Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilus galloprovincialis Caterina Ciacci a , Michele Betti a , Barbara Canonico a , Barbara Citterio b , Philippe Roch c , Laura Canesi d, * a DISUAN, Dipartimento di Scienze dell’Uomo, dell’Ambiente e della Natura, Italy b Dipartimento di Scienze Biomolecolari, Università ‘‘Carlo Bo” di Urbino, Italy c JRU Ecosystèmes Lagunaires, CNRS-Université de Montpellier 2, France d Dipartimento di Biologia, Università di Genova, Italy article info Article history: Received 8 February 2010 Accepted 13 May 2010 Available online 20 May 2010 Keywords: Bivalves V. splendidus LGP32 V. anguillarum Hemocytes Immune responses Cell signaling abstract In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated. In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguil- larum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytom- etry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Bivalves (such a mussels, clams, oysters...) can accumulate large numbers of bacteria as a consequence of their filter-feeding habit. In particular, Vibrio species are very abundant in coastal waters and are commonly isolated from edible bivalve tissues where they are accumulated (Wright et al., 1996) and where they can persist even after depuration processes (Pruzzo et al., 2005). Such a resistance to elimination is particularly evident for Vibrio spp. (such as Vibrio cholerae O1, Vibrio parahaemolyticus, Vibrio anguillarum)(Pruzzo et al., 2005). Persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bacte- ricidal activity of circulating hemocytes and hemolymph soluble factors, resulting from complex interactions between bacteria, bacterial components and host cells (Mitta et al., 2000; Canesi et al., 2002a). Host–pathogen interactions have been increasingly investi- gated in different bivalves, with the aim of understanding the path- ogenesis of diseases in cultured and wild populations of species susceptible to infection by certain Vibrio spp. and strains (Choquet et al., 2003; Lambert et al., 2003; Gay et al., 2004; Allam et al., 2006; Labreuche et al., 2006; Mateo et al., 2009). Some strains of Vibrio splendidus have been associated with the ‘summer mortali- ties’ syndrome of juvenile oysters Crassostrea gigas in France (Lac- oste et al., 2001; Gay et al., 2004). On the other hand, the mussel Mytilus is considered to be par- ticularly resistant to Vibrio infection, due to the presence of potent immune defence mechanisms. In Mytilus, studies involving differ- ent bacterial species and strains, heterologous cytokines and hor- mones, led to the identification of the role of conserved components of kinase-mediated transduction pathways, including cytosolic kinases (such as MAPKs-Mitogen Activated Protein Ki- 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.05.010 * Corresponding author. Address: Dipartimento di Biologia, Università di Genova, Corso Europa 26, 16132-Genova, Italy. Fax: +39 0103538267. E-mail address: Laura.Canesi@unige.it (L. Canesi). Journal of Invertebrate Pathology 105 (2010) 49–55 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip