Abstract:To understand the diet composition of wild yellowfin tuna (Thunnus albacores) and its ontogenetic variations, some surveys were conducted from February 2012 to January 2013 in the South China Sea. Both stomach content and stable isotope analysis were used to study the feeding ecology of yellowfin tuna. A total of 94 yellowfin tuna samples, ranging between 41 cm and 180 cm in fork length (FL), were measured according to the national standards on specifications for oceanographic survey (GB/T 12763.6–2007). Results showed that diet compositions of Thunnus albacores mainly consisted of Sthenoteuthis oualaniensis, Uroteuthis sp., Sepia sp., Decapterus sp., Gempylus, Alepisaurus, Brama japonica, Cypselurus sp., unidentified crustacean and fishes, etc. S. oualaniensis was the primary prey components of the diet, averaging 51.07% by percentage of Index of Relative Importance (IRI%). Decapterus was the second most important diet, with IRI of 39.81%. The percentage of empty stomach, mean stomach fullness index and diet compositions had significant monthly variation (P<0.05). The vacuity coefficient changed significantly in each group of FL and showed the upward trend (P<0.05). Mean stomach fullness index had significant monthly variation. With the group between 130 and 160 cm FL, Shannon-Weiner diversity index H¢ ranged between 0.05 to 1.18 and Pielou evenness index J varied from 0.10 to 0.32. The average trophic level of yellowfin tuna in the South China Sea was 3.54. In conclusion, the results improved the understanding of the yellowfin tuna biological characteristics and feeding habits, also provided some empirical data for marine ecosystem modeling in the South China Sea.

Abstract:The visceral white-spots disease is a serious bacterial disease in cage cultured large yellow croaker (Pseudosciaena crocea) in recent years in Zhejiang Province. The principal clinical signs of visceral white-spots disease among infected fish include multi-focal granulomas in the internal organs, especially in spleen and posterior kidney. In present study, the tissues including liver, spleen, kidney, heart, intestine and muscular tissues collected from infected large yellow croaker, P. crocea were examined histologically and ultrastructurally to elucidate the histological features after being infected by the bacterial disease. The collected diseased fish showed clinical signs of numerous gray, tan or white foci present in the spleen, liver or kidney internally without any external lesions. Histopathological analysis revealed severe degeneration and necrotic changes in multiple internal organs, including liver, spleen and kidney. Moreover, fibrosis in the liver tissue, proliferation of lymphocytes, infiltration of neutrophils and sedimentation of hemoferrin in spleen, and atrophy of glomeruli, deformation of renal tubules and amyloidosis in kidney were observed in this study. Furthermore, serious vacuolization, inflammatory cell infiltration and pathological nodules were observed. The results of ultrastructural analysis showed swelling and distortion of visceral cells in liver, spleen, and kidney. Rarefaction and vacuoles of the mitochondrial structure, disruption and disappearance of the mitochondrial cristae and condensation and margination of the chromatin were also observed in the liver, spleen and liver cells of diseased fish. In addition, large numbers of rod-shaped bacterial cells were packed in kidney and spleen. Our results clearly demonstrated the pathological changes in the cell or tissues among the diseased fish. The changes were characterized by serious visceral granulomas, and the release of toxic metabolites and loss of appetite leading to metabolic disorder and serious mortality of the fish. The study provided valuable data for understanding the pathogenesis of this bacterial pathogen in large yellow croaker.

Abstract:Turbot Scophthalmus maximus was introduced into China in 1992. Since then, it has become one of the most dominant mariculture industries. In recent years, however, a main bottleneck constraining its commercial development is the disease problem prevailing in the farms and causing significant mortalities. Ascites syndrome is a disease that is commonly found in larvae culture and grow-out period. The present study investigated the diversity and antimicrobial resistance of the pathogens isolated from bacterial ascetic disease in cultured turbot S. maximus in Shandong Province. In total, 27 bacterial pathogens were collected from different regions of the province between 2002 and 2010. These 27 strains of pathogens were identified through the use of 16S rDNA gene sequence analysis and their antibiotic resistance profiles were tested with 22 common antibiotics using the Kirby-Bauer disc diffusion methodology. The antimicrobial susceptibility data were used to identify correlations between antibiogram and the change of resistance. The results demonstrated that V. scophthalmi, E. tarda, V. anguillarum, V. harveyi, P. espejiana were the pathogenic strains associated with ascetic disease of cultured turbot. The major pathogenic strain was V. scophthalmi in Qingdao area and E. tarda in Weihai area, whereas all pathogenic strains were equally found in Yantai area. Five bacterial strains were resistant to Penicillin, Cephalosporins, Macrolides and T/S with resistance rates over 50%. In addition, the resistance rate to FFC was below 10%, and was little drug resistance in the long-term use, suggesting that FFC is a favorable antibacterial drug to prevent and treat the ascites disease in cultured turbot. Twenty-seven pathogenic bacteria formed 27 antibiogram types since all the strains had multiple antibiotic resistance. 74.1% of the strains were resistant to more than ten different types of antibiotics. In conclusion, the drug resistance rates among aquatic bacteria were very high, becoming the primary problem in the prevention of aquatic bacterial diseases. The results provide theoretical basis and reference for the epidemiological studies of turbot ascetic disease and its early prevention.

