Sexual Response of Male Drosophila to Honey Bee Queen Mandibular Pheromone: Implications for Genetic Studies of Social Insects

J Comp Physiol A (2017) 203:143–149 DOI 10.1007/s00359-017-1147-y

ORIGINAL PAPER

Sexual response of male Drosophila to honey bee queen mandibular pheromone: implications for genetic studies of social insects

Justin R. Croft1 · Tom Liu1 · Alison L. Camiletti1 · Anne F. Simon1 · Graham J. Thompson1

Received: 11 August 2016 / Revised: 31 December 2016 / Accepted: 9 January 2017 / Published online: 1 February 2017 © Springer-Verlag Berlin Heidelberg 2017

Abstract Honey bees secrete a queen mandibular phero- Keywords Chemical ecology · Social pheromones · Apis mone that renders workers reproductively altruistic and mellifera · Fertility signals · Social evolution drones sexually attentive. This sex-specific function of QMP may have evolved from a sexually dimorphic signaling mechanism derived from pre-social ancestors. If so, Introduction there is potential for pre-social insects to respond to QMP, and in a manner that is comparable to its normal effect on The essence of a eusocial breeding system is a reproduc- workers and drones. Remarkably, QMP applied to female tive division of labour, in which one or more individuals Drosophila does induce worker-like qualities [Camiletti monopolize reproduction, while the remaining majority et al. (Entomol Exp Appl 147:262, 2013)], and we here acts as reproductively altruistic helpers (Crespi and Yanega extend this comparison to examine the effects of bee pher- 1995). This reproductive coordination is predicted from omone on male fruit flies. We find that male Drosophila inclusive fitness theory (reviewed in Bourke 2011; Mar- melanogaster consistently orient towards a source of queen shall 2015), but the precise mechanism governing social pheromone in a T-maze, suggesting a recruitment response task specialization varies among species (Beshers and comparable to the pheromone’s normal effect on drones. Fewell 2001). The European honeybee Apis mellifera main- Moreover, exposure to QMP renders male flies more sex- tains its division of labour through queen and brood phero- ually attentive; they display intensified pre-copulatory mones that signal queen fecundity to her worker daughters behavior towards conspecific females. We can inhibit this (Le Conte and Hefetz 2008; Kocher and Grozinger 2011). sexual effect through a loss-of-olfactory-function mutation, Queen mandibular pheromone (QMP) is likely an evolu- which suggests that the pheromone-responsive behavioral tionarily honest signal (Gadagkar 1997; Keller and Nonacs mechanism is olfactory-driven. These pheromone-induced 1993; Oi et al. 2015; Peso et al. 2015) to which the work- changes to male Drosophila behavior suggest that aspects ers have been indirectly selected to respond by shutting off of sexual signaling are conserved between these two dis- their ovaries and otherwise adopting reproductively altruis- tantly related taxa. Our results highlight a role for Drosoph- tic roles within the colony (Butler and Fairey 1963; Hoover ila as a genetically tractable pre-social model for studies of et al. 2003; Backx et al. 2012). social insect biology. While this sterility-inducing function of queen pheromone is well understood (Mullen and Thompson 2015), it has a broader role within the honeybee society, and the Electronic supplementary material The online version of this function of QMP can vary with social context. For exam- article (doi:10.1007/s00359-017-1147-y) contains supplementary material, which is available to authorized users. ple, QMP can induce worker retinue formation, inhibit queen rearing and swarming, and excite congregating * Graham J. Thompson males (drones) to mate with virgin queens in flight (Sles- [email protected] sor et al. 2005; Free 1987). In this later context, QMP is 1 Biology Department, Western University, 1151 Richmond a mating attractant to which the males orient, and from Street, London, ON N6A 5B7, Canada which they anticipate sex (Gary 1962). As a consequence, a

