Abstract:Due to the increasing effects of anthropic activities and the climate change, there appears to be a rapid shift in the dominant species of fish in the Yellow Sea. Since the 1980s, Liparis tanakae has become the dominant species and now plays an important role in the dynamics of fishery resources in the Yellow Sea. In order to clarify the recruitment and the dynamics of L. tanakae population, we analyzed the inter-annual variations in the absolute fecundity, relative fecundity, egg diameter and gonadosomatic index of L. tanakae, based on the bottom trawl data collected by R/V “Beidou” in the central and southern Yellow Sea during 1985-1986 and 2011-2012. We found that the distribution of L. tanakae’s egg diameter changed from multi peak in 1985-1986 to double peak in 2011-2012, with an increase in the average value from (1.1±0.2) mm to (1.2±0.1) mm. The gonadosomatic index and condition factors measured in Jan, 2011 were lower than those measured in Jan, 1986, and there were no significant differences (P>0.05) between these two parameters and the absolute fecundity. The absolute fecundity (F) of L. tanakae was 9407-84209 eggs (37677±3625 eggs in average) found in 1985-1986, and the relative fecundity estimated with the body length (FL) and the body weight (FW) was 446-1981 eggs/cm and 18-157 eggs/g, respectively. All the values of the fecundity measured in 2011-2012 were lower than those measured in 1985-1986: the absolute fecundity was 2984-81614 eggs (24728±2822 eggs in average), FL was 114-1690 eggs/cm, and FW was 15-106 eggs/g. No significant differences (P>0.05) were detected in fecundity parameters between the two periods according to the covariance analysis. There was a positive power index correlation between F and the body length, and a positive linear correlation between F and the body weight. There was also a positive linear correlation between FL and the body length. In contrast, a negative linear correlation was found between FW and the body length. However, there was no significant correlation between FL, FW and the body weight. These results suggested that the L. tanakae population was relatively stable under the influence of multi-stressors in the Yellow Sea.

Abstract:Changjiang River estuary is the major economic offshore fishing water. Construction of Qingcaosha Reservoir may become unfavorable factors of juvenile growth and complementary resources in Changjiang Delta. This may eventually lead to the degradation of estuarine spawning grounds and abnormal biological community structure, and may restrict the sustainable use of fishery resources. Ecological characteristics of phytoplankton are important indicators of environmental quality assessment of fisheries. A multidisciplinary study on netz-phytoplankton assemblage structure was carried out in Qingcaosha Reservoir adjacent waters during February to November, 2012. This study identified 7 phyla, including 46 genera and 71 species, in which diatoms dominated, and Cyanophyta, Chlorophyta, Dinoflagellates, Chrysophyta, Cryptophyta and Euglenophyta were also found. Skeletonema spp. was prevailed cross-season in the whole investigation period. Temperate coastal species were the major ecotype, while fresh water species were occasional. Cell abundance decreased in turn from spring, summer, autumn to winter and the average value of spring and winter were 548.09 and 2.19 ind/ml, respectively. The major factor of daily migration was tidal impact, while the seasonal variation was affected by diluted water. Seasonal variation patterns in the survey area were discriminated by the cluster analysis of the phytoplankton assemblages. The change in seasonal runoff of the river and direction transformation of Changjiang diluted water was responsible for the variations. The canonical correspondence analysis (CCA) showed that the environmental factors drive the assemblage variation during all seasons.

