Variability of vibratory signals and mate choice selectivity in the southern green stink bug

Behavioural Processes 61 (2003) 131–142

Variability of vibratory signals and mate choice selectivity in the southern green stink bug b , M. Renou a,∗ , M. Virant-Doberlet b ˇ N. Miklas a , A. Cokl a

INRA Centre de Versailles, Unité de Phytopharmacie et Médiateurs Chimiques, route de St Cyr, F-78026 Versailles, France b NIB, Laboratory for Neurobiology, Veˇ cna pot 111, P.O. Box 141, SI-1001 Ljubljana, Slovenia Received 17 September 2002; received in revised form 27 November 2002; accepted 27 November 2002

Abstract The southern green stink bug, Nezara viridula, has a complex mate recognition system that implicates chemical and acoustic signals. To localise a sexual partner acoustically, a male and female alternate between the male courtship song (MCrS) and the female calling song type 1 (FCS1). Although previous research has revealed that both signals show geographical variability, until now no studies have explored the form of this variability. We analysed the temporal and spectral characteristics of MCrS and FCS1 pulse trains of males and females from a French and a Guadeloupe population. Pulse train duration of the MCrS varied within and between populations. Likewise, spectral and temporal parameters of FCS1 varied within and between the two populations. Although females did not show any behavioural responses to pre-recorded MCrS, males responded to pre-recorded FCS1 by emitting a higher number of MCrS per minute. Furthermore, males modulated the repetition rate of their MCrS pulse trains to match those of the FCS1. All males responded to FCS1 from French and Guadeloupe females despite the temporal differences in these songs; however, they responded with a shorter latency and a higher rate of MCrS/FCS1 pulse trains to the songs of females from their own population. In choice experiments with two alternatives, responses to FCS1 were inhibited when males received a simultaneous female call from another Pentatomidae sympatric species, Acrosternum hilare. We conclude that, although males prefer FCS1 from their own population, they recognise FCS1 from French and Guadeloupe N. viridula females as species-specific female calls. Variability of vibratory signals might play a role in mate choice. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Courtship behaviour; Nezara viridula; Vibratory communication; Geographical variability

1. Introduction Sexual communication is a major component of species recognition and reproduction. Although many insect species use one sensory channel, like olfaction or hearing, to locate and select a potential mate during the initial stages of their reproductive behaviour, other species rely on multimodal communication. For ∗ Corresponding author. Tel.: +33-1-30-83-32-32; fax: +33-1-30-83-31-19. E-mail address: [email protected] (M. Renou).

example, in the southern green stink bug, Nezara viridula (Heteroptera, Pentatomidae), sex pheromone and acoustic signals are implicated in a complex mate recognition system. The sex pheromone produced by mature males acts as a long range attractant to females (Aldrich et al., 1987; Borges et al., 1987; Brézot et al., 1994) enabling them eventually to reach the same plant. However, null or low level of catches to pheromone baited traps in the field, support the hypothesis that precise localisation of the male is not achieved by chemical cues. While on the same plant, males and females of N. viridula communicate

0376-6357/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0376-6357(02)00186-9

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both their presence and their receptivity via the plant substrate, producing four different species and sex ˇ specific songs (see Cokl et al., 2000, for a review). Two songs—which have been identified not only in all populations investigated thus far, but also in every mating pair—play a vital role in mate location. The female calling song (FCS) triggers male locomotion, search behaviour, and emission of the male courtship ˇ ˇ song (MCrS) (Ota and Cokl, 1991; Cokl et al., 1999). MCrS keeps female singing with a steady repetition ˇ rate of pulses (Cokl and Bogataj, 1982). Although males and females of geographically isolated populations of this cosmopolitan species share the same vibratory repertoire, the temporal parameters of songs differ substantially (Ryan et al., 1996; ˇ Cokl et al., 2000; Virant-Doberlet et al., 2000). Until now, however, no studies have focused on song variability within populations, and since early studies of the recognition by males of natural and artificial viˇ bratory signals (Cokl et al., 1978), less attention has been paid to the capacities of discrimination of song structure and parameter differences by mate partners. Courtship implicates the exchange of MCrS and female calling song type 1 (FCS1), and variability of both songs favours a high degree of discrimination through behaviour at this early step of the court. We hypothesised that from this time males and females are able to recognise sex and species of a potential mate, and can exercise a mate choice according to inter-individual differences of vibratory signals. To confirm this hypothesis we investigated (a) the interand intra-population variability of temporal and spectral characteristics of MCrS and FCS1 emitted by males and females of two separate populations, one from France and another from Guadeloupe, and (b) the selectivity of the behavioural responses of males and females to different natural vibratory signals produced, as in nature, on their host plants.

