Abstract:Flavor components of low-salt lactic acid fermented fish and traditional salted fish were compared to provide a theoretical basis for the new processing technology.The volatile compounds in the fresh fish(Otolithes ruber) with low-salt lactic acid fermentation method and traditional salted method were extracted by solid phase microextraction(SPME) and further analyzed and identified by gas chromatography-mass spectroscopy(GC-MS).The results showed that there were 80, 110 and 91 kinds of volatile compounds detected in the fresh fish, low-salt fermented dried fish and traditional salted-dried fish respectively.The contents of major volatile compounds in dry-salted fish products were aldehydes, alcohols and hydrocarbons.The major kinds of volatile components were only 17 and 21 in fresh fish and traditional salted-dried fish respectively, but there are 35 kinds in low-salt fermented dried fish.The volatile flavor components such as alcohols, aldehydes were increased a lot in low-salt fermented dried fish.The contents of amine substance in low-salt fermented did not exist.Not only the traditional salted fish flavor, but also fruity, floral, and winey flavors were increased significantly in low-salt fermented dried fish.The preparation for cured fish in low salt combined with compound lactic acid bacteria method, can not only shorten the curing time, but also enhance the flavor, prevent the amine production, and improve product quality and safety.

Abstract:After closed fishing had been implemented in the main river systems in China, it was carried out for the first time in Pearl River on April 1st, 2011.Guangdong black bream(Megalobrama hoffmanni) was one of the important economic fishes.For analyzing the effect of closed fishing on Megalobrama hoffmanni recruitment stock, the fish larvae were sampled by Jiang net at Zhaoqing section in lower reaches of Pearl River before(2006—2010) and after(2011—2012) closed fishing.The results showed that Megalobrama hoffmanni larvae usually occured from mid-April to mid-October, and duration was(189±11) days before closed fishing.And the peak density of Megalobrama hoffmanni larvae was from June to Auguest every year.After closed fishing, the starting time of Megalobrama hoffmanni larvae was without any major changes, but the end time was sooner than before closed fishing, and duration was a little shortened.The drift density of Megalobrama hoffmanni larvae increased obviously within the banned fishing periods from April to May, and the biomass and the relative abundance also rose greatly.For analyzing the effect of closed fishing on Megalobrama hoffmanni recruitment stock, the regression model was built, and the logarithmic equation was LgY=0.988Lnx-4.932(R²=0.365, P<0.01), and regression effect was significant.Based on the logarithmic equation, the fitted value of Megalobrama hoffmanni larvae was calculated, and the closed fishing effect was estimated combining actual monitored value at Zhaoqing sampling station.The increment of Megalobrama hoffmanni laevae caused by closed fishing was 9.43×10⁸ ind. in 2011 and 651.81×10⁸ ind. in 2012 respectively.The result indicates the present regime of closed fishing can effectively increase the Megalobrama hoffmanni recruitment stock based on the abundant discharge.

Abstract:Seaweed farming is an important form of carbon sink fisheries, which plays a significant role in ecological functions of carbon sink.In this paper, the yields and structures of Chinese and Zhejiang coastal algae farming were analyzed, and their carbon sink capacity was also estimated, based on the data of "Chinese Fishery Statistical Yearbook" from 1999 to 2012.The average annual production of algae in China was about 1 418 700 t.Chinese main marine algae were kelp, undaria, laver and gracilaria, which accounted for 60.29%, 7.92%, 5.67% and 5.39% respectively.Zhejiang's main marine algae were laver, kelp, Sargassum fusiforme and Enteromorpha prolifra, and their proportion was about 51.83%(laver), 27.73%(kelp), 12.72%(Sargassum fusiforme), and 1.27%(Enteromorpha prolifra).Although the portion of Zhejiang's marine algae production was only 2.55% out of total national production, the breeding structure was unique.In comparing with national data, certain kinds of Zhejiang's algae showed a higher yield, which had percentages of 83.00%(Enteromorpha prolifra), 68.29%(Sargassum fusiforme), and 23.53%(laver).Overall, Chinese marine algae carbon sink capacity demonstrates upward trend from 1999 to 2012.The highest carbon sink was up to 515 000 t in 2012, while the annual carbon sink capacity was 418 500 t.The annual average carbon sink of Chinese kelp was 264 500 t/a, which took up the highest proportion within all kinds of algae, followed by undaria 32 300 t/a, laver 22 400 t/a, and gracilaria 20 100 t/a.The annual algae carbon sink capacity of Zhejiang marine culture was 10 300 t, 2.47% of the corresponding national data.In order to meet the needs of the development of the low carbon economy, we suggest that natural carbon sink and its environment protection and management should be strengthened, and carbon fisheries should also be developed.In addition, technology and industry demonstration projects on carbon sink fisheries, marine biological carbon sink function and more potential of carbon sink fisheries basic scientific research should be carried out.

