1. Genetics and Genomics
Download icon

Firefly genomes illuminate parallel origins of bioluminescence in beetles

  1. Timothy R Fallon
  2. Sarah E Lower
  3. Ching-Ho Chang
  4. Manabu Bessho-Uehara
  5. Gavin J Martin
  6. Adam J Bewick
  7. Megan Behringer
  8. Humberto J Debat
  9. Isaac Wong
  10. John C Day
  11. Anton Suvorov
  12. Christian J Silva
  13. Kathrin F Stanger-Hall
  14. David W Hall
  15. Robert J Schmitz
  16. David R Nelson
  17. Sara M Lewis
  18. Shuji Shigenobu
  19. Seth M Bybee
  20. Amanda M Larracuente
  21. Yuichi Oba
  22. Jing-Ke Weng  Is a corresponding author
  1. Whitehead Institute for Biomedical Research, United States
  2. Massachusetts Institute of Technology, United States
  3. Cornell University, United States
  4. Bucknell University, United States
  5. University of Rochester, United States
  6. Chubu University, Japan
  7. Nagoya University, Japan
  8. Monterey Bay Aquarium Research Institute, United States
  9. Brigham Young University, United States
  10. University of Georgia, United States
  11. Arizona State University, United States
  12. Center of Agronomic Research, National Institute of Agricultural Technology, Argentina
  13. Centre for Ecology and Hydrology (CEH), United Kingdom
  14. University of California Davis, United States
  15. University of Tennessee HSC, United States
  16. Tufts University, United States
  17. National Institute for Basic Biology, Japan
Research Article
Cite this article as: eLife 2018;7:e36495 doi: 10.7554/eLife.36495
51 figures, 1 video, 26 tables, 39 data sets and 1 additional file

Figures

Geographic and phylogenetic context of the Big Dipper firefly, Photinus pyralis.

(A) P. pyralis males emitting their characteristic swooping ‘J’ patrol flashes over a field in Homer Lake, Illinois. Females cue in on these species-specific flash patterns and respond with their own species-specific flash (Lloyd, 1966). Photo credit: Alex Wild. Inset: male and female P. pyralis in early stages of mating. Photo credit: Terry Priest. (B) Cladogram depicting the hypothetical phylogenetic relationship between P. pyralis and related bioluminescent and non-bioluminescent taxa with Tribolium castaneum and Drosophila melanogaster as outgroups. Numbers at nodes give approximate dates of divergence in millions of years ago (mya) (Misof et al., 2014; Mckenna et al., 2015). Right: Dorsal and ventral photos of adult male specimens. Note the well-developed ventral light organs on the true abdominal segments 6 and 7 of P. pyralis and A. lateralis. In contrast, the luminescent click beetle, I. luminosus, has paired dorsal light organs at the base of its prothorax (arrowhead) and a lantern on the anterior surface of the ventral abdomen (not visible). (C) Empirical range of P. pyralis in North America, extrapolated from 541 reported sightings (Appendix 1.2). Collection sites of individuals used for genome assembly are denoted with circles and location codes. Cross hatches represent areas which likely have P. pyralis, but were not sampled. Diagonal hashes represent Ontario, Canada.

https://doi.org/10.7554/eLife.36495.003
Photinus pyralis genome assembly and analysis.

(A) Assembled Ppyr1.3 linkage groups with annotation of the location of known luminescence-related genes, combined with Hi-C linkage density maps. Linkage group 3a (box with black arrow) corresponds to the X chromosome (Appendix 1.6.4.1). (B) Fluorescence in situ hybridization (FISH) on mitotic chromosomes of a P. pyralis larvae. The telomeric repeats TTAGG (green) localize to the ends of chromosomes stained with DAPI (blue). 20 paired chromosomes indicates that this individual was an XX female (Appendix 1.13). (C) Genome schematic of P. pyralis mitochondrial genome (mtDNA). Like other firefly mtDNAs, it has a tandem repetitive unit (TRU) (Appendix 1.8). (D) mCG is enriched across gene bodies of P. pyralis and shows methylation levels that are at least two times higher than other holometabolous insects (Appendix 1.12). (E) Orthogroup (OGs) clustering analysis of genes with Orthofinder (Emms and Kelly, 2015) shows a high degree of overlap of the P. pyralis, A. lateralis, and I. luminosus genesets with the geneset of Tribolium castaneum. Numbers within curved brackets (colored by species) represent gene count from specific species within the shared orthogroups. Numbers with square brackets (black color) represent total gene count amongst shared orthogroups. OGs = orthogroups, *=Not fully filtered to single isoform per gene. See Appendix 4.2.1 for more detail. Intermediate scripts and species-specific overlaps are available as Figure 2—source data 1. (F) Assembly statistics for presented genomes. *=Tribolium castaneum model beetle genome assembly (Tribolium Genome Sequencing Consortium et al., 2008) **=Genome size estimated by FC: flow cytometry. P. pyralis n = 5 females (SEM) I. luminosus n = 5 males (SEM), A. lateralis n = 3 technical-replicates of one female (SD). ***=Complete (C), and Duplicated (D), percentages for the Endopterygota BUSCO (Simão et al., 2015) profile (Appendix 1.4, 2.4, 3.4, 4.1).

https://doi.org/10.7554/eLife.36495.005
A genomic view of luciferase evolution.

(A) The reaction scheme of firefly luciferase is related to that of fatty acyl-CoA synthetases. (B) Model for genomic evolution of firefly luciferases. Ranging from genome structures of luciferase loci in extant fireflies (top), to inferred genomic structures in ancestral species (bottom). Arrow (left) represents ascending time. Not all adjacent genes within the same clade are shown. (C) Maximum likelihood tree of luciferase homologs. Grey circles above gene names indicate the presence of peroxisomal targeting signal 1 (PTS1). Color gradients indicate the transcript per million (TPM) values of whole body in each sex/stage (grey to blue) and in the prothorax or abdominal lantern (grey to orange to green). Tree and annotation visualized using iTOL (Letunic and Bork, 2016). Prothorax and abdominal lantern expression values for I. luminosus are from whole prothorax plus head, and metathorax plus the two most anterior abdominal segments. Fluc = firefly luciferases, Eluc = elaterid luciferases, R/PLuc = rhagophthalmid/phengodid luciferases. (Appendix 4.3.2) Gene tree, gene accession numbers, annotation, and expression values are available as Figure 3—source data 1. (D) Synteny analysis of beetle luciferase homologs. Nine of the 14 A. lateralis PACS/ACS genes closely flank AlatLuc1 on scaffold 228, while 4 of the 13 P. pyralis PACS/ACS genes are close neighbors of PpyrLuc1 on LG1, with a further seven genes 2.4 Mbp and 39.1 Mbp away on the same linkage-group. Although the Luc1 loci in P. pyralis and A. lateralis are evidently derived from a common ancestor, the relative positions of the most closely related flanking PACS/ACS genes have diverged between the two species. IlumLuc was captured on a separate scaffold (Ilumi1.2_Scaffold13255) from its most most closely related PACSs (IlumPACS8, IlumPACS9) on Ilumi1.2_Scaffold9864, although three more distantly related PACS genes (IlumiPACS1, IlumiPACS2, IlumiPACS4) are co-localized with IlumLuc. In contrast, a different scaffold (Ilumi1.2_Scaffold9654) shows orthology to the firefly Luc1 locus. The full Ilumi1.2_Scaffold13255 was produced by a manual evidence-supported merge of two scaffolds (Appendix 3.5.4). Genes with a PTS1 are indicated by a dark outline, except for the genes with white interiors, which instead represent non-PACS/ACS genes without an identified homolog in the other scaffolds. Co-orthologous genes are labeled in the same color in the phylogenetic tree and are connected with corresponding color bands in synteny diagram. Genes and genomic regions are to scale (Scale bar = 25 Kbp). Gaps excluded from the figure are shown with dotted lines and are annotated with their length in square brackets. Scaffold ends are shown with rough black bars. MGST = Microsomal glutathione S-transferase, IMP = Inositol monophosphatase, PRNT = Polyribonucleotide nucleotidyltransferase. Figure produced with GenomeTools ‘sketch’ (v1.5.9) (Gremme et al., 2013). Figure production scripts available as Figure 3—source data 2.

https://doi.org/10.7554/eLife.36495.008
Figure 3—source data 1

Gene tree, gene accession numbers, annotation, and expression values for Figure 3C.

https://doi.org/10.7554/eLife.36495.009
Figure 3—source data 2

Bash scripts for Figure 3D figure production.

https://doi.org/10.7554/eLife.36495.010
Parallel evolution of elaterid and firefly luciferase.

(A) Ancestral state reconstruction recovers at least two gains of luciferase activity in bioluminescent beetles. Luciferase activity (top right figure key; black: luciferase activity, white: no luciferase activity, shaded: undetermined) was annotated on extant firefly luciferase homologs via literature review or inference via direct orthology. The ancestral states of luciferase activity within the putative ancestral nodes were then reconstructed with an unordered parsimony framework and a maximum likelihood (ML) framework (bottom left figure key; Appendix 4.3.3). Two gains (‘G’) of luciferase activity, annotated with black arrows and yellow stars, are hypothesized. These hypothesized gains occurred once in a gene within the common ancestor of fireflies, rhagophthalmid, and phengodid beetles, and once in a gene within the common ancestor of bioluminescent elaterid beetles. Scale bar is substitutions per site. Numbers adjacent to nodes represents node support. NEXUS and newick files available as Figure 4—source data 1 (B) Molecular adaptation analysis supports independent neofunctionalization of click beetle luciferase. We tested the molecular adaptation of elaterid luciferase using the adaptive branch-site REL test for episodic diversification (aBSREL) method (Smith et al., 2015) (Appendix 4.3.4). The branch leading to the common ancestor of elaterid luciferases (red star) was one of three branches (red and blue stars) recovered with significant (p<0.01) evidence of positive selection, with 35% of sites showing strong directional selection (ω or max dN/dS = 3.98), which we interpret as signal of the initial neofunctionalization of elaterid ancestral luciferase (EAncLuc) from an ancestor without luciferase activity. As the selected branches with blue stars are red-shifted elaterid luciferases (Oba et al., 2010a; Stolz et al., 2003), they may represent the post-neofunctionalization selection of a few key sites via sexual selection of emission colors. Specific sites identified as under selection using Mixed Effect Model of Evolution (MEME) and Phylogenetic Analysis by Maximum Likelihood (PAML) methods are described in Appendix 4.3.4. The tree and results from the full adaptive model are shown. Branch length, with the exception of the PpyrLuc1 branch which was shortened, reflects the number of substitutions per site. Numbers adjacent to nodes represents node support. Figure was produced with iTOL (Letunic and Bork, 2016). Gene tree, metadata, and coding nucleotide multiple sequence alignment available as Figure 4—source data 2.

https://doi.org/10.7554/eLife.36495.011
Figure 4—source data 1

NEXUS and Newick files for luciferase ancestral state reconstruction in Figure 4A.

https://doi.org/10.7554/eLife.36495.012
Figure 4—source data 2

Gene tree, metadata, and coding nucleotide multiple sequence alignment for Elaterid luciferase homolog branch selection test.

https://doi.org/10.7554/eLife.36495.013
Comparative analyses of firefly lantern expression highlight likely metabolic adaptations to bioluminescence.