2. Material and methods 2.1. Insects and plants Wild adults and larvae of N. viridula, were collected in the south of France on soybean (Glycine max L.), and in Guadeloupe on Cleome spinosa Jacq. Individuals issuing from these two populations were grown

in the laboratory. They were divided by sex just after the adult moult, kept in separate boxes (38 cm × 23 cm × 23 cm) according to their geographical origin and maintained at 22–26 ◦ C, 70–80% relative humidity and 16L:8D daily cycle. They were fed on a diet of roasted peanuts (Arachis hypogaea L.), sunflower seeds (Helianthus annus L.), green beans (Phaseolus vulgaris L.) and green soya (Vigna radiata L.). During stimulation with pre-recorded songs, male and female behaviour was monitored from animals standing on a bean plant. A different plant was used for each test. Bean plants were grown in the laboratory in pots 15 cm high and 10 cm in diameter. Plants used in experiments were 10–15 days old; the stem was 25–30 cm high and 2–3 mm in diameter. Each plant contained two stalks, 5–6 cm in length, located 2–5 cm below the tip, opposite one another, and each one bearing a leaf measuring 4–6 cm in both length and width. 2.2. Recording and analysis of songs To record and analyse vibratory songs we placed a male and a female, both virgin and sexually mature (at least 10 days of age following the final moult), on the membrane of a cone low-middle frequency loudspeaker WS 13 BF (Visaton GmbH, Haan, Germany; 8  impedance, 144 mm diameter and 40–6000 Hz bandpass). This procedure avoided signal distortion that occurs during transmission through plants (Michelsen et al., 1982). Bug vibratory emissions were then recorded and amplified by a Revox A-77 tape recorder (Revox, Regensdorf, Switzerland), digitised (6000 Hz sampling rate, 16-bit sample size, stereo) and stored on a computer through the input of a sound card (Sound Blaster AWE Gold 64, Creative, Singapore) using the CoolEditPro software (Syntrillium Software Corporation, AZ). All the experiments were conducted in a sound insulated chamber (Amplifon, Type E-large, Fa. Amplaid, Italy) between Hour 2 and Hour 11 of the photophase at ambient temperature, humidity and light. Songs were analysed using Sound Forge 4.0c software (Sonic Foundry Inc., Madison, WI). The temporal pattern of the FCS1 and the MCrS pulse train differ substantially from one another (see Fig. 1). The FCS1 is typically composed of short pulses followed by a long one. The MCrS pulse train is characterised

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Fig. 1. Temporal (left) and spectral (right) characteristics of the FCS1 and the MCrS of N. viridula.

by a sequence of short pulses, the rate of which increases until they fuse with each other in the last part of the pulse train. We determined dominant frequency peak (Frq) and pulse train duration (D) of MCrS and FCS1. The repetition time (RT = time between the beginning of a pulse train and the beginning of the following pulse train), and the inter-pulse train interval (IPI = time between the end of a pulse train and the beginning of the following one) were measured only in FCS1 because males produce MCrS pulse trains mainly in response to the FCS. 2.3. Vibratory stimuli Each stimulatory song was composed from elements of natural songs assembled by the use of the computer program (Sound Forge for Windows 95, version 4.0c; Sonic Foundry Inc.). The amplitude of the stimulation signals was adjusted to the level of the recorded signals. A vibratory stimulus was applied to a bean leaf via the tip of a 10-mm long plastic rod (3 mm in diameter) firmly fixed to the cone