Abstract:The distant hybridization of mollusk originated from oyster with 130 years' history.Moreover, both the prospects and problems for interspecific hybridization of oyster were summarized on the basis of the numerous attempts and recent research results.Generally, interspecific hybridization can generate transgressive hybrid phenotypes, which have extreme trait values exceeding the combined range of the parental species.Such as increasing growth rate, increasing harvest ability, increasing environmental tolerances, and increasing overall hardiness in culture conditions.Numerous attempts at hybridization in the genus Crassostrea have been reported, most of them suffer from several problems.In this review, the authors summarized up the past of works and pointed out the new aspects as follows:① Confirmation the taxonom of oyster;② Gametic compatibility:including one direction or two directions in fertilization and fertilized strength;③ Evaluating the phenotypic character;④ Genetic identification for hybrids;⑤ Heterosis examination;⑥ Analysis of the patterns of hybrid sterility;⑦ Alloploidy induction;⑧ Interspecific hybridization;⑨ Questions and prospects.Finally, we must indicate that the opinion of unviable hybrid larvae was inaccurate, and a lager number of hybrids were able to obtain in certain environments.Key issues in the interspecific hybridization required to carefully consider and carry out it in the genus Crassostrea.Trustingly, interspecific hybridization will be a good tool to improve phenotypic traits with modifying genes from two parents to the hybrids;and these were made as the excellent research materials for the molecular, cellular and evolutionary biology.

Abstract:To investigate the feeding characteristics of Oxyrrhis marina, Cyanobium sp.and Pleurochrysis dentata were used as food algae to culture O.marina respectively.The feeding processes, feeding rules and the color changes of cluture media of O.marina on two marine microalgae were analysed by the combined methods of microscopic examination, macro-observation and microscopic counting.The results showed that the feeding method of O.marina on Cyanobium sp.was wrapping the trichome sections slowly into the longitudinal groove, while the whole cell of P.dentata was incorporated into the longitudinal groove at the same time.The feeding rates of O.marina on two marine microalgae were 100% after inoculated by O.marina for 6 h, respectively.In static culture of O.marina, the color of culture media appeared pale pink to pink, whereas shaking culture media were not found.With the decrease of the initial density of O.marina(the cell density in Cyanobium sp.culture medium is 0.8×10⁴, 2.4×10³ and 0.8×10³ cells/mL repectively), the time when the population of O.marina reached the stationary phase required longer, namely, 3, 4 and 6 d respectively in culture media of Cyanobium sp., and the time when the population of Cyanobium sp.were fed up by O.marina required longer too, namely, 5, 10 and 15 d respectively.Also, with the decrease of the initial density of O.marina(the cell density in P.dentata culture medium was 3.9×10³, 2.4×10³ and 1.7×10³ cells/mL repectively), the time when the population of O.marina reached the stationary phase required longer, namely, 4, 5 and 12 d respectively in culture media of P.dentata, and the time when the population of P.dentata were fed up by O.marina required longer too, namely, 6, 7 and 13 d respectively.The experiment results showed that the feeding processes of O.marina on Cyanobium sp.and P.dentata were different for their different sizes and types and the color changes of culture media were affected by culture modes.Also the feeding of O.marina was affected by the kind of food alage and the initial concentration ratio of food alage to O.marina.During 15 day's culture period, Cyanobium sp.populations and P.dentata populations evolved to O.marina populations.

