Abstract:To analyze the mechanism and result of the zinc oxide nanoparticles(ZnO-NPs) injury to zebrafish intestine tissue,this paper has studied the effect of ZnO-NPs on zebrafish intestine tissue structure,antioxidant enzyme activity and apoptotic gene expression.Zebrafish were exposed to the different concentrations of ZnO-NPs(0,0.05,0.1,5,10,25,50 mg/L).The malondialdehyde(MDA),glutathione peroxidase(GSH-PX) and glutathione-s-transferase(GST) in the intestine tissues of zebrafish were measured by colorimetric method at the 4^th,24^th and 96^th hour.The mRNA expressions of Bcl-2, Bax,p53 and MDM2 in intestine tissues were also examined using quantitative real-time PCR assays.The change of anatomical structure in the intestine tissues of zebrafish was observed by Hematoxylin-Eosin(H-E)staining technique after zebrafish were exposed to ZnO-NPs for 7 d,15 d and 30 d.Compared with the control group,the experimental groups were shown as:the MDA content was increased;with a longer exposure time,the activity of GSH-PX and GST first increased and then decreased;the mRNA expression level of genes Bax/Bcl-2 ratio and p53 of experimental groups increased;with a longer exposure time and a higher concentration,the mRNA expression level of gene MDM2 first increased and then decreased.HE staining was observed in the experimental groups,changes in the structure of intestinal tissues were shown as the phenomena that the number of goblet cells and lymphocyte increased,goblet cells swelled,cytoplastic vacuolation,leukomonocyte increased and erosion of villi was observed in zebrafish intestine depending on the time and dose.The results suggest that the oxidative damage of ZnO-NPs to the zebrafish intestine was caused by the increase of oxidative stress,which made enzyme inactivating,induced the expression of cell apoptosis-related genes and caused the damage to histological structure of intestine.

Abstract:To study the role of AMH in gonadal development of Scatophagus argus,we cloned the full-length cDNA sequence of antik-Müllerian hormone (AMH) from S.argus using the RACE technique,which is 2 324 bp in length (GenBank no.:KP718479).The open reading frame (ORF) of this gene spanned a region of 1 631 bp and encoded 543 amino acids.AMH of S.argus shared 71.16% similarity in sequence with that of Lateolabrax japonicus,while only 29.83% similarity with that of Danio rerio.S.alignment analysis revealed that S.argus AMH is closely related to Perciformes AMH,indicating AMH is conserved during evolution.The protein peptide contains a signal peptide (1-28 amino acids),an AMH-N domain (69-426 amino acids),and a TGF-β conserved domain (444-543 amino acids).Results of Real-time PCR showed that the mRNA level of AMH in testis of adult S.argus is significantly higher than that in other organs. AMH expression was also detected in liver and ovary.In addition,the mRNA expression of testis AMH at the development stages of I,II and III was kept at a high level,but was significantly decreased at the development stage of IV.According to the results of H.E staining,spermatogenesis happened in these stages,indicating an important role of AMH in testis development and the sperm production.In the ovary, AMH expression level in Ⅰ,Ⅱ oocyte development stages is low,while in the Ⅲ,Ⅳ stages,when the oocytes are rapidly growing and yolk are gradually accumulating,the AMH level is high.The results demonstrated that AMH is involved in the development and function maintaining of oocyte.Taken together, AMH played a significant role in the development and function of both testis and ovary.