of a loudspeaker (45605BSA, Samsung Electronics, Seoul, Korea; 8  impedance, 40 mm diameter). Vibratory responses of stimulated bugs were recorded by a microphone cartridge D 3800 (AKG Acoustics, Vienna, Austria; 600  impedance, 22 mm diameter, 40–22,000 Hz), whose membrane was in contact with the bean plant stem; these responses were amplified, digitised and stored on a computer as described above. Bugs were stimulated with six different programs. Each program was composed of multiple pulse trains, played back in a continuous loop for the duration of the stimulation experiment. Pulse train amplitudes were normalised so that peaks were maintained at the same level. (1) The MCrS stimulatory program was synthesised from 34 individual pulse trains (Frq = 99 ± 7 Hz, D = 4.22 ± 1.18 s, IPI between 1.71 and 13.92 s) sampled from four different French males. (2) and (3) The stimulatory FCS1 programs were synthesised from natural songs of the

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French (FCS1-F) or Guadeloupe (FCS1-G) females. Different individuals’ pulse trains were used to make a stimulation sequence with natural RT of pulse trains. The FCS1-F sequence was composed of 16 pulse trains recorded from five French females (Frq = 107 ± 4 Hz; D = 0.99 ± 0.07 s; RT = 3.88 ± 0.12 s), and the FCS1-G sequence was composed of 30 pulse trains sampled from seven Guadeloupe females (Frq = 131 ± 13 Hz; D = 1.76 ± 0.44 s; RT = 7.06 ± 0.75 s). Peak amplitudes were normalised to the same level in both sequences. The main characteristics of the reconstituted pulse train sequences were not statistically different from the original songs (Student’s t-test). (4) The male rival song (MRS) stimulation program was composed of a sequence of 22 pulses recorded from four different N. viridula males (Frq = 86 ± 8 Hz; D = 0.19 ± 0.02 s; RT = 0.68 ± 0.11 s). Emission of MRS was obtained by introducing a second male to each of four different courting pairs.

(5) The FS-Acro (female song of the bug species Acrosternum hilare) stimulation program was composed of pulses (Frq = 82 ± 1 Hz, D = 0.66 ± 0.14 s, RT = 1.62 ± 0.15 s), recorded from one female at the University of California ˇ Riverside (Cokl et al., 2001). (6) Finally, an experimentally altered FCS1-F was produced in which the repetition rate of pulse trains was doubled. This program, the 2 × FS1-F, was constructed by copying and pasting a pulse train in the middle of each inter-pulse interval. The pulse duration and frequency characteristics of the 2 × FS1-F sequence did not differ from the FCS1-F, described above. In experiments in which a male had to choose between two songs, each of the two leaves of a bean plant was vibrated by a single program (Fig. 2). The two programs were applied simultaneously as follows: (a) In the FCS1-F/FCS1-F combination, the same FCS1-F program was applied to both leaves. Pulse trains were shifted in order to alternate the occurrence of pulses on left and right leaves. (b) In the FCS1-F/FCS1-G combination, the FCS1-F program was applied to one

Fig. 2. Setup used to stimulate a bug on a bean plant with vibratory songs and to record its vibratory emissions. L (left leaf), R (right leaf) and S (stem) indicate the recorded positions of bugs at the end of the experiment.

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leaf and the FCS1-G program was applied to the other leaf in counterbalanced fashion. The first pulse train of each program started at the same time on each leaf. (c) In the FCS1-F/MRS combination, one leaf was vibrated by FCS1-F and the other with MRS pulse trains both starting at the same time. (d) In the FCS1-F/FS-Acro combination, one leaf was vibrated with FCS1-F and the other with FS-Acro. The first pulse of each program started at the same time. 2.4. Experimental procedure For experiments in which only one song was presented, a bug was placed on the bean stem and, 2 min later, stimulation was applied to the leaf opposite the one on which it was standing for the remaining 13 min of the 15-min test. Vibratory responses of a stimulated bug were recorded during 15 min; its position on a plant was monitored 2 and 15 min after the start of the test. Each female and male was tested only once and, for each test, a different plant was used. Seven French females were tested in control situation (no vibratory stimulation), and eight French females were stimulated with pre-recorded MCrS. Twenty males (10 from French and 10 from Guadeloupe population) were tested without stimulation (control) during 15 min. Twenty-one males (10 from French and 11 from Guadeloupe populations) were stimulated with FCS1 from their own population. Then, 21 males (10 from French and 11 from Guadeloupe population) were stimulated with FCS1 from the other population. In stimulated males we monitored the following parameters: (a) Male position on the bean plant was defined as either on the left leaf (L), on the right leaf (R), or on the stem (S). (b) Song latency (in ms) was defined as the time between the start of the stimulation and the first emission of a MCrS. (c) Search latency (in ms) was defined as the time between the onset of stimulation and the onset of walking, usually while singing. (d) Search time (in s) was defined as the time between the beginning of the experiment and the arrival of the male on a loudspeaker.

