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Background Many deep‑sea invertebrates largely depend on chemoautotrophic symbionts for energy and nutrition, and some of them have reduced functional digestive tracts. By contrast, deep‑sea mussels have a complete digestive system although symbionts in their gills play vital roles in nutrient supply. This digestive system remains functional and can utilise available resources, but the roles and associations among gut microbiomes in these mussels remain unknown. Specifically, how the gut microbiome reacts to environmental change is unclear. Results The meta‑pathway analysis showed the nutritional and metabolic roles of the deep ‑sea mussel gut microbi‑ ome. Comparative analyses of the gut microbiomes of original and transplanted mussels subjected to environmental change revealed shifts in bacterial communities. Gammaproteobacteria were enriched, whereas Bacteroidetes were slightly depleted. The functional response for the shifted communities was attributed to the acquisition of carbon sources and adjusting the utilisation of ammonia and sulphide. Self‑protection was observed after transplantation. Conclusion This study provides the first metagenomic insights into the community structure and function of the gut microbiome in deep‑sea chemosymbiotic mussels and their critical mechanisms for adapting to changing environ‑ ments and meeting of essential nutrient demand. Keywords Gigantidas mussel, Metagenome, Nutritional role, Haima seep, In situ experiment Background The gut microbiome plays an essential role in nutrient *Correspondence: Pei‑Yuan Qian assimilation, converting photosynthesis-derived food email@example.com components into absorbable metabolites in most ani- Southern Marine Science and Engineering Guangdong Laboratory mals [1, 2]. Apart from photosynthesis, chemosynthesis (Guangzhou), Guangzhou 511458, People’s Republic of China Department of Ocean Science, The Hong Kong University of Science is a crucial process that enables animals to gain nutrition and Technology, Hong Kong, People’s Republic of China in deep-sea cold seep and hydrothermal vent ecosystems Center of Deep‑Sea Research, Institute of Oceanology, Chinese . These extreme habitats are characterised by darkness, Academy of Sciences, Qingdao, People’s Republic of China Research Center for the Oceans and Human Health, City University high hydrostatic pressure, and lack of photosynthesis- of Hong Kong Shenzhen Research Institute, Shenzhen 51807, People’s derived nutrients . Invertebrates, such as bathymodio- Republic of China line mussels and siboglinid tubeworms, have successfully Department of Biology, HADAL and Nordcee, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark colonised these hostile ecosystems and often formed © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Xiao et al. Animal Microbiome (2023) 5:17 Page 2 of 13 dense communities. The ecological success of deep-sea hypothesised that they can provide additional nutrient mussels and tubeworms relies on chemosynthetic symbi- supply to cold seep mussels with reduced symbiont func- onts fuelled by the simple reduction of molecules, such as tions due to environmental changes. We translocated G. methane and hydrogen sulphide, into organic compounds haimaensis mussels from a densely populated area in the that are passed from symbionts to the host. Compared Haima cold seep to a site 100 m away for 6 days to inves- with tubeworms, which have a degenerated digestive sys- tigate the contribution of the gut microbiome to deep- tem, mussels have a fully developed digestive system con- sea adaptation to changing environments (e.g. methane sisting of a mouth, a stomach, two digestive glands, an concentrations). We then performed 16S ribosomal RNA intestine, and other organs, although the gut is reduced (rRNA) gene amplicon and metagenomic sequencing to in size . The transmission electron microscope (TEM) compare the community structures and functional capa- image of a mussel stomach showed filled nutritional par - bilities of the gut microbial communities in the mussels ticles, and a stable isotope experiment detected a low in the original site with those in the translocated site after δ13C value in the gut [6, 7]. Bathymodiolus thermophi- an in situ transplantation experiment. This study aims to lus can ingest and assimilate free-living bacteria through shed light on the critical role of the gut microbiome in filter-feeding in a highly pressurised flow-through acrylic deep-sea mussels’ adaptation to environmental changes. aquarium . These observations clearly indicate that the digestive systems of deep-sea mussels have nutritional Methods and physiological functions. Transplantation experiment, sampling, measurement Deep-sea mussels have a mixotrophic diet