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Sender–receiver dynamics in leafhopper vibrational duetting
Animal Behaviour 114 (2016) 139e146
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Senderereceiver dynamics in leafhopper vibrational duetting Anka Kuhelj, Maarten de Groot 1, Andrej Blejec, Meta Virant-Doberlet* Department of Organisms and Ecosystems Research, National Institute of Biology, Ljubljana, Slovenia
a r t i c l e i n f o Article history: Received 16 September 2015 Initial acceptance 22 October 2015 Final acceptance 18 January 2016 Available online MS. number: 15-00804R Keywords: calling effort duet coordination mate searching rivalry behaviour signalling adaptability vibrational communication
A coordinated reciprocal exchange of acoustic signals (duetting) is common in arthropods relying on substrate-borne vibrational signalling. Communication between partners is under evolutionary pressures resulting from ecological and sexual selection and reciprocal effects arising from such dynamic interactions may influence the sender's and receiver's mating success. We investigated the influence of female reply duration on male mate-searching effort in the leafhopper Aphrodes makarovi in which the female reply is essential for successful location of the female. In a duet, the beginning of a female reply overlaps the end of the male call and males evaluate only the nonoverlapped duration of the female reply. In playback experiments we varied the duration of female replies within the natural range. The duration of a female reply was negatively correlated with the male calling effort. By increasing her reply duration a female may significantly reduce the male's direct and indirect costs associated with signalling and searching, thus, ultimately, affecting male reproductive success. Males showed high adaptability in signalling behaviour and when female replies were short, searching males shortened the last section of their advertisement calls. This strategy allows the nonoverlapped part of the female reply to be longer irrespective of its overall duration. Despite its deceptively simple form, vibrational duetting may entail more complex interactions than just temporal coordination. © 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Communication between partners is an essential part of reproductive behaviour (e.g. Shuster & Wade, 2003). Signals used in sexual communication enable identification (species, sex, condition, receptivity), as well as the location of a potential partner (Mendelson & Shaw, 2012; Wilkins, Seddon, & Safran, 2013), and therefore provide cues that are involved in mate choice and play an important role in promoting and maintaining reproductive isolation (Kraaijeveld, Kraaijeveld-Smit, & Maan, 2011; Ritchie, 2007). Sexual communication often involves a reciprocal exchange of signals between partners (Bailey, 2003; Hall, 2009; Lewis & Cratsley, 2008; Rodríguez & Barbosa, 2014). Duets are characterized by a predictable temporal association between the partners' airborne or substrate-borne acoustic signals, and temporal coordination between the initiating call and a reply is expressed in reply latency, as well as in alternation or overlapping of signals (Bailey, 2003; Hall, 2009). The coordinated exchange of airborne or substrate-borne acoustic signals has been described in many taxa including arthropods (Bailey, 2003; Uhl & Elias, 2011), amphibians (Emerson & Boyd, 1999), birds (Hall, 2009) and mammals
* Correspondence: M. Virant-Doberlet, Department of Organisms and Ecosystems Research, National Institute of Biology, Ve cna pot 111, SI-1000 Ljubljana, Slovenia. E-mail address: [email protected] (M. Virant-Doberlet). 1 Present address: Slovenian Forestry Institute, Ljubljana, Slovenia.
(Geissmann 2000). While duetting between partners appears to be relatively rare in airborne sound communication (Cooley & Marshall, 2001; Emerson & Boyd, 1999; Hall, 2009; Robinson & Hall, 2002), it is common in arthropods relying on vibrational signalling (Bailey, 2003; Boumans & Johnsen, 2015; Henry et al., 2013; Rodríguez & Barbosa, 2014). In arthropods, where partners establish only a temporary bond prior to copulation, the function of a duet has been associated with mate recognition and mate choice (Rodríguez & Barbosa, 2014), as well as with location of a partner (Derlink, Pavlov ci c, Stewart, & Virant-Doberlet, 2014; Legendre, Marting, & Cocroft, 2012; Polajnar et al., 2014). Duetting is a complex and dynamic interaction in which both partners modify their signals and behaviour according to the partner's reply (de Groot et al., 2012; Polajnar et al., 2014; Rodríguez & Barbosa, 2014; Rodríguez, Haen, Cocroft, & Fowler-Finn, 2012). Detailed studies of reciprocal effects arising from signal exchange that influence the sender's and receiver's individual mating success may improve our understanding of the function and evolution of duets and their role in sexual and ecological selection (Rodríguez & Barbosa, 2014; Wilkins et al., 2013). Furthermore, they may provide valuable insights into the mechanisms of animal communication in general (Bailey, 2003; Hall, 2009). Here, we investigated the role of the duration of a female reply in senderereceiver dynamics in the leafhopper Aphrodes makarovi.
http://dx.doi.org/10.1016/j.anbehav.2016.02.001 0003-3472/© 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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Sexual communication is based on a species-specific vibrational duet initiated by a male advertisement call to which a sexually receptive female responds and the beginning of a female reply overlaps with the last section of the male call (Fig. 1; Bluemel et al., 2014; de Groot et al., 2012). While in this species a higher calling rate increases the probability of the male locating the female (Kuhelj, de Groot, Pajk, Sim ci c, & Virant-Doberlet, 2015), it also has a detrimental effect on the male's survival, due to eavesdropping predators (Virant-Doberlet, King, Polajnar, & Symondson, 2011) and indirect costs arising from high energy expenditure (Kuhelj, de Groot, Pajk, et al., 2015). A reciprocal exchange of vibrational signals in A. makarovi differs from the majority of other duetting systems studied so far, in that female replies are often longer than male calls (Bluemel et al., 2014; de Groot et al., 2012). Furthermore, the duration of the female reply varies substantially (5e60 s). This communication system provided an ideal opportunity to explore in more detail the influence of the duration of the female reply on male signalling and searching behaviour (Rodríguez & Barbosa, 2014). We hypothesized (1) that male mate-searching effort (which includes advertising signalling and walking to locate the female) is correlated with the duration of the female reply and (2) that in order to optimize his searching behaviour the male has to adjust his signalling according to the duration of the female reply. Males have to trigger every female reply and they are able to evaluate only the nonoverlapped duration of the female signal which depends also on the duration of the initiating call (Kuhelj, de Groot, Blejec, & Virant-Doberlet, 2015). Moreover, during his approach to the female, the male is motionless while calling; walking associated with searching behaviour is limited to the period of the nonoverlapped female reply and shortly afterwards (Kuhelj, de Groot, Pajk, et al., 2015). Consequently, a longer nonoverlapped duration of the female reply should enable males to walk longer distances before stopping and calling again. We predicted that longer female replies would be associated with lower male calling effort and that when the female reply is short, males would shorten the duration of their calls in
order to obtain a longer nonoverlapped duration of the female reply. METHODS Study Species Aphrodes makarovi (Hemiptera, Cicadellidae) is a relatively large representative of the leafhopper family (males around 6.5 mm, females around 7.5 mm; Bluemel et al., 2014). This species is widely distributed over the Palaearctic and has also been introduced to North America. Aphrodes makarovi is a phloem feeder and has been found on various host plants and in different habitats, often syntopically with other members of this genus (Bluemel et al., 2014). Field population densities vary greatly between localities as well as between years (Derlink et al., 2014). Males increase their signalling space by moving from plant to plant (‘fly/jump-call’ strategy) and a reply from a sexually receptive female triggers a more localized search on a plant. The female reply is essential for successful mating, since the male does not approach the female if she does not respond. A female mates only once in her lifetime (Bluemel et al., 2014). In rivalry situations (trio: two males and one female) males produce masking signals that overlap either the latter part of the female reply or, less frequently, the call of another male (Kuhelj, de Groot, Pajk, et al., 2015; Appendix Fig. A1). Males of A. makarovi produce long and complex advertisement calls with a stereotyped structure composed of chirps and regularly repeated pulses (Bluemel et al., 2014; de Groot et al., 2012; Derlink et al., 2014; Fig. 1). The nonspecific and highly variable first section (makarovi element 0 (Me0)) is followed by species-specific elements Me1eMe3. The female reply is a series of regularly repeated pulses and there is no difference in the pulse repetition time between the last section of the male call (Me3) and the female reply. Both signals have broadband frequency characteristics; however, the dominant frequency of the male call usually lies between 100 and 300 Hz, while in the female reply most energy is
Frequency (kHz)
5 (a)
4 3 2 1
Velocity (relative)
+1 (b)
0
2s –1
Me1 Me0
Me2
Me3
Figure 1. Representative maleefemale duet in A. makarovi. The (a) spectrogram (FFT, window size 4096 samples, 50% overlap) and (b) the corresponding waveform are shown. Me0eMe3: elements in male advertisement call as described in Derlink et al., 2014.
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contained in the frequency range 500e1500 Hz (Bluemel et al., 2014; de Groot et al., 2012; Fig. 1). Collection and Maintenance Last-stage nymphs and young adult leafhoppers were collected using a sweep net in alfalfa, Medicago sativa, and stinging nettle, Urtica dioica, at various localities in Slovenia. In the laboratory, males and females were kept separated by sex and age in plastic boxes (38 26 cm and 27 cm high) containing up to 20 individuals at 23e28 C, 50e70% humidity and 15:9 h light:dark photoperiod. They were fed with cut plants placed in vials filled with water and replaced twice a week. Owing to morphological similarities between Aphrodes species, the species identity of males used in behavioural tests was determined prior to experiments by recording their vibrational signals (Derlink et al., 2014). Adult A. makarovi males used in behavioural trials were 3e4 weeks old (time after eclosion) and were put individually in plastic cups (volume 0.5 litres) 1 day before the start of experiments. Ethical Note The study does not involve endangered or protected species and no permits were required for the field collection. All experiments complied with national laws on animal experimentation. Leafhoppers were maintained under optimal conditions, handled with care and not harmed at any stage. Experimental Set-up For all behavioural tests we used an experimental set-up that enabled bilateral playback stimulation (described in de Groot, Cokl, & Virant-Doberlet, 2011; de Groot et al., 2012; Kuhelj, de Groot, Pajk, et al., 2015). The top of the nettle plant (height approximately 25 cm) was cut off and the bottom of the stem was inserted into a vial filled with water to prevent withering. All leaves, except the pair at the apex and another pair approximately 12 cm down the stem, were removed. Each of the lower leaves was attached to a separate vibration exciter and a new plant was taken every second day. We applied female vibrational replies (described below) to the leaf via the conical tip of the 5 cm metal rod (4 mm in diameter) screwed firmly into the head of a vibration exciter (Minishaker type 4810, Brüel & Kjær, Nærum, Denmark) and fixed to the tip of the leaf with Blu-tack. The vibration exciter was driven from the computer via a Sound Blaster X-Fi Surround 5.1 pro sound card (Creative, Singapore) by Cool Edit Pro2 (Syntrillium Software, Phoenix, AZ, U.S.A.). Female replies and male vibrational signals were registered from the reflective tape placed on the nettle stem 1 cm below the branching point of the lower leaves with a laser vibrometer (PDV-100, Polytech, GmbH, Waldbronn, Germany) and stored in a computer using the above-mentioned sound card and Cool Edit Pro2 software at the sampling rate 48 kHz and 16-bit resolution. We regularly checked that the frequency characteristics of playback signals recorded on the plant corresponded to the input signal by comparing spectral properties of the applied and recorded vibrational signals. Male behaviour together with recorded vibrational signals were simultaneously filmed with a 3CCD video camcorder (Canon DM XM2). Experimental Protocol A single male was placed on a leaf at the apex of the nettle plant and to induce him to call we played a prerecorded duet once.
141
Afterwards, the male received a manually triggered female reply (see below) to every advertisement call he made. To simulate a natural duet we timed the onset of each female reply with the typical decrease in the amplitude at the end of the male call, since this corresponds to the timing of the female response often observed in a natural A. makarovi duet (de Groot et al., 2011, 2012). Males are not able to detect a female reply while calling and in order to trigger an appropriate behavioural response of the male, a female reply has to continue immediately after the end of the initiating call (Kuhelj, de Groot, Blejec, et al., 2015). The amplitude of stimulation was adjusted to the level of natural male calls and female replies registered at the point of recording (male call: 0.3 mm/s; female reply: 0.2 mm/s). The side from which we applied the female reply changed randomly. The trials continued for 15 min after stimulation with a duet or until the male localized the vibration exciter, whichever came first. We tested males in two experimental series that each included 12 males (24 males altogether). In both series males were tested with the same four playback treatments in which we varied the duration of the female reply within the natural range of durations determined previously (Bluemel et al., 2014; de Groot et al., 2012). The female signal used in playbacks was chosen from the signal library and was composed of natural pulses assembled by Cool Edit Pro 2. This composed female reply and manual playback application trigger the same male signalling and searching behaviour as observed in natural mating behaviour (de Groot et al., 2012). In the control treatment F10 the duration of the female reply was 10.4 s which corresponded to the average nonoverlapped value. The F5 treatment represented a shorter-than-average female reply (duration 5.2 s), while two values (20.8 and 41.6 s) corresponded to the longer-than-average female responses (F20 and F41 treatments). Males were tested only once per day and each male was tested once with each female reply. The treatment order was randomized for each male. We monitored the following behavioural parameters: calling rate (number of calls/min of trial), duration of the replyecall interval (time between the end of the female reply and the beginning of the following male call), number of males searching (defined as leaving the apex of the nettle plant and walking during the female reply), number of males locating the source (i.e. vibrated leaf), searching time (time needed to locate the source after the onset of searching) and number of males producing masking signals. To get an insight into the dynamics of the exchange of male and female vibrational signals we measured the duration of the last section (Me3) within the calls (Fig. 1). To compare the effect of the duration of the female reply on male and female signalling effort, we calculated the cumulative calling duration needed to locate the female, which was defined as the sum of durations of all individual calls produced during the trial. In A. makarovi, male longevity is also negatively correlated with the cumulative calling duration (Kuhelj, de Groot, Pajk, et al., 2015). The corresponding cumulative signalling duration for females was obtained by summing the total durations of the playback replies.
Analyses We included in the analyses only males that produced advertisement calls since calling indicates that males were motivated to find a mate. Only males that located the source of the female reply were included in the analyses of searching time and cumulative signalling duration. We compared the numbers of males and values obtained in treatments with short and long female replies with the
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numbers and values obtained in the control F10 treatment (i.e. female reply of average duration). Since signalling and behavioural parameters were often not normally distributed (ShapiroeWilk test: P < 0.05) data were analysed with nonparametric tests. We compared values using the Wilcoxon matched-pairs signed-ranks test which takes into account male individual identity. One- and two-tailed Fisher's exact tests were used to compare the numbers of searching males, numbers of males locating the source and numbers of males producing masking signals. We used the Wilcoxon rank sum test to compare the durations of the replyecall intervals in males that located or failed to locate the source in the allotted time and the cumulative signalling durations for males and females. To analyse the dynamics of reciprocal exchange of male and female vibrational signals, males were divided into two categories according to their behaviour: males that called but did not search (calling males) and males that both called and searched for the source of the female reply (searching males). For each trial we took the Me3 duration of the first call in the trial as a reference, since males were not yet able to evaluate the duration of the female reply. Since Me3 durations in the first call did not differ significantly between treatments and male categories (Appendix Table A1), we determined the proportion of calls produced during the rest of the trial, in which the Me3 section was shorter than in the first call. For each male category within the treatment we determined whether the proportion of these calls deviated significantly from a random distribution (0.5) using a two-tailed, one-sample t test. All statistical analyses were conducted using R version 2.15.2 (R Development Core Team, 2012). RESULTS The proportion of searching males was high in all treatments (Fig. 2a). In comparison with duets that included the female reply of average duration (F10 treatment), the number of males locating the source of the shortest and the longest female replies was significantly lower (Fig. 2b). Males needed significantly more time to locate the source in the F5 and F20 treatments than the source of the average female reply (Wilcoxon signed-ranks test: F5: V ¼ 2, N ¼ 7, P ¼ 0.047; F20: V ¼ 1, N ¼ 7, P ¼ 0.031; Fig. 2c). In males that located the source, the calling rate decreased with increasing female reply duration (Wilcoxon signed-ranks test: F5: V ¼ 1, N ¼ 7, P ¼ 0.031; F20: V ¼ 26, N ¼ 7, P ¼ 0.047; F41: V ¼ 21, N ¼ 7, P ¼ 0.031; Fig. 2d). The duration of the replyecall interval did not differ between treatments (Wilcoxon signed-ranks test with Bonferroni correction) and searching males usually called 2e3 s after the end of the female reply (Appendix Fig. A2). In comparison with the F10 treatment, the cumulative calling duration needed to locate the source of the shorter-than-average female reply (F5 treatment) was significantly longer, while it was significantly shorter when males received the longest reply (Wilcoxon rank sum test: F5: W ¼ 99, N1 ¼ 14, N2¼8, P ¼ 0.002; F41: W ¼ 9, N1 ¼ 14, N2 ¼ 7, P ¼ 0.002; Fig. 3a). In contrast, when female replies were shorter than average, the cumulative signalling duration of the female replies in the F5 treatment did not differ from those in the F10 treatment, while it was significantly longer in both treatments with the longer-than-average female replies (F20 and F41 treatments; Wilcoxon rank sum test: F20: W ¼ 119, N1 ¼ 14, N2 ¼ 10, P ¼ 0.004; F41: W ¼ 82, N1¼ 14, N2 ¼ 7, P ¼ 0.015; Fig. 3b). Furthermore, while in duets with the average female reply, the cumulative signalling duration for males and females did not differ (Wilcoxon rank sum test: W ¼ 122, N1 ¼ N2 ¼ 14, P ¼ 0.280); it was significantly higher in males when the female reply was short (Wilcoxon rank sum test: W ¼ 62, N1 ¼ N2 ¼ 8, P ¼ 0.002) and significantly
higher in females when males received longer-than-average female replies (Wilcoxon rank sum test: W ¼ 24, N1 ¼ N2 ¼ 10, P ¼ 0.045 and W ¼ 0, N1 ¼ N2 ¼ 7, P ¼ 0.002 for 20.8 and 41.6 s reply, respectively). When males received the short (F5) and average (F10) female replies, in searching males, the proportion of calls with the Me3 section shorter than in the first call was significantly higher than random (Table 1). In contrast, in the presence of the longer-than-average female replies, the searching males did not consistently shorten the Me3 section. Although under our experimental conditions, in which the female reply was timed to the end of the male call, by shortening their calls males never achieved a longer nonoverlapped duration of the female reply, taken together these results suggest that males searching for the source of short replies adjusted their signalling according to the duration of the female reply. In all treatments, some males showed rivalry behaviour expressed as masking signals that overlapped the latter part of the female reply and the number of males producing these signals in treatments with longer-than-average female replies was significantly higher than in the F10 treatment (Fig. 4). DISCUSSION Communication between partners is under strong evolutionary pressures resulting from ecological and sexual selection (Maan & Seehausen, 2011; Mendelson & Shaw, 2012; Rodríguez et al., 2013), duetting being no exception (Bailey, 2003; Hall, 2009; Rodríguez & Barbosa, 2014). In agreement with our hypotheses, the duration of a female reply had profound effects on male matesearching behaviour and influenced the likelihood of finding the female. Overall, our results show the importance of female signals in senderereceiver dynamics which, ultimately, affects male reproductive success. In general, longer replies should provide males with better mate choice and searching cues (de Groot et al., 2011; Rodríguez & Barbosa, 2014; Rodríguez et al., 2012). The results of the present study show that a duet structure, in which the beginning of a female reply overlaps the end of the initiating male call, may offer males additional control over the reciprocal interaction. In A. makarovi, males obtain the information needed to increase their mating probability by evaluating the nonoverlapped duration of the female reply (Kuhelj, de Groot, Blejec, et al., 2015) and they can increase this duration irrespective of the latency and overall duration of the female reply by shortening the last section of their calls. In this species, it has been suggested that, in a natural mating sequence, the duration of a male call may be influenced by the duration of the previous female reply (de Groot et al., 2012). In the present study, males showed a surprisingly high adaptability in their signalling behaviour that requires from males that they retain the information at least about the duration of their last call and of the corresponding female reply and adjust the duration of the following call accordingly. The crucial role of the nonoverlapped duration of the female reply in the male's calling effort and reproductive success may also favour learning in the context of sexual behaviour. Learning and memory related to various behavioural contexts, including sexual behaviour, are well established in insects, although the available information is limited to only a few model systems (e.g. Dukas, 2006; Mery, 2013; Paur & Gray, 2011). Sexual selection on male phenotypes is often driven by female preference for more complex or longer signals (Ryan & KeddyHector, 1992) and in the treehopper Enchenopa binotata female preference is the strongest source shaping male vibrational signals (Rodríguez, Ramaswamy, & Cocroft, 2006; Sullivan-Beckers &
(a)
1 0.8 0.6 0.4 0.2 0
F10
N=
F5 22
20
*
0.8 0.6 0.4 0.2 0
F41 21
*
N=
F10
F5
F20
F41
14
8
10
7
F5 22
F20 21
F41 21
(d)
4
300
0
F10 20
N=
600
* *
Calling rate (per min)
Searching time (s)
900 (c)
F20 21
143
(b)
1
Proportion of calling males locating the female
Proportion of searching males
A. Kuhelj et al. / Animal Behaviour 114 (2016) 139e146
*
*
*
3
2
1
0 N=
F10
F5
F20
F41
14
8
10
7
Cumulative signalling duration (s)
Figure 2. The effect of duration of the female reply on signalling and searching behaviour of A. makarovi males. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. (a) Proportion of searching males; (b) proportion of calling males locating the source; (c) searching time; (d) calling rate in males locating the source. Proportions and values obtained in the control F10 treatment are shown in black. (a, b) Determined proportion (black or white circle) together with 95% confidence interval for proportions is shown. Asterisks indicate values that are significantly lower than in the F10 treatment (one-tailed Fisher's exact test: P < 0.05). (c, d) Box and whisker plots show the median (black or white line), the 25e75% interquartile range (boxes) and the lowest and the highest data points still within 1.5 times the interquartile range (whiskers). Asterisks indicate a significant difference from the F10 treatment (Wilcoxon signed-ranks test: P < 0.05). N ¼ number of males.
(a) 500
(b) 500
**
**
**
400
400
300
300
200
200
100
100
0 N=
F10
F5
F20
F41
14
8
10
7
0 N=
*
F10
F5
F20
F41
14
8
10
7
Figure 3. Cumulative signalling duration needed to locate the source of the female reply for (a) males and (b) females. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Values obtained in the control F10 treatment are shown in black. Box and whisker plots show the median (black or white line), the 25e75% interquartile range (boxes), the lowest and the highest data points still within 1.5 times the interquartile range (whiskers) and outliers (circles). Asterisks indicate values that differ significantly from the F10 treatment (Wilcoxon rank sum test: *P < 0.05; **P < 0.01). N ¼ number of males.
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Table 1 Proportion of advertisement calls with Me3 section shorter than in the first call in the trial Treatment
Proportion of calls with shorter Me3 section Calling males
F5 F10 F20 F41
Searching males
N
Mean
N
Mean
5 2 4 2
0.495 0.000a 0.337 0.875a
17 18 17 18
0.692* 0.701* 0.490 0.378
Duration of female reply: F10 treatment: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Calling males: males that called, but did not search for the source of a female reply. Searching males: males that both called and searched for the source of a female reply. N ¼ number of males. Only males that produced more than one call are included. Asterisks indicate values that deviate significantly from a random (0.5) distribution (two-tailed, one-sample t test: df ¼ N 1, *P < 0.05). a Not tested due to the small number of males.
Proportion of males producing masking signals
1
0.8 * * 0.6
0.4
0.2
0 N=
F10
F5
F20
F41
20
22
21
21
Figure 4. The proportions of A. makarovi males producing masking signals. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Proportion obtained in the control F10 treatment is shown in black. Determined proportion (black or white circle) together with 95% confidence interval is shown. Asterisks indicate values that are significantly different from the F10 treatment (two-tailed Fisher's exact test: P < 0.05). N ¼ number of males.
Cocroft, 2010). Females can express their preferences not only by selectively duetting with some males (Bailey, 2003; Rodríguez et al., 2006), but also by producing longer replies (Rodríguez & Barbosa, 2014; Rodríguez et al., 2012). In agreement with our hypothesis, short female replies resulted in higher male calling effort and increased searching time. Taken together, our results suggest that by increasing the duration of her replies from short to average, an A. makarovi female may, for no additional personal cost, significantly reduce the male's calling and searching costs. In this species vibrational calling imposes direct costs due to eavesdropping spiders (Virant-Doberlet et al., 2011), as well as indirect costs due to high energy expenditure (Kuhelj, de Groot, Pajk, et al., 2015); reduced costs may contribute more to a male's lifetime reproductive success. It has been suggested that, in natural duets, during a mating sequence A. makarovi females vary their reply duration according to the duration of the male call to which they are
responding (de Groot et al., 2012). Future studies should elucidate the extent of the observed large variation in female reply duration in this species (Bluemel et al., 2014; de Groot et al., 2012) related to mate choice (Rodríguez & Barbosa, 2014). However, contrary to our expectations, there was no overall beneficial effect of longer-than-average female replies on male searching behaviour. Moreover, producing longer-than-average replies means that the female would have to invest significantly more in signalling than the male. In addition, females may also be affected by direct costs due to eavesdropping predators (VirantDoberlet et al., 2011). Taken together, the results of the present and previous studies (de Groot et al., 2011, 2012; Kuhelj, de Groot, Pajk, et al., 2015) suggest that the durations of the male call and the female reply, the resulting costs and benefits and the processing of directional information in the central nervous system may be tightly coordinated. Neuronal mechanisms underlying vibrational directionality are virtually unexplored (Virant Doberlet, Cokl, & Zorovi c, 2006; Zorovi c, 2011) and we can only speculate that neuronal processing of long vibrational signals may €mer, 2014; Reinhold, 2011). However, be difficult (Hartbauer & Ro reduced success in locating the source of long female replies in the allotted time may also result from increased expression of rivalry behaviour, since males do not move while producing masking signals. The data obtained in the present study were not sufficient to reveal any correlation between the production of masking signals, success in locating the source and searching time. Unexpectedly, the results of the present study revealed behaviour that may underlie complex rivalry interactions. Males retained the information about the presence of a rival acquired from one presentation of a prerecorded duet at the beginning of each trial and produced more masking signals in the presence of long female replies. Alternative mating tactics have been documented in many acoustic communication systems (Gerhardt & Huber, 2002; Hammond & Bailey, 2003; Villarreal & Gilbert, 2014) including those based on vibrational signals (VirantDoberlet et al., 2014). Temporal variation in the social environment (e.g. level of competition and number of mating opportunities) over short timescales favours male behavioural plasticity (Bretman, Gage, & Chapman, 2011). Males of A. makarovi overlap female signals produced in reply to the call of another male (Kuhelj, de Groot, Pajk, et al., 2015) as well as the female reply received in response to their own calls (present study). While it is not yet clear whether long female replies offer some advantage to the eavesdropping rival males relying on satellite behaviour, it should be noted that masking signals are as energetically demanding to produce as advertisement calls (Kuhelj, de Groot, Pajk, et al., 2015). In addition, our results indicate that care should be taken when male signalling is induced by the playback of a maleefemale duet, since in some circumstances even a single presentation may be sufficient to induce the lasting expression of alternative mating tactics. In summary, our study shows that, although in insects duets have a superficially simple form, as in birds (Hall, 2009; Kovach, Hall, Vehrencamp, & Mennill, 2014; Templeton et al., 2013), vibrational duetting in insects entails more complex interactions than just temporal coordination. In A. makarovi mate-searching behaviour is associated with high plasticity in adapting male signalling behaviour to the duration of the female reply. Such attentiveness to the partner is usually associated with duets in birds (Hall, 2009; Kovach et al., 2014). The reciprocal exchange of vibrational signals is a highly complex interaction in which the female reply plays an important role, not only in male signalling and searching behaviour but also in the expression of rivalry tactics. Selective pressures driving the evolution of duetting are likely to
A. Kuhelj et al. / Animal Behaviour 114 (2016) 139e146
depend on various ecological and social interactions (Bailey, 2003; Hall, 2009), as well as on processes involved in sexual selection and reproductive isolation (Derlink et al., 2014; Rodríguez & Barbosa, 2014; Wilkins et al., 2013). Acknowledgments We thank our colleagues from the Department of Organisms and Ecosystems Research at the National Institute of Biology for their help with field work and maintaining leafhoppers in the laboratory. Funding was provided by the Slovenian National Research Agency (PhD fellowship 1000-11-310197 to A.K., research project J1-2181 and research programme P1-0255). We also thank the anonymous referees for their helpful comments.
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APPENDIX
5 Frequency (kHz)
(a) 4 3 2 1
Velocity (relative)
+1 (b)
0
0.5 s –1
Figure A1. Representative masking signal of an A. makarovi male. The (a) spectrogram (FFT, window size 1024 samples, 80% overlap) and (b) the corresponding waveform are shown.
Table A1 Durations of the Me3 section in the first male advertisement call in the trial for each treatment and male category
Reply - call interval (s)
40
Treatment
F5 F10 F20 F41
30
10
N=
Calling males
Searching males
N
Mean±SD
N
Mean±SD
5 2 4 3
9.6±1.9 9.5±0.1 8.8±1.6 8.8±3.2
17 18 17 18
10.0±1.8 9.6±1.9 10.1±2.4 9.6±2.1
Duration of female reply: F10 treatment: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Calling males: males that called but did not search for the source of a female reply. Searching males: males that both called and searched for the source of a female reply; this category includes males that were searching for, but did not locate, the source of the female reply in the allotted time, as well as males that located the source. N ¼ number of males in each category. Me3 durations in the first call did not differ significantly between treatments and male categories (KruskaleWallis test: c27 ¼ 2.706, P ¼ 0.911).
20
0
Duration of Me3 section in the first call (s)
F10
F5
F20
F41
18
17
17
18
Figure A2. The replyecall interval in searching A. makarovi males. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Values obtained in the control F10 treatment are shown in black. Box and whisker plots show the median (black or white line), the 25e75% interquartile range (boxes), the lowest and the highest data points still within 1.5 times the interquartile range (whiskers) and outliers (circles). N ¼ number of males.