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QMP-emitting queen may receive sperm from more than a using established Drosophila choice and courtship dozen drones on a single mating flight (Tarpy and Nielsen assays, and do so using two control lines and one olfac- 2002). From these observations, it is clear that Apis mel- tory mutant genotype that we expect to differ in their lifera queen mandibular pheromone can coordinate more responsiveness to olfactory cues. than one aspect of reproductive behavior (Brockmann et al. 1998). Like other Hymenopteran queen pheromones (Van Oystaeyen et al. 2014), QMP may have evolved through modification of fertility signals already present in pre-social Methods ancestors (Chapuisat 2014; Peso et al. 2015; Oliveira et al. 2015). Unlike other Hymenoptera studied thus far, how- Fly rearing and pheromone treatment ever, Apis mellifera queen mandibular pheromone appears to be relatively derived in its blended chemical composi- We reared all strains of Drosophila melanogaster under tion of volatile carboxylic acids (9-ODA and 9-HDA), aro- standard conditions (25 °C, 60% humidity and a 12 h matics (HOB and HVA), and other compounds, as opposed light: dark cycle) in an insect growth chamber (Caron to a composition of structurally simple linear or branched Inc., Marietta, OH) on a standard cornmeal diet as alkanes typical of ancestral ants, bees, and wasps (reviewed described in Camiletti et al. (2013). We used the Ore- in Oi et al. 2015). gon-R (Ore-R, Bloomington Stock Center #2376) strain Despite the apparent complexity of QMP, there is of flies as control, for which females have been shown emerging evidence that the taxon-specific qualities of this to respond to QMP in a worker-like manner (Camiletti 1 pheromone are, nonetheless, effective at suppressing repro- et al. 2016). In addition, we included the Orco mutant 1118 1 duction via ovary de-activation in un-related insects. Cami- strain in our trials, outcrossed 6 times in w . Orco letti et al. (2013) showed, for the first time, that female is homozygous for loss-of-function alleles at the major fruit flies (Diptera) exposed to a synthetic blend of QMP olfactory co-factor Orco locus (formerly Or83b; Larsson respond in a manner comparable to queenright worker bees. et al. 2004) and is accordingly non-responsive to a wide That is, they develop smaller ovaries that contain fewer range of olfactory stimuli (Steck et al. 2012). For all gen- 1 mature eggs, as if they were partially sterile. Moreover, this otypes—that is, Ore-R, Orco , and its genetic background 1118 response to ovary-inhibiting pheromone has a genetic com- control w —we synchronized adult emergence by ponent: Drosophila sitter flies (forS) are more responsive housing (for 24 h) a small reproductive population (n = 30 to the ovary-inhibiting pheromone than are rovers (forR), males and n = 30 females) in collection cages (60 mm; suggesting that the foraging gene may in some way medi- Diamed, Mississauga, Canada) fitted with nutrient (grape ate this response (Camiletti et al. 2014). These findings are juice and agar) plates. We then collected and transferred intriguing in that they suggest an underlying pheromone- day-old larvae to fresh food vials (28.5 × 95 mm, VWR responsive mechanism that is genetically variable, yet suffi- International, Radnar, PA) at a density of n = 30 larvae ciently preserved in flies and potentially other invertebrates per vial. Finally, we collected same-age, same-sex adult (Nayer 1963; Carlisle and Butler 1956; Sannasi 1969). virgins at eclosion (within 1 h), and reared males for 3 There is, therefore, potential to exploit the Drosophila days until sexually mature. To prepare filter papers prior model to uncover conserved genes, pathways, and neural to treatment, we first warmed (50 °C in water bath; to ren- circuits involved in ovary-suppression and potentially other der it liquid from a soft wax) and diluted a 500 mg stock fertility-linked traits (Camiletti and Thompson 2016). of synthetic QMP (Contech Ltd, Victoria, Canada) with Motivated by the prospect of harnessing Drosophila absolute ethanol into working aliquots of ~13 Qeq units. gene-finding tools toward questions relevant to honeybee For reference, 1 Qeq is roughly the amount produced by behavior and biology, we seek to test the effect of syn- a single living honey bee queen in 24 h. With bioaccu- thetic QMP on male Drosophila. We postulate that males mulation, the pheromone can persist in wax, resin, and might show drone-like behavior towards QMP. Specifi- cuticles within a colony at multi-queen equivalent doses cally, we test if male Drosophila respond to biologically (Hoover et al. 2003; Naumann et al. 1991). We loaded relevant doses of QMP by orienting themselves towards one full aliquot in a final volume of 20 µl onto individual a source of queen bee pheromone—i.e., ‘the queen’, or if pieces (200 mm2) of filter paper (grade 413; VWR Inter- males anticipate sex by increased mating effort towards a national, Radnar PA) that we air dried for 5 h to ensure conspecific female, or both. If so, we infer that the fertil- that ethanol was fully evaporated. For controls, we sim- ity signal inherent within Apis mellifera queen mandibu- ply added the equivalent volume of ethanol (no QMP) to lar pheromone and the ability to perceive it is evolution- filter paper. Each trial was conducted between 12:00 P.M. arily conserved between species from at least one social and 2:00 P.M. across multiple days at 23–25 °C, with and one pre-social insect order. We test both predictions even light and humidity above 30%.

