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Abstract Heimdallarchaeia is a class of the Asgardarchaeota, are the most probable candidates for the archaeal protoeukaryote ancestor that have been identified to date. However, little is known about their life habits regardless of their ubiquitous distribution in diverse habitats, which is especially true for Heimdallarchaeia from deep-sea environments. In this study, we obtained 13 metagenome-assembled genomes (MAGs) of Heimdallarchaeia from the deep-sea cold seep and hydrothermal vent. These MAGs belonged to orders o_Heimdallarchaeales and o_JABLTI01, and most of them (9 MAGs) come from the family f_Heimdallarchaeaceae according to genome taxonomy database (GTDB). These are enriched for common eukaryote-specific signatures. Our results show that these Heimdallarchaeia have the metabolic potential to reduce sulfate (assimilatory) and nitrate (dissimilatory) to sulfide and ammonia, respectively, suggesting a previously unappreciated role in biogeochemical cycling. Furthermore, we find that they could perform both TCA and rTCA pathways coupled with pyruvate metabolism for energy conservation, fix CO2 and generate organic compounds through an atypical Wood-Ljungdahl pathway. In addition, many genes closely associated with bacteriochlorophyll and carotenoid biosynthesis, and oxygen-dependent metabolic pathways are identified in these Heimdallarchaeia MAGs, suggesting a potential light-utilization by pigments and microoxic lifestyle. Taken together, our results indicate that Heimdallarchaeia possess a mixotrophic lifestyle, which may give them more flexibility to adapt to the harsh deep-sea conditions.

ABSTRACT Microorganisms are significant drivers of organic matter mineralization and are essential in marine biogeochemical cycles. However, the variations and influencing factors in prokaryotic communities from cold-seep sediments to the water column and the specific role of these microorganisms in biogeochemical cycles in the water column above cold seep remain unclear. Here, we investigated prokaryotic communities and their roles in nitrogen/sulfur cycling processes and conducted in situ dissolved organic matter (DOM) enrichment experiments to explore the effects of diverse sources of DOM on prokaryotic communities. Field investigations showed that the prokaryotic communities in the near-bottom water were more similar to those in the deep layer of the euphotic zone (44.60%) and at a depth of 400 m (50.89%) than those in the sediment (18.00%). DOM enrichment experiments revealed that adding dissolved organic nitrogen (DON) and phosphorus DOP caused a notable increase in the relative abundances of Rhodobacterales and Vibrionales, respectively. A remarkable increase was observed in the relative abundance of Alteromonadales and Pseudomonadales after the addition of dissolved organic sulfur (DOS). The metagenomic results revealed that Proteobacteria served as the keystone taxa in mediating the biogeochemical cycles of nitrogen, phosphorus, and sulfur in the Haima cold seep. This study highlights the responses of prokaryotes to DOM with different components and the microbially driven elemental cycles in cold seeps, providing a foundational reference for further studies on material energy metabolism and the coupled cycling of essential elements mediated by deep-sea microorganisms.IMPORTANCEDeep-sea cold seeps are among the most productive ecosystems, sustaining unique fauna and microbial communities through the release of methane and other hydrocarbons. Our study revealed that the influence of seepage fluid on the prokaryotic community in the water column is surprisingly limited, which challenges conventional views regarding the impact of seepage fluids. In addition, we identified that different DOM compositions play a crucial role in shaping the prokaryotic community composition, providing new insights into the factors driving microbial diversity in cold seeps. Furthermore, the study highlighted Proteobacteria as key and multifaceted drivers of biogeochemical cycles in cold seeps, emphasizing their significant contribution to complex interactions and processes. These findings offer a fresh perspective on the dynamics of cold-seep environments and their microbial communities, advancing our understanding of the biogeochemical functions in deep-sea environments.

Abstract 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.

Light and nitrogen availability are basic requirements for photosynthesis. Changing in light intensity and nitrogen concentration may require adaptive physiological and life process changes in phytoplankton cells. Our previous study demonstrated that two Thalassiosira species exhibited, respectively, distinctive physiological responses to light and nitrogen stresses. Transcriptomic analyses were employed to investigate the mechanisms behind the different physiological responses observed in two diatom species of the genus Thalassiosira. The results indicate that the congeneric species are different in their cellular responses to the same shifting light and nitrogen conditions. When conditions changed to high light with low nitrate (HLLN), the large-celled T. punctigera was photodamaged. Thus, the photosynthesis pathway and carbon fixation related genes were significantly down-regulated. In contrast, the small-celled T. pseudonana sacrificed cellular processes, especially amino acid metabolisms, to overcome the photodamage. When changing to high light with high nitrate (HLHN) conditions, the additional nitrogen appeared to compensate for the photodamage in the large-celled T. punctigera, with the tricarboxylic acid cycle (TCA cycle) and carbon fixation significantly boosted. Consequently, the growth rate of T. punctigera increased, which suggest that the larger-celled species is adapted for forming post-storm algal blooms. The impact of high light stress on the small-celled T. pseudonana was not mitigated by elevated nitrate levels, and photodamage persisted.

