Abstract:To investigate the effect of major morphometric traits of selective group of Trachinotus ovatus at different month ages on body weight,2 635 specimens at 1-month-old stage,4-month-old stage,7-month-old stage,10-month-old stage and 13-month-old stage were measured respectively in terms of total length(x₁),body length(x₂),body height(x₃)and weight(y).Correlation coefficients,path coefficients and determination coefficients of each trait to body weight were calculated through path analysis and multiple linear regression.Effects of each morphometric trait on body weight were also dissected and then the optimal regression equation was established.The results showed that the correlations among 4 traits ranged from 0.017 to 0.960,and all reached significant level(P<0.01)at different stages except that at 4-month-old stage.At 1-month-old stage and 4-month-old stage,the trait with strongest direct effect on body weight was the total length,but that at other stages was the body height.The result of determinant coefficient analysis was consistent with that of path analysis,and the fitness of equations at different stages was good(86.9%-90.6%).It indicated that the major morphometric traits affecting body weight were found and traits for selection should be considered at different stages,which provided technical parameters for selective breeding for T.ovatus.

Abstract:To investigate the applicability of microsatellite markers developed from Megalobrama amblycephala in M.pellegrini and find the molecular markers to identify the M.amblycephala,M.pellegrini and their reciprocal hybrids,90 pairs of M.amblycephala EST-SSR primers were synthesized for cross-species amplification on the genome DNA of M.pellegrini,and 26 pairs of microsatellite primers of M.amblycephala and M.pellegrini were screened for PCR amplification on the genome DNA of M.amblycephala,M.pellegrini and their reciprocal hybrids.The results revealed that 76 pairs(84.4%)of the primers were amplified successfully.Fifty pairs were screened from these 76 primers to test the polymorphism in M.pellegrini population and 13 markers were polymorphic with an average of 3.2 alleles per locus,the mean observed heterozygosity being 0.72 and the mean PIC being 0.42.The results of PCR amplification on the genome DNA of M.amblycephala,M.pellegrini and their reciprocal hybrids indicated that 4 primer pairs had no amplified products on M.amblycephala,and 2 primer pairs had no amplified products on M.pellegrini.Among the 20 loci which had effective amplified products,9 specific loci could identify M.amblycephala,M.pellegrini and the reciprocal hybrids,respectively.Combination of the other 17 specific loci also distinguished M.amblycephala,M.pellegrini and the reciprocal hybrids effectively.However,the reciprocal hybrids［M.amblycephala(♀)×M.pellegrini(♂),M.pellegrini(♀)×M.amblycephala(♂)］could not be identified through these markers used in this study.

Abstract:To investigate the effects of docosahexaenoic acid(DHA)on the expression profiles of B cell CLL/lymphoma-2(Bcl-2)in grass carp(Ctenopharyngodon idella)different tissues and adipocyte in vitro,the rapid amplification of cDNA ends(RACE)technique was used to clone the full-length cDNA of Bcl-2 gene of grass carp,and the real-time PCR(qRT-PCR)was employed in the analysis of gene expression pattern in different tissues and in the in-vitro cultured adipocyte of grass carp.The results revealed that the full-length Bcl-2 cDNA is 1 292 bp and contains a 5′-untranslated region with 133 bp and 3′-untranslated region with 784 bp.The open reading frame(ORF)has 375 nucleotides encoding 124 amino acids.The deduced amino acid sequences of Bcl-2 of grass carp displayed higher similarities with Cyprinus carpio(89.5%)and Danio rerio(91.1%),but lower identity to the other animals with the similarity ranging from 57.7% to 62.0%.The molecular weight of encoded protein was predicted at 14.14 ku with pIat 4.68.TheBcl-2 gene is widely expressed in heart,hepatopancreas,spleen,gills,kidney,muscle,intrapaneal fat body(IPF),brain,intestine and spermary.The highest expression level was observed in IPF,kidney,spermary and the lowest expression level was observed in muscle.The expression of other tissues was between IPF and muscle.After DHA treatment,the Bcl-2 expression was downregulated in the proliferation phase,but upregulated in the differentiation phase.This suggested the Bcl-2 was expressed widely,and DHA could regulate the expression of Bcl-2 gene.

