Abstract:Samples were collected in four seasons at the artificial reef area of Laizhou Bay in 2010~2011. Chemical oxygen demand (COD), dissolved inorganic nitrogen (DIN), dissolved active phosphate (DIP), total nitrogen (TN), total phosphorus (TP), and total organic carbon (TOC) were measured. Index including the ratio of N/P, organic pollution index A and nutrition index E were used to evaluate the quality of water in different years. The results showed that the content of DIN was significantly reduced in the second year, the content of DIP increased slightly, thus the N/P value decreased from 89.60 to 44.54 due to the changes of DIN and DIP, the result of T-test showed that there was a significant difference of DIN/DIP value in artificial reef area between 2011 and 2010 (P<0.05);However, there was no significant difference in control area(P>0.05). From these results, we can conclude that the artificial reefs can improve the quality of seawater. The result of nutrition index E indicated that the nutrition level of the sea water was relatively low, and there was no significant difference in artificial reef area and control area in 2010 and 2011; The investigation results showed that the organic pollution index A was less than 0 or 1, and there was no significant difference in artificial reef area and control area in 2010 and 2011. All these results indicated that the surveyed area were not polluted by organic material, and the quality of water was in good condition.

Abstract:Scientific management decision is of vital importance to improve the economic benefit of stock enhancement. Based on the fishing yield data and biological measurement of shrimp stock enhancement recapture in Laizhou Bay in 2011, we built a resource-economy model which includes resource, economy and decision components for policy simulation. If the opening date for fishing were delayed while the amount of juveniles and vessels were maintained, the total cost would decrease, the fishing income and social-economic benefit would first increase and then decrease, and there is an optimal opening date for fishing to maximize the benefit of enhancement. For example, when 738 million juveniles were released and fishing mortality coefficient was 0.03, the maximum social-economic benefit could be obtained if the opening date for fishing were October 8. If the amount of vessels is increased while the quantity of juveniles and the opening date were maintained, the total cost would increase, but an optimal number of vessel also existed, though more vessels may not mean greater benefit. The social-economic benefit is proportional to the amount of juvenile on the condition of opening date and quantity of fishing vessels unchanged.Management authority should set up reasonable opening date for fishing and optimum amount of juveniles and vessels to achieve the best effect of stock enhancement.

Abstract:The incubation period, hatching and deformity rate of the fertilized eggs and the survival activity index (SAI) of newly-hatched larvae of F1 Epinephelus moara (♀)×E. Septemfasciatus(♂) were examined at salinities of 5, 10, 15, 20, 25, 30 (natural seawater salinity), 35, 40 and 45. The results showed that the optimum salinity for hatching was in the range of 30~35. When the salinity was above 35 or below 30, the hatching rate decreased and the percentage of abnormal larvae increased. The optimum salinity for larval survival was between 30~35, and the SAI values were 33.3±0.8 and 24.1±0.2, respectively. When the salinity was above 30 or below 35, both of the larvae survival rates and the SAI values decreased.

Abstract:Full-length cDNA encoding common glycoprotein α subunit (CGα) was firstly cloned from half smooth tongue sole Cynoglossus semilaevis Günther by means of homology cloning and RACE PCR analyses. This sequence was submitted to GenBank with accession No. JQ364953. The whole cDNA length of tongue sole CGα gene was 685bp, including a 384bp open reading frame (ORF). The predicted mature tongue sole CGα consisted of 127 amino acids preceded by a signal peptide of 33 residues. The structure of mature CGα in tongue sole CGα was similar with those in the vertebrate CGα, including 10 conserved Cysteine residues and 2 N-linked glycosylation sites. Sequence comparison showed that tongue sole CGα mature peptide had higher identity to their orthologs of percomorphs (perciforms and pleuronectiforms; 60%~70% identity) than to cypriniformes (55%~60% identity). The CG(mRNA expression of tongue sole was detected in all the examined tissues by quantitative Real-Time PCR (qRT-PCR) method. The significant test analysis (P<0.05) for expression levels of tissues revealed that head kidney and spleen as well as spleen and liver did not differ significantly from each other. But other tissues showed significant different from each other. The highest expression level was in pituitary, followed by gill, kidney, muscle, ovary and brain; and lower level expression was found in other tissues including heart, head kidney, liver and spleen.

