Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage Susanne P.A. den Boer a, *, Jacobus J. Boomsma a , Boris Baer b,c a Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark b ARC Centre of Excellence in Plant Energy Biology, MCS Building M310, The University of Western Australia, 6009 Crawley, Australia c Centre for Evolutionary Biology, School of Animal Biology (MO92), The University of Western Australia, 6009 Crawley, Australia 1. Introduction Producing high quality ejaculates that remain viable during insemination is crucial for males to maximize their reproductive success (Garcı ´a-Gonza ´ lez and Simmons, 2005; Hunter and Birk- head, 2002), but how males actually influence the viability of their ejaculates remains unclear. In many species, males provide sperm with glandular secretions that are usually referred to as seminal fluid or seminal plasma, but details about the molecular composition of seminal fluid fractions are only available for a few insects such as fruit flies (Ravi Ram and Wolfner, 2007) and honeybees (Baer et al., in press; Collins et al., 2006) and several vertebrates including humans (Fung et al., 2004; Pilch and Mann, 2006). These seminal fluid components can affect both sperm cells and female physiology (reviewed by Chapman and Davies, 2004; Gillott, 2003; Poiani, 2006; Ravi Ram and Wolfner, 2007; Simmons, 2001) and at least some seminal fluid proteins have been predicted to enhance sperm viability and sperm survival (Baer et al., in press; Chapman and Davies, 2004). Females are also known to provide sperm with glandular secretions. In vertebrates, bovine oviduct secretions have been shown to affect sperm motility and viability (Abe et al., 1995; Satoh et al., 1995) and to enable sperm capacitation to increase fertilization success (King et al., 1994). Females of invertebrate species often possess specialized sperm storage organs (sometimes referred to as spermathecae) where sperm is kept between mating and egg fertilization (Eberhard, 1996; Simmons, 2001). These storage organs are often accompanied by glands and their secretions have been hypothesized to benefit the survival of stored sperm (Prokupek et al., 2008), although neither the molecular composition of these secretions nor their biological activity have been studied in great detail (but see Klenk et al., 2004; Koeniger, 1970; Lensky and Schindler, 1967). Investigating the mechanisms by which males and females affect sperm viability is particularly interesting in the eusocial Hymenoptera (the ants and some of the bees and wasps). Copulations and insemination are restricted to a single brief mating episode early in a queen’s life (Boomsma et al., 2005; Boomsma and Ratnieks, 1996). Males die during or shortly after copulating while queens store large amounts of sperm that remain viable over prolonged periods of time, sometimes for several decades (Keller, 1998; Pamilo, 1991). Reproductive success of males and queens is therefore likely to be correlated with both the quantity and the quality of the sperm cells that females are able to acquire and store (Cole, 1983). Male seminal fluid may thus be particularly important for sperm viability during the provisional storage of ejaculates in the female sexual tract prior to final storage. After transfer to the spermatheca, sperm might have to Journal of Insect Physiology 55 (2009) 538–543 ARTICLE INFO Article history: Received 11 November 2008 Received in revised form 13 January 2009 Accepted 23 January 2009 Keywords: Apis mellifera Ejaculate Seminal fluid Accessory glands Spermatheca Social insects ABSTRACT Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +45 35 32 13 41; fax: +45 35 32 12 50. E-mail address: spadenboer@bio.ku.dk (Susanne P.A. den Boer). Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.01.012