Abstract:Crustacean hyperglycemic hormone(CHH)is a peptide hormone originally identified in a crustacean neurosecretory complex,the X-organ/sinus gland complex,and located within the eyestalks.CHH is placed in the crustacean hyperglycemic hormone family,which also includes molt-inhibiting hormone(MIH),vitellogenesis-inhibiting hormone(VIH)or gonad-inhibiting hormone(GIH),mandibular organ-inhibiting hormone(MOIH)and insect ion transport peptide(ITP).CHH plays a significant role in the growth,molting and reproduction of crustaceans.By using degenerate primers which were designed according to the N-terminal amino acid sequences of CHH isolated by our lab and the sequences of homologous species CHH,a novel cDNA of crustacean hyperglycemic hormone gene(Ers-CHH,GenBank Accession No:JX485664)was cloned from Chinese mitten crab(Eriocheir sinensis).The full length cDNA consists of 585 bp with a 453 bp open reading frame,encoding 150 amino acids,containing a 30 amino acids signal peptide,and a 42 amino acids CHH precursor-related peptide,a 78 amino acids mature peptide.The mature peptide contains 6 conserved cysteines which formed three disulfide bonds C⁷-C⁴³、C²³-C³⁹、C²⁶-C⁵².There was an arthropod CHH/MIH/GIH neurohormones family signature in this domain.The three-dimensional structure prediction showed that Ers-CHH mature peptide consisted of 4 α-helixes,and not with the β-sheet structure.5 Prosite patterns,which were analyzed by a Swiss-PdbViewer(v4.0.4),are PS00005:Protein kinase C phosphorylation site(Ser⁶,Lys⁸);PS00006:Casein kinase Ⅱ phosphorylation site(Ser¹⁷,Glu²⁰);PS00008:N myristoylation site(Gly³⁸,Cys³⁹,Asn⁴²,Cys⁴³);PS00342:Microbodies C-terminal targeting signal(Ser¹⁷,Lys¹⁸,Leu¹⁹);PS01250:Arthropod CHH/MIH/GIH neurohormones family(Val³⁵,Cys³⁹,Arg⁴⁰,Asn⁴²,Cys⁴³,Tyr⁴⁴,Asn⁴⁶,Phe⁴⁹,Cys⁵²).The results of the structural model comparison show that the structure of Ers-CHH has high similarity with other CHHs and Ers-CHH.They have no β-sheet structure,except that CHH has 4α-helixes,MIH has 5α-helixes.Although α1 helix of MIH could not be found in CHHs,the position and the amino acid number of α2-α5 helixes in MIH molecular were same as the α1-α4 helixes in CHH.It might be able to interpret the function similarity between CHH and MIH.Multiple alignment results showed that Ers-CHH has the highest identity with Ptychognathus pusillus CHH(92%),and it also shared high identities with Neohelice granulata(86%)and Pachygrapsus marmoratus(72%).However,it showed lower identity with CHH from crayfishes,such as Nephrops norvegicus(49%)and Homarus gammarus(46%).Multiple alignments of Ers-CHH and MIH from other crustaceans showed that only six amino acid residues(Arg¹³,Asp²⁵,Asn²⁸,Arg³¹,Arg⁴⁰,Phe⁴⁹)in Ers-CHH sequence were same with MIH sequences.It may be helpful for further research on regulation mechanism of CHH in the process of crab growth and carbohydrate metabolism.