Abstract:In this study, the CASA (Computer Assisted Semen Analysis) was employed to determine how sperm quality changes during the spawning season. The sperm quality parameters, including motility ratio, concentration, volume, VSL (Straightline velocity) and VCL (Curvilinear velocity), were examined at four phases during 7-weeks from the beginning of sperm production to the end, with 2-week as an interval time. The results indicated that all the indexes have different varying patterns during the spawning season. The motility ratio changed from 83.4% to 36.9%, the sperm volume varied from 2.50ml to 2.23ml, the concentration decreased from 1.08×108 ind/ml to 0.46×108 ind/ml and the value of VSL/VCL was reduced from 0.69 to 0.35. The findings on different changing patterns and the amount of variations could contribute to turbot aquaculture directly either in broodstock breeding and its sperm application.

Abstract:Physiological and biochemical indices of blood from pond- and indoor tank-cultured Japanese flounder Paralichthys olivaceus were analyzed and compared in the present study. Four groups of fish at different body weights from pond-culture (PSJF, pond-cultured small Japanese flounder group and PBJF,pond-cultured big Japanese flounder group) and indoor tank-cultured (ISJF,indoor tank-cultured small Japanese flounder group and IBJF,indoor tank-cultured big Japanese flounder group) were sampled respectively, then 5 blood physiological indices and 13 serum biochemical indices were determined. Results revealed that the amount of red blood cells (RBC) of PSJF and PBJF were significantly lower than that of ISJF and IBJF (P < 0.05), and erythrocyte sedimentation rate (ESR), erythrocyte fragility (EOF), the amount of white blood cells (WBC), and lymphocyte proportion of leukocyte in blood were significantly higher in pond-culture fish (P < 0.05). Besides, serum glucose(GLU), total protein(TP), albumin(ALB), globulin(GLO), creatinine(CREA), cholesterol(CHOL), triglyceride(TG) concentrations and alkaline phosphatase(AKP) activity ofPSJF and PBJF were significantly higher than those in ISJF and IBJF (P < 0.05). However, serum lactate dehydrogenase activity of PSJF was significantly lower than ISJF (P < 0.05). Statistical analysis revealed no significant differences in serum chloride (Cl-) concentration, calcium (Ca2+) concentration, alanine aminotransferase (ALT) activity and aspartate aminotransferase (AST) activity among all the groups (P > 0.05). The results showed that pond-cultured Japanese flounder may have lower requirement for the respiratory metabolism than indoor tank-cultured Japanese flounder, whereas serum immunity level and nutrition metabolism level may be higher. These results may provide basic information on management of pond-cultured and indoor tank-cultured Japanese flounder.

Abstract:Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. HSP90 is identified in some crustaceans, however, few studies on the transcript of HSP90 gene in Chinese shrimp Fenneropenaeus chinensis in response to environmental pH and ammonia-N stresses have been reported. In the pH stress experiment, F. chinesis were exposed to marine water with the pH value of 7.0, 9.0 for 148h. During the ammonia-N stress experiment, F. chinensis were exposed to marine water with different concentration of ammonin-N for 96h. The gene expression profiling of HSP90 in different tissues (gill, hepatopancreas, muscle and haemocytes) after pH and ammonia-N stresses were analyzed by quantitative RT-PCR. The HSP90 gene expression in gill, muscle and haemocytes of Chinese shrimp increased when exposed to pH 7.0 and pH 9.0, but the time of the highest gene expression level is different. Obvious different expression of HSP90 gene in hepatopancreas exposed to pH 7.0 and pH 9.0 stress was observed. HSP90 transcripts increased to its highest level post-stress at 3h and then decreased in the pH 7.0 treatment, while in the pH 9.0 treatment the HSP90 gene expression was always higher than that in the control, which indicated the hepatopancreas of Chinese shrimp was the responsive organ to the high pH stress. When the concentration of ammonia-N in marine water was 6 mg/L, the gene HSP90 expression in gill and muscle reached its highest level about 5.46-fold and 1.55-fold respectively post-stress at 24h. HSP90 expression in hepatopancreas and heamocytes in all treatments increased to the maximum value in the range of 1.33~2.08 times and 2.20~5.45 times post-stress at 6h and 48h respectively. The results indicated that the hepatopancreas of Chinese shrimp is more sensitive to ammonia-N stress than other tissues, in which HSP90 expression was up regulated drastically at 6h. It is also illustrated that gill needs higher expression of HSP90 gene than other tissues for cell protection based on the results of the large fluctuation range of HSP90 gene expression in gill. When the concentration of ammonia-N was continuously higher than 2~6 mg/L, the HSP90 expression in all tested tissues was significantly decreased.

