Dietary Clostridium autoethanogenum protein has dose-dependent influence on the gut microbiota, immunity, inflammation and disease resistance of abalone Haliotis discus hannai.

Clostridium autoethanogenum protein (CAP) is an eco-friendly protein source and has great application potential in aquafeeds. The present study aimed to investigate the effects of dietary CAP inclusion on the anti-oxidation, immunity, inflammation, disease resistance and gut microbiota of abalone Haliotis discus hannai after a 110-day feeding trial. Three isonitrogenous and isolipidic diets were formulated by adding 0 % (control), 4.10 % (CAP4.10) and 16.25 % (CAP16.25) of CAP, respectively. A total of 540 abalones with an initial mean body weight of 22.05 ± 0.19 g were randomly distributed in three groups with three replicates per group and 60 abalones per replicate. Results showed that the activities of superoxide dismutase and glutathione peroxidase in the cell-free hemolymph (CFH) were significantly decreased and the content of malondialdehyde in CFH was significantly increased in the CAP16.25 group. The diet with 4.1 % of CAP significantly increased the activities of lysozyme and acid phosphatase in CFH. The expressions of pro-inflammatory genes such as tumor necrosis factor-α (tnf-α), nuclear factor-κb (nf-κb) and toll-like receptor 4 (tlr4) in digestive gland were downregulated, and the expressions of anti-inflammatory genes such as ß-defensin and mytimacin 6 in digestive gland were upregulated in the CAP4.10 group. Dietary CAP inclusion significantly decreased the cumulative mortality of abalone after the challenge test with Vibrio parahaemolyticus for 7 days. Dietary CAP inclusion changed the composition of gut microbiota of abalone. Besides, the balance of the ecological interaction network of bacterial genera in the intestine of abalone was enhanced by dietary CAP. The association analysis showed that two bacterial genera Ruegeria and Bacteroides were closely correlated with the inflammatory genes. In conclusion, the 4.10 % of dietary CAP enhanced the immunity and disease resistance as well as inhibited the inflammation of abalone. The 16.25 % of dietary CAP decreased the anti-oxidative capacity of abalone. The structure of the gut microbiota of abalone changed with dietary CAP levels.

Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5 h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

Characterization of the Aminosugar Biosynthetic and Regulatory Genes of Vicenistatin in Monodonata labio-Associated Streptomyces parvus SCSIO Mla-L010.

Vicenistatin (1) is a potent polyketide antitumor antibiotic composed of a 20-membered macrolactam core appended to a unique aminosugar, vicenisamine. In this study, vicenistatin was isolated and its biosynthetic gene cluster identified from Monodonata labio-associated Streptomyces parvus SCSIO Mla-L010. A set of five genes, vicC, vicD, vicE, vicF, and vicG, was confirmed to be involved in the biosynthesis of the aminosugar by gene inactivations. VicG was characterized as an N-methyltransferase that catalyzes the methylation of the 4'-amino group in the last step of the aminosugar biosynthetic pathway; the N-demethyl intermediate 4'-N-demethylvicenistatin (2) was isolated from the ΔvicG mutant strain. In addition, vicR1 was characterized as a positive pathway-specific regulatory gene. Notably, N-demethyl compound 2 was found to exert impressive antibacterial activities, with MIC values spanning 0.06-4 µg/mL, against a panel of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus, Gram-negative Helicobacter pylori, and mycobacterium Mycobacterium smegmatis and the fungal pathogen Candida albicans. Compound 2 was also found to display reduced cytotoxicities relative to vicenistatin, especially against noncancerous human cell lines.

TMKS8A, an antibacterial and cytotoxic chlorinated α-lapachone, from a sea slug-derived actinomycete of the genus Streptomyces.

TMKS8A (1), a new chlorinated α-lapachone derivative, along with five known related metabolites, A80915 C (2), SF2415B1 (3), chlorinated dihydroquinone 3 (4), SF2415B3 (5), and A80915 C (6), were identified from the culture extract of Streptomyces sp. TMKS8, which was isolated from a sea slug, Paromoionchis tumidus. The structure of 1 was determined by the analysis of NMR and MS spectral data, assisted by NMR chemical shift prediction using DFT-based calculation. The absolute configuration was determined to be R by comparison of experimental and calculated ECD spectra. Compound 1 displayed antimicrobial activity against Gram-positive bacteria with MIC values ranging from 6.25 to 12.5 µg ml-1 and cytotoxicity against murine leukemia P388 cells with IC50 9.8 µM.

