Identification, functional characterization and expression pattern of interferon-gamma (IFN-γ) and interferon-gamma receptor 1 (IFNGR1) in Nibea albiflora.

Interferon-gamma (IFN-γ) is an inflammatory cytokine that plays a crucial role in regulating both innate and cell-mediated immune responses by binding to a receptor complex made up of IFNGR1 and IFNGR2. In this study, the complete cDNA of IFN-γ and IFNGR1 from Nibea albiflora were cloned and functionally characterized (named NaIFN-γ and NaIFNGR1), whose complete cDNA sequences were 1593 bp and 2792 bp, encoding 201 and 399 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that the concluded amino acids sequences of NaIFN-γ and NaIFNGR1 shared high identity with their teleost orthologues including the IFN-γ signature and nuclear localization signal (NLS) motif in NaIFN-γ and FN Ⅲ domain in NaIFNGR1. Real-time PCR showed that NaIFN-γ and NaIFNGR1 constitutively expressed in all tested tissues, such as the head-kidney, spleen, liver, kidney, gill, muscle, blood, and intestine with the highest expression of NaIFN-γ and NaIFNGR1 appearing in the liver and gill, respectively. After experiencing stimulation with Polyinosinic-polycytidylic acid (Poly (I:C)), Vibrio alginolyticus (V. alginolyticus) or Vibrio parahaemolyticus (V. parahaemolyticus), NaIFN-γ and NaIFNGR1 mRNA were up-regulated with the time-dependent model. Due to the presence of a nuclear localization signal (NLS), the subcellular localization revealed that NaIFN-γ dispersed throughout the cytoplasm and nucleus. NaIFNGR1, as a member of Cytokine receptor family B, was primarily expressed on the cell membrane. When NaIFN-γ and NaIFNGR1 were co-transfected, their fluorescence signals overlapped on the membrane of HEK 293T cells indicating the potential interaction between IFN-γ and IFNGR1. The GST-pull-down results further showed that NaIFN-γ could directly interact with the extracellular region of NaIFNGR1, further confirming the affinity between IFN-γ and IFNGR1. Taken together, the results firstly demonstrated that the NaIFN-γ ligand-receptor system existed in N.albiflora and played a pivotal part in N.albiflora's immune response against pathogenic bacterial infections, which contributed to the better understanding of the role of IFN-γ in the immunomodulatory mechanisms of teleost.

Identification, functional characterization and immune response profiles of interleukin-10 in Nibea albiflora.

Interleukin-10 (IL-10) is an immunosuppressive cytokine, which plays a vital role in regulating inflammation for inhibiting the generation and function of pro-inflammatory cytokines in vivo or in vitro. In the present study, the full length cDNA of IL-10 was characterized from Nibea albiflora (named as NaIL-10) of 1238 base pairs (bp), containing a 5'-UTR (untranslated region) of 350 bp, a 3'-UTR of 333 bp and an open reading frame (ORF) of 555 bp (Fig. 1A) to encode 184 amino acid residues with a signal peptide at the N-terminus. The sequence analysis showed that NaIL-10 possessed the typical IL-10 family symbolic motif and conversed cysteine residues, similar to its teleost orthologues. Real-time PCR indicated that NaIL-10 had wide distribution in different healthy tissues, with a relatively high expression in immune-related tissues (head kidney, spleen, kidney, liver and gill). Significantly, up-regulations of NaIL-10 after infection against Vibrio parahaemolyticus, Vibrio alginolyticus and Poly I:C were also observed. Subcellular localization manifested that NaIL-10 mainly distributed in the cytoplasm unevenly and aggregately, and there was also a small amount on the cell membrane, indicating that NaIL-10 was secreted to the extracellular space as the known IL-10 homologous molecules. It could co-locate with IL-10 Rα on the membrane of HEK293T cells for their potential interaction, and GST pull-down and Co-IP studies certified the specific and direct interaction between NaIL-10 and NaIL-10 Rα, confirming that an IL-10 ligand-receptor system existed in N.albiflora. The expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, were dramatically inhibited in LPS-stimulated RAW264.7 macrophages pre-incubated with recombinant NaIL-10 protein, demonstrating its anti-inflammatory roles. Taken together, the results demonstrated the existence of IL-10 ligand-receptor system in N.albiflora for the first time, and indicated the suppressive function of NaIL-10 on pro-inflammatory cytokine expression in inflammatory response, which would be conducive to better comprehending the role of IL-10 in the immunomodulatory mechanisms of teleost.