Bathymodioline mussels dominate deep-sea methane seep and hydrothermal vent habitats and obtain nutrients and energy primarily through chemosynthetic endosymbiotic bacteria in the bacteriocytes of their gill. However, the molecular mechanisms that orchestrate mussel host–symbiont interactions remain unclear. Here, we constructed a comprehensive cell atlas of the gill in the mussel Gigantidas platifrons from the South China Sea methane seeps (1100 m depth) using single-nucleus RNA-sequencing (snRNA-seq) and whole-mount in situ hybridisation. We identified 13 types of cells, including three previously unknown ones, and uncovered unknown tissue heterogeneity. Every cell type has a designated function in supporting the gill’s structure and function, creating an optimal environment for chemosynthesis, and effectively acquiring nutrients from the endosymbiotic bacteria. Analysis of snRNA-seq of in situ transplanted mussels clearly showed the shifts in cell state in response to environmental oscillations. Our findings provide insight into the principles of host–symbiont interaction and the bivalves' environmental adaption mechanisms.

Abstract 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

The sediment-water interfaces of cold seeps play important roles in nutrient transportation between seafloor and deep-water column. Microorganisms are the key actors of biogeochemical processes in this interface. However, the knowledge of the microbiome in this interface are limited. Here we studied the microbial diversity and potential metabolic functions by 16S rRNA gene amplicon sequencing at sediment-water interface of two active cold seeps in the northern slope of South China Sea, Lingshui and Site F cold seeps. The microbial diversity and potential functions in the two cold seeps are obviously different. The microbial diversity of Lingshui interface areas, is found to be relatively low. Microbes associated with methane consumption are enriched, possibly due to the large and continuous eruptions of methane fluids. Methane consumption is mainly mediated by aerobic oxidation and denitrifying anaerobic methane oxidation (DAMO). The microbial diversity in Site F is higher than Lingshui. Fluids from seepage of Site F are mitigated by methanotrophic bacteria at the cyclical oxic-hypoxic fluctuating interface where intense redox cycling of carbon, sulfur, and nitrogen compounds occurs. The primary modes of microbial methane consumption are aerobic methane oxidation, along with DAMO, sulfate-dependent anaerobic methane oxidation (SAMO). To sum up, anaerobic oxidation of methane (AOM) may be underestimated in cold seep interface microenvironments. Our findings highlight the significance of AOM and interdependence between microorganisms and their environments in the interface microenvironments, providing insights into the biogeochemical processes that govern these unique ecological systems.

Abstract 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.

Blood pressure has a 24-hour repetitive and regular variation which shows circadian rhythm. Using the multivariate time series analysis method of vector autoregressive model, we could realize the simultaneous prediction for both systolic and diastolic blood pressures. We choose blood pressure from 6 AM to 10 AM in 3 weeks as an episode to construct a prediction model. Missing values were imputed by regression models. Subsequently, we defined segments as positive or negative segments according to blood pressure measurements. The predictions were accomplished by vector autoregressive model (VAR). Both positive and negative segments were randomly selected from each patient to summarize the effect of prediction models. In this study, the MAPE (Mean Absolute Percentage Error) of systolic blood pressure and diastolic blood pressure were both less than 10%, indicating that the VAR model was adaptable in predicting the blood pressure of hypertensive patients. Based on VAR, we could provide early warning to breakthrough of blood pressure thresholds. The sensitivity, specificity, and accuracy for patients in the training sets were 77.50%, 81.58 %, and 79.49% respectively, and the sensitivity, specificity, and accuracy for patients in the training sets were 76.92%, 80.00% and 78.43% respectively. This research took information of both systolic and diastolic blood pressures at the same time to establish the VAR models and enabled simultaneous prediction for systolic and diastolic blood pressure.

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.

