**4. Posttranscriptomics studies in** *Bathymodiolus azoricus*

In 2010 a high‐throughput sequencing and analysis of the gill tissue transcriptome from the deep‐sea hydrothermal vent mussel *B. azoricus* was reported by our group [50]. It represented the first tissue transcriptional analysis of a deep‐sea hydrothermal vent animal, using next generation sequencing technology, enabling the creation of a searchable catalog of genes that provided a direct method of identifying and retrieving vast numbers of novel coding sequences which could then be applied in gene expression profiling experiments, using quan‐ titative polymerase chain reaction (qPCR), from a nonconventional model organism [50]. It provided the most comprehensive sequence resource for identifying novel genes currently available for a deep‐sea vent organism, in particular, genes putatively involved in immune and inflammatory reactions in vent mussels. This first transcriptional analysis of gill tissues from the deep‐sea hydrothermal vent *B. azoricus* was organized as a searchable catalog of genes providing a direct method of identifying and retrieving vast numbers of novel coding sequences, which can be applied in gene expression profiling experiments. The assembled and annotated sequences were organized in a dedicated database, accessible through the website http://transcriptomics.biocant.pt/deepSeaVent [50].

With an unprecedented high number of gene sequences available from our transcriptomic data, we were able to tackle signaling pathways and compare gene expression profiles in a series of experiments aiming at better understanding innate immunity in animals physiologi‐ cally programmed to endure deep‐sea vent conditions. Responses to bacterial infections with different strains of *Vibrio* wound experiments, long‐term acclimatization in aquarium condi‐ tions and pressurization experiments with the hyperbaric chamber IPOCAMP [51] became the main focus of our research, setting thus the grounds for more in‐depth analyzes reveal‐ ing distinct gene expression profiles behind unique molecular relationships under which the regulation of gene transcription may be affected by biotic factors including microorganisms, the presence of endosymbiont bacteria and shell damage incurring in opportunistic infections or by abiotic factor as the hydrostatic pressure. The majority of the genes comprising four functional categories as described by Bettencourt et al. [50] and relating to immune recogni‐ tion, signaling transduction, transcription, and effector molecules mechanisms were analyzed by qPCR.

The long‐term aquarium maintenance of vent mussels to atmospheric pressure has long been central to our studies and proven to be a useful model to study unique molecular relation‐ ships under which the regulation of gene transcription may be affected by the gradual dis‐ appearance of endosymbiont bacteria from gill epithelia [20, 52]. Nonetheless, vent mussels from Menez Gwen hydrothermal vent site subsist for months at atmospheric pressure in aquarium conditions, in plain sea water or supplemented with methane and sulfide. This has allowed us to focus on developing experiments to investigate new physiological responses of vent mussels sustaining experimental challenges involving bacterial pathogens of the *Vibrio* genus, even in the absence of the characteristic high hydrostatic pressure found at deep‐sea vent sites and without methane and sulfide supplementation [21, 22].

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].

**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.

Pathogen in n: endosymbiontmediated responses inducing increased

PRRs a ity in hemocytes.

In 2010 a high‐throughput sequencing and analysis of the gill tissue transcriptome from the deep‐sea hydrothermal vent mussel *B. azoricus* was reported by our group [50]. It represented the first tissue transcriptional analysis of a deep‐sea hydrothermal vent animal, using next generation sequencing technology, enabling the creation of a searchable catalog of genes that provided a direct method of identifying and retrieving vast numbers of novel coding sequences which could then be applied in gene expression profiling experiments, using quan‐ titative polymerase chain reaction (qPCR), from a nonconventional model organism [50]. It provided the most comprehensive sequence resource for identifying novel genes currently available for a deep‐sea vent organism, in particular, genes putatively involved in immune and inflammatory reactions in vent mussels. This first transcriptional analysis of gill tissues from the deep‐sea hydrothermal vent *B. azoricus* was organized as a searchable catalog of

**4. Posttranscriptomics studies in** *Bathymodiolus azoricus*

Normal immunological state: hemocyte PRRs are primed by endosymbiont

MAMPs to steady-state levels.