Abstract:Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyses methylation of farnesoic acid (FA) to produce isoprenoid methyl farnesoate (MF) at the final step of the biosynthetic pathway. As a sesquiterpenoid analogue of the insect juvenile hormone (JH), MF has been suggested to play a vital role in regulating crustacean growth and reproduction. In this study, the full-length FAMeT cDNA (GeneBank accession number: KC192659) of P. trituberculatus is cloned, it contains a 201bp 5-untranslated region(5-UTR), a 318bp 3-untranslated region (3-UTR) and a 825bp opening reading frame, which encodes 274 amino acid residues. Alignment of deduced amino acid sequence with FAMeT amino acid sequences of other crustaceans revealed that it shares the highest identity with P. Pelagicus among the identities ranged from 75% to 97%. The amino acid residues consist of two copies of CF (CPAMD8/FAMeT) domain, which are the hallmark domain of FAMeT and are present in all crustacean FAMeTs. Realtime PCR was used to quantify the relative expression level of FAMeT in different tissues and molting stages in P. trituberculatus. The results showed that FAMeT was expressed in various tissues. The mRNA level of FAMeT was highest in Taoracic ganglia, followed by gill and mandibular organ (MO). During the molting process, the expression of FAMeT in MO increased to the maximum at D1 stage, then gradually decreased to the minimum at D4 stage. The results suggest that FAMeT plays an important role on molting regulation in P. trituberculatus.

Abstract:The chromosomes of Pacific abalone,Haliotis discus hannai,were examined by C-banding and fluorescence in situ hybridization(FISH)with human major rDNA(18S-5.8S-28S)as probes.The chromosome set of Pacific abalone was composed of 7 pairs of metacentric chromosomes,8 pairs of submetacentric chromosomes,and 3 pairs of metacentric/submetacentric chromosomes.Stable centromeric C-bands were present on 8 pairs of chromosome.Inter metaphase polymorphic telomeric C-bands and heteromorphic pericentric C-bands were found on 5-7 pairs and 3 pairs of chromosomes,respectively.In addition,4 major rDNA loci were mapped onto the telomere of four pairs of chromosomes(2S,7S,12S and 18L).These results provided new markers and characteristics to distinguish chromosomes in the genome of Pacific abalone,and would serve as basic data for further researches on chromosome evolution in abalone.

Abstract:In order to evaluate the hazard of chlorpyrifos to the cultured shrimp Litopenaeus vannamei,the lethal effect of the shrimp infected with white spot syndrome virus(WSSV)under the chlorpyrifos stress was tested,while the quantity of WSSV in the gill tissue and acetylcholinesterase acticity in the muscle tissue were analyzed.The 96 h median lethal concentration(LC₅₀)of chlorpyrifos to the shrimp was determined.The results showed that the 96 h LC₅₀ is 0.758 μg/L(0.521-0.987 μg/L),and as time prolonged,the values of LC₅₀ significantly declined,which showed a positive concentration-response relationship.On this basis,the stress concentration of chlorpyrifos of 0.2 μg/L was determined.After the shrimp were exposed to 0.2 μg/L chlorpyrifos for 4 days,they were infected with WSSV by injection.It showed that the cumulate mortality of chlorpyrifos-WSSV treatment(83.33?Ee4.7%)was significantly higher than that of ethanol-WSSV group(40.00?Ee0.9%).Meanwhile,the amount of WSSV was also determined by Real time Relative PCR at 48,72 and 96 h post injection.WSSV quantity of chlorpyrifos-WSSV treatment was about 4 times that of ethanol-WSSV control at 72 h.WSSV quantity of chlorpyrifos-WSSV treatment was significantly increased at 96 h,which was 4.9 times that of chlorpyrifos-WSSV treatment at 72 h,and 5.9 times that of ethanol-WSSV treatment at 96 h.The acetylcholinesterase(AchE)activity in shrimp muscle tissue under chlorpyrifos stress was 20% lower than that in control group.This showed that the proliferation rate of WSSV in shrimp increased rapidly under chlorpyrifos stress,which raised the mortality rate of the shrimp.

