Colour vision in the glow-worm Lampyris noctiluca (L.) (Coleoptera: Lampyridae): evidence for a green-blue chromatic mechanism
Sussex Centre for Neuroscience and School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
* Author for correspondence (e-mail: d.booth{at}sussex.ac.uk)
Accepted 21 April 2004
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Summary |
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Key words: Lampyris noctiluca, glow-worm, colour vision, phototaxis
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Introduction |
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In a classic series of neuroethological studies, Lall and colleagues
(Lall, 1981; Lall et al.,
1980
,
1988
) measured bioluminescence
and visual sensitivity in a range of North American firefly species. The
bioluminescence and the spectral sensitivity of the photoreceptors vary
according to the time after sunset, and hence light level, when the species is
normally active. Lampyrid emission spectra peak in the range 545575 nm.
Nocturnal species use shorter wavelengths than crepuscular fireflies
(Lall et al., 1980
), and peak
spectral sensitivity of the photoreceptors matches the spectral peak of the
bioluminescent emission. It is argued that this shift of spectra and spectral
sensitivities probably optimises detectability of bioluminescent signals
within species-specific photic niches. Possible reasons for the shift are: (i)
that under relatively high ambient illumination the detectability of the
bioluminescence is enhanced by their having spectra displaced from the
leaf-background (Seliger et al.,
1982a
), whereas under low illumination contrast with the
background is less important, or (ii) the communication system needs to
minimise receptor noise (i.e. photon-noise plus dark-noise). Dark-noise arises
from thermal isomerisation of photopigment which, at least in vertebrates, may
occur at a rate that increases with wavelength
(Rieke and Baylor, 2000
; but
see Koskelainen et al., 2000
).
As light intensity falls, the importance of dark-noise relative to
photon-noise increases, and this will favour a blue-shift by the
photopigment.
The presence of coloured filter pigments within the eyes of fireflies
(Cronin et al., 2000;
Lall et al., 1988
) somewhat
complicates the evolutionary interpretation of this communication system.
These filter pigments are thought to narrow the spectral sensitivity of the
receptors at the expense of light capture, a trade-off that might be expected
for the dusk-active species, but is more surprising for nocturnal species,
where one might expect the eyes to maximise light catch and minimise
dark-noise (Warrant,
1999
).
By comparison with measurements of bioluminescence and electrophysiology of
the eyes there are few studies of firefly behavioural action spectra. The
response of female Photinus pyralis is tuned to the emission spectrum
of the male (Lall and Worthy,
2000), and a general implication of the work on fireflies is that
the behaviour is achromatic and mediated by photoreceptors tuned to the
bioluminescence. However, a striking feature of behavioural responses in
fireflies and glow-worms is insensitivity to short wavelength light. Buck
(1937
) found that the firefly
Photinus pyralis responded to stimuli from 520700 nm, but that
wavelengths below 500 nm elicited no response, even when presented at 900
times the intensity of the attractive stimuli. He concluded that these insects
could not perceive the shorter wavelengths. Schwalb (1961) similarly reported
little response to blue light stimuli in the European glow-worm Lampyris
noctiluca. More recently, Lall and Worthy
(2000
) showed that female
Photinus pyralis fireflies failed to respond to any stimuli of
wavelength less than 480 nm, even when these were presented at 100 times the
intensity of longer wavelength stimuli. This characteristic sharp cut-off in
the behavioural response at short wavelengths has been attributed to the
effect of various long-pass filter pigments that have been described from the
eyes of several firefly species (Cronin et
al., 2000
; Lall et al.,
1988
).
An alternative to optical filtering to account for behavioural
insensitivity to short wavelengths is that the beetles have a chromatic
mechanism, involving spectral opponency between photoreceptor signals.
Although it is clear that the response to the bioluminescence is mediated by
the beetles' long (L) wavelength (sensitivity maximum >500 nm) receptors,
there are other spectral types of receptor as required for colour
vision. Electroretinogram (ERG) data from North American
(Lall et al., 1980) and
Japanese fireflies (Eguchi et al.,
1984
) show two sensitivity peaks, corresponding to L and UV
(<400 nm) receptors. In addition, a separate short (S) wavelength peak (c.
450 nm) was suggested by selective adaptation experiments on some species of
Photuris (Lall et al.,
1982
,
1988
) and three Japanese
species including Luciola lateralis
(Eguchi et al., 1984
). To date
the function of S photoreceptors has remained elusive. Lall
(1993
) postulates that they
may be involved in the initiation of bioluminescent flashing behaviour in
crepuscular fireflies, being used to detect when ambient light levels decline
to twilight intensity.
