Innate immune mechanisms in invertebrates: Insights into the Toll pathway, the Imd pathway, and the complement system

The innate immune system is the first line of defense against pathogens. This system is equipped with receptors, named Pattern Recognition Receptors (PRRs), that recognize a broad set of molecules present in pathogens, known as Pathogen-Associated Molecular Patterns (PAMPs). These receptors are present in many immune pathways, such as the Toll- or the Imd pathways, but also in other immune mechanisms as the complement system or lectins. The Toll- and the Imd pathways are pathways that trigger the production of antimicrobial peptides, whereas the complement system and lectins are involved in opsonization, phagocytosis and pathogen killing. This thesis aimed to investigate the evolution of innate immunity and, more specifically, the evolution of these pathways and systems in invertebrates. In order to fulfill this aim, I divided this thesis in two studies. First, I investigated the evolution of the Toll receptors (TLRs), which are the PRR involved in the Toll pathway; and second, I studied the presence and role of the aforementioned pathways and systems in the nemertean Lineus ruber, a member of the spiralian clade. Both studies combine in silico and wet-lab approaches in order to accomplish the aims. In my first study, I performed transcriptomic and genomic surveys in order to identify TLRs in 45 invertebrate species. The results show the presence of TLRs in 24 of these 45 species, being present in very variable numbers. Moreover, I performed phylogenetic analyses in order to reconstruct the evolution of TLR, showing that all metazoan TLRs originated from a single proto-TLR present in the planulozoan (cnidarian + bilaterian) last common ancestor. This gene later likely duplicated and diversified giving raise to TLRs that group in three clades. Further duplications and losses shaped the distribution of TLRs across the phylogeny, generating the extant diversity of TLRs in metazoans. Additionally, as TLRs are involved both in immunity and development, stage-specific transcriptomic analyses of four protostome species and in situ hybridization in the brachiopod Terebratalia transversa were performed, showing that TLRs are expressed in this species during ontogeny. In my second study, I investigated the presence and function of the Toll pathway, the Imd pathway, the complement system, and lectins in the nemertean Lineus ruber. Transcriptomic surveys in Lineus ruber show that components of these pathways and systems are present in this species. Moreover, in situ hybridization shows that lectins are expressed in the blood, the nervous system, and the gut. Additionally, in order to study the function of some of the components of these pathways and systems, I performed an immune challenging assay, in which Lineus ruber specimens were exposed to gram negative bacteria. Differential expression of TLRs, imd, C3-1, and lectins was tested, showing that all these genes, except for one TLR and imd, are upregulated upon gram-negative infection. The earliest immune response was detected at 6 hours of infection, with the upregulation of Lineus ruber TLRβ1 and TLRβ2. Additionally, a stronger upregulation of another TLR, TLRα3, occurred at 12 hours of infection, simultaneously to the upregulation of lectins. Upregulation of the complement gene C3-1 was first observed at 24h of infection. Altogether, the two studies that compose my thesis provide insights into how immunity functions in invertebrates and how this system has evolved