Abstract:The histology of gonadal development of Osteoglossum bicirrhosum which is a popular ornamental fish was studied by paraffin section technique and H.E staining method.The morphological characteristics and structures of ovary at stage Ⅱ-Ⅴ and testis at stage Ⅱ-Ⅵ were described.Moreover, the development of oocyte and spermatid were also described.The results showed that four months old ovaries were in stage Ⅱ, while the oocytes were in phase Ⅱ, and the nucleus occupied the half part of the cell.Eight months to two years old ovaries were in stage Ⅲ, most of the oocytes were in phase Ⅲ and egg macular and yolk granules could be observed;two to three years old ovaries were in stage Ⅳ, with the oocytes in phase Ⅳ which had yolk substance accumulated quickly and oolemma drape appeared.The ovary in stage Ⅴ was three years or more than three years old, and the oocytes in phase Ⅴ were full of yolk granules with the drape of oolemma disappeared.The primary spermatocyte in testis could be seen at the stage Ⅱ.Eight months to two years old testes were in stage Ⅲ, while the secondary spermatocyte appeared and the seminiferous tubules took shapes, and spermatids are full of the seminiferous tubules, primary spermatocyte, secondary spermatocyte and spermatids were seen at the stage Ⅳ, while the seminiferous tubules of void appeared.The testis in stage Ⅴ was three years old and more than three years old, the spermatozoa appeared and the void of seminiferous tubules became large.The testis highly vascularized and with connective tissue increased was also observed which was in stage Ⅵ.This study will provide the basic data for the artificial reproduction of O.bicirrhosum.

Abstract:In order to understand the muscle nutritional value and texture profile of I.punctatus reared in ponds and reservoir cages, the nutritional composition, the water holding capacity and texture profile of muscle in I.punctatus were investigated.The crude protein, crude fat, crude ash, and moisture of muscle in I.punctatus were assayed by biochemical analysis methods.The muscular contents of mineral elements were measured by inductively coupled plasma-atomic emission spectrometry(ICP-AES).The parameters of muscular texture profile, including hardness, springiness, cohesiveness, gumminess, chewiness, and resilience, were analyzed using a texture analyzer.Double compression was applied to construct the texture profile analyses(TPA) of raw fillets.The results showed that the rate of flesh content in I.punctatus reared in either pond or reservoir group was more than 65%.Flesh leaching loss increased significantly in the pond group(3.96%) compared with those in the reservoir group(3.35%).Compared to the I.punctatus in the pond group(1.73% and 17.19%, respectively), a significantly lower value in either drip loss or liquid loss was observed in the I.punctatus in the reservoir group(2.37% and 27.16%, respectively).The I.punctatus exhibited higher contents of crude protein and crude ash in the reservoir group(18.78% and 1.03%, respectively) than those of the pond group(16.69% and 0.90%, respectively).A significantly higher crude fat(3.90%) was found in the I.punctatus in the pond group.The I.punctatus reared either in pond or in reservoir had rich mineral elements content in muscle.Compared to the pond group(3 184, 1 787, 78.8, 246, 4.19 and 3.58 mg/kg, respectively), a significantly higher content of potassium(K), phosphorus(P), calcium(Ca), magnesium(Mg), zinc(Zn), and iron(Fe) was observed in the reservoir group(3 951, 2 325, 110, 312, 5.15 and 4.94 mg/kg, respectively).However, there were no significant differences in sodium(Na), copper(Cu), manganese(Mn), and chromium(Cr) between the pond group and the reservoir group.No selenium(Se) was detected in fish muscle of both groups.The calcium-phosphorus ratios of muscle in the pond group and the reservoir group were 1:22.6 and 1:21.1, respectively.The hardness, gumminess, and chewiness of muscle enhanced significantly in the I.punctatus in the reservoir group(2 721, 1 209 and 397 g, respectively) compared with those in the pond group(4 552, 1 738 and 578 g, respectively).The pond group(0.45 and 0.31, respectively) had significant higher resilience and cohesiveness than those of the reservoir group(0.38 and 0.24, respectively).Given the results, the muscle of I.punctatus showed higher water holding capacity in the pond group, whereas the I.punctatus muscle in the reservoir group contained higher protein, lower fat, richer mineral elements content, and higher hardness.