Comparative Transcriptome Analyses Provide New Insights into the Evolution of Divergent Thermal Resistance in Two Eel Gobies.

Adaptation to thermal conditions in tidal mudflats always involves tolerating frequent fluctuations and often extreme environmental temperatures. Regulation of gene expression plays a fundamental role in the evolution of these thermal adaptations. To identify the key gene regulatory networks associated with the thermal adaptation, we investigated the capability of cold tolerance, as well as the transcriptomic changes under cold stress in two mudflat inhabitants (Odontamblyopus lacepedii and O. rebecca) with contrasting latitude affinity. Our results revealed a remarkable divergent capacity of cold tolerance (CTmin: 0.61 °C vs. 9.57 °C) between the two gobies. Analysis of transcriptomic changes under cold stress unveiled 193 differentially expressed genes exhibiting similar expression profiles across all tissues and species, including several classic metabolic and circadian rhythm molecules such as ACOD and CIART that may represent the core cold response machinery in eel gobies. Meanwhile, some genes show a unique expression spectrum in the more cold-tolerant O. lacepedii suggesting their roles in the enhanced cold tolerance and hence the extreme thermal adaptations. In addition, a weighted gene co-expression network analysis (WGCNA) revealed a subset of metabolic hub genes including MYH11 and LIPT2 showing distinct down-regulation in O. lacepedii when exposed to cold stress which highlights the role of reduced energy consumption in the enhanced cold tolerance of eel gobies. These findings not only provide new insights into how mudflat teleosts could cope with cold stress and their potential evolutionary strategies for adapting to their thermal environment, but also have important implications for sound management and conservation of their fishery resources in a scenario of global climate warming in the marine realm.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

Chromosome-level genome assembly and population genomic analysis reveal evolution and local adaptation in common hairfin anchovy (Setipinna tenuifilis).

Understanding the genetic structure and the factors associated with adaptive diversity has significant implications for the effective management of wild populations under threat from overfishing and climate change. The common hairfin anchovy (Setipinna tenuifilis) is an economically and ecologically important pelagic fish species, spanning a broad latitudinal gradient along marginal seas of the Northwest Pacific. In this study, we constructed the first reference genome of S. tenuifilis using PacBio long reads and high-resolution chromosome conformation capture (Hi-C) technology. The assembled genome was 798.38 Mb with a contig N50 of 1.43 Mb and a scaffold N50 of 32.42 Mb, which were anchored onto 24 pseudochromosomes. A total of 22,019 genes were functionally annotated, which accounted for 95.27% of the predicted protein-coding genes. Chromosomal collinearity analysis revealed chromosome fusion or fission events in Clupeiformes species. Three genetic groups of S. tenuifilis were revealed along the Chinese coast using restriction site-associated DNA sequencing (RADseq). We investigated the influence of four bioclimatic variables as potential drivers of adaptive divergence in S. tenuifilis, suggesting that these environmental variables, especially sea surface temperature, may play important roles as drivers of spatially varying selection for S. tenuifilis. We also identified candidate functional genes underlying adaptive mechanisms and ecological tradeoffs using redundancy analysis (RDA) and BayeScan analysis. In summary, this study sheds light on the evolution and spatial patterns of genetic variation of S. tenuifilis, providing a valuable genomic resource for further biological and genetic studies on this species and other closely related Clupeiformes.

Lentibacillus daqui sp. nov., isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS110521T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu and was characterised by polyphasic taxonomy. This novel isolate grew in the presence of 0-20 % (w/v) NaCl, at pH 6.0-9.0 and 20-50 °C; optimum growth was observed with 8-10 % (w/v) NaCl, at pH 7.0 and 37 °C. A comparative analysis of the 16S rRNA gene sequence (1460 bp) of ZS110521T revealed that it displayed the highest similarity to Lentibacillus populi WD4L-1T (95.5 %), followed by Lentibacillus garicola SL-MJ1T (95.4 %) and Lentibacillus lacisalsi BH260T (95.2 %). ANI and dDDH values between ZS110521T and other strains of species of the genus Lentibacillus were less than 78 and 28 %, respectively. The predominant cellular fatty acids (> 10 %) of ZS110521T were anteiso-C17 : 0 (37.8 %), anteiso-C15 : 0 (28.1 %) and iso-C16 : 0 (15.5 %). The respiratory quinone was identified as menaquinone-7 (MK-7) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The polyphasic taxonomic data and the results of chemotaxonomic analysis confirmed that ZS110521T represents a novel species, for which the name Lentibacillus daqui sp. nov. is proposed. The type strain of this proposed species is ZS110521T (=CGMCC 1.19456T =JCM 35213T).

