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First published online May 1, 2006
Journal of Experimental Biology 209, 1944-1955 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02207
Color discrimination in the red range with only one long-wavelength sensitive opsin
1 Vision Group, Department of Cell and Organism Biology, Lund University,
Helgonavägen 3, S-22362 Lund, Sweden
2 Comparative and Evolutionary Physiology Group, Department of Ecology and
Evolutionary Biology, University of California, Irvine, CA 92697,
USA
* Author for correspondence (e-mail: guillermo.zaccardi{at}cob.lu.se)
Accepted 13 March 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: color vision, opsin, filter pigment, insect, butterfly, Heliconius erato
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Kelber et al., 2003). It
arises from the comparison of signals from at least two types of photoreceptor
with different spectral sensitivities. Depending on the number and
sensitivities of photoreceptor types, the spectral range of color vision
differs between species and is usually narrower than the total range of
vision. Most insects have a short wavelength (S) receptor sensitive in the
ultraviolet (UV) range, a medium wavelength (M) receptor sensitive in the blue
range and only one long wavelength (L) receptor sensitive in the green/yellow
range. In the case of the honeybee, the best studied example, these receptors
have peak sensitivities that correspond to 344 nm (S), 436 nm (M) and 544 nm
(L) (Peitsch et al., 1992).
Moths with a receptor set similar to that of the honeybee are incapable of
discriminating between lights of 590 and 630 nm by means of colors but instead
they used the intensity of the stimuli
(Kelber and Hénique,
1999).
The spectral sensitivity primarily depends on the visual pigment expressed
in the receptor. The three insect receptor types correspond to three major
clades of the insect opsin phylogenetic tree
(Fig. 1). In bees, moths and
butterflies, each ommatidium has six (or seven) receptors expressing L opsin,
and two receptors expressing either both M opsin, both S opsin or one M opsin
and one S opsin: Vanessa cardui
(Briscoe et al., 2003);
Danaus plexippus (Sauman et al.,
2005); Pieris rapae
(Arikawa et al., 2005;
Wakakuwa et al., 2004);
Manduca sexta (White et al.,
2003); Bombus terrestris
(Spaethe and Briscoe, 2005);
Apis mellifera (Wakakuwa et al.,
2005). Papilionid butterflies have evolved a fourth, red-sensitive
opsin (Fig. 1) expressed in
four receptors in a subset of ommatidia
(Arikawa, 2003), and behavioral
experiments have proved that this enables them to discriminate colors in the L
range, e.g. spectral lights of 590 and 640 nm wavelength
(Kelber and Pfaff, 1999).
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; Kong et al.,
1980). In butterflies (with the exception of papilionids) a
tapetum basal to the rhabdom reflects either broadband light (300–700
nm) as in Vanessa cardui (Briscoe
et al., 2003), or relatively narrow-band light (320–590 nm)
(Arikawa et al., 2005;
Douglas and Marshall, 1999;
Stavenga, 2002a) (G. Bernard,
personal communication). In the latter case, it can thus modify the spectral
sensitivity of the photoreceptors. However, the effect of a tapetum is limited
since it only affects the spectral composition of light during the second pass
through the retina, and most light absorption happens on the first pass, which
is before light reaches the tapetum.
Different filtering pigments associated with different photoreceptors that
express the same opsin can result in photoreceptors with different spectral
sensitivities that can be used for color vision. Additional photoreceptors
resulting from this kind of filtering would not extend the total spectral
sensitivity of the animal – that is defined by the sensitivities of the
opsin pigments – but it may extend the range of color vision (i.e. the
range of wavelengths that can be discriminated). The colored oil droplets
found in some birds had earlier been thought to play this role in color vision
(Walls, 1942;
King-Smith, 1969) but it is
now known that each colored oil droplet is associated with a specific opsin in
the receptor. They act as a cut-off filter, narrowing the spectral sensitivity
of the cones, rather than increasing spectral types
(Vorobyev, 2003). The same
principle applies to the lateral filtering pigments in the receptors of
Papilio xuthus (Arikawa et al.,
1999). However, in the eyes of the male small white butterfly,
Pieris rapae, the pale-red or deep-red pigment clusters that surround
the rhabdoms of different ommatidia act as long-pass filters, creating
receptors with peak sensitivity at 620 or 640 nm, but both contain the same
opsin (Qiu et al., 2002;
Qiu and Arikawa, 2003;
Wakakuwa et al., 2004). In
these cases, color discrimination could theoretically be extended by means of
filtering pigments but direct behavioral evidence confirming this is missing.
