Study On Microbial Community Structure And Functional Diversity From Sea Cucumber (Apostichopus Japonicus Selenka) Culture Ponds

Sea cucumber （Apostichopus japonicus Selenka） aquaculture has been one ofChina’s special seawater aquaculture industry, which mainly located along the coastof Shandong and Liaoning island. This industry developed rapidly, of which thenational aquaculture area has reached160thousand hectares, the output reached130thousand tons and the economic value been nearly20billion yuan in the year of2010.However, compared to the rapid development of this industry, basic research relatedto it is still relatively lagging behind. In this thesis, microbial community structure andfunctional diversity in sea cucumber culture pond environment, including the water,bottom mud and the shelter, were studied by using the16S rDNA PCR denaturinggradient gel electrophoresis （PCR-DGGE） and the BIOLOG method. In addition, theeffect of three bacterial strains （previously isolated from sea cucumber culture pond）supplemented in diets on growth performance and immune response of sea cucumberwas also investigated. The main results are detailed as follows:1Seasonal changes of microbial community structure in sea cucumber culturepondSeasonal changes of microbial community structure in water, bottom mud andshelter of sea cucumber （Apostichopus japonicus） cultural ponds were studied byPCR-DGGE technique, and meanwhile the relationships between them andenvironmental factors were detected by using the redundancy analysis （RDA） method.The results were concluded as follows:（1） In the water samples, phylogenetic analysisof the cloned bands showed that bacterial phylotypes were closely related toα-Proteobacteria, γ-Proteobacteria, Bacteroidetes, Cyanobacteria and some uncultured bacteria. Proteobacteria were the dominant bacteria with high relativeamount of49.3-72.1%in different seasons, among which α-Proteobacteria were thehighest. Cyanobacteria were only detected in summer season with a very low amount.Significant variance analysis revealed that γ-Proteobacteria exhibited significantseasonal changes in amount. Comparatively, the water microbial community structurewas the most complex in autumn, while the simplest in winter.（2） In the bottom mudsamples, phylogenetic analysis of the cloned bands showed that bacterial phylotypeswere closely related to α-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria,Bacteroidetes, Chloroflex, Actinobacteria and Sphingobacteria. Proteobacteria werethe dominant bacteria with high relative amount of49.6-55.1%in different seasons,among which γ-Proteobacteria were the highest. Chloroflex bacteria were onlyfound in spring and autumn seasons, of which the amount in spring was higher.Actinobacteria existed in all seasons except spring, among which the relative amountof Actinobacteria in summer was significantly higher than in winter. Comparatively,in the bottom mud, the microbial community structure in autumn was the mostcomplex.（3） In the shelter samples, phylogenetic analysis of the cloned bands showedthat bacterial phylotypes were closely related to α-Proteobacteria, γ-Proteobacteria,δ-Proteobacteria, ε-Proteobacteria, Flavobacteria, Bacteroidetes, Cyanobacteria,Actinobacteria and uncultured bacteria. Proteobacteria were the dominant bacteriawith high relative amount of54.6-60.2%in different seasons, among which α-Proteobacteria and γ-Proteobacteria were the highest. The amount ofFlavobacteria was also high, and that in spring and summer was just slightly lowerthan that of α-Proteobacteria. Cyanobacteria were not examined in spring, andActinobacteria were not detected in summer. There existed significant seasonalchanges in amount of γ-Proteobacteria, ε-Proteobacteria and Flavobacteria. In theshelter, the microbial community structure in autumn and winter was more complexthan that in spring and summer.（4） RDA analysis revealed that the criticalenvironmental factors influencing the seasonal changes of water, bottom mud andshelter microbial community structure were T, TN, NO₃^-N, PO₄^-P, NO₂^-N; PO₄^-P,NH₄^-N, NO₃^-N, TP; and TP, TN, TOC, SOM, NO₃^-N, respectively. In conclusion, seasonal changes of microbial community structure in water, bottom mud and shelterof sea cucumber culture pond were different, which were relatively more stable inbottom mud than those in water and shelter. The microbial community compositionwas very different in water, bottom mud and shelter, and that in the shelter was themost complex, while that in the water was the simplest. The seasonal changes ofmicrobial community structure in water, bottom mud and shelter of sea cucumberculture pond were all closely relevant to their environmental factors.2Seasonal changes of microbial community functional diversity in sea cucumberculture pondSeasonal variations of microbial community functional diversity in water, bottommud and shelter of sea cucumber （Apostichopus