| The gastrointestinal tract resides a complex ecosystem that is colonised by a large,diverse and dynamic collection of microorganisms.After millions of years of co-evolution with the host,the gut microbiota may be been defined as a component of the host.This microbial community plays a very importent role in the maintenance of normal gut function,such as protecting against pathogens,promoting intestinal cell proliferation,and digesting various macromolecules.Our understanding of the gut microbiota of animals is largely based on studies of mammals,the study of the gut microbiota of non-mammalian vertebrate is less.Fish have the highest species diversity among groups of vertebrates,with approximately 33,000 species,accounting for two thirds of vertebrates.Aquaculture is a fast-growing and rapidly expanding industry in the world.The microbial intestinal communities of fish are receiving increased attention for their relevance,among others,in a growing aquaculture industry.Currently aquaculture practices have shifted from extensive to super-intensive to elevate production at the expense of huge amounts of artificial feeds and degradation of aquatic environments.Poor culture management causes immunosuppression,stress and creates suitable conditions for the proliferation of opportunistic microbial pathogens(bacteria,viruses,and parasites)leading to infectious disease outbreaks.Control of bacterial and viral disease and enhancing the immunity of aquatic animals have become urgent needs in aquaculture production.Probiotics have been widely used in aquaculture.In the process of controlling fish diseases,it is a green and effective aquaculture strategy,which can replace traditional drugs and reduce the use of antibiotics.In this study,four kinds of marine fish(hairtail,mackerel,scophthalmus maximus and scophthalmus maximus)were selected as research objects to isolate and identify their intestinal flora;The relationship between gut microflora composition and food habits of four marine fishes was analyzed;The probiotics with certain antagonistic effects were screened,and the effects of probiotics on the growth and immune performance of Scophthalmus maximus were investigated.The following research results were obtained:1.Analysis of intestinal microbes of four marine fishes with different feeding habits showed that the diversity of intestinal microbiota of fishes was closely related to their feeding habitsFour types of marine fish with different feeding habits are selected in this part:Trichiurus japonicus,Scomberomorus niphonius,Scophthalmus maximus and Liza macropedis.The structure of gut flora of four types of fish is analyzed by 16S rRNA high-throughput amplification sequencing technology.The results showed that,at the phyla level,the dominant gut microflora of Trichiurus japonicus were Proteobacteria,Firmicutes,Bacteroidota and Cyanobacteria;the dominant microflora of Scomberomorus niphonius were Proteobacteria,Firmicutes,Bacteroidota and Verrucomicrobiota;The dominant bacteria of Scophthalmus maximus were Proteobacter,Firmicutes,Bacteroidota,Verrucomicrobiota,Cyanobacteria and Actinobacteria;the dominant bacteria phyla of Liza macropedis were Proteobacteria,Firmicutes,Spirochaetota,Actinobacteria and Bacteroidota.The results showed that the core microflora of the four marine fishes were Proteus and Firmicutes,accounting for 70.85～92.06%.However,the intestinal microflora structure of omnivorous fish(Liza macropedis)is quite different from that of other three types of carnivorous fish.Spirochaeta is only found in the intestinal microflora of the Liza macropedis,accounting for 16.08%.The analysis of subordination level showed that compared with the Trichiurus japonicus,Scomberomorus niphonius,Scophthalmus maximus,the abundance of Psychrophilic bacteria in the intestine of the Liza macropedis was 34.45%,significantly higher than that in the other three groups(p<0.05).Photobacterium has a very low content(<10-5)in Liza macropedis,while its abundance in Trichiurus japonicus,Scomberomorus niphonius and Scophthalmus maximus is 57.21%,45.0%and 1.97%respectively.However,the genus Brevinema and the species Clostridium sensu stricto 12 of Spirochaeta were only found in the Liza macropedis.The difference of food habits has an important impact on the composition of fish gut flora.The above research results show that the composition of intestinal flora of omnivorous Liza macropedis is significantly different from that of Trichiurus japonicus and Scomberomorus niphonius,and its abundance and diversity are higher than that of the latter.This result is similar to the research on intestinal flora diversity of mammals.The analysis shows that omnivorous fish are in a lower food chain position than carnivorous fish,so