Abstract:Largemouth bass (Micropterus salmoides) is one of the primary cultured species in China, with annual production of 350 000 tons. Growth is one of the most crucial economic traits of all aquaculture species, but the molecular mechanisms involved in growth of M. salmoides are poorly understood. Investigation of the correlations between single nucleotide polymorphism (SNP) and growth traits can provide candidate markers for molecular marker-assisted selection. Hypothesis-driven candidate gene association analysis is directed at genes that have clear roles in controlling special traits. Myosin heavy chain (MYH) is the main component of skeletal muscle cells and affects muscle growth and development. In order to study the relationship between DNA polymorphisms of MYH gene and growth traits of M. salmoides in this study, the sequence of MYH gene coding region was obtained by PCR amplification method, which was 9759 bp in length. MYH gene contained 37 exons and 36 introns and encoded a predicted protein of 1940 amino acids. Eight SNP mutations were identified (A-305G, G-558C, A-2784C, A-2816G, T-4765A, C-6206T, C-6811T and G-6935T) in M. salmoides MYH gene using direct sequencing method. Among them, four SNPs were located in exons and two synonymous mutations happened. Eight SNP loci of 430 randomly selected Youlu No.1 M. salmoides individuals were genotyped by SNaPshot method. Genetic structure was analyzed by Popgene32 software. The results showed that the average effective number (N_e), average expected heterozygosity (H_o) and average observed heterozygosity (H_e) of these SNPs was 1.635, 0.373 and 0.406, respectively. Chi-square test showed that three SNP loci (C-6206T, C-6811T and T-4765A) were in Hardy-Weinberg equilibrium state in the Youlu No.1 M. salmoides population. A-2784C and A-2816G formed two haplotypes (A and B), and three genotypes (AA, AB, and BB) were observed. A general linear model was established for correlation analysis between different genotypes at SNP loci of MYH gene and various growth traits using SPSS19.0 software. At the position of C-6811T, individuals with the CC genotype had signifcantly greater values for body weight or total length than those with the TT genotypes. The individuals with genotypes CC had a greater value than the individuals with the genotype CT and TT in body height and caudal peduncle length. Other seven SNP loci were not significantly correlated with growth traits. The growth-related C-6811T locus could be a useful candidate molecular marker for M. salmoides molecular marker-assisted selection.

Abstract:Twelve strains of pathogenic Vibrio parahaemolyticus isolated from Litopenaeus vannamei with acute hepatopancreatic necrosis disease (AHPND) were chosen, and their genetic characteristics were analyzed by the multilocus sequence typing method. Seven house-keeping genes (dnaE, gyrB, recA, dtdS, pntA, pyrC and tnaA) of V. parahaemolyticus were selected as target genes for PCR amplification and nucleic acid sequencing. The sequences of AHPND-causing V. parahaemolyticus in this study were uploaded to the PubMLST database for multi-alignment to determine the sequence type of each strain. MLST data of AHPND-causing V. parahaemolyticus from other areas were collected and the genetic evolution was analyzed by MEGA 5.0 and eBURST V3 software. AHPND-causing V. parahaemolyticus isolated from Guangdong province in 2014 belonged to the new sequence type ST1710(42, 134, 99, 79, 141, 41, 51). Compared with other sequence types in PubMLST database, it has the high homology with ST415 and ST975. The 9 STs of AHPND-causing V. parahaemolyticus were separated by eBURST into two doublets and five singletons. Phylogenetic tree analysis showed that the new sequence type ST1710 was similar to the singletons ST975. Our study is the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains in China using multilocus sequence typing, and enriches the PubMLST database and provides an insight into their evolutionary mechanisms.

Abstract:In oder to reconstruct the molecular phylogeny of Pinctada, partial sequences of 3 mtDNA fragments (12S rRNA, 16S rRNA, COⅠ) from 5 species of Pinctada (P. martensii, P. maxima, P. margaritifera, P. maculata, P. nigra) were amplified by polymerase chain reaction (PCR) and directly sequenced, and the samples were collected from Leizhou Peninsula. Base ratio and genetic distances were analyzed. At the same time, these sequences were compared with the published sequence of P. albina. Results showed that A+T contents of 3 mtDNA fragments from 5 species were higher than G+C obviously, in accordance with the other invertebrate mtDNA sequences. Data showed that the sequences of 3 mtDNA fragments had a high degree of saturation. Analysis showed that the molecular phylogenetic trees were basically constructed and well supported the traditional morphological taxonomy. In the relations of P. nigra and P. albina, 3 mtDNA fragments were very similar in nucleotide composition, genetic variability, genetic distance and molecular phylogenetic trees. So this suggests that P. nigra and P. albina represented two subspecies in the same species. About the taxonomy of P. maculata, the molecular phylogenetic trees showed that P.maculata was close to P. nigra and P. albina.Moreover, it revealed P. margaritifera and P. maxima were more primitive than the others. There was a little difference on the genetic relationship analysis about P. martensii by 3 mtDNA fragments, and no conclusion could be drawn from the existing data. It is necessary to do more study of molecular systems and morphological and anatomical structure.