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(e) MCrS rate (pulse train/min) was calculated as the number of MCrS pulse trains divided by the time interval between the beginning of the singing activity and the end of the test. In the second series of experiments we stimulated a male simultaneously with two songs (choice conditions). Because negative geotaxis might have influenced the results, we positioned the bean plants horizontally. Each male (N = 125) was placed on the base of the stem after stimulation had started. We recorded vibratory responses of the male and observed his position on the plant (L, R, S). The test was finished when the male had remained 10 min on S, or when it stayed 3 min either on L or R, whichever came first. Each male was tested twice, each time with a different stimulus combination, at least at 1-day interval. 2.5. Statistics To characterise the inter-individual variability within the Guadeloupe and French populations according to the intra-individual variation, we calculated the repeatability index (RI): RI =

inter-individual variance (inter-individual variance) +(intra-individual variance)

Inter-individual variability of FCS1 and MCrS parameters was analysed by ANOVA. Student’s t-tests were used to assess differences between songs of the two populations. Chi-square tests were used to compare differences in the number of bugs responding to one-side stimulation or in choice situation. An ANOVA was used to analyse response differences between males of the two populations.

3. Results 3.1. Variability of FCS1 and MCrS The spectral and temporal characteristics of pulse trains of the FCS1s and of the MCrS of the French and Guadeloupe populations are presented in Table 1 and Fig. 3. Guadeloupe females emitted FCS-1 pulse

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Table 1 Comparison of the characteristics of the FCS1 and the MCrS of Guadeloupe and French populations of N. viridula Song parameters

Guadeloupe population

t Test

French population

RI FCS1 Dominant frequency (Hz) Duration (s) Repetition time (s) Inter-pulse trains interval (s)

133.0 1.64 8.73 7.09

15.0 0.53 5.65 5.42

0.67 0.59 0.43 0.43

MCrS Dominant frequency (Hz) Duration (s)

128.0 ± 16.0 2.89 ± 0.91

0.12 0.44

± ± ± ±

RI ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

∗∗∗ ∗∗∗

108.0 1.10 4.58 3.49

± ± ± ±

3.4 0.30 1.39 1.34

0.12 0.19 0.25 0.20

103.0 ± 6.0 3.68 ± 0.91

0.10 0.10

Data are means ± S.D. of measures from nine Guadeloupe females (six pulse trains per female), five French females (five pulse trains per female), seven Guadeloupe males (six pulse trains per male), and eight French males (eight pulse trains per male). ∗∗∗ Significant difference (P < 0.001) (Student’s t-test).

Fig. 3. Distribution of the duration, inter-pulse train interval, repetition time and main frequency measured on MCrS and FCS1 pulse trains recorded in southern green stink bugs from Guadeloupe and France. Bars represent the range, circles represent the mean of the distribution, lines represent the median, and boxes the interval between the 25th and the 75th percentiles.

