J. Insect Physiol. Vol. 43, No. 10, pp. 965–971, 1997 1997 Elsevier Science LtdPergamon All rights reserved. Printed in Great Britain0022-1910/97 $17.00 + 0.00PII: S0022-1910(97)00034-6

The Effect of Flash Duration and Flash Shapeon Entrainment in Pteroptyx malaccae, aSynchronic Southeast Asian FireflyJONATHAN COPELAND,*‡ ANDREW MOISEFF†

Received 10 October 1996; revised 14 February 1997

Southeast Asian synchronic fireflies respond to stimulus flashes by phase-shifting theirendogenous oscillator. This is called ‘flash entrainment’. The releasers for entrainment werestudied by changing stimulus flash shape and duration in Pteroptyx malaccae. Stimulus flashshapes and durations were synthesized digitally and delivered by a field-portable computersystem. The computer also recorded male firefly flashes that were detected with a photometer.We found that the type of entrainment and its magnitude was influenced by the duration ofthe entrainment flash and by its shape. 1997 Elsevier Science Ltd. All rights reserved

Bioluminescence Entrainment Fireflies Synchrony

INTRODUCTION

Certain Southeast Asian fireflies show synchronousflashing (Buck, 1988). Mechanisms of synchrony havebeen studied in greatest detail in Pteroptyx malaccae andPteroptyx cribellata (Hanson, 1978, 1982; Buck et al.,1981a, b). In these species, synchrony was shown to useanticipatory mechanisms. Thus, the flashes of one fireflycould advance or delay the flashes of a second firefly,and this produced synchrony or maintained synchrony,even when individual free running periods were notidentical (Buck, 1988).

Hanson 1978, 1982 suggested that synchronic flashingin fireflies could be modelled as if it was controlled bya circadian rhythm-like mechanism. Like a circadianrhythm, flashes could be entrained by a ‘zeitgeber’ or apacer light (stimulus flash) that simulated a firefly flash.(Entrainment was defined as a steady state relationshipthat developed between a driving oscillator and a drivenoscillator.) Whereas, in circadian systems, an intervalwas matched in a steady-state relationship, in fireflysynchrony, both phase and interval could be matched.Two types of entrainment were shown for SoutheastAsian fireflies: one in which the period was constant (P.

*Department of Biology, Georgia Southern University, LB-8042,Statesboro, GA 30460, U.S.A.

†Department of Physiology and Neurobiology, University of Con-necticut, Storrs, CT 06269, U.S.A.

‡To whom all correspondence should be addressed. Fax: 011-1-912-681-0845.

965

cribellata) and one in which the period was variable(P. malaccae).

The photic releasers for the synchronic behavior ofSoutheast Asian fireflies have not yet been studied indetail. Here, we systematically changed stimulus shapeand stimulus duration, parameters not treated by currententrainment models, to see if entrainment would beinfluenced. In so doing, we used new technologies (notyet available when the Buck–Case–Hanson studies onsynchrony were carried out) to demonstrate that the dur-ation and the shape of the entrainment flash influencesthe type of entrainment shown and its magnitude.

MATERIALS AND METHODS

Pt. malaccae were collected on the Pontian River inPontian, Malaysia and transported to an air-conditionedlaboratory in Singapore and maintained on a natural lightcycle. Animals were housed individually in small plasticbags or in groups of 10–20 in 189-l bags. Each plasticbag contained a small ball of wet filter paper and a sliverof apple. Both were changed every other day.