Abstract:In recent years, Vibrio harveyi has become a main pathogen for mariculture animals. It caused serious diseases and mortalities of many aquaculture animals and caused huge economic loss worldwide. The isolation and identification of virulence factors of V. harveyi become research hotspot recently. The V. harveyi strain ML01 (CGMCC No. 11720) was isolated from diseased juvenile hybrid grouper Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂) suffering from severe skin ulcer. In this research, the virulence factors, including extracellular products (ECPs) and secretory proteins of strain ML01 were extracted and purified by cellophane plate and gel filtration techniques. Furthermore, extracellular products (ECPs) and secretory proteins were characterized and identified through toxicity tests, mass spectrometry MALDI-TOF/TOF, and molecular cloning methods. The results showed that ECPs of strain ML01 had the activity of gelatinase, amylase, lipase and caseinase, but no the activity of urease. The hemolysis to sheep red blood cells of ECPs was negative. The toxicity tests showed that ECPs of strain ML01 were lethal to zebrafish and the LD50 value was 19.55 μg/g body weight. Three major secretory proteins (P42, P36, and P31) corresponding to the molecular weight of 42 kDa, 36 kDa and 31 kDa on SDS-PAGE were purified from strain ML01. These proteins were identified as membrane proteins of OmpU, OmpN and a hypothetical protein of V. harveyi by mass spectrometry MALDI- TOF/TOF. Based on homology cloning techniques, the genes of proteins P42, P36 and P31 were amplified from the genome of strain ML01 and sequenced. The sequence alignment results showed that similarities of the open reading frames (ORFs) between strain ML01 and V. harveyi ATCC 33843 were 97.08% (p42), 100% (p36) and 99.67% (p31), while the similarities of peptide sequences were 99.71% (p46), 100% (p36) and 99.93% (p31), respectively. This research is of significance to further analysis of the pathogenic mechanism and the development of subunit vaccines of V. harveyi strain ML01.

Abstract:Superoxide dismutase (SOD), the first barrier of antioxidation, protects cells through the removal of excessive superoxide. However, little is known about the function of SOD genes in Odontobutis potamophila. In the present study, the rapid amplification of cDNA ends (RACE) approach and quantitative real-time PCR were used to clone the full-length cDNA of Mn-SOD and analyze its expression patterns. The full length of cDNA Mn-SOD was 1008 bp, including an open reading frame (ORF) of 678 bp, a 5¢-untranslated regions (UTR) of 15 bp and a 3¢UTR of 315 bp. Each 3¢UTR possessed a polyadenylation signal sequences of AATAAA and a poly A tail of 31 bp, a phenomenon that vertebrate usually has. It was speculated that the sequence encoded 225 amino acids. The Mn-SOD genes contains two conserved domains: the Sod_Fe_N (28-109) and Sod_Fe_C (116-219). The structure was similar with other species, which showed that the gene was conserved in the evolution. The homologous comparison with other species revealed that the Mn-SOD had the highest sequence identity with Oplegnathus fasciatus and Channa striatawhich, about 90.3 %. Quantitative real-time PCR was used to quantify the tissue-specific expression of Mn-SOD gene. Predominant expression of Mn-SOD was detected in muscle. Different developmental stages from embryo to larva had Mn-SOD gene expressed. Morula stage had the highest expression level. In addition, the expression levels of Mn-SOD mRNA in liver increased initially and then declined after the exposure of NaNO2. In gill, the mRNA expression levels of Mn-SOD fluctuated significantly. The findings of this study suggested that the Mn-SOD may play an important role in defending oxidative stress and cellular damage induced by nitrite, by detoxifying harmful reactive oxygen. Our study provides valuable reference to optimize the artificial breeding and rearing conditions of O.potamophila.

Abstract:A 10-week feeding experiment in indoor flowing-through water system was conducted to investigate the effects of dietary taurine, methionine and cysteine on growth and amino acid composition of Japanese seabass [mean initial weight (8.13±0.05) g]. A basal diet (as control) was formulated with 15% fish meal, 30% soybean meal, 13% corn gluten meal and 12% wheat gluten. The four experimental diets were supplemented with 1.0%, 2.0% taurine, 0.5% methionine and 0.5% cysteine (named as T-1, T-2, M-0.5 and C-0.5, respectively) to the basal diet to formulate isonitrogenous and isolipidic diets. Triplicate groups of 25 fish were fed to apparent satiation twice daily. During the experimental period, water temperature ranged in 23.2-28.8℃, and the dissolved oxygen content was approximately 7 mg/L. The results showed that the final body weight (FBW), specific growth rate (SGR), weight gain (WGR) and feed intake (FI) of T-1, T-2, M-0.5 and C-0.5 groups were significantly higher than those in the control group (P<0.05). Fish fed the diets with taurine, methionine and cysteine supplementation had significantly higher whole-body crude protein and crude lipid contents (P<0.05), while the whole-body moisture followed the opposite pattern. The taurine contents of liver and muscle in T-1 and T-2 groups were significantly higher than that in T-0 group (P<0.05), however, the taurine contents of liver and muscle in M-0.5 and C-0.5 groups showed no significant difference with T-0 group (P>0.05). Compared to the control, fish in T-1, T-2 and C-0.5 groups had higher liver essential amino acids and total amino acids contents (P<0.05), while these values were not significantly different in the M-0.5 group (P>0.05). The muscle essential amino acids content of the M-0.5 group was significantly higher than that of T-0 group (P<0.05), while fish in T-1, T-2 and M-0.5 groups had significantly higher muscle total amino acids contents than T-0 group (P<0.05). It is concluded that dietary taurine, methionine and cysteine could promote the growth of juvenile Japanese seabass under the experimental conditions, it can also improve the amino acids deposition in liver and muscle.