Solibacillus daqui sp. nov., isolated from high-temperature Daqu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS111008T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu, and was characterized by polyphasic taxonomy. This novel isolate grew in the presence of 0-5 % (w/v) NaCl, at pH 6.0-9.0 and 25-45 °C; optimum growth was observed with 1 % (w/v) NaCl, at pH 8.0 and 30 °C. A comparative analysis of the 16S rRNA gene sequence (1461 bp) of strain ZS111008T showed highest similarity to Solibacillus silvestris DSM12223T (96.7%), followed by Solibacillus cecembensis PN5T (96.6%) and Solibacillus isronensis AMCK01000046 (96.5%). The DNA G+C content of strain ZS111008T was 37.21 mol%. The respiratory quinone was identified as menaquinone-7 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and one unknown phospholipid. Lys was detected as the diagnostic diamino acid in the cell wall. Based on morphological characteristics, chemotaxonomic characteristics and physiological properties, strain ZS111008T represents a novel species of the genus Solibacillus, for which the name Solibacillus daqui sp. nov. is proposed. The type strain for this proposed species is ZS111008T (=CGMCC 1.19455T=JCM 35214T).

Molecular cloning, expression analysis and immune-related functional identification of tumor necrosis factor alpha (TNFα) in Sepiella japonica under bacteria stress.

Tumor necrosis factor α (TNFα), a cytokine mainly secreted by active macrophages and monocytes, causes hemorrhagic necrosis of tumor tissues, kills tumor cells, regulates inflammatory responses, and plays a crucial role in innate immunity. In this study, TNFα of Sepiella japonica (named as SjTNFα) was acquired, whose full-length cDNA was 1206 bp (GenBank accession no. ON357428), containing a 5' UTR of 185 bp, a 3' UTR of 137 bp and an open reading frame (ORF) of 1002bp to encode a putative peptide of 333 amino acids for constructing the transmembrane domain and the cytoplasmic TNF domain. Its predicted pI was 8.69 and the theoretical molecular weight was 44.72 KDa. Multiple sequence alignment and phylogenetic analysis showed that SjTNFα had the highest homology to Octopus sinensis, they fell into a unified branch and further clustered with other animals. Real-time PCR indicated that SjTNFα was widely expressed in all subject tissues, including spleen, pancreas, gill, heart, brain, optic lobe, liver and intestine, and exhibited the highest in the liver and the lowest in the brain. The relative expression of SjTNFα varied at the developmental period of juvenile stage, pre-spawning and oviposition in the squid, with the highest in the liver at the juvenile stage and oviposition, and in the optic lobe of pre-spawning. After being infected with Vibrio parahaemolyticus and Aeromonas hydrophila, the expression of SjTNFα in liver and gill were both upregulated with time, and the highest expression appeared at 24 h and 8 h in liver for different infection, and at 4 h in gill consistently. Cell localization showed that SjTNFα distributed on membrane of HEK293 cells because it was a type II soluble transmembrane protein. When HEK293 cells were stimulated with LPS of different concentrations, the NF-κB pathway was activated in the nucleus and the corresponding mRNA was transferred through the intracellular signal transduction pathway, resulting in the synthesis and release of TNFα, which made the expression of SjTNFα was up-regulated obviously. These findings showed that SjTNFα might play an essential role in the defense of S. japonica against bacteria challenge, which contributed to the understanding of the intrinsic immune signaling pathway of Cephalopoda and the further study of host-pathogen interactions.

Genome Sequencing Provides Novel Insights into Mudflat Burrowing Adaptations in Eel Goby Taenioides sp. (Teleost: Amblyopinae).

