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A cruise was conducted in the summer of 2023 from the Pearl River Estuary (PRE) to the adjacent waters of the Xisha Islands in the northern South China Sea (NSCS) to investigate the distribution, community structure, and assembly patterns of eukaryotic and prokaryotic phytoplankton using high-throughput sequencing (HTS) and microscopic observation. Dinophyta were the most abundant phylum in the eukaryotic phytoplankton community based on HTS, accounting for 92.17% of the total amplicon sequence variants (ASVs). Syndiniales was the most abundant order among eukaryotic phytoplankton, whereas Prochlorococcus was the most abundant genus within cyanobacteria. The alpha diversity showed the lowest values in the PRE area and decreased gradually with depth, while cyanobacteria exhibited higher alpha diversity indices in the PRE and at depths ranging from 75 m to 750 m. The morphological results were different from the data based on HTS. Diatoms (37 species) dominated the phytoplankton community, with an average abundance of 3.01 × 104 cells L-1, but only six species of dinoflagellate were observed. Spearman correlation analysis and redundancy analysis (RDA) showed that the distribution and community structure of phytoplankton were largely influenced by geographical location and environmental parameters in the NSCS. The neutral community model (NCM) and null model indicated that deterministic processes played a significant role in the assembly of eukaryotic phytoplankton, with heterogeneous selection and homogeneous selection accounting for 47.27 and 29.95%, respectively. However, stochastic processes (over 60%) dominated the assembly of cyanobacteria and undominated processes accounted for 63.44%. In summary, the formation of eukaryotic phytoplankton was mainly influenced by environmental factors and geographic location, but the assembly of cyanobacteria was shaped by both stochastic processes, which accounted for over 60%, and environmental selection in the NSCS. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Phages are found virtually everywhere, even in extreme environments, and are extremely diverse both in their virion structures and in their genomic content. They are thought to shape the taxonomic and functional composition of microbial communities as well as their stability. A number of studies on laboratory culture and viral metagenomic research provide deeper insights into the abundance, diversity, distribution, and interaction with hosts of phages across a wide range of ecosystems. Although most of these studies focus on easily accessible samples, such as soils, lakes, and shallow oceans, little is known about bathypelagic phages. In this study, through analyzing the 16S rRNA sequencing and viral metagenomic sequencing data of 25 samples collected from five different bathypelagic ecosystems, we detected a high diversity of bacteria and phages, particularly in the cold seep and hydrothermal vent ecosystems, which have stable chemical energy. The relative abundance of phages in these ecosystems was higher than in other three abyssal ecosystems. The low phage/host ratios obtained from host prediction were different from shallow ecosystems and indicated the prevalence of prophages, suggesting the complexity of phage-bacteria interactions in abyssal ecosystems. In the correlation analysis, we revealed several phages-bacteria interaction networks of potential ecological relevance. Our study contributes to a better understanding of the interactions between bathypelagic bacteria and their phages. [ABSTRACT FROM AUTHOR]

Cold seeps on the continental margins are characterized by intense microbial activities that consume a large portion of methane by anaerobic methanotrophic archaea (ANME) through anaerobic oxidation of methane (AOM). Although ANMEs are known to contain unique ether lipids that may have an important function in marine carbon cycling, their full lipidomic profiles and functional distribution in particular cold-seep settings are still poorly characterized. Here, we combined the 16S rRNA gene sequencing and lipidomic approaches to analyze archaeal communities and their lipids in cold seep sediments with distinct methane supplies from the South China Sea. The archaeal community was dominated by ANME-1 in the moderate seepage area with strong methane emission. Low seepage area presented higher archaeal diversity covering Lokiarchaeia, Bathyarchaeia, and Thermoplasmata. A total of 55 core lipids (CLs) and intact polar lipids (IPLs) of archaea were identified, which included glycerol dialkyl glycerol tetraethers (GDGTs), hydroxy-GDGTs (OH-GDGTs), archaeol (AR), hydroxyarchaeol (OH-AR), and dihydroxyarchaeol (2OH-AR). Diverse polar headgroups constituted the archaeal IPLs. High concentrations of dissolved inorganic carbon (DIC) with depleted δ13CDIC and high methane index (MI) values based on both CLs (MICL) and IPLs (MIIPL) indicate that ANMEs were active in the moderate seepage area. The ANME-2 and ANME-3 clades were characterized by enhanced glycosidic and phosphoric diether lipids production, indicating their potential role in coupling carbon and phosphurus cycling in cold seep ecosystems. ANME-1, though representing a smaller proportion of total archaea than ANME-2 and ANME-3 in the low seepage area, showed a positive correlation with MIIPL, indicating a different mechanism contributing to the IPL-GDGT pool. This also suggests that MIIPL could be a sensitive index to trace AOM activities performed by ANME-1. Overall, our study expands the understanding of the archaeal lipid composition in the cold seep and improves the application of MI using intact polar lipids that potentially link to extent ANME activities. [ABSTRACT FROM AUTHOR]

