Search

Coastal zones are among the world's most productive ecosystems. They store vast amounts of organic carbon, as 'blue carbon' reservoirs, and impact global climate change. Archaeal communities are integral components of coastal microbiomes but their ecological roles are often overlooked. However, archaeal diversity, metabolism, evolution, and interactions, revealed by recent studies using rapidly developing cutting-edge technologies, place archaea as important players in coastal carbon biogeochemical cycling. We here summarize the latest advances in the understanding of archaeal carbon cycling processes in coastal ecosystems, specifically, archaeal involvement in CO

Background: The objectives of this systematic review are to describe and summarize the suicide prevention approaches through offering social support, and to examine the efficacy of these interventions. Methods: Six electronic databses were searched. The inclusion criteria for the summary of intervention approaches were: (a) studies aimed at preventing suicide through approach(es) that offer social support directly; (b) suicide-related outcomes must be presented. The additional inclusion criteria for meta-analysis on the efficacy of these interventions included: (a) suicidal attempt or death was a outcome measure; (b) RCT design; and (c) using social support intervention as the main/only method. Findings: 17,645 articles were identified. In the 51 studies included in stage 1, the approaches were categorized into "Unidirectional" and "Reciprocal" based on the direction of support provided. Unidirectional support intervention were further classified as with and without interaction based on whether there were immediate interaction between support providers and receipients. For suicide attempt during a total of 10,095 person-years, there was a non-significant reduction in overall pooled odds ratio of 0·87 (95% CI: 0·69-1·11). However, the number of suicide during 14,834 person-years was significantly reduced in the intervention group (pooled odds ratio 0·48, 95% CI 0·30-0·77). Further sub-group analyses found that unidirectional interventions with interaction was significantly superior than those without interaction in preventing suicide (p=0·02), but not in preventing suicide attempt (p=0·70). Interpretation: Three types of approaches that provide support of different quality at different costs are identified. We recommend unidirectional intervention with interaction as a suicide prevention strategy for those with elevated suicide risk. Funding Statement: The authors stated: "There was no funding source for this study." Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was undertaken according to the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) guidelines.

Theionarchaea is a recently described archaeal class within the Euryarchaeota. While it is widely distributed in sediment ecosystems, little is known about its metabolic potential and ecological features. Here, we used metagenomics and metatranscriptomics to characterize 12 theionarchaeal metagenome-assembled genomes, which were further divided into two subgroups, from coastal mangrove sediments of China and seawater columns of the Yap Trench. Genomic analysis revealed that apart from the canonical sulfhydrogenase, Theionarchaea harbor genes encoding heliorhodopsin, group 4 [NiFe]-hydrogenase, and flagellin, in which genes for heliorhodopsin and group 4 [NiFe]-hydrogenase were transcribed in mangrove sediment. Further, the theionarchaeal substrate spectrum may be broader than previously reported as revealed by metagenomics and metatranscriptomics, and the potential carbon substrates include detrital proteins, hemicellulose, ethanol, and CO2. The genes for organic substrate metabolism (mainly detrital protein and amino acid metabolism genes) have relatively higher transcripts in the top sediment layers in mangrove wetlands. In addition, co-occurrence analysis suggested that the degradation of these organic compounds by Theionarchaea might be processed in syntrophy with fermenters (e.g., Chloroflexi) and methanogens. Collectively, these observations expand the current knowledge of the metabolic potential of Theionarchaea, and shed light on the metabolic strategies and roles of these archaea in the marine ecosystems.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A–E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

The combination of solution IR spectroscopy measurement, fast cooling crystallization experiments, conformational search, and molecular computation was investigated in detail in this study to explo...

