Study On Changes Of Bacterial Community In Sediment Environment Of Normal And Diseased Apostichopus Japonicas Culture Ponds

In this paper, the seasonal change of bacterial communities in the environment of notmalApostichopus japonicas culture ponds were analyzed using culture counting, and16SrRNA gene PCR-DGGE profile and the DGGE-Marker were prepared based on this. Atthe same time, the realationships between physical factors and the change of numbers ofseveral bacterial groups, including the heterotrophyic bacteria, Vibrio bacteria, nitrifyingbacteria, sulfur bacteria, nitrate-reducing bacteria, sulfate-reducing bacteria, in theenvironment of Apostichopus japonicas culture ponds were analyzed before and after thedisease occurance, preparation of DGGE, using culture counting, situ measurement and16S rRNA gene PCR-DGGE profile. Also, the responses of the bacterial community onthe sediment improver were studied through simulated experiments. The mainexperimental results as follows:1. Seasonal change of bacterial communities in the sediment environment ofApostichopus japonicas culture ponds and preparation of DGGE Marker. Sedimentsamples from several ponds in different season were selected and DNA was extracted.Unique primers for bacterial16SrRNAgene,8F and1492R, were used for PCR. The PCRproduct were cloned, RFLP and sequenced. The results showed that bacteria ofproteobacteria, planctomycetes, bacteroidetes and chloroflexi have a huge number. Inaddition to uncultured bacteria, proteobacteria bacteria was dominant flora in threeseasons except spring which the proportion in summer was about32%,in autumn theproportion of proteobacteria bacteria was about29%and bacteroidetes bacteria was21%,in winter the proportion of proteobacteria bacteria was about42%. As the dominantflora in spring the proportion of planctomycetes bacteria was about46%. Theproportion of type of γ, δ in proteobacteria bacteria was39%,32%.DGGE protocol wasoptimiaed and standardlized. Then DGGE marker was prepared. The results showed thata set of clear and simple DGGE marker was got including28predominant bacteria.2. The change of the numbers of several bacteria groups and the chemical-physicalfactors before and after the disease occurance in the environment of Apostichopusjaponicas culture ponds. Two diseased ponds and two normal ponds were selected andmornitored for40days before and after disease. Temperature, dissolved oxygen, salinity,pH were measured in situ the proportion. Water and sediment samples were collected for microbiological culture were sent to lab. The relationship between disease, physicalfactors and numbers of bacteria were analyzed.Results: The temperature range ofApostichopus japonicas culture ponds were26.55℃to29.98℃, with an average of28.27℃. The salinity range were25.83to31.31, with an average of28.69, the changetrend was not the same as temperature. The dissolved oxygen ranges were3.80mg/L to6.18mg/L, with an average of5.17mg/L. The PH ranges were7.94to8.90, with anaverage of8.36. Dissolved oxygen and PH were in line of “water quality standards”second-class standards. Microbiological culture results showed that the number ofbacteria was not increased significantly before disease, and the variation was not obvious.The conjecture was made combining the above two parts data analysis showed that,changes of physical factors may increase the sensitivity to the pathogenic bacteria eventhough the number of it was not significantly increased. So the changes of physicalfactors may be one cause of Apostichopus japonicas disease.3. Normal does （0.66g）of the sediment improver was added to the experimental group,but none to the control group. Sediment samples of each group were collected at day1,2,3,4,5,6,10,15respectively. Then, culture counting and by molecular biologicalmethods analysis were carried out.Results: The results showed the numbers of theheterotrophyic bacteria, nitrifying bacteria and sulfate-reducing bacteria in sediments ofdiseased ponds increased, but soon declined and kept at a low level in the experimentalgroup, and the rising amplitude significantly lower than those of control group. While,the numbers of Vibrials and sulfurizing bacteria sharply declined after adding ofsediment improver, significantly lower than those of control group. The quantityfluctuation range of the heterotrophyic bacteria were（1.27±0.66）×104^8.07±0.40×105CFU/g, the quantity fluctuation range of the Vibrio bacteria were（1.00±0）×102^6.87±0.83×103CFU/g, the quantity fluctuation range of the nitrifyingbacteria were （9.14±2.35）×103^2.32±0.51×105CFU/g, the quantity fluctuation range ofthe sulfur bacteria were （2.10±0.36）×102^3.60±0.9×103CFU/g, the quantity fluctuationrange of the nitrate-reducing bacteria were （1.50±0.24）×105^7.40±0.11×106cells/ml,the quantity fluctuation range of the sulfate-reducing bacteria were（2.40±0）×100^4.13±1.70×101cells/ml. The sequencing result of DGGE bands showed that thedominant bacteria in sea cucumber culture environment included bacteria in Chloroflexi,moraxellaceae, Caulobacteraceae and Aeromonadaceae in Proteobacteria, andBacillaceae, Lactobacillaceaein Firmicutes.Throughout the experiment the Acinetobacterwas present as the dominant bacterial, the dominancy of2species of Aeromonus wasobserved to decline after adding of sediment improver in the experimental group. Thehighest degree of similarity was83%between samples A5and A6, and the lowest was35.6%between samples A1and B8. The diversity indexes varied between2.5to3.5.[Conclusion] the sediment improver could adjust the numbers of different bacteria groups and decline the dominancy of some pathogenic bacteria in sediment environment,and may thus improve the quality of the pond sediment environment and protectApostichopus japonicas from disease.