FIREFLY FLASHING IS CONTROLLED BY GATING OXYGEN TO LIGHT-EMITTING CELLS
1
Department of Medical Microbiology, University of Wales College of
Medicine, Cardiff, UK
2
Department of Radiology, Dartmouth Medical School, Hanover NH 03755,
USA
*
Author for correspondence (e-mail:
mail{at}eprimaging.com
) at present address: College of Pharmacy, 2502 Marble NE, Albuquerque, NM
87131-5691, USA
Present address: USA Instruments Inc., Aurora, OH 44202, USA
Accepted June 5, 2001
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Summary |
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Key words: bioluminescence, oxygen gating, light-emitting cell, photocyte, firefly, Photinus pyralis
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Introduction |
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Here we provide experimental evidence supporting the most commonly
postulated theory of flash control, namely that the flash results from rapid
gating of oxygen (O2) to the light-emitting cells of the lantern
(photocytes). There is a great deal of circumstantial evidence supporting such
a control mechanism (Case and Strause,
1978; Wilson and Hastings,
1998
), including (1) firefly
bioluminescence absolutely requires oxygen (Reaction 2); (2) sequential
hypoxia and reoxygenation causes a `pseudoflash', simulating many features of
a genuine flash (Alexander,
1943
; Hastings and Buck,
1956
); (3) the tracheolar
system of the adult lantern (the structures supplying oxygen to insect cells)
has a unique structure compared to the larval form (Ghiradella,
1977
; Ghiradella,
1998
); and (4) the nerves
innervating the flash motor unit do not terminate upon the photocytes
themselves but rather the specialised tracheal end cells (Ghiradella,
1977
; Ghiradella,
1998
; Case and Strause,
1978
).
However, perhaps most importantly, only a few tens of milliseconds
(typically 40-60 ms) pass between the arrival of the nervous action potential
at the lantern and the start of light emission in vivo, with maximal
light emission occurring within 100-150 ms after action potential arrival (see
Fig. 1; Buck et al.,
1963). Only the reaction of
pre-formed luciferase-luciferyl-AMP complex (LL-AMP) with oxygen (Reaction 2)
occurs rapidly enough to be the point of control, with maximum light emission
occurring within 60 ms of mixing LL-AMP and oxygen in vitro (De Luca
and McElroy, 1974
). If however,
the reaction is instead initiated in vitro by mixing luciferin,
luciferase, ATP and oxygen (i.e. proceeding via Reaction 1 followed
by Reaction 2), a lag phase of 25 ms occurs before any light is emitted, and
maximal light is not emitted until 300 ms after mixing (De Luca and McElroy,
1974
). Hence these kinetics
dictate that any control mechanisms acting at points prior to the reaction of
LL-AMP with oxygen (e.g. via controlling ATP levels or luciferin
release from photocyte vesicles) would simply occur too slowly and can be
ruled out.
|
In vitro, the reaction of the LL-AMP complex with oxygen is rapid
and spontaneous, and although one could argue that in vivo this might
be subject to the repressive action of a cellular second messenger/binding
protein (not present in the in vitro experiments), with this
repression being rapidly and transiently lifted for the flash, no such
consensus sequences/binding sites, other than those for substrate carboxyl
group adenylation (Wood,
1995), were found after
extensive database searching (Altschul et al.,
1997
) using all known
coleopteran luciferase sequences (G. S. Timmins, unpublished results). [Note
that although no `control' sequences were found, a clear analogy between
coleopteran luciferase and bacterial thiophene oxidation protein sequences was
observed e.g. 45-AHIEVNITYAEY-56 for Photinus pyralis and
172-AHVEVNIDYPEY-183 for Bacillus halodurans. Given the similarities
in molecular structure between the site of benzothiazole luciferin oxidation
and thiophene, this may perhaps form a common oxidation and/or binding site,
arrived at through convergent evolution.]
