Pheromone evolution and sexual behavior in Drosophila are shaped by male sensory exploitation of other males Soon Hwee Nga, Shruti Shankara,b, Yasumasa Shikichic, Kazuaki Akasakad, Kenji Moric, and Joanne Y. Yewa,b,1 a Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604; bDepartment of Biological Sciences, National University of Singapore, Singapore 117546; cPhotosensitive Materials Research Center, Toyo Gosei Co., Ltd, Inzai-shi, Chiba 270-1609, Japan; and dShokei Gakuin University, Natori-shi, Miyagi 981-1295, Japan
Edited* by Edward A. Kravitz, Harvard Medical School, Boston, MA, and approved December 31, 2013 (received for review July 19, 2013)
Animals exhibit a spectacular array of traits to attract mates. Understanding the evolutionary origins of sexual features and preferences is a fundamental problem in evolutionary biology, and the mechanisms remain highly controversial. In some species, females choose mates based on direct benefits conferred by the male to the female and her offspring. Thus, female preferences are thought to originate and coevolve with male traits. In contrast, sensory exploitation occurs when expression of a male trait takes advantage of preexisting sensory biases in females. Here, we document in Drosophila a previously unidentified example of sensory exploitation of males by other males through the use of the sex pheromone CH503. We use mass spectrometry, high-performance liquid chromatography, and behavioral analysis to demonstrate that an antiaphrodisiac produced by males of the melanogaster subgroup also is effective in distant Drosophila relatives that do not express the pheromone. We further show that species that produce the pheromone have become less sensitive to the compound, illustrating that sensory adaptation occurs after sensory exploitation. Our findings provide a mechanism for the origin of a sex pheromone and show that sensory exploitation changes male sexual behavior over evolutionary time.
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sexual selection laser desorption/ionization male–male competition chiral pheromone
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exual selection is widely regarded as an important mechanism for the origin of new traits and species. Darwin first proposed that the elaboration of male secondary sexual traits is driven by female preferences (1, 2). This concept has been refined by models suggesting that females select male traits that indicate genetic quality or confer direct reproductive benefits (3– 7). In contrast, sensory exploitation occurs when expression of a male trait takes advantage of preexisting sensory biases in females (8). In this case, female preference does not coevolve with the male trait but rather precedes it. In one of the first examples documenting sensory exploitation, female Physalaemus coloradorum frogs were shown to prefer male calls that contain a low-frequency “chuck” component despite the absence of this feature in calls from conspecifics. The sensory bias for chucks was shown to have its mechanistic basis in the tuning properties of the inner ear, a physiological feature that predated the appearance of chucks (9). Similarly, female platyfish exhibit a preference for males with swordtails despite the absence of swordtails in male platyfish. Females consistently chose to spend more time with conspecific males exhibiting an artificially attached plastic sword (10). In both these examples, female preference predates expression of the trait. Sensory exploitation has since been documented for numerous other visual cues, across a diversity of taxa (11–14). In each case, females prefer traits that are not found naturally in their own species but appear in males of other species. Moreover, both the sensory bias and behavioral response to the trait already were present before expression of the trait.
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Pheromones are taste and olfactory cues that, in many species, play an important role in mate selection (15). As with courtship cues detected by other sensory modalities, pheromones are shaped by sexual selection and, thus, may exhibit enormous structural diversity and exquisite stereochemical specificity. In insects, exogenously secreted lipids advertise mating status, availability, and reproductive fitness (16). In some cases, male pheromones serve as a nuptial gift, thus providing direct reproductive benefits to females and offspring in the form of either nutritive or defensive compounds (17). Little is known, however, about the mechanisms underlying the diversification and the origin of chemical specificity. Here, we provide an example of a pheromone that has evolved from sensory exploitation. In Drosophila melanogaster, CH503 [formally, (3R,11Z,19Z)-3-acetoxy-11,19octacosadien-1-ol; Fig. 1A] functions as an antiaphrodisiac (18). The pheromone is secreted in the anogenital region, is transferred to females during mating, and suppresses courtship from males. Our findings indicate CH503 evolved from males exploiting the preexisting sensory biases of other males to gain mating advantage by limiting access to females. Moreover, the use of CH503 has altered male sexual behavior over evolutionary time such that males have adapted by becoming less sensitive to the pheromone. Results and Discussion Evolutionary Origin of CH503 Expression. To determine the evolu-
tionary origins of CH503, we examined eight species of Drosophila Significance How sexual features and preferences originate and evolve is one of the most important and contentious problems in evolutionary biology. In some species, females choose male traits that indicate genetic quality or confer benefits. Thus, male traits and female preferences are assumed to originate at the same time and to coevolve. In contrast, sensory exploitation occurs when males take advantage of females’ preexisting sensory biases. We show in Drosophila sensory exploitation of males by other males through the use of a pheromone to gain sexual advantage. Notably, sensory exploitation leads to male sensory adaptation. These findings provide a mechanism for the evolutionary origins of a pheromone and are a previously unidentified example of sensory exploitation between males. Author contributions: S.H.N., S.S., Y.S., K.A., K.M., and J.Y.Y. designed research; S.H.N., S.S., Y.S., K.A., K.M., and J.Y.Y. performed research; Y.S., K.A., and K.M. contributed new reagents/analytic tools; S.H.N., S.S., Y.S., K.A., K.M., and J.Y.Y. analyzed data; and S.H.N., K.A., K.M., and J.Y.Y. wrote the paper. The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. Freely available online through the PNAS open access option. 1
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1313615111/-/DCSupplemental.
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cVA 349.24 (3R,11Z,19Z)-CH503 (natural stereoisomer) OH
n-C 8H17 OAc
CH503 503.38
D. melanogaster D. simulans D. yakuba m/z
3 00
650
D. ananassae
D. sechelia
D. willistoni 501.37
405.32
471.43 503.39 485.40
cVA 349.25 407.26
515.42
D. mojavensis
D. virilis
559.34
487.39
577.37 499.45
515.42
471.41 443.38
563.35 551.34
507.28
415.37 3 00
m/z
65 0
m/z
30 0
6 50
B
37.3
2 3 5 1
4
32.5 D. simulans
6 7 8 32.7
37.6 D. yakuba 37.4
32.6 min.
0
Derivatized CH503 isomer and RT (min) 1. (3S,11Z,19Z) 34.8 5. (3R,11E,19Z) 2. (3R,11Z,19Z) 37.5 6. (3R,11Z,19E) 3. (3S,11E,19Z) 42.8 7. (3S,11E,19E) 4. (3S,11Z,19E) 44.7 8. (3R,11E,19E)
D. sechellia
80
24.4 46.7 48.8 68.3 74.8
D. ananassae 0
min.
80
Fig. 1. Characterization of CH503 expression in Drosophila. (A) Chemical structure of CH503 and representative UV-LDI mass spectra measured from the male anogenital region of different Drosophila species. Each spectrum is recorded from a single fly. Signals corresponding to the mass-to-charge ratio (m/z) for cVA (m/z 349.24) and CH503 (m/z 503.38) were detected in D. melanogaster, D. simulans, D. yakuba, D. sechellia, and D. ananassae. No signal for CH503 was detected from D. willistoni, D. mojavensis, or D. virilis. Potassium-bearing molecular compounds [M+K]+ constitute the major ion species in all cases. (B) The HPLC chromatogram shows distinct retention times (RT) for each of the eight synthetic CH503 stereoisomers following derivatization. HPLC analysis of derivatized CH503 isolated from D. simulans, D. yakuba, and D. sechellia reveals that (3R,11Z,11Z)-CH503 is the only expressed stereoisomer. The retention times for the major peaks are noted in each chromatogram. The compound isolated from D. ananassae has the same m/z and elemental composition as CH503 but a different structure.