Abstract:The objective of the present research was to investigate the effects of dry exposure on respiratory metabolism in different tissues of Exopalaemon carinicauda. In the trial, E. carinicauda were divided into two experimental groups, namely normal temperature (22-24℃) group and low temperature (4℃) group. Meanwhile, shrimp cultured in seawater were used as the control group. Activities of respiratory metabolism enzymes and content of lactate were measured under dry exposure challenges at 60 min, 90 min, 120 min and 180 min. The investigated enzymes included succinate dehydrogenase (SDH), cytochrome oxidase (CCO), fumarate reductase (FRD) and lactate dehydrogenase (LDH). SDH and CCO are the key enzymes of aerobic metabolism in animals, whereas FRD and LDH are the key enzymes of anaerobic metabolism in animals. In addition, the content of lactate is an indicator of anaerobic metabolism. The results showed that the activities of SDH and CCO in hepatopancreas, gill, and muscle in the normal temperature group decreased significantly than those in the control (P<0.05) with the processes of dry exposure. Activities of FRD, LDH and content of lactate in muscle increased initially and then decreased gradually. And the levels were significantly higher than those in the control (P<0.05). Activities of respiratory metabolism enzymes and content of lactate in low temperature group had no significant difference compared to those in the control group at 60 min. After that, the trends of the enzyme levels in the low temperature group were as similar as the normal temperature group. The changes of enzyme activities were tissue dependent. Activities of respiratory metabolic enzymes in hepatopancreas and gill were higher than those in muscle. The results suggested that anaerobic metabolism increased after dry exposure. By contrast, E. carinicauda can maintain the aerobic metabolism during the early stage of low temperature dry exposure. Furthermore, the hepatopancreas and gill might be more sensitive to dry exposure, whereas reduced temperature could increase the survival rate of E. carinicauda.

Abstract:In the present study, Fenneropenaeus chinensis were fed artificial diets supplemented with different concentrations of poly-β-hydroxybutyrate (PHB: 0, 0.5%, 1.0%, 2.5%, 5.0% and 10.0%) during the grow-out and spawning period. The goal was to investigate the effects of PHB on the reproductive performance of parental shrimps and thereafter the development of the larval F. chinensis. This study used the single factor concentration gradient method. After 192 days, the mortality rate, relative immune protectionrate (RPS), weight gain rate (WGR), specific growth rate, gonadal development cycle, fecundity, the number of nauplii hatched and the timing of various stages of metamorphosis were compared. The results indicated that the PHB had mixed effects on the fecundity of parental shrimps and growth performance of larva. Compared with control group, with the increase of PHB concentrations, the mortality rate of the experimental groups exhibited downward trend followed by increases. By contrast, the RPS had an opposite trend, with the lowest mortality rate in 2.0% PHB group; and the differences were significant compared to other groups (P<0.05) except for the 1.0% PHB group (P>0.05). The WGR of all the PHB supplemented groups was higher than the control group. Specifically, the WGR of the group supplied with 2.0% PHB was significantly higher than groups supplied with 0 and 5.0% PHB (P<0.05). But there was no significant difference in the gonadal development cycle among all groups (P>0.05). Fecundity and the number of nauplii hatched in the experimental groups were higher than the control group, but the significance was only found in the group supplemented with 2.0% PHB (P<0.05). In addition, there was no significant difference in the timing of the various stages of metamorphosis among all groups. Based on the results, it can be concluded that the PHB supplement could improve the fecundity and larval development in F. chinensis with the best dietary PHB concentration of 2.0%.