Cold seeps are oasis for the microbes in the deep-sea ecosystems, and various cold seeps are located along the northern slope of the South China Sea (SCS). However, by far most microbial ecological studies were limited to specific cold seep in the SCS, and lack of comparison between different regions. Here, the surface sediments (0–4 cm) from the Site F/Haima cold seeps and the Xisha trough in the SCS were used to elucidate the biogeography of microbial communities, with particular interest in the typical functional groups involved in the anaerobic oxidation of methane (AOM) process. Distinct microbial clusters corresponding to the three sampling regions were formed, and significantly higher gene abundance of functional groups were present in the cold seeps than the trough. This biogeographical distribution could be explained by the geochemical characteristics of sediments, such as total nitrogen (TN), total phosphorus (TP), nitrate (NO3−), total sulfur (TS) and carbon to nitrogen ratios (C/N). Phylogenetic analysis demonstrated that mcrA and pmoA genotypes were closely affiliated with those from wetland and mangroves, where denitrifying anaerobic methane oxidation (DAMO) process frequently occurred; and highly diversified dsrB genotypes were revealed as well. In addition, significantly higher relative abundance of NC10 group was found in the Xisha trough, suggesting that nitrite-dependent DAMO (N-DAMO) process was more important in the hydrate-bearing trough, although its potential ecological contribution to AOM deserves further investigation. Our study also further demonstrated the necessity of combining functional genes and 16S rRNA gene to obtain a comprehensive picture of the population shifts of natural microbial communities among different oceanic regions.

Microbial eukaryotes are key components of the marine food web, but their distribution in deep-sea chemosynthetic ecosystems has not been well studied. Here, high-throughput sequencing of the 18S rRNA gene and network analysis were applied to investigate the diversity, distribution and potential relationships between microbial eukaryotes in samples collected from two cold seeps and one trough in the northern South China Sea. SAR (i.e., Stramenopiles, Alveolata, and Rhizaria) was the predominant group in all the samples, and it was highly affiliated to genotypes with potential symbiotic and parasitic strategies identified from other deep-sea extreme environments (e.g., oxygen deficient zones, bathypelagic waters, and hydrothermal vents). Our findings indicated that specialized lineages of deep-sea microbial eukaryotes exist in chemosynthetic cold seeps, where microbial eukaryotes affiliated with parasitic/symbiotic taxa were prevalent in the community. The biogeographic pattern of the total community was best represented by the intermediate operational taxonomic unit (OTU) category, whose relative abundance ranged 0.01–1% within a sample, and the communities of the two cold seeps were distinct from the trough, which suggests that geographical proximity has no critical impact on the distribution of deep-sea microbial eukaryotes. Overall, this study has laid the foundations for future investigations regarding the ecological function and in situ trophic relationships of microbial eukaryotes in deep-sea ecosystems.