Abstract:Dosidicus gigas, jumbo flying squid, is widely distributed in the eastern Pacific Ocean, and has become one of the most important target species for Chinese squid jigging vessels. There is a great potential for exploitation due to the abundance of this species. The precise forecasting of hotspots could provide scientific guidance for squid jigging fleets. Hotspots of D. gigas are closely related to the abundance distribution which is affected by marine environmental factors including sea surface temperature and sea surface height. We established the suitability index of D. gigas based on either catch per unit fishing effort or fishing effort, with two data resources: the fishery data from Chinese squid jigging produced from 2008 to 2010, and the environmental data including real-time sea surface temperature and sea surface height obtained from remote sensing. Then we used the arithmetic average model to establish different habitat suitability index. We also used the established model based on the fishing catch and marine environmental data in 2011 to test the habitat suitability index model. We found that the suitability index based on fishing effort and environmental factors was consistent with the normal distribution (P<005). However, the suitability index based on catch per unit fishing effort and environmental factors was not significant (P>0.05). We hence used the habitat suitability index model based on fishing effort to forecast the hotspots of D. gigas. It was found that in the area where the habitat suitability index was higher than 0.6, the catch accounted for 48% of the total, the fishing effort occupied 46% of the total, and the CPUE ranged from 5.23 to 5.76 t/d. We also found that the forecasted hotspots of D. gigas were basically located in the actual fishing area, which indicated that this model could effectively forecast the hotspots of D. gigas.

Abstract:In order to study the migration, transformation, and the dilution mode of nutrients in the freshwater-seawater mixing plume at the Changjiang Estuary, we simulated environmental factors in the laboratory, such as salinity (S), temperature (T), pH, and dissolved oxygen (DO), and explored their influences on the silicon (SiO₃-Si) concentration in the mixing plume of the diluted Changjiang River water and the external seawater. Our results and their implications are: (1) when the external seawater was mixed with either filtered or unfiltered diluted Changjiang River water, the concentration of SiO₃-Si stabilized after 40 h. (2) The SiO₃-Si concentration in the freshwater-seawater mixing plume showed a clear negative correlation with the salinity, indicating that the SiO₃-Si concentration at the Changjiang Estuary was mainly affected by the physical mixing dilution. In the mixture of the external seawater and the diluted Changjiang River water, the concentration of SiO₃-Si showed no significant difference no matter whether the Changjiang River water was filtered, which suggested that the suspending particles in the water had no effect on the SiO₃-Si concentration in the freshwater-seawater mixing plume. (3) The concentration of SiO₃-Si slightly increased as the temperature of mixed water rose. This implied that there may be a seasonal change in the flow of SiO₃-Si from the diluted Changjiang River water to the offshore area of the East China Sea. (4) The SiO₃-Si concentration in the mixed water remained unchanged at pH 4-8, but altered when pH was 10. Simple explanations were that higher pH enhanced the SiO₃-Si desorption by suspending particles, or changes in the pH affected the form of SiO₃-Si. (5) The oxygen levels (anoxia or oxygen-enriched state) did not affect the SiO₃-Si concentration.

Abstract:Many studies have been conducted to investigate the pollution status of herbicides in rivers and groundwater; however, the pollution status of seawater is still unclear. In the present study, we established an ultra performance liquid chromatography with electrospray ionization tandem mass spectrometry (MS)method to determine 13 triazine herbicides and atrazine-desethyl in seawater. Samples were collected from the surface seawater of 43 stations in Laizhou Bay, China, filtrated and purified through the HLB solid phase extraction cartridges and separated by AcquityTM UPLC HSS C18 column (2.1 mm×100 mm, 1.8 μm), and then the analytes were confirmed and quantified using MS/MS system in multiple reaction modes. Only atrazine, prometryn, propazine, ametryn and atrazine’s metabolite atrazine-desethyl were detected, and the detection rates and average concentrations were 100% (31.3 ng/L), 97.7% (6.49 ng/L), 51.2% (1.57 ng/L), 100% (12.4 ng/L), and 93.0% (9.14 ng/L), respectively. The concentrations of the triazine herbicides in the bay were higher than those in the bay-mouth, which may be due to the different seawater exchange rates and terrigenous inputs. The concentrations in the western stations were higher than those in the eastern stations. The concentration of atrazine was significantly correlated with the concentration of atrazine-desethyl (R=0.336, n=43, P=0.034). The risk entropy of atrazine was less than 0.01 in all stations, and the ecological risk can be neglected theoretically.