This study examines the spectral selectivity of lampyrid behaviour using
behavioural tests as well as investigating the distribution of coloured filter
pigment in the European glow-worm Lampyris noctiluca. We present the
results of binary choice laboratory experiments where males were presented
with artificial light stimuli simulating the female bioluminescent signal. We
also describe the intraoccular filter pigments from the eye of Lampyris
noctiluca. L. noctiluca is a convenient subject because its signalling
behaviour allows binary choice experiments. The flightless female produces a
constant greenish glow from composite abdominal light organs, which attracts
the winged male (Tyler, 2002).
The species is active only after the end of dusk, when ambient light levels
are less than 1.7 lx (Dreisig,
1971
), placing them in the same nocturnal photic niche
(Lall et al., 1980
) as the
green-emitting North American Photuris fireflies. There is no flash
dialogue between the sexes.
We test for colour vision by a procedure first used in 1888 by Lubbock for
phototaxis in Daphnia, and since applied to phototactic responses in
various other animals (for a review, see
Kelber et al., 2003). The
logic is simple; first it is demonstrated that an animal is attracted to light
of a given spectral composition a (e.g. long wavelengths), and that
attractiveness increases with intensity. Next light with a different spectrum,
b (e.g. short wavelengths) is added to the original stimulus. If the
addition of b reduces the attractiveness of a we can
conclude that the animal has colour vision mediated by an antagonistic
interaction between the outputs of different spectral types of receptor
(Kelber et al., 2003
).
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Materials and methods |
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Experimental arena
Behavioural experiments were conducted in a plywood arena measuring 120 cm
longx80 cm deepx30 cm high, with a hinged mesh top. The floor of
the arena was marked out in a 20 cmx20 cm grid to enable the insects to
be tracked during each experimental run. The room was dark, but the arena was
illuminated with infra-red light (>690 nm), to which most insects are
insensitive (Goldsmith and Bernard,
1974). This light was provided by four 30 W luminaires, each
comprising an incandescent striplight behind infra-red filter Perspex. Two
were mounted externally on each of the long-axis walls of the arena such that
the illumination `windows' were flush with the interior. All cracks were
sealed to exclude stray light.
Experimental light stimuli were provided using two identical units mounted at one end of the arena. These were spaced 50 cm apart and 5 cm above the arena floor. The light sources each comprised a composite three-colour LED component with blue (peak 485 nm), green (peak 555 nm) and red (peak 640 nm) diodes focused into a common diffuser. Rotary controls controlled the intensity of each LED individually. The light output of each source was measured at 2 mm from the diffuser using a calibrated spectroradiometer (USB2000, Ocean Optics, BD, Duiven, The Netherlands).
Movements of the experimental insects within the arena were monitored with a gantry-mounted Sony AVC-D7CE CCD camera 1.2 m above the arena floor and facing downwards. A VCR (Panasonic) recorded all experimental runs.
Design of choice experiments
Individual male glow-worms were presented with a choice between two
different light stimuli. Animals were introduced singly into the arena through
a small aperture at the opposite end to, and facing, the two light stimuli. At
this point of entry these stimuli were equidistant from the insect. Each
choice test was replicated using separate runs with different males, with the
positions of the stimuli being set randomly to one of the two alternative
arrangements for each run. Departure of choices from an expected 1:1 ratio
were tested using two-tailed binomial tests
(Zar, 1984). Deliberately
stringent criteria were applied in assessing the males' responses. Only those
runs in which the male made close contact with a light source, or very
definite and repeated attempts to do so, were scored as a choice for that
particular signal. The run was aborted if there was no response within 2 min.
All experiments took place within 1.5 h of the onset of the dark period, since
we found that the insects tended to become very unresponsive after this, an
observation that seems to reflect their normal behaviour in the field
(Schwalb, 1961).
Cryosection and microscopy
A small number of male glow-worms were chloroformed and their heads
detached. As much of the hydrophobic cuticle as possible was removed from
around the eyes. The preparations were fixed for 1 h at room temperature in 4%
paraformaldehyde (in 0.2 mol l-1 phosphate-buffered saline at pH
7.2) under gentle agitation, and then cryoprotected by infusion in 30% sucrose
solution overnight in darkness at 5°C. The prepared eyes were then quickly
frozen and transferred to a Leica Microsystems (Milton Keynes, UK) CN3000
cryostat at 25°C, where 16 nm sections were taken. Freshly cut
sections were transferred immediately (within 5 min of cutting) to a Zeiss
(Welwyn Garden City, UK) Axiophot microscope for inspection and
photography.