Cold seep ecosystems are developed from methane-rich fluids in organic rich continental slopes, which are the source of various dense microbial and faunal populations. Extensive studies have been conducted on microbial populations in this unique environment; most of them were based on DNA, which could not resolve the activity of extant organisms. In this study, RNA and DNA analyses were performed to evaluate the active archaeal and bacterial communities and their network correlations, particularly those participating in the methane cycle at three sites of newly developed cold seeps in the northern South China Sea (nSCS). The results showed that both archaeal and bacterial communities were significantly different at the RNA and DNA levels, revealing a higher abundance of methane-metabolizing archaea and sulfate-reducing bacteria in RNA sequencing libraries. Site ROV07-01, which exhibited extensive accumulation of deceased Calyptogena clam shells, was highly developed, and showed diverse and active anaerobic archaeal methanotrophs (ANME)-2a/b and sulfate-reducing bacteria from RNA libraries. Site ROV07-02, located near carbonate crusts with few clam shell debris, appeared to be poorly developed, less anaerobic and less active. Site ROV05-02, colonized by living Calyptogena clams, could likely be intermediary between ROV07-01 and ROV07-02, showing abundant ANME-2dI and sulfate-reducing bacteria in RNA libraries. The high-proportions of ANME-2dI, with respect to ANME-2dII in the site ROV07-01 was the first report from nSCS, which could be associated with recently developed cold seeps. Both ANME-2dI and ANME-2a/b showed close networked relationships with sulfate-reducing bacteria; however, they were not associated with the same microbial operational taxonomic units (OTUs). Based on the geochemical gradients and the megafaunal settlements as well as the niche specificities and syntrophic relationships, ANMEs appeared to change in community structure with the evolution of cold seeps, which may be associated with the heterogeneity of their geochemical processes. This study enriched our understanding of more active sulfate-dependent anaerobic oxidation of methane (AOM) in poorly developed and active cold seep sediments by contrasting DNA- and RNA-derived community structure and activity indicators.

Cold seep ecosystems are developed from methane-rich fluids in organic rich continental slopes, which are the source of various dense microbial and faunal populations. Extensive studies have been conducted on microbial populations in this unique environment; most of them were based on DNA, which could not resolve the activity of extant organisms. In this study, RNA and DNA analyses were performed to evaluate the active archaeal and bacterial communities and their network correlations, particularly those participating in the methane cycle at three sites of newly developed cold seeps in the northern South China Sea (nSCS). The results showed that both archaeal and bacterial communities were significantly different at the RNA and DNA levels, revealing a higher abundance of methane-metabolizing archaea and sulfate-reducing bacteria in RNA sequencing libraries. Site ROV07-01, which exhibited extensive accumulation of deceased Calyptogena clam shells, was highly developed, and showed diverse and active anaerobic archaeal methanotrophs (ANME)-2a/b and sulfate-reducing bacteria from RNA libraries. Site ROV07-02, located near carbonate crusts with few clam shell debris, appeared to be poorly developed, less anaerobic and less active. Site ROV05-02, colonized by living Calyptogena clams, could likely be intermediary between ROV07-01 and ROV07-02, showing abundant ANME-2dI and sulfate-reducing bacteria in RNA libraries. The high-proportions of ANME-2dI, with respect to ANME-2dII in the site ROV07-01 was the first report from nSCS, which could be associated with recently developed cold seeps. Both ANME-2dI and ANME-2a/b showed close networked relationships with sulfate-reducing bacteria; however, they were not associated with the same microbial operational taxonomic units (OTUs). Based on the geochemical gradients and the megafaunal settlements as well as the niche specificities and syntrophic relationships, ANMEs appeared to change in community structure with the evolution of cold seeps, which may be associated with the heterogeneity of their geochemical processes. This study enriched our understanding of more active sulfate-dependent anaerobic oxidation of methane (AOM) in poorly developed and active cold seep sediments by contrasting DNA- and RNA-derived community structure and activity indicators. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fu, Liu, Wang, Lian, Cao, Wang, Sun, Wang and Li.)

