Microbiota of the Digestive Glands and Extrapallial Fluids of Clams Evolve Differently Over Time Depending on the Intertidal Position.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Alterins, a new family of marine antibacterial cyclolipopeptides.

Five strains of Pseudoalteromonas, isolated from oyster haemolymph, have exhibited antibacterial activity against several Gram-negative bacteria. Bioactive compounds have been identified in their cell-free supernatant and characterised as alterins, which are cyclolipopeptides comprising a heptapeptidic ring connected to a fatty acid chain. Using ultra-performance liquid chromatography-high-resolution mass spectrometry, this paper describes 37 structural analogues differing from each other by one or more amino acid residue, the length of the fatty acid chain, its hydroxylation and the presence of unsaturation.

Pseudoalteromonas ostreae sp. nov., a new bacterial species harboured by the flat oyster Ostrea edulis.

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA-DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1-2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7-8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas. In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

The marine intertidal zone shapes oyster and clam digestive bacterial microbiota.

Digestive microbiota provide a wide range of beneficial effects on host physiology and are therefore likely to play a key role in marine intertidal bivalve ability to acclimatize to the intertidal zone. This study investigated the effect of intertidal levels on the digestive bacterial microbiota of oysters (Crassostrea gigas) and clams (Ruditapes philippinarum), two bivalves with different ecological niches. Based on 16S rRNA region sequencing, digestive glands, seawater and sediments harbored specific bacterial communities, dominated by operational taxonomic units assigned to the Mycoplasmatales,Desulfobacterales and Rhodobacterales orders, respectively. Field implantation modified digestive bacterial microbiota of both bivalve species according to their intertidal position. Rhodospirillales and Legionellales abundances increased in oysters and clams from the low intertidal level, respectively. After a 14-day depuration process, these effects were still observed, especially for clams, while digestive bacterial microbiota of oysters were subjected to more short-term environmental changes. Nevertheless, 3.5 months stay on an intertidal zone was enough to leave an environmental footprint on the digestive bacterial microbiota, suggesting the existence of autochthonous bivalve bacteria. When comparing clams from the three intertidal levels, 20% of the bacterial assemblage was shared among the levels and it was dominated by an operational taxonomic unit affiliated to the Mycoplasmataceae and Spirochaetaceae families.

High hydrostatic pressure induced extraction and selective transfer of ß-phosvitin from the egg yolk granule to plasma fractions.

Egg yolk phosvitin is of particular interest due to its functional and biological properties. Recently, it was demonstrated that high hydrostatic pressure (HHP) (400 MPa for 5 min) induced the transfer of folic acid and phosvitin from the egg yolk granule to the plasma fraction. A granule fraction (Gin) produced by egg yolk centrifugation was pressure-treated at 400 and 600 MPa for 5 and 10 min, and centrifuged to generate granule fractions (GP1 to GP4) and plasmas (PP1 to PP4). Iron and phosphorus contents were also increased in PP1 to PP4 fractions, confirming the transfer of phosvitins from pressure-treated granule to plasma. Pressurization drastically improved phosvitin recovery in PP fractions, specifically at 600 MPa for 10 min, which had the highest value of phosvitin/100 mg of dry plasma at 33.3 ± 4.39 mg. Consequently, HHP represents an alternative approach for phosvitin transfer and recovery in the egg yolk soluble fraction.

Identification of A Novel Antibacterial Peptide from Atlantic Mackerel belonging to the GAPDH-Related Antimicrobial Family and Its In Vitro Digestibility.

The Atlantic mackerel, Scomber scombrus, is one of the most fished species in the world, but it is still largely used for low-value products, such as bait; mainly for crustacean fishery. This resource could be transformed into products of high value and may offer new opportunities for the discovery of bioactive molecules. Mackerel hydrolysate was investigated to discover antibacterial peptides with biotechnological potential. The proteolytic process generated a hydrolysate composed of 96% proteinaceous compounds with molecular weight lower than 7 kDa. From the whole hydrolysate, antibacterial activity was detected against both Gram-negative and Gram-positive bacteria. After solid phase extraction, purification of the active fraction led to the identification of 4 peptide sequences by mass spectrometry. The peptide sequence N-KVEIVAINDPFIDL-C, called Atlantic Mackerel GAPDH-related peptide (AMGAP), was selected for chemical synthesis to confirm the antibacterial activity and to evaluate its stability through in vitro digestibility. Minimal inhibitory concentrations of AMGAP revealed that Listeria strains were the most sensitive, suggesting potential as food-preservative to prevent bacterial growth. In addition, in vitro digestibility experiments found rapid (after 20 min) and early digestibility (stomach). This study highlights the biotechnological potential of mackerel hydrolysate due to the presence of the antibacterial AMGAP peptide.

Protective Efficacy of a Pseudoalteromonas Strain in European Abalone, Haliotis tuberculata, Infected with Vibrio harveyi ORM4.

