Molecular characterization, expression and antibacterial function of a macin, HdMac, from Haliotis discus hannai.

Macins are a family of antimicrobial peptides, which play multiple roles in the elimination of invading pathogens. In the present study, a macin was cloned and characterized from Pacific abalone Haliotis discus hannai (Designated as HdMac). Analysis of the conserved domain suggested that HdMac was a new member of the macin family. In non-stimulated abalones, HdMac transcripts were constitutively expressed in all five tested tissues, especially in hemocytes. After Vibrio harveyi stimulation, the expression of HdMac mRNA in hemocytes was significantly up-regulated at 12 hr (P < 0.01). RNAi-mediated knockdown of HdMac transcripts affected the survival rates of abalone against V. harveyi. Moreover, recombinant protein of HdMac (rHdMac) exhibited high antibacterial activities against invading bacteria, especially for Vibrio anguillarum. In addition, rHdMac possessed binding activities towards glucan, lipopolysaccharides (LPS), and peptidoglycan (PGN), but not chitin in vitro. Membrane integrity analysis revealed that rHdMac could increase the membrane permeability of bacteria. Meanwhile, both the phagocytosis and chemotaxis ability of hemocytes could be significantly enhanced by rHdMac. Overall, the results showed that HdMac could function as a versatile molecule involved in immune responses of H. discus hannai.

Identification, diversity, and evolution analysis of Commd gene family in Haliotis discus hannai and immune response to biotic and abiotic stresses.

The Commd (Copper Metabolism gene MURR1 Domain) family genes play crucial roles in various biological processes, including copper and sodium transport regulation, NF-κB activity, and cell cycle progression. Their function in Haliotis discus hannai, however, remains unclear. This study focused on identifying and analyzing the Commd genes in H. discus hannai, including their gene structure, phylogenetic relationships, expression profiles, sequence diversity, and alternative splicing. The results revealed significant homology between H. discus hannai's Commd genes and those of other mollusks. Both transcriptome quantitative analysis and qRT-PCR demonstrated the responsiveness of these genes to heat stress and Vibrio parahaemolyticus infection. Notably, alternative splicing analysis revealed that COMMD2, COMMD4, COMMD5, and COMMD7 produce multiple alternative splice variants. Furthermore, sequence diversity analysis uncovered numerous missense mutations, specifically 9 in COMMD5 and 14 in COMMD10. These findings contribute to expanding knowledge on the function and evolution of the Commd gene family and underscore the potential role of COMMD in the innate immune response of H. discus hannai. This research, therefore, offers a novel perspective on the molecular mechanisms underpinning the involvement of Commd genes in innate immunity, paving the way for further explorations in this field.

Halocynthiibacter halioticoli sp. nov., isolated from the viscera of abalone Haliotis discus hannai.

A Gram-stain-negative, rod-shaped, glide, non-flagellated, and facultatively anaerobic bacterial strain, designated as Z654T, was isolated from the gut of abalone Haliotis discus hannai from Rongcheng, Shandong province, China. Cells are 0.2-0.8 µm in width and 0.7-3.4 µm in length. Cells grew best at 30 °C (range, 15-37 °C), pH 7.0 (range, 6.0-8.5) and NaCl concentration of 2.0% (w/v) (range, 1-10%). According to the phylogenetic analysis of 16S rRNA gene sequence, the strain belongs to the genus Halocynthiibacter and the closest strain is Halocynthiibacter arcticus KCTC 42129 T (97.12%). The genome size of strain Z654T was 3,296,250 bp and the DNA G + C content was 54.2 mol%. The average nucleotide identity (ANI) scores and digital DNA-DNA hybridization (dDDH) scores with H. arcticus KCTC 42129 T were 70% and 14.6-18.2%, respectively. The predominant quinone was Q-10 and the major fatty acids were C18:0, C18:1 ω7c 11-methyl and summed feature 8. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, unidentified aminolipid and unidentifed lipids. Based on the phenotypic, phylogenetic and chemotaxonomic data, strain Z654T was considered to represent a novel species of the genus Halocynthiibacter, for which the name Halocynthiibacte halioticoli sp. nov., is proposed. The type strain is Z654T (= MCCC 1H00503T = KCTC 92003 T).

Ecological differences among hydrothermal vent symbioses may drive contrasting patterns of symbiont population differentiation.