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Testing male attraction to queen pheromone (orientation) for the first time. Second, we estimated over- in a T‑maze assay all mating effort—as measured by the graphical area under a time-by-intensity profile curve. Next, we estimated the To test if males are attracted to queen pheromone, we courtship index—simply, the proportion of time spent in used a T-maze apparatus (as described in Fernandez et al. courtship (Siegel and Hall 1979). Finally, we estimated 2014). Here, we allowed groups (n = 30) of males to accli- the low courtship intensity—the time a male spends court- mate on the bench top (2 h) before transferring them to the ing within the lower tier of intensity scores (1–2), and the maze chamber. The chamber contains left and right 10 cm high courtship intensity—the time a male spends courting vials covered with treated or untreated filter paper that is within the upper tier of intensity scores (4–6). For each of enclosed within endcaps. After 5 min, we scored their rest- the five measures, we tested for differences between treated ing distribution—first, as a proportion of individuals that and control male courtship effort using unpaired one-tailed had oriented to the left or right sides of the chamber, calcu- t tests, and applying a Benjamini–Hochberg correction lated as a preference index (PI; Min et al. 2013; Tully and for multiple (i.e., 5) testing (q value; Waite and Campbell Quinn 1985), and second, as a virtual frequency histogram 2006). of individual flies within the graded (±1 cm) chamber, as recorded by digital photographs. In each case, we compared the observed distribution of male flies against that expected Results under a null 50:50 (i.e., no preference) scenario. We repli- cated this assay for six independent groups of males (from There is a strong effect of queen bee pheromone on the different breeding stocks), and alternated the left and right choice made by male Drosophila, as evidenced by their dis- positions of QMP treatment with each trial. tribution within the T-maze. Ore-R flies consistently orient toward the pheromone, as indicated by a positive PI rela- Testing sex attraction under queen pheromone tive to untreated Ore-R controls (mean PI = 0.361 vs. 0.022; in a courtship arena t = 5.40, P < 0.001; Fig. 1). This effect of pheromone on 1 fly behavior is dependent on fly genotype; Orco mutants We tested if males adjust their courtship of conspecific show no preference for QMP and their PI is, therefore, not females upon exposure to queen pheromone. We scored different from zero (one-sample t test; mean PI = −0.016; the courting intensity of individual males using Manning’s t = 0.284, P = 0.783). Finally, white-eyed controls behave (1960) scheme, which reflects the stereotypical escalation of courtship intensity towards females: 1—orientation, 2— tapping, 3—singing, 4—licking, 5—attempted copulation, and 6—successful copulation. In instances where a male did not attend to the female, we assigned a score of zero. Here, we reared Ore-R flies as above, except to specifically motivate males towards mating, we housed them from eclosion as individuals in separate vials, but kept females housed together in single vials. After 3 days, or as courtship is expected to peak (Kosuda 1985), we transferred one CO2-anaesthetized male and female into a Lab-Tek™ tissue culture chamber (20 × 10 mm; Division Miles Laboratories Inc., Naperville) with filter paper that served as a courting arena. After a 10 min habituation period in the chamber, we video tracked courting behavior for a continuous 10 min using the Zeiss Zen Pro stereomicroscope software (Carl Zeiss Canada Ltd, Toronto), and did so for n = 22 QMP- treated and n = 22 control trials. From video playback, we recorded start and stop times (to the nearest 1 s) of each behavioral event. This combi- Fig. 1 Mean Preference Index for male flies as a function of geno- nation of information on frequency and duration of spe- type and pheromone treatment. A 50:50 distribution (no preference) would yield a PI of zero, and a fully biased distribution towards cific male behaviors allowed us to estimate four different 1118 courtship QMP+ would yield a PI of 1. Both the w and Oregon-R genotypes measures of mating effort. First, we estimated Orco1 latency show significant preference for queen pheromone, whereas —the time to initiate courtship (O’Dell 2003). mutants, and all untreated flies, showed no preference.Error bars In our case, we scored the time to reach a score of ‘1’ show ±SEM

