Toxicology assessment of deep-sea mining impacts on Gigantidas platifrons: A comparative in situ and laboratory metal exposure study.

Deep-sea toxicology is essential for deep-sea environmental impact assessment. Yet most toxicology experiments are conducted solely in laboratory settings, overlooking the complexities of the deep-sea environment. Here we carried out metal exposure experiments in both the laboratory and in situ, to compare and evaluate the response patterns of Gigantidas platifrons to metal exposure (copper [Cu] or cadmium [Cd] at 100 µg/L for 48 h). Metal concentrations, traditional biochemical parameters, and fatty acid composition were assessed in deep-sea mussel gills. The results revealed significant metal accumulation in deep-sea mussel gills in both laboratory and in situ experiments. Metal exposure could induce oxidative stress, neurotoxicity, an immune response, altered energy metabolism, and changes to fatty acid composition in mussel gills. Interestingly, the metal accumulating capability, biochemical response patterns, and fatty acid composition each varied under differing experimental systems. In the laboratory setting, Cd-exposed mussels exhibited a higher value for integrated biomarker response (IBR) while in situ the Cu-exposed mussels instead displayed a higher IBR value. This study emphasizes the importance of performing deep-sea toxicology experiments in situ and contributes valuable data to a standardized workflow for deep-sea toxicology assessment.

Integrated transcriptomic and metabolomic approaches reveal molecular response and potential biomarkers of the deep-sea mussel Gigantidas platifrons to copper exposure.

Metal pollution caused by deep-sea mining activities has potential detrimental effects on deep-sea ecosystems. However, our knowledge of how deep-sea organisms respond to this pollution is limited, given the challenges of remoteness and technology. To address this, we conducted a toxicity experiment by using deep-sea mussel Gigantidas platifrons as model animals and exposing them to different copper (Cu) concentrations (50 and 500 µg/L) for 7 days. Transcriptomics and LC-MS-based metabolomics methods were employed to characterize the profiles of transcription and metabolism in deep-sea mussels exposed to Cu. Transcriptomic results suggested that Cu toxicity significantly affected the immune response, apoptosis, and signaling processes in G. platifrons. Metabolomic results demonstrated that Cu exposure disrupted its carbohydrate metabolism, anaerobic metabolism and amino acid metabolism. By integrating both sets of results, transcriptomic and metabolomic, we find that Cu exposure significantly disrupts the metabolic pathway of protein digestion and absorption in G. platifrons. Furthermore, several key genes (e.g., heat shock protein 70 and baculoviral IAP repeat-containing protein 2/3) and metabolites (e.g., alanine and succinate) were identified as potential molecular biomarkers for deep-sea mussel's responses to Cu toxicity. This study contributes novel insight for assessing the potential effects of deep-sea mining activities on deep-sea organisms.

Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses.

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus-host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

Mosaic environment-driven evolution of the deep-sea mussel Gigantidas platifrons bacterial endosymbiont.

BACKGROUND: The within-species diversity of symbiotic bacteria represents an important genetic resource for their environmental adaptation, especially for horizontally transmitted endosymbionts. Although strain-level intraspecies variation has recently been detected in many deep-sea endosymbionts, their ecological role in environmental adaptation, their genome evolution pattern under heterogeneous geochemical environments, and the underlying molecular forces remain unclear. RESULTS: Here, we conducted a fine-scale metagenomic analysis of the deep-sea mussel Gigantidas platifrons bacterial endosymbiont collected from distinct habitats: hydrothermal vent and methane seep. Endosymbiont genomes were assembled using a pipeline that distinguishes within-species variation and revealed highly heterogeneous compositions in mussels from different habitats. Phylogenetic analysis separated the assemblies into three distinct environment-linked clades. Their functional differentiation follows a mosaic evolutionary pattern. Core genes, essential for central metabolic function and symbiosis, were conserved across all clades. Clade-specific genes associated with heavy metal resistance, pH homeostasis, and nitrate utilization exhibited signals of accelerated evolution. Notably, transposable elements and plasmids contributed to the genetic reshuffling of the symbiont genomes and likely accelerated adaptive evolution through pseudogenization and the introduction of new genes. CONCLUSIONS: The current study uncovers the environment-driven evolution of deep-sea symbionts mediated by mobile genetic elements. Its findings highlight a potentially common and critical role of within-species diversity in animal-microbiome symbioses. Video Abstract.