The hemolymph of healthy marine invertebrates is known to harbor antibiotic-producing bacteria belonging to the genus Pseudoalteromonas. Such strains are potential probiotics to control infectious diseases in aquaculture. In the present study, we screened a collection of Pseudoalteromonas strains isolated from the hemolymph of oyster and mussel for antimicrobial activity against Vibrio harveyi, a pathogenic species responsible for high mortality in abalone. Subsequently, the protective efficacy of the most active strain named hCg-6 was investigated in abalone culture faced with a Vibrio harveyi ORM4 infection. First, we have controlled the Pseudoalteromonas hCg-6 safety for abalone health. To that end, animals were immersed for 4 h in Pseudoalteromonas hCg-6 suspensions in seawater. The abalone viability was monitored and Pseudoalteromonas hCg-6 was tracked by quantitative-PCR in abalone hemolymph. After immersion, no abalone death occurred while the strain hCg-6 was significantly detected in hemolymph. Therefore, the strain hCg-6 was considered safe for abalone and evaluated for its ability to protect abalone against V. harveyi (injection of 1 × 103Vibrio per animal). A 4-h long immersion of abalone in a seawater suspension of Pseudoalteromonas hCg-6 (1 × 106 CFU mL-1) prior to infection with Vibrio harveyi significantly improved the abalone viability. Indeed, 15 days post infection, the hCg-6 treatment used increased the abalone survival rate from 16% in untreated animals to 40% in treated abalone. We hypothesized that Pseudoalteromonas hCg-6 antibacterial activity increased the hemomicrobiota shielding effect. In conclusion, Pseudoalteromonas hCg-6 is a promising anti-Vibrio strain for abalone culture.

Identification of Peptides Implicated in Antibacterial Activity of Snow Crab Hepatopancreas Hydrolysates by a Bioassay-Guided Fractionation Approach Combined with Mass Spectrometry.

Snow crab (Chionoecetes opilio) by-products are a rich source of biomolecules, such as lipids, proteins, and chitin, which have not been extensively investigated. This study aims to identify antibacterial peptides to enhance the value of C. opilio by-products. After hydrolysis of different component parts using Protamex®, and concentration by solid-phase extraction, the resulting fractions were tested for antibacterial activity against Escherichia coli, Listeria innocua, and Vibrio parahaemolyticus. Hepatopancreas was the only tissue to display antibacterial activity detected using this protocol. Four fractions obtained with and without enzymatic hydrolysis of hepatopancreas followed by SPE C18 fractionation and elution with 50 and 80% acetonitrile demonstrated bacteriostatic activity against L. innocua HPB13, from concentrations of 0.30 to 43.05 mg/mL of peptides/proteins. Eleven peptides sharing at least 80% amino acid homology with four antimicrobial peptides were identified by mass spectrometry. Two peptides had homology to crustin-like and yellowfin tuna GAPDH antimicrobial peptides belonging to the marine organisms Penaeus monodon and Thunnus albacares, respectively. Other peptide sequence homologies were also identified: Odorranain-C7 from the frog Odorrana grahami and a predicted antibacterial peptide in the Asian ladybeetle Harmonia axyridis. These active peptides may represent a novel group of bioactive peptides deserving further investigation as food preservatives.

Spotlight on Antimicrobial Metabolites from the Marine Bacteria Pseudoalteromonas: Chemodiversity and Ecological Significance.

This review is dedicated to the antimicrobial metabolite-producing Pseudoalteromonas strains. The genus Pseudoalteromonas hosts 41 species, among which 16 are antimicrobial metabolite producers. To date, a total of 69 antimicrobial compounds belonging to 18 different families have been documented. They are classified into alkaloids, polyketides, and peptides. Finally as Pseudoalteromonas strains are frequently associated with macroorganisms, we can discuss the ecological significance of antimicrobial Pseudoalteromonas as part of the resident microbiota.

The impacts of handling and air exposure on immune parameters, gene expression, and susceptibility to vibriosis of European abalone Haliotis tuberculata.

Wild or farmed abalone are regularly exposed to stressors, such as air exposure and handling. Immune and transcriptional responses as well as susceptibility to vibriosis of sexually mature or immature European abalone acclimated at 16 or 19 °C were determined following handling or air exposure. Hemocyte density and H2O2 production increased while hemocyte viability and phagocytic index decreased following handling. Air exposure induces a decrease of hemocyte density and phagocytic index. Measurement of the expression of genes implicated in general metabolic, immunological and stress responses in gills, foot-muscle and hemocytes by real time q-PCR suggested that both stressors lead to a metabolic rate depression, characterized by a general inhibition of transcription. Finally, following handling a Vibrio harveyi challenge enhances almost 100% mortality of sexually immature animals at 19 °C while it has been previously demonstrated that only mature are susceptible to vibriosis.