| Antibiotics have been widely used in antimicrobial drugs in aquaculture organisms or various infectious diseases in humans.With the increasing use and abuse of antibiotics,the use of antibiotic drugs has caused global environmental and health problems.As one of the most used antibiotics in aquaculture,sulfamethoxazole’s organic compounds are often detected in the marine environment and have become a major research hotspot as an emerging environmental pollutant.In recent years,the research on aquatic organisms of sulfamethoxazole has mainly focused on the toxic effects of fish,shrimp and cephalopods,but the toxic effects of the environmental pollutants sulfamethoxazole on Apostichopus japonicus and the intestinal flora and its metabolites have not been fully studied.In this study,the negative effects of sulfamethoxazole exposure on Apostichopus japonicus were investigated on the intestinal tissue structure,intestinal flora and functional characteristics of Apostichopus japonicus and its host metabolites.The main conclusions of this study are as follows:1.In order to investigate the effect of sulfamethoxazole on the growth of Apostichopus japonicus under stress,96 h LC50 toxicity experiments were performed on Apostichopus japonicus at different concentrations of 0mg/L,400mg/L,600mg/L,800mg/L,1000mg/L,1200mg/L,1400mg/L,1600mg/L and 1800mg/L.The results showed that the LC50 value of sulfamethoxazole for 96 hours was 1252.93mg/L.Combined with LC50 data,the concentrations of sulfamethoxazole stressed Apostichopus japonicus were set as control group(DZ: 0mg/L),low concentration group(SN: 1200ng/L),and high concentration group(SU: 1200ug/L).The results showed that at 35 days,there was a significant difference in the specific growth rate of Apostichopus japonicus in the SN group(P<0.05)compared with the DZ group,while there was no significant difference in the SU group.At 56 d,compared with the DZ group,there was a significant difference in the specific growth rate of Apostichopus japonicus in the SN group and the SU group(P<0.05),while the specific growth rate of Apostichopus japonicus in the SN group increased negatively2.H.E staining and microscopic examination methods were used to carry out the study of intestinal tissue of Apostichopus japonicus under sulfamethoxazole stress.The results showed that with the increase of sulfamethoxazole concentration,the thickness of the mucosal layer in the DZ group gradually decreased from(41.14±3.52μm)to SN group(32.34±9.65μm)and SU group(30.04±7.47μm),and the muscle layer also showed a downward trend with the increase of sulfamethoxazole concentration.Compared with the DZ group,the intestinal microvilli in the SN and SU groups became shorter and less,and the surface area of their absorption was also narrowed,thus showing edema or inflammatory response,which was not conducive to the digestion and absorption of Apostichopus japonicus intestinal tissue.3.Using 16 S rRNA high-throughput sequencing technology,the study of intestinal flora of Apostichopus japonicus with different concentrations of sulfamethoxazole was carried out,and the results showed that compared with the DZ group,the intestinal microbiota structure of SN and SU group showed significant differences,and the abundance and diversity of intestinal flora of Apostichopus japonicus in SU group showed a significant downward trend(P<0.05).At the phylum level,the dominant phylum of Apostichopus japonicus intestinal in the DZ,SN and SU groups were all proteobacteria,with a relative abundance higher than 64.5%,among which the subdominant phylum and the largest differential phylum of the DZ group were both verrucous microphylum,and the subdominant phylum and the largest differential phylum of the SN group were both phylum Bacteroides.At the genus level,the differentiating bacteria in the DZ group were Lactobacillales and Bacilli,and the differentiating bacteria in the SN and SU groups were lutibacter and Ralstonia,respectively.With the increase of sulfamethoxazole concentration,the intestinal flora of Apostichopus japonicus sinensis appeared monolithic(the relative abundance of Ralstocella was 81.6%).Based on the KEGG metabolic pathway database,a total of390 tertiary metabolic pathways were annotated,which could aggregate into 44 secondary metabolic pathways(P<0.05)and 6 primary metabolic pathways.The differential metabolic pathways of intestinal flora in the three groups of DZ,SN and SU groups were mainly reflected in metabolism,as follows: the DZ group focused on amino acid metabolism,the SN group was mainly based on membrane transport,lipid metabolismand amino acid metabolism,and the SU group was mainly based on carbohydrate metabolism,lipid metabolism,exogenous biodegradation and metabolism.Studies have shown that high concentrations of sulfamethoxazole in the environment affect the structural characteristics of the intestinal flora of Apostichopus japonicus spinosa,which is manifested as structural monolithography and dysfunction of the intestinal flora.4.The analysis of differential metabolites of Apostichopus japonicus intestinal flora under sulfamethoxazole stress by non-targeted metabolomics technology showed that 354 metabolites were identified,and compared with the DZ group,the differential metabolites of the SN group were mainly concentrated in L-threonine,L-glutamic acid,citric acid,arachidonic acid,Nervonic acid and docosapentaenoic acid,SU group differential metabolites are mainly concentrated in Nervonic acid,docosapentaenoic acid,Nervonic acid and docosapentaenoic acid.Based on the KEGG Pathway database annotation,the SN group differential metabolites are mainly enriched in the biosynthesis of unsaturated fatty acids,aminoacyl-tRNA biosynthesis and the Citrate cycle metabolic pathway.SU group differential metabolites are enriched into the Biosynthesis of unsaturated fatty acids metabolic pathway.The results showed that there were obvious differences in the intestinal metabolites of Apostichopus japonicus under different concentrations of sulfamethoxazole stress,and the metabolic pathways enriched into the body also changed significantly,and the results laid a theoretical foundation for the study of the correlation between Apostichopus japonicus intestinal flora and its metabolites under different concentrations of sulfamethoxazole.5.Correlation analysis of high-throughput sequencing data and metabolomics,screening of differential intestinal flora and host metabolites of Apostichopus japonicus under sulfamethoxazole stress.The results showed that the metabolites with significant differences were Glycoursodeoxycholic acid,oxidized glutathione oxidized,L-cystine,Diaminopimelic acid and different flora Ralstonia,Actinomyces,There are significant correlations between Pseudomonas,Cellvibrio,and Lutibacter.The study of the correlation between metabolites and intestinal flora provides a new research idea for understanding the role of microbiota in the expression and development of various Metabolites. |
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