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

Antennal RNAseq reveals odorant receptors with sex-biased expression in the common eastern firefly, Photinus pyralis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Antennal RNAseq reveals odorant receptors with sex-biased expression in the common eastern firefly, Photinus pyralis View ORCID Profile Sarah E. Lower , Samuel Pring , Hanh Tran , Katherine Martinson , Susan Deering , Mathew A. Price , Brian Vestal , View ORCID Profile Gregory M. Pask , Douglas B. Collins , Robert F. Mitchell doi: https://doi.org/10.1101/2025.03.21.644631 Sarah E. Lower 1 Department of Biology, Bucknell University , Lewisburg, PA 17837, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sarah E. Lower For correspondence: sel025{at}bucknell.edu Samuel Pring 1 Department of Biology, Bucknell University , Lewisburg, PA 17837, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hanh Tran 1 Department of Biology, Bucknell University , Lewisburg, PA 17837, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Katherine Martinson 1 Department of Biology, Bucknell University , Lewisburg, PA 17837, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Susan Deering 1 Department of Biology, Bucknell University , Lewisburg, PA 17837, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mathew A. Price 2 Department of Biology, University of Wisconsin Oshkosh , Oshkosh, WI 54901, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Brian Vestal 3 Center for Genes , Environment and Health, National Jewish Health, 1400 Jackson St, Denver, CO 80206, USA 4 Department of Biostatistics and Informatics, University of Colorado Denver , Aurora, CO 80045, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gregory M. Pask 5 Department of Biology, Middlebury College , Middlebury, VT 05753, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gregory M. Pask Douglas B. Collins 6 Department of Chemistry, Bucknell University , Lewisburg, PA 17837, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Robert F. Mitchell 2 Department of Biology, University of Wisconsin Oshkosh , Oshkosh, WI 54901, USA 7 Department of Entomology, The Pennsylvania State University, University Park , PA 16802, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Background 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, 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. Results 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 hind 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. Conclusions These findings increase known diversity of insect ORs in an understudied beetle family and suggest that bioluminescent fireflies use multimodal signals during mating. Background Fireflies, in the beetle family Lampyridae, are renowned for their nocturnal bioluminescent signals that adults use to recognize, find, and choose mates. However, some species lack light-producing organs as adults, are diurnal, and rely on volatile pheromones for mating [ 1 ]. Some species even use both dim glows and pheromones [ 2 – 4 ]. This mating signal diversity makes fireflies ideal for studying chemosensory evolution. While light signals are clearly important for mating in nocturnal species, less attention has been paid to chemical signals. Phylogenetic studies suggest that the ancestor of all fireflies was diurnal and non-luminescent and instead used pheromones for communication [ 5 – 7 ]. While all firefly larvae glow, supporting a single origin of bioluminescence early in firefly evolution, adult bioluminescence subsequently evolved either one or several times, with multiple secondary losses of light and reversion to pheromones occurring independently across firefly lineages. The evolutionary lability of mating signal mode in fireflies suggests that the molecular machinery for pheromone signaling and reception during mating remains, either functional or not, in light-using lineages. However, behavioral research using hexane-washed freeze-killed females suggests that at least close-range, cuticular chemical signals are important for species recognition only in non-luminescent species [ 8 ]. Fireflies, like other insects, likely detect odors primarily using their antennae, which are covered in sensilla that contain olfactory receptor neurons (ORNs; reviewed in [ 9 ]). Odor molecules enter through pores in the sensilla, and either diffuse through the sensillar lymph or are transported by proteins to the ORN dendrites where they bind odorant-gated ion channels that open and induce an action potential. The most common receptors mediating insect olfaction are the odorant receptors (ORs), which are heterotetrameric complexes of an odorant receptor co-receptor, Orco, and variable odorant receptor, OrX, the latter of which confers odorant specificity [ 10 , 11 ]. All ORs share a basic structure with seven transmembrane domains and an intracellular amino-terminus; however, OrXs show high copy number and sequence diversity across insect groups, while Orco is single-copy and conserved in sequence across all insect species examined to date [ 12 ]. High-throughput sequencing has facilitated the exploration of the molecular basis of chemosensation in diverse non-model insect lineages. Complete OR repertoires have now been annotated from many exemplar species within the largest insect orders, revealing species-specific OR diversity driven by rapid evolution [ 13 – 15 ]. For example, beetles have anywhere from tens to hundreds of functional ORs that are organized into seven major clades [ 14 ], with many copies departing from the canonical 5-exon (A,B,C,D,E) structure due to intron insertions and/or deletions. Nevertheless, even well-studied insect orders remain undersampled; for example, ORs from the diverse beetle infraorder Elateriformia, which includes fireflies, are described from only a single species, Agrilus planipennis (Buprestidae; [ 21 ]), which is a dietary specialist with a reduced OR repertoire. Here, we investigated OR evolution in Photinus pyralis , a well-studied firefly species for its nocturnal light-based mating displays (e.g., [ 16 – 18 ]). Using comprehensive manual annotation, we curated the full OR repertoire of P. pyralis and explored its diversification in structure and location throughout the genome. Further, using phylogenetic analysis, we investigated P. pyralis OR evolution relative to other beetle ORs. Finally, we examined OR expression using RNA sequencing of adult male and female antennae and hind legs. We found that P. pyralis hosts a large OR repertoire, with a dramatic Group 2B expansion found on 10 out of 11 linkage groups, including the putative sex chromosome. A third of ORs were significantly upregulated in male and female antennae as compared to legs, with a single OR, PpyrOR6, upregulated in male antennae as compared to female antennae. Taken together, these results provide a foundation for studying the evolution of olfaction in fireflies, and they suggest that males of light-using firefly species rely on olfaction for mating more than previously thought. Methods