Abstract:In the present study, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) genes were cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA 5' and 3' ends (RACE) in Scatophagus argus. The full-length of FSHR cDNA was 2538 bp, encoding a 702 amino acid protein and the full-length of LHR cDNA was 3315 bp, encoding a 722 amino acid protein. Both of them contain seven TM helix domains, a feature of glycoprotein hormone receptor (GHRs) family. Multiple sequence alignment shows that S. argus GtHRs have the highest homology with that of Dicentrarchus labrax. mRNA expression patterns showed that a high expression level of FSHR was detected at stage I in ovary, followed by an obvious decrease at stage Ⅱ, Ⅲ and IV. In the testis, the expression of FSHR gene increased gradually during early stages (stage I, Ⅱ and Ⅲ), and apparently reduced at stage IV. Our results indicates that according to H.E staining, sperm maturation started at stage Ⅲ, implying FSHR played an important role in early stages of the gonadal development, and LHR is vital for sperms/oocytes maturation and release as well as sperms.

Abstract:In order to research growth characteristics and the effect of morphological traits on body mass of Epinephelus fuscoguttatus at different month ages, data of total length (X₁), standard length (X₂), head length (X₃), body width (X₄), body depth (X₅), caudal peduncle depth (X₆), eye diameter (X₇), head length after eye (X₈) and body mass (Y) were collected at 3 months, 8 months and 13 months of age in this study. The morphological traits were analyzed by principal component and path analysis. The results showed that: (1) the correlation coefficients between all morphological traits at different age phases and body mass were extremely significant. And the correlation coefficients between each trait and body mass differed at different age development stages. (2) The first principal component reflected the growth and development of E. fuscoguttatus at different growth phases. The second principal component reflected the development of eye at different month ages. Thes third principal component was different and reflected the tail development at 3-month-old, body width development at 8-month-old and body height development at 13-month-old. (3) The trait with strongest direct effect on body mass was total length at all the age phases (0.742, 0.776 and 0.840). The path coefficients of four morphometric attributes (X₁, X₅, X₄, X₆) for 3-month body mass reached significant difference level. The path coefficients of X₁, X₄, X₆, and X₈ for 8-month body mass were also significant. The path coefficients of X₁, X₅, X₄ and X₂ for 13-month body mass were highly significant. The multivariate regression equation was Y_3-month-old=–36.774+3.992X₁+2.295X₅+2.759X₄+2.186X₆, Y_8-month-old=–64.569+7.020X₁+6.480X₄+6.754X₆+50.926X₈, and Y_13-month-old=–302.340+18.128X₁+17.688X₅–28.463X₄+5.928X₂. This result suggests that the vital traits that affected body mass at different growth phases were different. (4) The relation equation between standard length and body mass was W=0.0378L^2.873 (R²=0.9596) in 3-month to 13-month stages of E. fuscoguttatus, which demonstrated the standard length and body mass growth at similar velocity. We proposed that the total length was the target trait for selective breeding of E. fuscoguttatus at 3–13 month-old stages .This paper provides theoretical evidence and measure targets for breeding of E. fuscoguttatus in the early growth stage in aquaculture.

Abstract:Tyrosinase (Tyr) is the essential rate-limiting enzyme in melanogenesis. To study the relationship between Tyr gene and the formation of body color in Cyprinus carpio haematopterus, the black scales, silvery scales and fins were observed using microscope. Besides, we also obtained the full-length cDNA sequence of Tyr by rapid-amplification of cDNA ends (RACE) and examined the mRNA and protein expression levels of Tyr in different tissues using quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemical methods. The results showed that there were 3 kinds of pigment cells including melanophores, xanthophores and iridophores in different tissues, and the quantity, combination, and density of the chromatophores are varied. The full length cDNA of Tyr was 1717 bp (Genbank ID: No. KY305667), including 41bp at 5'-UTR, 71 bp at 3'-UTR, and a 1605 bp open reading frame encoded a peptide of 535 amino acids. Sequence analysis exhibited that Tyr from Yellow River carp had the highest amino acid similarity with Cyprinidae, while it had low similarity with mammalian and bird. And phylogenetic tree analysis indicated Yellow River carp has close relationship with Cyprinidae species like Cyprinus carpio, Cyprinus carpio Koi, Cyprinus carpio color and Danio rerio. qRT-PCR results revealed that Tyr mRNA was checked in all different tissues of Yellow River carp, among which the maximum level was detected in muscle, followed by black skin. In addition, Tyr also displayed a higher expression in the yellow skin, anal fin and caudal fin with abundant melanophores. But Tyr was not detected in the silvery skin lacking melanophores. The protein expression of Tyr in black skin, anal fin, caudal fin and silvery skin exhibited similar trend with Tyr mRNA. The immunohistochemical positive signals were detected in the melanophores and mucous cells of black skin. In conclusion, the expression level of Tyr is relevant to the abundance and location of melanophores, and could be associated with the body color formation of the Yellow River carp.