168 Organismal and Molecular Malacology

Earlier results from experimental exposures to *Vibrio splendidus*, *Vibrio alginolyticus*, *Vibro anguillarum*, and *Flavobacterium* sp. pointed at the immune discriminatory capacity of *B. azoricus* to distinguish different *Vibrio* strains, and at significant differences of immune gene expression levels between 12 and 24 h exposure times. These studies concluded that the immune gene transcriptional activity was modulated at two levels, i.e., over the course of time and according to the bacterial strain tested, suggesting thus, a selective response toward *Vibrio* spp. when vent mussels were experimentally challenged during 24 h [53, 54]. Additional experiments were carried out with *Vibrio diabolicus* aiming at the analysis of gene expression differences between distinct vent mussel populations from the hydro‐ thermal vent sites Menez Gwen (MG, 800 m depth) and Lucky Strike (LS, 1700 m depth) both located on the Mid‐Atlantic region, near the Azores islands. These comparative studies revealed unique immune transcriptional specificities at the gill, digestive gland, and mantle tissues level providing further evidence supporting different usage of transcription factors at the promoter region of immune genes possibly linked to the hydrothermal vent environ‐ ment Furthermore, Menez Gwen (MG) and Lucky Strike (LS) *B. azoricus* showed significant gene expression differences during *V. diabolicus* challenges over time demonstrating that immune genes are differentially expressed within the same mussel populations regardless of their hydrothermal vent origin suggesting thus site‐related tissue‐specific gene expres‐ sion patterns [55]. Moreover, these results also suggested different tissue tolerance to decompression and adaptation to atmospheric pressure not seen so far. Mantle tissues from LS mussels seemed unaffected by deep‐sea retrieval showing significantly higher levels of immune gene expressions as compared to MG mantle tissues. Thus, the decompression effect on the animal's internal organs may be evaluated by ways of its ability to respond, at the immune transcriptional level, to *V. diabolicus* challenges. For that reason, mantle tissues from LS animals appear to be decompression‐resistant and immune competent toward bac‐ terial challenges. On the other hand, the digestive gland revealed the most increased gene expression levels in MG animals showing how the tissue microenvironment is relevant to *in situ* immune responses. Gill immune transcriptional activity in both MG and LS mussels was not as significantly different as for the other tissues tested which may be attributed to the presence of endosymbiont bacteria in gill epithelia acting as a driving factor likely to affect host‐gene expression and the overall physiological statuses of MG and LS vent mus‐ sels while interacting with *V. diabolicus*. Even though gill tissues have been the main focus of most of our previous investigations in the deep‐sea vent mussel *B. azoricus*, the digestive gland and mantle tissues hold the potential for highlighting specific immune responses in tissues other than gills and how they can modulate the outcome of the animal's overall immune responses [55].

In addition to *ex vivo* experiments and *Vibrio* exposures to live vent mussels, we were able to carry out long‐term acclimatization experiments with vent mussels kept in aquaria and at atmospheric pressure. These experiments were devised to assess the effect of such prolonged aquarium conditions on immune and stress‐related reactions as mussels were gradually releasing their endosymbiont bacteria from gill bacteriocytes. These studies provided a basis for understanding the interactions between host‐immune and endosymbiont gene expressions during postcapture long‐term acclimatization in plain sea water and represented an ideal model for investigating *B. azoricus* immune genes transcriptional activity and symbiont bacte‐ ria prevalence, in view of changes in the availability of chemical‐based energy sources during acclimatization at atmospheric pressure. It also pointed out the relevance of gene expression studies while addressing the swift changes affecting metabolic adaptations and food intake fluctuations, whether induced by or as a result of the gradual loss of endosymbionts and subsequent presence of symbiont bacteria in the aquarium environment, altering thus the physiological homeostasis of *B. azoricus* [56]. These studies demonstrated that the transcrip‐ tional activity profiles for immune and bacterial endosymbiont genes followed a time‐depen‐ dent mRNA transcriptional pattern evidenced at 24 h, 1 week, and 3 weeks acclimatization. Furthermore, after 1 week acclimatization, vent mussels were under the influence of what appears to be a concomitant host‐immune and endosymbiont gene expression, possibly indi‐ cating a physiological transition point which induces higher levels of transcriptional activity [56]. Under such circumstances, survival of vent mussels will require immune gene repertoire switching involving the differential expression (DE) of recognition, signaling, transcription, and effector genes tied to environmental parameters and to the symbiotic relationships in *B. azoricus*. Metabolic adaptations and food intake changes, whether induced as a result of the gradual loss of endosymbionts and subsequent release in the aquarium environment, are likely to affect gene transcription activities and prevalence of symbionts in gill tissues [56–58].