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trains of significantly longer duration, as well as longer inter-pulse intervals, than French females (P < 0.001). Furthermore, FCS1 pulse trains recorded from Guadeloupe females were characterised by a higher dominant frequency, compared to those emitted by French females (P < 0.001). Guadeloupe males emitted MCrS pulse trains of significantly higher dominant frequency and shorter duration than French males (P < 0.001). Intra-populational variability was investigated by calculating the RI (see Table 1) and analysing inter-individual variability of the pulse trains parameters with ANOVA. The high value of the RI, together with significant differences between individuals for dominant frequency (P < 0.001), pulse train duration (P < 0.001), repetition time (P < 0.01) and inter-pulse interval (P < 0.01), indicate low intra-individual variation of the four FCS1-G parameters. In turn, the pulse trains of French females showed low RI, and no statistically significant differences were observed between females. The RI of MCrS pulse trains of Guadeloupe was low for the dominant frequency and high for the pulse duration. The latter value was close to that of the RI of the FCS1. RI differences for both parameters were confirmed by ANOVA analyses, which showed no significant differences (NS) between individuals’ dominant frequency but significant differences (P < 0.001) in pulse duration. Males of the French population showed low RI for the two investigated pulse train parameters. 3.2. Responses to one-side stimulation Females showed no vibratory, or locomotory, response to stimulation with the MCrS. In the absence or presence of vibratory stimulation, a similar percentage of females emitted FCS1 (28.6 and 37.5%, Chi-square test, NS). Only a minority of females moved on a plant between the 2nd and the 15th minute of the test, either in the control (no stimulus) (28.6%, N = 7), or stimulation (12.5%, N = 8) conditions. All males of the French or Guadeloupe populations responded with MCrS when stimulated by FCS1 of their own or alien population. By contrast, in the absence of stimulation, only 30% of French and 40% of Guadeloupe males emitted one or more MCrS pulse trains during the 15-min test. Male locomotion also

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increased significantly in response to FCS1 stimulation (Chi-square test, P < 0.001). When the results from both populations were combined, 87% of males changed position on the bean plant, compared to 48.4% of control males. In both populations, stimulation significantly increased the dominant frequency of MCrS pulse trains and at the same time it decreased inter-pulse intervals (Student’s t-test, P < 0.001 for both cases; Fig. 4). Pulse train duration was significantly shorter during stimulation in French males, but not in Guadeloupe males. Regardless of whether the FCS1 originated from their own or from the alien population, males of both populations responded by searching the female. Although, as Table 2 reveals, the average search latency did not differ significantly according to the origin of female signals (ANOVA, NS), males of both populations went significantly faster to a loudspeaker playing French FCS1, than with Guadeloupe FCS1 (ANOVA, P < 0.001). The number of MCrS pulse trains emitted by males varied with the origin of males, and with the origin of the FCS1 (Table 3). Whatever the origin of the FCS1, French males tended to emit more MCrS per minute than Guadeloupe males, but the difference was not significant (ANOVA, P = 0.054). Males of the two populations emitted more MCrS pulses per minute when they were stimulated with the French FCS1 than with those of the Guadeloupe FCS1 (ANOVA, P < 0.001). The dominant frequency and pulse duration of MCrS pulse trains did not vary with the origin of the female song. The inter-pulse intervals were longer when males responded to FCS1-G than to FCS1-F (ANOVA, P < 0.001). Because the repetition rates of the French and Guadeloupe types of FCS1 stimulation programs were different (16.1 and 8.3 FCS1/min, respectively), and this difference might be expected to contribute to the observed pattern of results, the ratio of the number of MCrS pulse trains emitted by a male per FCS1 pulse train may be a better index of performance. As Table 3 reveals, the ratio of MCrS/FCS1 is higher in Guadeloupe males when responding to their own FCS1 than when responding to French FCS1 but this same pattern did not obtain in French males. Finally, males of both populations responded with a shorter song latency to stimulation with FCS1 of their own population than of females of the alien population (ANOVA, P < 0.05; see Table 2).

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Fig. 4. Temporal parameters and main frequency of MCrS emitted by French and Guadeloupe males during control (no stimulus situation), or during stimulation with FCS1 from their own population ( ) or from an alien population ( ).

Table 2 Time spent by males of N. viridula to start to move (search latency) or to emit the first pulse train of MCrS (song latency) in response to FCS1 emitted from a loud speaker on a bean plant, and time needed to reach the loud speaker (search time) Male origin

France Guadeloupe

Search latency (ms)

Song latency (ms)

Search time (ms)

FCS1-F

FCS1-G

FCS1-F

FCS1-G

FCS1-F

FCS1-G

43 ± 49 52 ± 19

118 ± 171 56 ± 33

17.9 ± 18.71 36.2 ± 23.5

57.8 ± 55.5 31.5 ± 21.4

263 ± 281 406 ± 266

519 ± 286 701 ± 174

Data are means (N = 9 for French males, and 10 for Guadeloupe males) ± S.D.

