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

www.nature.com/scientificreports OPEN received: 27 July 2016 accepted: 09 November 2016 Published: 22 December 2016 Molecular characterization of firefly nuptial gifts: a multiomics approach sheds light on postcopulatory sexual selection Nooria Al-Wathiqui1,*, Timothy R. Fallon2,3,*, Adam South4, Jing-Ke Weng2,3 & Sara M. Lewis1 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. A powerful driver of evolutionary change, sexual selection is responsible for shaping myriad traits that impact reproductive success for individuals of each sex. It is now widely recognized that both intrasexual competition and intersexual choice continue to operate beyond the end of copulation1,2. These prolonged sexual interactions play out inside female reproductive tracts, and are accomplished through the action of diverse molecules manufactured by both sexes3. Polyandry results in temporal and spatial overlap of male ejaculates, and thus heightens selective pressure on males to maximize their own paternity success relative to that of their rivals1,4,5. One potential result is the elaboration of male reproductive glands that manufacture substances to increase relative reproductive success1,4. During copulation, males in diverse species deliver to females not only sperm, but a complex array of seminal products including proteins, hormones, nucleic acids, lipids, amino acids, carbohydrates and defensive compounds6,7. Known as endogenous nuptial gifts, these non-gametic components of male ejaculates can be transferred either via seminal fluid or within a sperm-containing package known as a spermatophore8,9. Among gift components, seminal fluid proteins (SFPs) have been characterized most extensively, particularly in humans and the fruit fly Drosophila melanogaster10. Male D. melanogaster transfer more than 200 distinct SFPs to their mates during copulation, and human seminal fluid contains approximately 100 different SFPs10–14. Despite the remarkable diversity and rapid evolution of SFPs15,16, the major protein classes are highly conserved, suggesting functional similarities across taxa even as distant as insects and mammals17,18. Interestingly, SFPs have been shown to dramatically alter female physiology and behavior once deposited inside the female reproductive tract. For instance, in D. melanogaster, SFPs reduce female receptivity to further mating, increase oogenesis and oviposition, alter female sperm storage and use, and change female feeding and 1 Department of Biology, Tufts University, Medford, MA, 02155, USA. 2Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142, USA. 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. 4Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, MA, 02115, USA. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.-K.W. (email: ) or S.M.L. (email: ) Scientific Reports | 6:38556 | DOI: 10.1038/srep38556 1 www.nature.com/scientificreports/ 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). Scientific Reports | 6:38556 | DOI: 10.1038/srep38556 2 www.nature.com/scientificreports/ 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. sleep patterns3,10,19. However, despite decades of research on nuptial gifts in select taxa, the detailed molecular mechanisms underlying how such gifts influence postcopulatory sexual selection remain largely unresolved. Transcriptomic studies of the male accessory glands (MAGs) that are responsible for manufacturing SFPs have been restricted primarily to Drosophila and other dipterans20–22. Although detailed anatomical descriptions of MAGs do exist for other taxa23, their glandular products remain poorly characterized. Additionally, sexual selection research shows a recognized bias toward male reproductive traits24. Thus, despite the central role that females play in postcopulatory sexual interactions, remarkably little is known about the products of female reproductive glands25–30. Understanding the role of nuptial gifts in the context of sexual selection will require comprehensive analyses interrogating the molecular composition of male nuptial gifts as well as secretions from female reproductive tissues that receive and process male gifts. Fireflies ar (...truncated)