that includes of environmental factors and onboard dissection heterotrophic and autotrophic nutritional processes, and The remotely operated vehicle (ROV) Haima 2 onboard the retained ability of filter-feeding affords them flex - R/V Haiyangdizhi 6 of Guangzhou Marine Geological ibility in using carbon sources and obtaining ecological Survey (China) was used in conducting in situ transplan- benefits. However, most previous studies on deep-sea tation, fixation experiment and sample collection in the mussels focused on the prominent trophic role of chem- Haima cold seep (~ 1400 m depth) in the South China osynthetic endosymbiotic bacteria and did not consider Sea. The mussels were transplanted from a dense mus - the function of the gut microbiome. The nutritional role sel bed to a peripheral site that was 100 m away from the of heterotrophy is poorly understood, and the adaptation original site and had no mussels for a 6 day experiment of the gut microbiome associated with the nutritional (May 2021). Drawstring net bags were used in the sample cycling in deep-sea mussels at genomic level has not been collection (Fig. 1). On May 23, mussels from the two sites explored. (original and transplantation sites) were cracked slightly Gigantidas haimaensis is a newly described species with an ROV manipulator arm and placed in separate of the deep-sea bathymodioline mussel from the Haima sampling chambers filled with ~ 12 L of in-house RNA cold seep in the South China Sea and houses methane- stabilising solution (700 g of ammonium sulphate, 40 mL oxidising bacteria (MOB) inside their gill epithelial cells of 0.5 M EDTA, 25 mL of 1 M sodium citrate and 935 mL . Symbiotic bacteria can capture bubble-forming gase- of distilled water; the pH was adjusted to 5.2). In situ fixa - ous methane advected to near-surface sediments in the tion can minimise the effect of sampling stress on genetic cold seep area for microbial oxidation. Depending on materials . Cold seep sediment porewater for geo- upflow rates, disturbance frequencies and other physical chemical parameters from both sites was sampled using factors, the total methane emission varies in active seep a pushcore sediment column, Rhizon MOM 2.5 mm areas , and these variations result in different meth - (a mean pore size of 0.15 μm, Rhizosphere Research ane concentrations around cold seep mussels. Cold seep Products No. 19.21.22F, Wageningen, the Netherlands, mussels were previously believed to use methane as the https:// www. rhizo sphere. com/ rhizo ns) and VACU- sole carbon and energy source, and they can adapt to a UETTE blood collection tubes (no additive tube, Greiner wide range of methane concentrations (0.7–33.7 µM) to bio-one, German), which were installed in Haima 2. All survive in extreme environments [11, 12]. The growth samples were stored in a cold room under 4 °C before and physiological conditions of cold seep mussels are on-land analysis. The concentrations of surface ammo - shaped by methane concentration . Notably, obser- nium and sulphide (2 cm below seafloor) were quickly vations from the gill indices and fluorescence in situ measured using a Dionex ICS-1100 ion chromatogra- hybridisation showed that Bathymodiolus azoricus exhib- phy system (Thermo Fisher Scientific, Poway, CA, USA) its a marked decrease in dry weight and total symbiont and methylene blue-SmartChem200 wet chemistry ana- abundance in the absence of methane . lyser (KPM Analytics, Westborough, MA, USA), respec- In this study, we found that the gut microbiome tively. The detection limits of ammonium and sulphide can assimilate nutrients from the genomic view and were 0.02 mg/L (RSD = 0.76%, n = 5) and 0.005 mg/L Xiao et al. Animal Microbiome (2023) 5:17 Page 3 of 13 Fig. 1 Illustration of transplantation experiment, in situ fixation, sample collection and dissection. Deep ‑sea mussels in net bags were translocated to a peripheral site and kept there for six days. Deep‑sea mussel samples were in situ fixed in transplantation site and original site by RNA stabilizing solution filled chamber, and were dissected into three parts (the stomach and gastrointestinal tract (GI) segments I and II) onboard immediately (RSD = 0.98%, n = 3), respectively. All parameters in sedi- conditions are provided in Additional file 1: Table S1. ments and porewaters were determined by the Analyzing Sample names were preceded by their place of origin and Testing Center of the Third Institute of Oceanog - (after or before transplantation), source individual (from raphy, Ministry of Natural Resources (Xiamen, China). 