The flash is therefore almost certainly controlled by regulating the access
of oxygen to the LL-AMP complex, most logically by control of oxygen entry to
the photocytes, perhaps by a mechanism analogous to (albeit faster than) the
spiracular control of discontinuous cyclic gas exchange (DCG) known to occur
in many insects (Lighton,
1996), or perhaps through
control of tracheolar fluid levels (Timmins et al.,
2000
). It has long been known
that oxygen supply to tissues in insects is mediated by changes in tracheolar
fluid levels, which result from changes in the balance of osmotic pressure of
the tracheolar fluid and intracellular milieu (Wigglesworth,
1935
). Indeed, Maloeuf
(Maloeuf, 1938
) proposed such
a mechanism of control of oxygen supply to the photocyte by controlling
tracheolar fluid levels some years ago, although Alexander (Alexander,
1943
) later proposed that the
tracheal end cell rather acts in the manner of a mechanical valve to control
oxygen access. Irrespective of the actual mechanism, in such a model light
emission during the dark phase of flashing would be repressed by restricting
oxygen access to the photocytes; these are richly endowed with mitochondria
around their internal periphery, adjacent to the photocyte plasma membrane
that abuts the oxygen-supplying tracheoles (Ghiradella,
1977
; Ghiradella,
1998
; Case and Strause,
1978
). The combination of
limiting oxygen supply, together with the oxygen consumption by these
mitochondria, could reasonably be expected to result in anoxia within the
centre of the photocytes and thereby inhibit light emission. This is supported
by the fact that the light-emitting subcellular structures, the photocyte
vesicles that contain the LL-AMP complex, are found at the centre of the
photocytes (Smalley et al.,
1980
). The flash of light
emission would therefore be achieved by transiently increasing the supply of
oxygen to the photocytes; their peripheral mitochondria would be unable to
consume all of this increased oxygen supply, so some would reach the photocyte
vesicles and result in light emission. The combination of oxygen consumption
by mitochondria and bioluminescence itself would consume the `pulse' of
increased oxygen, resulting in anoxia around the photocyte vesicles and
repression of light emission, ending the flash.
In this study we have confirmed this theory by first demonstrating that
increasing oxygen supply by normobaric hyperoxia (rapidly changing the
external gas stream to oxygen from air) can relieve this repression of light
emission, and more importantly, measuring the kinetics of this
hyperoxia-induced light emission. Secondly, we have measured pseudoflash
kinetics in fireflies in gas mixtures where the gas-phase diffusion
coefficient of oxygen varies to determine gas- and aqueous-phase barriers to
oxygen diffusion (Timmins et al.,
2000). Thirdly, we have used
direct electrical stimulation of firefly flashing by insertion of electrodes
into the lantern (Buck et al.,
1963
) and measured the delay
between electrical stimulation and peak light emission in a range of normoxic
gas mixtures, in which the gas-phase diffusion coefficient of oxygen varies.
From this, we have been able to provide constraints on the distance between
any such gas-phase gating structure and the photocytes. Finally, we use these
and previous data to provide the likely identity of the physical mechanism by
which rapid oxygen gating is achieved, namely modulation of tracheolar fluid
levels. The use of such an oxygen-gating control mechanism does not (to the
author's knowledge) occur elsewhere in animal biology, and has most likely
evolved via a series of sequential modifications of the pre-existing
mechanism through which insects regulate tissue oxygenation, via
changes in tracheolar fluid levels (Wigglesworth,
1935
).
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Materials and methods |
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Results and discussion |
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When samples that were not spontaneously flashing (prior to dusk, N=4) were studied, peak continuous hyperoxia-induced light emission due to changing the gas stream from air to oxygen was somewhat slower, taking 17-52 s (3 measurements on each sample, mean 31.6±10.7 s,) but still occurred, and upon changing the gas stream back to air, light emission similarly ended over a period of 10-30 s. A typical experiment is shown in Movie 3.
These observations, first reported by Kastle and McDermot (Kastle and
McDermot, 1910) and studied
extensively by Snell (Snell,
1932
) and Alexander
(Alexander, 1943
), directly
demonstrate that during the dark state, both between flashes and when not
spontaneously flashing at all, the repression of light emission can be lifted
simply by increasing the ambient oxygen concentration (by a factor of
approximately five) and hence increasing the supply of oxygen to the photocyte
by a similar factor. This in and of itself has been used to strongly support
the theory of oxygen control of bioluminescence. Additionally, the
overwhelming of spontaneous flashing at maximal hyperoxia further supports the
hypothesis of oxygen control, because if normal spontaneous flashing were
achieved by another mechanism that was independent of controlling oxygen
levels, then the spontaneous flashing should rather have been superimposed
upon the hyperoxia-induced continual bioluminescence. Analysis of the observed
kinetics of light emission is discussed later.