Abstract:The epidemic pathogen of Fenneropenaeus chinensis caused by white spot syndrome virus(WSSV)have brought great loss of the F.chinensis cultivation,no effective technical measures could control the WSSV epidemic so far.Improving the germplasm resources and cultivating the new varieties of F.chinensis are better measures to deal with this disease.The correlation among F.chinensis traits is an important parameter,as a result,it is very important to choose an appropriate genetic evaluation method in the analysis of correlation among F.chinensis traits.After testing the growth and white spot syndrome virus(WSSV)resistance of 130 F.chinensis families,we collected the F.chinensis growth and resistance data,and entered the date into database of aquatic animal breeding analysis and management software.According to the results of statistics software analysis,using the comprehensive index method to estimate growth and WSSV resistance breeding value of each F.chinensis family,we chose the five families each of the largest and the smallest growth breededing value,five families each of largest and smallest WSSV resistance breeding value.We detected the WSSV load of these broodstocks,50 d prawn and 170 d prawn of 20 families.The mean WSSV load of broodstock,50 d prawn and 170 d prawn of 20 families were 0.190 8,0.286 6 and 0.232 9 copies/ng DNA,respectively,which showed no significant difference(P>0.05).The correlation coefficients between WSSV-resistance breeding value and WSSV load of broodstock,50 d prawn and 170 d prawn were 0.021,0.463 and 0.185,respectively;and those between growth breeding value and WSSV load of broodstock,50 d shrimp and 170 d shrimp were 0.033,0.048 and 0.019,respectively.The results show that the relationships among growth breeding value,WSSV-resistance breeding value and the WSSV load of shrimp were not significant(P>0.05).

Abstract:We developed a new method using ultrasound-assisted degradation under alkaline condition for preparation of solution squid ink melanin.The squid ink melanin was mixed with NaOH solvent in brown glass tubes.The melanin-solvent suspension was treated by ultrasound for 1 h and then divided into three different molecular weight(Mw)fractions by membrane separation(below 10 ku,among 10-50 ku and over 50 ku).The result showed that the mean diameter of the squid ink melanin was decreased from 17.34 μm.to 1.467 μm after ultrasound process.UV,IR and solid¹³C NMR spectra indicated that the basic structure of melanin fraction was still retained after ultrasound process.The fraction with Mw below 10 ku showed more severe degree of oxidation on active groups(DHI and DHICA)than those above 10 ku.Further in vitro analysis showed soluble melanin fractions obtained in 0.5 and 1 mol/L NaOH,with Mw above 10 ku which had greater characteristic absorption peak of -OH and -NH structure exhibited much higher antioxidant potency.The IC₅₀ of these fractions(among 19-80 μg)on scavenging O^-₂· is more efficient than carnosine(IC₅₀=355 μg/mL.),a commercialized antioxidant.And they(IC₅₀ among 115-180 μg/mL)are as efficient as carnosine(IC₅₀ =110 μg/mL)on scavenging ·OH in free radical scavenge assay.In conclusion,our results showed that soluble squid melanin is a natural pigment and a new free radical scavenger with a promising prospect.The excellent solubility can increase absorption and enhance utilization rate in vivo,providing a potential prospect for squid ink melanin product as a natural antioxidant.

Abstract:The Three Gorges Dam is the largest hydroelectric power station project in the world.After the impounding of the Three Gorges Reservoir(TGR),its hydrological characteristics(water depth,water flow velocity and water temperature)markedly changed.Meanwhile,the external nutrients,e.g.terrestrial plant,debris,soil organic matter,come from the Yangtze River upstream and stagnate in the TGR after the impounding of the TGR.As no study was carried out investigating the comprehensive effects of the above factors on the fish community structure and their food web in the TGR,the stable carbon and nitrogen isotope(δ¹³C and δ¹⁵N)technology was applied in this study to evaluate the food web of fish and nutrient relationship caused by the seasonal variation in the main stream of the TGR from Wushan to Wanzhou(summer,July 2010;winter,December 2010),and the data of 2005 in summer were also provided to compare their changes.The results of δ¹³C indicated that the values of both particulate organic matter(POM)and periphytic algae samples were (-25.62‰)-(-23.72‰) and (-19.81‰)-(-19.47‰),respectively,and no significant variation of δ¹³C values was observed for primary producers(POM and periphytic algae)between summer and winter in 2010(P>0.05).However,as the POM and periphytic algae(endogenous nutrients)were the main basal carbon origin for the webs of the fish food,and the input of the exogenous nutrient substance was an important supplementary way for the consumers in TGR.In summary,an increasing trend of the δ¹³C values for consumers and the chain length for the webs of the fish food were obtained from 2005 to 2010.Our findings will be helpful to the management of the fish resources and the protection of the ecological restoration in the TGR.