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Senderereceiver dynamics in leafhopper vibrational duetting Anka Kuhelj, Maarten de Groot 1, Andrej Blejec, Meta Virant-Doberlet* Department of Organisms and Ecosystems Research, National Institute of Biology, Ljubljana, Slovenia
a r t i c l e i n f o Article history: Received 16 September 2015 Initial acceptance 22 October 2015 Final acceptance 18 January 2016 Available online MS. number: 15-00804R Keywords: calling effort duet coordination mate searching rivalry behaviour signalling adaptability vibrational communication
A coordinated reciprocal exchange of acoustic signals (duetting) is common in arthropods relying on substrate-borne vibrational signalling. Communication between partners is under evolutionary pressures resulting from ecological and sexual selection and reciprocal effects arising from such dynamic interactions may influence the sender's and receiver's mating success. We investigated the influence of female reply duration on male mate-searching effort in the leafhopper Aphrodes makarovi in which the female reply is essential for successful location of the female. In a duet, the beginning of a female reply overlaps the end of the male call and males evaluate only the nonoverlapped duration of the female reply. In playback experiments we varied the duration of female replies within the natural range. The duration of a female reply was negatively correlated with the male calling effort. By increasing her reply duration a female may significantly reduce the male's direct and indirect costs associated with signalling and searching, thus, ultimately, affecting male reproductive success. Males showed high adaptability in signalling behaviour and when female replies were short, searching males shortened the last section of their advertisement calls. This strategy allows the nonoverlapped part of the female reply to be longer irrespective of its overall duration. Despite its deceptively simple form, vibrational duetting may entail more complex interactions than just temporal coordination. © 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Communication between partners is an essential part of reproductive behaviour (e.g. Shuster & Wade, 2003). Signals used in sexual communication enable identification (species, sex, condition, receptivity), as well as the location of a potential partner (Mendelson & Shaw, 2012; Wilkins, Seddon, & Safran, 2013), and therefore provide cues that are involved in mate choice and play an important role in promoting and maintaining reproductive isolation (Kraaijeveld, Kraaijeveld-Smit, & Maan, 2011; Ritchie, 2007). Sexual communication often involves a reciprocal exchange of signals between partners (Bailey, 2003; Hall, 2009; Lewis & Cratsley, 2008; Rodríguez & Barbosa, 2014). Duets are characterized by a predictable temporal association between the partners' airborne or substrate-borne acoustic signals, and temporal coordination between the initiating call and a reply is expressed in reply latency, as well as in alternation or overlapping of signals (Bailey, 2003; Hall, 2009). The coordinated exchange of airborne or substrate-borne acoustic signals has been described in many taxa including arthropods (Bailey, 2003; Uhl & Elias, 2011), amphibians (Emerson & Boyd, 1999), birds (Hall, 2009) and mammals
* Correspondence: M. Virant-Doberlet, Department of Organisms and Ecosystems Research, National Institute of Biology, Ve cna pot 111, SI-1000 Ljubljana, Slovenia. E-mail address: [email protected] (M. Virant-Doberlet). 1 Present address: Slovenian Forestry Institute, Ljubljana, Slovenia.
(Geissmann 2000). While duetting between partners appears to be relatively rare in airborne sound communication (Cooley & Marshall, 2001; Emerson & Boyd, 1999; Hall, 2009; Robinson & Hall, 2002), it is common in arthropods relying on vibrational signalling (Bailey, 2003; Boumans & Johnsen, 2015; Henry et al., 2013; Rodríguez & Barbosa, 2014). In arthropods, where partners establish only a temporary bond prior to copulation, the function of a duet has been associated with mate recognition and mate choice (Rodríguez & Barbosa, 2014), as well as with location of a partner (Derlink, Pavlov ci c, Stewart, & Virant-Doberlet, 2014; Legendre, Marting, & Cocroft, 2012; Polajnar et al., 2014). Duetting is a complex and dynamic interaction in which both partners modify their signals and behaviour according to the partner's reply (de Groot et al., 2012; Polajnar et al., 2014; Rodríguez & Barbosa, 2014; Rodríguez, Haen, Cocroft, & Fowler-Finn, 2012). Detailed studies of reciprocal effects arising from signal exchange that influence the sender's and receiver's individual mating success may improve our understanding of the function and evolution of duets and their role in sexual and ecological selection (Rodríguez & Barbosa, 2014; Wilkins et al., 2013). Furthermore, they may provide valuable insights into the mechanisms of animal communication in general (Bailey, 2003; Hall, 2009). Here, we investigated the role of the duration of a female reply in senderereceiver dynamics in the leafhopper Aphrodes makarovi.
http://dx.doi.org/10.1016/j.anbehav.2016.02.001 0003-3472/© 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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Sexual communication is based on a species-specific vibrational duet initiated by a male advertisement call to which a sexually receptive female responds and the beginning of a female reply overlaps with the last section of the male call (Fig. 1; Bluemel et al., 2014; de Groot et al., 2012). While in this species a higher calling rate increases the probability of the male locating the female (Kuhelj, de Groot, Pajk, Sim ci c, & Virant-Doberlet, 2015), it also has a detrimental effect on the male's survival, due to eavesdropping predators (Virant-Doberlet, King, Polajnar, & Symondson, 2011) and indirect costs arising from high energy expenditure (Kuhelj, de Groot, Pajk, et al., 2015). A reciprocal exchange of vibrational signals in A. makarovi differs from the majority of other duetting systems studied so far, in that female replies are often longer than male calls (Bluemel et al., 2014; de Groot et al., 2012). Furthermore, the duration of the female reply varies substantially (5e60 s). This communication system provided an ideal opportunity to explore in more detail the influence of the duration of the female reply on male signalling and searching behaviour (Rodríguez & Barbosa, 2014). We hypothesized (1) that male mate-searching effort (which includes advertising signalling and walking to locate the female) is correlated with the duration of the female reply and (2) that in order to optimize his searching behaviour the male has to adjust his signalling according to the duration of the female reply. Males have to trigger every female reply and they are able to evaluate only the nonoverlapped duration of the female signal which depends also on the duration of the initiating call (Kuhelj, de Groot, Blejec, & Virant-Doberlet, 2015). Moreover, during his approach to the female, the male is motionless while calling; walking associated with searching behaviour is limited to the period of the nonoverlapped female reply and shortly afterwards (Kuhelj, de Groot, Pajk, et al., 2015). Consequently, a longer nonoverlapped duration of the female reply should enable males to walk longer distances before stopping and calling again. We predicted that longer female replies would be associated with lower male calling effort and that when the female reply is short, males would shorten the duration of their calls in
order to obtain a longer nonoverlapped duration of the female reply. METHODS Study Species Aphrodes makarovi (Hemiptera, Cicadellidae) is a relatively large representative of the leafhopper family (males around 6.5 mm, females around 7.5 mm; Bluemel et al., 2014). This species is widely distributed over the Palaearctic and has also been introduced to North America. Aphrodes makarovi is a phloem feeder and has been found on various host plants and in different habitats, often syntopically with other members of this genus (Bluemel et al., 2014). Field population densities vary greatly between localities as well as between years (Derlink et al., 2014). Males increase their signalling space by moving from plant to plant (‘fly/jump-call’ strategy) and a reply from a sexually receptive female triggers a more localized search on a plant. The female reply is essential for successful mating, since the male does not approach the female if she does not respond. A female mates only once in her lifetime (Bluemel et al., 2014). In rivalry situations (trio: two males and one female) males produce masking signals that overlap either the latter part of the female reply or, less frequently, the call of another male (Kuhelj, de Groot, Pajk, et al., 2015; Appendix Fig. A1). Males of A. makarovi produce long and complex advertisement calls with a stereotyped structure composed of chirps and regularly repeated pulses (Bluemel et al., 2014; de Groot et al., 2012; Derlink et al., 2014; Fig. 1). The nonspecific and highly variable first section (makarovi element 0 (Me0)) is followed by species-specific elements Me1eMe3. The female reply is a series of regularly repeated pulses and there is no difference in the pulse repetition time between the last section of the male call (Me3) and the female reply. Both signals have broadband frequency characteristics; however, the dominant frequency of the male call usually lies between 100 and 300 Hz, while in the female reply most energy is
Frequency (kHz)
5 (a)
4 3 2 1
Velocity (relative)
+1 (b)
0
2s –1
Me1 Me0
Me2
Me3
Figure 1. Representative maleefemale duet in A. makarovi. The (a) spectrogram (FFT, window size 4096 samples, 50% overlap) and (b) the corresponding waveform are shown. Me0eMe3: elements in male advertisement call as described in Derlink et al., 2014.
