Additional files for "Antennal RNAseq reveals odorant receptors with sex-biased expression in the common eastern firefly, Photinus pyralis"

With their charismatic nighttime flashes, fireflies are a classic organismal system for studying visual mating signal evolution. However, across their diversity, fireflies employ a variety of mating strategies that include both chemical and visual signals. While phylogenetic evidence points to a common ancestor that relied on long-range pheromones, current behavioral evidence suggests that light-dependent flashing fireflies do not use smell for mating. We tested this hypothesis by investigating the olfactory genetics of the nocturnal, light-using common eastern firefly, Photinus pyralis. In insects, odors are primarily detected by odorant receptor (OR) proteins embedded in the dendritic membranes of olfactory receptor neurons. If pheromones are part of mate signaling in light-using fireflies, then one or more OR genes should be upregulated in the antennae of the searching sex (males). We therefore annotated the complete suite of ORs from the genome of P. pyralis and measured expression of OR genes between the sexes.We identified 102 ORs in the Photinus pyralis genome, including the conserved single-copy Orco. Our phylogenetic analysis showed lineage-specific OR diversification in P. pyralis relative to other beetle species. Differential expression analysis of male and female antennae and back legs revealed that a subset of ORs are upregulated in antennae as compared to legs, suggesting a role in adult olfaction. Notably, PpyrOR6 was one of two genes, and the only OR, that was significantly upregulated between male and female antennae, suggesting a role in mating.These findings increase known diversity of insect ORs in an understudied beetle family and suggest that bioluminescent fireflies use multimodal signals during mating.Items included in this FigShare Repository are:S1.PpyrOR_Nucleotides_FINAL_2025_02_27.fastaNucleotide sequences for PpyrORs in fasta formatS2.PpyrOR_Nucleotides_FINAL_2025_02_27.gbNucleotide sequences for PpyrORs in genbank formatS3.PpyrOR_Protein_FINAL_2025_02_27.fastaAmino acid sequences for PpyrORs in fasta formatS4.PpyrOR_Protein_FINAL_2025_02_27.gbAmino acid sequences for PpyrORs in genbank formatS5.PpyrOR_Scaffolds_FINAL_2025_02_27.gffThe gff file of scaffolds used for assessing location and intron/exon structureS6.Queries_Agla-Apla-Hred-Nves-Pser-RdomORs.fastaBeetle ORs used as queries to identify putative PpyrORsS7.PpyrORs_Nves_Apla_Tc_functional_beetles_over100aa_n477_noPSE_noPAR_MUSCLE_no_ambigs_noisyclean_reordered_2025_03_04.fastaAlignment with Ppyr and other beetle ORs that generated the phylogeny in Figure 2S8.run_2.contree_rooted_2025_03_11.newickConsensus maximum likelihood phylogeny from IQtree, rooted in OrcoS9.renumbered_Ppyr_tx_2025_03_04.fnaTranscripts used for salmon analysis, edited to incorporate the gene models described in this study