1 3 146 J Comp Physiol A (2017) 203:143–149 as Ore-R flies, and are likewise biased in their distribu- the males that did court (had a score 1–6), exposure to tion towards the source of pheromone (mean PI = 0.334 queen pheromone did not significantly affect the mean vs. 0.039; t = 3.646, P = 0.004). This genotype-dependent time to initiate courtship (courtship latency, t = 1.44, df = response to pheromone is captured in a significant gene- 34, q = 0.126; Fig. 2b), but did increase the overall mating by-treatment interaction effect (from a two-way ANOVA; effort (area under a time-by-intensity profile curve, t = 2.25, F2, 30 = 6.47, P = 0.005). Our effort to capture the living df = 34, q = 0.040; Fig. 2c). Furthermore, exposure to pher- distribution of individual flies yields results that are con- omone did not increase the overall proportion of time spent sistent with the proportional calculation reflected in the PI; in courtship (courtship index, t = 1.00, df = 34, q = 0.162; Ore-R males orient towards the pheromone, regardless of Fig. 2d), nor did it affect the proportion of time spent by the actual polarity (left vs. right) of the apparatus (z score males orienting towards and tapping females (low intensity = −2.94, P = 0.003; see Electronic Supplementary Mate- score, t = 1.30, df = 34, q = 0.126; Fig. 2e). Pheromone- 1 rial). Likewise, Orco mutants do not deviate from a uni- exposed males did, however, spend a greater proportion of form distribution (z score = 0.168, P = 0.86) and, therefore, time licking, attempting to copulate, and copulating with appear indifferent to the pheromone. females (high intensity score, t = 2.29, df = 34, q = 0.040; There is a subtle but significant effect of queen phero- Fig. 2f), relative to untreated males. mone on fly courting behavior. From video-capture trails, we reconstructed the behavioral courtship profile of each male in the form of a score-by-time ethograph (Fig. 2a). Discussion First, the number of males showing non-zero courting scores was not statistically different between QMP-treated According to social signaling theory, the queen phero- and control trails (n = 19 vs. 17; χ2 = 0.61, P = 0.43). Of mones that coordinate reproductive roles within honey bee

Fig. 2 a Typical male courtship profile showing his courtship score time of trial, e Low courtship intensity—time spent in lower tier (0–2) (y axis) for each second of the observation period (x axis). From each of courtship intensity scores, and f High courtship intensity—time focal individual’s profile, we calculated mating effort in five unique spent in higher tier (4–6) of courtship intensity score. The uncor- but comparable ways: b Courtship latency—the time to initiate court- rected P values shown for c and f remain significant when adjusted ship (see text for full definitions),c Overall mating effort—the area for multiple testing (q value <0.05 in each case). Otherwise, NS non- under each individual male’s behavioral courting profile curve,d significant Courtship index—fraction of time courting (1–6) in relation to total

