Females of a tree cricket prefer larger males but not the lower frequency male calls that indicate large body size

Animal Behaviour 84 (2012) 137e149

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Females of a tree cricket prefer larger males but not the lower frequency male calls that indicate large body size Rittik Deb, Monisha Bhattacharya, Rohini Balakrishnan* Centre for Ecological Sciences, Indian Institute of Science, Bangalore, India

a r t i c l e i n f o Article history: Received 23 November 2011 Initial acceptance 4 January 2012 Final acceptance 3 April 2012 Available online 26 May 2012 MS. number: 11-00938R Keywords: carrier frequency female preference male morphology mating duration Oecanthus henryi repeatability spermatophore retention time tree cricket

In the tree cricket Oecanthus henryi, females are attracted by male calls and can choose between males. To make a case for female choice based on male calls, it is necessary to examine male call variation in the field and identify repeatable call features that are reliable indicators of male size or symmetry. Female preference for these reliable call features and the underlying assumption behind this choice, female preference for larger males, also need to be examined. We found that females did prefer larger males during mating, as revealed by the longer mating durations and longer spermatophore retention times. We then examined the correlation between acoustic and morphological features and the repeatability of male calls in the field across two temporal scales, within and across nights. We found that carrier frequency was a reliable indicator of male size, with larger males calling at lower frequencies at a given temperature. Simultaneous playback of male calls differing in frequency, spanning the entire range of natural variation at a given temperature, revealed a lack of female preference for low carrier frequencies. The contrasting results between the phonotaxis and mating experiments may be because females are incapable of discriminating small differences in frequency or because the change in call carrier frequency with temperature renders this cue unreliable in tree crickets. Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Darwin (1871, page 558) summarized the concept of sexual selection in the following lines: ‘The sexual struggle is of two kinds: in the one it is between individuals of the same sex, generally the males, in order to drive away or kill their rivals, the females remaining passive; whilst in the other, the struggle is likewise between the individuals of the same sex, in order to excite or charm those of the opposite sex, generally the females, which no longer remain passive, but select the more agreeable partners’. It has been clear since then that the idea of sexual selection works on two paradigms: maleemale competition and female choice. In the context of female choice, theory predicts that females will choose mates if mating is more costly to them (i.e. rate of reproduction is slower than in the opposite sex; Clutton-Brock & Vincent 1991) and choosiness will depend on the male’s capacity to provide resources to females or confer heritable benefits on offspring (Brown 1999). A large amount of work has been done on female mate choice in a number of taxa such as insects, fishes, frogs, birds and mammals (Bateson 1983; Andersson 1994). Among insects, true crickets and bush crickets have proven to be good model systems to study female mate choice, since most species have an elaborate acoustic * Correspondence: R. Balakrishnan, Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012, Karnataka, India. E-mail address: [email protected] (R. Balakrishnan).

communication system, diverse types of mating incentives for females (such as glandular feeding) and a female-dominated mating system (Gerhardt & Huber 2002). Generally, males of each species produce calls with a unique set of temporal and spectral features, which are used by females to distinguish between conspecific and heterospecific males (Otte 1992; Gerhardt & Huber 2002). Calls may also play a role in mate choice by acting as indicators of male quality, as females show phonotaxis to conspecific male calls (Walker 1957; Alexander 1967; Doherty & Hoy 1985; Brown et al. 1996; Greenfield 1997; Brown 1999; Gerhardt & Huber 2002), calls show high levels of intraspecific variation (Walker 1962; Wagner 1998; Brown 1999; Gerhardt & Huber 2002) and are energetically costly to produce (Prestwich & Walker 1981; Forrest 1991; Gerhardt & Huber 2002). There are certain prerequisites for establishing a case for female mate choice based on male calls. It is necessary to establish preferential female responses to particular call characteristics in the absence of other kinds of cues (Searcy & Andersson 1986; Brown 1999; Gerhardt & Huber 2002). For such preferences to evolve through sexual selection, females must discriminate among males on the basis of consistent call characters (Boake 1989; Brown 1999; Gerhardt & Huber 2002). It is also important to establish that such call features are reliable indicators of male phenotypic features that contribute to reproductive success (Brown 1999; Gerhardt & Huber

0003-3472/$38.00 Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2012.04.020

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2002). There have been studies exploring these different criteria outlined above but studies establishing a combination of all of these are rare (but see Brown et al. 1996). Female preferences for call features have been found in the direction of greater energy, such as higher call rates and longer call bout lengths, which are often positively correlated with body size (Gerhardt 1991; Ryan & Keddy-Hector 1992; Brown 1999; Gerhardt & Huber 2002). In Gryllus bimaculatus (a field cricket) it has been shown that body size is positively correlated with pulse repetition rate and females prefer the calls of larger males (Simmons 1987, 1988). Larger males of Scudderia curvicauda (a bush cricket) produced calls with more syllables per pulse, which were correlated with a relatively large spermatophore food gift (Tuckerman et al. 1993). Similar results were observed in Acheta domesticus; females preferred chirps with a greater number of pulses per chirp, which were indicative of larger males (Gray 1997). Females of Tettigonia cantans (a bush cricket) oriented to calls with lower frequency harmonics, an indicator of larger males, when played back from similar distances (Latimer & Schatral 1986; Latimer & Sippel 1987). Playback experiments on Eunemobius carolinus showed that female preference towards quieter males decreased with the relative intensity difference between two nearby males (Farris et al. 1997). In the tree cricket Oecanthus nigricornis, it was shown that females preferred calls with a carrier frequency corresponding to large males, but only if they had the opportunity to choose between calls that were broadcast simultaneously (Brown et al. 1996). The phonotaxis studies described above assume that females actually prefer larger males during mating. Many have corroborated this assumption through sequential or simultaneous mate choice experiments (Simmons 1986, 1995; Bateman et al. 2001) but a recent study in O. nigricornis (Bussière et al. 2005) has found contrary results. Hence this assumption needs to be tested further before drawing definitive conclusions. Moreover, in previous studies on true crickets, female preference has typically been examined at the two extreme ends of the male trait distribution, whereas the probability of a female encountering such a situation is low in nature (Brown 1999; Gerhardt & Huber 2002). Hence it may be more meaningful to examine female preference at finer differences of male trait values, which a female might encounter more often in the field. Female preference functions may also be different depending on the relative or absolute strength of the stimuli. Generally, in population-level studies, when one extreme trait is preferred over another a directional preference is inferred, or when there is no pattern a lack of preference is inferred. These inferences can be misleading as such patterns can also be generated by stabilizing or disruptive preferences (Wagner 1998; Gerhardt & Huber 2002). Although many of the studies establish a relationship between male morphology and call features and go on to test female preference based on them, rarely do they establish whether such call features are reliable indicators (but see Brown et al. 1996). Consistency of male call characters, although necessary for evolution of mate choice based on male call features (Searcy & Andersson 1986), has been the subject of discussion in only a few studies. Some have examined repeatability of various call traits whereas a few examined their heritability directly (Brown 1999; Gerhardt & Huber 2002). Not much is known about the repeatability of male call features in the field, where female choice actually occurs. Most studies have examined repeatability of call features in either laboratory-reared or laboratory-maintained animals (but see Nityananda & Balakrishnan 2008). Rearing and ad libitum feeding of animals in a controlled environment may influence call traits that are strongly environmentally influenced (Wagner & Hoback 1999; Holzer et al. 2003; Scheuber et al. 2003a, b, 2004) which in turn may provide an overestimate of the actual