OR identification We identified putative OR nucleotide sequences in the published Photinus pyralis genome assembly (GCA_008802855.1; [ 18 ]) using an exhaustive iterative tblastn strategy followed by manual curation [ 14 ]. Briefly, for searchability, we divided genomic scaffolds into 200 kb contigs, then we determined which contigs contained OR-like sequences using tblastn (e-value: 1.0; [ 19 ]). We used 547 ORs from six beetle species for tblastn search and subsequent annotation, selected to span the phylogeny of beetles and represent all beetle OR subfamilies: Anoplophora glabripennis , N = 132 [ 20 ]; Agrilus planipennis , N = 46 [ 21 ]; Hydroscapha redfordi , N = 39 [ 14 ]; Nicrophorus vespilloides , N = 84 [ 14 ]; Priacma serrata , N = 123 [ 14 ]; and Rhyzopertha dominica , N = 123 [ 22 ]. We imported promising contigs into Geneious Prime v. 2021.2 ( https://www.geneious.com ) for manual curation. We annotated exons and appropriate splice sites by repeated blastx (e-value: 1.0) of contig sequence against the database of known beetle ORs. We named ORs with missing C-terminal or N-terminal sequence, or including null mutations (pseudogenes), with suffixes CTE, NTE, and PSE, respectively. We then reiterated the process using newly-identified ORs as queries until no new ORs were identified. Finally, we compared ORs to those predicted from the most recent genome assembly (v1.3; GCF_008802855; [ 18 ]) and incorporated any additional sequence data from the predictions into our models. Where possible, we confirmed OR sequence and splice junctions using Illumina RNA sequencing reads from the antennae of one male (SEL534) and one female (SEL672) P. pyralis (see RNA extraction and sequencing below) mapped onto the contigs containing manually annotated ORs using the Geneious RNA read mapper (parameters: find novel introns up to 30,000 bp). To further support the annotation, we predicted transmembrane domains for all OR models, including full length, partial, and pseudogenized genes, using TOPCONS, a widely-used membrane topology prediction web server ( https://topcons.cbr.su.se/pred/ ; [ 23 ]). The TOPCONS algorithm combines topology predictions from five sub-methods into a single topology profile, and has been shown to be equally or more accurate for membrane topology prediction than other software [ 23 ]. To ensure that newly identified OR names were informative as possible, after removing stop codons and ambiguous amino acids, we compared all P. pyralis OR (PpyrOR) amino acid sequences, including CTE, NTE, and PSE sequences, with OR amino acid sequences from three other representative beetle species with well-curated OR annotations, excluding pseudogenes and sequences shorter than 100 amino acids: A. plannipennis , the sole elateriform relative of fireflies where genomic ORs have been described [ 14 , 24 ]; N. vespilloides , the closest polyphagan relative to Elateriformia with annotated ORs [ 14 ], and Tribolium castaneum , a model beetle, with group 5 ORs otherwise lacking from A . planipennis and N . vespilloides [ 14 , 25 ]. We built an initial phylogeny for naming PpyrORs by aligning these sequences with MUSCLE v. 5.3 (parameters: default; [ 26 ]), then trimming the resulting alignment to conserved regions using trimAL v. 1.5.0 (parameters: automated1; [ 27 ]), constructed a phylogeny using FastTree v. 2.1.11 (parameters: default; [ 28 , 29 ]). We visualized the resulting tree in Geneious Prime. To incorporate evolutionary and genomic location information, where possible we named PpyrORs sequentially according to their position in the phylogeny relative to known beetle ORs, and following gene order within tandem arrays. Phylogeny To more rigorously examine PpyrOR diversification and place it in context of potential OR function, we next constructed a maximum likelihood tree that included testing for a model of evolution and 13 additional beetle ORs with known ligands (Additional File 2: Table S1). Using Geneious Prime, we aligned amino acid sequences with MUSCLE 5.1 (parameters: algorithm PPP; [ 30 ]). We subsequently used Noisy v. 1.5.12.1 [ 31 ] on NGPhlyogeny.fr (default parameters; [ 32 ]) to identify conserved regions of the alignment. We then used the resulting trimmed alignment (477 sequences, alignment length: 755) to construct 50 maximum likelihood phylogenies with 1000 bootstraps in IQtree v. 1.6.12 (parameters: –bb 1000; [ 33 ]), rooted in the Orco clade. We visualized and annotated the tree with the best likelihood score with iToL v 6.9 [ 34 ]. We labeled major OR clades following established subfamily nomenclature [ 14 ]. OR genomic location and structure analyses To investigate how ORs diversified in both exon structure and location across the genome, we used a custom script to extract genomic coordinates and exon annotations from the GFF file of newly annotated P. pyralis ORs using R v. 4.4.1 [ 35 ]. We used the R packages ggtree v. 3.12.0 [ 36 ], ggplot2 v. 3.5.1 [ 37 ], and gggenes v. 0.5.1 [ 38 ] to visualize genomic position and exon structure relative to phylogeny. Scripts are available on GitHub: https://selower.github.io/Photinus_pyralis_ORs/ . RNA extraction and sequencing To characterize the OR expression, we isolated RNA from the antennae and hind legs of wild male (N= 4) and female (N = 3) P. pyralis captured during their active period (within 30 min of sunset) from May 28-30, 2019 in Brasstown, North Carolina (Additional File 2: Table S2). We maintained captive fireflies in individual polystyrene Drosophila vials with a piece of moist unbleached coffee filter paper on the bench at ambient light cycle and temperature until preservation in RNA later (Ambion; Millipore-Sigma #R0901; Burlington, MA, USA) following manufacturer instructions. One individual was briefly anesthetized with CO 2 for use in another experiment testing techniques for non-destructive sampling of surface hydrocarbons and returned to its vial for full recovery prior to preservation. We preserved tissues between 1420 and 1710 hours, representing 6.5-4.5 hours prior to onset of flashing activity in the wild, and within one week of capture. Preserved samples were stored at –80 °C until extraction. We extracted total RNA from dissected antennae and hind legs using TRIzol reagent (Invitrogen; ThermoFisher Scientific #15596026; Carlsbad, CA, USA) following manufacturer instructions, including the addition of glycogen during precipitation to increase RNA yield from the small tissues (GlycoBlue Coprecipitant, Invitrogen; ThermoFisher Scientific #AM9515). Each sample consisted of either both antennae or both hind legs from a single individual. After extraction, we treated samples for potential genomic DNA contamination using an RNA Clean-Up and Concentration Kit (Norgen Biotek Corp. #23600; Thorold, ON, Canada) following manufacturer’s instructions with optional on-column DNase incubation. We assessed sample yield using a Qubit 4.0 Fluorometer with RNA HS Assay (Invitrogen, ThermoFisher Scientific #Q32852). We sent samples with >100 ng of RNA to Novogene (Beijing, China) for quality assessment, followed by low-input Illumina library preparation and sequencing. Due to a previously undescribed hidden break in firefly 28S rRNA that results in a low RIN value (a known, but understudied, phenomenon in arthropods; e.g. [ 39 ]), rather than instituting an arbitrary RIN threshold, we assessed RNA quality by manually examining the Bioanalyzer (Agilent 2100; Santa Clara, CA, USA) traces to determine appropriateness for sequencing. All samples chosen for library preparation had a RIN of ≥ 4.8 (mean: 5.2 ± 0.2; Additional File 2: Table S3). Sequencing libraries were prepared at Novogene using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer recommendations. Samples were sequenced to at least 30 million PE 150 bp reads on an Illumina NovaSeq 6000. The raw reads are available on NCBI SRA (BioProject: PRJNA1196372). Differential Expression Analysis To obtain a transcript set that also included a complete set of ORs for differential expression analysis, we downloaded the most recent genome assembly (v1.3; GCF_008802855; [ 18 ]) and annotated transcripts from NCBI. We then identified the NCBI-annotated genomic locations that represented the manually-annotated ORs with reciprocal BLAST (parameters: blastn, e-value: 1e-5). Where there was not a one-to-one match between NCBI and manual annotations, we conducted further curation consisting of (i) splitting chimeric transcripts, where the complete open reading frame of another gene was included on the same transcript with an OR, and (ii) adding novel ORs – manually-annotated OR gene models that had no match in the NCBI annotations (Additional File 2: Table S1). To perform a differential expression analysis, we initially assessed raw RNAseq reads for quality using FastQC v. 0.11.9 [ 40 ], with output files concatenated for easier viewing using MultiQC v. 1.14 [ 41 ]. We trimmed low-quality reads using Trimmomatic v. 0.39 (default parameters; [ 42 ] and then identified and removed potential contaminant reads using Kraken2 v. 2.1.1 [ 43 ], using the PlusPF database that includes archaea, bacteria, viral, plasmid, human, protozoa, and fungal taxa (v. 5/17/2021). We used the cleaned reads for quasi-mapping and quantification to the curated P. pyralis GCF_008802855 mRNAs that included the additional manually-annotated ORs using Salmon v. 1.9.0 [ 44 ] with the genome as a decoy (parameters: –-validateMappings –-seqBias –-gcBias). We then imported the quantification into R v. 4.2.2 [ 35 ] and screened out genes with low expression by filtering the dataset to retain only genes that had a counts per million (CPM) value greater than one in at least three samples (i.e. the size of the smallest experimental group of interest in this study). Subsequently, we transformed the digital counts into continuous values using variance stabilizing transformation (VST) from the DESeq2 R package v. 1.36.0 [ 45 ]. We then verified that the major sources of variation in the data could be attributed to our experimental design rather than confounding factors using principal component analysis. To test for differential expression, we followed the general flow of the lmerSeq methodology and used linear mixed models with random effects to model the VST expression levels [ 46 ]. For fixed effects, we included binary sex, binary tissue type (leg vs. antennae), and the interaction between the two, while using a random intercept for each specimen to account for the paired nature of the data (hind legs and antennae were sampled from each individual). Additionally, we utilized the empirical Bayes procedure implemented in the REBEL R v. 0.1.0 [ 47 ] package to stabilize the random intercept variance estimates, thus minimizing the number of genes with singular fits that would have to be discarded due to unreliable p-values and regression coefficients. We then used linear contrasts of the regression coefficients to conduct the comparisons of interest, where t-statistics and their associated multiple-comparison-corrected p-values were extracted for inference. Significance was determined at FDR < 0.05. We assessed model fit by examining residuals versus fitted values for all ORs identified as differentially expressed. We used REBEL estimates of differential expression, approximately on the log 2 scale, and VST counts for subsequent plotting. The scripts and HTML of our data analysis are available on GitHub: https://selower.github.io/Photinus_pyralis_ORs/ . Results 101 ORs + Orco identified Using an exhaustive iterative blast search strategy with permissive e-value followed by manual curation, we found 101 ORs and the conserved Orco in the published Photinus pyralis genome assembly [ 18 ], Figure 1 ). Of these, one OR (PpyrOR8) had two potential splice forms, with mutually exclusive copies of the initial two exons. Two ORs were partial, missing the N-terminus (PpyrOR101NTE) or C-terminus (PpyrOR83CTE), while 15 had premature stop codons and were classified as pseudogenes (PSE, Additional File 2: Table S1). As expected, the majority of annotated ORs were predicted to have seven transmembrane domains (6.73 ± 0.76; TOPCONS; Additional File 1: Text S1, Additional File 2: Table S1). The number of ORs, rarity of alternative splice forms, presence of pseudogenes, and seven transmembrane domain structures are consistent with findings in other beetles [ 14 ]. Download figure Open in new tab Figure 1. Photinus pyralis odorant receptor numbers are in the range of beetles a) A common eastern firefly ( P. pyralis ) male (left) and female (right) mating in the lab. Photo: Lauren Shaffer. b) Numbers of P. pyralis odorant receptors (OR) and OR pseudogenes (PSE) compared to other beetle species used in this study. Code: the four-letter abbreviation used to name ORs in the phylogeny. Expansion of Group 2B ORs throughout the genome A maximum likelihood phylogeny comparing P. pyralis ORs with ORs from a subset of beetle species that span OR diversity shows that, in addition to Orco, P. pyralis has members of OR Groups 2A, 2B, 3, 4, and 6 ( Figure 2 ). Group 2B is particularly expanded in P. pyralis , with all the identified pseudogenes occurring in this clade. Group 2A ORs have the canonical OR structure, with distinct exons A,B,C,D, and E ( Figure 3 , Additional File 1: Text S2; Additional File 2: Table S4). All other ORs had introns inserted into the A exon, with ORs from Groups 2B, 3, 6, and 4 generally having fragmented A exons, sometimes fused to B exons. The median intron length of full-length ORs was 49 nucleotides (mean: 428 +/-1691, range: 38 – 25,152; Additional File 1: Text S3). None of the P. pyralis ORs were closely related to a beetle OR with a known ligand. Interestingly, the phylogeny did not mimic species relationships – A. planipennis ORs were generally not sister to P. pyralis ORs, except for Orco and OR1. The OR1 clade, consisting of AplaOR1 and PpyrOR1, though nested within the paraphyletic Group 2 clade, is divergent enough to warrant closer examination for classification. Download figure Open in new tab Figure 