Abstract:In this study, a full-length 2558 bp Sp-Sox14 cDNA sequence was obtained from Scylla paramamosain gonadal transcriptome database. The encoded protein consists of 427 amino acids and includes an HMG box domain. Phylogenetic tree analysis showed that Sp-Sox14 was closely clustered with Sox14 of other arthropods. Quantitative real-time PCR displayed that Sp-Sox14 had different expression levels in different tissues from mature male and female crabs with the highest expression in ovary and the heart of male crabs. During embryogenesis, Sp-Sox14 expression was the highest in eye-pigment formation stage. During the different stages of ovarian development, Sp-Sox14 was expressed at the highest level in previtellogenesis stage (O2) and which was significantly higher than other stages. During the testis development, the expression level of Sp-Sox14 was higher in mature sperm stage (T3) than in spermatocyte stage (T1) and spermatid stage (T2). It was speculated that Sp-Sox14 was involved in the early development of ovary and the process of sperm maturation. The whole mount in situ hybridization (WISH) results indicated that the positive signals of Sp-Sox14 were mainly distributed in the head and near the maxillipede at eye-pigment formation stage, and near the compound eyes at prehatching stage. A small number of Sp-Sox14 positive signals were still present in the head and near the appendages at hatching stage. The results of WISH implied that Sp-Sox14 was involved in the formation of the nervous organ as well as related to the occurrence of somite appendages.

Abstract:To obtain the genome sequence of Ostreid herpesvirus 1 (OsHV-1) infected Zhikong scallop (Chlamys farreri) in 2001 (ZK2001), and its phylogenetic relationship with the other reported variants, the genome DNA of the ZK2001 variant was firstly enriched through long-range PCR, and sequenced with Illumina Hiseq 2500 PE250 platform. Then the variation and phylogenetic relationship between ZK2001 and the other reported variants were analyzed. 8 scaffolds were obtained after assembly. Genome variation analysis indicated that there were 328 SNPs between ZK2001 and the reference genome. SNPs and insert/deletion were the main cause of genome differentiation. Phylogenetic inference indicated that ZK2001 variant was more closely related to OsHV-1 variants isolated from China than those from Europe. These results indicated that genetic differentiation had occurred as a result of geographic isolation. This study indicated genomic DNA of OsHV-1, which had been frozen for a long time, could be enriched with long-range PCR and used for high-throughput sequencing. The genomic data of different OsHV-1 variants will be necessary for further study of the genetic variation, evolution and phylogenetic analysis of the virus.

Abstract:In order to know the fish resource distribution and the variation of the biomass density, three acoustic surveys were conducted with a split-beam echo sounder (EY60 model, 200 kHz, Simrad) inside and outside the algae breeding zone of Baishawan in Nan'ao on Apr. 28, Dec. 2, 2015, and Apr. 19, 2016. The acoustic assessment results showed that the resource abundance density of three times inside and outside the algae breeding zone were 3.45/1.38×10⁵, 0.63/1.45×10⁵ and 3.67/2.98×10⁵ inds/n mile². Obviously, the biomass was higher in spring than in winter inside the algae breeding zone and the resource abundance density changed evidently as the seasons than outside the algae breeding zone. The highest resource abundance density was mainly in 2–6 m depth layer, 6–8 m depth layer took second place, and the upper and the bottom had the least. The biomass was affected by the depth of significant resources abundance density. Our research confirmed that during the period of algae breeding, the resource abundance density was higher inside than outside the algae breeding zone and changed evidently. It was important for the algal biology to change the biomass and distribution inside the algae breeding zone.

Abstract:On the basis of wild good broodstocks introduced from Poyang Lake (F₀ generation), the F₁ generation was obtained through population breeding from 2009 to 2011, and the passage from F₁ to F₂ was realized in 2011 through population breeding. In order to construct hypoxia tolerance strains of M. amblycephala, the Poyang Lake F₂ and “Pujiang No.1” F₉ broodstocks endured twice hypoxia stress in the summer and autumn in 2012, and 536 survival individuals were constructed as hypoxia-tolerant F₂ generation. In 2013, the F₂ parents (female 50, male 48) with great body weight and good body-shape were used to establish 24 F₃ family groups (22 groups of 2♀×2♂ and 2 groups of 3♀×2♂), a total of 100 F₃ families. The 100 F₃ families were cultured under hypoxia for one year from the 20th day post-hatching. By measuring the corresponding traits between growth and hypoxia tolerance, five fastest growing family groups (A2, A3, A18, A19 and A20) were selected; microsatellite analysis identified these rapidly growing individuals belonging to 20 families. Additionally, six faster growing groups (A4, A6, A17, A25, A27 and A28) belonging to 28 family were identified. Our results show that the growth performance of M. amblycephala was positively correlated with hypoxia-tolerance. Under long-time hypoxia stress culture at the first-year stage of F₃ families, the body weight of the families with strong hypoxia tolerance was significantly higher than that of the families with low hypoxia tolerance. The selected F₃ families with hypoxia resistant characteristics still maintained rapid growth rates at 2-year-old stage. The purpose of this study is to establish the hypoxia-tolerant F₃ generation for subsequent selection of new good breed of M. amblycephala.

