Abstract:NK-lysin is a cationic antimicrobial peptide mainly produced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and stored in cytolytic granules together with perforin and granzymes. NK-lysin is a member of saposin-like protein family and is orthologous with human granulysin. Mammalian NK-lysin is known to possess antibacterial and antitumor property. Homologues of NK-lysin have been identified in diverse organisms including human, pig, cattle, chicken, horse, water buffalo and teleost species. In fish, NK-lysin genes have been reported in five species, i.e. Japanese flounder (Paralichthys olivaceus), channel catfish (Ictalurus punctatus), zebrafish (Danio rerio), half-smooth tongue sole (Cynoglossus semilaevis) and large yellow croaker (Larimichthys crocea). In this study, we aimed to explore the structure and expression difference in tissues, and the NK-lysin (Cinkl) was identified from grass carp Ctenopharyngodon idella using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE), and primers were designed according to the conserved sequence of known fish NK-lysin genes. The expression of the Cinkl gene in different tissues was analyzed by real-time quantitative PCR. In order to explore the antibacterial activity of CiNkl, the recombinant expression vector was constructed by cloning its mature peptide into the prokaryotic expression plasmid pET-28b-MBP. The antibacterial analysis of recombinant protein was evaluated using agar disc diffusion. The full-length cDNA sequence of Cinkl was 768 bp, encoding 121 amino acids. Genomic DNA was 3 361 bp including 4 exons and 3 introns that was similar to zebrafish nklc and nkld genome. The multiple sequence alignment analysis showed that the CiNkl shared 57.98% and 63.03% identity with zebrafish Nklc and Nkld respectively. CiNkl possesses a Saposin B domain of members of saposin-like protein (SAPLIP) family and six conserved cysteins (Cys) residues that in mammals are known to form three intramolecular disulfide bonds essential to antimicrobial activity. Phylogenetic tree suggested that CiNkl is clustered closely with D. rerio Nklc and Nkld. Quantitative RT-PCR results showed that Cinkl mRNA was expressed in all tissues examined and highly in the spleen, with small amount in heart, gill, kidney, head kidney and weakly in skin, brain, liver and intestine. In addition, SDS-PAGE showed that the molecular mass of recombinant CiNkl expressed in host bacteria Rosetta (DE3) was approximately 57 ku. The recombinant protein purified by Ni-NTA His·Bind resin showed the antibacterial activity against Escherichia coli M15, Aeromonas hydrophila and Staphylococcus aureus. The results indicate that grass carp NK-lysin has potential immunomodulatory effects in fish innate immunity.

Abstract:The present study aimed to evaluate the effect of Astragalus polysacharide (APS) and Angelica sinensis polysaccharide (ASP) on the cell apoptosis of Carassius auratus induced by Aeromonas veronii. Negative control group, positive control group, APS group and APS group (four groups) were set up in this experiment. Experimental fish was infected with A. veronii. The changes of apoptosis rate and cell cycle in blood cells were determined by flow cytometer. The morphology of apoptosis cells were observed by fluorescence microscopy and transmission electron microscopy (TEM). The results showed that the blood cell apoptosis rate of the positive control group was significantly higher than those of two polysaccharide groups and negative control group after being infected with A. veronii. Polysaccharide groups could significantly reduce the apoptosis rate and APS group had more significant effect than ASP group. Karyopyknosis appeared in the liver cells and the head kidney lymphocytes of crucian carp. Besides,chromatic agglutination adjacent to the nuclear membrane and apoptotic bodies appeared after the infection. Compared with the negative control group, the A. veronii infection could significantly decrease the percentage of S/G2+M phase cells and increase sub-G1 phase cells in crucian carp blood cell cycle and the infection had an effect on inhibiting cell division and inducing apoptosis. Compared with the negative control group, the proportion of G0/G1 and sub-G1 cells in the polysaccharide group was significantly reduced, and the proportion of S/G2+M phase cells was significantly increased, which indicated that polysaccharide groups can promote cell division and inhibition of apoptosis. Based on the above results, we found that APS and ASP could effectively inhibit crucian carp apoptosis induced by A. veronii when the addition was 1%.