Abstract:Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is its main pathogen. A dynein regulation protein robl/LC7, which is likely to be secreted and to target host cell mitochondria, was found by the bioinformatics analysis on the whole genome sequence of N. seriolae. In order to determine the subcellular localization and study the preliminary function of robl/LC7 from N. seriolae, the gene cloning, eukaryotic expression plasmid construction, secreted protein identification, subcellular localization and apoptosis detection of robl/LC7 were carried out in this study. robl/LC7 was successfully cloned from N. seriolae and the eukaryotic expression plasmid of pEGFP- robl/LC7 and pcDNA- robl/LC7 were constructed. Mass spectrometry analysis of extracellular products from N. seriolae confirmed that robl/LC7 was a secreted protein. Subcellular localization of robl/LC7-GFP fusion protein discovered that the green fluorescence was distributed in the whole cell and did not co-localize with mitochondria. In addition, apoptosis assay revealed that apoptosis was induced by the over-expression of robl/LC7 in FHM cell. robl/LC7 from N. seriolae is a secreted protein which does not co-localize with mitochondria. It may help the host cell survival and immune escape of N. seriolae by participating the cell apoptosis regulation and may play an important role in the pathogenesis of N. seriolae.

Abstract:The transcriptome of Hyriopsis cumingii ventral mantle tissue whose nacre colors were purple and white was sequenced by a new generation of high throughput sequencing technology in order to find candidate genes involved in pearl color produced by H. cumingii. After quality control and assembly, the sequences acquired were blasted against NR, Swiss-Prot, COG, KOG, KEGG, GO and Pfam databases, and then cluster analysis were performed. In total, 72 800 581 bp of clean data, and 89 529 Unigenes were obtained, and 2644 genes were differentially expressed, among which 1323 were up-regulated and 1321 down-regulated. The Unigenes GO function in the transcriptome library was divided into 3 categories: molecular function, cellular component and biological process with 50 branches; it could be divided into 187 classes taking the KEGG database as a reference. Differentially expressed gene analysis revealed that some genes were related to pigment biosynthesis (such as xanthommatin, melanin and pteridine) and metal ion transportation. And there were 6 cytochrome P450, 3 cytochrome b561 and 1 cytochrome b560 genes. They might play important roles in the formation of pearl color of H. cumingii.

Abstract:In order to clarify the cause of sudden death that occurred in a shrimp (Marsupenaeus japonicus) farm in Weifang from July to August 2015, seven main shrimp pathogens including White spot syndrome virus (WSSV), Taura syndrome virus (TSV), Yellow head virus (YHV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Infectious myonecrosis virus (IMNV), Covert mortality nodavirus (CMNV) and Vibrio alginolyticus that causing acute hepatopancreatic necrosis disease (AHPND) were detected by Polymerase chain reaction (PCR) method. Meanwhile, histopathology of the diseased shrimp was observed. Furthermore, bacterial strains, isolated from the moribund shrimp and identified by the 16 SrDNA gene, were used for the evaluating of virulence by immersion challenge. The results showed that all the three shrimp samples gave strong positive PCR results (an amplicon of 941 bp) with primers WSSV 146 F2/R2, and two of three sample show weak positive results (an amplicon of 389 bp) with primers IHHNV 389 F/R and two of three sample show strong positive results (an amplicon of 167 bp) with primers CMNV-7 F2/R2. However the results of PCR amplification for the other four pathogens (TSV, YHV, IMNV and AHPND) show negative. Histopathology results show that there were inclusion body of WSSV in the epithelial tissue of stomach and gill, and the muscle fibres dispersed. Three dominant strains of bacterial microorganisms 2901, 2902, 2903 were isolated and 16 S rDNA sequencing indicated that they had higher similarity with Grimontia indica (99%)、Alteromonas sp. (99%) and Vibrio alginolyticus (100%) respectively. Challenge test show that LC₅₀ of bacteria 2901, 2902, 2903 were 9.8×10⁷ CFU/mL, 1.1×10⁸ CFU/mL and 2.3×10⁸ CFU/mL, which means the bacterial microorganisms had lower virulence and may not be the main pathogen of shrimp. Comprehensive analysis suggested that sudden death of M.japanicus might be caused by coinfection of WSSV, IHHNV and CMNV, in which WSSV infection might play a key role. The results of this study could provide strong support for clarifying the cause of outbreak death in shrimp M. japonicus.

