| Gyrodactylus is a kind of pathogenic microorganism which parasitize on the skin and gills of fish.The pathogenic microorganisms mainly lead to a high fatality rate of host and cause huge economic losses to the aquaculture industry by serious secondary bacterial infection with a large scale of parasitism.Meanwhile,a large number and variety of bacteria colonize on the skin of animals,and they are closely related to the parasites in their vicinity.Mass studies have shown that there is a certain interaction between parasites and bacterial communities in the same microecological environment during long-term coevolution,which may determine the normal course of parasite infection to a certain degree.Thus,in this study,16 S r RNA amplicon high-throughput sequencing technology was used to confirm that Gyrodactylus infection can have a significant impact on the microflora of goldfish tail fin skin.Meanwhile,a combination of antibiotics(levofloxacin combined with neomycin)was selected to change the bacterial community on the goldfish tail fin skin,and it was further found that the treatment could significantly reduce the number of Gyrodactylus infection.Finally,we analyzed the difference of microbial structure on the goldfish caudal fin skin after the combined treatment of levofloxacin and neomycin,in order to provide a theoretical basis for fine regulation of host microecosystem.Mainly the following results are obtained:1.The composition of skin microflora is changed significantly by Gyrodactylus infectionIn this study,healthy goldfish(200)and unhealthy goldfish(40)infected with Gyrodactylus were mixed.The bacteria on the goldfish caudal fin skin were sampled when the infection rate exceeded 80%.The degree of infestation was divided into three levels:severe infestation(the number of Gyrodactylus more than 40 in 40 x optical microscope single field),mild infestation(the number of Gyrodactylus less than 10 in 40 x optical microscope single field)and healthy group.The results obtained were as follows:(1)At the generic level,there were 159 endemic outs in the severely infected group compared with the other groups,and the number of outs in the severely infected group was significantly increased(p <0.05);(2)β-diversity analysis showed that the microbial community composition in the severely infected group changed significantly;(3)At phylum level,the results showed that the abundance of Firmicutes increased significantly in the severely infected group(p <0.05);At genus level,it showed that the abundance of Delftia and Ralstonia increased significantly in the severely infected group(p <0.05),the abundance of Phenylobacterium and Sphingopyxis decreased significantly(p <0.05).2.Treatment with antibiotics alters the skin microflora and reduces Gyrodactylus infectionTwelve antibiotics were used to treat the infected goldfish for 72 h,and eleven antibiotics were selected which did not affect the growth and reproduction of the Gyrodactylus.Then,according to the plate coating and absolute quantitative results,the antibiotics that can change the microflora on the surface of the tail fin of goldfish were screened out,including neomycin,levofloxacin,neampicillin,ceftazidine and oxytetracycline hydrochloride.Different antibiotics are used in combination according to the antibacterial effect difference.Based on this,the healthy goldfish were treated with antibiotics immersion 72 h,and then severely infected goldfish were introduced 48 h.The results showed that the average number of Gyrodactylus in the combined treatment group of levofloxacin and neomycin was 0.50±1.22 per fish.In the control group,the combined treatment group of penicillin and ceftazidime,and the oxytetracycin treatment group,the average number of Gyrodactylus per fish reached 16.00±16.65,19.17±9.32 and 18.33±4.71,respectively.These results indicated that the number of Gyrodactylus in levofloxacin and neomycin combined treatment group was significantly decreased compared with other groups(p <0.05).3.Change of microflora on skin of goldfish under the selective pressure of levofloxacin and neomycinIn order to reveal the reason of the decrease in the number of Gyrodactylus,we researched the changes of the skin microflora of goldfish treated with levofloxacin and neomycin.The results obtained were as follows:(1)The significant difference was found in the β diversity between levofloxacin and neomycin combined treatment and control group,but the combination treatment of penicillin and ceftazidime which did not interfere with the Gyrodactylus infection had no significant difference in the β diversity of goldfish skin bacteria compared with the control group;(2)LEf Se analysis showed that Flavobacterium and Candidatus Megaira were the characteristic bacteria after 96 h of combined treatment with levofloxacin and neomycin;(3)After combined treatment with levofloxacin and neomycin,the relative abundance and absolute abundance of Flavobacterium were significantly higher than those in other groups(p <0.05),the relative abundance of Candidatus Megaira was significantly lower than that of other groups(p <0.05),but its absolute abundance did not change significantly.In conclusion,the severe infection of Gyrodactylus could significantly change the microbial structure on the skin of the tail fin of goldfish,leading to a significant increase in the relative abundance of secondary infection-related bacteria.Correspondingly,the altered skin microflora of goldfish treated with levofloxacin and neomycin for 96 h also significantly interfered with the infestation number of the Gyrodactylus.Further high-throughput analysis showed that antibiotic combinations that significantly altered the skin microflora of goldfish were able to interfere with the ability of Gyrodactylus infection,which was characterized by high abundance of Flavobacterium and low abundance of Candidatus Megaira.This study provides data reference for revealing the changes of the microecological environment in the process of Gyrodactylus infestation,and provides research direction for the ecological control strategies of the following Gyrodactylus infestation. |
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