Abstract:Mosquitofish(Gambusia affinis)is a globe invasive species. In order to analyze why they can invade so quickly all over the world, 11 polymorphic microsatellite loci were isolated and employed to examine paternity patterns in 755 samples of 53 families from 10 locations in 3 rivers basins. After sequence genetic analysis of the multiplex SSR PCRs, we got the best fullsib family and analyzed the phylogenetic relationship. We found that there are multiple inseminations in G. affinis. The female in the same womb can reproduce a number of the juveniles, up to 72, with different males, even up to 9. The max fullsib is 41, but the breeder's size isn't the max even not bigger than others. At the same time the max size of the breeder isn't the female which reproduce the largest number of the juveniles. The phylogenetic tree shows that the evolution was far away the sea. The 10 populations can be obviously divided into 3 clusters. The conclusion can be drawn that the key reason of the invasion is G. affinis multiple inseminations. It is suspected that the fertilized frequency isn't proportional to the size of female. Their gene flows rapidly specifically when they were in the migratory distance.

Abstract:The northern snakehead (Channa argus) is extensively distributed throughout rivers and lakes of China. With overexploitation and water eco-environment deterioration, the resources of C. argus are severely affected. To examine genetic structure and the demographic history of the northern snakehead, we collected 212 specimens from 8 locations for generating mitochondrial DNA complete control region sequences. Result showed that the length of control region is about 907 bp. The genetic diversity in southern China is higher than that in the north with boundary line 34°N. The factors that warm and wet climate in the south China and dry and cold climate in the north China, and that the rapid uplift of Qinling Mountain aggravated differentiation of the east Asian monsoon, may have an important influence on genetic diversity. The phylogenetic tree showed a shallow genealogy and high genetic differentiation and significant level of genetic structure which behaved in different drainages. This indicated that geographical barriers led to recent genetic differentiation. The demographic history of 7 populations of China displayed that the northern snakehead experienced population rapid growth in the interglacial period of the middle Quaternary. Then, the demography declined during the last glacial period. With climatic warming in the Holocene, a slight population growth happened. The Dongting Lake population also experienced a population expansion in the interglacial period of the middle Quaternary. The glacial-interglacial cycles in the Quaternary, especially from the middle Quaternary, caused pronounced climatic fluctuations and had an important influence on the northern snakehead populations. Information on genetic diversity and genetic structure will have implications for the management of fisheries and conservation efforts.

Abstract:White spot syndrome virus (WSSV), Taura syndrome virus (TSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) are the three major pathogenic viruses responsible for the epidemic diseases threatening Pacific white leg shrimp farming industry. We use the PCR detection methods recommended by World Organisation for Animal Health to investigate the epidemiology of these three kinds of diseases in Jiangsu province. 1436 Pacific white leg shrimp samples were detected in the year from 2015 to 2016. Results showed the positive rate of WSSV infection was 17.20%, 0% in TSV infection and 39.48% in IHHNV infection where shrimp samples were collected from Nanjing, Dongtai, Dafeng, Ganyu, and Sheyang. And all five IHHNV strains detected belong to the infectiontype after gene sequence analysis, which were close to the Korean strain. Our investigation aims to clarify the epidemic stiutation of WSSV, TSV and IHHNV in Jiangsu province, identify IHHNV strain type via sequence analysis. The result has a high reference value for diseases prevention and treatment of shrimp farming industry.

Abstract:Fish behavior data are quite important to water pollution monitoring and these kinds of data sets imply the fish healthy conditions. To acquire fish behavior data, we present a multiple-view based fish swimming 3D trajectory reconstruction technique. In this technique, we use three cameras to observe an aquarium from three different aspects and employ foreground detection and tracking methods to reconstruct 2D fish swimming trajectories from these three cameras. Based on the multiple-view 3D reconstruction theories, we fuse these three 2D fish swimming trajectories to achieve a 3D fish swimming trajectory. Experiments show that our method can accurately reconstruct the 3D swimming trajectory of a single fish in a cubic aquarium and get the real-time 3D position and the swimming speed of the fish. This method needs neither expensive experimental equipment nor strict installation requirement to make cameras vertically face the aquarium faces. However, it can acquire accurate 3D fish swimming trajectories in real-time.

Abstract:Belanger's croaker (Johnius belangerii) is a fundamental fish species of family Sciaenidae which usually has high gastric vacuous rate during 24 hours trawl surveys making the diet analysis difficult to continue. However, it plays an important role in the predator-prey relationship in the food web of Ma'an Archipelago, East China Sea. In this study, we use visual inspection with DNA barcoding technology combined previous collected data to make more comprehensive analysis towards its diet compositions and realistic prey-predator relationships. We amplified mitochondrial cytochrome oxidase subunit I (COI) using fish and invertebrates universal primers in sampled fish stomach contents which could not be identified by morphological observation. Our results indicated that using DNA barcoding could successfully identify those partially digested food compositions species, such as Solenocera crassicornis, Alpheus distinguendus, Palaemon macrodactylus, Alpheus japonicus, Acetes chinensis, Sebastiscus marmoratus. We found belanger's croaker mostly feeds on shrimps (frequency of occurrence, 45%) and crabs (23%), some juvenile fishes (27%) occasionally and among them S. crassicornis, P. macrodactylus, Portunus sanguinolentus, Oratosquilla oratoria had not been observed in summer investigation ever before. In conclusion, the accurate and sufficient diet analysis is the precondition of feeding habit research which could contribute to reasonable suggestions of the management of island reef area ecosystem and fisheries forecasting.