repeatability values. It is also important to measure repeatability across various temporal scales as it has been shown that repeatability values of traits measured in short gaps can be higher than those measured at longer gap durations (Bell et al. 2009). The issue that is evident from the above discussion is the lack of observations in the field. Hence we carried out a study encompassing both observations on male traits from the field and laboratory experiments to examine female choice for reliable acoustic indicators of male quality (Searcy & Andersson 1986). In this study, we examined whether certain call features can act as reliable indicators of male morphology (body size) as well as overall genetic quality (fluctuating asymmetry, FA, of morphological traits). Body size was chosen, as in multiple studies (discussed above) it has been shown that females prefer calls of larger males. We chose FA as it is thought to be an estimate of developmental stability and hence a good indicator of genetic quality (Thornhill 1992; Graham et al. 2010). We examined the repeatability of male call traits in the field to understand natural call variation and to make the estimations relevant to the actual scenario of female choice. Repeatability was measured at two temporal scales: across nights and within nights to get an estimate of the effect of temporal gaps. Repeatability of a trait can be affected by recent and past environmental effects which will show up only if both the temporal scales are considered. Furthermore, from the point of view of mate sampling, a trait needs to be highly repeatable within a night to be a useful indicator. We also examined the repeatability of sound pressure level (SPL), which is an important call feature and is often preferred by females (Brown 1999; Gerhardt & Huber 2002) but has rarely been examined in true crickets. Female preference for male call characters that were highly repeatable and indicators of male quality were tested in the laboratory using call playback. Rather than using the values at the extremes of the male trait distribution, we examined female preference at finer intervals. The preference function was examined at SPLs close to the hearing threshold of the female as well as at the mean male calling SPL, corresponding to different distances at which a female may make decisions in the field. This was done as the auditory tuning curve of a female broadens with increase in SPL; hence at the threshold the curve can be much tighter than at mean male calling SPL (Gerhardt & Huber 2002; Kostarakos et al. 2008, 2009). At threshold, male call carrier frequency can be outside the female’s hearing range. Thus at two different SPLs we might expect differences in female preference. Female preference for reliable acoustic indicators of male body size assumes that females actually prefer larger males to smaller males and mating with larger males is beneficial for females (Simmons 1986; Eggert & Sakaluk 1994; Brown et al. 1996; Vahed 1998; Bateman et al. 2001; Bussière et al. 2004). Hence we started by examining the assumption of female preference for larger males using a sequential mating experiment. A sequential design was adopted to avoid the effect of maleemale competition on female choice. The tree cricket Oecanthus henryi Chopard was chosen as the study system for a number of reasons. In the genus Oecanthus, males attract females by calling and mating is female dominated (Walker 1957). Male call features change with change in ambient temperature (Metrani & Balakrishnan 2005) which makes female choice based on call traits an interesting issue. Previous work on O. henryi (Mhatre et al. 2011), using a no-choice paradigm, has shown that females in this species solve the problem of senderereceiver matching in the frequency domain by being broadly tuned over the natural range of male calling carrier frequencies. Hence, female choice in O. henryi, using long-distance acoustic cues, especially male call carrier frequency, is of particular interest.

R. Deb et al. / Animal Behaviour 84 (2012) 137e149

METHODS Study Species Oecanthus henryi is abundantly found in arid scrubland of southern India, including the region in and around Bangalore, in the bushes of Hyptis suaveolens. Adult animals emerge twice a year, in February and September, and survive for 3e4 months. Field observations and animal collection for laboratory experiments, morphological and call feature measurements were carried out at two field sites. The first site was a field outside Gandhi Krishi Vigyan Kendra, Bangalore, Karnataka, India (site 1); the other site was a field in a village named Ullodu (near Bangalore; 13
380 270 N, 77
420 00 E; site 2). Marking, Collection and Rearing Mating and phonotaxis experiments Phonotaxis and mating experiments were performed with adult virgin females. Female nymphal instars of O. henryi for phonotaxis experiments were caught in October 2010 whereas for the mating experiment the animals (female nymphs and adult calling males) were collected between October and November 2011 from field site 2. Female nymphs and adult males were maintained individually in cylindrical plastic boxes (8 cm in diameter, 5 cm height), with perforated lids. Water and food (apple pieces) were provided ad libitum. The animals were maintained on a 12:12 h light:dark cycle at room temperature which varied between 18
C and 24
C (within the natural range of temperature variation in the field). Experiments were performed with females 2e3 weeks after their final moult. Repeatability and morphological correlation Wild male O. henryi adults were captured and marked on their hindlimbs with unique colour codes using a nontoxic paint marker (Edding 780, Edding, St Albans, U.K.). Capturing and marking were carried out in the field after which the animals were immediately released at the same location. This was done for 15 days and then a gap of 2 days was given before the start of observations and recordings. All field observations were carried out on marked individuals. If any new individuals were found during the observations they were immediately captured and marked. Bushes on which animals were found were also marked using coloured flags so that the same animal could be recorded repeatedly within and across nights. For morphological measurements all the marked animals were collected after we had recorded their calls and were preserved in 70% alcohol at 4
C. Sequential Presentation of Different Sized Males to Females Experimental set-up The experiment was conducted indoors in an anechoic chamber. The set-up consisted of two glass petri dishes (diameter: 10 cm; height: 2 cm) placed one on top of another to create a transparent arena 4 cm in height. The base of the petri dish placed at the bottom was covered by host plant leaves. The height and the area of the arena were such that the male and female tree crickets could roam freely and perform all the mating activities. For each trial the male was released inside the arena first, by lifting the top petri dish. Once the male was acclimatized and calm, the female was released in a similar way. The trials were conducted in complete darkness and an IR-sensitive video camera (Sony DCRHC96E, Sony Corporation, Japan) was used to record the movements of the animals. Sequential choice experiments were conducted, where each female was exposed to a male on the first night and a new male on the next night. The female had the choice of

