Analysis Of Gut Microflora In Early Developmental Stage And Its Relationship With Immunity In Common Carp, Cyprinus Carpio

The gut of fishes harbored a large number of bacteria,which were abundant and dynamically changed with various factors such as host genotype,intestinal microenvironment,developmental stage,feeding habit,and external environmental factors,etc.,and affected the nutrition,immunization and development of fishes.Common carp(Cyprinus carpio)was one of the most important freshwater fish and was also an important protein source.However,some bacterial diseases such as explosive hemorrhagic disease had become increasing common as breeding density increased,which caused damages to aquaculture.The research studied the composition,origin and diversity of gut microbia in larval common carp,and the changes in the expression of immune related genes in intestine and spleen after immersion challenge with Aeromonas hydrophila by NGS technology.Targeted embryonic and larval common carp for experimental materials,we studied the composition of the gut microflora and its relations to microflora in pond water and feedstuff by choosing the fertilized egg stage,the yolk-sac stage,one,three,fourteen,twenty-one,twenty-eight,forty-two,and sixty-three day after initial feeding(d.a.f.).The results showed that the composition and diversity of the gut microflora changed with the growth,and there were some developmental stage specific intestinal microflora,although Proteobacteria,Cyanolacteria,Firmicutes and Actinobacteria existed in all the samples and the relative abundance varied with different samples.The predominant genus changed with the developmental stage and there was no dominant genus existed in all stages.The predominant species in common carp was different with the environmental samples including the pond water and the feedstudff,and although there was one core bacteria species in common carp,the relative abundance of the common species showed very significant difference in different samples(one-way ANOVA,P<0.0001).The results of the Alpha diversity index showed that the trendency of the diversity and the richness in common carp samples were undulating with the development.The biggest diversity was at 1 d.a.f.,and the smallest diversity was at 63d.a.f..The biggest richness was at 3 d.a.f.,and the smallest richness was at 28 d.a.f..The diversity index of fish gut microflora was lower than the pond water samples,but higher than the feed samples.The results of the Anosim(analysis of similarity)showed that there were obvious variation between any two developmental stages although the difference was not significant(R≥0.889,P=0.1),except 28 and 42 d.a.f.(R=0.111)and42 and 63 d.a.f.(R=0.444).There were obvious changes between the fish gut and the corresponding pond water samples(R=1).The analysis of microflora in fertilized egg(CCE)and the pond water in the same stage(WE)showed that 66.5%of the OTUs in CCE existed in WE,and the valid reads of the OTUs accounted for 95.9%of all valid reads.Which suggested that the pond water might offer the bacteria for the fertilized egg of common carp.The further analysis of NMDS(Non-metric Multidimensional scaling)and PCoA(Principal Co-ordinates Analysis)showed that there was higher similarities between the pond water samples and the fish samples,especially the fish samples of early developmental stages(before and 1 d.a.f.)compared with the feedstuff samples.Targeted the common carp,crucian carp(Carassius auratus)and grass carp(Ctenopharyngodon idella)polycultured in the same pond for four months for experimental materials,we studied the composition of gut microflora in common carp,and the differences with crucian carp and grass carp,as well as the differences with the pond water and pond sediment.The results showed that the dominant phylum were Proteobacteria,Firmicutes,Bacteroidetes,Tenericutes and Fusobacteria,and the preponderant genus were Aeromonas and Lactococcus in the intestine of common carp.The dominant phylum were Proteobacteria,Firmicutes and Bacteroidetes,and the preponderant genus were Aeromonas and Cetobacterium in the intestine of crucian carp.The dominant phylum were Proteobacteria,Fusobacteria,Bacteroidetes,Firmicutes and Spirochaetae,and the preponderant genus was Bacteroides in the intestine of grass carp.There were 212 kinds of core gut microbia,which were abundant although there were some individual differences between the juvenile common carp.The core microbial flora in crucian carp and grass carp was 103 and 57 respectively,and there were 22 kinds of shared microflora in common carp,crucian carp and grass carp.The relative abundance of the specific and common core microbial flora varied with different samples.The results of the Alpha diversity index showed that the diversity of fish gut microflora was lower than that of the pond sediment and the pond water samples,and the diversity and the richness in common carp was higher than that in crucian carp and grass carp.The further analysis showed that the gut microbia of omnivorous common carp was more similar to the omnivorous crucian carp compared to the herbivorous grass carp.The differences of the intestine contents and mucus were not notable in the healthy fishes.The Tree_bar and Hcluster_tree analysis showed that the mucus of common carp and crucian carp samples clustered together,and then clustered to the intestinal contents of common carp and crucian carp successively,meanwhile,the intestinal mucus and contents of grass carp clustered together and located in another branch of the evolutionary tree.The results demonstrated that there were obvious differences in common carp of 6 months old and the larval common carp of 63 d.a.f.although the difference was not significant(Anosim,R=1,P=0.1),and there were obvious different microbial flora in pond water of the two developmental stages.The diversity and the richness of the gut microflora in 6 months old common carp were bigger than that in the common carp of 63 d.a.f.,although the diversity of the microbial flora in polycultured pond water was smaller than that in the pond water of 63 d.a.f..Targeted the common carp of 42 d.a.f.for experimental materials,we studied the variation of the gut microflora and the expression of immune related genes of intestine and spleen,and the relationship of them after the immersion challenge with A.hydrophila,the pathogenic bacteria of the explosive hemorrhagic disease.The larval common carp accommodated to the lab environment for 2 weeks before the experiments,and the density of the A.hydrophila