Molecular characterization of firefly nuptial gifts: a multi-omics approach sheds light on postcopulatory sexual selection

Abstract

Postcopulatory sexual selection is recognized as a key driver of reproductive trait evolution, including the machinery required to produce endogenous nuptial gifts. Despite the importance of such gifts, the molecular composition of the non-gametic components of male ejaculates and their interactions with female reproductive tracts remain poorly understood. During mating, male Photinus fireflies transfer to females a spermatophore gift manufactured by multiple reproductive glands. Here we combined transcriptomics of both male and female reproductive glands with proteomics and metabolomics to better understand the synthesis, composition and fate of the spermatophore in the common Eastern firefly, Photinus pyralis. Our transcriptome of male glands revealed up-regulation of proteases that may enhance male fertilization success and activate female immune response. Using bottom-up proteomics we identified 208 functionally annotated proteins that males transfer to the female in their spermatophore. Targeted metabolomic analysis also provided the first evidence that Photinus nuptial gifts contain lucibufagin, a firefly defensive toxin. The reproductive tracts of female fireflies showed increased gene expression for several proteases that may be involved in egg production. This study offers new insights into the molecular composition of male spermatophores, and extends our understanding of how nuptial gifts may mediate postcopulatory interactions between the sexes.

Figure 1. Nuptial gift formation, transfer and fate in Photinus fireflies.

(a) During mating the male spermatophore (stained here with rhodamine B) moves through the ejaculatory duct (Ej) into the female’s bursa copulatrix (B). Several male glands contribute to the spermatophore, including the paired spiral glands (SpAG), and other accessory glands (OAG; long accessory gland not shown). (b) Spiral accessory glands (SpAG) manufacture the major portion of the spermatophore, which is visible as a dark structure edged with serrated scales; seminal vesicle (SV) stores sperm rings that get packaged into the spermatophore before transfer. (c) After transfer, sperm released from the tip of the spermatophore enter the female spermatheca (Spt), the sperm storage organ; the clear spermatophore sheath is visible (originally published in ref. 34). (d) The rest of the spermatophore moves into the spermatophore-digesting gland (SDG) where it disintegrates over the next 2–3 d). Scale bars are 500 µm (a,b) and 50 µm (c,d).

Figure 2

Distributions of gene ontology categories for P. pyralis genes up-regulated in males’ other accessory glands (OAGs) and spiral accessory glands (SpAGs), both compared to thorax for: (a) males whose mating status was unknown, and (b) males that had mated within the previous 2 h.

Figure 3. Comparison of differences in gene expression (log2 fold change) for annotated sequences co-expressed in other accessory glands (OAGs) and spiral accessory glands (SpAGs) of P. pyralis males.

Figure 4. SDS-PAGE gel of soluble protein extract from a single P. pyralis male spermatophore with BLUEstain™ Protein MW ladder.

Right-hand numbers indicate gel sections excised for proteomic analysis. Proteomic data from individual gel sections is available online (PRIDE Supplementary Data).

Figure 5

Positive ion mode extracted-ion-chromatograms (EIC) of the diacetylated lucibufagin [M + H]⁺ exact mass from an LC-HRAM-MS analysis of (A) P. pyralis male body (with posterior abdominal segments removed) and (B) P. pyralis male spermatophore. The difference in retention time is expected as these samples were run on different C18 liquid chromatography columns. The retention times of these features did match after retention time alignment (MetaboLights Supplementary Data).