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Results |
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Next we tested the effect of adding blue (max=485 nm)
light to G2, leaving the dimmer green, G1, unchanged
(Fig. 1B). The addition of blue
reduced the attractiveness of G2 below that of G1 (20:3, G1:G2+blue;
P=0.0005).
However, when a red component (max=640 nm) was added to
G2, with the green stimulus intensity kept constant
(Fig. 1C), there was no
aversive effect (0:9, G1:G2+red; P=0.004).
Filter pigments in the eye
Male L. noctiluca have a band of bright yellow filter pigment
present in the frontal and dorsal regions of the compound eye. This pigment
appears to lie between the inner and outer parts of the retina (i.e. the
crystalline cones and the photoreceptor microvilli), much like that of the
nocturnal American firefly Photuris versicolor
(Cronin et al., 2000). The
filter pigment was absent from ventral regions of the eye.
Fig. 2A shows a single vertical
section taken from the lateral part of the eye, while the yellow pigment is
similarly absent from frontal ventral regions
(Fig. 2B).
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Discussion |
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Nonetheless, there is compelling evidence from previous studies that the
spectral tuning of visual sensitivity to bioluminescence in lampyrids is
accomplished by species-specific pairings of an L photoreceptor with an
overlying long-pass filter pigment (Cronin
et al., 2000; Lall et al.,
1988
).
The use of colour vision by these North American species would not negate the significance of such systems in the visual ecology of the Lampyridae; rather we propose the existence of another component that may operate synergistically with the tuned photoreceptor/filter pigment pairs. The relative contribution of each component to sexual signalling behaviour, however, may differ between species occupying divergent photic niches.
The possibility of colour vision in the Lampyridae has been mooted
(Lall et al., 1982), but until
now there has been no direct evidence for chromatic opponency. There are a
number of possible explanations as to why colour vision was not found.
Firstly, our study animal, L. noctiluca, allows simple binary choice
experiments that are more difficult in species that have a flash dialogue
between the sexes. Moreover, few experiments have measured behavioural, rather
than electrophysiological, responses to spectral lights. A notable exception
was the recent measurement of behavioural action spectra in the
twilight-active firefly Photinus pyralis
(Lall and Worthy, 2000
). While
this experiment was not designed to test for opponency between L and S
receptors, the data presented do suggest that P. pyralis females can
perceive light at shorter wavelengths (<480 nm) but do not respond to
it.
Blue photoreceptors and yellow filters: a paradox?
Male glow-worms fly at light intensities at which humans are colour blind.
Colour vision in dim light is problematic due to photon-noise
(Land and Osorio, 2003). The
yellow (blue-absorbing) pigment apparently overlying blue photoreceptors in
the glow-worm eye presents an intriguing paradox because the resultant
reduction in photon catch would tend to restrict green-blue colour vision
still further.
However, the yellow filter pigment in L. noctiluca is not evenly
distributed across the visual field (Fig.
2). Pigmentation is most pronounced in the frontal-dorsal region,
and is reduced, or absent, in the ventraland dorsal-most portions. Although
their precise flight patterns have not been studied, male glow-worms are
believed to mate-search by flying low over the vegetation
(Tyler, 2002), so the ventral
retina probably plays a vital role in the initial stage of mate location. In
our experiments the males were not overflying the light stimuli, and in most
cases the choice between stimuli was made as the male walked along the arena
floor. It is difficult to explain how the ventral-most portions of the eyes
might have been used to compare the two signals in this situation.
Until we have resolved the position of the yellow pigments in the optical
pathway with respect to the short-wavelength photoreceptors we cannot be
certain to what extent the pigments screen the receptors, even in those
ommatidia where the former occur. Lampyrids have a three-tiered retina
(Hariyama et al., 1998;
Horridge, 1969
) in which a
distal rhabdomere overlies the main bundle of rhabdomeres, with a single,
small, basal rhabdomere close to the basement membrane. It is therefore
conceivable that at least some rhabdomeres protrude through the filter
pigment.