japonicus） cultural ponds wereexamined using the BIOLOG technique, and the relationships between them andenvironmental factors were also detected by using the redundancy analysis （RDA）method. The results showed that the amount of total and different types of carbonsources utilized by water, bottom mud and shelter microbes in sea cucumber culturalponds varied seasonally, which was higher in spring, summer and autumn whencompared to the winter, respectively. The main types of carbon sources utilized werepolymers by water microbes, polymers, carbohydrates, carboxylic acids and aminoacids by sediment microbes, and polymers, carbohydrates, amino acids and amides byshelter microbes, respectively. Principal Component Analysis indicated that carbonmetabolism functional diversity of water, bottom mud and shelter microbes in seacucumber cultural ponds varied significantly over the seasonal courses. For watermicrobes,15categories of carbon sources were significantly related to the principalcomponents, among which10were significantly related to the principal component1and belonged to the polymers, carbohydrates, carboxylic acids, amino acids andamines. For bottom mud microbes,18categories of carbon sources were significantlyrelated to the principal components, among which13were significantly related to theprincipal component1and belonged to carbohydrates, carboxylic acids and aminoacids. For shelter microbes,22categories of carbon sources were significantly related to the principal components, among which22were related to the principal component1and belonged to the polymers, carbohydrates, carboxylic acids and amino acids.Significant seasonal changes were detected for all microbial diversity indexesincluding Shannon, McIntosh, Simpson and S-E in water, bottom mud and shelter ofsea cucumber cultural ponds, but differences were observed in seasonal variationsbetween all microbial diversity indexes. RDA analysis revealed that the criticalenvironmental factors influencing the seasonal changes of water, bottom mud andshelter microbial community functional diversity were TP, NO₃^-N, TN, PO₄^-P; TP,NO₃^-N, PO₄^-P; and SOM, NO₃^-N, TN, respectively. In conclusion, seasonal changesof microbial community functional diversity were marked and different in water,bottom mud and shelter of sea cucumber cultural ponds, which all correlated wellwith environmental factors.3Studies on microbial community structure in different types of shelters of seacucumber culture pondMicrobial community structures in different types of shelters includingearthenware, polyethylene mesh and plastic pipe in sea cucumber （Apostichopusjaponicus） cultural ponds were studied by PCR-DGGE technique, and therelationships between them and environmental factors were also investigated by usingthe redundancy analysis （RDA） method. The results were concluded as follows:（1） Inboth the earthenware and polyethylene mesh shelters, bacterial phylotypes wereclosely related to α-Proteobacteria, γ-Proteobacteria, Sphingobacteria, Chloroflex,Bacteroidetes, Cyanobacteria and Flavobacteria. While in the plastic pipe shelter,bacterial phylotypes were closely related to α-Proteobacteria, γ-Proteobacteria,Sphingobacteria, Chloroflex, Bacteroidetes, Actinobacteria and Flavobacteria.（2）Flavobacteria were the first dominant bacteria with relative amount of more than45%in each of three types of shelter, for which, however, no significant differences wereobserved between different types of shelter. Proteobacteria were the second dominantbacteria in all shelters, and the relative amount of Proteobacteria was the lowest inearthenware （20.7%） and the highest in polyethylene mesh （34.7%）, respectively. In addition, γ-Proteobacteria were the dominant bacteria in both the earthenware andplastic pipe shelters, while α-Proteobacteria were dominant in polyethylene meshshelter. There were significant differences for amount of Proteobacteria in differenttypes of shelter, in which the amount of α-Proteobacteria in polyethylene mesh（16.2%） was higher than that in earthenware （9.5%） and plastic pipe （6.4%）, while theamount of γ-Proteobacteria in plastic pipe （28.3%） was higher than in earthenware（11.2%） and polyethylene mesh （13.2%）. The relative amount of Bacteroidetespresented a trend of decrease in shelters of earthenware, polyethylene mesh andplastic pipe, and that in earthenware （6.0%） was significantly higher than that inplastic pipe （0.4%）. Cyanobacteria had a similar changing trend with Bacteroidetes,which however were not detected in samples of plastic pipe. Conversely,Actinobacteria were only observed in plastic pipe shelter samples. Sphingobacteriaand Chloroflex both showed no significant changing in amount between differenttypes of shelters.