Enzymes which are highly expressed (HE), differentially expressed (DE), and annotated as enzymes via InterProScan are shown in the Venn diagrams for their respective species. Those genes in the intersection of the two sets which are within the same orthogroup (OGs) as determined by OrthoFinder are shown in the table. Many-to-one orthology relationships are represented by bold orthogroups and blank cells. See Appendix 4.2.2 for more detail. *=genes of previously described function. Underlying expression quantification and Venn analysis available on FigShare: (DOI: 10.6084/m9.figshare.5715151)

https://doi.org/10.7554/eLife.36495.014
Figure 5—source data 1

Table of Figure 5 highly expressed, differentially expressed, orthogroup overlapped genes.

https://doi.org/10.7554/eLife.36495.015
An expansion in the CYP303-P450 family correlates with lucibufagin content.

(A) Hypothesized lucibufagin biosynthetic pathway, starting from cholesterol. (B) LC-HRAM-MS multi-ion-chromatograms (MIC) showing the summation of exact mass traces for the [M + H]+ of 11 lucibufagin chemical formulas ± 5 ppm, calibrated for run-specific systematic m/z error (Appendix 4—table 9). Y-axis upper limit for P. pyralis adult hemolymph and larval body extract is 1000x larger than other traces. Arrows (blue/teal) indicate features with high MS2 spectral similarity to known lucibufagins. Sporadic peaks in A. lateralis body, and I. luminosus thorax traces are not abundant, preventing MS2 spectral acquisition and comparison, but do not match the m/z and RT of P. pyralis lucibufagins (Appendix 4.6). (C) Maximum likelihood tree of CYP303 family cytochrome P450 enzymes from P. pyralis, A. lateralis, T. castaneum, and D. melanogaster. P. pyralis shows a unique CYP303 family expansion, whereas the other species only have a single CYP303. Circles represent node bootstrap support >60%. Branch length measures substitutions per site. Pseudogenes are annotated with the greek letter Ψ (Appendix 1.10.1; 4.2.4). (D) Genomic loci for P. pyralis CYP303 family genes. These genes are found in multiple gene clusters on LG9, supporting origin via tandem duplication. Introns >4 kbp are shown.

https://doi.org/10.7554/eLife.36495.016
Figure 6—source data 1

CYP303 multiple sequence alignment and gene tree for Figure 6C.

https://doi.org/10.7554/eLife.36495.017
Appendix 1—figure 1
Detailed geographic distribution map for P. pyralis.

P. pyralis sightings (red circles show county centroided reports) in the United States and Ontario, Canada (diagonal hashes). The World Wildlife Fund Terrestrial Ecoregions (Olson et al., 2001; World Wildlife Fund, 2017) are also shown (colored shapes). The P. pyralis sighting dataset shown is identical to that used to prepare Figure 1B.

https://doi.org/10.7554/eLife.36495.020
Appendix 1—figure 2
P. pyralis aedeagus (male genitalia).

(A) Ventral and (B) side view of a P. pyralis aedeagus dissected from specimens collected on the same date and locality as those used for PacBio sequencing. Note the strongly sclerotized paired ventro-basal processes (‘mickey mouse ears’) emerging from the median process, characteristic of P. pyralis (Green, 1956).

https://doi.org/10.7554/eLife.36495.021
Appendix 1—figure 3
Luminescence of P. pyralis eggs.

(A) Photograph under ambient light of ~1 day post-deposition P. pyralis eggs. (B) Photograph of self-luminescence of ~1 day post-deposition P. pyralis eggs. Both photographs taken with a NightOwl LB98 cooled CCD luminescence imager (Berthold Technologies, USA). Luminescence was not visible to the dark-adapted eye.

https://doi.org/10.7554/eLife.36495.022
Appendix 1—figure 4
Gregarious predation of young P. pyralis larvae on a live Lumbricus terrestris.

Both P. pyralis larvae (red arrows), and Enchytraeus albidus (yellow arrows), were observed to feed on the paralyzed earthworms.

https://doi.org/10.7554/eLife.36495.023
Appendix 1—figure 5
Gregarious predation of 3rd-4th instar P. pyralis larvae on a live Lumbricus terrestris.
https://doi.org/10.7554/eLife.36495.024
Appendix 1—figure 6
Genome scope kmer analysis of the P. pyralis short read library.

(A) Linear and (B) log plot of a kmer spectral genome composition analysis of the ‘8369’ P. pyralis Illumina short-read library from a single P. pyralis XO adult male (Appendix 1.5.1; Appendix 4—table 1) with jellyfish (v2.2.9; parameters: -C -k 35) (Marçais and Kingsford, 2011) and GenomeScope (v1.0; parameters: Kmer length = 35, Read length = 100, Max kmer coverage = 1000) (Vurture et al., 2017). len = inferred haploid genome length, uniq = percentage non-repetitive sequence, het = overall rate of genome heterozygosity, kcov = mean kmer coverage for heterozygous bases, err = error rate of the reads, dup: average rate of read duplications. These results are consistent with the genome size of a XO male, when possible systematic error of kmer spectral analysis and flow cytometry genome size estimates is considered. The heterozygosity is somewhat low when compared to some other arthropods.

https://doi.org/10.7554/eLife.36495.025
Appendix 1—figure 7
PFGE of P. pyralis HMW DNA used for PacBio sequencing.

Lane 1 was used for further library prep and sequencing, Lanes 2–5 represent separate batches of P. pyralis HMW DNA that was not used for PacBio sequencing. Lane 1 was used as it had the highest DNA yield, and an equivalent DNA size distribution to the other samples.

https://doi.org/10.7554/eLife.36495.026
Appendix 1—figure 8
Subread length distribution for P. pyralis PacBio RSII sequencing.

Figure produced with SMRTPortal (v2.3.0.140936, Pacific Biosciences, 2017) by aligning all PacBio reads from data from the 61 SMRT cells against Ppyr1.3 using the RS_Resequencing.1 protocol with default parameters. Subread length unit is basepair (bp).

https://doi.org/10.7554/eLife.36495.027
Appendix 1—figure 9
Blobplot of Illumina short-insert reads aligned against the Ppyr1.2 reference.

Coverage shown represents mean coverage of reads from the Illumina short-insert library (Sample name 8369; Appendix 4—table 1), aligned against Ppyr1.2 using Bowtie2 with parameters (--local). Scaffolds were taxonomically annotated as described in Appendix 1.6.4.2.

https://doi.org/10.7554/eLife.36495.028
Appendix 1—figure 10
Blobplot of P. pyralis PacBio reads aligned against Ppyr1.2.

Coverage shows represents mean coverage of reads from the PacBio library (Sample name 1611; Appendix 4—table 1). The reads were aligned using SMRTPortal v2.3.0.140893 with the ‘RS_Resequencing.1’ protocol with default parameters. Scaffolds were taxonomically annotated as described in Appendix 1.6.4.2.

https://doi.org/10.7554/eLife.36495.029
Appendix 1—figure 11
Venn diagram representation of blobtools taxonomic annotation filtering approach for Ppyr1.2 scaffolds.

(A) The blue set represents scaffolds which have >10.0 coverage in both Illumina and PacBio libraries. (B) The red set represents scaffolds which had either genes on repeats (non simple or low-complexity) annotated. (C) The green set represents scaffolds with suspicious taxonomic assignment (Non ‘Arthropod’ or ‘no-hit’). Outside A, B, and C, represents low-coverage, unannotated scaffolds. Ppyr1.3 consists of the intersection of A and B, minus the intersection of C. All linkage groups (LG1-LG10) were annotated as ‘Arthropod’ by blobtools, and captured in the intersection between A and B but not set C.

https://doi.org/10.7554/eLife.36495.030
Appendix 1—figure 12
Mitochondrial genome of P. pyralis.

The mitochondrial genome of P. pyralis was assembled and annotated as described. Note the firefly specific tandem-repeat-unit (TRU) region. Figure produced with Circos (Krzywinski et al., 2009).

https://doi.org/10.7554/eLife.36495.031
Appendix 1—figure 13
P. pyralis P450 gene phylogenetic tree.

Neighbor-joining phylogenetic tree of 165 cytochrome P450s from P. pyralis. Four pseudogenes and one short sequence were removed. The P450 clans have colored spokes (CYP2 clan brown, CYP3 clan green, CYP4 clan red, Mito clan blue). Shading highlights different families and family clusters within the CYP3 clan. The tree was made using Clustal Omega at EBI (European Bioinformatics Institute, 2017) with default settings. The resulting multiple sequence alignment is available on FigShare (DOI: 10.6084/m9.figshare.5697643). The tree was drawn with FigTree v1.3.1 using midpoint rooting.

https://doi.org/10.7554/eLife.36495.033
Appendix 2—figure 1
Genome scope kmer analysis of the A. lateralis short-insert genomic library.

(A) Linear and (B) log plot of a kmer spectral genome composition analysis of the ‘FFGPE_PE200’ A. lateralis Illumina short-insert library (Appendix 2.5; Appendix 4—table 1) with jellyfish (v2.2.9; parameters: -C -k 35) (Marçais and Kingsford, 2011) and GenomeScope (v1.0; parameters: Kmer length = 35, Read length = 100, Max kmer coverage = 1000) (Vurture et al., 2017). len = inferred haploid genome length, uniq = percentage non-repetitive sequence, het = overall rate of genome heterozygosity, kcov = mean kmer coverage for heterozygous bases, err = error rate of the reads, dup: average rate of read duplications. These results are consistent when considering the possible systematic error of kmer spectral analysis and flow cytometry genome size estimates. The heterozygosity is lower than that measured for P. pyralis, possibly reflecting the long-term laboratory rearing in reduced population sizes of A. lateralis strain Ikeya-Y90.

https://doi.org/10.7554/eLife.36495.037
Appendix 2—figure 2
Blobplot of A. lateralis Illumina reads aligned against Alat1.2.

Coverage shown represents mean coverage of reads from the Illumina short-insert library (Sample name FFGPE_PE200; Appendix 4—table 1), aligned against Alat1.2 using Bowtie2. Scaffolds were taxonomically annotated as described in Appendix 2.5.2.

https://doi.org/10.7554/eLife.36495.038
Appendix 3—figure 1
I. luminosus aedeagus (male genitalia).

