Identification of adherent microbiota in the stomach and intestine of emperor red snapper (Lutjanus sebae Cuvier) using 16S rDNA-DGGE.

摘要

The overall purpose of the present study was to analyse the autochthonous stomach and gut microbiota in emperor red snapper Lutjanus sebae using 16SrDNA-DGGE. The investigation of the autochthonous bacterial community in the stomach compared with data found in the gut is highly relevant, as the pH is reported to be between 3.0 and 4.5 in the stomach and between 7.0 and 9.0 in the intestine of salmonid even though the pH data of emperor red snapper are not available. Five healthy emperor red snappers (241.9+or-48.7 g) were randomly collected from a marine cage (lengthxwidthxheight, 4 mx2 mx2 m respectively) in Xincun Harbor (Sanya City, Hainan Province, China) on 13 July 2005. The water temperature was 30 degrees C and salinity was 33%. The fish were fed an extruded diet (crude protein, 45% dry matter; crude lipid, 8.5% dry matter; and moisture, 8.2%, Guangdong Evergreen Feed Company, Zhanjiang, China) to satiation for at least 4 months with three meals per day at 08.00, 13.00 and 18.00 hours. Sampling was performed using a tuck net at 15.00 hours, and a total of five fish were killed by blow to the head. The fish were placed on ice, individually sealed in sterile plastic bags and examined at the laboratory within 12 h. The GI tract was removed from each individual with sterile dissecting instruments and divided into two sections: the stomach and the gut. After examining both sections, it was observed that Band 1 was most closely related to Stenotrophomonas spp., while bands 4, 8 and 9 showed the highest similarity to uncultured bacterium clone SZB12 (accession no. AM176836.1), uncultured bacterium clone P4D1-497 (accession no. EF509734.1) and uncultured bacterium clone MSFC 3M20 (DQ447821.1) respectively. These uncultured bacteria were first isolated from mangrove sediment, human tracheal aspirates of patients and the regenerative enclosed life support module simulator air system (unpublished data, NCBI) respectively. Although bands 5, 7, 10 and 12 were identified as Photobacterium damselae ssp. damselae (95% similarity), Stenotrophomonas maltophilia strain ISSDA-424 (83% similarity), Vibrio aestuarianus (90% similarity) and Vibrio olivaceus (85% similarity), respectively, these identification of adherent microbiota strains might represent new bacterial species as their sequence similarity values fell below 97%, which is the recommended cut-off used to delineate new bacterial species. As the current study presents new information on the gut microbiota in fish, some general information is therefore presented on strains with >95% similarity to sequences deposited in the NCBI database.