Abstract:To breed the Litopenaeus vannamei that could resist low temperature during production cycle, this study investigated the heterosis advantage in breeding program and compareds the growth performance and genetic diversity between two populations under the low temperature conditions. One population, namely the Selected Population (SP), was bred for three consecutive years in our aquatic laboratory, whereas the other one, the Hybrid Population (HP), came from the hybridization of the Selected Population and the Introduced Population. The results showed that the growth performance of HP was significantly higher than SP. The average body weight of HP and SP shrimps were (13.18±3.65) g and (12.20±3.14) g respectively, and the coefficient of variation were 27.69% and 25.74%, respectively. The average body weight and other measurable traits in HP were higher than those in SP. The results of One-way ANOVA indicated that there were significant differences (P<0.001) between HP and SP in both body weight and TASW. The specific growth rate (SGR) and absolute growth rate (AGR) in HP were (5.09±0.61) %/d and (0.26±0.60) g/d, respectively. By comparison, in SP group, the SGR was (4.94±0.57) %/d and the AGR was (0.24±0.63) g/d, significantly lower (P<0.01) than the HP group. The results of genetic diversity analysis showed that the Na of HP (Na=7.9) was slightly higher than SP (Na=7.6). The average polymorphic information content (PIC) of HP and SP was similar as 0.63 and 0.62, respectively, both belong to the relatively high polymorphism level. The average observed heterozygosity (Ho) of HP and SP was 0.492 and 0.483, respectively, and the expected heterozygosity (He) was 0.675 and 0.663, respectively. Both Ho and He in HP were higher than that in SP, suggesting that HP had higher genetic diversity abundant compared to the SP. The analysis of genetic differentiation indicated that the genetic differentiation was significant between HP and SP, with the genetic differentiation index (Fst) between HP and SP was 0.1556. The results of this study provides a genetic background and production experience of L. vannamei for breeding low temperature breed and crossbreeding technique.

Abstract:To analyze the genetic effects and inbred degrees of 3 families of Exopalaemon carinicauda inbred line, 33 microsatellite loci were selected for genetic analysis. Composition of alleles of each line was analyzed. Number of allele (N), heterozygosity (H), polymorphism information content (PIC), and mean rate of homozygote were calculated. According to gene frequencies of 33 microsatellites, genetic distances between these lines were estimated. In additon, the dendrograms were obtained based on genetic distances. The results indicatedthat 72 alleles were detected at the 33 loci with an average number of allele of 2.758. The estimation of average N, H and PIC of the whole population was 2.758, 0.294, 0.272, respectively. The average N in three families were 2.258, 0.226, 0.214, respectively. The average H in three families were 2.469, 0.283, 0.300, respectively. The average PIC in three families were 2.219, 0.268, 0.207, respectively. And 7 loci could be used as special gene loci for genetic detection. Cluster analysis showed that Line A and B were gathered first, and then merged with Line H. In conclusion, this study revealed the pattern of genetic variation at 33 microsatellite loci in three inbred families of E. carinicauda. And the results indicated that inbred families have reached a certain degree of inbreeding. Some loci could be used as special gene loci for genetic detection and identification of independent genetic groups. This study provided important information for genetic analysis and germplasm conservation in E. carinicauda.

Abstract:Immune response of Litopenaeus vannamei fed with biofilm and planktonic cells of Bacillus cereus was studied by evaluating the growth rate, disease resistance and intestinal microflora. The biofilm and planktonic cells of B.cereus (PC465) were added to original feed as biofilm feed and planktonic feed respectively. Shrimps were maintained in 24 buckets with similar lighting and position in three groups. Biofilm group and planktonic group were fed with the concentration of 108 CFU/g of either biofilm or planktonic cells added feed for 15 d. Shrimps were challenged with white spot syndrome virus (WSSV) after 17 days post feeding in both experimental diet groups. Then a reverse gavage challenge with Vibrio parahaemolyticus has been tested on day 22. The growth rate of shrimp was evaluated on day 1, 5, 10, 15. The diversity and composition of bacterial communities of shrimp intestine were assessed by high throughput sequencing of 16S rDNA on day 1, 5, 10, 15, 20 and 25. The results showed that the growth rate and final weight of the biofilm group and the planktonic group were higher than the control group. The intestinal microflora of L.vannamei mainly consisted of Proteobacteria, while Bacteroidetes, Actinobacteria, Firmicutes, Verrucomicrobia, Cyanophyta, with the content of Proteobacteria accounting for about 94%. The proportion of Firmicutes in the shrimp intestine of the biofilm group (0.45%) and planktonic group (0.93%) was higher than the control group (0.02%). Proteobacteria mainly included Vibrio, Photobacterium, Pseudoalteromonas, etc. The proportion of Vibrio in the biofilm group, planktonic group and control group was 34.65%, 39.27%, and 58.00%, respectively. The cumulative mortalities of the biofilm group, planktonic group and control group challenged with WSSV were 80%, 77% and 92%, respectively. The cumulative mortalities of the biofilm group, planktonic group and control group challenged with V. parahaemolyticus were 61.3%, 75% and 77.3%, respectively. It is concluded that diet supplemented with B. cereus can change the intestinal microflora of L.vannamei, improve the disease resistance and enhance growth. The biofilm fed shrimps were more resistant to virus Vibrio and pathogens compared to that with planktonic fed and the control shrimps.

