© 2001 by The Society for Integrative and Comparative Biology
Regulation of Chorusing in the Vibrational Communication System of the Leafhopper Graminella nigrifrons1
1 Department of Biology, Centre College, Danville, Kentucky 40422
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Male Graminella nigrifrons participate in alternating choruses. Vibrational calls emitted by males consist of three sections (S1, S2, and S3) that differ in pattern of amplitude modulation. In this study we examined the response of single males to synthetic choruses and to isolated call components to gain insight into the regulation of chorus structure. Males initiated calls primarily during the silent periods within synthetic choruses. In all 15 trials the number of overlapping calls and the duration of overlap was significantly less than expected if males call at random. Playback of S2, S3, or random noise while males emitted S1 caused males to interrupt calling, whereas males continued to call when S1 or no signal was played. In a related experiment, we played S2 or no signal while males were beginning to emit the S1, S2, or S3 phase of their calls. In response to this playback the duration of S1 and S3 was reduced, but the duration of S2 was not affected. These results suggest that an inhibitory-resetting mechanism may result in alternation of calls in this leafhopper.
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INTRODUCTION |
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The evolution of communication systems is strongly influenced by the mode of communication used by animals and the environment through which signals are transmitted (Wiley and Richards, 1982
; Römer, 1992; Endler, 1993; Robisson et al., 1993). Thus, knowledge of signal properties and how the environment may facilitate or constrain transfer of information has been an important guide in theoretical and empirical research. Acoustic signals are transmitted rapidly over great distances and the environment influences signal fidelity in ways that are often predictable. Thus, sexual selection acting on acoustic signals and receiver mechanisms at long distances is possible and has led to male-male competition and female preference in a broad array of insect and vertebrate taxa (Andersson, 1994; Greenfield, 1994). The most conspicuous outcome of such selection is chorusing. Although chorusing is sometimes cooperative (Greenfield, 1994), it is often a reflection of competition among males for mating opportunities and consists of males that either overlap or alternate calls with neighboring males. In contrast, vibrational communication is sometimes viewed as an alternative mode of communication which reduces the likelihood of eavesdropping by natural enemies or sexual competitors (Henry, 1994). The enemies hypothesis is supported by studies of certain Neotropical katydids that have shifted from acoustic to vibrational communication in response to bat predation (Belwood and Morris, 1987). The competition hypothesis suggests that males avoid vibrational interactions or have little potential to interact because of the limited range of their signals. Thus, chorusing should be a rare or absent component of their mating system.
Although the possible involvement of vibrational signals in male-male competition or female preference has received little study (Ott, 1994), vibrational signals have been associated with interactions among males in a few species of planthoppers (Delphacidae) and leafhoppers (Cicadellidae) (Ichikawa, 1982; Heady et al., 1986; Claridge and de Vrijer, 1994). These signals are often referred to as aggressive or rivalry signals. They differ structurally from advertisement signals which elicit response calls from females (see below) and are emitted when males are in close proximity or engaged in physical aggression. For example, Ichikawa (1982) found that male rice brown planthoppers (Nilaparvata lugens) reared at high density emit aggressive calls that appear to suppress calling activity of males reared at low density. However, experiments designed to isolate aggressive signals as the cause of calling suppression and the relationship between aggressive signaling and mating success were not conducted (see Ott, 1994). More recently, however, studies of the leafhopper Graminella nigrifrons show that competition for mating opportunities involves chorusing (R.E.H. and T.L.M., unpublished data).
G. nigrifrons is one of the most common leafhoppers found in the eastern United States (Whitcomb et al., 1987). This small insect (3.5–4.5 mm) feeds on many annual and perennial grasses, but it is especially well adapted to exploit annuals, including many grain crops (Whitcomb, 1987; Hunt and Nault, 1990). Although field or laboratory studies have not determined the relationship between population density and the potential for males and/or females to interact, monitoring of male signaling in urban landscapes indicates that interactions such as those reported here are common (R.E.H. and T.L.M., unpublished data).
This study examines how individual male Graminella nigrifrons interact with recordings of other males. Our goal was to determine whether vibrational signals in the absence of other stimuli (physical or visual) would result in chorusing. We also report on a series of playback experiments that contribute to an understanding of how chorus structure is regulated in this leafhopper.
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METHODS |
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G. nigrifrons were reared indoors on oats and maize at 24–26°C on a 15:9 light:dark cycle following procedures described by Hunt and Nault (1991)
. Males were housed separately from females and used in experiments 1–2 wk after adult eclosion.
