Establishment Of Caenorhabditis Elegans Model For Selecting Probiotics Candidates To Control Vibrio Anguillarum And Mechanism Preliminary Study And Identification Of Novel Species

With the development of mariculture industry and the continuous expansion of intensive aquaculture,aquaculture diseases are on the rise.Vibriosis,caused by Vibrio anguillarum is one of the most serious diseases because of its high prevalence and explosiveness.Aquaculture household has relied largely on prophylactic use of antibiotics of control V.anguillarum leading to imbalance of normal flora in the intestinal tract of aquatic organisms and a growing concern over the human health and safety due to the drug residues in aquatic organisms.Thus,probiotics which is safe and heathy were widely considered to be a potential alternative to dietary antibiotics.Probiotics often lack pertinence and effectiveness due to the different sources of separation.The paper attempts to isolate and screen indigenous microorganisms from the intestinal tracts of healthy oysters and bass,aiming to isolate potential probiotics that can better adapt to the intestinal environment of aquatic animals and play an effective promoting role.Selecting of the isolates with well-targeted and probiotic properties from a large number of bacteria is critical problem in developing effective probiotics.In present study,the marine probiotics controling V.anguillarum SDUM002245 were screened from the oyster and bass intestines in vitro and in vivo,and the mechanism of action was preliminarily explored.Finally,five novel marine bacteria were identified using polyphasic which enriched the microbial resources.The following results were demonstrated:(1)Strain ML1206 is capable of controling V.anguillarum SDUM002245 with significant probiotic properties.A total of 41 strains of marine bacteria were isolated from the intestinal contents of wild oysters and bass,of which 18 strains were isolated from oysters and 23 from bass.Hemolytic experiments showed that 21 strains showed obvious hemolytic activity,while the other 20 did not secreted hemolysis.Next,an V.anguillarum SDUM002245 infecting C.elegans model and C.elegans protecting from V.anguillarum SDUM002245 infecting screen model have been successfully established.The infection concentration is 1.2×108 CFU/mL and five good protection isolates(ML1206,ML1229,YLY05,YLY09 and YLY28)offered higher than 20%protection.Combined with the acid and bile salt tolerance experiment,strains ML1206,YLY05 and YLY09 all showed resistant abilities to both acid and bile salt resistant abilities and there is no relationship between acid tolerance and bile salt tolerance.After final in vivo screening,strain ML1206,which is capable of controling V anguillarum SDUM002245 with significant probiotic properties,was selected as potential marine probiotics for fuether study.(2)The taxonomic status of strain ML1206 was clarified and there were no side effects on vital signs of C.elegans.Strain ML1206 was proposed for the name Planococcus maritimus by molecular biology,16S rDNA sequencing and phylogenetic analysis.The physiological indexes testing of C.elegans showed strain ML1206 could significantly prolong the lifespan of C.elegans(18.7%elongation),but has no adverse effects on pumping rate,body size,brood size and exercise capacity.In addition,the acute oral toxicity test found that the strain was safe and non-toxic.(3)The mechanism of potential marine probiotic ML1206 against V.anguillarum SDUM002245 to protect C.elegans was preliminarily studied by transmission electron microscopy,transcriptome analysis and qPCR technology.In present study,the colonization of strain ML 1206 and SDUM002245 in the intestine of C.elegans was examined.The colonization level of strain SDUM002245 in the intestine of C.elegans remained at approximately 2.5×104 CFU/mL/worm.It was also found that strain ML 1206 could not be attached to the nematode intestinal epithelial cells but could significantly control the colonization level of V.anguillarum SDUM002245.These data indicated that intestinal colonization of SDUM002245 may be the major factor causing the death of C.elegans and strain ML 1206 has effect on colonization of nematode intestine by strain SDUM002245.In addition,the RNA-seq results showed that strain ML1206 protection group changed the 506 genes expression,of which 342 genes were up-regulated and 164 were down-regulated.Through GO annotation,12 representative nematode defense genes were screened for qPCR verification.Compared with the control group,antibacterial peptide(ilys-3)and detoxification related genes(ugt-22,cyp-35A3 and cyp-14A3)in ML1206 protection group were highly expressed,with statistical significance.These genes each showed the same tendency as them observed in the RNA-seq experiments.(4)Polyphasic studies on 5 bacterial strains.Strain NS08T represents a novel species belonging to Flavobacteriaceae and Aquimarina,named Aquimarina celericrescens sp.nov.which was isolated from coastal seawater of Xiaoshi Island,Weihai.Strain N53T represents a novel species belonging to Rhodobacteraceae and Roseovarius,named Roseovarius salinarum sp.nov.,which was isolated from a marine solar saltern at Wendeng.Strain w01T represents a novel species belonging to Flavobacteriaceae and Aquimarina,named Aquimarina sediminis sp.nov.,which was isolated from coastalsediment of Xiaoshi Island,Weihai.Strain PX7T represents a novel species belonging to Rhodobacteraceae and Primorskyibacter,named Primorskyibacter marinus sp.nov.,which was isolated from coastalsediment of Xiaoshi Island,Weihai.Strain YLY08T represents a novel species belonging to Rhodobacteraceae and Oceaniglobus,named Oceaniglobus ichthyenteri sp.nov.,which was isolated from the gut microflora of sea bass(Dicentrarchus labrax L.).