Abstract:Microsporidia Enterocytozoon hepatopenaei (EHP) was detected using probe based real-time PCR from 442 shrimp coming from 5 farmed Litopenaeus vannamei populations in Tianjin, Zhejiang, and Shandong Province. The biological length and body weight of shrimp were measured individually. The Rohrer’s ponderal index (PI, W/L3) originally used in medical science was introduced in this study to establish the fuction of body weight (W) and biological length (L) of shrimp. The results showed that the PI of 4 EHP positive populations with an average biological length at (5.37±1.19) cm was calculated as (5.19±0.26)× 10–3 g/cm3, while that of the EHP negative population with an average biological length was (7.96±0.51)×10–3 g/cm3. After adjusting of the difference of PI caused by biological length according to the function PI=a×L(b–3), the PI of EHP positive populations was about (70.5±8.7)% of that of the EHP negative populations at the same biological length. It indicated that the average body weight of shrimp in an EHP positive population was about 30% smaller than that of shrimp with same size in biological length in an EHP negative population. The coefficient of variation (CV) of biological length and body weight in EHP positive populations was (2.39±0.93) times and (2.05±0.86) times of those in the EHP population, respectively, suggesting significant size variation in the EHP positive populations. The deviation ratio of actual body weight to body weight estimated on biological length in the EHP positive populations was 2.34 to 3.45 times of that in the EHP negative population, indicating the shrimp with same size in the EHP positive populations had greater fluctuation in body weight.

Abstract:The pharmacokinetics, tissue distribution and elimination of sulfadiazine (SD) and trimethoprim (TMP) in mud crab (Scylla paramamosain) were investigated in order to provide scientific data to support its safe use. SD and TMP were orally administrated at a single dose of SD (83.3 mg/kg) and TMP (16.7 mg/kg) at temperature of (27±1) ℃ and seawater salinity of 10. The hemolymph concentration-time curve for SD and TMP were described by a two-compartment model with first order absorption. Peak hemolymph concentration (Cmax) for SD and TMP was 49.56 mg/L and 2.79 mg/L, respectively, and area under curve (AUC) for SD and TMP was 1417.6 mg/L·h and 82.7 mg/L·h, respectively. Of the tissues analyzed, the hepatopancreas was the tissue with the highest SD and TMP concentrations, and Cmax was 59.36 mg/kg and 74.82 mg/kg, respectively. Thus, a large amount of TMP was accumulated in the hepatopancreas, while only a small amount of TMP entered blood circulation. In gill, peak concentration for SD and TMP was 51.89 mg/kg and 42.58 mg/kg, respectively, and elimination half-life was 23.28 h and 25.29 h, respectively. The drug concentration in gill tissue was relatively high and the elimination rate was fast, suggesting crab gill plays a role in drug elimination. In muscle, peak concentration for SD and TMP was 44.95 mg/kg and 10.09 mg/kg, respectively, and elimination half-life was 25.09 h and 35.08 h, respectively. According to SD and TMP elimination and the maximum residue limit of 0.1 mg/kg for SD and 0.05 mg/kg for TMP, the theoretical withdrawal time of SD/TMP in muscle and hepatopancreas, was 290.6 h/302.8 h and 340.4 h/377.0 h, respectively.

Abstract:In order to better manage and protect the genetic resources of Corbicula fluminea in Hongze Lake, molecular marker (mitochondrial cytochrome oxidase subunit I gene sequence, COI) and morphometric parameters (wet weight, shell length, width and height) were used to investigate the differences in genetic diversity and morphological characteristics of two populations with different morphology in color: black and yellow. Morphological analysis showed that the morphological parameters were significantly different between two populations. Using PCR amplification and sequencing, 614 bp of COI sequences were obtained. The base composition of COI sequences were very similar between black and yellow populations, in which the A+T content was higher than the G+C content. Seven haplotypes were found among 28 black individuals; the haplotype and nucleotide diversity were 0.794 and 0.04274, respectively. In parallel, 5 haplotypes were found among 30 yellow individuals, with the haplotype and nucleotide diversity being 0.607 and 0.02825, respectively. The genetic distance varied between 0.002 and 0.091 among all the COI haplotypes. Molecular phylogenetic trees were constructed using neighbor- joining and maximum parsimony, suggested that the COI gene haplotypes clustered into two clades. Analysis of molecular variance (AMOVA) based on COI sequences suggested that 78.26% variation occurred within the population, and a lower percentage of 21.74% variation occurred between two populations, and there was significant genetic differentiation in both populations (Fst=0.21736, P<0.01). The present results suggested that black and yellow C.fluminea populations in Hongze Lake should be protected and managed as two independent units in genetic resource.

Abstract:In this study, we evaluated the accumulation of heavy metals in organisms, and discussed its effect on the quality and food safety of the shellfish. The status of heavy metal pollution was analyzed in the seawater, sediments and organisms in the major shellfish culture area of Shangdong Province in August 2014. The contents of heavy metals in the water followed the order As>Cu>Cr>Pb>Cd>Hg; the order in sediments was Cr>Pb>Cu>As>Cd>Hg; in organisms, it was Cu>As>Cd>Cr>Pb>Hg. It was found that the contents of heavy metals in the seawater met the second seawater quality standard, and those in sediments complied with the first marine sediment quality standard. We also found that the contents of Hg, Cd, Pb and Cr in seashells were below the limit according to the standard for the toxic substance in the non-environmental pollution aquatic products. The level of Cu was lower than the limit according to the first marine biological quality standard. The enrichment coefficients of heavy metals in the shellfish followed the order Cu>Cd>Hg>Pb>Cr>As. There accumulation of heavy metals in the shellfish was moderate. Based on the evaluation of the heavy metal pollution in the shellfish, we concluded that the food safety risk of the shellfish in the major culture area of Shangdong Province was relatively low. However, we highly recommend the removal of internal organs of the shellfish before consumption.