Amblyopinae is one of the lineage of bony fish that preserves amphibious traits living in tidal mudflat habitats. In contrast to other active amphibious fish, Amblyopinae species adopt a seemly more passive lifestyle by living in deep burrows of mudflat to circumvent the typical negative effects associated with terrestriality. However, little is known about the genetic origin of these mudflat deep-burrowing adaptations in Amblyopinae. Here we sequenced the first genome of Amblyopinae species, Taenioides sp., to elucidate their mudflat deep-burrowing adaptations. Our results revealed an assembled genome size of 774.06 Mb with 23 pseudochromosomes anchored, which predicted 22,399 protein-coding genes. Phylogenetic analyses indicated that Taenioides sp. diverged from the active amphibious fish of mudskipper approximately 28.3 Ma ago. In addition, 185 and 977 putative gene families were identified to be under expansion, contraction and 172 genes were undergone positive selection in Taenioides sp., respectively. Enrichment categories of top candidate genes under significant expansion and selection were mainly associated with hematopoiesis or angiogenesis, DNA repairs and the immune response, possibly suggesting their involvement in the adaptation to the hypoxia and diverse pathogens typically observed in mudflat burrowing environments. Some carbohydrate/lipid metabolism, and insulin signaling genes were also remarkably alterated, illustrating physiological remolding associated with nutrient-limited subterranean environments. Interestingly, several genes related to visual perception (e.g., crystallins) have undergone apparent gene losses, pointing to their role in the small vestigial eyes development in Taenioides sp. Our work provide valuable resources for understanding the molecular mechanisms underlying mudflat deep-burrowing adaptations in Amblyopinae, as well as in other tidal burrowing teleosts.

Amblyopinae Mitogenomes Provide Novel Insights into the Paraphyletic Origin of Their Adaptation to Mudflat Habitats.

The water-to-land transition is one of the most important events in evolutionary history of vertebrates. However, the genetic basis underlying many of the adaptations during this transition remains unclear. Mud-dwelling gobies in the subfamily Amblyopinae are one of the teleosts lineages that show terrestriality and provide a useful system for clarifying the genetic changes underlying adaptations to terrestrial life. Here, we sequenced the mitogenome of six species in the subfamily Amblyopinae. Our results revealed a paraphyletic origin of Amblyopinae with respect to Oxudercinae, which are the most terrestrial fishes and lead an amphibious life in mudflats. This partly explains the terrestriality of Amblyopinae. We also detected unique tandemly repeated sequences in the mitochondrial control region in Amblyopinae, as well as in Oxudercinae, which mitigate oxidative DNA damage stemming from terrestrial environmental stress. Several genes, such as ND2, ND4, ND6 and COIII, have experienced positive selection, suggesting their important roles in enhancing the efficiency of ATP production to cope with the increased energy requirements for life in terrestrial environments. These results strongly suggest that the adaptive evolution of mitochondrial genes has played a key role in terrestrial adaptions in Amblyopinae, as well as in Oxudercinae, and provide new insights into the molecular mechanisms underlying the water-to-land transition in vertebrates.

Comparative Transcriptomic Analyses Propose the Molecular Regulatory Mechanisms Underlying 1,8-Cineole from Cinnamomum kanehirae Hay and Promote the Asexual Sporulation of Antrodia cinnamomea in Submerged Fermentation.

Antrodia cinnamomea is a valuable edible and medicinal mushroom with antitumor, hepatoprotective, and antiviral effects that play a role in intestinal flora regulation. Spore-inoculation submerged fermentation has become the most efficient and well-known artificial culture process for A. cinnamomea. In this study, a specific low-molecular compound named 1,8-cineole (cineole) from Cinnamomum kanehirae Hay was first reported to have remarkably promoted the asexual sporulation of A. cinnamomea in submerged fermentation (AcSmF). Then, RNA sequencing, real-time quantitative PCR, and a literature review were performed to predict the molecular regulatory mechanisms underlying the cineole-promoted sporulation of AcSmF. The available evidence supports the hypothesis that after receiving the signal of cineole through cell receptors Wsc1 and Mid2, Pkc1 promoted the expression levels of rlm1 and wetA and facilitated their transfer to the cell wall integrity (CWI) signal pathway, and wetA in turn promoted the sporulation of AcSmF. Moreover, cineole changed the membrane functional state of the A. cinnamomea cell and thus activated the heat stress response by the CWI pathway. Then, heat shock protein 90 and its chaperone Cdc37 promoted the expression of stuA and brlA, thus promoting sporulation of AcSmF. In addition, cineole promoted the expression of areA, flbA, and flbD through the transcription factor NCP1 and inhibited the expression of pkaA through the ammonium permease of MEP, finally promoting the sporulation of AcSmF. This study may improve the efficiency of the inoculum (spores) preparation of AcSmF and thereby enhance the production benefits of A. cinnamomea.