2. Group 2 ORs expanded in Photinus pyralis A maximum likelihood phylogeny comparing P. pyralis ORs (Ppyr; shaded labels) to complete OR repertoires from Agrilus planipennis (Apla), Nicrophorus vespilloides (Nves), Tribolium castaneum (Tcas). Also included are ORs with known ligands from other beetle species: Megacyllene caryae (Mcar), Ips typographus (Ityp), Holotrichia parallela (Hpar), and Rhynchophorus ferrugineus (Rfer). Size of circles at nodes corresponds to bootstrap values and clades with less than 70% bootstrap support are collapsed into polytomies. One clade of Group 5A Tcas ORs is depicted as a triangle for visual clarity. OR subfamily (group) identity follows Mitchell et al. 2020. P. pyralis has members of Groups 2, 3, 4, and 6. PpyrOR1 is in a divergent clade with AplaOR1 within the paraphyletic Group 2 ORs, suggesting that OR1s require further consideration for classification. PpyrORs are divergent from beetle ORs with known function. Download figure Open in new tab Figure 3. Variation in OR exon structure in Photinus pyralis a) Example exon/intron structures of full-length PpyrORs. We selected the shortest (starting nucleotide to ending nucleotide) OR from each structure type for display (see Additional File 1: Supplementary Text S2 for the full display). All structures are variants of the canonical five-exon (A, B, C, D, E) structure in beetles (14). b) The number of ORs in each OR group, colored by structure as in panel (a). Group 2B has the most structural diversity. c) Exon structures on the PpyrOR phylogeny. Colored circles at tip correspond to structures in (a). Phylogeny from Figure 2 , trimmed to only include full-length PpyrORs. ORs are found on every linkage group (LG) in the P. pyralis genome assembly. However, not every OR group is present on every LG. Group 2B has the greatest distribution throughout the genome, as it is found on every LG except for LG10 ( Figure 4 ), with the most occurrences on LG4. This expansion includes one Group 2B OR on LG3a, the putative P. pyralis X chromosome. In contrast, Groups 2A and 3 are the most restricted, each found on only a single LG (LG1 and LG10, respectively). All other groups, except for Orco on LG10, are located on at least 2 LGs. While LG4 has the highest absolute number of ORs, most of which are from OR group 2B, LG3b hosts the highest OR group diversity, with members of Groups 2A, 2B, 4, and 6. Download figure Open in new tab Figure 4. PpyrORs diversified across linkage groups a) Position of full length, partial, and pseudogenized (PSE) PpyrORs on linkage groups (LG, white bars), colored by OR group. Points are vertically jittered for visualization. b) Number of ORs on each LG, colored by OR group as in (a). LG4 has the highest number of ORs, dominated by 2B ORs. LG3b hosts the most OR diversity (four OR groups). Pseudogenized ORs are on six out of 10 LGs. Group 3 and 2A ORs are limited to a single linkage group, while Group 2B ORs are on every linkage group except LG10, including the sex chromosome (LG3a). c) ORs have diversified across linkage groups (LGs). Colored circles at tips correspond to LGs. Phylogeny is identical to that in Figure 2 , trimmed to only include PpyrORs. Some OR groups diversified on the same linkage group (e.g. Group 3 ORs), while others are distributed throughout the genome (e.g. Group 2B). A subset of ORs are upregulated in adult antennae Pseudoalignment of the PE 150 bp Illumina RNA sequence reads from antennae and hind legs of wild-caught signaling P. pyralis (4 male, 3 female; 41.1 +/-6.6 million reads per sample after cleaning, Additional File 2: Tables S2, S3) yielded 13,340 genes with a CPM > 1 in at least 3 samples, including 45 of the 101 manually-annotated PpyrORs (Orco, 41 full length ORs, and 3 OR pseudogenes). The remaining ORs may be expressed in other tissues, at different times, or in different life stages. Principal components analysis (PCA) of the variance stabilized counts revealed that tissue (PC1) and sex (PC2) accounted for 57.1% of the variance in gene expression (45.6% and 11.5%, respectively; Additional File 1: Text S4; Additional File 2: Table S5). Thus, our experimental variables accounted for the major axes of variation in gene expression in our dataset. Unknown factors at capture such as infection status, age, and mating status may have contributed to the remaining PCs. All 45 ORs that passed filtering had non-zero expression in all tissues of interest. 32 and 36 ORs were significantly upregulated in male and female antennae, respectively, as compared to legs ( Figures 5 and 6 , Additional File 1: Text S5, S6, Additional File 2: Table S6). 30 of these were upregulated in the antennae of both sexes relative to hind legs, while two were significantly upregulated only in male antennae relative to male hind legs (PpyrOR67, OR100), and six were significantly upregulated only in female antennae relative to female hind legs (PpyrOR15, OR23, PpyrOR28, OR59PSE, OR64, OR81). There were 10 differentially expressed genes between male and female hind legs, none of them ORs. Download figure Open in new tab Figure 5. OR expression is antenna-biased Volcano plots of fold-change versus significance in (a) male antennae versus hind legs, (b) female antennae versus hind legs, (c) male versus female antennae, (d) male versus female hind legs. Inset: Orange points show genes that are significantly upregulated, while blue points show genes significantly downregulated, while grey shows genes that did not reach significance. Black outlines indicate ORs. 32 and 36 ORs are upregulated in males and females, respectively. Orco is significantly upregulated in the antennae of both sexes. PpyrOR6 is the next most highly expressed OR in male antennae (a) and the only differentially expressed gene between male and female antennae (c). A subset of ORs are significantly upregulated in the antennae of only one sex – PpyrOR67 and 100 are significantly upregulated in males, but not in females (a, b) and PpyrOR15, 23, 28, 59PSE, 64, and 81 are significantly upregulated in females, but not in males (a, b). Download figure Open in new tab Figure 6. OR expression patterns across the phylogeny a) Normalized (VST) counts for each OR on the phylogeny presented in Figure 2 , trimmed to only Photinus pyralis ORs. Bars at right show the OR group designation. M: Male, F: Female, An: Antennae, Le: Hind legs. ORs with blank counts did not pass initial filtering (CPM > 1 in at least 3 samples). b) Boxplots of normalized (VST) counts for each tissue by sex combination for six exemplar ORs, listed in order of placement in the phylogenetic tree (top to bottom): PpyrOR100 (significantly upregulated in antennae vs legs in males only), PpyrOR11 (significantly upregulated in antennae relative to legs in both sexes), PpyrOR81 (significantly upregulated in antennae relative to legs in females only), PpyrOR6 (significantly upregulated in male vs female antennae), and Orco (the most highly expressed OR in antennae). Black jittered points show values for each sample. Blue boxplots show males, while