Asgard is a recently discovered superphylum of archaea that appears to include the closest archaeal relatives of eukaryotes1–5. Debate continues as to whether the archaeal ancestor of eukaryotes belongs within the Asgard superphylum or whether this ancestor is a sister group to all other archaea (that is, a two-domain versus a three-domain tree of life)6–8. Here we present a comparative analysis of 162 complete or nearly complete genomes of Asgard archaea, including 75 metagenome-assembled genomes that—to our knowledge—have not previously been reported. Our results substantially expand the phylogenetic diversity of Asgard and lead us to propose six additional phyla that include a deep branch that we have provisionally named Wukongarchaeota. Our phylogenomic analysis does not resolve unequivocally the evolutionary relationship between eukaryotes and Asgard archaea, but instead—depending on the choice of species and conserved genes used to build the phylogeny—supports either the origin of eukaryotes from within Asgard (as a sister group to the expanded Heimdallarchaeota–Wukongarchaeota branch) or a deeper branch for the eukaryote ancestor within archaea. Our comprehensive protein domain analysis using the 162 Asgard genomes results in a major expansion of the set of eukaryotic signature proteins. The Asgard eukaryotic signature proteins show variable phyletic distributions and domain architectures, which is suggestive of dynamic evolution through horizontal gene transfer, gene loss, gene duplication and domain shuffling. The phylogenomics of the Asgard archaea points to the accumulation of the components of the mobile archaeal ‘eukaryome’ in the archaeal ancestor of eukaryotes (within or outside Asgard) through extensive horizontal gene transfer. Comparative analysis of 162 genomes of Asgard archaea results in six newly proposed phyla, including a deep branch that is provisionally named Wukongarchaeota, and sheds light on the evolutionary history of this clade.

Background Previous studies have shown that curcumin derivatives can improve the fatty degeneration of liver tissue that occurs in nonalcoholic fatty liver disease (NAFLD). However, the specific mechanism for that improvement remains unclear. We examined whether the curcumin derivative galangin could reduce the fatty degeneration of liver tissue in mice with NAFLD by inducing autophagy, from the perspective of both prevention and treatment. Methods C57BL/6J mice were randomly assigned to a prevention group (given galangin and a HFD simultaneously) or a treatment group (given galangin after being fed an HFD). The prevention group was treated with galangin (100 mg/kg/d) or an equal volume of normal saline (NS) while being fed an HFD. Some mice were treated with an autophagy inhibitor (3-methyladenine, 3-MA; 30 mg/kg/biwk, i.p.) while being fed an HFD and galangin. HepG2 cells were cultured in DMEM medium containing both free fatty acids and galangin. Results Galangin was found to reduce the fatty degeneration of liver tissue induced by eating an HFD at both the prevention and treatment levels, and that effect might be related to an enhancement of hepatocyte autophagy. Inhibition of autophagy by 3-MA blocked the protective effect of galangin on hepatic steatosis. At the cellular level, galangin reduced lipid accumulation and enhanced the level of hepatocyte autophagy. Conclusion In vitro and in vivo studies showed that galangin cannot only improve pre-existing hepatic steatosis but also prevent the development of stenosis by promoting hepatocyte autophagy.

In recent years, the hadal trenches have been recognized as biological hot spots for deep sea researchers. Due to high hydrostatic pressure, low temperatures, high salinity and low nutrients, the m...

Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.

The 1-cyseine peroxiredoxin (Prx6) is an importantly antioxidant enzyme that protects cells from oxidative damage caused by excessive production of reactive oxygen species (ROS). In this study, we described the molecular characteristics of the noble scallop Chlamys nobilis peroxiredoxin 6 (designed as CnPrx6), immune responses and DNA protection activity of the recombinant protein. The complete ORF (696 bp) of CnPrx6 encoded a polypeptide (25.5 kDa) of 231 amino acids, harboring a conserved peroxidase catalytic center (41PVCTTE46) and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. The deduced amino acid sequence of CnPrx6 shared a relatively high amino acid sequence similarity (more than 50%). The qRT-PCR revealed that the CnPrx6 mRNA was constitutively expressed in all examined tissues, with the highest expression observed in adductor. Upon immunological challenge with Vibrio parahaemolyticus, lipopolysaccharides (LPS) and polyinosinic-polycytidylic acid (Poly I:C), the expression level of CnPrx6 mRNA was significantly up-regulated (P