Abstract:The optimal proliferation conditions of infectious spleen and kidney necrosis virus(ISKNV) in CPB cells with synchronous inoculation method were confirmed through detecting the viral copy numbers.The virus which proliferated with different FBS concentration,amount of inoculated virus,cell state,and viral harvest time was harvested and then DNA was extracted for qPCR detection.Results showed that all of the above factors had obvious influence on the ISKNV proliferation in vitro.It would obtain the highest amount of virus(2.54×10⁸ copies/mL) under the conditions that the MOI was 0.2,the virus was inoculated to the CPB cells cultured for 48 h at the middle-log phase and cultured with L-15 medium containing 6% fetal bovine serum and harvested after culturing at 28℃ for 9 days.The average cost of viral yield per yuan and per day was further analyzed.The results indicated that if the culture medium cost was only considered,it would obtain the highest viral yield(4.24×10¹¹ copies) per yuan as the above method;however,if it was as far as possible to shorten the time of cell culture and virus culture for saving time cost,it would obtain the highest viral yield per day(3.64×10⁷ copies) under the conditions that the MOI was 4,virus was inoculated to the CPB cells cultured for 24 h at the early-log phase and cultured with L-15 medium containing 10% fetal bovine serum and harvested after culturing at 28℃ for 4 days.In conclusion,this paper will provide the theoretical basis for ISKNV vaccine production with low cost through the comprehensive viral yield analysis of the medium cost and culture time cost.

Abstract:The establishment of marine mollusks permanent cell lines is one of the scientific problems that has not been conquered.The main reasons are lacking appropriate culture medium and cell factors.In this study,we investigate the effects of vertebrate cell factors on primary cell culture and biomineralization ability of mantle cells from the pearl oyster Pinctada fucata.Mantle tissues of 3-year-old pearl oysters were selected to culture the primary cells.Epidermal growth factor (EGF),endothelial cell growth supplement (ECGS),insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) were added separately into the culture medium.The effects were evaluated by comparing the cell viability,adherence capacity,migration ability and expression levels of four matrix protein genes (pif80, n16, msi7 and accbp).According to the results,the cell viability,adherence and migration ability were significantly improved by EGF.The expression levels of pif80, n16 and accbp all increased under the stimulation of this factor.After ECGS treatment,the adherence and migration abilities of primary cells were improved,and the expression levels of pif80, n16 and msi7 were increased significantly.The cell viability,adherence,migration ability as well as the expression levels of msi7 were increased by IGF-1 treatment.However the expression level of pif80, n16 and accbp decreased after cells were treated by this factor.After bFGF treatment,the cell viability and adherence capacity were improved.The expression levels of pif80, n16 and accbp were also increased by this factor.These results suggested that vertebrate cell factors may be used as cell culture supplements,which were able to increase cell viability and biomineralization ability of mantle cells in Pinctada fucata.This study therefore may provide some theoretical references for the establishment of marine mollusks permanent cell lines.

Abstract:In 2012,a green rot disease incidence occurred in a Pyropia haitanensis culture farm located in Putian,Fujian Province.The present study was carried out to investigate the pathogenic agent of green rot disease.The combination approach of biochemical tests and mutilocus sequences analysis was used to identify the bacterial isolate,and the artificial infection experiment and histopathological observation were performed to determine the pathogenecity of the isolate.A dominant bacterial strain assigned as X5 was isolated from the diseased blades using 2216E medium plate.In artificial infection experiment on the P.haitanensis blades,X5 was able to cause the same characteristic histopathology of green rot disease seen in natural infections:small yellow greenish spots appeared over the blades,then gradually developed into large green holes,and finally leading to collapse of the whole blade.By infection with X5 at 10⁴-10⁸ cfu/mL,the infected P.haitanensis blades were found to have lesion area rates from 10% to 100%.These results indicated that X5 was the etiological agent of green spot disease of P.haitanensis.X5 was gram-negative,short rod,non-flagellum bacterium wit size of 0.6 μm×0.4 μm-1.0 μm×0.4 μm.It grew well at temperatures from 4 to 42℃,salinities from 50 to 150 and pH from 6 to 10.The optimum growth condition was 16℃,50 salinity and pH 7.X5 was resistant to metronidazole,clindamycin and penicillin,while susceptible to 25 antibiotics.It was identified as a member of the genus Vibrio by Biolog and API ID-32E.Based on phylogenetic analysis of multilocus sequences (16S rRNA, rpoA,recA,yprH),X5 was showed to be highly related with V.casei,V.litoralis and V.rumoiensis,with the evolutionary distance of 0.095-0.108.These results indicate that X5 might be a novel species of the genus Vibrio or a novel subspecies of V.casei,V.litoralis or V.rumoiensis.The present study indicates that a Vibrio sp.strain can cause green rot disease in P.haitanensis,which will support the study on P.haitanensis epidemiology and disease control of P.haitanensis.