Tamlana haliotis sp. nov., isolated from the gut of the abalone Haliotis rubra.

A Gram-stain-negative, aerobic, non-motile, yellow-pigmented rod-shaped and alginate-degrading bacterium, designated B1N29T, was isolated from the gut of the abalone Haliotis rubra obtained in Weihai, China. Strain B1N29T was found to grow at 4-35 ℃ (optimum, 25 ℃), at pH 6.5-9.0 (optimum, 7.0-7.5) and in the presence of 0.5-9% (w/v) NaCl (optimum, 2%). Cells were positive for oxidase and catalase activity. The 16S rRNA-based phylogenetic analysis revealed that the nearest phylogenetic neighbors of strain B1N29T were Tamlana carrageenivorans KCTC 62451T (98.2%) and Tamlana agarivorans KCTC 22176T (97.7%). Based on the phylogenomic analysis, the average nucleotide identity (ANI) values between strain B1N29T and the neighbor strains were 79.2 and 79.0%, respectively; the digital DNA-DNA hybridization (dDDH) values between strain B1N29T and its two closest neighbors were 22.8 and 23.0%, respectively. Menaquinone-6 (MK-6) was detected as the sole respiratory quinone. The dominant cellular fatty acids were iso-C15:0, iso-C17:0 3-OH, anteiso-C15:0 and iso-C15:1 G. The polar lipids included phosphatidylethanolamine, one aminophospholipid, seven aminolipids and five unidentified lipids. Based on the phylogenetic and phenotypic characteristics, strain B1N29T is considered to represent a novel species of the genus Tamlana, for which the name Tamlana haliotis sp. nov. is proposed. The type strain is B1N29T (= KCTC 72683T = MCCC 1H00394T).

Cohaesibacter intestini sp. nov., isolated from the intestine of abalone, Haliotis discus hannai.

A Gram-stain-negative, rod-shaped and facultative anaerobic strain named YE-B6T was isolated from the intestine of abalone, Haliotis discus hannai. The reactions of oxidase and catalase were both positive. Strain YE-B6T could grow at 10-37 °C (optimum, 28-33 °C), at pH 7-9 (optimum, pH 7) and in salinity of 0-8 % NaCl (w/v; optimum, 2 %). The 16S rRNA gene sequence of strain YE-B6T had maximum sequence similarities to Cohaesibacter celericrescens H1304T (98.0 %), Cohaesibacter marisflavi DQHS21T (97.4 %), Cohaesibacter haloalkalitolerans JC131T (97.4 %) and Cohaesibacter gelatinilyticus CL-GR15T (97.3 %). Results of phylogenetic analysis showed that it was affiliated to the genus Cohaesibacter within the order Rhizobiales in the Alphaproteobacteria. The respiratory quinone of strain YE-B6T was Q-10. The cellular fatty acids mainly comprised C18 : 1 ω7c and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, two unidentified aminolipids, two unidentified polar lipids and one unidentified phospholipid. The G+C content of strain YE-B6T was 55.6 mol%. Based on its genotypic, physiological and biochemical characteristics, strain YE-B6T represented a novel species within the genus Cohaesibacter, for which the name Cohaesibacterintestini sp. nov. is proposed. The type strain of the new species is YE-B6T (=MCCC 1A13131T=KCTC 62716T).

Lottiidibacillus patelloidae gen. nov., sp. nov., isolated from the intestinal tract of a marine limpet and reclassification of Bacillus taeanensis as Maribacillus taeanensis gen. nov., comb. nov.

