The Effects Of Bdellovibrio-and-like Organisms And Lactobacillus On Gut Microbiota Of Turbot (Scophthalmus Maximus) And Abalone (Haliotis Discus Hannai Ino)

With the increasing pursuit of health requirement,the study of probiotics in food,especially the research on regulations of probiotics in gastrointestinal tract(GIT),has been become more and more popular in recent years.Probiotics are live micro-organisms or their products,if the live micro-organisms or their products are supplied to their hosts,they will show health beneficial modification and balance to the GIT of their hosts.In aquaculture,many studies showed that probiotics were the better choice to curb the remaining problems that antibiotics abuse caused,and they were gradually becoming a substitute for antibiotics.However,the regulatons fuction of Bdellovibrio and like organisms(BALOs)and lactic acid bacteria(LAB)on cold water rearing turbot(Scophthatmus maximus)and abalone(Haliotis discus hannai Ino)remain unanswered,especially at the production level.In the present study,experiments were designed at the production level to explore the effects of LAB and BALOs that were fed with different methods on intestinal flora structure using high throughput sequencing(HTS)and denaturing gradient gel electrophoresis(DGGE)technique.The main research contents and results are follows:1.Denaturing gradient gel electrophoresis(DGGE)was conducted to explore the effects of BALOs strain BDWO3 that was fed by 60 d pouring water on microbiota in juvenile turbot rearing water.The effects of BALOs strain BDWO3 on rearing water quality,total cultivable bacteria counts(TCBC)and total cultivable vibrio counts(TCVC)both in wearing water and intestines,survival rates etc.were also investigated.The results showed that compared with control,BDW03 had no significant impacts on rearing water quality(P>0.05),while it significantly reduced(P<0.05)the TCBC and TCVC both in rearing water and intestines.At the end of 60 d rearing water experiment,BDW03 significantly improved turbot survival rates,percentage of final length gain and weight gain and average daily weight gain(ADWG).Furthermore,DGGE profiles results in rearing water revealed that BDW03 simplified bacterial community structures with time.The sequencing of disappeared DGGE bands revealed that two disappeared bands belonged to vibrios,and the others belonged to uncultured bacteria.2.HTS technique was conducted to explore the effects of BALOs strain BDWO3 that was fed by 60 d pouring water on microbiota in turbot guts.The effects of BALOs strain BDWO3 on rearing water quality,TCBC and TCVC both in rearing water and guts,growth performance etc.were also explored.Results demonstrated BDW03 had no significant impacts on rearing water quality(P>0.05),but it significantly reduced(P<0.05)the TCBC and TCVC both in rearing water and guts.Moreover,HTS results showed BDW03 reduced Shannon index,ACE index and Chao1 index in turbot guts of treated group.The number of genera that BDW03 had negative control in turbot guts was 8,among them,6 genera were Gram-positive bacteria.However,the percentage that BDW03 had positive control effects in turbot guts flora was 46.6%(7 genera),among this,26.7% was Gram-negative bacteria,and 20.0% was Gram-positive bacteria.At the end of the experiment(day 60),BDW03 had significantly improved turbot survival rates,final length,final weight,weight gain and ADWG.3.In this section,to explore whether BALOs had the similar effects on other cold water wearing species or not,DGGE technique was employed to study the effects of BALOs that was fed by 90 d pouring water on gut microbiota in juvenile abalone guts of other cold water rearing species.The effects of BALOs on main rearing water quality,TCBC and TCVC both in wearing water and intestines,survival rates etc.were also studied.Results showed that BALOs had no significant impacts on abalone rearing water quality(P>0.05),while it significantly reduced(P<0.05)TCBC and TCVC both in abalone rearing and guts.On day 90,compared with control,BALOs significantly improved survival rate,percentage of length gain and weight gain,and ADWG(P<0.05).Pearson correlation results between growth