Abstract:The morphological characteristics and distribution of chromatophores, as well as body color changes during early life history(0~40dph) of Acanthopagrus schlegelii were observed and photographed．The larval melanophores occurred firstly at blastopore colsure stage. Larval xanthophores occurred at olfactory vesicle stage，and iridophores at 6dph on the upper side of eye, then on the abdominal cavity (28dph) and around the opercle (32dph). Melanophores appeared as spots, then formed dendrites，and the number of cells with dendrites increased during the ontogenetic process. At 28dph, the larval melanophores were substituted by adult spotted melanophores. Xanthophores coocurred with the melanophores during the early ontogeny, then obscured due to the overlapping by melanophores with no obvious morphological changes during the entire study. Melanin aggregation at 6~8 segements from the tail in newly-hatched larvae became a special characteristics of this species. At 28dph, the larval body color developed into the pattern formation stage, and firstly three stripes formed, then seven stripes at 40dph. And from then on, the body color was similar to that of the adult, which suggests that the mode pattern was already formed.

Abstract:The metaphase chromosomes of Nibea albiflora(Richardson)and Nibea japonica were obtained from kidney tissue by the method of PHA and colchicines injection, and the karyotype of these two fishes were analyzed. The results showed that both fishes had 48 telocentric chromosomes in the diploid, and the karyotype formula was 2n=48t, NF=48. The relative length of chromosomes ranged from 3.06±0.20 to 5.94±0.33 in N. albiflora, and from 2.66±0.14 to 5.68±0.22 in N. japonica. Karyotype data of Sciaenidae species in previous reports were also collected including N. diacanthus, N. miichthioide and Pseudosciaena crocea. The clustering analysis was carried out for karyotype evolutionary distance among N. albiflora, N. japonica, N. diacanthus, N. miichthioide,and P. crocea. The results showed that the relationship between N. albiflora and N. diacanthus, and between N. japonica and N. miichthioide, were relatively close based on karyotype evolutionary distance, and the largest karyotype evolutionary distance (0064 8) were found between N. miichthioide and P. crocea. Results from the clustering analysis were consistent with the phylogeny based on the morphology of these species.

Abstract:Development and distribution of mucous cells in the digestive tracts (oropharyngeal cavity, esophagus, stomach, and intestine) of larval and juvenile (0~28 dph) Epinephelus malabaricus (Bloch & Schneider) were observed and analyzed by the method of histological sections and AB-PAS histochemical staining techniques. Mucous cells of E. malabaricus were classified into Ⅰ~Ⅳ types based on different staining and three morphological shapes (cystic, pear shaped and cup shaped). The result showed that mucous cells first appeared in the oropharyngeal cavity in 3 dph larvae of E. malabaricus. The number of mucous cells before 12dph were few and mainly type Ⅱand cystic. The number of mucous cells increased rapidly since 12~15 dph, and the spatial distribution patterns of mucous cells in larval digestive tract were basically formed. There were more type Ⅲ and type Ⅳ mucous cells than type I and type Ⅱ after this period. In the oropharyngeal cavity and esophagus, mucous cells were mainly cystic and pear shaped in the early samples. The mucous cells in stomach and intestine were cystic cells, then, pear shaped cells gradually increased, ultimately, goblet cells were the majority. There are significant morphological differences between anterior esophagus and anterior intestinal, and they are both the main distribution locations of mucous cells.

Abstract:The effects of temperature and salinity on the survival and growth of larval Hexagrammos otakii Jordan et Starks were studied. It showed that the newly hatched larvae and 10 dph larvae of H. otakii Jordan et Starks could survive and grow at temperature of 4~16℃, with the survival rate of more than 84%, while the survival rate was below 50% at 20℃. Under gradual salinity change, the survival rate was more than 90% at salinity of 10~30.The survival rate was zero when the salinity was 5 and below, and the ability of adapting to high salinity was also poor. Under sudden salinity change,the survival rate was more than 95% at salinities of 25 and 30, and the survival rate was 56%~75% at salinity of 10~20. The most suitable temperature range is 12~16℃，and the most suitable salinity range is 25~30 for H. otakii Jordan et Starks.

