**3.** *Ex vivo* **experiments with gill tissues: new insights into tissue immune specificity toward microorganisms**

As mytilid species, the deep‐sea vent mussel *B. azoricus* exhibits large lamellated gills (ctenidia) arranged as numerous filament structures stacked together through ciliary junctions. Each filament is organized in two coalescent epithelial cell sheets overlaying a central lumen where hemocytes can be found. The thinness of gill filaments allows for the visualization of live hemocytes through the epithelium, and to monitor hemocyte motility directly under light microscopy [19]. Bivalve mollusk gills assume thus a strategic importance at the interface between the external milieu and the internal body cavities of the animal where contact with microorganisms is inevitable during feeding processes inasmuch as host defense responses may incur from interactions with infective pathogens during normal filtration [36–39]. For this reason, a number of typical cellular and humoral immune reactions are likely to take place in gill tissues and observable as hemocyte proliferation and phagocytosis, the activation of immune signaling pathways, and the activation of genes involved in immune, antioxidant and antibacterial responses against invading bacterial pathogens or the presence of metal toxicants [36–39].

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].

affinities toward microbial molecules or to live bacteria [19].

**specificity toward microorganisms**

164 Organismal and Molecular Malacology

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

**3.** *Ex vivo* **experiments with gill tissues: new insights into tissue immune** 

As mytilid species, the deep‐sea vent mussel *B. azoricus* exhibits large lamellated gills (ctenidia) arranged as numerous filament structures stacked together through ciliary junctions. Each filament is organized in two coalescent epithelial cell sheets overlaying a central lumen where To further test the gill's ability to mount immune reactions, a series of *ex vivo* experiments have been performed using gill tissues freshly dissected from vent mussels and subjected to short‐term incubations in tissue culture well‐plates and under different experimental set‐ tings. Different stimuli were carried out to demonstrate the expression of genes in gill tissues exposed to a mixture of endosymbionts previously obtained from gill extracts, and *V. parahaemolyticus*, in comparison with sterile sea water incubations (**Figure 1**). Differential gene expression results indicated that exposure to methanotrophic and thiotrophic endosymbiont preparations led to general upregulation of genes involved in immune recognition reactions, without the addition of hemolymph, while gill incubations with endosymbiont extract and to which hemolymph serum (hemocyte free) was added led to an opposite effect, resulting in a lower expression of immune recognition genes. These results contrasted with incuba‐ tions performed with *V. parahaemolyticus* or with control sterile sea water. In this case, higher levels of gene expression were achieved when hemolymph was added to gill tissues incu‐ bated with *V. parahaemolyticus* or control sterile sea water (**Figure 1**). *Ex vivo* experiments as described bring evidence supporting a yet uncharacterized effect of hemolymph and its humoral constituents over endosymbionts, likely controlling immune gene expression of its host *B. azoricus*. This prompted the question of whether the host gill tissue would be able or not to recognize endosymbiont as self‐particles and to which extent the host immune system does not disturb the acquisition of endosymbionts by horizontal transfer during the host larval stages. Moreover, the permanence and survival of endosymbionts within gill tissue would require a fitted control over the host immune system, acting on the transcriptional reg‐ ulation of immune genes and at the level of pattern recognition receptors (PRRs) expressed by cells of the host innate immune system to detect microbial‐associated molecular patterns (MAMPs) present on the surface of microorganisms [40, 41]. This endosymbiont effect over the host immune system would likely require the presence of hemolymph and its humoral constituents, as demonstrated by the gill *ex vivo* experiments (**Figures 1** and **2**). Since the gill tissue does grow over the mussel life's time we also probed different gill sections to deter‐ mine levels of immune gene expression along the anterior‐posterior axis, notably the "bud‐ ding zone" on the posterior end, considered as the youngest section of the gill and through which endosymbionts are believed to make their entry. It was found that vent mussel gene expressions were markedly lower than other gill tissue sections, suggesting a thigh mecha‐ nism of transcriptional regulation of host genes in the presence of endosymbiont bacteria in the gill budding zone.

**Figure 1.** *Ex vivo* experiments performed with dissected gills. Gill fragments were incubated with *V. parahaemolyticus* and with an enriched preparation of endosymbionts freshly obtained from gill homogenates and gradient centrifugation. The effect of hemolymph humoral factors was tested by incubating gill fragments with and without hemolymph in the presence of *V. parahaemolyticus* and endosymbiont mixture. Results were compared to control incubations with plain sterile sea water. Gene expression was performed by qPCR targeting the immune recognition genes Aggrecan, C‐type lectin, C‐type lectin immune receptor, immune lectin receptor, lipopolysaccharide‐binding protein‐bactericidal/ permeability‐increasing protein, peptidoglycan recognition protein, and serine protease inhibitor‐2 [50]. Incubations performed with endosymbiont preparations distinctively induced immune recognition genes in the absence of hemocyte‐free hemolymph.

As filter feeders living most of their lives attached to a substrate, bivalves are exposed to constant biologically available pollutants over an extended period of time [42–44]. They have been studied as biological models to assess the impact of pollution in the environment and used as a biomonitoring "tool" due to their capacity of bioaccumulating high concentrations of trace metals, mostly in soft tissues such as gills and digestive gland [45–47]. The large sur‐ face of the gills and their involvement in gas exchange and feeding processes bring bivalves to constant and intimate contact with their environment where pathogens may also find their route of entry and encounter the bivalve first‐line immune defense reactions.

