| In this study,four earthworm farms with sludge,cow manure,distiller’s lees+cow manure and straw+chicken manure as bait were selected as the research objects.Based on fluorescence quantitative PCR and Illumina high-throughput sequencing technology,the structure of 13 ARGs and bacterial communities including tetM,tetO,tetT,tetB,tetC,tetL,tetX,tetZ,SUl 1,floR,ermB,blaTEM and intI 1 were analyzed in different culture backgrounds.The aims were to explore the evolutionary characteristics of ARGs under different culture backgrounds and their relationship with bacterial community structure,and to clarify the mechanism of intestinal processes in the evolution of ARGs.The main research results are as follows:1.The bacterial abundance of vermiculture decreased by 47.71%to 81.89%compared with bait under different culture backgrounds.The total abundance of ARGs in vermiculture decreased by 80.60%,14.45%and 45.43%under cow manure,wine grains+cow manure and straw+chicken manure,respectively,while increased by 38.28%under sludge culture background.The absolute abundance of vermicast intI 1 decreased by 79.00%,27.29%and 89.29%in sludge,distiller’s lees+cow manure and straw+chicken manure,respectively.However,in the background of cow manure breeding,the total abundance of ARGs was up to 1119.98%.Overall,the total abundance of ARGs were as high as 1.58×108～1.51×1010(copies/g).2.In terms of the composition characteristics of ARGs,differences exsited in bait,intestinal wall,egg and worm castings under different breeding backgrounds.Based on principal component analysis(PCA),the ARGs composition of vermiculture and intestinal wall were more similar under different breeding backgrounds,but the ARGs composition of vermiculture and bait was more similar under the background of distiller’s lees and cow manure.In general,SUI 1,floR,tetM,tetX,tetZ and blaTEM constituted the dominant ARGs of vermiculas.SUI 1,floR and tetX were prevalent in all culture backgrounds,and the highest abundance reached 109,109 and 108 copies/g,respectively.In addition to shared dominant ARGs,dominant ARGs in bait included tetC,tetO,tetX,ermB,etc.,while dominant ARGs in intestinal wall included tetX,tetZ,tetM,blaTEM,etc.Compared with the dominant ARGs in bait,the composition of vermicast and ARGs in intestinal wall were more similar.The intestinal wall mightcontribute more to the formation of vermicomical ARGs.3.The culture background had influences on the abundance and composition of ARGs in the same sampling site.The total ARGs abundance of bait,intestinal wall,intra-,extracaand worm fecal samples in different culture backgrounds were 1.49×109～1.10×1010,1.47×108～4.78×109,2.05×109～1.01×1010,7.67×108～2.31×l010、2.88×108-l.51×1010copies/g.SUl 1,floR,tetC,ermB,tetX,and tetO constituted different bait advantages ARGs,SUl 1,floR,and tetX were shared advantages ARGs,and the bait ARGs are similar under the background of cow manure,distillers lees and cow manure culture.floR,SUl 1,tetX,tetZ and tetM constituted different intestinal wall dominant ARGs,SUl 1,floR and blaTEM are dominant ARGs,and intestinal wall ARGs were similar under sludge and cow manure culture background.floR,tetM,tetL.tetX,SUl 1,tetZ and so on constituted different intraand extra-egg advantage ARGs,among which SUl 1,floR,tetZ is intra-egg sample sharing advantage ARGs,SUl 1,tetB was extra-egg sample sharing advantage ARGs,SUl 1,tetB was extra-egg sample sharing advantage Args.The composition of ARGs in egg is similar in sludge,cow dung and wine lees+cow dung culture,while the composition of ARGs in egg was similar in sludge and cow dung culture.floR,tetM,tetL,tetX,SUl 1,tetZ and so on constituted the dominant ARGs of different vermiculture,SUl 1,floR,tetX were shared dominant ARGs,and the composition of vermiculture ARGs was similar under the background of sludge and cow manure culture.4.The bacterial community structure in the intestinal process of earthworms changed dramatically.Under the background of sludge,cow manure,lees+cow manure,straw+chicken