Abstract:CyHV-2 (Cyprinid herpesvirus 2) is a double-stranded DNA virus first isolated from goldfish, Carassius auratus (L). It is classified as a member of the genus Cyprinivirus, which includes carp pox (CyHV-1), koi herpesvirus (CyHV-3) and anguillid herpesvirus-1 (AngHV-1). It has an icosahedral shape with an average diameter of 100–110 nm. CyHV-2 can cause approximately 50% to 100% mortalities in cultured goldfish when water temperature is between 15℃ and 25℃. Therefore, rapid and precise detection of CyHV-2 is needed to prevent and control disease outbreak. In this study we established a droplet digital PCR (ddPCR) method used for accurate quantification of CyHV-2 DNA. The ddPCR method was compared with the quantitative real-time PCR (qPCR) in the aspect of the sensitivity, reproducibility, specificity and practical application. In terms of detecting CyHV-2 DNA, the sensitivity of the ddPCR assays was 20-fold lower than qPCR. The correlation coefficient (R2) obtained from linear regression analysis showed a good linearity of amplification for both ddPCR (R2=0.994) and qPCR (R2=0.994) assays. A positive correlation (R2=0.989) was observed between ddPCR and qPCR assays. To determine the same dilution series of CyHV-2 DNA, the expected numbers of DNA copies calculated by qPCR was 10-fold higher than the number of copies determined by ddPCR. CyHV-2 DNA reproducibility determined by ddPCR was found to be significantly more stable than by qPCR. The ddPCR assay had no cross-reaction with other similar fish herpesviruses, including CyHV-3, CCV (Channel catfish virus)、STIV (Soft-shelled turtle iridovirus) and EHNV (Epizootic hematopoietic necrosis virus). By contrast, the specificity of ddPCR was consistent with qPCR. Therefore, the ddPCR method was proven to be more precise than qPCR. This new absolute quantitation tool will be useful to standardize quantitative detection of CyHV-2 DNA.

Abstract:Notch Pathway is a very conservative signaling pathway in evolution, through the interaction between Notch receptor and the ligand DSL protein which conveys signals to regulate cell proliferation, differentiation, apoptosis, and determine the fate of cells. Rhopilema esculentum belongs to cnidaria, it is an ideal model organism to study cnidaria asexual propagation because of the mature artificial breeding and aquaculture technology. The cDNA cloning and expression analysis of Notch were first performed in the R. esculentum based on 454 GS-FLX sequencing technique by RT-PCR and RACE method. The results showed that the full-length cDNA of Notch was 6768 bp, containing 5¢- untranslated region (UTR) of 90 bp, an open reading frame (ORF) of 6066 bp and 3¢- untranslated region (UTR) of 612 bp which contains a polyadenylation signals of AATAAA. SMART analysis showed that R. esculentum Notch, as a secretory protein, included a putative signal peptide of 21 amino acid residues; its mature peptide included 2000 amino acid residues and consisted of 37 structure domains, which includes 26 EGF-like domains, 6 ANK domains, and a transmembrane domain except 3 Notch / Lin-12(NL) structure domain, a NOD and a NODP domains which only exist in Notch family. The homology and phylogenetic analysis showed that the amino acid sequence similarity between R.esculentum Notch and Cnidaria Nematostella vectensis amino acid was 39%; by contrast, compared with invertebrates and vertebrates, R. esculentum shares 35%-37% and 34%-38% respectively, in the similarity of the amino acid. Phylogenetic tree of the Notch based on neighbor-joining method showed that R.esculentum was clustered with Cnidaria N. vectensis. Quantitative real-time PCR analysis revealed that the expression of Notch transcript was detected in all four developmental stages, with the highest level in scyphistoma stage and the lowest in strobili stage. The expression level of scyphistoma stage was 1.85 -fold of that in strobili. The results indicate that Notch gene is involved in the regulation of asexual reproduction of R.esculentum.

