Toll-like receptors (TLRs) are conserved membrane-bound receptors that function in animal innate immunity. When bound to secreted microbial ligands, TLRs initiate a signaling cascade that results in the transcription of several genes including interferons and anti-microbial peptides. Ctenophores, one of the oldest metazoan lineages, encode TLRs as well as TLR pathway proteins such as IRF and AP-1. However, they lack some canonical TLR associated proteins including MyD88 and NF-κB. Beyond sequencing data, little is known about the localization or functional conservation of the TLR pathway in ctenophores. This research proposal provides an avenue to address these uncertainties in the ctenophore Mnemiopsis leidyi. Using in situ hybridization, the tissue level distribution of TLRs can be observed in adult ctenophores. Likewise, RNA-seq can be used to measure differential gene expression after immune stimulation. Finally, immunofluorescence will allow nuclear translocation of IRF to be observed to determine its role in ctenophore immunity. This research will enhance our understanding of the physiology of ctenophore innate immunity as well as provide insight into the evolution of early animal immunology.