Abstract:In order to study the effect of aeration on vertical distribution of the ions in overlying and interstitial waters in aquaculture systems, we constructed 8 plexiglass microcosms paved with aquaculture sediment, including 4 tests with continuous aeration and 4 controls without aeration. Intact overlying and interstitial waters were collected by Peeper (pore water equilibriums) devices on the days of 0, 1, 4 and 7 respectively, before and after initiating the aeration. The ions of NH₄⁺-N, NO₃^--N, NO₂^--N, PO₄^3--P and SO₄^2--S were measured by miniaturized photometrical methods using a microplate reader. The result showed that one week aeration didn’t significantly change the vertical distribution of NH₄⁺-N in interstitial and overlying waters, but it greatly increased the concentration of NO₃^--N in overlying and 0–2 cm interstitial waters. The highest average concentration of NO₂^--N presented in surface layer waters before the aeration, while it peaked in the surface sediment interstitial waters at the depth of 1 cm during the aeration. The aeration promoted the adsorption and immobilization of PO₄^3--P in the sediment, greatly decreased the concentration of PO₄^3--P in overlying and 0–2 cm interstitial waters. The concentration of SO₄^2--S in overlying and 0–2 cm interstitial waters was significantly increased by oxidizing reducible sulfur in biological and chemical path. The physicochemical property of the overlying water on 1 d, 4 d, 7 d was greatly changed, which distanced farthest away from the control group, suited at the bottom of the PCA image. On the contrary, it didn’t distinctively chang the property of the sediment interstitial waters, which suited at the left site of the PCA image. In conclusion, aeration conditions can elevate oxidative ions of NO₃^--N, NO₂^--N and SO₄^2--S, and reduce the ions of PO₄^3--P which can cause eutrophication. It greatly changes the physicochemical property of the overlying water and surface sediment interstitial waters, which is an applicable method to control aquaculture pond water quality and remediate sediment.

Abstract:Nocardia seriolae is the main pathogen of fish nocardiosis, which is a chronic systemic granuloma disease of fish. A gene of protein tyrosine phosphatase (PTP) was found by analyzing the whole genome sequence of N. seriolae, and bioinformatics analysis showed that the PTP gene mayencode a secreted protein which could possibly target host cell mitochondria. In this study, the gene cloning, subcellular localization, over-expression and mitochondrial membrane potential detection were carried out. The results showed that the protein PTP was identified in the extracellular products of N. seriolae, which confirmed that PTP was a secreted protein. Subcellular localization of PTP-GFP fusion proteins were evenly distributed in the whole cell of FHM cells, and did not coincide with the distribution of mitochondria, which indicated that the protein PTP was not targeted at mitochondria. Typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were found when PTP protein was expressed in FHM cells by both subcellular localization and over-expression studies. The mitochondrial membrane potential was significantly damaged in FHM at 48h after the transfection of pcDNA-PTP, which confirmed that PTP was a bacterial protein which can likely induce cell apoptosis. The gene cloning and preliminary function study of PTP from N. seriolae have laid the foundation for further research on the gene and for promoting the understanding of the pathogenic mechanism of N. seriolae.