Measurement(s) metagenome assembled genomes Technology Type(s) metagenome sequencing and genome binning Sample Characteristic - Organism microorganism Sample Characteristic - Environment marine cold seep biome Sample Characteristic - Location South China Sea

A cold seep is one of the typical deep-sea chemical energy ecosystems and a hotspot for studying unique life processes and biogeochemical cycles in the deep sea. Macrofauna, which is one of the most important components of the cold seep ecosystem, has not been thoroughly studied. We examined the macrofauna community at Site F using images collected in 2016 by an imaging and laser profiling system and biological samples collected in 2020 and 2021 by TV grab and a remotely operated vehicle. In total, 41 species were found. The overall number of macrofauna identified at Site F (20,000 m2) reached 252,943 individuals, and the biomass reached 726.15 kg by dry weight. As the dominant species, Gigantidas platifrons and Shinkaia crosnieri reached their highest densities of 629 and 396 individuals/m2, respectively. The comparisons between different stations revealed that the diversity and density, even the biomass of dominant species, were much higher in the south than in the north at Site F in 2020. Correlation analysis showed that methane had a positive effect on macrofauna density. Compared with S. crosnieri, G. platifrons seems to be more adapted to the harsh cold seep environment. Methane consumption rates of the dominant species show that macrofauna are important in influencing seafloor methane fluxes. Our findings provide valuable insights into the ecology, community structure, and biota-environment interaction in the cold seep at Site F.

IntroductionMarine metal contamination caused by deep-sea mining activities has elicited great concern from both social and scientific communities. Among the various metals deep-sea organisms might encounter, cadmium (Cd) is a widely detected metal that in very small amounts is nonetheless capable of severe toxicity. Yet due to both remoteness and technical challenges, insights into the effects of metal exposure resulting from mining activities upon deep-sea organisms are limited.MethodsHere, we investigated Cd’s toxicological effects on deep-sea mussels of Gigantidas platifrons exposed to 100 or 1000 g/L of Cd for 7 days; an integrated approach was used that incorporated proteomics and metabolomics along with traditional approaches (metal concentrations, metal subcellular distribution, and anti-oxidative and immune-related biochemical indexes).Results and DiscussionResults showed that Cd exposure caused significant Cd’s accumulation in mussel gills and redistribution of Cd among subcellular compartments, with cellular debris being the primary binding site. Although anti-oxidative enzymes activities (superoxide dismutase and catalase) were not significantly altered in mussel gills of both exposed groups, the markedly increased level of glutathione S-transferase detected via proteomic technique clearly evinced that deep-sea mussels suffered from oxidative stress under Cd exposure. Besides, altered activities of acid phosphatase and alkaline phosphatase assayed by traditional methods along with the predominant presence of largely altered immune-related proteins detected by proteomic data strongly revealed an immune response of deep-sea mussels elicited by Cd. In addition, results of proteomics combined with those of non-targeted metabolomics demonstrated that Cd could exert toxicity by disrupting cytoskeleton structure, ion homeostasis, and primary metabolisms of energy, lipid, and nucleotide in deep-sea mussels. As demonstrated in this study, proteomics and metabolomics can be used in tandem to provide valuable insights into the molecular mechanisms of deep-sea organisms’ response to Cd exposure and for helping to discover potential biomarkers for application during deep-sea mining assessments.

Confocal Raman microscopy (CRM) is a powerful tool for biological research, which can provide information regarding the composition and distribution of biomolecules in an in situ, label-free, non-destructive manner and with high spatial resolution. Sample preservation is often an unavoidable step, especially for symbiotic deep-sea samples. Moreover, protocols for the preservation of samples for CRM have not been established and specific effects of different preservation methods on biomolecules have not been studied for relevant samples. In this study, we used deep-sea mussel Gigantidas platifrons, an ideal model in the study of deep-sea symbiosis and investigated the effect of four common preservation methods on the results of CRM imaging and signals. The methods included snap-freeze (SF), SF followed by rapid fixation in methanol (SF-MeOH), 2.5% glutaraldehyde and 2% paraformaldehyde fixation (SF-GP), and 4% paraformaldehyde and alcohol fixation (PS-PA). The results of this study indicate that SF was the most effective method for the comprehensive analysis of the biomolecular composition although the sectioning success rate was relatively low. Moreover, SF-MeOH was found to be effective when SF is not sufficient in obtaining good morphology in sections, or when the effect of chemical bonding on the composition of biomolecules upon SF-MeOH can be neglected. Finally, SF-GP and PS-PA were found to be the most effective methods considering the overall morphological observation. However, they were less suitable for metabolic studies. We believe our results can provide guidance for further studies of Raman on symbiotic deep-sea biological samples. It is of great importance for the wide application of Raman technique.