orange boxplots show females. Conditions that are significantly different are indicated by bars, where * = p < 0.05. See Additional File 1: Figure S5 for boxplots of all significantly differentially expressed ORs. PpyrOR6 shows sex-biased expression Two genes were significantly upregulated in male as compared to female antennae (Additional File 2: Table S6). One of these was an OR – PpyrOR6 (Estimate: 2.71, p < 0.05; Figure 5 , 6). The other gene was uncharacterized. No ORs were significantly differentially expressed between male and female hind legs. Discussion Here we have identified, for the first time, a comprehensive OR repertoire for a firefly species. Like comparative studies with other beetle ORs, we found lineage-specific diversification in P. pyralis , with evidence of gene duplication both in tandem arrays and across the genome. Further, we have shown that ∼38% of ORs are upregulated in adult antennae as compared to hind legs, including a single OR that is significantly upregulated in male antennae. Manual annotation yields a comprehensive OR repertoire The total number of ORs in P. pyralis is within the expected range for beetles [ 14 ]. Manual curation was effective – we annotated 75 more gene models than using a conservative ortholog search strategy (e-value: 10 -5 , N = 26 ORs + Orco; [ 48 ]), and 65 more than NCBI’s automated annotation (N = 36). By annotating ORs in the genome assembly and subsequently using RNAseq to measure expression, we discovered that fewer than 50% of all ORs are detectable in our RNAseq data. This reinforces previous studies demonstrating that OR repertoire derived from RNA sequencing are significant underestimates of total OR repertoire in beetles (e.g. [ 21 , 22 ]). We recommend that future studies of OR diversity should seek to incorporate both genome and transcriptome data for OR discovery. ORs diversify in place and around the genome Previous studies in beetles have noted a pattern of lineage-specific OR diversification with the addition of data from a representative species from each new major group [ 14 ]. Similarly, our data showed that there has been clade-specific OR diversification in fireflies, particularly in Group 2B. PpyrOR1 is in a divergent clade with AplaOR1 within the paraphyletic Group 2 ORs, suggesting that OR1s require further consideration for classification. Three OR groups were not present, and we presume them lost in P. pyralis (Groups 1, 5, and 7). Absence of Group 5A ORs was expected, since they appear to have evolved as late as the Bostrichidae [ 22 ], and elateriforms, which include fireflies, diverged before that split [ 24 ]. However, Group 5B ORs were absent in P. pyralis , which is surprising given their presence in the buprestid beetles (e.g. A. plannipennis ). This may represent a novel loss in the P. pyralis lineage. In contrast, ORs in Groups 1 and 7 appear to be absent in A. planipennis as well as P. pyralis , which are also elateriforms – evidence that loss of these OR groups happened early in elateriform evolution. Some ORs occurred in tandem arrays on a single linkage group, such as the Group 3 ORs on LG10, suggesting processes like unequal crossing over playing a role in their duplication. In contrast, Group 2B ORs have proliferated throughout the genome, to every linkage group but LG10, suggesting that long-range duplication events, such as TE-mediated transposition, have occurred. Future work will examine repetitive DNA content with respect to OR genomic location. Implications of expression pattern for OR function A previous study found no behavioral evidence for light-using fireflies employing close-range chemosensation during mating [ 8 ]. In our study one of only two genes that were differentially upregulated in male as compared to female antennae was an OR, PpyrOR6. Because P. pyralis males land near females and scramble to approach during mating [ 17 ], male antennae are usually the first sensory organ directed in contact with a female. Males tap their antennae over the dorsal surface of the female prior to mating, suggesting a potential role of ORs in detecting female-specific compounds, such as cuticular hydrocarbons (CHCs), at close-range. CHCs provide species identity, sex, and mating status information in other insect species [ 49 – 52 ] and several compounds have been described for fireflies [ 8 , 53 – 55 ]. Close-range chemosensory signals may also function in predator escape, for instance, when Photuris firefly females mimic Photinus females to lure Photinus males for a meal [ 56 ]. Though many CHCs are large molecules, they can be detected at distance without any direct contact [ 57 ]. PpyrOR6 may therefore be involved in avoiding predation by sensing odorants from these male-specialist predators. Eight ORs were found upregulated in antennae relative to hind legs in one sex, but not the other. PpyrOR67 and 100 were upregulated in male antennae relative to male legs, but not in female antennae relative to female legs. Despite no significant upregulation between males and females in antennae, it is possible that these ORs are involved in male-specific functions during mating. PpyrORs 15, 23, 28, 59PSE, 64, and 81 were upregulated in female antennae relative to female legs, but not in male antennae relative to male legs. However, like PpyrORs 67 and 100, they were not significantly differentially expressed between female and male antennae. These genes may have female-specific biological functions, such as selecting oviposition sites to avoid harmful microbes [ 58 ]. Notably, we annotated PpyrOR59 as a pseudogene. However, during our annotation process, comparison of RNAseq reads with the reference genome suggested the potential for segregating null alleles of other ORs, e.g., PpyrOR23 (Additional File 2: Table S1). It is possible that a functional version of PpyrOR59 will be found by sequencing additional individuals. Alternatively, PpyrOR59 could be recently pseudogenized. 44% of the full OR repertoire did not pass filtering steps in RNAseq analysis, meaning that they were poorly or not expressed in our samples. These ORs are likely expressed at other times or in other life stages. P. pyralis spend 1-2 years in their soil-dwelling larval stage where they hunt soft bodied invertebrate prey. Chemosensation likely plays a significant role in larval ecology, which involves navigating low-light environments with a rudimentary larval visual system, including living underground in winter and summer, and nocturnal activity [ 59 , 60 ]. Taken together, our results indicate that P. pyralis males may use olfaction for conspecific or heterospecific species recognition during mating. As we sampled only two tissues from adults, future studies could gather evidence for potential function by examining OR expression across additional tissues, times of day, parts of the season, and life stages. Because phylogenetic relationships did not yield clues as to potential ligands, future behavioral and neurophysiological studies will seek to functionally characterize these