**2. Innate immunity in** *Bathymodiolus azoricus*

last few years, a series of investigations, aimed at characterizing molecular indicators of adaptation processes of which components of the host defense system has received most attention. This study reviews recent advances on the characterization of molecules and genes participating in immune reactions, using *in vivo* and *ex vivo* models, to elucidate cellular and humoral defense mechanisms in vent mussels and the strategies they have

**Keywords:** innate immunity, deep‐sea hydrothermal vents, chemosynthetic ecosystems, long‐term acclimatization, host‐symbionts interactions, endosymbionts, differential gene expression, transcriptomics, mollusc bivalve, hydrothermal vent mussel,

Deep‐sea hydrothermal vents were discovered 40 years ago in the Galapagos Rift [1] reveal‐ ing for the first time, to the amazement of the scientific community, unusual life forms that have developed unique biochemical adaptations to high temperatures and toxic chemical nature of vent surrounding, otherwise harmful to life as we know it on the surface of the planet [2–5]. The animals dwelling around the vent sites exhibit high productivity and thus must cope with the seemingly deleterious physical and chemical conditions, while develop‐ ing surprisingly successful strategies to withstand adverse environmental conditions, includ‐ ing environmental microbes and mechanical stress whether due to animal predation or from

At such depths and in the absence of light, life is thriving in chemosynthesis‐based ecosys‐ tems where most abundant marine invertebrates have developed mutualistic relationships with chemosynthetic bacteria. These symbiotic interactions are believed to play a crucial role in the survival of hydrothermal vent animals, driving their transcriptional activities, and their successful adaptation strategies to subsist under extreme environmental conditions. They essentially rely on the establishment of endosymbiosis relationships between vent animals

Deep‐sea vent mussels of the *Bathymodiolus* genus are dominant members at hydrothermal vents and cold seep habitats. These mussels have the peculiarity of sheltering both endosymbi‐ otic sulfide‐oxidizing and methane‐oxidizing bacteria in their gills [9–13], supporting thus their endurance within this type of environment. *Bathymodiolus azoricus* is also the dominant species in deep‐sea hydrothermal vents in the Azores region and is well adapted to extreme conditions particularly to toxic concentrations of heavy metals, acidic pH, and absence of light [14–17].

In an attempt to understand physiological reactions of animals normally set to endure extreme conditions, in deep‐sea environments, our laboratory has undertaken, for the last 6 years, a series of investigations aimed at characterizing molecular indicators of adaptation processes of which components of the immune and stress‐related systems have received most of our attention [18, 19]. Central to our studies is the long‐term maintenance of vent mussels to atmospheric pressure proven to be a useful model to study unique molecular relationships under which the regulation of gene transcription may be affected by aquaria conditions and

and sulfur‐oxidizing (SOX) or methane‐oxidizing (MOX) bacteria [9–13].

adopted to survive under extreme environments.

*Bathymodiolus azoricus*

162 Organismal and Molecular Malacology

deep‐sea volcanic eruptions [6–8].

**1. Introduction**

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell‐mediated and humoral reactions to over‐ come the pathogens that naturally occur in the marine environment [27]. Growing interest in deep‐sea vent biology has turned the vent mussel *B. azoricus* into a model organism cen‐ tered on research activities based on the premise that vent mussels clearly have need for an immune system to overcome microbial challenges in their natural surroundings. For this rea‐ son, our research strategies have been focused on the molecular characterization of molecules participating in immune reactions, using *in vivo* and *ex vitro* models, to elucidate cellular and humoral defense mechanisms in vent mussels and its survival strategies under extreme environments. As for other bivalves, the innate immune system of *B. azoricus* is based on cel‐ lular constituents and soluble hemolymph (blood) factors, which play a prominent role in protecting the animals against invading microorganisms. The circulating hemocytes or blood cells are mostly found in the hemolymph and extrapallial fluid. They are responsible for cell‐ mediated defense reactions such as phagocytosis and the activation of a variety of cytotoxic reactions including the release of lysozomal enzymes and antimicrobial peptides [28–30]. Moreover, the generation of highly reactive oxygen intermediates (ROIs) and nitric oxide also plays an important defense role against pathogens [30–33]. Besides their decisive role in protecting the host from microbial assaults, bivalve hemocytes have also been implicated in other important physiological functions, including nutrient transport, digestion, wound heal‐ ing and shell regeneration and/or mineralization, and excretion [34]. In addition, the hemo‐ lymph serum contains humoral defense factors such as lectins and cytokine‐like molecules that are directly and indirectly involved in the killing of pathogens and in mediating cell‐cell interactions, respectively. Lectins are important mediators of cellular reactions and exhibit opsonin properties, which facilitate the phagocytosis [35–39]. The hemolymph also contains antibacterial factors and lysozomal components that ensure, along with hemocyte phagocytic and cytotoxic processes, the clearance of pathogenic bacteria [38, 39]. Using a combination of light microscopy and staining procedures, three major hemocyte types are discernible in the extrapallial fluid and hemolymph of *B. azoricus*. The most abundant type was identified as granulocyte readily recognizable by their cytoplasmic granules [19]. They appear fairly homogeneous in size and showing a characteristic crescent, or half‐moon shape morphol‐ ogy upon adherence to glass slides and before migratory movements. Granulocytes spread well onto the glass surface averaging 30–40 μm in length. In contrast, hyalinocytes presented smoother cytoplasm, i.e., a nongranular appearance due to a lower amount of cytoplasmic granules noticeable under phase contrast and differential interference contrast visualizations [19]. A third less common hemocyte type was also observed. They correspond to hemoblast‐ like cells and presented a spherical shape appearance with higher nucleus to cytoplasm ratio when compared to granulocytes and hyalinocytes [19]. *In vitro* phagocytic assays carried out with *B. azoricus* hemocytes revealed that 70% of the hemocytes containing more than two zymosan particles were granulocytes and to a lesser extent the percentage of phagocytic cells corresponding to hyalinocytes was 23%. In contrast, the percentage of hemoblasts containing ingested zymosan particles was 5–7%, the lowest revealed in our studies [19].

Along with hemocytes studies, we began to tackle signaling pathways putatively involved in the mediation of cellular responses in the presence of *Vibrio* spp. It was demonstrated that compounds of microbial origin could trigger detectable phosphorylation events in *B. azoricus* hemocyte extracts and likely involving the activation of different classes of mitogen‐activated protein kinases (MAPKs). When challenged with a marine bacterium, *Vibrio parahaemolyticus* or a nonmarine bacterium, *Bacillus subtilis*, to stimulate hemocytes, cellular proteins were dif‐ ferently phosphorylated as demonstrated in Western blotting experiments using the MAPK/ ERK, p38, and JNK rabbit polyclonal antibodies. Moreover, the differences seen in phosphor‐ ylation patterns could be attributed to inherent properties of the bacterial strain used, differ‐ ences in the mechanisms of binding to hemocytes, or differential activation of cell membrane receptors and signaling pathways, resulting in different patterns of protein phosphorylation. Western blotting analyses suggest that *B. azoricus* hemocytes display receptors with binding affinities toward microbial molecules or to live bacteria [19].