Outbreaks of scyphozoan Aurelia coerulea and Nemopilema nomurai in the coastal sea of China are managed in recent years because they have severely jeopardized local socioeconomic development and ecological health. In this study, we propose specific strategies to control these blooms based on the different physio-ecological characteristics of their polyps, which can produce medusae by strobilation. High densities of A. coerulea polyps can survive chronically on the surfaces of some artificial constructions submerged in harbors or bays, China. Through buddings, they can resist the invasion of biofouling organisms and proliferate on the surfaces of some fouling organisms (e.g., ascidians, and bryozoans). However, N. nomurai polyps have not been recorded in natural environment. The in situ experiments found that polyps on settling plates fail to survive via podocysts due to severe biofouling invasion and post-strobilated degeneration in late spring and summer. As a result, the population size following is strongly dependent on the sexual recruitment of medusae during late summer and autumn. Therefore, we suggest that the reasonable governance strategy is to manage polyp populations together with biofouling organisms for A. coerulea blooms, however, with a focus on the medusa stage (particularly young medusae) to decrease the sexual reproduction in N. nomurai blooms. Accordingly, massive occurrences of A. coerulea in Qingdao Middle Port, China were alleviated by eliminating polyps and biofouling organisms on the undersurfaces of floating docks and then brushing the surfaces with modified alloprene paints. Some applicable control measures, including resource utilization of N. nomurai medusae and more severe and earlier summer fishing moratoriums, were used to possibly help restrain outbreaks of N. nomurai in Chinese coastal waters.

ABSTRACT 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.

Single-cell RNA-sequencing (scRNA-seq) is the genomic approach that directly examines gene expression information from individual cells and provides a higher resolution of cellular differences than bulk RNA-seq. In the past decade, with the rapid development of microfluid, barcoding, and next-generation sequencing (NGS) technologies, scRNA-seq has revolutionized transcriptomic studies and gained great success and broad prospects. However, compared with the wide use in the few model animals, the application of scRNA-seq in marine organisms is limited due to the high cost of early scRNA-seq and the immature of single-cell methods for marine organisms. Recently, with the increase of genomic data, maturation of scRNA-seq platform and downstream bioinformatics algorithms, the scRNA-seq has been successfully applied in several marine model animals with great success, which demonstrated that the scRNA-seq could be the ideal and powerful tool to extend our understanding of marine organisms’ evolutionary and physiological processes and their adaptation to ecological niches. Here, we review the state-of-the-art improvements of single-cell sequencing techniques and new studies that apply single-cell methods to marine organisms. We also summarize the opportunities and challenges scientists may face in further single-cell research and propose several appealing prospects that may benefit from the combination of single-cell techniques and marine organisms.

Abstract Background Takayasu arteritis (TA) is a rare disease, lacking convenient and feasible biomarkers to identify disease activity. We aimed to evaluate the value of complements in distinguishing active TA. Methods Consecutive patients were enrolled from the prospective East China TA cohort from April 2008 to June 2019. Patients were divided into two groups according to their baseline Kerr score. The value of complements and other biomarkers in identifying disease activity were analysed with cluster analysis, ROC curves, and combined tests. An independent group of patients from July 2019 to December 2019 were employed to validate the results. Results Of the enrolled 519 patients, 406 (72.2%) cases were identified as active disease. Higher erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), interleukin-6 (IL-6), and complement 3 (C3) levels were observed in the active group. Elevated C3 (≥ 1.085 g/L) had a high value to identify active TA with a sensitivity of 69.9%, specificity of 66.7%, and AUC of 0.715. Combining the CRP (≥ 10.65 g/L; sensitivity, 50.7%; specificity, 82.4%) and C3, the sensitivity could be improved to 85.1% in parallel test and the specificity could be improved to 94.1% in serial test. Validation was further performed to confirm the value of C3 for disease activity assessment. The accuracy of the parallel test of CRP and C3 in external validation with independent 53 TA cases was 72.73% with the AUC of 0.721. Conclusion Elevated C3 could effectively evaluate the disease activity of TA, and C3 combining with CRP could further improve the disease activity evaluation.