gene expression differences during *V. diabolicus* challenges over time demonstrating that immune genes are differentially expressed within the same mussel populations regardless of their hydrothermal vent origin suggesting thus site‐related tissue‐specific gene expres‐ sion patterns [55]. Moreover, these results also suggested different tissue tolerance to decompression and adaptation to atmospheric pressure not seen so far. Mantle tissues from LS mussels seemed unaffected by deep‐sea retrieval showing significantly higher levels of immune gene expressions as compared to MG mantle tissues. Thus, the decompression effect on the animal's internal organs may be evaluated by ways of its ability to respond, at the immune transcriptional level, to *V. diabolicus* challenges. For that reason, mantle tissues from LS animals appear to be decompression‐resistant and immune competent toward bac‐ terial challenges. On the other hand, the digestive gland revealed the most increased gene expression levels in MG animals showing how the tissue microenvironment is relevant to *in situ* immune responses. Gill immune transcriptional activity in both MG and LS mussels was not as significantly different as for the other tissues tested which may be attributed to the presence of endosymbiont bacteria in gill epithelia acting as a driving factor likely to affect host‐gene expression and the overall physiological statuses of MG and LS vent mus‐ sels while interacting with *V. diabolicus*. Even though gill tissues have been the main focus of most of our previous investigations in the deep‐sea vent mussel *B. azoricus*, the digestive gland and mantle tissues hold the potential for highlighting specific immune responses in tissues other than gills and how they can modulate the outcome of the animal's overall

In addition to *ex vivo* experiments and *Vibrio* exposures to live vent mussels, we were able to carry out long‐term acclimatization experiments with vent mussels kept in aquaria and at atmospheric pressure. These experiments were devised to assess the effect of such prolonged aquarium conditions on immune and stress‐related reactions as mussels were gradually releasing their endosymbiont bacteria from gill bacteriocytes. These studies provided a basis for understanding the interactions between host‐immune and endosymbiont gene expressions during postcapture long‐term acclimatization in plain sea water and represented an ideal model for investigating *B. azoricus* immune genes transcriptional activity and symbiont bacte‐ ria prevalence, in view of changes in the availability of chemical‐based energy sources during acclimatization at atmospheric pressure. It also pointed out the relevance of gene expression studies while addressing the swift changes affecting metabolic adaptations and food intake fluctuations, whether induced by or as a result of the gradual loss of endosymbionts and subsequent presence of symbiont bacteria in the aquarium environment, altering thus the physiological homeostasis of *B. azoricus* [56]. These studies demonstrated that the transcrip‐ tional activity profiles for immune and bacterial endosymbiont genes followed a time‐depen‐ dent mRNA transcriptional pattern evidenced at 24 h, 1 week, and 3 weeks acclimatization. Furthermore, after 1 week acclimatization, vent mussels were under the influence of what appears to be a concomitant host‐immune and endosymbiont gene expression, possibly indi‐ cating a physiological transition point which induces higher levels of transcriptional activity [56]. Under such circumstances, survival of vent mussels will require immune gene repertoire switching involving the differential expression (DE) of recognition, signaling, transcription, and effector genes tied to environmental parameters and to the symbiotic relationships in *B. azoricus*. Metabolic adaptations and food intake changes, whether induced as a result of

immune responses [55].

