ARTICLE IN PRESS

Proc. R. Soc. B 1 doi:10.1098/rspb.2008.0375 65 2 66 3 67 4 68 5 The Coolidge effect, individual recognition 69 6 70 7 and selection for distinctive cuticular signatures 71 8 72 9 in a burying beetle 73 10 1,* 1,† 1,2 ¨ 1 74 11 Sandra Steiger , Ragna Franz , Anne-Katrin Eggert and Josef K. Muller 75 12 1Department of Evolutionary Biology and Ecology, Faculty of Biology, University of Freiburg, 76 13 Hauptstrasse 1, 79104 Freiburg, Germany 77 14 2Behavior, Ecology, Evolution, and Systematics (BEES) Section, Department of Biological Sciences, 78 15 Illinois State University, Normal, IL 61790-4120, USA 79 16 80 The ability to recognize individuals is an important aspect of social interactions, but it can also be useful to 17 81 avoid repeated matings with the same individual. The Coolidge effect is the progressive decline in a male’s 18 82 propensity to mate with the same female combined with a heightened sexual interest in new females. 19 83 Although males that recognize previous partners and show a preference for novel females should have a 20 84 selective advantage as they can distribute sperm evenly among the females they encounter, there are few 21 85 invertebrate examples of the Coolidge effect. Here we present evidence for this effect in the burying beetle 22 86 Nicrophorus vespilloides and examine the mechanism underlying the discrimination between familiar and 23 87 novel mates. Burying beetles feed and reproduce on vertebrate carcasses, where they regularly encounter 24 88 conspecifics. Males showed greater sexual interest in novel females (virgin or mated) than in females they 25 89 had inseminated before. The application of identical cuticular extracts allowed us to experimentally create 26 90 females with similar odours, and male responses to such females demonstrated that they use female 27 91 cuticular patterns for discrimination. The chemical analysis of the cuticular profile revealed greater inter- 28 92 individual variation in female than in male cuticular patterns, which might be due to greater selection on 29 93 females to signal their individual identity. 30 94 31 Keywords: burying beetles; Nicrophorus; Coolidge effect; individual recognition; cuticular lipids; 95 32 identity signals 96 33 97 34 98 35 99 1. INTRODUCTION (bees: Barrows 1975; amphibians: Donovan & Verrell 36 100 Although the cost of mating to males has long been 1991; reptiles: Tokarz 1992; flies: Wcislo 1992, O¨ deen & 37 101 regarded as negligible, recent evidence shows that mating Moray 2008; beetles: Arnaud & Haubruge 1999; fishes: 38 102 and sperm production may generate non-trivial costs Kelley et al. 1999) have been shown to avoid mating or 39 103 (Dewsbury 1982; Van Voorhies 1992; Olsson et al. 1997; re-mating with familiar individuals. 40 104 41 Preston et al. 2001), promoting the evolution of prudent The Coolidge effect per se has received no attention in 105 42 ejaculate allocation or even male reluctance to mate invertebrates with the exception of one recent study on 106 43 (Wedell et al. 2002). If the value of a female to a male snails (Koene & Ter Maat 2007). Given the number of 107 44 decreases with his increasing mating investment to that studies that have examined mate choice in insects, it is 108 45 individual female, males are expected to avoid re-mating surprising that there have been no investigations of the 109 46 with the female in favour of other reproductive opportu- Coolidge effect per se in this group. Researchers may 110 47 nities (Wedell et al. 2002; Pizzari et al. 2003). The frequently have assumed that the Coolidge effect requires 111 48 ‘Coolidge effect’, defined as a decline in the propensity of a more complex neural processing than invertebrates are 112 49 male to copulate repeatedly with the same female capable of Koene & Ter Maat (2007). However, some 113 50 combined with a heightened sexual interest in novel social insects have proved capable of individual recog- 114 51 females (Wilson et al. 1963; Dewsbury 1981), can be a nition (Tibbetts 2002, 2004; D’Ettorre & Heinze 2005), 115 52 mechanism to distribute sperm more evenly. The Cool- and avoiding matings with one’s previous mate may not 116 53 idge effect was first observed in rats (Beach & Jordan necessarily require individual recognition. Female crickets 117 54 1956) and has since been demonstrated in a number of Gryllus sigillatus use self-referencing (marking males with 118 55 other mammals (see Dewsbury 1981) and birds (Pizzari their individual specific chemical signature) to avoid 119 56 et al. 2003). Similarly, animals of many different groups re-mating with previous mates (Ivy et al. 2005). Numer- 120 57 ous insects have evolved other mechanisms of discrimi- 121 58 nation against previously mated individuals that have 122 59 * Author for correspondence ([email protected]). 123 † typically been interpreted as adaptations to sperm 60 Present address: Division of Zoo Animals, Exotic Pets and Wildlife, 124 Vetsuisse Faculty, University of Zurich, Winterthurerstaße 260, 8057 competition. The so-called ‘antiaphrodisiacs’ are male- 61 Zu¨rich, Switzerland. produced chemicals that are transferred to females 125 62 126 Electronic supplementary material is available at http://dx.doi.org/10. during mating and discourage further matings by these 63 1098/rspb.2008.0375 or via http://journals.royalsociety.org. females (e.g. Happ 1969; Kukuk 1985; Peschke 1987; 127 64 128 Received 18 March 2008 1 This journal is q 2008 The Royal Society Accepted 23 April 2008

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9 ARTICLE IN PRESS 2 S. Steiger et al. Coolidge effect in burying beetle