Characterization of Insulin-like Peptide (ILP) and Its Potential Role in Ovarian Development of the Cuttlefish Sepiella japonica.

The insulin-like peptide (ILP) family is well known for regulating reproduction in invertebrates, while its role in mollusks remains largely unknown. In this study, we first isolated and characterized the ILP gene in the cuttlefish Sepiella japonica. The full-length SjILP cDNA obtained was 926 bp and encoded a precursor protein of 161 amino acids. The precursor protein consisted of a signal peptide, a B chain, a C-peptide, and an A chain. It possessed the typical features of ILP proteins, including two cleavage sites (KR) and eight conserved cysteines. To define the function of SjILP, the expression of SjILP in different tissues and ovarian development stages were analyzed using qRT-PCR. SjILP was mainly expressed in the ovary, and its gene expression correlated with ovarian development. Furthermore, silencing SjILP using RNA interference (RNAi) dramatically decreased the expression levels of four ovarian-development-related genes (vitellogenin1, vitellogenin2, cathepsin L1-like, and follistatin). These data suggest the critical role of SjILP in the regulation of ovarian development in S. japonica.

Constructing a Defined Starter for Multispecies Vinegar Fermentation via Evaluation of the Vitality and Dominance of Functional Microbes in an Autochthonous Starter.

Mature vinegar culture has usually been used as a type of autochthonous starter to rapidly initiate the next batch of acetic acid fermentation (AAF) and maintain the batch-to-batch uniformity of AAF in the production of traditional cereal vinegar. However, the vitality and dominance of functional microbes in autochthonous starters remain unclear, which hinders further improvement of fermentation yield and production. Here, based on metagenomic (MG), metatranscriptomic (MT), and 16S rRNA gene sequencings, 11 bacterial operational taxonomic units (OTUs) with significant metabolic activity (MT/MG ratio >1) and dominance (relative abundance >1%) were targeted in the autochthonous vinegar starter, all of which were assigned to 4 species (Acetobacter pasteurianus, Lactobacillus acetotolerans, L. helveticus, Acetilactobacillus jinshanensis). Then, we evaluated the successions and interactions of these 11 bacterial OTUs at different AAF stages. Last, a defined starter was constructed with 4 core species isolated from the autochthonous starter (A. pasteurianus, L. acetotolerans, L. helveticus, Ac. jinshanensis). The defined starter culture could rapidly initiate the AAF in a sterile or unsterilized environment, and similar dynamics of metabolites (ethanol, titratable acidity, acetic acid, lactic acid, and volatile compounds) and environmental indexes (temperature, pH) of fermentation were observed as compared with that of autochthonous starter (P > 0.05). This work provides a method to construct a defined microbiota from a complex system while preserving its metabolic function. IMPORTANCE Complex microorganisms are beneficial to the flavor formation in natural food fermentation, but they also pose challenges to the mass production of standardized products. It is attractive to construct a defined starter to rapidly initiate fermentation process and significantly improve fermentation yield. This study provides a comprehensive understanding of vital and dominant species in the autochthonous vinegar starter via multi-omics, and designs a defined microbial community for the efficient fermentation of cereal vinegar.

Metabolite-Based Mutualistic Interaction between Two Novel Clostridial Species from Pit Mud Enhances Butyrate and Caproate Production.