Abstract:Based on the aggregation experiments of feature-reef trough,the occurrence frequency of Sparus Macrocephalus larvae around artificial fish reefs was studied with different structures and at different background velocities.The highest frequency of fish occurrence,space volume of flow velocity of <0.7 times and<0.8 times to background were considered as indexes of wake vortex range.The space volume with vertical upwelling velocity >0.1 time and >0.2 time of the upwind horizontal velocity was considered as the index of upwelling range.Based on Fluent with a turbulence model named LES(Large Eddy Simulation),a numerical trough was built and four unit-reef assemblages as an example were adopted to analyze the difference and merits of various assemblages’ flow effect.The result showed it was appropriate to place 20-30 unit reefs(cube,3 meters of side length)as a 5-point symmetric mode,and 1-2 times reef distance in reef area,as this could not only bring about synergistic effect of reefs,but also optimize the regulation range of unit-reef.

Abstract:Symplectoteuthis oualaniensis is a pelagic squid species widely distributed in the Indo-Pacific region,especially in the South China Sea,S.oualaniensis is the dominant species and therein there exists abundant biomass in the central and southern deep sea.It is necessary to carry out the exploitation and assessment of S.oualaniensis resources to promote the development of the fishery resources of the South China Sea.Due to the low population densities in natural conditions,resource survey by using squid jigging machine or pelagic trawl is ineffective.Although fisheries acoustics is a faster and more efficient way to assess the resources,because of insufficient data on biology and acoustic characteristics,it(when used alone)is not a perfect method.Therefore,the aim of this study is to explore a reasonable and effective assessment method and provide theoretic basis for reasonable utilization.This study proposes a model which combines light falling-net and acoustic survey by using a split-beam echo-sounder(Simrad EY60,120 kHz).Based on the acoustic data and biological data obtained from light falling-net conducted in the South China Sea from April to May in 2011,S.oualaniensis distribution,population structures,as well as method of acoustic survey were analyzed.The results showed that the effects of light trapping squids were obvious and the squid was mainly distributed at depths of 0-100 m.The average mantle length range was 10.4 to 14.2 cm,and the corresponding range of average target strength was -58 to -60.7 dB.Based on an analysis of collected data,the relationship TS=21.23logML-82.48 was obtained for S.oualaniensis at 120 kHz,with TS in dB and ML in centimeters.Our results indicated that this method is feasible and could provide important theoretical support for the squid resource assessment.

Abstract:Beak is one of the most important hard tissues of Cephalopoda which is usually used in fishery biology and biomass estimation.According to the 2 916 samples of short-fin squid Illex argentinus collected by Chinese squid jigging vessels from January to March in 2010 in Southwest Atlantic Ocean,the morphologic parameters were measured and the growth characteristics of beak were studied.The result of principal component analysis of twelve morphologic indices showed that upper hood length(UHL),upper crest length(UCL),upper wing length(UWL),lower hood length(LHL),lower crest length(LCL)and lower wing length(LWL)could be used to represent the length growth features of beak of each dome,and the ratios of HL to CL,RL to RL,RW to WL,LWL/CL and WL/CL could be used as indicators of entire growth for beak(upper beak and lower beak)growth,and the each dome growth,the entire beak growth impacted by sex,gonad maturity and individual size were analyzed by the analysis of variance(ANOVA)and least-significant difference(LSD).The results indicated that the UHL,UCL,UWL,LHL,LCL and LWL were closely related to sex,and for females or males,the UHL,UCL,UWL,LHL,LCL and LWL were closely related to gonad maturity and mantle length,however the ratios of HL/CL,RL/CL,RW/CL,LWL/CL,WL/CL were nearly constant without changing with sex,gonad maturity or different mantle length with the ratios of 78.04%,22.46%,17.28%,81.41% and 23.07% for upper beak and 50.69%,43.31%,1.13%,144.98% and 93.68% for lower beak respectively.This study suggested that there are significant effects of individual size on beak morphology of I.argentinus in the Southwestern Atlantic Ocean.

