**5. Host‐endosymbiont interactions: implications for host immunity and defense mechanisms against bacterial pathogens**

The transcriptome sequencing of gill tissues from the mussel *B. azoricus* revealed a set of genes of bacterial origin, providing a functional insight into the microbial vent community [71]. The transcripts supported a metabolically active microbiome and a variety of bacterial mechanisms and pathways, among which the fixation of carbon, the use of nitrate as a terminal acceptor of electrons and oxidation of sulfur and methane. The bacterial genes ensued from this sequenc‐ ing work were deemed relevant to evaluate the influence of abiotic and biotic environmental conditions on *B. azoricus* transcriptional activity and also potentially useful to assess sym‐ biont density differences in vent animals originated from distinct hydrothermal vent sites, respectively, to their environmental settings [72]. Keeping in line with the assumption that geographically distinct vent mussels will adopt different physiological statuses in relation to their environmental conditions, we also surmised that the relative abundance of methano‐ trophic and sulfide oxidizing endosymbiotic bacteria would differ between the Menez Gwen and Lucky Strike mussels as previously reported by other researchers [12, 58, 73]. We hypoth‐ esized that geographically distinct *B. azoricus* individuals may be experimentally traced back to their original hydrothermal vent sites based on their bacterial transcriptional activity and bacterial gill densities at the time animals were retrieved from the shallower Menez Gwen and deeper Lucky Strike vent sites. A taxonomical structure of the vent mussel gill's microbiome was also assessed to determine the bacterial community composition of gill tissue from MG and LS mussels to infer the symbiont densities differences between animals from both vent sites. Results from the ribosomal RNA amplicon sequencing of the V6 hypervariable regions, by massive parallel 454 pyrosequencing, indicated that the percentage of sequences obtained was from endosymbiont bacteria at nearly the same proportion between Menez Gwen and Lucky Strike samples. Moreover, comparative analyses based on BLAST searches in the RDP database, using the 16S rRNA OTU sequences, revealed that the thiotrophic endosymbiont represented 90% of all the sequences and methanotrophic endosymbiont almost 5% of the sequences from vent mussel samples originated from the distinct Menez Gwen and Lucky Strike hydrothermal vent fields [72].

While the majority of our experiments using live vent mussels were performed shortly after their retrieval from the Menez Gwen hydrothermal vent, long‐term studies with vent mussels acclimatized to atmospheric pressure conditions have hardly been addressed until recently. As above‐mentioned, long‐term acclimatization experiments in aquarium systems have allowed us to study the expression of bacterial symbionts genes, particularly methanotrophic and thiotrophic bacteria, over time of acclimatization while their mussel host is faced with drastic physiological challenges, metabolic adaptations, and food intake changes in an effort to adapt to an aquarium environment at atmospheric pressure and without supplementation

**Figure 6.** Expression plots across the five experimental conditions, "3 months"; "IPOCAMP"; "cage"; "*Vibrio,*" and "Wound," representing differential gene expression analyses using EdgeR. The top‐scoring BLAST hit for each of the

genes exemplified is shown on top of the respective expression plot.

174 Organismal and Molecular Malacology

of methane and sulfur [56]. The physiological adaptation to aquarium environment is likely to be aggravated by the expelling of endosymbionts into the aquarium environment, progres‐ sively emptying the gill tissue of its autotrophic bacteria, essential for the host vent mussel nutritional sustenance. Long‐term aquarium acclimatization represents thus a model study to investigate the presence and maintenance of symbiotic associations between chemosynthetic bacteria and vent animals, which depend on controlled cell‐cell communication between host and endosymbionts and the role of the host immune system [56, 74].