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rejecting or mating with the male. The trials had a cutoff time of 1200 s, within which if the female did not mount the male the pair was removed from the arena. The experiments were conducted at ambient temperature (range 24.5e25.5
C) which was close to the mean temperature of 25
C in the field. The temperature of the test arena was continuously monitored during the experiments with a Temperature meter HTC1 (Shanghai Xiang Electric Co., Ltd, Yueqing City, Zhejiang Province, China). Before we started the experiments, the animals were acclimatized for 30 min in the experimental room. The experiments were conducted between 1900 and 2200 hours, the peak activity time for these animals. Morphological measurements and sorting A total of 140 adult calling males were collected from the field. These males were cold anaesthetized by placing them at 4
C for 2 min. Body length of these males was measured using a Leica Stereo Zoom Microscope (M 165C, Leica Microsystems GmbH, Wetzlar, Germany). Pictures of all the males were taken using a digital camera (Leica DFC 290, Leica Microsystems GmbH, Wetzlar, Germany) attached to the microscope. These images were analysed using the software ImageJ version 1.43 (National Institutes of Health, U.S.A.). All the body measurements were performed at least 2 days prior to the experiment. Males that had body lengths greater than a mean þ SD of 11.49 þ 0.59 mm were chosen as big males and males with body lengths below 11.49  0.59 mm were chosen as small males. This was decided using the male body length distribution generated from males collected for morphological correlations (N ¼ 56). A total of 80 males were thus chosen out of which 40 were large males and 40 were small males. Female body lengths were also measured under the microscope after the experiment. We weighed the males and females that were used for the mating experiment 30 min before and immediately after the experiment using a Sartorius weighing balance (CP 124S, sensitivity: 0.1 mg; Sartorius AG, Goettingen, Germany). Experimental design and analysis The experiment was designed as a sequential choice experiment where 40 females were tested. Females were randomly divided into two groups of 20 each. One group of 20 females was presented with large males (L) whereas the other group of 20 females was given small males (S) as their mate on the first night. Ten females were randomly selected from the group of 20 females that mated with a large male on the first night and were given a large male again on the second night (LL), whereas the other 10 females were given a small male on the second night (LS). Similarly 10 females were randomly selected from the group of 20 females that mated with a small male on the first night and were given a large male on the second night (SL), whereas the other 10 females were given a small male on the second night (SS). We used this two-level nested experimental design as the virgin females may mate with males indiscriminately and hence discrimination may not be picked up at the first mating. Such a design also helped us to tease out the effect of previous mating on female preference. Females that accepted the males and mated were allowed to complete the mating, remove the spermatophore and feed on it. Once the process was completed a gap of 5 min was given before the females and males were collected for weighing. The mating video was divided into four segments for the analysis. The (1) duration between first antennation and female mounting, (2) total duration of female mounted on the male before the first dismount, (3) total duration of female mounted on the male (excluding the gaps where the female dismounted) until the end of mating and (4) total duration of spermatophore attachment to the female body were measured. The first two measures were considered as criteria of female preference. Total mount duration was

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measured as the proxy for mating benefit obtained by the females through mounting whereas the total spermatophore attachment duration was taken as a proxy for amount of sperm transferred. A box and whiskers plot was generated for all the parameters using the median values and quartile ranges. To determine whether the females had a preference for male size, a Welch two sample t test was performed for the first-level mating for all the measurements. For the second-level mating an ANOVA or generalized linear model (GLM) was performed where the information regarding its first mating was also included (i.e. whether it mated with a large male or a small male on the first night). This was performed to check whether the females are more discriminating in the second mating as well as whether prior mating with a large or small male influences the discrimination pattern. ANOVA was carried out when the variance was homoscedastic and the distribution was normal, whereas GLM was run when these assumptions were violated leading to overdispersion. Pairwise Tukey HSD tests were performed to check whether each treatment was different from another as ANOVA and GLM provide a general overall result. Correlations between male body size and male body weight, male body size and decrease in male body weight caused by mating, male body size and increase in female body weight after mating were also examined. Morphological Measurements All the morphological measurements were performed on wildcaught males whose calls had been recorded in the field. Two characters were chosen as indicators of male quality: body size and FA. The animals consisted of two groups: one group of 26 animals which had all the call characters measured except SPL and another group of 30 individuals which had only SPL measurements. All body size measurements were made using a Leica Stereo Zoom Microscope (M 165C, Leica Microsystems GmbH, Wetzlar, Germany). Twelve morphological characters were measured to get an estimate of body size, out of which nine were linear measurements (body length, maximum width of the pronotum, minimum width of the pronotum, pronotum length, elytra length, elytra breadth, length of femora, breadth of femora, length of tibiae) and three were area measurements (area of pronotum, mirror area, harp area). Pictures of all these characters were taken using a digital camera (Leica DFC 290, Leica Microsystems GmbH, Wetzlar, Germany) attached to the microscope. These images were analysed using the software ImageJ version 1.43 (National Institutes of Health, U.S.A.). All except the body length and pronotum characters were measured for both left and right sides of the body, but their average was used for further analysis of body size. If any specimen was found to have lost a body part it was rejected from the study. An index of body size was generated using principal components analysis to summarize the variation of all the morphological characters. Next, an index of FA was generated for both the groups by calculating the differences between the left and right measurements of elytral length, elytral breadth, harp area, mirror area, length of femora, length of tibia and breadth of femora. Each value was size scaled by dividing by the mean and then checked for actual FA, which is that the trait has a normal distribution with a mean of zero. Morphological characters for both the groups that satisfied the above criteria (elytral length, harp area, femoral length and tibial length and femoral breadth) were chosen to carry out principal components analysis. Correlations between indices of body size and FA with call features were performed. To measure the predictability of male morphological phenotypes from the call structure, a multiple regression analysis was performed. The dependent variables were the index of body size and FA, and call features (temporal and