A. Kuhelj et al. / Animal Behaviour 114 (2016) 139e146
contained in the frequency range 500e1500 Hz (Bluemel et al., 2014; de Groot et al., 2012; Fig. 1). Collection and Maintenance Last-stage nymphs and young adult leafhoppers were collected using a sweep net in alfalfa, Medicago sativa, and stinging nettle, Urtica dioica, at various localities in Slovenia. In the laboratory, males and females were kept separated by sex and age in plastic boxes (38 26 cm and 27 cm high) containing up to 20 individuals at 23e28 C, 50e70% humidity and 15:9 h light:dark photoperiod. They were fed with cut plants placed in vials filled with water and replaced twice a week. Owing to morphological similarities between Aphrodes species, the species identity of males used in behavioural tests was determined prior to experiments by recording their vibrational signals (Derlink et al., 2014). Adult A. makarovi males used in behavioural trials were 3e4 weeks old (time after eclosion) and were put individually in plastic cups (volume 0.5 litres) 1 day before the start of experiments. Ethical Note The study does not involve endangered or protected species and no permits were required for the field collection. All experiments complied with national laws on animal experimentation. Leafhoppers were maintained under optimal conditions, handled with care and not harmed at any stage. Experimental Set-up For all behavioural tests we used an experimental set-up that enabled bilateral playback stimulation (described in de Groot, Cokl, & Virant-Doberlet, 2011; de Groot et al., 2012; Kuhelj, de Groot, Pajk, et al., 2015). The top of the nettle plant (height approximately 25 cm) was cut off and the bottom of the stem was inserted into a vial filled with water to prevent withering. All leaves, except the pair at the apex and another pair approximately 12 cm down the stem, were removed. Each of the lower leaves was attached to a separate vibration exciter and a new plant was taken every second day. We applied female vibrational replies (described below) to the leaf via the conical tip of the 5 cm metal rod (4 mm in diameter) screwed firmly into the head of a vibration exciter (Minishaker type 4810, Brüel & Kjær, Nærum, Denmark) and fixed to the tip of the leaf with Blu-tack. The vibration exciter was driven from the computer via a Sound Blaster X-Fi Surround 5.1 pro sound card (Creative, Singapore) by Cool Edit Pro2 (Syntrillium Software, Phoenix, AZ, U.S.A.). Female replies and male vibrational signals were registered from the reflective tape placed on the nettle stem 1 cm below the branching point of the lower leaves with a laser vibrometer (PDV-100, Polytech, GmbH, Waldbronn, Germany) and stored in a computer using the above-mentioned sound card and Cool Edit Pro2 software at the sampling rate 48 kHz and 16-bit resolution. We regularly checked that the frequency characteristics of playback signals recorded on the plant corresponded to the input signal by comparing spectral properties of the applied and recorded vibrational signals. Male behaviour together with recorded vibrational signals were simultaneously filmed with a 3CCD video camcorder (Canon DM XM2). Experimental Protocol A single male was placed on a leaf at the apex of the nettle plant and to induce him to call we played a prerecorded duet once.
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Afterwards, the male received a manually triggered female reply (see below) to every advertisement call he made. To simulate a natural duet we timed the onset of each female reply with the typical decrease in the amplitude at the end of the male call, since this corresponds to the timing of the female response often observed in a natural A. makarovi duet (de Groot et al., 2011, 2012). Males are not able to detect a female reply while calling and in order to trigger an appropriate behavioural response of the male, a female reply has to continue immediately after the end of the initiating call (Kuhelj, de Groot, Blejec, et al., 2015). The amplitude of stimulation was adjusted to the level of natural male calls and female replies registered at the point of recording (male call: 0.3 mm/s; female reply: 0.2 mm/s). The side from which we applied the female reply changed randomly. The trials continued for 15 min after stimulation with a duet or until the male localized the vibration exciter, whichever came first. We tested males in two experimental series that each included 12 males (24 males altogether). In both series males were tested with the same four playback treatments in which we varied the duration of the female reply within the natural range of durations determined previously (Bluemel et al., 2014; de Groot et al., 2012). The female signal used in playbacks was chosen from the signal library and was composed of natural pulses assembled by Cool Edit Pro 2. This composed female reply and manual playback application trigger the same male signalling and searching behaviour as observed in natural mating behaviour (de Groot et al., 2012). In the control treatment F10 the duration of the female reply was 10.4 s which corresponded to the average nonoverlapped value. The F5 treatment represented a shorter-than-average female reply (duration 5.2 s), while two values (20.8 and 41.6 s) corresponded to the longer-than-average female responses (F20 and F41 treatments). Males were tested only once per day and each male was tested once with each female reply. The treatment order was randomized for each male. We monitored the following behavioural parameters: calling rate (number of calls/min of trial), duration of the replyecall interval (time between the end of the female reply and the beginning of the following male call), number of males searching (defined as leaving the apex of the nettle plant and walking during the female reply), number of males locating the source (i.e. vibrated leaf), searching time (time needed to locate the source after the onset of searching) and number of males producing masking signals. To get an insight into the dynamics of the exchange of male and female vibrational signals we measured the duration of the last section (Me3) within the calls (Fig. 1). To compare the effect of the duration of the female reply on male and female signalling effort, we calculated the cumulative calling duration needed to locate the female, which was defined as the sum of durations of all individual calls produced during the trial. In A. makarovi, male longevity is also negatively correlated with the cumulative calling duration (Kuhelj, de Groot, Pajk, et al., 2015). The corresponding cumulative signalling duration for females was obtained by summing the total durations of the playback replies.