Abstract:The exhibition of growth and the induction of metallothioneins of Chlorella sp. exposed to different kinds of zinc salts were investigated. The biomass, heat-stable proteins and Zn-MTs were measured when the Chlorella sp. were exposed to four kinds of Zn²⁺ (including zinc chloride, zinc acetate, zinc gluconate, zinc citrate) at the concentrations of 5, 10, 20, 50 and 100 μmol/L. Results showed that the growth of Chlorella sp. was not significantly inhibited when the zinc concentrations were less than 5 and 10 μmol/L for zinc citrate and zinc chloride, respectively. But the growth was significantly inhibited at the concentrations of 5 μmol/L for each of zinc acetate and zinc gluconate, and the algal biomass rapidly decreased with the increases of Zn²⁺ concentrations for the two zinc salts. The results also showed that the amounts of heat-stable proteins significantly increased in Chlorella sp. exposed to the four kinds of zinc salts compared with that of the control group (without adding any zinc salt), and the largest amount of heat-stable proteins was obtained when the algae were exposed to zinc gluconate at 50 μmol/L for 3 days, producing 34.5 mg of heat-stable proteins per gram of wet algae mud, which was 3.3 times higher than that of the control group. And the largest amount of Zn-MTs was induced under the inducing condition.

Abstract:In order to investigate the expression characteristics of IL-8 during the lipopolysaccharide (LPS)-induced inflammatory process in grass carp (Ctenopharyngodon idella), recombinant grass carp IL-8 (rgcIL-8) was generated in prokaryotic expression system, and an anti-rgcIL-8 polyclonal antibody was prepare by immunizing mice with the purified rgcIL-8 alternately via intraperitoneal and subcutaneous injections. The polyclonal antibody was validated for specificity and titer by Western blotting and enzyme-linked immunosorbent assay (ELISA), and was used to detect IL-8 in immune-related tissues from healthy and/or LPS-stimulated grass carp by flow cytometry analysis. Results showed that the antibody titer peaked at 40 000. Flow cytometry analysis confirmed that IL-8 protein was expressed constitutively in all tested tissues of healthy fish, with higher levels in head kidney and gill. Moreover, the expression of IL-8 protein was found to be significantly up-regulated by LPS stimulation in all tested fish tissues, the protein levels peaked in intestine, liver and trunk kidney at 4 h, in head kidney and spleen at 12 h, and in thymus at 24 h following LPS stimulation, respectively. Our results provide further evidence that IL-8 is a key player in host inflammatory responses. Therefore, it is proposed that an elevated expression level of IL-8 protein could be used as a marker for monitoring the early inflammation, and be considered as a risk indicator for inflammatory diseases in aquaculture.

Abstract:This paper conducted a cross of white crucian carp (Carassius auratus cuvieri) (♀)×red crucian carp(Carassius auratus red var.) (♂) (WR), and the nutritional composition and quality of muscles from WR and its parents were investigated and analyzed by biochemical analysis methods. The results show that the moisture content of WR (71.00%) was significantly lower than that of its parents (WCC:75.60%, RCC:75.50%), while the protein content of WR (17.70%) was higher than that of RCC (17.00%) and was much higher than that of WCC (14.80%). Fifteen kinds of amino acids were found in muscles from WR and its parents. Both total amino acids and total essential amino acids contents of WR (15.87% and 6.55%) were higher than those of RCC (15.52% and 6.46% respectively), and were significantly higher than those of WCC (13.13% and 5.27% respectively). In particular, the total flavor amino acids content of WR (6.26%) was higher than that of RCC (6.07%), and was much higher than that of WCC (5.29%). Taken together, the results provided evidences for proving the advantages of WR in nutritional value and taste, and provided the theoretical supports for its application in production.