Stimuli were synthesized digitally and delivered usinga 12-bit digital-to-analog converter operating with a sam-ple rate of 1 kHz. The wave form (shape and amplitude)of stimulus flashes could be adjusted independently, ascould the timing between flashes. Smoothly varying‘Sine-like’ flashes were produced by calculating onecycle of an offset cosine:

y = 0.5 × (1 − cos(w)), for 0 # w # 2p

966 JONATHAN COPELAND AND ANDREW MOISEFF

Flash intensity and duration were modified by scalingthe amplitude and duration of this wave form. Other arbi-trary wave forms were synthesized by specifying theamplitude and time of arbitrary inflection points. Theregions between these inflection points were connectedby linear interpolation. A correction factor was appliedto the wave forms to account for the nonlinear propertiesof the light emitting diodes. During this study, we restric-ted ourselves to five stimulus shapes (Fig. 1): normal(which mimicked the bimodal normal Pt. malaccaeflash), sine-like, rectangular, blink, and ramp. Animalswere tested individually in the small clear plastic bags.A green LED, positioned 10 cm from the bag, deliveredthe stimulus.

Firefly flash activity was detected with a Hanson-typephotometer (Buck and Buck, 1968) that used an RCA931 phototube. The photometer’s output was digitized(12-bit analog-to-digital converter, sampled at 1 kHz)and stored for subsequent analysis. Each experimentaltrial lasted 1 min (Fig. 2). During the first 8–10 s, nostimulus flashes were produced, and the free-runningfirefly flashes were recorded. After this time, stimulusflashes (of a selected shape) were presented at a 1200-ms interval, which is approximately the normal fireflyflash interval. The flash activity of the firefly was con-tinually monitored throughout the trial.

The digitized photometer records were reviewed asraster displays with the width of the raster set to the inter-val of the stimulus flash presentation. The stimulusoccurred at the beginning of each raster line. Using sucha display, a constant free-running interflash intervalwould appear as a signal that drifted across the displayat a rate proportional to the difference between theinterflash interval and the stimulus repetition rate. In con-trast, signals that were entrained to the stimulus would

FIGURE 1. Stimulus shapes used were (A) normal (which mimickedthe bimodal normal P. malaccae flash); (B) sine-like; (C) rectangular;

(D) blink; and (E) ramp.

appear at similar locations in successive raster lines andthus be aligned vertically.

When displayed in this way, entrainment of the fire-fly’s flashes was easily visualized. Responses werequantified with a temporal resolution to within 1 ms.

RESULTS

Open-loop entrainment

The entrainment paradigm was open-loop because thestimulus was produced at a fixed interval, irrespective ofthe response of the animal (see Discussion) (but the ani-mal actually closes the loop to effect the entrainment).During the first 8–10 s of each experimental trial, werecorded the firefly’s free-running interflash interval (firstfive lines of Fig. 2). During the remaining 50–52 s ofeach trial, we presented stimulus flashes of selectedshapes at repetition rates close to the individual’sinterflash interval.

The occurrence of entrainment and whether it occurredthrough phase-advance or phase-delay mechanisms werealso evident with this plot. In the example shown (Fig.2), the firefly’s free-running interflash interval wasapproximately 1250 ms (T = 22°C). We delivered allstimuli at a repetition rate of 1200 ms, and so the free-running flashes drifted to the left in the display. Thefirefly shortened its interflash interval by almost 50 msper stimulus flash (phase-advance mechanism) until itwas entrained with the stimulus flashes (firefly flashesoccurred at similar locations in successive raster lines).

Once steady-state entrainment was attained, it wasclassified as either Lead or Lag synchrony, according towhether the firefly’s flash preceded (Lead) or followed(Lag) the entrainment flash when in a steady state. Fig.2 illustrates Lead synchrony since the first flash of thetwo flash signal preceded the stimulus flash, which,although not visible, began at the left most edge of eachraster line.

