Distribution And Interrelation Of The Biological Pollutants In The Gut And Living Environment Of Freshwater Shrimp

In recent years,with the increasingly rapid development of aquaculture in China,the pollution of aquaculture has worsened,resulting in the deterioration of aquaculture environment.This has attracted more and more attention.Antibiotics play an irreplaceable role in modern aquaculture,however,long-term abuse of antibiotics can induce the microorganism harboring antibiotics resistant genes(ARGs)in aquatic animals.If ARGs are released into the environment,they will not only cause potential genetic contamination to the culture environment and its surroundings,could also be transferred into pathogenic microorganisms via mobile genetic elements such as plasmids,transposons and integrons,so that spread among the environment,animals and humans through various pathways,which pose a potential worldwide human health risk.In this study,shrimp samples were obtained from six freshwater shrimp farms in Jiangsu province.Sediment and pond water from each shrimp pond were also sampled.Based on the bacterial 16S rRNA amplification sequencing and high-throughput metagenomic sequencing,along with bioinformatics and statistical analyses,we studied the distribution characteristics of microbial community structure in the gut and living environment of shrimp systematically and comprehensively.In addition,the diversity and abundance of ARGs in water,shrimp gut and sediment samples were further analyzed,the correlations among the ARGs,bacterial communities and MGEs were revealed and the relationship between ARGs and antibiotic/environment factors(TN,TP,TOC,DO,T,pH)also were examined.On the other hand,the abundance and diversity of human pathogenic bacteria in shrimp gut and its living environment were analyzed.Significant differences of the potential pathogens compositions among these different samples were observed.The correlation of environmental factors and the potential pathogens,as well as co-occurrence of potential pathogens and ARGs were further demonstrated.The detailed results of this study are listed as follows:(1)Based on the high-throughput sequencing of 16S rRNA gene amplicons,Proteobacteria was found to be the most abundant bacterial group in gut samples(89.68%±1.21%)followed by Bacteroidetes and Firmicutes.The most abundant OTU was assigned to the genus Dongia,and 57 genera were defined as the shared main gut bacterial population(constituting 38.00?99.48%of the total genera in each of the 40 shrimp gut samples).The result also indicated that temperature(p=0.004),TP(p=0.010)and DO(p=0.007)significantly contribute to the bacterial community-environment relationship.(2)Based on high-throughput metagenomics sequencing,60,102 and 67 ARGs were detected in shrimp gut,pond water and sediment samples,which were dominated by bacitracin and multidrug resistance types.Efflux pump and target modification were the predominant resistance mechanisms in all samples.It was found that the ARG sequences of the gut samples showed an average identity of 54.89%to their best hits in the ARDB database,which were higher than those in both the pond water and sediment samples,and the vast majority of the genes from the sediment microbiome were previously uncharacterized novel ARGs.MGEs and ARGs,it was found that Aeromonas,Yersinia and Clostridium XlVb were significantly correlated with the distribution of the ARGs.Besides,the relative abundance of ARGs was positively correlated with plasmids(R2=0.8852),integrons(R2=0.8763)and ISs(R2=0.8907).Pearson correlation analysis demonstrated that sulfadiazine and methyl benzyl oxygen pyrimidine were positively correlated with most of ARGs including multidrug,aminoglycoside,?-lactam,chloramphenicol and M-L-S five resistance types,Meanwhile,significant correlations were observed between tetracyclines(oxytetracycline and aureomycin)and multidrug resisitant type(mexF,mexW and acrB).(3)35 genera and 59 species of human pathogenic bacteria were detected in all samples by comparing with human pathogenic bacteria sequences.The abundance of the detectable potential pathogenic bacteria in the pond water(0.07?9.00%)were higher than those in the shrimp gut and sediment samples,Aeromonas were commonly shared among all samples,which accounted for 0.76?99.20%of potential pathogenic bacteria.Potential pathogenic bacteria in shrimp gut have high similarities with those in pond water.Exploring the relationship between the major potential pathogenic bacteria in shrimp gut and environmental factors indicated that temperature,TP and DO were significantly correlated with the distribution of the major potential pathogenic bacteria.Network explored ARGs-Pathogens co-occurrence patterns and indicated that numerous potential pathogenic bacteria carry various ARGs.In conclusion,this study comprehensively and systematically revealed the bacterial community compositions in shrimp guts and their aquaculture environments(pond water and sediment),examined the diversities and abundances of the biological contaminants including ARGs and pathogenic bacteria and their correlation in three different samples,evaluated the potential threats to human health posed by biological contaminants in the guts and living environments of shrimp,and provided scientific theoretical basis for the risk assessment and prevention of the potential pathogens and ARGs in the living environments of shrimp.