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for production of the pheromone CH503 and tested whether males of these species respond to CH503 as an antiaphrodisiac. We first used UV laser desorption/ionization mass spectrometry (UV-LDI MS) to analyze the chemical profiles of the male anogenital region in other species. UV-LDI MS revealed signals matching the expected molecular weight for CH503 in the anogenital region of D. melanogaster, Drosophila simulans, Drosophila yakuba, Drosophila sechellia, and Drosophila ananassae (Fig. 1A). A signal for cis-vaccenyl acetate (cVA), another known antiaphrodisiac, also was found in these species (19). In contrast, no signal for CH503 could be detected from the anogenital region of Drosophila willistoni, Drosophila mojavensis, or Drosophila virilis (Fig. 1A). To determine the double-bond geometries and absolute configuration of CH503 from melanogaster group flies, chemical derivatization and high-performance liquid chromatography (HPLC) separation were used to compare the retention times of the derivative of naturally occurring CH503 with synthetic standards of the eight possible derivatized stereoisomers. Each of the derivatized stereoisomers could be differentiated based on their distinct retention times (Fig. 1B). D. melanogaster previously was shown to express (3R,11Z,19Z)-CH503 [hereafter abbreviated as (R,Z,Z)-CH503] (20). HPLC analysis of derivatized thin-layer chromatography-purified fractions revealed a single peak corresponding to (R,Z,Z)-CH503, indicating that as with D. melanogaster, D. simulans, D. yakuba, and D. sechellia express a single stereoisomer (Fig. 1B). Although a compound with a mass signal corresponding to (R,Z,Z)-CH503 was observed by UV-LDI MS analysis in D. ananassae, the retention time did not match any of the eight stereoisomers. Taken together, chemical profiling with UV-LDI MS and HPLC reveals that all tested drosophilids of the melanogaster subgroup express (R,Z,Z)-CH503. In contrast, other drosophilids representing outgroup taxa from Sophophora (D. willistoni, D. ananassae) and Drosophila (D. virilis, D. mojavensis) do not express (R,Z,Z)-CH503 or any other stereoisomer. The phylogenetic distribution of the trait suggests a single origin in the melanogaster group of Drosophila. Conserved Function of CH503 as an Antiaphrodisiac. We next tested whether the function of CH503 as an antiaphrodisiac is conserved across the different species. Socially isolated virgin males were placed with a virgin female perfumed with various amounts of CH503. Surprisingly, all Drosophila species tested suppressed courtship initiation in a dose-dependent manner in response to CH503, although the pheromone is produced only by a subgroup of these species. Male courtship behavior was significantly inhibited in D. melanogaster, D. simulans, D. yakuba, and D. sechellia, all species that produce CH503 (Fig. 2A). The latency to courtship initiation also increased significantly in a dose-responsive manner to increasing amounts of CH503 (Fig. S1). Notably, D. ananassae, D. willistoni, D. mojavensis, and D. virilis, none of which expresses CH503, also responded to the compound by suppressing courtship behavior and delaying courtship initiation (Fig. 2B, Fig. S1, and Table S1). These findings indicate that the behavioral response to CH503 predates the expression of CH503. We hypothesize that males of the ancestral species that gave rise to the melanogaster subgroup used sensory exploitation to inhibit courtship from male competitors. To test whether production of CH503 might provide an advantage in male–male competition for access to females, we examined the effect of introducing (R,Z,Z)-CH503 into the mating system of species that do not produce the pheromone. D. ananassae and D. virilis males were given a choice of mating with CH503perfumed or solvent-perfumed females. In both species, males showed a significant aversion to courting the former (Fig. 2E). Thus, the use of a potent antiaphrodisiac by males could significantly shift courtship choice of rival males. By limiting access to mated females, male producers of the pheromone might potentially gain a mating advantage by reducing sperm competition. PNAS | February 25, 2014 | vol. 111 | no. 8 | 3057
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A
**** **** ****
****
****
**
****
ns
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**
40
0
0 83 166 249
20 0 83 249 415
0 83 249 415
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60
0 83 166 249
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80
****
ns **
**
ns 0 1245 1660 2075
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Dana
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Dmoj
Dvir
0 83 249 415
0 83 249 **** 415 ****
Dsim
Dyak
Dsec
**
****
Dana
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Dmoj
Dvir
20 0
****
***
ns
****
0 83 249 **** 415 ****
0 83 249 **** 415 ****
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20
40
0 83 249 415
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**** 0 83 249 415
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40
60
*
0 249 332 *** 415 ****
*
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** **
60
80
0 83 415 830
*
80
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ns
D 100Response to (S,Z,Z)-CH503
100 Courtship initiation (%)
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ns
ns ** Dmel
Response to (S,Z,Z)-CH503
Courtship choice D. virilis ns
****
0.8 0.6 0.4 0.2 0.0 Control
250ng TB-CH503
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250ng (R,Z,Z)CH503
D. melanogaster ns **
****
1.0
1.0 Courtship vigor
D. ananassae ns
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1.0 Courtship vigor
0 580 700 830
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40
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Response to (R,Z,Z)-CH503 100
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100
0 83 415 830
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83ng TB-CH503
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*
0.8 0.6 0.4 0.2 0.0
83ng 83ng Control 83ng Control 83ng TB(S,Z,Z)- (R,Z,Z)- (S,Z,Z)CH503 CH503 CH503 CH503
Fig. 2. Comparative analysis of the behavioral response to natural CH503 and an artificial stereoisomer. (A and B) Courtship assays reveal that the natural pheromone (R,Z,Z)-CH503 functions as an antiaphrodisiac both in species that produce the natural pheromone (A) and in species that do not (B). Doses (in nanograms) indicated represent the approximate amount on the cuticular surface following perfuming. The courtship initiation percentage for each dose is compared with the percentage from control trials and statistically assessed using a Holm–Bonferroni-corrected Fisher exact test. Dana, D. ananassae; Dmel, D. melanogaster; Dmoj, D. mojavensis; Dsec, D. sechellia; Dsim, D. simulans; Dvir, D. virilis; Dwil, D. willistoni; Dyak, D. yakuba; ns, nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 24–36 for each dose. (C and D) A dose-dependent response to the artificial stereoisomer (S,Z,Z)-CH503 is observed in all tested species. In species that produce CH503 (C), the artificial stereoisomer is more potent than the natural pheromone. In species that do not produce CH503 (D), the artificial stereoisomer and the natural pheromone are effective over a similar range of doses. Statistical analysis was performed as in A. (E) In courtship choice assays, D. ananassae and D. virilis males spent a greater amount of time courting nonperfumed females over (R,Z,Z)-CH503–perfumed females. D. melanogaster males preferentially courted nonperfumed females over females perfumed with (S,Z,Z)-CH503. In the presence of both stereoisomers, males preferentially courted females perfumed with the natural pheromone. No effect on courtship was observed when females were perfumed with an equivalent dose of a CH503 analog (TB-CH503). The ends of the box plot represent the spread of data between the 25th (bottom) and the 75th percentile (top), whereas the horizontal line represents the median and + represents the mean. Courtship vigor within each pairing was compared using a Wilcoxon rank-sum test. ****P < 0.0001; **P = 0.0032; *P = 0.0397; n = 21–29.
Adaptive Response to Sensory Exploitation. It is well established that the biological activity of pheromones can be highly dependent on the stereostructure (21). To determine whether the stereostructure of CH503 is important for its function as an antiaphrodisiac, we tested the effect of a synthetic stereoisomer of CH503, (S,Z,Z)-CH503, a stereoisomer not known to be expressed naturally by drosophilids (Fig. S2). Unexpectedly, D. melanogaster, D. simulans, D. yakuba, and D. sechellia exhibited stronger courtship suppression and a longer latency to initiate courtship in the presence of the artificial stereoisomer compared with the natural pheromone at equivalent doses (Fig. 2C, Fig. S2, and Table S2). When D. melanogaster males were given a choice of females perfumed with (S,Z,Z)-CH503 or a nonperfumed female, males preferred to court the latter (Fig. 2E). Even in the presence of a female perfumed with an equivalent amount of the wild-type stereoisomer, (R,Z,Z)-CH503, males continued to show a stronger aversion to the unnatural pheromone (Fig. 2E). The disparity between the strength of the natural vs. unnatural pheromone is particularly striking in D. simulans, in which nearly 15 times the 3058 | www.pnas.org/cgi/doi/10.1073/pnas.1313615111
amount of the natural pheromone is needed to achieve the same level of courtship inhibition produced by the artificial stereoisomer. In contrast, males of D. ananassae, D. willistoni, D. virilis, and D. mojavensis exhibited an equivalent or greater responsiveness to the wild-type form of CH503 compared with the artificial stereoisomer (Fig. 2D, Fig. S2, and Table S2). Thus, species that produce the pheromone exhibit a weaker behavioral response to the natural compound, whereas nonproducers of the pheromone are more sensitive to the pheromone (Fig. 3). The lowered sensitivity exhibited by CH503-producing species to the natural pheromone may be the result of sensory adaptation or habituation from exposure to the pheromone from conspecifics. However, adaptation likely is not a contributing factor because males were individually housed during the late pupal stage and remained isolated until testing at 4–5 d old. Furthermore, the response of D. melanogaster males collectively raised in groups was not significantly different from that of individually housed flies (Fig. S3). Thus, even in the presence of heightened preexposure to CH503, the differential sensitivity exhibited by Ng et al.