Abstract:To investigate the relationship between the developmental capacity and biochemical parameters of egg in Takifugu obscurus,the basic components,phosphatase activity,malate dehydrogenase activity,sialic acid,amino acid content in unfertilized eggs of T.obscurus were measured by means of biochemical analysis,also fatty acids composition were measured by means of GC-MS,and the correlation between components and hatching rates were analysed.Results of experiments showed that activity of acid phosphatase,sialic acid content and the hatching rate were significantly correlated,alkaline phosphatase and hatching rate were significantly negative correlated.C14:0 and hatching rate were significantly negative correlated,and showed a decline trend.ARA,DHA and hatching rates were significantly correlated,and both of them showed a tendency of first increasing and then decreasing with the hatching rate.The content of n-3 fatty and the ratio of DHA and EPA and the ratio of EPA and ARA all had significant correlation with the hatching rate.The results demonstrated that phosphatase,sialic acid,fatty acid and egg hatching rate have correlation,also when the activity of ACP were in the range of 68.045,141.038,the activity of AKP in the range of 2.298-7.241, the content of SA in the range of 9.081-19.973, the content of C14:0 in the range of 0.485-0.658, the content of ARA in the range of 1.371-2.498, the content of DHA in the range of 13.838-18.469,all of them could be the references of which hatching rates were no less than 50%.Accordingly these references could reflect the developing value of T.obscurus' egg in breeding production,which could also be used as reference index for embryo and objective evaluation of the quality of its egg.

Abstract:Lipid and protein oxidation can lead to the quality decline,off flavours,texture deterioration,loss of nutritional value and functional properties decrease in meat and meat products,which is an important reason for its quality deterioration.Recently,the use of synthetic antioxidants is under increasing scrutiny due to the health risks associated with such compounds.So,search for natural antioxidants as alternatives to synthetic ones is of great concern among researchers.Protein hydrolysates,a potent alternative,have been shown to inhibit lipid oxidation in pork meat system.However,few studies have attempted to link the hydrolysates of by-product of aquatic products and their antioxidant effects to the inhibition of oxidation of lipid and protein in cooked pork products.The objective of this study was to evaluate the inhibitory effect of the 6h hydrolysates and FractionⅡ(1-4 ku) of Scomberomorus niphonius skin on lipid and protein oxidation and colour deterioration in cooked pork patties during chilled storage.Six cooked pork patties samples including control,2.0% 6 h hydrolysates,1.0% FractionⅡ,1.5% FractionⅡ,2.0% FractionⅡ and 0.02% BHA(positive control) were stored at 4℃ for 8 d.During the chilled storage period,thiobarbituric acid-reactive substances(TBARS),peroxide value(PV),carbonyl content,sulfhydryl content,pH value were measured and sensory evaluation was performed.The results showed that the S.niphonius 6 h hydrolysates and FractionⅡ could lower TBARS and PV value and decrease the carbonyls formation and reduce the sulfhydryl loss of cooked patties compared with control,of which the FractionⅡwas more effective than 6 h hydrolysates during storage time.Furthermore,the study demonstrated that enzyme-hydrolyzed S.niphonius skin protein and FractionⅡ could retard the peroxidation of lipids and protein oxidation,and maintain the mince bright red color,and inhibit sour and rancid flavor,improving overall sensory evaluation value.During the whole storage time,molecular weight in 1-4 ku can be more effective in the inhibition of cooked meat fat and protein oxidation that occurred and had a better capability of maintaining the quality and extending shelf life.The TBARS value,PV value,carbonyl content,total sulphur content and pH value were 1.39 mg/kg,1.57 meq/kg,8.27 nmol/kg,49.6 nmol/kg protein and 6.35 in 2.0% treatment groups at refrigerating for 8 days;the effect is better than any other groups in keeping the quality of the cooked meat,and the same results were got by sensory evaluation.So,antioxidative capability present in Alcalase-hydrolysed S.niphonius skin protein can be attributed,at least in part,to its radical scavenging capability.Short peptides in the 1-4 ku range of S.niphonius skin hydrolysates possessed the strongest hydroxyl radical quenching effect and therefore,seemed to play a major role in keeping the balance of oxidative and antioxidative in meat systems.Further research is needed to purify the specific peptides responsible for the antioxidant activity of the hydrolysates,and to explore their amino acid sequences which will allow a better understanding of the peptide structure and mechanism relationship in muscle foods.

