AMER.ZOOL., 41:1222±1228 (2001)

Regulation of Chorusing in the Vibrational Communication System of the Graminella nigrifrons1

RANDY E. HUNT2 AND THOMAS L. MORTON3 Department of Biology, Centre College, Danville, Kentucky 40422

SYNOPSIS. Male participate in alternating choruses. Vibra- tional 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 signi®cantly 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 alter- nation of calls in this leafhopper.

INTRODUCTION ing. Although chorusing is sometimes co- The evolution of communication systems operative (Green®eld, 1994), it is often a is strongly in¯uenced by the mode of com- re¯ection of competition among males for munication used by and the envi- mating opportunities and consists of males ronment through which signals are trans- that either overlap or alternate calls with mitted (Wiley and Richards, 1982; RoÈmer, neighboring males. In contrast, vibrational 1992; Endler, 1993; Robisson et al., 1993). communication is sometimes viewed as an Thus, knowledge of signal properties and alternative mode of communication which how the environment may facilitate or con- reduces the likelihood of eavesdropping by strain transfer of information has been an natural enemies or sexual competitors important guide in theoretical and empirical (Henry, 1994). The enemies hypothesis is research. Acoustic signals are transmitted supported by studies of certain Neotropical rapidly over great distances and the envi- katydids that have shifted from acoustic to ronment in¯uences signal ®delity in ways vibrational communication in response to that are often predictable. Thus, sexual se- bat predation (Belwood and Morris, 1987). lection acting on acoustic signals and re- The competition hypothesis suggests that ceiver mechanisms at long distances is pos- males avoid vibrational interactions or have sible and has led to male-male competition little potential to interact because of the and female preference in a broad array of limited range of their signals. Thus, cho- and vertebrate taxa (Andersson, rusing should be a rare or absent component 1994; Green®eld, 1994). The most conspic- of their mating system. uous outcome of such selection is chorus- Although the possible involvement of vi- brational signals in male-male competition or female preference has received little 1 From the Symposium Vibration as a Communi- cation Channel presented at the Annual Meeting of the study (Ott, 1994), vibrational signals have Society for Integrative and Comparative Biology, 3±7 been associated with interactions among January 2001, at Chicago, Illinois. males in a few species of planthoppers 2 Present address of Randy E. Hunt is Department (Delphacidae) and (Cicadelli- of Biology, Indiana University Southeast, New Alba- ny, Indiana 47150; E-mail: [email protected] dae) (Ichikawa, 1982; Heady et al., 1986; 3 Present address of Thomas L. Morton is P.O. Box Claridge and de Vrijer, 1994). These signals 152, Wilmore, Kentucky 40390. are often referred to as aggressive or rivalry 1222 REGULATION OF CHORUSING 1223