Abstract:This study investigated the resistance and related physiological changes in response to high water temperature of the new breed (Gaokang No.1) sea cucumber Apostichopus japonicus for a period of 3 years, using ecological, histological and digestive enzyme assays. The results showed that for year 2013, 2014 and 2015, “Gaokang No.1” sea cucumber entered into aestivation at the aestivation temperature (AT) of 29.13℃, 30.47℃ and 29.68℃, respectively, and finished aestivation at the released aestivation temperature (RAT) of 24.55℃, 24.94℃ and 24.16℃, correspondingly. While the AT of the sea cucumber in the non-selected group was 27.08℃, 28.61℃ and 27.93℃, and the RAT was 21.33℃, 21.83℃ and 22.06℃, respectively. Compared with the non-selected sea cucumber, the aestivation critical temperature (ACT) of “Gaokang No.1” breed increased by 1.89℃, and aestivation lifted critical temperature (ALCT) increased by 2.81℃. Hence, the average aestivation period was shortened about 26.7 days a year. The intestine of sea cucumber was severely degraded after aestivation, with characteristics of atrophy, reduced length and width, dark color, and no food in the intestine. Also the intestinal chorionic abundance decreased and the intestinal ridge height was reduced to about 140 μm. The respiratory tree became atrophy in size. By comparison, when the aestivation released, sea cucumber restored to the normal intestinal length; the intestinal chorionic abundance increased significantly and the intestinal ridge height increased to about 640–660 μm. In addition, the amylase and trypsin activities tended to decrease as the water temperature increased for both “Gaokang No.1” breed and the non-selested sea cucumber. Both enzyme activities increased significantly as the aestivation released. However, the enzyme activities were higher among “Gaokang No.1” breed compared to the non-selested sea cucumber under the same rearing temperature. Compared to the non-selested sea cucumber, the “Gaokang No.1” breed can tolerate high temperature, therefore extended the production period for nearly a month. The results indicated the “Gaokang No.1” sea cucumber is a better breed in terms of high temperature resistance and shows improved survival rate and productivity. The study provides a technical reference to health management and seed production in sea cucumber culture.

Abstract:Selenium is an important trace element to maintain the normal physiological function of an organism. It plays important roles in regulating the balance of redox environment and improving the immunity. Appropriate amount of exogenous selenium can enhance the immune function of aquatic animals, improve ability to resist disease, and rich selenium content in aquatic product. In current study, juvenile sea cucumber (Apostichopus japonicus), with body weight of (60.75±0.32) g, were cultured in tanks of each 150 cm × 120 cm × 60 cm for 4 weeks with water temperature of 16-18℃. They were fed diets containing selenium-enriched yeast at the dose of 0, 0.20 mg/kg, 0.40 mg/kg, 0.80 mg/kg, 1.60 mg/kg, 3.20 mg/kg and 6.40 mg/kg. Survival rate was determined and different enzyme activities were assayed in order to evaluate the influence of different levels of selenium inclusion on biological indices related to growth, digestion and immunity. Selenium enrichment was detected by hydride generation-atomic fluorescence spectrometry (HG-AFS) in sea cucumber. The results showed that the survival of sea cucumber (up to 100%) and activities of digestive and immune enzymes were improved by appropriate concentration of selenium supplementation with the maximum activities of protease (35.13 U/mg prot) and amylase (0.51 U/mg prot), 2.45 and 2.07 times higher than the control group. The maximum activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), alkaline phosphatase (AKP) and acid phosphatase (ACP) were observed in the group fed the diet containing selenium concentration of 0.80-1.60 mg/kg diet, and they were significantly higher than those in the other groups. Furthermore, selenium contents in the sea cucumber tissues were increased at different levels. The organic selenium concentration was several times higher than inorganic selenium, indicating that enrichment of organic selenium is the main way into the body. In conclusion, the diet of selenium concentration from 0.80 mg/kg to 1.60 mg/kg fed in sea cucumber was the most appropriate amount. This study enriches the nutrition research of sea cucumber and provides a frame reference for sea cucumber’s healthy, efficient and sustainable breeding.

Abstract:In recent years, with the expansion of sea cucumber (Apostichopus japonicus Selenka) aquaculture, the demand for seaweed as its diet is increasing, resulting in the continuous increase in the price of macroalgae, i.e. sargassum. Therefore, researchers are seeking for an alternative plant based diet with large quantity and relatively low cost that can feed sea cucumber. In this study, formulated diets containing tuber or stem powder of sweet potato with 6 different concentrations (10%, 20%, 30%, 40%, 50%, 60%) were fed to sea cucumbers [weight (25.6±1.4) g] to study the effects of two feedstuffs on growth and non-specific immune enzyme activities of sea cucumber. The basal diet (16% crude protein and 5% crude lipid) was used as control. The results showed that during a 50-days experiment, the average body weight of sea cucumber fed with tuber powder of sweet potato increased as time went on. At the end of the experiment, 20% of tuber powder inclusion significantly improved the specific growth rate (SGR) compared with the control (P<0.05), and the SGR of 10% and 30% tuber powder inclusion was not significantly different from the control (P>0.05). By contrast, the SGR of sea cucumber fed with stem powder of sweet potato in 10% group was not significantly different from the control (P>0.05), while other groups were significantly lower than the control (P<0.05). The catalase(CAT), acid phosphatase(ACP) and alkaline phosphatase(AKP) of sea cucumber fed with tuber powder of sweet potato of 10% and 20% groups were significantly higher than the control (P<0.05). The ACP of sea cucumber fed with stem powder of sweet potato of 10% group was significantly higher than the control (P<0.05), while the lysozyme(LZM), CAT, AKP and total superoxide dismutase(T-SOD) of this group was not significantly different from the control (P>0.05). These results suggested that 30% tuber powder or 20% stem powder (or less) could improve the growth and non-specific immune enzyme activities of sea cucumber.