Different corneal filters in different ommatidia found in tabanid flies and
grasshoppers have the same potential to create new receptor types
(Kong et al., 1980;
Lunau and Knüttel,
1995).
The ability to discriminate colors in the red range seems to be very
useful. It can increase the number of flower species that can be distinguished
and facilitate the finding of better hosts for larvae. This seems to be the
case in Papilio butterflies. These animals use color vision not only
when foraging for flowers (Kelber and
Pfaff, 1999; Kinoshita et al.,
1999) but also when making decisions about where to oviposit
(Kelber, 1999). Color
discrimination in the L range enables them to choose the optimal host for
their offspring. The same probably applies to Pieris butterflies
(Scherer and Kolb, 1987;
Kelber, 2001;
Weiss and Papaj, 2003).
All species investigated so far within the third butterfly family,
Nymphalidae, have only three opsin genes
(Sauman et al., 2005;
Briscoe and Bernard, 2005) but
more than three receptor types have been reported from several species
(Bernard, 1979;
Kinoshita et al., 1997;
Stavenga et al., 2001) (for a
review, see Briscoe and Chittka,
2001). The sensitivity curves recorded from the nymphalid
butterflies Polygonia c-aureum and Sasakia charonda show
clearly that the long-wavelength cut-off of all L receptors coincide, whereas
peak sensitivities differ by as much as 50 nm. This can only result from
filtering, not from multiple opsins
(Kinoshita et al., 1997).
We have chosen to investigate whether color vision extends into the L
range, in two species of the nymphalid family, Vanessa atalanta
(Linnaeus 1758; subfamily Nymphalinae) and Heliconius erato (Linnaeus
1758; subfamily Heliconiinae). Both species have red areas on their wings,
thus color discrimination in the red range could be useful for mate detection
as well. In H. erato only three receptor types have been described so
far, with 
max at 370, 470 and 570 nm
(Fig. 2)
(Struwe, 1972). However,
electroretinograms (Bernhard et al.,
1970; Swihart and Gordon,
1971) and electrophysiological recordings in the brain
(Swihart, 1972) give
indications that a second L receptor (max at 620 nm) may
exist. It has long been known that Heliconius uses color to find food
(Swihart, 1971), and both
color and polarized light cues are used by males in choosing mates
(Jiggins et al., 2001;
Sweeney et al., 2003). V.
atalanta has three receptors with max at 360, 470 and
530 nm similar to the congener V. cardui
(Fig. 2)
(Briscoe et al., 2003) (G.
Bernard, personal communication). We have performed behavioral experiments,
characterized the opsin genes and their expression in the retina, and studied
the eye glow and lateral filtering pigments. In short, we prove, for the first
time using behavioral studies, that an insect with only S, M and L opsin
pigments can discriminate colors in the red range. This is not explained by
the sensitivities of the opsins alone and it is probably due to the shift in
the receptors' sensitivity caused by the presence of lateral filtering
pigments.
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Materials and methods |
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Behavioral experiments
The experiments with H. erato were performed in an indoor cage (2
mx1.60 mx2.8 m) constructed from metal pipes and covered with
gauze except for the ceiling that was made of a thin plastic sheet. The cage
was illuminated by 26 fluorescent tubes (Osram Biolux 965, 65 W; München,
Germany) distributed around and above the cage. The intensity of the
illumination was approximately 100 cd m–2 in the center of
the cage. The temperature was 30°C, and the light regime was set at 12
h:12 h light:dark. The experiments with V. atalanta were performed in
a smaller cage (75 cmx50 cmx60 cm) illuminated by four fluorescent
tubes (Philips TLD 965 18 W; Eindhoven, The Netherlands). The light intensity
in the center of the cage was roughly the same as in the big cage. The light
regime was 20 h:4 h light:dark, and the temperature between 20 and
25°C.
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Training and test
The butterflies were fed for the first time between 6 h and 8 h after they
emerged. A drop of sugar water was placed on the feeder illuminated with the
positive color (+); the other feeder (–) was always kept empty. Each
butterfly was grasped by the wings and the proboscis was unrolled with a thin
needle until it touched the sugar solution. Immediately the butterfly started
to drink. This procedure was repeated twice a day. After 3–4 days, the
animals flew towards the apparatus by themselves and were able to find the
rewarded color (+). From this moment, the choices were registered. Each time
an animal approached and touched a feeder, either with its proboscis or with a
tarsus, a choice was registered. If an animal touched the feeder more than
once during an approach, only the first touch was counted. In further data
analysis, we only included animals that made a minimum number of 15 choices
with each intensity combination, thus in total at least 60 choices.