Abstract:The technique of measuring fish appetite is the key to accurate automatic feeding. As light attenuates quickly in the water, visual observation is not an effective method to measure the fish appetite in the turbid water body of fish farms. Recent studies have shown that the feeding activity of fish can be measured directly by monitoring sounds emitted by the fish during a feeding period. In our study, we used an acoustic monitoring platform that was composed of a programmable filter and a standard measuring hydrophone, with bandwidth from 1 Hz to 470 kHz and with free field voltage sensitivity of -217 dB (reference level 1 V/μPa) under 40 kHz. Tilapias in the laboratory pond were fed by hand, while the feeding sound of Tilapia was recorded for the analysis of its feeding activity. The feeding sound with the frequency ranging from 0 to 6 kHz could be distinguished from background noises. The appetite of Tilapias faded after a period of feeding, resulting in the decrease in feeding activity and the subsequent change in the acoustic power. The acoustic power had a significant positive correlation with the feeding activity. Therefore efficient feeding strategies can be established, based on the appetite feedback (generated by the moving average of acoustic power) together with growth models and feeding models.

Abstract:In this study we investigated the pharmacokinetics and elimination of enrofloxacin (ENRO) and its metabolite ciprofloxacin (CIP) in Paralichthys olivaceus. After the oral administration of a single dose at 80 mg/kg•bw, the time courses of concentrations of ENRO and CIP in fish tissues were measured with high-performance liquid chromatography and a fluorescence detector. At 0.25 h, the muscle retained the lowest level of ENRO residues among five tissues (See below). The elimination half-life of ENRO in the five tissues was in the order: gills＞liver＞kidney＞blood＞muscle. ENRO in muscle was eliminated fastest and had the lowest elimination half-life of 67.759 h; hence in the 12th day after the drug withdrawal, ENRO in the muscle was no longer detectable. At 0.25 h, the concentration of CIP residues in different tissues was in the order: liver＞kidney＞blood, while CIP could not be detected in the muscle and gills at the same time. In blood, liver and kidney, ENRO was still detectable in the 22nd day after the drug withdrawal; whereas CIP could not be detected in these tissues since the 7th day. These results indicated that the metabolism and elimination of ENRO in P. olivaceus was relatively slow. We also found that the degradation of ENRO into CIP occurred only in a short period after the ENRO administration. Obviously the elimination of CIP in flounder was much faster than that of ENRO.

Abstract:This study evaluated the effects of vitamin A supplementation on the reproduction and offspring quality of tongue sole Cynoglossus semilaevis. Broodstock (females, average weight 1.58 kg; males, average weight 0.2 kg) were fed with one of the three diets (Ac, control, no vitamin A supplementation; Al, 20 IU/g vitamin A as retinol acetate; and Ah, 100 IU/g vitamin A as retinol acetate) for 10 weeks at water temperature of 19-25℃. Results showed that female gonad mature rates (GMA) in the Al and Ah groups were higher than that of the Ac group. The relative fecundity in the Al group was the highest (1.57×10⁵ ind./kg), while that in the Ac group was the lowest (1.00×10⁵ ind./kg). Hatching and fertilization rate in the Al and Ah groups were significantly higher than those in the Ac group (P<0.05). Buoyant eggs rate, larvae length, larvae survival rate and survival activity index of larvae (SAI) were the highest in the Al group. The dietary vitamin A supplementation increased the abnormal larvae ratio, but the effect was not significant (P>0.05). Vitamin A content in the tissues and eggs and activity of the superoxide dismutase (SOD) in serum were significantly increased with the increasing dietary vitamin A level (P<0.05). In addition, compared to those in the control group, vitamin C levels in the eggs were significantly higher in the two treatment groups (P<0.05); and the oxidation resistance of the eggs increased in the two treatment groups. The levels of n-3HUFA, n-6PUFA and PUFA in the eggs in the Al and Ah groups were all higher than those in the Ac group. These results demonstrated that the proper amount of vitamin A supplemented in the diet may promote the development of broodstock gonads and improve the quality of the eggs and larvae. The broodstock in the Al group (20 IU/g) had the best reproductive performance compared to those in the Ac and Ah groups．

