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Integrative and Comparative Biology 2004 44(3):220-224; doi:10.1093/icb/44.3.220
© 2004 by The Society for Integrative and Comparative Biology
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Is the Firefly Flash Regulated by Calcium?1

Albert D. Carlson2,1
1 Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, New York 11794


   SYNOPSIS
TOP
SYNOPSIS
INTRODUCTION
 DISCUSSION
 References
 
The very different courtship flashes of Photuris versicolor and Photuris lucicrescens males mirror the pattern of neural impulses produced by their brain. Their lanterns luminescence very differently, however, in response to direct, electrical stimulation. Whereas P. lucicrescens lanterns glow in response to high frequency, continuous electrical stimulation, those of P. versicolor produce only rapid, triple-pulsed flashlettes that resemble, but are not identical to, their courtship flashes. In addition, the exposed lantern tissue of P. versicolor males, when immersed in firefly saline high in potassium and calcium ions, scintillates with hundreds of photocytes flashing in random fashion. P. lucicrescens male lanterns, so treated, only glow. Tests of P. versicolor lanterns with salines of different composition suggest that calcium ions are essential in producing this intense, long lasting scintillation response and are therefore possibly implicated in the final stages of flash control in this species.


   INTRODUCTION
TOP
SYNOPSIS
 INTRODUCTION
DISCUSSION
 References
 
The males of two fireflies of the genus Photuris possess widely different courtship flashes. P. versicolor produces a triple-pulsed flash while P. lucicrescens produces a long, slowly building crescendo flash (Fig. 1). A possible explanation of why these fireflies differ is that the flashes are shaped by the neural impulses generated in the brain that eventually impinge on the lantern tissue. The neural bursts produced by the nervous system of these two fireflies do indeed differ as expected. Recordings of the nerve impulses that arrive in the lantern of P. versicolor reveal three short bursts that clearly trigger its courtship flash (Fig. 2) (Christensen and Carlson, 1981Go). The neural burst triggering the crescendo flash of P. lucicrescens is a long, slowly developing burst (Fig. 3) (Carlson et al., 1982Go). It appeared, therefore, that it was the brain that determined the overall shape of the male courtship flash, while the lantern acted in a passive manner responding precisely to its neural input. We assumed that it was possible to shape any kind of flash by simply adjusting the frequency and duration of the stimulus activating the lantern nerves.



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  		FIG. 1. Photomultiplier recordings of spontaneous courtship flashes of Photuris male fireflies. A. Twinkling, multi-peaked courtship flash of P. versicolor. B. Crescendo courtship flash of P. lucicrescens. Recording intensities are not equivalent; P. lucicrescens flash is much brighter than P. versicolor flash and the peak is not shown. Calibration, 1 sec. Data from Carlson (1981)Go. (In this and all following figures the methods may be found in the articles cited)

 


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  		FIG. 2. Lantern neural potentials that trigger the spontaneous courtship flash of P. versicolor male. Photomultiplier output (upper trace). Action potential volleys recorded from anterior lantern segment (middle trace) and posterior lantern segment (lower trace). Calibration, 100 ms. From Carlson (1981)Go

 


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  		FIG. 3. Lantern neural potentials triggering a spontaneous crescendo flash of a P. lucicrescens male. Action potential burst recorded from anterior lantern segment (upper trace) and photomultiplier output (lower trace). From Carlson et al. (1982)Go. The flash shape differs from that shown in Figure 1 because of the prominent light oscillations that often occur as the flash begins its slow buildup and the reduced amplification of the light to show the entire flash

 
Whereas direct stimulation of the lantern of P. lucicrescens had the result we expected, namely, a 1-sec burst of impulses of increasing frequency produced a continuously rising glow that plateaued at the highest frequency and was rapidly extinguished upon stimulus cessation (Fig. 4), the same stimulus delivered to the P. versicolor lantern produced instead a rapid, multi-pulsed flash sequence that was extinguished before the stimulation stopped (Fig. 5). The lantern of this species responds to continuous electrical stimulation of its ventral nerve cord with its ganglion intact with 3 or 4 rapid flashlettes (Fig. 6). Its posterior lantern responds in virtually identical fashion when stimulated directly even when its ganglion, which resides in the anterior segment, has been removed by severing the nerves between the two segments (Fig. 6). The observation that under direct, continuous stimulation the posterior lantern segment can produce triple-pulsed flashes appears to rule out the role of the ganglion in shaping this flash response. The triple-pulsed flashes induced by neural bursts from the brain that trigger the courtship flash and those triple-pulsed flashes induced by direct, continuous electrical stimulation of the lantern do appear to differ physiologically as shown by their different responses to temperature. Whereas the spontaneous courtship flashes show a typical Q10 close to 2, the stimulated flashes are much less temperature sensitive with a Q10 around 1.5 (Fig. 7) (Carlson, 1981Go).