129 Carlson & Schlein 1991; Andersson et al. 2003) which (b) General procedures of behavioural experiments 193 130 may also help the original male to avoid re-mating with Experiments that involved observation of copulatory 194 131 the same female. behaviour were conducted during the last 2 hours before 195 132 This study investigated the occurrence and underlying the end of the light phase. Males emit pheromones and mate 196 133 mechanism of the Coolidge effect in the burying beetle with attracted females during this period both in our own 197 134 Nicrophorus vespilloides. Males of this species show clear Q2 (unpublished data) and a northern German population near 198 135 evidence of sperm depletion after several days spent with Bielefeld (Mu¨ller & Eggert 1987; Eggert & Mu¨ller 1989). 199 136 multiple females (Eggert 1990). To a male of this species, Matings were observed in small plastic containers (8!5! 200 137 re-mating with a female may thus entail a twofold cost: 6 cm) with a moistened plaster bottom. To minimize 201 138 time that could have been spent seeking out other females disturbance immediately prior to an observation, males 202 139 and sperm that could have been more efficiently used to were transferred to observation chambers 3 hours before 203 140 inseminate another female. Burying beetles reproduce on the onset of an experiment. Over the course of an experiment, 204 141 vertebrate carcasses, frequently in groups with several males remained in the same chamber while females were 205 142 females (Pukowski 1933;Mu¨ller et al. 1990, 2007). When introduced and removed. In each trial, a male was presented 206 143 females leave the carcass early, or males have only limited with a female and either mated with her or did not mate 207 144 time on a carcass, males may benefit from distributing (see below), then the female was removed and the male 208 145 sperm evenly among the available females. Even without remained alone for 5 min before the next female (either the 209 146 reproductive resources, males mate more frequently when same one or a different one) was introduced into the chamber. 210 147 held with multiple females than with a single one (Eggert For each encounter, we recorded the time to mating defined 211 148 1990). This effect could arise if males respond more as the time from the beetles’ first physical contact to the actual 212 149 readily to females other than their last mate. coupling of genitalia. Females were removed from the 213 150 In burying beetles, cuticular contact pheromones chamber as soon as the first mating ended. If the pair did 214 151 contain information about breeding and nutritional status, not mate within 5 min (300 s), we removed the female, 215 152 sex and species (Whitlow 2003; Steiger et al. 2007), and scoring a time to mating of 300 s. 216 153 could play an important role in recognition processes like Our study was designed to determine the readiness 217 154 nestmate recognition (Mu¨ller et al. 2003). To date, there is of males to mate, and we interpret a long time to mating 218 155 no information about the role of cuticular substances in as low male sexual interest, since matings are initiated by 219 156 interactions between the sexes. We tried to address males, who approach and mount females prior to mating. In 220 157 whether males are capable of discriminating between order to avoid the potentially confounding effects of the 221 158 222 their last mate and other females on the basis of cuticular females’ response to male attempts, we excluded trials in 159 223 substances. Following the experimental protocol of Ivy which females clearly tried to avoid copulations by struggling 160 224 et al. (2005), we tested whether males transfer their own and attempting to leave during male mating attempts (17 out 161 225 chemical substances to females or learn cues from the of 198 trials). 162 chemical profile of their females to recognize previous 226 163 mates. If males transfer substances, they should treat the 227 164 mate of a close male relative like they would treat their own (c) The effect of female novelty on male 228 165 previous mate, provided that there is genetic variation in mating behaviour 229 166 chemical compositions and the cuticular patterns of close Our first experiment was designed to test for a Coolidge 230 167 relatives are similar to each other. If males rely on female effect in N. vespilloides males. Each experimental male 231 168 specific cues, they should instead exhibit reduced sexual (nZ20) was presented with the same female four times. 232 169 interest when presented with a close female relative of their The females were chosen randomly; they were virgin females 233 170 own previous mate. To confirm the importance of that had not encountered any males prior to the experiment. 234 171 cuticular extracts in male discrimination of females, we In his fifth encounter, each male was presented with a novel 235 172 observed male responses to females, whose cuticular unmated female to test for increased sexual interest. To 236 173 patterns we had manipulated, to be similar by applying assess possible effects of overall physical exhaustion of males 237 174 identical cuticular extracts. We examined the similarity of from the effort of mating on time to mating in successive 238 175 the cuticular pattern among relatives to find evidence that encounters, we established a control group in which males 239 176 genetic similarity between individuals is correlated with (nZ10) were presented with novel unmated females in all 240 177 similarity of cuticular patterns. five successive encounters. In an additional experiment, 241 178 males (nZ19) were presented with the same female in all 242 179 five successive encounters. 243 180 244 181 2. MATERIAL AND METHODS 245 182 (a) Origin and maintenance of experimental animals (d) Recognition of familiar females 246 183 Experimental animals were the first-generation offspring of (i) Female mating status 247 184 beetles collected from carrion-baited pitfall traps in the field The following experiments were meant to reveal the cues 248 185 in June and July 2006. The field site was a deciduous forest males use to recognize a previous mate. In a first experiment 249 186 near Freiburg in southwestern Germany (488000 N, we tested whether female mating status influences male 250 187 078510 E). Beetles were maintained in temperature-controlled time to mating. As in the above experiment, males 251 188 chambers at 208C under a 16 L : 8 D photoperiod. Groups of were presented with the same female four times. In a fifth 252 189 up to six same-sex siblings were kept in small transparent encounter we introduced either a novel virgin female (nZ17) 253 190 plastic containers (10!10!6 cm) with moist peat and were or a novel mated female (nZ20). Mated females were taken 254 191 fed freshly killed mealworms twice a week. All experimental from other trials in which they had all encountered a male 255 192 specimens were between 20 and 60 days of age. four times. 256

Proc. R. Soc. B

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9 ARTICLE IN PRESS Q1 Coolidge effect in burying beetle S. Steiger et al. 3