Abstract:In order to study the effect of donor and host mussels on the quality of non-nucleated pearls in freshwater pearl mussel (Hyriopsis cumingii),the purple strain F₅ of H.cumingii was chosen as materials.Before grafting the saibos into mantles,the growth traits (shell length,shell height,shell width and body weight) of donor and host mussels were respectively recorded,and the inner shell color parameters including L,a,b,dE of donor mussels were measured.At the 18th month after grafting,the specific growth rate and inner shell color of the host mussels were measured.Besides,the color,size,roundness,luster and weight of pearls were noted.The results showed that there was no significant correlation with the growth traits of the donor mussels and the non-nucleated pearl quality (P>0.05),and that extremely significant correlation existed between inner shell color of the donor and pearls color (P<0.01).Moreover,the greater the dE value of the donor mussels is,the higher percentage of purple pearl the mussel can produce,whereas,a higher percentage of white pearl was yielded when dE value of the donor mussels turns out to be lower.Specific growth rate of the host mussels had highly significantly positive correlation with the pearl size,luster and pearl yield (P<0.01).The degree of correlation:body weight> shell length> shell width> shell height.Specific growth rate of the host mussels is anremarkably correlated with pearl roundness (P<0.01).The degree of correlation:shell length> body weight> shell width> shell height.No significant correlation between the inner shell color of the host mussels and the non-nucleated pearl quality was found (P>0.05).In consideration of all the correlations above,it can be concluded that the pearl color is probably improved by improving the inner shell color of the donor mussels,and that non-nucleated pearl quality (size,luster,roundness,pearl yield) can be greatly enhanced by promoting growth traits of the host mussels.

Abstract:To explore the correlation between the mechanism changes in texture quality of post-harvested commercial bivalve molluscs and the physiological properties changes of muscle protein,a trial study of scallop (Patinopecten yessoensis) edible muscles was carried out focusing on myofibrillar (Mf) protein,and ATPase activities were taken as indicators to explore the stability of myofibrillar protein extracted from adductor (A-Mf) and mantle (M-Mf).Comparisons and analysis between the extracted Mfs from adductor muscle and mantle were made,including the solubility,viscosity,the influences of I,pH and temperature on the ATPase stability of Mfs,and their ATPase inactivation characteristics.The results showed that:(1) A-Mf and M-Mf presented similarity in solubility and both pI appeared around 5.Viscosity analysis showed that the thermal stability of A-Mf was higher than that of M-Mf.(2) Compared with Mg²⁺-ATPase,Ca²⁺-ATPase can more accurately indicate the stability of A-Mf and M-Mf which was the same as vertebrate fishes.(3) The Ca²⁺-ATPase activities of adductor and mantle Mfs had the common characteristics,for example,the highest activity was in the neutral pH;the difference between A-Mf and M-Mf was that the highest Ca²⁺-ATPase activity of the former was at low ionic strength (I=0.2) whereas the latter was at high ionic strength (I=0.5);the influence of ionic strength on the A-Mf was not obvious whereas M-Mf was affected by the ionic strength obviously which showed the better stability at low ionic strength.(4) The inactivation characteristics of Ca²⁺-ATPase showed that both the adductor and mantle Mfs had a significant correlation (R²=0.818 1,0.843 6 and R²= 0.9887,0.9557) with the ionic strength and temperature,the best stability of A-Mf and M-Mf were in the range of pH 7.0,and acid conditions damaged Ca²⁺-ATPase activity more obviously than alkaline conditions.