CH503 producing species non-CH503 producing species
sensory trap induced sensory adaptation (14). Interestingly, we did not observe sensory adaptation to cVA. Responsiveness to the courtship inhibition properties of the pheromone showed no correlation with whether the species produced the pheromone (Table S3). The multiple functions of cVA as an aggregation cue (26) and aphrodisiac for females (27) likely prevent males from completely losing sensitivity to the pheromone despite the costs of courtship suppression. The robust behavioral response of melanogaster subgroup species to (S,Z,Z)-CH503 suggests future opportunities for sensory exploitation. A subtle change in stereochemistry due to allelic variations in genes underlying pheromone production might allow for expression of a stereoisomer variant that would circumvent male adaptation. Behavioral experiments are consistent with this prediction: use of the unnatural pheromone results in significantly greater courtship aversion, even in the presence of the natural pheromone. It will be interesting to consider whether persistent use of (S,Z,Z)-CH503 over the long term may contribute to physiological desensitization and also result in disadvantages for the population, such as lowered mating frequency, resulting in reduced offspring fitness (28). Several mechanisms underlying the evolution of antiaphrodisiacs have been proposed, arguing that chemical diversity in pheromone structures arises as a result of male–female cooperation (23, 29), male–female sexual conflict (30), or male–male competition (31). Our study indicates that sensory exploitation is another mechanism for the evolution of antiaphrodisiacs and traits used in male–male competition. A similar phenomenon whereby synthetic nonnatural stereoisomers elicited a behavioral response stronger than that of the natural pheromone was reported previously in the German cockroach, indicating that sensory exploitation may underlie the evolution of other insect pheromone systems (32). These findings contrast with an alternative mechanism shown in Nasonia wasps, in which novel pheromone compounds appear before a preexisting response (33). Currently, the chemosensory receptor(s) and underlying neural circuits mediating CH503 detection are unknown. Once they are identified in D. melanogaster, it will be possible to correlate evolution of the receptor(s) structure with behavioral responses of various species and in this way, to understand the underlying neurophysiological mechanisms. Furthermore, determining
D. simulans
0.20
CH503
D. sechellia 0.24
(R,Z,Z)-CH503 Courtship suppression index: 0.14 to 0.19 0.20 to 0.25 0.26 to 0.32 0.33 to 0.38
0.39 to 0.44 0.45 to 0.50 0.51 to 0.56 0.57 to 0.64
0.64 to 0.69 0.70 to 0.75 0.76 to 0.82
D. melanogaster 0.14 melanogaster group
D. yakuba 0.34
Sophophora
D. ananassae 0.76 willistoni group
D. willistoni
repleta group
D. mojavensis
0.54
0.63
Drosophila
D. virilis
virilis group 50
40 30 20 10 Divergence time (million years)
0.59
0
Fig. 3. Evolution of CH503 expression and the behavioral response to CH503. CH503 expression (left) and the behavioral response to natural CH503 (right) are mapped onto the Drosophila phylogeny using a linear parsimony model. The values at the branch termini (right phylogram) reflect the courtship suppression index, a measure of the strength of courtship suppression induced by the natural pheromone compared with the artificial stereoisomer. All tested species respond to both stereoisomers. However, species that produce CH503 respond more weakly to the natural pheromone. Hatch marks indicate the likely point of origin of each trait.
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D. melanogaster to the natural and artificial pheromone remained unchanged. Courtship suppression in the presence of (R/S,Z,Z)-CH503 might simply be the result of aversion to a foreign chemical or masking of endogenous female pheromones. To address this possibility, males of all species were tested with females perfumed with an equivalent dose of modified (S,Z,Z)-CH503 or (R,Z,Z)-CH503 analogs bearing either two triple bonds (instead of two double bonds) or a single double bond only. In each case, no significant courtship suppression was observed (Tables S1 and S2; Fig. 2E). These findings indicate that first, melanogaster subgroup males have evolved partial resistance to the antiaphrodisiac effects of the natural pheromone, and second, the behavioral response is specific to the stereostructure of (R/S,Z,Z)-CH503. Our findings support the hypothesis that in Drosophila, an antiaphrodisiac pheromone evolved as a result of sensory exploitation. All tested species, regardless of their endogenous pheromone expression, suppressed courtship in response to the natural pheromone, (R,Z,Z)-CH503. Because (R,Z,Z)-CH503 originates only in the melanogaster subgroup, response to the pheromone predates expression of the pheromone. In contrast to previous findings that identified attractive superstimulants, these results show that preexisting sensory systems for highly aversive cues also may be exploited and that sensory exploitation also takes place between males. In D. melanogaster, multiple pheromones, such as cVA and (7Z)-tricosene, and accessory gland proteins are used to suppress female mating frequency (22–25). We speculate that initial use of CH503 helped reinforce courtship inhibition from rival males because, in contrast to other male-transferred cues, CH503 persists on female cuticles for at least 10 d (18). The low vapor pressure of the pheromone and its behavioral potency would place strong selective pressure for males to become less sensitive to the pheromone to benefit from increased mating opportunities. Our results are consistent with this prediction: members of the (R,Z,Z)CH503–expressing melanogaster subgroup have adaptively evolved to become less sensitive to the natural pheromone. In this way, a trait that originated from sensory exploitation drives the evolution of sensory pathways. A similar phenomenon occurs in Goodeidae splitfin fish, where a visual cue originating as a female
molecular markers for the relevant sensory pathways will provide a way to map the evolutionary origins of predisposed sensory biases and the shaping of pheromone structural specificity. Materials and Methods Drosophila Stocks and Husbandry. Flies were obtained from the Bloomington Stock Centre and Ehime University Stock Center, and maintained on cornmeal agar food at 25 °C, 60% humidity, with a 12:12-h light:dark cycle. D. mojavensis were wild-caught from Las Bocas, Sonora, Mexico (in March 2009) by W. J. Etges (University of Arkansas) and raised on standard cornmeal agar food supplemented with banana (∼110 g per 20 half-pint bottles) and cactus powder (∼2.3 g per 20 half-pint bottles). Adult flies used in courtship assays were isolated at the pupal stage or collected within 4 h after pupal eclosion. Male virgins were raised individually in a test tube containing 2 mL of cornmeal agar food, whereas females were kept in groups of 10 per vial. For grouped-male conditions, adult males were collected at the pupal stage and kept in groups of 10 per vial for 7 d. Flies were tested at the following ages, corresponding to sexual maturity: D. melanogaster and D. simulans, 4–7 d; D. sechellia and D. yakuba, 5– 7 d; D. ananassae, 7 d; D. virilis and D. mojavensis, 10 d; and D. willistoni, 15 d. Pheromone Profiling of Drosophila Species Using UV-LDI MS Analysis. UV-LDI MS analysis and the procedures for preparing the flies were described in detail previously (18). Measurements were performed on a Q-Star Elite (AB SCIEX) orthogonal time-of-flight mass spectrometer equipped with an intermediate pressure oMALDI2 source and an N2 laser (λ= 337 nm, 40-Hz repetition rate, 200-μm beam diameter, 3-ns pulse duration). Ions were generated in a buffer gas environment by using 2 mbar of N2. Individual flies were attached to a coverslip with adhesive tape and mounted onto a custom-built sample plate. During data acquisition, the anogenital region was irradiated for 30 s, corresponding to 1,200 laser shots. For each species, 10–15 socially naïve male flies were measured. Mass accuracy for the mass spectrometer was ∼20 ppm. More details about instrumentation conditions and analytical parameters important for the chemical imaging of insects are provided in ref. 34. Purification of CH503 from Drosophila Species. Purification of CH503 by thin layer chromatography (TLC) was performed using the conditions described by Yew et al. (18). Approximately 800–1,000 males and females of D. melanogaster, D. simulans, D. yakuba, D. sechellia, and D. ananassae were extracted with hexane (10 mL) for 20 min at room temperature. The extract was concentrated to dryness using a stream of N2. The residue was dissolved in hexane before separation by TLC. TLC separation was performed on glassbacked silica gel plates (10 × 10 cm, coated with 0.2 mm of silica gel 60; Merck) using a running solvent consisting of hexane/diethyl ether/acetic acid (66:33:1, each by volume). The major component of the isolated fraction was confirmed as CH503 by Direct Analysis in Real Time (DART) mass spectrometry. The DART ion source was operated in positive-ion mode with helium gas, with the gas heater set to 200 °C. The glow discharge needle potential was set to 3.5 kV. Electrode 1 was set to +150 V, and electrode 2 (grid) was set to +250 V. HPLC Analysis. The separation of eight stereoisomers was determined using a previously described method of derivatization with (1R,2R)- or (1S,2S)-2(2,3-anthracenedicarboximido)cyclohexanecarboxylic acid and HPLC (20, 35). HPLC separation was performed using a Tosoh DP-8020 pump equipped with a Rheodyne 7125 sample injector, Jasco FP-920 fluorescence detector, and column heater (Cryocool CC100 II). Data analysis was performed with Chromatocorder 21 (System Instruments). Synthesis of CH503 Analogs. CH503 analogs (R)- and (S)-3-acetoxy-11,19octacosadiyn-1-ol (triple-bond or TB-CH503), and (3R,11Z)- and (3S,11Z)-3acetoxy-11-octacosen-1-ol (11Z)-dihydro-CH503 were synthesized according to published procedures (36).