257 (ii) Self-referent cues versus female-specific cues some time to recover from the pentane application. In the 321 258 Based on our previous knowledge about the role of cuticular experimental group, each male (nZ12) was presented with 322 259 hydrocarbons in social interactions between individuals, it is the same extract-treated female four times. In the fifth 323 260 reasonable to assume that the recognition of mating partners encounter, a new unfamiliar female was introduced that had 324 261 is also mediated by these chemical cues. Therefore, we been treated with the same extract. In the control group, 325 262 carried out a second experiment to learn whether males males (nZ10) were presented with the same pentane-treated 326 263 transfer chemical substances from their own cuticle during female in the first four encounters and with a novel unfamiliar 327 264 mating and use self-referent cues to recognize females (as pentane-treated female in the fifth. 328 265 appears to be the case in crickets, where females initiate 329 266 matings and transfer cuticular chemicals, Ivy et al. 2005)or 330 (f ) Cuticular patterns of inbred families: 267 whether they learn their mates’ chemical signature (their 331 chemical analysis 268 individual hydrocarbon pattern). To obtain individuals that 332 Five brothers from each of the five different inbred families 269 were more similar genetically than full sibs (‘inbred’ 333 (families A, B, C, D and G, each of which resulted from a 270 individuals, inbreeding coefficient F Z0.25), we subjected 334 A different brother–sister pairing) and five sisters also from 271 F offspring of field-collected beetles to one generation of 335 1 each of the five different inbred families (families B, D, E, F 272 full-sib mating and allowed them to rear offspring. Individuals 336 and G) were killed by freezing at K278C for 15 min. They 273 in the resulting F were thus the product of brother–sister 337 2 were then thawed for 30 min at room temperature, placed 274 pairings, and siblings in this population were more closely 338 individually in flasks with 3 ml n-pentane (greater than 99%, 275 related than full sibs produced by unrelated parents 339 Fluka, Switzerland), and shaken for 15 min on an orbital 276 (coefficient of relatedness, rZ0.6 for our individuals and 340 shaker for extraction. The extract was then transferred to a 277 rZ0.5 for ordinary full sibs). As in the first experiment, each 341 clean vial and reduced by evaporation using a stream of 278 experimental male was presented with the same female in four 342 gaseous nitrogen until approximately 0.1 ml remained. 279 successive encounters. In the fifth encounter, one of the 343 Samples were quantified on an HP 6890 gas chromatograph 280 following types of females was introduced to the male: 344 with a split/splitless injector (3008C, automatic sampling, 281 (i) familiar females (nZ20) that had previously mated with 345 injection of 1 ml). We used a fused silica column (DB-1, 282 the same male, (ii) novel females (nZ20) that had previously 346 30 m!0.25 mm ID, 0.25 mm, J&W Scientific, Folsom, 283 mated with a different unrelated male, (iii) novel females 347 Canada) with a helium flow of 1 ml minK1. The oven 284 (nZ16) that had previously mated with the male’s inbred 348 temperature was programmed as follows: 2 min at 358C, to 285 brother, and (iv) novel females (nZ21) that were inbred 349 1008C at a rate of 208 minK1, to 3008Cat68C minK1, 25 min 286 sisters of the male’s original mate. To avoid any confounding 350 at 3008C. The flame ionization detector was run with 40 ml 287 effects of inbreeding avoidance, we never presented females 351 H minK1 and 450 ml air minK1. In a previous study (Steiger 288 to related males. If males use self-referent cues, their res- 2 352 et al. 2007), 88 peaks out of 91 regularly occurring peaks 289 ponse to females inseminated by their inbred brother should 353 were identified. For the current study, the 40 peaks with the 290 resemble their response to their own previous mate. If males 354 largest area were chosen for integration (see electronic 291 learn their mate’s chemical features, their response to their 355 supplementary material for chemical identity of the 40 292 previous mate’s inbred sister should resemble their response 356 peaks). One of the males (MC3) had to be excluded from 293 to the previous mate herself. 357 the analysis because the extract was contaminated. 294 358 295 (e) Masking of cuticular substances with 359 296 concentrated extracts (g) Statistical analyses 360 297 The experiment was designed to test the hypothesis that Statistical analyses were performed using SPSS v. 15. We 361 298 cuticular substances are involved in the recognition process. used a repeated measures ANOVA to compare the same 362 299 To this end, we attempted to experimentally manipulate male’s behaviour in successive matings and a one-way 363 300 female surface chemicals to create pairs of females with ANOVA to compare the behaviour of males from different 364 301 similar odour. We first extracted females individually in treatment groups in their fifth encounter with a female. To 365 302 pentane for 15 min. These females came from our inbred meet the criterion of homogeneity of variances, we log 366 303 group that resulted from full-sib matings (see previous transformed all data prior to analysis. 367 304 experiment). The extracts of four inbred sisters were For the analysis of chemical data, the total peak area of the 368 305 combined and their combined extract was completely 40 peaks of each individual was standardized to 100% and 369 306 reduced by evaporation under a stream of gaseous nitrogen multivariate analyses were performed. Because peak areas 370 307 and dissolved in 40 ml pentane. We applied 20 ml of the extract represent compositional data, they were transformed to 371 308 to each of the two live virgin females by spreading small logcontrasts (Aitchinson 1986). To assess the similarity of 372 309 droplets of it evenly over the elytra, pronotum and the the pattern of individuals within and between families, a 373 310 exposed part of the dorsal side of the abdomen. In effect, we cluster analysis was performed using the PAM procedure 374 311 applied a concentration of substances that should have been (partitioning around medoids; Kaufman & Rousseeuw 1990) 375 312 equivalent to amounts found on two females rather than one of the R package with the chord distance as the distance 376 313 to increase the probability that the experimental odour would index. The average silhouette width that provides an 377 314 conceal the females’ actual individual chemical pattern. Live evaluation of clustering validity was used to select an 378 315 females were used because males give up mating attempts appropriate number of clusters (Kaufman & Rousseeuw 379 316 when they experience difficulties with intromission of their 1990). In addition, a discriminant analysis (DA) was 380 317 aedeagus into the genital tract of dead females. To control for performed to determine whether females of different families 381 318 any effects of the solvent, a separate group of females was could be discriminated on the basis of their cuticular profile. 382 319 treated with 20 ml of pentane only. We started a trial 20 min To reduce the number of variables prior to the DA, we first 383 320 after applying the extracts to allow the experimental females performed a principal component analysis (PCA). 384

Proc. R. Soc. B

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9 ARTICLE IN PRESS 4 S. Steiger et al. Coolidge effect in burying beetle