Lall et al. (1988) found
that in the nocturnal firefly Photuris frontalis near-UV and blue
sensitivity was most acute in the dorsal-frontal region of the eye, which is
where our results suggest the yellow pigment is most dense. Further, this
species did not exhibit the expected attenuation of spectral sensitivity
(S
) below 500 nm, which is expected from the spectral properties of
the yellow filter pigments. Given that the short-wavelength photoreceptors
almost certainly play a part in flight orientation
(Kelber, 1999
; Lall,
1993
,
1994
), the primary function of
these filter pigments in nocturnal lampyrids may be more concerned with
shielding the sensitive eye from skylight than with signal discrimination. In
this regard the distinction should be made between nocturnal species, such as
L. noctiluca, and the crepuscular North American fireflies: the
latter possess filter pigments that are quite different in both spectral
absorbance properties and location within the eye
(Cronin et al., 2000
), and it
has already been suggested that the role played by filter pigments may differ
between nocturnal and crepuscular species
(Lall et al., 1988
).
Nocturnal colour vision and detection of bioluminescence
Until recently there has been little work on colour vision of insects at
low light intensities (but see Menzel,
1981; Rose and Menzel,
1981
). Photon noise limits vision at low intensities
(Land and Nilson, 2002
), and
because colour vision is based on a comparison of two receptor signals, and
these differences are relatively small, one might expect colour to be of
little use at night. However recent work demonstrates that the nocturnal hawk
moth Deilephila elpenor (Kelber
et al., 2002
), sees colour of (model) flowers at starlight
intensities (10-310-4 cd m-2).
Lampyris noctiluca is also strictly nocturnal, but the problems of
locating flowers by reflected starlight and bioluminescence are different. The
bioluminescent emission of the female glow-worm (3.8 cd m-2 at 555
nm) is a brighter target. Under such conditions one might assume that colour
vision is less limited by noise than by reflected light. The female's colour
is indeed visible to humans from several metres
(Tyler, 2002
), as were the
test stimuli used in our experiments. However this fact alone does not explain
why L. noctiluca uses colour vision. First, the female's light is
likely to subtend a smaller angle than the receptive field of a single
photoreceptor, so its effective intensity is lower than 3.8 cd m-2.
At a range of 1 m a 3 mm spot subtends c. 0.35° whereas the acceptance
angle (
) of the photoreceptors (at 50% max sensitivity) is at
least 2°. Assuming
=2°, the effective brightness of the
female is <10% (7.5%) that of an extended source, although this
disadvantage is offset by the relatively low f-number of beetle
superposition eyes (Land and Nilsson,
2002
). More important, photon noise will be higher in the
S-receptor than the L-receptor. Thus S-receptor noise will dominate any
LS chromatic signal, in effect giving colour vision performance
expected for effective intensity experienced by the S-receptor not
the L-receptor.
Conclusions
The matching of visual sensitivity and bioluminescent signalling to
ecological constraints in the Lampyridae remains an excellent case study of
the coevolution of signals and sensors. There is convincing evidence that
interspecific differences in signal wavelength are adaptations to different
light environments or `photic niches'
(Cronin et al., 2000;
Lall et al., 1980
;
Seliger et al., 1982b
).
Coloured filter pigments have been found in every species so far examined,
with spectral absorbance properties that imply a role in tuning spectral
sensitivity to match the species' bioluminescence
(Cronin et al., 2000
;
Lall et al., 1988
). Our
results show that in addition to possessing intraocular filter pigments,
L. noctiluca is able to distinguish light signals on the basis of
chromaticity.
Glow-worm colour vision is probably attributable to an opponent interaction
between long and short-wavelength photoreceptors. The use of colour vision is
not consistent with the notion that under low ambient illumination photon
capture alone limits detection of bioluminescent signals by lampyrids
(Seliger et al., 1982b). Were
this the case an achromatic mechanism would be best. The presence of colour
vision implies that even in their nocturnal photic niche the insects need to
discriminate between bioluminescence and background noise (such as reflections
of moonlight from water droplets or wet leaves), or possibly between species
(although in England there is only one).
Further work is required to understand the visual ecology of luminescent lampyrids, and to establish the extent to which colour vision is used for bioluminescent signal discrimination in other members of the Lampyridae. For example, studies are needed to establish whether the balance between colour vision and achromatic contrast-enhancement strategies differs between the nocturnal and crepuscular fireflies. A key question is whether glow-worm phototaxis is based purely on a chromatic signal, or if there is also an achromatic component to this behaviour.
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Acknowledgments |
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