Abstract:Effects of three feeding models on growth rates throughout the whole growth cycle,gonad yield and gonad quality at harvest of the sea urchin Strongylocentrotus intermedius were studied.Six-month-old sea urchins were selected and fed on three diets kelp(E₁),kelp+mussel(E₂),maize+mussel(E₃)to harvest.The test diameter and live body weight of sea urchins were measured every two months.The gonad weight,gonad moisture content and gonad color were measured at the end of the experiment.Results showed that:the feeding model significantly affected(P<0.01)the test diameter and live body weight of the sea urchin during its growth stage.The test diameter and live body weight performed as:E₂>E₁>E₃ among feeding models.The feeding model significantly affected most specific growth rates(SGRs)of the test diameter and live body weight(P<0.01),which ranked as:E₂≥E₁>E₃.The feeding model significantly affected the gonad yield and gonad quality at harvest(P<0.05 or P<0.01).E₂［female:(7.58±1.21)g,male:(7.74±1.95)g］was the optimal model for the gonad wet weight,followed by E₁［female:(4.50±1.20)g,male:(4.87±1.02)g］.E₃［female:(2.66±1.02)g,male:(2.32±0.75)g］was the worst model.E₂(female:15.07?Ee1.84%,male:15.83?Ee3.01%)was the optimal model for the gonad index,followed by E₁ (female:11.54?Ee4.94%,male:11.84?Ee3.70%)and E₂(female:12.48?Ee4.33%,male:12.78?Ee3.73%).E₂ and E₁ were the optimal models for the gonad moisture content and gonad color,while E₃ was the weakest.Effects of the feeding model on male or female sea urchins were similar.The result suggested that,S.intermedius can grow good test diameter by feeding kelp only.Animal protein can significantly improve the growth rate and gonadal production of S.intermedius.Kelp+mussel model can be applied to improve the culture efficiency of the sea urchin.Maize had no beneficial effect on the promotion of the growth,gonad yield and quality of the sea urchin.Even if animal protein was added,maize+mussel can hardly be an alternative feeding model in aquaculture of S.intermedius.

Abstract:The experiment was carried out to investigate the effects of intermittent food-deprivation on growth,digestive enzyme activities and some serum biochemical indices in the rice-field eel,Monopterus albus.A 64-day growth trial with five feeding regimes was conducted using the live tubificid worms(Limnodrilus hoffmeisteri)as the food at(25±1)℃.Groups of fish were deprived of food for 1,2,4,8 day(s)and then followed by satiation re-feeding for the corresponding day(s),respectively(i.e.S1F1,S2F2,S4F4 and S8F8 groups).The fish fed to satiation daily throughout the experiment served as the control group(i.e.C).The results showed that,deprived fish had significantly lower specific growth rates(SGR),relative weight gains and final body weights,but significantly higher actual feeding rates,than that of the control fish(P<0.05).Moreover,the fish in S1F1 group had significantly higher feed conversion efficiency(FCE)than those of any other groups(P<0.05).It indicated that the partial growth compensation was elicited by various extents of intermittent food-deprivation,and that the fish of S1F1 group showed the best growth compensation.The protease activity of stomach in the S1F1 group was significantly higher than that in the S2F2 group(P<0.05),and the protease activities of stomach in both of the two groups were significantly higher than those in other groups(P<0.05).There was no difference in amylase activities of liver,stomach,foregut and hindgut among fish in all groups(P>0.05).In the liver and hindgut,the S1F1 group had significantly higher lipase activities than other groups of the control,the S4F4 or the S8F8(P<0.05).In all groups of the deprived fish,the serum total protein contents and glucose contents were slightly higher and lower than that in the control group,respectively,however,there was no significant difference among the five treatments(P>0.05).There was no significant difference in serum total cholesterol level between the control group and S1F1 group(P>0.05),and the serum total cholesterol levels in these two groups were significantly higher than that of any other group(P<0.05).In contrast,among the groups of S2F2,S4F4 or S8F8,the serum total cholesterol level gradually descended with the more serious intermittent food-deprivation.These results suggested that the growth compensation in the rice-field eel could be achieved partially through the enhancement of the activities of some of digestive enzymes,food utilization efficiency,and the physiological adaptability of digestion,and the desirable growth compensation could be elicited through mild intermittent food-deprivation that incorporates one-day of feed-deprivation with one-day of re-feeding.