385 250 250 449 386 450 387 451 a 388 200 200 452 389 453 390 454 391 150 b 150 455 392 456 393 a, b 457 100 100 394 a, b 458 time to mating (s) 395 time to mating (s) 459 396 b 460 50 a 50 b 397 a 461 398 462 399 463 0 0 400 first second third fourth fifth previous novel female sister of 464 female female mated to previous 401 order of presented females 465 402 brother female 466 Figure 1. Time to mating in males in sequential encounters 403 Figure 2. Time to mating in males with different females 467 with females. The first four encounters were between the 404 in their fifth mating encounter. Males were presented with one 468 same male–female pair, while the fifth encounter involved a 405 of the following females: (i) the previous mate (nZ20), (ii) a 469 novel female. Back-transformed meanGs.e. are presented. Z 406 novel female (n 20), (iii) an inbred brother’s previous mate 470 Different letters indicate a significant difference (repeated Z Z 407 (n 16), and (iv) the inbred sister of the previous mate (n 21). 471 measures ANOVA, within-subject contrasts (correction after Back-transformed meanGs.e. are presented. Different letters 408 Z ! 472 Bonferroni), n 18, p 0.05). indicate a significant difference ( post hoc Bonferroni, p!0.05). 409 473 410 474 411 3. RESULTS 475 (a) The effect of female novelty on male 412 250 476 413 mating behaviour 477 414 In the first experiment, time to mating varied significantly 478 415 with the number of encounters (repeated measures 200 479 416 ANOVA of log-transformed times, nZ18, d.f.Z4, 480 417 FZ6.31, p!0.001; figure 1). Time to mating increased 481 418 continuously over the course of the first four encounters 150 482 419 (with the same female), but only the pairwise comparisons 483 420 between the first and third or fourth exposure were 484 100 421 significant. When males were exposed to a novel virgin 485

422 female in the fifth encounter, they mated significantly time to mating (s) 486 423 faster than in the third or fourth exposure to the first 487 50 424 female and as fast as in their first encounter with a female 488 425 (figure 1). 489 426 Our results provided no indication that physical 0 490 427 exhaustion caused males to increase their time to mating female treated female treated 491 428 after multiple copulations. When males were exposed a with female extract with pentane 492 429 new virgin female each time, time to mating remained presented female 493 Z 430 short and did not differ between the encounters (n 9; Figure 3. Time to mating in males in their fifth mating 494 431 back-transformed mean and meanGs.e. for time to encounter. After four encounters with the same female, a 495 432 mating: 17.06, 20.96 and 13.89 s; repeated measures novel female was offered in the fifth. Familiar and novel 496 433 ANOVA, d.f.Z4, FZ0.88, pZ0.49). Most of these males females were either treated with the same female cuticular 497 434 mated with all five females (meanGs.e.: 4.4G0.2), extract (nZ12) or treated with pentane (nZ10). Back- 498 G 435 whereas males encountering the same female five times, transformed mean s.e. are presented. 499 436 copulated an average of three times (nZ19; meanGs.e.: 500 437 2.9G0.1), significantly less (t-test, tZ6.23, p!0.001). 501 438 (ii) Self-referent cues versus female-specific cues 502 439 Time to mating in a male’s fifth encounter was signifi- 503 440 (b) Recognition of familiar females cantly affected by features of the females he encountered 504 441 (i) Female mating status (ANOVA, d.f.Z3, FZ7.46, p!0.001; figure 2). Males 505 442 Female mating status had no influence on male mating took less time to mate with novel mated females than with 506 443 behaviour. Time to mating did not differ significantly their own previous mate, even when the female’s previous 507 444 between trials in which the novel female was a virgin or a mate was the male’s inbred brother. When males 508 445 mated female (back-transformed mean and meanGs.e. for encountered a close relative of their previous mate (inbred 509 446 time to mating; virgin female (nZ16): 32.13, 45.22, sister), time to mating was intermediate and not signi- 510 447 22.82 s; mated female (nZ19): 23.64, 31.31, 17.85 s; ficantly different from those observed with novel females or 511 448 t-test, tZ0.70, pZ0.49). previous mates (figure 2). 512

Proc. R. Soc. B

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9 ARTICLE IN PRESS Q1 Coolidge effect in burying beetle S. Steiger et al. 5

513 577 514 cluster 6 578 515 579 males females cluster 5 516 580 517 cluster 4 581 518 582 519 cluster 3 583 520 584 FF1 FF2 FF3 FF4 FF5 FE1 FE2 FE3 FE4 FE5 FB1 FB2 FB3 FB4 FB5 521 FD1 FD2 FD3 FD4 FD5 FG1 FG2 FG3 FG4 FG5 cluster 2 585 MB1 MB2 MB3 MB4 MB5 MC1 MC2 MC4 MC5 MA1 MA2 MA3 MA4 MA5 MD1 MD2 MD3 MD4 MD5 MG1 MG2 MG3 MG4 MG5 522 586 523 cluster 1 587 524 588 525 Figure 4. Visual representation of the cluster analysis (PAM) based on 40 cuticular substances of individuals from different 589 526 families. Different colours represent different clusters. (M, males; F, females; A–G, different families; 1–5, different individuals 590 of a family). 527 591 528 592 (c) Masking of cuticular substances with 6 529 concentrated extracts 593 530 5 594 When males were presented in a fifth encounter with a 4 531 female treated with the same surface extract as the female 595 532 3 596 with whom the males had interacted previously, males 2 533 took significantly longer to mate than the males of the 597 534 1 598 control group (t-test, tZ2.77, pZ0.016; figure 3). This 535 0 599 difference was not due to an overall reduction in the 536 –1 600

