Probing P. pyralis reproductive tissue gene expression profiles by RNAseq
To elucidate gene expression in specific male accessory glands that manufacture nuptial gifts as well as in the female tissues that receive and process such gifts, we used RNAseq to assemble a transcriptome of P. pyralis reproductive tissues. We successfully demultiplexed a total of 320,271,148 reads into 18 separate libraries, each containing an average of 17,792,841 sequences. All libraries were assembled into a de novo transcriptome containing 47,131 contigs with an average contig length of 1159 bp. Gene expression patterns indicated strong tissue-specificity, and biological coefficient of variation analysis based on normalized read counts demonstrated the expected clustering of biological replicates within each P. pyralis male tissue (Supplementary Fig. 1).
Differential gene expression in male reproductive glands
To examine P. pyralis differential gene expression in the spiral accessory glands and other accessory glands, we identified transcripts that showed a log2 fold change (logFC) ≥ 2 in these reproductive glands compared to male thorax, and also showed a false discovery rate (FDR) ≤ 0.01. Our transcriptome analysis identified 3294 putative transcripts that were significantly up-regulated in the major accessory glands compared to male thorax (Supplementary Table 1). Both types of male accessory glands showed similar gene ontology (GO) functional categories (Molecular Function, Level III), including peptidases and peptidase regulators, metabolic processes, structural proteins, transmembrane transport and signal transduction (Fig. 2a). In other male accessory glands, 11.5% of the transcripts had functions related to peptidase and peptidase regulators, compared to only 4.8% of genes in the spiral accessory glands (Fig. 2a).
To gain insight into differentiated function between these male glands, we first identified sequences that were significantly up-regulated with LogFC ≥ 10 in either male spiral accessory glands or other accessory glands compared to thorax, then identified sequences that were significantly differentially expressed between the two male gland types (LogFC ≥ 2; FDR ≤ 0.01). Comparison of GO functional categories for this subset of uniquely expressed genes confirmed that other accessory glands were mainly enriched in peptidase and peptidase regulator activities (Table 1).
We further characterized differences between male spiral and other accessory glands by comparing expression levels of sequences co-expressed in both tissues (Fig. 3; Supplementary Table 2). The 14 annotated genes that were up-regulated in males’ other accessory glands compared to spiral accessory glands were predicted to be involved in general cellular processes. The 13 genes up-regulated in male spiral accessory glands compared to other male accessory glands (Fig. 3) included a homolog to a metalloprotease, a disintegrin and metalloproteinase with a thrombospondin motif (ADAMTS; DN15036_c0_g1_i8).
Effects of mating on male gene expression
We also examined reproductive gene expression in P. pyralis males 2 h after mating, a time when they are actively manufacturing new spermatophores. We identified 206 sequences in the spiral accessory glands and 253 sequences in the other accessory glands that were up-regulated in each tissue compared to thorax and contained a secretion signal (Supplementary Table 1). Of these, 402 were uniquely expressed in only one type of male accessory gland. In comparison to other males (Fig. 2a), the other accessory glands of recently mated males showed an increase in metabolic processes (Fig. 2b), particularly purine and cysteine metabolism, while the spiral accessory glands of recently mated males showed an increase in transmembrane transport function (Fig. 2b), primarily amino acid transporters.
Protein composition of the firefly nuptial gift
To examine the composition of P. pyralis nuptial gifts, we dissected a spermatophore from a mated female immediately after copulation, separated solubilized proteins on a SDS-PAGE gel (Fig. 4), and examined protein composition by digestion of proteins into peptides followed by nano LC-HRAM-MS/MS proteomic analysis. Combined with transcriptome data from P. pyralis male accessory glands and fat body, this approach allowed us to identify 425 proteins that were transferred to females in the male spermatophore. Of these, 208 were annotated by identifying homologs in other organisms using Blast2go and InterProScan (Supplementary Table 4). Based on our male transcriptome results, we were also able to determine the putative anatomical production site for 68 of these spermatophore proteins (Table 2; Supplementary Table 3). As the spermatophore is extracellular, proteins that are packaged into the spermatophore presumably must first be secreted, though this is not the only possible mechanism of spermatophore incorporation. To identify protein products detected in the spermatophore that may be secreted, we performed an in silico prediction of signal peptide sequences. This analysis revealed that many of the proteins identified via proteomics and that were associated with differentially expressed transcripts do indeed contain predicted signal peptides (Tables 2 and 3; Supplementary Table 3).