(A) Dorsal and (B) ventral view of an Ignelater luminosus aedeagus, dissected from the same batch of specimens used for linked-read sequencing and genome assembly. The species identity of this specimen was confirmed as I. luminosus by comparison of the aedeagus to the keys of Costa and Rosa (Costa, 1975; Rosa, 2007; Rosa, 2010).

https://doi.org/10.7554/eLife.36495.042
Appendix 3—figure 2
Genome scope kmer analysis of the I. luminosus linked-read genomic library.

(A) Linear and (B) log plot of a kmer spectral genome composition analysis of the ‘1610_IlumiHiSeqX’ I. luminosus Illumina linked-read library (Appendix 2.5; Appendix 4—table 1) with jellyfish (v2.2.9; parameters: -C -k 35) (Marçais and Kingsford, 2011) and GenomeScope (v1.0; parameters: Kmer length = 35, Read length = 138, Max kmer coverage = 1000) (Vurture et al., 2017). Before analysis, 10x Chromium barcodes were trimmed off Read1 using cutadapt (v1.8; parameters: -u 23) (Martin, 2011). vlen = inferred haploid genome length, uniq = percentage non-repetitive sequence, het = overall rate of genome heterozygosity, kcov = mean kmer coverage for heterozygous bases, err = error rate of the reads, dup: average rate of read duplications. These results are consistent when considering the possible systematic error of kmer spectral analysis and flow cytometry genome size estimates. The heterozygosity is higher than that measured for P. pyralis and A. lateralis. The read error rate for this library is also significantly higher than the P. pyralis and A. lateralis results, possibly highlighting the difference in raw read error rate between HiSeq2500 and HiSeqX sequencing, or is possibly an artifact of the Chromium library.

https://doi.org/10.7554/eLife.36495.043
Appendix 3—figure 3
Blobtools plot of Ilumi1.0.

Coverage shown represents mean coverage of reads from the HiSeqX Chromium library sequencing (Sample name 1610_IlumiHiSeqX; Appendix 4—table 1), aligned against Ilumi1.0 using Bowtie2 with parameters (--local). Scaffolds were taxonomically annotated as described in Appendix 3.5.2.

https://doi.org/10.7554/eLife.36495.044
Appendix 3—figure 4
Self alignment of the Ilumi1.1_Scaffold13255 right-edge extending long MinION read.

Alignment performed in in Gepard (Krumsiek et al., 2007). Note the large (10 kbp+) tandem repetitive region.

https://doi.org/10.7554/eLife.36495.045
Appendix 3—figure 5
Diagram of manual scaffold merges between Ilumi1.1 and Ilumi1.2.

Diagram of the manual merge of Ilumi1.1_Scaffold13255 with Ilumi1.1_Scaffold11560 between I. luminosus genome assembly versions Ilumi1.1 and Ilumi1.2. This merge was supported by: (1) The putative missing first exon of IlumPACS4 being present on the right edge of Ilumi1.2_Scaffold11560. (2) The right edge of Ilumi1.1_Scaffold13255, and the right edge of Ilumi1.1_Scaffold11560, having anti-parallel versions of a homologous complex tandem repeat. See Figure 3 in the maintext for explanation of presented genes.

https://doi.org/10.7554/eLife.36495.047
Appendix 3—figure 6
Mitochondrial genome of I. luminosus.

The mitochondrial genome of I. luminosus was assembled and annotated as described. in the Appendix 3.10. Figure produced with Circos (Krzywinski et al., 2009).

https://doi.org/10.7554/eLife.36495.050
Appendix 4—figure 1
Venn diagram of P. pyralis, A. lateralis, I. luminosus, T. castaneum, and D. melanogaster orthogroup relationships.

Orthogroups were calculated between the PPYR_OGS1.1, AQULA_OGS1.0, ILUMI_OGS1.2, genesets, and the T. casteneum and D. melanogaster filtered Uniprot reference proteomes using OrthoFinder(Emms and Kelly, 2015). See Appendix 4.2.1 for description of clustering method. OGs = Orthogroups, OGS = Official gene set, *=Not completely filtered to single peptide per gene. Figure produced with InteractiVenn (Heberle et al., 2015). Intermediate scripts and species specific overlaps are available as Figure 2—source data 1.

https://doi.org/10.7554/eLife.36495.055
Appendix 4—figure 2
DNA and tRNA methyltransferase gene phylogeny.

Levels and patterns of mCG in P. pyralis are corroborated by the presence of de novo and maintenance DNMTs (DNMT3 and DNMT1, respectively). Notably, P. pyralis possesses two copies of DNMT1, and 3 copies of DNMT3, in contrast to a single copy of DNMT1 and DNMT3 in the firefly Aquatica lateralis. The evolutionary history was inferred by using the Maximum Likelihood method with the LG + G (five gamma categories) (Le and Gascuel, 2008). Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016). Size of circles at nodes corresponds to bootstrap support (100 bootstrap replicates). Branch lengths are in amino acid substitutions per site. T. castaneum = Tribolium castaneum, D. melanogaster = Drosophila melanogaster, N. vespilloides = Nicrophorus vespilloides. The multiple sequence alignment and phylogenetic topology are available on FigShare (10.6084/m9.figshare.6531311).

https://doi.org/10.7554/eLife.36495.056
Appendix 4—figure 3
Detection of DNA methylation using CpG[O/E].
https://doi.org/10.7554/eLife.36495.057
Appendix 4—figure 4
Intron-exon structure of beetle luciferases.

(A) Intron-exon structure of P. pyralis and A. lateralis Luc1 and Luc2 from Ppyr1.3 and Alat1.3, and IlumLuc from Ilumi1.2. Between fireflies and click-beetles, the structure of the luciferase genes are globally similar, with seven exons, similar intron lengths, and identical splice junction locations (Appendix 4—figure 5). The intron-exon structure of IlumLuc is consistent with the reported intron-exon structure of Pyrophorus plagiophthalamus luciferase (Velez and Feder, 2006).

https://doi.org/10.7554/eLife.36495.058
Appendix 4—figure 5
Multiple sequence alignment of firefly luciferase genes.

MAFFT (Katoh and Standley, 2013) L-INS-i multiple sequence alignment of luciferase gene nucleotide sequences from PpyrOGS1.1 and AlatOGS1.0 demonstrates the location of intron-exon junctions (bolded blue text) is completely conserved amongst the four luciferases. Exonic sequence is capitalized, whereas intronic sequence is lowercase.

https://doi.org/10.7554/eLife.36495.059
Appendix 4—figure 6
Preliminary maximum likelihood phylogeny of luciferase homologs.

A preliminary maximum likelihood tree was reconstructed from a 385 amino acid multiple sequence alignment, generated via a BLASTP and orthoDB search using P. pyralis luciferase as query (e-value: 1.0 × 10−60). Members of the clade that includes both known firefly luciferase and CG6178 of D. melanogaster (bold) are defined as luciferase co-orthologous genes (highlighted in gray), and were selected and used for the independent maximum likelihood analysis in Figure 3C (Appendix 4.3.2). Branch length represents substitutions per site. Genes found from this study are indicated in blue. Lampyridae Luc1-type and Luc2-type luciferases are highlighted in yellow-green and green. Rhagophthalmidae and Phengodidae luciferases are highlighted in lime-green. Elateridae luciferases are highlighted in yellow. Genbank accession numbers of luciferase orthologs genes are indicated after the species name. OrthoDB taxon and protein IDs of luciferase co-orthologs are indicated after species name. Bootstrap values are indicated on the nodes. The genes from Coleoptera are indicated as purple strip. Grey closed circles indicate genes that have PTS1.

https://doi.org/10.7554/eLife.36495.060
Appendix 4—figure 7
Amino acid variation at sites recovered in selection analysis.

Amino acid variation of extant Elaterid luciferases (Clade D ‘Eluc’ subset; Figure 3) at all sites recovered via both the MEME and PAML-BEB selection analysis (Appendix 4—table 5). Site numbering relative to IlumLuc. Figure produced with seqkit (Shen et al., 2016) and WebLogo(v3.6.0) (Crooks et al., 2004).

https://doi.org/10.7554/eLife.36495.063
Appendix 4—figure 8
Maximum likelihood gene tree of the combined adenylyl-sulfate kinase and sulfate adenylyltransferase (ASKSA) orthogroup.

Peptide sequences from P. pyralis, A. lateralis, I. luminosus, T. castaneum, and D. melanogaster were clustered (orthogroup # 698), multiple sequence aligned, and refactored into a species rooted maximum likelihood tree, via the OrthoFinder pipeline (Appendix 4.2.1). As this is a genome-wide analysis where bootstrap replicates would be computationally prohibitive, no bootstrap replicates were performed to evaluate the support of the tree topology. PTS1 sequences were predicted from the peptide sequence using the PTS1 predictor server (Neuberger et al., 2017). Figure produced with iTOL (Letunic and Bork, 2016).

https://doi.org/10.7554/eLife.36495.065
Appendix 4—figure 9
ML tree and gene expression levels of opsin genes.
https://doi.org/10.7554/eLife.36495.066
Appendix 4—figure 10
Positive mode MS1 total-ion-chromatogram (TIC) of P.pyralis adult hemolymph LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.067
Appendix 4—figure 11
Negative mode MS1 total-ion-chromatogram (TIC) of P. pyralis adult hemolymph LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.068
Appendix 4—figure 12
Positive mode MS1 total-ion-chromatogram (TIC) of P. pyralis larval whole body minus two posterior segments LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.069
Appendix 4—figure 13
Negative mode MS1 total-ion-chromatogram (TIC) of P. pyralis larval whole body minus two posterior segments LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.070
Appendix 4—figure 14
Positive mode MS1 total-ion-chromatogram (TIC) of A. lateralis adult hemolymph LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.071
Appendix 4—figure 15
Negative mode MS1 total-ion-chromatogram (TIC) of A. lateralis adult hemolymph LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.072
Appendix 4—figure 16
Positive mode MS1 total-ion-chromatogram (TIC) of A. lateralis larval whole body LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.073
Appendix 4—figure 17
Negative mode MS1 total-ion-chromatogram (TIC) of A. lateralis larval whole body extract LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.074
Appendix 4—figure 18
Positive mode MS1 total-ion-chromatogram (TIC) of I. luminosus mesothorax +abdomen extract LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.075
Appendix 4—figure 19
Negative mode MS1 total-ion-chromatogram (TIC) of I.luminosus mesothorax + abdomen extract LC-HRAM-MS data.

Figure produced using MZmine2 (Pluskal et al., 2010).

https://doi.org/10.7554/eLife.36495.076
Appendix 4—figure 20
Positive mode MS2 spectra of (A) diacetylated lucibufagin [M + H]+ and (B) dipropylated lucibufagin [M + H]+.
https://doi.org/10.7554/eLife.36495.077
Appendix 4—figure 21
MS2 spectral similarity network for P.pyralis adult hemolymph lucibufagins.

