Distribution Characteristics Of Antibiotic Resistance Genes And Changes Of Bacterial Community In Zero-water Exchange Biofloc Culture Of Shrimp

The extensive use of antibiotics renders them largely residual in the environment,thus leading to the proliferation of resistant bacteria（ARB）and antibiotic resistance genes（ARGs）.In recent years,ARGs have been regarded as emerging contaminants and have attracted public attention.The water environment is more susceptible to the pollution by ARGs released from several sources,such as agricultural runoff,domestic sewage discharge,and livestock wastewater discharge.In addition,ARGs were also enriched in aquaculture environments due to inappropriate use of antibiotics in aquaculture earlier.Numerous studies have shown that different species of ARGs have been spread in aquaculture environments.Previous studies have focused on the distribution characteristics and dynamic changes of ARGs in the traditional aquaculture mode.However,the transfer of ARGs in the biofloc culture with zero water exchange remains unknown.The aims of this study are to reveal the relationship between ARGs,bacterial communities and environmental factors in the zero-water exchange biofloc culture system,and to provide the prospect of further research in related fields in the future.In this study,In order to explore the feasibility of removing and controlling the spread of ARGs by the fishing oxidant trichloroisocyanuric acid（C3Cl3N3O3）in the aquaculture water environment,we applied real-time quantitative PCR to monitor and determine the ARGs concentrations in the offshore source water,storage water of sedimentation tank and reserved water after oxidation and disinfection for 29 d.The final concentration of trichloroisocyanuric acid used for oxidation and disinfection was 40 mg·L^-1.In addition,real-time fluorescence quantitative PCR was used to track and monitor the distribution and change characteristics of major ARGs such as flo R,sul1,sul2 and tet X in the water environment and shrimp intestine during the breeding process,and the composition and structure of flora in the breeding environment and shrimp intestine and the change characteristics at different breeding stages were analyzed with high throughput;Redundancy analysis and Pearson correlation analysis were used to explore the relationship between flora,ARGs and environmental factors.The following results are obtained:1.The offshore aquaculture source water is the main source of ARGs in the shrimp zero-exchange intensive aquaculture environment;among them,the offshore source water,the reservoir water and the stand by water for pond culture after disinfection treatment have the highest concentration of sulfonamide resistance genes;Pool sedimentation and strong chlorine oxidative disinfection treatment can significantly reduce the content of common ARGs in the water body,and the removal amount can reach 0.72 log.In addition,the content of chemical oxygen demand（COD）in water was reduced from 9.2 to 22.2 mg·L^-1 to≤0.8 mg·L^-1,indicating that reducing substances in water would affect the oxidation effect of oxidants.Therefore,suitable types of oxidants should be selected for different water environments,or several different oxidants should be used in combination to improve the removal effect of fishing oxidants on ARGs.2.The results showed that the total concentration of ARGs in the water body increased first and then decreased during the whole aquaculture period.The concentration range of ARGs in the aquaculture water was 10⁵～10⁶copies·m L-1,of which the dominant ARGs was sul2,and the average concentration was 6×10⁵copies·m L^-1,accounting for 86.7%of the total ARGs;the concentration of ARGs in bioflocs generally showed an increasing trend,and its concentration was basically stable at the order of magnitude of 10⁸ copies·m L^-1,which was higher than that of the water in aquaculture ponds.The dominant ARGs were sul2,with an average concentration of 1.7×10⁸ copies·m L^-1,accounting for 75.8%of the total ARGs;the concentration of ARGs in the intestines of shrimp at the middle and late stages of culture gradually increased,The concentration of ARGs in the intestinal samples was107～10⁸ copies.g^-1,none of the four ARGs were not detected in the early stage,Three ARGs including flo R,sul1 and sul2 were detected in the middle and late stage,among which the dominant ARGs was sul2,with a concentration value of 1.5×10⁸copies·m L^-1.The study shows that there may be the transfer of ARGs between aquaculture water,biological flocs and shrimp intestines.It is speculated that ARGs present a process of tranfer pathway and enrichment from aquaculture water to bioflocs and then to the shrimp intestines.3.In the shrimp zero water exchange biological floc culture system,with the progress of the culture cycle,the bacterial concentration in the culture water body is2.0×10⁶～4.9×10⁷ CFU·m L^-1;The concentration of bacteria in biological flocs is6.5×10⁹～6.5×10¹⁰CFU·m L^-1;The concentration of bacteria in shrimp intestine was2.3×10⁸～7.3×10⁹ CFU·g^-1;The bacterial concentration in aquaculture water,biological flocs and shrimp intestine showed an increasing trend.Compared with the initial stage,the bacterial concentration in the middle and late stage increased by 1～2orders of magnitude.In addition,the bacterial concentration in biological flocs is 2～3orders of magnitude higher than that in aquaculture water.Proteus is the dominant flora in the culture environment;In the middle and later stage,the Chao index of aquaculture water body and biological floc is about 950 and 960 respectively.It can be seen that the diversity of bacteria and microorganisms in aquaculture water body and biological floc is similar in this stage;The microbial diversity in shrimp intestine was lower than that in aquaculture water and biological flocs;The relative abundances of Proteobacteria and actinomycetes in aquaculture water,biological flocs and shrimp intestine gradually decreased,while those of flocculus and chlorocampylobacter gradually increased.4.The concentration of water quality factors such as suspended solids（SS）,total inorganic nitrogen（TIN）,active phosphate(PO₄^3-),total nitrogen（TN）and chemical oxygen demand（COD）in the water samples increased t throughout the culture period.RDA analysis,Pearson correlation analysis and linear regression analysis showed that,environmental factors such as ammonia nitrogen,nitrite nitrogen,actived phosphate were positively correlated with ARGs,Bacteroidetes,Actinomycetes and Proteobacteria;Pearson correlation analysis showed that ARGs were positively correlated with Planktophyta and Bacteroidetes（P<0.01）.That the results suggest that some environmental factors and bacterial populations may promote the proliferation and spread of ARGs in the rearing environment of aquaculture.