Z,Z)-CH503 was performed by gently vortexing live females in a glass vial coated with 2–100 μg of CH503, as previously described (18, 38). The lowest dose tested was 83 ng, corresponding to the approximate amount expressed by males from laboratory Canton-S stocks (37). The same procedures were used for perfuming control flies, except that the vials were coated with hexane alone and the solvent was allowed to evaporate before vortexing. Courtship assays were performed at 23.3 °C, 60% humidity. Perfumed female flies were decapitated and placed in 16 × 9-mm or 35 × 10-mm courtship chambers containing moistened filter paper to maintain humidity. Control trials with hexane-perfumed females were conducted in parallel with experimental trials. For D. sechellia and D. mojavensis, live females were necessary to induce male courtship behavior. Males were aspirated into the chambers, and features of courtship behavior [latency to initiate courtship, orienting, wing extension and vibration, attempted copulation, and copulation (for live females)] were scored for 30 min. Courtship initiation percentage is defined as the number of trials in which males displayed continuous courtship behavior for at least 1 min. For D. mojavensis, 20 s was used as the threshold because of the rapid copulation time (usually between 1 and 2 min). Courtship latency is defined as the amount of time elapsed between aspiration of the male into the chamber and the first display of courtship behavior sustained for at least 1 min. When no courtship is observed during the 30-min trial, the maximum score of 1,800 s is given. Two-Choice Courtship Behavior Assays. A single CH503-perfumed female and a single hexane-perfumed female were decapitated and placed 10–15 mm apart in a 35 × 10-mm courtship chamber. A socially naïve male then was aspirated into the chamber, and courtship behavior was scored for 30 min. Courtship vigor was calculated by normalizing the amount of time spent displaying courtship behaviors toward one target to the total time males spent courting either target. Trials in which courtship lasted for less than 10 s were not considered. Statistical Analysis. Courtship percentages were compared using a Holm– Bonferroni corrected Fisher exact test. For courtship latencies, a Kruskall– Wallis test followed by a Wilcoxon rank sum test was applied. All analyses were performed using GraphPad Prism 6. Mapping of CH503 Expression and Behavioral Response onto the Phylogeny. Character mapping was performed with Mesquite 2.75 (39) using a linear parsimony model. Presence or absence of CH503 expression was treated as an unordered character state. The behavioral response to natural CH503 was represented in terms of a courtship suppression index. The index is a measure of the behavioral response elicited by the natural pheromone (R,Z,Z)-CH503 relative to the artificial stereoisomer (S,Z,Z)-CH503, and was calculated as follows: RCP , ðRCP + SCP Þ where RCP is the courtship initiation percentage for (R,Z,Z)-CH503 and SCP is the courtship initiation percentage for (S,Z,Z)-CH503 at the lowest or second lowest effective dose. To determine whether there is significant phylogenetic signal from the behavioral response to the natural pheromone, the terminal taxa of the tree were shuffled 10,000 times. The likelihood of the character distribution was greater than expected by chance alone (P = 0.017), indicating that the courtship suppression index exhibits significant phylogenetic signal. Drosophila phylogeny was reconstructed based on information from FlyBase (40, 41).
Single-Fly Courtship Behavior Assay. (R,Z,Z)- and (S,Z,Z)-CH503 were synthesized previously by Mori et al. (37). Perfuming of females with synthetic (R/S,
ACKNOWLEDGMENTS. We thank D. P. Araujo, Y. N. Chiang, J. Chin, J. Y. Chua, Q. L. Koh, W. C. Ng, A. Pirkl, M. Rozenbaum, K. Su, and K. J. Tan for technical support and helpful suggestions and W. J. Etges for D. mojavensis flies. Fly illustrations were drawn by J. Y. Chua. R. Meier, S. Pletcher, and K. Dreisewerd provided critical comments on the manuscript. J.Y.Y. is supported by a Singapore National Research Foundation fellowship and a fellowship from the Alexander von Humboldt research foundation.
1. Darwin C (1859) On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life (John Murray, London). 2. Darwin C (1871) The Descent of Man, and Selection in Relation to Sex (John Murray, London). 3. Fisher RA (1930) The Genetical Theory of Natural Selection (Clarendon, Oxford). 4. Kirkpatrick M (1982) Sexual selection and the evolution of female choice. Evolution 36(1):1–12. 5. Zahavi A (1975) Mate selection—a selection for a handicap. J Theor Biol 53(1):205–214.
6. Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds: A role for parasites? Science 218(4570):384–387. 7. Andersson M (1986) Evolution of condition-dependent sex ornaments and mating preferences—sexual selection based on viability differences. Evolution 40(4):804–816. 8. Ryan MJ (1998) Sexual selection, receiver biases, and the evolution of sex differences. Science 281(5385):1999–2003. 9. Ryan MJ, Fox JH, Wilczynski W, Rand AS (1990) Sexual selection for sensory exploitation in the frog Physalaemus pustulosus. Nature 343(6253):66–67.
