Study On The Succession Of Microflora In Apostichopus Japonicus During The Early Developmental Stages And The Screening Of Potential Probiotics

In this paper,the changes of intestinal microflora and the influence of environmental?aquaculture inlet water and feeds?microflora on the intestinal microflora of Apostichopus japonicus at early developmental stages was studied.At the same time,the preliminary screening of potential probiotics from strains isolated from intestine of sea cucumber larvae and juveniles was also performed.The aim of this study was to analyze the formation and sucession regulation of the intestinal microflora in sea cucumber and provide a basis on the screening of probiotics used on aquaculture of sea cucumber.The main results were as follows:1.Conventional bacteriological culture techniques and molecular identification methods were used to reveal the composition and structure of the intestinal microflora in sea cucumber?Apostichopus japonicus?larvae at different developmental stage and the aquacultural environment?aquaculture inlet water and feeds?microflora,and analyse the correlation between them.The results of bacterial culture from sea cucumber larvae showed that culturable bacteria occurred after larvae mouth-developed and feeding.From auricularia larvae to doliolaria larvae,the number of culturable bacteria in gastrointestinal?GI?tract were low?10¹ CFU/ind?.After one-month old,the culturabal bacteria in GI tract were increased significantly,reaching the highest level?10⁸ CFU/g?in four-month-old juvenile.The results of culturable vibrio quantity in the intestine of sea cucumber larvae showed similar trend at different developmental stages,and the proportion of vibrio in total culturable bacteria in sea cucumber juvenile was from 2.2%to 77.3%.The results of bacterial culture from environment showed that there was no siginificant change of bacteria content in the aquaculture inlet water,and with the change of ingredients in feeds there was significant difference in the bacterial content in the feeds from different early developmental stages of sea cucumber.Significant correlation of bacteria count was showed between intestine of sea cucumber larvae and feeds.65 dominant strains isolated from aquaculture system were identified by sequencing their 16S rDNA for alignment with EzBioCloud database,and classified into 14 genera and 43 species.The results of statistical analysis on the strain distribution in larvae and environment showed that Halomonas alkaliantarctica was the communal dominant strain in auricularia larvae and doliolaria larvae.In one-month-old juvenile,Vibrio atlanticus and Vibrio natriegens from both feeds and water were greatly colonized in intestine,followed by Bacillus tequilensis from feeds only.In two-month-old juvenile,Vibrio alginolyticus was the communal dominant strain in intestine and feeds.From two-month old to four-month old,Vibrio fortis was the communal dominant strain in their intestine.In six-month-old juvenile,Bacillus siamensis from both feeds and water were greatly colonized in GI tract.The correlation analysis showed that in the early developmental stages,the correlation of microflora between larvae and the aquaculture water was higher,and with the further development of sea cucumber the correlation of microflora between larvae and feeds increased gradually.2.High throughput sequencing was used to futher explore the the composition and structure of the microflora in sea cucumber?Apostichopus japonicus?larvae at different developmental stage and the aquacultural environment?aquaculture inlet water and feeds?.From zygotes to doliolaria,the total abundance of Proteobacteria,Bacteroidetes and Cyanobacteria remained above 90%.From one-month-old to four-month-old juvenile,with the increase of the total abundance of Cyanobacteria,Planctomycetes and Verrucomicrobia,the proportion of Proteobacteria decreased greatly,reaching the lowest level in the intestine of four-month-old juvenile,and the proportion of Bacteroidetes was still at a low level?0.91%³.73%?.In the six-month-old juveniles,Proteobacteria dominated again in the intestinal microflora,while the other bacteria in phylum level decreased.In family level,Pseudoalteromonadaceae dominated in zygotes;Flavobacteriaceae dominated in gastrula;from gastrula to doliolaria,the abundance of Rhodobacteraceae was increasing and at a high level;the proportion of Alteromonadaceae was relatively high?14.73%?in auricularia;the proportion of Vibrionaceae was relatively high?18.62%?in auricularia either,reaching the highest level?78.25%?in six-month-old juveniles;Planctomycetaceae and Verrucomicrobiaceae dominated in juveniles from one-month-old to six-month-old period,with abundance28.27%⁴0.22%,9.56%²1.37%respectively.The correlation analysis of microflora between larvae intestine and environment showed that at the early stages of sea cucumber,higher similarity was found between intestinal microflora and inlet water microflora,and with the development of larvae the intestinal microflora was influenced greatly by the microflora from feeds.In family level,the bacteria from inlet water and feeds played a more complicated role in the intestinal microflora of sea cucumber.The abundance of Rhodobacteraceae in larvae was mainly affected by the inlet water,as positive correlation of its abundance changes from auricularia stage to four-month-old stage was discovered between intestinal microflora of larvae and inlet water bacterial community.The abundance of Flavobacteriaceae between larvae?auricularia and doliolaria?and inlet water was more closely related.In addition,the abundance of Vibrionaceae and Planctomycetaceae in larvae was mainly affected by the feeds,for the abundance of Vibrionaceae in auricularia and six-month-old juveniles was relatively higher than that in other stages,so as the abundance of Vibrionaceae in feeds from the same stage.The proportion of Planctomycetaceae in larvae from doliolaria to four-month-old was at relatively high level,and during the same period?except four-month-old stage?its abundance in feeds was higher than that in inlet water.3.27 strains isolated from sea cucumber at early developmental stages were screened for potential probiotics by pathogen antagonistic test,enzyme production ability testing,safety evaluation,physiological and biochemical and molecular identification.2 strains were obtained for its inhibitory effect on pathogenic bacteria Pseudoalteromonas nigrifaciens and Vibrio splendidus.The strain YB-ZS₅-D isolated from one-month-old juvenile of sea cucumber had strong inhibitory effect on Pseudoalteromonas nigrifaciens and Vibrio splendidus,and inhibitory effect of the strain YB-ZS₁₀-A isolated from six-month-old juvenile was stronger on Pseudoalteromonas nigrifaciens than its on Vibrio splendidus.The results of ability of enzyme external secretion was showed that the external secretion ability of amylase was equally between two strains with the enzymatic hydrolysis circle 22²5 mm,and the strain YB-ZS₅-D possessed a stronger ability of Caseinase external secretion than the strain YB-ZS₁₀-A.After immersion with different concentration?10⁷ CFU/mL?10⁸CFU/mL?10⁹ CFU/mL?of the two potential probiotics,the viability of sea cucumber was good and none of them died during the experiment,which indicated that the strain YB-ZS₅-D,YB-ZS₁₀-A were harmless to sea cucumber.As the strain YB-ZS₅-D was superior to the strain YB-ZS₁₀-A in the ability of antagonism to pathogenic bacteria,enzyme production and safety,the strain YB-ZS₅-D was selected for further research on bacterial identification and effect evaluation.Based on the physiological and biochemical characteristics and 16S rDNA sequence analysis,strain YB-ZS₅-D was identified as Bacillus tequilensis.The result of immersion experiment with different concentration?10³ CFU/mL,10⁴ CFU/mL,10⁵ CFU/mL,10⁶ CFU/mL?of strain YB-ZS₅-D showed that Bacillus tequilensis YB-ZS₅-D added to aquacultural water could significantly increased the specific growth rate?SGR?of Apostichopus japonicas,and the activity of trypsin?TRY?and amylase?AMS?in intestine,and the acid phosphatase?ACP?,alkaline phosphatase?AKP?and superoxide dismutase?SOD?in coelomic fluid.In view of the effect evaluation result and cost,the approperate addition concentration of Bacillus tequilensis YB-ZS₅-D was 10³ CFU/mL.