Ethology

Interactions Between Multiple Forms of Nuptial Feeding in the Wood sylvestris (Bosc): Dual Spermatophores and Male Forewings Pavol Prokop*, & Michael R. Maxwellà

* Department of Biology, University of Trnava, Trnava, Slovakia Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia à Department of Mathematics and Natural Sciences, National University, La Jolla, CA, USA

Correspondence Abstract Michael R. Maxwell, Department of Mathematics and Natural Sciences, National Nuptial feeding is widespread in , with many species showing one University, 11255 North Torrey Pines Road, form of feeding. In the wood cricket , the male may La Jolla, CA 92037, USA. provide multiple forms of feeding during an encounter: two kinds of E-mail: [email protected] edible spermatophores (microspermatophore and macrospermatophore) and forewing secretions. We examined the roles and interactions of the Received: August 16, 2007 spermatophores and forewing exposure in the mating sequence of this Initial acceptance: January 14, 2008 Final acceptance: July 4, 2008 species. The small microspermatophore was not found to contain sperm, (S. Forbes) whereas the larger macrospermatophore contained sperm. In mating tri- als, the microspermatophore may be transferred to the female early in doi: 10.1111/j.1439-0310.2008.01562.x the trial. Transfer of the microspermatophore was not a necessary prere- quisite to the subsequent transfer of one or more sperm-filled macro- spermatophores. Forewing exposure increased male mating success, as males with exposed forewings were more successful in transferring the macrospermatophore than males with experimentally covered fore- wings, both in terms of occurrence of successful transfer and the num- ber of macrospermatophores transferred. Male mating success was very low when the male’s forewings were covered and when the male did not transfer a microspermatophore. The sperm-filled macrospermato- phore may have nutritional value, as females eventually consumed all transferred macrospermatophores, and males consumed all rejected mac- rospermatophores. Somewhat unexpectedly, this study casts doubt on the role of the forewings in nuptial feeding. Although males with exposed forewings were more successful in macrospermatophore trans- fer, females actually palpated these males’ forewings less. We posit the alternative hypothesis that the forewing secretions play a role in chemi- cal communication to the female (e.g., signaling male quality), possibly instead of female nourishment.

male, glandular secretions by the male, edible sper- Introduction matophores, and even the male’s body parts Nuptial feeding, a male’s provision of nourishment (reviewed in Gwynne 1997; Vahed 1998, 2007). to his mate, occurs in many (Thornhill & Research over the past few decades points to male Alcock 1983; Zeh & Smith 1985; Parker & Simmons mating benefits via nuptial feeding, as well as 1989; Simmons & Parker 1989; Boggs 1995; Vahed potential male paternal investment and reproductive 2007). This nourishment has been observed in vari- benefits and costs to the female (Gwynne 1997; ous forms including food items captured by the Vahed 1998, 2007).

Ethology 114 (2008) 1173–1182 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 1173 Nuptial Feeding in the Wood Cricket P. Prokop & M. R. Maxwell

For many species, males provide a single form of opening. The female may eat the microspermato- nuptial feeding (Gwynne 1997; Vahed 1998). In phore, and then palpate liquid secretions on the dor- some insects, however, males offer multiple modes sal surface of the male’s right forewing (tegmen), of nuptial feeding. On the one hand, males may which overlaps and covers the left forewing when present different gift items to successive females. the male is not stridulating. This behavior is similar Male scorpionflies, for example, may offer either to female palpations of male metanotal secretions in dead prey or salivary secretions to a other gryllids (e.g., Ono et al. 1995; Brown 1997; female (reviewed in Sauer et al. 1998). Alterna- reviewed in Gwynne 1997). After palpations, the tively, a male may provide two or more forms of female may remount the male, who then passes a feeding during an encounter with a female. Exam- larger macrospermatophore to the female’s genital ples include the sagebrush cricket Cyphoderris strepi- opening. The macrospermatophore is approx. 1 mm tans, wherein the female may consume the in diameter, being 3· wider than the microspermato- spermatophylax (gelatinous component of the sper- phore (Gabbutt 1954; Campan & Demai 1983). The matophore) as well as portions of the male’s hind female typically eats the macrospermatophore after wings (Dodson et al. 1983; Eggert & Sakaluk 1994; it has been attached to her genital opening for a per- Gwynne 1997). Similarly, a female of the striped iod. The male may pass multiple macrospermato- ground cricket Allonemobius fasciatus may eat the phores to the female, and the female may perform spermatophore in addition to feeding on a glandular multiple bouts of palpations on the male’s forewing. spur on the male’s hind tibia (Mays 1971; Bidochka Interactions between the transfer of the dual sper- & Snedden 1985). matophores and the forewing secretions remain Multiple forms of nuptial feeding within a species unclear in this species, as well as their influences on raise intriguing questions. The male faces decisions male mating success. We address these questions regarding energy and nutrient allocation between experimentally. First, we examine the roles of the the different forms. For the female, multiple offer- microspermatophore and macrospermatophore. We ings broaden information relevant to mate choice, ask whether both contain sperm, as spermless micro- while simultaneously broadening the arena for sex- spermatophores have been found in the gryllids ual conflict regarding male investment and poten- Laupala spp. (Shaw & Khine 2004; deCarvalho & tial manipulation (Arnqvist & Rowe 2005; Vahed Shaw 2005). Through mating trials, we ask whether 2007). Nevertheless, the interaction between differ- successful transfer of the microspermatophore is ent modes of nuptial feeding has received relatively necessary for successful transfer of the macrosperma- little attention. In this study, we examine the roles tophore, as suggested by Gabbutt (1954) and Mays of the forewings and edible spermatophores in the (1971). Second, we examine the influence of expo- mating behavior of the wood cricket Nemobius syl- sure of the male’s forewings on mating success. We vestris.InN. sylvestris, females palpate secretions on experimentally cover the forewings of males, and the males’ forewings (Richards 1952, 1953; Gabbutt ask how this treatment affects mating success. 1954). This species is also notable for having two kinds of spermatophores: small ‘microspermato- Methods phores’ and larger ‘macrospermatophores.’ Such dual spermatophores have been recently studied in Rearing and Mating Trial Protocol other gryllids (e.g., Shaw & Khine 2004; deCa- rvalho & Shaw 2005). In early July 2006, subadult wood crickets were col- The mating sequence of N. sylvestris is complex, lected from leaf litter in mixed -pine woodland consisting of stridulation by the male, the passage of near Trnava, Slovakia (4837¢ N and 1758¢ E). two kinds of edible spermatophores to the female, Crickets were briefly anaesthetized with CO2, sexed, and the female’s palpations of the male’s forewing and group-reared in single-sex containers housed at secretions (Richards 1952, 1953; Gabbutt 1954; Mays room temperature (approx. 20C) and exposed to 1971; Campan & Demai 1983; Dombrowski & Dam- natural photoperiod. Crickets were fed ad libitum bach 1994). After attraction via male stridulation, with crushed dog food, oat flakes, fresh fruit, and tactile exchange occurs between the sexes including dry Daphnia sp. Each rearing container contained antennation of the male by the female (Gabbutt pieces of paper to serve as concealment, and several 1954; Campan & Demai 1983). If the female is water reservoirs consisting of wet cotton placed in receptive, the male typically passes a small micro- Petri dishes. Crickets were checked daily for adult spermatophore, affixing it to the female’s genital molting. New adults were identified as belonging to