their flora diversity is more abundant.As Scophthalmus maximus is a cultured fish,its food structure is relatively simple,so its gut flora abundance and diversity are low.In a word,the composition of intestinal flora of omnivorous Liza macropedis is quite different from that of carnivorous Trichiurus japonicus and Scomberomorus niphonius,and its abundance and diversity are higher than that of carnivorous.This result is similar to that of mammalian intestinal flora.The analysis shows that omnivorous fish are at the bottom of the food chain compared with carnivorous fish,so their flora diversity is more abundant.As Scophthalmus maximus is a cultured fish,its diet is relatively simple,so its intestinal flora abundance and diversity are low.2.Isolation and screening of culturable intestinal microflora of 15 wild marine fish species and the polyphasic taxonomy of three new strains of bacteria.It was found that the function of extracellular biomass degrading enzymes secreted by intestinal microorganisms of different fish species was related to fish feeding habits.The intestinal microflora of 15 species of marine fish were isolated and screened by using a variety of media and culture methods.The plates were separated with 2216E,2YT and PY.The biochemical characteristics of the isolated aerobic and anaerobic bacteria were analyzed by shaking flask culture and static culture.Secondly,644 strains of bacteria were isolated from 19 batches of marine fish intestinal flora.According to the source of each strain,the structure of the isolates from the intestinal tract of marine fish was analyzed.The results showed that,at the phyla level,the intestinal microflora of marine fish was mainly composed of Proteobacteria,Firmicutes,Actinobacteria and Bacteroidota.Due to the influence of the environment,habits,heredity and feeding habits of marine fish,the proportion of each bacterium in fish varies greatly.According to the analysis,the proportion of Proteobacteria in Liza macrolepis is 88%,while that in Scomberomorus niphonius is only 55%.It is speculated that this may be related to the food habits of fish.The enzyme activity of the strains screened from Trichiurus japonicus and Liza macrolepis was preliminarily analyzed.The results showed that 49.5%of the strains isolated from the intestine of Trichiurus japonicus had the function of secreting and producing biological enzymes,among which casein,lipase and amylase were the most abundant,which would help fish digest and absorb various foods and provide nutrition for the growth of fish.However,the number of strains with enzymatic activity in the intestinal microflora of Liza macrolepis was only 25.7%,and the enzymes produced were mainly lipase,protease and amylase producing bacteria.The difference of microbial flora in the intestines of the two species of fish may be related to the omnivorous characteristics of the former,which feeds on fish,while the latter,which feeds on debris and algae.It can be seen that the food habits of fish are related to the composition and function of intestinal flora.Finally,7 new strains were identified(similarity lower than 98.7%)by PCR amplification and comparison.Three new strains were selected for polyphasic taxonomy.Based on the analysis of the physiological and biochemical,genetic,and chemical characteristics of the strains,their classification status was determined.The strain Co35T elongs to a new strain of Aeromicrobium of Actinobacteria,named Aeromicrobium piscarium.Strains 29W222T and 2943T both belong to the genus Fulvivirga of Bacteroidetes and are named Fulvivirga marina and Fulvivirga sediminis respectively.3.Screened four probiotic antagonistic bacteria from fish intestinal bacteria and analyzed their physiological characteristics.With 644 strains of fish intestinal bacteria isolated in the previous experiment as the starting strains,and Acinetobacter baumannii,Staphylococcus aureus,Escherichia coli,Aeromonas hydrophila,Vibrio parahaemolyticus and Vibrio harveyi as the test bacteria,97 strains with certain antagonistic properties were screened through the antagonistic test.Based on the results of in vitro hemolysis test,drug sensitivity test and re-screening of antagonistic bacteria,four probiotics DE11(Bacillus amyloliquefaciens),DN23(Bacillus aerius),XS13(Citricoccus nitrophenolicus)and XS43(Bacillus altudinis)with better antagonistic effect and no hemolysis reaction in vitro were selected for further study.Through the study of fermentation characteristics,the growth time of strain DE11,DN23 and XS43 has entered the stable growth period in 24 hours,while strain XS13 has entered the stable growth period after 48 hours,and the growth is slow.The optimum growth temperature of strain DE11 was 45℃,that of strain DN23 was 