Abstract:The stress,strain and deformation after the hook in the stress play a decisive role on the mechanical properties and structural stability of the hook.The pros and cons of the mechanical properties of the hook directly affect the capture effectiveness of the target species.It is beneficial to improving the hook design and material selection to understand the mechanical properties (eg.tension and deformation) of tuna longline hook.There are some advantages for the circle hooks,such as reducing the sea turtle by catch rate,high survival rate after capture release,low escaping rate,etc..There is a trend that circle hook replaces the ring hook and J hook.The deformation or fracture of hook will make longline fishing invalid.Therefore,the hook should have enough stiffness and strength when the hook is designed and the material is determined.Mechanical analyses are carried out on two kinds of typical hooks (ring hook and circle hook) using finite element analysis method (FEM) by ANSYS.The distribution of equivalent stress,strain and deformation of hook are obtained,and the possible failure condition and the failure type are also analyzed.Intensity examination and optimization design are made on hookstructure based on material mechanics theory,compare with hook's data obtained in the tensile test.Results showed:(1) With the same specifications,ring hook'stensile strength is higher than that of circle hook by 15.80%-29.40%,and the max. deformation rate in X and Y axis of ring hook is smaller than that of circle hook by 3.00% and 0.21% respectively;(2) Hook's main stress is undertaken by the bend of the hook (83.00%-86.00%),and there is a stress concentration in the hole of hook eye;(3) Flattening the bends surface (flattening rate is 15%),increasing the hook circular arc of the bending curvature appropriately (the radius increased from 14 mm to 16 mm),connection of hook eye and shank added with arc transition can avoid stress concentration and enhance the strength of the hook by 4.60%-10.80%.This study suggests that (1) Circle hook is the eco-friendly hook type;(2) Its tensile strength meets the requirement of the fishing;and (3) It can be used in the longlining.

Abstract:Shellfish provide an important environmental service by filtering a large amount of suspended organic matter,which could otherwise lead to the organic enrichment of sediments and the related impact.The impact of shellfish on the water column nutrient concentration(nitrogen,phosphorus)and dissolved carbon distribution was investigated during maturity and seedling period at a mussel farm (Mytilus edulis) of Gouqi Island.In order to understand the effect of mussel culture on water health and dissolved carbon pattern during the maturity and seedling periods,we studied the concentration of dissolved carbon,nitrogen and phosphorus on the spatial scales and the change in distribution pattern on time scales.The result illustrates that the concentrations of nitrogen and phosphorus in zones are higher than those in controls in maturity stage,in particular the nitrogen (P<0.05).The emissions and filter-feeding of mussel lead to the damage of the water health.On spatial scales,the distribution pattern of nitrogen,phosphorus in summer is contrary to the autumn.The water was purified by the emissions and filter-feeding of mussel.From the perspective of ecological effects,the concentrations of dissolved inorganic carbon in culture zone surface water are significantly lower than in control zones (P<0.05).Nitrogen-rich resulted in dissolved organic carbon in an unstable state.Therefore,from the perspective of dissolved organic carbon,the mussel culture was regarded as carbon source.Based on the above,mussel culture has a different effect on water health in maturity and seedling periods,and it needs further discussion about whether the mussel culture has carbon sink function or not.