170 Organismal and Molecular Malacology

The geographic proximity to the nearby hydrothermal vent fields, in the Azores region, gave our laboratory a positional advantage for earning first insight into immediate physiological responses comprising both cellular and humoral responses of freshly collected mussels from different hydrothermal vents, which upon arrival, are acclimatized to our aquarium system, LabHorta [23].The maintenance of live mussels from the shallower vent field, Menez Gwen, became thus a key factor in gaining knowledge into the physiology of vent animals including the study of evolutionary conserved immune, inflammatory and stress‐related factors com‐ monly found in other marine bivalves [18–22].

Taking advantage of the LabHorta facility, comparisons studies were made possible, with live vent mussels subjected to *V. parahaemolyticus* infection, wound injury, hyperbaric pres‐ surization, and 3 months acclimatization (**Figure 4**). These experiments allowed for the char‐ acterization of the differential activation of signaling pathways and the relative quantification of immune genes expressed during each type of stimulation. Differential gene expression results indicated that the four experimental conditions tested were distinctively inducing the immune genes of vent mussels to different levels of transcriptional activity of which the immune and signal transduction genes showed the highest expressions (**Figure 5**).

Of the four challenging conditions *V. parahaemolyticus* infections resulted in the highest num‐ ber of genes with higher level of expression during this comparison study based on qPCR and selected genes targeting immune recognition, signal transduction, transcription, and synthesis of effector molecules processes (**Figure 5**). Also, cross‐talk between signaling path‐ ways may occur in *B. azoricus* individuals subjected to *Vibrio* infections, wound responses, and hyperbaric stimulations, i.e., same immune or pro‐inflammatory signaling molecules may serve different signaling pathways whether they are conspicuously more expressed or not during such experiments. Cleary, the activation of signaling pathways involved in *Vibrio* infections was distinct from that of wound and hyperbaric reactions and thus conferring the animal model presented here with the physiological versatility to cope with deep‐sea hydro‐ thermal vent environments. These experiments were important to elucidate the molecular mechanisms under which, physiological responses to bacterial infections, would responses, hyperbaric stimulations and long‐term maintenance in aquaria conditions, may be involved in *B. azoricus* adaptation processes whether in deep‐sea vent environments or at atmospheric pressure. However, in‐depth analysis of different signaling genes and pathways involved in such experimental challenges remained fragmentary and elusive.

One the most common goals of RNA Sequencing (RNA‐Seq) profiling is to identify genes or molecular pathways that are differentially expressed (DE) between two or more biological conditions [59–63]. Changes in expression can then be associated with differences in phys‐ iological reactions, providing clues for further investigation into potential mechanisms of action [64, 65]. In order to gain additional insight into the different signaling genes involved in *Vibrio* infection, wound response, long‐term acclimatization, and hyperbaric repressur‐ ization, we sequenced the full transcriptome of gill tissues from each of these experimental challenges to which deep‐sea vent mussels were subjected and compared their differential

**Figure 4.** Schematic representation of immune signaling activation. After initial events characterized by immune recognition and stress‐related reactions, signal transduction pathways are induced into transmitting a series of protein phosphorylation events, through the intracellular milieu, which ultimately result in the translocation of transcription factors into the nucleus that initiates the transcription of genes encoding immune effector molecules, here represented as lysozyme, metallothionein, and ferritin.

gene expression levels with that of gene expression in animals immediately retrieved from the vent sites with the help of acoustically trigged cages that were recovered at the sea sur‐ face. Transcript sequences for the five cDNA libraries were obtained from the Illumina RNA‐ sequencing platform and *de novo* assembly of RNA‐Seq transcripts performed with Trinity [66, 67] followed by differential expression (DE) analyses using the edgeR package [68–70]. DE results were presented as Heatmaps clusters (transcriptional cluster report for edgeR DE analysis). The advantage of Heatmaps is that it can display the expression pattern of the genes across all the RNA samples. Visualization of the results is aided by clustering together genes that have correlated expression patterns [68].

