© 2004 by The Society for Integrative and Comparative Biology
Flash Precision at the Start of Synchrony in Photuris frontalis1
1 Department of Biology and Applied Coastal Research Laboratory, Georgia Southern University, Statesboro, Georgia 30460-8042
2 Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269-4156
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SYNOPSIS |
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Synchronous flashing occurs in certain species of Southeast Asian and North American fireflies. Most Southeast Asian synchrony involves stationary congregating fireflies, but North American synchrony occurs in flying fireflies that do not congregate. Southeast Asian synchrony is usually continuous, but North American synchrony is interrupted. Photuris frontalis, the only member of the North American genus Photuris to synchronize, shows an intermittent synchrony. This involves synchronization and repeated re-synchronizations while in flight. The precision that occurs at the start of synchrony was studied in Ph. frontalis using caged fireflies and photometry. Barrier experiments (using two fireflies) or flash entrainment experiments (using one LED and one firefly) were performed to measure the temporal precision of the first entrained flash. In both cases, the first entrained flash was close to unison synchrony (phase = 1.0) and showed little variability. The behavioral implications of the ability to synchronize with the first entrained flash are not known, but it might facilitate male-male interactions during brief, transient encounters such as maintaining distance between closely flying males in search of females.
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INTRODUCTION |
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The modern study of synchrony in fireflies dates from 1968, when John and Elisabeth Buck used cine photography and photometry to demonstrate that a certain number of Southeast Asian firefly species flash in rhythmic synchrony (Buck and Buck, 1968
, 1976). They showed that flashes in the congregation of fireflies varied ±13 to 15 msec when the average interflash interval was 560 msec. Buck and Buck (1968) used the coefficient of variation (V = standard deviation/mean period) to compare the firefly's flashing rhythm with the precision of other rhythmically occurring animal behaviors, e.g., North American non-synchronic rover fireflies, the call of the whippoorwill, song of the snowy tree cricket, human heartbeat, human fingertap. The rhythmically repetitive flashing of the synchronic Southeast Asian fireflies was found to have a V = 0.6 to 1.4 whereas the other behaviors ranged between 3.5 and 11. The North American rover fireflies Photinus pyralis and Photuris versicolor had a V of 11 and 7 respectively (Buck and Buck, 1968). Thus, Southeast Asian synchrony was considered to be very precise biological timing (summarized in Buck, 1988).
We used the approaches developed by Buck and Buck (1968) and later by Hanson, Case, Buck, and Buck (Hanson et al., 1971; Hanson, 1978; Buck et al., 1981a, b) to establish that two species of North American fireflies also flash synchronically. Synchrony was defined as concurrent rhythmic group flashing (Buck, 1988).
We used videography, photometry, computer-shaped LED flash, and flash entrainment experiments to show that flashes of the North American firefly Photinus carolinus were synchronic (fixed phase rhythmic group flashing) (Copeland and Moiseff, 1995) and described some of the mechanisms underlying its discontinuous synchrony (an interflash interval that is insensitive to exogenous pulses of light, an interburst interval that is phase-sensitive to exogenous light pulses) (Moiseff and Copeland, 1995; Moiseff et al., 1999). A few years later, we discovered that the North American firefly Photuris frontalis is synchronic (Moiseff and Copeland, 2000) and described some of the mechanisms underlying its intermittent synchrony (initial inhibition, only phase delay responses to exogenous pulses [Moiseff and Copeland, 2000; Copeland et al., 2000]).
Ph. frontalis is the only member of the genus Photuris to show synchrony. The male species-specific flash code involves a single flash repeated rhythmically every 0.6 to 1.0 sec (depending upon temperature) (Fig. 1 top). At irregular intervals, pauses (indicated by the arrow) occur in this flash pattern. Flashing males fly into proximity (30 cm) and flash together repeatedly and rhythmically at the species-specific interval, i.e., synchronously, for several flash cycles (Fig. 1B bottom). Then, one firefly will stop flashing while the other continues flashing. Subsequently the stopped firefly begins to flash, again synchronously. Because pauses and resumptions are characteristic of both species-specific rhythmic solo flashing and the synchronic flashing of two or more males, the synchrony of Ph. frontalis has been termed an intermittent synchrony (Moiseff and Copeland, 2000). It is different from the continuous synchrony of the Southeast Asian firefly Pt. malaccae (Buck and Buck, 1968) in which the males are stationary in trees and flash continuously all evening once they start.