The intra-host composition of horizontally transmitted microbial symbionts can vary across host populations due to interactive effects of host genetics, environmental, and geographic factors. While adaptation to local habitat conditions can drive geographic subdivision of symbiont strains, it is unknown how differences in ecological characteristics among host-symbiont associations influence the genomic structure of symbiont populations. To address this question, we sequenced metagenomes of different populations of the deep-sea mussel Bathymodiolus septemdierum, which are common at Western Pacific deep-sea hydrothermal vents and show characteristic patterns of niche partitioning with sympatric gastropod symbioses. Bathymodiolus septemdierum lives in close symbiotic relationship with sulfur-oxidizing chemosynthetic bacteria but supplements its symbiotrophic diet through filter-feeding, enabling it to occupy ecological niches with little exposure to geochemical reductants. Our analyses indicate that symbiont populations associated with B. septemdierum show structuring by geographic location, but that the dominant symbiont strain is uncorrelated with vent site. These patterns are in contrast to co-occurring Alviniconcha and Ifremeria gastropod symbioses that exhibit greater symbiont nutritional dependence and occupy habitats with higher spatial variability in environmental conditions. Our results suggest that relative habitat homogeneity combined with sufficient symbiont dispersal and genomic mixing might promote persistence of similar symbiont strains across geographic locations, while mixotrophy might decrease selective pressures on the host to affiliate with locally adapted symbiont strains. Overall, these data contribute to our understanding of the potential mechanisms influencing symbiont population structure across a spectrum of marine microbial symbioses that occupy contrasting ecological niches. IMPORTANCE Beneficial relationships between animals and microbial organisms (symbionts) are ubiquitous in nature. In the ocean, microbial symbionts are typically acquired from the environment and their composition across geographic locations is often shaped by adaptation to local habitat conditions. However, it is currently unknown how generalizable these patterns are across symbiotic systems that have contrasting ecological characteristics. To address this question, we compared symbiont population structure between deep-sea hydrothermal vent mussels and co-occurring but ecologically distinct snail species. Our analyses show that mussel symbiont populations are less partitioned by geography and do not demonstrate evidence for environmental adaptation. We posit that the mussel's mixotrophic feeding mode may lower its need to affiliate with locally adapted symbiont strains, while microhabitat stability and symbiont genomic mixing likely favors persistence of symbiont strains across geographic locations. Altogether, these findings further our understanding of the mechanisms shaping symbiont population structure in marine environmentally transmitted symbioses.

The Role of Diversity in Mediating Microbiota Structural and Functional Differences in Two Sympatric Species of Abalone Under Stressed Withering Syndrome Conditions.

Withering syndrome (WS) is a gastro-intestinal (GI) infectious disease likely affecting all abalone species worldwide. Structural and functional changes in abalone GI microbiotas under WS-stressed conditions remain poorly investigated. It is unclear if interspecific microbiota differences, such as the presence of certain microbes, their abundance, and functional capabilities, may be involved in the occurrence of this disease. Bacterial microbiotas of healthy Haliotis fulgens and Haliotis corrugata are mainly composed by Tenericutes, Proteobacteria, Fusobacteria, and Spirochaetes. We previously reported species-specific structural and functional profiles of those communities and suggested that they are of consequence to the different susceptibility of each species to WS. Here, we address this question by comparing the structure and function of healthy and dysbiotic microbiota through 454 pyrosequencing and PICRUSt 2, respectively. Our findings suggest that the extent to which WS-stressed conditions may explain structural and functional differences in GI microbiota is contingent on the microbiota diversity itself. Indeed, microbiota differences between stressed and healthy abalone were marginal in the more complex bacterial communities of H. corrugata, in which no significant structural or functional changes were detected. Conversely, significant structural changes were observed in the less complex bacterial microbiota of H. fulgens. Moreover, structural alterations led to a significant downregulation of some metabolic activities conducted by GI bacteria. Accordingly, results suggest that gastro-intestinal bacterial diversity appears to be related with both the health of abalone and the etiology of WS.

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.

Withering syndrome induced gene expression changes and a de-novo transcriptome for the Pinto abalone, Haliotis kamtschatkana.