Abstract:Feed processing technology was a main factor which affects the quality of feed particle and its utilization by cultured animals. In this study, we compared two feed produced with different processing technologies in terms of their physical properties, their effects on the growth of turbot juvenile, and their effects on feed utilization and the aquaculture environment. Two kinds of diets (D1 and D2) were fed to turbot juvenile Scophthalmus maximus with initial weight of 16 g for 64 d. D2 showed significantly higher values than D1 (P<0.05) in the average diameter of feed pellets, the average weight of 100 feed pellets, and the water stability, but also showed significantly lower values regarding the water absorption, the bulk density, and the settling velocity (P<0.05). The weight gain rate, specific growth rate, feed intake rate, and protein efficient rate of turbot juvenile fed with D2 were all significantly higher than the D1 group (P<0.05). The apparent digestibility coefficient (ADC) of dry matter and crude protein of D2 group was also significantly higher than D1 group (P<0.05). However there were no differences in ADCs of crude lipid and total phosphorus between the two groups (P>0.05). The nitrogen and phosphorus levels in the aquarium increased significantly after 18 h feeding. The nitrite nitrogen per liter water produced by one kilogram of D2 fish was significantly higher than that produced by D1 fish (P<0.05); whereas the nitrate nitrogen and the total nitrogen produced by D2 were significantly lower (P<0.05). The labile phosphate and total phosphate produced by D2 group were also significantly lower than those of D1 group (P<0.05). The results suggested that the physical properties of feed pellets, the feed utilization, and the aquaculture environment could be all affected by different processing techniques.

Abstract:Clones are defined as groups of genetically identical fish. It is unclear whether clonal fish have identical reactions to toxicants. To address this question we used the static test method to investigate the acute toxicity of Hg²⁺ to clonal and common Japanese flounder, Paralichthys olivaceus. The clonal and common Japanese flounder in the experiments were produced by Beidaihe Central Experiment Station, with the average body length of (12.38±1.13) cm and the average body weight of (17.97±5.79) g. Fully aerated and filtered seawater [temperature (21±1.2)℃, pH 8.1-8.3, salinity 31.0, and hardness 6200 mg/L] was used in the tests. The results showed that the clonal Japanese flounder treated with high concentration of Hg² first became anxious, rose to the water surface, and swam around in the aquarium, before they eventually stayed still at the bottom of the aquarium. The majority of common Japanese flounder treated with high concentration of Hg² remained at the bottom all the time, except for only a few that occasionally swam to the surface. As the poisoning aggravated over the time, the common Japanese flounder began to rotate, swam rapidly or jumped up and down when touched by a glass rod; in the contrast, clonal Japanese flounder swam slowly around the aquarium regardless of the touch. Both the clonal and common Japanese flounder eventually stopped breathing and died with rigid bodies. At 24, 48 and 96 h, the median lethal concentrations of Hg² to clonal Japanese flounder were 1.50, 0.86, and 0.84 mg/L respectively. The values of common Japanese flounder were 1.93, 1.04, and 0.90 mg/L respectively. These results suggested that the clonal Japanese flounder was more sensitive to Hg² than the common Japanese flounder. A trend was observed in all groups of clonal Japanese flounder that they mainly died during the 8 hours starting from the occurrence of the first death; however, the death of the common Japanese flounder occurred evenly during the whole experiment.

Abstract:In this study we compared the reproductive performance of female parents, growth of these filial generations, survival, and yield of six inbreeding generations (F1-F6) of full-sib families of Portunus trituberculatus. The statistical methods applied in the study were single factor analysis of variance (One-Way ANOVA), least significant difference (LSD), multiple comparison analysis, and analysis of covariance. We set up the reproduction between two full-sib inbreeding families (F3-F6), Pedigree A (F6) and B (F3), at different genetic purification degree. There are four experimental groups, of which there are two inbreeding groups F66 (A♀×A♂) and F33 (B♀×B♂) and two hybrid groups F63 (A♂×B♀) and F36（B♂×A♀). By comparing the growth of filial generations of four experimental groups during the outdoor periods, we found that at 80-day, 100-day, 120-day and 150-day, different characters of the same hybrid group had different heterosis at the same growth stage, and the same character of the same experimental group also had different heterosis at different growth stages. In inbreeding groups, different characters had the same growth trend as F33 > F66. As for the growth rate, the hybrid groups showed overall heterosis (-3.50% to 19.47%) with different heterosis levels in individual characters. In terms of survival, hybrid groups showed significant heterosis compared to inbred groups with a heterosis rate of 24.8%, and the overall heterosis of 15.99% in the yield associated with survival. Family A benefited more from the hybridization than Family B regarding both the growth characters and the survival, which was consistent with the results of early growth study in the four sub-generation experimental groups. Genetic differences between Family A and B were the basis of potential heterosis and improvements in characters.