Gigantidas platifrons is one of the most dominant deep-sea mussels in cold seeps ecosystems in the South China sea. Studies have shown that deep-sea mussels are a gonochoristic species, however, little is known about the molecular mechanisms of sex determination in G. platifrons. In this study, RNA-seq and WGBS methylation analysis were performed on adult G. platifrons gonads to identify potential sex-related genes and generate a comprehensive analysis of sex determination in deep-sea mussels. A total of 5923 genes were identified as differentially expressed between the ovaries and testes, of which 2711 were female-biased and 3212 were male-biased. Among them, 161 genes may participate in the sex determination, and we found that DMRT2 may play an important role in male sex determination, and FOXL2, Wnt7, and β-catenin may have impact on female sex determination. Moreover, common expression patterns were found in majority of the sex-related genes such as FOXL2, β-catenin, and genes in SOX family, suggesting the sex determination mechanisms of mussels in different habitats were conserved. The 5mC levels of transcription start sites (TSS2K) were significantly higher in sex-related genes than other DEGs in both gonads. Positive correlation was observed between sex-related genes expression and methylation in male, however, the effects of the DNA methylation on gene expression were complex in female. In short, we argue that sex determination mechanisms of deep-sea mussel G. platifrons is mainly controlled by genetic, and the methylation may have a regulation role on male sex determination or differentiation.

Marine Heatwaves (MHWs) events have been increasing, causing severe impacts on marine ecosystems and aquaculture. In this study, the effects of temperature on changes in sediment dissolved oxygen (DO) and hydrogen sulfide (H2S) concentrations and their effects on the behavioral and physiological responses of a bivalve were investigated by simulating different sudden summer temperature change conditions. The results showed that temperature was an important factor affecting DO consumption and H2S release in sediments, and sediment type also played a key role. At higher temperatures, DO was consumed more rapidly and H2S release increased in sediments, and the DO was consumed and H2S released in sandy sediments was less than in silty sand sediments. The response of Manila clam’s exercise behavior under environmental pressure was also rapid. The excavation index of the Manila clam decreased with the DO consumption, indicating that the Manila clam could alter its burial depth and move toward the sediment-water interface when stressed. Compared with phenol oxidases (PO) activity being activated under experimental conditions, succinate concentrations only increased slightly at 32°C, and did not surpass the threshold indicative of anaerobic metabolism. However, when toxic substances such as H2S start to accumulate, they may damage the immune system and tissues of the Manila clam, thereby affecting its future survival. Therefore, when temperatures are high for long periods in summer, it is necessary to take timely action to prevent and guard against harm caused by DO and H2S to aquaculture organisms.

Objective: To investigate whether contrast-enhanced ultrasound (CEUS) is helpful for assessing the disease activity of Takayasu arteritis (TA).Methods: Eighty-four patients with TA were examined with ultrasound (US) and CEUS. Intima-media thickness (IMT) of both sides of the common carotid artery was measured for each patient. Semiquantitative analyses of contrast enhancement within the arterial wall were performed with a visual interpretation scale. Disease activity was determined by one experienced physician based on Physician Global Assessment criteria.Results: Carotid CEUS revealed that 6 arteries showed no enhancement (7.1%, 6/84) while 78 arteries showed homogeneous enhancement within the thickened wall (92.9%, 78/84). Grade 1 enhancement was noted in 40 patients, and grade 2 in 38 patients. IMT of the active group was significantly thicker than that of the inactive group (2.4 ± 0.9 mm vs. 1.8 ± 0.5 mm, P = 0.001) on B-mode US, and slightly correlated with ESR (r = 0.344, P < 0.05) and CRP (r = 0.261, P < 0.05). Grade 2 enhancement was observed more in the active patients (61.7% vs. 41.3%, P = 0.001) on CEUS.Conclusions: CEUS is a convenient and non-invasive imaging modality that is useful for evaluating disease activity in TA patients by assessing the vascularization within the carotid wall.

The biomass and estimated production, feeding rate of chaetognaths were studied in the Yellow Sea. Sagitta crassa, Sagitta enflata, Sagitta bedoti, and Sagitta nagae were found during the study period, and S. crassa and S. nagae were the dominant species. The mean chaetognath biomass was in the range of 2.56 - 5.57 mg DM m-3, and S. crassa and S. nagae comprised 49 - 90 and 10 - 33%, respectively. The geographical distribution pattern was differed between different species. The annual average estimated feeding rate and production of chaetognaths was evaluated as 55.24 mg C m-3 year-1 and 15.90 mg C m-3 year-1, respectively. The body length of Sagitta species less than 9 - 11 mm had relatively high feeding rate in August, September, and December because of their relatively high total biomass, while the body length with 8 - 16, 11 - 15, and 6 - 16 mm groups had relatively high feeding rate in March, May, and June, respectively. The average feeding pressure on zooplankton biomass and production by chaetognaths could be evaluated as 1.07 and 18.95%, respectively, and the biomass and production of small copepods, and nauplius and copepodite of large copepods would be seriously impacted during whole year.