(A) MS2 similarity network produced with the MZmine2 MS2 similarity search module. Nodes represent MS2 spectra from the initial dataset, whereas edges represent an MS2 similarity match between two MS2 spectra. Thickness/label of the edge represents the number of ions matched between the two MS2 spectra. (B) Table of matched ions between diacetylated lucibufagin (m/z: 533.2385 RT:15.1), and core (unacetylated) lucibufagin (m/z: 449.2171 RT:10.8 min). MS1 adducts and complexes of the presented ions were manually removed.

https://doi.org/10.7554/eLife.36495.078
Appendix 5—figure 1
Mitochondrial genome of Apocephalus antennatus.

The mitochondrial genome of A. antennatus was assembled and annotated as described in the Appendix 5.2, and taxonomically identified as described in Appendix 5.3. Figure produced with Circos (Krzywinski et al., 2009).

https://doi.org/10.7554/eLife.36495.084
Appendix 5—figure 2
Photinus pyralis viruses and endogenous viral-like elements.

(A) Phylogenetic tree based in MAFFT alignments of predicted replicases of Orthomyxoviridae (OMV) ICTV accepted viruses (green stars), new Photinus pyralis viruses (underlined) and tentative OMV-like virus species (black stars). ICTV recognized OMV genera: Quaranjavirus (orange), Thogotovirus (purple), Issavirus (turquoise), Influenzavirus A-D (green). Silhouettes correspond to host species. Asterisk denote FastTree consensus support >0.5. Question marks depict viruses with unidentified or unconfirmed host. (B) Phylogenetic tree of OMV proposed and recognized species in the context of all ssRNA (-) virus species, based on MAFFT alignments of refseq replicases. Photinus pyralis viruses are portrayed by black stars. (C) Phylogenetic tree of ICTV recognized OMV species and PpyrOMLV1 and 2. Numbers indicate FastTree consensus support. (D) Genetic distances of concatenated gene products of OMV depicted as circoletto diagrams. Proteins are oriented clockwise in N-HA-PB1-PB2-PA order when available. Sequence similarity is expressed as ribbons ranging from blue (low) to red (high). (E) Genomic architecture, predicted gene products and structural and functional domains of PpyrOLMV1 and 2. (F) Virus genomic noncoding termini analyses of PpyrOLMV1 and 2 in the context of ICTV OMV. The 3’ and 5’ end, A and U rich respectively, partially complementary sequences are associated to tentative panhandle polymerase binding and replication activity, typical of OMV. (G) 3D renders of the heterotrimeric polymerase of PpyrOMLV1 based on Swiss-Expasy generated models using as template the Influenza A virus polymerase structure. Structure comparisons were made with the MatchAlign tool of the Chimera suite, and solved in PyMOL. (H) Conserved functional motifs of PpyrOLMV1 and 2 PB1 and related viruses. Motif I-III are essential for replicase activity of viral polymerase. (I) Dynamic and prevalent virus derived RNA levels of the corresponding PpyrOMLV1 and 2 genome segments, determined in 24 RNA libraries of diverse individuals/developmental stages/tissues and geographic origins. RNA levels are expressed as normalized TPM, heatmaps were generated by Shinyheatmap. Values range from low (green) to high (red). (J) Firefly EVEs (FEVEs) identified in the P. pyralis genome assembly mapped to the corresponding pseudo-molecules. A 15 Kbp region flanking nucleoprotein like FEVES are depicted, enriched in transposable elements. Representative products of a putative PB2 FEVE are aligned to the corresponding protein of PpyrOMLV 2.

https://doi.org/10.7554/eLife.36495.085
Appendix 5—figure 3
Pairwise identity of OMLV viral proteins amongst identified OMLV viruses.
https://doi.org/10.7554/eLife.36495.086

Videos

Video 1
A Photinus pyralis courtship dialogue.
https://doi.org/10.7554/eLife.36495.004

Tables

Appendix 1—table 1
P. pyralis RNA sequencing libraries.

N: number of individuals pooled for sequencing; Sex/stage: M = male, F = female, A = adult, L = larva, L1 = larva 1 st instar, L4 = larvae fourth instar, E13 = 13 days post fertilization eggs; Tissue: H = head, PA = lantern abdominal segments, FB = abdominal fat body, T = thorax, OAG = other accessory glands, SD = spermatophore digesting gland/bursa, SG = spiral gland, SC = spermatheca, p=dissected photophore, E = egg, WB = whole body.

https://doi.org/10.7554/eLife.36495.032
Library nameSource*SRA IDNSex/stageTissueLibrary type
8175 Photinus pyralis male head (adult) transcriptomeSRA1SRR21038481M/AH
8176 Photinus pyralis male light organ (adult) transcriptomeSRA1SRR21038491M/APA
8819 Photinus pyralis light organ (larval) transcriptomeSRA1SRR21038671LPA
9_Photinus_sp_1_lanternSRA2SRR35214241M/APAStrand-specific. Ribo-zero
Ppyr_FatBody_1SRA3SRR38837566M/AFB
Ppyr_FatBody_2SRA3SRR38837576M/AFB
Ppyr_FatBody_3SRA3SRR38837666M/AFB
Ppyr_FatBody_MatedSRA3SRR38837674M/AFB
Ppyr_FThoraxSRA3SRR38837683F/AT
Ppyr_MThorax_1SRA3SRR38837696M/AT
Ppyr_MThorax_2SRA3SRR38837706M/AT
Ppyr_MThorax_3SRA3SRR38837716M/AT
Ppyr_OAG_1ASRA3SRR38837726M/AAG
Ppyr_OAG_1BSRA3SRR38837736M/AAG
Ppyr_OAG_2SRA3SRR38837586M/AAG
Ppyr_OAG_MatedSRA3SRR38837594M/AAG
Ppyr_SDGBursaSRA3SRR38837603F/ASD
Ppyr_SG_MatedSRA3SRR38837614M/ASG
Ppyr_SpermathecaSRA3SRR38837623F/ASC
Ppyr_SpiralGland_1SRA3SRR38837636M/ASG
Ppyr_SpiralGland_2SRA3SRR38837646M/ASG
Ppyr_SpiralGland_3SRA3SRR38837656M/ASG
Ppyr_Lantern_1ASRR63454536M/AP
Ppyr_Lantern_2SRR63454546M/AP
Ppyr_Lantern_3SRR63454466M/AP
Ppyr_EggsSRR63454477E13EStrand-specific
Ppyr_LarvaeSRR63454454L1WBStrand-specific
Ppyr_wholeFemaleSRR63454491F/AWBStrand-specific
Ppyr_wholeMaleSRR63454521M/AWBStrand-specific
TF_VA2017_3pooled_larval_lanternSRR73455803L4P
  1. *SRA1 = NCBI BioProject PRJNA289908 (Sander and Hall, 2015); SRA2 = NCBI BioProject PRJNA321737 (Fallon et al., 2016); SRA3 = NCBI BioProject PRJNA328865 (Al-Wathiqui et al., 2016).

    Parent of eggs and larvae with data from this study.

  2. This study.

Appendix 1—table 2
Annotated repetitive elements in P. pyralis.
https://doi.org/10.7554/eLife.36495.034
Repeat classFamilyCountsBases% of assembly
DNAAll122551383646858.14
Helitrons3506893081001.97
LTRAll28860114016482.42
Non-LTRAll52107177443203.76
LINE48983167634993.56
SINE12411396370.03
Unknown interspersed69651114197097730.1
Complex tandem repeats1039523527960.50
Simple repeat4822423721830.50
rRNA4491615170.034
Appendix 1—table 3
Photinus pyralis genome Experiment.com crowdfunding donors (https://experiment.com/projects/illuminating-the-firefly-genome).
https://doi.org/10.7554/eLife.36495.035
Liliana BachrachDoug FambroughBenjamin LowerLuis CunhaJoshua Guerriero
Atsuko FishTom AlarNoreen HuefnerDavid EsopiJohn Skarha
Rutong XieRichard HallZachary MichelJack HynesKeith Guerin
Nathan ShanerJoe DoggettJoe T. BambergMichael McGurkPureum Kim
Sara LewisMark LewisLauren SolomonPeter BerxMilo Grika
Jing-Ke WengSarah SanderDr. Husni ElbaheshMatt GrommesDaniel Zinshteyn
Peter RodenbeckDaniel BearKathryn LarracuenteColette DedynTom Brekke
Larry FishDon SalvatoreMatthew CichockiFlorencia SchlampEdoardo Gianni
Amanda LarracuenteEmily DavenportMarcel BruchezMarie LowerCindy Wu
Hunter LowerTed SharpeRobert UncklessMichael R. McKainChristina Tran
Allan KleinmanDavid PlunkettArvid ÅgrenBen PfeifferEric Damon Walters
Misha KoksharovTim FallonMargaret S ButlerKathryn KehoGeoffrey Giller
Sarah ShekherEdward GarrityYasir Ahmed-BraimahJenny WayfarerFahd Butt
Jared LeeHuaping MoRuth Ann GrissomDarby ThomasChristophe Mandy
Raphael De CockTimGTomáš PluskalEmily Hatas
Linds FallonJan ThysGenome GalaxyRichard Casey
Grace LiFrancisco Martinez GascoDustin GreinerWilliam Nicholls
Appendix 2—table 1
Aquatica lateralis RNA sequencing.