Abstract:Biological accumulation and release of heavy metals in Ruditapes philippinarum have been observed in previous studies. However the specific process in individual tissues remained unclear. In this study, we used two-compartment bio-concentration model to evaluate the kinetic parameters of the biological accumulation and release of seven heavy metals (Pb, Zn, Cu, Cd, Cr, Hg, and As) in different tissues and organs of Ruditapes philippinarum, including visceral mass, gill, foot part and mantle tissues. In the experiments R. philippinarum was exposed to water containing all seven heavy metals. The uptake rate constant (k1), elimination rate constant (k2), bio-concentration factor (BCF), biological half-lives (B1/2), and equilibrium concentration in vivo (CAmax) of heavy metals were determined with non-linear curve fitting method. The results were summarized below: (1) Different heavy metals were accumulated with a preference in major organs and tissues of R. philippinarum. The CAmax of Cu, Cd, As and Pb in R. philippinarum followed the order: visceral mass > foot part > gill > mantle tissues; the CAmax of Cr: visceral mass > gill > foot part > mantle tissues; Hg (CAmax): foot part > mantle tissues > gill > visceral mass; Zn (CAmax): foot part > gill > mantle tissues > visceral mass. (2) BCFs of seven heavy metals in R. philippinarum were significantly different (P<0.05). Cu was the most accumulated heavy metal in R. philippinarum with a BCF range of 1514.0-4818.0, followed by Cd and Hg with a BCF range of 104.5-975.4 and 255.9-447.4, respectively. As, Cr, Pb and Zn were accumulated at a lower level with the BCF values below 300. The weakest accumulation was observed for As, and the BCF values in foot part, gill and mantle tissues were 56.0, 53.1, and 47.4 respectively. The BCF of Zn in visceral mass was 61.9. (3) After 25 days, concentrations of heavy metals in the major organs and tissues of R. philippinarum were still at high levels, which indicated R. philippinarum had weak ability to release heavy metals. Although the two-compartment kinetic model efficiently described the accumulation of heavy metals in R. philippinarum with most fitting indicators (R) above 0.9, it was not suitable for the simulation of the release phase, for example, the R values of Pb and Cu were only 0.110 and 0.230 respectively.

Abstract:As an immuno-potentiator Schizochytrium limacinum was widely used in aquaculture, however its impact in the farming of sea cucumber Apostichopus japonicus Selenka has not been reported. The purpose of this study is to evaluate the effects of dietary S. limacinum on the growth, immunity and the digestion enzyme activities of sea cucumber A.japonicus, and therefore to determine its optimal portion in the feeding diet.In the 8-week experiment, three groups of sea cucumber(with initial average weight of 12.5±2.0g) were fed with basic diet implemented with different portions of S. limacinum: 0.5% (Group B), 1.25% (Group C), and 2.0% (Group D).The control group (A)was fed with the same basal diet only. The results showed that the specific growth rate (SGR) was significantly higher in Group C and D compared to the control group (P<0.05). Compared to the control, Groups C and D also showed significantly higher activities in terms of alkaline phosphatase (AKP),acid phosphatase(ACP), lysozyme (LZM), superoxide dismutase (SOD), phagocytic activity, and intestinal amylase (P<0.05).When fed with 1.25% S.limacinum (Group C), the sea cucumber exhibited significantly higher respiratory burst activity in coelomic fluid and higher activity of intestinal protease than the control group (P<0.05). In Group B, the activities of AKP, LZM and intestinal amylase were significantly higher than those of the control group(P<0.05). During the feeding experiment, all the treated sea cucumbers were healthy. Based on the facts above, our conclusions are: (1) Basic diet with the addition of 1.25% S. limacinum effectively enhances the growth and the immunity of sea cucumber;(2) Long-term administration of S.limacinum should be safe in sea cucumber farming.