While marine bivalves living in sandy, rocky intertidal, and shallow subtidal environments may rely on well‐established humoral and cellular immune reactions to counteract pathogenic microorganisms, a new level of molecular intricacy may be seen between endosymbiont‐bearing An Insightful Model to Study Innate Immunity and Stress Response in Deep-Sea Vent Animals: Profiling the Mussel... http://dx.doi.org/10.5772/68034 167

**Figure 2.** *Ex vivo* experiments performed with dissected gills. As in **Figure 1**, gill fragments were incubated with distinct bacterial stimulants: an enriched methanotrophic bacterial preparation; an enriched thiotrophic bacterial preparation, a mixture of methanotrophic and thiotrophic bacterial preparations, *Vibrio parahaemolyticus* and *Photobacteria* bacterium. Results were compared to control incubations with plain sterile sea water. Gene expression was performed by qPCR targeting same immune recognition genes as in **Figure 1**. Separate methanotrophs and thiotrophs preparations induced higher levels of immune gene expressions when compared to a mixture of the two endosymbiont bacteria preparations. Incubations using *Vibrio parahaemolyticus* resulted in drastic downregulation of immune recognition genes.

bivalves living in anaerobic and sulfide‐rich environments and the pathogenic microorgan‐ isms they encounter. These natural molecular interactions would account for the role of endosymbionts in modulating the host immunity by controlling the transcriptional activ‐ ity of immune genes. Bivalve associations with chemoautotrophic endosymbionts are now well known and widely distributed across a range of different chemosynthetic environments, including deep‐sea hydrothermal vents (*Bathymodiolus* spp., *Calyptogena* sp.); gas seeps, mud volcanoes, and petroleum seeps (*Bathymodiolus* spp., *Calyptogena* sp.); whale and wood falls (*Idas* spp., *Adipicola* spp., *Vesicomya* sp., *Axinodon* sp.); and shallow water anoxic sediments mediated by sulfate reduction (*Solemya* spp., Codaki spp., *Anodontia* spp., *Lucina* spp.) [7].

As filter feeders living most of their lives attached to a substrate, bivalves are exposed to constant biologically available pollutants over an extended period of time [42–44]. They have been studied as biological models to assess the impact of pollution in the environment and used as a biomonitoring "tool" due to their capacity of bioaccumulating high concentrations of trace metals, mostly in soft tissues such as gills and digestive gland [45–47]. The large sur‐ face of the gills and their involvement in gas exchange and feeding processes bring bivalves to constant and intimate contact with their environment where pathogens may also find their

**Figure 1.** *Ex vivo* experiments performed with dissected gills. Gill fragments were incubated with *V. parahaemolyticus* and with an enriched preparation of endosymbionts freshly obtained from gill homogenates and gradient centrifugation. The effect of hemolymph humoral factors was tested by incubating gill fragments with and without hemolymph in the presence of *V. parahaemolyticus* and endosymbiont mixture. Results were compared to control incubations with plain sterile sea water. Gene expression was performed by qPCR targeting the immune recognition genes Aggrecan, C‐type lectin, C‐type lectin immune receptor, immune lectin receptor, lipopolysaccharide‐binding protein‐bactericidal/ permeability‐increasing protein, peptidoglycan recognition protein, and serine protease inhibitor‐2 [50]. Incubations performed with endosymbiont preparations distinctively induced immune recognition genes in the absence of

While marine bivalves living in sandy, rocky intertidal, and shallow subtidal environments may rely on well‐established humoral and cellular immune reactions to counteract pathogenic microorganisms, a new level of molecular intricacy may be seen between endosymbiont‐bearing

route of entry and encounter the bivalve first‐line immune defense reactions.

hemocyte‐free hemolymph.

166 Organismal and Molecular Malacology

Our recent results from *ex vivo* gill tissue experiment proven to be a valuable system for the study of tissue‐specific immune responses where the thin epithelial cell layers of gill filaments would make it possible to signal pathogen‐sensing directly through gill epithelia and affect‐ ing adjacent methanotrophic or thiotrophic endosymbionts which in turn would functionally prime host immune cells, the hemocytes, into altering their transcriptional activity (**Figure 3**). The endosymbiont immunomodulatory effect on the host immune system, as discussed in detail further below, is still under current investigations by our research team as the complex‐ ity of host‐endosymbiont interactions in the deep‐sea vent mussel *B. azoricus* remains to be

Normal immunological state: hemocyte PRRs are primed by endosymbiont MAMPs to steady-state levels.

Pathogen in n: endosymbiontmediated responses inducing increased PRRs a ity in hemocytes.

**Figure 3.** Hypothetical model representing the host‐endosymbiont‐mediated immune responses against pathogens. In a normal immunological state, hemocytes PRRs are being sensitized by host‐endosymbiont interactions allowing the vent mussel immune system to remain active and tolerant to the presence of MOX and SOX bacteria. Upon interacting with extracellular pathogens, host‐symbiont interactions are altered and incur in higher endosymbiont genes transcriptional activity [74] and subsequently affecting host hemocytes by triggering its immune repertoire via PRRs activation.

fully understood. This model is consistent with the hypothesis that innate immune receptors are required to promote long‐term colonization by microbiota. This emerging perspective chal‐ lenges current paradigms in immunology and suggests that PRRs may have evolved, in part, to mediate the bidirectional cross‐talk between microbial symbionts and their hosts [48, 49].