Abstract:Chromosome karyotyping is the main content in cytogenetic study. Manual analysis, image systemic analysis and software analysis are the most commonly used methods in chromosome karyotyping. But all three methods have their own disadvantage, which are large measurement error, high quality metaphase image requirement and uncontrollable detail error, respectively. In order to obtain accurate and convenient chromosome karyotype analysis method, the introduced salt-tolerant varieties, Barbus capito, was used as experimental fish. The metaphase chromosomes of B. capito were obtained from head kidney tissue by the method of PHA and colchicine injection and then the chromosome samples were prepared by air-drying technique. We presented a method for chromosome karyotyping by combining two software E-ruler and Photoshop, and compared the measure and karyotyping results from the regular method. The results showed that there is no significant difference in the measurement of stretched straight chromosomes by E-ruler software and conventional methods, however, the former is more accurate in the measurement of curved chromosome. Photoshop software can quickly and conveniently complete the chromosome, because it can accurately judge the location of centromere by eliminating the background of karyotype. It can be concluded that E-ruler and Photoshop combining method achieved much more accurate and clear karyotyping results. As an accurate and convenient method, this new method can also be used in chromosome analysis of other fish species and even other organisms. The results showed that the chromosome ploidy of B. capito was diploid, and no gender-related heterotypical chromosomes were found. The karyotype formula was: 2n=100=12m+38sm+38st+12t, NF=150. Compared with other species from Barbinae family, B. capito has similar karyotypes to several domestic species, which shows the karyotype evolutionary characters.

Abstract:Zebrafish and macrophages were used as in vivo and in vitro infection models to evaluate the pathogenetic characteristics of Streptococcus agalactiae piscine strain (GD201008-001) and human strain (A909). iTRAQ and LC-MS/MS technology was used to identify differentially expressed proteins in GD201008-001 and A909, and provide new clues for exploring the pathogenic mechanism of S. galactiae strains isolated from different hosts. The pathogenetic characteristics of GD201008-001 and A909 were evaluated by analyzing LD₅₀ to zebrafish and phagocytosis of macrophages. Cellular proteins were extracted from cultures of GD201008-001 and A909, and labelled with iTRAQ reagent. Differentially expressed proteins were identified with LC-MS/MS. The mass spectrometry data was analyzed by Mascot2.2 and Proteome Discoverer1.4, and then subjected to biological information analysis of GO (gene ontology) and KEGG pathway. The result of pathogenicity difference analysis indicated that the virulence of GD201008-001 was higher than that of A909. Differentially expressed proteins were identified in S. galactiae piscine strain compared with human strain. A total of 368 differentially expressed proteins were revealed (P<0.05), among which 193 proteins were up-regulated (ratio>1.5) and 175 proteins were down-regulated (ratio<0.667) in GD201008-001. Bioinformatics analysis predicted that these proteins are mainly involved in 26 biological functions and 14 pathways. It is speculated that ClpX, GlmS and CpsIVK may play an important role in the pathogenicity of GD201008-001 and A909. These results may shed light on the pathogenic mechanism of S. galactiae strains isolated from different hosts.

Abstract:Based on 364 ovarian samples of North Pacific albacore tuna (Thunnus alalunga) collected in the north Pacific waters (29°08'–41°08' N, 163°50'–144°19' W) from October 2013 to February 2014, using histological analysis, the present study describes oocyte and ovary developmental stage of albacore tuna. The results show that North Pacific albacore is a species of the batch spawning type due to different development stages of oocytes found in the same ovaries. Histologically, the oocyte development of North Pacific albacore can be divided into six phases, and ovarian development into six periods. GSI (gonad somatic index) increases with the mature period during the stages I–V, and decreases in the stage VI. The spawning peak is from the middle December to early January, the GSI increases with the decreasing of latitude but no significant difference can be found. The present study can provide biological information for the stock assessment and sustainable development of north Pacific albacore fisheries, by analyzing the growth characteristics of albacore ovaries.