N: number of individuals pooled for sequencing; Sex/stage: M = male, F = female, A = adult, L = larva, L = larvae, E = Eggs, p=Pupae, P-E = Pupae early, P-M = Pupae middle, P-L = Pupae late; Tissue: H = head, La = dissected lantern containing cuticle, photocyte layer and reflector layer, H = head, B = Thorax, plus abdomen excluding lantern containing segments. W = whole specimen. AEL = After egg laying.

https://doi.org/10.7554/eLife.36495.039
Library nameLabelSRA IDNSex/
Stage
TissueLibrary type
R102L6_idx13BdM1DRR1192641M/ABIllumina paired-end, non-stranded specific, PolyA
R128L1_idx25BdM2DRR1192651M/ABIllumina single-end, non-stranded specific, PolyA
R128L2_idx27BdM3DRR1192661M/ABIllumina single-end, non-stranded specific, PolyA
R102L6_idx15HeF1DRR1192673F/AHIllumina paired-end, non-stranded specific, PolyA
R128L1_idx22HeF2DRR1192683F/AHIllumina single-end, non-stranded specific, PolyA
R128L2_idx23HeF3DRR1192693F/AHIllumina single-end, non-stranded specific, PolyA
R102L6_idx12HeM1DRR1192702M/AHIllumina paired-end, non-stranded specific, PolyA
R128L1_idx20HeM2DRR1192712M/AHIllumina single-end, non-stranded specific, PolyA
R128L2_idx21HeM3DRR1192722M/AHIllumina single-end, non-stranded specific, PolyA
R102L6_idx16LtF1DRR1192735F/ALaIllumina paired-end, non-stranded specific, PolyA
R128L1_idx06LtF2DRR1192745F/ALaIllumina single-end, non-stranded specific, PolyA
R128L2_idx12LtF3DRR1192755F/ALaIllumina single-end, non-stranded specific, PolyA
R102L6_idx14LtM1DRR1192765M/ALaIllumina paired-end, non-stranded specific, PolyA
R128L1_idx05LtM2DRR1192775M/ALaIllumina single-end, non-stranded specific, PolyA
R128L2_idx19LtM3DRR1192785M/ALaIllumina single-end, non-stranded specific, PolyA
R128L2_idx15WAF1DRR1192791F/AWIllumina single-end, non-stranded specific, PolyA
R128L1_idx16WAF2DRR1192801F/AWIllumina single-end, non-stranded specific, PolyA
R128L2_idx18WAF3DRR1192811F/AWIllumina single-end, non-stranded specific, PolyA
R128L1_idx11WAM1DRR1192821M/AWIllumina single-end, non-stranded specific, PolyA
R128L2_idx13WAM2DRR1192831M/AWIllumina single-end, non-stranded specific, PolyA
R128L1_idx14WAM3DRR1192841M/AWIllumina single-end, non-stranded specific, PolyA
R102L6_idx4Egg1DRR11928519.6 mg
(~30–50)
E
~6 hr AEL
WIllumina paired-end, non-stranded specific, PolyA
R128L1_idx01Egg2DRR11928621.6 mg
(~30–50)
E
~7 d AEL
WIllumina single-end, non-stranded specific, PolyA
R102L6_idx5Lrv1DRR1192871LWIllumina paired-end, non-stranded specific, PolyA
R128L1_idx03Lrv2DRR1192881LWIllumina single-end, non-stranded specific, PolyA
R128L2_idx04Lrv3DRR1192891LWIllumina single-end, non-stranded specific, PolyA
R128L1_idx07PpEMDRR1192901M/P-EWIllumina single-end, non-stranded specific, PolyA
R128L2_idx10PpLFDRR1192911F/P-LWIllumina single-end, non-stranded specific, PolyA
R128L1_idx09PpMFDRR1192921F/P-MWIllumina single-end, non-stranded specific, PolyA
R128L2_idx08PpMMDRR1192931M/P-MWIllumina single-end, non-stranded specific, PolyA
R102L6_idx7PpEFDRR1192941F/P-EWIllumina paired-end, non-stranded specific, PolyA
R102L6_idx6PpLMDRR1192951M/P-LWIllumina paired-end, non-stranded specific, PolyA
Appendix 2—table 2
Annotated repetitive elements in A. lateralis.
https://doi.org/10.7554/eLife.36495.040
Repeat classFamilyCountsBases% of assembly
DNAAll229064732635938.06
Helitrons9304666790.051
LTRAll59499233919562.57
Non-LTRAll151788503948535.55
LINE151788503948535.55
SINE000
Unknown interspersed4509349999895811.01
Complex tandem repeats295332370.004
Simple repeat15526566567570.73
rRNA000
Appendix 3—table 1
Sequence of the I. luminosus luciferase cluster splitting complex tandem repeat.
https://doi.org/10.7554/eLife.36495.046
Repeat nameRepeat unit lengthRepeat unit sequence
Ilumi.complex.repeat.1~100 bpTGGTACGAACTATACACGTATACTCAAATCTAATT
GTGATACAGCAAAGTAATAATGCAGCATTGTTTGCC
GCTCTATACTGCGATTTTATAGTGGT
Appendix 3—table 2
I. luminosus RNA-Seq libraries.
https://doi.org/10.7554/eLife.36495.048
Library nameSRA IDNSexTissueNotes
Pyrophorus_luminosus_headSRR63398351M*Prothorax and head (lantern containing)Illumina RNA-Seq
Prothorax_A3SRR63398341M*Prothorax and head
(lantern containing)
BGISEQ-500 RNA-Seq
Thorax_A3SRR63398331M*Mesothorax and metathoraxBGISEQ-500 RNA-Seq
Abdomen_A3SRR63398321M*Abdomen
(lantern containing)
BGISEQ-500 RNA-Seq
Prothorax_A4SRR63398311M*Prothorax and head
(lantern containing)
BGISEQ-500 RNA-Seq
Thorax_A4SRR63398301M*Mesothorax and metathoraxBGISEQ-500 RNA-Seq
Abdomen_A4SRR63398381M*Abdomen
(lantern containing)
BGISEQ-500 RNA-Seq
  1. *Gender inferred. See Appendix 3.3 for a discussion on this inference.

Appendix 3—table 3
Annotated repetitive elements in I. luminosus.
https://doi.org/10.7554/eLife.36495.049
Repeat classFamilyCountsBases% of assembly
DNAAll158853712218438.45
Helitrons3441398630.016
LTRAll23433113415771.35
Non-LTRAll151788503948534.75
LINE97703400528404.75
SINE000
Unknown interspersed75720615958726918.93
Complex tandem repeats49768489920.1
Simple repeat10891444399670.52
rRNA000
Appendix 4—table 1
Genomic sequencing library statistics.

ID: NCBI BioProject or Gene Expression Omnibus (GEO) ID. N: Number of individuals used for sequencing. Date: collection date for wild-caught individuals. Locality: GSMNP: Great Smoky Mountains National Park, TN; MMNJ: Mercer Meadows, Lawrenceville, NJ; IY90: laboratory strain Ikeya-Y90; MAPR: Mayagüez, Puerto Rico. Tissue: Thr: thorax; WB: whole-body; Type: SI: Illumina short insert; MP: Illumina mate pair; PB: Pacific Biosciences, RSII P6-C4; HC: Hi-C; BS: Bisulfite; CH: 10x Chromium; ONT: Oxford Nanopore MinION R9.4. Reads: PE: paired-end, CLR: continuous long read. Number: number of reads. Cov: Mode of autosomal coverage (mode of putative X chromosome, LG3a, coverage), determined from mapped reads with QualiMap (v2.2). ND: Not Determined. Insert size: Mode of insert size after alignment (orientation: FR: forward, RF: reverse), determined from mapped reads with QualiMap. Contamination: Percent contamination as estimated by kraken v1.0.

https://doi.org/10.7554/eLife.36495.052
LibrarySRA IDNDateLocalitySexTissueTypeReadsNumberCovInsert
size (Ori)
Contamination
Photinus pyralis
8369*SRR6345451/
SRR2127932
16/13/11GSMNPMThrSI101 × 101 PE203,074,23098 (49)354 bp (FR)0.28
8375_3 KSRR634544816/13/11GSMNPMThrMP101 × 101 PE101,624,630212155 bp (RF)2.63
8375_6 KSRR634545716/13/11GSMNPMThrMP101 × 101 PE23,564,45654889 bp (RF)3.36
83_3 KSRR634545036/13/11GSMNPMThrMP101 × 101 PE121,757,858132247 bp (RF)0.79
83_6 KSRR634545536/13/11GSMNPMThrMP101 × 101 PE17,905,70014877 bp (RF)1.38
1611_PpyrPB1SRX344487047/9/16MMNJMWBPBCLR-PB3,558,20138 (21)7 Kbp3.5
1704SRR634545627/9/16MMNJMWBHC80 × 80 PE93,850,923NDNDND
1705GSE10717717/9/16MMNJMWBBS150 SE113,761,746~16x§NDND
Aquatica lateralis
FFGPE_PE200DRR1192961N/AIY90FWBSI126 × 126 PE561,450,68672180 bp (FR)ND
FFGPE_PE800DRR119297WBSI126 × 126 PE218,830,95020476 bp (FR)ND
FFGMP_MPGFDRR119298WBMP101 × 101 PE358,601,808312300 bp (RF)ND
Ignelater luminosus
1610_Ilumi
HiSeqX#
SRR63398371MAPRMWBCH151 × 151 PE408,838,92799339 bp (FR)ND
1706_Ilumi
HiSeq2500#
SRR6339836WBCH150 × 150 PE145,250,48048334 bp (FR)ND
18_lib1SRR6760567ONTCLR824,248~2x2984
  1. *Mean of three sequencing lanes

    Mean of two sequencing lanes

  2. Mean subread (PacBio) or read (Oxford Nanopore) length after alignment

    §Estimate from quantity of mapped reads

  3. #Same library, different instruments

    Inferred from specimens collected at the same time and locality

Appendix 4—table 2
Assembly statistics
https://doi.org/10.7554/eLife.36495.053
AssemblyLibrariesAssembly
scheme
Assembly*
/measured**
genome size
(Gbp)
Scaffold/
Contig (#)
Contig
NG50***
(Kbp)
Scaffold
NG50***
(Kbp)
BUSCO
statistics
Ppyr0.1-PBPacBio (61 RSII SMRT cells)Canu (no polishing)721/42225986/
25986
8686C:93.8%[S:65.2%,D:28.6%],F:3.3%,M:2.9%
Ppyr1.1Short read
Mate Pair
PacBio
MaSuRCA +
redundancy
reduction
473/4228065/
8285
193.4202C:97.2% [S:88.8%, D:8.4%],
F:1.9%, M:0.9%
Ppyr1.2ShortPpyr1.1+
Phase Genomics
scaffolder
(in-house)
473/4222535/
7823
193.450,607C:97.2% [S:88.8% ,D:8.4%],F:1.9%, M:0.9%
PacBio
Hi-C
Ppyr1.3Short read
Mate Pair
PacBio
Ppyr1.2
+Blobtools
+ manual
filtering
472/4222160/
7533
192.549,173C:97.2% [S:88.8%, D:8.4%],
F:1.9%, M:0.9%
Alat1.2Short read
Mate Pair
ALLPATHS-LG920/9407313/
36467
38673C:97.4% [S:96.2%, D:1.2%],
F:1.8%, M:0.8%
Alat1.3Short read
Mate Pair
Alat1.2+Blobtools
+ manual
filtering
909/9405388/
34298
38670C:97.4% [S:96.2%, D:1.2%],
F:1.8%, M:0.8%
Ilumi1.0Linked-readSupernova845/76491560/
105589
31.6116.5C:93.7% [S:92.3%, D:1.4%],
F:4.3%, M:2.0%,
Ilumi1.2Linked
read+
nanopore
Ilumi1.0+Blobtools+ Pilon indeland gappolishing. Manual scaffolding842/76491305/
105262
34.5115.8C:94.8% [S:93.4%, D:1.4%],F:3.5%, M:1.7%
  1. *Calculated from genome assembly file with ‘seqkit stat’