spectral) were chosen as the independent variables. Predictability of morphology by SPL measurements was examined separately as the samples were nonoverlapping. Repeatability Measurement Call recording and analysis All recordings were made in the field between 1900 and 2200 hours (peak calling period for O. henryi). Male calls were recorded on a solid-state audio recorder (Marantz PMD-660, Kanagawa, Japan) using a Sennheiser (Wennebostel, Germany) MKH20 P48 microphone (frequency range 20 Hz to 20 kHz). All calls were recorded at a sampling rate of 44.1 kHz and were saved as .wav (uncompressed) files. Recordings were made at a distance of 20 cm from the males. The ambient temperature during recording was also noted using a Testo 110 Precision Thermometer (Testo Ltd, Hampshire, U.K.) at a similar distance from the males. SPL of the calls of individual males was measured in the field at 20 cm from the males using a Bruel and Kjaer Sound Level Meter, Type 2231 (Bruel & Kjaer, Naerum, Denmark) with a ½00 microphone, Type 4155 (20 Hze20 kHz). These animals call by raising their wings perpendicular to the body, so all the recordings and SPL measurements were taken perpendicular to the wing position from the back of the animal. The recorded calls were later observed using Spectra Plus-Professional edition version 3.0a (Pioneer Hill Software, Poulsbo, WA, U.S.A.) and analysed using custom-written programs (Chandra Sekhar Seelamantula, Electrical Engineering, Indian Institute of Science) in MATLAB version 6.5 (MathWorks, Natick, MA, U.S.A.). We analysed 75e80 chirps for each animal and calculated the mean of each call character. Temporal features measured include the chirp period, chirp duration, syllable period, syllable duration, average number of syllables per chirp and chirp duty cycle (Fig. 1). In addition, the carrier frequency of the call was measured (Fig. 1). Temperature dependence was checked for all the call characters using linear regression. The following call characters showed significant temperature dependence: chirp period (b ¼ 0.061, P < 0.05, R2 ¼ 0.75), chirp duration (b ¼ 0.011, P < 0.05, R2 ¼ 0.41), syllable period (b ¼ 0.0009, P < 0.05, R2 ¼ 0.79), syllable duration (b ¼ 0.0005, P < 0.05, R2 ¼ 0.6353) and carrier frequency (b ¼ 97.78, P < 0.05, R2 ¼ 0.88). Duty cycle was calculated after temperature regression of chirp period and chirp duration. SPL (P ¼ 0.42) and average number of syllables per chirp (P ¼ 0.89) showed no relationship with temperature. Repeatability analysis Repeatability was measured using the field recordings at two timescales: across nights and within nights. Across-night repeatability was measured for all the call features for 24 animals across 24 nights where each animal was located on three nights. Similarly, within-night repeatability for all the call characters was measured on another set of 24 animals, where each animal was revisited three times a night, at intervals of 1 h. Call recordings and SPL measurements were made for all the marked animals located. The call features that showed temperature dependence were corrected to the mean temperature (26.8
C for across-night recordings and 28.7
C for within-night recordings) as the temperature distribution did not differ significantly from normality (ShapiroeWilks test: P ¼ 0.279). This was done to make the comparisons independent of the effect of temperature. As repeatability is measured in an ANOVA framework, these values were then tested for normality (ShapiroeWilks test) and homoscedasticity (Bartlett’s test). All the regressed call features as well as SPL and average number of syllables per chirp were normally distributed and their variances were homoscedastic.

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Repeatability was measured using the formula (Lessells & Boag 1987):

  r ¼ s2A = s2 þ s2A where ‘r’ is repeatability, s2A is the between-groups variance component and s2 is the within-groups variance component derived from a one-way analysis of variance. Significance of the F value from the ANOVA was checked. For both within- and across-night repeatability, standard errors and confidence intervals were calculated (Nakagawa & Schielzeth 2010). Statistical differences between within- and across-night repeatabilities for all call features were checked using 84% confidence interval overlap which approximates a significance level of a ¼ 0.05 (Payton et al. 2003). Playback of Synthetic Calls to Females Experimental set-up Experiments were conducted indoors in a test arena. The test arena consisted of two loudspeakers (Creative SBS 240, Creative Technology Ltd, Singapore) placed 120 cm apart, and at a height of 60 cm from the ground on concrete stands (Mhatre et al. 2011). In a straight line between the two speakers a stripped-down branch of H. suaveolens was placed vertically. Similar branches of H. suaveolens were placed horizontally to form a bridge connecting the two speakers and the vertical branch in the middle (see Mhatre et al. 2011 for details). Hyptis suaveolens branches were used to make the set-up natural for O. henryi females. The ground, surrounding walls and the speaker stands were all covered with acoustic foam

(Monarch tapes and foams Ltd, Bangalore, India) to minimize echoes (see Mhatre et al. 2011 for details). For each trial an animal was released at the base of the central branch. The trials were conducted in complete darkness and an IR-sensitive video camera (Sony DCR-TRV 17E, Sony Corporation, Japan) was used to record the movements of the animals. Choice experiments were conducted, in which both the speakers played simultaneously. The animal had a choice of approaching either of the two speakers. The trials had a cutoff time of 120 s, within which if the animal reached either of the speakers it was scored as a positive responder. The experiments were conducted at 25
C which was the mean temperature in the field. The room was either heated with a room heater or cooled with an air conditioner to maintain the desired temperature. The temperature of the test arena was continuously monitored during the experiments with a Testo 110 Precision Thermometer (Testo Ltd, Hampshire, U.K.). Before we started the experiments, the animals were acclimatized for 1 h to the temperature in the experimental room. The experiments were conducted between 1900 and 2200 hours, the peak activity time for these animals. Acoustic stimuli The experiments were performed to determine whether O. henryi females can discriminate between frequencies, despite being broadly tuned to a range of frequencies (2.5e4.5 kHz; Mhatre et al. 2011). Females were provided with a choice of stimuli where the temporal pattern of both the calls was the same but the carrier frequencies were varied. A set of eight combinations was prepared. The frequencies were chosen from the distribution obtained from a set of field recordings of naturally calling O. henryi males, the data having being regressed to 25
C (b ¼ 97.78, P < 0.05, R2 ¼ 0.88), the mean

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temperature. A normal distribution was obtained from this set of field recordings with a mean of 2911 Hz (rounded off to 2900 Hz) and standard deviation of 122 Hz (rounded off to 125 Hz). The combinations tested were: mean versus þ1 SD (M-P1), mean versus þ2 SD (M-P2), mean versus 1 SD (N1-M), mean versus 2 SD (N2-M), þ1 SD versus 1 SD (N1-P1) and þ2 SD versus 2 SD (N2-P2). Two controls were carried out: in the first one, the same stimuli (M-M) were played from both speakers at the same intensity (either 45 dB or 61 dB) to examine motivation for phonotaxis to songs at mean natural frequency at the temperature of testing. In the second control, one stimulus was played 6 dB louder than the other (45 versus 51 dB or 61 versus 67 dB), to examine the localizing ability of females in the presence of two simultaneously presented sound sources, where one source (the louder) was expected to be more attractive than the other. The different carrier frequencies were obtained by multiplying the envelope of a single representative syllable with a sine wave of the desired frequency. This envelope was extracted from a natural call recording. To obtain the temporal pattern the syllable was then repeated at the appropriate syllable repetition rate (59 Hz) to form a chirp, which was in turn repeated at the appropriate chirp repetition rate (1.57 Hz) to form the call. The syllable period was 16.8 ms with the duration being 11.0 ms. The chirp period was 633.7 ms with the chirp duration being 250.8 ms.