Metagenomic explorations of the Earth's biosphere enable the discovery of previously unknown bacterial lineages of phylogenetic and ecological significance. Here, we retrieved 11 metagenomic-assembled genomes (MAGs) affiliated to three new monophyletic bacterial lineages from the seawater of the Yap Trench. Phylogenomic analysis revealed that each lineage is a new bacterial candidate phylum, subsequently named Candidatus Qinglongiota, Candidatus Heilongiota, and Candidatus Canglongiota. Metabolic reconstruction of genomes from the three phyla suggested that they adopt a versatile lifestyle, with the potential to utilize various types of sugars, proteins, and/or short-chain fatty acids through anaerobic pathways. This was further confirmed by a global distribution map of the three phyla, indicating a preference for oxygen-limited or particle-attached niches, such as anoxic sedimentary environments. Of note, Candidatus Canglongiota genomes harbor genes for the complete Wood- Ljungdahl pathway and sulfate reduction that are similar to those identified in some sulfate-reducing bacteria. Evolutionary analysis indicated that gene gain and loss events, and horizontal gene transfer (HGT) play important roles in shaping the genomic and metabolic features of the three new phyla. This study presents the genomic insight into the ecology, metabolism, and evolution of three new phyla, which broadens the phylum-level diversity within the domain Bacteria.

Polymorphism occurs in nonsteroidal anti-inflammatory drug flufenamic acid (FFA) and brings about differences in physical and chemical properties among various polymorphs. Form I and form III of FFA were characterized by powder X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. The mole fraction solubility of FFA form I and form III was measured in (water + acetonitrile) and (water + 2-propanol) binary mixed solvents with different solvent compositions using a static method. It was observed that the solubility trends varied in the two mixed solvent systems with an increase in the fraction of the organic solvent. The experimental solubility data were correlated with the solvent composition or/and temperature using the modified Apelblat equation, the Jouyban–Acree model, and the CNIBS/R–K model. It was found that the calculated solubility was consistent with the experimental data; thus, the established model was suitable for both mixed solvent systems and the predicted s...

Aquaculture ecosystem has become a hotspot of antibiotics resistance genes (ARGs) dissemination, owing to the abuse of prophylactic antibiotics. However, it is still unclear how and to what extent ARGs respond to the increasing antibiotic pollution, a trend as expected and as has occurred. Herein, a significant sediment antibiotic pollution gradient was detected along a drainage ditch after decades of shrimp aquaculture. The increasing antibiotic pollution evidently promoted the diversities and tailored the community structures of ARGs, mobile genetic elements (MGEs), virulence factors and pathogens. The profiles of ARGs and MGEs were directly altered by the concentrations of terramycin and sulphadimidine. By contrast, virulence factors were primarily affected by nutrient variables in sediment. The pathogens potentially hosted diverse virulence factors and ARGs. More than half of the detected ARGs subtypes non-linearly responded to increasing antibiotic pollution, as supported by significant tipping points. However, we screened seven antibiotic concentration discriminatory ARGs that could serve as independent variable for quantitatively diagnosing total antibiotic concentration. Co-occurrence analysis depicted that notorious aquaculture pathogens of Vibrio harveyi and V. parahaemolyticus potentially hosted ARGs that confer resistance to multiple antibiotics, while priority pathogens for humankind, e.g., Helicobacter pylori and Staphylococcus aureus, could have harbored redundant virulence factors. Collectively, the significant tipping points and antibiotic concentration-discriminatory ARGs may translate into warning index and diagnostic approach for diagnosing antibiotic pollution. Our findings provided novel insights into the interplay among ARGs, MGEs, pathogens, virulence factors and geochemical variables under the scenario of increasing antibiotic pollution.