    **Measured via flow cytometry of propidium iodide stained nuclei. See Appendix 1.4, 2.4, 3.4.

  2. ***Calculated with QUAST (v4.5) (Gurevich et al., 2013), parameters ‘-e --scaffolds --est-ref-size X --min-contig 0’ and the measured genome size for ‘est-ref-size’

Appendix 4—table 3
Comparison of BUSCO conserved gene content with other insect genome assemblies
https://doi.org/10.7554/eLife.36495.054
SpeciesGenome version (NCBI assemblies)NoteGenome BUSCO
(endopterygota_odb9)
Protein geneset BUSCO
(endopterygota_odb9)**
Drosophila melanogasterGCA_000001215.4
Release 6
Model insectC:99.4%[S:98.7%,D:0.7%],
F:0.4%,M:0.2%,n:2442
C:99.6%[S:92.8%,D:6.8%],
F:0.3%,M:0.1%,n:2442
Tribolium castaneumGCF_000002335.3
Release 5.2
Model beetleC:98.4%[S:97.9%,D:0.5%],
F:1.2%,M:0.4%,n:2442
C:98.0%[S:95.8%,D:2.2%],
F:1.6%,M:0.4%,n:2442
Photinus pyralis*Ppyr1.3*North American fireflyC:97.2%[S:88.8%,D:8.4%],
F:1.8%,M:1.0%,n:2442
C:94.2%[S:84.0%,D:10.2%],
F:1.2%,M:4.6%,n:2442
Aquatica lateralis*Alat1.3*Japanese fireflyC:97.4%[S:96.2%,D:1.2%],
F:1.8%,M:0.8%
C:90.0%[S:89.1%,D:0.9%],
F:3.2%,M:6.8%,n:2442
Nicrophorus vespilloides (Cunningham et al., 2015)GCF_001412225.1
Release 1.0
Burying beetleC:96.8%[S:95.3%,D:1.5%],
F:2.1%,M:1.1%,n:2442
C:98.7%[S:69.4%,D:29.3%],
F:0.8%,M:0.5%,n:2442
Agrilus planipennis (Poelchau et al., 2015)GCF_000699045.1
Release 1.0
Emerald Ash Borer beetleC:92.7%[S:91.8%,D:0.9%],
F:4.6%,M:2.7%,n:2442
C:92.1%[S:64.1%,D:28.0%],
F:4.5%,M:3.4%,n:2442
Ignelater luminosus*Ilumi1.2Puerto Rican bioluminescent click beetleC:94.8%[S:93.4%,D:1.4%],
F:3.5%,M:1.7%,n:2442
C:91.8%[S:89.8%,D:2.0%],
F:4.4%,M:3.8%,n:2442
  1. *=This report, **=Protein genesets downloaded from the NCBI Genome resource associated with the mentioned assembly in the 2nd column, or in the case of D. melanogaster, and T. castaneum, protein genesets were produced from Uniprot Reference Proteomes which had been heuristically filtered down to ‘canonical’ isoforms with a custom script and BLASTP against the D. melanogaster, T. castaneum, Apis mellifera, Bombyx mori, Caenorhabditis elegans, and Anopheles gambiae protein genesets associated with their more recent genome assembly on NCBI. See Appendix 4.2.1 for more detail.

Appendix 4—table 4
Results of PAML branch x sites analysis.

Proportion indicates the proportion of sites in each site class (0, 1, 2a, 2b). Site classes 0 and 1 are those in the constrained and neutral classes, respectively. 2a are sites that were constrained on the background branches, but are either neutral (H0) or in the selective class (HA) on the foreground branches. 2b are sites that were neutral on the background branches, but are either neutral (H0) or in the selective class (HA) on the foreground branches.

https://doi.org/10.7554/eLife.36495.061
HypothesisSite class:012a2blnL
 H0: no selectionproportion0.620.140.180.04−15888.16
background ω0.1210.121
foreground ω0.12111
 HA: selectionproportion0.710.150.110.02−15833.50*
background ω0.1210.121
foreground ω0.1213.253.25
  1. *significant (LRT: 9.32, df = 1)

Appendix 4—table 5
Sites identified as under selection on foreground branches using both Bayes Empirical Bayes (BEB) and Mixed Effects Model of Evolution (MEME).
https://doi.org/10.7554/eLife.36495.062
Site numberingMEME2PAML-BEB
 MSAIlumLucIlumLuc site AA1αβ+LRTEpisodic selection p-value# branchesBEB site class probabilityBEB significance
 2828M0.986*
 3434K0.4723.54.10.06030
 4141Q0.5
 4644V034.50.04850
 4947I0.93792.43.80.06920
 5048G0.573332.34.80.042700.836
 7270N0.553333.13.10.099800.776
 7775M0.964*
 8583A0.962*
 8987K0.958*
 9997W0.598
 105103V0.446.84.30.054900.768
 118116C0.33333.17.40.01091
 122120G0.82
 146144L0.3412.84.90.0390
 147145G0.753333.65.90.02360
 172170A0.698
 189185F0.534
 223219L0.507
 226222T1.4429.64.80.042700.889
 234230I1.139.63.10.099100.613
 279275A0.559
 290286N0.92333340.0640
 315311L0.6929.55.10.036200.884
 329325L0.766
 337333P0.2613.36.30.01980
 341337C0.812
 365361L0.587.64.40.05200.912
 369365T0.216.86.60.016900.843
 379375R0.932
 383379E02.84.10.05940
 389385Q0.792
 398394P0.961999.24.50.0500.951*
 401397S0.617
 406402N0.585.53.70.074500.949
 423419S0.671574.64.70.04300.569
 432428E02.93.10.09991
 441437Y1.4339.34.20.057300.912
 478474V010.36.90.013910.646
 502498Y0.51790.44.90.039300.583
 508504R0.519
 528524N02.23.60.07720
 541537Q01999.210.40.00241
 542538L0.56686.30.01970
 550542T0.743332.94.30.05410
  1. 1 = amino acid. 2=All recovered sites in a single partition with a p+ value of 1.000.

Appendix 4—table 6
Highly expressed (HE), differentially expressed (DE), non-enzyme annotated (NotE), lantern genes whose closest relative in the opposite species is also HE, DE, NotE. BSN-TPM = between sample normalized TPM.
https://doi.org/10.7554/eLife.36495.064
P. pyralis ID (OGS1.1)Predicted functionPpyr expression rankPpyr
BSN-TPM
OrthogroupAlat expression rankAlat
BSN-TPM
A. lateralis ID (OGS1.0)
 PPYR_04589Fatty-acid binding protein170912OG0000524231943AQULA_005253
 PPYR_04589Fatty-acid binding protein170912OG0000524810464AQULA_005257
 PPYR_04589Fatty-acid binding protein170912OG0000524108520AQULA_005259
 PPYR_05098Peroxisomal biogenesis factor 11 (PEX11)154005OG0001490263294AQULA_005466
 PPYR_14966Octopamine binding secreted hemocyanin342353OG0000369213658AQULA_008529
 PPYR_11733MFS transporter superfamily421853OG0000980841335AQULA_012209
 PPYR_07633Reticulon561556OG00047641091123AQULA_005090
 PPYR_09394lysosomal Cystine Transporter871098OG0000847691494AQULA_009474
 PPYR_08979PF03670 Uncharacterised protein family114860OG0003009340411AQULA_012099
 PPYR_05852Vacuolar ATP synthase 16 kDa subunit118836OG0001039287475AQULA_001418
 PPYR_11443RNA-binding domain superfamily134782OG00042681221108AQULA_003174
 PPYR_02465Peroxin 13189581OG0001667196710AQULA_010288
 PPYR_06160V-type ATPase, V0 complex209543OG0000381541251AQULA_000400
 PPYR_11300Mitochondrial outer membrane translocase complex232509OG0004557402349AQULA_004355
 PPYR_08174PF03650 Uncharacterised protein family249475OG0000647163836AQULA_009867
 PPYR_04602Leucine-rich repeat domain superfamily262459OG0004508378373AQULA_004134
 PPYR_01678MFS transporter superfamily264458OG0000347455302AQULA_002485
 PPYR_08192PF03650 Uncharacterised protein family271453OG0000647163836AQULA_009867
 PPYR_13497Mitochondrial substrate/solute carrier285438OG0004402379372AQULA_003680
 PPYR_08917LysM domain superfamily315398OG0002035483278AQULA_002396
 PPYR_04424Domain of unknown function (DUF4782)332379OG00074471296101AQULA_013946
 PPYR_08278Protein of unknown function DUF1151348365OG0001306430325AQULA_000628
 PPYR_13261Major facilitator superfamily404309OG0000410158862AQULA_007558
 PPYR_14848Homeobox-like domain superfamily - Abdominal-B-like413304OG0001849737186AQULA_000483
 PPYR_11623GNS1/SUR4 family446281OG0008603308449AQULA_009341
 PPYR_01828TLDc domain490250OG0002035483278AQULA_002396
 PPYR_03449Innexin533230OG0000992619219AQULA_013430
 PPYR_05702Sulfate permease family543225OG0007205396357AQULA_013064
 PPYR_05993V-type ATPase, V0 complex, 116 kDa subunit family579210OG0000381541251AQULA_000400
 PPYR_04179Haemolymph juvenile hormone binding protein606202OG0002916879152AQULA_011187
 PPYR_08298Peroxisomal membrane protein (Pex16)623198OG0007339395358AQULA_013536
 PPYR_06294Homeobox-like domain superfamily - Abdominal-B-like627197OG0001849737186AQULA_000483
 PPYR_05397PDZ superfamily773164OG0006975367379AQULA_012321
 PPYR_12625Homeobox domain796160OG0002661139595AQULA_008665
 PPYR_08494Armadillo-type fold846152OG0001600986133AQULA_008183
 PPYR_09217Haemolymph juvenile hormone binding protein853151OG0001089441316AQULA_003304
 PPYR_01677MFS transporter superfamily1234108OG0000347455302AQULA_002485
Appendix 4—table 7
Putative lucibufagin compounds from LC-HRAM-MS of P. pyralis adult hemolymph.

