| The Antarctic notothenioids evolved in the Antarctic from a single benthic and temperate primitive ancestor adapted to the extreme cold and seasonal dark environment,and now the dominant fish fauna occupying all depth of the water columns of the freezing Southern Ocean.The intestinal and mucus microflora of Antarctic notothenioids,coevolved with the cold-adapted hosts,many unique composition and function.So far,there are few studies have been conducted on characterizing the microbiome associated with intestinal and mucus in Antarctic notothenioids.Researches on the relationship among the microbiota of intestine and mucus,and its relationship with environmental microbiology.To this aim,we carried out the following studies:We first studied the composition of intestinal microbes in 20 Chionodraco hamatus specimen captured from 2010 to 2018,near the Davis Station and Ross sea Station(74°55' S,163°45' E),respectively,and compared with that of surrounding seawater through high-throughput 16 S r RNA gene sequencing.We found: 1)The number of OTU's classified to the genus level was 443 in the C.hamatus intestinal microbiota and 609 in the water samples,respectively.The ? diversity analysis,Shannon indices and Simpson diversity estimate all show that the seawater contained had greater mean richness and diversity than that of the intestine,which are significantly different in composition.The Proteobacteria,Tenericutes,and Firmicutes are dominant in the fish gut,while Proteobacteria,Cyanobacteria and Bacteroidetesare are the three dominant ones in the seawater.2)The intestinal microbial composition of C.hamatus,Cyprinus carpio,Oncorhynchus mykiss and the Ross Sea water were compared.? diversity,non-metric multidimensional scaling analysis(NMDS)show that the fish gut flora and water microbial community structure are separately clustered,and the Antarctic fish gut exhibiting lower species-level diversity than common carp,which is different from each other,indicating that the intestinal microorganisms were host dependent are different from that of the surrounding water.3)The C.hamatus gut microbiota may be influenced by season,gut microbiota are clearly different on species composition among the February 2016,December 2017 and January 2018 samples.4)Function prediction from metagenome indicate abundant KEGG pathways in energy production and metabolic functions,genetic information processing and environmental information processing,suggestive of adaptation to the cold environment.We then studied the intestinal and mucus microbiomes of some notothenioid species,collected from different locations,times,with or without LPS injection or high temperature stimuli.The locations included the Zhongshan Station,Ross sea Station and the Great Wall Station in Antarctica from 2017 to 2018.Result showed: 1)the gut microbiome structure of C.hamatus and T.bernacchii in Zhongshan station and Ross sea are different with those of N.coriiceps and N.rossii from the Great Wall Station,and the species abundance of intestinal and mucus microorganisms in different geographical locations is significantly different at genus level.Pseudomonas and Ralstonia are relatively high in notothenioids gut and mucus of Ross sea and Zhongshan station,while Aliivibrio,Photobacterium and Cetobacterium are abundant in the intestinal tracts of N.coriiceps and N.rossii in the Great Wall Station,and Colwellia and Psychromonas are more abundant in the mucus of N.coriiceps and N.rossii.2)Whether the control group or the experimental group(high temperature stimulating group and LPS group),the gut and mucus microorganisms are obviously divided into separate communities between the Ross sea,Zhongshan Station and Great Wall Station,indicating higher influence of geographical location on the distribution of microorganisms over the high temperature and LPS stimuli.3)The microbial communities of the fish intestines from both the Ross sea and the Zhongshan Station collections in early December 2017,late January 2018 and early February 2018 are different in both of C.hamatus and T.bernacchii,suggesting seasonal influence of gut microbial community.4)12 hours after LPS injection,T.bernacchii intestinal microbial diversity initially increased,then gradually decreased to about 72% of the control group after 72 hours.Microbial diversity of intestinal and mucus microorganisms also decreased in N.coriiceps and N.rossii from the Great Wall station after LPS injection,and temperature treatment with the compositions of intestinal microbial community changed greatly.The N.rossii intestinal flora diversity is only 45% of the normal group when treated with combination of high temperature and LPS,and mucus diversity is about 67% of the normal group,which indicate that LPS injection and high temperature stimulate can reduce the diversity of intestinal and mucus microorganisms,and the two combined stimulation can reduce the microbial diversity significantly.When put T.bernacchii in the air,the gut microbiology of T.bernacchii is more sensitive than mucus,and gut diversity is significantly increased when exposed to the air,while mucus microorganisms were not great change,which suggests that gut microbes may play an important role in host immune system.5)KEGG functional annotation after C.hamatus,T.bernacchii intestinal and mucus microbial metagenomics sequencing,the results of gut microbes function mainly enriched in the Oxidative phosphorylation and Neurodegenerative diseases,mucus microbial function mainly enriched in Environmental Information Processing,such as Calcium signaling pathways,c GMP-PKG signaling pathways,suggests that energy production and conservation and to the environment information processing are important in the Antarctic notothenioids to adapt to the cold Antarctic environment.We investigated 38 sea water samples from different depths ofthe Ross sea collected between January and February 2018.And found that: 1)there are 45 phyla of microbiomes in Ross sea,the most abundant phyla are Proteobacteria,Bacteroidetes,Firmicutes,Cyanobacteria,while Actinobacteria,Epsilonbacteraeota,Tenericutes,Patescibacteria,and Rokubacteria are represented by a relatively minor portion.2)The microbial communities of different depths differ.In the upper water of less than 50 meters,Cyanobacteria,Proteobacteria and Bacteroidetes were the most abundant phyla,while in the deeper water of over than 1000 meters,Proteobacteria becomes most abundant one,followed with Firmicutes,Bacteroidetes and Actinomycetes,suggest that water depth has an effect on the microbial community composition.3)Biomass of some bacteria are positively correlated with environmental parameters while others are negatively correlated,in which fluorescence,conductivity,temperature,density,depth and salinity affect the distribution of microbiota.On the phylum level,Cyanobacteria is significantly positive correlated with fluorescence and oxygen(P ? 0.001)and negatively with salinity.Chloroflexi and Acidobacteria positively correlate with density and salinity,while Patescibacteria,Spirochaetes and Chlamydiae are negatively correlated with fluorescence,oxygen and temperature.On the genus level,Bacteroides and Lactobacillus were significant negative with fluorescence,and Lactobacillus,Treponema?2 and Romboutsia were significant negative with oxygen,Norank?f??norank?o??Chloroplast,SAR92?clade were significant positive with fluorescence and oxygen and significant negative with conductivity and depth.Norank?f??Muribaculaceae was significant positive correlation with depth and salinity,and negative with fluorescence,which suggested that environment physicochemical factors have important effects on the distribution of the Ross Sea microorganisms.4)Compared the sediment and water of Ross Sea water from the same area in the Ross Sea.? diversity result revealed that the microbiome community diversity of the bottom water is more than sediment,and Proteobacteria is dominant on the phylum level,and the next phyla are Bacteroidetes,Firmicutes and Actinobacteria in the Ross Sea bottom water samples.And in the sediment microbiology,the Bacteroidetes and Proteobacteria are the most,the two phyla reached over 91% of the total number.And the NMDS result showed obviously different between the bottom water and the sediment microbiology from the species level to the family level,which suggested that the source of samples has important influence on the composition of microorganisms.At last,we studied the microbial communities of waters from Arctic and Antarctic including Zhongshan Station.1)A total of 733 OTUs and 237 genera were detected from samples collected from five locations of Zhongshan Station.The locations differ at the OUT level,while little difference is detected on the phylum level.Proteobacteria,Bacteroidetes and Cyanobacteria achieved the total 98.44% microbiology.On the genus level,Flavobacterium and Psychrobacter were dominant genera,each of them achieved more than 20%.2)Comparison of the microbial community structure between Zhongshan Station and the upper waters of the Ross sea,showed that the upper waters of the Ross sea have higher microbial diversity than those of Zhongshan Station,and the abundance of the major bacterial is different.Flavobacterium and Psychrobacter were most abundant in the Zhongshan Station,and norank?f??norank?o??Chloroplast and Polaribacter?1 were abundant in the upper level of the Ross sea.? diversity analysis showed the microbial structure is different on OUT level between the two sites.3)In the Antarctic and Arctic seawaters,Antarctic the phyla Proteobacteria,Bacteroidetes and Cyanobacteria are ubiquitously and abundantly present.Proteobacteria is the dominant phylum of waters from both polar regions,and Cyanobacteria is most abundant in the Southern Ocean,which may be important in maintaining the balance of Antarctic Marine ecosystems.The microbial compositions of Antarctic(western Antarctic peninsula,Ross Sea upper water and Zhongshan Station)are significantly different from Arctic(Arctic Ocean and Norway Svalbard),suggests that geographical locations has important influence on the microbiota.4)KEGG functional annotation on the metagenomes of microbiota from the Zhongshan Station enrichment was in the Biosynthesis of amino acids and Carbon metabolism,suggesting that the seawater of Zhongshan Station maybe play an important role in the carbon cycling pathway. |
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