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In fireflies, the precision of the species-specific rhythm has been measured (Lloyd, 1966
; Buck and Buck, 1968; Buck and Case, 1986), but the precision at the beginning of synchrony in any s firefly has not. This is because most synchronic fireflies show a continuous synchrony where the start and resumption of synchrony are infrequent and can only be indirectly studied through flash entrainment. The goal of this study was to assess the precision of initial synchronous flash timing in an intermittently synchronic firefly. An understanding of the precision of synchronization after an initial inhibitory period may shed some light on the behavioral function of synchrony. It could also promote an understanding of the neural control of the flash in Ph. frontalis and could play a role in our understanding of brain function in fireflies and other insects.
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MATERIALS AND METHODS |
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Ph. frontalis males were collected in a maritime forest on Skidaway Island, Georgia over three field seasons. They were maintained individually in 9 cm petri dishes at 20–23°C on a 14:10 light:dark cycle. Darkness occurred at 20:30, which was similar to the natural light cycle. Each petri dish was lined with wet filter paper and apple shavings were provided ad lib.
Two types of experiments were carried out: barrier experiments and flash entrainment experiments. In both, flashes were recorded from a freely walking firefly contained in an isolated 9 cm diameter petri dish cage using a side-window RCA-9611 photomultiplier tube. Two photomultiplier tubes were used in barrier experiments with one photomultiplier tube looking into each chamber. One photomultiplier tube was used for flash entrainment experiments. Fireflies were tested between 20:30 and 1:00 hours. Photomultiplier output was digitized (1,000 samples/sec) and processed off-line using customized software (A.M.) to determine flash timing and precision.
In the barrier experiment, each firefly was housed separately in a 9 cm petri dish and placed in a dark rectangular 40 l x 28 w x 20 h cm chamber. Petri dishes were separated by at least 15 cm. The chamber was closed on 5 sides and open on the sixth. The open ends of the chambers faced each other. A single removable opaque divider separated the two chambers. When this was removed, the fireflies could see each other. In each trial the flashing was recorded for one minute by photometry and digitized using DTVee software customized by A.M.
In the one minute entrainment experiments, the spontaneous flashing of an isolated male was recorded for the first ten seconds, then, for the remaining 50 seconds, he was exposed to the rhythmic flashing of a green LED, as in the work of Hanson et al. (1971). An LED was used because the interflash interval of an individual isolated firefly showed some variability and because we compounded this "noise" by using two fireflies in the barrier experiment. The LED's flash contour was shaped to counterfeit the species-specific flash of Ph. frontalis. The LED provided a precise, rhythmic, and repetitive stimulus (entrainment) pulse, and the firefly flashed synchronically with this counterfeit flash. The LED's interflash interval was set to be just less than the firefly's spontaneous interflash interval because only phase advance flashes were reliably seen.
These observations (Fig. 2) were then displayed as a phase plot (Fig. 3). Phase is the time difference between two related events in a cyclical system, e.g., the time in a standard (pacer) flash cycle in which an exogenous flash occurs. The phase plot normalizes the data so that the relative temporal relationships between two fireflies (or an LED and a firefly) can be determined without concern for the exact time intervals, which may differ between individual subjects at different temperatures.
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1. Sometimes (flashes 41–47) the phase locking was not constantAll experiments were done in a darkened room. Firefly flash and locomotion were observed using IR illumination that did not effect the phototube.
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RESULTS |
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Caged Ph. frontalis flashed rhythmically (solo or synchronic) while they walked or were standing still. They also walked or stood while not flashing. Their behavior was similar during flash entrainment. They could not be viewed during the barrier experiments because of the chambers' placement. During the barrier experiments, they could not be viewed.
Barrier experiments
Figure 2A illustrates a typical experiment. The barrier was lifted after 10 sec, at the 13th flash (arrow) whereupon one firefly (the one whose flashes were recorded on the lower trace) stopped flashing almost immediately. This initial inhibition lasted 16.5 sec and then the bottom firefly's flashing resumed (Fig. 2A, B). These flashes were closely phased with those of the other firefly. Then, the bottom firefly's flashing stopped (1 sec) and started again (Fig. 2A–B). Such resynchronizations are seen in fireflies in the field.