We first trained both species to discriminate a yellow or red (590 nm or 620 nm) and a blue (440 nm) color, to determine whether the animals used color vision at all. The animals that were able to discriminate these colors were then trained to a second pair of wavelengths. Animals did not survive long enough to be trained to three wavelength combinations. Therefore, H. erato that were trained to two combinations of long wavelengths were not initially trained with blue and yellow. These H. erato were first trained to 620 nm (+) vs 590 nm. When they reached the minimum number of choices, the same individuals were trained to 620 nm (+) vs 640 nm. V. atalanta were first trained to 620 nm (+) vs 440 nm, and once the animals reached the minimum number of choices, the same individuals were trained to 620 nm (+) vs 590 nm. Only one animal at a time was allowed to visit the apparatus. Each butterfly was allowed to drink for 1–2 s and then gently forced to leave for a new choice. Between choices the intensities and/or the position of the stimuli were changed in a pseudorandom way. Five different ratios of the physical intensities of +/– were used: 0.01, 0.1, 1, 10 and 100 (i.e. +100 times less intense than –, +10 times less intense than –, equal intensities and +10 times more intense than the –, +100 times more intense than –, respectively), but only three or four of them were used with one wavelength combination. This was achieved by changing the intensities of both the rewarded and the unrewarded stimulus. This schema resulted in an average of 10 choices per animal daily.
The performance of each animal with each intensity combination was
evaluated separately by comparing the numbers of choices this animal made with
the critical value corresponding to two-tailed significance levels (
)
of 0.05 for a binomial proportion of P=0.5 [equal number of choices
for each color; Rohlf and Sokal (Rohlf and
Sokal, 1995), p. 107, Table Q].
Light microscopy
Under daylight illumination the heads of 10-day old H. erato were
severed in two halves and the pieces were put in 4% PFA (paraformaldehyde) in
0.1 mol l–1 phosphate-buffered saline (pH 7.2) for 1 h. The
eyes were dehydrated in an alcohol series (50%, 75%, 96%, 100%), finally
immersed in 100% acetone and embedded in Epon resin (Agar Scientific, Agar
Scientific Ltd, Essex, UK) After hardening the resin at 60°C for 48 h the
eyes were cut laterally in 10 µm thick sections using a microtome and
mounted on a slide. In this way the ommatidia that in the living animal
pointed sideways, pointed directly at the observer in the microscope. Some
H. erato were dark adapted by keeping them in total darkness for 30
min at room temperature and then for 20 min in total darkness at 4°C.
After that, the head of the animal was cut off under red light (produced by
attaching a 660 nm cut-off filter to the tip of a light guide connected to a
Schott KL 2500 lamp) in order to avoid light adapting the eyes. V.
atalanta butterflies were captured locally around Lund (Sweden) and were
treated in the same way as the light adapted H. erato.
Eye glow
At least four dark-adapted H. erato and V. atalanta eyes
were photographed with a Nikon camera connected to a modified epi-illumination
microscope. In this instrument, the incident light applied to the eye is
channeled by the facet lens and crystalline cone into the light-guiding
rhabdoms (see Land, 1984;
Miller and Bernard, 1968;
Stavenga, 2002b). When the
dark-adapted eye is illuminated with strong light and observed from the same
direction (orthodromic illumination), a bright eye-glow is seen for a few
seconds. Light reaching the ommatidial tapetum is reflected and guided back
through the rhabdom. When not absorbed there, it leaves the eye again and is
then observable as the eye glow. The butterflies were restrained by waxing the
thorax and head to a support but were otherwise alive and intact. The animals
were oriented such that pictures could be taken in the frontal-lateral part of
the eye. The eye glow was photographed by leaving the shutter of the camera
open and delivering flashes of between 0.1 s and 0.5 s with intervals of 10 s.
In this way, the eye glow could be photographed without the pupil closing. As
a light source we used a xenon arc lamp that supplied
1.4x10–4 W cm–2. The objective lens
was a Zeiss Luminar 25 mm (3.5/A 0.15). To study the spectral sensitivity of
the pupil response, we illuminated the eye with lights of different
wavelengths obtained by means of interference filters (680 nm, 650 nm, 620 nm,
590 nm; 10 nm bandwidth) and observed pupil closure.