exactly between and at the same elevation as the two speakers (see Mhatre et al. 2011 for details). Each animal was tested at the two SPLs (45 dB and 61 dB SPL) on 2 consecutive days. The order of exposure to the two SPLs was randomized. On a given night, the order of exposure to seven of the eight combinations was random. The stimulus combination of M-M with a 6 dB difference was always played last, so as not to affect the results of the main experiment, in which intensity was held constant between any pair of stimuli, and only frequency was varied. To determine whether females had a preference for any frequency among the call combinations presented, a Pearson chisquare test was conducted. The animals could approach either of the two speakers in the arena with equal probability since they were equidistant from the junction. The null hypothesis was that the animals showed no preference for either stimulus (P ¼ 0.5). A total of 26 animals were tested. All statistical analyses were performed using the software R version 2.13 (R Development Core Team, University of Auckland, New Zealand). RESULTS Female Preference for Male Body Size In the mating experiments, mounting latency was significantly shorter (in the first mating: Fig. 2a, Table 1) in the females that mated with large males (L) than in those that mated with small males (S). In the second mating also, similar results were found for the females that mated with large males (LL and SL) and those that mated with small males (LS and SS; Fig. 2a; ANOVA results: Table 1). The latency to mount in the second mating was significantly influenced by the first mating (LL and LS versus SL and SS; Fig. 2a; ANOVA result: Table 1). The effect of the first mating became evident from the results of pairwise Tukey HSD tests, which revealed that if females mated with larger males in their first mating, they took longer to mount a smaller male on the second mating (SS versus LS: P ¼ 0.04; Fig. 2a, Table 1). Such an effect was

Experimental design and analysis The animals were tested at two intensities: 45 dB SPL (near threshold intensity) and 61 dB SPL (the mean SPL at 20 cm from a calling male). The intensities chosen were based on field observations and previous experiments (R. Deb, unpublished data). The SPL broadcast from each speaker was adjusted before every trial with the help of a Brüel and Kjær microphone (Type 4155) and integrating Sound Level Meter (Type 2231) (Brüel & Kjær Sound & Vibration Measurement A/S, Denmark) using the fast integration, RMS and flat response settings. The measurement of the SPL was carried out at the junction of the horizontal and vertical branches, which was thus

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SL

SS

Treatments Figure 2. Distributions of durations of the various components of the mating behaviour shown as box plots for all the treatments. The box plots show the median and 25th and 75th percentiles; the whiskers indicate the values within 1.5 times the interquartile range and the circles are outliers. (a) Time from first antennation to mounting, (b) Time from first mount to dismount, (c) Total mount duration, (d) Spermatophore retention time. Width of the boxes reflects sample sizes. L: females mated with large males in the first mating (N ¼ 20 females); S: females mated with small males in the first mating (N ¼ 20 females); LL: females mated with large males both in first and second matings (N ¼ 10 females); LS: females mated with large males in the first mating and small males in the second mating (N ¼ 10 females); SL: females mated with small males in the first mating and large males in the second mating (N ¼ 10 females); SS: females mated with small males both in first and second matings (N ¼ 10 females).

R. Deb et al. / Animal Behaviour 84 (2012) 137e149

143

Table 1 Test statistics for the mating experiment Tests

Time from first antennation to mounting (s)

L versus S (1st mating) Welch t test LL & SL versus LS & SS (2nd mating) ANOVA/GLM LL & LS versus SL & SS (2nd mating) ANOVA/GLM Tukey HSD test (2nd mating) SL versus LL LS versus LL SS versus LL LS versus SL SS versus SL SS versus LS

t¼31.17

Statistic

df 28.05

F¼358.14

1,37

F¼12.16

1,37

Time from first mount to first dismount (s)

Total mount duration (s)

Spermatophore retention time (s)

P

Statistic

df

P

Statistic

df

P

Statistic

df

P

<0.01

t¼10.85

23.28

<0.01

t¼15.95

29.65

<0.01

t¼13.66

28.34

<0.01

<0.01

t¼7.40

<0.01

t¼10.69

<0.01

t¼10.75

0.6

t¼0.49

0.62

0.001

t¼0.52

0.18 <0.01 <0.01 <0.01 <0.01 0.038

0.6 <0.01 <0.01 <0.01 <0.01 0.99

0.99 <0.01 <0.01 <0.01 <0.01 0.94

t¼1.97

<0.01

0.056

0.99 <0.01 <0.01 <0.01 <0.01 0.84

Significant P values are written in bold.

not evident if the female was exposed to a larger male on the second mating (SL versus LL; Fig. 2a, Table 1). All the other treatments were significantly different (P < 0.01) indicating a preference for larger males in the second mating (Fig. 2a, Table 1). The duration of all other mating components (duration of female mounted on the male before the first dismount, total duration of female mounted on the male and total duration of spermatophore attachment to the female body) was significantly longer in the first mating (Fig. 2b, c, d, Table 1) in the females that mated with large males (L) than in those that mated with small males (S). In the second mating also, similar results were found (Fig. 2b, c, d; GLM results: Table 1). There were no effects of the first mating on the second mating for these mating components (LL and LS versus SL and SS; Fig. 2b, c, d; GLM results: Table 1). Pairwise Tukey HSD tests revealed that all treatments in the second mating for these components of the mating process were significantly different from each other (P < 0.01), except SL versus LL and SS versus LS (Fig. 2b, c, d, Table 1). These results corroborated the GLM result stated above. Male body size and male body weight were highly positively correlated (r ¼ 0.84, t78 ¼ 13.84, P < 0.01; Fig. 3a). Male body weight was also significantly positively correlated with the decrease in their body weight after mating (r ¼ 0.42, t78 ¼ 4.05, P < 0.01; Fig. 3b). Weight gained by the females after mating was also significantly positively correlated with body weight of the males (r ¼ 0.415, t78 ¼ 4.03, P < 0.01; Fig. 3c). Morphology and Call Characters Body size and call characters The first principal component for the first group of animals explained 80% of the variance and was taken as a measure of overall body size (Table 2). The first principal component of the second group of animals explained 70% of the variance and was also taken as a measure of overall body size (Table 2). The distribution of body size for both the groups did not deviate significantly from normality (ShapiroeWilk normality test: Group 1: P ¼ 0.19; Group 2: P ¼ 0.12). Chirp period, syllable period, syllable duration and carrier frequency were all significantly correlated with body size (Table 3, Fig. 4b, c, d). Chirp period, syllable period and syllable duration were positively correlated, whereas carrier frequency was negatively correlated with body size (Table 3, Fig. 4a). Chirp duration, chirp duty cycle, average number of syllables per chirp and SPL were not significantly correlated with body size (Table 3).