Abstract:With the development of modern agriculture, pesticide is extensively used in agricultural production. The herbicide as an important part of pesticide has also been widely used, causing significant environmental and food safety concerns. Currently, effect of herbicide contamination on the aquatic food product is not well understood. Furthermore, detection of herbicide residues in aquatic products is lack of efficient multi-residue detection method. This study established a reliable method for determining 16 herbicide residues in animal origin aquatic products. The method involves gel permeation chromatography- solid phase and extraction-gas chromatography-mass spectrometry. The sample was extracted using dichloromethane and ethyl acetate mixed solution, followed by a clean-up step using gel permeation chromatography. The product was then purified by solid phase extraction column. Results were determined by monitoring selected ion and quantified by external marker method. Good linearity was observed in the range of 4-200 μg/L in pendimethalin and cyanizine, whereas other herbicides showed good linearity in the range of 2-100 μg/L, with correlation coefficient higher than 0.9970 in both cases. The quantitation limit for propazine, atrazine, terbutylazine, simazine, desmetryn, prometryn, ametryn and metolachlor was 1.0 μg/kg. The quantitation limit for acetochlor, alachlor, butachlor, pretilachlor, cyprazine and simetryn was 1.5 μg/kg, and the quantitation limit for pendimethalin and cyanizine was 2.0 μg/kg. Average recovery rates ranged in 67.2%-103.0% and relative standard deviation of the method was 5.1%-9.5%. The method had good reproducibility and high recovery rate. In terms of residue detection, the precision and accuracy can meet the testing requirements, and the method could be used for determining 16 herbicide residues in aquatic products. The established method was applied to detect herbicides in sea cucumber, oyster, scallop etc. The results showed that ametryn had the highest detection rate in these samples and followed by atrazine, terbutylazine, simetryn and acetochlor. This study established the method for simultaneous detection of herbicide residues in aquatic products and built the foundation for further study of the impact of herbicide pollution on the quality and safety of aquatic products.

Abstract:The effect of careshell on growth and survival of Scapharca broughtonii was investigated in Sanggou Bay from April 6 to October 26,2015. S. broughtonii with initial mean weight of 160 g were exposed to the environment with four doses of Careshell: 0 (control, C0), 80 g(C1), 160 g(C2) and 320 g(C3) respectively. The growth indices (shell length, shell width, shell height, wet weight) of S. broughtonii were measured and the number of deaths recorded in every 40-day period. The experiment lasted 200 days. Growth and mortality rates were calculated in each period. The results showed that the growth indices of S. broughtonii for C3 group were significantly higher than those of the control group C0 (P<0.05), but the difference with C2 and C1 was not significant (P>0.05). The growth rates of shell length, shell width, shell height and wet weight for C3 during the experimental period were, (0.34±0.01)%/d, (0.46±0.02)%/d, (0.39±0.01)%/d and (1.11±0.002)%/d, respectively, which were significantly higher than those of C0 (P<0.01). On the average, the indices during the experimental period were increased by 17.9%, 20.3%, 18.1% and 16.7% respectively, which were significantly higher than those of C0 (P<0.01). The mortality rate of C3 group (19.6±5.6)% was lower than that of the control group C0 (26.1±2.5)%, but the difference was not significant (P>0.05)

Abstract:n teleost, thyroid hormones (THs) regulate growth, development, and reproduction, under the modulation of the hypothalamus-pituitary-thyroid (HPT) axis. Recent research has implicated that multiple endocrine disturbing chemicals (EDCs) in the environment can interfere with the HPT axis of teleost through different pathways. In fish, the corticotropin-releasing hormone (CRH) produced in the hypothalamus stimulates the synthesis and secretion of the thyroid stimulating hormone (TSH) in the pituitary, which further increases the synthesis and secretion of extrathyroidal L-thyroxine (T4) in the thyroid. T4 binds to the transthyretin (TTR) in the plasma and is transported to the peripheral tissue. 3,3¢,5-triiodo-L-thyronine (T3), the most active thyroid hormone, is formed by T4 deiodination, and acts mainly by binding to the specific nuclear thyroid hormone receptors (TRs). On the basis of introducing the regulatory mechanisms of the HPT axis and the dynamic changes of THs in teleost, this study summarized the commonly-used indexes indicating the interference effects of EDCs on the HPT axis, in the aspects related to the synthesis, transportation, transformation, metabolism, and clearance of THs. These indexes involve the histological structure of thyroid, mRNA expressions of crh gene in the hypothalamus and tshβ in the pituitary, mRNA expressions of nis, tg, tpo genes in the thyroid, TTR levels in the plasma, iodothyronine deiodinases levels and mRNA expressions of ugt1ab and sult1st5 genes in the peripheral tissues, TRs levels in the target tissue and THs levels in the plasma or the whole body homogenesis of fish. Furthermore, research emphasis and directions in this field include the development of biomarkers for the HPT axis, the selection of exposure time and dose of EDCs for in vivo tests, the establishment of evaluation systems combining both in vivo and in vitro tests, and the integration of indicators at both the microscopic and macroscopic levels, are also prospected in this study to provide more theoretical references for related investigation.