Abstract:A feeding trial was conducted to evaluate the effects of dietary methionine levels on growth performance, body composition of GIFT(Oreochromis niloticus) [ initial body weight of(66.76±2.29) g].Six isonitrogenous(32.09% crude protein) and isoenergetic(17.82 kJ/g gross energy) semipurified diets were formulated with the graded methionine levels(0, 0.3%, 0.6%, 0.9%, 1.2% and 1.5% dry diet, respectively), using gelatin, fishmeal, peanut meal and crystalline amino acids mixtures as the main dietary protein sources.Amino acid pattern in diet is to simulate the amino acid pattern found in the whole body protein of GIFT except for methionine.The six trial diets were determined to contain methionine of 0.26%, 0.55%, 0.85%, 1.14%, 1.44% and 1.73% dry diet, respectively.Each diet was randomly assigned to triplicate groups of 25 fish and fed to apparent satiation by hand thrice daily(8:00, 13:00 and 16:00) for 60 days.The results showed that the weight gain rate(WGR), specific growth rate(SGR), protein efficiency ratio(PER) and protein deposition rate(PDR) initially increased with increasing dietary methionine levels, but then decreased.Conversely, the feed conversation ratio(FCR) first decreased and then increased.The WGR, SGR, PER were all the highest(361.91%, 2.73%/d and 2.53% respectively) when the dietary methionine level was 1.14%.At the same time, the FCR was the lowest(1.23).The PDR was the highest(47.22%) when the dietary methionine level was 1.44%.The hepatosomatic index(HSI) and viscerosomatic index(VSI) of GIFT first decreased with increasing dietary methionine levels, but then increased(P<0.05), yet the condition factor(CF) had no significant differences(P>0.05).The tendency of crude protein content in muscle was similar to WGR and SGR with increasing dietary methionine levels(P<0.05), while the crude lipid in muscle first increased with increasing dietary methionine levels and then leveled off.And there were no significant differences of crude protein in whole body, moisture and the ash in whole body and muscle(P>0.05).With the increase of dietary methionine level, the histidine, serine and cystine in muscle had no significant differences(P>0.05), at the same time, the all other amino acids contents, gross of essential amino acid(ΣEAA), gross of total amino acid(ΣTAA) in muscle first decreased, then increased.With the WGR, SGR, PER, PDR, FCR as evaluation indicators, quadratic regression analysis showed that the optimum dietary methionine level of GIFT was 1.13%-1.16% of diet(dry matter basis) or 3.52%-3.61% dietary protein with cystine level at 0.30%.

Abstract:The experiment was conducted to determine the effect of dietary carbohydrate levels(CBH) and starvation on the growth performance, biochemical indices in plasma and liver/muscle glycogen content of the grouper.Total 300 fish were randomly distributed into 5 treatments with triplicate groups of 20 fish(initial weight:35±0.28 g) in each treatment.Three isonitrogenous and isoenergetic diets were formulated containing three carbohydrate levels(35% C_H, 21% C_M, 7% C_L), combined with starvation group(S) and refeeding group(R, starving 4W+refeeding 4W, 21% carbohydrate in the diet).Experiment lasted 8W.14 fish in each treatment were selected and injected with 300 mg glucose per 100 g body weight for glucose tolerance test(GTT) after the end of experiment and blood sampling at 0, 1, 3, 6, 12, 24, 48 h.The results showed that the weight gain and specific growth rate increased with dietary carbohydrate level increase but no significant difference(P>0.05), and the feed coefficient rate of C_L group was the highest(P<0.05).Plasma glucose levels significantly increased in 1-3 h and then decreased in C_H, C_M and C_L(P<0.05).Plasma glucose levels in C_M returned to the initial values within 6 h after being injected and initial level of plasma glucose content in C_H and C_L were obtained within 12 h.Plasma insulin content in C_H, C_M and C_L decreased at 1 h and then increased, but insulin level of C_L group significantly decreased at 3 h(P<0.05).Glycogen content in liver and plasma triglyceride levels were significantly increased in C_H, C_M and C_L within 1 h after injection(P<0.05).C_M, S and R groups presented persistent hyperglycemia after being injected and C_M restored initial level quickly within 6 h.Plasma insulin levels of C_M and R groups significantly decreased within 1 h(P<0.05), but that of R group continued to rise within 12 h.Plasma triglyceride levels of C_M were higher than those of S and R groups within 0 to 6 h(P<0.05).The results of GTT indicate that proper carbohydrate level in diet(21%) and continuous feeding can significantly improve glucose tolerance of the grouper.