Cold seeps on the continental margins are characterized by intense microbial activities that consume a large portion of methane by anaerobic methanotrophic archaea (ANME) through anaerobic oxidation of methane (AOM). Although ANMEs are known to contain unique ether lipids that may have an important function in marine carbon cycling, their full lipidomic profiles and functional distribution in particular cold-seep settings are still poorly characterized. Here, we combined the 16S rRNA gene sequencing and lipidomic approaches to analyze archaeal communities and their lipids in cold seep sediments with distinct methane supplies from the South China Sea. The archaeal community was dominated by ANME-1 in the moderate seepage area with strong methane emission. Low seepage area presented higher archaeal diversity covering Lokiarchaeia, Bathyarchaeia, and Thermoplasmata. A total of 55 core lipids (CLs) and intact polar lipids (IPLs) of archaea were identified, which included glycerol dialkyl glycerol tetraethers (GDGTs), hydroxy-GDGTs (OH-GDGTs), archaeol (AR), hydroxyarchaeol (OH-AR), and dihydroxyarchaeol (2OH-AR). Diverse polar headgroups constituted the archaeal IPLs. High concentrations of dissolved inorganic carbon (DIC) with depleted δ 13 C DIC and high methane index (MI) values based on both CLs (MI CL ) and IPLs (MI IPL ) indicate that ANMEs were active in the moderate seepage area. The ANME-2 and ANME-3 clades were characterized by enhanced glycosidic and phosphoric diether lipids production, indicating their potential role in coupling carbon and phosphurus cycling in cold seep ecosystems. ANME-1, though representing a smaller proportion of total archaea than ANME-2 and ANME-3 in the low seepage area, showed a positive correlation with MI IPL , indicating a different mechanism contributing to the IPL-GDGT pool. This also suggests that MI IPL could be a sensitive index to trace AOM activities performed by ANME-1. Overall, our study expands the understanding of the archaeal lipid composition in the cold seep and improves the application of MI using intact polar lipids that potentially link to extent ANME activities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, He, Liang, Zheng, Xiao, Zeng, Zhou, Yao, Chen, Zhu, Zhao, Zheng and Zhang.)

Reducing conditions with elevated sulphide and methane concentrations in ecosystems such as hydrothermal vents, cold seeps or organic falls, are suitable for chemosynthetic primary production. Understanding processes driving bacterial diversity, colonization and dispersal is of prime importance for deep-sea microbial ecology. This study provides a detailed characterization of bacterial assemblages colonizing plant-derived substrates using a standardised approach over a geographic area spanning the North-East Atlantic and Mediterranean. Wood and alfalfa substrates in colonization devices were deployed for different periods at 8 deep-sea chemosynthesis-based sites in 4 distinct geographic areas. Pyrosequencing of a fragment of the 16S rRNA-encoding gene was used to describe bacterial communities. Colonization occurred within the first 14 days. The diversity was higher in samples deployed for more than 289 days. After 289 days, no relation was observed between community richness and deployment duration, suggesting that diversity may have reached saturation sometime in between. Communities in long-term deployments were different, and their composition was mainly influenced by the geographical location where devices were deployed. Numerous sequences related to horizontally-transmitted chemosynthetic symbionts of metazoans were identified. Their potential status as free-living forms of these symbionts was evaluated based on sequence similarity and monophyly with demonstrated symbionts. Results suggest that some free-living forms of metazoan symbionts or their close relatives, such as the epsilonproteobacterium associated with the shrimp Rimicaris exoculata, are efficient colonizers of plant substrates at vents and seeps.

Pockmarks are crater-like depression on the seafloor associated with hydrocarbon ascent through muddy sediments in continental shelves around the world. In this study, we examine the diversity and distribution of benthic microbial communities at shallow-water pockmarks adjacent to the Middle Adriatic Ridge. We integrate microbial diversity data with characterization of local hydrocarbons concentrations and sediment geochemistry. Our results suggest these pockmarks are enriched in sedimentary hydrocarbons, and host a microbial community dominated by Bacteria, even in deeper sediment layers. Pockmark sediments showed higher prokaryotic abundance and biomass than surrounding sediments, potentially due to the increased availability of organic matter and higher concentrations of hydrocarbons linked to pockmark activity. Prokaryotic diversity analyses showed that the microbial communities of these shallowwater pockmarks are unique, and comprised phylotypes associated with the cycling of sulfur and nitrate compounds, as well as numerous know hydrocarbon degraders. Altogether, this study suggests that shallow-water pockmark habitats enhance the diversity of the benthic prokaryotic biosphere by providing specialized environmental niches. [ABSTRACT FROM AUTHOR]