Retention time and m/z values are not calibrated to the other samples.

https://doi.org/10.7554/eLife.36495.079
Assigned ion identityIon typeChemical formulaExpected M/zMeasured M/zM/z error* (ppm)Retention time (mins)Feature area (arb)
Core lucibufagin isomer 1[M + H]+C24H33O8449.2175449.2171−0.897.96.7E + 05
Core lucibufagin isomer 2""“”“”""“”9.31.1E + 07
Monoacetylated lucibufagin isomer 1""C26H35O9491.2281491.2277−0.8110.24.2E + 07
Core lucibufagin isomer 3""C24H33O8449.2175449.2171−0.8910.81.7E + 07
Monoacetylated lucibufagin isomer 2""C26H35O9491.2281491.2277−0.8111.41.1E + 06
Monoacetylated lucibufagin isomer 3""“”“”""“”11.91.8E + 07
Monoacetylated lucibufagin isomer 4""“”“”""“”13.02.7E + 08
Monoacetylated lucibufagin isomer 5""“”“”""“”13.26.0E + 07
Monoacetylated lucibufagin isomer 6""“”“”""“”14.56.2E + 06
Diacetylated lucibufagin isomer 1""C28H37O10533.2387533.2385−0.3715.14.0E + 09
Diacetylated lucibufagin isomer 2""“”“”""“”15.41.9E + 09
Monoacetylated, mono propylated lucibufagin isomer 1""C29H39O10547.2543547.2542−0.1817.01.5E + 07
Monoacetylated, mono propylated lucibufagin isomer 2""“”“”""“”17.42.8E + 08
Monoacetylated, mono propylated lucibufagin isomer 3""“”“”""“”17.71.2E + 08
Dipropylated lucibufagin isomer 1""C30H41O10561.2700561.2695−0.8918.91.4E + 08
Dipropylated lucibufagin isomer 2""“”“”""“”19.53.9E + 07
Dipropylated lucibufagin isomer 3""“”“”""“”19.81.8E + 08
Appendix 4—table 8
Putative lucibufagin compounds from LC-HRAM-MS of P. pyralis larval partial body extracts.

Retention time and m/z values are not calibrated to the other samples. *=m/z error and expected m/z extrapolated from ions with similar m/z, and chemical formula predicted from resulting extrapolated m/z. **=Likely chemical formula cannot be determined due to many possible chemical formula from the expected m/z.

https://doi.org/10.7554/eLife.36495.080
Assigned ion identityIon typeChemical formulaExpected m/zMeasured m/zm/z error (ppm)Retention time (mins)Feature area (arb)
Core lucibufagin isomer 2[M + H]+C24H33O8449.2175449.2215+8.99.158.5E + 06
Monoacetylated lucibufagin isomer 1“”C26H35O9491.2277491.2326+9.910.041.2E + 07
UnknownunknownC28H39O10*535.2543*535.2592+9.1*12.401.6E + 07
UnknownunknownC24H38NO6*436.2695*436.2735+9.1*13.302.2E + 07
UnknownunknownC27H45N2O8*525.3173*525.3221+9.1*13.351.3E + 08
UnknownunknownC24H40NO7*454.2799*454.2840+9.1*13.731.3E + 07
Diacetylated lucibufagin isomer 1[M + H]+C28H37O10533.2387533.2426+7.314.931.7E + 09
Diacetylated lucibufagin isomer 2[M + H]+“”“”533.2426+7.315.163.5E + 08
UnknownUnknownC29H46NO8*536.3216*536.3256+7.3*16.574.1E + 07
UnknownUnknownUnknown**563.2854*563.2896+7.3*16.801.3E + 07
UnknownUnknownC26H31O7455.2056455.2097+9.1*17.225.8E + 07
Dipropylated lucibufagin isomer 3UnknownC30H41O10561.2700561.2738+6.719.532.0E + 09
Dipropylated lucibufagin isomer 4UnknownC30H41O10561.2700561.2738+6.719.822.2E + 08
Appendix 4—table 9
Putative lucibufagin [M + H]+ exact masses adjusted for instrument run specific systematic m/z error (Figure 6B).

Used for multi-ion-chromatogram (MIC) traces in Figure 6B.

https://doi.org/10.7554/eLife.36495.081
Chemical formulaPredicted exact massExact mass adjusted to
P. pyralis hemolymph data (+0.6 ppm)
Exact mass adjusted to
P. pyralis partial larval body data (+9.9 ppm)
Exact mass adjusted to
A. lateralis hemolymph data (+1.6 ppm)
Exact mass adjusted to
A. lateralis larval body data
(+1.1 ppm)
Exact mass adjusted to
I. luminosus thorax data (+0.6 ppm)
C24H33O8449.2175449.2178449.2219449.2182449.2180449.2178
C24H38NO6*436.2699436.2702436.2742436.2706436.2704436.2702
C24H40NO7*454.2804454.2807454.2849454.2811454.2809454.2807
C26H31O7455.2069455.2072455.2114455.2076455.2074455.2072
C26H35O9491.2281491.2284491.2330491.2289491.2286491.2284
C27H45N2O8*525.3175525.3178525.3227525.3183525.3181525.3178
C28H37O10533.2386533.2389533.2439533.2395533.2392533.2389
C28H39O10*535.2543535.2546535.2596535.2552535.2549535.2546
C29H39O10547.2543547.2546547.2597547.2552547.2549547.2546
C29H46NO8*536.3223536.3226536.3276536.3232536.3229536.3226
C30H41O10561.2699561.2702561.2755561.2708561.2705561.2702
  1. *=Chemical formula assigned for structurally unclear putative lucibufagins

Appendix 4—table 10
Relative quantification of A. lateralis features identified by lucibufagin MS2 similarity search
https://doi.org/10.7554/eLife.36495.082
Assigned identityM/zChemical formulaRT (mins)Similarity score# of ions matchedA. lateralis feature area (arb)P. pyralis feature area (arb)
Unknown460.2462C22H38NO7P*; C25H29N7O2*15.274.10E + 11347.04E + 080.00E + 00
""657.2229N.D.12.019.50E + 11296.13E + 07""
""414.2043N.D.18.071.20E + 11255.61E + 06""
""381.2176C23H28N2O3*15.773.80E + 11181.22E + 08""
""476.1839N.D.15.933.80E + 11169.87E + 06""
""456.2148N.D.192.30E + 11145.03E + 06""
""351.228N.D.19.422.60E + 11131.56E + 07""
""479.1948N.D.19.832.20E + 11121.11E + 07""
  1. *Determined with Sirius (MS2 analysis), and MZmine2 (isotope pattern analysis).

