The Effects Of Sulfamethazine On Gut Microbiota,Antibiotic Resistance Genes And Endocrine System On Marine Medaka(Oryzias Melastigma)

The excessive use of antibiotics in marine aquaculture had accelerated the accumulation and spread of antibiotic resistance genes(ARGs)in coastal area,which had a certain impact on the ecosystem and human health.Antibiotics residues could be absorbed by the fish intestines,which could cause gut microbiota dysbiosis,lead to the dissemination of ARGs,interfere with the endocrine system and affect the physiological health of the host with a certain ecological risk.In this study,sulfamethazine(SM2),a typical veterinary antibiotic commonly applied in aquaculture,was used to study the biological effects on marine model organisms marine medaka(Oryzias melastigma)by waterborne exposure at 10 μg/L and 100 μg/L exposure concentrations.By analyzing the bioconcentration and metabolism of SM2 in fish intestines,the time and dose-dependent bioaccumulation relationships were established;by exploring the effects of SM2 on gut microbiota and fish endocrine system,the response differences and interplay between gut microbiota and endocrine system were demonstrated;by analyzing the distribution characteristics of ARGs in fish intestines,the driving factors for the enrichment and spread of ARGs were clarified.This research could provide a comprehensive view of understanding the biological effects of antibiotics on marine fish,and provide scientific basis for the scientific use of antibiotics in marine aquaculture and the establishment of environmental quality standards.The main research results were as follows:(1)Studies on the bioaccumulation and metabolism of SM2 in the intestine of marine medaka showed that in the low concentration groups,bioconcentrations of SM2 were increased with the exposure duration.While in the high concentration groups,bioconcentrations of SM2 were affected by its metabolism and decreased significantly with the exposure duration.The antimicrobial effects of SM2 on gut microbiota and the biotransformation and degradation of SM2 by microbial community were the main factors influencing the time/dose-dependent bioaccumulation relationships of SM2 in the intestine of marine medaka.In addition,the accumulation patterns of SM2 in the intestines of male and female fish were different,but there was no significant gender difference in the bioconcentrations of SM2 in the intestines.(2)Studies on the effect of SM2 on gut microbiota of marine medaka showed that SM2 exposure had led to a significant enrichment of opportunistic pathogens in fish intestines and the ecological stability of the gut microbial community structure had been weakened.Among them,β-Proteobacteria,y-Proteobacteria,Flavobacteria and Fusobacteria were main biomarkers,and were associated with gut microbiota dysbiosis and fish immune responses.In addition,the responses of gut microbiota were sexdependent,female fish were more sensitive to SM2 exposure,the main affected bacteria including Vibrio,Shewanella,Ruegeria,and Propionigenium responded differently in low and high exposure concentration groups.While the effects of SM2 on gut microbiota of male fish were relatively weak,only high exposure concentration groups were affected.(3)The study on the distribution of ARGs in gut microbiota of marine medaka found that horizontal gene transfers(HGTs)were the main driving factor of ARGs dissemination in gut microbiota,and mobile genetic events(MGEs)were important vectors for HGTs of ARGs.The induction of multidrug resistance efflux pump by SM2 and the co-selection of non-corresponding types of resistance genes were the main factors affecting the distribution and spread of ARGs in fish intestines.By combining the results of SM2 bioaccumulation and metabolism,the correlation between the structure and function of gut microbiota and distribution characteristics of ARGs in female fish,it was clear that the time and dose-dependent bioaccumulation relationships significantly affects the responses of microbial community to SM2 stress.In addition,the microbial metabolism might be the downstream results of the expression of ARGs and served as a tolerance mechanism in response to SM2 stress.Besides,under the long-term and low-concentration exposure of SM2,its bioaccumulation in fish intestines was accompanied by a significant up-regulation of microbial metabolic activity and significant enrichment of some ARGs,indicating that the long-term low-concentration exposure environment of SM2 would effectively promote the accumulation and diffusion of ARGs in fish intestines,and pose a certain ecological risk to the host and the surrounding environment.(4)Studies on the expressions of HPG endocrine axis-related genes and the secretion of gonadal steroid hormones of marine medaka showed that SM2 could significantly affect the reproductive system of marine medaka by feminizing the male fish and reducing the fertility of female fish,which led to a significant reduction of the survival rates and quality of the offspring.Besides,the impact of SM2 on the HPG endocrine axis showed significant gender differences.Affected by the higher levels of estrogen in female fish to compensate for SM2 endocrine interference and the difference of sex hormone receptor binding in female and male fish,SM2 had a more significant effect on HPG endocrine axis of male fish,reflecting that the sex-driven fish endocrine system has different sensitivity to SM2 exposure.In addition,the sensitivities of gut microbiota and endocrine systems of marine medaka to SM2 exposure were gender-relevant.The possible mechanisms of action included the differences of SM2 bioaccumulation and metabolism in different organs,the sex-dependent endocrine system in fish and the absorption and metabolism of steroid hormones by gut microbiota.