ORs, particularly PpyrOR6. P. pyralis hosts a relatively large OR repertoire P. pyralis maintains a relatively large OR repertoire as compared to buprestids, a related beetle group that also relies primarily on visual signals. This could be because P. pyralis uses chemosensation in the adult stage for mate recognition or other activities, or in other life stages. In other beetles, OR number correlates with dietary breadth [ 21 ]. While larval P. pyralis are considered earthworm specialists with limited evidence for adult Photinus feeding [ 61 , 62 ], suggesting some dietary specialization, P. pyralis has also been found nectaring on milkweed [ 63 ]. It is also possible that this large OR repertoire is a result of phylogenetic inertia, i.e., ORs in P. pyralis represent a repertoire similar in size from a common ancestor. This hypothesis is particularly intriguing as Photinus corruscus , a diurnal, exclusively pheromone-using firefly, and P. pyralis diverged around 25 million years ago [ 64 ]. While P. pyralis is known primarily for visual bioluminescent mating signals, it is possible that P. corruscus was predisposed for evolution of a volatile mating pheromone due to the molecular machinery in place, and perhaps even in use, in their likely lighted common ancestor [ 5 , 6 ]. Both light and pheromone signals are used for mating by the glow-worm firefly Lamprohiza splendidula [ 4 ] and are also presumed in other species with neotenic females (e.g., Pyrocoelia rufa , [ 2 ]). Our results suggest that simultaneous and/or sequential use of light and pheromones in firefly courtship is more widespread than previously considered. New firefly genomes (e.g. [ 65 – 68 ]) becoming available will facilitate the future examination of OR repertoire relative to primary mating signal modality across firefly diversity. Declarations Ethics approval and consent to participate Photinus pyralis is an unregulated invertebrate and is listed as of “Least Concern” by IUCN. All specimens were collected with landowner permission (see Acknowledgements). Consent for publication Not applicable. Availability of data and materials Annotation, alignment, and phylogeny files are available on Figshare: (DOI:10.6084/m9.figshare.28365455). Illumina RNAseq data has been deposited at NCBI SRA (BioProject: PRJNA1196372). Code for analysis and figure generation is available on GitHub: https://selower.github.io/Photinus_pyralis_ORs/ . Competing interests The authors declare that they have no competing interests. Funding This study was funded by the University of Wisconsin Oshkosh (to RFM), and Bucknell University (C. Graydon and Mary E. Rogers Fellowship to SEL; Scadden Research Fellowship to SEL, GP, DC), and the National Science Foundation (IOS-2035286 to SEL, DC and IOS-2035239 to GP). Author Contributions SEL conceived, designed, and coordinated the study, acquired data and funding, analyzed and interpreted the data, and took a primary role in drafting and revising the manuscript. SP analyzed and interpreted expression data, drafted relevant parts of the methods, and revised the manuscript. KM analyzed genomic location, interpreted the data, drafted relevant parts of the methods, figure legends, and results, and revised the manuscript. SD conducted transmembrane domain analysis and interpretation, drafted relevant parts of the methods and results, and revised the manuscript. HT collected specimens and extracted RNA, assembled transcriptomes and annotated ORs, drafted the introduction and relevant methods sections, and revised the manuscript. MP annotated ORs and revised the manuscript. BV consulted on RNAseq analysis methods and interpretation, including writing scripts and running data, drafted relevant sections of the methods, and revised the manuscript. GP acquired funding, and revised the manuscript. DC acquired funding, and revised the manuscript. RM annotated ORs, consulted on phylogeny construction and interpretation, and substantially revised the manuscript. Additional files Additional Files ● 1. Supplementary text for ORs in Ppyr ● 2. Supplementary tables for ORs in Ppyr Data files on Figshare (DOI: 10.6084/m9.figshare.28365455) ● S1.PpyrOR_Nucleotides_FINAL_2025_02_27.fasta ○ Nucleotide sequences for PpyrORs in fasta format ● S2.PpyrOR_Nucleotides_FINAL_2025_02_27.gb ○ Nucleotide sequences for PpyrORs in genbank format ● S3.PpyrOR_Protein_FINAL_2025_02_27.fasta ○ Amino acid sequences for PpyrORs in fasta format ● S4.PpyrOR_Protein_FINAL_2025_02_27.gb ○ Amino acid sequences for PpyrORs in genbank format ● S5.PpyrOR_Scaffolds_FINAL_2025_02_27.gff ○ The gff file of scaffolds used for assessing location and intron/exon structure ● S6.Queries_Agla-Apla-Hred-Nves-Pser-RdomORs.fasta ○ Beetle ORs used as queries to identify putative PpyrORs ● S7.PpyrORs_Nves_Apla_Tc_functional_beetles_over100aa_n477_noPSE_noPA R_MUSCLE_no_ambigs_noisyclean_reordered_2025_03_04.fasta ○ Alignment with Ppyr and other beetle ORs that generated the phylogeny in Figure 2 ● S8.run_2.contree_rooted_2025_03_11.newick ○ Consensus maximum likelihood phylogeny from IQtree, rooted in Orco ● S9.renumbered_Ppyr_tx_2025_03_04.fna ○ Transcripts used for salmon analysis, edited to incorporate the gene models described in this study Acknowledgments The authors would like to thank Staurolite Farm for permission to collect, Lourenço Martins and Margot Popecki for collection assistance, and Lauren Shaffer for photos. Thank you as well to Anqi Zhang for early work with the P. pyralis transcriptome. Many thanks to Jeremy Dreese and Mike Harvey for smooth administration of Bucknell’s High Performance Computing Cluster, BisonNet (NSF #1659397). Heartfelt appreciation to the cohort, coaches, and staff at the Cold Spring Harbor Scientific Writing Retreat 2024 for support and feedback. References 1. ↵ Lower SE , Pask GM , Arriola K , Halloran S , Holmes H , Halley DC , et al. Identification of a Female-Produced Sex Attractant Pheromone of the Winter Firefly , Photinus corruscus Linnaeus (Coleoptera: Lampyridae). J Chem Ecol [Internet] . 2023 Mar 15 [cited 2023 Mar 23]; Available from : doi: 10.1007/s10886-023-01417-2 OpenUrl CrossRef 2. ↵ Ohba N . Flash Communication Systems of Japanese Fireflies . Integrative and Comparative Biology . 2004 ; 44 ( 3 ): 225 – 33 . OpenUrl CrossRef PubMed 3. Stanger-Hall KF , Lower SES , Lindberg L , Hopkins A , Pallansch J , Hall DW . The evolution of sexual signal modes and associated sensor morphology in fireflies (Lampyridae , Coleoptera). Proc R Soc B . 2018 Jan 31; 285 ( 1871 ): 20172384 . OpenUrl PubMed 4. ↵ Novák M , Jakubec P . Beyond the glow: proving the unity of pheromones and bioluminescent signals in glow-worm firefly courtship . Zoological Journal of the Linnean Society . 2024 Apr 11; zlae045 . 5. ↵ Stanger-Hall KF , Lloyd JE , Hillis DM . Phylogeny of North American fireflies (Coleoptera: Lampyridae): Implications for the evolution of light signals . Molecular Phylogenetics and Evolution . 