Male playback signals were constructed from recordings of calls emitted by five individual males (exemplars) (Fig. 1). Each call was recorded by resting a BSR-X5H phonograph cartridge on a leaf near a male (ca. 2 cm). Signals were amplified 100-fold using a Stanford Research Systems Model 560 preamplifier and recorded on a Sony DTC 700 or DTC A7 digital tape recorder (DAT) at 48,000 samples sec–1. Recordings were transferred to a Macintosh computer equipped with a Digidesign Audiomedia II soundboard. Editing of calls was done using Alchemy (Passport Designs) software. Band-limited white noise (0.0–20.0 kHz) was created using SoundEdit Pro (Macromedia) software. It was then subjected to a –40.0 dB low-pass filter with the cut-off frequency set at 0.8 kHz using Alchemy software. This procedure resulted in band-limited noise that covers the frequency range of male signals (Hunt et al., 1992). Playback signals were fed from the computer to the DAT recorder and played into a leaf using a Brüel and Kjaer Model 4810 mini-shaker connected to the headphone jack of the DAT recorder. Each call was filtered using Q10 (Waves) software to compensate for the –12 dB/octave rolloff associated with the minishaker.
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We determined whether chorusing could be elicited solely by calls of other males by playing recordings of male calls to individual males and recording their response. The five exemplar calls ranged in length from 8–32 sec. We added 40 sec of silence to each call to provide adequate time for a male to respond. The sequence of exemplars used within each block (replicate) of the experiment was randomized. Although the response of each male during a trial represents an independent test with an associated P value (see below), we used a randomized complete block design so that we could detect possible changes in the response of males over the course of the experiment which lasted several weeks. For each trial (N = 15, 5 exemplars x 3 blocks) we positioned a male about 2 cm from the minishaker. After the male emitted a call he was removed and the output from the DAT recorder to the minishaker was adjusted so that the exemplar had a volume level equal to, or slightly below that of the calling male. A similar adjustment was made for each exemplar. We then placed a different male on the leaf and immediately played the appropriate exemplar recording. The playback signal was transmitted simultaneously to the minishaker and to a second DAT recorder. The second recorder monitored the playback on one channel and the trial events on the other channel, thus allowing easier measurement of overlap between the experimental male and the playback stimulus. A trial ended when the individual flew or when the observer grew weary (ca. 1 hr). An acceptable trial required a sample size of at least six calls which included four calls from the male and two playback calls.
A sampled randomization test was used to determine whether the number and overlap time of calls emitted by a male interacting with the playback stimulus was significantly less than the random expectation. The test was implemented using a program developed by one of us (T.L.M). This program calculated descriptive statistics for each sequence: average call length, total number of overlapping calls (NUMBER) and the total time of call overlap (TIME). We determined the time of call overlap of individual i by individual j by subtracting the starting time of individual j from the ending time of individual i (see Brush and Narins, 1989). We then randomized the starting times of each call emitted by a male within the total time of the trial while avoiding the overlap of calls by the same individual. The randomization was repeated 10,000 times for each trial. We compared each value of NUMBER and TIME to the appropriate matching distribution from the randomization output. The probability of receiving a value higher than or equal to the observed value was the number of values less than or equal to the observed value divided by the total number of values or randomizations. P values were considered significant at alpha = 0.05.
We conducted two playback experiments designed to determine the effect of manipulated calls on the regulation of male calling. Preliminary observations suggested that males emitting the S1 phase of their call often cease calling if another male begins to call. In the first experiment we played 30 sec of isolated and looped S1, S2, S3, band-limited noise, or no signal (silence) to individual males (N = 75) as they began to emit S1 of their call (five treatments x five exemplars of each signal type x three blocks). Treatment and exemplar sequences were randomized within each block. Each experimental male was scored as a "shutdown" (i.e., a positive response) if he ceased calling for the duration of playback (30 sec). Data were analyzed using Ryan's multiple comparison procedure for proportions.
In a second experiment we assessed the response of males (N = 60) to silence (control) or a stimulus played while they were emitting each of their call sections. We used S2, looped for 30 sec, as the stimulus because of its effectiveness in shutting down calling (see Results). To avoid pseudoreplication (see Kroodsma et al., 2001), the experimental design included four treatments x 5 exemplars x three blocks with the sequence of treatments and exemplars randomized within each block. However, the full power of this design could not be implemented in the data analysis (e.g., analysis using mixed-model ANOVA) because of lack of independence in the duration of call sections. Therefore, we measured the duration of each section of an experimental male's call and analyzed treatment effects using Friedman's test for related samples.
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RESULTS |
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Preliminary analyses showed that males interacting with recordings of male calls tended to limit their calling to the 40.0 sec period of silence between playback calls, thus avoiding overlap. Eight of the 15 and 12 of the 15 trials were significantly different from the randomized distribution for NUMBER and TIME, respectively. Closer analysis of the trials revealed that many of the overlaps were initiated by the playback file and not by the experimental male, so we eliminated playback initiated overlaps by omitting from the analysis that section of the playback call which overlapped an already calling male. Experimental individuals from this reanalysis averaged 0.73 (±SD = ±1.03, N = 15) overlapping calls and 5.73 sec (±SD = ±8.66, N = 15) of overlapping time per trial for the five exemplar calls. All 15 trials were significantly different from the random distribution for NUMBER and TIME (Table 1).