Abstract:Expression of channel catfish TLR5 and TLR5S genes in the head kidney, intestine, liver and spleen were analyzed by quantitative real time PCR method at 0 h, 12 h, 24 h, 48 h, 72 h and 7 d after infection with Edwardsiella tarda, Aeromonas hydrophila, Streptococcus iniae and channel catfish hemorrhage reovirus (CCRV), respectively. The results showed that TLR5 and TLR5S mRNAs were largely up-regulated by E. tarda and S. iniae and the most significant increase of TLR5S gene expression occurred in head kidney 12h after being challenged with S. iniae, which was 132-fold higher than the PBS control. After infection with A. hydrophila, TLR5 and TLR5S showed up regulation at 12h and 24h and obvious down-regulation at 48h to 7d post-infection. TLR5 and TLR5S genes expression were suppressed by infection with CCRV in most tissues. In spleen, the expression of TLR5 was 0.017-fold while TLR5S was only 0.01-fold compared with PBS control. Among the four immune-related different tissues, the expression of TLR5 and TLR5S showed significant up-regulation in intestine and head kidney. For example, after being challenged with E. tarda and S. iniae, at time point of 12h, the expression of TLR5 increased 50.4-fold and 14.8-fold respectively in intestine, and the expression of TLR5S increased 52.8-fold and 132-fold respectively in head kidney. All data suggested that TLR5 and TLR5S were involved in the immune response of channel catfish against the intracellular bacterial and virus pathogen in a tissue-specific and pathogen-specific manner, and further confirmed that both of them played significant roles in the channel catfish innate immunity.

Abstract:The full-length cDNA sequence of interferon regulatory factor 7 (IRF-7) of tongue sole Cynoglossus semilaevis was cloned by rapid amplification cDNA end (RACE) method. The full-length cDNA of C. semilaevis IRF-7 (CsIRF-7) is 1957bp, containing a 48 bp 5′-untranslated region (5′-UTR), 1302 bp open reading frame (ORF), and a 607 bp 3′-UTR. The putative amino acid sequence of CsIRF-7 contains 433 amino acid residues. Through protein sequence alignment of IRF-7 in vertebrates,we found that the IRF-7 contains three completely conserved residues across all species,which are DNA-binding domain (DBD), IRF associated domain (IAD) and serine-rich domain (SRD). In contrast to IRF-7 of vertebrates other than fish, the DBD which is highly conserved has five tryptophan repeats, while CsIRF-7 and other fish IRF-7s have only four tryptophans with the second tryptophan residue missing. The deduced amino acid sequence of CsIRF-7 exhibited a high-level of homology with other fish, and shared the highest similarity (83.8%) and identity (73.6%) with the IRF-7 of Paralichthys olivaceus. The CsIRF-7 shared a low-level of homology with mammalia and aves. Phylogenetic analysis revealed that the C. semilaevis IRF-7 was clustered into IRF-7 subgroup with fish IRF-7 and formed the IRF-3 subfamily with IRF-3 group and other vertebrates IRF-7. Quantitative real-time method was used to detect the CsIRF-7 mRNA expression in different tissues. The results showed that CsIRF-7 was expressed in all tested tissues including liver, kidney, spleen, gill, intestine, blood cells, and heart. The high level expression of CsIRF 7 is detected in the intestine, heart, kidney, and spleen, while a lower-level expression in the liver, gill, and blood cells. These results will be helpful for further studies on functions of antiviral and antibacterial of CsIRF-7 gene.

Abstract:The elimination of florfenicol in Paralichthys olivaceus (T&S) was studied by oral administration. The concentrations of florfenicol in P. olivaceus (T&S) were determined by HPLC. The results showed that: 1）After oral administration at a dose of 50mg/kg for five consecutive days, the concentration of florfenicol in the tissues are ranked as: blood>visceral mass>muscle. The drug concentration in tissues stabilized after 6d, but florfenicol concentration in liver and kidney at 10d and 14d showed rebound phenomenon. 2）T1/2 were 32.09～40.73h , which showed the faster elimination and the less residual amount of florfenicol in the body. However, residual concentrations in the kidney and liver were more obvious. 3) To explore the development of off-drug period, considering edible parts and MRL standard(0.1 μg /g), under the culture conditions of P.olivaceus: average temperature of 16~18℃, off-drug period ≥18d, the product of temperature and time is no less than 324 degree•days.