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26. Bartelt RJ, Schaner AM, Jackson LL (1985) cis-Vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster. J Chem Ecol 11(12):1747–1756. 27. Kurtovic A, Widmer A, Dickson BJ (2007) A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446(7135):542–546. 28. Priest NK, Galloway LF, Roach DA (2008) Mating frequency and inclusive fitness in Drosophila melanogaster. Am Nat 171(1):10–21. 29. Ayasse M, Paxton RJ, Tengö J (2001) Mating behavior and chemical communication in the order Hymenoptera. Annu Rev Entomol 46:31–78. 30. Andersson J, Borg-Karlson AK, Wiklund C (2004) Sexual conflict and anti-aphrodisiac titre in a polyandrous butterfly: Male ejaculate tailoring and absence of female control. Proc Biol Sci 271(1550):1765–1770. 31. Estrada C, Schulz S, Yildizhan S, Gilbert LE (2011) Sexual selection drives the evolution of antiaphrodisiac pheromones in butterflies. Evolution 65(10):2843–2854. 32. Eliyahu D, Mori K, Takikawa H, Leal WS, Schal C (2004) Behavioral activity of stereoisomers and a new component of the contact sex pheromone of female German cockroach, Blattella germanica. J Chem Ecol 30(9):1839–1848. 33. Niehuis O, et al. (2013) Behavioural and genetic analyses of Nasonia shed light on the evolution of sex pheromones. Nature 494(7437):345–348. 34. Yew JY, Soltwisch J, Pirkl A, Dreisewerd K (2011) Direct laser desorption ionization of endogenous and exogenous compounds from insect cuticles: Practical and methodologic aspects. J Am Soc Mass Spectrom 22(7):1273–1284. 35. Imaizumi K, Terasima H, Akasaka K, Ohrui H (2003) Highly potent chiral labeling reagents for the discrimination of chiral alcohols. Anal Sci 19(9):1243–1249. 36. Shikichi Y, Shankar S, Yew JY, Mori K (2013) Synthesis and bioassay of the eight analogues of male pheromone CH503 (3-acetoxy-11,19-octacosadien-1-ol) of the fruit fly Drosophila melanogaster. Biosci Biotechnol Biochem 77(9):1931–1938. 37. Mori K, Shikichi Y, Shankar S, Yew JY (2010) Pheromone synthesis. Part 244: Synthesis of the racemate and enantiomers of (11Z,19Z)-CH503 (3-acetoxy-11,19-octacosadien1-ol), a new sex pheromone of male Drosophila melanogaster to show its (S)-isomer and racemate as bioactive. Tetrahedron 66(35):7161–7168. 38. Billeter JC, Atallah J, Krupp JJ, Millar JG, Levine JD (2009) Specialized cells tag sexual and species identity in Drosophila melanogaster. Nature 461(7266):987–991. 39. Maddison WP, Maddison DR (2011) Mesquite: A modular system for evolutionary analysis (http://mesquiteproject.org), Version 2.75. 40. Marygold SJ, et al.; FlyBase consortium (2013) FlyBase: Improvements to the bibliography. Nucleic Acids Res 41(Database issue, D1):D751–D757. 41. Powell JR (1997) Progress and Prospects in Evolutionary Biology: The Drosophila Model (Oxford Univ Press, New York).
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10. Basolo AL (1990) Female preference predates the evolution of the sword in swordtail fish. Science 250(4982):808–810. 11. McClintock WJ, Uetz GW (1996) Female choice and pre-existing bias: Visual cues during courtship in two Schizocosa wolf spiders (Araneae: Lycosidae). Anim Behav 52(1):167–181. 12. Jones IL, Hunter FM (1998) Heterospecific mating preferences for a feather ornament in least auklets. Behav Ecol 9(2):187–192. 13. Burley NT, Symanski R (1998) “A taste for the beautiful”: Latent aesthetic mate preferences for white crests in two species of Australian grassfinches. Am Nat 152(6): 792–802. 14. Garcia CM, Ramirez E (2005) Evidence that sensory traps can evolve into honest signals. Nature 434(7032):501–505. 15. Wyatt TD (2003) Pheromones and Animal Behavior: Communication by Smell and Taste (Cambridge Univ Press, Cambridge, UK). 16. Howard RW, Blomquist GJ (2005) Ecological, behavioral, and biochemical aspects of insect hydrocarbons. Annu Rev Entomol 50:371–393. 17. Gwynne DT (2008) Sexual conflict over nuptial gifts in insects. Annu Rev Entomol 53: 83–101. 18. Yew JY, et al. (2009) A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila. Curr Biol 19(15):1245–1254. 19. Brieger G, Butterworth FM (1970) Drosophila melanogaster: Identity of male lipid in reproductive system. Science 167(3922):1262. 20. Shikichi Y, et al. (2012) Pheromone synthesis. Part 250: Determination of the stereostructure of CH503 a sex pheromone of male Drosophila melanogaster, as (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol by synthesis and chromatographic analysis of its eight isomers. Tetrahedron 68(19):3750–3760. 21. Mori K (2007) Significance of chirality in pheromone science. Bioorg Med Chem 15(24):7505–7523. 22. Ejima A, et al. (2007) Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate. Curr Biol 17(7):599–605. 23. Scott D (1986) Sexual mimicry regulates the attractiveness of mated Drosophila melanogaster females. Proc Natl Acad Sci USA 83(21):8429–8433. 24. Ferveur JF, Sureau G (1996) Simultaneous influence on male courtship of stimulatory and inhibitory pheromones produced by live sex-mosaic Drosophila melanogaster. Proc Biol Sci 263(1373):967–973. 25. Chen PS, et al. (1988) A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster. Cell 54(3):291–298.
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PNAS | February 25, 2014 | vol. 111 | no. 8 | 3061
Edited* by Edward A. Kravitz, Harvard Medical School, Boston, MA, and approved December 31, 2013 (received for review July 19, 2013)
Animals exhibit a spectacular array of traits to attract mates. Understanding the evolutionary origins of sexual features and preferences is a fundamental problem in evolutionary biology, and the mechanisms remain highly controversial. In some species, females choose mates based on direct benefits conferred by the male to the female and her offspring. Thus, female preferences are thought to originate and coevolve with male traits. In contrast, sensory exploitation occurs when expression of a male trait takes advantage of preexisting sensory biases in females. Here, we document in Drosophila a previously unidentified example of sensory exploitation of males by other males through the use of the sex pheromone CH503. We use mass spectrometry, high-performance liquid chromatography, and behavioral analysis to demonstrate that an antiaphrodisiac produced by males of the melanogaster subgroup also is effective in distant Drosophila relatives that do not express the pheromone. We further show that species that produce the pheromone have become less sensitive to the compound, illustrating that sensory adaptation occurs after sensory exploitation. Our findings provide a mechanism for the origin of a sex pheromone and show that sensory exploitation changes male sexual behavior over evolutionary time.
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sexual selection laser desorption/ionization male–male competition chiral pheromone
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| supernormal stimulus |
S
exual selection is widely regarded as an important mechanism for the origin of new traits and species. Darwin first proposed that the elaboration of male secondary sexual traits is driven by female preferences (1, 2). This concept has been refined by models suggesting that females select male traits that indicate genetic quality or confer direct reproductive benefits (3– 7). In contrast, sensory exploitation occurs when expression of a male trait takes advantage of preexisting sensory biases in females (8). In this case, female preference does not coevolve with the male trait but rather precedes it. In one of the first examples documenting sensory exploitation, female Physalaemus coloradorum frogs were shown to prefer male calls that contain a low-frequency “chuck” component despite the absence of this feature in calls from conspecifics. The sensory bias for chucks was shown to have its mechanistic basis in the tuning properties of the inner ear, a physiological feature that predated the appearance of chucks (9). Similarly, female platyfish exhibit a preference for males with swordtails despite the absence of swordtails in male platyfish. Females consistently chose to spend more time with conspecific males exhibiting an artificially attached plastic sword (10). In both these examples, female preference predates expression of the trait. Sensory exploitation has since been documented for numerous other visual cues, across a diversity of taxa (11–14). In each case, females prefer traits that are not found naturally in their own species but appear in males of other species. Moreover, both the sensory bias and behavioral response to the trait already were present before expression of the trait.