The Figure 2A data can also be displayed as a phase plot (Fig. 3). In Figure 3, the interflash interval of the top trace of Figure 2 was used as a standard. When the fireflies were not in visual contact (barrier in place), each firefly maintained its own constant, individual interflash interval. The phase relationship (a difference) between the two constant, but independent interflash intervals produces a non-zero slope, as was observed (Fig. 3, prior to arrow). The initial inhibition began when the barrier was removed (Fig. 3, arrow), the initial inhibition began after three additional flashes (indicated by the filled circles). During the initial inhibition, the top trace's firefly flashed steadily while the bottom trace's firefly stopped flashing completely (Figs. 2 and 3). Then, synchronous flashing (phase 0.8 to 1.0) occurred (starting with flash 37, phase = 0.93). When synchrony occurred, the bottom trace's firefly (Fig. 2) flashed 47 to 76 msec before the top firefly. During synchronous flashing, sometimes the phase was constant (e.g., flashes 37 to 41, 59 to 68, 68 to 75) and sometimes drifted (flashes 41 to 47). Toward the end of this trial (e.g., flashes 68 to 75), the synchrony was at unison or close to it. In Figures 2 and 3, all but two flashes from the bottom trace's firefly occurred before the standard firefly's flash, i.e., had a phase lead.
To define the precision at the beginning of synchrony, 35 trials were carried out using 12 pairs of fireflies (Fig. 4). The first flash of the previously inhibited firefly was at a phase near 1.0 (range = 0.8 to 1.45, mean = 1.02 ± 0.13) (Fig. 4). When converted to time, these phases correspond to temporal deviations of 0 to 113 msec from a perfect unison with the reference flash which had an average interflash interval of 750 msec. The mean phase difference was not significantly different from a phase of 1.0 (t-test, P > 0.05). In these fireflies, there was no difference in the number of exogenous stimulus flashes that led or lagged the standard flash during synchrony.
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Flash entrainment experiments
The flashing LED produced an almost immediate cessation of spontaneous firefly flashes. In all cases (N = 37 trials, 8 fireflies), an inhibition then occurred. The duration of the initial inhibition ranged from 500 msec to 28 sec.
The precision of the timing of the first flash to occur after the initial inhibition was measured with the LED's interflash interval as the standard. Most first flashes after initial inhibition occurred at a phase of 0.9–1.0 (Fig. 5), and these were mostly lead flashes (mean = 0.97 ± 0.09). When converted to time, these phases ranged from 0 to 90 msec from a perfect unison with the standard flash. The mean phase was not significantly different from a phase of 1.0 (t-test, P > 0.05). This indicated that there was no consistent lead flash behavior.
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DISCUSSION |
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Comparison of synchronizers
In the North American synchronic firefly Ph. frontalis, the first flash after the initial inhibition occurred at an average phase difference of only 0.97 (Fig. 4) or 1.02 (Fig. 5), i.e., at an average of 23 msec before and 15 msec after the driver, respectively. These intervals represent 3.06% and 2.00% of the cycle time of a 750 msec spontaneous interflash interval, and compare favorably with the steady state synchrony in Southeast Asian fireflies (Buck and Buck, 1968
; Buck, 1988).
The precision of the first synchronic flash with an artificial driver is not known for the Southeast Asian synchronizers Pt. malaccae and Pt. cribellata. Additionally, no initial inhibition was seen in either Southeast Asian species that were studied in depth (Buck and Buck, 1968; Buck et al., 1981a, b). In Pt. malaccae, flash entrainment began with a gradual change in interflash interval until steady state entrainment was achieved. If steady state entrainment is considered the beginning of synchrony, the start of synchrony could not be determined with any reliability. In Pt. cribellata, unlike Pt. malaccae, the entrainment occurred after one or two interflash intervals. Again unlike Pt. malaccae, the entrained firefly's interflash interval did not change. In Pt. cribellata, the phase was reported, but the precision at the start of synchrony was not (Buck et al., 1981a, b; Hanson, 1978). Phase changed with stimulus interval, and unison synchrony (phase = 1.0) could only be achieved when the firefly's spontaneous interflash interval was identical to the stimulus interflash interval.
In the field Ph. frontalis males flashes synchronously with nearby males though their flight paths are independent (Moiseff and Copeland, 2000). This is in stark contrast with Oriental congregational fireflies that flash synchronously while stationary (Buck, 1988) or hovering (Buck and Buck, 1978). However, the males of both Pt. malaccae and Pt. cribellata joining a swarm tree often flash synchronously with the males already perched (Buck and Buck, 1978). A few flash cycles of synchronous flashing of an unidentified New Guinea Highlands firefly have been filmed while they flew and flashed discontinuously like a synchronic moving cloud (Buck, 1988), and a North American firefly P. carolinus is known to fly and synchronize discontinuously (Copeland and Moiseff, 1995), a characteristic feature of this synchrony (Copeland and Moiseff, 1995).