1 to 4) and tissue name (W, for the stomach; P, GI seg- Once these mussels were onboard the research ves- ment I; and C, GI segment II). sel, eight mussel specimens (four individuals each from the original and transplantation groups) were immedi- Taxonomic profiling ately immersed in RNAlater (Thermo Fisher Scientific, Taxonomic profiling was performed using metagenomic Waltham, MA, USA) on ice. Their visceral mass was reads. Raw reads were firstly trimmed with Trimmo - carefully dissected through the stomach and gastrointes- matic v0.39  for the removal of adaptors and low- tinal tract (GI) segments I and II (Fig. 1, [5, 16]) for total quality reads. Trimmed reads were then taxonomically DNA extraction. The dissected tissues were transferred assigned using Kaiju v1.8.2  in greedy mode, and the to RNAlater, incubated at 4 °C overnight, frozen with liq- E-value cutoff was 0.05. The Kaiju employed the refer - uid nitrogen and stored at − 80 °C until sequencing. ence genomes of archaea, bacteria and viruses from the NCBI RefSeq database to remove eukaryotic reads in this DNA extraction, 16S rRNA sequencing and metagenomic study. Successfully mapped reads of every tissue sample sequencing were used for taxonomic classification. The read count Tissue samples fixed in RNAlater were submitted to per sample is listed in Additional file 1: Table S2. Novogen Bioinformatics Institute (Beijing, China) for DNA extraction, library preparation and sequencing. The Alpha and beta diversity analyses V3–V4 regions of 16S rRNA genes were sequenced with Alpha and beta diversity analyses were based on 16S an Illumina NovaSeq-PE250 platform to generate 50,000 rRNA amplicon data. Amplicons were processed using reads per sample. The library for metagenome sequenc - the QIIME2 v2021.8 bioinformatics tool  and aligned ing was paired-end sequenced on an Illumina NovaSeq to amplicon sequence variants (ASVs) for the classifi - 6000 instrument with a 350 bp insert size. Approxi- cation of bacterial or archaeal 16S rRNA gene with the mately 10 Gb of reads were generated for each tissue. SILVA rRNA database (v132). Amplicons were sub-sam- Two sequencing approaches were not evenly applied to pled to an even read depth of 4000 according to the read- every tissue, given the sample quality. Details of sample ing sequences of ASVs (19 samples were used for further Xiao et al. Animal Microbiome (2023) 5:17 Page 4 of 13 study). Rarefy in the R package vegan v2.5-7  was carbohydrate-active enZYmes (CAZymes), including gly- used. Microbial diversity indices were determined using coside hydrolases (GHs), polysaccharide lyases (PLs), gly- abundance-based coverage estimators (ACE), Chao1, cosyltransferases (GTs) and carbohydrate esterases (CEs) Richness, Shannon and Simpson values. The R script in . The KEGG pathway was further reconstructed using vegan v 2.5-7 was used. The original and transplantation the KEGG Mapper (https:// www. genome. jp/ kegg/ map- groups were compared using an independent samples per/ recon struct. html). Subsequently, the Fisher’s exact t-test (t-test) in R. Population distance was calculated on test with Bonferroni correction by the R script was used the basis of the Bray–Curtis similarity index, and dataset to evaluate significant differences between the anno - rank order was examined by nonmetric multi-dimen- tated KEGG numbers of transplantation and original sional scaling (nMDS) for the ordination of similarity groups and prevent the influence of metagenome size. data. KEGG orthologs with a number larger or smaller than the compared group and the adjusted p value (< 0.05) Microbial metagenome assembly, identification were considered to be influenced significantly after and annotation transplantation. Trimmed reads were assembled into contigs with the SPAdes genome assembler v3.14.1 with meta option and Histology and fluorescent in situ hybridisation k-mer values of 21, 33, 55, 77, 99 and 127 [21, 22]. Bacte- Dissected gut samples of G. haimaensis were fixed with rial contigs from metagenomes were isolated on the basis 4% PFA at 4 °C overnight. Specimens were then washed of taxonomic kingdom against the NonRedundant (NR) three times with ice-cold PBS, dehydrated in 75% etha- database with Autometa v2.0.2 . The length cutoff was nol and stored at − 20 °C until use. The specimens were 100. Protein-coding genes were predicted from bacterial embedded in Epredia histoplast paraffin (Thermofisher) contigs with Prodigal v2.6.3 . The protein dataset of by using a Revos tissue processor (Thermo Scientific). each tissue was predicted by the functional Clusters of The standard program was used, and the specimens were Orthologous Groups (COG) using DIAMOND BLASTp mounted and embedded with a HistoStar embedding v2.0.2 . We conducted principal component analysis station (Thermo Scientific). Thin tissue sections (5 µM (PCA) based on the relative abundance of COG catego- thick) were cut using an HM325 microtome (Thermo Sci - ries in each tissue sample by R package ggplot2 v3.3.6 entific). For