Pit mud microbial consortia play crucial roles in the formation of Chinese strong-flavor baijiu's key flavor-active compounds, especially butyric and caproic acids. Clostridia, one of the abundant bacterial groups in pit mud, were recognized as important butyric and caproic acid producers. Research on the interactions of the pit mud microbial community mainly depends on correlation analysis at present. Interaction between Clostridium and other microorganisms and its involvement in short/medium-chain fatty acid (S/MCFA) metabolism are still unclear. We previously found coculture of two clostridial strains isolated from pit mud, Clostridium fermenticellae JN500901 (C.901) and Novisyntrophococcus fermenticellae JN500902 (N.902), could enhance S/MCFA accumulation. Here, we investigated their underlying interaction mechanism through the combined analysis of phenotype, genome, and transcriptome. Compared to monocultures, coculture of C.901 and N.902 obviously promoted their growth, including shortening the growth lag phase and increasing biomass, and the accumulation of butyric acid and caproic acid. The slight effects of inoculation ratio and continuous passage on the growth and metabolism of coculture indicated the relative stability of their interaction. Transwell coculture and transcriptome analysis showed the interaction between C.901 and N.902 was accomplished by metabolite exchange, i.e., formic acid produced by C.901 activated the Wood-Ljungdahl pathway of N.902, thereby enhancing its production of acetic acid, which was further converted to butyric acid and caproic acid by C.901 through reverse ß-oxidation. This work demonstrates the potential roles of mutually beneficial interspecies interactions in the accumulation of key flavor compounds in pit mud. IMPORTANCE Microbial interactions played crucial roles in influencing the assembly, stability, and function of the microbial community. The metabolites of pit mud microbiota are the key to flavor formation of Chinese strong-flavor baijiu. So far, researches on the interactions of the pit mud microbial community have been mainly based on the correlation analysis of sequencing data, and more work needs to be performed to unveil the complicated interaction patterns. Here, we identified a material exchange-based mutualistic interaction system involving two fatty acid-producing clostridial strains (Clostridium fermenticellae JN500901 and Novisyntrophococcus fermenticellae JN500902) isolated from pit mud and systematically elucidated their interaction mechanism for promoting the production of butyric acid and caproic acid, the key flavor-active compounds of baijiu. Our findings provide a new perspective for understanding the complicated interactions of pit mud microorganisms.

Insulin-like Peptide Receptor (ILPR) in the Cuttlefish Sepiella japonica: Characterization, Expression, and Regulation of Reproduction.

Insulin-like peptide receptor (ILPR) can effectively regulate ovarian development in invertebrates, but its effect in cuttlefish has not been reported. We isolated and characterized a ILPR gene from Sepiella japonica, referred to as SjILPR. This gene displayed significant homologies to Octopus bimaculoides ILPR, and contained all typical features of insulin receptors and tyrosine kinase domain structure. SjILPR is expressed in all detected tissues, with the highest expression in the ovary. During ovarian development stages, its expression levels in the ovary, pancreas, and liver were correlated to the female reproductive cycle. After the silencing of SjILPR in vivo, comparative transcriptome analysis identified 4314 differentially expressed genes (DEGs) in the injected group, including 2586 down-regulated genes and 1728 up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that 832 DEGs were assigned to 222 pathways, many pathways of which were related to gonadal development. Four down-regulated genes relevant to ovarian development (Vitellogenin 1, Vitellogenin 2, Cathepsin L1-like, and Follistatin) were selected to confirm the accuracy of RNA-seq data by qRT-PCR. These results showed that SjILPR might regulate ovarian development to control reproduction by affecting the expression of the relevant genes in female S. japonica.

Mining the Factors Driving the Evolution of the Pit Mud Microbiome under the Impact of Long-Term Production of Strong-Flavor Baijiu.