Abstract:Portunus trituberculatus belongs to the class Crustacea, family Portunidae. It is an economically valued crab species. With the rapid expansion and intensification of Portunus trituberculatus culture, various problems such as diseases, bad quality of larvae and less natural resources, have showen and become serious, causing economic losses. These have become the limiting factors in the sustainable development of this program. At last, these problems are resolved by new varieties obatined by selective breeding. In October 2005, 1261 individuals were collected from four wild geographic populations distributed in the Yalvjiangkou, Laizhou Gulf, Zhoushan, and Haizhou Gulf . Firstly, we established fundamental stock using the four populations. Secondly we estimated the effective population size for core breeding population of P. trituberculatus, and setup breed maintenance mode. Population selection was carried out at a seed reservation rate of 5%. The new variety “Huangxuan No. 1” was selected successfully in 2010, after selection for five-generations successively. “Huangxuan No. 1” showed better economic traits such as fast growth and high survival rate. It showed a body weight increase of 20.12% and survival rate increase of 32.00% respectively, compared with the natural population. With support from the government fishery authority and crab farmers, YSFRI has made significant efforts to popularize the “Huangxuan No. 1” in Weifang, Rizhao, Qingdao and Yantai in Shandong province, Cangzhou in Hebei province, and Ningbo in Zhejiang province. The accumulated extension area during 2010—2012 was up to 6,000 ha covered by the new variety. Average yield increased 30%.The main farming model for crab is “crab,shrimp,clam and fish” ecological culture “Huangxuan No. 1” was approved by China National Aquaculture Variety Approval Committee as a new variety for aquaculture in 2012 (registered No.: GS-01-002-2012).

Abstract:The total RNA extracted from the hemocyte sample of Portunus trituberculatus was used to amplify the DNA sequence encoding an open reading frame for C-type lectin like-domain protein (noted CTLD) by RT-PCR. The sequence was then cloned into pMD18-T vector and was sequenced. The recombinant plasmid was digested by BamH I and Xho I. The target gene was subsequently inserted into the pET-32a(+) vector，which was also digested with the corresponding restriction endonuclease. The recombinant plasmid p32a-CTLD was transformed into Escherichia coli BL21(DE3) and then induced by isopropylthio-β-D-galactoside (IPTG) .The fusion protein was purified through His-Band resin chelating chromatography and Amicon Ultra centrifugal filter devices，and agglutination activity was assayed finally. Results showed that the CTLD recombinant plasmid was successfully obtained by double enzyme identification. The inserted fragment was confirmed correctly by sequencing. SDS-PAGE and western-blot analysis showed that the fusion protein was successfully expressed. The fusion protein of about M39600 was successfully induced, which was identified by SDS-PAGE and western blot analysis. For the agglutination assay, no obvious agglutination was detected on the rabbit erythrocytes, while the agglutination titer of the mice erythrocytes was 22. No obvious agglutination was observed when Vibrio alginolyticus，V. harueyi ，V. anguillarum， Staphylococcus aureus were incubated with the recombinant protein. However, mass agglutination was observed after E. coli was incubated with the recombinant protein for 1h. This study provides a good foundation for further studies on the function of C-type lectin.

Abstract:This paper mainly reports on the study on hybridization (K♀×C♂) of two different geographic populations of Scapharca broughtonii from Korea (K) and China (C). Microscopic observations on the fertilization and early embryonic development stained by fluorescent dyes Hoechst 33258 were done by fluorescence microscope. The fertility rate and embryonic malformation rate were counted respectively. The result showed that the sperm could smoothly enter the eggs and activate meiotic division, then the male and female pronucleus formed after two polar body were released in succession. The two pronuclei then moved to each other to fuse into the zygote, which subsequently started the mitosis and early embryonic development. Most embryos developed into D-larvae successfully. The fertility rate and embryonic malformation rate were 92% and 23.5% respectively. The relative high malformation rate was probably attributed to polyspermy which disrupted chromosome separation.

