_In vivo_ emission and time-resolved spectra of firefly _Luciola praeusta_ Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae) have been recorded. The emission spectrum shows the FWHM value for this particular species to be 55 nm, which is significantly narrower than the _in vivo_ half widths reported till now. The time-resolved spectrum reveals that a flash, of duration about a hundred milliseconds, is in fact composed of a number of microsecond pulses. This result suggests that the speed of the enzyme-catalysed chemiluminescence reaction in the firefly for the emission of light is much faster than is believed to be

Anurup Gohain Barua

Gauranga Dhar Baruah

Nayanmoni Saikia

Simanta Hazarika

English

Nature Precedings

 1 
Bioluminescence emissions of firefly Luciola praeusta 
Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae) 
 
 
 
Anurup Gohain Barua,a,* Simanta Hazarika,a Nayanmoni Saikiaa and 
Gauranga Dhar Baruahb 
 
 
 
 
aDepartment of Physics, Gauhati University, Guwahati-781014, India 
bDepartment of Physics, Dibrugarh University, Dibrugarh-786004, India 
 
 
 
 
 
 
 
*Corresponding author; E-mail: agohainbarua@yahoo.com 
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Abstract 
In vivo emission and time-resolved spectra of firefly Luciola praeusta 
Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae) have been 
recorded. The emission spectrum shows the FWHM value for this particular 
species to be 55 nm, which is significantly narrower than the in vivo half 
widths reported till now. The time-resolved spectrum reveals that a flash, of 
duration about a hundred milliseconds, is in fact composed of a number of 
microsecond pulses. This result suggests that the speed of the enzyme-
catalysed chemiluminescence reaction in the firefly for the emission of light 
is much faster than is believed to be. 
 
Keywords Firefly; emission spectrum; FWHM; time-resolved spectrum; 
pulse-width; pulse-duration  
 
 
 
 
 
 
 
 
 