attractiveness of females treated with female cuticular function 2 (23.6 %) 537 –2 601 extract: in the first mating encounter, there was no 538 –3 602 difference between the time to mating in the treatment 539 –4 603 and control (back-transformed mean and meanGs.e. for –6 –4 –2 0 2468 540 604 time to mating; female with surface extract (nZ12): function 1 (60.3 %) 541 605 25.74, 33.98, 19.50 s; female with pentane (nZ10): 542 Figure 5. DA based on the relative amount of cuticular 606 31.60, 46.09, 21.67 s; t-test, tZK0.45, pZ0.66). 543 hydrocarbons of five N. vespilloides families (each with 5 607 females). Envelopes represent 95% confidence ellipses. Filled 544 608 squares, family B; filled circles, family D; triangles, family E; 545 (d) Cuticular patterns of inbred families: 609 open squares, family F; open circles, family G. 546 chemical analysis 610 547 The average silhouette width was maximal when six In a cross validation (leave-one-out cross validation), the 611 548 clusters were produced. The visual representation of the value of correct classifications was reduced to 68.0%, but 612 549 six clusters reveals three features of the cuticular pattern this compares to a value of 20% correct classification 613 550 (figure 4): male and female beetles were well separated and expected by chance. 614 551 fell into different clusters (with the exception of one 615 552 female, FF2). Males were assigned to only two different 616 553 clusters, females to five, suggesting greater variation of 4. DISCUSSION 617 554 cuticular composition among females than males. This The results of our study provide unambiguous evidence 618 555 result was supported by the Nei indices for male and for the Coolidge effect in burying beetles N. vespilloides. 619 556 female substances, which showed that male chemical Male sexual interest declined over the course of several 620 557 profiles were more similar than females (all possible repeat encounters with the same female and was renewed 621 558 combinations were calculated within one sex: meanG when males encountered a novel female. This effect 622 559 s.d.; male: 0.94G0.04; female: 0.89G0.10; t-test, was not caused by a preference for virgin females: 623 560 d.f.Z574, tZ7.31, p!0.001). In addition, the five female male responses to novel mated and novel virgin females 624 561 clusters were not equivalent to the five families. However, were similar. 625 562 at least three sisters of each family fell into the same Our experimental manipulation of female chemical 626 563 cluster. To examine if the female families can be separated features documents the role of cuticular substances in the 627 564 on the basis of the cuticular pattern, a DA was performed. discrimination between familiar and novel mates. When a 628 565 Prior to DA a PCA was used to reduce the number of male encountered a novel female that had been coated 629 566 variables (40 substances). This produced eight principal with the same cuticular substances as his previous mate, 630 567 components with eigenvalues of more than 1, explaining he acted as if this female was his previous mate. 631 568 91.75% of the total variance. The DA performed on the We could also show that males do not simply leave some 632 569 eight principal components significantly differentiated the of their own substances on the female to mark them as 633 570 chemical profiles of the female deriving from different Q3 previous mates. Males did not treat females mated by their 634 2 571 families (Wilks’ l!0.015, c32Z73.13, p!0.001). Three inbred brothers as different from virgin females or novel 635 572 discriminant functions added significantly to the discrimi- females mated by unrelated males. The application of 636 573 nation between groups, with the first explaining 60.3%, extracts suggested that males instead learn their mates’ 637 574 the second 23.6% and the third 9.1% of the total variation individual specific cues during mating and discriminate 638 575 (the first two functions are shown in figure 5). Most against similar scents in their subsequent mating behaviour. 639 576 females (88.0%) were correctly assigned to their groups. Consistent with this interpretation, male responses to their 640

Proc. R. Soc. B

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9 ARTICLE IN PRESS 6 S. Steiger et al. Coolidge effect in burying beetle

641 previous mate’s close relative (inbred sister) were inter- conditions, kept in the same containers and substrate and 705 642 mediate to their response to their previous mate and novel fed the same diet. 706 643 females. This indicates that inbred sisters were similar but Our results provide the first clear demonstration of the 707 644 still somewhat different from each other in their cuticular Coolidge effect and definitive evidence of its underlying 708 645 cues, such that males could sometimes but not always mechanism in an insect. In burying beetles, their complex 709 646 recognize them as different individuals. This interpretation social lives may have been selected for the ability to 710 647 is compatible with the result of our chemical analysis: some discriminate between individuals, which may have facili- 711 648 females from the same family were assigned to the same tated the Coolidge effect. Generally, the risk of re-mating 712 649 cluster, while others were not, which means that at least with a previous mate will increase if a male has continued 713 650 some of the inbred sisters were more similar to each other access to the same female, and preferring alternative mates 714 651 than to females from other inbred families. The result of the will only benefit a male if such alternatives are actually 715 652 cluster analysis was also consistent with that of the DA, available. Thus, we would expect the Coolidge effect only 716 653 in which approximately 70% of the individuals were if male–female associations are somewhat stable in time 717 654 correctly assigned to their family. and space, and if there is some clumping of females. The 718 655 Cuticular substances play a fundamental role in insect carcass as an essential, but rare, ephemeral and unpre- 719 656 communication, especially in recognition systems (Singer dictable resource required for feeding and reproduction 720 657 1998; Howard & Blomquist 2005). Many species studied can temporarily cause such clumping (Pukowski 1933). 