Analyses We included in the analyses only males that produced advertisement calls since calling indicates that males were motivated to find a mate. Only males that located the source of the female reply were included in the analyses of searching time and cumulative signalling duration. We compared the numbers of males and values obtained in treatments with short and long female replies with the
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numbers and values obtained in the control F10 treatment (i.e. female reply of average duration). Since signalling and behavioural parameters were often not normally distributed (ShapiroeWilk test: P < 0.05) data were analysed with nonparametric tests. We compared values using the Wilcoxon matched-pairs signed-ranks test which takes into account male individual identity. One- and two-tailed Fisher's exact tests were used to compare the numbers of searching males, numbers of males locating the source and numbers of males producing masking signals. We used the Wilcoxon rank sum test to compare the durations of the replyecall intervals in males that located or failed to locate the source in the allotted time and the cumulative signalling durations for males and females. To analyse the dynamics of reciprocal exchange of male and female vibrational signals, males were divided into two categories according to their behaviour: males that called but did not search (calling males) and males that both called and searched for the source of the female reply (searching males). For each trial we took the Me3 duration of the first call in the trial as a reference, since males were not yet able to evaluate the duration of the female reply. Since Me3 durations in the first call did not differ significantly between treatments and male categories (Appendix Table A1), we determined the proportion of calls produced during the rest of the trial, in which the Me3 section was shorter than in the first call. For each male category within the treatment we determined whether the proportion of these calls deviated significantly from a random distribution (0.5) using a two-tailed, one-sample t test. All statistical analyses were conducted using R version 2.15.2 (R Development Core Team, 2012). RESULTS The proportion of searching males was high in all treatments (Fig. 2a). In comparison with duets that included the female reply of average duration (F10 treatment), the number of males locating the source of the shortest and the longest female replies was significantly lower (Fig. 2b). Males needed significantly more time to locate the source in the F5 and F20 treatments than the source of the average female reply (Wilcoxon signed-ranks test: F5: V ¼ 2, N ¼ 7, P ¼ 0.047; F20: V ¼ 1, N ¼ 7, P ¼ 0.031; Fig. 2c). In males that located the source, the calling rate decreased with increasing female reply duration (Wilcoxon signed-ranks test: F5: V ¼ 1, N ¼ 7, P ¼ 0.031; F20: V ¼ 26, N ¼ 7, P ¼ 0.047; F41: V ¼ 21, N ¼ 7, P ¼ 0.031; Fig. 2d). The duration of the replyecall interval did not differ between treatments (Wilcoxon signed-ranks test with Bonferroni correction) and searching males usually called 2e3 s after the end of the female reply (Appendix Fig. A2). In comparison with the F10 treatment, the cumulative calling duration needed to locate the source of the shorter-than-average female reply (F5 treatment) was significantly longer, while it was significantly shorter when males received the longest reply (Wilcoxon rank sum test: F5: W ¼ 99, N1 ¼ 14, N2¼8, P ¼ 0.002; F41: W ¼ 9, N1 ¼ 14, N2 ¼ 7, P ¼ 0.002; Fig. 3a). In contrast, when female replies were shorter than average, the cumulative signalling duration of the female replies in the F5 treatment did not differ from those in the F10 treatment, while it was significantly longer in both treatments with the longer-than-average female replies (F20 and F41 treatments; Wilcoxon rank sum test: F20: W ¼ 119, N1 ¼ 14, N2 ¼ 10, P ¼ 0.004; F41: W ¼ 82, N1¼ 14, N2 ¼ 7, P ¼ 0.015; Fig. 3b). Furthermore, while in duets with the average female reply, the cumulative signalling duration for males and females did not differ (Wilcoxon rank sum test: W ¼ 122, N1 ¼ N2 ¼ 14, P ¼ 0.280); it was significantly higher in males when the female reply was short (Wilcoxon rank sum test: W ¼ 62, N1 ¼ N2 ¼ 8, P ¼ 0.002) and significantly
higher in females when males received longer-than-average female replies (Wilcoxon rank sum test: W ¼ 24, N1 ¼ N2 ¼ 10, P ¼ 0.045 and W ¼ 0, N1 ¼ N2 ¼ 7, P ¼ 0.002 for 20.8 and 41.6 s reply, respectively). When males received the short (F5) and average (F10) female replies, in searching males, the proportion of calls with the Me3 section shorter than in the first call was significantly higher than random (Table 1). In contrast, in the presence of the longer-than-average female replies, the searching males did not consistently shorten the Me3 section. Although under our experimental conditions, in which the female reply was timed to the end of the male call, by shortening their calls males never achieved a longer nonoverlapped duration of the female reply, taken together these results suggest that males searching for the source of short replies adjusted their signalling according to the duration of the female reply. In all treatments, some males showed rivalry behaviour expressed as masking signals that overlapped the latter part of the female reply and the number of males producing these signals in treatments with longer-than-average female replies was significantly higher than in the F10 treatment (Fig. 4). DISCUSSION Communication between partners is under strong evolutionary pressures resulting from ecological and sexual selection (Maan & Seehausen, 2011; Mendelson & Shaw, 2012; Rodríguez et al., 2013), duetting being no exception (Bailey, 2003; Hall, 2009; Rodríguez & Barbosa, 2014). In agreement with our hypotheses, the duration of a female reply had profound effects on male matesearching behaviour and influenced the likelihood of finding the female. Overall, our results show the importance of female signals in senderereceiver dynamics which, ultimately, affects male reproductive success. In general, longer replies should provide males with better mate choice and searching cues (de Groot et al., 2011; Rodríguez & Barbosa, 2014; Rodríguez et al., 2012). The results of the present study show that a duet structure, in which the beginning of a female reply overlaps the end of the initiating male call, may offer males additional control over the reciprocal interaction. In A. makarovi, males obtain the information needed to increase their mating probability by evaluating the nonoverlapped duration of the female reply (Kuhelj, de Groot, Blejec, et al., 2015) and they can increase this duration irrespective of the latency and overall duration of the female reply by shortening the last section of their calls. In this species, it has been suggested that, in a natural mating sequence, the duration of a male call may be influenced by the duration of the previous female reply (de Groot et al., 2012). In the present study, males showed a surprisingly high adaptability in their signalling behaviour that requires from males that they retain the information at least about the duration of their last call and of the corresponding female reply and adjust the duration of the following call accordingly. The crucial role of the nonoverlapped duration of the female reply in the male's calling effort and reproductive success may also favour learning in the context of sexual behaviour. Learning and memory related to various behavioural contexts, including sexual behaviour, are well established in insects, although the available information is limited to only a few model systems (e.g. Dukas, 2006; Mery, 2013; Paur & Gray, 2011). Sexual selection on male phenotypes is often driven by female preference for more complex or longer signals (Ryan & KeddyHector, 1992) and in the treehopper Enchenopa binotata female preference is the strongest source shaping male vibrational signals (Rodríguez, Ramaswamy, & Cocroft, 2006; Sullivan-Beckers &
(a)
1 0.8 0.6 0.4 0.2 0
F10
N=
F5 22
20
*
0.8 0.6 0.4 0.2 0
F41 21
*
N=
F10
F5
F20
F41
14
8
10
7
F5 22
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F41 21
(d)
4
300
0
F10 20
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* *
Calling rate (per min)
Searching time (s)
900 (c)
F20 21
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Proportion of calling males locating the female
Proportion of searching males
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*
*
*
3
2
1
0 N=
F10
F5
F20
F41
14
8
10
7
Cumulative signalling duration (s)
Figure 2. The effect of duration of the female reply on signalling and searching behaviour of A. makarovi males. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. (a) Proportion of searching males; (b) proportion of calling males locating the source; (c) searching time; (d) calling rate in males locating the source. Proportions and values obtained in the control F10 treatment are shown in black. (a, b) Determined proportion (black or white circle) together with 95% confidence interval for proportions is shown. Asterisks indicate values that are significantly lower than in the F10 treatment (one-tailed Fisher's exact test: P < 0.05). (c, d) Box and whisker plots show the median (black or white line), the 25e75% interquartile range (boxes) and the lowest and the highest data points still within 1.5 times the interquartile range (whiskers). Asterisks indicate a significant difference from the F10 treatment (Wilcoxon signed-ranks test: P < 0.05). N ¼ number of males.
(a) 500
(b) 500
**
**
**
400
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0 N=
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Figure 3. Cumulative signalling duration needed to locate the source of the female reply for (a) males and (b) females. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Values obtained in the control F10 treatment are shown in black. Box and whisker plots show the median (black or white line), the 25e75% interquartile range (boxes), the lowest and the highest data points still within 1.5 times the interquartile range (whiskers) and outliers (circles). Asterisks indicate values that differ significantly from the F10 treatment (Wilcoxon rank sum test: *P < 0.05; **P < 0.01). N ¼ number of males.