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  		FIG. 4. Effect of electrical stimulation of anterior lantern segment on the lantern of a decapitated P. lucicrescens male. Light intensity recorded by photomultiplier in arbitrary units (upper trace) and electrical stimulation of lantern (lower trace). Stimulus parameters: 1 ms pulses delivered for 955 ms at: A, 56 Hz; B, 80 Hz; C, 110 Hz. From Carlson et al. (1982)Go

 


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  		FIG. 5. Effect of 10 sec direct, continuous stimulation of the anterior lantern segment of a decapitated male P. versicolor with its ganglion intact. Stimulus frequency, 50 Hz. Calibration, 2 sec. From Carlson (1981)Go

 


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  		FIG. 6. Flashes produced by continuous electrical stimulation of a decapitated P. versicolor male. A. Stimulation of ventral nerve cord at level of third abdominal ganglion (stimulus frequency, 140 Hz). B. Direct electrical stimulation of the posterior lantern segment with its ganglion intact (stimulus frequency, 170 Hz). C. Direct electrical stimulation of the posterior lantern segment with possible input from its ganglion removed (stimulus frequency, 185 Hz). Photomultiplier recording on upper trace and electrical stimulation on lower (A and B) or middle (C) trace. Diagram of male showing nervous system. Removal of the possible influence of the ganglion of posterior lantern produced by transection of the nerves between the anterior and posterior lantern segments. The lantern ganglion (AG7) lies in the anterior lantern segment. From Carlson (1981)Go

 


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  		FIG. 7. Effect of temperature on the frequency of the first two peaks of the spontaneous courtship flash (triangles, n = 46), and the stimulated flash (circles, n = 41) of the male P. versicolor. From Carlson (1981)Go

 
The luminescent responses of the lanterns of these two species also differ in another significant manner. Whereas perfusion of the exposed lantern tissue of P. lucicrescens with a saline high in potassium with calcium induces merely a glow, the exposed lantern tissue of P. versicolor produces a spectacularly different response. High potassium saline containing calcium induces in the lantern of the latter species intense scintillation composed of the rapid, tiny flashes of hundreds of photocytes that can persist for over an hour (Fig. 8) (Carlson, 1967Go). Alternating treatments with high potassium and high sodium salines while stimulating the ventral nerve cord can produce massive scintillation alternating with coordinated flashing (Fig. 9) (Carlson, 1967Go).



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  		FIG. 8. Effect solution composed of 0.16 M KCL and 0.002 M CaCl2 saline (scintillation saline) on induction of scintillation. A. Glow of exposed lantern tissue 16 sec prior to immersion in saline. B. Scintillation 26 sec after immersion. C. Scintillation 231 sec after immersion. D. Scintillation 30 sec after addition of distilled water. Time line 1 mark/sec. From Carlson (1967)Go

 


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  		FIG. 9. Three successive periods of scintillation and flashing of exposed lantern tissue of P. versicolor male by applying alternating periods of saline composed of 0.16 M KCl and 0.002 M CaCl2 saline and 0.16 M NaCl and 0.002 M CaCl2 saline. Note that high concentration of potassium induces scintillation while high concentration of sodium suppresses scintillation and induces coordinated flashes. A. 5 ms after high potassium saline applied. B. 17 sec after high sodium saline applied. C. 30 sec after high potassium saline reapplied. D. 50 sec after high sodium saline reapplied. E. 81 sec after high potassium saline reapplied. F. 62 sec after high sodium saline reapplied. Electrical stimulation of the nerve cord of 15 vs., 20 ms duration, 0.5 Hz. Photomultiplier recording top trace, time base middle trace 1 mark/sec, electrical stimulation of nerve cord bottom trace. From Carlson (1967)Go

 
It therefore appears that the lantern tissues of the P. versicolor and P. lucicrescens fireflies possess different coupling physiology between their nerves and the endorgan complex containing the tracheal end cells, the tracheolar cells and the photocytes. I propose that calcium ions may play a significant role in coupling the stimulus from the nerves to the flash control system in the lantern of P. versicolor. The evidence upon which this is based is suggested by experiments studying the induction of the persistent and long term scintillation response on exposed lantern tissue of P. versicolor males with salines of different ionic composition (Table 1: See lines 1 & 2 on effect of calcium concentration and lines 6, 7, 8, & 9 on effect of potassium concentration). Only saline solutions high in potassium and calcium ions induce this persistent scintillation in a majority of the lanterns tested. As calcium ions are reduced in high potassium the scintillation response is less strong and when calcium is replaced with magnesium it fails completely (Table 1: See line 5 on effect of replacement of calcium with magnesium).