Ethology 114 (2008) 1173–1182 ª 2008 The Authors 1174 Journal compilation ª 2008 Blackwell Verlag, Berlin P. Prokop & M. R. Maxwell Nuptial Feeding in the Wood Cricket subweekly cohorts by means of white paint marked water to create a diluted sperm solution (Laird et al. on the legs. Paint patterns changed every 4–5 d, so 2004). The entire glass slide was searched under a adult age is known to 4–5 d. New adults were main- compound microscope (400· total magnification) for tained in the single-sex containers. the presence of sperm cells. The presence of sperm Virgin adults were used in mating trials at 8–15 d cells was checked for macrospermatophores using post-molting. Mating trials were conducted between similar methods in 10 trials. To get macrospermato-

19 and 28 July 2006. To control for potential diur- phores, males were anaesthetized with CO2 at the nal effects on spermatophore production (e.g., appearance of the trial’s first (five trials), second deCarvalho & Shaw 2005), each mating trial started (three trials) or third (two trials) macrospermato- at 10:00 and finished at 16:00. Each mating trial phore on the surface of the male’s genitals. Each consisted of a male and female, paired in a glass macrospermatophore was removed with fine twee- mating arena (15 · 8 · 15 cm) with a circular zers and examined under the microscope as opening at the top covered with fine mesh. Each described above. arena contained fresh paper on the bottom, a fresh moist cotton wool and fresh fruit (Prunus sp.). Forewing Manipulations Mating pairs were observed continuously by the experimenter (P. Prokop) for 6 h, and their behav- We conducted 92 trials to examine the effect of ior was recorded. Overall, 107 mating trials were covering the forewing secretions on mating behav- conducted: 15 for inspection of spermatophore con- ior. At each trial, we weighed the virgin male and tent and 92 for experimental manipulation of the female (to 0.001 g), anaesthetized them with CO2, males’ forewings. Ten mating trials were typically and measured their pronotum width with digital conducted simultaneously, with the experimenter calipers (to 0.01 mm). Each male was then ran- observing the trials and recording the occurrence domly assigned to one of the three treatments: con- (to 1 min) of the following behaviors: female trol (n = 28), wing (n = 35), and pronotum mounting and palpation of males’ forewings, pro- (n = 29). Males in the control treatment were left duction, transfer, attachment, and consumption of intact. For males in the wing treatment, we spermatophores. In contrast to gland-feeding crick- brushed water-based wax (distributed by AV TRAD- ets (e.g., Bidochka & Snedden 1985; Brown 1997; ING, Vra´ble, Slovakia) onto the dorsal surface of Fedorka & Mousseau 2002), palpations by female the right forewing. The wax is a liquid mixed with N. sylvestris typically last for a few seconds (Gabbutt water; after application, the water quickly evapo- 1954; Mays 1971). We, therefore, recorded the rates, leaving a uniform covering on the wing. number of separate palpation bouts within a trial Males receiving this wax treatment could still strid- (i.e., palpation bout = mounting by a female and ulate. Males in the pronotum treatment served as a palpating male’s body), rather than total time spent sham treatment: the water wax was brushed onto palpating. Simultaneous trials were visually isolated the pronotum only. from each other by the placement of paper parti- tions between the mating arenas. At the conclusion Data Analysis of each trial, all contents were removed from each mating arena, crickets were returned to their Pronotum width, a body part of fixed measurement, housing containers, and the arena was cleaned with was used to quantify adult size (cf. Andrade & water. Mason 2000). There was a significant correlation between body mass and pronotum width for both males (r = 0.71, p < 0.001, n = 92) and females Spermatophore Content (r = 0.66, p < 0.001, n = 92). Body condition on the We checked for the presence of sperm cells in micro- day of a mating trial was calculated as the residuals spermatophores and macrospermatophores in 15 tri- of regression of body mass on pronotum width. Male als. For five trials, a virgin male was paired with a and female conditions were similar across treatments virgin female. At the moment of microspermato- (anova for males: F2,89 = 0.57, p > 0.50; anova for phore transfer, the female was anaesthetized with females: F2,89 = 1.83, p > 0.10). All statistical tests CO2, and the microspermatophore was removed are two-tailed and calculated with statistica (Stat- from the female’s genital surface with fine tweezers. Soft, Inc 2001, Version 6; StatSoft, Inc., Tulsa, OK, The microspermatophore was placed on a glass slide, USA). Mean values are presented with standard shredded with a sharp pin, and mixed with a drop of errors (SE).