721 658 to date have complex chemical profiles that could provide On any carcass, suitable for reproduction or not, potential 722 659 cues for individual discrimination. In N. vespilloides,at mates may be available for a limited time only. The losers 723 660 least 91 major substances contribute to cuticular pattern of aggressive interactions on carcasses, subordinate males 724 661 (Steiger et al. 2007). If males can distinguish only two (Bartlett 1988) and females (Mu¨ller et al. 1990), leave the 725 662 concentrations of each compound, there are 291 possible carcass early (Bartlett 1988;Mu¨ller et al. 1990, 2007; 726 663 combinations. We do not have any information about Scott & Williams 1993). Male or female intruders may 727 664 male abilities to differentiate between concentrations of leave quickly after unsuccessful attempts to take over the 728 665 individual compounds, but it appears that burying beetle carcass from the original residents (Trumbo 1990). In 729 666 cuticular patterns could easily contain sufficient infor- both the situations, males may have limited opportunities 730 667 mation to allow for discrimination between individuals. to mate with particular females and may benefit from 731 668 Many mammals use scent to distinguish between spreading sperm evenly. 732 669 conspecific individuals (see references in Thom & Hurst The Coolidge effect wears off quickly, and is non- 733 670 2004) and so do a number of crustaceans (see references existent approximately 30 min after an initial mating ( J. K. 734 671 in Gherardi & Tiedemann 2004) and insects (Barrows Q4 Mu¨ller, unpublished data). Our experiments were not 735 672 1975; Breed 1981; D’Ettorre & Heinze 2005; Widemo affected by this short duration because males re-encoun- 736 673 2006). In insects, the role of cuticular substances, tering the same female always did so within 5–10 min after 737 674 specifically hydrocarbons, has frequently been inferred their previous contact. If the loss of sexual interest was of 738 675 based on inter-individual or inter-colony variation. longer duration, it could potentially interfere with 739 676 Providing definitive evidence of the role of cuticular frequent matings used by males to maximize their 740 677 substances in mate or colony member recognition requires paternity on carcasses. Dominant males increase their 741 678 experimental manipulations, such as stripping dead paternity with the dominant female through repeated 742 679 individuals of cuticular substances and reapplying extracts matings during carcass burial and preparation, approxi- 743 680 to abolish and restore specific response behaviours (e.g. mately 70 during the first 24 hours (Mu¨ller & Eggert 744 681 Wedell & Tregenza 1997), treating dummies with extracts 1989). The short-term loss of male sexual interest in his 745 682 (Akino et al. 2004) or applying extracts, fractions of previous mate may actually function in part to allow him 746 683 extracts or solid-phase cuticular hydrocarbons from one to space out matings evenly during the oviposition period 747 684 individual to another live individual to manipulate the to allow for optimal fertilization success. 748 685 response to the second individual (Lahav et al. 1999; Male N. vespilloides perceive individual differences in 749 686 Torres et al. 2007). In the context of discrimination the cuticular signatures of individual females and use them 750 687 between individuals, direct application of concentrated to discriminate between familiar and unfamiliar females. 751 688 cuticular extracts in order to mask cuticular compositions This constitutes individual recognition sensu Beecher & 752 689 (as in this study) can be a useful method to provide Bekoff (1981) and Dale et al. (2001) because each female 753 690 unequivocal evidence of the role of cuticular substances. individual in our population can be discriminated from 754 691 A study of the responsiveness of male bees (Lasioglossum every other individual on the basis of a unique set of cues. 755 692 zephyrum) to novel females after an initial 10 min encounter However, although males use individual specific cuticular 756 693 with a first female showed that as the relationship between information, they do so simply to discriminate between 757 694 two females increased, males increasingly failed to dis- two ‘classes’ (Tibbetts & Dale 2007) or ‘heterogeneous 758 695 tinguish them, indicating that the cues produced by females subgroups’ (Barrows et al. 1975) of individuals, familiar 759 696 have a genetic basis (Smith 1983). Similarly, this study females and unfamiliar females. This has led some authors 760 697 suggests that males may be confused about female identity to classify systems like this as cases of ‘binary discrimi- 761 698 when encountering close relatives (inbred sisters) of their Q5 nation’ (Gherardi & Thienemann 2004) instead of true 762 699 original mate, which also suggests that genetic effects are individual recognition. 763 700 important. Diet and other environmental factors can Interestingly, our study revealed higher variation in the 764 701 significantly affect chemical cues produced by animals chemical composition of the female than the male cuticle. 765 702 (Thom & Hurst 2004), but in our study, there was little This suggests that females may be under stronger selection 766 703 to no variation in environment or diet, since all females in for individual distinctiveness. In Polistes wasps, there is 767 704 the study had been reared under identical laboratory evidence that complex social behaviour can select for 768