Abstract:According to the gene sequences, the lectins in the genome of Chinese shrimp Fenneropenaeus chinensis, could be assorted into seven kinds of genes. In order to explore the expression differences among the seven lectins after WSSV challenge, real-time quantitative PCR was used to detect the expression profiles of these lectins at different time after WSSV challenge in Chinese shrimp. The results showed that, of the seven lectins, LT1(DQ167572.1), LT2(EU834289.1), LT3(EU834292.1) and LT5(EU834290.1) displayed hepatopancreas-specific expression profiles, and were WSSV-induced genes, while the other genes were mainly expressed in gills, intestines and muscles, and were non-WSSV-induced genes. The expression profiles of seven detected genes at different time after WSSV challenge were different with each other. Additionally, the analysis of variance (ANOVA) indicated that no significant difference was found at the expression level of seven lectins in gills, intestines and muscles, except hepatopancreases. These results showed that the biology function of the seven lectins were different from each other in Chinese shrimp F. chinensis.

Abstract:Feeding trials were conducted to investigate the effect of dietary protein and amino acid level on the growth of juvenile sea cucumber Apostichopus japonicus at 4.5~4.8 g. The experiments on juvenile sea cucumbers were conducted over 70 days using an artificial feed, mainly composed of fish meal, Sargassum thumbergii and lees (by products from wine and beer production). The content of protein in artificial feed was 14.7%, 17.7%, 19.1% and 21.5% respectively. The results showed that the weight gain and feed digestibility increased with the protein content in the diet. The optimum protein content was 21.5%, resulting in the highest weight gain and feed digestibility. Based on a second growth experiment over a 40 day period, during which five different feed formulas were tested, it was found that the weight gain was maximal when the diet was rich in the following amino acids: lysine, arginine, glycine and methionine. It suggested that lysine, arginine, glycine and methionine are important amino acids for the growth of sea cucumbers.

Abstract:In the present study, accumulation and elimination processes of two metals(Cu,Cd) in Crassostrea plicatula were simulated by using the two-conpartment kinetic model. A semi-static system was maintained throughout 36d exposure period in different concentrations of heavy metals. Kinetic parameters of bio-concentration were obtained from the two-conpartment kinetic model by nonlinear curve fitting for the two periods respectively, including uptake rate constant(k1), elimination rate constant(k2), bioconcentration factor(BCF), and biological half-life(B1/2). It was found that the metal concentration and elimination were well fittted to the two-conpartment model by using the goodness-of-fit test. The results indicated that: (1) In the uptake period, the uptake rate constant and bioconcentration factor of heavy metals generally decreased with the increase of heavy metal concentration in ambient seawater, and maximal content in the organism(CAmax)at the theoretic equilibrium increased with the increase of heavy metal concentration in ambient seawater; (2) During the elimination period, the uptake rate constant of heavy metals and the B1/2 of Cu in C. plicatula were decreased with the increase of heavy metal concentration in ambient seawater (uptake period), and the B1/2 of Cd in C. plicatula had no obvious trend with the exposure concentration; (3)The Cu bioaccumulation ability of C. plicatula was higher than Cd; (4)The bioaccumulation ability of large-size C. plicatula was stronger than small-size ones, indicating that the differences in size of C. Plicatula had no significant effect on the elimination of heavy metals .

Abstract:The Zhikong scallop spats’ (1.177～2.017 mm in shell length) diurnal rhythm of filtration rate were determined using static water method. For spats, the microalgae density was 3.0×104cell/ml and the temperature was 24±0.5℃, and for veliger, the microalgae density was 1.0×104cell/ml and temperature was 18±0.5 ℃. We measured the filtration rate of the veliger and spat at 17∶00，21∶00，01∶00，05∶00，09∶00，and 13∶00. Analysis showed that the filtration rate of the veliger and spat had similar diurnal rhythm. They had the lowest filtration rate(veliger: 0.002 6 ml/h•ind ,spat: 0.123 1 ml/h•ind) in the daytime(9∶00~13∶00) and the highest one(veliger: 0.025 8 ml/h•ind;spat: 0.509 6 ml/h•ind) in the night(21∶00~1∶00). There were significant difference between the highest and the lowest value（P<0.01）.