Characterizing habitats and species distribution is important to understand the structure and function of cold seep ecosystems. This paper develops a deep learning model for the fast and accurate recognition and classification of substrates and the dominant associated species in cold seeps. Considering the dense distribution of the dominant associated species and small objects caused by overlap in cold seeps, the feature pyramid network (FPN) embed into the faster region-convolutional neural network (R-CNN) was used to detect large-scale changes and small missing objects without increasing the number of calculations. We applied three classifiers (Faster R-CNN + FPN for mussel beds, lobster clusters and biological mixing, CNN for shell debris and exposed authigenic carbonates, and VGG16 for reduced sediments and muddy bottom) to improve the recognition accuracy of substrates. The model’s results were manually verified using images obtained in the Formosa cold seep during a 2016 cruise. The recognition accuracy of the two dominant species, e.g., Gigantidas platifrons and Munidopsidae could be 70.85 and 56.16%, respectively. Seven subcategories of substrates were also classified with a mean accuracy of 74.87%. The developed model is a promising tool for the fast and accurate characterization of substrates and epifauna in cold seeps, which is crucial for large-scale quantitative analyses.

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.

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.

Summary: 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.

Abstract Seamounts are undersea mountains rising abruptly from the sea floor and interacting dynamically with underwater currents. They represent unique biological habitats with various microbial community structures. Certain seamount bacteria form conspicuous extracellular iron oxide structures, including encrusted stalks, flattened bifurcating tubes, and filamentous sheaths. To extend our knowledge of seamount ecosystems, we performed an integrated study on population structure and the occurrence of magnetotactic bacteria (MTB) that synthesize intracellular iron oxide nanocrystals in sediments of a seamount in the Mariana volcanic arc. We found Proteobacteria dominant at 13 of 14 stations, but ranked second in abundance to members of the phylum Firmicutes at the deep-water station located on a steep slope facing the Mariana-Yap Trench. Live MTB dwell in biogenic sediments from all 14 stations ranging in depth from 238 to 2,023 m. Some magnetotactic cocci possess the most complex flagellar apparatus yet reported; 19 flagella are arranged in a 3:4:5:4:3 array within a flagellar bundle. Phylogenetic analysis of 16S rRNA gene sequences identified 16 novel species of MTB specific to this seamount. Together the results obtained indicate that geographic properties of the seamount stations are important in shaping the bacterial community structure and the MTB composition.

Deep-sea hydrothermal vents in the western Pacific are increasingly explored for potential mineral extraction. The study of the composition of the food web plays an important guiding role in the ecological protection and restoration of potential mining areas. The general picture of the nutritional sources of species should be established to assess the potential impacts of future mining activities on the biological composition and food sources. To provide basic information, we analyzed the carbon and nitrogen stable isotope ratios of the dominant macrofauna (mussels, commensal scale worms, crustaceans, gastropods, and vestimentiferans) at three different sites in the PACManus hydrothermal area and the Desmos caldera. The δ13C ratio was significantly different between species: mussels and commensal scale worms showed lighter δ13C ratios, whereas crustaceans showed heavier ratios. In terms of δ15N, mussels had the lowest values and the crustaceans had the highest values. By taking into account these stable isotope signatures, we were able to develop inferences of the food sources for vent community organisms. We found that the food web was based on various species of chemoautotrophic bacteria. Mussels appeared to rely primarily on sulfur-based endosymbionts, which use the Calvin-Benson-Bassham (CBB) cycle and RuBisCO form I as the CO2-fixing enzyme. Commensal polychaetes mostly obtained their nutrition from their hosts. Crustacean species were omnivorous, feeding on chemosynthetic bacteria, sedimentary debris, or even animals according to the local environment. In contrast, gastropods relied mainly on symbiotic bacteria with some supplementary consumption of detritus. Vestimentiferans obtained food from symbiotic bacteria using the RuBisCO form II enzyme in the CBB cycle and may have several symbionts using different fixation pathways. Although most macrofauna relied on symbiotic chemoautotrophic bacteria, our study suggested a closer trophic relationship between animals. Therefore, to evaluate the potential impacts of deep sea mining, it is necessary to study the cascade effects on the food web of the whole ecosystem. Before exploiting deep-sea resources, further systematic investigations concerning the protection of deep-sea ecosystems are necessary.