The spiral accessory glands were identified as the production site for two serine peptidases. One of these, a transcript with homology to the peptidase Snake (DN10938_c0_g1_i1), showed a LogFC of 9.8 compared to male thorax (Table 2; Supplementary Table 3), which is within the top 8% of differentially expressed genes in this male gland. Snake is a member of the protease cascade that leads to the activation of the Toll pathway, which is important for Drosophila embryonic development and immune response activation55. Another peptidase (DN8730_c0_g1_i1) showed homology to trypsin 1; with a LogFC of 8.5, this transcript is in the top 15% of most differentially expressed genes compared to male thorax (Table 2).
Proteomics also confirmed the presence in the P. pyralis spermatophore of several male reproductive proteins apparently manufactured by other male accessory glands (Table 2; Supplementary Table 3). Among the peptidases, one transcript (DN14826_c0_g1_i1; LogFC = 3.5 compared to male thorax) showed significant similarity to Neprylisin 11 from Tribolium castaneum (Table 2). Another transcript showed homology to Neprylisin 2 (DN12753_c1_g1_i4; LogFC = 10.8 compared to male thorax). Neprylisins are membrane-bound zinc metalloproteases that are responsible for the activation/inactivation of peptide hormones and neuropeptides56.
We also investigated the transcriptome of male fat body, an insect tissue possessing high metabolic and protein biosynthetic activity. The proteomics dataset of the P. pyralis male spermatophore contained several proteins that appear to be synthesized in male fat body (Table 2; Supplementary Table 3). One was a cysteine protease, Cathepsin L11 (DN10232_c0_g1_i1; LogFC = 2.2 compared to male thorax), a lysosomal endopeptidase that can be secreted and interact with structural proteins, such as collagen and fibronectin57.
Metabolomic analysis of the firefly nuptial gift
To examine the small molecule composition of the P. pyralis spermatophore, we conducted an LC-HRAM-MS metabolomic analysis aimed at elucidating compounds specifically enriched in the spermatophore compared to extracts from the male body with the posterior abdomen excised. In an untargeted metabolomic analysis, we noted several mass features exclusively present or present at significantly higher abundance in the spermatophore extract. However, these mass features did not match any compounds in the KEGG Database, suggesting they may represent specialized metabolites yet to be identified (MetaboLights Supplementary Data).
Using a targeted metabolomic analysis, we determined that a known firefly defense compound, lucibufagin C, was present in both the spermatophore and male body. Lucibufagins have previously been shown to be a major class of anti-predator defense compounds in Photinus fireflies58,59. In the positive ion mode extracted ion chromatograms (EICs), both tissues showed a large peak characteristic of lucibufagin C, as well as a smaller second peak likely representing a different isomer of diacetylated lucibufagin (Fig. 5). This targeted analysis also identified P. pyralis pterin, a high-abundance compound of unknown function previously purified from P. pyralis60. The identity of lucibufagin C and P. pyralis pterin in the spermatophore was confirmed by comparison of retention time, exact mass, and MS/MS fragmentation spectra between male body and spermatophore. These compounds were among the most abundant mass features detected in the male body extract (Supplementary Fig. 3), and were identified without authentic chemical standards, as the feature retention time, exact mass, isotopic pattern, and fragmentation spectra were consistent with their respective structural identities.
