The Toxicity Of Abamectin To Chinese Mitten Crab And The Screening Of Its Degrading Bacteria

Avermectins?AVMs?,belonging to macrolides antibiotics,do not have the antibacterial effect,but have high insecticidal activity.As a widely used antiparasitic drug,it is also been used in aquaculture to treat diseases,which are caused by Gyrodactylus and Dactylogyrus,etc.AVMs were used in Eriocheir sinensis aquaculture to remove wild fish and parasites.However,aquatic animals were more sensitive to it,and the residual AVMs affected greatly the growth and quality of aquatic animals.In recent years,microbial remediation,as the main method in biological remediation,has gradually developed into one of the effective methods for treating pesticide residues in the environment.In this paper,AVM was used to investigate the toxic effect on E.sinensis.An AVM-degrading strain AVM-2 was isolated from the bottom mud of E.sinensis culture pond,and the degradation efficiency of the strain was studied.The specific results are as follows:?1?The method of periodically changing the test solution every 24 h was used to perform the acute toxicity test of AVM on E.sinensis.In this study,the LC₅₀ of AVM to E.sinensis at 24,48,72 and 96 h was determined as 21.875,13.403,9.108 and 7.1mg/L,respectively,and the safe concentration was 1.51 mg/L.Chronic toxicity experiments were performed at concentrations of 1/2,1/5 and 1/10 of 96 h-LC50,as3.55,1.42 and 0.71 mg/L.The activities of superoxide dismutase?SOD?,catalase?CAT?,phenol oxidase?PO?and malondialdehyde?MDA?,and the transcriptional expression changes of related genes were detected at the 1,3,6,10 and 15 d post AVMs stress.After 15 d of AVMs stress,the hepatopancreas of E.sinensis was observed through HE stain.The results showed that,the enzyme activity and gene transcription of SOD,CAT,PO in hepatopancreas of E.sinensis after AVMs challenge showed a tendency of first increase and then recover.The activities of SOD,CAT and PO in the low and medium concentration groups peaked on the 6 d,while reach the maximum at 3 d in the high concentration group,reflecting the dose-time dependence.The content of MDA accumulated with the increase of stress time,and the accumulation amount in the high concentration group was larger.After 15 d of AVMs stress,the hepatopancreas of E.sinensis were proved to be damaged through HE stain.The number of B cells decreased and vacuoles appeared.The basement membrane of the hepatopancreas thickened and separated from columnar epithelial cells.In the high-concentration group,the volume of vacuoles increased,and some contents appeared in the lumen.The results showed that the hepatopancreas of E.sinensis were under oxidative stress and tissue damage after AVMs stress.In addition,there was a dose-effect relationship between AVMs and the degree of hepatopancreas injury.?2?The sediment of E.sinensis culture ponds polluted with AVMs for a long time was added sterilized saline to suspend and naturally precipitate and to the AVMs-containing basal salt medium for four cycles of domestication and culture.The enriched culture solution was diluted and then cultured on the AVMs concentration gradient screening medium plate.The single colonies growing on the plates were selected for purification,and a candidate strain of AVM-degrading bacteria AVM-2was isolated and screened.Morphological observation revealed that the stain AVM-2was round,slightly raised,milky white and smooth on LB solid medium.Using transmission electron microscope,the strain AVM-2 was rod shaped with flagellate.Gram's staining show AVM-2 was Gram-negative bacterium.Physiological and biochemical identification showed that the strain was positive for nitrate reduction,but indole,V·P test,ONPG,sucrose,raffinose,sorbitol,citrate and xylose were all negative.Combined with 16s r DNA gene sequence analysis,AVM-2 was concluded to be one kind of Ochrobatrum sp.The degradation efficiency of Ochrobatrum sp AVM-2 was increased with the increase of the inoculation amount when the concentrations of Ochrobatrum sp.AVM-2 was low.When the inoculation concentration of Ochrobatrum sp.AVM-2 was 4×10⁶ CFU/m L,the degradation efficiency was the highest,reaching 32.1%within 72 h after cultured at 30?.As the bacterial concentration increased,the degradation rate of the strain tended to be stable.