Paralvinella hessleri is a terrebellomorphy polychaetae worm lives on the hydrothermal vents in the Okinawa trough and Mariana Back-Arc basin. We report the complete mitochondrial genome of P. hessleri, which is 16,978 bp in length, containing 13 protein-coding genes, two ribosomal proteins, and 22 tRNAs. The gene order is identical to that of reported terebellids. The overall base composition is AT-biased. Our results of P. hessleri mt genome may provide useful genetic information for phylogenetic and population connectivity studies of the deep-sea alvinellid worms.

Deep-sea mussels of the genus Bathymodiolus are numerically dominant macrofauna in many cold seep and hydrothermal vent ecosystems worldwide, and they depend on organic carbon produced by symbionts present in the epithelial cells of the gills. Although Bathymodiolus platifrons represents typical methanotrophic endosymbiosis, our understanding of molecular mechanisms of methane oxidization and carbon fixation is still in its infancy. Moreover, the laboratory maintenance of B. platifrons and the symbiont abundance dynamics during maintenance has not been reported. In the present study, we report the first systematic identification and phylogenetic analysis of three subunits of methane monooxygenase (pmoA, pmoB, and pmoC) obtained from the endosymbiotic bacteria found in B. platifrons. The coding sequences (CDS) of the three genes in the B. platifrons endosymbiont were 750, 1,245, and 753 bp, encoding 249, 414, and 250 amino acids, respectively. Sequence alignment and phylogenetic analysis revealed that the symbiont of B. platifrons belongs to the type I methanotrophs. In order to clarify the impact of environmental methane on symbiont abundance, a 34-day laboratory maintenance experiment was conducted in which B. platifrons individuals were acclimatized to methane-present and methane-absent environments. Symbiont abundance was evaluated by calculating the relative DNA content of the methane monooxygenase gene using quantitative real-time PCR. We found that symbiont quantity immediately decreased from its initial level, then continued to gradually decline during maintenance. At 24 and 34 days of maintenance, symbiont abundance in the methane-absent environment had significantly decreased compared to that in the methane-present environment, indicating that the maintenance of symbionts relies on a continuous supply of methane. Our electron microscopy results validated the qPCR analysis. This study enriches our knowledge of the molecular basis and the dynamic changes of the methanotrophic endosymbiosis in B. platifrons, and provides a feasible model biosystem for further investigation of methane oxidization, the carbon fixation process, and environmental adaptations of deep-sea mussels.

Elucidating the genetic mechanisms of adaptation to the hydrothermal vent in organisms at genomic level is significant for understanding the adaptive evolution process in the extreme environment. We performed RNA-seq on four different tissues of a vent crab species, Austinograea alayseae, producing 725,461 unigenes and 134,489 annotated genes. Genes related to sensory, circadian rhythm, hormone, hypoxia stress, metal detoxification and immunity were identified. It was noted that in the degenerated eyestalk, transcription of phototransduction related genes which are important for retinal function was greatly reduced; three crucial neuropeptide hormones, one molt-inhibiting and two crustacean hyperglycemic hormone precursors were characterized with conserved domains; hypoxia-inducible factor 1 and two novel isoforms of metallothioneins in the vent crabs were discovered. An analysis of 6,932 orthologs among three crabs A. alayseae, Portunus trituberculutus and Eriocheir sinensis revealed 19 positive selected genes (PSGs). Most of the PSGs were involved in immune responses, such as crustins and anti-lipopolysaccharide factor, suggesting their function in the adaptation to environment. The characterization of the first vent crab transcriptome provides abundant resources for genetic and evolutionary studies of this species, and paves the way for further investigation of vent adaptation process in crabs.

The impact of transient wind events on an established zooplankton community was observed during a field survey in a coastal region off northern Norway in May 2002. A transient wind event induced a coastal jet/filament intrusion of warm, saline water into our survey area where a semi-permanent eddy was present. There was an abrupt change in zooplankton community structure within 4-7 days of the wind event, with a change in the size structure, an increase in lower size classes less than 1 mm in equivalent spherical diameter (ESD) and a decrease in larger size classes greater than 1.5 mm in ESD. The slope of zooplankton biovolume spectra changed from -0.6 to -0.8, consistent with the size shifting towards smaller size classes. This study shows that even well established zooplankton communities are susceptible to restructuring during transient wind events, and in particular when wind forcing induces horizontal currents or filaments.