Abstract:Fish models of intestinal inflammation are still fairly scarce though dozens of such models have been established in other animals.In this study, we aimed to develop a TNBS-induced enteritis model in grass carp.To induce the intestinal inflammation, 0.25 mL TNBS/ethanol solution(at a concentration of 2.5% TNBS in 50% ethanol) was instilled into the intestine of each fish(70±5 g) via the anus at a water temperature of 28°C.An equal volume of 0.7% physiological saline was instilled as control.After TNBS enema, the intestinal inflammation was assessed by examining the degree of body injury, and the intestinal histopathological characteristics, and by analyzing myeloperoxidase(MPO) activities and gene expression patterns of inflammatory cytokines, IL-1β, IL-8 and TNF-α, in different intestinal segments.The pathological results indicated that all fish survived during the whole observation period following TNBS treatment, and one day after TNBS enema, serious congestion and fester in intestinal mucosa were observed, and abundant inflammatory cells infiltrated into intestinal tissues.On the third day, severe abdominal dropsy occurred, large amounts of inflammatory cells were recruited at the damaged site, and healing of intestinal ulcers appeared.On the seventh day, the number of inflammatory cells decreased gradually, while that of goblet cells increased.Afterwards the abdominal dropsy reduced, the goblet cells densely arranged in the intestinal mucus layer.On the twenty-first day after TNBS enema, the fish recovered nearly into normal conditions, and only a few inflammatory cells could be found.In addition, our results showed that TNBS administration induced similar changes in MPO activities between different intestinal segments.MPO activities in the intestine remarkably increased after TNBS treatment, peaked on the first day.Subsequently, the activities decreased rapidly.On the seventh day, there was no significant difference between the TNBS-induced and the control groups.This pattern in MPO activity appeared to be consistent with those results obtained from the histopathological observations.Moreover, we also found that, after TNBS enema, the mRNA expression patterns of inflammatory cytokine genes, IL-1β, IL-8 and TNF-α, showed roughly the same trend as those of MPO activities.Our results also showed that different intestinal segments exhibited significant differences in inflammatory response, and TNBS induced more severe inflammatory signs in the 3^rd and 4^th segments than the 1^st and 2^nd ones.Overall, these results suggest that we have developed a reliable and reproducible TNBS-induced enteritis model in grass carp.This grass carp model will not only extend our understanding in pathological mechanisms involved in intestinal inflammation of teleost fish, but also provide a tool to screen novel drugs for the treatment of inflammatory diseases in grass carp and other aquacultural animals.

Abstract:The differences between female and male turbot in morphology and growth were compared using total length/body length(TL/BL), body depth/body length(BD/BL) and total length/body depth(TL/BD) as well as body weight.The comparison results of morphology showed that although TL/BL, BD/BL and TL/BD of the two groups presented respective dynamic changes at different growth stages, the differences of the three proportion indexes did not exist or were not significant except that the differences of BD/BL and TL/BD of 33 months of age were significant, namely, TL/BL, BD/BL and TL/BD between female and male groups were fluctuating simultaneously with time series of growth when the initial weight of the two groups were same or similar.The comparison results of the two groups, growth showed that female groups showed later starting point(14.7), inflection point(24.0) and ending point(33.2), heavier weight at inflection(1 443.56 g) and maximum month gain(103.10 g), longer fast-growing period time interval(18.4 months) and fast-growing body weight intervals(1 666.88 g) and heavier month gain(90.39 g) during fast-growing period than those of male groups.Furthermore, the instantaneous growth rates of female groups was higher than those of male group during months 13.1-33.0.The differences of instantaneous growth rates of the two groups showed an increasing trend with accretion of body weight differences during development.The findings suggest that the differences of the two groups in growth are significant and the differences in morphology are not significant.

