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Vibrio aestuarianus is a bacterium related to mass mortality outbreaks of the Pacific oyster, Crassostrea gigas in Europe. In this study, the role of different planktonic substrates (phytoplankton cells, marine aggregates and chitin fragments) in mediating V. aestuarianus 02/041 infection of oysters was evaluated by controlled infection experiments. It was shown that phytoplankton cells and, to a greater extent, marine aggregates, significantly promote V. aestuarianus 02/041 intake by C. gigas maintained under stressful conditions in the laboratory. Such intake is associated with higher concentration of the pathogen in the bivalve hemolymph and compromised health status of infected oysters. In contrast, chitin particles do not play a significant role as transmission vector for V. aestuarianus 02/041 infecting its bivalve host. Interestingly, incorporation into marine aggregates foster extracellular proteases (ECPs) activity and a higher expression of bacterial virulence genes, that are potentially involved in bivalve infection. Results from this study contribute to elucidate transmission patterns of V. aestuarianus 02/041 to C. gigas that may be useful for the development of efficient measures to prevent and control oyster disease outbreaks. [ABSTRACT FROM AUTHOR]

Summary: In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio–bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators. [ABSTRACT FROM AUTHOR]

Recent results for Vibrio cholerae interactions with bivalves and chitin-containing substrates are reviewed. Chitin, composed of β-1,4-linked N-acetylglucosamine residues, is one of the most abundant biopolymers in nature and the most abundant in the marine environment. V. cholerae connection to chitin is a well known phenomenon and one of the best documented examples of a successful bacteria-substrate interaction, affecting both the lifestyle of the microorganisms and natural system functioning. In sea water, vibrios can be entrapped by filter-feeding invertebrates that sieve suspended food particles from the aquatic environment. Persistence of bacteria in bivalves depends on different factors including their sensitivity to the bactericidal activity of hemolymph. Both V. cholerae survival inside bivalves and chitin colonization depend on cell surface properties, with contribution of different ligands (e.g. chitin-binding proteins, MSHA). The role of these ligands in interactions with human surfaces is discussed and evidence is presented supporting (i) the link between vibrio persistence in the environment (both inside bivalves and adhering to chitin-containing surfaces) and infection of the human host, and (ii) the hypothesis that virulence mechanisms of bacteria that have environmental reservoirs represent a consequence of adaptive mechanisms to the environment. [ABSTRACT FROM AUTHOR]

Marine bivalves are widespread in coastal environments and, due to their filter-feeding habit, they can accumulate large numbers of bacteria thus acting as passive carriers of human pathogens. Bivalves possess both humoral and cellular defence mechanisms that operate in a co-ordinated way to kill and eliminate infecting bacteria.Vibriospecies are very abundant in coastal waters and are commonly isolated from edible bivalves tissues where they can persist after depuration processes in controlled waters. Such observations indicate that vibrios are regular components of bivalve microflora and that the molluscs can represent an important ecological niche for these bacteria. Here we tried to summarize data on the interactions between vibrios and bivalve haemolymph; the available evidence supports the hypothesis that persistence of bacteria in bivalve tissues depends, at least in part, on their sensitivity to the bactericidal activity of the haemolymph. Results obtained with anin vitromodel ofVibrio choleraechallenged againstMytilus galloprovincialishaemocytes indicate that bacterial surface components, soluble haemolymph factors and the signalling pathways of the haemocyte host are involved in determining the result of vibrio–haemolymph interactions. [ABSTRACT FROM AUTHOR]

Marine bivalves accumulate large amounts of bacteria from the environment (mainlyVibrionaceaeand coliforms). Although persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating haemocytes and haemolymph soluble factors, the mechanisms involved in bacteria–host cell interactions in these invertebrates are largely unknown. In the musselMytilus, differences in interactions between haemocytes and differentEscherichia coliandVibrio choleraestrains[ E. coliMG155, a wild-type strain carrying type 1 fimbriae, and its unfimbriated derivative, AAEC072Δ fim;V. choleraeO1 El Tor biotype strain N16961, carrying the mannose-sensitive haemagglutinin (MSHA), and its MSHA mutant] lead to differences in bactericidal activity in the presence of serum. Here we show that different bacteria induced distinct patterns of phosphorylation of mitogen-activated protein kinases (MAPKs), in particular of the stress-activated MAPKs involved in the immune response. Differences in phosphorylation of PKC-like proteins were also observed. The results support the hypothesis that, like in mammalian host cells, different bacteria can modulate the signalling pathways of mussel haemocytes. The lower anti-bacterial activity towards the mutantE. colistrain and wild-typeV. choleraecompared with wildE. colimay result from a reduced capacity of activating MAPKs. Moreover, the mutantV. choleraestrain that was the most resistant to the haemocyte bactericidal activity induced downregulation of cell signalling and showed the strongest effect on lysosomal membrane stability, evaluated as a marker of bivalve cell stress. These data suggest that certain bacteria could evade the bactericidal activity of mussel haemocytes through disruption of the host signalling pathways. [ABSTRACT FROM AUTHOR]