FIG. 1. Typical call emitted by a male G. nigrifrons. The call is composed of three main sections: section 1 (S1), 2 (S2), a transitional phase, and 3 (S3) (refer to text). S1 is composed of a series of low amplitude pulses, whereas sections 2 and 3 are composed of repeated chirps. S2 and S3 chirps differ in the number of lower amplitude pulses that precede the major pulse (see Hunt et al., 1992). signals. They differ structurally from ad- such as those reported here are common vertisement signals which elicit response (R.E.H. and T.L.M., unpublished data). calls from females (see below) and are This study examines how individual emitted when males are in close proximity male Graminella nigrifrons interact with re- or engaged in physical aggression. For ex- cordings of other males. Our goal was to ample, Ichikawa (1982) found that male determine whether vibrational signals in the rice brown planthoppers (Nilaparvata lu- absence of other stimuli (physical or visual) gens) reared at high density emit aggressive would result in chorusing. We also report calls that appear to suppress calling activity on a series of playback experiments that of males reared at low density. However, contribute to an understanding of how cho- experiments designed to isolate aggressive rus structure is regulated in this leafhopper. signals as the cause of calling suppression and the relationship between aggressive sig- METHODS naling and mating success were not con- G. nigrifrons were reared indoors on oats ducted (see Ott, 1994). More recently, how- and maize at 24±26ЊC on a 15:9 light:dark ever, studies of the leafhopper Graminella cycle following procedures described by nigrifrons show that competition for mating Hunt and Nault (1991). Males were housed opportunities involves chorusing (R.E.H. separately from females and used in exper- and T.L.M., unpublished data). iments 1±2 wk after adult eclosion. G. nigrifrons is one of the most common Male playback signals were constructed leafhoppers found in the eastern United from recordings of calls emitted by ®ve in- States (Whitcomb et al., 1987). This small dividual males (exemplars) (Fig. 1). Each insect (3.5±4.5 mm) feeds on many annual call was recorded by resting a BSR-X5H and perennial grasses, but it is especially phonograph cartridge on a leaf near a male well adapted to exploit annuals, including (ca. 2 cm). Signals were ampli®ed 100-fold many grain crops (Whitcomb, 1987; Hunt using a Stanford Research Systems Model and Nault, 1990). Although ®eld or labo- 560 preampli®er and recorded on a Sony ratory studies have not determined the re- DTC 700 or DTC A7 digital tape recorder lationship between population density and (DAT) at 48,000 samples secϪ1. Recordings the potential for males and/or females to in- were transferred to a Macintosh computer teract, monitoring of male signaling in ur- equipped with a Digidesign Audiomedia II ban landscapes indicates that interactions soundboard. Editing of calls was done us- 1224 R. E. HUNT AND T. L. MORTON ing Alchemy (Passport Designs) software. dividual ¯ew or when the observer grew Band-limited white noise (0.0±20.0 kHz) weary (ca. 1 hr). An acceptable trial re- was created using SoundEdit Pro (Macro- quired a sample size of at least six calls media) software. It was then subjected to a which included four calls from the male and Ϫ40.0 dB low-pass ®lter with the cut-off two playback calls. frequency set at 0.8 kHz using Alchemy A sampled randomization test was used software. This procedure resulted in band- to determine whether the number and over- limited noise that covers the frequency lap time of calls emitted by a male inter- range of male signals (Hunt et al., 1992). acting with the playback stimulus was sig- Playback signals were fed from the com- ni®cantly less than the random expectation. puter to the DAT recorder and played into The test was implemented using a program a leaf using a BruÈel and Kjaer Model 4810 developed by one of us (T.L.M). This pro- mini-shaker connected to the headphone gram calculated descriptive statistics for jack of the DAT recorder. Each call was each sequence: average call length, total ®ltered using Q10 (Waves) software to number of overlapping calls (NUMBER) compensate for the Ϫ12 dB/octave rolloff and the total time of call overlap (TIME). associated with the minishaker. We determined the time of call overlap of We determined whether chorusing could individual i by individual j by subtracting be elicited solely by calls of other males by the starting time of individual j from the playing recordings of male calls to individ- ending time of individual i (see Brush and ual males and recording their response. The Narins, 1989). We then randomized the ®ve exemplar calls ranged in length from starting times of each call emitted by a male 8±32 sec. We added 40 sec of silence to within the total time of the trial while each call to provide adequate time for a avoiding the overlap of calls by the same male to respond. The sequence of exem- individual. The randomization was repeated plars used within each block (replicate) of 10,000 times for each trial. We compared the experiment was randomized. Although each value of NUMBER and TIME to the the response of each male during a trial rep- appropriate matching distribution from the resents an independent test with an associ- randomization output. The probability of ated P value (see below), we used a ran- receiving a value higher than or equal to the domized complete block design so that we observed value was the number of values could detect possible changes in the re- less than or equal to the observed value di- sponse of males over the course of the ex- vided by the total number of values or ran- periment which lasted several weeks. For domizations. P values were considered sig- each trial (N ϭ 15, 5 exemplars ϫ 3 blocks) ni®cant at alpha ϭ 0.05. we positioned a male about 2 cm from the We conducted two playback experiments minishaker. After the male emitted a call he designed to determine the effect of manip- was removed and the output from the DAT ulated calls on the regulation of male call- recorder to the minishaker was adjusted so ing. Preliminary observations suggested that the exemplar had a volume level equal that males emitting the S1 phase of their to, or slightly below that of the calling call often cease calling if another male be- male. A similar adjustment was made for gins to call. In the ®rst experiment we each exemplar. We then placed a different played 30 sec of isolated and looped S1, S2, male on the leaf and immediately played S3, band-limited noise, or no signal (si- the appropriate exemplar recording. The lence) to individual males (N ϭ 75) as they playback signal was transmitted simulta- began to emit S1 of their call (®ve treat- neously to the minishaker and to a second ments ϫ ®ve exemplars of each signal type DAT recorder. The second recorder moni- ϫ three blocks). Treatment and exemplar tored the playback on one channel and the sequences were randomized within each trial events on the other channel, thus al- block. Each experimental male was scored lowing easier measurement of overlap be- as a ``shutdown'' (i.e., a positive response) tween the experimental male and the play- if he ceased calling for the duration of play- back stimulus. A trial ended when the in- back (30 sec). Data were analyzed using REGULATION OF CHORUSING 1225