The mud cellar creates a unique microenvironment for the fermentation of strong-flavor baijiu (SFB). Recent research and long-term practice have highlighted the key roles of microbes inhabiting pit mud in the formation of SFB's characteristic flavor. A positive correlation between the quality of SFB and cellar age was extracted from practice; however, the evolutionary patterns of pit mud microbiome and driving factors remain unclear. Here, based on the variation regularity analysis of microbial community structure and metabolites of samples from cellars of different ages (∼30/100/300 years), we further investigated the effects of lactate and acetate (main microbial metabolites in fermented grains) on modulating the pit mud microbiome. Esters (50.3% to 64.5%) dominated the volatile compounds identified in pit mud, and contents of the four typical acids (lactate, hexanoate, acetate, and butyrate) increased with cellar age. Bacteria (9.5 to 10.4 log10 [lg] copies/g) and archaea (8.3 to 9.1 lg copies/g) mainly constituted pit mud microbiota, respectively dominated by Clostridia (39.7% to 81.2%) and Methanomicrobia (32.8% to 92.9%). An upward trend with cellar age characterized the relative and absolute abundance of the most predominant bacterial and archaeal genera, Caproiciproducens and Methanosarcina. Correlation analysis revealed significantly (P < 0.05) positive relationships between the two genera and major metabolites. Anaerobic fermentation with acetate and lactate as carbon sources enhanced the enrichment of Clostridia, and furthermore, the relative abundance of Caproiciproducens (40.9%) significantly increased after 15-day fed-batch fermentation with lactate compared with the initial pit mud (0.22%). This work presents a directional evolutionary pattern of pit mud microbial consortia and provides an alternative way to accelerate the enrichment of functional microbes. IMPORTANCE The solid-state anaerobic fermentation in a mud cellar is the most typical feature of strong-flavor baijiu (SFB). Metabolites produced by microbes inhabiting pit mud are crucial to create the unique flavor of SFB. Accordingly, craftspeople have always highlighted the importance of the pit mud microbiome and concluded by centuries of practice that the production rate of high-quality baijiu increases with cellar age. To deepen the understanding of the pit mud microbiome, we determined the microbial community and metabolites of different-aged pit mud, inferred the main functional groups, and explored the forces driving the microbial community evolution through metagenomic, metabolomic, and multivariate statistical analyses. The results showed that the microbial consortia of pit mud presented a regular and directional evolutionary pattern under the impact of continuous batch-to-batch brewing activities. This work provides insight into the key roles of the pit mud microbiome in SFB production and supports the production optimization of high-quality pit mud.

Blautia liquoris sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese strong-flavour liquor.

A novel Gram-positive, non-motile, non-flagellated, strictly anaerobic, non-spore-forming and dumbbell-shaped, coccoid- or chain-shaped bacterium, designated strain LZLJ-3T, was isolated from a mud fermentation cellar which has been used for the production of Chinese strong-flavour liquor for over 100 years. Strain LZLJ-3T grew at 20-40 °C (optimum, 37 °C), at pH 6.0-8.0 (optimum, pH 8.0) and with NaCl concentrations up to 1 % (w/v; optimum, 0 %). Phylogenetic trees established based on 16S rRNA gene sequences showed that strain LZLJ-3T belonged to the genus Blautia of the family Lachnospiraceae, with the highest sequence similarity to Blautia stercoris GAM6-1T (91.7 %) and Blautia faecicola KGMB01111T (91.7 %). Comparative genome analysis showed that the orthologous average nucleotide identity (OrthoANI) and genome-to-genome distance (GGD) values between strain LZLJ-3T and B. stercoris GAM6-1T were respectively 69.1 and 22.9 %; the OrthoANI and GGD values between strain LZLJ-3T and B. faecicola KGMB01111T were respectively 70.86 and 36 % . The DNA G+C content of strain LZLJ-3T genome was 42.1 mol%. The predominant celluar fatty acids (>10 %) of strain LZLJ-3T were C16 : 0 FAME (27.9 %), C14 : 0 FAME (17.6 %) and C16 : 0 DMA (13.0 %). Arabinose, glucose and maltose could be utilized by strain LZLJ-3T as sole carbon sources for growth, with weak utilization of raffinose and l-fucose. API ZYM analysis gave positive reactions with α-galactosidase, ß-galactosidase, α-glucosidase and ß-glucosidase. The major end product of glucose fermentation was acetic acid. Based on the results of phenotypic, genotypic and phylogenetic analyses, strain LZLJ-3T is considered to represent a novel species of Blautia, for which the name Blautia liquoris sp. nov. is proposed. The type strain is LZLJ-3T (=KCTC 25163T=CGMCC 1.5299T=JCM 34225T).

Novisyntrophococcus fermenticellae gen. nov., sp. nov., isolated from an anaerobic fermentation cellar of Chinese strong-flavour baijiu.