Abstract:Mannose receptor (MR) is a member of C-lectin family. It is mainly expressed on the surface of macrophage and immature dendritic cells. It has been shown that MR not only plays a significant role in innate immune responses, but also initiates acquired immune responses through processing and presenting antigens to activate T cells. In this study, we cloned and characterized pfMR of Chinese yellow catfish (Pelteobagrus fulvidraco). The expression profiles of pfMR mRNA in different tissues of P. fulvidraco were assessed by real-time quantitative reverse transcription PCR (qRT-PCR). To investigate the role of pfMR participating in the antibacterial infection, yellow catfish head kidney macrophages were infected with Edwardsiella ictaluri and competition binding test of mannan to MR was carried out. The phylogenetic tree analysis showed that MR of P. fulvidraco, Megalobrama amblycephala, Ctenopharyngodon idella, Danio rerio, and Oreochromis niloticus formed a clade. The mRNA of pfMR was expressed in all detected tissues with higher level in the kidney, spleen and muscle, and lowest expression in the blood. The expressions of pfMR, IL-1β and TNF-a mRNAs were all increased in cultured primary head-kidney macrophages after E. ictaluri infection. The infection of E. ictaluri also resulted in the over-expression of large number of superoxide anion (O₂^-·) and nitric oxide (NO) in the macrophages. The infection of E. ictaluri could significantly activate the production of O₂^-·at 30 min post treatment, while the amount of NO in the macrophage was significantly elevated at 12 h post treatment. Mannan significantly inhibited the engulfed GFP-E. ictaluri by macrophages through competition binding to MR. The engulfed GFP-E. ictaluri was significantly reduced by addition of EDTA, and restored by addition of Ca²⁺. The present study indicated that MR in head kidney macrophages of yellow catfish mediated phagocytosis of E. ictaluri and it was Ca²⁺-dependent.

Abstract:Marine biofilms play a crucial role in the settlement of plantigrades of the mussel Mytilus coruscus, but the effect of Vibrio derived from different sources on the settlement of plantigrades of the mussel M. coruscus remains unknown. Ten marine Vibrio species were isolated from natural biofilms and the intestine of the wild adult mussel M. coruscus and identified to investigate the relationship between these biofilms and mussel planitigrade settlement. The results showed that Vibrio biofilm cell densities increased with the increasing initial cell density. All test Vibrio biofilms can significantly promote mussel settlement, and the percentage of plantigrade settlement ranged from 17% to 67%. The inducing activities of these bacterial biofilms were significantly correlated to the microbial biofilm density, and the correlation coefficient in V. crassostreae ECSMB14106 was the highest (0.8992). In addition, the inducing activity of these bacterial strains was not correlated to the source of Vibrio. This study showed that marine Vibrio of the different sources induced mussel settlement and the finding is important for understanding the potential molecular mechanism of the biofilms mediating the settlement process in this species.

Abstract:In order to study the correlation between HcTyr gene and shell nacre colour, genomic sequence fragments were amplified according to cDNA fragments of HcTyr gene. Regarding the detected SNP, the genotype and the polymorphic information (PIC) of 144 Hyriopsis cumingii were analyzed, and the correlation between SNP and shell nacre colour-related traits were further analyzed using Chi-square test. Moreover, the location of HcTyr gene on gene map was also studied. The results showed that 7 of the 8 detected SNP in HcTyr gene were significantly associated with shell nacre colour (L, a and dE). Haplotype analysis revealed that the frequency of three major predominant haplotypes (Ⅱ, Ⅲ, Ⅳ) in the white strain was significantly higher than that in the purple strain. And the other two major predominant haplotypes (Ⅴ and Ⅶ) showed significantly higher frequency in the purple strain. The haplotypes were further validated in commercial cultured populations and the results showed that Ⅳ and Ⅶ can be considered as predominant haplotypes for white strain and purple strain respectively. The results suggest that HcTyr could be candidate shell nacre colour related genes for H. cumingii, and some polymorphic loci in this gene could be potential genetic markers for molecular breeding. Furthermore, we mapped HcTyr gene on LG16 of the previous linkage map, which will lay the foundation for the further study of the molecular mechanisms about this gene.