Biodiversity exploration of Formosa Ridge cold seep in the South China Sea using an eDNA metabarcoding approach.

The Formosa Ridge, also named Site F, is an active cold seep marine ecosystem site that has been studied since it was discovered on the continental slope of the northeast South China Sea (SCS). However, few studies have focused on the eukaryotic diversity at Site F. Environmental DNA (eDNA) technology is a non-invasive method applied in biodiversity surveys with a high species detection probability. In the present study, we identified multi-trophic biodiversity using eDNA metabarcoding combined with multiple ribosomal RNA gene (rDNA) markers. We detected 142 phytoplankton, 90 invertebrates, and 64 fish species by amplifying the 18S rRNA gene V4 region, the 18S rRNA gene V9 region, and the 12S rRNA gene. The results elucidated dissimilar trends of different assemblages with depth. The diversity of phytoplankton and invertebrate assemblages markedly decreased with depth, whereas little change was observed within the fish assemblage. We comprehensively assessed the relationship between the three assemblages and environmental factors (temperature, salinity, depth, dissolved oxygen, and chlorophyll a). These factors strongly impacted on phytoplankton and invertebrates, but only slightly on fish. We inferred the finding might be due to fish having a strong migration capacity and wide distribution. This study indicates that eDNA metabarcoding with multiple markers is a powerful tool for marine biodiversity research that is able to provide technical support and knowledge for resource management and biodiversity protection efforts.

Variation in epibiotic bacteria on two squat lobster species of Munidopsidae.

The relationships between epibiotic bacteria on deep-sea hosts and host lifestyle factors are of particular interest in the field of deep-sea chemoautotrophic environmental adaptations. The squat lobsters Shinkaia crosnieri and Munidopsis verrilli are both dominant species in cold-seep ecosystems, and they have different distributions and feeding behaviors. These species may have evolved to have distinct epibiotic microbiota. Here, we compared the epibiotic bacterial communities on the M. verrilli carapace (MVcarapace), S. crosnieri carapace (SCcarapace), and S. crosnieri ventral plumose setae (SCsetae). The epibiotic bacteria on SCsetae were dense and diverse and had a multi-layer configuration, while those on MVcarapace and SCcarapace were sparse and had a monolayer configuration. Chemoautotrophic bacteria had the highest relative abundance in all epibiotic bacterial communities. The relative abundance of amplicon sequence variant 3 (ASV3; unknown species in order Thiotrichales), which is associated with sulfide oxidation, was significantly higher in SCsetae than MVcarapace and SCcarapace. Thiotrichales species seemed to be specifically enriched on SCsetae, potentially due to the synthetic substrate supply, adhesion preference, and host behaviors. We hypothesize that the S. crosnieri episymbionts use chemical fluxes near cold seeps more efficiently, thereby supporting the host's nutrient strategies, resulting in a different distribution of the two species of squat lobster.

Hologenome analysis reveals independent evolution to chemosymbiosis by deep-sea bivalves.

BACKGROUND: Bivalves have independently evolved a variety of symbiotic relationships with chemosynthetic bacteria. These relationships range from endo- to extracellular interactions, making them ideal for studies on symbiosis-related evolution. It is still unclear whether there are universal patterns to symbiosis across bivalves. Here, we investigate the hologenome of an extracellular symbiotic thyasirid clam that represents the early stages of symbiosis evolution. RESULTS: We present a hologenome of Conchocele bisecta (Bivalvia: Thyasiridae) collected from deep-sea hydrothermal vents with extracellular symbionts, along with related ultrastructural evidence and expression data. Based on ultrastructural and sequencing evidence, only one dominant Thioglobaceae bacteria was densely aggregated in the large bacterial chambers of C. bisecta, and the bacterial genome shows nutritional complementarity and immune interactions with the host. Overall, gene family expansions may contribute to the symbiosis-related phenotypic variations in different bivalves. For instance, convergent expansions of gaseous substrate transport families in the endosymbiotic bivalves are absent in C. bisecta. Compared to endosymbiotic relatives, the thyasirid genome exhibits large-scale expansion in phagocytosis, which may facilitate symbiont digestion and account for extracellular symbiotic phenotypes. We also reveal that distinct immune system evolution, including expansion in lipopolysaccharide scavenging and contraction of IAP (inhibitor of apoptosis protein), may contribute to the different manners of bacterial virulence resistance in C. bisecta. CONCLUSIONS: Thus, bivalves employ different pathways to adapt to the long-term co-existence with their bacterial symbionts, further highlighting the contribution of stochastic evolution to the independent gain of a symbiotic lifestyle in the lineage.

