Toll-like receptors (TLRs) are evolutionarily conserved sensors of pathogen-associated molecular patterns. The chromosomal organization and post-duplication diversification of the TLR repertoire in Lethenteron reissneri remain incompletely characterized. We combined chromosome-level genome screening with phylogenetic, genomic organization, and transcriptional analyses. We identified 21 TLR and TLR-like genes, including expansions of the TLR2, TLR5, TLR7, TLR14, and TLR21 subfamilies, but detected no orthologs of vertebrate TLR1, TLR4, TLR6, TLR9, or TLR10. Two atypical TIR-only genes, designated TLR-like-a and TLR-like-b, formed a distinct phylogenetic group. Chromosomal mapping revealed local paralog clusters alongside members distributed across separate chromosomes. RT-qPCR revealed treatment-associated transcriptional changes in peripheral blood leukocytes after LPS or Poly(I:C) administration, with paralogs differing in response magnitude and timing. Together, these results characterize a distinctive TLR repertoire in L. reissneri and show that duplicated members differ in protein architecture and stimulus-associated transcription, adding comparative evidence on the early evolution of vertebrate innate immunity.
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