manure,the number of ASVs shared by vermicompost and bait were 85,130,618 and 74,respectively,and the relative abundance in bait were 10.91%,15.59%,82.11%and 3.07%,respectively.The relative abundance in vermicompost were 32.34%,14.39%,79.02%and 21.04%,respectively.The number of ASVs shared by intestinal wall with bait were 40.146,63,71,and the relative abundance in intestinal wall were 3.15%,22.96%,42.13%,6.13%,and the relative abundance in bait were 49.57%,35.21%,89.23%,and 21.04%,respectively.The number of ASVs shared by intestinal wall and vermicomecia were 101,331,68,and 268,respectively,and the relative abundance in intestinal wall were 73.32%,52.67%,91.78%,77.01%,and the relative abundance in vermicomecia were 41.82%,65.85%,58.51%,and 49.39%,respectively.Some of the bacteria in the bait adapted to the intestinal environment and colonized in the intestinal tract after passing through the intestinal tract process of the earthworm,while others were eliminated in the intestinal tract.Therefore,the bacterial abundance in the worm droppings changed differently in the end,and the bacteria in the worm droppings were more closely related to the intestinal wall in general.Proteobacteria.Bacteroidetes and Actinobacteria were the dominant bacterial communities in four different cultures.Compared with bait.the abundance of Proteobacteria in intestinal wall and vermiculture increased significantly in the four cultures.5.The culture background had an effect on the bacterial community structure of the same sampling site.Proteobacteria,Bacteroidetes,Actinobacteria and Firmicutes are the dominant bacteria in intestinal wall,eggs and vermicomstools under different culture backgrounds,but their abundance was different.For example,Bacteroidetes accounted for 10.77%in the intestinal wall under the background of cow manure cultivation,but only 2.08%,0.52%and 3.03%in the intestinal wall under the background of sludge,lees+cow manure,straw+chicken manure.Firmicutes accounted for 14.39%of eggs under straw and chicken manure culture,but only 4.99%,0.31%and 0.1 5%of eggs under sludge,cow dung and wine lees+cow dung culture,respectively.Actinobacteria accounted for 15.94%of vermiculture in the context of cow manure cultivation,but only 2.31%,7.41%and 13.98%of vermiculture in the context of sludge,distiller’s lees+cow manure and straw+chicken manure,respectively.There were also significant differences in bacterial community composition at genus leve in the intestinal wall,egg in the four cultures.Aeromonas was the most dominant genera under different cultures,and its relative abundance ranges were 49.58%-88.34%,9.12%-44.29%,0.25%～88.31%and 3.23%-29.45%,respectively.Based on principal coordinate analysis(PCoA),it was found that the bacterial communities of bait,intestinal wall,egg and vermicompost were different in different breeding backgrounds,and the bacterial communities of intestinal wall and egg still had a certain convergence under the four breeding backgrounds.6.The results of co-occurrence network analysis showed that the potential hosts of ARGs in different culture backgrounds were significantly different or similar.Under the background of sludge,cow manure,distillers lees+cow manure,straw+chicken manure,the number of potential hosts of ARGs shared by worm manure and bait were 10,2,35,7,respectively,and the total abundance of phase in worm manure were 36.73%,0.43%,18.90%,15.30%,respectively.The total relative abundance in bait were 3.23%,0.03%,8.05%,0.48%;The number of potential hosts of ARGs shared by vermiculture and intestinal wall were 11,14,8,30,respectively,and the total phase abundance in intestinal wall were 46.54%,29.31%,91.70%,41.64%,respectively,and the total relative abundance in vermiculture were 38.39%,5.54%,16.61%,27.51%.The results showed that the bait host had little effect on the formation of ARGs from vermiculture in most cultures,and the intestinal wall ARGs host might contributed more to the formation of ARGs from vermiculture than the bait. |