Abstract:Gracilaria bailinae, the genus Gracilaria, is a commercially important species of red seaweeds, and is currently farmed in the ponds along the coast of south China. It is commonly used as the food source in abalone aquaculture and employed for the extraction of agar as a raw material. However, the optimum growth conditions of G. bailinae are unclear. This study examined the physiochemical responses of G. bailinae to different levels of temperature, light and salinity to determine the suitable environmental conditions for the growth of G. bailinae. Thalli of G. bailinae were cultured under different conditions of temperatures (18℃, 23℃, 28℃, 33℃, and 38℃), salinities (13, 18, 23，28, 33, and 38), and light intensities (3000 lx, 6000 lx, 9000 lx, and 12000 lx), and then the growth and biochemical components of the algal thalli were measured. The results showed that the optimum temperature for the growth of G. bailinae is in the range of 23-33℃. Under this temperature range, G. bailinae exhibited higher relative growth rate, higher contents of soluble protein and phycobiliprotein, and lower activity of peroxidase compared with other temperatures. The optimum salinity for the growth of G. bailinae is between 18-28, and G. bailinae displayed a higher tolerance to low salinity than to high salinity. The optimum light intensity for the growth of G. bailinae is 6000 lx. These results suggest that it is essential to choose suitable sea area or pond and season for successful mariculture.

Abstract:Vibrio harveyi is widely distributed in the marine estuarine environments and has been widely recognized as a primary pathogen of many commercially cultured invertebrate species over the world. Breeding season of spring, 2011, the outbreak of mass mortality of mysis of Fenneropenaeus chinensis L. occurred in a hatchery in Lianyungang, Jiangsu Province. In this study, we isolated and identified the pathogenic bacteria, analyzed the pathogenicity of isolated strains, and detected the presence of virulence genes of isolated strains. Dominant bacteria isolated from the diseased mysis were identified based on morphological characteristics, physiological and biochemical characteristics, sequences similarity and phylogenicity analysis of the 16S rRNA and gyrB genes. The phylogenetic trees were constructed using the neighbor-joining method, and pathogenicity of the tested strain was analyzed by bath challenge experiment on postlarvae F. chinensis and P. japonicus, and virulence genes of isolated strains were detected by a specific PCR assay. The characteristics of identified strains (kx1-kx4) consist with V. harveyi, and the 16S rRNA and gyrB genes of the tested strains exhibited high similarity with V. harveyi. Two strains (kx1 and kx2) were clustered with V. harveyi, which was supported by a high bootstrap value. The tested strain (kx1) was lethal to the healthy postlarvae F. chinensis and P. japonicus with the LD₅₀ of 2.0×10⁶ CFU/ml and 7.0×10⁵ CFU/ml, respectively. The genes (luxR, toxR, vhhA, vhhB and pap6) existed in these four strains with gene fragments were 679 bp, 390 bp, 1324 bp, 216 bp and 355 bp, respectively. These results suggested that the diseased mysis of F. chinensis were infected by V. harveyi, and the isolated four strains have the same virulence genes, which can be used as the biomarkers to detect pathogenic V. harveyi.

Abstract:The levels of chondroitin sulfate in Oncorhynchus keta are low, and the traditional method to separate and purify chondroitin sulfate primarily focus on cartilage. The present study was conducted to establish a new method to effectively obtain chondroitin sulfate from the low content raw materials. Chondroitin sulfate was prepared and isolated from the fish head through compound protease digestion and D958 resin adsorption. We also evaluated the effects of pH value of enzymatic hydrolysate, adsorption temperature and time on purification using the desorption capacity of chondroitin sulfate as an index. The results showed that the optimum purification conditions using D958 resin were as follows: the pH of enzymatic hydrolysate was 6.0, and the absorption temperature and time were 4℃ and 3 h respectively. The desorption rate was up to 87.66% under the optimum conditions. Chondroitin sulfate was effectively purified by the resin adsorption method. We have developed a key method to separate and purify chondroitin sulfate from low content raw material, which pave a new pathway for the utilization of wastes from O. keta processing.

