located in Wenzhou, with its northern border along the river mouth of the Oujiang River estuary and its southern border along the Feiyun River estuary. A beach reclamation project and a series of marine engineering construction efforts will inevitably lead to changes in the local nekton community. Therefore, we aimed to carry out a survey of the nekton resources along the Oufei shoal in the adjacent waters and to evaluate its community structure, to provide baseline data for observing trends of change. This information is of great significance for evaluating and restoring ecological health of the area. Data were obtained from a fisheries resources survey carried out near Oufei Beach in May 2013. A series of statistical methods including population encounter incidence (PIE), variance ratio (VR), test, association coefficient (AC), percentage of co-occurrence (PC), point correlation coefficients (), Ochiai index, functional group classification, and trophic level constituents combining the means of niches were used to analyze the relationships among the major marine nekton species. Our results showed a population encounter incidence of 0.65. The maximum population encounter incidence was observed in , at 0.244, and the minimum population encounter incidence was observed in Leptochelia gracilis formed a species pair with a maximum value of 0.305, while formed a species pair with a minimum value of 0.003. There were significant positive correlations between the major nekton community species. Most species pairs had weak positive association or were unrelated, indicating that species had independent distributions. According to feeding habit, the major nekton species can be divided into five functional groups; in this survey, the omnivorous group formed the most numerous of these, while the phytophagous group was not represented by any species. Based on the relationship among the functional groups, the trophic levels for major nekton species varied from the second to fourth trophic levels, with the third and fourth trophic levels as the key trophic levels in our study. A combination of the above methods yields a comprehensive analysis of the major nekton adjacent to Oufei Beach, showing functional relationships between different populations. An in-depth analysis and study of interspecies relationships in this habitat is valuable as a scientific basis for protecting fisheries resources, developing breeding programs, and building management strategies. Our study has demonstrated that the ecosystem structure in Oufei Beach is mature, and that the community has the capacity to tolerate some degree of environmental change. However, the number of major nekton and the type of resources in the area have been heavily affected by environmental degradation of the area. To realize sustainable development of fisheries resources in this area, greater emphasis should be placed on the regeneration and maintenance of these resources.

Abstract:(Chlorophyceae, Order Volvocales), a freshwater green microalgal species, has commercial value owing to its ability to accumulate high concentrations of astaxanthin (up to 5% of dry weight). Astaxanthin (3,3'-dihydroxy--carotene-4,4'-dione) is a red ketocarotenoid which has many important biological functions, including antioxidant activity, regulation of immune responses, and disease resistance, and has the potential for application in the aquacultural, nutraceutical, pharmaceutical, and cosmetic industries. has a distinctive lifecycle as it exhibits a green motile stage and a red non-motile resting stage called an aplanospore. In general, astaxanthin accumulation in is restricted to the aplanospore stage. Astaxanthin is accumulated in extra-plastidic lipid vesicles as a secondary carotenoid, and it is believed to be synthesized in response to oxidative stress in the red aplanospore stage under unfavorable environmental conditions such as high light, temperature, and salinity, or low nutrient availability. Several enzymes such as lycopene -carotene ketolase (Bkt) are involved in the astaxanthin bios-y-n-thesis pathway in -carotene from lycopene, and CrtR-B and Bkt cat-alyze further steps leading to astaxanthin synthesis. Changes in expression of the genes encoding these three enzymes can critically affect the biosynthesis and accumulation of astaxanthin in . In addition, various ant-i-oxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) also have important protective effects for combating oxidative stress under unfavourable environmental conditions. We aimed to explore the effect of salt stress on astaxanthin accumulation in , by examining the mechanism of astaxanthin synthesis and the relationship between the different antioxidant mechanisms in at high salinity. We examined its growth rate; astaxanthin content; gene expression levels; SOD, CAT, and GSH-Px activities; and malondialdehyde (MDA) content at four salinity levels (0.04 mol/L, 0.08 mol/L, 0.12 mol/L, and 0.16 mol/L) over three timescales (3 days, 6 days, and 9 days). Our results showed that the density of decreased under increasing salinity over different periods of time, while its mortality rate and aplanospore proportion increased with increasing salt stress concentration by the ninth day of stress. Asta-xanthin content, gene expressions increased over time with increasing salinity. SOD, CAT, and GSH-Px activities and MDA content also increased in comparison to grown at the control level of 0 mol/L NaCl, and significantly increased at the 0.12 mol/L NaCl level ( gene expressions were lower during the early and mid-stages of salt stress (e.g., on the third and sixth days of observation), and increased by the ninth day. Meanwhile, SOD, CAT, and GSH-Px activities and MDA content were higher during early and mid-stage stress, but were lower by the ninth day. These results suggest that salt stress can improve astaxanthin accumulation over time in at the appropriate level of salt stress, despite its negative effects on growth. Astaxanthin synthesis in is promoted mainly through an increase in the transcription level of astaxanthin synthesis-related enzyme genes under salt stress, and the antioxid-ant activities of astaxanthin and antioxidant enzymes complement one other to protect from oxidative damage under salt stress. This study provides a new insight into the astaxanthin synthesis and antioxidant mechanisms in .

Abstract:is the summer dominant species of planktonic copepod in Zhoushan waters. We evaluated the effects of two kinds of unicellular algae food- -on the nutritional value of using biochemical methods. The aim of this paper was to provide theoretical information to evaluate the nutritional value and large-scale cultivation of . The results indicated that the crude protein content (wet weight) of Chlorella sp. (5.21±0.98)% was significantly higher than that of the control group (2.44±0.63)% (<0.05). The lower crude protein content of the control group may have been due to the poorer growth conditions, less food availability, and higher energy consumption under natural conditions. After feeding on , the crude fat content (0.25±0.03)% (wet weight) increased significantly ( sp. (0.15±0.03)%, most likely as the fatty acid content of sp.. The content of crude protein and crude fat in sp.; therefore, it was possible that consumed protein to supplement the energy required for growth due to the inadequate fat content in this food. The changes in content (wet weight) of total sugar and ash were not significant. Nine essential amino acids (EAA) and 8 non-essential amino acids (NEAA) of were detected in this study. The content (dry weight) of EAA in was (47.56±0.04)% to (49.84±0.07)%, EAA:NEAA was approximately 5:5, higher than that of , which may be beneficial for the growth of this fish. Twenty-one kinds of fatty acids in were determined. The content (dry weight) of DHA and EPA in the control group (9.77%) was lower than the group that fed on sp. (21.51%), but higher than the group that fed on (8.40%). Therefore, the proportion of DHA and EPA in , and could better meet the needs of juvenile fish growth. Overall, the results indicated that the nutritional value of was higher under artificial cultivation conditions and had potential value for the live food economy.