Abstract:The column-type net enclosure aquaculture (NEA) is a new ecological ocean aquaculture mode. It is mainly composed of piles and net panels. More and more attention has been paid to NEA, because it is a new aquaculture mode which has big aquaculture area, large space for fishes,more natural environment, and more ecological high quality of food. NEA is still in its developing stage, many kinds of technical problems still need to be solved.The net system is regarded as the key component which controls the safety of the whole NEA system. In this paper, based on lumped mass point and clustering mesh method, the mechanical properties of unit net of a column-type NEA engineering in current were studied using computer numerical simulation. The distribution characters of tension force in twines, the offset characters of net nodes and the load characters at fixing points were investigated. The effect of net width and fixing mode on the net hydrodynamics was discussed as well. The results showed that the maximum tension force of unit net of the pile-column type net enclosure in current mainly occurred at the both sides of the top and bottom belly lines of net panel, and was proportional to the net width.The maximum offset of the net panel mainly appeared at the upper and lower positions, and was proportional to the net width as well. Regarding the force on fixing points, the largest forces on the top and bottom fixing points of net panel were observed in this study and they were much higher than that of the middle fixing points.The research found that the forces at the fixing points decreased with the increasing of the number of fixing points, and the maximum tension force in twine and the maximum offset of net nodes declined rapidly too. When the number of fixing points exceeds a certain number, the maximum tension force and offset will no longer drop significantly. Thus, there was a critical number of fixing points, which was found to be 4 or 5 in this paper. The results achieved in this paper pointed out the potential dangerous positions of the net panel and showed the rules of influence on the force and deformation of net panel. The results may give guidance to the design and installation of the column-type NEA engineering.

Abstract:As a key species in the Antarctic ecosystem, the spatial and temporal dynamics of Antarctic krill's fishing ground is becoming important issues for the role effects of krill's oceanic ecosystem function and resource management.Using multi-year Chinese krill fishery scientific data by month group,combining three spatial factors-longitude, latitude and offshore distance, the optimal point pattern model of monthly krill distribution and the variance analysis results were obtained for the krill fishery in the northern Antarctic Peninsula based on the spatial point pattern method, and the possible potential fishing grounds were predicted based on the proposed optimal point pattern model. The results were: (1) In December, the greatest distance of fishing grounds to shore (45 km) can be found. Subsequently, the fishing grounds were gradually approaching coastline of the South Shetland Islands. By March the fishing grounds were at the least distance to the Antarctic Peninsula shoreline. After March, fishing ground gradually retreated from the coastal area, in April and May distance of fishing grounds to shore maintained about 13 km. (2) The variance analysis of the optimal point pattern model indicated the explained rates of variance to the model in December, January, March and April were 59%, 60%, 57% and 68%, respectively. Spatial factors can fit the distribution of krill fishing ground very well in these months. But in February and May, the explained rate of the variance to spatial point pattern model reduced to 38% and 32%, respectively. (3) Fishing grounds were located in the northern King George Island during austral summer (December to January).In February, a secondary fishing ground was formed around the Livingston Island, and the third fishing ground occurred in the middle of the Bransfield Strait.During March-May fishing ground distributed along in the Bransfield Strait paralleled to the Antarctic Peninsula. In conclusion, the offshore distance of the fishing ground coincided with the extension and retreating of the sea ice boundary in the northern Antarctic Peninsula. The point pattern model of krill fishery based on spatial factors can well fit the temporal and spatial evolution of krill fishing ground in most months. Combined with the catch per fishing unit (CPUE), the point pattern model can be used as a potential technique to explore the hotspot of krill aggregation.

Abstract:A 12-week experiment was conducted to evaluate the effects of dietary soy lecithin on ovary development, gonadal histology and vitellogenin mRNA expression of swimming crab. 2×2 double-factor random block design was used to arrange 0% or 4% soy lecithin for mated or unmated female swimming crab, respectively. The results indicated that crabs fed the diets containing 4% soy lecithin had significantly higher gonadosomatic index (GSI), oocyte diameter, the levels of serum vitellogenin and sex steroids than those fed the unsupplemented with soy lecithin diets. Hepatopancreatic vitellogenin mRNA expression increasesed with dietary soy lecithin increasing from 0% to 4%. The results of hepatosomatic index (HSI) showed a reverse trend with GSI. Mated female crabs exhibited higher hepatopancreatic vitellogenin mRNA expression than unmated female crabs. Furthermore, the mated female crabs had higher ovarian yolk granules in number and size than those unmated female crabs. Mating treatment did not affect HSI, serum vitellogenin (VG) and progesterone (PG) contents. The interaction of dietary soy lecithin and mating on serum progesterone (PG) and estradiol (E₂) contents was identified. In conclusion, the dietary soy lecithin and mating stimulated had a positive effect on ovarian development in swimming crab broodstock, and mating stimulated had a more beneficial effect on promoting oocyte maturation.