Reducing conditions with elevated sulfide and methane concentrations in ecosystems such as hydrothermal vents, cold seeps or organic falls, are suitable for chemosynthetic primary production. Understanding processes driving bacterial diversity, colonization and dispersal is of prime importance for deep-sea microbial ecology. This study provides a detailed characterization of bacterial assemblages colonizing plant-derived substrates using a standardized approach over a geographic area spanning the North-East Atlantic and Mediterranean. Wood and alfalfa substrates in colonization devices were deployed for different periods at 8 deep-sea chemosynthesis-based sites in four distinct geographic areas. Pyrosequencing of a fragment of the 16S rRNA-encoding gene was used to describe bacterial communities. Colonization occurred within the first 14 days. The diversity was higher in samples deployed for more than 289 days. After 289 days, no relation was observed between community richness and deployment duration, suggesting that diversity may have reached saturation sometime in between. Communities in long-term deployments were different, and their composition was mainly influenced by the geographical location where devices were deployed. Numerous sequences related to horizontally-transmitted chemosynthetic symbionts of metazoans were identified. Their potential status as free-living forms of these symbionts was evaluated based on sequence similarity with demonstrated symbionts. Results suggest that some free-living forms of metazoan symbionts or their close relatives, such as Epsilonproteobacteria associated with the shrimp Rimicaris exoculata, are efficient colonizers of plant substrates at vents and seeps. [ABSTRACT FROM AUTHOR]

Cold seeps are oasis for the microbes in the deep-sea ecosystems, and various cold seeps are located along the northern slope of the South China Sea (SCS). However, by far most microbial ecological studies were limited to specific cold seep in the SCS, and lack of comparison between different regions. Here, the surface sediments (0-4 cm) from the Site F/Haima cold seeps and the Xisha trough in the SCS were used to elucidate the biogeography of microbial communities, with particular interest in the typical functional groups involved in the anaerobic oxidation of methane (AOM) process. Distinct microbial clusters corresponding to the three sampling regions were formed, and significantly higher gene abundance of functional groups were present in the cold seeps than the trough. This biogeographical distribution could be explained by the geochemical characteristics of sediments, such as total nitrogen (TN), total phosphorus (TP), nitrate (NO 3 - ), total sulfur (TS) and carbon to nitrogen ratios (C/N). Phylogenetic analysis demonstrated that mcr A and pmo A genotypes were closely affiliated with those from wetland and mangroves, where denitrifying anaerobic methane oxidation (DAMO) process frequently occurred; and highly diversified dsr B genotypes were revealed as well. In addition, significantly higher relative abundance of NC10 group was found in the Xisha trough, suggesting that nitrite-dependent DAMO (N-DAMO) process was more important in the hydrate-bearing trough, although its potential ecological contribution to AOM deserves further investigation. Our study also further demonstrated the necessity of combining functional genes and 16S rRNA gene to obtain a comprehensive picture of the population shifts of natural microbial communities among different oceanic regions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jiang, Jing, Liu and Du.)

Microbial eukaryotes are key components of the marine food web, but their distribution in deep-sea chemosynthetic ecosystems has not been well studied. Here, high-throughput sequencing of the 18S rRNA gene and network analysis were applied to investigate the diversity, distribution and potential relationships between microbial eukaryotes in samples collected from two cold seeps and one trough in the northern South China Sea. SAR (i.e., Stramenopiles, Alveolata, and Rhizaria) was the predominant group in all the samples, and it was highly affiliated to genotypes with potential symbiotic and parasitic strategies identified from other deep-sea extreme environments (e.g., oxygen deficient zones, bathypelagic waters, and hydrothermal vents). Our findings indicated that specialized lineages of deep-sea microbial eukaryotes exist in chemosynthetic cold seeps, where microbial eukaryotes affiliated with parasitic/symbiotic taxa were prevalent in the community. The biogeographic pattern of the total community was best represented by the intermediate operational taxonomic unit (OTU) category, whose relative abundance ranged 0.01-1% within a sample, and the communities of the two cold seeps were distinct from the trough, which suggests that geographical proximity has no critical impact on the distribution of deep-sea microbial eukaryotes. Overall, this study has laid the foundations for future investigations regarding the ecological function and in situ trophic relationships of microbial eukaryotes in deep-sea ecosystems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Huang, Wang, Liu and Jing.)

Keywords: anaerobic methanotrophs; methane; anaerobic oxidation of methane; cold seeps; calyptogena EN anaerobic methanotrophs methane anaerobic oxidation of methane cold seeps calyptogena N.PAG N.PAG 1 02/15/21 20210211 NES 210211 In the published article, there was an error in affiliation 3. Instead of "Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou, China", it should be "Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, China." Anaerobic methanotrophs, methane, anaerobic oxidation of methane, cold seeps, calyptogena. [Extracted from the article]