Abstract:In order to establish TaqMan Real-Time PCR for detection of GCRV genotype Ⅰ, the primers were designed according to conservative region of S6 as follows:normal primers designed as P₁ and P₂ with predicted product size of 661 bp, real time primers designed as F₁ and F₂ with predicted product size of 159 bp, and a probe.Standard curve(Y=-3.389X+38.076) was generated between the cycle threshold(C_t) and standard plasmid(PVAX1-S6) with 10-fold serial dilutions.The results showed that there was a good linear relationship between C_t value and template concentration with a detection range from 3.9×10⁷ to 3.9×10¹ copies/μL, and the correlation coefficient reached 0.992 while the detection limit of this method was 4 copies/μL for plasmid template.This assay had a specific detection of the GCRV genotype Ⅰ, and had no detection signals to GCRV genotype Ⅱ, Ⅲ and other pathogens.16 suspected grass carp hemorrhage specimens were tested, and 2 more positive samples were detected using this established assay than normal RT-PCR.The developed TaqMan Real-Time PCR detection method for the GCRV genotype Ⅰ with high efficiency, specificity, sensitivity and repeatability is available for clinical rapid detection and quantitative analysis of the GCRV genotype Ⅰ.

Abstract:In this study, three multiplex PCR panels were established using microsatellite markers developed from our laboratory by optimizing annealing temperature, concentration of primers and times of cycle.A breeding group "JD-01" was analyzed using the three multiplex PCR panels.The results showed:the average number of alleles and effective number of alleles were 12.111 and 7.408 respectively.The average of polymorphism information content(PIC) were 0.846.The average of observed heterozygosity(H_o) and expected heterozygosity(H_e) was 0.825 and 0.868, respectively.The Shannon's diversity index was 2.165.By using Cervus 3.0 software, we verified parentage assignment that the pedigree relationship was known in 9 families of large yellow croaker.As a result, the accuracy rate of parentage assignment was 100% by using three multiple PCR panels.The development of microsatellite multiple PCR system provides a high efficiency technical method for genetic diversity analysis, parentage assignment and marker-assisted family management in large yellow croaker.

Abstract:Mitochondrial D-loop sequences were analyzed to study the genetic structure of the sea cucumber Apostichopus japonicus.In this study, 71 individuals from three natural populations were collected from Dalian, China(CD), Rajin, North Korea(KN) and Vladivostok, Russia(RV).447-465 bp D-loop sequences were obtained using PCR amplification.The mean content of A+T(59.2%) was higher than G+C.The proportion of polymorphic sites was 24.24%, in which parsimony informative sites were 53.Forty haplotypes were identified from 71 samples, among which 7 haplotypes were shared by three populations.Genetic diversity parameters showed high genetic diversity among populations.The fixation indices(F_ST) of analysis of molecular variance(AMOVA) within populations was 93.04%, which indicated that the genetic differentiation of the three populations was weak or only moderately differentiated.The genetic distances among populations were 0.037 4(KN and RV) and 0.042(CD and RV).The molecular phylogenetic tree was constructed with the D-loop sequences of Qingdao(QD) and Weihai(WH) from GenBank with NJ method using software MEGA 4.0.Populations of QD and WH clustered into a small branch, then with CD, while KN and RV clustered into the other branch.The results were consistent with their geographical isolation mode.