performance(final weight and length)and bacteria density(TCBC and TCVC)both in rearing water and guts showed that abalone final weight and final length had significantly negative correlation(P<0.05)with TCBC and TCVC in the treated groups.Furthermore,DGGE profile of abalone guts showed that the percentage of disappeared or undetectable DGGE bands in treated group was 22.22 %(6/27).Among those disappeared or undetectable sequenced bands: two bands belonged to vibrios,the other sequenced bands were Serratia sp.,Alteromonas sp.,Sphingomonas sp.and Psychrobacter fozii.4.In this part,LAB strains were isolated from Guangzhou pickles which were randomly brought from local market.Their characteristics included hemolysis,producing biogenic amine(histamine,tyramine and phenylethylamine),antimicrobial activity against 39 different sources of pathogens,acid tolerance,bile tolerance,?-galactosidase activity,antibiotics resistance,etc.were investigated.Results showed that none of the tested LAB isolates induced hemolysis on sheep blood agar(?-hemolytic)and produced no biogenic amine(histamine,tyramine and phenylethylamine).14 isolated LAB strains had different antimicrobial activity against 39 different pathogens,among them,P1,P2 and P11 had the strongest antimicrobial activity,34/39,33/39 and 33/39,respectively.Moreover,P2(cell-free supernatants)exerted the strongest growth inhibitory effects on the 13 tested pathogens(“+++”,diameter of inhibition zone>15 mm),which was the highest growth inhibitory rate of all.After those characterizations,P1,P2 and P11 were screened out for further characteristics.Results of acid tolerance and bile tolerance of P1,P2 and P11 demonstrated that P2 strains showed acid tolerance at pH value 2.5,the other two strains didn't.P2 and P11 showed bile tolerance at bile density of 0.5%,1.0% and 1.5%,but P1 didn't.As to ?-galactosidase activity,P2 showed the highest ?-galactosidase activity among P1,P2 and P11.Considering antibiotic resistance of six antibiotics(chloramphenicol,erythromycin,tetracycline,cefalotin,ciprofloxacin and gentamicin),P2 showed the highest susceptible rate with 100%,P11 and P1 with 67% and 50%,respectively.Analysis of 16 S rDNA via RDP classifier and NCBI BLAST demonstrated that P2 was identified as Lactobacillus salivarius(identity of 99%).5.HTS technique was conducted to explore the effects of lactic acid bacteria(LAB)strain P2 that was fed by mixed with diet on microbiota in juvenile turbot guts.The effects of LAB strain P2 on rearing water quality,TCBC and TCVC both in wearing water and turbot guts,growth performance etc.were also explored.Results demonstrated that compared with control,P2 had no significant impacts on rearing water quality(P>0.05)in test;It also had no significant impacts(P>0.05)on TCBC and TCVC in turbot rearing water in test.However,it significantly reduced(P<0.05)the TCBC and TCVC in treated turbot guts.Except percentage of the final length gain,P2 had significantly improved survival rates,final weight,final length,percentage of weight gain and ADWG(P<0.05).Furthermore,HTS results showed that P2 reduced Shannon index,ACE index and Chao1 index in turbot guts of the test.What's more,the percentage that P2 had positive control effects in turbot guts flora was 60.0%(9/15),among this percentage,33.3% was Gram-negative bacteria,and 26.7% was Gram-positive bacteria.6.The comparison of the effects of BALOs strain BDW03 and LAB strain P2 on turbot showed that after 60-days rearing of pouring BALOs in water and mixing P2 with diet,the percentage of BALOs and LAB that had positive control on Gram-negative bacteria in turbot guts was 33.3% and 26.7%,respectively,which was higher than those of Gram-positive bacteria that was 26.7% and 20.0%,respectively.Moreover,BALOs and LAB had significantly improved survival rates,final weight,final length,percentage of weight gain in test(P<0.05),those results from BALOs were also demonstrated in abalone experiment.The different effects of BALOs and LAB on turbot guts were that the colonization capacity of P2 was higher than BALOs in turbot guts,and LAB contributed to the higher abundance of unclassified phylum flora than BALOs in test turbot guts.