haematoxylin and eosin (HE) staining, tissue . Protein sequences derived from the same tissue slices were dewaxed and stained with HE in accordance after or before transplantation were combined for the with the standard protocol. After staining, the sections production of a gut functional database based on the were dehydrated and sealed with neutral balsam. Images stomach, GI segment I and II. Thus, six combined pro - were captured using an AxioScan slide scanner (Zeiss). tein sets (three for each group) were generated. The CD- In histology and fluorescent in situ hybridisation HIT v4.8.1 [27, 28] with the setting “-c 0.95” was used (FISH), the tissue sections were initially dewaxed in to cluster a large set of proteins and remove those with accordance with the standard protocol and washed three sequence similarity exceeding 95%. In functional annota- times with PBST (1 × PBS, 0.1% Tween 20) for 5 min tion, protein genes clustered by CD-HIT were searched each. Hybridisation with three 16S rRNA probes was against the NCBI NR database with BLASTp version performed using EUB338-FITC , Alf968-Cy3  2.10.0+ , and the E-value cutoff was 1e-5. The pur - and Gamma42a-Cy5  probes. The protocol described pose was to generate a primary catalogue of gut microbi- by Halary et al.  was used. After hybridisation and ome genes for deep-sea cold seep mussels. The resultant . washing, the sections were labelled with 4′,6-diamid- xml file from NR hits was predicted in OmicsBox version ino-2-phenylindole (DAPI, Sigma-Aldrich) and finally 2.0.36 , and the Gene Ontology (GO) database (ver- mounted on a Prolong glass antifade mounting medium sion 2022.03) was searched. The GO item distributions (Invitrogen) and imaged with a Nikon AX confocal for biological process (BP), cellular components (CC) microscope. and molecular functions (MF) were visualised. All six combined gene sets (without clustering) were searched Results and discussion against the GhostKOALA database through BLAST and Microbial composition of the gut microbiome GHOSTX searches in the Kyoto Encyclopedia of Genes Analyses of metagenomic DNA sequences of 22 speci- and Genomes (KEGG) website (http:// www. kegg. jp/ mens, including stomach and GI segments I and II from blast koala/, ). Gene sets were searched against the original and transplantation sites, detected 10 major Pfam databases with HMMER v3.3  and dbCAN microbial phyla (Fig. 2a, Additional file 1: Note S1) and meta server (http:// bcb. unl. edu/ dbCAN2/). We per- 18 major classes (Fig. 2b, constituting > 1% in any tissue). formed searches on DIAMOND and eCAMI to identify Proteobacteria was the most abundant phylum in the gut Xiao et al. Animal Microbiome (2023) 5:17 Page 5 of 13 microbiome at the original cold seep site. Alphaproteo- Histology analysis of the digestive system of G. haim- bacteria from Proteobacteria dominated most stomach aensis showed that segment II of the GI tract was pri- and GI segment II tissues (average = 33.65%), followed by marily empty, whereas GI segment I contained apparent Gammaproteobacteria (average = 21.9%). The gut micro - contents (Fig. 3a). Subsequent FISH analyses showed bial community structure differed from that of the gill in that the G. haimaensis GI-segment I content contained G. haimaensis, where the gammaproteobacterial endos- digested food particles (Fig. 3b). Notably, the FISH sig- ymbiont constituted ~ 77% of the total microbial com- nals in the gut also indicated that bacterial populations munity (unpublished data) and that of digestive glands in existed in the gut GI track of G. haimaensis. shallow-water mussels, where Proteobacteria is not the most abundant phylum [37, 38]. These results suggested Essential functions of the gut microbiome the major trophic function of Proteobacteria in the gills The de novo assembly results of metagenomic reads of deep-sea mussels. Proteobacteria remained dominant and the annotation rates of all protein sets correspond- in the mussel gut after transplantation, showing a mod- ing to taxa were described in detail in Additional file 1: erate increase in abundance (68.06% at the original site Note S2. Deep-sea bathymodioline mussels have reduced vs. 73.23% at the transplantation site on average). How- digestive systems , but they are still capable of taking ever, Gammaproteobacteria became the most dominant up food through ingestion . In the present study, the class in all tissues, followed by Alphaproteobacteria, and gut microbiome of the cold seep mussel G. haimaensis the relative abundance of Bacteroidetes declined in the exhibited broad and diverse metabolic functions (Fig. 4a), transplantation group. PCA score plot showed a sepa- such as digesting carbohydrates, proteins and lipids, ration between the original and transplantation groups and participated