Toward a behavioral function for synchrony
In Ph. frontalis, the precision of the first synchronic flashes and the pauses in synchronous flashing might only be an epiphenomenon reflecting the neurophysiological characteristics of the species-specific CNS network that controls synchronic flashing and thus not have any identifiable behavioral function. Alternatively, the precision and the inhibition could have a behavioral (evolutionary) function. Unfortunately, the behavioral function of synchrony has not been determined for any firefly species but at least eight hypotheses have been proposed (summarized in Buck, 1988).
We suggest that the male-male activity of synchrony in Ph. frontalis serves to spatially separate the flying flashing males. Flying Ph. frontalis males do not congregate in the field (Moiseff and Copeland, 2000) and caged males do not congregate in the lab (J.C., unpublished data). When ten flashing Ph. frontalis males were placed in a 30 cm diameter clear cage, the males flashed, synchronized, walked, stood still, but did not congregate. They appeared to be separated in the cage and without any discernable spatial order. In another observation, two flashing males, placed in two 23 cm long glass tubes, flashed while stationary or while they walked up and down the tube, seldom remaining in proximity. In fact, synchrony occurred more regularly when the fireflies were distant than when they were close (J.C., unpublished data). Buck and Buck (1968, 1978) reported that synchronizing Pt. malaccae males were separated by 10 to 15 cm. At closer distances, synchronizing males repelled each other aggressively.
The purpose of spatial separation in Ph. frontalis is unclear. If it were greater than the female's spatial resolution (at the male's patrolling elevation) (Moiseff and Copeland, 2000) two fireflies could be discriminated. The separation could allow a male sole access to a female, enabling the next stage of mating behavior to take place, as may be the case in Pt. malacccae. (Buck and Buck, 1978, 1980). Synchrony and spacing might be important for Ph. frontalis males because females are extremely rare at our Ph. frontalis study site (J.C., unpublished data).
The precision at the start of synchrony might result from a male firefly flying, synchronizing, and re-synchronizing. Flying might mean that there is a need to synchronize quickly. A previously inhibited male would benefit by flashing precisely at the start of synchrony because precision means that synchrony starts immediately. If synchrony is an enabler, what ever it is enabling can quickly begin. It may be significant that flash entrainment is rapid in flying Ph. frontalis and more gradual in stationary Pt. malaccae (Copeland and Moiseff, 2004).
In initial inhibition, one of a synchronic firefly pair continues to flash. This could be a form of photic male competition similar to that seen in Photinus fireflies (Buck, 1937; Carlson and Copeland, 1988; Cratsley and Lewis, 2003). A flashing male is visible to a receptive female and a non-flashing male is invisible, so this male interaction could serve as a competition.
Possible neurophysiological directions
Initial inhibition at the start of synchrony might contribute to our understanding of how the firefly's brain controls flash behavior. Although much is known about the firefly's lantern and its activity as a neuroeffector (Buck and Case, 1961; Case and Buck, 1963; Carlson, 1968; Christensen and Carlson, 1983), little is known of how the CNS controls flash.
The flash is triggered by a ventral nerve cord volley whose origin is probably the head (Case and Buck, 1963; Buck and Buck, 1978). If the ventral cord burst continues to be recorded during initial inhibition, peripheral inhibition of flashing becomes a possibility. If the ventral cord burst stops during initial inhibition, either the flash pacemaker continuously cycles and the cord bursts are gated or the pacemaker runs intermittently. Thus, Ph. frontalis may be useful in the study of the neural control of synchrony, the involvement of the firefly's brain, and the neural control of the flash.
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ACKNOWLEDGMENTS |
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We thank Dr. V. J. Henry, Director, Applied Coastal Research Laboratory and U. Sterling for providing lab space, Dr. A. D. Carlson for his critique of the manuscript, and the helpful comments of an anonymous reviewer. Supported by the Graduate School, Georgia Southern University, and the Research Foundation, University of Connecticut.
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FOOTNOTES |
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1 From the Symposium Flash Communication: Fireflies at Fifty presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2003, at Toronto, Canada.
2 E-mail: Copeland{at}GeorgiaSouthern.edu
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References |
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