Table 3 Rate of emission of MCrS (in pulse trains per minute), and ratios between the number of pulse trains of MCrS emitted and the number of FCS1 received during response, in males of the French and Guadeloupe populations, unstimulated (control), or stimulated with FCS of French or Guadeloupe females Stimulus

French males Guadeloupe males Data are means ± S.D.

Rate of emission of the MCrS (pulse trains/min)

MCrS/FCS1 ratios

Control

FCS1-France

FCS1-Guadeloupe

FCS1-France

FCS1-Guadeloupe

0.5 ± 0.3 0.8 ± 0.8

10.4 ± 3.5 5.4 ± 2.0

11.0 ± 3.0 6.5 ± 2.5

0.65 ± 0.21 0.45 ± 0.15

0.64 ± 0.33 0.65 ± 0.23

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Fig. 5. Percentages of singing males (French population) during control and during different stimulation situations (upper histogram) and number of MCrS pulse trains emitted per minute during control or during stimulation (lower histogram). Histogram bars with different letters are statistically different (Chi-square test for the percentage of males, Student’s t-test for the number of MCrS per minute; P < 0.05 in both cases).

3.3. Responses in choice situation We found no significant difference (NS) between uni- and bi-lateral stimulation in the number of males which responded to stimulation, in the mean rate of MCrS pulse trains (Fig. 5), and in the male position on the plant (Fig. 6). On the other hand, the values of these same parameters were significantly lower (Chi-square test, P < 0.01) in control experiments (compare Figs. 5 and 6). Although unilateral stimulation with 2 × FCS1-F decreased significantly the number of responding males (P < 0.05), the rate of the response remained similar (NS; see Fig. 6); moreover, attraction to the vibrated leaf was of the same level (Fig. 6).

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French males responded equally to FCS1-F and FCS1-G, whether each song was presented unilaterally or to a different leaf in the choice experiments. We found no significant difference (NS) in the number of responding males to unilateral stimulation with FCS1-F or FCS1-G (see Fig. 5). Although when the two different songs were presented simultaneously the number of responding males decreased significantly, compared to unilaterally presented FCS1-F (Chi-square test, P < 0.05), it remained in the range of the values obtained by unilateral stimulation with FCS1-G (NS). The rate of MCrS pulse trains was not significantly (NS) different when FCS1-G was presented unilaterally or simultaneously with FCS1-F on the other leaf. Unilateral stimulation with FCS1-F evoked a significantly higher rate of responding than either unilateral stimulation with FCS1-G (Student’s t-test, P < 0.001), or bilateral FCS1-F/FCS1-G stimulation (P < 0.05; see Fig. 5). The percentage of males arriving to the vibrated leaf was not significantly different in unilateral stimulation with FCS1-F or FCS1-G and in bilateral presentation of both songs (Fig. 6). In paired stimulation, males did not show a preference either for one side, or for the song of one population. In tests with the MRS presented unilaterally, MRS had no effect on male singing activity and orientation. Indeed, when unilateral stimulation with MRS was compared to the control condition (no stimulation), no significant differences (NS) were found in the number of males emitting MCrS, in the MCrS rate (Fig. 5) or in the number of males reaching the leaf (Fig. 6). Similarly, MRS stimulation depressed responding in choice tests. Comparing bilateral (MRS/FCS1-F) and unilateral (FCS1-F) stimulations, we found a significantly (Chi-square test, P < 0.05) lower number of males responding, and a significantly lower repetition rate of MCrS pulse trains, in bilateral stimulation conditions. Nevertheless, the repetition rate of MCrS pulse trains was higher when compared with males responding to MRS pulses (P < 0.01). The percentage of males moving to the leaves was not statistically different in bilateral MRS/FCS1-F and unilateral FCS1-F stimulations, and, in bilateral stimulation, 78% of males stayed on the leaf vibrated by FCS1-F. We also stimulated males with a female song of A. hilare (FS-Acro) presented on one leaf, or simultaneously with FCS1-F at the other leaf. No male emitted