in carbon, sulphur and nitrogen cycles (Additional file 1: Fig. S1a), while different parts of tissue in the deep-sea ecosystem. Moreover, bacteria in differ - and further considering the two treatment groups didn’t ent phyla were found to co-exist in the gut. Their diverse display a clear separation in functional gene structure metabolic capabilities revealed cooperative and competi- (Additional file 1: Fig. S1b and c). These results suggested tive associations between Proteobacteria (predominantly that the transplantation experiment caused changes in Gammaproteobacteria and Alphaproteobacteria) and functional genes. Bacteroidetes. Fig. 2 Taxonomic analysis of gut microbiome after and before transplantation. The relative abundance of major bacterial phylum a and major class b (constituting > 1% in any tissue) based on metagenomic data. Sample names were preceded by their place of origin (after or before transplantation), source individual (from 1 to 4) and tissue name ( W, for the stomach; P, GI segment I; and C, GI segment II) Xiao et al. Animal Microbiome (2023) 5:17 Page 6 of 13 Fig. 3 Histology and fluorescent in situ hybridisation analysis of the Gigantidas haimaensis digestive track. a The HE staining of G. haimaensis digestive track, crosssection. The GI tract of G. haimaensis is surrounded by adipocytes (ab) and oocytes (oo). Inserts a1 and a3 show the cross‑sections of GI‑segment II, while a2 shows the cross‑section of GI‑segment I. b The FISH analysis of GI content with FITC labelled EUB338 (Green), Cy3 labelled Alf968 (Yellow), and Cy5 labelled Gamma42a (Red) probes Gut microbes play a primary role in acquiring energy mannose residue, and α- and β-mannose-rich algal poly- from complex and diverse polysaccharides. Bacteroides saccharides are common in marine systems . Over- are considered efficient glycan degraders along with other all, Bacteroides were found to participate in carbon and bacteria. The degradation function of Bacteroides is often energy cycling by breaking down carbohydrates and pro- accomplished by the polysaccharide utilisation locus teins in deep-sea mussel’s guts with divergent organo- (PUL) gene cluster, and bacteria transport oligosaccha- trophic capacities. rides across the outer membrane for further depolymeri- After surpassing primary degradation, monosaccha- sation by starch utilisation system (Sus; ) (Fig. 4b). rides can be rapidly consumed by the microbiota in the This unique function helps other gut bacteria that cannot gut for pyruvate and subsequent ATP production via the transport long-chain polysaccharides across membranes. Embden–Meyerhof–Parnas (EMP) pathway, Entner– PULs contain the homologues of susC, susD and compo- Doudoroff (ED) pathway or pentose phosphate (PP) nents of CAZyme families, including GTs, GHs, PLs and pathway. Gammaproteobacteria, Alphaproteobacteria CEs. A total of 2113 hits of CAZyme families were identi- and Bacteroidetes had complete EMP and PP cycles. In fied in the dbCAN database and contained 153 types in our metagenomic data, Gammaproteobacteria and Bac- this study. CAZyme families were particularly abundant teroidetes encoded KDPG aldolase (Eda), which is the key and diverse in GI segment I. About 41% of CAZyme enzyme in the ED pathway . annotations came from Bacteroides and contained 116 In mussels from the original site, 26.6–50.9% (aver- types. These annotations covered 22 GTs, 73 GHs, 9 PLs age = 32.5%) of the contigs matched the gene family and 12 CEs. Specific PULs determined which metabolic annotation from the Pfam database, including diverse niches Bacteroides occupied. Enzyme families (i.e. GH26, proteases and lipases. Digesting amino acids requires GH51, GH67, GH89, GH97, GH110, GH123, GH125 and interconversion steps and consumes large amounts of CE7) were uniquely identified in Bacteroides. Among energy. Thus, amino acids degraded by proteases are gen - these families, GH89 (α-N-acetylglucosaminidases) erally less considered efficient energy sources than carbo - degrades mucins for cross-feeding interactions and ena- hydrates . Limited dietary fat can reach GI, where gut bles complex microbial populations to inhabit mucosal microorganisms produce triglyceride lipases to degrade layers . The prominent sources of sulphate are sul - long-chain triglycerides, phospholipase and phospho- phated glycans, which are mostly accessible from mucin lipids . Numerous identified lipases provide a vital provided by hosts [41, 42]. GH125 acts on α-linked role in homeostasis. GDSL-like lipase or acylhydrolase Xiao et al. Animal Microbiome (2023) 5:17 Page 7 of 13 Fig. 4 a Overview of meta‑pathway of Gigantidas haimaensis mussel’s gut microbiome in carbon, sulphur and nitrogen cycles. b Overview of the Bacteroides starch utilisation system (Sus) in this study. IM Inner