TABLE 1. In all trials the number of overlapping calls (A) and the amount of time overlapped (B) between experimental males and looped playback of recorded males was signi®cantly less than the random expec- tation (see text for further explanation).

(A) Number of overlapping calls (P value)

Exem- plar Block 1 Block 2 Block 3 1 0 (0.0000) 1 (0.0061) 0 (0.0021) 2 0 (0.0000) 2 (0.0000) 2 (0.0090) 3 0 (0.0010) 3 (0.0350) 1 (0.0103) 4 0 (0.0031) 0 (0.0002) 0 (0.0219) 5 2 (0.0138) 0 (0.0059) 0 (0.0225) (B) Amount of time overlapped in seconds (P value) FIG. 2. Percentage of males that ceased calling (shut- Exem- down) while emitting S1 of their call in response to plar Block 1 Block 2 Block 3 playback of S1, S2, S3, noise, or silence. Percentages 1 0 (0.0000) 2 (0.0007) 0 (0.0021) followed by the same letter are not signi®cantly dif- 2 0 (0.0010) 14 (0.0000) 14 (0.0040) ferent, Ryan's test, P Ͻ 0.05. 3 29 (0.0034) 10 (0.0339) 14 (0.0363) 4 0 (0.0031) 0 (0.0002) 0 (0.0219) 5 3 (0.0002) 0 (0.0059) 0 (0.0225) NUMBER and TIME, respectively. Closer analysis of the trials revealed that many of Ryan's multiple comparison procedure for the overlaps were initiated by the playback proportions. ®le and not by the experimental male, so In a second experiment we assessed the we eliminated playback initiated overlaps response of males (N ϭ 60) to silence (con- by omitting from the analysis that section trol) or a stimulus played while they were of the playback call which overlapped an emitting each of their call sections. We used already calling male. Experimental individ- S2, looped for 30 sec, as the stimulus be- uals from this reanalysis averaged 0.73 cause of its effectiveness in shutting down (ϮSD ϭϮ1.03, N ϭ 15) overlapping calls calling (see Results). To avoid pseudorepli- and 5.73 sec (ϮSD ϭϮ8.66, N ϭ 15) of cation (see Kroodsma et al., 2001), the ex- overlapping time per trial for the ®ve ex- perimental design included four treatments emplar calls. All 15 trials were signi®cantly ϫ 5 exemplars ϫ three blocks with the se- different from the random distribution for quence of treatments and exemplars ran- NUMBER and TIME (Table 1). domized within each block. However, the Most males ceased calling when S2, S3, full power of this design could not be im- or noise was played while they were emit- plemented in the data analysis (e.g., analy- ting S1 of their call. Signi®cantly fewer sis using mixed-model ANOVA) because males ceased calling in response to play- of lack of independence in the duration of back of S1 and no males ceased calling call sections. Therefore, we measured the when no signal was played (Fig. 2). duration of each section of an experimental Playback of S2 while males emitted S1, male's call and analyzed treatment effects S2, or S3 had a signi®cant effect on the using Friedman's test for related samples. duration of call components and the total call duration (Fig. 3A±D). Three main pat- RESULTS terns are apparent. First, all males ceased Preliminary analyses showed that males calling when S2 was played while they interacting with recordings of male calls emitted their S1 (Fig. 3B, C). Second, the tended to limit their calling to the 40.0 sec duration of S3 was greatly reduced when period of silence between playback calls, S2 was played while males emitted their thus avoiding overlap. Eight of the 15 and S1, S2, or S3 (Fig. 3C). Third, the duration 12 of the 15 trials were signi®cantly differ- of S2 was not apparently affected when S2 ent from the randomized distribution for was played while they emitted S2 (Fig. 3B). 1226 R. E. HUNT AND T. L. MORTON