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Fig. 6. Final position of male N. viridula of the French population on a bean plant after stimulation with single or paired vibratory stimulation. Males were released on the stem and challenged with a single song or a combination of the FCSs of their own population (FCS1-F), of the Guadeloupe population (FCS1-G), the MRS, or the female song of A. hilare (FS-Acro). Data are percentages of males on left leaf (L), right leaf (R), or stem (Stem). Number in brackets indicate number of replicates. In paired stimulations, the position of each stimulus was alternated, but data were collapsed to draw the figure such that L corresponds to FCS1.

MCrS in response to FS-Acro. Simultaneous stimulation with FS-Acro and FCS1-F increased the percentage of singing males to 46%; however, this value was significantly lower when compared to that obtained in unilateral stimulation with FCS1-F (100%, Chi-square test, P < 0.01) or with FCS1-F/MRS paired stimulation (64%, Chi-square test P < 0.05). The repetition rate of MCrS pulse trains increased significantly in FCS1-F/FS-Acro, compared to unilateral FS-Acro, but it remained significantly below the value obtained by FCS1-F one-side stimulation (Fig. 5, Student’s t-test, P < 0.01). Only 15% of males stimulated simultaneously with FS-Acro and FCS1-F moved to the vibrated leaves; the majority of them remained on the stem. This percentage was not different from the percentage measured in control experiments (Chi-square test, NS).

4. Discussion The first aim of this paper was to assess the variability of the songs emitted by female and male N. viridula during courtship. Because MCrS and FCS1 were recorded from bugs singing on a membrane of a loudspeaker, they were not subjected to distortion that occurs during transmission through plants (Michelsen et al., 1982) and which affects commu-

nication in N. viridula (Miklas et al., 2001). Thus, temporal and spectral characteristics of songs of the French and Guadeloupe populations were compared at their origin. Dominant frequency of the MCrS of males of both populations and pulse train duration in the French males did not vary consistently between individual males within population. However, French MCrSs could be distinguished from those of Guadeloupe males by both pulse train duration, and dominant frequency. In our experiment, females did not exhibit any conspicuous behaviour in response to pre-recorded MCrS. Whereas MCrS is emitted mainly during the pre-mating duet, we never observed direct vibratory responses, nor orientation behaviour, of the females to a source of male vibrations. Nonetheless, the MCrS might have less visible effects on female beˇ haviour. For example, Cokl and Bogataj (1982) have shown that MCrS pulses increase the stability of the FCS1 repetition rate in singing females. The absence of locomotory or singing responses from females prevented us from investigating how differences in male songs could determine female specific preferences. The spectral and temporal parameters measured on the FCS1 pulse trains of N. viridula females varied both within and between French and Guadeloupe populations. Thus, we were able to distinguish not only between females of the two populations, as has been ˇ shown previously (Kon et al., 1988; Cokl et al., 2000),

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but also between females of the same population. Male responses to female songs have been described in the context of male substrate-borne directionality mediˇ ˇ ated by FCS (Ota and Cokl, 1991; Cokl et al., 1999). Together with previously described reactions we have demonstrated that FCS1 increases the rate of emission of MCrS pulse trains, and modifies MCrS spectral and temporal characteristics. When males responded to FCS1 from the two populations they emitted MCrS pulse trains with the same dominant frequency and pulse train duration, but with different repetition times and inter-pulse intervals. This modulation of the MCrS temporal parameters is probably caused by the temporal tuning of the males’ MCrS pulse trains to female song. That is, during a courtship duet, males modulate the inter-pulse interval of their MCrS so that the MCrS pulse train is inserted between two FCS1 pulse trains. Thus, the number of MCrS emitted per minute depends primarily on the repetition rate of FCS1 stimulation. During search behaviour a male alternatively (a) stops and waits for a call to orient himself toward the female and (b) walks while emitting MCrS pulse trains. Thus, the time to reach the singing female also depends on the repetition rate of emitted MCrS which, during alternation, synchronises with the repetition rate of FCS1. The repetition rates of the French types of FCS1 was higher compared to that of the Guadeloupe type. Correspondingly, in our experiment, we observed a shorter search time when males oriented to French FCS1 than to Guadeloupe FCS1. This synchronisation of the male songs to female songs might increase the efficiency of the duet as a mate localisation mechanism. No rivalry singing has been described by female southern green stink bugs and one can expect that in natural conditions several females concurrently emit their calling songs on same plant. In this case males are challenged with relevant vibratory sensory inputs coming from different sides as well as, at least in standing wave conditions, with distortion of the temporal, spectral and amplitude structures of those signals. In such situations, we might wonder whether interferences between the two signals prevent recognition of the signals by male, and whether males can discriminate the two signals. In our experiments on a natural host plant, stimulation with the same vibratory pulses from two sides of the same plant