Effect of stimulus duration

Our instrumentation gave us the ability to control allaspects of the shape and timing of the stimulus flashes.We investigated whether entrainment was affected bystimulus duration. The results from five different firefliesstimulated with rectangular waves of different durationare shown in Table 1 (animal A is shown in Fig. 3). MaleP. malaccae could be entrained to stimulus flashes of 10–1000 ms. When the stimulus flash duration was changed,the nature of the synchrony changed. Lag synchrony waselicited by longer duration ( $ 160 ms) stimulus flashes,whereas Lead synchrony was elicited by shorter duration( , 160 ms) stimulus flashes. Similar results wereobtained using ramp stimuli. The results of one experi-ment are shown in Fig. 3. Again, Lead synchronyoccurred at shorter stimulus durations ( , 160 ms) andLag synchrony occurred at longer stimulus durations ($ 160 ms). When the duration of sine-like waves was

967ENTRAINMENT IN PTEROPTYX MALACCAE

FIGURE 2. Flash entrainment was studied with a portable-computer-based photometric system. Records read from left to rightand top to bottom. The record begins with spontaneous flashes from a single Pteroptyx malaccae male. After 10 s, when theframe broadens (arrow), an 80 ms rectangular stimulus occurred at the beginning of each sweep. Half way down the recordon the right, the firefly’s flash occurred just before the next stimulus flash and a steady state was achieved. This was called

Lead synchrony. T = 22°C.

changed, a linear change occurred in the value of theLead synchrony (Fig. 4A), and the slope of this curvewas significantly different from that of the rectangularwave (P , 0.05) (see below).

Effect of stimulus shape

The species-specific display flash of P. malaccae con-sisted of two symmetrical flashes that occurred at aninterval of 60 ms (Fig. 2, lines 1–5). Each flash took25 ms to go from base to peak and 25 ms to go frompeak to base. The amplitude of the first flash was lowerthan that of the second flash. This two-flash signal wasrepeated at a set interflash interval [850 ms for MalaysianP. malaccae at 28°C, Buck and Buck (1978); 930 ms forMalaysian P. malaccae, Hanson (1978); here, 1250 msfor Malaysian P. malaccae at 22°C].

Our instrumentation allowed us to synthesize flasheswith almost any arbitrary shape. However, since we wereinterested in the effect of edges, i.e. sharp light–darktransitions, we chose five wave-form shapes that variedin the rate of rise of the stimulus and the number andlocation of stimulus edges. Normal-like wave and sine-like wave were similarly shaped, except that normal-likehad bimodal peaks (first smaller than the second) like thedisplay flash of P. malaccae. The pulses of both weredifferentiated versions of the rectangular stimulus pulse,but had slower rise times and fall times and no sharpedges. Rectangular had two sharp edges, one at stimuluson and another at stimulus off. Blink had one edge (at

stimulus on) and then a slow ramp down until the stimu-lus went off. Ramp had a slow ramp on and a sharp edgeat stimulus off.

When an 80-ms-duration blink stimulus and an 80-msramp stimulus were compared within the same firefly (N= 4 fireflies) (Table 2, animals A–D), the entrainmentshown following blink stimulus was always significantlygreater than that of ramp stimulus (t-test, P , 0.01).Sine-like stimulus pulses produced entrainment that wasdifferent from that produced by rectangular pulses (t-test,P , 0.05) and showed a different rate of decrease inLead entrainment when stimulus duration was increased(Fig. 4A–B). Stimulation with a normal-like stimulus wasnot significantly different from a stimulus with rectangu-lar waves at the same duration (Table 2, Animal I, Fig.4C). At stimulus durations greater than 80 ms (Fig. 4A–B), normal-like pulses produced smaller Leadentrainments than rectangular pulses, but paired compari-sons were not made.

DISCUSSION

The control of synchronous flashing in P. malaccaehad previously been studied by Hanson, Buck, Buck, andCase (Buck, 1988). Using single pulses and repetitivestimulation, they used a square-wave flash stimulus toconstruct phase-response curves. A stimulus flash wouldmake the next firefly flash occur faster (phase-advance)or slower (phase-delay), depending upon the phase when

968 JONATHAN COPELAND AND ANDREW MOISEFF

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969ENTRAINMENT IN PTEROPTYX MALACCAE

FIGURE 3. Effect of stimulus duration on flash entrainment. Rec-tangular wave was used with one firefly and ramp stimulus with asecond firefly. Entrainment changed from Lead to Lag as the stimulusduration increased. The ramp stimulus showed a linear change withduration, but the rectangular wave showed only slight change after

160 ms. T = 23°C.

the stimulus occurred in the firefly’s cycle. When repeti-tive stimulus flashes were provided, a steady stateentrainment could be achieved that could be close to uni-son [see Hanson (1982), fig. 4.9–10].