2007 Oct; 45 ( 1 ): 33 – 49 . OpenUrl CrossRef PubMed Web of Science 6. ↵ Martin GJ , Branham MA , Whiting MF , Bybee SM . Total evidence phylogeny and the evolution of adult bioluminescence in fireflies (Coleoptera: Lampyridae) . Molecular Phylogenetics and Evolution . 2017 Feb 1; 107 : 564 – 75 . OpenUrl CrossRef PubMed 7. ↵ Powell GS , Saxton NA , Pacheco YM , Stanger-Hall KF , Martin GJ , Kusy D , et al. Beetle bioluminescence outshines extant aerial predators . Proc R Soc B . 2022 Jul 27; 289 ( 1979 ): 20220821 . OpenUrl PubMed 8. ↵ South A , LeVan K , Leombruni L , Orians CM , Lewis SM . Examining the Role of Cuticular Hydrocarbons in Firefly Species Recognition . Ethology . 2008 ; 114 ( 9 ): 916 – 24 . OpenUrl CrossRef Web of Science 9. ↵ Zufall F Pask GM , Ray A . Chapter 6 – Insect Olfactory Receptors: An Interface between Chemistry and Biology . In: Zufall F , Munger SD , editors. Chemosensory Transduction [Internet] . Academic Press ; 2016 [cited 2024 Dec 19]. p. 101 – 22 . Available from: https://www.sciencedirect.com/science/article/pii/B9780128016947000068 10. ↵ Wang Y , Qiu L , Wang B , Guan Z , Dong Z , Zhang J , et al. Structural basis for odorant recognition of the insect odorant receptor OR-Orco heterocomplex . Science . 2024 Jun 28; 384 ( 6703 ): 1453 – 60 . OpenUrl CrossRef PubMed 11. ↵ Zhao J , Chen AQ , Ryu J, del Mármol J. Structural basis of odor sensing by insect heteromeric odorant receptors . Science . 2024 Jun 28; 384 ( 6703 ): 1460 – 7 . OpenUrl CrossRef PubMed 12. ↵ Soffan A , Subandiyah S , Makino H , Watanabe T , Horiike T . Evolutionary Analysis of the Highly Conserved Insect Odorant Coreceptor ( Orco ) Revealed a Positive Selection Mode , Implying Functional Flexibility. Journal of Insect Science [Internet] . 2018 Nov 1 [cited 2024 Nov 26]; 18 ( 6 ). Available from: https://academic.oup.com/jinsectscience/article/doi/10.1093/jisesa/iey120/5236977 13. ↵ Engsontia P , Sangket U , Chotigeat W , Satasook C . Molecular Evolution of the Odorant and Gustatory Receptor Genes in Lepidopteran Insects: Implications for Their Adaptation and Speciation . J Mol Evol . 2014 Aug 1; 79 ( 1 ): 21 – 39 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Mitchell RF , Schneider TM , Schwartz AM , Andersson MN , McKenna DD . The diversity and evolution of odorant receptors in beetles (Coleoptera) . Insect Mol Biol . 2020 Feb; 29 ( 1 ): 77 – 91 . OpenUrl CrossRef PubMed 15. ↵ Gautam S , McKenzie S , Katzke J , Hita Garcia F , Yamamoto S , Economo EP . Evolution of odorant receptor repertoires across Hymenoptera is not linked to the evolution of eusociality . Proceedings of the Royal Society B: Biological Sciences . 2024 Sep 25; 291 ( 2031 ): 20241280 . OpenUrl PubMed 16. ↵ Lloyd JE . Studies on the flash communication system in Photinus fireflies . 1966 ; 17. ↵ Vencl FV , Carlson AD . Proximate Mechanisms of Sexual Selection in the Firefly Photinus pyralis (Coleoptera: Lampyridae) . Journal of Insect Behavior . 1998 Mar 1; 11 ( 2 ): 191 – 207 . OpenUrl 18. ↵ Fallon TR , Lower SE , Chang CH , Bessho-Uehara M , Martin GJ , Bewick AJ , et al. Firefly genomes illuminate parallel origins of bioluminescence in beetles . Waterhouse R, Tautz D, editors. eLife . 2018 Oct 16; 7 : e36495 . OpenUrl PubMed 19. ↵ Altschul SF , Gish W , Miller W , Myers EW , Lipman DJ . Basic local alignment search tool . Journal of Molecular Biology . 1990 Oct 5; 215 ( 3 ): 403 – 10 . OpenUrl CrossRef PubMed Web of Science 20. ↵ Mitchell RF , Hall LP , Reagel PF , McKenna DD , Baker TC , Hildebrand JG . Odorant receptors and antennal lobe morphology offer a new approach to understanding olfaction in the Asian longhorned beetle . J Comp Physiol A . 2017 Feb 1; 203 ( 2 ): 99 – 109 . OpenUrl PubMed 21. ↵ Andersson MN , Keeling CI , Mitchell RF . Genomic content of chemosensory genes correlates with host range in wood-boring beetles (Dendroctonus ponderosae , Agrilus planipennis, and Anoplophora glabripennis). BMC Genomics . 2019 Sep 2; 20 ( 1 ): 690 . OpenUrl PubMed 22. ↵ Oppert B , Muszewska A , Steczkiewicz K , Šatović-Vukšić E , Plohl M , Fabrick JA , et al. The Genome of Rhyzopertha dominica (Fab .) ( Coleoptera: Bostrichidae): Adaptation for Success. Genes . 2022 Mar; 13 ( 3 ): 446 . OpenUrl PubMed 23. ↵ Tsirigos KD , Peters C , Shu N , Käll L , Elofsson A . The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides . Nucleic Acids Research . 2015 Jul 1; 43 ( W1 ): W401 – 7 . OpenUrl CrossRef PubMed 24. ↵ McKenna DD , Shin S , Ahrens D , Balke M , Beza-Beza C , Clarke DJ , et al. The evolution and genomic basis of beetle diversity . PNAS . 2019 Dec 3; 116 ( 49 ): 24729 – 37 . OpenUrl Abstract / FREE Full Text 25. ↵ Engsontia P , Sanderson AP , Cobb M , Walden KKO , Robertson HM , Brown S . The red flour beetle’s large nose: An expanded odorant receptor gene family in Tribolium castaneum . Insect Biochemistry and Molecular Biology . 2008 Apr 1; 38 ( 4 ): 387 – 97 . OpenUrl CrossRef PubMed Web of Science 26. ↵ Edgar RC . MUSCLE: multiple sequence alignment with high accuracy and high throughput . Nucleic Acids Research . 2004 Mar 1; 32 ( 5 ): 1792 – 7 . OpenUrl CrossRef PubMed Web of Science 27. ↵ Capella-Gutiérrez S , Silla-Martínez JM , Gabaldón T . trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses . Bioinformatics . 2009 Aug 1; 25 ( 15 ): 1972 – 3 . OpenUrl CrossRef PubMed Web of Science 28. ↵ Price MN , Dehal PS , Arkin AP . FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix . Molecular Biology and Evolution . 2009 Jul 1; 26 ( 7 ): 1641 – 50 . OpenUrl CrossRef PubMed Web of Science 29. ↵ Price MN , Dehal PS , Arkin AP . FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments . Poon AFY, editor. PLoS ONE . 2010 Mar 10; 5 ( 3 ): e9490 . OpenUrl CrossRef PubMed 30. ↵ Edgar RC . Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny . Nat Commun . 2022 Nov 15; 13 ( 1 ): 6968 . OpenUrl CrossRef PubMed 31. ↵ Dress AW , Flamm C , Fritzsch G , Grünewald S , Kruspe M , Prohaska SJ , et al. Noisy: Identification of problematic columns in multiple sequence alignments . Algorithms Mol Biol . 2008 Dec; 3 ( 1 ): 7 . OpenUrl CrossRef PubMed 32. ↵ Lemoine F , Correia D , Lefort V , Doppelt-Azeroual O , Mareuil F , Cohen-Boulakia S , et al. NGPhylogeny.fr: new generation phylogenetic services for non-specialists . Nucleic Acids Research . 2019 Jul 2; 47 ( W1 ): W260 – 5 . OpenUrl CrossRef PubMed 33. ↵ Nguyen LT , Schmidt HA , von Haeseler A , Minh BQ . IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies . Molecular Biology and Evolution . 2015 Jan 1; 32 ( 1 ): 268 – 74 . OpenUrl CrossRef PubMed 34. ↵ Letunic I , Bork P . Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool . Nucleic Acids Research . 2024 Apr 13; gkae268 . 35. ↵ R Core Team . R: A language and environment for statistical computing [Internet] . Vienna, Austria : R Foundation for Statistical Computing ; 2022 . Available from: https://www.R-project.org/ 36. ↵ Yu G , Smith DK , Zhu H , Guan Y , Lam TTY . ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data . Methods in Ecology and Evolution . 2017 ; 8 ( 1 ): 28 – 36 . OpenUrl CrossRef 37. ↵ Wickham H . ggplot2: elegant graphics for data analysis . Second edition. Cham : Springer international publishing ; 2016 . 