Abstract:Antarctic fish species have a very long life history and are slow-growing. Age determination of Antarctic fish is more difficult than fish species outside the Antarctic area because of unique biological characteristics. However, age determination of Antarctic fish is the basis of fishery stock assessment. Recently, some calcified structures, such as scales, fin rays, vertebrae and otoliths became the main materials for age determination of Antarctic fish. This review compares different methods and materials of Antarctic fish age determination, as well as analyzes their merits and deficiencies to provide the references for further studying Antarctic fish species. The results showed that, compared to otolith, scales and fin rays were seldom used to age determination of Antarctic fish. Length frequency distribution could not be suitable to age older Antarctic fishes because of the long life history. Otolith weight could erroneously estimate the age of Antarctic fishes because of typical individuals occurring. Because fin rays are easily damaged, vertebrae are difficult to collect, the age of some Antarctic fish species without scales were frequently estimated based on otolith sections and verified by scales as well. Meanwhile, some disadvantages, such as no unified age determination protocol, strong subjectivity and lack of background knowledge of early life history, etc. limit the accuracy and precision of ageing Antarctic fish species. Otolith microchemistry and microstructure will be used extensively for studying early life history of Antarctic fish.

Abstract:In order to study the population genetic structure and germplasm resources of the Sipunculus nudus. mtDNA control region sequences of S. nudus of six geographic populations along the coast of Beibu Gulf (Beihai, Zhanjiang, Qinzhou, Fangchenggang, Danzhou of China and Haiphong of Vietnam) were analyzed. Results showed that a total of 93 control region sequences with 107 variable sites and 85 haplotypes were obtained, and an evident AT-skew was found in mtDNA control region sequences of S. nudus; the haplotype diversity (H_d) and nucleotide diversity (P_i) were 0.998 and 0.018 89 respectively. The NJ (Neighbor Joining) tree base on haplotype showed that the six populations presented no obvious branches, and all haplotypes were randomly distributed in the haplotype network. The range of fixation index (F_st) of different populations was -0.018 13-0.028 05, and the genetic differentiation among populations was not obvious. The results of AMOVA revealed that the genetic variance mainly occurred within populations (99.74%). In terms of the negatively selective neutrality test and mismatch distribution of pairwise, we could suggest that a population expansion occurred in S. nudus, and the estimated time was about 1 710 000 years ago. The results indicated that there was high genetic diversity in S. nudus and existed gene exchange among populations, and the estimated population expansion could occur during the Early Pleistocene.

Abstract:To study the hydrolysis activity of chitosanase, in this report, the coding sequence of chitosanse from Bacillus amyloliquefaciens HZ-1510 was cloned, and glutathione S-transferase (GST) fused-chitosanase was expressed and purified from Escherichia coli. Furthermore, we analysed the signal peptide, amino acid sequence and its three-dimensional structure. In the end, its chitosan hydrolysis activity was measured. The chitosanase gene consisted of an open reading frame of 837 bp which encoded a protein of 279 amino acids with predicted molecular weight of 31.45 ku. The protein contained a signal peptide with a cleavage site located between the 36th and 37th amino acids. The deduced amino acid sequence homology analysis of the chitosanase revealed that it belonged to GH46 family. The recombinant chitosanase has been purified to homogeneity by using only GST column chromatography. The optimal pH and temperature for the chitosan hydrolysis activity was 5.5 and 55°C, respectively. The enzyme activity was increased in the presence of Mn²⁺, Ca²⁺ and Mg²⁺. However, Fe³⁺, Ag⁺, Cu²⁺, Ba²⁺ and K⁺ could inhibit its activity. Furthermore, its activity could be increased in the presence of 0.1 mmol/L Zn²⁺ while it was inhibited by 2.0 mmol/L Zn²⁺. This study explored the optimal condition for hydrolytic activity of chitosanase and established a theoretical basis for its industrial application.