Insights into symbiotic interactions from metatranscriptome analysis of deep-sea mussel Gigantidas platifrons under long-term laboratory maintenance.

Symbioses between invertebrates and chemosynthetic bacteria are of fundamental importance in deep-sea ecosystems, but the mechanisms that enable their symbiont associations are still largely undescribed, owing to the culturable difficulties of deep-sea lives. Bathymodiolinae mussels are remarkable in their ability to overcome decompression and can be maintained successfully for an extended period under atmospheric pressure, thus providing a model for investigating the molecular basis of symbiotic interactions. Herein, we conducted metatranscriptome sequencing and gene co-expression network analysis of Gigantidas platifrons under laboratory maintenance with gradual loss of symbionts. The results revealed that one-day short-term maintenance triggered global transcriptional perturbation in symbionts, but little gene expression changes in mussel hosts, which were mainly involved in responses to environmental changes. Long-term maintenance with depleted symbionts induced a metabolic shift in the mussel host. The most notable changes were the suppression of sterol biosynthesis and the complementary activation of terpenoid backbone synthesis in response to the reduction of bacteria-derived terpenoid sources. In addition, we detected the upregulation of host proteasomes responsible for amino acid deprivation caused by symbiont depletion. Additionally, a significant correlation between host microtubule motor activity and symbiont abundance was revealed, suggesting the possible function of microtubule-based intracellular trafficking in the nutritional interaction of symbiosis. Overall, by analyzing the dynamic transcriptomic changes during the loss of symbionts, our study highlights the nutritional importance of symbionts in supplementing terpenoid compounds and essential amino acids and provides insight into the molecular mechanisms and strategies underlying the symbiotic interactions in deep-sea ecosystems.

Metabolism Interactions Promote the Overall Functioning of the Episymbiotic Chemosynthetic Community of Shinkaia crosnieri of Cold Seeps.

Remarkably diverse bacteria have been observed as biofilm aggregates on the surface of deep-sea invertebrates that support the growth of hosts through chemosynthetic carbon fixation. Growing evidence also indicates that community-wide interactions, and especially cooperation among symbionts, contribute to overall community productivity. Here, metagenome-guided metatranscriptomic and metabolic analyses were conducted to investigate the taxonomic composition, functions, and potential interactions of symbionts dwelling on the seta of Shinkaia crosnieri lobsters in a methane cold seep. Methylococcales and Thiotrichales dominated the community, followed by the Campylobacteriales, Nitrosococcales, Flavobacteriales, and Chitinophagales Metabolic interactions may be common among the episymbionts since many separate taxon genomes encoded complementary genes within metabolic pathways. Specifically, Thiotrichales could contribute to detoxification of hydroxylamine that is a metabolic by-product of Methylococcales. Further, Nitrosococcales may rely on methanol leaked from Methylococcales cells that efficiently oxidize methane. Elemental sulfur may also serve as a community good that enhances sulfur utilization that benefits the overall community, as evidenced by confocal Raman microscopy. Stable intermediates may connect symbiont metabolic activities in cyclical oxic-hypoxic fluctuating environments, which then enhance overall community functioning. This hypothesis was partially confirmed via in situ experiments. These results highlight the importance of microbe-microbe interactions in symbiosis and deep-sea adaptation. IMPORTANCE Symbioses between chemosynthetic bacteria and marine invertebrates are common in deep-sea chemosynthetic ecosystems and are considered critical foundations for deep-sea colonization. Episymbiotic microorganisms tend to form condensed biofilms that may facilitate metabolite sharing among biofilm populations. However, the prevalence of metabolic interactions among deep-sea episymbionts and their contributions to deep-sea adaptations are not well understood due to sampling and cultivation difficulties associated with deep-sea environments. Here, we investigated metabolic interactions among the episymbionts of Shinkaia crosnieri, a dominant chemosynthetic ecosystem lobster species in the Northwest Pacific Ocean. Meta-omics characterizations were conducted alongside in situ experiments to validate interaction hypotheses. Furthermore, imaging analysis was conducted, including electron microscopy, fluorescent in situ hybridization (FISH), and confocal Raman microscopy (CRM), to provide direct evidence of metabolic interactions. The results support the Black Queen Hypothesis, wherein leaked public goods are shared among cohabitating microorganisms to enhance the overall adaptability of the community via cooperation.