A taxonomic study was carried out on strain SA5d-4T, which was isolated from a marine limpet (Patelloida saccharina lanx [Reeve, 1855]) collected from intertidal rocks in Xiamen, China. Strain SA5d-4T was aerobic, Gram-positive, lacked flagellum, non-motile, filamentous, formed a slightly-yellowish colony, and non-sporulating. The strain grew optimally at 28 °C, at pH values 7.0-8.0, and in the presence of 1-2% (w/v) sodium chloride. The major cellular fatty acids identified were iso-C15:0, iso-C17:0ω10c, and iso-C17:0. The dominated respiratory quinone was menaquinone-7. The major phospholipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C content was 35.3 mol%, calculated from a draft genome sequence. Phylogenetic analysis based on the full-length 16S rRNA gene sequence showed that strain SA5d-4T belongs to a new genus within the family Bacillaceae, and this gene shares 95.6% similarity with that from Bacillus taeanensis BH030017T, 95.2% with Bacillus algicola KMM 3737T, 95.1% with Bacillus alkalinitrilicus ANL-iso4T, 94.9% with Bacillus hwajinpoensis SW-72T, and 94.6% with Anaerobacillus alkalidiazotrophicus MS6T. Whole genome phylogenetic analyses indicated that strain SA5d-4T formed a monophyletic branch with B. taeanensis BH030017T. The average nucleotide identity between strain SA5d-4T and B. taeanensis BH030017T was 69.6%. Based on polyphasic taxonomic characteristics, strain SA5d-4T represents a novel species of a new genus, for which the name Lottiidibacillus patelloidae gen. nov., sp. nov., is proposed with the type strain SA5d-4T (= MCCC 1A11654T = KCTC 33831T). Based on phylogenetic analyses, B. taeanensis should be transferred to a new genus, named Maribacillus, as Maribacillus taeanensis comb. nov., with type strain BH030017T (= KCTC 3918T = DSM 16466T).

The Saposin-Like Protein AplD Displays Pore-Forming Activity and Participates in Defense Against Bacterial Infection During a Multicellular Stage of Dictyostelium discoideum.

Due to their archaic life style and microbivor behavior, amoebae may represent a source of antimicrobial peptides and proteins. The amoebic protozoon Dictyostelium discoideum has been a model organism in cell biology for decades and has recently also been used for research on host-pathogen interactions and the evolution of innate immunity. In the genome of D. discoideum, genes can be identified that potentially allow the synthesis of a variety of antimicrobial proteins. However, at the protein level only very few antimicrobial proteins have been characterized that may interact directly with bacteria and help in fighting infection of D. discoideum with potential pathogens. Here, we focus on a large group of gene products that structurally belong to the saposin-like protein (SAPLIP) family and which members we named provisionally Apls (amoebapore-like peptides) according to their similarity to a comprehensively studied antimicrobial and cytotoxic pore-forming protein of the protozoan parasite Entamoeba histolytica. We focused on AplD because it is the only Apl gene that is reported to be primarily transcribed further during the multicellular stages such as the mobile slug stage. Upon knock-out (KO) of the gene, aplD- slugs became highly vulnerable to virulent Klebsiella pneumoniae. AplD- slugs harbored bacterial clumps in their interior and were unable to slough off the pathogen in their slime sheath. Re-expression of AplD in aplD- slugs rescued the susceptibility toward K. pneumoniae. The purified recombinant protein rAplD formed pores in liposomes and was also capable of permeabilizing the membrane of live Bacillus megaterium. We propose that the multifarious Apl family of D. discoideum comprises antimicrobial effector polypeptides that are instrumental to interact with bacteria and their phospholipid membranes. The variety of its members would allow a complementary and synergistic action against a variety of microbes, which the amoeba encounters in its environment.

Identification and characterization of molluscan caveolin-1 ortholog from Haliotis discus discus: Possible involvement in embryogenesis and host defense mechanism against pathogenic stress.

Caveolins are principal membrane proteins of caveolae that play a central role in signal transduction, substrate transport, and membrane trafficking in various cell types. Numerous studies have reported the crucial role of caveolin-1 (CAV1) in response to invading microbes; yet, very little is known about molluscan CAV1. In this study, we identified and characterized CAV1 ortholog from the disk abalone, Haliotis discus discus (HdCAV1). The cDNA sequence of HdCAV1 is 826 bp long and encodes a 127-amino acid polypeptide. Characteristic caveolin superfamily domain (Glu3 - Lys126) and two possible transmembrane domains (Cys48 - Tyr67 and Ile103 - Phe120) were identified in the HdCAV1 protein. Homology analysis revealed that HdCAV1 shared higher identity (>47%) with molluscans, but lower identity with other species. Phylogenetic tree constructed by the neighbor-joining (NJ) method revealed a distinct evolutionary pathway for molluscans. Transcriptional analysis by SYBR Green qPCR showed the highest expression of HdCAV1 mRNA in late veliger stage, as compared to that in other embryonic developmental stages of disk abalone. In adult animals, gill tissue showed highest HdCAV1 transcript levels under normal physiological condition. Stimulations with two bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), viral hemorrhagic septicemia virus, and two pathogen-associated molecular patterns (LPS and poly I:C) significantly modulated the expression of HdCAV1 transcripts. Collectively, these data suggest that CAV1 plays an important role in embryogenesis and host immune defense in disk abalone.