Our study was initiated to provide further understand-ing of the mechanisms of entrainment (synchrony). Thus,we exploited the availability of high speed portable com-puters and data acquisition hardware to develop a systemthat could deliver computer-synthesized stimulus flashesand record the flash activity of individual fireflies withspeed and accuracy. This could not have been achievedwith simple waveform generation equipment or when anincandescent bulb was used to produce stimulus flashes.

We found that stimulus duration and stimulus shapeaffected the nature of the entrainment shown by P. mal-accae. This effect could not be predicted from the pre-viously existing models for synchrony (Hanson, 1978;Buck et al., 1981a, b).

In preliminary studies, Hanson (personalcommunication) found that stimulus duration could effectentrainment of P. malaccae, and our observations are inagreement with Hanson’s. As stimulus flash duration wasincreased, entrainment changed from Lead to Lag (Table1). This effect was seen using a rectangular stimulus (twoedges) and a ramp stimulus (one edge at stimulus off).Other stimuli were not tested over the same stimulusrange, but the amount of Lead entrainment also decreasedfor a sine-like stimulus when the stimulus duration wasincreased (Fig. 4A).

A synchronously flashing Southeast Asian firefly willshow a phase-response curve to repeated light presen-tation (Buck, 1988). (An exogenous timing flash comingin just before the firefly’s flash will shorten the nextcycle. The same timing flash coming in just after thefirefly’s flash will lengthen the firefly’s next cycle. In thisway, by speeding up or slowing down his cycle, a maleP. malaccae comes into synchrony with the

FIGURE 4. Effects of flash shape on entrainment. Response of singlefirefly to rectangular (Fig. 4A–C) and sine-like stimulus flashes (Fig.4A) at different stimulus durations and a comparison to normal stimu-lus flashes (P. malaccae simulation) at the same duration. (A), (B),

and (C) are from three different fireflies, respectively.

congregation.) This suggests that there must be precisetiming cues present in the entrainment signal. Our obser-vation that magnitude and the sign of the entrainmentchanges as the duration of the stimulus changes (Table1) supports this suggestion. (In the field, where there aremultitudes of fireflies, it is not clear exactly how theinterflash interval is set.) The changes that occur aresmall, and they might not have been detected were wenot using a rapid stimulation-recording system with accu-racy that was less than 1 ms.

970 JONATHAN COPELAND AND ANDREW MOISEFF

TABLE 2. Effect of stimulus shape on flash entrainment X (±SD)

Animal Lead/Lag entrainment (X ± N n PSD)

Ramp stimulus compared to blink stimulusA − 23.3 ± 7.7 17 1 , 0.01

− 126.8 ± 53.2 53 3B − 31.8 ± 11.9 34 1 , 0.01

− 56.9 + 5.0 116 5C − 48.4 ± 1.5 100 4 , 0.01

− 72.8 ± 5.4 71 3D − 66.1 ± 9.8 175 7 , 0.01

− 72.9 ± 3.1 63 3E − 24.1 ± 7.6 23 1F − 30.3 ± 5.4 87 3G − 31.8 ± 11.9 34 1H − 54.3 ± 14.0 150 5

Rectangular stimulus compared to normal stimulusI − 49.1 ± 20.2 95 5 NS

− 46.0 ± 8.5 265 13J − 29.3 ± 4.2 277 10K − 38.8 ± 11.8 213 7L − 46.8 ± 6.4 89 5M − 60.0 ± 20.4 12 1

All stimulus flashes are presented at a duration of 80 ms.N = number of entrainment intervals measured.n = number of 60-s trials.Animals A–D, I: t-test.− = Lead.Animals A–E in Table 2 are different from A–E in Table 1.