Abstract:Based on the morphological measure ment and biochemical analysis,this study was conducted to compare the morphology,hepatosomatic index (HSI) and biochemical composition between wild-caught (WC) and pond-reared (PR) juvenile Eriocheir sinensis.The results showed that:the significant differences were found on the morphology of WC and PR.There were four of dominated principal component (i.e.PC1-PC4) for both females and males.PC1 contributed more significantly to the principal component analysis than the other PC,and PC1 of both gender consist of 16 and 19 main variables,respectively.Among the 36 morphological indices,there were 25 and 22 indices with significant differences between WC and PR,for males and females.Four to five indices were further selected to establish discriminant equation,and those equations had more than 95% of accuracy for distinguishing WC and PR.WC had the higher HSI,tissue total lipids,muscular 16:0,18:1n9 and 20:4n6 levels,but the lower moisture contents,muscular 18:0,20:5n3 and 22:6n3 levels than PR.Therefore,the effective identification methods are established based on their morphology and biochemical composition between WC and PR.

Abstract:Vitamin E (VE) plays a key role in reproduction,metabolism,antioxidant function and immunity of fish.For the research of effects of dietary VE on tolerance of Epinephelus moara for low salinity stress,five levels of VE supplementation were designed as 0,20,40,80 and 160 mg/kg (A,B,C,D,E in turn) in this experiment.Juvenile E.moara were fed for 56 days,and the real supplementation of VE was tested.Then,low salinity (12) stress experiment lasted for 6 h,different indexes of experimental fish were tested,serum biochemical indexes:cortisol (COR),glucose (GLU),lactic acid (LD);serum antioxidant:superoxide dismutase (SOD),catalase (CAT),glutathione peroxidase (GSH-Px) activities,total antioxidant capacity (T-AOC) and malondialdehyde (MDA);as well as gill Na⁺/K⁺-ATPase (NKA),Ca²⁺-ATPase activities.The results showed that serum COR concentration was increased by supplementing VE in diet.And after low salinity stress,serum COR concentration in every groups increased.There was no huge difference among serum GLU concentration in different groups.And after low salinity stress,serum GLU concentration in groups A,B,C increased significantly.Serum LD concentration in groups C,D,E increased in feeding experiment,then it increased in groups A,B and decreased in groups C,D,E after low salinity stress.Maximum of serum COR concentration was after stress A (32.74±1.53) ng/mL,minimum was before stress E(19.06±3.88) ng/mL;maximum of GLU concentration was after stress A (16.46±0.99) mmol/L,minimum was before culture O (7.90±0.34) mmol/L;maximum of LD concentration was before stress C(1.94±0.15) mmol/L,minimum was after stress E(1.30±0.06) mmol/L.The results of serum COR,GLU,and LD suggested that supplementation of VE would enhance anti-stress ability of E.moara,and the best range of supplementation of VE was 47.524-134.566 mg/kg (B-D).Maximum of SOD activity was before stress C (105.29±9.07) U/mL,minimum was after stress A(11.23±2.30) U/mL;maximum of CAT was before culture O (4.09±0.17) U/mL,minimum was after stress A (0.35±0.10) U/mL;maximum of GSH-Px was before stress E (972.58±55.35) U,minimum was after stress A (47.90±10.64)U;minimum of MDA was before stress D(33.48±2.34) nmol/mL,maximum was after stress A (101.79±7.79) nmol/mL.The supplementation of VE were close relative to serum SOD,CAT,and GSH-Px activities,T-AOC,MDA concentration,of which range was 91.378-178.924 mg/kg (C-E) that could strengthen antioxidant function of E.moara.VE can enhance the NKA activity.When the supplementation of VE was 91.378 mg/kg (C),low salinity stress had little effect on NKA activity.The range of supplementary VE which was 91.378-178.924 mg/kg (C-E),appeared to result in the activation of the Ca²⁺-ATPase activity.This study suggested that diets supplemented with VE can enhance vitality and anti-stress ability of body.In summary,analysis of biochemical,antioxidant and ion-regulatory indicators estimated that the best range of supplementation of VE was 91.378-134.566 mg/kg (C-D).