Abstract:Based on the triangle artificial reef that was deployed in the project of reef construction at Xiaoshidao by Weihaixigang Aquatic Products Company, an equilateral triangle artificial reef was designed in the present study. The side length, width, weight of this artificial reef is 3.3 m, 2 m and 8.1 t, respectively. According to the wave and current condition at Shandong Province, 5 current velocities and 8 wave conditions were set. The maximum force, coefficient of maximum static friction, coefficient of anti-rolling, and coefficient of anti-slide at different test conditions were calculated. With the increment of wave period and wave height, the maximum forces acting on the reef increased. The maximum forces also increased gradually with the increase of current velocity; and the relationship between maximum force and velocity was expressed as Fmax=4.146v2. The results of stability calibration showed that the reef slided when the water depth reached 15 m and the wave height reached 6 m. The reef kept stable on the sea floor in which the water depth is 15 m and the current velocity is 1 m/s. To validate the calculation results, an observation test of the stability of this reef was carried out. The results of the observation test and those from calculation matched. The stability of the equilateral triangle artificial reef met the requirement and it is suitable to use in Shandong.

Abstract:Aeromonas is one of the pathogens causing skin ulcer syndrome in cultured Apostichopus japonicas. It is important to monitor the population of these bacteria in the culture environment for disease control. Unfortunately, current real-time quantitative PCR detection method is mostly based on the pure culture and cannot truly reflect the bacteria population in the sediment environment. Therefore, we established a real-time fluorescent quantitative PCR method to improve the pathogen detection. The simulative sediment samples were made by adding known-concentration bacteria into the sterilyzed sediment; the DNA extraction method was selected by comparing three improved DNA extraction methods; genus-specific primers were selected based on the GyrB gene of Aeromonas. Meanwhile, PCR reaction conditions and systems were optimized and a standard curve based-on extracted sediment DNA was utilized to examine the bacteria in the sediment. The results indicated that the modified lysozyme-SDS gentle lyse method and genus-specific primers (IAF and IAR based on GyrB gene of Aeromonas) were efficient and unique to detect Aeromonas. In addition, the standard curve was set up by using the extracted DNA from the simulated sediment samples as template. The new real-time fluorescent quantitative PCR protocol was highly sensitive and specific to detect different species of Aeromonas from the sediment with the quantification limit of 103 CFU/g. The coefficient of variation fell between 0.21% and 0.80% by statistical analysis (less than 5%), which demonstrated the good repeatability of the method. Thus, we have established a highly sensitive, specific, and reproducible method to measure Aeromonas in sediment environment of aquaculture even with low quantification.

Abstract:Vibrio splendidus is a deleterious pathogenic bacterium for most marine animals, and it causes great losses in aquaculture industry. To develop a quantitative detection method of V. splendidus is important because its pathogenicity is closely related to the population density. To develop a rapid SYBR Green I real-time fluorescence quantitative PCR method, a pair of specific primers were designed according to V. splendidus gyrB gene to determine the specificity and sensitivity. The real-time PCR amplification conditions were optimized. Recombinant plasmid containing gyrB gene of V. splendidus was constructed and used to establish the standard curve. The detection limit and reproducibility were calculated. A 251 bp fragment was amplified from chromosomal DNA, but no positive reaction was detected in 9 other bacteria species using conventional PCR, which indicated that the primer pair has good intra-species specificity and inter-species commonality. The standard curve was y=-3.338x+37.67; the correlation coefficient was 0.999 and the amplification efficiency was 0.99, indicating a good linear relationship between initial templates and CT values. The melting curve had only one specific peak when annealing temperature was 62℃. The detection limit of the assay was 20 copies per reaction. The results indicated that the established SYBR Green I real-time fluorescence quantitative PCR method for V. splendidus had high specificity, sensitivity and repeatability, which may help V. splendidus diagnosis and epidemiology.