Ethology 114 (2008) 1173–1182 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 1175 Nuptial Feeding in the Wood Cricket P. Prokop & M. R. Maxwell

initially crawled away from the males, which might Results increase the chance of the macrospermatophore becoming dislodged. The males pursued the females, Spermatophore Content knocking their heads against the females’ bodies, We failed to find sperm cells in any of the five mi- presumably to prevent the females from prematurely crospermatophores examined. In contrast, we found removing the macrospermatophores (cf. Bidochka & sperm cells in all 10 macrospermatophores: first Snedden 1985). In the 28 trials where the male pro- transferred in the trial (n = 5), second transferred duced a macrospermatophore but did not transfer it (n = 3), and third transferred (n = 2). to the female, the male always ate it (x Æ SE time to macrospermatophore consumption after its produc- tion = 43 Æ 6 min, n = 28). General Mating Behavior Successful transfer of the microspermatophore was Early courtship behavior, such as the male’s calling not a necessary prerequisite to successful transfer of song, antennation of the male by the female, and the macrospermatophore. Of the 64 trials in which a mounting by the female (Gabbutt 1954; Campan & microspermatophore was transferred to the female, Demai 1983), occurred in all 92 experimental trials. one or more macrospermatophores were transferred Outcomes of the trials are summarized in Fig. 1. in 44 trials (69%). Of the 28 trials in which a micro- Of the 92 trials, males transferred a microsperma- spermatophore was not transferred, one or more tophore to the females in 64 trials. Because of its macrospermatophores were transferred in 11 trials small size, actual production and consumption of the (39%). Similarly, palpation was not a necessary pre- microspermatophore was difficult to observe consis- requisite to macrospermatophore transfer. Palpation tently, so we only report microspermatophore trans- preceded macrospermatophore transfer in 15 of 37 fer. No more than one microspermatophore was trials (41%). When palpation did not occur, macro- transferred during a trial, and males did not pursue spermatophore transfer occurred in 40 of 55 trials the females after transfer. Palpation of the males’ (73%). forewings occurred in 37 trials. With regard to the large macrospermatophores, males produced at least Forewing Manipulations and Mating Success one in 83 trials, and successfully transferred at least one in 55 trials. A total of 73 macrospermatophores Males with exposed forewings (control and prono- were transferred: 40 males transferred exactly one tum treatments) most commonly transferred a macrospermatophore, 12 males transferred two, and microspermatophore, received no palpation from the three males transferred three. Females consumed female, and transferred one or more macrospermato- macrospermatophores only after successful transfer phores (57.14% and 34.48% of control and prono- and attachment. The females ate all of the 73 macro- tum trials, respectively; Fig. 1a, b). On the contrary, spermatophores that were transferred and attached the most common outcome for males with covered to them. forewings (wing treatment) was not transferring a Latencies to produce and transfer spermatophores microspermatophore, receiving palpation, while not were similar across treatments. Overall, x Æ SE time transferring a macrospermatophore (34.28% of wing from beginning of trial to microspermatophore trials; Fig. 1c). The link between microspermato- transfer = 58 Æ 6 min (n = 64), x Æ SE time from phore and macrospermatophore transfer was signifi- beginning of trial to the first macrospermatophore cantly affected by treatment (chi-square test: production = 77 Æ 8 min (n = 83), x Æ SE time from v2 = 35.7, df = 7, p < 0.001; Table 1). For control beginning of trial to the first macrospermatophore and pronotum males, macrospermatophore transfer transfer = 83 Æ 6 min (n = 55). Significant differ- occurred with high probability, regardless of micro- ences in these times were not detected across treat- spermatophore transfer (Fisher’s exact test, control ments (mancova, with treatments as categorical and treatment: p = 0.286; Fisher’s exact test, pronotum male and female conditions as covariates: treatment treatment: p = 0.633; Table 1). In contrast, males

Wilk’s k = 0.74, F6,62 = 1.65, p > 0.1; male and with covered forewings that did not pass a micro- female conditions, Wilk’s k = 0.89 and 0.98, F3,31 = spermatophore were particularly unsuccessful in 1.26 and 0.20, p > 0.3 and p > 0.8, respectively). macrospermatophore transfer (Fisher’s exact test: After transferring the first macrospermatophore, p = 0.015; Table 1). the male aggressively pursued the female for several We examined the influences of the male’s forewing minutes in 23 of 55 trials. In these cases, the females treatment, male condition, and female condition on

