Study On The Ultrastructure Of Digestive Tract And The Diversity Of Microbial Community In Tadpole

Rana clamitans belongs to the Amphibia, Anura, Ranidae, Rana, which are common aquatic animals with its embryonic and larval development in water. They are native to the United States and so far have been introduced to many provinces in China. In this paper, by using histological and T-RFLP methods, we have studied the ultrastructure and microbial community diversity of digestive tract in Rana clamitans tadpoles, and 500 mg/L sodium chloride had affected the research findings, dramatically. The main results are as follows:1. Ultrastructure of the midgut of Rana clamitans tadpoles were observed using transmission electron microscope. The results showed that the normal structure of the digestive tract midgut can be divided into four layers, from inner to outer:mucous, submucous, muscularis and adventitia. Mucosal epithelial layer had microvillus. Muscular layer was mainly composed of absorptive cells, with irregular intercellular space between cells and no close connection. At the same time, a small number of goblet cells can be observed among absorptive cells. There were a lot of neat rows of mitochondria, ribosomes, hovering folding endoplasmic reticulum and a small amount of lipid droplets in the absoptive cells. Muscular layer was underdeveloped. There were two inner circular muscle layers and one outer longitudinal muscle layer.2. Ultrastructure of midgut of Rana clamitans tadpoles exposed to 500 mg/L sodium chloride were observed using transmission electron microscope. The results showed that the epithelial absorptive cells of the midgut of Rana clamitans tadpoles exposed to sodium chloride shrinked and condensed. Meanwhile, the number of mitochondria and free ribosomes obviously declined but lipid droplets increased significantly in cytoplasm. Near the lipid droplets the peroxisome appeared. It showed that sodium chloride caused significant damage in the midgut of Rana clamitans tadpole.3. Study the digestive tract microflora of Rana clamitans tadpoles using T-RFLP technology. The results showed that the numbers of T-RFs were different with four different restriction enzymes and different T-RF represents different taxa. In the control group, the maximum T-RFs with the restriction enzymes HaeⅢ were detected and the minimum T-RFs with restriction enzymes MspⅠ were detected. Compared to the control group exposed to sodium chloride, the maximum T-RFs with the restriction enzymes Msp1 was detected and the minimum T-RFs with restriction enzymes AluⅠ were detected. The above four different restriction enzymes applied in this trial is a good tool to analyze the effect on diversity of microbial community in digestive tract of the Rana clamitans tadpoles exposed to sodium chloride.4. Study the effect on structure and abundance of digestive tract microflora of Rana clamitans tadpoles exposed to sodium chloride using T-RFLP technology. The results showed that, under the HaeⅢ digestion, the relative content of T-RF15 was significantly reduced in sodium chloride treated group, under HindP1Ⅰ digestion, the relative content of T-RF92 and T-RF117 were significantly reduced in sodium chloride treated group, under AluⅠ digestion, the relative content of T-RF53 and T-RF97 were significantly reduced in sodium chloride treated group, under MspⅠ digested, the relative content of T-RF97 and T-RF117 were significantly reduced in sodium chloride treated group, these bacteria are sensitive to sodium chloride. Under the HaeⅢ digestion, the relative content of T-RF293 was significantly increased in sodium chloride treated group, under Alul digestion, the relative content of T-RF72 was significantly increased in sodium chloride treated group, under MspⅠ digestion, the relative content of T-RF90 and T-RF72 was significantly increased in sodium chloride treated group, these bacteria are more tolerant to sodium chloride. In addition, there were appearance and disappearance of T-RF fragments. T-RFLP technique can reveal the influence of sodium chloride on the gastrointestinal microbial community diversity of tadpoles, which can provide a theoretical reference on sodium chloride’s toxicology for tadpoles.5. Using white-blue plaque selection, we studied microbial colony in the digestive tract of the Rana clamitans tadpole. It showed that there were Paludibacter, Ilyobacter, Bacteroides, Fusobacterium and Shewanella in the digestive tract of the Rana clamitans tadpole. Sodium chloride had significantly inhibited the growth of Ilyobacter and Paludibacter. But sodium chloride promoted the growth of Fusobacterium and Shewanella, which were not sensitive to sodium chloride.The results show that 500 mg/L sodium chloride helped to observe intestinal submicroscopic structure of tadpoles and test microbial community types. But we found early Rana clamitans tadpoles were sensitive to sodium chloride and sodium chloride had a certain toxic effects on early Rana clamitans tadpoles. It could provide experimental reference and theoretical foundation for further research on characteristics of microbial diversity in the different period of amphibians and protection for aquatic wildlife by means of studing on the ultrastructure and the characteristics of the microbial community diversity of digestive tract in Rana clamitans tadpoles.