    N.D., Not determined

Appendix 5—table 1
Best hits from BLASTP of PpyrOMLV proteins against the NCBI database
https://doi.org/10.7554/eLife.36495.087
Genome
segment
Size (nt)Gene
product (aa)
Best hitBest hit
taxonomy
Query coverE valueIdentity
PpyrOMLV1-PB12510801 PB1Wuhan Mothfly VirusOrthomyxoviridae83%0.051%
PpyrOMLV1-PA2346754 PAHubei earwig
virus 1
Orthomyxoviridae98%4.00E-13735%
PpyrOMLV1-HA1667526 HATjuloc virusOrthomyxoviridae91%9.00E-2525%
PpyrOMLV1-PB22517804 PB2Hubei earwig virus 1Orthomyxoviridae91%3.00E-11831%
PpyrOMLV1-N1835562 NHubei earwig virus 1Orthomyxoviridae93%8.00E-7430%
PpyrOMLV2-PB12495802 PB1Hubei orthomyxo-
like virus 1
Orthomyxoviridae93%0.048%
PpyrOMLV2-PA2349762 PAHubei earwig virus 1Orthomyxoviridae98%1.00E-10731%
PpyrOMLV2-HA1668525 HAWellfleet Bay virusOrthomyxoviridae82%3.00E-4026%
PpyrOMLV2-PB22506801 PB2Hubei earwig virus 1Orthomyxoviridae96%3.00E-8627%
PpyrOMLV2-N1738528 NHubei earwig virus 1Orthomyxoviridae95%6.00E-8232%
Appendix 5—table 2
InterProScan domain annotation of PpyrOMLV proteins.
https://doi.org/10.7554/eLife.36495.088
Genome productAnnotationStartEndLengthDatabaseIdInterPro IDInterPro name
PpyrOMLV1
-PB1
Flu_PB148752705PFAMPF00602IPR001407RNA_pol_PB1_
influenza
RDRP
_SSRNA
330529200PROSITE_
PROFILES
PS50525IPR007099RNA-dir_pol_
NSvirus
PpyrOMLV2
-PB1
Flu_PB154766713PFAMPF00602IPR001407RNA_pol_PB1_
influenza
RDRP
_SSRNA
337539203PROSITE_
PROFILES
PS50525IPR007099RNA-dir_pol_
NSvirus
PpyrOMLV1
-PB2
Flu_PB213421409PFAMPF00604IPR001591RNA_pol_PB2_
orthomyxovir
PpyrOMLV2
-PB2
Flu_PB213415403PFAMPF00604IPR001591RNA_pol_PB2_
orthomyxovir
PpyrOMLV1
-HA
SignalP-noTM11919SIGNALP_
EUK
SignalP-noTMUnintegrated
Baculo
_gp64
108432325PFAMPF03273IPR004955Baculovirus_
Gp64
PpyrOMLV2
-HA
SignalP-noTM12121SIGNALP_
EUK
SignalP-noTMUnintegrated
Baculo
_gp64
66426361PFAMPF03273IPR004955Baculovirus_
Gp64
PpyrOMLV1
-PA
Flu_PA66373674PFAMPF00603IPR001009RNA-dir_pol_
influenzavirus
PpyrOMLV2
-PA
Flu_PA66774074PFAMPF00603IPR001009RNA-dir_pol_
influenzavirus
 PpyrOMLV1
-PB1
flu NP-like94459366SUPER
FAMILY
SSF161003Unintegrated
PpyrOMLV2
-PB1
flu NP-like363483121SUPER
FAMILY
SSF161003Unintegrated
Appendix 5—table 3
Total reads mapped to PpyrOMLV genome segments from P. pyralis RNA-Seq datasets.
https://doi.org/10.7554/eLife.36495.089
SRR
3883773
SRR
3883772
SRR
3883758
SRR
3883771
SRR
3883770
SRR
3883769
SRR
3883768
SRR
3883767
SRR
3883765
SRR
3883764
SRR
3883763
SRR
3883762
Ppyr
OMLV1 HA
115412160048812021992848
Ppyr
OMLV1 NP
03210141005230001201460
Ppyr
OMLV1 PA
325609500306105100660
Ppyr
OMLV1 PB1
23642208048200006691464
Ppyr
OMLV1 PB2
5194015220319200106696
Ppyr
OMLV2 HA
1244426612454247549382210232710
Ppyr
OMLV2 NP
295262751446629965324205572741067
Ppyr
OMLV2 PA
12882167240204971815850838
Ppyr
OMLV2 PB1
9115757226787687457146493
Ppyr
OMLV2 PB2
550576747131110228572173728
SRR
3883761
SRR
3883760
SRR
3883759
SRR
3883757
SRR
3883756
SRR
3883766
SRR
2103867
SRR
2103849
SRR
2103848
Ppyr
_larvae
Ppyr
_Female
Ppyr
_eggs
Ppyr
OMLV1 HA
05782686700001664782615586
Ppyr
OMLV1 NP
028903647020064452166562
Ppyr
OMLV1 PA
0124026260000126436929564
Ppyr
OMLV1 PB1
246003160720002824714415952
Ppyr
OMLV1 PB2
0188028480000648256210568
Ppyr
OMLV2 HA
132362354633728643190415000
Ppyr
OMLV2 NP
322482250148219651127432000
Ppyr
OMLV2 PA
14936234222131755497000
Ppyr
OMLV2 PB1
29904168180632296190000
Ppyr
OMLV2 PB2
4990625623094225796000
Appendix 5—table 4
FPKM of reads mapped to PpyrOMLV genome segments from P. pyralis RNA-Seq datasets.
https://doi.org/10.7554/eLife.36495.090
SRR
3883773
SRR
3883772
SRR
3883758
SRR
3883771
SRR
3883770
SRR
3883769
SRR
3883768
SRR
3883767
SRR
3883765
SRR
3883764
SRR
3883763
SRR
3883762
Ppyr
OMLV1 HA
19.100.320.056.460.000.1130.690.050.000.084.0769.54
Ppyr
OMLV1 NP
10.370.000.005.210.000.0016.660.000.000.002.2432.61
Ppyr
OMLV1 PA
6.460.060.002.740.000.007.620.020.000.131.4611.52
Ppyr
OMLV1 PB1
8.530.040.045.570.000.0718.950.000.000.009.0723.72
Ppyr
OMLV1 PB2
4.500.100.004.030.050.007.290.030.000.001.4211.16
Ppyr
OMLV2 HA
16.130.367.415.152.316.8019.680.901.050.394.8817.84
Ppyr
OMLV2 NP
17.360.796.965.442.577.4821.270.528.872.015.2424.36
Ppyr
OMLV2 PA
2.210.254.172.071.193.892.410.300.490.210.7314.58
Ppyr
OMLV2 PB1
2.730.181.371.950.731.401.780.122.301.442.018.10
Ppyr
OMLV2 PB2
1.180.101.031.811.312.342.560.342.631.812.3611.88
SRR
3883761
SRR
3883760
SRR
3883759
SRR
3883757
SRR
3883756
SRR
3883766
SRR
2103867
SRR
2103849
SRR
2103848
Ppyr_ larvaePpyr_
Female
Ppyr_ eggs
Ppyr
OMLV1 HA
0.0018.290.080.2123.440.000.000.000.0015.8974.25104.49
Ppyr
OMLV1 NP
0.008.370.000.0916.000.000.040.000.005.6245.2740.24
Ppyr
OMLV1 PA
0.002.810.000.0512.100.000.000.000.008.6325.0545.85
Ppyr
OMLV1 PB1
0.049.660.000.0728.830.040.000.000.0017.8944.9770.96
Ppyr
OMLV1 PB2
0.003.910.000.0515.050.000.000.000.004.0615.9646.51
Ppyr
OMLV2 HA
0.437.680.9519.309.389.741.024.948.950.000.000.00
Ppyr
OMLV2 NP
0.977.340.8216.0912.196.071.103.008.470.000.000.00
Ppyr
OMLV2 PA
0.322.100.175.734.283.091.230.971.450.000.000.00
Ppyr
OMLV2 PB1
0.631.920.113.883.271.400.341.632.680.000.000.00
Ppyr
OMLV2 PB2
1.061.900.165.884.162.080.340.961.350.000.000.00
Appendix 5—table 5
FEVE hits from BLASTX of PpyrOMLV PB1.
https://doi.org/10.7554/eLife.36495.091
ScaffoldStartEndStrandId with PpOMLVE valueCoverageFEVE
Ppyr1.2_LG11278732312786796(-)56.30%8.22E-5039.10%EVE PB1 like-1
Ppyr1.2_LG11301664713016120(-)56.30%8.22E-5039.10%EVE PB1 like-2
Ppyr1.2_LG13470148034701560(+)37.00%2.88E-2626.70%EVE PB1 like-3
Ppyr1.2_LG13470156234701774(+)37.60%2.88E-2630.20%EVE PB1 like-3
Ppyr1.2_LG13470180134702214(+)45.30%2.88E-2634.00%EVE PB1 like-3
Ppyr1.2_LG13509464535095094(+)28.10%2.15E-109.50%EVE PB1 like-4
Ppyr1.2_LG13511008435109956(-)53.50%2.37E-144.40%EVE PB1 like-5
Ppyr1.2_LG13511021435110107(-)75.00%2.37E-1414.70%EVE PB1 like-5
Ppyr1.2_LG13511034735110213(-)42.60%2.37E-142.90%EVE PB1 like-5
Ppyr1.2_LG15003146450031330(-)64.40%1.18E-0910.00%EVE PB1 like-6
Ppyr1.2_LG15003149850031457(-)71.40%1.18E-0911.60%EVE PB1 like-6
Ppyr1.2_LG15061313050612921(+)49.40%3.71E-114.90%EVE PB1 like-7
Ppyr1.2_LG15067321150673621(+)38.50%1.03E-129.70%EVE PB1 like-8
Ppyr1.2_LG15120846451207634(-)77.20%056.40%EVE PB1 like-9
Ppyr1.2_LG15120939951208467(-)68.50%053.60%EVE PB1 like-9
Ppyr1.2_LG15120955651209398(-)71.70%039.20%EVE PB1 like-9
Ppyr1.2_LG16187168261872158(+)31.10%2.84E-2336.00%EVE PB1 like-10
Ppyr1.2_LG16187215861872319(+)46.30%2.84E-2328.30%EVE PB1 like-10
Ppyr1.2_LG16187235561872456(+)41.20%2.84E-2327.00%EVE PB1 like-10
Ppyr1.2_LG16193052861930205(-)38.00%3.58E-2730.90%EVE PB1 like-11
Ppyr1.2_LG16193068661930504(-)63.60%3.58E-2735.90%EVE PB1 like-11
Ppyr1.2_LG16803899968039073(+)60.00%7.73E-126.60%EVE PB1 like-12
Ppyr1.2_LG16803907268039314(+)40.70%7.73E-125.00%EVE PB1 like-12
Ppyr1.2_LG16803928968039330(+)64.30%7.73E-128.00%EVE PB1 like-12
Ppyr1.2_LG16812882068129008(+)51.50%1.89E-064.90%EVE PB1 like-13
Ppyr1.2_LG23454581434545680(-)58.70%3.84E-067.20%EVE PB1 like-14
Ppyr1.2_LG23454616934545801(-)52.80%1.16E-3134.10%EVE PB1 like-14
Appendix 5—table 6
FEVE hits from BLASTX of PpyrOMLV PB2.
https://doi.org/10.7554/eLife.36495.092
ScaffoldStartEndStrandId with PpOMLVE valueCoverageFEVE
Ppyr1.2_LG15031386950314219(+)82.10%6.91E-5448.30%EVE PB2 like-1
Ppyr1.2_LG15031421650315016(+)82.40%1.92E-14257.90%EVE PB2 like-1
Ppyr1.2_LG15031577250315002(-)89.10%9.97E-14560.60%EVE PB2 like-1
Ppyr1.2_LG15870740358706942(-)52.60%6.19E-4235.80%EVE PB2 like-2
Appendix 5—table 7
FEVE hits from BLASTX of PpyrOMLV PA.
https://doi.org/10.7554/eLife.36495.093
ScaffoldStartEndStrandId with PpOMLVE valueCoverageFEVE
Ppyr1.2_LG13497739234977231(-)48.10%7.73E-073.50%EVE PA like-1
Ppyr1.2_LG16205228962052023(-)28.70%8.92E-117.10%EVE PA like-2
Ppyr1.2_LG16211707762116811(-)28.70%1.22E-107.10%EVE PA like-3
Ppyr1.2_LG16211749362117101(-)26.30%1.22E-108.60%EVE PA like-3
Ppyr1.2_LG16812234868122440(+)77.40%3.40E-0615.70%EVE PA like-4
Appendix 5—table 8
FEVE hits from BLASTX of PpyrOMLV NP
https://doi.org/10.7554/eLife.36495.094
ScaffoldStartEndStrandId with PpOMLVE valueCoverageFEVE
Ppyr1.2_LG1181303181404(+)79.40%7.01E-0917.90%EVE NP like-1
Ppyr1.2_LG110294251029568(+)93.80%9.59E-2127.40%EVE NP like-2
Ppyr1.2_LG120278602027438(-)35.50%3.00E-2130.80%EVE NP like-3
Ppyr1.2_LG13656832436568551(+)42.10%8.99E-117.20%EVE NP like-4
Ppyr1.2_LG15287725652877086(-)68.40%3.87E-1514.60%EVE NP like-5
Ppyr1.2_LG15992741459927271(+)93.80%5.60E-2026.40%EVE NP like-6
Ppyr1.2_LG31720434617204122(-)46.70%7.60E-137.10%EVE NP like-7
Ppyr1.2_LG33163534431635030(-)35.80%3.30E-0810.00%EVE NP like-8
Ppyr1.2_LG35017582150175922(+)79.40%7.01E-0917.90%EVE NP like-9
Ppyr1.2_LG42781168127811758(+)38.50%3.22E-132.50%EVE NP like-10
Ppyr1.2_LG42781185327812179(+)39.00%3.22E-1310.90%EVE NP like-10

Data availability

Genomic assemblies (Ppyr1.3, Alat1.3, and Ilumi1.2), associated official geneset data, a SequenceServer BLAST server, and a JBrowse genome browser are available at www.fireflybase.org (see Appendix 6.2). Raw genomic and RNA-Seq reads for P. pyralis, A. lateralis, and I. luminosus, are available under the NCBI/EBI/DDBJ BioProjects PRJNA378805, PRJDB6460, and PRJNA418169 respectively. Raw WGBS reads can be found on the NCBI Gene Expression Omnibus (GSE107177). Mitochondrial genomes for P. pyralis and I. luminosus and A. antennatus are available on NCBI GenBank with accessions KY778696, MG242621, and MG546669. The complete genome of Entomoplasma luminosum subsp. pyralis is available on NCBI GenBank with accession CP027019. The viral genomes for Photinus pyralis orthomyxo-like virus 1 & 2 are available on NCBI Genbank with accessions MG972985-MG972994. LC-MS data is available on MetaboLights (Accession MTBLS698). Other supporting datasets are available on FigShare (Appendix 6).

The following data sets were generated
  1. 1
  2. 2
    Entomoplasma luminosum isolate NJ-2016 chromosome, complete genome
    1. Timothy R Fallon
    2. Sarah E Lower
    3. Megan Behringer
    4. Jing-Ke Weng
    (2018)
    Publicly available at the NCBI/EBI/DDBJ Genbank database (accession no: CP027019).
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
The following previously published data sets were used
  1. 1
  2. 2
    Transcriptomes of 10 North American firefly species
    1. Sarah E Lower
    2. David W Hall
    (2015)
    Publicly available at NCBI BioProject (accession no.PRJNA289908).
  3. 3

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)