Bivalves present the largest and most diversified repertoire of Toll-like receptors in the animal kingdom, suggesting broad-spectrum pathogen recognition in marine waters.

Toll-like receptors (TLRs) are the most widespread class of membrane-bound innate immune receptors, responsible of specific pathogen recognition and production of immune effectors though the activation of intracellular signaling cascades. The repertoire of TLRs was analyzed in 85 metazoans, enriched on molluscan species, an underrepresented phylum in previous studies. Following an ancient evolutionary origin, suggested by the presence of TLR genes in Anthozoa (Cnidaria), these receptors underwent multiple independent gene family expansions, the most significant of which occurred in bivalve molluscs. Marine mussels (Mytilus spp.) had the largest TLR repertoire in the animal kingdom, with evidence of several lineage-specific expanded TLR subfamilies with different degrees of orthology conservation within bivalves. Phylogenetic analyses revealed that bivalve TLR repertoires were more diversified than their counterparts in deuterostomes or ecdysozoans. The complex evolutionary history of TLRs, characterized by lineage-specific expansions and losses, along with episodic positive selection acting on the extracellular recognition domains, suggests that functional diversification might be a leading evolutionary force. We analyzed a comprehensive transcriptomic dataset from Mytilus galloprovincialis and built transcriptomic correlation clusters with the TLRs expressed in gills and in hemocytes. This evidenced the implication of specific TLRs in different immune pathways, as well as their specific modulation in response to different biotic and abiotic stimuli. We propose that, in a similar fashion to the remarkable functional specialization of vertebrate TLRs, the expansion of the TLR gene family in bivalves attends to a functional specification motivated by the biological particularities of these organisms and their living environment.