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  		TABLE 1. Effect of high K solutions containing calcium on induc tion of scintillation in exposed lantern of P. versicolor male (Modi fied from Carlson, 1967)

 

   DISCUSSION
TOP
SYNOPSIS
 INTRODUCTION
 DISCUSSION
References
 
The luminescent responses of the two Photuris firefly species described here, namely P. versicolor and P. lucicrescens, differ in very fundamental ways. Their courtship flashes differ significantly in that P. lucicrescens produces a long, slow crescendo flash and P. versicolor emits its light as a group of 3 or 4 rapid flickers. This might be explained by the pattern of brain generated neural bursts that initiate luminescence and this appears to be the case in P. lucicrescens. The discovery that even continuous electrical stimulation of the nerve cord or lantern of P. versicolor induces only a few rapid flickers reveals a fundamental difference in the lantern physiology of these two closely related species. It is surprising that continuous electrical stimulation of P. versicolor fails entirely to induce luminescence after the initial flickers. This may signal that something occurs in the end organ complex beyond the nerves to block neural action. The complex end organ composed of tracheolar cells, tracheal end cells and photocytes could provide numerous sites for failure, but the scintillation response of the exposed lantern to salines containing potassium and calcium suggests that the failure may lie at the most fundamental level, namely the photocytes themselves. This conclusion is supported by the observation that the scintillation appears to be uncoordinated with tiny points of light appearing in total disorder. If scintillation were controlled by the end organ complexes one might anticipate that it should show some patterning in the highly structured P. versicolor lantern.

The most parsimonious explanation of the scintillation effect is that high K+ saline depolarizes the nerve terminals, which in the presence of Ca2+ ions, causes a dump of the transmitter octopamine that is known to be the output of these nerves (Christensen et al., 1983Go; Carlson and Jalenak, 1986Go; Carlson and Evans, 1986Go). But perfusion of the exposed lantern tissue of the P. versicolor male with octopamine does not induce scintillation, only a glow. It is unlikely that the nerves of these two Photuris firefly species show entirely different physiology. That is, that the nerves of P. lucicrescens respond without failure to continuous electrical stimulation but those of P. versicolor do not. It therefore appears that some fundamental difference in neural physiology cannot explain the difference in luminescent response in these two species.

In the adult firefly lantern the lantern nerves do not terminate on the photocytes, the cells that produce the light, as they do in the larva. Instead, the nerves terminate between the tracheolar cells and a new cell in the adult lantern, the tracheal end cell (Smith, 1963Go). There is evidence that the nerves actually synapse on the tracheolar cells because vesicular profiles appear to align the membranes of the nerve terminals and those cells (Case and Strause, 1978Go). How the final coupling between excitation by the nerves and luminescence by the photocytes occurs is not known, however. There seems to be a step missing in this process that calcium channels could possibly fill. To contemplate the possible role of calcium in the initiation of flashes at the end organ level its action would most likely be controlled by calcium channels. Calcium channels are found in nearly all cells and there are some types that might be activated and inactivated sufficiently rapidly to explain flash kinetics in the P. versicolor lantern.

"Voltage-gated Ca (calcium) channels are found in every excitable cell. Indeed, I feel they define excitable cells." (Hille, 2001Go, p. 97)

"All members of this broader superfamily of voltage-gated Na, K, and Ca channels have steeply voltage-dependent gates that open with a delay in response to membrane depolarization. They shut rapidly after repolarization and show some form of inactivation during maintained depolarization. By controlling the flow of Ca2+ into the cytoplasm, they can regulate a host of Ca2+ dependent intracellular events." (Hille, 2001Go, p. 97)

If calcium channels are indeed involved in control of the firefly flash, a number of observations on their activity in cell physiology may be relevant.

  1. Calcium channels translate electrical signals into chemical signals. By controlling the flow of Ca2+ into the cytoplasm, they can regulate a host of Ca2+-dependent intracellular events. (Hille, 2001Go, p. 98)
  2. Calcium channels are known to open in response to high K+ solutions. (Hille, 2001Go, p. 97)
  3. Calcium channels differ in voltage dependence, inactivation rate, ion selectivity and pharmacology. (Hille, 2001Go, p. 128)
  4. "A good rule of thumb is that Ca2+ acts locally in the vicinity of the channels that deliver it." (Neher, 1998Go)
  5. In the adult photocyte, mitochrondria are densely localized in a differentiated zone immediately adjacent to the photocyte cell membrane that abuts the tracheolar cell membrane (Beams and Anderson, 1955Go; Kluss, 1958Go; Smith, 1963Go). It is possible that interaction of Ca2+ ions with the mitochondria may initiate the light reaction.
  6. Ca2+ has also been implicated in activating nitric oxide synthetase (NOS), which has already been shown to be involved in the control of the firefly flash. (Newby and Henderson, 1990Go)