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Most males ceased calling when S2, S3, or noise was played while they were emitting S1 of their call. Significantly fewer males ceased calling in response to playback of S1 and no males ceased calling when no signal was played (Fig. 2).
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Playback of S2 while males emitted S1, S2, or S3 had a significant effect on the duration of call components and the total call duration (Fig. 3A–D). Three main patterns are apparent. First, all males ceased calling when S2 was played while they emitted their S1 (Fig. 3B, C). Second, the duration of S3 was greatly reduced when S2 was played while males emitted their S1, S2, or S3 (Fig. 3C). Third, the duration of S2 was not apparently affected when S2 was played while they emitted S2 (Fig. 3B).
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DISCUSSION |
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Our finding that individual males avoid overlapping calls with recordings of males provides strong evidence that vibrational cues alone initiate chorusing behavior. Also, calls exchanged among males are the same as those which elicit female response calls (R.E.H., unpublished data). In other species of leafhoppers and planthoppers vibrational interactions between or among males sometimes involve signals distinctly different than mating signals (i.e., aggressive or rivalry signals) and interactions are often elicited by or accompany physical aggression (Ichikawa, 1982
; Heady et al., 1986; see Claridge and de Vrijer, 1994; Ott, 1994 for review). These studies, however, did not employ experimental procedures to isolate the effect of vibrational signals in eliciting various types of interactions among males.
Why do male G. nigrifrons alternate calls? Call alternation may result from cooperative or competitive interactions (see Greenfield, 1994). Cooperative interactions include those where overlap reduces female response to all signalers due to the degradation of call structure or inappropriate timing of signal components. In contrast, interactions may be viewed as competitive if females show a preference related to the timing of male signals or in cases where signal timing facilitates the assessment of competitors or allows a male to interlope in courtship. Chorusing in G. nigrifrons is most likely a competitive strategy involved in courtship disruption, whereas cooperative behavior can be excluded because female response does not decline significantly when overlapped male calls are played (R.E.H. and T.L.M., unpublished data).
Although there are no apparent structural differences between calls emitted by G. nigrifrons males in the context of mating and chorusing, it is possible that structural differences within the male call have multiple functions. Playback of isolated S2 and S3 are equally effective in eliciting female responses when played at levels approximating a nearby male (Hunt et al., 1992). In the present study the amplitude of playback signals also approximated that of a nearby male. Under these conditions playback of S2 to males that were emitting S1 resulted in the highest percentage shutdown, although not significantly different than S3 or noise. Before concluding that S2 and S3 are functionally identical, their influence on males and females when played at amplitudes that simulate distant males should be investigated. Furthermore, the influence of random noise on male calling suggests that other environmental factors (e.g., wind generated vibrations) may be important in regulating calling behavior.
Our finding that males interrupt call emission in response to S2, S3, and noise while they are emitting S1, but not S2, suggests a general mechanism that results in call alternation in G. nigrifrons. S1 may represent a "ready to call phase" similar to that proposed for the frog Eleutherodactylus coqui (Narins, 1992). Following this rule a male would proceed to call unless another male is calling. Thus, calling by another male or possibly environmental noise, detected during S1 inhibits call emission until the channel is clear. Inhibitory-resetting mechanisms have been described in detail for several species of anurans and Orthoptera and often explain the regulation of chorusing behavior (Greenfield, 1994). Lack of inhibition during S2 suggests a neural constraint on call shutdown, thus a commitment to call, or possibly the inability to detect some types of signals because of masking or neural or biomechanical mechanisms (see Greenfield, 1994). However, lack of inhibition is not absolute because males do interrupt S2 emission and begin to search in response to female response calls that they emit between chirps that comprise S2 (Hunt et al., 1992). Further studies are required to more clearly define temporal constraints on call suppression and promotion in G. nigrifrons.
In addition to males ceasing call emission when signals are played during S1 of their call, they also reduce the duration of their S3 when S2 is played. Their sensitivity to signals while emitting S1 and S3, but not S2, leads us to predict that individual males may reduce their call rate and the total duration of their calls as chorus size increases.
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ACKNOWLEDGMENTS |
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We thank Robin Lacour for laboratory assistance. This study was funded by a National Research Initiative Competitive Grants Program grant (9401839) to R.E.H. and an Undergraduate Biological Sciences Education Initiative, Howard Hughes Medical Institute grant to Centre College.
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FOOTNOTES |
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1 From the Symposium Vibration as a Communication Channel presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3–7 January 2001, at Chicago, Illinois.
2 Present address of Randy E. Hunt is Department of Biology, Indiana University Southeast, New Albany, Indiana 47150; E-mail: rhunt01{at}ius.edu
3 Present address of Thomas L. Morton is P.O. Box 152, Wilmore, Kentucky 40390
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References |
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