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Pheromones are taste and olfactory cues that, in many species, play an important role in mate selection (15). As with courtship cues detected by other sensory modalities, pheromones are shaped by sexual selection and, thus, may exhibit enormous structural diversity and exquisite stereochemical specificity. In insects, exogenously secreted lipids advertise mating status, availability, and reproductive fitness (16). In some cases, male pheromones serve as a nuptial gift, thus providing direct reproductive benefits to females and offspring in the form of either nutritive or defensive compounds (17). Little is known, however, about the mechanisms underlying the diversification and the origin of chemical specificity. Here, we provide an example of a pheromone that has evolved from sensory exploitation. In Drosophila melanogaster, CH503 [formally, (3R,11Z,19Z)-3-acetoxy-11,19octacosadien-1-ol; Fig. 1A] functions as an antiaphrodisiac (18). The pheromone is secreted in the anogenital region, is transferred to females during mating, and suppresses courtship from males. Our findings indicate CH503 evolved from males exploiting the preexisting sensory biases of other males to gain mating advantage by limiting access to females. Moreover, the use of CH503 has altered male sexual behavior over evolutionary time such that males have adapted by becoming less sensitive to the pheromone. Results and Discussion Evolutionary Origin of CH503 Expression. To determine the evolu-
tionary origins of CH503, we examined eight species of Drosophila Significance How sexual features and preferences originate and evolve is one of the most important and contentious problems in evolutionary biology. In some species, females choose male traits that indicate genetic quality or confer benefits. Thus, male traits and female preferences are assumed to originate at the same time and to coevolve. In contrast, sensory exploitation occurs when males take advantage of females’ preexisting sensory biases. We show in Drosophila sensory exploitation of males by other males through the use of a pheromone to gain sexual advantage. Notably, sensory exploitation leads to male sensory adaptation. These findings provide a mechanism for the evolutionary origins of a pheromone and are a previously unidentified example of sensory exploitation between males. Author contributions: S.H.N., S.S., Y.S., K.A., K.M., and J.Y.Y. designed research; S.H.N., S.S., Y.S., K.A., K.M., and J.Y.Y. performed research; Y.S., K.A., and K.M. contributed new reagents/analytic tools; S.H.N., S.S., Y.S., K.A., K.M., and J.Y.Y. analyzed data; and S.H.N., K.A., K.M., and J.Y.Y. wrote the paper. The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. Freely available online through the PNAS open access option. 1
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1313615111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1313615111
cVA 349.24 (3R,11Z,19Z)-CH503 (natural stereoisomer) OH
n-C 8H17 OAc
CH503 503.38
D. melanogaster D. simulans D. yakuba m/z
3 00
650
D. ananassae
D. sechelia
D. willistoni 501.37
405.32
471.43 503.39 485.40
cVA 349.25 407.26
515.42
D. mojavensis
D. virilis
559.34
487.39
577.37 499.45
515.42
471.41 443.38
563.35 551.34
507.28
415.37 3 00
m/z
65 0
m/z
30 0
6 50
B
37.3
2 3 5 1
4
32.5 D. simulans
6 7 8 32.7
37.6 D. yakuba 37.4
32.6 min.
0
Derivatized CH503 isomer and RT (min) 1. (3S,11Z,19Z) 34.8 5. (3R,11E,19Z) 2. (3R,11Z,19Z) 37.5 6. (3R,11Z,19E) 3. (3S,11E,19Z) 42.8 7. (3S,11E,19E) 4. (3S,11Z,19E) 44.7 8. (3R,11E,19E)
D. sechellia
80
24.4 46.7 48.8 68.3 74.8
D. ananassae 0
min.
80
Fig. 1. Characterization of CH503 expression in Drosophila. (A) Chemical structure of CH503 and representative UV-LDI mass spectra measured from the male anogenital region of different Drosophila species. Each spectrum is recorded from a single fly. Signals corresponding to the mass-to-charge ratio (m/z) for cVA (m/z 349.24) and CH503 (m/z 503.38) were detected in D. melanogaster, D. simulans, D. yakuba, D. sechellia, and D. ananassae. No signal for CH503 was detected from D. willistoni, D. mojavensis, or D. virilis. Potassium-bearing molecular compounds [M+K]+ constitute the major ion species in all cases. (B) The HPLC chromatogram shows distinct retention times (RT) for each of the eight synthetic CH503 stereoisomers following derivatization. HPLC analysis of derivatized CH503 isolated from D. simulans, D. yakuba, and D. sechellia reveals that (3R,11Z,11Z)-CH503 is the only expressed stereoisomer. The retention times for the major peaks are noted in each chromatogram. The compound isolated from D. ananassae has the same m/z and elemental composition as CH503 but a different structure.
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for production of the pheromone CH503 and tested whether males of these species respond to CH503 as an antiaphrodisiac. We first used UV laser desorption/ionization mass spectrometry (UV-LDI MS) to analyze the chemical profiles of the male anogenital region in other species. UV-LDI MS revealed signals matching the expected molecular weight for CH503 in the anogenital region of D. melanogaster, Drosophila simulans, Drosophila yakuba, Drosophila sechellia, and Drosophila ananassae (Fig. 1A). A signal for cis-vaccenyl acetate (cVA), another known antiaphrodisiac, also was found in these species (19). In contrast, no signal for CH503 could be detected from the anogenital region of Drosophila willistoni, Drosophila mojavensis, or Drosophila virilis (Fig. 1A). To determine the double-bond geometries and absolute configuration of CH503 from melanogaster group flies, chemical derivatization and high-performance liquid chromatography (HPLC) separation were used to compare the retention times of the derivative of naturally occurring CH503 with synthetic standards of the eight possible derivatized stereoisomers. Each of the derivatized stereoisomers could be differentiated based on their distinct retention times (Fig. 1B). D. melanogaster previously was shown to express (3R,11Z,19Z)-CH503 [hereafter abbreviated as (R,Z,Z)-CH503] (20). HPLC analysis of derivatized thin-layer chromatography-purified fractions revealed a single peak corresponding to (R,Z,Z)-CH503, indicating that as with D. melanogaster, D. simulans, D. yakuba, and D. sechellia express a single stereoisomer (Fig. 1B). Although a compound with a mass signal corresponding to (R,Z,Z)-CH503 was observed by UV-LDI MS analysis in D. ananassae, the retention time did not match any of the eight stereoisomers. Taken together, chemical profiling with UV-LDI MS and HPLC reveals that all tested drosophilids of the melanogaster subgroup express (R,Z,Z)-CH503. In contrast, other drosophilids representing outgroup taxa from Sophophora (D. willistoni, D. ananassae) and Drosophila (D. virilis, D. mojavensis) do not express (R,Z,Z)-CH503 or any other stereoisomer. The phylogenetic distribution of the trait suggests a single origin in the melanogaster group of Drosophila. Conserved Function of CH503 as an Antiaphrodisiac. We next tested whether the function of CH503 as an antiaphrodisiac is conserved across the different species. Socially isolated virgin males were placed with a virgin female perfumed with various amounts of CH503. Surprisingly, all Drosophila species tested suppressed courtship initiation in a dose-dependent manner in response to CH503, although the pheromone is produced only by a subgroup of these species. Male courtship behavior was significantly inhibited in D. melanogaster, D. simulans, D. yakuba, and D. sechellia, all species that produce CH503 (Fig. 2A). The latency to courtship initiation also increased significantly in a dose-responsive manner to increasing amounts of CH503 (Fig. S1). Notably, D. ananassae, D. willistoni, D. mojavensis, and D. virilis, none of which expresses CH503, also responded to the compound by suppressing courtship behavior and delaying courtship initiation (Fig. 2B, Fig. S1, and Table S1). These findings indicate that the behavioral response to CH503 predates the expression of CH503. We hypothesize that males of the ancestral species that gave rise to the melanogaster subgroup used sensory exploitation to inhibit courtship from male competitors. To test whether production of CH503 might provide an advantage in male–male competition for access to females, we examined the effect of introducing (R,Z,Z)-CH503 into the mating system of species that do not produce the pheromone. D. ananassae and D. virilis males were given a choice of mating with CH503perfumed or solvent-perfumed females. In both species, males showed a significant aversion to courting the former (Fig. 2E). Thus, the use of a potent antiaphrodisiac by males could significantly shift courtship choice of rival males. By limiting access to mated females, male producers of the pheromone might potentially gain a mating advantage by reducing sperm competition. PNAS | February 25, 2014 | vol. 111 | no. 8 | 3057
EVOLUTION
A
**** **** ****
****
****
**
****
ns
***
**
40
0
0 83 166 249
20 0 83 249 415
0 83 249 415
Dsec
60
0 83 166 249
***
*** 0 580 830 1000
Dyak
80
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ns **
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ns 0 1245 1660 2075
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ns
D 100Response to (S,Z,Z)-CH503
100 Courtship initiation (%)
Courtship initiation (%)
ns
ns ** Dmel
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Courtship choice D. virilis ns
****
0.8 0.6 0.4 0.2 0.0 Control
250ng TB-CH503
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D. melanogaster ns **
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1.0
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D. ananassae ns
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1.0 Courtship vigor
0 580 700 830
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40
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C
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60
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Courtship initiation (%)
80
Response to (R,Z,Z)-CH503 100
****
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100
0 83 415 830
Response to (R,Z,Z)-CH503
A
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Fig. 2. Comparative analysis of the behavioral response to natural CH503 and an artificial stereoisomer. (A and B) Courtship assays reveal that the natural pheromone (R,Z,Z)-CH503 functions as an antiaphrodisiac both in species that produce the natural pheromone (A) and in species that do not (B). Doses (in nanograms) indicated represent the approximate amount on the cuticular surface following perfuming. The courtship initiation percentage for each dose is compared with the percentage from control trials and statistically assessed using a Holm–Bonferroni-corrected Fisher exact test. Dana, D. ananassae; Dmel, D. melanogaster; Dmoj, D. mojavensis; Dsec, D. sechellia; Dsim, D. simulans; Dvir, D. virilis; Dwil, D. willistoni; Dyak, D. yakuba; ns, nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 24–36 for each dose. (C and D) A dose-dependent response to the artificial stereoisomer (S,Z,Z)-CH503 is observed in all tested species. In species that produce CH503 (C), the artificial stereoisomer is more potent than the natural pheromone. In species that do not produce CH503 (D), the artificial stereoisomer and the natural pheromone are effective over a similar range of doses. Statistical analysis was performed as in A. (E) In courtship choice assays, D. ananassae and D. virilis males spent a greater amount of time courting nonperfumed females over (R,Z,Z)-CH503–perfumed females. D. melanogaster males preferentially courted nonperfumed females over females perfumed with (S,Z,Z)-CH503. In the presence of both stereoisomers, males preferentially courted females perfumed with the natural pheromone. No effect on courtship was observed when females were perfumed with an equivalent dose of a CH503 analog (TB-CH503). The ends of the box plot represent the spread of data between the 25th (bottom) and the 75th percentile (top), whereas the horizontal line represents the median and + represents the mean. Courtship vigor within each pairing was compared using a Wilcoxon rank-sum test. ****P < 0.0001; **P = 0.0032; *P = 0.0397; n = 21–29.