Presumably, the loss of endosymbiont induces a dramatic change in host gene expression profiles especially if endosymbiont genes exert some transcriptional control over host gene expression. For this reason, acclimatization studies have been instrumental to further our understanding of *B. azoricus* immune system. These studies have provided insights into physiological principles underlying mechanisms of adaptation to aquarium conditions at sea level pressure while taking advantage of the remarkable capacity of vent mussels to survive well decompression once brought to surface [21, 22, 56]. Furthermore, these studies have allowed analyses using immune challenged mussels comparatively to acclimatized control mussels, maintained under aquarium conditions. In view of our previous experiments per‐ formed with live gill tissues and postcapture immune gene expression studies in *B. azoricus* acclimatized to atmospheric pressure, the presence of endosymbiont bacteria is now being under investigation as a driving factor under which host‐immune genes may transcription‐ ally be modulated and reciprocally endosymbiont genes may transcriptionally be modulated by the host [53–56]. Moreover, the impact of aquarium acclimatization on *B. azoricus* immune responses and its capacity to react to *V. diabolicus* challenges was recently evaluated dur‐ ing recurrent incubations with *V. diabolicus* during short periods of time, followed by clean sea water incubations allowing animals to depurate and subsequently be reexposed to the same load of *V. diabolicus* over a period of 3 weeks acclimatization experiment [74]. As pre‐ viously described, we found a time‐dependent immune gene response in *B. azoricus* tied to the endosymbiont presence inside the vent mussel gills. The vent mussel's immune defense capabilities were affected by the gradual loss of symbiont bacteria suggesting a symbiont‐ mediated defense mechanism under which the transcriptional regulation of host immune genes is directly affected by symbiont density and/or activity. The host‐immune system‐ endosymbiont interactions were actively higher during the first week of acclimatization as a result of *Vibrio* exposures, demonstrating the ability of *B. azoricus* to increase the transcrip‐ tion of immune genes while endosymbiont gene expression also correlated with an increased symbiotic metabolism and prevalence. A synergistic response was proposed to counteract the presence and potential infection by *V. diabolicus* bacterium while modulating *B. azoricus* immune defenses‐endosymbiont interactions to an extant, which host‐immune and endosym‐ biont genes are mutually reliant during the first weeks of acclimatization [74]. The evidence presented suggests successful *V. diabolicus* recognition prompting immune genes to increase their levels of transcriptional activity particularly for genes involved in the Toll‐like receptor signaling [75, 76] and apoptosis‐related pathways [77] during first day of acclimatization in aquarium environments. In agreement with this, *B. azoricus* is presented as a suitable model to study molecular interactions involving host‐mediated immune recognition events and adap‐ tation mechanisms, to mitigate apoptosis harmful effects induced by *Vibrio* exposure against which, endosymbionts were prompted to increase their transcriptional activity, evocative of a possible protection role to the host [74]. This work brings to light other questions relating to how the host‐immune system regulates the symbiont population within their gills and con‐ versely symbionts avoid being recognized and eliminated by the host. These topics are being further investigated in our group and focused on finding and characterizing the molecular mechanisms underlying the establishment (recognition and acquisition) and functioning of symbiosis between deep‐sea vent mussel *B. azoricus* and the methanotrophic and thiotrophic bacteria (gene expression, energy metabolism, regulation of symbiont population).

Interestingly, the study of intricate associations with chemosynthetic symbiont‐bacteria living in the gills of deep‐sea vent animals led us to the conception of a new pathogenesis model system based on an unconventional host‐symbiont model system. This new marine invertebrate model system, as for the ecotoxicological model *Mytilus* spp. [78] relies, instead, on its unique host‐immune‐symbiont bacteria interactions believed to play a crucial role in counteracting infectious pathogens. The establishment of invertebrate host pathogen systems may serve as suitable and useful models to study pathogenicity. The molecular mechanisms through which pathogens are able to colonize and overtake host's immune system, particu‐ larly during the initial phase of infection when molecular recognition of MAMPs is occurring, as the pathogen defines its route of entry, are expected to reveal new molecular strategies that could help developing new therapies in aquaculture diseases. Using the deep‐sea vent mussel *B. azoricus* as an alternate invertebrate model system to study pathogenesis brings a new perspective into the search for new drug targets that could directly interfere with patho‐ gen recognition processes and/or with *in situ* inflammatory process where immune cells (i.e., hemocytes) and cytokine‐like molecules are being mobilized. Indeed, in such host‐endosym‐ biont model systems, the role of endosymbiont‐derived molecules could have an important influence in mediating pathogenesis and in counteracting the deleterious effect of pathogens on the host immune system. From an experimental approach, several genera of bacterial fish pathogens may be used in *B. azoricus*, as infectious agents, e.g., *Vibrio*, *Flavobacterium*, *Pseudomonas*, *Aeromonas*, *Streptococcus*. Host and endosymbiont gene expression profiles may be studied during infection experiments carried with a given bacteria and genes that are markedly upregulated or downregulated further analyzed and their cDNA sequences deter‐ mined by traditional sequencing methods.

Particular attention should be given to genes whose encoded proteins are participating in signal transduction pathways directly influencing the outcome of immune effector molecules, as antibacterial peptides; immune recognition lectins and antioxidant products such as super‐ oxide dismutase, ferritin, metalloproteinases, metallothioneins, and heat shock proteins [26]. Synergistic effects resulting from interactions between host immune and endosymbiont activ‐ ity, in counteracting infectious pathogens, may now be studied at the molecular level, for future therapies design, targeting key steps during pathogen infection processes, for instance, host recognition events; production of the anti‐inflammatory factor TNF‐alpha and cytokine‐ like growth factors; enhancement of antibacterial molecules synthesis.