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1 Introduction 
Bioluminescence is an enchanting process in which living organisms 
convert chemical energy into light. Fireflies are common organisms 
exhibiting this process. The enzyme luciferase catalyzes the bioluminescence 
reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an 
electronically excited oxyluciferin species. Visible light is emitted during 
relaxation of excited luciferin to its ground state. The emission of light by 
fireflies has been of considerable interest to naturalists and biochemists due 
to the complicated chemical reactions involved, and to electro-optical 
physicists due to the desire to generate laser light by efficient chemical 
means. It has been of interest in biomagnetics, even, due to the effect of 
magnetic fields on enzymatic activities (Iwasaka and Ueno 1998). 
 The spectral distribution of bioluminescence has been the subject of 
numerous investigations. Existence of distinct groups of bands in a few 
species of fireflies has also been reported (Iwasaka and Ueno 1998, Biggley 
et al 1967, Bora and Baruah 1991). In this report, we have presented, of 
firefly Luciola praeusta, an in vivo emission spectrum, where the full width at 
half maximum (FWHM) value has come out to be significantly narrow. This 
value has been consolidated by another spectrum of the firefly emitting 
continuous light under the influence of ethyl acetate. It is worthwhile to 
mention here that fireflies of this species emit flashes of light from their 
abdominal lanterns. Fireflies have a remarkable flash communication system 
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involving precisely timed, rapid bursts of bioluminescence. Females of a 
firefly species were shown to discriminate between males on the basis of 
variation in the flash rate of male patterns (Branham and Greenfield 1996). It 
has been reported (Venel and Carlson 1998) that female Photinus pyralis 
fireflies prefer flashes of greater intensity and precedence  suggesting that 
flash ‘synchronisation’ is a competitive display. Nitric oxide (NO), a 
ubiquitous signaling molecule, has been discovered to play a fundamental 
and novel role in controlling the firefly flash (Trimmer et al 2001), while it 
has been suggested that the firefly flash could be regulated by calcium (Buck 
et al 1963). It is worthwhile to mention here that the term flash has been used 
synonymous with the term pulse till now. The duration of a single pulse/flash 
has been reported to very from about 70 milliseconds (Branham and 
Greenfield 1996) to a few hundred milliseconds (Barry et al 1979, Saikia et al 
2001, Carlson 2004). The time-resolved spectrum presented in this 
communication is clearly in disagreement with these values. 
2 Materials and Methods 
 The emission spectrum was recorded in an Ocean Optics HR2000 Series 
High-Resolution Fiber Optic Spectrometer. The experiments were conducted 
during early evening to midnight hours, local time. Prior to the experiment, 
the spectrometer was calibrated with the known lines of iron from an arc, and 
tested against the sodium yellow line. A single firefly was collected just 
before the experiment from the Gauhati University campus, and kept 
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immobile in a cotton plug with its light organ positioned towards the entrance 
face of the fiber. In vivo emission spectra of fifty specimens, both male and 
female, of the firefly species have been recorded in this way. For recording 
the continuous glow of the firefly, we kept it in a 1.5 ml capacity micro 
centrifuge plastic tube of length 4 cm. One end, of opening diameter 3 mm, 
of the tube was attached to the entrance face of the optical fiber in the 
spectrometer. The other end, of diameter 1 cm, was filled with cotton dipped 
in ethyl acetate. It was observed that the flashing rate of the firefly rapidly 
decreased. After about a minute, a constant glow appeared from the last 
segment in the abdomen of the firefly which spread to the other light emitting 
segment in about 3 minutes. A black patch in the middle of the upper 
segment of the lantern finally gave way to the glow in 5 to 6 minutes.  Ten 
emission spectra of fireflies emitting this kind of continuous light were 
recorded. The experiments were performed at laboratory temperatures 260C - 
310C. Because of the very low intensity of the emitted light, the integration 
time of the spectrometer had to be increased to 3000 ms, which resulted in 
the appearance of the system noise (Fig. 1a and Fig. 1b).  
 The experimental set up for recording time-resolved pulses of the firefly 
is shown in Fig. 2. The outside of the wooden firefly chamber was blackened, 
while the inside was painted white. We interposed a ‘high pass’ (DC 
blocking) filter between the anode of the Dumont 6364 photomultiplier tube 
and the succeeding electronics. Since the time constant, which is the product 
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of resistance (R) and Capacitance (C), of the high pass filter should be higher 
than the width of the pulse to be recorded, we used different RC values from 
500 ms to 150  µs to confirm the result. Tektronix TDS 520A digital storage 
oscilloscope was used to record the pulses. Time-resolved spectra of five 
specimens were recorded in this way. The emission and time-resolved 
experiments, with arrangements described above, could be easily reproduced, 
if the flashing fireflies are available. 
3 Results and Discussion  
 The peak wavelength and the full width at half maximum (FWHM) in all 
the emission spectra of the firefly  recorded in trapped as well as ethyl 
acetate-affected conditions and shown, one each, in Fig. 1a and Fig. 1b 
respectively  have been found to be remarkably constant. The position of 
the peak wavelength has been observed at 562 nm, that is, in the yellow 
region. The wavelength spread clearly shows that this particular firefly 
species emits in the green and yellow region, with the weak red sector lying 
outside the halfwidth upto 670 nm. The FWHM value has been measured to 
be 55 nm. If we leave aside the outrageously small values reported by 
Coblentz 1912 (for example, the Photinus pyralis FWHM value was reported 
to be as low as 333 Ǻ!), this is the smallest of all the in vivo half width values 
of different species of fireflies published till now. From spectroscopic point 
of view, this small value implies that out of the luciferin molecules excited to 
the oxyluciferin state of this firefly species, about 50 percent occupy levels in 
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that state narrower in spacing compared to that of the other species. It has 
been proposed that different fireflies emit in slightly different spectral regions 
due to slightly different enzyme structure (Seliger et al 1964); different 
FWHM values could also be due to this fact. The narrowest half width, by the 
way, has been 64 nm measured for firefly species Photinus consimilis and 
Photinus umbratus (Biggley et al 1967). The asymmetric nature of the 
intensity profile is in agreement with earlier investigations. The half width at 
half maximum (HWHM) value for the lower half is 25 nm, while the same 
for the upper half is 30 nm. No discrete bands are observed in the spectrum. It 
could be mentioned here that approximately equal in vitro FWHM values 
have been reported, for example in the green-emitting luciferase of the 
Japanese firefly Pyrocoelia miyako (Viviani et al 2001).    
 The time-resolved spectrum of the firefly, shown in Fig. 3, exhibits 
striking similarity with the output of a multimode laser. The spectrum 
presented here has revealed that the duration of a pulse is a couple of 
microseconds! A survey of literature indicates that this is the first report of a 
bioluminescence system emitting microsecond pulses. On a bigger scale (Fig. 
4) it is evident that a flash, consisting of a number of microsecond pulses, is 
of duration about 100 ms, and consists of about 30,000 pulses. From studies 
of similar spectra of five such specimens, it can be concluded that the flashes 
are separated from one another by a few hundred ms. We have found that the 
flashes, on an average, are repeated after 800 ms, and have noted the 
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minimum separation between two flashes as 150 ms. The event recorded in 
Fig. 4 shows the average separation of 800 ms between flashes. The time-
resolved spectra appear to be noisy because of the very low energy of the 
signal. The signal to noise ratio is approximately 1.8. Of course, the signals 
recorded were not the advertisement ones emitted by fireflies for courtship 
and mating; these could be lightly described as SOS signals sent by them!  
 It has been proposed (Saikia et al 2001) that the time-resolved spectrum 
of the firefly can be considered as the manifestation of oscillating chemical 
reactions, the so-called BZ reactions (Belousov 1959, Zhabotinsky 1964). 
The oscillatory nature of the time-resolved spectrum in our work (Fig. 3) also 
points towards this direction. The characteristics of the pulses suggest that the 
speed of the chemiluminescence reaction must be remarkably high. The 
challenge at the moment is to record both the emission as well as time-
resolved spectra in a natural environment for finding out (a) whether the 
wavelength spread remains the same as in the ‘trapped’ condition in the 
laboratory, and (b) by what amount the flash duration and flash repetition rate 
vary from specimen to specimen.  
Acknowledgment 
This work was supported by University Grants Commission, India, Major 
Research Project F.31-15/2005 (SR). We thank Dr. L. Ballantyne, School of 
Agricultural and Wine Sciences, Charles Sturt University, Australia, and Dr. 
P. Mukhupadhyay, Coleoptera section, Zoological Survey of India, Kolkata, 
 9 
for identifying the firefly species of the specimens collected. We are grateful 
to Professors K. Boruah and P. K. Boruah, Departments of Physics and 
Instrumentation, respectively, Gauhati University, for allowing us to use the 
facilities in the UHE Cosmic Ray Research Laboratory for the recording of 
time-resolved spectra. 
References 
Barry J D,  Heitman J M and Lane C R 1979 Time-resolved spectrometry of 
in vivo firefly bioluminescence emissions; J. Appl. Phys. 50 7181-7184 
Belousov B P 1959 A periodic reaction and its mechanism; Compilation abs. 
radiation med. 147 145 
Biggley W H, Lloyd J E and Seliger H H 1967 The spectral distribution of 
firefly light II; J. Gen. Physiol. 50 1681-1692 
Bora L and Baruah G D 1991 Bioluminescence emission of few species of 
fireflies; Indian J. Phys. 65B 551-557 
Branham M A and Greenfield M D 1996 Flashing males win mate success; 
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Buck J, Case J F and Hansen F E 1963 Control of flashing in fireflies III. 
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Carlson A D 2004 Is the firefly flash regulated by calcium?; Integr. Comp. 
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Iwasaka M and Ueno S 1998 Bioluminescence under static magnetic fields; J. 
Appl. Phys. 83 6456-6458 
Saikia J, Changmai R and Baruah G D 2001 Bioluminescence of fireflies and 
evaluation of firefly pulses in light of oscillatory chemical reactions; 
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Seliger H H, Buck J B, Fastie W G and McElroy W D 1964 The spectral 
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T, Qazi S and Zayas R M 2001 Nitric oxide and the control of firefly 
flashing; Science  292 2486-2488 
Venel F D and Carlson A D 1998 Proximate mechanisms of sexual selection 
in the firefly Photinus pyralis (Coleoptera: Lampyridae); J. Ins. Behav. 11 
191-207 
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is a key residue for bioluminescence spectra determination in beetle 
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Captions to Figures 
 11 
Fig. 1a. Emission spectrum of firefly Luciola praeusta. The peak wavelength 
appears at 562 nm and the half width is of the value 55 nm.  
 