Proc. R. Soc. B

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9 ARTICLE IN PRESS Q1 Coolidge effect in burying beetle S. Steiger et al. 7

769 variation in traits used in individual recognition (Tibbetts Dale, J., Lank, D. B. & Reeve, H. K. 2001 Signaling 833 770 2004) and similarly, signals of individual identity may individual identity versus quality: a model and case studies 834 771 facilitate stable joint-breeding associations of burying with ruffs, queleas, and house finches. Am. Nat. 158, 835 772 beetles on carcasses, which are more common in females 75–86. (doi:10.1086/320861) 836 773 than males (Mu¨ller et al. 2007). Being individually D’Ettorre, P. & Heinze, J. 2005 Individual recognition in ant 837 774 recognizable may also benefit female burying beetles in queens. Curr. Biol. 15, 2170–2174. (doi:10.1016/j.cub. 838 2005.10.067) 775 the context of mating. Female reproduction depends on 839 Dewsbury, D. A. 1981 Effects of novelty on copulatory 776 the amount of fertile sperm they have available for 840 behavior: the Coolidge effect and related phenomena. 777 841 fertilization, and sperm degenerate after prolonged Psychol. Bull. 89, 464–482. (doi:10.1037/0033-2909. 778 storage in the spermatheca (Eggert 1992). When 89.3.464) 842 779 encounters between a particular male and female are Dewsbury, D. A. 1982 Ejaculate cost and male choice. 843 780 brief or infrequent, an even distribution of male sperm Am. Nat. 119, 601–610. (doi:10.1086/283938) 844 781 through the Coolidge effect may benefit females because it Donovan, A. & Verrell, P. A. 1991 The effect of partner 845 782 increases the probability that they receive sufficient fertile familiarity on courtship success in the salamander Desmog- 846 783 sperm to ensure fertilization of their egg clutch. nathus ochrophaeus. J. Herpetol. 25,93–95.(doi:10.2307/ 847 784 1564800) 848 We thank Martin Woywood for helpful assistance with R 785 Eggert, A.-K. 1990 Chemische Kommunikation beim 849 and cluster analysis. We are grateful to Volker Nehring for Totengra¨ber Necrophorus vespilloides Herbst (Coleoptera: 786 stimulating discussion and Allen J. Moore and Patrizia 850 Silphidae): Pheromonabgabe als alternative Fortpflan- 787 d’Ettorre for their helpful comments on the manuscript. We 851 788 also thank Laura Fillinger, Irena Ivankovic and Corinna zungstaktik der Ma¨nnchen. PhD thesis, University of 852 789 Mummelthei for preliminary experiments and Klaus Peschke Bielefeld, Bielefeld, Germany. 853 790 for research support. The study was supported by a PhD Eggert, A.-K. 1992 Alternative male mate-finding tactics in 854 burying beetles. Behav. Ecol. 3, 243–254. (doi:10.1093/ 791 grant from the German National Academic Foundation to 855 beheco/3.3.243) 792 S.S. and by a grant from the Deutsche Forschungs- 856 gemeinschaft to J.K.M. (Mu 1175/3-1). Eggert, A.-K. & Mu¨ller, J. K. 1989 Mating success of 793 857 pheromone-emitting Necrophorus males: do attracted 794 females discriminate against resource-owners? Behaviour 858 795 110, 248–259. (doi:10.1163/156853989X00493) 859 796 REFERENCES Gherardi, F. & Tiedemann, J. 2004 Chemical cues and 860 797 Aitchinson, J. 1986 The statistical analysis of compositional data: binary individual recognition in the hermit crab Pagurus 861 798 monographs in statistics and applied probability. London, longicarpus. J. Zool. Lond. 263, 23–29. (doi:10.1017/ 862 799 UK: Chapman and Hall. S0952836904004807) 863 800 Akino, T., Yamamura, K., Wakamura, S. & Yamaoka, R. Happ, G. M. 1969 Multiple sex pheromones of mealworm 864 801 2004 Direct behavioral evidence for hydrocarbons as beetle Tenebrio molitor L. Nature 222, 180–181. (doi:10. 865 nestmate recognition cues in Formica japonica (Hymenop- 802 1038/222180a0) 866 tera: Formicidae). Appl. Entomol. Zool. 39, 381–387. 803 Howard, R. W. & Blomquist, G. J. 2005 Ecological, 867 (doi:10.1303/aez.2004.381) 804 behavioral, and biochemical aspects of insect hydrocar- 868 Andersson, J., Borg-Karlson, A. K. & Wiklund, C. 2003 bons. Annu. Rev. Entomol. 50, 371–393. (doi:10.1146/ 805 Antiaphrodisiacs in pierid butterflies: a theme with 869 annurev.ento.50.071803.130359) 806 variation!. J. Chem. Ecol. 29, 1489–1499. (doi:10.1023/ 870 Ivy, T. M., Weddle, C. B. & Sakaluk, S. K. 2005 Females 807 A:1024277823101) 871 use self-referent cues to avoid mating with previous 808 Arnaud, L. & Haubruge, E. 1999 Mating behaviour and male 872 mates. Proc. R. Soc. B 272, 2475–2478. (doi:10.1098/ 809 mate choice in Tribolium castaneum (Coleoptera, Teneb- 873 rspb.2005.3222) rionidae). Behaviour 136, 67–77. (doi:10.1163/156853 810 Kaufman, L. & Rousseeuw, P. J. 1990 Finding groups in data: 874 999500677) 811 an introduction to cluster analysis. New York, NY: Wiley. 875 Barrows, E. M. 1975 Individually distinctive odors in an 812 Kelley, J. L., Graves, J. A. & Magurran, A. E. 1999 876 813 invertebrate. Behav. Biol. 15, 57–64. (doi:10.1016/S0091- 877 6773(75)92079-9) Familiarity breeds contempt in guppies: male guppy 814 878 Barrows, E. M., Bell, W. J. & Michener, C. D. 1975 fish increase their reproductive success by mating with 815 879 Individual odor differences and their social function in unfamiliar females. Nature 401, 661–662. (doi:10.1038/ 816 insects. Proc. Natl Acad. Sci. USA 7, 2824–2828. (doi:10. 44314) 880 817 1073/pnas.72.7.2824) Koene, J. M. & Ter Maat, A. 2007 Coolidge effect in pond 881 818 Bartlett, J. 1988 Male mating success and paternal care in snails: male motivation in a simultaneous hermaphrodite. 882 819 Nicrophorus vespilloides (Coleoptera: Silphidae). Behav. BMC Evol. Biol. 7, 212. (doi:10.1186/1471-2148-7-212) 883 820 Ecol. Sociobiol. 23, 297–304. (doi:10.1007/BF00300576) Kukuk, P. 1985 Evidence for an antiaphrodisiac in the sweat 884 821 Beach, F. A. & Jordan, L. 1956 Sexual exhaustion and bee Lasioglossum (Dialictus) zephyrum. Science 227, 885 822 recovery in the male rat. Quart. J. Exp. Psychol. 8, 656–657. (doi:10.1126/science.3969557) 886 Lahav, S., Soroker, V. & Hefetz, A. 1999 Direct behavioral 823 121–133. (doi:10.1080/17470215608416811) 887 evidence for hydrocarbons as ant recognition discrimina- 824 Beecher, M. D. & Bekoff, M. 1981 Individual recognition and 888 tors. Naturwissenschaften 86, 246–249. (doi:10.1007/ 825 social relationships. J. Theor. Biol. 88, 589–593. (doi:10. 889 1016/0022-5193(81)90285-X) s001140050609) 826 890 Breed, M. D. 1981 Individual recognition and learning of Mu¨ller, J. K. & Eggert, A.-K. 1987 Effects of carrion- 827 891 queen odors by worker honeybees. Proc. Natl Acad. Sci. independent pheromone emission by male burying beetles 828 USA 78, 2635–2637. (doi:10.1073/pnas.78.4.2635) (Silphidae: Necrophorus). Ethology 76, 297–304. 892 829 Carlson, D. A. & Schlein, Y. 1991 Unusual polymethyl Mu¨ller, J. K. & Eggert, A.-K. 1989 Paternity assurance by 893 830 alkenes in tsetse-flies acting as abstinon in Glossina helpful males: adaptations to sperm competition in 894 831 morsitans. J. Chem. Ecol. 17, 267–284. (doi:10.1007/ burying beetles. Behav. Ecol. Sociobiol. 24, 245–249. 895 832 BF00994332) (doi:10.1007/BF00295204) 896

Proc. R. Soc. B

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9 ARTICLE IN PRESS 8 S. Steiger et al. Coolidge effect in burying beetle