membrane; OM Outer membrane; A‑ G: starch utilisation system (Sus) locus A‑ G. c Overview of starch and sucrose metabolism‑related genes (more details in Additional file 1: Table S5) with positive influence (red) after transplantation. Disaccharides are marked by green. Other abbreviations are provided in Additional file 1: Table S6, and the figure was created with BioRender.com (https:// app. biore nder. com/) participates in lipid homeostasis and signalling and is short-chain FAs (SCFAs) as end-products together with detected in the gut . This enzyme has an activated carbon dioxide and hydrogen . The utilisation of these serine site near the N-terminus, and this site can bind gaseous by-products results from cross-feeding amongst different substrates compared with other lipase-activated different gut microbiota taxa rather than host absorption, sites at the conserved pentapeptide centre . Certain thereby improving the overall efficiency of metabolism glycerol-reducing bacteria in the GI, such as Proteobac- . Hydrogen is routinely recycled through acetogen- teria, reduce glycerol into 1,3-propanediol, which is an esis, methanogenesis and sulphate reduction, whereas efficient hydrogen sink . the recycling of carbon dioxide occurs due to the first The anaerobic metabolism of the gut microbiome two processes . Gammaproteobacteria and Alphapro- through the digestion of the dietary substrates generates teobacteria had a nearly complete Wood–Ljungdahl Xiao et al. Animal Microbiome (2023) 5:17 Page 8 of 13 Community shifts and function influences pathway. Chemolithotrophic Gammaproteobacteria in after transplantation the gut used methane monooxygenase (MMO) to oxidise A total of 21 samples had amplicon sequences and gener- methane and then utilised the RuMP and serine path- ated 5,033 ASVs, of which 572 (11.4%) ASVs overlapped ways to fix carbon. Alphaproteobacteria utilised the CBB between transplantation and original ASV (Additional pathway. Sulphate reduction is the most efficient way of file 1: Fig. S2). The lack of shared taxonomy between hydrogenotrophs. Meta-pathway analysis indicated that the two groups suggests compositional shifts after the Gammaproteobacteria, Alphaproteobacteria and Bacte- transplantation of deep-sea mussels. Alpha and beta roidetes have a complete cycle of assimilatory sulphate diversity analyses were performed on the basis of the reduction. Gammaproteobacteria underwent typical rarefied 16s rRNA ASV table. The median ACE, Chao1, dissimilatory sulphate reduction. Gammaproteobacte- Richness, Shannon and Simpson values in the trans- ria and Alphaproteobacteria had an accomplished cycle plantation group were lower than those in the original of the Sox system. The microbial nitrogen cycle is envi - group. ACE, Richness and Chao1 indices decreased sig- ronmentally essential, and nitrogen obtained through nificantly (p < 0.05; Fig. 5a). The nMDS analysis based on filter-feeding is an important component of nutri - the Bray–Curtis dissimilarity distance illustrated the sig- tional requirements for cold seep mussels . Genes nificant community difference between the two groups for dissimilatory nitrate reduction, nitrification and (ANOSIM r = 0.2711, p = 0.012; Fig. 5b). The relative denitrification were found in Gammaproteobacteria. Alp- abundance values of different bacterial taxa in the gut haproteobacteria had functions of dissimilatory nitrate microbiota were highly sensitive. These gut microbiotas reduction and nitrification. Bacteroidetes contributed to can shift and interact with the environment and quickly assimilatory nitrate reduction. Gammaproteobacteria, adapt and respond to environmental stressors . After Alphaproteobacteria and Bacteroidetes also had urease the translocation of deep-sea mussels 100 m away from that hydrolyses urea to ammonia (and generates C O ) to the cold seep mussel bed, the abundance of symbionts in be used for protein metabolism. Urea or glutamine syn- the gill decreased according to the ratio between eukary- thase serves as a major alternative ammonia detoxifica - otes to prokaryotes (unpublished data), implying that tion pathway to maintain ammonia homeostasis. Overall, the methane concentration declined after transplanta- the meta-pathway analysis provided genomic evidence tion . Community structures and functions in the of the potential nutritional roles of carbon, sulphur and gut shifted even under a short experimental time. The nitrogen acquired by heterotrophy, and such information elevation of Gammaproteobacteria in the intestinal com- was previously hypothesised only by laboratory measure- munity of the transplanted mussels demonstrated their ments or isotope labelling experiments. essential role in methane utilisation as methanotrophic Microbe–microbe interactions can be beneficial (like bacteria in the cold-seep area. The relative abundance cross-feeding mentioned above) or adverse . The of Bacteroidetes decreased, which may indicate a loss of type VI secretion system (T6SS) contributes to the dis- dietary polysaccharides in the digestive tract. A decline tribution of diverse antibacterial effectors. These toxic in the alpha diversity index implies the declined tendency effectors target multiple activities, such as phospholi - of microbial diversity and richness, and the Bray–Curtis pases, peptidoglycan hydrolases, nucleases and mem- distance reflects the separation between two groups of brane pore-forming proteins, to conciliate interbacterial samples. These findings are consistent with short-term conflict and competition [54, 55]. The T6SS translocates and long-term stress exposure experiments, such as fast- resources (effectors) directly into adjacent bacteria, host ing [60–62]. cells or extracellular milieu  and inhibits them. Pro- On functional influences under transplantation (Addi - teobacterial T6SS has conserved proteins. While TssR tional file 1: Note S3), GO categories, including processes was not present in proteobacterial T6SS loci but in Bacte- related to carbon metabolism, signalling and transport, roides sp. in this study, so it was likely to serve as a novel were found to exclusively exist in either the transplanta- transmembrane function in Bacteroides sp., as previously tion or original group. Detailed gene numbers of six com- reported by Coyne and Comstock . Rhs-family toxins bined datasets can be found in Additional file 1: Table S7, are common effectors attached to the VgrG spike struc - and the summed number is displayed in Fig. 6a. Positively ture of T6SS, and the diversification of this combination influenced genes were assigned to carbohydrate, nitro - determines bacterial coexistence . Bacteroides sp. and gen and sulphur metabolism and other functional path- Escherichia coli in our study were found to encode dis- ways on the basis of the KEGG pathway reconstruction tinct Rhs element Vgr protein that competes to dominate (Fig. 6b). All KO results of the two pooled datasets can the niche in deep-sea mussel’s gut. be found in Additional file 1: Table S5. An overview of Xiao et al. Animal Microbiome (2023) 5:17 Page 9 of 13 Fig. 5 Alpha‑ and beta‑ diversity. a ACE, Chao1, Richness, Shannon, and Simpson values of the transplantation group (green) is lower than the original group (red). *p value < 0.05. b Bay‑ Curtis dissimilarity distance is illustrated by the nonmetric multi‑ dimensional scaling (nMDS) plot of the ASV matrix. These analyses were performed after applying the different read depths to 4000 based on read sequences of ASVs and square Root Transformation (Sqrt) positively influenced genes in starch and sucrose metabo - through functional investigations and blasting against lism pathways is illustrated in Fig. 4c. public databases. In this study, functional shifts occurred The mechanisms of assisting the symbiont to minimise in many nutritional, transport and element cycle- challenges in deep-sea invertebrates can be determined related pathways. The GI segment I of deep-sea Xiao et al. Animal Microbiome (2023) 5:17 Page 10 of 13 Fig. 6 a Categories in biological process (BP), cellular components (CC) and molecular functions (MF) that only existed in either the transplantation group or the original group. Detailed gene numbers of each tissue dataset can be found in Additional file 1: Table S7. b KO ID under essential metabolism and transduction pathways that are positively influenced after transplantation by Fisher’s exact test. The histogram shows KEGG annotation hits, and the gene number in the bracket indicates the amount of influenced KO in a specific pathway Xiao et al. Animal Microbiome (2023) 5:17 Page 11 of 13 mussels had numerous CAZymes, but both annotation gut microbiome may have a high level of response to hits and overall categories of CAZymes in the gut micro- host pressure, specifically using bacterial conjunction biome decreased (153 vs 60) after transplantation pos- belonging to a large type IV secretion system to share sibly because of the loss of Bacteroidetes. Carbohydrate antibiotic resistance genes. The activator of the transfers metabolism-related genes are summarised in Fig. 6b, (tra) was identified as exclusive BP in the original group. showing a high number of genes that break down disac- These genes are encoded by the plasmid and can control charides to supplement carbon sources under transplan- the expression of its conjugal gene cluster to sense and tation conditions. Deep-sea mussels can uptake various respond to periods of host stress . resources of inorganic nitrogen from the environment Demonstrated from our results, the mechanisms that through