In the abalone and Candidatus Xenohaliotis californiensis (Ca. Xc) system, the Ca. Xc bacterium infects abalone digestive tissues and leads to extreme starvation and a characteristic "withering" of the gastropod foot. First identified in black abalone in California after an El Niño event, withering syndrome (WS) has caused large declines in wild black and captive white abalone on the northeastern Pacific coast, but disease resistance levels are species-, and possibly population-specific. This study compared gene expression patterns in the digestive gland of Ca. Xc-exposed and unexposed (control) Pinto abalone (Haliotis kamtschatkana), a particularly susceptible species. Lab-induced Ca. Xc infections were followed over 7 months and RNAseq was used to identify differential gene expression. Exposed Pinto abalone showed distinct changes in expression of 68 genes at 3 and 7 months post-infection relative to those in control animals. Upregulation of an orexin-like receptor (which is involved in feeding signaling) and a zinc peptidase-like region (many amino peptidases are zinc peptidases) in animals infected for 7 months indicates that animals with Ca. Xc infection may be starving and upregulating processes associated with feeding and digestion. Other groups of differentially expressed genes (DEGs) were upregulated or downregulated across control and exposed individuals over the 7-month experiment, including DEG groups that likely correspond to early disease state and to general stress response of being held in captivity. No patterns emerged in genes known to be involved in molluscan immune response, despite this being an expectation during a 7-month infection; digestion-related genes and unannotated DEGs were identified as targets for future research on potential immune response to WS in abalone.

Algibacter onchidii sp. nov., a symbiotic bacterium isolated from a marine invertebrate.

A novel symbiotic bacterium, designated strain XY-114T, was isolated from the cerata of an Onchidium marine invertebrate species collected in the South China Sea. Strain XY-114T was an aerobic, Gram-stain-negative, non-motile and short rod-shaped bacterium (0.5-0.8 µm wide and 1.0-1.5 µm long) without flagellum. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain XY-114T belonged to the genus Algibacter with the highest similarity of 97.2 % to the closest phylogenetic relative Algibacter aestuarii KYW371T. Cells grew at 15-37 °C (optimum, 30 °C), at pH 5.5-9.0 (optimum 7.0-8.0) and at NaCl concentrations of 0.5-5.0 % (w/v; optimum 1.5-3.0 %). The major fatty acids (>10 %) were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The predominant polar lipid was phosphatidylethanolamine. The predominant respiratory quinone was MK-6. Flexirubin-type pigments were absent. The genome size of strain XY-114T was 3.4 Mbp, with 34.9 mol% of DNA G+C content. The average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values between strain XY-114T and A. aestuarii KYW371T were 74.5 %, 17.0±1.8 % and 73.9 %. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that strain XY-114T represents a novel species of the genus Algibacter, for which the name Algibacter onchidii sp. nov. is proposed. The type strain is XY-114T (=KCTC 72217T=MCCC 1K03606T).

Muricauda onchidii sp. nov., isolated from a marine invertebrate from South China Sea, and transfers of Flagellimonas algicola, Flagellimonas pacifica and Flagellimonas maritima to Muricauda algicola comb. nov., Muricauda parva nom. nov. and Muricauda aurantiaca nom. nov., respectively, and emended description of the genus Muricauda.

An aerobic, Gram-stain-negative, rod-shaped and non-motile strain (XY-359T) was isolated from the mouth of a marine invertebrate Onchidium species from the South China Sea. It grew at pH 6.0-8.5 (optimum, pH 7.5), at 15-37 °C (optimum, 30 °C) and in the presence of 0.5-4.5 % (w/v) NaCl (optimum, 2.5 %). It could not hydrolyse Tweens 20, 40, 60 or 80 and no flexirubin-type pigments were produced. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, six unidentified phospholipids and two unidentified polar lipids. The major fatty acids were iso-C17:0 3-OH, iso-C15:1 G and iso-C15:0 3-OH. The respiratory quinone was MK-6. Strain XY-359T showed the greatest degree of 16S rRNA sequence similarity to Flagellimonas algicola AsT0115T (96.54 %), followed by Muricauda flava DSM 22638T (96.27 %). Phylogenetic analysis based on 16S rRNA gene sequences and 31 core genes indicated that strain XY-359T belongs to the genus Muricauda. The genome size of strain XY-359T was 4 207 872 bp, with 39.1 mol% of DNA G+C content. The average nucleotide identity and digital DNA-DNA hybridization values between strain XY-359T and F. algicola AsT0115T were 74.58 % and 18.5 %, respectively, and those between strain XY-359T and M. flava DSM 22638T were 74.2 % and 18.3 %. The combined phenotypic, chemotaxonomic and phylogenetic data suggest that strain XY-359T represents a novel species of the genus Muricauda, for which the name Muricauda onchidii sp. nov. is proposed. The type strain is XY-359T (=MCCC 1K03658T =KCTC 72218T). Moreover, based on the proposal of nesting Spongiibacterium and Flagellimonas within Muricauda by García (Validation List No. 193) and the analyses of phylogenetic trees and average amino acid identities in this study, the transfers of F. algicola, F. pacifica and F. maritima to the genus Muricauda as Muricauda algicola comb. nov., Muricauda parva nom. nov. and M. aurantiaca nom. nov., respectively, are proposed, with an emended description of the genus Muricauda.