Abstract:Ctenopharyngodon idella and Pelteobagrus fulvidraco are considered as two economically important aquaculture species in China with the largest production. The sediment-water interface in aquatic systems is a bridge connecting bottom sediments with the overlying water column. The transition zone between oxygenated overlying water and anoxic, or anaerobic, sediments forms over a gradient of just a few millimeter sand and can easily be disturbed by mechanical mixing (bioturbation). To determine the seasonal variance of nitrogen and phosphorus exchange in different aquaculture model ponds and explore the relationship between nitrogen and phosphorus fluxes and environment factors of overlying water in two polyculture systems, C. idella and P. fulvidraco aquaculture models ponds, we studied by in situ Observation device of nutrient flux in sediment-water interface from April to October of 2013. In situ experiments, benthic fluxes of nutrients at the sediment-water interface were measured, and its related factors were analyzed. During our experiment, nitrate, nitrite and TN were taken up by sediment in the beginning of the culture period (from Apr. to Jun.), while it changed to release from sediments to water in the middle and later stages of the culture period. Meanwhile, fluxes of PO₄^3- and TP presented the similar trends. Results also showed that the differences of concentration variation range of NO₂^--N, NO₃^--N and PO₄^3--P were not significant between the two aquaculture models, however there were significant differences of NH₄⁺-N, TN and TP fluxes at sediment-water interface. Otherwise, there was significantly positive relationship between nitrite and nitrate fluxes and DO concentration, while there was negative relationship between NH₄⁺-N and PO₄^3--P fluxes and DO concentration. Elevated temperature can promote different forms of nitrogen and phosphorus fluxes in aquacultural ponds.

Abstract:To study the morphological characteristics of white spots in the carapace of Portunus trituberculatus, a computer vision-based recognition method was developed in this paper with an accuracy of 100%. The molting process of thirty crabs from sea area[ body weight (2.8±0.56) g] was tracked to investigate the growth changes of white spots in the carapace of P. trituberculatus. Results showed that the number and relative positions of white spots had no significant changes after molting, however, the white spot area and its distribution area grew with the carapace. After molting, the white spot area increased by 89.33%±8.61%, the distribution area increased by 90.51%±7.95%, and the carapace area increased by 94.66%±8.26%. P. trituberculatus from North Korea, Liaoning and Zhejiang were collected to compare six parameters related to white spots trait in the carapace, and results showed significant geographical differences. The white spot traits of North Korea had the features of small spots, small distribution area and accounted for a low proportion of the carapace area. The traits of Liaoning had the features of small spots, larger distribution area and accounted for a high proportion of the carapace area. While the traits of Zhejiang had the features of big spots, small distribution area and accounted for a low proportion of the carapace area. This study developed the recognition method based on computer vision for white spots in the carapace of P. trituberculatus, and illustrated the growth features of this trait, and revealed significant geographical differences among three populations.

Abstract:Nuclear factor E2-related factor 2 (Nrf2) plays a key role in the regulation of respiratory burst which can produce amount of reactive oxygen species (ROS). In the present report, we have cloned and analyzed the Nrf 2gene from grass carp (Ctenopharyngodon idella), generated its polyclonal antibody, and characterized its role in regulation of ROS. The obtained cDNA of Nrf2 gene was 1994 bp, containing an open reading frame with 1782 bp encoding 593 amino acids. The amino acid sequences of Nrf2 from grass carp were highly identical with those from common carp (Cyprinus carpio) with 87% similarity. Nrf2 from grass carp contained six typical Neh (Nrf2-Epoxy chloropropane (ECH) homology) domains, indicating it was highly conserved in evolution. The qRT-PCR and Western blot analysis showed that Nrf2 was expressed in all eight detected tissues. The total antioxidant capacity of CIKs were up-regulated but ROS generated from CIK cells were down-regulated and the mRNAs of Nrf2 and its downstream gene (GST and HO-1) were all up-regulated when CIK was exposed to the ROS inducer, tert-Butylhydroquinone (tBHQ). Similarly, the expression of Nrf2 protein was also increased and it was translocated in the nucleus of CIK exposed to tBHQ. In conclusion, evolutionary conserved Nrf2 was ubiquitously expressed in the tissues of grass carp and it is involved in down-regulating the generation of ROS in CIK cells via up-regulating the expression of itself and its downstream antioxidant genes, so as to regulate respiratory burst.