Fig. 1b. Emission spectrum of the firefly Luciola praeusta under the 
influence of ethyl acetate. The peak wavelength and FWHM values are the 
same as in Fig. 1a. 
 
Fig. 2. Experimental arrangement to record time-resolved spectra of the 
firefly.  
 
Fig. 3. Time-resolved pulses of the firefly, showing the relaxation oscillation. 
The duration of a pulse has come out to be approximately 2 microseconds. 
Before the application of the signal, the noise level was approximately 15 
mV, which got amplified to about 20 mV after the firefly began flashing in 
the chamber. To be absolutely on the safe side, we have considered the pulses 
only if they are above the trigger level, i.e. 32 mV, shown by the arrow on the 
right ordinate in the oscilloscope screen. 
 
Fig. 4. Time-resolved pulses of the firefly, on a larger scale, showing the 
flash. In the figure, the duration of a flash appears to be about 100 ms, while 
the three flashes are separated from one another by approximately 800 ms.  
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Bioluminescence of fireflies and evaluation of firefly pulses in light of oscillatory chemical reactions;

Carlson A D

Time-resolved spectrometry of in vivo firefly bioluminescence emissions;

Bioluminescence emissions of firefly _Luciola praeusta_ Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae)

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Bioluminescence emissions of firefly _Luciola praeusta_ Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae)

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