Multiple regression analysis taking call features (excluding SPL) as independent variables and body size as the dependent variable was significant (R2 ¼ 0.66, F7, 18 ¼ 5.066, P ¼ 0.002). Syllable duration (t ¼ 3.98, P ¼ 0.0005) and carrier frequency (t ¼ 2.35, P ¼ 0.03) were significant factors explaining the variation in size. An ANOVA on the regression model confirmed the observation (syllable duration: F1, 18 ¼ 14.47, P ¼ 0.001; carrier frequency: F1, 18 ¼ 16.75, P ¼ 0.0007). Regression analysis followed by ANOVA on SPL and body size revealed no significant relationship (t ¼ 0.769, R2 ¼ 0.027, F1, 28 ¼ 0.5921, P ¼ 0.448) between them. FA and call characters The first principal component of the first group of animals explained 61% of the variance and was taken as a measure of FA (Table 2). The first principal component of the second group of animals explained 62% of the variance and thus it was also taken as a measure of FA (Table 2). The distribution of FA for both the groups did not deviate significantly from normality (ShapiroeWilk normality test: Group 1: P ¼ 0.053; Group 2: P ¼ 0.051). FA was not significantly correlated with any of the call features (Table 3). Body size and FA were not significantly correlated (Group 1: r ¼ 0.011, P ¼ 0.95; Group 2: r ¼ 0.10, P ¼ 0.58). Multiple regression analysis taking call features as independent variables and FA as the dependent variable was not significant (R2 ¼ 0.41, F7, 18 ¼ 1.815, P ¼ 0.14). Regression analysis on SPL and FA revealed no significant relationship (t ¼ 0.287, R2 ¼ 0.002, F1, 28 ¼ 0.08234, P ¼ 0.776). Carrier frequency and syllable duration of the call act as predictors of male body size. Repeatability of Call Features All the examined call features except syllable period showed significant across-night repeatability (Table 4). The repeatability values of the significant temporal call features ranged between 0.28 and 0.38 (Table 4). Carrier frequency had a high repeatability value of 0.71. The repeatability of SPL was moderate (0.34) and similar to that of some of the temporal features. Carrier frequency of the call thus turned out to be the most repeatable character across nights. Temporal features and SPL had much lower repeatability than carrier frequency (Table 4). Confidence intervals (CI; Table 4) revealed that except for carrier frequency all other call features showed comparable repeatability values (overlap in 84% CI).

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R. Deb et al. / Animal Behaviour 84 (2012) 137e149

Male body weight (mg)

38 (a) 34 30 26 22 18 10

11

12

13

14

Male body size (cm) 4 Male weight loss (mg)

3.5

(b)

3 2.5 2 1.5 1 0.5 0 18

20

22

24

26

28

30

32

34

36

38

40

34

36

38

40

Male body weight (mg)

Female weight gain (mg)

4

(c)

3.5 3 2.5 2 1.5 1 0.5 0 18

20

22

24

26

28

30

32

Male body weight (mg) Figure 3. Correlations between (a) male body size and male body weight, (b) male body weight and loss in male body weight after mating, (c) male body weight and gain in female body weight after mating.

All the examined song features showed significant within-night repeatability (Table 4). The repeatability values of all temporal call features ranged between 0.22 and 0.37 (Table 4). Carrier frequency had a high repeatability of 0.67. The within-night repeatability value of SPL (0.92) was much higher than that across nights (0.34). Temporal characters had much lower repeatability values than SPL and carrier frequency (Table 4). Confidence intervals for all other call features except carrier frequency and SPL were comparable, that is the 84% CI showed overlap of values. The standard errors were comparable for all the call features across and within nights (except SPL). In the pairwise comparisons between across nights and within nights, all the call features (except SPL) had overlapping 84% CIs, that is, they were not significantly different from each other. Carrier frequency had consistently high

repeatability values both across and within nights (Table 4). Carrier frequency was thus the only character that was both a significant predictor of male body size and highly repeatable within and across nights. Female Preference for Call Frequency At 45 dB SPL, for all the six pairs of frequency combinations, there was no significant preference for either carrier frequency (chi-square test: P > 0.07 for all the pairs of frequency combinations; Fig. 5a). At 61 dB SPL, females did not show significant preference between the two carrier frequencies presented in five of six treatments (P > 0.2 for the five pairs of frequency combinations; Fig. 5b). When presented with the pair of carrier frequencies at the extremes of the

R. Deb et al. / Animal Behaviour 84 (2012) 137e149 Table 2 Loadings of the morphological characters on the first principal components and variation accounted for by these components Characters

Body size PC 1 (Group 1)

Body size PC 1 (Group 2)

Body length Maximum pronotum width Minimum pronotum width Pronotum length Pronotum area Elytra length Elytra breadth Harp area Mirror area Femora length Tibia length Femora breadth Proportion of variance

0.328 0.054

0.171 0.051

0.033

0.028

0.068 0.143 0.359 0.147 0.368 0.622 0.291 0.320 0.026 0.801

0.060 0.129 0.375 0.155 0.376 0.701 0.234 0.303 0.012 0.707

FA PC1 (Group 1)

FA PC1 (Group 2)

0.117

0.003

0.172

0.134

0.224 0.047 0.950 0.611

0.310 0.097 0.935 0.62

distribution (N2-P2), females showed a significant preference for the higher frequency (c224 ¼ 8.1667, P ¼ 0.007; Fig. 5b). When the response to pairs of carrier frequencies at the two SPLs (45 and 61 dB) were checked for consistency at the individual level, it was found that there were no significant differences in the response patterns between the two SPLs for all the treatments (P > 0.05 for all the six pairs of combinations). There was also no consistent individual preference for the mean frequency between the two SPLs (P > 0.05). All animals responded with positive phonotaxis in all control trials and were able to locate the playback speaker. At 61 versus 67 dB SPL, females showed a preference for the louder speaker (c2 ¼ 6.55, P < 0.05). At 45 versus 51 dB SPL, there was no significant preference for the louder speaker (c2 ¼ 1.09, P > 0.05). DISCUSSION Female Preference for Larger Males Although there was no rejection of any male (big or small) by the females, discrimination was evident in all the stages of the mating process. Females mounted larger males faster than smaller males both on the first and second mating. Female first mount and total mount duration were longer for larger males in both first and second matings. In contrast to our results, Bateman et al. (2001) and Bussière et al. (2004) showed a first mating advantage in G. bimaculatus and O. nigricornis, respectively, as virgin females were indiscriminate in choosing larger or smaller males. Also, in our sequential design, we did not find any influence of the first mating Table 3 Correlation of call features with index of body size and index of FA Call features

Chirp period Chirp duration Syllable period Syllable duration Carrier frequency Chirp duty cycle Average number of syllables per chirp Sound pressure level

Body size index

FA index

Correlation coefficient

P

Correlation coefficient

P

0.49 0.09 0.57 0.67 L0.56 0.31 0.06

0.01 0.65 0.002 0.0003 0.002 0.12 0.76

0.10 0.36 0.27 0.02 0.08 0.26 0.24

0.64 0.07 0.19 0.92 0.69 0.20 0.25

0.45

0.05

0.78

0.14

Significant values are written in bold.