Ethology 114 (2008) 1173–1182 ª 2008 The Authors 1176 Journal compilation ª 2008 Blackwell Verlag, Berlin P. Prokop & M. R. Maxwell Nuptial Feeding in the Wood Cricket

(a) % of trials Macro transferred (3) 10.71% Palpation (3) Micro Macro not transferred (0) 0.00% transferred (23) No palpation (20) Macro transferred (16) 57.14% Control Macro not transferred (4) 14.29% (n = 28) Macro transferred (1) 3.57% Palpation (3) Micro not Macro not transferred (2) 7.14% transferred (5) Macro transferred (2) 7.14% No palpation (2) Macro not transferred (0) 0.00%

(b) % of trials Macro transferred (4) 13.79% Palpation (8) Micro Macro not transferred (4) 13.79% transferred (23) No palpation (15) Macro transferred (10) 34.48% Pronotum Macro not transferred (5) 17.24% (n = 29) Macro transferred (1) 3.45% Palpation (1) Micro not Macro not transferred (0) 0.00% transferred (6) 13.79% No palpation (5) Macro transferred (4) Macro not transferred (1) 3.45%

(c) % of trials Macro transferred (4) 11.43% Palpation (8) Micro Macro not transferred (4) 11.43% transferred (18) Macro transferred (7) 20.00% No palpation (10) Wing Macro not transferred (3) 8.57% (n = 35) Macro transferred (2) 5.71% Palpation (14) Micro not Macro not transferred (12) 34.28% transferred (17) Macro transferred (1) 2.86% No palpation (3) Macro not transferred (2) 5.71%

Fig. 1: Transfer of microspermatophore, occurrence of palpation, and transfer of one or more macrospermatophores for the three forewing treat- ments (92 trials in total). Numbers of trials showing a certain behavior are given in parentheses. Sequential percentages greater than 60% are indi- cated by heavy arrows. For each treatment, the most common outcome is boxed. (a) Control treatment (28 trials), (b) pronotum treatment (29 trials), and (c) wing treatment (35 trials). the occurrences of microspermatophore transfer, tophore (Table 2; x Æ SE female condition with mi- palpation, macrospermatophore production, and crospermatophore transfer = 0.001 Æ 0.001 residual macrospermatophore transfer (Table 2). Forewing values, n = 64; x Æ SE female condition without mi- treatment showed significant effects on microsperma- crospermatophore transfer = )0.002 Æ 0.001 residual tophore transfer, palpation and macrospermatophore values, n = 28). Regarding palpation, males of the transfer (Table 2). With regard to microspermato- wing treatment were significantly more likely to phore transfer, males of the wing treatment were sig- receive palpation than control and pronotum males nificantly less successful than control and pronotum [wing: 22 of 35 males, control: 6 of 28 males, prono- males [wing: 18 of 35 males, control: 23 of 28 males, tum: 9 of 29 males; post hoc subdivision of contingency pronotum: 23 of 29 males; post hoc subdivision of table, Zar (1984): v2 with Yates’ correction = 10.6, contingency table, Zar (1984): v2 with Yates’ correc- df = 1, p < 0.01]. Regarding macrospermatophore tion = 7.4, df = 1, p < 0.01]. Females in better transfer, males of the wing treatment were signifi- condition were more likely to receive a microsperma- cantly less successful than control and pronotum

Ethology 114 (2008) 1173–1182 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 1177 Nuptial Feeding in the Wood Cricket P. Prokop & M. R. Maxwell

Table 1: Microspermatophore transfer and macrospermatophore 3 transfer by forewing treatment. Overall, chi-square test for microsper- b matophore · macrospermatophore · treatment: v2 = 35.7, df = 7, p < 0.001. Results of the Fisher exact tests appear below each treat- 2 ment table. Microsperm: presence or absence of microspermatophore transfer, Macrosperm: presence or absence of macrospermatophore transfer 1 (mean ± SE) a a # palpation bouts Control Pronotum Wing (n = 28) (n = 29) (n = 35) 0 Control Pronotum Wing Macrosperm Macrosperm Macrosperm Treatment

Microsperm Yes No Yes No Yes No Fig. 2: Number of palpation bouts on male forewings by females. Dif- Yes 19 4 14 9 11 7 ferent letters denote significant differences based on Tukey post hoc No 32 51 314tests (a vs. b, p < 0.001). p = 0.286 p = 0.633 p = 0.015 n = 42; the Mann–Whitney U = 302.0, p > 0.8). In this subset of 57 control and pronotum trials, the males [wing: 14 of 35 males, control: 22 of 28 males, occurrence of palpation by the females was not asso- pronotum: 19 of 29 males; post hoc subdivision of con- ciated with successful macrospermatophore transfer. tingency table, Zar (1984): v2 with Yates’ correc- When palpation occurred (15 trials), at least one tion = 7.9, df = 1, p < 0.01]. macrospermatophore was transferred in nine trials Analysis of actual number of palpation bouts con- (60%); when palpation did not occur (42 trials), at firms more palpation with males of the wing treat- least one macrospermatophore was transferred in ment (ancova: treatment F2,87 = 14.53, p < 0.0001; 32 trials (76%; Fisher’s exact test: p > 0.3). male and female condition, F1,87 = 0.005 and 3.48, Analysis of actual number of macrospermato- p > 0.9 and p = 0.065, respectively; Fig. 2). Females phores produced and transferred points to a disad- in relatively poor condition showed a non-significant vantage for males with covered forewings (wing tendency to perform more palpation (p = 0.065); treatment). The number of macrospermatophores correlation between number of palpation bouts and produced and transferred significantly differed female condition was weak, negative, and non-sig- between treatments (Fig. 3; mancova, overall effect nificant (r = )0.12, p = 0.25, n = 92). Female condi- of treatment: Wilk’s k = 0.82, F4,172 = 4.58, p < 0.01; tion failed to significantly differ between treatments effect of treatment on macrospermatophore