Gene expression in the female reproductive tract
To determine how specific tissues might process the spermatophore and interact with male reproductive proteins, we examined differential gene expression in the reproductive tract of P. pyralis females relative to thorax, although the single replicate available for female tissues meant that we could not test for statistical significance. However, using the more stringent criteria of a LogFC ≥ 3 and FDR < 0.01, we identified numerous highly expressed genes in different portions of the female reproductive tract (Table 3).
The female bursa copulatrix initially receives the male spermatophore, which is then moved into spermatophore-digesting gland where the spermatophore is degraded over several days. In the combined spermatophore-digesting gland and bursa tissues, we found 80 transcripts that were up-regulated compared to female thorax, of which 33 were annotated (Table 3). Four sequences showed homology to peptidases, including one sequence (DN8737_c0_g1_i1; LogFC = 4.9 compared to female thorax) with homology to angiotensin-converting enzyme, a zinc-metallopeptidase.
We also examined gene expression in the female spermatheca, where male sperm are stored prior to fertilization, and identified 80 up-regulated genes (39 annotated) in this female reproductive tissue (Table 3). As in the spermatophore-digesting gland and bursa, a sequence (DN8737_c0_g1_i1; LogFC = 6.2 compared to female thorax) with homology to angiotensin-converting enzyme was up-regulated in the female spermatheca, along with five other peptidases (Table 3). Another peptidase showed homology to Neprylisin 2.
Home » Guide » Arthropods (Arthropoda) » Hexapods (Hexapoda) » Insects (Insecta) » Beetles (Coleoptera) » Water, Rove, Scarab, Long-horned, Leaf and Snout Beetles (Polyphaga) » Series Elateriformia » Click, Firefly and Soldier Beetles (Elateroidea) » Fireflies (Lampyridae) » Lampyrinae » Lucidotini » Photinus » pyralis Group (Photinus pyralis Group) » Photinus pyralis
Species Photinus pyralis
Classification · Other Common Names · Explanation of Names · Size · Identification · Range · Habitat · Food · Life Cycle · Works Cited
Species pyralis (Photinus pyralis)
Other Common Names
Big Dipper Firefly
Explanation of Names
Photinus pyralis (Linnaeus 1767)
pyralis = 'of fire'
Large for a Photinus. Blackish-brown finely, densely rugose (wrinkled) elytra, side margins and suture of elytra yellow. Pronotal disk pinkish with a black spot. Pronotum convex. Underside: Ventral abdominal segments six and seven large and occupied by light organ in male. Abdominal sternites of male have distinct (2). Female flightless (3), or "seldom" flies, as it does have normal wings (4).
Flash is distinctive: male hovers about two feet (0.6 m) above ground, then drops vertically, gives single prolonged flash as is ascending, then flash diminishes (2). Flashing occurs at dusk, earlier in evening than most other fireflies.
e US (NY-FL to NE-TX) (5)
Meadows and edges of woodlands, including lawns, suburbs.
Adult does not feed, larvae predaceous on insect larvae, slugs, snails(3).
Eggs are laid on moist soil. Larvae take two summers to complete growth, overwintering twice, pupate in (spring?) in chambers in moist soil (3).
|1.||Revision of the Nearctic species of Photinus (Lampyridae: Coleoptera).|
Green, J.W. 1956. Proceedings of the California Academy of Sciences 28: 561–613.
|2.||A Manual of Common Beetles of Eastern North America|
Dillon, Elizabeth S., and Dillon, Lawrence. 1961. Row, Peterson, and Company.
|4.||The Common Insects of North America|
Lester A. Swan, Charles S. Papp. 1972. Harper & Row.
|5.||Studies on the flash communication system in Photinus fireﬂies.|
Lloyd, J.E. 1966. Museum of Zoology, University of Michigan 130: 1-95.
Contributed by Cotinis on 14 July, 2006 - 12:16pm
Additional contributions by Bbarnd, Mike Quinn, v belov
Last updated 1 July, 2017 - 10:27pm