Abstract:In order to investigate the genetic structure and variability of Scapharca broughtonii，thus to make proposals for the species culture and breeding，four geographical populations of S. broughtonii were collected from Shandong Penglai（PL），Shandong Huangdao（HD），Jiangsu Qiansandao（QSD）in China，and Tongying in Korea（KTY） respectively，and the genetic variation was analyzed based on ITS nucleotide sequences of rDNA. PCR amplification products were sequenced and aligned subsequently. The 486bp and 541bp necleotide sequences of ITS-1 and ITS-2 respectively were obtained（primers and insertion/deletion sites included）. The results showed that though the genetic variation of ITS-1 and ITS-2 was relatively high，the base composition of them was stable.The genetic diversity of the four populations was rich, which indicated that the germplasm resource of the species was in good condition. The genetic diversity of HD population was the richest.Also, the number of variation sites of ITS-1 and ITS-2 were the largest in HD population. There were many neighbor common variation sites in HD and QSD populations. In this study, we calculated the genetic distances between individuals and constructed the molecular phylogenetic tree by using minimum evolution method by MEGA 4.0. The results showed that the nucleotides in one species' genome had different variation frequency, and variation of nucleotides segment couldn't represent the genetic variation of the whole genome. Fifty-five blood clams did not cluster obviously based on the ITS-1 segment base sequences. However, 57 blood clams can be clustered into two catigories completely, one as HD and QSD population, the other as KTY and PL populations. The information from nucleotides variation of ITS-2 is similar to the conclusion from multivariate morphomertrics and molecular marker analysis carried out by our research group in the past. The genetic diversity of HD population is very rich, while the KTY population showed relatively low variation. There were certain genetic differences between each population, so that cross- and selective-breeding may be tested according to their biological characteristics.

Abstract:The Yellow River estuary is a major fishery and spawning ground in the Bohai Sea. With the decrease of Yellow River runoff, the salinity of water in the estuary became higher, thus many researches focused on the study of ecological water requirement. In this experiment, we used the local dominant species Mactra veneriformis as target organism to detect the influence of high salinity stress on ingestion and growth by semi-static water. The results showed that, with the increase of salinity, the ingestion rate and daily growth rate of shell length and weight decreased. The ingestion rate increased firstly under sudden changes in salinities, and then increased. Through the One-Way ANOVA analysis, the thresholds of ingestion and growth were obtained as 26～28 and 22～24, respectively. Finally,the threshhold of the high-salt stress on ingestion and growth of Mactra veneriformis was determined as 22～24.

Abstract:In order to explore the effect of environment on the nacre color of shells, we cultured a group of genetically identical oyster Pinctada martensii in four different areas: Li'an in Hainan Province, Liusha in Guangdong Province, Beihai and Fangcheng in Guangxi Province. One year later, the shell colors of P.martensii in the four areas were measured using CSE-1 Imaging and Color-Measuring System, and the Lab values were recorded. The results showed that the oysters cultured in different areas exhibited different shell colors. Specifically, the oysters of Beihai, Fangcheng and Li'an were quite close in shell colors, having higher lightness and green and yellow tone, while the oysters of Liusha showed significant color difference in shells to those of the other three areas, demonstrating relatively lower lightness and green and yellow tone, quite close to white color.

Abstract:With the unique advantages of high sensitivity, equipment independence, and rapid procedure, loop-mediated isothermal amplification (LAMP) has been extensively developed to detecting various pathogens. The substances of enzyme, dNTP, and other complex organic molecules in LAMP mixture are subjected to a variety of chemical changes under room temperature, many of which are quite unacceptable in terms of storage and transportation. In order to solve this problem, we tried to develop a lyophilization technology for the protection of the LAMP mixture with the addition of trehalose as a stabilizer. The predictive effects of freeze-drying and different concentrations of trehalose (5%, 10% and 15%) in LAMP mixture during storage under temperature of 4℃, 25℃ and 37℃ on amplification activity of LAMP were analyzed systematically. The non-freeze-dried reagents without trehalose were also stored at the same temperature as controls. Our results showed that the lyophilizing LAMP reagents with 15% trehalose exhibited the greatest performance. Furthermore, at 4℃, the half-life of freeze-dried mixtures with 15% trehalose (w/v) was 112.3d, which was 6.5-fold of the control. At the temperature of 25℃, the half-life of treated reagents was 36.2d, which was 8.7-fold higher than control. Even under the temperature of 37℃, the half-life was 6.2d, which was 2-fold of the control. We report here the cost effective, simple developing procedure of a freeze-drying LAMP reagent which will promote the widespread use of LAMP in the field.