was 1×10~8 bacteria/ml.The first immension challenge lasted 40 minutes,and the first time to draw materials was at the third day after the first stimulation.The second immension challenge carried out at the seventh day after the first stimulation and the immension time also lasted 40 minutes,and the second time to draw materials was at 1 hour after the second stimulation.The results of the treatment samples(the samples at the third day after the first stimulation named CCe3 and the samples at one hour after the second stimulation named CCe7)and the control samples(the samples at the third day after the first stimulation named CCc3 and the samples at one hour after the second stimulation named CCc7)showed that the composition of the gut bacteria in the larval common carp changed after the immune stimulation.The preponderant phylum were Proteobacteria and Fusobacteria in CCc3,CCe3 and CCe7.The ratio of the relative content of Fusobacteria and proteobacteria slightly declined after the stimulation.Although the content of Cetobacterium changed a little after the stimulation,the relative content of Cetobacterium sp.ZOR0034 declined after the first stimulation and then rised after the second stimulation,and the relative content of Cetobacterium sp.ZOR0034 in treatment groups was lower than the contrast groups.The relative content of Vibrio cholera increased after the stimulation.There were 22 kinds of core gut microbiota and the relative abundance of the OTUs were high in all the samples.The results of the Alpha diversity index showed that the diversity and the richness of the gut microflora raised gradually in CCc3,CCe3 and CCe7,but was lower than CCc7.The cluster analysis of all samples revealed that the stimulated samples flocked together,and the NMDS and Pco A analysis demonstrated that the treated samples flocked together,the control samples of CCc3-1 and CCc7 flocked together,and the control samples of CCc3-2 and CCc3-3 flocked together.Samples of CCe3,CCe7,CCc3 and CCc7successively clustered,and there were obvious differences between the CCc7 and CCe7although the difference was not significant(Anosim,R=1,P=0.1).The results of the intestine and spleen transcriptome obtained by the NGS technology were comparatively analysed and annotated with the published genome of common carp,and the results showed that there were many differentially expressed genes(DEGs)in the intestine and spleen samples after the stimulation.PCA analysis demonstrated that the expressed genes of intestine and spleen were different,and the expressed genes of them changed after the stimulation.There were nineteen and nine immunological genes expressed differently in CCie3(the treatment samples of intestine at the third day after the first stimulation)and CCie7(the treatment samples of intestine at one hour after the second stimulaiton)compared to CCic(the control samples of intestine).There were sixteen and seventeen immunological genes expressed differently in CCse3(the treatment samples of spleen at the third day after the first stimulation)and CCse7(the treatment samples of spleen at one hour after the second stimulation)compared to CCsc(the control samples of spleen).The results of GO terms showed that there were differences of the DEGs carried out different biological function.The results of KEGG enrichment analysis revealed that there were some pathways related to the immunological genes that differently expressed.And the most pathways including antigen processing and presentation,NOD-like receptor signaling pathway,complement and coagulation cascades,inflammatory mediator regulation of TRP channels,RIG-I-like receptor signaling pathway,etc.were innate immunity related,and only Fc epsilon RI signaling pathway was adaptive immunity related.The results above suggested that the gut microflora of the common carp might vary gently and continuously in each stage of development during the early developmental period,and the environment of pond water might provide the bacteria for common crap fry and could have some influences on the variation of gut microflora of the larval common crap.The selective pressure of the host contributed more to the establishment of gut microflora of the larval fish.The existence of core gut microbe declated that some kinds of bacteria suited the intestinal microenvironment of fish and were effective on the fish although gut microflora was affected by many factors.There were some differences in the gut microflora of common carp,crucian carp and grass carp,which were not significantly.The results suggested that feeding habit was also an important factor influenced the gut microflora by comparing the gut microflora of crucian carp and grass carp with the common carp.There were some kinds of core gut microbia in common carp,as well as in crucian carp and grass carp.There were some kinds of core gut microbia in common carp,crucian carp and grass carp.The component and the variety of the gut microflora in fish samples were different with the microflora in pond water and sediment samples.All of these results suggested that the selective pressure of host contributed more to the gut microbia.The differences of the gut microflora in intestinal contents and mucus of healthy common carp were not notable.The gut microflora was still in variation after 63 d.a.f.,and the influences of the environmental factors were less than the factors of host itself.The composition of gut microbia of larval common carp and the expression of some genes in intestine and spleen could be affected by the immersion challenge with pathogenic bacteria.Relatively,the genes involved in the pathways related to innate immunity were more than that in the pathways related to adaptive immunity.The number of the pathways were less in general,suggested that the response of larval common carp to pathogenic bacteria was not so strong as juvenile and adult fish,or the limitation of the reference genome influenced the functional localization of the DEGs.The variation of the gut microflora and the differential expression of the immune related genes in intestine and spleen of stimulated common carp might influence each other,but the causality of them was still unclear.In a word,the results of the research could be conducive to learn the change rules of the gut microflora with the development.The resules also could be supportive to understand the relationship of gut microflora to the development of the immune system and the immune response of fishes,and then be effective to the growth and disease prevention of the common carp.