The Enhanced Immune Protection in Small Abalone Haliotis diversicolor Against a Secondary Infection With Vibrio harveyi.

In recent years, more and more studies have shown that early pathogenic bacterial infection in invertebrates can enhance immunity and significantly reduce mortality when reinfected with the same pathogen. There are mechanisms to explain this phenomenon, but they are relatively few. In addition, dose-dependent primary infection is also associated with increased immunity. In the present study, the initial infection dose and mortality of abalone Haliotis diversicolor after reinfection with Vibrio harveyi were recorded, and the mechanism of immune enhancement was investigated by the transcriptomic response of abalone after two successive stimuli with V. harveyi. Priming with different concentrations of pathogen can enhance immunity; however, higher concentration is not always better. Compared with the first exposure, more genes were up-regulated after the second exposure. Among the commonly expressed genes, the immune related genes were significantly or persistently highly expressed after two infections and included pattern recognition receptors as well as immune effectors, such as toll-like receptors, perlucin 4, scavenger receptor class B-like protein, cytochrome P450 1B1-like, glutathione S-transferase 6, lysozyme and so on; in addition, these immune-related genes were mainly distributed in the pathways related to phagocytosis and calcium signaling. Among the specifically expressed genes, compared with the first infection, more genes were involved in the immune, metabolic and digestive pathways after the second infection, which would be more conducive to preventing the invasion of pathogens. This study outlined the mechanism of immune enhancement in abalone after secondary infection at the global molecular level, which is helpful for a comprehensive understanding of the mechanism of immune priming in invertebrates.

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).

Endosymbionts of Metazoans Dwelling in the PACManus Hydrothermal Vent: Diversity and Potential Adaptive Features Revealed by Genome Analysis.

Deep-sea hydrothermal vent communities are dominated by invertebrates, namely, bathymodiolin mussels, siboglinid tubeworms, and provannid snails. Symbiosis is considered key to successful colonization by these sedentary species in such extreme environments. In the PACManus vent fields, snails, tubeworms, and mussels each colonized a niche with distinct geochemical characteristics. To better understand the metabolic potentials and genomic features contributing to host-environment adaptation, we compared the genomes of the symbionts of Bathymodiolus manusensis, Arcovestia ivanovi, and Alviniconcha boucheti sampled at PACManus, and we discuss their environmentally adaptive features. We found that B. manusensis and A. ivanovi are colonized by Gammaproteobacteria from distinct clades, whereas endosymbionts of B. manusensis feature high intraspecific heterogeneity with differing metabolic potentials. A. boucheti harbored three novel Epsilonproteobacteria symbionts, suggesting potential species-level diversity of snail symbionts. Genome comparisons revealed that the relative abundance of gene families related to low-pH homeostasis, metal resistance, oxidative stress resistance, environmental sensing/responses, and chemotaxis and motility was the highest in A. ivanovi's symbiont, followed by symbionts of the vent-mouth-dwelling snail A. boucheti, and was relatively low in the symbiont of the vent-periphery-dwelling mussel B. manusensis, which is consistent with their environmental adaptations and host-symbiont interactions. Gene families classified as encoding host interaction/attachment, virulence factors/toxins, and eukaryotic-like proteins were most abundant in symbionts of mussels and least abundant in those of snails, indicating that these symbionts may differ in their host colonization strategies. Comparison of Epsilonproteobacteria symbionts to nonsymbionts demonstrated that the expanded gene families in symbionts were related to vitamin B12 synthesis, toxin-antitoxin systems, methylation, and lipopolysaccharide biosynthesis, suggesting that these are vital to symbiont establishment and development in EpsilonproteobacteriaIMPORTANCE Deep-sea hydrothermal vents are dominated by several invertebrate species. The establishment of symbiosis has long been thought to be the key to successful colonization by these sedentary species in such harsh environments. However, the relationships between symbiotic bacteria and their hosts and their role in environmental adaptations generally remain unclear. In this paper, we show that the distribution of three host species showed characteristic niche partitioning in the Manus Basin, giving us the opportunity to understand how they adapt to their particular habitats. This study also revealed three novel genomes of symbionts from the snails of A. boucheti Combined with a data set on other ectosymbiont and free-living bacteria, genome comparisons for the snail endosymbionts pointed to several genetic traits that may have contributed to the lifestyle shift of Epsilonproteobacteria into the epithelial cells. These findings could increase our understanding of invertebrate-endosymbiont relationships in deep-sea ecosystems.

