Molecular Ecology Of Intestinal Bacteria In Large Yellow Croaker(Larimichthys Crocea)

Large yellow croaker(Larimichthys crocea),also known as yellow croaker,belonging to Osteichthyes class,Perciformes order,Sciaenidae family and Larimichthys genus,is one of the most important marine farming fish in China.Intestine microbes are important part of their host and are praised as “external organ of the host”.Understanding the community structure of intestinal microbes,dynamic change,and the influencing factors will promote the sustainable and healthy development for large yellow croaker industry.In the present study,for the first time,we investigated the bacterial community structure in large yellow croaker intestine during different developing period,the corresponding aquaculture water,and the feed in the same period,and the relationship between them using four different method: the traditional isolating and cultivating,the culture-independent PCR-DGGE,the clone libraries constructing,and the next generation high throughput sequencing.The results were as follows:1.Significant difference of bacterial community structure was found in large yellow croaker guts among different development stages.The intestine bacteria diversity in early developing stage of large yellow croaker was higher than that of one year old fish.Fusobacteria was mainly in the one year old fish intestine,while ?-Proteobacteria mainly resided in intestine of the early developing fish;Bacteroidetes was not abundant in intestine of early developing fish and one year old fish.The result from clone libraries constructing showed that Firmicutes was abundant in the early developing(26 days old)intestine,while few in one year old gut sample.However,the high-throughput sequencing showed that Firmicutes was dominant in one year old gut sample,and few in 12 days old gut sample.Planctomycetes was found in early developing gut sample,but was absent in one year old gut sample.?-Proteobacteria were present in all samples,but the members of this class were different.Cyanobacteria/Chloplast were found in all samples,and were more abundant in early developing gut samples.?-proteobacteria and Actinobacteria were present as minor groups in all samples.Tenericutes and Spirochaetae were also detected in different developing gut samples.Further analysis of the high throughput sequencing showed that Pseudoalteromonas accounted for 73.7 % in early developing gut samples,but was absent in one year old gut sample.Clostridium and Lactococcus were dominant in one year old gut sample,accounting for 59.4 %and 33.7 % respertively.However,Clostridium was few,and Lactococcus was absent in 12 days old gut sample.2.Isolating and cultivating results: The mediums TSA,Macconkey and TCBS were used for isolating intestineal bacteria from one year old gut sample.About 1469,688 and 97 bacterial colonies were obtained from TSA,Macconkey and TCBS,respectively.Different morphological colonies were randomly selected and purified,and finaly,63,40 and 10(a total of 113)purified isolates were obtained from the three mediums respectively.The 113 isolates were analysed by RFLP and sequencing.After removing the unsuccessful sequences,95 of 113 isolates were analysed and were divided into 6 OTUs.Sequences analysis showed that Photobacterium was dominant,accounting for 69.5 %,and the unclassified Firmicutes was the second dominant group(20.0 %),and Vibrio was also detected,it account for 10.5 %.3.The bacteria genetic diversity was high in large yellow croaker culture water.?-Proteobacteria were most dominant(accounting for 67.9 %).?-Proteobacteria,Bacteroidetes and Firmicutes were the second abundant groups(accounting for 13.1 %,13.1 % and 6.6 %,respectively).?-Proteobacteria,?-Proteobacteria,Planctomycetes,Chloroflexi,Chlorobi and OP11 were also present as minor groups.4.Sequences from feed sample were clustered into 3 major groups,in which?-Proterbacteria are the most predominant group,accounting for about 84.4 % of the sampled clones;Flavobacteria bacteria are the second dominant groups,and accounted for about 10.9 %.There are a small number of Fusobacteria bacteria,accounting for about 4.7 %.Psychrobacter sp.are the most abundant members within the ?-Proterbacteria,and Photobacterium are the second abundant members.5.Three different kinds of samples,the large yellow croaker gut samples,the aquaculture water samples,and the feed samples,breed environment specific bacterial flora,respectively.DGGE profiles showed that different kinds of samples could be clustered together separately.The bacterial composition was different among different samples even if the samples were from the same stage.Bacteria diversity in aquaculture water sample was most abundant,the feed sample came next,and the bacteria diversity in fish gut sample was the lowest.Proteobacteria were the common dominant bacteria in the three kinds of samples.?-Proteobacteria were most dominat in both feed and gut samples.Bacteroidetes were also common in all of the three kinds of samples,but it was not so abundant in aquaculture samples as in gut and feed samples.Planctomycetes,?-Proteobacteria and Firmicutes were common bacteria in gut,aquaculture water and feed samples.Many bacteria detected in aquaculture water were not detected in feed or gut samples.