Abstract:An 8-week feeding trial and a 24-hour starvation trial were conducted to evaluate the effects of three different carbohydrate sources on growth performance, feed utilization and glucose metabolism key enzyme activities of large yellow croaker. Three isonitrogenous and isoenergetic diets (about 48% crude protein and 12% crude lipid) were formulated to supplement with three different carbohydrate sources, namely glucose, wheat starch and dextrin, respectively. Each diet was randomly assigned to feed three replicate groups of 50 juvenile large yellow croakers with the initial weight of (8.51±0.02) g twice daily to apparent satiation. After the end of the feeding trial, the fish was continuously starved for 24 h and samples were taken at 0, 1, 3, 5, 7, 9, 11, 24 h, respectively. The results indicated that fish fed the diets containing wheat starch and dextrin had significantly higher weight gain rate (WGR) and specific growth rate (SGR) than those fed the diet containing glucose, moreover, feed conversion ratio (FCR) of these two diets was significantly lower than that of diet containing glucose. Fish fed the diet containing dextrin had significantly higher hepatosomatic index (HSI) than those fed the other diets. However, survival rate (SR), viscerosomatic index (VSI) and condition factor (CF) were not significantly affected by three different carbohydrate sources. Serum glucose (GLU) concentration of the diet containing glucose and wheat starch significantly increased after starving 1 h and glucose diet group presented high serum glucose levels which lasted for at least 10 h. However, serum glucose concentration of the diet containing wheat starch significantly decreased to initial level and didn't display high serum glucose level. Serum glucose concentration of the dextrin diet group continuously increased as time went on, peaked at 11 h, and high serum glucose level lasted for at least 4 h. The concentration of serum insulin and hepatic glycogen were significantly affected by diets supplemented with three different carbohydrates. The results indicated that the increasing of glucokinase (GK) can be induced by wheat starch. While serum glucose concentration increased after feeding trial, the activities of gluconeogenic key enzymes such as glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) didn't decrease. The activities of pyruvate kinase (PK) were significantly influenced by the dietary glucose and wheat starch. The results demonstrated that the ability of utilizing structure-complicated polysaccharide (such as wheat starch and dextrin) was higher than that of utilizing monosaccharide (such as glucose). Not only the serum glucose regulation but also the activities of glycolysis and gluconeogenic key enzymes were affected by different dietary carbohydrate sources.

Abstract:The embryonic chromosomes of hybrid triploid loach (4n♀×2n♂), Misgurnus anguillicaudatus, were examined by C-banding and fluorescence in situ hybridization (FISH).The characteristics of C-band of hybrid triploid loach were discussed for the first time, so as to provide the basis for identifying chromosomes accurately.Meanwhile, the number and position of ribosomal 5.8S+28S rDNA in the metaphase chromosomes of hybrid triploid loach embryos were studied.The results of C-banding in embryonic chromosomes of hybrid triploid loach show that, C-bands were classified into types of terminals and centromeres, but no middle type of arm was observed.The type of terminals were located on terminal of short arm of M1, whereas, compared with the type of centromeres, bigger and more obvious C-band was found on terminal of short arm of M1.While the other M chromosomes and SM chromosomes and T chromosomes only have centromeric C-band.The results of FISH show that, ribosomal 5.8S+28S rDNA was clearly localized in the region of terminal of middle centromere (M) of the metaphase chromosome of the hybrid triploid loach.Three hybrid signals could be detected in metaphase chromosomes of hybrid triploid loach.The results further confirmed that the hybrid triploid loach is a genetic triploid with three sets of chromosomes from the aspect of molecular cytogenetics.

Abstract:To study the pathogenicity of Yersinia ruckeri to rainbow trout (Oncorhynchus mykiss), this paper established a pathological model of enteric redmouth disease (ERM) and developed the corresponding scoring system of the clinical signs and histopathology. 43 healthy O. mykiss with the average weight of 12 g were randomly divided into 5 groups: three experimental groups (n=30) , control group (n=10)and sentinel group (n=3). The fish in three experimental groups were intraperitoneally injected with Y. ruckeri at a concentration of 2.0×10⁶, 2.0×10⁷ and 2.0×10⁸ CFU/mL, respectively. The clinical signs and pathological changes of intestine, liver, spleen and kidney in the infected fish were determined. Bacteriological test was also used to evaluate the effect of the infection model. The challenged fish presented different degrees of death in 72 h and showed some clinical signs such as red mouth, anal swelling, different degrees of congestion in fins (pectoral, pelvic, anal fins), obvious blood spots in lateral line and abdomen. Histopathology indicated that the infiltration, hyperemia and hemorrhage of cells in the liver, spleen, kidney and intestine were observed. Degeneration and necrosis were found in the liver cells, intestinal epithelial cells, renal tubular epithelial cells and other parenchymal cells. In spleen, the number of lymphocytes decreased and the red blood cells heaped up to death. The results showed that the best disease model could be constructed by Y. ruckeri at a concentration of 2.0×10⁷ CFU/mL for O. mykiss and the clinical signs were significant with little differences and the disease was in a slow progress. The pathological model of the enteric red mouth disease was successfully established by intraperitoneal injection of 0.1 mL Y. ruckeri at a concentration of 2.0×10⁷ CFU/mL for O. mykiss juveniles with the body weight of about 12 g.