Adaptive Response to Sensory Exploitation. It is well established that the biological activity of pheromones can be highly dependent on the stereostructure (21). To determine whether the stereostructure of CH503 is important for its function as an antiaphrodisiac, we tested the effect of a synthetic stereoisomer of CH503, (S,Z,Z)-CH503, a stereoisomer not known to be expressed naturally by drosophilids (Fig. S2). Unexpectedly, D. melanogaster, D. simulans, D. yakuba, and D. sechellia exhibited stronger courtship suppression and a longer latency to initiate courtship in the presence of the artificial stereoisomer compared with the natural pheromone at equivalent doses (Fig. 2C, Fig. S2, and Table S2). When D. melanogaster males were given a choice of females perfumed with (S,Z,Z)-CH503 or a nonperfumed female, males preferred to court the latter (Fig. 2E). Even in the presence of a female perfumed with an equivalent amount of the wild-type stereoisomer, (R,Z,Z)-CH503, males continued to show a stronger aversion to the unnatural pheromone (Fig. 2E). The disparity between the strength of the natural vs. unnatural pheromone is particularly striking in D. simulans, in which nearly 15 times the 3058 | www.pnas.org/cgi/doi/10.1073/pnas.1313615111
amount of the natural pheromone is needed to achieve the same level of courtship inhibition produced by the artificial stereoisomer. In contrast, males of D. ananassae, D. willistoni, D. virilis, and D. mojavensis exhibited an equivalent or greater responsiveness to the wild-type form of CH503 compared with the artificial stereoisomer (Fig. 2D, Fig. S2, and Table S2). Thus, species that produce the pheromone exhibit a weaker behavioral response to the natural compound, whereas nonproducers of the pheromone are more sensitive to the pheromone (Fig. 3). The lowered sensitivity exhibited by CH503-producing species to the natural pheromone may be the result of sensory adaptation or habituation from exposure to the pheromone from conspecifics. However, adaptation likely is not a contributing factor because males were individually housed during the late pupal stage and remained isolated until testing at 4–5 d old. Furthermore, the response of D. melanogaster males collectively raised in groups was not significantly different from that of individually housed flies (Fig. S3). Thus, even in the presence of heightened preexposure to CH503, the differential sensitivity exhibited by Ng et al.
CH503 producing species non-CH503 producing species
sensory trap induced sensory adaptation (14). Interestingly, we did not observe sensory adaptation to cVA. Responsiveness to the courtship inhibition properties of the pheromone showed no correlation with whether the species produced the pheromone (Table S3). The multiple functions of cVA as an aggregation cue (26) and aphrodisiac for females (27) likely prevent males from completely losing sensitivity to the pheromone despite the costs of courtship suppression. The robust behavioral response of melanogaster subgroup species to (S,Z,Z)-CH503 suggests future opportunities for sensory exploitation. A subtle change in stereochemistry due to allelic variations in genes underlying pheromone production might allow for expression of a stereoisomer variant that would circumvent male adaptation. Behavioral experiments are consistent with this prediction: use of the unnatural pheromone results in significantly greater courtship aversion, even in the presence of the natural pheromone. It will be interesting to consider whether persistent use of (S,Z,Z)-CH503 over the long term may contribute to physiological desensitization and also result in disadvantages for the population, such as lowered mating frequency, resulting in reduced offspring fitness (28). Several mechanisms underlying the evolution of antiaphrodisiacs have been proposed, arguing that chemical diversity in pheromone structures arises as a result of male–female cooperation (23, 29), male–female sexual conflict (30), or male–male competition (31). Our study indicates that sensory exploitation is another mechanism for the evolution of antiaphrodisiacs and traits used in male–male competition. A similar phenomenon whereby synthetic nonnatural stereoisomers elicited a behavioral response stronger than that of the natural pheromone was reported previously in the German cockroach, indicating that sensory exploitation may underlie the evolution of other insect pheromone systems (32). These findings contrast with an alternative mechanism shown in Nasonia wasps, in which novel pheromone compounds appear before a preexisting response (33). Currently, the chemosensory receptor(s) and underlying neural circuits mediating CH503 detection are unknown. Once they are identified in D. melanogaster, it will be possible to correlate evolution of the receptor(s) structure with behavioral responses of various species and in this way, to understand the underlying neurophysiological mechanisms. Furthermore, determining
D. simulans
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Fig. 3. Evolution of CH503 expression and the behavioral response to CH503. CH503 expression (left) and the behavioral response to natural CH503 (right) are mapped onto the Drosophila phylogeny using a linear parsimony model. The values at the branch termini (right phylogram) reflect the courtship suppression index, a measure of the strength of courtship suppression induced by the natural pheromone compared with the artificial stereoisomer. All tested species respond to both stereoisomers. However, species that produce CH503 respond more weakly to the natural pheromone. Hatch marks indicate the likely point of origin of each trait.