Agaribacterium haliotis gen. nov., sp. nov., isolated from abalone faeces.

A marine bacterium, named strain feces2T, was isolated from the excreted faeces of an abalone, Haliotis discus hannai. The bacterium was Gram-stain-negative, rod-shaped and had a polar flagellum. It formed a white, small and crater-like colony on an agar plate, and had the capability of degrading agar. Activity of oxidase was positive and that of catalase was negative. Strain feces2T grew at 16 to 40 °C with an optimum of 28-30 °C. The nearly full-length 16S rRNA gene of strain feces2T had the greatest sequence similarity of 92.9 % with Marinibactrumhalimedae Q-192T, followed by of 92.8 % with Teredinibacterturnerae T7902T. Phylogenetic analysis indicated that strain feces2T belonged to the family Cellvibrionaceae, representing an independent clade with an uncultured bacterium clone NEP3-15 (98 % sequence similarity of 16S rRNA gene) derived from the phycosphere of Enteromorphaprolifera. The respiratory quinone was ubiquinone Q-8. The predominant fatty acids consisted of summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C16 : 0 and summed feature 3 (C16 : 1 ω6c/C16 : 1ω7c). The polar lipids were identified as phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified amino phospholipid and four unknown lipids. The genomic DNA G+C content was 50.5 mol%. On the basis of polyphasic characterizations, strain feces2T represented a novel species and a novel genus in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria, for which the name Agaribacteriumhaliotis gen. nov., sp. nov. is proposed, with the type strain being feces2T (=MCCC 1A11750T=KCTC 52708T). [corrected].

Tenacibaculum haliotis sp. nov., isolated from the gut of an abalone Haliotis discus hannai.

A Gram-stain-negative, non-flagellated, gliding, non-spore-forming bacterial strain, designated RA3-2T, was isolated from the gut of an abalone (Haliotis discus hannai) collected from the sea around Jeju island, South Korea, and subjected to a polyphasic taxonomic study. RA3-2T grew optimally at 20 °C and in the presence of 2.0-3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences indicated that RA3-2T fell within the clade comprising the type strains of species of the genus Tenacibaculum, clustering with the type strains of Tenacibaculum soleae, Tenacibaculum ovolyticum and Tenacibaculum dicentrarchi; showing 16S rRNA gene sequence similarity values of 96.2-96.8 %. The novel strain exhibited 16S rRNA gene sequence similarity values of 93.5-96.9 % to the type strains of the other species of the genus Tenacibaculum. RA3-2T contained MK-6 as the predominant menaquinone and iso-C15 : 0 and iso-C15 : 0 3-OH as the major fatty acids. The major polar lipids of RA3-2T were phosphatidylethanolamine, two unidentified lipids, one unidentified aminophospholipid and one unidentified glycolipid. The DNA G+C content of RA3-2T was 31.7 mol%. The differential phenotypic properties, together with the phylogenetic data, revealed that RA3-2T is separated from other species of the genus Tenacibaculum with validly published names. On the basis of the data presented, RA3-2T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum haliotis sp. nov. is proposed. The type strain is RA3-2T (=KCTC 52419T=NBRC 112382T).

A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone.

Shewanella intestini sp. nov., isolated from the intestine of abalone, Haliotis diversicolor.

A Gram-stain-negative, rod-shaped bacterium with a singular polar flagellum, designated as strain XMDDZSB0408T, was isolated from the intestine of adult abalone, Haliotis diversicolor. Activity of oxidase was positive and catalase activity was negative. It could grow at salinities from 3 to 6 % NaCl (w/v), and pH 7-9. It had the highest sequence similarity of <96.0 % with all previously established species for the complete 16S rRNA gene (1531 bp). The results of phylogenetic analysis indicated that the strain was affiliated to the genus Shewanella and closely related to Shewanella gaetbuli TF-27T and Shewanella japonica KMM 3299T (95.8 % sequence similarity), Shewanella electrodiphila MAR441T (95.6 %), Shewanella pacifica KMM 3597T (95.4 %), Shewanella donghaensis LT17T (95.3 %) and Shewanella olleyana ACEM 9T (94.7 %). The respiratory quinones were MK-7, Q-8, Q-7, MK-8, Q-7 (H4) and Q-6. The predominant fatty acids consisted of C16:0, summed feature 3 (comprised of C16 : 1ω7c/C16 : 1ω6c), C18 : 0, summed feature 8 (comprised of C18 : 1ω7c/C18 : 1ω6c), C12 : 0 and C14 : 0. The polar lipids were identified as phosphatidylethanolamine (PE), a glycolipid (GL), a phospholipid (PL) and one unidentified lipid (L). The DNA G+C content was 41.4 mol% calculated from the draft genome sequence. On the basis of its polyphasic taxonomic properties, strain XMDDZSB0408T represented a novel species, for which the name Shewanella intestini sp. nov. was proposed, with the type strain XMDDZSB0408T (=KCTC 52125T=MCCC 1A01895T).