Abstract:To obtain a continuous monoclonal Oreochromis niloticus macrophage cell line, macrophages were isolated and purified from peritoneal cavity of O. niloticus. The O. niloticus macrophage cell line was established and EBV infection identification, electron microscopy, telomerase activity assay, carcinogenicity evaluation, karyotype analysis, and molecular biology identification were then carried out. The results showed that EBV had been integrated into O. niloticus peritoneal macrophages and stably expressed. After 30 generations of stable passage, the cell line still maintained a good proliferative state. The cell line surface was not smooth and had obvious blunt round protrusion and slender pseudopodia, which was the typical morphology performance of the macrophages. Telomerase activity of this cell line was significantly higher than that of untreated macrophages (P<0.05), but the difference was not significant with HeLa cells, and the cell line was not carcinogenic, which indicated that immortalized cell lines were successfully constructed. Karyotype analysis showed that the cell line had 44 chromosomes, and the karyotype formula was 2 n=2 x=44=4 sm+17 st+1 t. PCR detection revealed that CD33, and CD205 transcripts were found in this cell line, all of which were markers of monocytes/macrophages. 18S rRNA detection showed that the cell line was from O. niloticus macrophages. Immortalized tilapia macrophage cell lines were successfully established. The establishment of this cell line laid the foundation for the further study of the immunological function of peritoneal macrophages in O. niloticus and also provided a tool for studying the mechanism of high protection rate of O. niloticus Streptococcus HSP70-peptide vaccine.

Abstract:In order to evaluate the changes of the bacterial communities in the intestine of spotted knifejaw (Oplegnathus punctatus) at different healthy levels and intervened with florfenicol, and to find out the relationship with environmental factors, the Illumina HiSeq PE250 high-throughput technique was used to analyze the bacterial community diversity and structure of the fish intestine for healthy, sub-healthy, typical dark body disease (DBD) infected and oral florfenicol intervened individuals, as well as the culture water and pellet feed. The results showed that bacterial diversity in the culture water was higher than that of intestine and pellet feed. The dominant bacterial communities in intestine were Proteobacteria, Firmicutes and Tenericutes, at different health levels and with florfenicol intervention, and the proportions of corresponding operational taxonomic unit (OTU) number to all the OTU in the samples were above 85%. It could affect the ranking order of the top 20 dominant bacterial species in intestine when the DBD occurred, with the relative abundance of Vibrio in Proteobacteria increasing significantly. In addition, the DBD symptoms aggravated gradually, with the increase of Vibrio abundance. Feeding with florfenicol could reduce the Vibrio abundance from 60.33% to 1.29% in the intestine of diseased fish, and also change its structure of bacterial community to a greater degree, which proved that florfenicol was effective in treating DBD. It was also found that the culture water and pellet feed had a certain influence on intestine bacterial community, and the effect of the culture water was higher than the pellet feed. The present results would provide a reference basis and technical support for the health culture, disease control and microecological study in spotted knifejaw.

Abstract:To investigate the difference of the compositional and functional structures of the intestinal microflora between healthy and diseased Litopenaeus vannamei, we screened the intestinal bio-indicators to evaluate the health status of the host, and assess the functional redundancy of the intestinal microflora in response to disease. Based on the Illumina high-throughput sequencing technology, we compared the intestinal microflora between healthy and diseased L. vannamei, and analyzed the functional profiles through the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). In addition, we estimated the correlation between the intestinal microflora structures and predicted the functional compositions. The results showed that the occurrence of disease was associated with significant changes of the bacterial community structures and functional compositions, and there was no significant change in bacterial diversity. Compared with the intestinal microflora of the healthy L. vannamei, the relative abundances of Actinobacteria, Planctomycetes and Verrucomicrobia significantly decreased in the diseased L. vannamei, whereas, the Gammaproteobacteria and Bacteroidetes exhibited an opposite pattern. In addition, 16 sensitive indicator families were screened, and their occurrences can indicate the health status of the host very well. Furthermore, the abundance of pathways involved in Vibrio spp. infection significantly increased in the diseased L. vannamei; in contrast, the pathways for antibacterial and immunity, such as lysosome and peroxisome pathways, markedly decreased in diseased shrimp. Notably, there was a significant and positive association in compositional and functional composition between the gut microbiota community, indicating a low functional redundancy of the shrimp gut bacterial community. The occurrence of disease was associated with the change of the intestinal microflora, which subsequently disrupts the bacterial-mediated functions. Further, this study provides sensitive bio-indicators for evaluating the host health status.