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Table 1 Proportion of advertisement calls with Me3 section shorter than in the first call in the trial Treatment
Proportion of calls with shorter Me3 section Calling males
F5 F10 F20 F41
Searching males
N
Mean
N
Mean
5 2 4 2
0.495 0.000a 0.337 0.875a
17 18 17 18
0.692* 0.701* 0.490 0.378
Duration of female reply: F10 treatment: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Calling males: males that called, but did not search for the source of a female reply. Searching males: males that both called and searched for the source of a female reply. N ¼ number of males. Only males that produced more than one call are included. Asterisks indicate values that deviate significantly from a random (0.5) distribution (two-tailed, one-sample t test: df ¼ N 1, *P < 0.05). a Not tested due to the small number of males.
Proportion of males producing masking signals
1
0.8 * * 0.6
0.4
0.2
0 N=
F10
F5
F20
F41
20
22
21
21
Figure 4. The proportions of A. makarovi males producing masking signals. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Proportion obtained in the control F10 treatment is shown in black. Determined proportion (black or white circle) together with 95% confidence interval is shown. Asterisks indicate values that are significantly different from the F10 treatment (two-tailed Fisher's exact test: P < 0.05). N ¼ number of males.
Cocroft, 2010). Females can express their preferences not only by selectively duetting with some males (Bailey, 2003; Rodríguez et al., 2006), but also by producing longer replies (Rodríguez & Barbosa, 2014; Rodríguez et al., 2012). In agreement with our hypothesis, short female replies resulted in higher male calling effort and increased searching time. Taken together, our results suggest that by increasing the duration of her replies from short to average, an A. makarovi female may, for no additional personal cost, significantly reduce the male's calling and searching costs. In this species vibrational calling imposes direct costs due to eavesdropping spiders (Virant-Doberlet et al., 2011), as well as indirect costs due to high energy expenditure (Kuhelj, de Groot, Pajk, et al., 2015); reduced costs may contribute more to a male's lifetime reproductive success. It has been suggested that, in natural duets, during a mating sequence A. makarovi females vary their reply duration according to the duration of the male call to which they are
responding (de Groot et al., 2012). Future studies should elucidate the extent of the observed large variation in female reply duration in this species (Bluemel et al., 2014; de Groot et al., 2012) related to mate choice (Rodríguez & Barbosa, 2014). However, contrary to our expectations, there was no overall beneficial effect of longer-than-average female replies on male searching behaviour. Moreover, producing longer-than-average replies means that the female would have to invest significantly more in signalling than the male. In addition, females may also be affected by direct costs due to eavesdropping predators (VirantDoberlet et al., 2011). Taken together, the results of the present and previous studies (de Groot et al., 2011, 2012; Kuhelj, de Groot, Pajk, et al., 2015) suggest that the durations of the male call and the female reply, the resulting costs and benefits and the processing of directional information in the central nervous system may be tightly coordinated. Neuronal mechanisms underlying vibrational directionality are virtually unexplored (Virant Doberlet, Cokl, & Zorovi c, 2006; Zorovi c, 2011) and we can only speculate that neuronal processing of long vibrational signals may €mer, 2014; Reinhold, 2011). However, be difficult (Hartbauer & Ro reduced success in locating the source of long female replies in the allotted time may also result from increased expression of rivalry behaviour, since males do not move while producing masking signals. The data obtained in the present study were not sufficient to reveal any correlation between the production of masking signals, success in locating the source and searching time. Unexpectedly, the results of the present study revealed behaviour that may underlie complex rivalry interactions. Males retained the information about the presence of a rival acquired from one presentation of a prerecorded duet at the beginning of each trial and produced more masking signals in the presence of long female replies. Alternative mating tactics have been documented in many acoustic communication systems (Gerhardt & Huber, 2002; Hammond & Bailey, 2003; Villarreal & Gilbert, 2014) including those based on vibrational signals (VirantDoberlet et al., 2014). Temporal variation in the social environment (e.g. level of competition and number of mating opportunities) over short timescales favours male behavioural plasticity (Bretman, Gage, & Chapman, 2011). Males of A. makarovi overlap female signals produced in reply to the call of another male (Kuhelj, de Groot, Pajk, et al., 2015) as well as the female reply received in response to their own calls (present study). While it is not yet clear whether long female replies offer some advantage to the eavesdropping rival males relying on satellite behaviour, it should be noted that masking signals are as energetically demanding to produce as advertisement calls (Kuhelj, de Groot, Pajk, et al., 2015). In addition, our results indicate that care should be taken when male signalling is induced by the playback of a maleefemale duet, since in some circumstances even a single presentation may be sufficient to induce the lasting expression of alternative mating tactics. In summary, our study shows that, although in insects duets have a superficially simple form, as in birds (Hall, 2009; Kovach, Hall, Vehrencamp, & Mennill, 2014; Templeton et al., 2013), vibrational duetting in insects entails more complex interactions than just temporal coordination. In A. makarovi mate-searching behaviour is associated with high plasticity in adapting male signalling behaviour to the duration of the female reply. Such attentiveness to the partner is usually associated with duets in birds (Hall, 2009; Kovach et al., 2014). The reciprocal exchange of vibrational signals is a highly complex interaction in which the female reply plays an important role, not only in male signalling and searching behaviour but also in the expression of rivalry tactics. Selective pressures driving the evolution of duetting are likely to
A. Kuhelj et al. / Animal Behaviour 114 (2016) 139e146
depend on various ecological and social interactions (Bailey, 2003; Hall, 2009), as well as on processes involved in sexual selection and reproductive isolation (Derlink et al., 2014; Rodríguez & Barbosa, 2014; Wilkins et al., 2013). Acknowledgments We thank our colleagues from the Department of Organisms and Ecosystems Research at the National Institute of Biology for their help with field work and maintaining leafhoppers in the laboratory. Funding was provided by the Slovenian National Research Agency (PhD fellowship 1000-11-310197 to A.K., research project J1-2181 and research programme P1-0255). We also thank the anonymous referees for their helpful comments.
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APPENDIX
5 Frequency (kHz)
(a) 4 3 2 1
Velocity (relative)
+1 (b)
0
0.5 s –1
Figure A1. Representative masking signal of an A. makarovi male. The (a) spectrogram (FFT, window size 1024 samples, 80% overlap) and (b) the corresponding waveform are shown.
Table A1 Durations of the Me3 section in the first male advertisement call in the trial for each treatment and male category
Reply - call interval (s)
40
Treatment
F5 F10 F20 F41
30
10
N=
Calling males
Searching males
N
Mean±SD
N
Mean±SD
5 2 4 3
9.6±1.9 9.5±0.1 8.8±1.6 8.8±3.2
17 18 17 18
10.0±1.8 9.6±1.9 10.1±2.4 9.6±2.1
Duration of female reply: F10 treatment: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Calling males: males that called but did not search for the source of a female reply. Searching males: males that both called and searched for the source of a female reply; this category includes males that were searching for, but did not locate, the source of the female reply in the allotted time, as well as males that located the source. N ¼ number of males in each category. Me3 durations in the first call did not differ significantly between treatments and male categories (KruskaleWallis test: c27 ¼ 2.706, P ¼ 0.911).
20
0
Duration of Me3 section in the first call (s)
F10
F5
F20
F41
18
17
17
18
Figure A2. The replyecall interval in searching A. makarovi males. Duration of female reply: control treatment F10: 10.4 s; F5 treatment: 5.2 s; F20 treatment: 20.8 s; F41 treatment: 41.6 s. Values obtained in the control F10 treatment are shown in black. Box and whisker plots show the median (black or white line), the 25e75% interquartile range (boxes), the lowest and the highest data points still within 1.5 times the interquartile range (whiskers) and outliers (circles). N ¼ number of males.