Polaribacter haliotis sp. nov., isolated from the gut of abalone Haliotis discus hannai.

A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated RA4-7T, was isolated from the gut of an abalone (Haliotis discus hannai) collected from the sea around Jeju island, South Korea, and subjected to a polyphasic taxonomic study. RA4-7T grew optimally at 25 °C, at pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that RA4-7T represented a member of the genus Polaribacter. RA4-7T exhibited 16S rRNA gene sequence similarity values of 97.64 and 97.23 % to Polaribacter atrinae WP25T and Polaribacter dokdonensis DSW-5T, respectively, and of 93.83-96.99 % to the type strains of the other species of the genus Polaribacter. RA4-7T contained MK-6 as the predominant menaquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0 3-OH and iso-C15 : 0as the major fatty acids. The major polar lipids detected in RA4-7T were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of RA4-7T was 30.5 mol% and its DNA-DNA relatedness values with the type strains of P. atrinae and P. dokdonensis were 16 and 11 %, respectively. Differential phenotypic properties, in combination with its phylogenetic and genetic distinctiveness, revealed that RA4-7T is separated from species of the genus Polaribacter with validly published names. On the basis of the data presented, RA4-7T represented a novel species of the genus Polaribacter, for which the name Polaribacterhaliotis sp. nov. is proposed. The type strain is RA4-7T (=KCTC 52418T=NBRC 112383T).

Pseudofulvibacter gastropodicola sp. nov., isolated from a marine conch and emended descriptions of the genus Pseudofulvibacter Yoon et al. 2013 and Pseudofulvibacter geojedonensis.

A Gram-stain-negative, aerobic, rod-shaped and non-motile marine bacterium, designated MEBiC08749T, was isolated from a marine conch collected at Samcheok Port in the East Sea, Korea (also known as the Sea of Japan). 16S rRNA gene sequence analysis revealed that strain MEBiC08749T shared highest similarity with Pseudofulvibacter geojedonensis YCS-9T (94.9 %). Growth was observed at 5-31 °C (optimum, 25 °C), at pH 6.0-8.5 (optimum, pH 7.5) and with 0-6 % (w/v) NaCl (optimum, 2.5 %). The predominant cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH, summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c) and summed feature 9 (comprising 10-methyl C16 : 0 and/or iso-C17 : 1ω9c). The DNA G+C content was 30 mol%. The only respiratory quinone was menaquinone-6 (MK-6). Phosphatidylethanolamine, two unidentified lipids and two unidentified aminolipids were detected as major polar lipids. Several phenotypic characteristics, such as production of acetoin, activity of cystine arylamidase, utilization of some carbohydrates, organic acids and amino acids, differentiated strain MEBiC08749T from P. geojedonensis KCTC 23884T. On the basis of data from this polyphasic taxonomic study, strain MEBiC08749T should be classified as representing a novel species in the genus Pseudofulvibacter, for which the name Pseudofulvibacter gastropodicola sp. nov. is proposed. The type strain is MEBiC08749T ( = KCCM 43046T = JCM 30369T). Emended descriptions of the genus PseudofulvibacterYoon et al. 2013 and of Pseudofulvibacter geojedonensis are also given.

Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod.

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita.

Identification and expression analysis of immune-related genes linked to Rel/NF-κB signaling pathway under stresses and bacterial challenge from the small abalone Haliotis diversicolor.

Inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB), and Akirin2 are all important members of Rel/NF-κB signaling pathway, which plays a pivotal role in regulating the innate immune response of vertebrates and invertebrates. In this study, the IκB (SaIκB) and Akirin2 (SaAkirin2) cDNAs of small abalone Haliotis diversicolor were cloned and characterized. The full length cDNA of SaIκB and SaAkirin2 were 1748 bp and 1452 bp respectively, encoding a protein of 401 aa and 187 aa respectively. A conserved degradation motif (DS56GIYS60) and six ankyrin repeats were identified in the SaIκB by SMART analysis. Meanwhile, a typical nuclear localization signal (NLS) was found at the N-terminal region of the SaAkirin2 protein. Also, the mRNA expression level of SaIκB, SaAkirin2, and AbNF-κB were detected by quantitative real-time PCR. The results revealed that all these three genes were ubiquitously expressed in 7 selected tissues. The expression level of SaIκB in gills was higher than that in other tissues (P < 0.05) while the expression level of AbNF-κB was significantly higher in hepatopancreas and haemocytes. The highest expression level of SaAkirin2 was detected in hepatopancreas, followed by mantle. The mRNA expression levels in either gills or haemocytes of SaIκB, SaAkirin2, and AbNF-κB were significantly up-regulated (P < 0.05) post thermal stress, hypoxia exposure, thermal plus hypoxia stress and the injection of Vibrio parahaemolyticus. These results indicated that these three NF-κB signaling pathway-related genes are involved in response to bacterial infection and play essential roles in response to thermal and hypoxia stress.

A galectin with quadruple-domain from red abalone Haliotis rufescens involved in the immune innate response against to Vibrio anguillarum.

Galectins are proteins that recognize and bind specifically ß-galactosidase residues, playing important roles in the innate immune response of vertebrates and invertebrates. The cDNA of a tandem repeat galectin from the red abalone Haliotis rufescens cDNA (HrGal) was cloned and characterized using rapid amplification of cDNA end technique. The full-length cDNA of HrGal was 2471 bp, with a 5' terminal untranslated region (UTR) of 131 bp, a 3' UTR of 672 pb, and an open reading frame (ORF) of 1668 bp encoding a polypeptide of 556 amino acid. The ORF contains four domains carbohydrate recognition (CRD) with typical conserved motifs, which are important for carbohydrate recognition, and it appear to posses neither a signal peptide nor a transmembrane domain. The deduced amino acid sequence and the multi-domain organization of HrGal were highly similar to those described for other tandem repeat galectins of invertebrate organisms. Quantitative real time PCR analyses indicated that HrGal mRNA was highly expressed in hemocytes and gills tissues. The temporal expression of HrGal mRNA in hemocytes challenged to Vibrio anguillarum was time-dependent, showing u-regulation at 32 h post challenge. The results suggest that HrGal may be involved in the immune innate response against bacterial infection.

Antibacterial activity and immune responses of a molluscan macrophage expressed gene-1 from disk abalone, Haliotis discus discus.

The membrane-attack complex/perforin (MACPF) domain-containing proteins play an important role in the innate immune response against invading microbial pathogens. In the current study, a member of the MACPF domain-containing proteins, macrophage expressed gene-1 (MPEG1) encoding 730 amino acids with the theoretical molecular mass of 79.6 kDa and an isoelectric point (pI) of 6.49 was characterized from disk abalone Haliotis discus discus (AbMPEG1). We found that the characteristic MACPF domain (Val(131)-Tyr(348)) and transmembrane segment (Ala(669)-Ile(691)) of AbMPEG1 are located in the N- and C-terminal ends of the protein, respectively. Ortholog comparison revealed that AbMPEG1 has the highest sequence identity with its pink abalone counterpart, while sequences identities of greater than 90% were observed with MPEG1 members from other abalone species. Likewise, the furin cleavage site KRRRK was highly conserved in all abalone species, but not in other species investigated. We identified an intron-less genomic sequence within disk abalone AbMPEG1, which was similar to other mammalian, avian, and reptilian counterparts. Transcription factor binding sites, which are important for immune responses, were identified in the 5'-flanking region of AbMPEG1. qPCR revealed AbMPEG1 transcripts are present in every tissues examined, with the highest expression level occurring in mantle tissue. Significant up-regulation of AbMPEG1 transcript levels was observed in hemocytes and gill tissues following challenges with pathogens (Vibrio parahemolyticus, Listeria monocytogenes and viral hemorrhagic septicemia virus) as well as pathogen-associated molecular patterns (PAMPs: lipopolysaccharides and poly I:C immunostimulant). Finally, the antibacterial activity of the MACPF domain was characterized against Gram-negative and -positive bacteria using a recombinant peptide. Taken together, these results indicate that the biological significance of the AbMPEG1 gene includes a role in protecting disk abalone through the ability of AbMPEG1 to initiate an innate immune response upon pathogen invasion.