Abstract:The residue of tetracycline antibiotics including oxytetracycline(OTC), tetracycline(TC), doxycycline(DC) and chlorotetracycline(CTC) in carps，eels, shrimps and sea cucumbers were determined with the optimized method of high performance liquid chromatography(HPLC) coupled with fluorescence detection(FL). The target compounds were extracted with acidic EDTA-Mcllvaine buffer solution,then enriched and cleared up using a hydrophilic-lipophilic balance(HLB) SPE cartridge,followed by separation by the C18 RP-HPLC column with imidazole buffer-methanol. The limit of quantification of tetracycline antibioitics in samples were 20, 20, 40, 40 μg/kg for TC, OTC, CTC and DC respectively. The average recovery for all the compounds were 70.5%~100%. The inter-variation coefficients were under 10.0%，and intra-variation coefficients were under 15.0%. The method is rapid, sensitive, accurate, reliable and can be applied in the analysis of antibiotics residues in aquatic products.

Abstract:Gas chromatography coupled with solid-phase extraction was developed for determination of tefluthrin, bifenthrin, fenpropathrin, cyhalothrin, permethrin, cyfluthrin, cypermethrin, fenvalerate, tau-fluvalinate, and deltamethrin residues in surface sediment from aquaculture area. The pyrethroids were extracted with petroleum ether-acetone(V/V, 3∶1), followed by sulfide removal by copper powder, and purification on a carbon-florisil SPE column, then analyzed by gas chromatography with electron capture detector(GC-ECD). The linear range of tefluthrin was 2.5～100 ng/ml and the other nine prrethroids were 5～200 ng/ml(r≥0.996 7). When spiked with 2μg/kg, 20μg/kg, 40μg/kg , the average recoveries ranged from 72.8% to 107% and their relative standard deviations（RSD）were 2.75%~11.3%. The limits of detection(LOD) and quantitation( LOQ ) for all pyrethroid were in the range of 0.01～0.1μg/kg（wet weight） and 1～2μg/kg（wet weight） respectively. This method can be applied in the detection of pyrethroid residues in aquaculture area．

Abstract:Based on environmental survey in March, May, August, October, 2011 and May, August, October from 1997 to 2010, the status of nitrogen and phosphorous pollution and its variations in shellfish culture areas of Jiaozhou Bay were analyzed and assessed. It was found that: (1) Nitrogen and phosphorus pollution in shellfish culture areas of Jiaozhou Bay was more serious in 2011. Concentrations of these nutrients met the national standard in March only, but were above the limit in all other months, when DIN concentrations were 11.11% to 100% and phosphate (PO4-P) concentrations were 33.33% to 66.67% higher above the limit. (2) DIN and PO4-P in survey areas showed obvious seasonal variations. The maximum and minimum values appeared in October and March, respectively. The mean pollution index (Pi) of DIN and PO4-P were 1.35 and 0.93 respectively, indicating more serious pollution by nitrogen. (3) Spatial variation was also found for DIN and PO4-P in Jiaozhou Bay, with much higher concentrations of nutrients in the western and eastern regions of shellfish culture area but lower in the central region. The ranking of monthly DIN and PO4-P eutrophication was August>March>May>October. (4) Relatively serious eutrophication was found in the survey areas, with 44.44%~100% sampling stations were in eutrophication. The ranking of average monthly eutrophication index values was August>October>May>March. (5) In the four months in 2011, the N/P ratio of most seawater samples was between 20.96 and 43.22, which was above the Redfield ratio, with only few exceptions in May, indicating that PO4-P may be the main limiting factor for phytoplankton growth. (6) Pollution index of nitrogen and phosphorus showed obvious interannual variation. Pi of DIN was the highest in 2008 and the lowest in 1997 and 2000, and Pi of PO4-P was the highest in 1997 and the lowest in 2011. (7) The causes of seawater eutrophication were very complex, but two main factors were identified in the aquaculture areas of Jiaozhou Bay. One was the large amounts of nitrogen, phosphorus and other nutrient inputs from the rivers, and the other was the shellfish aquaculture self pollution. In recent years, although the nitrogen and phosphorus pollution in shellfish culture areas of Jiaozhou Bay showed no obvious deterioration, eutrophication is still serious, which may contribute to more frequent algal blooms.