Bacterial Resource Mining From Larviculture System Of Litopenaeus Vannamei And Primary Genome Analysis Of Vibrio Alginolyticus Strains

In this study,water samples and larvae were collected from Litopenaeus vannamei hatcheries in the coastal area of South China,and the rearing bacterioplankton structure was analyzed by high-throughput sequencing.Heterotrophic bacteria were isolated and identified from water and larvae,and potential probiotics were screened and drug resistance of Vibrio isolates was detected.The whole genomes of seven V.alginolyticus strains were sequenced and analyzed.These studies provide basis for bacterial resource mining,prevention and control of vibriosis in shrimp larviculture system.Firstly,the bacterioplankton structures in 42 ponds from nine shrimp hatcheries(Guangdong: DH,GL,HD,HY,RH;Guangxi: HT,ZK,ZZ;Hainan: HC)were analyzed by Illumina high-throughput sequencing.The results showed that the geographical locations of hatcheries,larval stage,and healthy state of larvae all had obvious influence on the bacterioplankton structure.Bacteroidetes and α-Proteobacteria were the most dominant or sub-dominant bacteria in all the rearing water,with the exception of postlarvae 5 stage of GL group.At family level,Rhodobacteriaceae,Flavobacteriaceae,Haliscomenobacteriaceae and Microbacteriae were the dominant families in the aquatic microbiota,among which,Rhodobacteraceae existed predominantly in all the ponds,with relative abundance of 22%-49%.At level of dominant genera,Marivita,Phaeobacter,Ruegeria,Donghicola,Phaeodactylibacter,Pseudoalteromonas and Epibacterium were commonly found in normal ponds,which can be used as candidate indicators for healthy larviculture ponds,while Vibrio may be used as indicator for diseased ponds.Secondly,bacterial strains were isolated from rearing water and larvae and were identified preliminarily by 16 S r RNA gene sequencing.Potential probiotics of non-Vibrio were screened based on the characteristics of extracellular enzyme and the antagonistic activity against luminescent vibrio.A total of 231 non-Vibrio bacteria were isolated and preserved in this study.Proteobacteria isolates were the most common(94 strains),and most of which were Pseudoalteromonas.The second most isolates were Firmicutes(67 strains),and most of them belonged to Bacillus,while Bacteroides isolates(46 strains)all belonged to the family Flavobacteriaceae,and Meridianimaribacter strains were the most one.Of the 24 Actinobacteria isolates,half were Microbacterium.Two potential probiotics,including Pseudoalteromonas sp.HD-L11 and Epibacterium sp.CX-W13,which could inhibit luminescent vibrio and belonged to the dominant microflora in rearing water were selected,of which HD-L11 also had strong protease and amylase activities.Furthermore,with 16 S r RNA and HSP60 gene sequencing,46 strains of Vibrio were identified,and their sensitives to 17 kinds of drugs were examined by disc diffusion method.The results showed that V.alginolyticus was the most dominant vibrio in larviculture water.Drug resistance analysis showed that the resistance rate of the 46 strains to furazolidone,cefixime,ampicillin and amoxicillin reached more than 50%,while the resistance to erythromycin and ciprofloxacin was low(<5%),and no resistance to chloramphenicol.The proportion of strains resistant to ≥4 and ≥7 antibiotics was 63% and 15%,respectively.Finally,using Pac Bio sequencing technology,the whole genomes of 7 strains of V.alginolyticus were subjected to sequencing,annotation and collinear analysis.The results showed that all the 7 strains had two circular chromosomes,ch1 and ch2,and 3 strains had 1-2 plasmids.The genome size was 5.20-5.33 Mb,and the GC content was 44.61%-44.74%;The predicted coding genes were 4531-4768;A total of 3030-3069 genes were obtained functional annotations of COG;2643-2675 genes were obtained functional annotations of KEGG;2740-2799 genes were obtained functional annotations of GO;4474-4711 genes were obtained functional annotations of Refseq;3937-4029 genes were obtained functional annotations of Pfam;and 1517-1541 genes were obtained functional annotations of TIGRFAMs.The COG function annotation shows that among the known genes,amino acid transport and metabolism genes are the most,followed by transcription-related protein genes.Collinearity analysis showed that the 7 strains could be divided into two categories,and there were frequent inversion and deletion of homologous fragments among the strains.