Bacterial diversity in the clarki ecotype of the photosynthetic sacoglossan, Elysia crispata.

Few studies have examined the bacterial communities associated with photosynthetic sacoglossan sea slugs. In this study, we determined the bacterial diversity in the clarki ecotype, Elysia crispata using 16S rRNA sequencing. Computational analysis using QIIME2 revealed variability between individual samples, with the Spirochaetes and Bacteroidetes phyla dominating most samples. Tenericutes and Proteobacteria were also found, among other phyla. Computational metabolic profiling of the bacteria revealed a variety of metabolic pathways involving carbohydrate metabolism, lipid metabolism, nucleotide metabolism, and amino acid metabolism. Although associated bacteria may be involved in mutually beneficial metabolic pathways, there was a high degree of variation in the bacterial community of individual slugs. This suggests that many of these relationships are likely opportunistic rather than obligate and that many of these bacteria may live commensally providing no major benefit to the slugs.

Bivalent Metal-Chelating Properties of Harzianic Acid Produced by Trichoderma pleuroticola Associated to the Gastropod Melarhaphe neritoides.

Harzianic acid is a secondary metabolite of Trichoderma, structurally belonging to the dienyltetramic acid subgroup of the tetramic acids. Biological activities of harzianic acid are of great interest for its antimicrobial and plant growth-promoting activities, which might be related to its chelating properties. In the present work harzianic acid, isolated from cultures of a strain of Trichoderma pleuroticola associated to the gastropod Melarhaphe neritoides, was studied as a complexant agent of a number of biologically relevant transition metals (i.e., Zn2+, Fe2+, Cu2+, and Mn2+), using UV-VIS, potentiometry, MS and NMR techniques. Our findings show the coordination capacity of harzianic acid toward the above cations through the formation of neutral or charged complexes in a variable ratio depending on the metal and pH conditions.

A novel alphaproteobacterium with a small genome identified from the digestive gland of multiple species of abalone.

We identified an alphaproteobacterium in the digestive gland of the abalone species Haliotis discus hannai. This phylotype dominated our 16S rRNA clone libraries from the digestive gland of H. discus hannai. Diversity surveys revealed that this phylotype was associated with H. discus hannai and also in another host species, H. gigantea. Whole genome phylogenies placed this bacterium as a new member affiliated with the family Rhodospirillaceae in Alphaproteobacteria. Gene annotation revealed a nearly complete glycolysis pathway but no TCA cycle, but the presence of anaerobic ribonucleoside-triphosphate reductase and oxygen-insensitive NAD(P)H-dependent nitroreductase, which show the genomic potential for anaerobic metabolism. A large cluster of genes encoding ankyrin repeat proteins (ANK) of eukaryotic-like repeat domains and a large gene set for the flagellar system were also detected. Alginate-binding periplasmic proteins and key genes responsible for alginate assimilation were found in the genome, which could potentially contribute to the breakdown of the host's alginate-rich macroalgal diet. These results raise the possibility that this novel alphaproteobacterium is a widespread member of the abalone microbiome that may use polysaccharides derived from its host's macroalgal diet.

Abalone Withering Syndrome Disease Dynamics: Infectious Dose and Temporal Stability in Seawater.