Here we present examples of expression plots for some of the most DE genes across the five different experimental conditions referred to as "cage," animals freshly collected with acousti‐ cally triggered cages, from the bottom of the deep‐sea vent floor; "3 months," same animals as in "cage" acclimatized for 3 months in aquaria environment at 1 atm; "Vibrio," same animals as in 3 months exposed to *V. parahaemolyticus*; "Wound," same animals as in 3 months with shell injury caused by mechanical abrasion to expose the mantle; "IPOCAMP," same animals as in 3 months subjected to 80 bar hydrostatic pressure for 72 h. The top‐scoring BLAST hit for each of the gene exemplified is shown on top of the respective expression plot (**Figure 6**).

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 173

**Figure 5.** Comparative gene expression profiles from vent mussels subjected to 3 months acclimatization in aquaria at atmospheric pressure; *Vibrio parahaemolyticus* exposures; wound injury, and repressurization in the IPOCAMP chamber. Results are presented as relative expression folds calculated by qPCR and targeting immune genes from recognition, signaling transduction, transcription and effector functional gene categories as defined in Bettencourt et al. [50].

gene expression levels with that of gene expression in animals immediately retrieved from the vent sites with the help of acoustically trigged cages that were recovered at the sea sur‐ face. Transcript sequences for the five cDNA libraries were obtained from the Illumina RNA‐ sequencing platform and *de novo* assembly of RNA‐Seq transcripts performed with Trinity [66, 67] followed by differential expression (DE) analyses using the edgeR package [68–70]. DE results were presented as Heatmaps clusters (transcriptional cluster report for edgeR DE analysis). The advantage of Heatmaps is that it can display the expression pattern of the genes across all the RNA samples. Visualization of the results is aided by clustering together genes

**Figure 4.** Schematic representation of immune signaling activation. After initial events characterized by immune recognition and stress‐related reactions, signal transduction pathways are induced into transmitting a series of protein phosphorylation events, through the intracellular milieu, which ultimately result in the translocation of transcription factors into the nucleus that initiates the transcription of genes encoding immune effector molecules, here represented

Here we present examples of expression plots for some of the most DE genes across the five different experimental conditions referred to as "cage," animals freshly collected with acousti‐ cally triggered cages, from the bottom of the deep‐sea vent floor; "3 months," same animals as in "cage" acclimatized for 3 months in aquaria environment at 1 atm; "Vibrio," same animals as in 3 months exposed to *V. parahaemolyticus*; "Wound," same animals as in 3 months with shell injury caused by mechanical abrasion to expose the mantle; "IPOCAMP," same animals as in 3 months subjected to 80 bar hydrostatic pressure for 72 h. The top‐scoring BLAST hit for each of the gene exemplified is shown on top of the respective expression plot (**Figure 6**).

that have correlated expression patterns [68].

as lysozyme, metallothionein, and ferritin.

172 Organismal and Molecular Malacology

Comparison of DE across the five experiment revealed interesting correlations as for "cage" and "3 months" mussels indicating that vent mussels endured well aquarium conditions for as long as 3 months, as demonstrated by similar levels of gene expression. *Vibrio* infec‐ tions and IPOCAMP pressurization also showed clustering patterns of gene expression which would seem to indicate that once mussels are acclimatized to atmospheric pressure, repressurization stimulus is impacting vent mussels in similar ways as in *V. parahaemolyticus* challenges, suggesting thus the occurrence of stress‐related reactions in both types of stimula‐ tions. The expression pattern seen for wound injury was particularly distinct as compared to the other four experimental conditions. Wound injury seemed to affect drastically the vent mussel transcriptional activity which some of its genes were severely downregulated prob‐ ably due to the damaging effect caused by the mechanical abrasion and direct exposure of the

**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.

mantle to the aquarium environment. Taken together these experiments proven to be insight‐ ful in demonstrating the contrasting behavioral expression of given important physiological transcripts such as the peptidoglycan recognition and LBP‐BPI proteins, both involved in innate immune responses, when vent mussels are met with distinct environment factors.