Abstract:The acute effects of ammonia exposure on selected immunological and metabolic parameters in juvenile red claw crayfish,Cherax quadricarinatus as well as the post-exposure recovery were studied by using Real-time PCR technique and biochemical assays.The crayfish were first exposed to ammonia for 3 d,then half of them were sampled and the other half were moved into aerated tap water for a 7 d post-exposure recovery.The results showed that a 3 d ammonia exposure had significant impact on the activities of ACP,AKP and SOD in the muscle of juvenile C.quadricarinatus.As ammonia concentration increased,the activities of these enzymes significantly decreased.At the highest concentration(16 mg/L),they decreased to 76%,68% and 62% of the control,respectively.The mRNA expression of mitochondrial manganese superoxide dismutase(mMnSOD),cytosolic MnSOD(cMnSOD),extracellular copper/zinc SOD(exCu/ZnSOD)also showed a decreasing trend following increased ammonia concentration.In contrast,the mRNA expression of catalase(CAT),glutathione peroxidase(GPX)and glutathione transferase(GST)in muscle remained unchanged compared to the control.Dissolved protein and triglycerides in hepatopancreas significantly decreased as ammonia concentration increased.At the highest concentration,they decreased to 72% and 59% of the control.AST showed a noble elevation which was 134% of the control in 12 mg/L group.After a 7 d post-exposure recovery,ACP and AKP activities returned to the normal level in ammonia treatments.On the contrary,the activities of SOD and GPX in all ammonia treatments were significantly higher than the control.The expression of antioxidant genes was higher than the control in all ammonia treatments.Metabolic parameters showed no differences between all groups.The results indicate that high levels of ammonia might suppress activities and gene expression patterns of immune-related enzymes,leading to a loss of defence mechanism.The crayfish which had been exposed to ammonia for 3 d could not fully recover after a 7 d post-exposure recovery and oxidative stress still existed in the muscle in C.quadricarinatus.

Abstract:The correlation between hemolysis time and concentration of saponin(2.5,5,12.5,25,50 and 125 mg/L)was analyzed via microscope observation.In vitro hemolysis rate of turbot blood cells in different concentrations of saponin(2.5,5,12.5,25 and 50 mg/L)at 20 and 4 ℃,for different time duration were tested respectively.To investigate the toxicity of saponin extracted from Quillaja saponaria to turbot(Scophthalmus maximus),median lethal concentration(LC50)was calculated by immersing the turbot in sea water with different concentrations of saponin.In addition,the lactic dehydrogenase(LDH)activities in serum of turbot after bathing in saponin seawater solution(0,5,25 and 45 mg/L)were analyzed.The results indicated that the correlation between hemolysis time of erythrocytes and saponin concentration showed a logarithmic negative correlation(R²=0.98),and that saponin could also induce hemolysis to leukocytes of turbot.Saponin in concentration of 50 mg/L could result in 100% hemolysis at 20 ℃ for 5 min,in contrast,only 42.2% hemolysis was observed at 4 ℃.The 24 h LC₅₀ of saponin for S.maximus was 64.85 mg/L.In this report,from cell level,the evaluation of the toxicity of saponin to blood cells for hemolysis was made,which will theoretically support its safe application in aquaculture.

