| Litopenaeus vannamei also known as South American white shrimp,has the advantages of a short culture cycle,a wide range of salinity,high yields,is currently one of the world’s most important farmed shrimp species,farming production accounted for 53%and 34%of the world and China’s total crustacean production,respectively.With the development of intensive shrimp farming and the deterioration of the ecological environment,a number of environmental stressors,highly virulent bacteria or viruses have led to frequent outbreaks of shrimp diseases,causing huge losses to the shrimp farming industry.Vibriosis,aeromonas and streptococcal diseases are particularly common among bacterial diseases.Of these,Vibrio is considered to be a constant threat to shrimp farming.is a group of Gram-negative bacteria that are widely distributed in aquaculture waters and are highly pathogenic to both aquatic organisms and humans.In this study,a strain of Vibrio alginolyticus was isolated from a shrimp aquaculture system,and the inhibition of Vibrio alginolyticus by antibiotics and micro-ecological agents was investigated.The attenuation of Vibrio alginolyticus in different models of shrimp culture was analysed in relation to water quality,shrimp growth and microbial community structure.(1)A dominant strain of Vibrio alginolyticus was isolated and purified from a shrimp culture system in Yantai,Shandong Province,China,and identified as Vibrio alginolyticus by morphology and 16Sr DNA sequence analysis.(2)The results showed that the minimum inhibitory concentration(MIC)of enrofloxacin and acetylmethane were 0.31 mg/m L and 1.25 mg/m L respectively,and were more sensitive to enrofloxacin.The MICs of enrofloxacin and acetylmethane were diluted 3,10,20 and 50 times,respectively.Regeneration of Vibrio alginolyticus was observed after 6h and 4h in all acetylmethalin and low concentration enrofloxacin(1/10 MIC,1/20 MIC and 1/50 MIC)experimental groups,respectively,only 1/3 MIC enrofloxacin had a stable inhibitory effect on vibrio alginolyticus,with the number of Vibrio alginolyticus below 100 CFU/m L after 15h.(3)The inhibition effect of two micro-ecological preparations,Bacillus and nitrifying compound bacterium,on Vibrio alginolyticus was investigated.The results showed that the inhibition effect of Nitrobacter on vibrio alginolyticus was higher than that of Bacillus at the same dosage(p<0.05).The 7.0%(V/V)and 10.0%(V/V)Bacillus groups showed higher decay rates than the other experimental groups at 72h with 95.40%and 98.94%decay rates of Vibrio alginolyticus respectively,a minimum value of 2.7×10~3CFU/m L of Vibrio alginolyticus occurred in the 10.0%(V/V)group at 336h.Compared with antibiotics,the inhibitory effect of nitrifying bacteria against Vibrio alginolyticus was more stable and there was no regeneration.(4)0.5%(V/V),2%(V/V),5%(V/V),10%(V/V)pre-filmed polyurethane fillers and 2%(V/V)un-filmed polyurethane fillers were added to a suspension containing 10~9CFU/m L of Vibrio alginolyticus.The results showed that the 10.0%(V/V)group required only 120h to reduce the Vibrio alginolyticus count to the 1000CFU/m L,un-filmed polyurethane fillers group reduced the number of Vibrio alginolyticus to 10~3orders of magnitude within 192h.This was followed by an increase in Vibrio alginolyticus at 336h.The polyurethane sponge filler pre-filled with nitrifying microorganisms has a stable inhibitory effect on Vibrio alginolyticus,which is related to the volume of the biological filler and with better inhibition than the free nitrifying bacterial solution.(5)Three shrimp aquaculture systems were established:nitrifying bacterial preparation(N1),biofloc(B1)and built-in pre-mounted biofilm filler(P1),while the aquaculture systems without Vibrio alginolyticus were set up as control groups(N0,B0 and P0)to investigate the number of Vibrio alginolyticus,water quality indexes and the growth of Litopenaeus vannamei in different culture systems.The results showed that the peak concentrations of ammonia and nitrite in the control group were lower than those in the experimental group,and the differences in peak concentrations of ammonia and nitrite nitrogen in the experimental groups N1,B1 and P1 were significant(p<0.05).The rate of decline in Vibrio alginolyticus was fastest in P1,which reduced the number to 1000 CFU/m L within 72 h,with a decay rate of 99.50%,followed by N1,which reduced the number to 1000 CFU/m L within 96 h,with a decay rate of 99.80%,and B1,which took 144 h to reach 1000 CFU/m L,with a decay rate of 99.90%.The survival rate and final body weight of shrimp varied between systems,with P1>N1>B1.(6)High-throughput sequencing was used to analyze bacterial community structure diversity in different culture systems.Alpha diversity indicated that the microbial community diversity was relatively high in all systems;Beta diversity results indicate that the similarity of microbial communities in the same aquaculture system is high,and there are significant differences in microbial communities among different aquaculture systems.The similarity of microbial community was higher in the same culture system,and the difference was significant in different culture systems.Less variability between samples at 10 d and 20 d within each system.Proteobacteria,Bacteroidetes,Planctomycetes and Chloroflexi are the dominant phyla.Rhodobacterales(2.32%-30.76%),Flavobacterales,Pseudomonas,Cytophagales and Rhizobiales were the dominant genus,with Micavibrionales and Bdellovibrionales were found in samples obtained at both 10 and 20 d.The relative abundance of Micavibrionales in the culture systems of each experimental group tended to decrease,while the relative abundance of Bdellovibrionales in the PA1,PB1 and B1 samples tended to increase,and N1 was vice versa. |
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