The Anniversary Variation Of The Number Of Several Bacterial Groups And Rapid Detection Method In The Environment Of Apostichopus Japonicus Culture Ponds

With the fast development of Apostichopus japonicus aquaculture, there is agrowing disease problem. As the integral part of the ecosystem in Apostichopusjaponicus culture ponds, bacteria and physicochemical indexes are bound up with thesea cucumbers health. Therefore, it is crucial to detect the anniversary variations ofphysic-chemical indexes and culturable bacteria in the environment of Apostichopusjaponicus culture ponds. Besides, it is necessary to detect and quantify the number ofknown pathogens, such as Aeromonas and Vibrio parahaemolyticus, in theaquaculture environment rapidly in order to warn the accurance of the disease and totake active action in time. In this paper, the numbers of several bacterial groups andphysicochemical indexes in the water and sediment samples were tested inApostichopus japonicus culture ponds regularly, and the rapid detecting methods forthe major pathogens were established. The main contents and results were as follows:1. From April2012to April2013, the dynamic variation of4culturable bacteriagroups (heterotrophic bacteria, vibrios, nitrifying bacteria, sulphur bacteria) and4physicochemical indexes (temperature, pH, salinity, dissolved oxygen) in water ofApostichopus japonicus culture ponds in Jimo, Shandong province were monitored,besides,6culturable bacteria groups (heterotrophic bacteria, vibrios, nitrifyingbacteria, nitrate-reducing bacteria, sulphur bacteria, sulphurate-reducing bacteria) and6physicochemical indexes (temperature, pH, redox potential, sulfide, organic carbon,total nitrogen) in the sediment were detected. Meanwhile, the correlation betweendifferent bacteria groups and physicochemical environmental indexes in sedimentwere analyzed. The result showed that the numbers of heterotrophic bacteria andvibrios in water varied in the ranges of7.10×102cfu/mL~5.80×103cfu/mL,1.00×101cfu/mL~3.60×102cfu/mL, respectively. The numbers of nitrifying bacteria and sulphurbacteria were varied in the ranges of1.08×102cfu/mL~6.09×104cfu/mL,1.25×101cfu/mL~5.11×103cfu/mL, respectively, with the same variable trend with thetemperature. Numbers of heterotrophic bacteria, vibrios, nitrifying bacteria, nitrate-reducing bacteria, sulphur bacteria and sulphurate-reducing bacteria in sediment were varied in the ranges of1.16×104cfu/g~9.62×104cfu/g,1.16×102cfu/g~9.01×103cfu/g,4.20×103cfu/g~7.48×104cfu/g,4.80×103cfu/g~5.85×104cfu/g,5.50×102cfu/g~4.45×103cfu/g and9.30×101cfu/g~8.38×102cfu/g, respectively. pH, redox potential, sulfidewere varied in the ranges of7.10~7.61,-42.18~-65.06mV and36.9~89.7mg/kg,respectively. pH、salinity、dissolved oxygen in water were varied in the ranges of7.35~8.31，30.5~37.6，5.7~9.31mg/L.According to the correlation analysis, water temperature showed a significantnegative correlation with dissolved oxygen, while showed a significant positivecorrelation with the heterotrophic bacteria, vibrios, nitrifying bacteria and sulphurbacteria; pH showed a significant positive correlation with salinity, dissolved oxygenshowed negative correlation with heterotrophic bacteria. Redox potential showed asignificant positive correlation with pH in the sediment, however, both of themshowed an extreme significant negative correlation with sulfide. The sulfide showedan extreme significant positive correlation with heterotrophic bacteria, sulphurate-reducing bacteria. The nitrifying bacteria，showed an extreme significant negativecorrelation with sulphur bacteria and nitrate-reducing bacteria. The total nitrogenshowed significant correlation with nitrifying bacteria. There are also somecorrelations between different bacteria groups:1) the heterotrophic bacteria showed asignificant correlation with nitrifying bacteria and sulphurate-reducing bacteria;2) thenitrifying bacteria showed an extreme significant positive correlation withsulphurate-reducing bacteria;3) the vibrios showed a positive correlation withheterotrophic bacteria and nitrifying bacteria;4) the nitrate-reducing bacteria showeda negative correlation with nitrifying bacteria;5) the sulphurate-reducing bacteriashowed a negative correlation with sulphur bacteria.2. To establish the real-time fluorescent quantitative PCR method to reflect therelatively true number of Aeromonas in sediment environment of aquaculture basedon GyrB gene, the simulative sediment samples were made by addingknown-concentration bacteria into the sterilyzed sediment. The real-time fluorescentquantitative PCR method for testing Aeromonas in the sediment was developed bycomparing3improved DNA extraction methods, selecting suitable specific-primers,choosing the genes based on which the standard curve based-on sediment DNA wasestablished. The sensitivity, specificity and repeatability of the new method weredemonstrated. The result indicated that modified lysozyme-SDS gentle lyse methodgenus-specific primers (IAF and IAR) was the best for detecting Aeromonas, while the standard curve was set up by using the extracted DNA from the simulatedsediment samples as template. The new real-time fluorescent quantitative PCRprotocol was proved to be highly sensitive, specific for detecting different species ofAeromonas from the sediment, with the quantification limit down to103cfu/g. Thecoefficient of variation was between0.21%and0.80%by One-way ANOVA (lessthan5%), which demonstrated the good repeatability of the method.3. The real-time fluorescent quantitative PCR method based on toxR gene fortesting Vibrio parahaemolyticus in the sediment was also developed by establishingstandard curve based-on DNA extracted from simulative sediment. The method had ahigh repeatability as a result of all the coefficient of variation (CV) values between0.31%and0.92%, besides, sensitivity and specificity were demonstrated well, withthe quantification limit down to102cfu/g.