Abstract:To study the effects of acute stress reaction on iron concentration and iron homeostasis in Megalobrama amblycephala, we stimulated stress response by intraperitoneal injection of cortisol. The iron content in serum and liver was determined by spectrophotometer, and the iron homeostasis related gene (hepcidin & transferrin) expression were detected by quantitative real-time PCR at 0, 2, 4, 8, 10, 12, 24 and 48 h after cortisol injection. The results showed that the content of serum cortisol immediately increased significantly after injection, almost 10 folds compared with the control group, and gradually decreased backward. The content of serum iron in experimental group is higher than that in control group at 4, 8, 10 and 12 hours post injection (hpi), but the content of liver iron led to the opposite results. Hepcidin mRNA expression in liver rose remarkably, reaching the maximum at 8 hpi and 10 hpi, respectively, and then descended to the normal level. The transferrin showed similar expression pattern, and reached the peak at 10 hpi. The result indicated that large release of intracellular iron into body fluid led to increased risk of pathogenic infection and proliferation in vivo. Hepcidin and Tf were involved in regulation of iron metabolism and inflammatory response under stress in M. amblycephala and may play important roles in iron homeostasis and innate immune system.

Abstract:To investigate the toxic effect of lead on male accessory gland function in the freshwater crab Sinopotamon henanense, the function of accessory gland was investigated firstly, acrosome reaction induction rate was high up to 78% under the combination of calcium ionophore A23187 and accessory gland homogenate, it implied that accessory gland could adjust the action of sperm acrosome reaction. Then the effects of Pb on the accessory gland function in sperm acrosome reaction induction and oxidative stress were investigated after 3, 5 and 7 d, exposed to different Pb concentrations (0, 3.675, 7.35, 14.70, 29.40 and 58.80 mg/L). The results showed that the acrosome reaction induction rate was deceased significantly when the accessory gland was taken from crabs which was treated by Pb. Meanwhile, the reactive oxygen species (ROS) content and protein carbonylation (PCO) content were increased in a concentration- and time-dependent manner in accessory gland, however, DNA-protein crosslinks rate (DPC) level had no significant change. Our findings documented that Pb deceased the function of accessory gland in sperm acrosome reaction induction, which was associated with the increasing of ROS leading to protein oxidative damage.

Abstract:In order to investigate the relationship between the high temperature resistance and variation of phytohormones under high temperature environment, thalli of Pyropia haitanensis, Zhedong No. 1, collected from Xiangshan, Ningbo were used as experimental materials. And a method using high performance liquid chromatography coupled with mass spectrometry in selective reaction mode was developed, to analyse the variations of 11 kinds of phytohormones of P. haitanensis under different high temperature environments. The results showed that there were totally 7 kinds of phytohormones detected in the P. haitanensis under different high temperature environments, including N₆-(2-isopentenyl) adenosine, N₆-(2-isopentenyl)adenine, trans-zeatinriboside, indole-3-acetic acid, jasmonic acid, salicylic acid and methyl jasmonate. At the different high temperature 20, 25, 28 and 35°C, the contents of methyl jasmonate and jasmonic acid were increased as the stimulated temperature rose. While the contents of N₆-(2-isopentenyl) adenosine, N₆-(2-isopentenyl) adenine, trans-zeatinriboside and indole-3-acetic acid in high temperature group were lower than those of control group and showing the decreased trends. When at the different heat shock time, as the stimulated time increased, the contents of N₆-(2-isopentenyl) adenosine, N₆-(2-isopentenyl)adenine, trans-zeatinriboside, indole-3-acetic acid were reduced, while the contents of salicylic acid and methyl jasmonate were increased by comparing to that of control group. However, the content of jasmonic acid was increased, then decreased at 6h to that of control group. In the recovery procedure, the contents of indole-3-acetic acid, jasmonic acid, salicylic acid and methyl jasmonate were decreased, then increased, finally decreased again. For N₆-(2-isopentenyl) adenosine, N₆-(2-isopentenyl)adenine and trans-zeatinriboside, the contents of them were reduced gradually. Under high-temperature stress, the stress responses of P. haitanensis are co-regulated by positive growth regulator phytohormones and stress phytohormones to maintain its growth. However, the defense capacity of P. haitanensis is limited and it might be difficult for algae to repair its damage caused by high-temperature stress.

Abstract:In order to grasp the influences of nutrients and sea water reuse on growth and toxin productivty of the Alexandrium minutum, single factor experiments were used to observe the influences of the concentrition of nitrogen (NaNO₃), phosphorus (NaH₂PO₄), trace element (FeCl₃, Na₂EDTA, CuSO₄, Na₂MoO₄, ZnSO₄, CoCl₂, MnCl₂), vitamin (Vitamin B₁₂, Vitamin H, Vitamin B₁), carbon (NaHCO₃) and the style of sea water (new or reuse) on the growth, toxin content (μmol/L) and toxin constitution of Alexandrium minutum C4. The results showed that nitrogen and phosphorus had a significant effect on the total toxin content (μmol/L), while others had non-significant effect (μmol/L); nitrogen, phosphorus, trace element and carbon had a significant effect on the ratio of GTX1/4(GTX1+GTX4) to total toxin content(GTX1+GTX2+GTX3+GTX4), while vitamin and sea water reuse had non-significant effect on that ratio. There was a positive correlation between the concentration of toxin and nitrogen when the nitrogen concentrition was between 0 and 883 μmol/L, and then toxin content stayed invariant with the increase of nitrogen. However, with the increase of phosphorus concentrition from 0 to 145.2 μmol/L, the toxin content increased at first, then reduced,and stayed invariant in the end.The optimal conditions for the toxin production were nitrogen 883 μmol/L, phosphorus 18.15 μmol/L, trace element one half of that in the f/2 medium and carbon free.

Abstract:The catfish, Silurus asotus, is an important commercial fish in East Asia, and it is widely distributed throughout rivers and freshwater lakes of China.Unfortunately, population quantities of S.asotus decreased sharply in recent years because of overfishing and habitat fragmentation. Genetic diversity and population genetic structure are important aspects of the population genetics of fish, and provide essential information for understanding local adaptation and dispersal patterns, and for clarifying the genetic variation. Cytochrome b (Cytb) has a moderate evolutionary rate and a clear evolutionary pattern, suitable for the studies of phylogenetic evolution at the intra- and inter-specific levels. To identify the genetic diversity and population history among geographic populations of S.asotus in different water systems in China, including Yangtze River, Pearl River, Liao River and Southeast coastal rivers, and clarify its population demographic history and genetic structure, the genetic diversity, structure, differentiation, and molecular variance were analyzed using DNAsp5.10.01 and Arlequin 3.11. In the present study, 1110 bases of mtDNA Cytb gene were obtained from 216 of S. asotus collected in the different water systems in China from 2011 to 2012. Generally, there exists high diversity between haplotype (H_d=0.948±0.009) and nucleotide (P_i=0.017 99±0.000 55) in all populations of the catfish, suggesting that the populations were relatively large and stable. In addition, seventy-eight haplotypes, Hap45 was shared in individuals of all populations. Phylogenetic analysis of the MEGA5 was conducted to determine the relationships between S.asotus haplotypes, and detect discernible groups related to geography, and these analyses distinguished four inclusive clades. Moreover, the median-joining network was similar to the topology of the phylogenetic tree with 78 haplotypes, and revealed obivously four lineages in phylogeographic pattern. The results of neutrality tests on S.asotus data set of the four lineages, combined with the nuimodal mismatch distribution, indicated recent population expansion of S. asotus on large spatial scales in the period of late Pleistocene (0.04-0.05 Ma).

Abstract:Raft aquaculture is one of the popular farm modes for growing aquatic product in the ocean. However, tough conditions in the open ocean often impose severe damages on the raft systems. Therefore, comprehensive studies on the mechanics of the raft systems exposed to waves will greatly give help to optimizing raft system design and construction. This paper focuses on the mechanics of a typical long-line raft lug aquaculture system under wave conditions. In this paper, the numerical simulation was used for this study. The raft system was assumed as a flexible structure which was composed of nodes connected with springs. Wave forces on the rope and floating balls were calculated using Morison equations. Second order differential equations were established based on Newton's second law. The equations were resolved using fourth-order Runge-kutta method to find out the inner tension force and displacement of each element. It was found that free-weight and system angle have an effect on long-line rope aquaculture facility. Long-line displacement is negatively related with free-weight. The displacement of long-line decreased as free-weight increased.The force of long-line, back anchor rope, float ball rope and lifting rope increased as free-weight increased. But the force of front anchor rope decreased as free-weight increased. Float ball pattern had little impact on the system's movement and force. Anchor rope force and the displacement of long-line had positively correlated relationship with system angle at 0° to 60°. They increased as the angle increased. Anchor rope force and the displacement of long-line had negative correlated relationship with angle at 60° to 90°. They decreased as the system angle increased. The force of long-line, float ball rope, the lifting rope affected by the system angle is small. The results have important significance on evaluating the safety of the raft culture system. Deep-water long-line rope aquaculture facility belongs to the typical flexible multi degree of freedom offshore structures, and this research results can provide the reference for designing deep-water long-line rope aquaculture facility and safety evaluation. But the hydrodynamic characteristics of the deep-water long-line rope aquaculture facility are related to its structure and environmental factors, therefore we need specific conditions and specific analysis in the raft system designing and safety assessment.