Toll like receptors and their evolution in the lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts for zoonotic trematodes

One of the major evolutionarily conserved pathways in innate immunity of invertebrates is the toll-like receptor (TLR) pathway. However, little is known of the TLR protein family in gastropod molluscs despite their role in the transmission of human diseases, especially the common lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts of zoonotic trematodes. Using comparative genomics and gene prediction approaches utilising the freshwater snail Biomphalaria glabrata genome as a reference ten putative TLR proteins were identified in both R. auricularia and L. stagnalis. Phylogenetic analyses revealed that unlike other molluscs the lymnaeid species also possessed class 1 TLRs, previously thought to be unique to B. glabrata. Gene duplication events were also seen across the TLR classes in the lymnaeids with several of the genes appearing to exist as potential tandem elements in R. auricularia. Each predicted TLR was shown to possess the typical the leucine-rich repeat extracellular and TIR intracellular domains and both single cysteine clusters and multiple cysteine clusters TLRs were identified in both lymnaeid species. Principle component analyses of 3D models of the predicted TLRs showed that class 1 and 5 proteins did not cluster based on similarity of structure, suggested to be potential adaptation to a range of pathogens. This study provides the first detailed account of TLRs in lymnaeids and affords a platform for further research into the role of these proteins into susceptibility and compatibility of these snails with trematodes and their role in transmission.