Isolation,Identification And Community Structure Analysis Of Bacteria In The Clam Meretrix Petechialis

The clam Meretrix petechialis is one of the important commercial molluscs in the coastal areas of China.There are abundant endosymbiotic bacteria in clams,and the composition and changes of these endosymbiotic bacterial communities have certain effects on the host ability to against pathogens colonization and the resistance to stress.In this study,endosymbiont bacteria in the gill tissue of M.petechialis were isolated,purified and identified by pure culture method,and the species and quantity of these endosymbiont bacteria were counted and analyzed.16 S r RNA amplificon sequencing was performed to analyze the bacterial community structure in the gills and hepatopancreas of M.petechialis under different health states.Our results found the bacterial species abundance variations in different health states and in different tissues,and we also found 4 specific bacterial groups in the clams under different health states.In addition,the earlier screened Arcobacter MP43,a potential pathogen,was used to challenge M.petechialis and to evaluate its potential pathogenic ability and effect on immunity to this clam.The main results of this study are as follows:1.Pure culture method was used to isolate and purify the endosymbiont bacteria in the gill of M.petechialis.The 16 S r RNA genes of all of the obtained strains were sequenced to determine their classification.The total 215 strains were classified into four phyla: Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria.The major genera were Alternomonas,Nautella,Staphylococcus and Pseudomonas,which were isolated up to 20,18,15 and 13 strains respectively.The species and abundance of endosymbiont microorganisms in the gill can provide references for the further exploration of endosymbionts in this clam.2.The bacterial community structures in the gill and hepatopancreas of M.petechialis under different healthy states were analyzed by 16 S r RNA amplicon sequencing.The results of cluster analysis and species annotation of the obtained OTU data showed the different bacterial composition for this clam in different health states and in different tissues.Proteobacteria is the most abundant phylum in 4 sample groups.In healthy clams,the abundance of Actinobacteria,Firmicutes and Bacteroidetes were higher in hepatopancreas than their abundance in gill.Arcobacter is the genus group with the highest bacterial abundance in gill tissues,while Rhodococcus is the genus group with the highest bacterial abundance in hepatopancreas.In the diseased clams,the species of bacteria in the gill tissues were far less than that in the hepatopancreas at the genus level,and the genus Endozoicomonas occupied an extremely high abundance in the gill tissues,while the genus group with the highest abundance in the hepatopancreas was Acinetobacter.There was no significant difference in the bacterial Alpha diversity and Beta diversity in the hepatopancreas between healthy and diseased clams,but 4 significantly specific bacterial groups were found between these two groups,which can be used as reference indicators for clam health.3.Through the artificial infection experiment of Arcobacter MP43 in clams at different temperature and different bacterial concentration,there was no significant difference in the mortality between the control group and the experimental group,indicating that strain MP43 was not directly pathogenic to clams.At the same time,the expression of immune-related genes in the hepatopancreas showed the clams had no obvious immune response and the vibrio load of hepatopancreas had no significant change after MP43 challenge.The results showed that the Arcobacter MP43 strain was not directly pathogenic to M.petechialis,and could not cause the mass mortality of this clam.In this study,the structure of the endosymbiont bacterial community was studied by pure culture method and 16 S r RNA amplification sequencing,and the pathogenicity of a potential pathogen was evaluated.The above research results provide reference for the study of endosymbiosis microflora of clams,discover the potential microbial reference of the healthy state of clams,and provide ideas for the screening and identification of bivalve mollusc pathogenic bacteria.