(see ‘Methods’), and ancova interaction between production: F2,91 = 5.91, p < 0.01; effect of treatment female condition · treatment showed no significant on macrospermatophore transfer: F2,91 = 5.42, effect (F2,75 = 1.94, p > 0.1). As a subset of these p < 0.01). Males in the wing treatment did not data, we examined trials in which males’ forewings necessarily produce fewer macrospermatophores were exposed (control and pronotum, n = 57 trials), than control males, but they transferred fewer but failed to detect a difference in female condition macrospermatophores than control and pronotum between trials with and without palpation (x Æ SE males (Fig. 3). Neither male condition (Wilk’s female condition with palpation = )0.0006 Æ 0.0018 k = 0.99, F2,86 = 0.52, p > 0.6) nor female condition residual value, n = 15; x Æ SE female condition (Wilk’s k = 0.99, F4,172 = 0.2, p > 0.8), which were without palpation = )0.0006 Æ 0.0011 residual value, defined as covariates, significantly influenced these

Table 2: Effects of forewing treatment (control, pronotum, and wing), male condition, and female condition on four dependent variables: transfer of microspermatophore (yes ⁄ no), occurrence of palpation (yes ⁄ no), production of macrospermatophore (yes ⁄ no), and transfer of macrospermato- phore (yes ⁄ no). Four separate multiple logistic regression tests conducted

Transfer microsperm Palpation Produce macrosperm Transfer macrosperm

Ind. variable Wald’s v2, p Wald’s v2, p Wald’s v2, p Wald’s v2,p Treatment 9.53, p < 0.01 12.13, p < 0.01 3.04, NS 10.08, p < 0.01 Male condition 0.07, NS 0.00, NS 2.53, NS 0.07, NS Female condition 4.46, p < 0.05 1.02, NS 1.18, NS 1.71, NS

NS: p > 0.05.

Ethology 114 (2008) 1173–1182 ª 2008 The Authors 1178 Journal compilation ª 2008 Blackwell Verlag, Berlin P. Prokop & M. R. Maxwell Nuptial Feeding in the Wood Cricket

Macrosperm. produced Discussion Macrosperm. transferred This study elucidates several aspects of the mating 3 system of the wood cricket N. sylvestris. We found a sperm cells in the macrospermatophore, but not 2 ab in the microspermatophore. Transfer of the micro- b spermatophore was not a necessary precursor to c c successful transfer of the macrospermatophore. 1 (mean ± SE) d Macrospermatophore transfer occurred in the absence of microspermatophore transfer, whereas micro- # Macrospermatophores 0 spermatophore transfer was not always followed by Control Pronotum Wing macrospermatophore transfer. Exposure of the fore- Treatment wings (control and pronotum treatments) increased

Fig. 3: Number of macrospermatophores produced (dark bars) and male success in terms of microspermatophore and number of macrospermatophores transferred (open bars). Different macrospermatophore transfer. An interaction between letters denote significant differences based on Tukey post hoc tests forewings and spermatophores was detected, as males (a vs. b, p < 0.01; c vs. d, p < 0.05). with covered forewings (wing treatment) were signifi- cantly less successful in macrospermatophore transfer results. For trials in which the males’ forewings were when they failed to transfer a microspermatophore. exposed (control and pronotum, n = 57 trials), the number of transferred macrospermatophores failed Role of the Dual Spermatophores: to differ between trials with and without palpation Microspermatophore and Macrospermatophore (x Æ SE transferred macrospermatophores with pal- pation = 1.5 Æ 0.2, n = 15; x Æ SE transferred mac- Our study is the first to examine the contents of the rospermatophores without palpation = 1.8 Æ 0.1, microspermatophore of N. sylvestris. We failed to find n = 42; the Mann–Whitney U = 283.5, p > 0.9). sperm in the microspermatophore. We note that For trials with macrospermatophore transfer, Gerhardt (1921) referred to ‘pseudospermatophoren’ in this species, although it is not clear whether he overall x Æ SE attachment duration of the first equated this with the microspermatophore (Gabbutt macrospermatophore was 25 Æ 2 min (n = 55). Attachment duration of the first macrospermato- 1954). Furthermore, Campan & Demai (1983) assert phore failed to differ among treatments (ancova, that the smaller first spermatophore does not contain with treatments as categorical and male and female sperm, but they do not describe sample size or meth- ods of inspecting the spermatophore. condition as covariates: treatment F2,50 = 0.07, In N. sylvestris, the apparently spermless microsper- p > 0.9, male and female condition F1,50 = 0.25 and 0.43, p > 0.6 and p > 0.5, respectively). Inclusion of matophore does not appear to be a necessary prere- the occurrence of palpation as a categorical variable quisite to the transfer of the sperm-filled did not significantly change this result. Looking at macrospermatophore. Transfer of the macrosperma- trials in which males’ forewings were exposed (con- tophore occurred in the absence of microspermato- trol and pronotum) and in which a macrospermato- phore transfer, whereas the transfer of the phore was transferred (n = 41 trials), attachment microspermatophore was not always followed by duration of the first macrospermatophore failed to transfer of the macrospermatophore. differ between trials with and without palpation Microspermatophores and macrospermatophores have been recently described in gryllids of the genus (x Æ SE duration with palpation = 28 Æ 6 min, (Shaw & Khine 2004; deCarvalho & Shaw n=9; x Æ SE duration without palpation = Laupala 2005; Mendelson & Shaw 2006). The microsperma- 24 Æ 3 min, n = 32; the Mann–Whitney U = 109.5, p > 0.2). For these 41 trials, attachment duration of tophores of at least two species, Laupala cerasina and the first macrospermatophore failed to significantly Laupala pacifica, are similar to those of the wood differ between trials with and without transfer of the cricket N. sylvestris: the microspermatophore is microspermatophore (x Æ SE duration with micro- roughly one-third the diameter of the macrosperma- tophore, and the microspermatophore does not spermatophore transfer = 24 Æ 3 min, n = 33; x Æ SE duration without microspermatophore transfer = contain sperm (Shaw & Khine 2004; deCarvalho & Shaw 2005). males transfer multiple 28 Æ 6 min, n = 8; the Mann–Whitney U = 107.5, Laupala p > 0.4). microspermatophores, yet their function remains

Ethology 114 (2008) 1173–1182 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 1179 Nuptial Feeding in the Wood Cricket P. Prokop & M. R. Maxwell unclear (Shaw & Khine 2004; deCarvalho & Shaw 2003; Vahed 2007). The present study does not 2005). strongly support this hypothesis for the microsper- Hypotheses concerning the role of the microsper- matophore alone, given that failure to transfer a mi- matophore include male fertilization success and crospermatophore per se did not prevent eventual female benefits (Gwynne 1997; Vahed 1998, macrospermatophore transfer by the male. 2007). The microspermatophore may increase the In N. sylvestris, female and male behaviors suggest male’s fertilization success by (1) manipulating the that the larger sperm-filled macrospermatophore has female’s reproductive behavior, and ⁄ or (2) prolong- nutritional value, despite the lack of a large sperma- ing sperm transfer. Chemical substances in the tophylax found in other orthopterans (Gwynne microspermatophore may make the female more 1997). Females consumed every macrospermato- likely to accept the male’s sperm, may shorten the phore that they accepted. Importantly, males always latency to lay eggs, or may make the female less consumed macrospermatophores that were rejected likely to mate with subsequent males (Loher & by females. Male body condition, however, failed to Dambach 1989; Simmons 2001; Vahed 2007). With significantly affect the production of the macrosper- regard to the female’s acceptance of the male, males matophore, and female body condition failed to in the present study generally transferred macrosper- affect macrospermatophore transfer. We emphasize matophores regardless of whether they transferred a that stronger tests of the nutritional value of the microspermatophore (Table 1). Males with covered macrospermatophore would involve manipulations forewings, however, were significantly less likely to of male and female feeding regimes, as well as transfer a macrospermatophore if they did not examinations of female lifespan and fecundity. transfer a microspermatophore. The present study cannot address the manipulation of female oviposi- Forewing Manipulations and Interactions with the tion or remating behavior. In the sagebrush cricket Dual Spermatophores (Cyphoderris strepitans), females that feed on the males’ hind wings delay remating when compared Males with exposed forewings (control and prono- with females that are prevented from feeding (John- tum treatments) were more successful in microsper- son et al. 1999). With regard to the notion of pro- matophore transfer and macrospermatophore longing sperm transfer, it seems unlikely that the transfer than males with covered forewings (wing female’s consumption of the microspermatophore treatment). An interaction between forewing treat- prolongs the attachment of the macrospermato- ment and the spermatophores was detected in that phore. In N. sylvestris, the microspermatophore is male mating success (macrospermatophore transfer) transferred and consumed quickly by the female, was particularly low for males that had covered fore- averaging 25 min before the transfer of the wings and had failed to transfer a microspermato- macrospermatophore. Furthermore, the transfer of phore. Thus, male mating success increases when the microspermatophore did not affect macrosperma- the forewings are exposed. Failing that, providing a tophore attachment duration in the present study. microspermatophore appears to increase male suc- The female may derive direct benefits from nutri- cess. Either the microspermatophore or the forewing ents in the microspermatophore, although we note secretions may provide direct benefits (e.g., nutri- that the microspermatophore makes a small meal tion) or indirect benefits (e.g., mate assessment (approx. 0.3 mm diameter). Under this hypothesis, information) to the female before sperm transfer one would expect females in relatively poor condi- (Loher & Dambach 1989; Simmons 2001; Kokko tion to be more likely to receive a microspermato- et al. 2003; Vahed 2007). phore. The present study, however, does not Somewhat unexpectedly, this study casts doubt on strongly support this hypothesis, as females in better the role of the forewings in nuptial feeding. condition were more likely to receive the microsper- Although males with exposed forewings were more matophore. We note that our measure of female successful in macrospermatophore transfer, females feeding condition reflects quantity of food eaten. actually palpated these males’ forewings less. Palpa- A stronger test of the nutritional benefit hypothesis tion by the female was not strongly associated with would involve experimental manipulation of food male acceptance, either in terms of the occurrence quantity and quality, as the microspermatophore of macrospermatophore transfer or the number of may contain certain key nutrients. Female indirect macrospermatophores transferred. In trials with benefits include acquiring information about male males with exposed wings, palpation occurred in quality via the microspermatophore (Kokko et al. only 9 of 41 trials (22%) with macrospermatophore

Ethology 114 (2008) 1173–1182 ª 2008 The Authors 1180 Journal compilation ª 2008 Blackwell Verlag, Berlin P. Prokop & M. R. Maxwell Nuptial Feeding in the Wood Cricket transfer. Similarly, Gabbutt (1954) reports palpation than males with covered wings, so access to the fore- in 19 of 52 trials (36%) with macrospermatophore wing secretions may lead to more total sperm trans- transfer. When the males’ forewings were covered in ferred. Additionally, females accruing direct benefits the present study, palpation by females was signifi- via ingestion of the secretions remain a possibility, cantly more frequent, and the actual number of pal- but the present study cannot address the secretions’ pation bouts during the trials was greater with effects on female lifespan or fecundity. covered forewings. The higher incidence of female The female gaining indirect benefits via forewing palpation of covered forewings suggests searching secretions is a viable hypothesis, regardless of behavior by the females, wherein they were whether she ingests the secretions or merely senses attempting to locate or sample the forewing secre- them. By sampling the secretions, the female may tions. gain information about the male’s quality. We posit Hypotheses concerning the role of the forewings that the female might sample the secretions by in N. sylvestris require a consideration of the proxi- means other than palpation, based on two lines of mate behaviors involved. First, we acknowledge that evidence. First, the occurrence of palpation per se did our experimental covering of the forewings might not predict mating success in the present study, as have affected stridulation by the males. Although we discussed above. Second, we observed antennation did not perform acoustic recordings of the males, we of the male by the female throughout the mating observed stridulation by every male in all treat- trial, as noted by previous authors (Gabbutt 1954; ments. Furthermore, Richards (1952, 1953) and Campan & Demai 1983). During these sweeps of the Gabbutt (1954) present data that suggest that close- antennae, the female may be sampling the forewing range stimuli in addition to stridulation are impor- secretions via olfaction. The possible nutritional and tant for mating: a small number of males (four) were informational roles of the forewing secretions and able to mate successfully, despite lacking functional microspermatophore warrant further investigation in forewings. Second, the present study echoes earlier this species. doubts on the role of the forewing secretions as nup- tial feeding. The female wood cricket typically Acknowledgements brushes the male’s secretions with her labial palps, which has lead previous authors to question how We thank Bill Brown, Tagide deCarvalho, Karim much ‘feeding’ actually occurs (Richards 1952, 1953; Vahed and anonymous reviewers for valuable com- Gabbutt 1954; Mays 1971). Further work, such as ments. Huda Makhluf assisted with translation of radiolabeling substances in the forewing secretions, French articles. The University of Trnava provided would help to determine ingestion of the secretions logistical support during this study. by the females. If female wood crickets ingest the males’ forewing Literature Cited secretions, then hypotheses about the secretions’ function include male fertilization success as well as Andrade, M. C. B. & Mason, A. C. 2000: Male condition, direct and indirect benefits to the females. As dis- female choice, and extreme variation in repeated mat- cussed for microspermatophores, increasing male fer- ing in a scaly cricket, Ornebius aperta (: Gryl- tilization success can involve manipulating the lidae: Mogoplistinae). J. Behav. 13, 483—497. female’s behavior (e.g., mate acceptance) and pro- Arnqvist, G. & Rowe, L. 2005: Sexual Conflict. Princeton longing sperm transfer, as in other orthopterans with Univ. Press, Princeton. glandular or wing feeding (Hohorst 1937; Morris Bidochka, M. J. & Snedden, W. A. 1985: Effect of nuptial et al. 1989; Eggert & Sakaluk 1994; Brown 1997; feeding on the mating behaviour of female ground Johnson et al. 1999; Bussie`re et al. 2005; reviewed crickets. Can. J. Zool. 63, 207—208. in Gwynne 1997). Regarding mate acceptance, pal- Boggs, C. L. 1995: Male nuptial gifts: phenotypic conse- quences and evolutionary implications. In: Insect pation by the female was not strongly associated Reproduction (Leather, S. R. & Hardie, J., eds). CRC with the occurrence of macrospermatophore transfer Press, Boca Raton, pp. 215—242. or the number of macrospermatophores transferred. Brown, W. D. 1997: Courtship feeding in tree crickets Regarding sperm transfer, attachment duration of increases insemination and female reproductive life the first macrospermatophore failed to differ across span. Anim. Behav. 54, 1369—1382. treatments, and was not affected by the occurrence Bussie`re, L. F., Clark, A. P. & Gwynne, D. T. 2005: Pre- of palpation. We note that males with exposed wings ferred males are not always good providers: female transferred significantly more macrospermatophores