heterotrophic and autotrophic feeding strategies enable deep-sea mussels to respond to environmen- to prevent nitrogen limitation during growth . Their tal shifts were as follows: deep-sea mussels manage to nitrogen assimilation rate is not affected by methane con - maintain a balance of gut bacteria by competitive exclu- centration, although it is considered the determinant of sion that allows autotrophic Proteobacteria, especially mussel abundance and condition . After transplan- chemoautotrophic Gammapreoteobacteria, to dominate tation, the number of nitrite reductase (nirB) increased, the gut microbiota. Such a shift in microbial community whereas glutamate dehydrogenase (gdhA), glutamate structure improves metabolism by swiftly adjusting the synthase (gltB and gltS) and ferredoxin-nitrate reductase number of metabolic enzymes to balance nutrient sup- (NarB) decreased; thus, nitrite trends to generate ammo- plementation and further stimulate transport and signal- nia instead of nitrate in the gut. When the ammonium ling systems to work against inter- or intra-competition. concentrations drop, mussel tissues prefer to have more glutamate synthase, based on isotope measurement , Conclusion and this activity occurs in the original site (23.8 mg/L). In this study, we illustrated the microbial community When the environmental ammonium concentration is structure and primary microbial gene catalogues and higher in the transplantation site (37.4 mg/L), the assimi- investigated changes in bacterial composition and func- lation of ammonium inhibits nitrate reduction , and tions in response to inadequate environmental methane ammonium becomes a major end-product . These supply for the deep-sea cold seep mussel G. haimaen- results indicated that the gut microbiome is sensitive to sis. As a result of technical limitations, functional gene nitrogen shifts, whose concentration may affect mus - analysis only highlighted the beneficial and adverse sel growth. A decrease in thiosulphate reductase was microbe–microbe interactions in the gut on the class detected after transplantation, and it was coincident with level. In general, the gut microbiomes of deep-sea mus- a higher sulphide concentration in the transplantation sels are functionally versatile and facilitate inter-bacterial site (0.8 mg/L at the transplantation site and 0.13 mg/L at associations by adjusting the unique metabolic pathway the original site), which suggests that mussels can obtain to acquire necessary energy and elements. When sym- more sulphide from the surrounding environment. bionts were deprived, competitive exclusion occurred The gene counts of lantibiotic biosynthesis protein and altered microbial diversity and structure in the gut (NisB) increased in the transplantation group, which of deep-sea mussels as an adaptation strategy. These gut showed an enhanced dehydration efficiency of prenisin microbes can also integrate carbon, nitrogen and sul- during antimicrobial activity. This protein catalyses the phur source utilisation and immune-related activities in dehydration of specific serine and threonine residues. response to such stress. These findings provide the first u Th s, the peptide attached to the fully modified lanti - metagenomic insights into the gut microbiome and its biotic can abolish antimicrobial activity, suggesting a changes during deep-sea mussel in situ transplantation self-protection role against increased inter-species com- experiment. petition . The sporulation of spore formers is a cell density-dependent response to nutrient deprivation, Supplementary Information leading to the production of sporulation sensor kinase The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s42523‑ 023‑ 00238‑8. B (kinB) protein in the transplantation group during ini- tial sporulation . Motile bacteria have an adaptive Additional file 1. Supplementary Information. mechanism to compare temporal surrounding chemical conditions and can swim in response to chemical gradi- Acknowledgements ents . This adaptive strategy is mediated by methyl - The authors wish to thank the crew of R/V Haiyangdizhi 6 and the operation transferase (CheR) and methylesterase (CheB) , both team of ROV Haima 2 for their technical support in collecting samples during showed a higher number of genes in the transplantation the HYDZ6‑202102 cruise. We would like to especially thank Jun Tao, Yi Yang and Yitao Lin for their assistance during sample collection, Qisinuo Yin for his group than in the original group. By contrast, the original Xiao et al. 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Animal Microbiome – Springer Journals
Published: Mar 11, 2023
Keywords: Gigantidas mussel; Metagenome; Nutritional role; Haima seep; In situ experiment
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