Metagenome sequencing and 768 microbial genomes from cold seep in South China Sea.

Cold seep microbial communities are fascinating ecosystems on Earth which provide unique models for understanding the living strategies in deep-sea distinct environments. In this study, 23 metagenomes were generated from samples collected in the Site-F cold seep field in South China Sea, including the sea water closely above the invertebrate communities, the cold seep fluids, the fluids under the invertebrate communities and the sediment column around the seep vent. By binning tools, we retrieved a total of 768 metagenome assembled genome (MAGs) that were estimated to be >60% complete. Of the MAGs, 61 were estimated to be >90% complete, while an additional 105 were >80% complete. Phylogenomic analysis revealed 597 bacterial and 171 archaeal MAGs, of which nearly all were distantly related to known cultivated isolates. In the 768 MAGs, the abundant Bacteria in phylum level included Proteobacteria, Desulfobacterota, Bacteroidota, Patescibacteria and Chloroflexota, while the abundant Archaea included Asgardarchaeota, Thermoplasmatota, and Thermoproteota. These results provide a dataset available for further interrogation of deep-sea microbial ecology.

The complete mitochondrial genome of Asteroschema tubiferum (Ophiuroidea: Euryalida: Asteroschematidae).

We describe the first mitochondrial genome of a brittle star Asteroschema tubiferum Matsumoto 1911 in family Asteroschematidae. The mitogenome was sequenced and assembled using next-generation sequencing technology, and were 16,361 bp in size with 37 genes containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. The phylogenetic tree was constructed based on 13 protein-coding mitochondrial genes of A. tubiferum and 26 species in the phylum Echinodermata by RAxML, which showed that it was mostly related to the species in Family Gorgonocephalidae. These results could provide a novel insight to the phylogeny of Ophiuroidea.

Aequorivita iocasae sp. nov., a halophilic bacterium isolated from sediment collected at a cold seep field in the South China Sea.

A moderately halophilic bacterium, designated strain KX20305T, was isolated from sediment collected from a cold seep field in the South China Sea. Cells of strain KX20305T were Gram-stain-negative, rod-shaped, non-motile, facultatively anaerobic, oxidase- and catalase-positive, and grew optimally at 25-30 °C, pH 6.0-8.0 and with 3-6 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain KX20305T grouped with members of the genus Aequorivita, including Aequorivita aquimaris D-24T (98.3 % sequence similarity), Aequorivita vladivostokensis KMM 3516T (98.1 %) and Aequorivita echinoideorum CC-CZW007T (97.5 %). Genome sequencing of strain KX20305T revealed a genome size of 3.35 Mb and a DNA G+C content of 38.71 mol%. Genomic average nucleotide identity (orthoANI) values of strain KX20305T with A. aquimaris D-24T, A. vladivostokensis KMM 3516T and A. echinoideorum JCM 30378T were 83.8, 81.7 and 75.4 %, respectively, while in silico DNA-DNA hybridization (GGDC) values for strain KX20305T with these strains were 27.2, 25.0 and 19.6 %, respectively. The major fatty acids of strain KX20305T were iso-C15 : 0, iso-C17 : 0 3-OH and 10-methyl C16 : 0/iso-C17 : 1 ω9c. The predominant respiratory quinone was menaquinone-6 (MK-6). The polar lipids mainly comprised phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids. Based on comparative analysis of phylogenetic, phylogenomic, phenotypic and chemotaxonomic characteristics, strain KX20305T represents a novel species of the genus Aequorivita, for which the name Aequorivita iocasae sp. nov. is proposed. The type strain is KX20305T (=KCTC 82699T=MCCC 1K06238T=JCM 34635T).

Adaption to hydrogen sulfide-rich environments: Strategies for active detoxification in deep-sea symbiotic mussels, Gigantidas platifrons.