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did not prevent the male from recognizing the FCS1. When males were stimulated bilaterally with two different vibratory stimuli in which the pulses did not alternate, the number of MCrS per minute decreased. This lower rate does not necessarily implicate impaired recognition of the FCS, but rather the difficulty of alternating MCrS pulse trains with female pulse trains. Male responsiveness to female calling varied across the two populations. All males responded to French and Guadeloupe FCS1 in spite of the temporal differences in those songs. When stimulation was unilateral, males emitted MCrS pulses and oriented themselves to FCS1, whatever the geographical origin of the female. When, however, FCS1 was presented bilaterally, males did not orient preferentially to the FCS1 from one particular population, but they still responded with MCrS pulses. Nonetheless, some parameters of male responsiveness depended on the geographical origin of the FCS1. Males responded with a shorter song latency and with a higher rate of MCrS/FCS1 pulses to the FCS1 from their own population. Furthermore, FCS1-G was less attractive to French males than FCS1-F. Taken together, these results indicate that, although males recognise the female signal from French and Guadeloupe populations as a species specific female call of a potential sexual partner, they show a preference for the FCS1 of their own population. Although males did not respond to MRS, they responded to bilateral stimulation with FCS1–F/MRS by emitting MCrS pulses and orienting to FCS. Males are, thus, able to discriminate between female and male acoustic signal, and are clearly selecting the female signal. On the other hand, when males were stimulated by the call of another Pentatomidae species (A. hilare), all their behaviour, whether acoustic or locomotory, was inhibited. Indeed, males did not respond at all to FCS1 from their own species if they received simultaneously the A. hilare FS. Interestingly, although N. viridula and A. hilare are sympatric in California ˇ and can be found on the same host plants (Cokl et al., 2001), no hybrids between both species have ever been found. Our results suggest the presence of a sensory exclusion mechanism, based on the inhibition of male orientation behaviour by the song of the other species, which could insure reproductive isolation between the two species.

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Recognition and discrimination of a sexual partner are central mechanisms in reproduction. Mate choice and conspecific recognition are advantageous only if they occur in an early step of the courtship. In N. viridula, vibratory communication enables male and female to meet on a plant through the reciprocal exchanges of vibratory signals. FCS appears to be an important component of the mate recognition system. The ability of males to discriminate between different songs, and to orient when several sources compete for attention, indicates that this behaviour constitutes a refined mechanism for mate recognition. Acknowledgements We thank Viktor Triler for dedicated technical assistance. We are indebted to Prof. Dr. Karen Hollis for critical reading of the manuscript and helpful comments on it. We thank two anonymous referees for helpful criticisms and comments. The first author was supported by a grant from the French Ministry for Education and Research. This research was supported by the Slovene Ministry for Education, Science and ˇ Sports (Program 0105-0503 Cokl). References Aldrich, J.R., Lusby, W.R., Kochansky, J.P., Lockwood, J.A., 1987. Pheromone strains of the cosmopolitan pest, Nezara viridula (Heteroptera: Pentatomidae). J. Exp. Zool. 244, 171–175. Borges, M., Jepson, P.C., Howes, P.E., 1987. Long-range mate location and close-range courtship behaviour of the green stink bug, Nezara viridula and its mediation by sex pheromones. Entomol. Exp. Appl. 44, 205–212.

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