（3） RDA analysis found that the critical environmental factorsresulting in the difference of microbial community structures in shelters ofearthenware, polyethylene mesh and plastic pipe were TN, PO₄^-P, NO₃^-N and NH₄^-N.In conclusion, microbial community structure was different in different types ofshelters of earthenware, polyethylene mesh and plastic pipe in sea cucumber culturepond, and comparatively that in earthenware and polyethylene mesh was much similarto each other, but both were very different from that in plastic pipe. The microbialcommunity structures in shelters of earthenware, polyethylene mesh and plastic pipeof sea cucumber culture pond were all closely relevant to their environmental factors.4Studies on microbial community functional diversity in different types ofshelters of sea cucumber culture pondMicrobial community functional diversity in different types of shelters includingearthenware, polyethylene mesh and plastic pipe in sea cucumber （Apostichopusjaponicus） cultural ponds were examined using the BIOLOG method, and therelationships between them and environmental factors were also detected by using theredundancy analysis （RDA） method. The results showed that the amount of total and different types of carbon sources utilized by earthenware, polyethylene mesh andplastic pipe shelter microbes in sea cucumber cultural ponds varied, which was thehighest in plastic pipe and the lowest in earthenware. And the amount of differenttypes of carbon sources utilized by polyethylene mesh or plastic pipe shelter microbeswas significantly higher than that utilized by earthenware shelter microbes. The maintypes of carbon sources utilized were polymers, carbohydrates and amino acids byplastic pipe shelter microbes; polymers, carbohydrates and carboxylic acids bypolyethylene mesh shelter microbes; and polymers and carboxylic acids byearthenware shelter microbes, respectively. Principal Component Analysis indicatedthat carbon metabolism functional diversity of shelter microbes in sea cucumbercultural ponds varied significantly among earthenware, polyethylene mesh and plasticpipe shelters. And this difference was presented by Principal Component1, to which13categories of carbon sources were significantly related which belonged to thepolymers （3categories）, carbohydrates （4categories）, amino acids （4categories）,carboxylic acids （1category） and amines （1category）. Significant differences weredetected for microbial diversity indexes including McIntosh and Simpson, except forShannon and S-E, among earthenware, polyethylene mesh and plastic pipe shelters ofsea cucumber cultural ponds. And McIntosh index was significantly higher inpolyethylene mesh and plastic pipe than that in earthenware. Simpson index wassignificantly higher in plastic pipe than in earthenware. RDA analysis revealed thatthe critical environmental factors resulting in the difference of shelter microbialcommunity functional diversity in earthenware, polyethylene mesh and plastic pipewere TN, PO₄^-P and NO₃^-N. In conclusion, microbial community functionaldiversities were different in different types of shelters of earthenware, polyethylenemesh and plastic pipe in sea cucumber cultural ponds, which was closely relevant tothe environmental factors.5Effects of probiotics supplemented in diets on growth performance andimmune response in sea cucumberThe present study assessed the growth performance and immune response in the juvenile sea cucumber Apostichopus japonicus by two potential probiotic strains,Paracoccus marcusii DB11and Bacillus baekryungensis YD13. The fresh cells ofDB11and YD13were respectively added to sea cucumber basal feed at three doses of1×10⁴,1×10⁶and1×10⁸CFU/g in diet, and administered orally to A. japonicus （initialmean wet weight5.34±0.11g）. After a60-day feeding trial with probioticsupplemented and unsupplemented control diets, A. japonicus fed with DB11at110⁸cfu/g, with YD13at110⁴and110⁶cfu/g, all exhibited significantly bettergrowth performance in weight gain rate and specific growth rate as compared to thecontrol （P<0.05）. Five non-specific immune parameters including superoxidedismutase （SOD）, catalase （CAT）, lysozyme （LSZ）, acid phosphatase （ACP） andalkaline phosphatase （ALP） activities were measured to evaluate the immune responsein different tissues （coelomic fluid, respiratory tree, intestine and body wall） of A.japonicus to supplemented DB11or YD13. Results showed that the enhancement ofimmune enzyme activities in different tissues of A. japonicus were the greatest whenfed with DB11at110⁸cfu/g and with YD13at110⁶cfu/g, respectively. Theimmune response in A. japonicus to supplement DB11and YD13was highlytissue-specific, and the immune stimulation intensity was coelomic fluid>respiratorytree>intestine>body wall, indicating that the coelomic fluid plays a key role in thenon-specific immunity of A. japonicas. To conclude, the probiotics, Paracoccusmarcusii DB11and Bacillus baekryungensis YD13, could improve the growth andimmune enzyme activities of A. japonicas so that they both have potential applicationprospect in sea cucumber aquaculture.