| In recent years,earthworm conversion of cow manure has become an important way of livestock and poultry manure utilization.While reducing the total amount of livestock manure,it can also produce economically valuable products such as earthworms and vermicompost.However,there are large amounts of heavy metals and antibiotic resistance gene residues in livestock and poultry manure,which may be transferred to earthworms or vermicompost along with the conversion process of earthworms,further increasing the risk of ARGs entering the food chain,and ultimately threatening human health and life safety.This has become a major problem affecting the technology used by earthworms to convert cow manure.Therefore,as for the issue of unclear migration rules,driving factors,and transmission mechanisms of ARGs during the conversion of cow manure by earthworms,this study comprehensively analyzed the transformation process of"cow manure,earthworm,substrate and vermicompost"and the behavioral fate,migration mechanisms,and driving factors of ARGs in different intestinal segments of the earthworm gut,in order to discover the mechanisms that different factors affect the changes of ARGs during the conversion process of earthworms.（1）The relative abundance of tet-ARGs in 15 earthworm farms is the highest(10-6～10-1 copies/16S copies),while ARGs closely related to human health(i.e.,blaamp C,bla OXA-1,and bla TEM-1)are also commonly detected in earthworm farms.After earthworm transformation,the detection frequency of ARGs in cow manure decreased,but the six target genes,tet Q,blaamp C,bla TEM-1,qnr S,cfr,and fex A,showed an upward trend.Although the content of ARGs in cow manure samples from different farms varies greatly,the diversity and abundance of ARGs in all earthworm compost samples after earthworm transformation are similar;Among these investigated earthworm farms,the earthworm farms that added vinegar as an adjunct had the best removal effect on ARGs,with the total ARGs abundance reaching10-1 copies/16S copies before treatment and decreasing to 10-3 copies/16S copies after treatment.Earthworm transformation has effectively reduced some ARGs in cow manure,but there are still large amounts of ARGs in earthworms and earthworm compost(up to 10-1 and 10-2 copies/16S copies,respectively).Overall,earthworm transformation can accelerate the reduction of ARGs abundance in cow manure,which can reduce its environmental risks to some extent.（2）A total of 377 ARGs subtypes in 24 categories were detected in the intestines of earthworms cultured with cow manure,with the highest detection rates of multidrug resistance genes（42.4%）and vancomycin（14.3%）,followed by macrolide-linkamide-streptomycin（MLS,8.7%）,tetracyclines（8.6%）,and aminoglycosides（7.7%）.Compared with before feeding cow manure,the total ARGs abundance in the foregut and midgut increased by 28.9%and 30.2%,respectively,while the total ARGs abundance in the hindgut of earthworms decreased by 53.7%.Among all intestinal segments of earthworms,Actinobacteria has the highest abundance,followed by Proteobacteria,Chloroflexi,and Firmicutes.From the foregut to the midgut and hindgut,the abundance of Proteobacteria gradually increases,and the abundance of Bacteroides in the hindgut of earthworms is higher than that in the foregut and midgut.In addition,the type,abundance,and mobility of ARGs in earthworm dung have decreased compared to cow manure.（3）Under different Cu concentrations,the cumulative total abundance of ARGs showed a trend of CF（without Cu added）<T3(high concentration,1000 mg·kg-1)<T1(low concentration,100 mg·kg-1)<T2(medium concentration,500 mg·kg-1).Compared to the control group without earthworms,the total abundance of ARGs in the CF treatment group with earthworms increased from 5.28×10-1 reduced to4.33×10-1 copies/16S copies.The addition of low and medium concentration Cu significantly increased the total abundance level of ARGs in the sample（P<0.05）.By studying the changes of different types of ARGs at different time stages,it was found that low and high concentration Cu treatments（T1,T3）increased the abundance of tet-ARGs,erm-ARGs,and str-ARGs during earthworm transformation,while the abundance of tet-ARGs and str-ARGs in medium concentration Cu treatments（T2）decreased.Redundancy analysis（RDA）showed that p H significantly affected the relative abundance of ARGs during earthworm transformation（P<0.05）,and TOC,TN,and Cu also played an important role in the growth and decline of ARGs.（4）The distribution of antibiotic resistant genomes in the foregut,midgut,and hindgut of earthworms was significantly altered by transformation of cow manure:the abundance of ARGs in the foregut and midgut increased,while the abundance of ARGs in the hindgut decreased.Compared with the copper free group（CK）,the total ARG abundance in the foregut,midgut,and hindgut of earthworms under copper stress decreased by 0.7±4.7%,3.4±2.6%,and 3.5±39.2%,respectively.A similar trend was also observed in metal resistance genes（MRGs）.This indicates that the content of Cu in cow manure has no significant impact on the composition of ARGs and MRGs in the intestinal tract of earthworms.However,copper in cow manure(100 and 300 mg·kg-1)significantly increased the abundance of ARGs and MRGs in earthworm dung. |
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