Abstract:Fish mucosa and their commensal microbiota serve as the first biological bulwark against invading pathogens. The purpose of this study is to study the implications on tilapia health status of the associated commensal microorganisms. Illumina sequencing was used to study the difference between the microbiota of mucosal surfaces from healthy tilapia, live and dying tilapia after Streptococcus agalactiae infection. The results showed that there were several genera dominated on tilapia skin and gill surfaces, i.e., Truepera, Thiobacillus, Arcobacter, Marinomonas, and Vibrio. There was no significant difference between the skin and gill microbiota of the live tilapia after Streptococcus agalactiae infection and those of the initial tilapia sample. A decreased bacterial diversity was shown in the skin and gill microbiota of the dying tilapia after the pathogen infection, and the genera of Arcobacter, Pseudoalteromonas, Marinomonas, Pseudomonas and Vibrio were decreased, while the genus of Streptococcus dominated in the skin and gill microbiota of the dying tilapia after the pathogen infection, with the relative abundance of 55.30%±1.24%. The results suggested that commensal microbiota of tilapia skin and gill surface could be related to fish health.

Abstract:Coproporphyrinogen-Ⅲ oxidase (CPOX) is a key enzyme in the synthesis of porphyrins, which plays an important role in the formation of pigment. cDNA of Meretrix meretrix CPOX (MmCPOX) was cloned by SMART RACE techniques, then the bioinformatics and expression profiles in different tissues and developmental stages were analyzed. The results indicated that the full length cDNA of MmCPOX gene was 1490 bp, containing a 1173 bp opening reading fame(ORF) encoding 390 amino acids. The molecular weight of MmCPOX was 12.04 ku and pI was 5.05. It was also predicted that protein MmCPOX had a transmembrance region and a Coprogen-oxidas region. Comparisons of amino acid sequence, MmCPOX was highly homologous with Aplysia californica and shares 64.5% similarity. The result of qRT-PCR showed that MmCPOX gene was expressed in all stages, and significantly increased from D-shaped larva stage (P<0.01). MmCPOX was expressed in all seven tissues, in which the expressions in mantle and blood were significantly higher than those of other tissues (P<0.01), indicating that it was related to synthesis of hematoporphyrin and formation of shell porphyrin. Considering different shell colors, the relative expressions of mantle in red color, dark fringe and thin checkered shells, were significantly different from the black and white shells (P<0.05), suggesting that MmCPOX gene was responsible for the formation of red and brown shell color.

Abstract:In this study, we used the side scan sonar system to investigate and evaluate the two artificial stone reef areas in A and B in the sea area of the Mayi Island in Jinzhou Bay, Dalian. The acoustic method was applied to the estimation of stone reef volume. The purpose was to explore the new estimation method of stone reef volume to improve the accuracy of the calculation results, improve work efficiency. The data mining of the sonar data was carried out by using the data processing software and the computer-aided technology. The data of the stone reef were extracted and the volume of the stone reef was estimated by the function curve. The results show that the side scan sonar image can clearly reflect the distribution of stone reefs in the seabed, and the scouring and silting degree of the stone reef can be judged according to the image. The geometric relationship and the related technical means are used to estimate the volume of the stone reef. Through several different angles of detection or with other auxiliary software errors were minimized. In the A reef area, the volume of the reef is estimated by the integral section method and the relative error is close to 16% compared with the actual volume of the reef area. The B reef area is tested and evaluated. The relative error between the evaluated volume and actual volume of the stone reff is close to 16%. The estimated volume of the two artificial fish reef areas is very different from the actual volume. Although the physical environment and the size of the reefs are different in the two reef areas, the relative error is close to and less than 20%. The integral curve is verified rationality. Therefore, under the influence of many uncertain factors, this method can estimate the volume of stone reefs relatively accurately, which can provide support and basis for the construction, management and evaluation of artificial reef area.

Abstract:Pyropia haitanensis is a sessile marine macroalgae that inhabits the intertidal zone. High light was the main environmental stressor of P. haitanensis during low tides and the key factor affecting yield. To explore the molecular mechanisms of high light tolerance, gene expression analysis was the first step. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression levels of six housekeeping genes [18S rRNA (18S), ubiquitin-conjugating enzyme (UBC), actin (ACT), β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] were examined by the quantitative real-time PCR in samples corresponding to different high light stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (Ct) method and by three different software packages: geNorm, Bestkeeper and NormFinder. The results showed that the Ct value of UBC had the least variation, while that of 18S obviously changed under different light intensities treatments. The analysis of geNorm also exhibited that the most stable housekeeping genes were UBC and EF2. The analysis of both Bestkeeper and NormFinder exhibited that stability measure M of UBC was the lowest (0.044 and 0.80, respectively) among six candidate genes. Thus the most stable housekeeping gene is UBC. Based on the above results, it is proposed that the most appropriate internal control gene for expression analyses under high light stress of P. haitanensis is UBC.