Comparative analysis of 162 (nearly) complete genomes of Asgard archaea, including 75 not reported previously, substantially expands the phylogenetic and metabolic diversity of the Asgard superphylum, with six additional phyla proposed. Phylogenetic analysis does not strongly support origin of eukaryotes from within Asgard, leaning instead towards a three-domain topology, with eukaryotes branching outside archaea. Comprehensive protein domain analysis in the 162 Asgard genomes results in a major expansion of the set of eukaryote signature proteins (ESPs). The Asgard ESPs show variable phyletic distributions and domain architectures, suggestive of dynamic evolution via horizontal gene transfer (HGT), gene loss, gene duplication and domain shuffling. The results appear best compatible with the origin of the conserved core of eukaryote genes from an unknown ancestral lineage deep within or outside the extant archaeal diversity. Such hypothetical ancestors would accumulate components of the mobile archaeal ‘eukaryome’ via extensive HGT, eventually, giving rise to eukaryote-like cells.

Nibea, an economical marine fish, is generally fed on trash fish (the low‐value fish), which can cause high feed costs and waste pollutions in high‐density aquaculture. To assess the effect of formulated diet on the gut microbiota in Nibea coibor and Nibea diacanthus, denaturing gradient gel electrophoresis, clone libraries analysis and Illumina sequencing of the 16S ribosomal RNA gene were used in this study. Two Nibea fishes were both dominated by Gammaproteobacteria (especially Photobacterium) and shared a set of gut microbiota, including Bacilli, Mollicutes, Alphaproteobacteria and Fusobacteriia. Statistical analyses revealed that formulated diet led to lower feed conversion ratio (p

Fishes represent the highest diversity of vertebrates; however, our understanding of the compositions and functions of their gut microbiota is limited. In this study, we provided the first insight into the gut microbiota of the herbivorous fish Siganus canaliculatus by using three molecular ecology techniques based on the 16S rRNA genes (denaturing gradient gel electrophoresis, clone library construction, and highthroughput Illumina sequencing), and the Illumina sequencing technique is suggested here due to its higher overall coverage of the total 16S rRNA genes. A core gut microbiota of 29 bacterial groups, covering >99.9% of the total bacterial community, was found to be dominated by Proteobacteria and Firmicutes in fish fed three different diets with/without the supplementation of Ulva pertusa and non-starch polysaccharide (NSP) enzymes (cellulase, xylanase, and β-glucanase). Diverse potential NSP-degrading bacteria and probiotics (e.g., Ruminococcus, Clostridium and Lachnospiraceae) were detected in the intestine of the fish fed U. pertusa, suggesting that these microorganisms likely participated in the degradation of NSPs derived from U. pertusa. This study supports our previous conclusion that U. pertusa-based diets are suitable for the production of S. canaliculatus with lower costs without compromising quality.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 ( Sp -TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp -TRAf6 has highest identity of 88% with Pt -TRAF6 from Portunus trituberculatus , while the similarity of Sp -TRAF6 with other crustacean sequences was 54–55%. RT-PCR analysis indicated that Sp -TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp -TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp -TRAF6. The qRT-PCR results showed that silencing Sp -TRAF6 gene could inhibit Sp ALF1, Sp ALF2, Sp ALF5 and Sp ALF6 expression in hemocytes, while inhibit Sp ALF1, Sp ALF3, Sp ALF4, Sp ALF5 and Sp ALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of Sp ALFs gene expression indicate that Sp -TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain.

Special innate immune mechanisms against pathogens are developed in marine invertebrates such as mud crab, which is also an economically important aquaculture species in many coastal countries. Hemolymph is a critical site in host immune response, but its source of microorganisms is less known. In this study, we provided a detailed investigation of the microorganisms inhabiting various body sites of healthy mud crabs, including hemolymph, midgut, gill, subcuticular epidermis and hepatopancreas. By using fluorescence microscopy and high-throughput sequencing of the bacterial 16S rRNA genes, various abundances and kinds of microorganisms were observed in the healthy mud crabs, of which some are potential pathogens to mud crab and human. The SourceTracker analysis and oral injection experiment confirm the hypothesis that hemolymph microorganisms are derived from the digestive systems of invertebrates with open circulatory systems, indicating that these microorganisms play vital roles in crab immune response. Moreover, physiological differences (gut length), behavioral characteristics (foraging behavior), diet preferences (herbivory), and/or sex hormones (testosterone) possibly determine the unique features of the crab-associated microbiota for both sexes. These findings also contribute to the development of appropriate microbial immunoenhancers, which has potential applications for improving quality and yield during crab aquaculture.