Pseudoflash kinetics for five samples were obtained using helium and
nitrogen as carrier gases (Timmins et al.,
2000), with delays to peak
pseudoflash intensity of 0.249±0.061 s and 0.154±0.05 s in
nitrogen and helium carrier gases, respectively. The pseudoflash occurs due to
accumulation of the LL-AMP complex within the photocytes (Reaction 1) caused
by the inhibition of Reaction 2 by anoxia (sample held in gas stream of He or
N2); after rapid changing to a normoxic gas stream, the delay until
peak pseudoflash intensity represents the time taken for diffusion of oxygen
from the spiracle to the photocyte. These values are substantially lower than
those obtained for larval Pyrearinus termitilluminans (1.95 s and
1.49 s, respectively), and indicate that the adult firefly has an enhanced
system for oxygen supply compared to the larval light organ of P.
termitilluminans (Timmins et al.,
2000
).
Effects of modulating gas-phase diffusion on kinetics of light
emission in electrically stimulated fireflies
In order to provide constraints upon the gas-phase distance between the
photocytes and any gating structure responsible for this control of oxygen
supply (and hence to determine its possible nature), we studied the kinetics
of light emission in electrically stimulated fireflies held in normobaric,
normoxic gas mixtures of varying binary diffusion coefficients. These were 21
kPa O2 in 80 kPa of either He, N2 or SF6,
with binary diffusion coefficients of 7.91, 2.19 and
1.00x10-5 m2s-1, respectively, at 310 K
(Chang, 1987). The delay time
t, taken for peak flash intensity after stimulation, was measured
(Fig. 2) and this and the ratio
of delay times in either O2/He and O2/N2, or
in O2/He and O2/SF6 gas mixtures,
tN2/O2/tHe/O2
and
tSF6/O2/tHe/O2,
respectively, were calculated and the data presented in
Table 1. The greater the
distance from the gating structure to the photocytes, the more this value will
increase from unity. It can be seen from
Table 1 that the mean ratio of
delay time in low-diffusivity gas/high-diffusivity gas mixture in the same
specimen was 0.997±0.022, implying either that a gas-phase gating
structure must be close to the photocyte or that gas-phase gating is not
important in flash control. The time taken for gas-phase diffusion from such a
structure to the photocyte must therefore be less than the observed
experimental error, approximately 2 ms, corresponding to an r.m.s. distance in
the gas phase from the gating structure to the photocyte of about 300 µm.
This is substantially less than the average gas-phase distance from the
spiracles to the photocyte (see later) and so these cannot be sites of
gas-phase oxygen gating. However, the distance from the tracheal end cell to
the photocyte is within this value (Ghiradella,
1977
; Ghiradella,
1998
; Case and Strause,
1978
) and so these measurements
do not discount its prior assignment as the potential oxygen gating structure
(Alexander, 1943
), although
this is now thought not to be the case.
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Calculation of gas- and aqueous-phase diffusional barriers for oxygen
supply to photocytes
By analysing the delay required for peak pseudoflash intensity in different
gas mixtures, it is possible to calculate the r.m.s. pathlengths of diffusion
in the gaseous and aqueous phases (Timmins et al.,
2000), and this leads to
values of 2.21±0.24 mm and 27.4±5 µm, respectively. These
values are in agreement with overall morphology of the firefly lantern
(Ghiradella, 1977
; Ghiradella,
1998
; Case and Strause,
1978
). During the period of
anoxia prior to initiation of the pseudoflash, the levels of fluid in the
tracheoles will be minimised, decreasing the barrier against oxygen diffusion
to the photocytes they supply (Wigglesworth et al.,
1935
). This presumably occurs
through the osmotic mechanism proposed by Wigglesworth, in which intracellular
accumulation of metabolites from anaerobic metabolism during hypoxia increases
the intracellular osmotic potential, with diffusion of fluid from the
tracheoles into the cells being driven by this change in osmotic pressure.
Thus the pathlength of aqueous-phase diffusion measured in these experiments
approaches the minimum possible dictated by lantern morphology and physiology,
although that occurring during flashing may be smaller.
Analysis of the delay to maximum light emission in hyperoxia-induced
glowing is more complicated than the transient pseudoflash, as the former
represents attainment of a new steady state. However, the long times indicate
the barrier to diffusion occurs primarily in the aqueous phase, with the time
to maximal glowing in spontaneously flashing and non-flashing samples (10.2
and 31.6 s) implying r.m.s. distances of diffusion in the aqueous phase of
approximately 120 and 220 µm, respectively (Boag,
1969). [From type `B' boundary
conditions for planar geometry, with 99% maximal diffusion into photocyte at
dt/l2=2.0, where l is diffusion
pathlength; pp. 170-171.] These must represent the extent of fluid filling the
terminal portions of the tracheoles during the period between flashes and
during non-flashing periods, respectively, as these are much greater than the
r.m.s. aqueous-phase diffusion distance measured in pseudoflash experiments
(such values are within the typical length of tracheoles in a variety of
insect tissues, i.e. 200-300 µm, although some filling of the smaller
trachea supplying the tracheoles may also occur). The large differences in the
length of the aqueous-phase diffusional barrier dependant upon oxygen supply
also provide a hint as to how oxygen supply to the photocyte might be gated,
and hence provide the mechanism of flash control.