Abstract:A parasite was isolated from Coilia nasus collected from Yangtze River.Morphological(light,scanning electron microscopes) and molecular(28S rRNA amplification)methods were used to identify the species of the isolates.Their typical characteristics:haptor not clearly distinguished from body proper,provided with 4 pairs of heteromorphic clamps.Two clamps on one side are larger than other six clamps.Narrow posterior appendage with 2 pairs of sharp hooks and egg with long filament.The results of sequence alignment showed 100% homology to 28s of Heteromazocraes hexacantha.Neighbor-joining methods of phylogenetic tree also showed that the isolates were in the same clade with H.hexacantha,These results suggested that this parasite is Heteromazocraes lingmueni, and its 28S rRNA was measured for the first time in this study.

Abstract:Edwardsiella tarda is widely distributed in aquatic environments and can be pathogenic to a broad range of hosts.It is currently one of the most important fish pathogens that affect the aquaculture industries worldwide.Though the pathogenesis of E.tarda is poorly understood for the present,previous studies have shown that several factors may contribute to the virulent mechanisms of E.tarda.The adhesive properties such as fimbrial protein precursor A(fimA)and fimbrial protein precursor B(fimB),glutamate decarboxylase isozyme(gadB),citrate lyase ligase(citC)were observed to be the virulence genes that correlated with the mortality of infected fish.In this study,we investigated the prevalence distribution of the four virulence-associated genes and that of E.tarda isolated from flounder(Paralichthys olivaceus)and turbot(Scophthalmus maximus),and established the methods of dulplex PCR and LAMP that will detect E.tarda.Four pairs of primers were designed according to the published nucleotide sequence of virulence genes(fimA,fimB,gadB and citC)for screening the virulence genes of pathogenic E.tarda,and the methods of dulplex PCR and LAMP that will detect E.tarda were established using fimA and gadB genes as molecular marker.The results showed fimA,fimB,gadB and citC genes were detected simultaneously in 10 pathogenic strains of pathogenic E.tarda,and 240,217,171 and 119 bp gene fragments from chromosomal DNA of E.tarda could be amplified,and no positive reaction was detected in 4 other control strains;the PCR primers designed by fimA and gadB genes could detect E.tarda at a level of as low as 3.0×10³ CFU/mL using dulplex PCR method;the LAMP primers designed by fimA gene could detect E.tarda at a level of as low as 30 CFU/mL within 60 min under isothermal condition at 65 ℃ using the LAMP detection system.The green amplified products were observed directly by naked-eye in the reaction tube by addition of SYBR Green Ⅰ,and negative reaction(no amplified bands and with orange color)was detected in 4 kinds of control pathogenic bacteria,including V.anguillarum,V.harveyi,V.damsela and Aeromonas salmonicida.These methods could be used as the rapid diagnosis of the disease caused by E.tarda in aquaculture.

Abstract:Singapore grouper iridovirus(SGIV)is a major viral pathogen which can result in heavy economic losses in grouper aquaculture.SGIV is a large DNA virus containing 162 open reading frames(ORFs),among which the ORF050,a tumor necrosis factor receptor like gene,may play possible roles in the virus immune evasion.In this study,the gene of SGIV ORF050 was cloned,and the recombinant vectors containing full length gene or the four Cysteine-rich Domain(CRD)deletion mutants were constructed,respectively.RT-PCR and drug inhibition assay demonstrated that SGIV ORF050 is an immediate-early(IE)gene.Intracellular localization revealed that SIGV ORF050 is distributed in the cytoplasm evenly and gathered around the nucleus.The localization changed when the first CRD was deleted,which presented punctated distribution in the cytoplasm.The cytopathic effect(CPE)was no changed significantly in SGIV infected ORF050-expressing fish cells compared with control cells.In addition,real-time quantitative PCR(qRT-PCR)analysis showed that there was not significant change of SGIV MCP gene transcription in ORF050-expressing fish cells,suggesting that ORF050 might not have much effect on SGIV replication.Overexpressing ORF050 could enhance grouper TNF and TNFR transcription levels in SGIV infected fish cells,revealing that SGIV may regulate the host TNF/TNFR signaling pathway to evade the host immune attack via ORF050.