In conclusion, the observations described in the introduction suggest the following:

  1. In the lantern tissue of P. versicolor fireflies, high K+ saline appears to depolarize some component of the firefly light organ.
  2. High K+ saline containing Ca2+ induces massive, uncoordinated scintillation of photocytes, whereas high Na+ saline containing Ca2+ allows the photocytes to produce a coordinated flash.
  3. There is a fundamental difference between the physiology of the two firefly species and this difference appears to exist at the level of the photocytes and it may involve Ca2+ channels.
  4. The Ca2+ channels, if present, appear to be L-Type or High Voltage Activating (HVA) channels. That is, they open in response to strongly depolarizing potentials.
  5. The results of these experiments suggest, but certainly do not prove, that one step in flash control in the P. versicolor lantern involves depolarization of at least one of the intervening cell types.

Further experiments will be necessary to conclusively demonstrate the possible role that Ca2+ channels may play in the control of the firefly flash.

  1. The role of such calcium channel blockers as Mg2+, Ni2+, Cd2+, or Co2+ should be investigated to demonstrate that they block the scintillation effect of calcium.
  2. If calcium channels are conclusively shown to be present, determine what type of channels are involved using appropriate blocking agents.
  3. Patch-clamp photocytes and tracheal end cells to study their channel activity in the presence of high K+ saline.
  4. Use calcium imaging dyes and confocal imaging to examine the dynamics of Ca2+ in lantern coupling cells.


   ACKNOWLEDGMENTS
 
I thank the anonymous reviewers for very helpful comments and suggestions on the previous version of this paper. The research reported in this paper was supported, in part, by grants from the National Science Foundation.


   FOOTNOTES
 
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. Back

2 E-mail: acarlson{at}notes1.cc.sunysb.edu Back


   References
TOP
SYNOPSIS
 INTRODUCTION
 DISCUSSION
 References
 
Beams, H. W., and E. Anderson. 1955. Light and electron microscope studies on the light organ of the firefly (Photinus pyralis). Biol. Bull, 109:355-393.

Carlson, A. D. 1967. Induction of scintillation in the firefly. J. Insect Physiol, 13:1031-1038.[CrossRef]

Carlson, A. D. 1981. Neural control of the male Photuris versicolor firefly flash. J. Exp. Biol, 92:165-172.[Abstract/Free Full Text]

Carlson, A. D., J. Copeland, and R. Shaskan. 1982. Flash communication between the sexes of the firefly, Photuris lucicrescens. Physiol. Entomol, 7:503-514.

Carlson, A. D., and M. Jalenak. 1986. Release of octopamine from the photomotor neurones of the larval firefly lanterns. J. Expt. Biol, 122:453-457.[Free Full Text]

Carlson, A. D., and P. D. Evans. 1986. Inactivation of octopamine in larval firefly light organs by a high-affinity uptake mechanism. J. Expt. Biol, 122:369-385.[Abstract/Free Full Text]

Case, J. F., and L. G. Strause. 1978. Neurally controlled luminescent systems. In P. J. Herring (ed.), Bioluminescence in action, pp. 331–366. Academic Press London, New York, San Francisco.

Christensen, T. A., and A. D. Carlson. 1981. Synmetrically organized dorsal unpaired median (DUM) nerones and flash control in the male firefly, Photuris versicolor. J. Exp. Biol, 93:133-147.[Abstract/Free Full Text]

Christensen, T. A., T. G. Sherman, R. E. McCaman, and A. D. Carlson. 1983. Presence of octopamine in firefly photomotor neurons. Neuroscience, 9:183-189.[CrossRef][ISI][Medline]

Hille, B. 2001. Ion channels of excitable membranes. 3rd Ed. Sinauer Assoc., Massachusetts.

Kluss, B. C. 1958. Light and electron microscope of observations on the photogenic organ of the firefly, Photuris pennsylvanica, with special reference to the innervation. J. Morphol, 103:159-186.[CrossRef]

Neher, E. 1998. Vesicle pools and Ca2+ microdomains. New tools for understanding their roles in neurotransmitter release. Neuron, 20:389-399.[CrossRef][ISI][Medline]

Newby, A. C., and A. H. Henderson. 1990. Stimulus-secretion coupling in vascular endothelial cells. Ann. Rev. Physiol, 52:661-674.[CrossRef][ISI][Medline]

Smith, D. S. 1963. The organization and innervation of the luminescent organ in a firefly, Photuris pennsylvanica (Coleoptera). J. Cell Biol, 16:323-359.[Abstract/Free Full Text]


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