1 3 J Comp Physiol A (2017) 203:143–149 147 and other social Hymenopteran societies may be derived multi-component blend that mimics the natural bee, phero- from fertility signals of pre-social taxa (Chapuisat 2014; mone presumably has the best potential to capture additive Peso et al. 2015; Oliveira et al. 2015; Oi et al. 2015). Our and synergistic effects among the individual pheromone results provide two observations consistent with this idea. components, as it does in bees (Hoover et al. 2003; Sles- First, we show that D. melanogaster males are attracted to sor et al. 2005; Brockmann et al. 2006). We have not yet honeybee queen mandibular pheromone. This is evident by tested single components of QMP on Drosophila, but we orientation of Ore-R males toward the source of pheromone predict that 9-ODA ((2E)-9-oxodecenoic acid) may elicit within a T-maze chamber. This behavior pattern suggests the strongest single-component attraction and aphrodisiacal that male Drosophila, despite their phylogenetic position effect on male flies, as it appears to inApis mellifera drones and pre-social biology, can respond to a eusocial honey bee (Brockmann et al. 2006; Free 1987). fertility signal. We can inhibit this response through a loss- We here suggest that males, as for females (Camiletti of-olfactory-function mutation, which suggests the behav- et al. 2016), perceive the pheromone through olfactory neu- ioral response is mediated by olfaction. Second, exposure rons, but we do not yet know which neurons are specifically of male flies to queen pheromone increases the aspects of excited by this or related pheromones, or whether neurons their reproductive effort towards conspecific females. Pher- are functionally segregated between males and females. omone-exposed males spent more time courting females at Current work suggests that neurons associated with olfac- ‘high intensity’ (licking, attempting to copulate, and copu- tory receptors Or49b, Or56a, and Or98a may specifically lating), but QMP did not otherwise obviously affect male be necessary for female detection of QMP (Camiletti et al. fly behavior. These two conspicuous behavioral responses 2016). If this response is dependent on olfactory neural cir- from D. melanogaster—attraction and mating intensity—to cuitry, then it may be possible to exploit a neural map of a bee pheromone is potentially significant, because it mim- Drosophila (Chiang et al. 2011) and in vivo calcium imag- ics the fertility signal’s normal effect on male bees. Like ing (Masuyama et al. 2012) to infer a detailed connectome drones (Slessor et al. 2005; Free 1987; Brockmann et al. associated with the fly’s response to QMP. A first-genera- 2006; Butler and Fairey 1964), male flies orient towards, tion neural map of the aphrodisiac (male) or anti-ovarian and display increased intensity to court with, pheromone- (female) response to queen bee pheromone, even from emitting ‘queens’. This basic comparison between fly and Drosophila, could compliment the emerging framework bee behavior to a single pheromone suggests a conserved for understanding how honey bees and other social animals mechanism for sexual signaling between social and pre- modulate social information to regulate personal reproduc- social insect orders. tion (Robinson et al. 2008; Sokolowski 2010; Hofmann The preference index is widely applied to T-maze behav- et al. 2014; Thompson and Richards 2016). ioral assays (Min et al. 2013; Suh et al. 2004; Tully and Scoring Drosophila courtship behavior is well estab- Quinn 1985) and provides a normalized measure of vari- lished, and includes schemes that rank courtship progres- ance in dispersion. In our case, Ore-R flies showed a range sion and intensity along an ordinal scale (Hall 1994; Ruedi in PI-value from near zero (untreated control populations) and Hughes 2008). We predicted that queen bee phero- to a PI-value of ~0.35 (pheromone treated population). This mone might induce a comparable response from male flies range is consistent with comparable studies (e.g., Min et al. as it does from drones—namely to increase motivation for 2013) that score the behavior of small (~20–50 individuals) sex. We found that male Drosophila can augment aspects populations of D. melanogaster and indicates a moderate of their mating effort, but our evidence for an aphrodisiac level of attraction that is well above baseline. This attrac- effect was subtle. Specifically, we found that pheromone- tion to pheromone is, however, a function of genotype, as exposed males tended to prolong the more ‘intense’ aspects olfactory deficient mutants showed unbiased PI-values. of their courting effort towards females—that is, they tend 1 This null result for Orco mutants is consistent with our to spend longer courting at higher intensities (high intensity no-pheromone controls (blank filter paper vs. evaporated mating score) than simply courting for longer (courtship ethanol; not shown) and with our prediction that flies index). This subtle yet biologically relevant time-by-inten- strongly deficient in function for olfactory sensory neurons sity effect of pheromone on Drosophila courting behavior would not likely detect the pheromone. Given that Orco, would not be apparent if we had used only standard mat- by any name (Vosshall and Hansson 2011), plays a con- ing progression metrics. Taken together, the behavioral served and essential role in insect olfaction (Krieger et al. response of male flies to bee pheromone that we observe 2003; Larsson et al. 2004), we reason that the Drosophila in this study is striking for two reasons. First, Drosophila response to QMP is at least partly dependent on olfaction. would never normally be exposed to a bee pheromone, and Here, we follow precedent (Camiletti et al. 2013, 2014, thus, their conspicuous response is unlikely to be adaptive, 2016) and use a naturalistic blend of synthetic pheromone at least not specifically adaptive to Apis mellifera QMP. to induce changes to fly reproductive phenotype. Using a Instead, the response suggests a conserved mechanism

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