From Krogh's equation of diffusion in a planar model (applicable to supply
by tracheoles where length >> area), one can define the value of any
particular barrier to oxygen diffusion in terms of the gradient in
Po2 required to maintain a given oxygen flux
through that barrier
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The even greater tracheolar fluid length in non-spontaneously flashing
specimens (approximately 220 µm) implies that during periods when flashing
does not occur, an even greater barrier to oxygen diffusion to the photocytes
is maintained. Anoxia within the central portions of the photocytes (to
maintain repression of light emission from the photocyte vesicles) is a
function both of controlling oxygen diffusion by the length of the tracheolar
fluid and of the oxygen consumption by mitochondria in the periphery of the
photocytes (adjacent to the tracheoles). Hence, increasing the length of the
tracheolar fluid when spontaneous flashing does not occur (i.e. most of the
time) will allow a decrease in mitochondrial respiration during this time, and
this may be important in minimising the overall energetic cost of this
mechanism. The requirement for a short period of time between physical
stimulation and competency for light emission observed by Case and Buck (Case
and Buck, 1963), during which
atypical light emission (dim, localised glowing, blushing and a final flash)
can occur, may in part result from the time taken to induce a reduction in
tracheolar fluid length from approximately 220 to 120 µm, i.e. changing
from a `resting' to a `primed' state. However, and perhaps more importantly,
during this time one might reasonably expect temporary mismatches between
increasing oxygen supply (by decreasing tracheolar fluid levels) and
increasing mitochondrial consumption (to maintain anoxia within the photocyte
centre between flashes), which may result in this atypical light emission.
Thus, a system of controlling oxygen access to the photocytes by modulating tracheolar fluid levels is in agreement with the calculated diffusional barriers from fireflies in vivo. Since it occurs by changes in the length of the aqueousphase diffusional pathlength, it is also consistent with the observation that alteration of gas phase diffusion coefficients had no measurable effect upon flash kinetics in electrically stimulated fireflies.
A possible mechanism for modulating tracheolar fluid levels to
control flashing
It has been argued that the repression of firefly bioluminescence is
achieved by regulating oxygen access to the photocytes, and shown that the
gating of oxygen access to photocytes by modulation of tracheolar fluid levels
between values that have been directly measured in vivo provides a
mechanism for controlling oxygen access to the photocytes. We also know that
adult firefly lantern tracheoles have a uniquely strengthened structure
(Ghiradella, 1977; Ghiradella,
1998
), that the nerves
stimulating the flash motor unit terminate upon the tracheal end cells and not
the photocytes, and that these cells show morphological features
characteristic of cells adapted for active transport of ions and fluid; indeed
the foremost and lattermost observations led Ghiradella (Ghiradella,
1977
; Ghiradella,
1998
) to conclude that some
sort of osmotic mechanism might be involved in controlling the flash. Maloeuf
(Maloeuf, 1938
) observed that
injection of a hypertonic solution into fireflies resulted in continual and
prolonged glowing, and interpreted this as showing that a decrease in
tracheolar fluid levels (due to the resultant increased osmotic pressure in
the insect abdomen) resulted in an increased transport of oxygen to the
light-emitting cells. Maloeuf suggested that a direct change in photocyte
osmotic potential would modulate tracheolar fluid levels and therefore control
the flash (although later studies showing a lack of direct photocyte
innervation make this less likely; Ghiradella,
1977
; Ghiradella,
1998
; Case and Strause,
1978
). This mechanism was later
argued against by Alexander (Alexander,
1943
) who, although concurring
that modulation of tracheolar fluid levels occurred, and was important in a
`secondary regulatory nature rather than being involved in the flash
itself', rather suggested that the primary mechanism of controlling the
flash through oxygen supply to the photocytes was the tracheal end cell acting
as a mechanical gas-phase valve; however, this has been discounted (Case and
Strause, 1978
).