145

on the subsequent mating except for the latency in mounting. This is in contrast with the results obtained in G. bimaculatus by Bateman et al. (2001) who showed that mated females show higher spermatophore retention time for subsequent bigger males. Other empirical studies have also shown that the quality of a previously encountered male influences the female’s decision (Gibson & Langen 1996). Females in O. henryi thus show preference for larger males irrespective of their own mating status. Females that mated with larger males gained more direct benefit in terms of more weight gain after mating. Larger males lost more body weight from mating than smaller males and females gained more body weight by mating with larger males. This result can be attributed to either more glandular food gifts and/or larger spermatophores provided by the larger males. The fact that larger males provide larger food gifts and/or larger spermatophores has also been shown in various studies (Walker & Gurney 1967; Simmons 1986; Brown et al. 1996; De Luca & Morris 1998; Brown 1999; Johnson et al. 1999). In contrast to these, Bussière et al. (2005) and Brown (2008) found no correlation between male size and nuptial gifts in O. nigricornis. Simmons (1986) and Bateman et al. (2001) showed in G. bimaculatus that females retain the spermatophore of larger males longer than smaller males. Simmons (1986) also showed that longer retention time leads to more sperm transfer. Concordant with these studies, we observed similar results where females retained the spermatophore of larger males longer both in the first and the second mating. Calls as an Indicator of Male Size and Symmetry Brown et al. (1996) showed that syllable duration was an indicator of male size in O. nigricornis. They found that older and larger males produced shorter syllables. In contrast, in O. henryi, we found a positive correlation of chirp period, syllable period and syllable duration with size. Syllable duration was a predictor of size and larger males called with longer rather than shorter syllable durations. A negative correlation of male size with carrier frequency has often been observed and attributed to the mechanism of sound production: the larger the resonator, the lower the frequency at which it resonates (Brown 1999; Gerhardt & Huber 2002). Larger males have been shown to produce lower frequency calls in several species of katydids, moths and anurans (Brown 1999; Gerhardt & Huber 2002) and also in O. nigricornis (Brown et al. 1996). In the case of O. henryi, we found a negative correlation of carrier frequency with body size in concordance with previous studies and carrier frequency was also a predictor of male size. In mole crickets, Scapteriscus sp. (Forrest & Green 1991), SPL showed a positive correlation with body size and moisture quantity in the ground. In O. henryi, however, we found no significant relationship between SPL and male size and conclude that SPL is not a predictor of male size. Earlier work by Brown et al. (1996) did not find any significant relationship between call features and FA in O. nigricornis. Ryan et al. (1995) examined the cricket frog Acris crepitans and found no significant relationship between FA and call features. In concordance with previous studies, FA did not show significant correlation with any of the call features in O. henryi and none of the call features were significant predictors of FA. In contrast to these results, Simmons (1995) found that larger, older male G. bimaculatus were more symmetric. Repeatability of Call Characters Previous work by Hedrick (1988) showed that male calling bout length of Gryllus integer was highly repeatable (0.85) and 74% heritable. Brown et al. (1996) examined within-night repeatability of call characters in O. nigricornis to determine whether they were

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R. Deb et al. / Animal Behaviour 84 (2012) 137e149

Index of body size (PC1)

4

4

(a)

3

3

2

2

1

1

0

0

-1

-1

-2

-2

(b)

-3 -3 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 0.015 Frequency (kHz) 4

0.017

0.019 0.021 Syllable period (s)

0.023

0.025

4 (c)

3

3

2

2

1

1

0

0

-1

-1

-2

-2

-3 0.35

0.55

0.75

0.95

1.15

(d)

-3 0.008

0.01

0.012

0.014

0.016

Syllable duration (s)

Chirp period (s)

Figure 4. Correlation between male song features and body size. (a) Carrier frequency, (b) syllable period, (c) chirp period, (d) syllable duration. Only those features that showed significant correlation with body size are shown.

reliable predictors of male morphology using laboratory-fed and laboratory-reared animals. It was found that male calls were highly repeatable for carrier frequency (0.98), pulse duration (0.99) and pulse period (0.96). Since many of these call features may be strongly influenced by immediate body condition (Wagner & Hoback 1999; Holzer et al. 2003; Scheuber et al. 2003a; Hunt et al. 2005) feeding and rearing in the laboratory may have biased the repeatability values. In the case of O. henryi, we found that the repeatability values measured in the field for all temporal features were low both across and within nights. Low repeatability of temporal characters in O. henryi was consistent with a field study on a tropical katydid Mecopoda elongata (Nityananda & Balakrishnan 2008). In this study a temporal character (chirp period), when measured across nights, was found to have no repeatability (0.117). The study by Brown et al. (1996) is the only

study in which repeatability of carrier frequency was examined. The repeatability value was found to be high (0.98). In our study also, repeatability of carrier frequency was high, considering both the timescales (0.71 and 0.67). For female mate choice, SPL is an important criterion as females prefer louder males and directional preferences for other call traits can be traded off by an increase in SPL (Gerhardt & Huber 2002). To date no study has examined the repeatability of SPL in true crickets and bush crickets. We found that repeatability of SPL was moderate (0.34) and comparable with the temporal features when measured across nights, but was very high (0.92) when measured within a night. Low repeatability of a trait means higher level of intraindividual variability with respect to interindividual variability, and vice versa (Falconer & McKay 1996). It is known that call features such as chirp

Table 4 Across-night and within-night repeatability of call features Call features Chirp period Chirp duration Duty cycle Syllable period Syllable duration Carrier frequency Sound pressure level Average number of syllables per chirp

Across Within Across Within Across Within Across Within Across Within Across Within Across Within Across Within

Repeatability

Significance of F

SE

Confidence limit (95%)

Confidence limit (84%)