Many marine bivalves are regarded as healthy foods due to their high carotenoid content. Only plants and microorganisms have natural carotenoids biosynthesis ability, hence, animals such as bivalves must obtain carotenoids from their diets. Due to the filter-feeding behavior of bivalves, they have high diversity of gut microbes. However, the relationship between gut microbes and carotenoids has not been explored in mollusks. In the present study, the interaction between gut microbes and carotenoids in two polymorphic noble scallop Chlamys nobilis, golden scallops (designated GG) and brown scallops (designated BW), were studied. The gut of GG and BW showed statistically different bacteria communities. Results from 16S rRNA gene sequencing and qPCR analysis revealed that the gut of GG had significantly higher relative abundance of carotenoids-producing bacteria Brevundimonas, compared with BW. Moreover, HPLC-MS analysis showed that isolate Brevundimonas could produce astaxanthin. The current findings are very useful as they could form the basis for future studies in determining the relationship between gut microbiota and carotenoids absorption in bivalves.

Previous studies have shown that curcumin derivatives can improve the fatty degeneration of liver tissue that occurs in nonalcoholic fatty liver disease (NAFLD). However, the specific mechanism for that improvement remains unclear. We examined whether the curcumin derivative galangin could reduce the fatty degeneration of liver tissue in mice with NAFLD by inducing autophagy, from the perspective of both prevention and treatment.C57BL/6J mice were randomly assigned to a prevention group (given galangin and a HFD simultaneously) or a treatment group (given galangin after being fed an HFD). The prevention group was treated with galangin (100 mg/kg/d) or an equal volume of normal saline (NS) while being fed an HFD. Some mice were treated with an autophagy inhibitor (3-methyladenine, 3-MA; 30 mg/kg/biwk, i.p.) while being fed an HFD and galangin. HepG2 cells were cultured in DMEM medium containing both free fatty acids and galangin.Galangin was found to reduce the fatty degeneration of liver tissue induced by eating an HFD at both the prevention and treatment levels, and that effect might be related to an enhancement of hepatocyte autophagy. Inhibition of autophagy by 3-MA blocked the protective effect of galangin on hepatic steatosis. At the cellular level, galangin reduced lipid accumulation and enhanced the level of hepatocyte autophagy.In vitro and in vivo studies showed that galangin cannot only improve pre-existing hepatic steatosis but also prevent the development of stenosis by promoting hepatocyte autophagy.

Marine bacteria produce many bioactive compounds, including carotenoids. However, the quality of bacterium carotenoids is relatively unknown. Therefore, in this study, a novel carotenoids-producing bacterium Brevundimonas scallop Zheng & Liu was isolated from Chlamys nobilis. The genome of the isolate was analyzed, carotenoid compounds were screened using HPLC-MS and the carotenoid production in B. scallop was monitored. The results revealed that the genome of B. scallop contained a carotenoid synthesis gene cluster, which involved in astaxanthin and hydroxy-astaxanthin biosynthesis. The 2,2′-dihydroxy-astaxanthin was the major carotenoid produced by B. scallop. The optimum culture condition for the highest carotenoids production (1303.62 ± 61.06 µg/g dry cells) for B. scallop was at temperature and salinity of 20 °C and 3% salt, respectively, in 10 g/L glucose as carbon source. The results showed the B. scallop is a new carotenoids resource in marine bivalve, which has an excellent antioxidative activity and potential industrial use.