The deep-sea mussel Gigantidas platifrons is a representative species that relies on nutrition provided by chemoautotrophic endosymbiotic bacteria to survive in both hydrothermal vent and methane seep environments. However, vent and seep habitats have distinct geochemical features, with vents being more harsh than seeps because of abundant toxic chemical substances, particularly hydrogen sulfide (H2S). Until now, the adaptive strategies of G. platifrons in a heterogeneous environment and their sulfide detoxification mechanisms are still unclear. Herein, we conducted 16S rDNA sequencing and metatranscriptome sequencing of G. platifrons collected from a methane seep at Formosa Ridge in the South China Sea and a hydrothermal vent at Iheya North Knoll in the Mid-Okinawa Trough to provide a model for understanding environmental adaption and sulfide detoxification mechanisms, and a three-day laboratory controlled Na2S stress experiment to test the transcriptomic responses under sulfide stress. The results revealed the active detoxification of sulfide in G. platifrons gills. First, epibiotic Campylobacterota bacteria were more abundant in vent mussels and contributed to environmental adaptation by active oxidation of extracellular H2S. Notably, a key sulfide-oxidizing gene, sulfide:quinone oxidoreductase (sqr), derived from the methanotrophic endosymbiont, was significantly upregulated in vent mussels, indicating the oxidization of intracellular sulfide by the endosymbiont. In addition, transcriptomic comparison further suggested that genes involved in oxidative phosphorylation and mitochondrial sulfide oxidization pathway played important roles in the sulfide tolerance of the host mussels. Moreover, transcriptomic analysis of Na2S stressed mussels confirmed the upregulation of oxidative phosphorylation and sulfide oxidization genes in response to sulfide exposure. Overall, this study provided a systematic transcriptional analysis of both the active bacterial community members and the host mussels, suggesting that the epibionts, endosymbionts, and mussel host collaborated on sulfide detoxification from extracellular to intracellular space to adapt to harsh H2S-rich environments.

microRNAs facilitate comprehensive responses of Bathymodiolinae mussel against symbiotic and nonsymbiotic bacteria stimulation.

Bathymodiolinae mussels are dominant species in cold seeps and hydrothermal vents and could harbor endosymbionts in gill bacteriocytes. However, mechanisms underlying the symbiosis have remained largely undisclosed for years. In the present study, the global expression pattern of immune-related genes and miRNAs were surveyed in Gigantidas platifrons during bacterial challenges using enriched symbiotic methane oxidation bacteria MOBs or nonsymbiotic Vibrio. As a result, multiple pattern recognition receptors were found differentially expressed at 12 h and 24 h post bacteria challenges and distinctly clustered between stimulations. Dozens of immune effectors along with signal transducers were also modulated simultaneously during MOB or Vibrio challenge. A total of 459 miRNAs were identified in the gill while some were differentially expressed post MOB or nonsymbiotic bacteria challenge. A variety of immune-related genes were annotated as target genes of aforesaid differentially expressed miRNAs. As a result, biological processes including the immune recognition, lysosome activity and bacteria engulfment were suggested to be dynamically modulated by miRNAs in either symbiotic or nonsymbiotic bacteria challenge. It was suggested that G. platifrons mussels could maintain a robust immune response against invading pathogens while establishing symbiosis with chemosynthetic bacteria with the orchestra of immune-related genes and miRNAs.

Biochemical and metabolic responses of the deep-sea mussel Bathymodiolus platifrons to cadmium and copper exposure.

Greater interest in commercial deep-sea mining has been accompanied by mounting environmental concerns, including metal contamination resulting from mining activities. However, little is known about the toxic effects of metal exposure on deep-sea life. Given its ability to accumulate metals from the surrounding environment, its wide distribution at both vents and seeps, and its high abundance, the deep-sea mussel Bathymodiolus platifrons could serve as an ideal model to investigate the toxicological responses of deep-sea organisms to metal exposure. Here, we evaluated metal accumulation, traditional metal-related biomarkers, namely acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase, catalase, reduced glutathione, metallothioneins, and malondialdehyde, as well as metabolic profiles in the gills of B. platifrons after a 7-day exposure to copper (100 µg/L), cadmium (500 µg/L), or copper-plus-cadmium treatments (100 µg/L Cu and 500 µg/L Cd). Metal exposure concentrations selected in this study can be found in deep-sea hydrothermal environments. Metal exposure resulted in significant metal accumulation in the gills of the mussel, indicating that B. platifrons has promise for use as an indicator of deep-sea metal pollution levels. Traditional biomarkers (AKP, ACP, and measured antioxidants) revealed cellular injury and oxidative stress in mussels following metal exposure. Metabolic responses in the three treatment groups indicated that metal exposure perturbed osmoregulation, energy metabolism, and nucleotide metabolism in mussels, in a response marked by differentially altered levels of amino acids, hypotaurine, betaine, succinate, glucose 6-phosphate, fructose 6-phosphate, guanosine, guanosine 5'-monophosphate, and inosine. Nevertheless, several uniquely altered metabolites were found in each treatment exposure group, suggesting dissimilar modes of toxicity between the two metal types. In the Cd-exposed group, the monosaccharide D-allose, which is involved in suppressing mitochondrial ROS production, was downregulated, a response consistent with oxidative stress in Cd-exposed B. platifrons. In the Cu-exposed group, the detected alterations in dopamine, dopamine-related, and serotonin-related metabolites together suggest disturbed neurotransmission in Cu-exposed B. platifrons. In the Cu-plus-Cd group, we detected a decline in fatty acid levels, implying that exposure to both metals jointly exerted a negative influence on the physiological functioning of the mussel. To the best of our knowledge, this is the first study to investigate changes in metabolite profiles in Bathymodiolus mussels exposed to metal. The findings reported here advance our understanding of the adverse impact of metal exposure on deep-sea life and can inform deep-sea mining assessments through the use of multiple biomarkers.