Grazing and metabolism of Euphausia pacifica in the Yellow Sea were studied from September 2006 to August 2007. Euphausia pacifica is a selective-feeding omnivore and grazing rates among different months were monitored using a Coulter Counter and batch culture feeding experiments. Euphausia pacifica mainly grazed microzooplankton in August and September, which resulted in an increase in chlorophyll a concentration. Oxygen consumption rate of E. pacifica was 38.7-42.5 μmol O2 g(-1) DW h(-1) in March, which was four times higher than the oxygen consumption rates in September and December. The vigorous metabolism of E. pacifica in March consumed 3.1% of body carbon daily, which is likely related to its high reproduction and grazing rate. Respiration and metabolism of E. pacifica in September and December were similar and were lower. O:N ratio of E. pacifica was the highest (17.3-23.8) in March when spawning activity occurred and when food was abundant. The energetic source of E. pacifica during September and December was mostly protein from eating a carnivorous diet, including such items as microzooplankton. Euphausia pacifica was found in cold water at the bottom of the Yellow Sea in summer and autumn and maintained a low consumption status. O:N ratios of E. pacifica in March, September, and December were negatively correlated with SSTs and no significant correlation was found between O:N ratios and chlorophyll a concentration. Seawater temperature is clearly the most important parameter influencing the metabolism of E. pacifica.

BACKGROUND: Despite their species abundance and primary economic importance, genomic information about copepods is still limited. In particular, genomic resources are lacking for the copepod Calanus sinicus, which is a dominant species in the coastal waters of East Asia. In this study, we performed de novo transcriptome sequencing to produce a large number of expressed sequence tags for the copepod C. sinicus. RESULTS: Copepodid larvae and adults were used as the basic material for transcriptome sequencing. Using 454 pyrosequencing, a total of 1,470,799 reads were obtained, which were assembled into 56,809 high quality expressed sequence tags. Based on their sequence similarity to known proteins, about 14,000 different genes were identified, including members of all major conserved signaling pathways. Transcripts that were putatively involved with growth, lipid metabolism, molting, and diapause were also identified among these genes. Differentially expressed genes related to several processes were found in C. sinicus copepodid larvae and adults. We detected 284,154 single nucleotide polymorphisms (SNPs) that provide a resource for gene function studies. CONCLUSION: Our data provide the most comprehensive transcriptome resource available for C. sinicus. This resource allowed us to identify genes associated with primary physiological processes and SNPs in coding regions, which facilitated the quantitative analysis of differential gene expression. These data should provide foundation for future genetic and genomic studies of this and related species.

Abstract Background Copepods are highly diverse and abundant, resulting in extensive ecological radiation in marine ecosystems. Calanus sinicus dominates continental shelf waters in the northwest Pacific Ocean and plays an important role in the local ecosystem by linking primary production to higher trophic levels. A lack of effective molecular markers has hindered phylogenetic and population genetic studies concerning copepods. As they are genome-level informative, mitochondrial DNA sequences can be used as markers for population genetic studies and phylogenetic studies. Results The mitochondrial genome of C. sinicus is distinct from other arthropods owing to the concurrence of multiple non-coding regions and a reshuffled gene arrangement. Further particularities in the mitogenome of C. sinicus include low A + T-content, symmetrical nucleotide composition between strands, abbreviated stop codons for several PCGs and extended lengths of the genes atp6 and atp8 relative to other copepods. The monophyletic Copepoda should be placed within the Vericrustacea. The close affinity between Cyclopoida and Poecilostomatoida suggests reassigning the latter as subordinate to the former. Monophyly of Maxillopoda is rejected. Within the alignment of 11 C. sinicus mitogenomes, there are 397 variable sites harbouring three 'hotspot' variable sites and three microsatellite loci. Conclusion The occurrence of the circular subgenomic fragment during laboratory assays suggests that special caution should be taken when sequencing mitogenomes using long PCR. Such a phenomenon may provide additional evidence of mitochondrial DNA recombination, which appears to have been a prerequisite for shaping the present mitochondrial profile of C. sinicus during its evolution. The lack of synapomorphic gene arrangements among copepods has cast doubt on the utility of gene order as a useful molecular marker for deep phylogenetic analysis. However, mitochondrial genomic sequences have been valuable markers for resolving phylogenetic issues concerning copepods. The variable site maps of C. sinicus mitogenomes provide a solid foundation for population genetic studies.