Abstract:Chemokines are a large group of small proteins involved in migration, activation, and differentiation of leukocytes and are key regulators of the immune response and inflammatory response. In this study, we cloned and sequenced the cDNA of two CC chemokines (CCL4 and CCL19) from swordtail fish (X. helleri). The ORF-length of CCL4 is 294 nucleotides (nt) encoding a protein of 97 amino acids (aa), and the ORF-length of CCL19 is 333 nucleotides (nt) encoding a protein of 110 amino acids (aa). Quantitative real-time RT-PCR (qRT-PCR) analysis revealed both two CC chemokines were constitutively expressed in all tissues examined. The CCL4 was highly expressed in the spleen, lowly in the muscle and intestine; the CCL19 had the highest expression in the spleen and the relatively lower level in the heart. After challenge with bacteria, the CCL4 and CCL19 shared the same expression trend. In the spleen and liver, the mRNA level increased remarkably at 12 h, which suggested that the two CC chemokines may be involved in the X. helleri defense against A. hydrophlia infection. In summary, this study demonstrates the basic profiles of two CC chemokines in X. helleri and chemokines take an important part in inflammatory and immune responses to bacteria. Together, our results also indicate that the X. helleri could be utilized as a useful fish model for the research of fish diseases.

Abstract:In order to understand the expressions of capsular polysaccharide synthetic gene of Streptococcus agalactiae (GBS) isolated from Oreochromis niloticus (Nile tilapia) and their effects on capsular sialic acid content and bacterial pathogenicity, capsular polysaccharide synthetic gene cpsE, cpsK and neuA of GBS from O. niloticus were cloned in the study. The expression levels of these genes at different temperature were detected by qPCR. Capsular sialic acid content was detected by colorimetric method. The mortality rate of O. niloticus infected with GBS cultured at different temperature was analyzed by artificial challenge test. The results showed that amino acid sequence analysis encoded by cpsE, cpsK and neuA of GBS had conservative enzyme active sites which were essential for the synthesis of capsular polysaccharide. CpsE and NeuA of GBS isolated from O. niloticus shared high homology with those of GBS isolated from human and other fish species (>97%). The identities of cpsK of GBS isolated from O. niloticus with those of other fish species (serotype Ia and Ib) and human (serotype Ia, Ib and II~IX) were 56%–100% and 27%–100%, respectively. The expression levels of cpsK and neuA of GBS cultivated at different temperature were consistent with that of capsular sialic acid content. Capsular sialic acid content and the mortality rate of tilapia infected with GBS cultivated at 28 and 34 ℃ were increased with the rise of temperature. The mortality rate of tilapia after artificial challenge at 22 ℃ was the lowest though the highest capsular sialic acid content was observed. The research suggested that the expression levels of CpsK and NeuA played an important role in the process of sialylation of capsular polysaccharide. Higher capsular sialic acid content of GBS cultivated at low temperature may provide protection for it to stay in the host. However, the expressions of some important virulence factors besides capsular sialic acid might be necessary for a strong bacteria pathogenicity for GBS cultivated at higher temperature.

Abstract:To obtain SNP mutations of mitochondrial genome in Portunus trituberculatus, a total of 179 primer pairs were designed, nearly covering the whole mitochondrial genome in this study. Of the 179 primer pairs, 89 got the expected specific amplification fragments by regular PCR. Then, 22 of 89 amplification fragments were found to contain the SNPs, by testing of HRM technology (High resolution melting curve). Though sequencing, 24 SNPs were obtained in the 22 amplification fragments. The results by statistical analyzing demonstrated that SNP frequency in mitochondrial genome of P. trituberculatus was 0.15/100 bp, in which the conversion mutation rate was 79.1%, 16.6% for transvesion mutation rate, 79.1% for mutation rate of C to T (or G to A), 8.3% for the mutation rate of G to T (or C to A), and 4.16 % for the mutation rate of G to C and A to T, respectively. Thus, the mutation of C to T (containing the type of G to A) is the most common mutation type. Additionally, the distribution of 24 SNP mutation loci was not even in the whole mitochondrial genome. The number of SNPs was the greatest in COX1 gene region, and the second is in D-LOOP and ND1 genes. However, no SNPs were found in some regions, such as tRNA, 12S rRNA. Our findings will provide an analysis tool that may help further researches in genetic diversity of germplasm resources and enhancement in P. trituberculatus.