Abstract Vibrio coralliilyticus has emerged as a coral pathogen of concern throughout the Indo-Pacific reef. The interest towards understanding its ecology and pathogenic potential has increased since V. coralliilyticus was shown to be strongly virulent also for other species; in particular, it represents a serious threat for bivalve aquaculture, being one of the most important emerging pathogen responsible for oyster larval mortalities worldwide. V. coralliilyticus has a tightly regulated temperature-dependent virulence and it has been related to mass mortalities events of benthic invertebrates also in the temperate northwestern Mediterranean Sea. However, no data are available on the effects of V. coralliilyticus in the mussel Mytilus galloprovincialis , the most abundant aquacultured species in this area. In this work, responses of M. galloprovincialis to challenge with V. coralliilyticus (ATCC BAA-450) were investigated. In vitro, short term responses of mussel hemocytes were evaluated in terms of lysosomal membrane stability, bactericidal activity, lysozyme release, ROS and NO production, and ultrastructural changes, evaluated by TEM. In vivo, hemolymph parameters were measured in mussels challenged with V. coralliilyticus at 24h p.i. Moreover, the effects of V. coralliilyticus on mussel early embryo development (at 48 hpf) were evaluated. The results show that both in vitro and in vivo, mussels were unable to activate immune response towards V. coralliilyticus , and that challenge mainly induced lysosomal stress in the hemocytes. Moreover, V. coralliilyticus showed a strong and concentration-dependent embryotoxicity. Overall, the results indicate that, although M. galloprovincialis is considered a resistant species to vibrio infections, the emerging pathogen V. coralliilyticus can represent a potential threat to mussel aquaculture. Graphical abstract Image 1 Highlights • Evaluation of Mytilus immune responses to the emerging pathogen V. coralliilyticus. • In vitro and in vivo responses and developmental effects were measured. • Hemocyte lysosomal stress was observed, but no induction of immune parameters. • Strong and concentration-dependent effects on embryo development. • Mytilus is unable to mount an efficient immune response towards V. coralliilyticus. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes. In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis . The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis ; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios. [ABSTRACT FROM AUTHOR]

In bivalve molluscs, cell-mediated immunity is carried out by circulating hemocytes, resembling the monocyte/macrophage lineage of vertebrates, that can kill the microbes through phagocytosis and various cytotoxic reactions. Previous data demonstrated that activation of MAPKs (Mitogen Activated Protein Kinases) is involved in the response of mussel hemocytes (Mytilus galloprovincialis Lam.) to bacterial challenge. In this work, the possibility that modulation of components of tyrosine kinase-mediated cell signalling may participate in the activation of mussel hemocytes was investigated. Cell pre-treatment with the macrophage activator IFNγ significantly increased the bactericidal activity of mussel hemocytes towards E. coli. Human recombinant IFNγ stimulated tyrosine phosphorylation of different members of STAT-like proteins (Signal Transducers and Activators of Transcription), as evaluated by Western blotting of hemocyte protein extracts with specific anti-phospho-STAT antibodies. A similar increase in phosphorylation of immunoreactive STATs was observed in hemocytes incubated with E. coli, this indicating that tyrosine phosphorylation of STAT-like members represents a physiological step in hemocyte activation. IFNγ lead to persistent phosphorylation of immunoreactive STAT1, a transcription factor that plays a critical role in innate immunity towards Gram negative bacteria in mammalian systems; moreover, hemocyte pretreatment with IFNγ significantly increased bacteria-induced STAT1 phosphorylation, whereas IFNα did not. IFNγ also transiently affected the phosphorylation state of different MAPKs. The extent and time course of MAPK phosphorylation induced by IFNγ were distinct from those elicited by either IFNα or bacterial challenge. Overall, the results indicate that the hemocyte function can be modulated by heterologous cytokines and bacterial signals that act in concert through tyrosine kinase-mediated transduction pathways converging on STAT- and MAPK-like members. [Copyright &y& Elsevier]