897 Mu¨ller, J. K., Eggert, A.-K. & Dressel, J. 1990 Intraspecific Thom, M. D. & Hurst, J. L. 2004 Individual recognition by 961 898 brood parasitism in the burying beetle Necrophorus scent. Ann. Zool. Fenn. 41, 765–787. 962 899 vespilloides (Coleoptera: Silphidae). Anim. Behav. 40, Tibbetts, E. A. 2002 Visual signals of individual identity in 963 900 491–499. (doi:10.1016/S0003-3472(05)80529-9) the wasp Polistes fuscatus. Proc. R. Soc. B 269, 1423–1428. 964 Mu¨ller, J. K., Eggert, A.-K. & Elsner, T. 2003 Nestmate 901 (doi:10.1098/rspb.2002.2031) 965 recognition in burying beetles: the “breeder’s badge” as a Tibbetts, E. A. 2004 Complex social behaviour can select 902 966 cue used by females to distinguish their mates from male for variability in visual features: a case study in Polistes 903 967 intruders. Behav. Ecol. 14, 212–220. (doi:10.1093/beheco/ wasps. Proc. R. Soc. B 271, 1955–1960. (doi:10.1098/rspb. 904 14.2.212) 2004.2784) 968 905 Mu¨ller, J. K., Braunisch, V., Hwang, W.& Eggert, A.-K. 2007 Tibbetts, E. A. & Dale, J. 2007 Individual recognition: it is 969 906 Alternative tactics and individual reproductive success in good to be different. Trends Ecol. Evol. 22, 529–537. 970 907 natural associations of the burying beetle, Nicrophorus (doi:10.1016/j.tree.2007.09.001) 971 908 vespilloides. Behav. Ecol. 18, 196–203. (doi:10.1093/ Tokarz, R. R. 1992 Male mating preference for unfamiliar 972 909 beheco/arl073) females in the lizard, Anolis sagrei. Anim. Behav. 44, 973 ¨ 910 Odeen, A. & Moray, C. M. 2008 Drosophila melanogaster 843–849. (doi:10.1016/S0003-3472(05)80580-9) 974 911 virgins are more likely to mate with strangers than familiar Torres, C. W., Brandt, M. & Tsutsui, N. D. 2007 The role of 975 flies. Naturwissenschaften 95, 253–256. (doi:10.1007/ 912 cuticular hydrocarbons as chemical cues for nestmate 976 s00114-007-0314-3) 913 recognition in the invasive Argentine ant (Linepithema 977 Olsson, M., Madsen, T. & Shine, R. 1997 Is sperm really so humile). Insect. Soc. 54, 363–373. (doi:10.1007/s00040- 914 cheap? Costs of reproduction in male adders, Vipera berus. 978 915 007-0954-5) 979 Proc. R. Soc. B 264, 455–459. (doi:10.1098/rspb.1997. Trumbo, S. T. 1990 Reproductive benefits of infanticide in a 916 0065) 980 biparental burying beetle Nicrophorus orbicollis. Behav. 917 Peschke, K. 1987 Male-aggression, female mimicry and 981 Ecol. Sociobiol. 27, 269–274. (doi:10.1007/BF00164899) female choice in the rove beetle, Aleochara curtula 918 Van Voorhies, W. A. 1992 Production of sperm reduces 982 (Coleoptera, Staphylinidae). Ethology 75, 265–284. 919 nematode life-span. Nature 360, 456–458. (doi:10.1038/ 983 Pizzari, T., Cornwallis, C. K., Lovlie, H., Jakobsson, S. & 920 360456a0) 984 Birkhead, T. R. 2003 Sophisticated sperm allocation in 921 Wcislo, W. T. 1992 Attraction and learning in mate-finding 985 male fowl. Nature 426, 70–74. (doi:10.1038/nature02004) 922 by solitary bees, Lasioglossum (Dialictus) figueresi Wcislo 986 Preston, B. T., Stevenson, I. R., Pemberton, J. M. & Wilson, 923 987 K. 2001 Dominant rams lose out by sperm depletion. and Nomia triangulifera Vachal (Hymenoptera: Halicti- 924 Nature 409, 681–682. (doi:10.1038/35055617) dae). Behav. Ecol. Sociobiol. 31, 139–148. (doi:10.1007/ 988 925 Pukowski, E. 1933 O¨ kologische Untersuchungen an BF00166347) 989 926 Necrophorus F. Z. Morphol. O¨ kol. Tiere 27, 518–586. Wedell, N. & Tregenza, T. 1997 Definitive evidence for 990 927 (doi:10.1007/BF00403155) cuticular pheromones in a cricket. Anim. Behav. 54, 991 928 Scott, M. P. & Williams, S. M. 1993 Comparative 979–984. (doi:10.1006/anbe.1997.0500) 992 Wedell, N., Gage, M. J. G. & Parker, G. A. 2002 Sperm 929 reproductive success of communally breeding burying 993 competition, male prudence and sperm-limited females. 930 beetles as assessed by PCR with randomly amplified 994 Trends Ecol. Evol. 17, 313–320. (doi:10.1016/S0169- 931 polymorphic DNA. Proc. Natl Acad. Sci. USA 90, 995 5347(02)02533-8) 932 2242–2245. (doi:10.1073/pnas.90.6.2242) 996 Singer, T. L. 1998 Roles of hydrocarbons in the recognition Whitlow, S. 2003 Recognition in burying beetles (Nicrophorus 933 997 systems of insects. Am. Zool. 38, 394–405. spp., Silphidae, Coleoptera). PhD thesis, University of 934 Smith, B. H. 1983 Recognition of female kin by male bees Freiburg, Freiburg, Germany. 998 935 through olfactory signals. Proc. Natl Acad. Sci. USA 80, Widemo, F. 2006 Male–male pheromone signalling in a 999 936 4551–4553. (doi:10.1073/pnas.80.14.4551) lekking Drosophila. Proc. R. Soc. B 273, 713–717. (doi:10. 1000 937 Steiger, S., Peschke, K., Francke, W. & Mu¨ller, J. K. 2007 1098/rspb.2005.3379) 1001 938 The smell of parents: breeding status influences cuticular Wilson, J. R., Kuehn, R. E. & Beach, F. A. 1963 Modification 1002 939 hydrocarbon pattern in the burying beetle Nicrophorus in sexual behavior of male rats produced by changing 1003 940 vespilloides. Proc. R. Soc. B 274, 2211–2220. (doi:10.1098/ stimulus female. J. Comp. Physiol. Psychol. 56, 636–644. 1004 941 rspb.2007.0656) (doi:10.1037/h0042469) 1005 942 1006 943 1007 944 1008 945 1009 946 1010 947 1011 948 1012 949 1013 950 1014 951 1015 952 1016 953 1017 954 1018 955 1019 956 1020 957 1021 958 1022 959 1023 960 1024

Proc. R. Soc. B

RSPB 20080375—30/4/2008—12:51—CHANDRAN—299268—XML – pp. 1–9