Abstract:In order to screen out lymphocystis disease resistance-related SSR markers in Chinese flounder population, 102 individuals were used(56 disease susceptible and 46 disease resistant individuals) in the present study.Firstly, two gene pools were constructed using 15 susceptible individuals and 15 resistant individuals, respectively.The two gene pools were scanned using 178 pairs of microsatellite primers.Secondly, the differential bands amplified in gene pools were verified using 30 individuals which were used to construct the gene pools for the first time.Lastly, a second verification for 102 individuals was performed to verify the difference significance of the differential bands identified in the first verification.Results of BSA analysis demonstrated that some differential bands were amplified using four pairs of primers(scaffold440_22585, scaffold826_5003, scaffold703_4284 and scaffold185_597).The first verification indicated that the differential bands are significantly different in the SSR markers scaffold826_5003(P=0.023) and scaffold185_597(P_178bp=0.028, P_173bp=0.009).And in the second verification, the difference was extremely significant in the totally 102 individuals with the SSR marker scaffold185_597, the frequency of differential bands in disease resistant individuals and disease susceptible individuals was 60.9% and 14.3%, respectively(P=0.001<0.01).The differential bands amplified from ten disease resistant individuals using primer scaffold185_597 were cloned and sequenced.Sequence alignment by BLAST confirmed that the bands were fragments of microsatellite marker scaffold185_597 published by lab for aquatic genomics and cell engineering in Yellow Sea Fisheries Research Institute, homology of which was up to 96%.The present study has shown that the microsatellite marker scaffold185_597 may be associated with lymphocystis disease resistance in Japanese flounder(Paralichthys olivaceus), providing some basis for the molecular marker-assisted selection in Chinese flounder.

Abstract:The effects of perfluorooctane sulfonate(PFOS) exposure on hepatic Cu/Zn-SOD and stress-related gene expression in swordtails(Xiphophorus helleri) were studied in order to screen the sensitive biomarkers induced oxidative stress in vivo.We cloned and analyzed the swordtail fish Cu/Zn-SOD cDNA of full sequence.Swordtails were randomly divided into five groups, with one control group and four experimental groups(3.5, 7.0, 14.0 and 28.0 mg/L PFOS).Parallel experimental groups were also established.The Cu/Zn-SOD, HSP70-1, HSP70-2 and GST mRNA expression levels in fish livers were determined after 24 h, 48 h, 96 h, and 14 d exposure.The X.helleri Cu/Zn-SOD cDNA was cloned, which had 794 bp nucleotides, encoding 154 amino acids.The comparison among the similarity nucleotide sequence and amino acid sequence of Cu/Zn-SOD in other related fishes showed that nucleotide and amino acid sequence similarity were 77%-83% and 79%-88%, respectively.After exposure to high concentrations of PFOS, a variety of stress-related genes in X.helleri liver changed in different degrees.The expression of Cu/Zn-SOD mRNA in the dose-effect was not significant by RT-PCR, and with no correlation between the SOD activity and the expression of Cu/Zn-SOD mRNA.It may be other types of SOD gene that play more important role under the PFOS exposure.With the exposure time to increase continuously, Cu/Zn-SOD gene changes were consistent with the SOD activity and the expression of HSP70-1 mRNA.Thus HSP70 by increasing the level of swordtail fish SOD protected the body from oxidative damage.HSP70-2 mRNA was more sensitive than the levels of HSP70-1 mRNA expression.The changes of HSP70-2 were not relevant with Cu/Zn-SOD mRNA expression.The underlying mechanism needed further study.GST mRNA expression was constantly increasing, which is related with the Nrf2 signal transduction pathways, and it may also be a form of defense in fish when PFOS entered the body.Swordtails in vivo experiments showed that PFOS could induce oxidative stress in the liver.The expression of GST mRNA and HSP70-2 mRNA could act as sensitive biomarkers of PFOS exposure.