Withering syndrome (WS) is a chronic bacterial disease that affects numerous northeastern Pacific abalone Haliotis spp. The causative agent of WS is an obligate intracellular Rickettsiales-like bacterium (WS-RLO) that remains unculturable, thereby limiting our understanding of WS disease dynamics. The objectives of our study were to (1) determine the temporal stability of WS-RLO DNA outside of its abalone host in 14°C and 18°C seawater, (2) develop a standardized protocol for exposing abalones to known concentrations of WS-RLO DNA, and (3) calculate the dose of WS-RLO DNA required to generate 50% infection prevalence (ID50) in the highly cultured red abalone Haliotis rufescens. The WS-RLO stability trials were conducted in October 2016, February 2017, and June 2017. A quantitative PCR (qPCR) analysis was used to quantify bacterial DNA for 7 d in seawater collected at an abalone farm in southern California, where the pathogen is now endemic. For all trials and temperature treatments, WS-RLO DNA was unstable in seawater for longer than 2 d. To determine an ID50, groups of uninfected juvenile red abalone were subjected to 3-h bath exposures with four concentrations of WS-RLO at 0, 103 , 104 , and 105 DNA copies/mL. Abalone feces were tested biweekly for the presence of WS-RLO DNA, and abalone tissues were sampled 9 weeks postinfection for histological and qPCR analyses. The ID50 results indicated that our protocol was successful in generating WS-RLO infections; a pathogen dose of 2.3 × 103 DNA copies/mL was required to generate a 50% infection prevalence in red abalone tissue. These findings are critical components of disease dynamics that will help assess WS transmission risk within and among abalone populations and facilitate appropriate management and restoration strategies for both wild and cultured abalone species in WS-endemic areas.

Aeromonas spp. Isolated from Pacific Abalone (Haliotis discus hannai) Marketed in Korea: Antimicrobial and Heavy-Metal Resistance Properties.

Antimicrobial and heavy-metal resistance of 29 Aeromonas spp. (Aeromonas hydrophila n = 9, Aeromonas enteropelogenes n = 14, Aeromonas veronii n = 3, Aeromonas salmonicida n = 2, and Aeromonas sobria n = 1) isolated from Pacific abalone marketed in Korea were analyzed. All isolates were found to be resistant against ampicillin. High level of resistant to cephalothin (86%), rifampicin (73%), imipenem (42%), and oxytetracycline (35%) were also detected. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) index ≥ 0.2. The PCR assays implied the presence of qnrS, qnrB, qnrA, tetB, tetA, aac (3')- IIa, aac(6')-Ib, aphAI-IAB, blaCTX, blaTEM, and intI1 genes among 76%, 28%, 14%, 17%, 3%, 3%, 41%, 10%, 41%, 28%, and 66% of the isolates, respectively. Class 1 integron gene cassette profiles aadA1(3%) and aadA2 (3%) were also identified. Lead (Pb) resistance was the highest (69%) among 5 heavy metals tested, whereas 38%, 27%, and 20% of the isolates were resistant to Cadmium (Cd), Chromium (Cr), and Copper (Cu), respectively. Heavy-metal resistance genes, CopA, CzcA, and merA were positive in 83%, 75%, and 41% of the isolates, respectively. In conclusion, observed genotypic and phenotypic resistance profiles of Aeromonas spp. against antimicrobials and heavy metals reveal the ability of serving as a source of antimicrobials and heavy-metal-resistant traits.

New polyketides and diterpenoid derivatives from the fungus Penicillium sclerotiorum GZU-XW03-2 and their anti-inflammatory activity.

Two new compounds, named penisclerotiorin A (1) and diaporthein C (8), and a new natural product, penidepsidone A (4), together with five known compounds (2, 3, 5-7) were isolated from the fungus Penicillium sclerotiorum GZU-XW03-2. Their structures were assigned using spectroscopic methods, quantum chemical calculations, and single-crystal X-ray diffraction analysis. Penisclerotiorin A (1) that belongs to the highly oxidized diphenyl ether is rare found in natural sources, and it was the sixth example of highly oxidized diphenyl ether analogues in natural sources. Penidepsidone A (4) is a new natural product and no any NMR spectral data were reported to date, in this paper, we firstly used the NMR calculations to confirm the intact structure by comparison of the experimental NMR data. Diaporthein C (8) represents the third example of pimarane diterpenes bearing a double bond at C-8 and C-9. In the bioassays, all of the isolates (1-8) were tested for their anti-inflammatory effects on the production of nitric oxide in lipopolysaccharide-induced microglial cells (RAW 264.7 cells). Compounds 2, 3 and 6 showed potent anti-inflammatory effects than the positive control (indomethacin, IC50, 24.0 µM) with IC50 values of 11.52, 8.13 and 21.27 µM, respectively.