| Earthworms living in the soil, in which closely contact with various microorganisms. How can earthworms defend the pathogens with a innate immune system? In our research, with E.coli O157:H7 as a stress factor, we used the second generation sequencing technology (Illumina, pair-end) and the isobaric tags for relative and absolute quantitation (iTRAQ) to detected the immuno-related genes and proteins of the Amynthas heterochaetus. We also used the methods of T-RFLP (Terminal Restriction Fragment Length Polymorphism) and 16S rRNA gene clone library to analyzed the intestinal bacterial community shifts of the Amynthas heterochaetus, which stressed on E.coli O157:H7. The following are the results:(1) We built the first transcriptome database and proteome profile of the wild type earthworm in Asia, and the functional annotation of which was also completed. Based on the gene function annotation (Nr, NCBI non-redundant protein sequences), we found the protein coding sequence of Amynthas heterochaetus with high similarity to the Capitella teleta (28%) and Helobdella robusta (20.1%), and lower similarity to Crassostrea gigas (8.2%), Lottia gigantea (4.2%), Aplysia californica (3.6%), which belong to Mollusca.(2) With comparative transcriptome analysis, proteomics analysis and the conjoint analysis, we found 130 genes and proteins in Amynthas heterochaetus related to ubiquitination.51 of them with significantly change of expression quantity were annotated. We also found 22 genes and proteins in Amynthas heterochaetus related to complement system.17 of them with significantly change of expression quantity were annotated. The results prove the ubiquitination and complement system paly an important role in the immunoreaction of Amynthas heterochaetus.(3) We analyzed the number change of the bacterial community and E.coli O157:H7 in the passage of ingesta through the digestive tract of Amynthas heterochaetus, in which, the bacterial community structure shift of ingesta also be detected. We found the number of E.coli O157:H7 decreased because of the physical digestion of crop and gizzard at the beginning of which entered the digestive tract. In the intestinal digestion, the number decrease of E.coli O157:H7 was mainly observed in earthworm foregut. The bacterial community in midgut was observed a regulation to the number or proportion of the E.coli O157:H7:when the number of E.coli O157:H7 less than 104 CFU, which was increased; when the number of E.coli O157:H7 more than 104 CFU, which was decreased. In hindgut, the bacterial community structure and the total number of which maintained stable. The Pseudomonadaceae and Clostridiaceae performed antagonism to the E.coli O157:H7. The number change of them were observed inducible.(4) We fed Amynthas heterochaetus in substrates which with different bacterial community, and analyzed the bacterial number change and community structure shift of ingesta in the passage of throught the earthworm gut. We observed the crop and gizzard selectived decreased the proportion of Gram-negative bacteria in the ingesta bacterial community. This selectived decrease of crop and gizzard may be relate to the cell wall component of bacteria. Homeostasis mechanism of the intestinal bacterial community in Amynthas heterochaetus was observed, which could maintained the stability of bacterial community structure. The selectived decrease of crop and gizzard and the homeostasis mechannism of intestinal bacterial community result the Normalized Effect of the Amynthas heterochaetus digestive tract, which could normalized the diferent bacterial communities to the similar community structure.In summary, the ecological immune system of Amynthas heterochaetus was constituted by natural immune system and synergistic immune of intestinal bacterial community. The ubiquitination and complement system paly an important role in the immunoreaction of Amynthas heterochaetus, and the synergistic immune of intestinal bacterial community could help the host to reduce the number of pathogen. The transcriptome database and proteome profile of Amynthas heterochaetus are the reference of the metabolism, reproduction and immune research. The homeostasis mechannism of intestianl bacterial community and the Normalized Effect of the Amynthas heterochaetus digestive tract could be the theoretical foundation of the earthworm adaptive mechanism research. |
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