Dynamics Of Bacterial Communities Associated With Yesso Scallop(Patinopecten Yessoensisis) Larvae And In The Water Of Larval-rearing Tanks, And Selection Of Potential Probiotics

Bacteria are an important component of the aquatic ecosystem. In this study, dynamics of bacterial communities associated with Yesso scallop(Patinopecten yessoensisis) larvae and in the water of larval-rearing tanks were studied during five different larval development periods(zygote S1/W1, trochophora S2/W2, D-shaped larvae S3/W3, umbo larvae S4/W4, juvenile scallop S5/W5) by culture-dependent and high throughput sequencing methods. At the same time, potential probiotics were screened based on enzyme production ability, antagonistic activity against pathogen, hemolytic activity and safety for scallop larvae of the isolates.Overall, 106760 and 96619 optimized sequences were obtained from larvae and water of larval-rearing tanks by Miseq high-throughput sequencing. Sequences were compared in 16 S rRNA gene database, larval sequences were classified as 4 phyla, 7 classes, 15 orders, 21 families, 31genera; water’s sequences were classified as 9 phyla, 17 classes, 33 orders, 50 families, 83 genera. For larvae associated bacterial community, Proteobacteria was predominant phylum. At genus level, Pseudomonas was dominant genus in stages of S1, S2 and S5, followed by Photobacterium and Aeromonas in S1 stage, Serratia, Stenotrophomonas and Acinetobacter in S2 stage, Shewanella and Pseudoalteromonas in S5 stage. However, Pseudoalteromonas became the dominant in stages of S3 and S4, followed by Photobacterium, Pseudomonas, Aliivibrio and Shewanella in S3 stage, Pseudomonas, Aliivibrio, Shewanella, Psychromonas and Psychrobacter in S4 stage. For bacterial community in the water of larval-rearing tanks, Proteobacteria and Bacteroidetes were predominant phylum. At genus level, Pseudoalteromonas and Sulfitobacter was dominant genus in W1 W4, W5 stages, followed by Alteromonas, Phaeobacter, Roseobacter in W4, Roseobacter and Alteromonas in W5. However, Vibrio became the dominant in W2 stage, followed by Pseudoalteromonas, Sulfitobacter, Roseobacter and Aliivibrio. Glaciecola was dominant in W3 stage, followed by Roseobacter, Sulfitobacter, Alteromonas and Polaribacter. The results show that scallop larvae and water contained rich bacterial resource, and the diversity of bacteria in water was higher than that in larvae. There existed various degree differences in both larval and water bacterial communities.Three hundred bacterial strains were isolated from larvae and water. Polymerase chain reaction and sequencing of partial 16 S rRNA gene of the 300 strains were performed. Results showed that all isolates from larvae belonged to 2 phyla, 3 classses, 8 orders, 12 families, 16 genera and 39 species; all isolates from water belonged to 2 phyla, 3 classses, 6 orders, 9 families, 10 genera and 22 species. At phylum level, Proteobacteria was the most abundant and Firmicutes subdomiant. At genus level, Vibrio and Pseudoalteromonas was dominant genus in stages of S1, S2 and S3, followed by Planococcus in S1 stage, Sulfitobacter and Bacillus in S2 stage, and Planococcus and Alteromonas in S3 stage. However, Pseudoalteromonas became the dominant in stages of S4 and S5, Vibrio was the second dominant genus in S4 and S5, followed by Bacillus and Alteromonas in S4, Bacillus and Pontibacillus in S5 stage. The first dominant genus in water of five development was Pseudoalteromonas, followed by Alteromonas and Vibrio in W1, Vibrio and Alteromonas in W2, Vibrio and Alteromonas in W3 stage; Alteromonas and Bacillus in W4 stage, Vibrio and Pseudophaeobacter in W5 stage. The results show that scallop larvae and water contained rich culture bacterial resource, and the diversity of culture bacteria in larvae was higher than that in water. There were various degree differences in both larval and water bacterial communities.Two strains were screened based on isolates producing a variety of enzymes(amylase, protease and cellulase) and having antagonistic activity against pathogen Vibro splendidus. Similarity analysis of 16 S rRNA gene sequences indicated that the bacterial strain 4W25 had 100.0% similarity to Pseudoalteromonas espejiana and strain 3S16 had 99.8% similarity to Bacillus aerophilus. Hemolysis was tested in 2 strains and the results showed that strain 4W25 did not secrete hemolysin, without potential pathogenicity. Using 24-well plate assays, 9d larvae were challenged by strain 4W25 at 1×106cells/mL final concentration for 48 h, confirming that 4W25 strain was safe for scallop larvae.