Toxicity Of Florfenicol On Portunus Trituberculatus And Optimization Of Administration Scheme

Florfenicol（FLR）is one of the most commonly used antibiotics in aquaculture.It is a fluorine-containing antibacterial analogue of chloramphenicol.According to monitoring,the content of FLR in China’s coastal waters can reach 42 ng·L^-1.In the process of aquaculture,FLR has been widely used in aquatic animal disease control in China because of its wide antibacterial spectrum and low price.However,with the unreasonable use and abuse of FLR in aquaculture and the lack of understanding of the safe dose range and drug withdrawal period of FLR,FLR remains in aquatic animals or aquaculture water environment,which poses a potential threat to human health and the lives of other organisms in the water.This study first evaluated the toxic effect of FLR on Portunus trituberculatus from the molecular level of m RNA and mi RNA,and then explored the metabolic law of FLR in P.trituberculatus through the pharmacokinetics of FLR in P.trituberculatus,so as to provide a theoretical basis for guiding the standardized use of FLR in aquaculture in the later stage.The research results are as follows:1.Study on the toxic effect of FLR on P.trituberculatusUnder the acute stress of FLR(80 mg·kg^-1)in the treatment group,the hepatopancreas of P.trituberculatus stressed for 3h and 12h were taken as the experimental group（FLR-3h,FLR-12h）and the cosolvent DMSO control group（C）for histopathological section observation and transcriptome analysis.The results of histopathological sections showed that with the increase of FLR stress time,the degree of hepatopancreatic tissue injury gradually increased,including the changes of the morphology and structure of basement membrane,lumen and epithelial cells.It is speculated that high-dose FLR will have some toxic and side effects on the important detoxification and metabolic organ（hepatopancreatic gland）of P.trituberculatus.Transcriptome sequencing showed that a total of 498,601,504 clean reads were obtained from 9 libraries,with a data volume of 74.77 G,the Q20 of 97.40～97.76%,and the Q30 of 92.89～93.64%.After that,the high-quality sequences were compared with the reference genome,and a total of 441,591,209reads were aligned to the genome,accounting for 88.57%.In this study,the number of differentially expressed genes between DMSO control group and 3h FLR high dose group,DMSO control group and 12h FLR high dose group,3h FLR high dose group and 12h FLR high dose group were 3152,2935 and 665 respectively.Eight differential genes were screened for q RT-PCR,and the quantitative results were consistent with the transcriptome sequencing results,indicating that the transcriptome sequencing data were reliable.The results of GO and KEGG showed that the differential genes were mainly concentrated in immune and apoptosis related pathways such as cytochrome P450,metabolic pathway and drug metabolic enzymes.It was speculated that P.trituberculatus would produce stress and immune response after FLR stress,and eliminate the toxic and side effects on itself through apoptosis or metabolism.2.Correlation analysis between m RNA and mi RNA of P.trituberculatus under FLR stress and its validation in vitroIn order to further analyze the transcriptome effect formation and regulation mechanism of P.trituberculatus hepatopancreas under FLR stress,this study explored whether the differentially expressed mi RNA negatively regulated the differential genes screened in the transcriptome through mi RNA and m RNA association analysis,and verified the screened mi RNA target genes in vitro by double luciferase test.The results of mi RNA expression profile data showed that a total of 144 mi RNAs were detected,including 25 known m RNAs and 119 newly predicted m RNAs.The results of difference analysis showed that 35 and 31 mi RNAs were differentially expressed between DMSO control group and 3h FLR high-dose group,DMSO control group and12h FLR high-dose group respectively.Due to the difference between 3h FLR high-dose group and 12h FLR high-dose group,the expression of mi RNA had not reached the analysis standard,so no further analysis was made.Eight differential mi RNAs were screened for q RT-PCR,and the quantitative results were consistent with the results of mi RNA omics sequencing,indicating that the mi RNA omics sequencing data were reliable.According to the functional enrichment analysis of GO and KEGG,the predicted target genes are involved in a variety of biological processes,including some immune related pathways（such as endocytosis,JAK-STAT signal pathway and apoptosis pathway）and exogenous substance metabolism related pathways（such as cytochrome P450 metabolism and ABC transporter）.The above results show that mi RNAs participate in FLR metabolism in P.trituberculatus through various ways,which provides a theoretical basis for further study on the regulation mechanism of mi RNA in FLR metabolism in P.trituberculatus.Based on the results of m RNA and mi RNA association analysis,mi R-34 and p53 target protein 1 target gene pairs,mi R-263b and Cytochrome c target gene pairs related to apoptosis,detoxification metabolism and stress immunity were selected for in vitro validation.The results showed that only when mi R-263b was co transfected with cytochrome c-3’UTR wt,the expression of luciferase was significantly down regulated（p<0.001）,indicating that mi R-263b could directly inhibit the expression of Cytochrome c at the cell level in vitro.However,mi R-34 can not directly inhibit the expression of p53 target protein 1,and there is no targeted regulation relationship between them.3.Pharmacokinetic comparison and residue elimination of different concentrations of FLR in P.trituberculatusAfter intramuscular injection of FLR at doses of 20 mg·kg^-1,40 mg·kg^-1 and 80 mg·kg^-1,the content of FLR in hemolymph of P.trituberculatus was determined by high performance liquid chromatography（HPLC）.Using DAS2.0 pharmacokinetic software analyze the blood concentration of FLR in P.trituberculatus in order to fit the curve equation of pharmacokinetics,compare the pharmacokinetic differences in the hemolymph of P.trituberculatus under the condition of different doses of FLR administration,and optimize the administration scheme.The results showed that the hemolymph of P.trituberculatus injected with different doses of FLR accorded with the two compartment open model.The metabolic trend of different doses of FLR in the hemolymph of P.trituberculatus was first slowly increased and then decreased.Under the condition of intramuscular injection in the low dose group,medium dose group and high dose group,the elimination half-life of FLR in the hemolymph of P.trituberculatus was 21.128 h,21.277 h and 20.112 h respectively.The elimination half-life of FLR in the hemolymph of P.trituberculatus was almost the same in the low dose group and medium dose group.The elimination half-life of the high dose group changed slightly,and the elimination half-life was independent of the drug dose.According to the results of histopathological observation and correlation analysis of m RNA and mi RNA levels after intramuscular injection of 80 mg·kg^-1FLR in the early stage,it was found that the toxic and side effects of FLR on P.trituberculatus were time-dependent.Therefore,it is suggested that the dosage of 40 mg·kg^-1 intramuscular injection should be administered several times every 12 h,so that FLR can be maintained above0.8μg·m L^-1in P.trituberculatus,which can effectively treat the common bacterial diseases of P.trituberculatus.The maximum residue limit of FLR in national animal food is 0.1 mg·kg^-1.However,when the dosage is 40 mg·kg^-1,the residual content of FLR drug is not lower than 0.1mg·kg^-1 within 72 h.Therefore,it is not possible to give a recommended withdrawal period,which will be further discussed as a research topic in the next stage.This paper has 22 figures,29 tables and 135 references.