Hadal biosphere represents the deepest part of the ocean with water depth >6,000 m. Accumulating evidence suggests the existence of unique microbial communities dominated by heterotrophic processes in this environment. However, investigations of the microbial diversity and their metabolic potentials are limited because of technical constraints for sample collection. Here, we provide a detailed metagenomic analysis of three seawater samples at water depths 5,000–6,000 m below sea level (mbsl) and three surface sediment samples at water depths 4,435–6,578 mbsl at the Yap Trench of the western Pacific. Distinct microbial community compositions were observed with the dominance of Gammaproteobacteria in seawater and Thaumarchaeota in surface sediment. Comparative analysis of the genes involved in carbon, nitrogen and sulfur metabolisms revealed that heterotrophic processes (i.e., degradation of carbohydrates, hydrocarbons, and aromatics) are the most common microbial metabolisms in the seawater, while chemolithoautotrophic metabolisms such as ammonia oxidation with the HP/HB cycle for CO2 fixation probably dominated the surface sediment communities of the Yap Trench. Furthermore, abundant genes involved in stress response and metal resistance were both detected in the seawater and sediments, thus the enrichment of metal resistance genes is further hypothesized to be characteristic of the hadal microbial communities. Overall, this study sheds light on the metabolic versatility of microorganisms in the Yap Trench, their roles in carbon, nitrogen, and sulfur biogeochemical cycles, and how they have adapted to this unique hadal environment.

Symbiotic microorganisms have been found in the hemolymph (blood) of many aquatic invertebrates, such as crabs, shrimp, and oysters. Hemolymph is a critical site in the host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies using selective media (e.g., thiosulfate-citrate-bile salts-sucrose [TCBS], 2216E, and LB) for enumerating and isolating microbial cells. However, doubts remain about the “true” representation of the microbial abundance and diversity of symbiotic microorganisms in hemolymph, particularly for uncultivable microorganisms, which are believed to be more abundant than the cultured microorganisms. To explore this, we developed a culture-independent cell extraction method for separating microbial cells from the hemolymph of three aquatic invertebrates ( Scylla paramamosain [mud crab], Litopenaeus vannamei [whiteleg shrimp], and Crassostrea angulata [Portuguese oysters]) involving filtration through a 5-μm-pore-size mesh filter membrane (the filtration method). A combination of the filtration method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. More than 2.6 × 10 4 cells/ml of microbial cells dominated by Escherichia-Shigella and Halomonas , Photobacterium and Escherichia-Shigella , and Pseudoalteromonas and Arcobacter were detected in the hemolymph of Scylla paramamosain , Litopenaeus vannamei , and Crassostrea angulata , respectively. A parallel study for investigating the hemolymph microbiomes by comparing the filtration method and a culture-dependent method (the plate count method) showed significantly higher microbial abundances (between 26- and 369-fold difference; P < 0.05) and less biased community structures of the filtration method than those of the plate count method. Furthermore, hemolymph of the three invertebrates harbored many potential pathogens, including Photobacterium , Arcobacter , and Vibrio species. Finally, the filtration method provides a solution that improves the understanding of the metabolic functions of uncultivable hemolymph microorganisms (e.g., metagenomics) devoid of host hemocyte contamination. IMPORTANCE Microorganisms are found in the hemolymph of invertebrates, a critical site in the host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies. However, doubts remain about the “true” representation of the hemolymph microbiome. This study developed a culture-independent cell extraction method that could separate microbial cells from the hemolymph of three aquatic invertebrates ( S. paramamosain , L. vannamei , and C. angulata ) based on filtration through a 5-μm-pore-size mesh filter membrane (the filtration method). A combination of the filtration method with fluorescence microscopy and a high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. Our results demonstrate that the hemolymph of aquatic invertebrates harbors a much higher microbial abundance and more distinct microbial community composition than previously estimated. Furthermore, this work provides a less biased solution for studying the metabolic functions of uncultivable hemolymph microbiota devoid of host hemocyte contamination.

As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, β-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca2+-dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d -fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain.

Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high-biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment.