Behav Ecol Sociobiol (2012) 66:1333–1340 DOI 10.1007/s00265-012-1388-2

ORIGINAL PAPER

Rhythmic male reproductive behavior controls timing of courtship and mating in Laupala cerasina

Tagide N. deCarvalho & Daniel J. Fergus & Rayna C. Bell & Kerry L. Shaw

Received: 21 November 2011 /Revised: 31 January 2012 /Accepted: 9 July 2012 /Published online: 25 July 2012 # Springer-Verlag 2012

Abstract In many organisms, mating behavior occurs at a observed and thus male rhythm alone appears to be respon- particular time of day, which may be important for avoiding sible for the timing of mating. Furthermore, when courtship mate competition or interspecific mating. Crickets of the is initiated later in the day, males produce fewer nuptial gifts Hawaiian genus Laupala exhibit an unusually protracted and increase nuptial gift production rate while delaying courtship in which males produce a series of nuptial gifts mating, suggesting that the number of gifts a female prior to the species-typical time of mating. Mating time is receives is important to male reproductive success. one of several rhythmic behaviors that have diverged among closely related Laupala species, which exhibit an extremely Keywords Laupala . Mating behavior . Reproductive high speciation rate. Mating rhythm may reflect direct se- isolation . Nuptial gift . Circadian rhythm . Courtship lection on male and/or female sexual receptivity or the pleiotropic consequence of selection on other rhythmic behaviors. To examine the role of sexual rhythmicity in Introduction Laupala cerasina, we characterized the time boundaries or “circadian gate” of courtship and mating, as well as female Sexual behaviors such as mate searching, courtship, and phonotactic response to male song. We also examined which copulation often occur during a very narrow window of time sex is responsible for mating rhythmicity by phase-shifting during the day, the so-called “circadian gate” (Pittendrigh males relative to the female photophase. Our results dem- and Skopik 1970). This temporal regulation of mating is onstrate that mating behavior is gated by the end of the light related to the circadian rhythm of sexual ability or recep- phase. Time limits to female mating receptivity were not tiveness of one or both sexes. Insects, in particular, have been the focus of study of the contribution of each sex to the Communicated by N. Wedell : : mating rhythm. For example, in the noctuid moth, T. N. deCarvalho (*) D. J. Fergus K. L. Shaw Spodoptera littoralis, both sexes are synchronized in their Department of Biology, University of Maryland, daily rhythm of receptivity. Females release pheromones at a College Park, MD 20742, USA e-mail: [email protected] certain time of day and males have a corresponding rhythm to their responsiveness to pheromones (Silvegren et al. R. C. Bell 2005). Furthermore, both sexes are restricted to only mating Department of Ecology and Evolutionary Biology, at this particular time, which was demonstrated by pairing Cornell University, Ithaca, NY 14853, USA males and females out of phase. In contrast, only females are responsible for the daily rhythm of mating activity in the Present Address: fruit fly genus Drosophila (Sakai and Ishida 2001). Through T. N. deCarvalho the study of behavioral mutants, Drosophila melanogaster Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218, USA females have been shown to display circadian gates to mating receptivity while males courted females throughout Present Address:: the day. D. J. Fergus K. L. Shaw In most crickets, activities that are necessary for pair Department of Neurobiology and Behavior, Cornell University, formation such as male calling song and female locomotory Ithaca, NY 14853, USA response to song (phonotaxis) have a daily cycle and appear 1334 Behav Ecol Sociobiol (2012) 66:1333–1340 to set the time for mating (Walker 1983), although mating is responsible for the mating rhythm, this would rule out the able to occur at any time of the day if the opportunity arises hypothesis that mating rhythmicity is due to indirect selec- (Nowosielski and Patton 1963;Loher1974;Zuk1987). tion on other male rhythmic behaviors. We performed an Crickets of the genus Laupala may be an exception, exhib- experiment in which males were shifted out of phase both iting mating behavior that appears to be restricted by a relatively earlier and later to the female light cycle to exam- circadian gate, which is preceded by unusually protracted ine whether the timing of mating activity is cued by the courtship of several hours (Shaw and Khine 2004; female clock. We also use the results of the circadian gate deCarvalho and Shaw 2005; Fergus et al. 2011). It is known and phase-shift experiments to evaluate how pair establish- that male song, which serves as a long distance signal ment time affects male nuptial gift production. Because the initiating pair formation, shows daily or diel rhythmicity donation of a series of nuptial gifts increases insemination (Danley et al. 2007). After establishing physical contact, success (deCarvalho and Shaw 2010), the length of the courting males transfer a series of nuptial gifts to females courtship bout (and thus the number of micros transferred) in the form of spermless “micro” spermatophores via copu- may be important to male reproductive success. lation (Shaw and Khine 2004), which are produced at reg- ular intervals. At the end of courtship, males produce and transfer a single, sperm-containing “macro” spermatophore, Methods which is considered the mating event (Shaw and Khine 2004; deCarvalho and Shaw 2005). In the field, mating L. cerasina were collected at Kalopa State Park, Hawaii has been observed to occur within a relatively narrow time (20°02′ N, 155°26′ W) in 2005. All crickets were housed period, despite large variation in the onset of courtship at 20 °C on a 12:12-h light–dark cycle in 120-ml-plastic (Shaw and Khine 2004; deCarvalho and Shaw 2010). cups with damp Kimwipes. Crickets were fed ad libitum However, whether a true circadian gate for mating time Fluker’s Cricket Feed, which was treated with Methyl exists in Laupala has not been investigated. Furthermore, Paraben (USB Corporation) to inhibit mold growth. Males the time restriction to mating may reflect limits of male and females were housed separately until they reached ability to produce macros, female receptivity, or a combina- sexual maturity. Breeding pairs were housed together in a tion of the two. single cup to allow mating and oviposition into the moistened Mating rhythmicity in many animals likely reflects selec- Kimwipe. Cricket nymphs were housed under the same tive pressures on these behaviors to occur at a favorable time conditions as the parental individuals and reared to sexual with respect to social or environmental conditions. In maturity. Virgin laboratory-reared males and females were Laupala, the timing of macro production and transfer (i.e., used for the following experiments. mating) could reflect selection on males to inseminate females at a particular time that is favorable for male–male competi- Circadian gate experiment tion or female reproductive physiology. Alternatively, the presence of a mating rhythm in Laupala may be the indirect In order to examine the effect of pair establishment time on by-product of selection on another rhythmic behavior, such as sexual activity, sexually mature pairs were placed in plastic male calling song rate, that has a shared genetic basis. For Petri dishes with moist Kimwipes at Zeitgeber time (ZT): 0, example, the timing of mating and age of reproduction are 6, 8, 10, and 11.75 (ZT 0 0 lights on; ZT 12 0 lights off). linked via pleiotropy in the melon fly, Bactrocera cucurbitae Pair establishment times were spaced closer toward the end (Miyatake 2002). Establishing that a circadian gate exists for of the photophase because the onset of courtship typically Laupala mating and determining the role of each sex in its occurs later in the day (Shaw and Khine 2004). The first expression will provide insight into the selection pressures four groups were observed under fluorescent light until the acting on the timing of mating and how such pressures might end of the light phase at hour 12. The group established at have led to the reproductive asynchrony observed between ZT 11.75 was observed under red-light during the entire species (Danley et al. 2007;Fergusetal.2011). dark phase by experimenter or video-recorder (Sony DCR- In this study, we characterize the boundaries of the court- SR300 digital camcorder). Pairs that began micro produc- ship and mating behavior circadian gate in Laupala cera- tion in the dark phase were allowed to continue courtship sina. By establishing pairs at different times, we test the under fluorescent light in the light phase and macro produc- hypothesis that L. cerasina has a limit to the time at which tion time was noted (to the hour). All pairs that failed to courtship and mating occur. To assess whether the timing of engage in courtship or mating were re-established early in sexual behavior corresponds to temporal fluctuations in the light phase on the following day to assess whether the female response to song, we examine the daily rhythm of lack of sexual activity was based on time or male/female female phonotaxis. We also investigate whether one sex is incompatibility. We used only pairs that succeeded in mat- responsible for the mating rhythm. If females alone are ing for our analyses. Behav Ecol Sociobiol (2012) 66:1333–1340 1335

To assess the change in sexual activity levels due to pair preliminary study to assess female phonotaxis behavior establishment time, a Fisher’s exact test was used to com- throughout the day. Field and laboratory observations dem- pare the proportion of pairs that engaged in courtship onstrate that females are sexually responsive between (scored when males produced and transferred one or more 3 weeks to 3 months post final moult. Mature females micros) and successful mating (scored when males pro- ranging in age from 35 to 70 days post final moult were duced and transferred the macro) between treatment groups. assessed for daily variation in phonotaxis behavior. The The circadian gates of courtship and mating were estimated average age in each group was 46 to 49 days post final by examining 1-h time bins and designating the time range moult, with comparable age distributions across groups. that contained greater than 90 % of the total pairs engaged in Individuals were marked on the back and pronotum with each activity. non-toxic acrylic white paint for identification purposes. To assess the effect of pair establishment time on sper- Playback experiments were run in a 47-cm circular arena matophore production, a one-way ANOVA followed by a within an anechoic chamber at 20ºC (see Shaw and Herlihy Tukey–Kramer corrected multiple comparisons test was 2000). Songs were generated digitally by a personal com- used to compare micro number and macro production time puter using custom software; a pulsed, sinusoidal tone was between treatment groups. played back through a 16-bit digital/analogue converter (hardware and software: Tucker-David Technologies, Phase-shift experiment Gainesville, FL). To prevent aliasing, the acoustic output was filtered at 10 kHz using a Krohn-Hite filter (model In order to examine the influence of each sex on the mating 3322). The pulse amplitude envelope had a rise time of rhythm, we performed two experiments. The first experi- 10 ms and a fall time of 30 ms. The song was broadcast ment consisted of two treatments groups of males shifted out from either the left or right speaker (8.5 cm, Radio Shack of phase relative to the light phase of the female LD cycle, model no. 40-1218). The pulse rate, pulse duration, and 5 h earlier and 5 h later. Both treatment groups were housed carrier frequency were 2.5 pulses per second, 40 ms, and in incubators for a minimum of 14 days prior to the mating 5 kHz, respectively, corresponding to the pulse rate prefer- experiment on their respective light cycles. A control group ences of L. cerasina at Kalopa Park. of unmanipulated males was housed in the same incubator We have no a priori knowledge of the timing of female as females. On the day of the experiment, individuals were phonotactic activity; therefore, we evaluated female respon- held in the incubators until the onset of the female light siveness at four evenly spaced time points corresponding to phase (ZT 0). Pairs were placed in plastic Petri dishes with ZT 1, 7, 13, and 19. Eight experimental, and four control moist Kimwipes within 15 min of ZT 0. Interactions were groups were designated, each consisting of five females. observed through mating, or until the end of the female light Each group of females performed one ZT trial per day in a phase. Micro production, macro production, and macro randomized order, with a day of rest between trials. We transfer behaviors were recorded. conducted 12 trials each day (two experimental and one Because late-shifted males were paired within their own control group at each of the four ZT times), for a total of dark phase (their own ZT 19) in the first experiment, an 7 days to complete the experiment. The time to complete a additional experiment was performed to test how pair estab- trial for one group was approximately 12 min (see below). lishment at the beginning of late-shifted male light phase Therefore groups were tested from 15 min prior to the target would affect the time of macro production. Late-shifted ZT to 30 min past the target ZT in a randomized order. We males were placed together with females at late-shifted male used groups of females in order to estimate the proportion of ZT0 (female ZT 5). Experimental methods follow the pre- females that show acoustic response at a give ZT. Estimating vious phase-shift experiment. this response from phonotactic trials on single females We used male macro production time as a measure of would have greatly extended the number of days necessary mating rhythmicity. A one-way ANOVA followed by a for these round the clock experiments, making the experi- multiple comparisons test with Tukey–Kramer adjustment ment infeasible. was used to test differences in macro production time be- In a given ZT trial, the acoustic stimulus was randomized tween groups. A Fisher’s exact test was used for male pair- to the right or left speaker. A control group received identi- wise comparisons of macro transfer success (i.e., mating cal conditions but lacked the acoustic stimulus. One hour success) between groups. before the trial, the five females in each group were placed into a single plastic specimen cup (120 ml). Five minutes Phonotactic response before the start of the experiment, the cup with females was placed top-down in the center of the arena, equidistant from To assess whether females display a reproductive rhythm either speaker, to acclimate to the arena. The experiment independent of mating interactions, we conducted a began by raising the cup remotely via a monofilament line 1336 Behav Ecol Sociobiol (2012) 66:1333–1340 attached to the top. Movement toward the acoustically active engaged in micro production during the dark phase and the speaker was used to quantify female responsiveness. During rest began courtship in the light phase of the following day. the 5-min trial, a female was scored “responsive” if she All ZT 11.75 pairs produced macros within ZT 8-10 the entered a 10-cm zone (the scoring area) in front of the active following day. We estimated the lower end of the gate by speaker. In control trials, a female was scored “responsive” if only using activity levels of the ZT 11.75 group, while all of she entered the scoring area (the “active” speaker was desig- the groups were used to estimate the upper end of the gate. nated at random each trial, although no sound was played). The courtship gate spanned from ZT 16 to ZT 10 (Fig. 1). The response of a given group was scored as the proportion of We found that the time of pair establishment had an effect the five females that responded to the active speaker. After on spermatophore production. There was a significant dif- each trial, females were returned to their individual cups and ference in the number of micros produced among groups the arena was cleaned with a moist sponge. (Fig. 1; F3, 48094.24, p<0.0001); micro number significant- To account for the range of female ages in the phonotaxis ly decreased as pairs were established later in the light phase trials, we tested for a correlation between age and respon- (Table 1; Tukey–Kramer HSD: alpha00.05). There was also siveness to acoustic stimuli. We excluded control individu- a significant difference among groups in macro production als from this analysis because they were not exposed to time (Fig. 1; F3, 31041.43, p<0.0001); macro production acoustic stimuli. Females ranged in age from 35 to 71 days time was significantly later as pairs were established later in and responsiveness ranged from zero (no responses through- the light phase (Table 1; Tukey–Kramer HSD, alpha00.05). out the experiment) to four (response at all four time points). To determine whether female responsiveness varied across Phase-shift experiment the four time points, we used a repeated measures mixed model that included time as a fixed effect, individual and Macro production time differed significantly among all 0 group as random effects, and the interaction terms time × groups (Fig. 2,Table2; F3, 39 186.89, p<0.0001). If individual, and time × group, as random effects (PROC females alone are responsible for the mating rhythm, we GLIMMIX in SAS 9.2, SAS Institute, Cary, NC). expected to observe the same macro production time among the phase-shifted groups and the control group (approxi- mately female ZT 8) because only the males were phase- Results shifted. Conversely, if males alone are responsible, we expected macro production 5 h earlier by the early-shifted Circadian gate experiment males (female ZT 3) and 5 h later by the late-shifted males (female ZT 13) when paired at the beginning of their own The circadian gate of mating spans approximately 4 h; the light phase. Alternatively, if both sexes are responsible for ends of the gate were estimated by bins of 1-h width in mating rhythm, we expected to observe treatment groups which 90 % or more of the total pairs (n063) engaged in displaying either intermediate macro production times or a macro transfer. The “lower” end of the gate was ZT 8 and significant reduction in mating success. Relative to the “upper” end was ZT 11. Prior to the end of the gate, there average macro production time for the control group, the was a decline in sexual activity; significantly fewer pairs early-shifted group produced macros 3 h 58 min earlier engaged in mating in the ZT 10 group compared to the ZT (female ZT 4:04; Table 2) and the late-shifted group pro- 8 group (Table 1; one-tailed Fisher’s exact test, p00.005). duced macros approximately 1 h 15 min later, on average We observed courtship activity within both phases, dem- (female ZT 9:17; Table 2). Late-shifted males that were onstrating that courtship has a wide circadian gate of ap- paired with females at the beginning of the male light phase proximately 14 h. Three pairs of the ZT 11.75 group (n011) produced macros 4 h 58 min later than the average time of

Table 1 Percentage of pairs that engaged in courtship (transfer of one macro production time (h:min) are presented for each group. Zeitgeber or more micros) and percentage of pairs that mated (transferred macro) time (ZT) 00lights on during the light phase. Mean±standard error of micro number and

Pair establishment time % courtship % mating Micro number Macro production time

ZT 0 100 % (11/11) 100 % (11/11) 8.0±0.36 8:14±00:08 ZT 6 100 % (13/13) 100 % (13/13) 5.6±0.25 8:54±00:06 ZT 8 84.6 % (11/13) 69.2 % (9/13) 2.9±0.43 8:52±00:07 ZT 10 57.1 % (8/14) 14.3 % (2/14) 0.8±0.21 10:56±00:06 ZT 11.75 0 % (0/12) 0 % (0/12) –– Behav Ecol Sociobiol (2012) 66:1333–1340 1337

ZT 11.75 ZT 10 ZT 8 ZT 6 ZT 0

Late-shifted expt 2 Late-shifted Early-shifted Control 0 2468101214161820 22 24

Zeitgeber Time 08910111213462 57 13 14 15

Fig. 1 Micro and macro production times over 24 h for males paired Control Zeitgeber Time with females at five Zeitgeber times (ZT) 0, 6, 8, 10, and 11.75. Each row consists of data from a single pair and each dot represents the Fig. 2 Micro and macro production times of males shifted relative to production time of a spermatophore (small dot 0 micro; large dot 0 the female/control male light cycle. Males were paired with females at macro). Only pairs that successfully produced and transferred macro- the onset of the female photophase, except late-shifted experiment 2 spermatophores are shown. For ZT 11.75 pairs, micros produced only males, which were paired at the onset of their own photophase. Each during the dark phase are plotted row consists of data from a single pair and each dot represents the production time of a spermatophore (small dot 0 micro; large dot 0 macro). Black and white boxes represent the dark and light phases of the control group (female ZT 13:00, Table 2). There was no females/control males significant difference in mating success between groups (two-tailed Fisher’sexacttests,p>0.05) and mating oc- responsiveness at ZT 7 was significantly higher than all curred approximately 45 min to 1 h after macro production other time periods (p<0.05); all other contrasts were non- (Table 2). significant (p>0.05).

Phonotactic response Discussion Groups of females in the acoustic treatments were signifi- cantly more responsive than were groups of females in The time of mating follows a daily rhythm in many animals, control trials (group responses were averaged across trials; often related to rhythmicity in the receptivity or physiolog- Mann Whitney U032.0, p00.004; Fig. 3). We did not find a ical ability of one or both sexes. In most crickets, the significant relationship between female age and responsive- specific time of mating corresponds to the timing of female ness to acoustic stimuli (r0−0.09, p>0.1, df038). Neither of phonotaxis and male calling, which in turn affects the time the interaction terms was significant in our repeated meas- of pair establishment (Loher 1989). Previous work sug- ures mixed model of acoustic response (log-likelihood ratio gested that Laupala also possess species-typical mating tests, p>0.05). Therefore we removed these terms to sim- times, with mating preceded by an unusually extensive plify the final model. The final model revealed a significant courtship involving serial micro donation (Shaw and effect of time on female acoustic responsiveness (F3, 1120 Khine 2004; deCarvalho and Shaw 2005;Fergusetal. 5.01, p00.0032). Post hoc pairwise tests adjusted for mul- 2011). We found that micro production has a wide circadian tiple comparisons using Tukey–Kramer indicated that gate. However, the production of the single sperm- 1338 Behav Ecol Sociobiol (2012) 66:1333–1340

Table 2 Mean±standard error of macro production and transfer time for each group

Treatment Pair establishment time N Macro production time N Macro transfer time

Control Female light phase 13 8:02±00:12 12 8:57±00:10 Early-shifted Female light phase 10 4:04±00:08 9 (1) 5:03±00:09 Late-shifted Female light phase 9 9:17±00:29 8 10:00±00:27 Late-shifted Late-shifted male light phase 11 13:00±00:11 9 (2) 13:59±00:13

Times are presented in Zeitgeber time of the female light phase. Lower sample sizes for macro transfer times compared to macro production times reflect that certain transfer observations were not precise (therefore dropped from the analyses) and that some males failed to transfer macros. Numbers in parentheses represent transfer failures. Macro production and transfer times differed significantly between all treatment groups (Tukey– Kramer HSD tests alpha00.05) containing macro is restricted to relatively few hours at the due to advanced age would likely lead us to underestimate end of the light phase (Fig. 1). This is unlike other crickets therelativedifferencebetweenthepeakandnon-peak in which both sexes will mate at any time when the oppor- responses. tunity arises (Nowosielski and Patton 1963; Loher 1974; Although macro production has a gate, it was delayed to Zuk 1987). Our preliminary assessment of rhythmicity in a certain degree when pairs were established later in the female phonotaxis behavior showed that females display a light phase (Fig. 2), which may serve to facilitate greater significant peak in phonotactic response corresponding to micro donation. Our data demonstrate that macro production ZT 7. While we lack the experimental resolution to test for a time is based on the time of the male photophase and that more precise overlap, peak phonotaxis appears to roughly females do not appear to play a substantial role in this correspond to the beginning of the circadian gate of the timing, due to their receptivity to macro transfer at any time macro. This peak responsiveness cannot be explained by in the experimental design. Males adjust both micro number variation in female age as we did not observe systematic and to a lesser extent, macro production time, based on variation in response among females due to variation in age. when they begin courtship, which suggests that both factors Females were in a relatively advanced, albeit reproductively are important to male reproductive success. active, age for first mating, which might make them more Although macro production is gated by the end of the responsive relative to younger females. However, enclosure light phase, we found that macros were produced at signif- data from the wild suggest that L. cerasina remain repro- icantly later times between each successive treatment group ductively active for at least 44 days (Turnell and Shaw in the circadian gate experiment. This adjustment of macro unpublished). Regardless, increased overall responsiveness production suggests that males delay mating. Further evi- dence to support this idea is the short courtship duration by 1.0 some of the pairs established at ZT 8 and ZT 10, indicating * Experimental (Song) that at least some males are physiologically able to produce Control (No Song) 0.8 a macro within 1 to 2 h after pairing with females. However, pairs established at ZT 6 court for approximately 3 h.

0.6 Therefore, the later time at which macros are produced in the ZT 6 group compared to the ZT 0 group does not reflect

0.4 a physiological constraint on how long it takes males to form a macro. Rather, these data suggest that males are delaying macro production for another purpose, such as

Proportion Responding 0.2 increasing micro donation. This may indicate that micro

0.0 number is important to male reproductive success. Micro transfer has been demonstrated to increase sperm uptake by 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 the female (deCarvalho and Shaw 2010), therefore the num- Zeitgeber Time ber of micros transferred could affect the degree of sperm transfer enhancement. Fig. 3 The proportion of females responding to acoustic stimuli at four Zeitgeber times. The black and white dots represent the mean propor- Although males are able to delay macro production to a tion of females scored as “responsive” with and without acoustic certain extent, late pair establishment has a negative conse- stimuli, respectively. Error bars indicate standard error of the means quence for the allocation of nuptial gifts and mating. The at each point. The significantly greater responsiveness of experimental negative relationship between the time of establishment and females at ZT 7 relative to the other times is indicated by an asterisk. The length of black and white boxes represents the duration of dark and micro number demonstrates that males are not able to pro- light phases duce as many micros when they pair with females later in Behav Ecol Sociobiol (2012) 66:1333–1340 1339 the day. We have observed in the current study and previous produced 5 h later than control males. The 1-h difference in experiments (Shaw and Khine 2004; Fergus et al. 2011) that macro production time between the control males and 5-h the rate of micro production increases towards the end of the late-shifted males when set up at the start of the female light photophase. Males appear to reach the upper limit on micro phase indicates an important role for external cues, as well as production rate when established at ZT 6 (Fig. 2). This endogenous circadian timing. This difference in the time of upper limit of micro production rate together with the limit macro production may reflect a rapid resetting of the internal on the timing of macro production prevents late paired clock by early light cues. males from producing the same number of micros as males Knowing the sex responsible for the time of mating that have paired early in the photophase. Therefore, if micro (macro transfer) lends insight into the selection pressure number is important to male reproductive success, there for the mating period. Because macro timing is primarily may be selection on males to attract females early in the male-limited, we can rule out the hypothesis that the time of day in order to supply an adequate number of micros. mating is a consequence of female receptivity for macro The results from the male phase-shift experiment dem- transfer. Instead, there may be selection on males to produce onstrate that once pairs are established, the time of mating in macros at a certain time of day due to female phonotaxis and L. cerasina is a function of male diel rhythmicity in macro locomotory activity patterns or intra-sexual competition production. Males that were shifted out of phase from the (e.g., sperm competition). Laboratory experiments demon- female light cycle produce and transfer macros based on the strate that females will readily remate the day after their first timing of their own light cycle, rather than on that of the mating (deCarvalho and Shaw 2010) and anecdotal evi- female’s light cycle. Under the male regulated timing hy- dence from the field suggests that females oviposit during pothesis, males shifted 5 h earlier than the females were the dark phase (Turnell and Shaw, unpublished), thus a male expected to produce macros 5 h earlier than controls (i.e., may benefit by delaying macro transfer until as late as both groups producing macros at their respective male ZT possible in the light phase to prevent his partner from mating 8). We observed that early-shifted males produced macros with another male before she begins oviposition. Therefore, earlier than controls, consistent with the expectation of male a male’s sperm would gain exclusive access to his partner’s regulated timing. The four rather than 5 h shift between eggs until the next day; this effect is referred to as the first early-shifted and control male macro production time (ex- male advantage (Calos and Sakaluk 1998). A behavioral perimental male ZT 9 instead of ZT 8) likely reflects the strategy that relies on timing may be employed by L. cera- difference in pair establishment time. Early-shifted males sina males because of a lack of first male sperm precedence. were established with females later in the males’ own light Preliminary data suggest there may be sperm-mixing instead phase (male ZT 5) relative to male controls (male ZT 0), (Turnell and Shaw, unpublished data), as in many other which likely delayed their macro production as observed in crickets (Simmons 2001). However, uncovering the selec- the circadian gate experiment (Fig. 2). Conversely, males tion pressure(s) for males to produce a macro towards the that were shifted later than controls by 5 h and established at end of the day may not explain why L. cerasina males do the female ZT 0 produced their macros significantly later not produce a macro after dark despite female willingness to than control males, but only by approximately 1 h (the mate at this time, as demonstrated by the clock-shifting implications of this are discussed below). However, late- experiment. shifted males established at the beginning of their own light Alternatively, the timing of mating could be a pleiotropic phase produced their macros approximately 5 h later than effect of selection on the timing of other behaviors. control males, suggesting that macro production does not Divergence in selection on reproductive age or other rhyth- include a female-timing component. Therefore, we conclude mic behaviors such as song pulse rate could play a role in that the time of macro production and transfer is primarily the species-specific rhythmicity of macro production in based on the male light cycle. Laupala. However, while pleiotropy could explain diver- The timing of male macro production appears to be under gence in mating time, it