Abstract:Polypeptides from sea cucumber cooking water were prepared by neutral protease, pepsin and trypsin,and the antioxidant activity of the polypeptides was compared with synthetic antioxidant TBHQ by measuring the scavenging capacities to DPPH•,•OH and O-2•,and the relative molecular mass was assayed. The results showed that scavenging capacities of TBHQ and polypeptides prepared by three proteases on free radicals were enhanced with the increase of sample concentration. The corresponding IC50 values of polypeptide from sea cucumber cooking water prepared by neutral protease to •OH，O-2•and DPPH• free radicals were 8.565 mg/ml, 6.658 mg/ml and 2.015 mg/ml respectively, and it had better scavenging ability to O-2•than TBHQ.The molecular weight of polypeptides from sea cucumber cooking water prepared by neutral protease, pepsin and trypsin was respectively less than 2 500,3 500 and 1 000D， analyzed by liquid chromatography.

Abstract:The purpose of this research was to establish an optimized system to analyze Apostichopus japonicus proteomics. The protein extraction method, loading methods, sample volume and focusing condition in two-dimensional gel electrophoresis (2-DE) were optimized to obtain best results. Several methods of protein preparation (TCA-acetone precipitation treatment, Tris-HCl treatment, Tris-HCl acetone treatment and pure water treatment) were applied to extract total protein from A. japonicus intestine. The most effective profiles with the most electrophoretic protein spots were obtained by the TCA-acetone precipitation treatment. The 2-DE electrophoretic analysis in intestine achieved an optimized map by loading without buffer solution treatment at the sample volume of 25 μg， The pH of ampholytes range from 4 to 6.5. The samples were electrophoresed for 1000 V and 3 hours， and the gels were immersed in 2.5 ml equilibration buffer and equilibrated for 30 min. The system optimization of protein two-dimensional electrophoresis would provide supports in sea cucumber proteomics study.

Abstract:Sipunculus nudus juveniles at average body weight of 15.49±0.03 mg were fed with seven isonitrogenous (45% crude protein) and isolipidic (9% crude lipid) diets containing different carbohydrate (glucose, sucrose, dextrin, tapioca starch, potato starch, corn starch, or gelatinized corn starch) for 8 weeks to investigate the effects of different dietary carbohydrate (CBH) sources on their growth, survival and body composition. The treatment fed with gelatinized corn starch showed the highest weight gain (WG) and specific growth rate (SGR), and were significantly higher than the other CBH diets (P<0.05). However, the dietary CBH sources had no significant effects on survival rate of S. nudus (P>0.05). The results of body composition analysis showed that different CBH sources had significant effects on body moisture and body protein (P<0.05). The S. nudus fed with glucose had the highest body moisture and the lowest body protein, which were significantly different from the others (P<0.05). No significant differences were observed in body lipid content and body ash content of S. nudus among dietary treatments with different CBH (P>0.05). It could be concluded that the ability for S. nudus to utilize macromolecular carbohydrates was better than that to utilize small molecule carbohydrates, and pre-gelatinization of corn starch significantly improved corn starch utilization in S. nudus based on the experiment results.

Abstract:A new method for rapid determination of lead in fishery seawater was developed by using highly sensitive conductive carbon black paste electrode (CCBPE) as the working electrode. CCBPE showed many attractive electroanalytical properties, such as low background current, large electrochemical active area, good accumulation ability and so on. The heavy metal lead showed sensitive response at the CCBPE with anodic stripping voltammetry mode. Some important factors, including the supporting electrolyte, pH, accumulation potential, accumulation time, interference substances, etc. were studied. Under the optimized conditions, a detecting limit of 0.1 μg/L was obtained. Based on the results, a new method for determining lead in seawater was developed. The new method was used to determine the lead concentration in real seawater samples collected from nearshore site in Qingdao. It was found that the test data were in good agreement with that obtained by atomic absorption spectrometry, indicating that the simple, rapid, mercury-free and cost-effective method had a great potential value for application.