All of these disparate observations are most convincingly integrated into
one mechanism by which the access of oxygen to the photocyte is gated by
modulation of tracheolar fluid levels, as hypothesised by Malouef (Malouef,
1938), but with changes in
fluid levels being actively brought about by nervous stimulation of the cells
that are innervated, the tracheal end cells. This nervous stimulation is
postulated to result in rapid and transient fluid uptake into the tracheal end
cell and the tracheolar cell that ensheathes the tracheoles, probably by
movement of an ion across a membrane (analogous is the case for
Ca2+ activation of muscle contraction). The morphology of both the
tracheolar and tracheal end cells appears to be classically adapted to sodium
pumping (Ghiradella, 1977
;
Ghiradella, 1998
), and so this
may well be the ion utilised. This increases tracheolar cell osmotic pressure
and results in the absorption of fluid from the tracheoles into the tracheolar
cell (and perhaps, hence, the tracheal end cell), with the uniquely
strengthened structure of these tracheoles (Ghiradella,
1977
) being required to
withstand the lateral forces due to the large and rapid changes in pressure.
The decreased barrier against oxygen diffusion to the photocytes is now
sufficiently low to allow transient oxygenation of the photocyte interior, and
light emission. Fig. 2A,B shows
diagrams of the postulated mechanism, and Movie 4 provides a time course of
this mechanism. Rapid ending of increased tracheal end cell osmotic pressure
reverses the process, and results in an increase in tracheolar fluid level,
raising the barrier against oxygen diffusion to the photocytes, resulting in
internal anoxia and repression of light emission.
A key question is whether such a mechanism could operate with the speed
required in order to be compatible with the observed kinetics of light
emission, i.e. is there sufficient time for the movement of a length of
tracheolar fluid of approximately 100 µm within the time period between
arrival of the nervous action potential at the lantern and light emission
(typically 40-60 ms)? It would appear that firefly tracheoles are highly
permeable to water (Ghiradella,
1998), and this may result
from the presence of specific water-channel proteins, aquaporins, known to
occur in other insect tracheoles (Pietrantonio et al.,
2000
). Such high permeability
is a fundamental requirement for the operation of such a mechanism. Since the
transport of tracheolar fluid into the tracheolar cell will presumably occur
axially along the strengthened wall of the tracheole, the diffusional path
length of water from inside the tracheole into the tracheolar cell (resulting
from the change in osmotic potential) will only be of the order of
approximately 10 µm, requiring approximately 25x10-6 s,
with bulk flow along the tracheole (which is not restricted by diffusion)
being responsible for most of the required length of movement of fluid. Thus,
such a mechanism could indeed operate sufficiently rapidly to be consistent
with the observed kinetics of light emission in vivo.
The evolution of such a mechanism for the rapid control of light emission
(as opposed to the slower acting mechanism in the larval form, presumably
acting via control of the reaction of luciferin with ATP; Case and
Strause, 1978) can therefore be
envisaged as having merely required progressive modifications to one of the
preexisting mechanisms by which insects control tissue oxygen supply, rather
than the less likely concept of development of an entirely novel mechanism for
gating oxygen access. Since present-day selection by females for more rapidly
flashing males (in Photinus consimilis) is known to occur (Branham
and Greenfield, 1996
), a
similar selection process could feasibly have provided the evolutionary
pressure required for its initial development and refinement. It is also
interesting to speculate that the active control of tracheolar fluid length in
modulating oxygen supply to tissues, as opposed to that passively caused by
intracellular accumulation of metabolites from anaerobic metabolism during
hypoxia as proposed by Wigglesworth (Wigglesworth,
1935
), might be more
widespread in insects.
Finally, it is also worth noting that an analogous system of oxygen control
by using an aqueous-phase diffusion barrier of variable length at the ends of
a predominantly gas-phase oxygen supply system, although more slowly acting,
has evolved to control the oxygen supply to the symbiotic bacteroids in legume
nodules. These must meet the requirement for oxygen for aerobic energy
metabolism (nitrogen fixation is highly endergonic) whilst avoiding
oxygen-induced destruction of the bacterial enzyme nitrogenase, in the face of
an oxygen supply that can vary widely, as soil oxygen concentrations greatly
vary due to flooding (Witty and Minchin,
1998). Thus, similar
mechanisms to achieve control of cellular oxygen concentrations would appear
to have evolved in both animal and plant kingdoms, which is an interesting
demonstration of convergent evolution.
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Acknowledgments |
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Footnotes |
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References |
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