Immune responses in Bathymodiolus azoricus upon Vibrio challenges: approaches to characterize mussel survival strategies and physiological adaptations in deep sea extreme environments

The deep-sea environment is excellent source for investigating and improving our knowledge in the field of marine biology of organisms living in extreme habitats and their relationships with chemosynthetic-based ecosystems. Along with the study of chemosynthetic-based ecosystems the necessity to understand immune system function in deep-sea hydrothermal animals such as in the mussel Bathymodiolus azoricus, has emerged gradually. Indeed, these mussels live and thrive in extreme environments as the deep-sea hydrothermal vents. B. azoricus species has become an interesting model to characterize the survival strategies and physiological adaptations in these environments. At such depths and under hydrothermal physico-chemical conditions, their immune system is constantly challenged by foreign vent compounds and microorganisms (e.g. bacteria). However, these immune responses in B. azoricus upon Vibrio challenges are not thoroughly understood. Vibrio bacteria are frequently pathogens in the aquatic environment that cause a high mortality in bivalves. This thesis was conceived and achieved with the aim of better understanding the immune response in B. azoricus mussels after several experimental challenges with different Vibrio strains. The core of this thesis consisted of elucidating signaling gene pathways and proteins involved in mussel defenses during bacterial stimuli. In this way, the thesis methods were undertaken using several molecular biology techniques, highlighting the qPCR. This technique was used: in gills from B. azoricus mussels infected with suspension of different marine bacterial pathogens (Chapter I); in comparative studies of gene expression and protein analyses between deep-sea hydrothermal vent mussel B. azoricus and the shallow-water mussel Mytilus galloprovincialis (Chapter II) and in an immune response tissue-specificity study in different tissues, and between Menez Gwen and Lucky Strike B. azoricus populations, after exposure to Vibrio diabolicus bacterium (Chapter III). Chapter I present a study on differential immune system responses in deep-sea mussels submitted to different bacterial strains, consisting of Vibrio splendidus, Vibrio alginolyticus, or Vibrio anguillarum and a pool of these bacteria as well as Flavobacterium bacterium. This study investigated the discriminatory capabilities of the B. azoricus immune responses. Results demonstrated a general gene expression pattern, decreasing from 12 h to 24 h post-infections. Among the animals tested, Flavobacterium is the bacterium inducing the highest gene expression level in 12 h post-infection animals, whereas the 24 h infected animals with V. splendidus showed a greater gene expression levels. SDS-PAGE analysis also pointed at protein profiles differences between 12 h and 24 h, particularly evident for proteins of 18-20 KDa molecular mass. Multivariate analyses demonstrated that immune genes, as well as, experimental infections, clustered in discrete groups in accordance with gene expression pattern induced by the marine bacteria tested. Chapter II presents a comparative immune responses study in two bivalve species from distinct marine habitats. The two species are the deep-sea hydrothermal vent mussel B. azoricus and the shallow-water mussel M. galloprovincialis. Both species were exposed to V. splendidus, V. alginolyticus, V. anguillarum and to a mixture of these mentioned Vibrio strains. The aim of this study was to evaluate the features of signaling pathways in different bivalve species. Gene expression results revealed that these bivalve species exhibited significant expression differences between 12 h and 24 h post-challenge times, as well as between bacteria tested. V. splendidus bacterium induced the strongest gene expression level in the two bivalve species, whereas the NF-kB and Aggrecan genes were the most differentially expressed in B. azoricus and M. galloprovincialis. HPLC- ESI-MS/MS analyses resulted in different peptide sequences in gill tissues from B. azoricus and M. galloprovincialis, suggesting that naive animals present differences, at protein synthesis level, in their natural environment. Chapter III was focused on the immune responses tissue-specificity using gill, digestive gland and mantle tissues from MG and LS B. azoricus populations challenged with V. diabolicus bacterium. Results demonstrated a significantly tissue-specific gene expression in both B. azoricus populations. However, MG mussels showed gill and digestive gland gene expression levels remarkably higher than LS mussels. In mantle tissue, most genes were down-regulated in MG mussels and up-regulated in LS animals.