1 p. (Use R). 38. ↵ Wilkins D , Kurtz Z . gggenes: Draw Gene Arrow Maps in “ggplot2” [Internet] . 2023 [cited 2024 Dec 18]. Available from: https://cran.r-project.org/web/packages/gggenes/index.html 39. ↵ DeLeo DM , Pérez-Moreno JL , Vázquez-Miranda H , Bracken-Grissom HD . RNA profile diversity across arthropoda: guidelines, methodological artifacts, and expected outcomes . Biology Methods and Protocols . 2018 Jan 1; 3 ( 1 ): bpy012 . OpenUrl 40. ↵ Andrews S . FastQC: A Quality Control Tool for High Throughput Sequence Data [Internet] . 2010 . Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 41. ↵ Ewels P , Magnusson M , Lundin S , Käller M . MultiQC: summarize analysis results for multiple tools and samples in a single report . Bioinformatics . 2016 Oct 1; 32 ( 19 ): 3047 – 8 . OpenUrl CrossRef PubMed 42. ↵ Bolger AM , Lohse M , Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data . Bioinformatics . 2014 Aug 1; 30 ( 15 ): 2114 – 20 . OpenUrl CrossRef PubMed Web of Science 43. ↵ Wood DE , Lu J , Langmead B . Improved metagenomic analysis with Kraken 2 . Genome Biology . 2019 Nov 28; 20 ( 1 ): 257 . OpenUrl CrossRef PubMed 44. ↵ Patro R , Duggal G , Love MI , Irizarry RA , Kingsford C . Salmon provides fast and bias-aware quantification of transcript expression . Nat Methods . 2017 Apr; 14 ( 4 ): 417 – 9 . OpenUrl CrossRef PubMed 45. ↵ Love MI , Huber W , Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biology . 2014 Dec 5; 15 ( 12 ): 550 . OpenUrl CrossRef PubMed 46. ↵ Vestal BE , Wynn E , Moore CM . lmerSeq: an R package for analyzing transformed RNA-Seq data with linear mixed effects models . BMC Bioinformatics . 2022 Nov 16; 23 ( 1 ): 489 . OpenUrl CrossRef PubMed 47. ↵ Wynn E , Vestal B , Moore C . REBEL: Repeated measures Empirical Bayes differential Expression analysis using Linear mixed models [Internet] . 2025 . Available from: https://github.com/ewynn610/REBEL 48. ↵ Tran H . Identification of Odorant Receptor Genes in the Antennal Transcriptome of the Common Eastern Fireflies, Photinus pyralis [Internet] [Honors Thesis] . [Lewisburg, PA]: Bucknell University ; 2020 . Available from: https://digitalcommons.bucknell.edu/honors_theses/532 49. ↵ Ginzel MD , Blomquist GJ , Millar JG , Hanks LM . Role of Contact Pheromones in Mate Recognition in Xylotrechus colonus . J Chem Ecol . 2003 Mar 1; 29 ( 3 ): 533 – 45 . OpenUrl CrossRef PubMed 50. Martin SJ , Helanterä H , Drijfhout FP . Evolution of species-specific cuticular hydrocarbon patterns in Formica ants . Biological Journal of the Linnean Society . 2008 ; 95 ( 1 ): 131 – 40 . OpenUrl CrossRef Web of Science 51. Snellings Y , Herrera B , Wildemann B , Beelen M , Zwarts L , Wenseleers T , et al. The role of cuticular hydrocarbons in mate recognition in Drosophila suzukii . Sci Rep . 2018 Mar 22; 8 ( 1 ): 4996 . OpenUrl PubMed 52. ↵ Sun Q , Haynes KF , Zhou X . Temporal Changes in Cuticular Hydrocarbons During Worker-Reproductive Transition in the Eastern Subterranean Termite (Blattodea: Rhinotermitidae) . Annals of the Entomological Society of America . 2021 Sep 1; 114 ( 5 ): 643 – 53 . OpenUrl 53. ↵ Shibue K , Goto Y , Shibue T , Ohba N . Analysis of Sex-Attractant Pheromones of Firefly Pyrocoelia oshimana by Gas Chromatography Mass Spectrometry . Analytical Sciences . 2000 ; 16 ( 9 ): 995 – 6 . OpenUrl 54. Shibue K , Goto Y , Kawashima I , Shibue T . Chemical Analysis of Surface Hydrocarbons in Fireflies by Direct Contact Extraction and Gas Chromatography-Mass Spectrometry . Analytical Sciences . 2004 ; 20 ( 12 ): 1729 – 31 . OpenUrl PubMed 55. ↵ Ming QL , Lewis SM . Mate Recognition and Sex Differences in Cuticular Hydrocarbons of the Diurnal Firefly Ellychnia corrusca (Coleoptera: Lampyridae) . ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA . 2010 ; 103 ( 1 ): 128 – 33 . OpenUrl CrossRef 56. ↵ Lloyd JE . Occurrence of Aggressive Mimicry in Fireflies . The Florida Entomologist . 1984 ; 67 ( 3 ): 368 – 76 . OpenUrl 57. ↵ Brandstaetter AS , Endler A , Kleineidam CJ . Nestmate recognition in ants is possible without tactile interaction . Naturwissenschaften . 2008 Jul 1; 95 ( 7 ): 601 – 8 . OpenUrl CrossRef PubMed Web of Science 58. ↵ Stensmyr MC , Dweck HKM , Farhan A , Ibba I , Strutz A , Mukunda L , et al. A Conserved Dedicated Olfactory Circuit for Detecting Harmful Microbes in Drosophila . Cell . 2012 Dec 7; 151 ( 6 ): 1345 – 57 . OpenUrl CrossRef PubMed Web of Science 59. ↵ McLean M , Buck J , Hanson FE . Culture and Larval Behavior of Photurid Fireflies . The American Midland Naturalist . 1972 ; 87 ( 1 ): 133 – 45 . OpenUrl 60. ↵ De Cock R , Matthysen E . Do Glow-Worm Larvae (Coleoptera: Lampyridae) Use Warning Coloration? Ethology . 2001 ; 107 ( 11 ): 1019 – 33 . OpenUrl CrossRef 61. ↵ Williams FX . Notes on the Life-History of Some North American Lampyridæ . Journal of the New York Entomological Society . 1917 ; 25 ( 1 ): 11 – 33 . OpenUrl 62. ↵ Crespi BJ , Choe JC Lloyd JE . Firefly mating ecology, selection and evolution . In: Crespi BJ , Choe JC , editors. The Evolution of Mating Systems in Insects and Arachnids [Internet] . Cambridge : Cambridge University Press ; 1997 [cited 2021 Dec 21]. p. 184 – 92 . Available from: https://www.cambridge.org/core/books/evolution-of-mating-systems-in-insects-and-arachnids/firefly-mating-ecology-selection-and-evolution/8C7671F14AB3F25E094A248F20AE9F3F 63. ↵ Faust L , Faust H . The Occurrence and Behaviors of North American Fireflies (Coleoptera: Lampyridae) on Milkweed , Asclepias syriaca L. cole . 2014 Jun; 68 ( 2 ): 283 – 91 . OpenUrl 64. ↵ Höhna S , Lower SE , Duchen P , Catalán A . Robustness of Divergence Time Estimation Despite Gene Tree Estimation Error: A Case Study of Fireflies (Coleoptera: Lampyridae) . Systematic Biology . 2024 Nov 13; syae065 . 65. ↵ Tzi-Yuan Wang , King-Siang Goh , Liang-Jong Wang , Li-Ling Wu , Feng-Yu Wang , Yu-Wei Wu . Long-Read Genome Sequencing of Abscondita cerata (Coleoptera: Lampyridae), the Endemic Firefly of Taiwan . Zoological Studies . 2023 Jan 1;–( 62 ). 66. Fu X , Meyer-Rochow VB , Ballantyne L , Zhu X . An Improved Chromosome-Level Genome Assembly of the Firefly Pyrocoelia pectoralis . Insects . 2024 Jan; 15 ( 1 ): 43 . OpenUrl PubMed 67. Fu X , Zhu X . Key homeobox transcription factors regulate the development of the firefly’s adult light organ and bioluminescence . Nat Commun . 2024 Mar 5; 15 ( 1 ): 1736 . OpenUrl PubMed 68. ↵ Catalán A , Gygax D , Rodríguez-Montes L , Hinzke T , Hoff KJ , Duchen P . Two novel genomes of fireflies with different degrees of sexual dimorphism reveal insights into sex-biased gene expression and dosage compensation . Commun Biol . 2024 Jul 27; 7 ( 1 ): 1 – 12 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted March 25, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Lower , Samuel Pring , Hanh Tran , Katherine Martinson , Susan Deering , Mathew A. Price , Brian Vestal , Gregory M. Pask , Douglas B. Collins , Robert F. 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