A Toll-like receptor identified in Gigantidas platifrons and its potential role in the immune recognition of endosymbiotic methane oxidation bacteria.

Symbiosis with chemosynthetic bacteria is an important ecological strategy for the deep-sea megafaunas including mollusks, tubeworms and crustacean to obtain nutrients in hydrothermal vents and cold seeps. How the megafaunas recognize symbionts and establish the symbiosis has attracted much attention. Bathymodiolinae mussels are endemic species in both hydrothermal vents and cold seeps while the immune recognition mechanism underlying the symbiosis is not well understood due to the nonculturable symbionts. In previous study, a lipopolysaccharide (LPS) pull-down assay was conducted in Gigantidas platifrons to screen the pattern recognition receptors potentially involved in the recognition of symbiotic methane-oxidizing bacteria (MOB). Consequently, a total of 208 proteins including GpTLR13 were identified. Here the molecular structure, expression pattern and immune function of GpTLR13 were further analyzed. It was found that GpTLR13 could bind intensively with the lipid A structure of LPS through surface plasmon resonance analysis. The expression alternations of GpTLR13 transcripts during a 28-day of symbiont-depletion assay were investigated by real-time qPCR. As a result, a robust decrease of GpTLR13 transcripts was observed accompanying with the loss of symbionts, implying its participation in symbiosis. In addition, GpTLR13 transcripts were found expressed exclusively in the bacteriocytes of gills of G. platifrons by in situ hybridization. It was therefore speculated that GpTLR13 may be involved in the immune recognition of symbiotic methane-oxidizing bacteria by specifically recognizing the lipid A structure of LPS. However, the interaction between GpTLR13 and symbiotic MOB was failed to be addressed due to the nonculturable symbionts. Nevertheless, the present result has provided with a promising candidate as well as a new approach for the identification of symbiont-related genes in Bathymodiolinae mussels.

A glimpse of deep-sea adaptation in chemosynthetic holobionts: Depressurization causes DNA fragmentation and cell death of methanotrophic endosymbionts rather than their deep-sea Bathymodiolinae host.

Bathymodiolinae mussels are typical species in deep-sea cold seeps and hydrothermal vents and an ideal model for investigating chemosynthetic symbiosis and the influence of high hydrostatic pressure on deep-sea organisms. Herein, the potential influence of depressurization on DNA fragmentation and cell death in Bathymodiolinae hosts and their methanotrophic symbionts were surveyed using isobaric and unpressurized samples. As a hallmark of cell death, massive DNA fragmentation was observed in methanotrophic symbionts from unpressurized Bathymodiolinae while several endonucleases and restriction enzymes were upregulated. Additionally, genes involved in DNA repair, glucose/methane metabolism as well as two-component regulatory system were also differentially expressed in depressurized symbionts. DNA fragmentation and programmed cell death, however, were rarely detected in the host bacteriocytes owing to the orchestrated upregulation of inhibitor of apoptosis genes and downregulation of caspase genes. Meanwhile, diverse host immune recognition receptors were promoted during depressurization, probably enabling the regain of symbionts. When the holobionts were subjected to a prolonged acclimation at atmospheric pressure, alternations in both the DNA fragmentation and the expression atlas of aforesaid genes were continuously observed in symbionts, demonstrating the persistent influence of depressurization. Contrarily, the host cells demonstrated certain tolerance against depressurization stress as expression level of some immune-related genes returned to the basal level in isobaric samples. Altogether, the present study illustrates the distinct stress responses of Bathymodiolinae hosts and their methanotrophic symbionts against depressurization, which could provide further insight into the deep-sea adaptation of Bathymodiolinae holobionts while highlighting the necessity of using isobaric sampling methods in deep-sea research.