DISCUSSION Our ®nding 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 vi- brational interactions between or among males sometimes involve signals distinctly different than mating signals (i.e., aggres- sive or rivalry signals) and interactions are often elicited by or accompany physical ag- gression (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 co- operative or competitive interactions (see Green®eld, 1994). Cooperative interactions include those where overlap reduces female response to all signalers due to the degra- dation of call structure or inappropriate tim- ing of signal components. In contrast, in- teractions 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 coopera- tive behavior can be excluded because fe-

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FIG. 3. A) Duration of S1 of male calls in response to silence (control) or playback of S2 while males were emitting S1, S2, or S3 (Friedman's test: chi-square ϭ 22.9, df ϭ 3, P Ͻ 0.001). B) Duration of S2 of male calls in response to silence (control) or playback of S2 while males were emitting S1, S2, or S3 (Friedman's test: chi-square ϭ 31.8, df ϭ 3, P Ͻ 0.001). C) Du- ration of S3 of male calls in response to silence (con- trol) or playback of S2 while males were emitting S1, S2, or S3 (Friedman's test: chi-square ϭ 28.58, df ϭ 3, P Ͻ 0.001). D) Duration (all sections) of male calls in response to silence (control) or playback of S2 while males were emitting S1, S2, or S3 (Friedman's test: chi-square ϭ 30.42, df ϭ 3, P Ͻ 0.001). REGULATION OF CHORUSING 1227 male response does not decline signi®cantly sponse calls that they emit between chirps when overlapped male calls are played that comprise S2 (Hunt et al., 1992). Fur- (R.E.H. and T.L.M., unpublished data). ther studies are required to more clearly de- Although there are no apparent structural ®ne temporal constraints on call suppres- differences between calls emitted by G. ni- sion and promotion in G. nigrifrons. grifrons males in the context of mating and In addition to males ceasing call emis- chorusing, it is possible that structural dif- sion when signals are played during S1 of ferences within the male call have multiple their call, they also reduce the duration of functions. Playback of isolated S2 and S3 their S3 when S2 is played. Their sensitiv- are equally effective in eliciting female re- ity to signals while emitting S1 and S3, but sponses when played at levels approximat- not S2, leads us to predict that individual ing a nearby male (Hunt et al., 1992). In males may reduce their call rate and the to- the present study the amplitude of playback tal duration of their calls as chorus size in- signals also approximated that of a nearby creases. male. Under these conditions playback of S2 to males that were emitting S1 resulted ACKNOWLEDGMENTS in the highest percentage shutdown, al- We thank Robin Lacour for laboratory though not signi®cantly different than S3 or assistance. This study was funded by a Na- noise. Before concluding that S2 and S3 are tional Research Initiative Competitive functionally identical, their in¯uence on Grants Program grant (9401839) to R.E.H. males and females when played at ampli- and an Undergraduate Biological Sciences tudes that simulate distant males should be Education Initiative, Howard Hughes Med- investigated. Furthermore, the in¯uence of ical Institute grant to Centre College. random noise on male calling suggests that other environmental factors (e.g., wind gen- REFERENCES erated vibrations) may be important in reg- Andersson, M. 1994. Sexual selection. Princeton Uni- ulating calling behavior. versity Press, Princeton, New Jersey. Our ®nding that males interrupt call Belwood, J. J. and G. K. Morris. 1987. Bat predation and its in¯uence on calling behavior in neotropical emission in response to S2, S3, and noise katydids. Science 238:64±67. while they are emitting S1, but not S2, sug- Brush, J. S. and P. M. Narins. 1989. Chorus dynamics gests a general mechanism that results in of a neotropical amphibian assemblage: Compar- call alternation in G. nigrifrons. S1 may ison of computer simulation and natural behav- iour. Anim. Behav. 37:33±44. represent a ``ready to call phase'' similar to Claridge, M. F. and P. W. F. de Vrijer. 1994. 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