would not explain why males are both endogenous and exogenous control. This is evident restricted to when they can produce macros, unlike other from the behavior of late-shifted males established at the crickets. Future work on the genetic basis of behavioral beginning of the female light phase. These late-shifted rhythms in Laupala will lend insight into whether there is males produced macros 1 h later than control males, al- direct selection on the timing of spermatophore production though both groups experienced the onset of light and or if the patterns in this behavior are a product of indirect female interactions at the same time on the day of the selection. experiment. If exogenous cues alone controlled the timing This work also sheds light on whether mating rhythmic- of macro production, we would see no difference between ity plays a role in the community dynamics of sympatric these two groups, while a complete endogenous regulation species of Laupala. Danley et al. (2007) suggest that the would have resulted in macros of the late shifted group being significant difference in the timing of courtship and mating 1340 Behav Ecol Sociobiol (2012) 66:1333–1340 between L. cerasina and Laupala paranigra could act as a Danley PD, deCarvalho TN, Fergus DJ, Shaw KL (2007) Reproductive premating barrier between these two species. When both asynchrony and the divergence of Hawaiian crickets. Ethol 113:1125–1132 species are established at the onset of the light phase, the deCarvalho TN, Shaw KL (2005) Nuptial feeding of spermless sper- average mating time of L. paranigra is approximately 2.5 h matophores in the Hawaiian swordtail cricket, Laupala pacifica later than L. cerasina (Fergus et al. 2011). However, in the (Gryllidae: Trigonidiinae). Naturwissenshaften 92:483–487 present study, the mating circadian gate of L. cerasina deCarvalho TN, Shaw KL (2010) Elaborate courtship enhances sperm transfer in the Hawaiian swordtail cricket, Laupala cerasina. extended to the end of the light phase, which overlaps with Anim Behav 79:819–826 the latest time in the distribution of L. paranigra mating Fergus DF, deCarvalho TN, Shaw KL (2011) Genetically regulated activity (Fergus et al. 2011). Furthermore, L. cerasina fe- temporal variation of novel courtship elements in the Hawaiian – male mating receptivity does not appear to have a gate, cricket genus Laupala. Behav Genet 41:607 614 Loher W (1974) Circadian control of spermatophore formation in which suggests that the time of day would not preclude cricket Teleogryllus commodus Walker. J Insect Physiol female L. cerasina from accepting spermatophores from L. 20:1155–1172 paranigra males. In the male phase-shift study, L. cerasina Loher W (1989) Temporal organization of reproductive behavior. In: females accepted macros throughout an 11-h period (ZT 4– Huber F, Moore TE, Loher W (eds) Cricket behavior and neuro- biology. Cornell University Press, Ithaca, pp 83–113 ZT 15 of their own light cycle) and micros in every hour of Miyatake T (2002) Circadian rhythm and time of mating in Bactrocera the photophase except two (ZT 16 and 17). If L. paranigra cucurbitae (Diptera: Tephritidae) selected for age at reproduction. females have a similarly wide window of receptivity, it Heredity 88:302–306 would overlap with the time that L. cerasina males produce Nowosielski JW, Patton RL (1963) Studies on circadian rhythm of the house cricket, Gryllus domesticus L. J Insect Physiol 9:401– macros. Therefore, it does not appear that a shift in male 410 macro timing would be a strong premating barrier to repro- Pittendrigh C, Skopik SD (1970) Circadian systems, V. The driving duction between these two species. However, Loher (1989) oscillation and the temporal sequence of development. Proc Nat – suggests that female locomotion, not spermatophore recep- Acad Sci USA 65:500 57 Sakai T, Ishida N (2001) Circadian rhythms of female mating activity tivity, appears to be temporally constrained. If female mate governed by clock genes in Drosophila. Proc Nat Acad Sci USA searching has a circadian rhythm that corresponds to male 98:9221–9225 calling or spermatophore production, this may reduce the Shaw KL, Herlihy DP (2000) Acoustic preference functions and song probability that females encounter males of other species. variability in the Hawaiian cricket Laupala cerasina. Proc R Soc B 267:577–584 Female phonotaxis is achieved through locomotion. Shaw KL, Khine AH (2004) Courtship behavior of the Hawaiian Interestingly, the results of our experiment suggest that L. cricket Laupala cerasina: males provide spermless spermato- cerasina female locomotory response to song may peak phores as nuptial gifts. Ethol 110:81–95 around macro production time. Direct investigation of Silvegren G, Lofstedt C, Rosen WQ (2005) Circadian mating activity and effect of pheromone pre-exposure on pheromone response inter-specific differences in the timing of female mate rhythms in the moth Spodoptera littoralis. J Insect Physiol searching behavior would benefit our understanding of these 51:277–286 dynamics. Simmons LW (2001) Sperm competition and its evolutionary consequences in the insects. Princeton University Press, Princeton Acknowledgments We thank Kevin Oh for the statistical consulta- Walker T (1983) Diel patterns of calling in nocturnal Orthoptera. In: tion, and Brian Coyle and Daniel Gorelick for their helpful comments Gwynne DT, Morris GK (eds) Orthopteran mating systems: sex- on the manuscript. ual competition in a diverse group of insects. Westview, Boulder, pp 45–72 Wang Q, Zeng W, Chen L, Li JS, Yin X (2002) Circadian reproductive References rhythms, pair-bonding, and evidence for sex-specific pheromones in Nadezhdiella cantori (Coleoptera: Cerambycidae). J Insect Behav 15:527–539 Calos JB, Sakaluk SK (1998) Paternity of offspring in multiply-mated Zuk M (1987) The effects of gregarine parasites, body size, and time of female crickets: the effect of nuptial food gifts and the advantage day on spermatophore production and sexual selection in field of mating first. Proc R Soc B 265:2191–2195 crickets. Behav Ecol Sociobiol 21:65–72