0.384 0.350 0.283 0.220 0.280 0.260 0.120 0.370 0.370 0.310 0.710 0.670 0.340 0.920 0.370 0.290

0.001 0.002 0.012 0.039 0.012 0.019 0.340 0.001 0.001 0.0006 0.001 0 0.002 0 0.002 0.01

0.130 0.132 0.134 0.134 0.134 0.134 0.130 0.131 0.131 0.133 0.082 0.092 0.127 0.027 0.131 0.134

0.1140.653 0.0780.625 0.0050.560 0.0620.493 0.0000.556 0.0230.534 0.1540.385 0.1010.643 0.1050.606 0.0380.591 0.5480.888 0.4840.863 0.0810.604 0.8630.976 0.0980.641 0.0110.566

0.1950.572 0.1590.541 0.0880.477 0.0260.414 0.0850.475 0.0650.455 0.0690.309 0.1800.560 0.1800.560 0.1170.503 0.5910.829 0.5370.803 0.1560.524 0.8810.959 0.1800.560 0.0960.484

Across-night repeatability values are indicated as ‘Across’ and within-night repeatability values are indicated as ‘Within’.

R. Deb et al. / Animal Behaviour 84 (2012) 137e149

147

(a) 20

15

10

No. of responders

5

0

*

(b) 20

15

10

5

0

M-P1

M-P2

N1-M N2-M Stimulus pair

N1P1

N2-P2

Figure 5. Female response to simultaneous playback of pairs of calls differing in carrier frequency at (a) 45 dB SPL and (b) 61 dB SPL (N ¼ 26 females). Black bars ( ) indicate response to the lower frequency call in the stimulus pair while the grey bars ( ) indicate response to the higher frequency call. Asterisk (*) indicates significant differences between the number of responders towards higher and lower frequency for a stimulus pair. M-P1: mean versus þ1 SD; M-P2: mean versus þ2 SD; N1-M: 1 SD versus mean; N2-M: 2 SD versus mean; N1-P1: 1 SD versus þ1 SD; N2-P2: 2 SD versus þ2 SD.

period change with age, which could create excess variance (Ritchie et al. 1995). The repeatability values can be higher or lower depending on how age affects the two variances (within an individual and between individuals). Since the animals were of unknown age, we cannot rule out the effect of age on these repeatability values. As temperature-corrected values for all temperature-dependent call features were used for calculation of repeatability, temperature-dependent changes cannot be an explanation for the variation observed. The relatively low repeatability of the temporal call features suggests that there are significant environmental effects on the traits and they should have low heritability (Boake 1989; Falconer & McKay 1996). The high repeatability of carrier frequency means that it is consistent, less dependent on condition and is likely to have high heritability. Inconsistency between within-night and across-night repeatability values for SPL may be caused by various environmental effects. Such effects include recent conditions (e.g. feeding on a particular night) affecting the adult animals (Simmons & Zuk

1992; Wagner & Hoback 1999; Holzer et al. 2003; Scheuber et al. 2003a, 2004) or past conditions affecting the nymphs and hence the adult’s energy reserve (Scheuber et al. 2003b). SPL, which showed high repeatability within a night but low repeatability across nights, is probably dependent on recent conditions (e.g. feeding on that night). On the other hand, animals produced other call characters at a similar level of consistency over different temporal scales, probably using body fat as an energy reserve, which is independent of present condition. Female Preference for Male Call Carrier Frequency Brown et al. (1996) found that O. nigricornis females showed preference for the calls of lower carrier frequency (indicators of large body size), given a choice of male calls differing in carrier frequency. They, however, did not find a preference for lower or higher frequencies when the calls were presented sequentially or when calls differed only in pulse duration (although pulse duration

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was a reliable indicator of age and body size). Consistent with this study, Mhatre et al. (2011) found, using a no-choice paradigm, that female O. henryi do not show a preference for any particular carrier frequency within the natural range of male carrier frequencies. In contrast to Brown et al. (1996), we did not find a female preference for lower male call carrier frequencies at either SPL value (45 dB and 61 dB). Hence it is unlikely that females are using carrier frequency as a cue to locate larger males. The lack of preference for carrier frequency, even though it is a reliable indicator of male size, may be attributed to the physiology and ecology of the study system. In O. henryi, carrier frequency tends to change with temperature (Metrani & Balakrishnan 2005) and females in this species are broadly tuned to carrier frequency (Mhatre et al. 2011). Thus, two males calling at two different carrier frequencies may indicate merely that they are at different local temperatures in the field, which makes the issue of using frequency as a reliable indicator of size a complex one. This is in contrast to the situation in other groups, such as field crickets, in which carrier frequency varies far less with temperature and could serve as a more reliable indicator of size. It is also possible that the males are spaced far apart such that multiple males are not within the hearing range of an individual female, so that there is no selection pressure to choose between simultaneously presented carrier frequencies. In our population-level analysis, we found a significant female preference for higher frequency in the N2-P2 treatment at 61 dB SPL. This pattern of higher frequency preference was observed only when the two extremes of the male call distribution were presented. The probability of a female encountering such a situation (two simultaneously calling males with frequencies at the two ends of the distribution) is likely to be low in the field, so we do not believe that this will have major consequences, that is, that females will end up approaching smaller males with high probability. Hence, the trend we observed at 45 dB and the significant preference at 61 dB SPL for N2-P2 might be better explained by physiology. The females of this tree cricket species are broadly tuned (Mhatre et al. 2011) and the male call carrier frequency distribution is very narrow (standard deviation: 125 Hz) so it is possible that females can actually discriminate between frequencies only when the two frequencies are from the extreme ends of the distribution. Frequency differences are usually perceived as sound level differences (Gerhardt & Huber 2002). The significant preference that we found for the higher frequencies at 61 dB SPL at the two ends of the distribution could be because the higher frequency is perceived as being significantly louder owing to an asymmetric auditory tuning curve (Gerhardt & Huber 2002; Kostarakos et al. 2008, 2009). The best frequencies for recognition and for localization of a call in a cricket female might also be different, resulting in a mismatch between the male call spectrum and female frequency tuning curve (Kostarakos et al. 2008, 2009). This could result in greater preference for values at one end of the male carrier frequency distribution, in this case for higher frequencies. General Conclusion Females in the tree cricket O. henryi showed discrimination against smaller males during mating, not via outright rejection but in the form of shorter mating durations and shorter spermatophore retention times. However, females did not preferentially approach the lower frequency calls, which are reliable indicators of large males. These results suggest that in O. henryi, calling song may be used largely to locate conspecific males, whereas mating preferences may be based on nonacoustic, possibly chemosensory, cues. We speculate that such a strategy may allow females to maximize both direct and indirect benefits of mating.

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