Abstract:To study the germplasm resources and genetic diversity of the wild population of Hippocampus japonicus, the mitochondrial DNA control region fragments were obtained from wild populations by PCR amplification. The homologous sequences of other Syngnathidae fishes from GenBank were also included in this study. PCR amplification products of 557–558 bp CR fragments were obtained, and the average contents of A, T, G and C were 34.3%, 29.7%, 14.1%, 21.9%, respectively. A total of 16 polymorphic nucleotide sites were detected, which defined 16 haplotypes. The variation level was low as H was 0.70±0.02 and π was 0.0032±0.0021. With the aid of the homologous sequences retrieved from GenBank, phylogenetic trees were constructed based on neibour-joining (NJ), maximum-likelihood (ML) and Bayesian inference (BI) methods to build phylogenetic relationships within the Syngnathidae fishes. The phylogenic analysis showed that the topology of phylogenetic trees were almost consistent with each other, although the NJ tree and the ML/BI tree were somewhat different. The phylogenic analyses of CR gene sequences were also consistent with morphological taxonomy. Based on the results, several conclusions were drawn as follows: (1) CR is an appropriate marker for the seahorse species identification and population genetic analysis; (2) CR is highly divergent among different Syngnathidae fishes and could be an appropriate marker for molecular systematic studies. This study is expected to provide important information for the protection and utilization of H. japonicus resources in China.

Abstract:As assessment of genetic structure of population is the key point of aquatic animal breeding, the selection of sample size and loci number is the first step of correct evaluation of population genetic structure. Here, we solved the problem from an analysis of genetic diversity of three breeding groups of Macrobrachium rosenbergii at 60 polymorphic microsatellite loci. The results show that selection of different sample sizes and loci numbers both had a great effect on the genetic diversity index. The sample size was positively correlated with the average number of alleles and the average effective number of alleles, and the sample size was moderate and highly correlated with heterozygosity and Nei's genetic diversity index, respectively. Only when the sample size exceeds 30, they tend to be stable. When we select the loci, we found the different polymorphism information contents also directly affect the accuracy of the genetic parameters. When the loci number was 5–25, there was a significant difference in the genetic diversity index(P<0.05). When the loci number was more than 25, the genetic parameters changed little(P>0.05). So, we suggested that when microsatellite markers were used to estimate genetic diversity of population as far as possible to choose high polymorphism information content and we can't consider sample gender, meanwhile the minimum loci number and sample size are needed to exceed 25 and 30, respectively.

Abstract:Synaptonemal complex protein 3 (SCP3) is a meiosis-specific component of the synaptonemal complex essential for gametogenesis. In vertebrates, SCP3 protein is specifically localized in primary spermatocytes and many researchers have used it as a molecular marker to identify and separate primary spermatocytes from all types of germ cells. To measure whether SCP3 can mark any specific germ cells in invertebrates, we cloned a 1 033 bp full-length cDNA of Chlamys farreri SCP3 (Cf-SCP3) that contains a 726 bp open reading frame encoding 241 amino acids with one conserved Cor1 domain and two coiled-coil domains. Then we synthesized the DIG-labeled RNA probe, expressed the Cf-SCP3 recombinant protein and generated the anti-Cf-SCP3 polyclonal antibody. In situ hybridization and immunohistochemistry observations showed that Cf-SCP3 transcripts and Cf-SCP3 protein were specifically expressed in primary spermatocytes and unfertilized eggs, while no expression was detected in any other spermatogenic cells or ovarian cells of C. farreri. Thus, it suggested that the Cf-SCP3 protein might participate in meiosis of gametogenesis as a component of the synaptonemal complex like that in vertebrates and could be used as a candidate molecular marker for identifying the primary spermatocytes in germ cells differentiation study of C. farreri.