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

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, <i>Photinus pyralis. </i>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 <i>P</i>.<i> pyralis</i> and measured expression of OR genes between the sexes.We identified 102 ORs in the <i>Photinus pyralis </i>genome, including the conserved single-copy Orco. Our phylogenetic analysis showed lineage-specific OR diversification in <i>P. pyralis</i> 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.<br>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萤火虫凭借其极具魅力的夜间闪光行为，成为研究视觉求偶信号演化的经典模式生物系统。然而，在其多样的类群中，萤火虫演化出涵盖化学信号与视觉信号的多种求偶策略。系统发育证据表明，萤火虫的共同祖先依赖长距离信息素进行通讯，但当前行为学证据显示，依赖发光闪光的萤火虫并不会通过嗅觉完成求偶。 我们以夜行性、依靠发光的普通东部萤火虫（*Photinus pyralis*）为研究对象，对其嗅觉遗传学展开探究，以此验证上述假说。在昆虫体内，气味主要由嵌入嗅觉受体神经元树突膜的气味受体（odorant receptor, OR）蛋白所识别。若依赖发光的萤火虫在求偶过程中使用信息素作为通讯信号，那么搜寻配偶的雄性个体的触角中，应当存在一种或多种OR基因的表达上调现象。因此，我们对*P. pyralis*的基因组中全部OR基因家族进行了注释，并检测了不同性别间OR基因的表达水平。 我们在*Photinus pyralis*的基因组中鉴定出102个OR基因，其中包含保守的单拷贝气味受体共受体（Orco）。系统发育分析显示，相较于其他甲虫类群，*P. pyralis*的OR基因发生了谱系特异性扩张。对雌雄个体的触角及后足开展差异表达分析后发现，相较于后足，触角中有一类OR基因呈现表达上调，提示这些基因在成虫嗅觉过程中发挥功能。值得注意的是，PpyrOR6是雌雄触角间存在显著表达上调的两个基因之一，且是其中唯一的OR基因，这表明该基因可能参与求偶行为。 本研究的发现拓展了昆虫OR基因家族在一类尚未被充分研究的甲虫类群中的已知多样性，并提示发光萤火虫在求偶过程中使用多模态信号。 本FigShare存储库包含的数据集如下： S1. PpyrOR_Nucleotides_FINAL_2025_02_27.fasta：FASTA格式的PpyrOR核苷酸序列文件 S2. PpyrOR_Nucleotides_FINAL_2025_02_27.gb：GenBank格式的PpyrOR核苷酸序列文件 S3. PpyrOR_Protein_FINAL_2025_02_27.fasta：FASTA格式的PpyrOR氨基酸序列文件 S4. PpyrOR_Protein_FINAL_2025_02_27.gb：GenBank格式的PpyrOR氨基酸序列文件 S5. PpyrOR_Scaffolds_FINAL_2025_02_27.gff：用于评估基因座及内含子/外显子结构的支架序列GFF文件 S6. Queries_Agla-Apla-Hred-Nves-Pser-RdomORs.fasta：用于鉴定推定PpyrOR的甲虫OR基因查询序列文件 S7. PpyrORs_Nves_Apla_Tc_functional_beetles_over100aa_n477_noPSE_noPAR_MUSCLE_no_ambigs_noisyclean_reordered_2025_03_04.fasta：包含PpyrOR及其他甲虫OR基因的比对文件，用于生成图2中的系统发育树 S8. run_2.contree_rooted_2025_03_11.newick：基于IQtree构建的一致性最大似然系统发育树文件，以Orco为外类群完成根化 S9. renumbered_Ppyr_tx_2025_03_04.fna：用于Salmon转录组定量分析的转录本序列文件，已编辑以整合本研究中描述的基因模型