Seasonal Changes Of Bacterial Community Composition In Two Culture Area And Digestive Diverticula Of Scallops, Chlamys Farreri

The seasonal changes of bacterial community composition in two different scallop culture model, Sanggou Bay (kelp-scallop poly-culture model) and Shazikou (scallop single-culture model) was analyzed. 5 L water samples from 0.5 m and 3 m depth was collected from May to Nov. 2009 in Sanggou Bay and May 2009 to April 2010(except Feb.) in Shazikou. The water samples were filtered by sterile micro-filtration membranes to collect the micro-organisms with the size of 3μm to 0.22μm. Total DNA of the samples were extracted by CTAB method, the bacterial 16S rDNA V3-V5 variable sequence was amplified, and separated by denaturing gradient gel electrophoresis (DGGE) techniques. The DGGE fingerprint was analyzed by Quantity One and SensiAnsys. The results as follows:1) In Sanggou Bay, DGGE gels got a total of 231 detectable bands with 26 different positions. The number of bands per sample varied between 14 and 23 (mean 19.75). High similarities of bacterial composition (73%~91%) were found between 0.5m and 3m depth. The most abundant bacterial composition was found in August. The monthly samples were grouped into 3 seasonal types: spring (May), early summer and late autumn (June, July and November), and late summer to early fall (August to October) based on the UPGMA cluster analysis. The 11 dominant DGGE bands were successfully cloned and sequenced, showing high similarities (91~99%) with sequences logged on NCBI (http://www.ncbi.nlm.nih.gov/). Among the11 bands, five belonged to Proteobacteria (3 sequences were belonged toα-Proteobacteria, the others belonged toβ-Proteobacteria andγ-Proteobacteria respectively), 3 of the sequences belonged to Bacteroidetes and the remainder belonged to Actinobacteria. 10 sequences of the11 cloned sequences showed high similarities to sequences of uncultured bacteria logged on NCBI.2) In Shazikou, DGGE gels got a total of 198 detectable bands in 36 different positions. The number of bands per sample varied between 15 and 23 (mean 18). The most abundant bacterial composition was found in January, and March is least. Based on the the UPGMA cluster analysis, the monthly samples were grouped into 2 seasonal types: spring (March to May), summer to autumn (June to January). The 20 dominant DGGE bands were successfully sequenced, showing high similarities (94~ 100%) with sequences logged on NCBI. Among 20 bands, 35% belonged toα-Proteobacteria, 10% belonged toβ-Proteobacteria andγ-Proteobacteria respectively, 25% belonged to Bacteroidetes, 15% belonged to Actinobacteria, and only 5% belonged to Verrucomicrobia. 16 sequences showed high similarities to sequences of uncultured bacteria logged on NCBI.3) In order to study the seasonal changes of bacterial community composition in digestive diverticula of scallops C. farreri, 10 scallops were collected from Shazikou sea area every month since June 2009 to June 2010 (except Feb.). Total DNA in digestive diverticulas were extracted by SDS method, the bacterial 16S rDNA V3~V5 variable sequence was amplified and separated by DGGE techniques. There were 11 electrophoric bands in different positions in DGGE image. The number of bands in different months were similar, 9 bands existed in all 12 months, indicated the bacterial community composition in digestive diverticula were relatively stable. 9 dominant DGGE bands were successfully sequenced, showing high similarities (97%~99%) with sequences logged on NCBI. Among 9 bands, 33.33% belonged to Actinobacteria, 22.22% belonged toδ-Proteobacteria, 22.22% belonged to Firmicute, and 11.11% belonged toβ-Proteobacteria and Cyanobacteria respectively.4) 28 bacterial strains were isolated from digestive diverticula of health scallops C. farreri by conventional culture method, and were identified by 16S rDNA sequence. The results showed that the isolates mainly belonged to 4 genus: Vibrio, Tenacibaculum, Colwellia and Ruegeria. 3 different genus bacteria (E8: Vibrio, E14: Ruegeria, E19: Tenacibaculum ) were studied for infection experiment. Each scallop was injected 1.7×10~6 bacteria. The serum samples were collected at l d, 3 d, 5 d, 7 d, 9 d, and ACP, AKP and SOD activity were examined, and mortalities were recorded simultaneously. The results showed that, the mortality of E8 group were higher than the other controls (5%). For the group E8, the activity of ACP gradually decreases first, then increased sharply. SOD activity rose up frist, then declined , finally increased again; For E14 group, there were no significant differences of ACP and SOD activity between experimental groups and control throughout the experiment; For E19, the activity of ACP dropped after rising, and significantly higher than controls at 3 d (P<0.05); the SOD activity presented a similar trend, there were no significant differences (P >0.05) of AKP during the experiment.