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D. melanogaster to the natural and artificial pheromone remained unchanged. Courtship suppression in the presence of (R/S,Z,Z)-CH503 might simply be the result of aversion to a foreign chemical or masking of endogenous female pheromones. To address this possibility, males of all species were tested with females perfumed with an equivalent dose of modified (S,Z,Z)-CH503 or (R,Z,Z)-CH503 analogs bearing either two triple bonds (instead of two double bonds) or a single double bond only. In each case, no significant courtship suppression was observed (Tables S1 and S2; Fig. 2E). These findings indicate that first, melanogaster subgroup males have evolved partial resistance to the antiaphrodisiac effects of the natural pheromone, and second, the behavioral response is specific to the stereostructure of (R/S,Z,Z)-CH503. Our findings support the hypothesis that in Drosophila, an antiaphrodisiac pheromone evolved as a result of sensory exploitation. All tested species, regardless of their endogenous pheromone expression, suppressed courtship in response to the natural pheromone, (R,Z,Z)-CH503. Because (R,Z,Z)-CH503 originates only in the melanogaster subgroup, response to the pheromone predates expression of the pheromone. In contrast to previous findings that identified attractive superstimulants, these results show that preexisting sensory systems for highly aversive cues also may be exploited and that sensory exploitation also takes place between males. In D. melanogaster, multiple pheromones, such as cVA and (7Z)-tricosene, and accessory gland proteins are used to suppress female mating frequency (22–25). We speculate that initial use of CH503 helped reinforce courtship inhibition from rival males because, in contrast to other male-transferred cues, CH503 persists on female cuticles for at least 10 d (18). The low vapor pressure of the pheromone and its behavioral potency would place strong selective pressure for males to become less sensitive to the pheromone to benefit from increased mating opportunities. Our results are consistent with this prediction: members of the (R,Z,Z)CH503–expressing melanogaster subgroup have adaptively evolved to become less sensitive to the natural pheromone. In this way, a trait that originated from sensory exploitation drives the evolution of sensory pathways. A similar phenomenon occurs in Goodeidae splitfin fish, where a visual cue originating as a female
molecular markers for the relevant sensory pathways will provide a way to map the evolutionary origins of predisposed sensory biases and the shaping of pheromone structural specificity. Materials and Methods Drosophila Stocks and Husbandry. Flies were obtained from the Bloomington Stock Centre and Ehime University Stock Center, and maintained on cornmeal agar food at 25 °C, 60% humidity, with a 12:12-h light:dark cycle. D. mojavensis were wild-caught from Las Bocas, Sonora, Mexico (in March 2009) by W. J. Etges (University of Arkansas) and raised on standard cornmeal agar food supplemented with banana (∼110 g per 20 half-pint bottles) and cactus powder (∼2.3 g per 20 half-pint bottles). Adult flies used in courtship assays were isolated at the pupal stage or collected within 4 h after pupal eclosion. Male virgins were raised individually in a test tube containing 2 mL of cornmeal agar food, whereas females were kept in groups of 10 per vial. For grouped-male conditions, adult males were collected at the pupal stage and kept in groups of 10 per vial for 7 d. Flies were tested at the following ages, corresponding to sexual maturity: D. melanogaster and D. simulans, 4–7 d; D. sechellia and D. yakuba, 5– 7 d; D. ananassae, 7 d; D. virilis and D. mojavensis, 10 d; and D. willistoni, 15 d. Pheromone Profiling of Drosophila Species Using UV-LDI MS Analysis. UV-LDI MS analysis and the procedures for preparing the flies were described in detail previously (18). Measurements were performed on a Q-Star Elite (AB SCIEX) orthogonal time-of-flight mass spectrometer equipped with an intermediate pressure oMALDI2 source and an N2 laser (λ= 337 nm, 40-Hz repetition rate, 200-μm beam diameter, 3-ns pulse duration). Ions were generated in a buffer gas environment by using 2 mbar of N2. Individual flies were attached to a coverslip with adhesive tape and mounted onto a custom-built sample plate. During data acquisition, the anogenital region was irradiated for 30 s, corresponding to 1,200 laser shots. For each species, 10–15 socially naïve male flies were measured. Mass accuracy for the mass spectrometer was ∼20 ppm. More details about instrumentation conditions and analytical parameters important for the chemical imaging of insects are provided in ref. 34. Purification of CH503 from Drosophila Species. Purification of CH503 by thin layer chromatography (TLC) was performed using the conditions described by Yew et al. (18). Approximately 800–1,000 males and females of D. melanogaster, D. simulans, D. yakuba, D. sechellia, and D. ananassae were extracted with hexane (10 mL) for 20 min at room temperature. The extract was concentrated to dryness using a stream of N2. The residue was dissolved in hexane before separation by TLC. TLC separation was performed on glassbacked silica gel plates (10 × 10 cm, coated with 0.2 mm of silica gel 60; Merck) using a running solvent consisting of hexane/diethyl ether/acetic acid (66:33:1, each by volume). The major component of the isolated fraction was confirmed as CH503 by Direct Analysis in Real Time (DART) mass spectrometry. The DART ion source was operated in positive-ion mode with helium gas, with the gas heater set to 200 °C. The glow discharge needle potential was set to 3.5 kV. Electrode 1 was set to +150 V, and electrode 2 (grid) was set to +250 V. HPLC Analysis. The separation of eight stereoisomers was determined using a previously described method of derivatization with (1R,2R)- or (1S,2S)-2(2,3-anthracenedicarboximido)cyclohexanecarboxylic acid and HPLC (20, 35). HPLC separation was performed using a Tosoh DP-8020 pump equipped with a Rheodyne 7125 sample injector, Jasco FP-920 fluorescence detector, and column heater (Cryocool CC100 II). Data analysis was performed with Chromatocorder 21 (System Instruments). Synthesis of CH503 Analogs. CH503 analogs (R)- and (S)-3-acetoxy-11,19octacosadiyn-1-ol (triple-bond or TB-CH503), and (3R,11Z)- and (3S,11Z)-3acetoxy-11-octacosen-1-ol (11Z)-dihydro-CH503 were synthesized according to published procedures (36).
Z,Z)-CH503 was performed by gently vortexing live females in a glass vial coated with 2–100 μg of CH503, as previously described (18, 38). The lowest dose tested was 83 ng, corresponding to the approximate amount expressed by males from laboratory Canton-S stocks (37). The same procedures were used for perfuming control flies, except that the vials were coated with hexane alone and the solvent was allowed to evaporate before vortexing. Courtship assays were performed at 23.3 °C, 60% humidity. Perfumed female flies were decapitated and placed in 16 × 9-mm or 35 × 10-mm courtship chambers containing moistened filter paper to maintain humidity. Control trials with hexane-perfumed females were conducted in parallel with experimental trials. For D. sechellia and D. mojavensis, live females were necessary to induce male courtship behavior. Males were aspirated into the chambers, and features of courtship behavior [latency to initiate courtship, orienting, wing extension and vibration, attempted copulation, and copulation (for live females)] were scored for 30 min. Courtship initiation percentage is defined as the number of trials in which males displayed continuous courtship behavior for at least 1 min. For D. mojavensis, 20 s was used as the threshold because of the rapid copulation time (usually between 1 and 2 min). Courtship latency is defined as the amount of time elapsed between aspiration of the male into the chamber and the first display of courtship behavior sustained for at least 1 min. When no courtship is observed during the 30-min trial, the maximum score of 1,800 s is given. Two-Choice Courtship Behavior Assays. A single CH503-perfumed female and a single hexane-perfumed female were decapitated and placed 10–15 mm apart in a 35 × 10-mm courtship chamber. A socially naïve male then was aspirated into the chamber, and courtship behavior was scored for 30 min. Courtship vigor was calculated by normalizing the amount of time spent displaying courtship behaviors toward one target to the total time males spent courting either target. Trials in which courtship lasted for less than 10 s were not considered. Statistical Analysis. Courtship percentages were compared using a Holm– Bonferroni corrected Fisher exact test. For courtship latencies, a Kruskall– Wallis test followed by a Wilcoxon rank sum test was applied. All analyses were performed using GraphPad Prism 6. Mapping of CH503 Expression and Behavioral Response onto the Phylogeny. Character mapping was performed with Mesquite 2.75 (39) using a linear parsimony model. Presence or absence of CH503 expression was treated as an unordered character state. The behavioral response to natural CH503 was represented in terms of a courtship suppression index. The index is a measure of the behavioral response elicited by the natural pheromone (R,Z,Z)-CH503 relative to the artificial stereoisomer (S,Z,Z)-CH503, and was calculated as follows: RCP , ðRCP + SCP Þ where RCP is the courtship initiation percentage for (R,Z,Z)-CH503 and SCP is the courtship initiation percentage for (S,Z,Z)-CH503 at the lowest or second lowest effective dose. To determine whether there is significant phylogenetic signal from the behavioral response to the natural pheromone, the terminal taxa of the tree were shuffled 10,000 times. The likelihood of the character distribution was greater than expected by chance alone (P = 0.017), indicating that the courtship suppression index exhibits significant phylogenetic signal. Drosophila phylogeny was reconstructed based on information from FlyBase (40, 41).
Single-Fly Courtship Behavior Assay. (R,Z,Z)- and (S,Z,Z)-CH503 were synthesized previously by Mori et al. (37). Perfuming of females with synthetic (R/S,
ACKNOWLEDGMENTS. We thank D. P. Araujo, Y. N. Chiang, J. Chin, J. Y. Chua, Q. L. Koh, W. C. Ng, A. Pirkl, M. Rozenbaum, K. Su, and K. J. Tan for technical support and helpful suggestions and W. J. Etges for D. mojavensis flies. Fly illustrations were drawn by J. Y. Chua. R. Meier, S. Pletcher, and K. Dreisewerd provided critical comments on the manuscript. J.Y.Y. is supported by a Singapore National Research Foundation fellowship and a fellowship from the Alexander von Humboldt research foundation.
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