28℃,and that of strain XS13 and XS43 was 35℃.The optimum growth pH of strain DE11 was 5.0;strain DN23 can adapt to the wide pH range of pH6～9;the optimal growth pH of strain XS13 and XS43 was 7.0 and 6.0 respectively.In vitro gastrointestinal simulation test showed that strains XS13 and XS43 showed better bile salt tolerance.The second was strain DN23.It can be seen from the pH test that the strains DE11,DN23 and XS43 all belong to Bacillus spp.and have good stress resistance characteristics,so they can show different degrees of antagonistic characteristics in a wide pH range.However,strain XS13 had certain antagonistic properties only at pH 7-9.Finally,the interaction of these four strains was investigated by plate confrontation test.The results showed that there was a certain affinity between strains XS13 and DN23,and between strains DN11 and DN23.Further research on mixed probiotic preparations can be considered in the future.4.Based on the application of three probiotic antagonistic bacteria to cultured Scophthalmus maximus,it was found that the three bacteria could promote the growth of fish and improve the immune function of fish.In this part,the strains DN23(Bacillus aerius),XS13(Citricoccus nitrophenolicus)and XS43(Bacillus altitudini)are added to the daily feed of Scophthalmus maximus respectively to investigate the effect of screening probiotics on the growth of Scophthalmus maximus.The results showed that the three strains could promote the growth of fish,and the body weight gain,body weight gain rate and specific growth rate of XS13 group and XS43 group were significantly higher than those of the control group(p<0.05).It can be seen from the activity indexes of immune enzymes that DN23 group can effectively increase the indexes of superoxide dismutase(TSOD)and lysozyme(LZM)in fish and can reduce the contents of malondialdehyde(MDA),acid phosphatase(ACP)and alkaline phosphatase(LCP)in fish,but it has little effect on the growth indexes of fish.The immune enzyme activity indexes of strain XS43(Bacillus altitudinis)were slightly different from those of the control group,but the difference was not significant.Combined with the challenge test,the three antagonistic bacteria(DN23,XS43 and XS13)used in the test can effectively improve the ability of fish to resist the pathogenic bacteria Aeromonas hydrophila.It can be seen that the three probiotics have significantly improved the immunity of fish.Through the analysis of microbial diversity,it was found that with the growth of Scophthalmus maximus,the abundance of microorganisms in the aquaculture water and the intestine of Scophthalmus maximus were significantly increased.T-test analysis showed that the abundance of Firmicutes and Desulfobacteria in the experimental group was significantly increased compared with the control group(p<0.05).The analysis shows that the addition of strain XS43 has played a certain role in promoting the increase of the abundance of intestinal flora Firmicutes.At the same time,with the growth of Scophthalmus maximus,the excretion in the water body gradually increases,and the increase of organic matter content also promotes the increase of anaerobic bacteria abundance in the fish body.Finally,the gene of Scophthalmus maximus liver tissue was predicted and analyzed by transcriptome sequencing technology.It was found that the immune related pathways of Scophthalmus maximus liver in XS43 group compared with the control group included calcium signaling pathway,C-type lectin receptor signaling pathway,NOD-like receptor signaling pathway Cell adhesion molecules,Toll like receptor signaling pathway and mTOR signaling pathway have up-regulated expression.These pathways are related to the intrinsic immune modulation of fish and the bypass activation pathway of complement,and have a direct relationship to improve the immunity of fish.At the same time,the pathways related to the transcription and replication of DNA in the nucleus also showed up regulation.These pathways include MAPK signaling pathway,FoxO signaling pathway,mTOR signaling pathway,RIGI-like receptor signaling pathway Cytosolic DNA sensing pathway and cellular senescence pathway.The results showed that strain XS43 could activate the expression of immune related genes in Scophthalmus maximus and enhance its immune regulation.In this study,three probiotics with good antagonism and high safety were obtained through isolation and screening of intestinal flora of wild marine fish.The results of feeding experiments showed that the three strains had significant effects on improving the immunity of turbot.It provides a theoretical basis for the application and development of these three probiotics in the future. |
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