Abstract:White spot syndrome virus(WSSV) caused serious disease and economic losses in the world, becoming a limiting factor in sustainable devolpment of shrimp industry.Shrimp cell culture is a simple and rapid tool to study the pathogenic mechanism of the virus and to determine the viral susceptibility to the cells.In this research, primary culture cell from lymphoid organ of greasyback shrimp(Metapenaeus ensis) has been established by using an improved 1.5×L-15 cell culture medium.The cell cytopathic effects(CPE) were observed, as the primary cell monolayer inoculated with WSSV.The results showed that the migration of lymphoid cells from the explants was initiated at 3 h after seeding, and a 80% confluent cell monolayer was formed within 24-36 h and remained viable for over 20 days.Within 24 h post inoculation, apparent CPE was observed in the primary culture cells.The infected cells initially exhibited shrinkage or became aggregated, while the network structure between the cells disappeared.Finally, the most infected cells rounded up and then detached from the culture dishes.Under fluorescence microscope, Hoechst 33342 staining showed that the nuclei of lymphoid cell enlarged and deformed with a deeper color, 48 h after inoculation with WSSV.Under electron microscope, a large number of clustered viral particles were founded in the affected nuclei, while the organelles were pushed to the cell edge, and the outlines of the cell membranes were vague.The study illustrated that the cultured lymphoid cells had apparent CPE as inoculated with WSSV.This phenomenon proved that WSSV could infect and multiply within the primary culture cells.

Abstract:Tyrosine-3-monooxygenase/Tryp-tophane-5-monooxygenase activator protein(14-3-3ζ protein) is one of the isoform of 14-3-3 proteins which are highly conserved small acidic proteins.Several studies have shown that 14-3-3 proteins can bind many signal proteins, such as kinase and phosphatase, and play crucial roles in many biological processes including regulation of signaling and cell apoptosis.In this study, the full length cDNA of 14-3-3ζ from Haliotis diversicolor was cloned for the first time by SMART-RACE and named Hd14-3-3ζ.Its full-length cDNA sequence was 3 010 bp, with an open reading frame of 819 bp encoding a protein of 272 amino acids.The Real-time Quantitative PCR results showed that Hd14-3-3ζ was detected in all examined tissues and with the highest expression level in hepatopancreas and haemocytes.When under the stress of hypoxia, the expression level of Hd14-3-3ζ in haemocytes of exposed groups showed a significant up-regulation at 4 h, but in gill it happened at 24 h and 96 h.Under thermal stress, in haemocytes, it showed no significant difference between control and thermal groups but it was up-regulated significantly at 96 h in gill.Under thermal & hypoxia stresses, it showed no significant difference between control and exposed groups in haemocytes.However, in gill, it was up-regulated significantly at 4 h, 24 h and 96 h.After Vibrio parahaemolyticus challenge, the expression of Hd14-3-3ζ reached a significant level at 6 h in haemocytes and reduced to the normal level at 12 h, and then reached the highest significant level at 24 h.In gill, the significant expression level appeared at 3 h and 24 h and there was no difference between other time phases.All these results indicate that Hd14-3-3ζ may play important roles in immunoreaction of H.diversicolor.

Abstract:In order to track the target bacteria effectively, fluorescein isothiocyanate(FITC) was used as the marker to study the effect of labeling concentrations(0.2, 2, 20, 50, 100, 200 μg/mL) and time(1, 15, 30, 60, 120, 240 min) on the fluorescence intensity of V.anguillarum labeled FITC.In the meanwhile, the stability of fluorescence labeled on V.anguillarum stored at different temperatures(25, 4, -20, -70 ℃) and different time(24, 48, 96, 192 h) were compared.The possible change of fluorescence intensity was monitored when V.anguillarum labeled FITC was inactivated by formaldehyde.Also the fluorescence quenching effects of different intensity natural light(0, 150, 1 500, 25 000 lx) indoors and the effects of repeated freezing and thawing at -20 and -70 ℃ on V.anguillarum labeled FITC were analyzed.The results showed that the optimal concentration of FITC was in the range of 20 to 100 μg/mL and the optimal time was in the range of 60 to 120 min.The fluorescent signal from V.anguillarum labeled FITC was more stable when it was stored at -20 and -70 ℃ than those stored at 4 and 25 ℃.The stability of fluorescent signal from formaldehyde inactivated V.anguillarum labeled FITC was better than that from the control, and no difference was found in the quenching effect in different intensity of natural lighting.After 3 times of freezing and thawing, the fluorescent signal from V.anguillarum labeled FITC frozen at -70 ℃ was more stable.The results above provided useful technical parameters when FITC was used as a marker to label the marine bacteria.