Abstract:An 8-week feeding trial was conducted to evaluate the effects of different dietary protein and wheat starch levels on growth performance,hepatic metabolic enzymes activities,hematological characteristics,glycogen content and digestive enzyme activities in large yellow croaker.Six experimental diets were formulated to contain two protein levels(41% and 46%) and three wheat starch levels(10%,20% and 30%).Each diet was randomly assigned to 3 replicates of 50 juvenile large yellow croaker[initial weight approximately(14.84±0.16)g].The results indicated that weight gain rate and specific growth rate were not significantly affected by the interaction of dietary protein and wheat starch levels(P>0.05).When the dietary protein level was the same,dietary wheat starch levels had no significant effect on WGR and SGR(P>0.05).However,the interaction of dietary protein and wheat starch levels had significant effects on phosphofructokinase,pyruvate kinase,fructose-1,6-bisphosphatase(P<0.05),but there were no significant influences on glucokinase,glucose-6-phosphatase,phosphoenolpyruvate in liver(P>0.05).When the dietary protein level was 41%,glucokinase,phosphofructokinase and fructose-1,6-bisphosphatase were significantly affected by dietary wheat starch levels(P<0.05),and fructose-1,6-bisphosphatase significantly decreased with increase of dietary wheat starch levels,but had no significant effects on pyruvate kinase,glucose-6-phosphatase,phosphoenolpyruvate in liver(P>0.05).When dietary protein level was 46%,glucokinase,phosphofructokinase,pyruvate kinase and fructose-1,6-bisphosphatase were significantly affected by wheat starch levels(P<0.05),and glucokinase,phosphofructokinase,pyruvate kinase increased with dietary wheat starch levels increasing from 20% to 30%.The results indicated that,when the dietary wheat starch level is high,large yellow croaker has the ability to maintain blood glucose content by adjusting the hepatic glycogen,glycolysis and gluconeogenic key enzymes activities with increase of dietary wheat starch level.However,under condition of low dietary protein level,the growth performance was improved with increase of dietary wheat starch level,but blood glucose content was also increasing with increase of dietary wheat starch level.

Abstract:To carry out accurately the parentage determination on different families in Japanese flounder and select the microsatellite DNA markers with higher parental probabilities of exclusion,in this study,10 mature females and 10 mature males chosen from the selection and breeding founder were used to produce 10 full-sib families of Japanese flounder.30 individuals from each family separately raised were random selected to construct the family population whose pedigree structure was known.200 individuals from the families raised together were random selected to construct the mixed population whose pedigree structure was unknown.A set of 48 microsatellite DNA markers distributed evenly on 24 linkage groups were selected from the second genetic linkage maps of Japanese flounder,furthermore,2 markers were located on each linkage group.The results showed that 10 microsatellite DNA markers took on the higher parental probabilities of exclusion and richer genetic polymorphism based on the genetic analysis of family population,and their range for Excl 1 and Excl 2 was from 0.655 to 0.719 and from 0.792 to 0.837,respectively.The combined probabilities of exclusion gradually increased as the number of markers identified increased.It reached 100% when 8 microsatellite DNA markers were jointly used.The results of parentage determination of mixed population by using these markers demonstrated that 13 females and males contributed to the reproduction in total.To some extent,there were differences in the number of offspring from the combination of different parents.Moreover,significant differences did not exist in each genetic statistical data between the parental and progeny populations.The research demonstrated that these microsatellite DNA markers selected with higher parental probabilities of exclusion can effectively perform the parentage determination on family in Japanese flounder,therefore,they are designated as candidate markers to develop joint breeding by combining the family selection with DNA molecular marker in future.