A Gram-stain-negative, coccus-shaped, obligately anaerobic, non-motile and non-spore-forming bacterium, designated strain JN500902T, was isolated from the mud in a fermentation cellar used continuously over 30 years for Chinese strong-flavour baijiu production. Colonies were white, circular, convex and smooth-edged. Growth was observed at 20-40 °C (optimum, 37 °C), at pH 5.0-10 (optimum, pH 7.5), with 0-2 % (w/v) NaCl and with 0-4 % (v/v) ethanol. The Biolog assay demonstrated positive reactions of strain JN500902T in the metabolism of l-fucose and pyruvate. The predominant cellular fatty acids (>10 %) consisted of C16 : 0 and C14 : 0. The major end metabolites of strain JN500902T were acetic acid and ethanol when incubated anaerobically in liquid reinforced clostridial medium. Acetate was the major organic acid end product. The complete genome size of strain JN500902T was 3 420 321 bp with 3327 identified genes. The G+C content was 43.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain JN500902T with the family Lachnospiraceae, having low sequence similarity (92.8 %) to the nearest type strain, Syntrophococcus sucromutans DSM 3224T and forming a clearly distinct branch. Core genome phylogenetic analysis of the isolate and 134 strains belonging to the family Lachnospiraceae also revealed that strain JN500902T was well-separated from other genera of this family as a monophyletic clade. The average nucleotide identity and amino acid identity values between strain JN500902T and 134 Lachnospiraceae strains were less than 74 and 65 %, respectively. Considering its polyphasic characteristics, strain JN500902T represents a novel genus and species within the family Lachnospiraceae, for which the name Novisyntrophococcus fermenticellae gen. nov., sp. nov. is proposed. The type strain is JN500902T (=CICC 24502T=JCM 33939T).

Characterization of the complete mitochondrial genome of Macrotocinclus affinis (Siluriformes; Loricariidae) and phylogenetic studies of Siluriformes.

The vertebrate mitochondrial genome is typically circular molecules made up of 14,000 to 16,000 bp, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (12 s rRNA and 16 s rRNA) and a control region. Compared with nuclear DNA, mitochondrial DNA has a higher mutation rate, so it is one of the most effective and reliable molecular markers in fish phylogeny. Macrotocinclus affinis was the only species in Macrotocinclus (it was classified as Otocinclus in the past) and currently lacks genetic information. Most of the current researches are based on the mitochondrial Cytb gene and RAG1 and RAG2 nuclear genes to study the phylogenetic analysis of Siluriformes. So, the study provides the characteristic features of the Macrotocinclus affinis mitochondrial genome and this is the first time that the phylogenetic relationship of Siluriformes has been reconstructed based on COI. We aimed to sequence the entire mitochondrial genome of Macrotocinclus affinis using conventional PCR techniques and to clarify its phylogenetic status in Siluriformes by using the COI sequence of mitochondria. In this study, we sequenced the whole mitochondrial genome of this species yielding a 16,632 bp circular assembly composed of the typical vertebrate mitochondrial features. It contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a putative control region, and one origin of replication on the light-strand. The overall base composition includes A (30.07%), T (24.43%), C (29.43%) and G (16.01%). The genome composition is A + T biased (54.5%), and exhibits AT-skew (0.1036) and GC-skew (-0.2962). Moreover, the 13 PCGs encode 3850 amino acids in total. The result of the phylogenetic tree supports Macrotocinclus affinis has a closest relationship with Otocinclus cf. hoppei far. These results will help to understand the characteristics of the mitochondrial genome of Macrotocinclus affinis and provide molecular basis for the evolutionary relationship of Loricariidae.

Similarities and differences of oligo/poly-saccharides' impact on human fecal microbiota identified by in vitro fermentation.

The dietary supplementation of prebiotics is considered a promising strategy for the modulation of gut microbiota. Due to the wide variety of animal models and tremendous inter-individual variability from human investigations, the prebiotic effect of fibers is often difficult to compare between studies. Here, the effects of 11 dietary fibers on human fecal microbiota were studied using an in vitro human fecal fermentation model under well-controlled conditions. All fibers showed positive regulatory effects on short chain fatty acids (SCFAs) and several beneficial bacteria, including Parabacteroides distasonis and Bifidobacterium spp. Cultures supplemented with xylo-oligosaccharide and konjac flour showed the highest SCFAs. According to regulatory effects, fibers were divided into three groups, with 13 indicator OTUs (operational taxonomic units) identified. Fecal microbiota regulated by isomalto-oligosaccharide and chitosan-oligosaccharide were similar to fructo-oligosaccharide and inulin outputs. As a supplement to in vivo studies, our results comprehensively summarized the similarities and distinctiveness of fibers in regulating fecal microbiota structures. KEY POINTS: • Fibers were divided into three groups based on the regulatory effects in microbiota. • Thirteen indicator OTUs were identified using pairwise comparisons. • Fiber similarities and distinctive traits in regulating microbiota effect were identified.