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choice and male investment in tree crickets. Behav. Mays, D. L. 1971: Mating behaviour of nemobiine Ecol. 16, 255—259. crickets Hygronemobius, Nemobius and Campan, M. & Demai, F. 1983: Le comportement sexuel (Orthoptera: ). Fla. Entomol. 54, 113—126. de Nemobius sylvestris (Orthoptera: Gryllidae). Biol. Mendelson, T. C & Shaw, K. L. 2006: Close-range acous- Behav. 8, 185—204. tic signaling and mate choice in Hawaiian crickets deCarvalho, T. N. & Shaw, K. L. 2005: Nuptial feeding of (Gryllidae: Laupala). Behav. Ecol. Sociobiol. 59, spermless spermatophores in the Hawaiian swordtail 770—776. cricket, Laupala pacifica (Gryllidae: Triginodiinae). Morris, G. K., Gwynne, D. T., Klimas, D. E. & Sakaluk, S. Naturwissenschaften 92, 483—487. K. 1989: Virgin male mating advantage in a primitive Dodson, G. N., Morris, G. K. & Gwynne, D. T. 1983: acoustic insect (Orthoptera: Haglidae). J. Insect Behav. Mating behaviour of the primitive orthopteran genus 2, 173—185. Cyphoderris (Haglidae). In: Orthopteran Mating Ono, T., Hayakawa, F., Matsuura, Y., Shiraishi, M., Systems: Sexual Competition in a Diverse Group of Yasui, H., Nakamura, T. & Marakawa, M. 1995: Insects (Gwynne, D. T. & Morris, G. K., eds). Westview Reproductive biology and function of multiple mating Press, Boulder, pp. 305—318. in the mating system of a tree cricket, Truljalia Dombrowski, A. & Dambach, M. 1994: Ein tegminales hibinonis (Orthoptera, Podoscritinae). J. Insect Behav. Druesenfeld beim Maennchen der Waldgrille (Nemobius 8, 813—824. sylvestris) und seine Bedeutung beim Paarungsverhal- Parker, G. A. & Simmons, L. W. 1989: Nuptial feeding in ten. Verh. Dtsch. Zool. Ges. 87, 238. insects: theoretical models of male and female interests. Eggert, A. K. & Sakaluk, S. K. 1994: Sexual cannibalism Ethology 82, 3—26. and its relation to male mating success in sagebrush Richards, T. J. 1952: Nemobius sylvestris in S. E. Devon. crickets, Cyphoderris strepitans (Haglidae: Orthoptera). Entomologist 85, 83—87. Anim. Behav. 47, 1171—1177. Richards, T. J. 1953: Nemobius sylvestris (F.) (Orthopt., Fedorka, K. M. & Mousseau, T. A. 2002: Material and Gryllidae): a correction and some further notes. Ento- genetic effects of female multiple mating and polyan- mologist 86, 133—134. dry. Anim. Behav. 64, 361—367. Sauer, K. P., Lubjuhn, T., Sindern, J., Kullmann, H., Gabbutt, P. D. 1954: Notes on the mating behaviour of Kurtz, J., Epplen, C. & Epplen, J. T. 1998: Mating Nemobius sylvestris. Anim. Behav. 2, 84—88. system and sexual selection in the scorpionfly Panorpa Gerhardt, U. 1921: Neue Studien u¨ ber Copulation und vulgaris (Mecoptera: Panorpidae). Naturwissenschaften Spermatophoren von Grylliden und Locustiden. Acta 85, 219—228. Zool. 2, 293—327. Shaw, K. L. & Khine, A. H. 2004: Courtship behavior in Gwynne, D. T. 1997: The evolution of edible ‘‘sperm the Hawaiian cricket Laupala cerasina: males provide sacs’’ and other forms of courtship feeding in crickets, spermless spermatophores as nuptial gifts. Ethology katydids and their kin (Orthoptera: ). In: The 110, 81—95. Evolution of Mating Systems in Insects and Arachnids Simmons, L. W. 2001: Sperm Competition and its Evolu- (Choe, J. & Crespi, B., eds). Cambridge Univ. Press, tionary Consequences in the Insects. Princeton Univ. Cambridge, pp. 110—129. Press, Princeton. Hohorst, W. 1937: Die Begattungsbiologie der Grylle Oec- Simmons, L.W. & Parker, G. A. 1989: Nuptial feeding in anthus pellucens Scolpi. Z. Morphol. Oekol. Tiere 32, insects: mating effort versus paternal investment. 227—275. Ethology 81, 332—343. Johnson, J. C., Ivy, T. M. & Sakaluk, S. K. 1999: Female StatSoft, Inc. 2001: STATISTICA Data Analysis Software remating propensity contingent on sexual cannibalism System, Version 6. StatSoft, Inc., Tulsa. Available at: in sagebrush crickets, Cyphoderris strepitans: a mechanism http://www.statsoft.com. of cryptic female choice. Behav. Ecol. 10, 227—233. Thornhill, R. & Alcock, J. 1983: The evolution of insect Kokko, H., Brooks, R., Jennions, M. D. & Morley, J. mating systems. Harvard Univ. Press, Cambridge. 2003: The evolution of mate choice and mating biases. Vahed, K. 1998: The function of nuptial feeding in Proc. R. Soc. Lond. B (Suppl. 6) 270, 653—664. insects: a review of empirical studies. Biol. Rev. 73, Laird, G., Gwynne, D. T. & Andrade, M. C. B. 2004: 43—78. Extreme repeated mating as a counter-adaptation to Vahed, K. 2007: All that glisters is not gold: sensory bias, sexual conflict? Proc. R. Soc. Lond. B 271, sexual conflict and nuptial feeding in insects and spi- S402—S404. ders. Ethology 113, 105—127. Loher, W. & Dambach, M. 1989: Reproductive behav- Zar, J. H. 1984: Biostatistical Analysis, 2nd edn. Prentice- iour. In: Cricket Behaviour and Neurobiology (Huber, Hall, Englewood Cliffs. F., Moore, T. E. & Loher, W., eds). Cornell Univ. Press, Zeh, D. W. & Smith, R. L. 1985: Paternal investment by Ithaca, pp. 43—82. terrestrial arthropods. Am. Nat. 25, 785—805.

Ethology 114 (2008) 1173–1182 ª 2008 The Authors 1182 Journal compilation ª 2008 Blackwell Verlag, Berlin