Insight into the microbial communities associated with first larval stages of Mytilus galloprovincialis: Possible interference by estrogenic compounds.

The microbiota, the host-associated community of microbes, play important roles in health status and whole body homeostasis of all organisms, including marine species. In bivalves, the microbiota composition has been mainly investigated in adults, whereas little information is available during development. In this work, the microbiota composition of the first larval stages of Mytilus galloprovincialis was evaluated by 16S rRNA gene-based profiling, at 24 and 48 hours post fertilization in comparison with those of eggs and sperm. The main genera detected in both larvae (Vibrio, Pseudoalteromonas, Psychrobium, Colwellia) derived from eggs. However, a clear shift in microbiota was observed in developing larvae compared to eggs, both in terms of core microbiome and relative abundance of different genera. The results provide a first insight into the composition of the microbial communities associated with gametes and early larvae of mussels. Moreover, the impact on larval microbiome of estrogenic chemicals that potentially affect Mytilus early development, 17ßestradiol-E2, Bisphenol A-BPA and Bisphenol F-BPF (10 µg/L), was investigated. Exposure to estrogenic chemicals leads to changes in abundance of different genera, with distinct and common effects depending on the compound and larval stage. Both potential pathogens (Vibrio, Arcobacter, Tenacibaculum) and genera involved in xenobiotic biotransformation (Oleispira, Shewanella) were affected. The effects of estrogenic compounds on larval microbiome were not related to their developmental effects: however, the results address the importance of evaluating the impact of emerging contaminants on the microbiota of marine invertebrates, including larval stages, that are most sensitive to environmental perturbations.

Whole-Genome Enrichment Provides Deep Insights into Vibrio cholerae Metagenome from an African River.

The detection and typing of Vibrio cholerae in natural aquatic environments encounter major methodological challenges related to the fact that the bacterium is often present in environmental matrices at very low abundance in nonculturable state. This study applied, for the first time to our knowledge, a whole-genome enrichment (WGE) and next-generation sequencing (NGS) approach for direct genotyping and metagenomic analysis of low abundant V. cholerae DNA (<50 genome unit/L) from natural water collected in the Morogoro river (Tanzania). The protocol is based on the use of biotinylated RNA baits for target enrichment of V. cholerae metagenomic DNA via hybridization. An enriched V. cholerae metagenome library was generated and sequenced on an Illumina MiSeq platform. Up to 1.8 × 107 bp (4.5× mean read depth) were found to map against V. cholerae reference genome sequences representing an increase of about 2500 times in target DNA coverage compared to theoretical calculations of performance for shotgun metagenomics. Analysis of metagenomic data revealed the presence of several V. cholerae virulence and virulence associated genes in river water including major virulence regions (e.g. CTX prophage and Vibrio pathogenicity island-1) and genetic markers of epidemic strains (e.g. O1-antigen biosynthesis gene cluster) that were not detectable by standard culture and molecular techniques. Overall, besides providing a powerful tool for direct genotyping of V. cholerae in complex environmental matrices, this study provides a 'proof of concept' on the methodological gap that might currently preclude a more comprehensive understanding of toxigenic V. cholerae emergence from natural aquatic environments.

Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32.

Marine bivalves can accumulate large numbers of bacteria, in particular Vibrio species, whose persistence in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors. The interactions between vibrios and hemolymph have been investigated, in particular in bivalve species susceptible to infection by certain Vibrio spp. and strains. In this work, the effects of two bivalve pathogens, Vibrio splendidus LGP32 (V.s.) and Vibrio aestuarianus 01/032 (V.a.), isolated from oyster mortality outbreaks, on the hemocytes of Mytilus galloprovincialis were investigated. In vitro, V.s., but not V.a., induced a dramatic decrease in lysosomal membrane stability-LMS in the hemocytes; both vibrios induced a moderate lysozyme release, with V.s. > V.a.. The V.s.-induced decrease in LMS was mediated by activation of PI-3Kinase, as shown by use of different kinase inhibitors. TEM analysis showed rapid internalization of both vibrios; however, V.s. lead to cellular and lysosomal damage and was able to survive within the hemocytes, whereas significant killing of V.a. was observed. In vivo, in mussels challenged with either vibrio and sampled at 6, 24 and 96 h post-injection, transient decreases in hemocyte LMS and progressive increases in serum lysozyme activity were observed, with V.s. > V.a.. Moreover, whereas V.a. was efficiently cleared from hemolymph, V.s. showed significant growth, that was maximal at 24 h p.i. when lowest LMS values were recorded in the hemocytes. Both vibrios also induced significant decreases in LMS in the digestive gland, again with V.s. > V.a.. The results indicate distinct interactions between mussel hemocytes and the two vibrio strains tested. The effects of V.s. may be due to the capacity of this strain to interfere with the signaling pathways involved in hemocyte function, thus escaping the bactericidal activity of the host cell, as observed for certain mammalian pathogens. Although V.s. is considered not pathogenic to Mytilus, this vibrio strain can affect the lysosomal function at the cellular and tissue level, thus leading to stressful conditions.

A general role for surface membrane proteins in attachment to chitin particles and copepods of environmental and clinical vibrios.

AIMS: To investigate the role of surface membrane proteins (MP) to promote attachment to chitin particles and copepods of different environmental and clinical vibrios. METHOD AND RESULTS: The role of surface MP to promote attachment to chitin particles and the copepod Tigriopus fulvus was investigated in several environmental and clinical Vibrio strains by inhibition test methods. Attachment to both substrates was significantly inhibited by homologous MP treatment in all strains and percentages of inhibition were comparable with the ones observed with N-acetyl glucosamine (GlcNAc). Sarkosyl-insoluble MP extracted from tested strains were added to chitin particles either in the presence or in the absence of GlcNAc and the fraction bound to chitin in both conditions was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitin-binding proteins (CBP) defined as Sarkosyl-insoluble MP that bound chitin in the absence of GlcNAc but did not in the presence of the sugar were isolated in all strains. CONCLUSION: CBP are common in both environmental and clinical Vibrio strains and they have an important general role in mediating cell interactions with chitin-containing surfaces. SIGNIFICANT AND IMPACT OF THE STUDY: The role of CBP should be taken into account when investigating environmental persistence of aquatic vibrios.