Molecular analyses of the gill symbiosis of the bathymodiolin mussel Gigantidas platifrons.

Although the deep-sea bathymodiolin mussels have been intensively studied as a model of animal-bacteria symbiosis, it remains challenging to assess the host-symbiont interactions due to the complexity of the symbiotic tissue-the gill. Using cold-seep mussel Gigantidas platifrons as a model, we isolated the symbiont harboring bacteriocytes and profiled the transcriptomes of the three major parts of the symbiosis-the gill, the bacteriocyte, and the symbiont. This breakdown of the complex symbiotic tissue allowed us to characterize the host-symbiont interactions further. Our data showed that the gill's non-symbiotic parts play crucial roles in maintaining and protecting the symbiosis; the bacteriocytes supply the symbiont with metabolites, control symbiont population, and shelter the symbiont from phage infection; the symbiont dedicates to the methane oxidation and energy production. This study demonstrates that the bathymodiolin symbiosis interacts at the tissue, cellular, and molecular level, maintaining high efficiency and harmonic chemosynthetic micro niche.

Nitrincola iocasae sp. nov., a bacterium isolated from sediment collected at a cold seep field in the South China Sea.

A novel bacterium, designated strain KXZD1103T, was isolated from sediment collected at a cold seep field of the Formosa Ridge in the South China Sea. Cells were Gram-stain-negative, facultatively anaerobic, motile, oxidase- and catalase-positive, and grew optimally at 28 °C, pH 6.0-pH 7.0 and in the presence of 1-3 % (w/v) NaCl. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c) and C16 : 0. The major respiratory ubiquinone was Q-8. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Analysis of 16S rRNA gene sequences revealed that strain KXZD1103T grouped with members of the genus Nitrincola, with Nitrincola lacisaponensis 4CAT (98.1 % sequence similarity) and Nitrincola schmidtii R4-8T (97.7 %) as its closest neighbours. Genome sequencing revealed a genome size of 4.17 Mb and a DNA G+C content of 50.1 %. Genomic average nucleotide identity values for strain KXZD1103T with the type strains within the genus Nitrincola ranged from 71.0 to 75.7 %, while the in silico DNA-DNA hybridization values for strain KXZD1103T with these strains ranged from 16.1 to 21.6 %. On the basis of the results of phylogenetic, phenotypic and chemotaxonomic analyses, strain KXZD1103T is considered to represent a novel species of the genus Nitrincola, for which the name Nitrincola iocasae sp. nov. is proposed. The type strain is KXZD1103T (=KCTC 72678T=MCCC 1K04283T).

Identification and characterization of endosymbiosis-related immune genes in deep-sea mussels Gigantidas platifrons.

Deep-sea mussels of the subfamily Bathymodiolinae are common and numerically dominant species widely distributed in cold seeps and hydrothermal vents. During long-time evolution, deep-sea mussels have evolved to be well adapted to the local environment of cold seeps and hydrothermal vents by various ways, especially by establishing endosymbiosis with chemotrophic bacteria. However, biological processes underlying the establishment and maintenance of symbiosis between host mussels and symbionts are largely unclear. In the present study, Gigantidas platifrons genes possibly involved in the symbiosis with methane oxidation symbionts were identified and characterized by Lipopolysaccharide (LPS) pull-down and in situ hybridization. Five immune related proteins including Toll-like receptor 2 (TLR2), integrin, vacuolar sorting protein (VSP), matrix metalloproteinase 1 (MMP1), and leucine-rich repeat (LRR-1) were identified by LPS pull-down assay. These five proteins were all conserved in either molecular sequences or functional domains and known to be key molecules in host immune recognition, phagocytosis, and lysosome-mediated digestion. Furthermore, in situ hybridization of LRR-1, TLR2 and VSP genes was conducted to investigate their expression patterns in gill tissues of G. platifrons. Consequently, LRR-1, TLR2, and VSP genes were found expressed exclusively in the bacteriocytes of G. platifrons. Therefore, it was suggested that TLR2, integrin, VSP, MMP1, and LRR-1 might be crucial molecules in the symbiosis between G. platifrons and methane oxidation bacteria by participating in symbiosis-related immune processes.