Abstract:Two strains of nitrosobacteria with high ammonia transformation rate were isolated by enrichment and selective culture from bio-flocs of shrimp culture ponds. Sequences of 16S rRNA indicated that both strains belonged to the genus Halomonas sp., while strain 2011072708 was identified as H. alimentaria with 99% similarity, and strain 2011072709 as H. shengliensis with 100% similarity. Ammonia transformation rates of the two strains were compared under different temperatures, salinities, pH, and C/N ratios. The ammonia transformation rate of strain 2011072708 reached the highest level at 37 ℃, 30~40, pH 8, and C/N ratio of 28, while that of strain 2011072709 reached the maximum at 27~42 ℃, 40~50, pH 6, and C/N ratio of 21. The present study showed high ammonia transformation rates in both nitrosobacteria strains isolated from bio-flocs. Strain H. shenliensis (2011072709) had better adaptability to temperature, salinity, pH, and C/N ratio than H. alimentaria (2011072708), and may be more suitable for application in bio-flocs technology.

Abstract:An analytical method based on liquid phase of fluorescence detector combined with UV detector has been developed for the simultaneous determination of sixteen polycyclic aromatic hydrocarbons (PAHs) in marine sediments. After being extracted with isopropanol, the extract was distilled with HLB column, then performed on HPLC with a Waters PAH C18 column (4.6 mm×250 mm, 5 μm), separated by gradient elution using a mixture of acetonitrile and water as mobile phase. The calibration curves were linear with correlation coefficient over 0.999. The average recoveries from spiked marine sediments at the 5 μg/kg and 10 μg/kg concentrations ranged from 72.6%~117%(n=6) with relative standard deviations less than 10％, and limits of detection(LOD) were 0.2~4.9 μg/kg. Some PAHs were found in six marine sediment samples collected from Dalian and Yantai areas. This method was validated to be accurate and sensitive for the analysis and risk assessment of PAHs in marine sediments.

Abstract:The analysis of aluminium (Al) in the seaweed is usually according to GB/T 23374-2009 which is in fact the method for measuring total Al. However, most of the Al in the seaweed exists in organic forms which can not be absorbed by biological organism, and the toxic soluble ionic Al (Ⅲ) is very low. So it is urgent to establish a method for the Al speciation analysis in the seaweed. In the present research, the liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS) was used to determine the Al (Ⅲ) in the seaweed. The samples were extracted by the eluent (0.040mol/L LiOH,0.006 mol/L PDCA and 0.090 mol/L CH3COOH) and shaken under 70 ℃ for 2 h, and then the extract solution was centrifuged and filtered. After separation by CS5A column, the Al (Ⅲ) was tested by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The concentration of standard Al solutiors (Ⅲ) were 5.0mg/kg and50.0mg/kg, and the average recoveries were all above 90.8%, and the relative standard deviations were all below 4% (n=5). This method was successfully used in the analysis of real samples and proved to be very accurate and reliable, which may provide important technical support for drawing up the agricultural professional standard.

Abstract:Chlamys farreri was cultured in HgCl2 solution at different concentrations for 35 days, then transferred to natural sea water and be purified for 30days. Sampling was done at different time to detect total mercury and MeHg content in the tissue. Accumulation and purification of mercury and MeHg in the tissue was studied. The mercury solution was not toxic to the scallops when the concentration was lower than 2μg/L. Exposure concentration and accumulation of Hg in scallops showed a direct relationship.Kahle double-box model was used in the experiment design, and data was processed by hyperbolic toxicity kinetic model. The results showed that the accumulation and depuration of mercury and MeHg in the tissue of C. farreri was in line with the law of two-compartment kinetic model; total mercury accumulation in C. farreri showed the highest value at 35d, while the MeHg accumulation in C. farreri was delayed, and the highest peak was found at 40d. Comparing the biphasic kinetics parameters of mercury and MeHg accumulation and depuration in the tissue of C. farreri, it was found that the BCF of total mercury in the tissue of C. farreri was generally greater than the BCF of MeHg. The BCF and biological half-life of Hg ang MeHg in C. farreri correlated with exposure concentration.