There could be more than one type of timing cue pro-vided by the stimulus flash. The ‘on’ and ‘off’ of a stimu-lus (rectangular, blink, ramp) could serve as two inde-pendent timing cues to pace the synchrony. This issupported by the changes in synchrony that seem to bestimulus duration-dependent. The integration of the lightoutput over a short period of time (normal-like, sine-like)could also act as a timing cue as well.

Stimulus shape made a difference in the magnitude ofthe entrainment as well (Table 2). When similar rec-tangular and sine-like stimuli were compared (two edgesvs. no edges), there were significant differences betweenthe two (t-test, P , 0.05) (Fig. 4A–B). When blink andramp (both with one edge) were compared at the same80-ms duration (Table 2), entrainment occurred at differ-ent delays (t-test, P , 0.05). There were no apparentdifferences in entrainment when the entrainment pro-duced by rectangular flashes (two edges) and normalflashes (no edges) were compared (Table 2, animal D;Fig. 4C).

Perhaps the number of edges present in a rectangularstimulus mimics the number of edges present in the fire-fly’s normal display flash. P. malaccae has a display flashthat consists of two very short (50 ms) pulses that occurwith only 20 ms between flashes. The flash seems to be asingle flash to human observers (although the photometryreveals that it is two). Perhaps each short duration ‘nor-mal’ flash is interpreted as an edge by the firefly and,thus, the two flashes of the species-specific flash eachprovide timing cues that lead to entrainment.

Our observations expand the findings of previous stud-ies concerning the effects of the phase of the timing pulseon flash entrainment (Hanson, 1978, 1982; Buck et al.,1981a, b; Buck, 1988). Stimulus duration and shape arealso important factors that control flash entrainment.Also, existing models make the assumption that synch-rony occurs as if there were a continuously open windowthrough which steady-state photic stimulus driving leadsto entrainment. These models assume that the system isopen-loop, with the stimulus flash occurring regardlessof what the firefly does. However, we have tried closed-loop stimulation, in which the firefly flash triggers theentrainment flash, and find that the previously narrowrange of entrainment shown by P. malaccae (Hanson,1978, 1982) becomes larger when closed-loop stimu-lation is used (Moiseff and Copeland, unpublishedobservation). Also, Soucek and Carlson (personalcommunication) have proposed that the firefly is onlysusceptible to modulation by a photic stimulus during asmall phase of its cycle. All these factors should be takeninto consideration when we develop new models for Sou-theast Asian firefly synchrony.

REFERENCES

Buck J. (1988) Synchronous flashing of fireflies II. Quarterly Reviewof Biology 63, 263–281.

Buck J. and Buck E. (1968) Mechanism of rhythmic synchronousflashing of fireflies. Science 159, 1319–1327.

Buck J. and Buck E. (1978) Toward a functional interpretation ofsynchronous flashing by fireflies. American Naturalist 112, 471–593.

971ENTRAINMENT IN PTEROPTYX MALACCAE

Buck J., Buck E., Hanson F. E., Case J. F., Mets L. and Atta G. J.(1981a) Control of flashing in fireflies IV. Free run pacemaking ina synchronic Pteroptyx. Journal of Comparative Physiology 144,277–286.

Buck J., Buck E., Case J. F. and Hanson F. E. (1981b) Control offlashing in fireflies V. Pacemaker synchronization in Pteroptyx cri-bellata. Journal of Comparative Physiology 144, 287–298.

Hanson F. E. (1978) Comparative study of firefly pacemakers. Feder-ation Proceedings 37, 2158–2164.

Hanson, F. E. (1982). Pacemaker control of rhythmic flashing of fire-

flies. In Cellular Pacemakers, ed. D. Carpenter, Vol. 2, pp. 81–100. Wiley, New York.

Acknowledgements—This work was supported by National ScienceFoundation grant BNS-9208708. We thank Dr Ivan Polunin and MrsFam Siew Yin for logistical support in the field. We thank Dr A.D.Carlson for his critique of the manuscript.