| The clam Meretrix petechialis is one of the important economic aquaculture shellfish in China.With the expansion of high-density farming mode and the deterioration of the ecological environment,mass mortality events often occurred during the process of clam culture,which have become the primary restriction for the development and sustainability of the clam culture industry.Vibrio parahaemolyticus is an opportunistic pathogen causing mass mortality of cultured clams in summer.In present study,we analyzed the bacterial load variation in different individuals in the same vibrio dose challenge using the hepatopancreas tissue.The correlation between the host bacterial load and the artificial vibrio dose of the environment seawater was investigated.We also study the diversity and dynamics of hepatopancreas microbiota during the infection process by 16 s amplicon sequencing.Our results revealed several stage-specific indicator species by LEfSe analysis during vibrio disease progression.In addition,the changes of the immune related genes expression were analyzed after vibrio infection.The main results are as follows:1.The vibrio load changes both in host hepatopancreas tissue and environment seawater under vibrio challenge was investigated by TCBS plate account.The bacterial growth and reproduction in the seawater showed a trend of increase,maintenance and decrease process during 24 hours.The maintenance time of vibrio effective concentration in seawater account for over 1/3 of the time in the 24-hour cycle,which was able to meet the vibrio concentration requirements in experiment design.Then,vibrio immersion experiments were carried out to simulate vibrio challenge in natural environment.It was found that bacterial load in clam hepatopancreas was sharply increased in day 1 and then decreased rapidly in day 3 after challenge,and then kept on a low level.In the different vibrio dose challenge experiments,a significant positive correlation was found between the bacterial load and the vibrio dose in the seawater(Spearman's ?=0.899,P=0.000)in the initially phase,and no significant difference in bacterial load among different groups in the middle-late stage with a lower level of 0-205 CFU/mg.In addition,high concentrations of vibrio stimulation can lead to higher clam mortality.The results provide references for the study on vibrio load during infection and resistance evaluation in the clam M.petechialis.2.We used 16 S rDNA amplicon sequencing technology to analyze the microbial community structure and diversity in the hepatopancreas microbiota changes to vibrio challenge.It revealed that there were significant microbial diversity composition differences between healthy and infected clam.During the course of the vibrio challenge,the highest relative abundance of vibrio(14.9%±8.8%)was found at 1 DPI,which was in agreement with the vibrio colony counts.However,the relative abundance of vibrio decreased at the subsequent days and kept on a low level.A significant increase in the Alpha diversity was observed at 3 DPI(P < 0.05),followed by a distinct decline at 4 and 6 DPI(P < 0.05).The low abundance and diversity microbial population at 6 DPI has been found to coincide with the outbreak of death event.It has also been found that the stability of these microflora may have crucially affected the health status of clams during vibrio infection,and our survey further revealed stage-specific indicator species by LEfSe analysis.3.Bcl-2 family proteins play key roles in apoptosis,which proteins can be divided into two types pro-apoptotic and anti-apoptotic proteins.Here one pro-and two anti-apoptotic genes expression pattern were investigated under vibrio infection.The pro-apoptotic gene MpBax was cloned.The full-length of MpBax cDNA sequence is 1309 bp,containing a 615 bp open reading frame(ORF),coding a protein of 204 amino acids,and has highest similarity with Tegillarca granosa(66%).MpBax was expressed at the highest level in the gill,followed by the foot and hepatopancreas.The cDNA of the anti-apoptotic gene Bcl-xL contains a 558 bp open reading frame,encoding 185 amino acids,and has a similarity to the Philippine clams up to 70%.The cDNA of the anti-apoptotic gene Bcl-w contains a 698 bp open reading frame,encoding 252 amino acids,and has a similarity to the Philippine clams up to 70%.MpBax expression was down-regulated,then gradually recover to baseline,and the low point of expression appeared at 8 DPI under vibrio infection.On the contrary,the expression of MpBcl-xL and Bcl-w gene was up-regulated after stimulation.The expression pattern of these pro-and anti-apoptotic genes is consistent with its potential function.Conclusion: In this paper,the relationship between bacteria load in clam hepatopancreas tissue and in environment seawater was investigated under the challenge of vibrio.Meanwhile,the microbial community structure and diversity in the hepatopancreas microbiota changes to vibrio challenge was studied,and the stage-specific indicator species during the infection process was discovered.Finally,the expression pattern of one pro-and two anti-apoptotic genes response to infection was understood.Taken together,our results provide references for better understand the immune mechanism of clam response to bacterial challenge,and provide useful information for the diagnosis,prediction and prevention of disease outbreak in clam aquaculture. |
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