PCR, cloning and sequencing
The H. erato opsins were isolated by the polymerase chain reaction
(PCR) using a combination of degenerate and gene-specific primers. cDNA
template was prepared from RNA extracted from whole head tissue (Trizol;
Gibco-BRL, Gaithersburg, MD, USA) and synthesized using the Marathon cDNA
Amplification Kit (BD Biosciences Clontech, Mountain View, CA, USA). PCR
products were ligated into the pGEM-T Easy cloning vector (Promega, Madison,
WI, USA) and plasmids were screened by EcoRI digestion for inserts of
the correct size. Plasmid DNA was cycle sequenced using the Big Dye Terminator
3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the
core sequencing facilities at the University of California, Irvine. In this
initial screen, we sequenced more than 100 clones, and then designed opsin
gene-specific primers to use in a multiplex PCR. To ensure that all opsins had
indeed been recovered, approximately 120 additional plasmids were screened
using multiplex PCR in which three pairs of opsin gene-specific primers were
combined in a single PCR reaction. Only those plasmids that did not amplify
with those primers were then sequenced.
Phylogenetic analysis
To identify the homology of the cloned H. erato opsins, we
conducted a BLAST search and downloaded a total of 41 full-length arthropod
opsin genes from GenBank including representatives of all available insect
orders (six total), as well as chelicerates and crustaceans. Only first and
second nucleotide positions were used as third positions were saturated
(A.D.B.,, unpublished observation). A gene tree was reconstructed using the
neighbor-joining algorithm with Tamura-Nei distance, heterogeneous rates among
lineages and complete deletion of gaps as implemented in MEGA 3.0
(Kumar et al., 2004).
Robustness of the tree was assessed by bootstrap analysis (1000
replicates).
Cryosectioning
The adult butterflies were placed at 4°C for 1 h before being killed by
a swift severing of the head with a scalpel. Subsequently, the head was cut in
half. The eyes were fixed in 4% PFA in 1x phosphate-buffered saline (pH
7.2) for 2 h to 4 h at room temperature and stepped through a sucrose gradient
(10%, 20%, 30%). Then the tissue was cryostat sectioned into 8–12 µm
slices at –18°C and placed on a slide. The eyes were oriented such
that the ommatidia that in the living animal pointed directly to the front,
pointed directly at the observer.
Riboprobe synthesis and in situ hybridization
Starting with 1 µg of purified PCR product (amplified from plasmid DNA),
digoxigenin-UTP-labeled RNA probes (riboprobes) complementary to the mRNAs of
the visual pigments were synthesized by using a DIG RNA labeling kit (Roche
Diagnostics, Mannheim, Germany). After the synthesis, the riboprobes were
precipitated with 4 mol l–1 LiCl and 100% ethanol. A dot blot
procedure was used to quantify the amount of riboprobe. Typically, 10 ng/µl
of riboprobe was obtained after this procedure.
The slides with the sections were then incubated in hybridization buffer
(0.3 mmol l–1 NaCl, 2.5 mmol l–1 EDTA, 20
mmol l–1 Tris-HCl, pH 8.0, 50% formamide, 10% dextran
sulfate, 100 g ml–1 yeast tRNA and 1x Denhart's medium)
(Sakamoto et al., 1996) in a
humid chamber for 30 min at 60°C. The labeled probe was diluted in the
hybridization buffer (1:75), corresponding to approximately 0.013 ng of probe
per ml of hybridization buffer. The sections were incubated in the diluted
probe overnight at 55–60°C in a humid chamber and then washed with
2x, 1x and 0.1x standard saline citrate and 0.1% Tween 20,
for 10 min each. The probes were identified in the histologic sections by
incubation with an anti-digoxigenin alkaline phosphatase-conjugated antibody
(Boehringer Mannheim), diluted in 1x phosphate buffer plus Tween 20
(1:1000) for 2 h. The probes were detected by a colorimetric reaction produced
by Nitro Blue tetrazolium (5-bromo-4-chloro-3-indolylphosphate) and 10% Tween
20 in alkaline phosphatase developing solution. An Axioskop microscope (Zeiss,
Thornwood, NY, USA) equipped with an AxioCam Hrc digital camera (Zeiss) was
used to collect images. Image data were recorded in ZeissVision 3.1 software
on a personal computer at 2,060x2,600 pixel resolution.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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)], it is
more sensitive to light of 590 nm than to light of 620 nm. If both lights have
the same physical intensity, 590 nm is thus supposed to look brighter for
V. atalanta than 620 nm. The fact that the choice frequencies
massively change with the relative intensities of the stimuli clearly
disproves the use of color vision.
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A similar behavioral result was reported in Papilio aegeus
(Kelber and Pfaff, 1999). In
this study it was shown that Papilio butterflies could discriminate
colors in the red range (590 nm vs 620 nm). In Papilio, this
ability is based on duplicated L opsins that these animals possess
(Arikawa, 2003). In contrast,
only one L opsin has so far been found in the compound eyes of
Heliconius (Hsu et al.,
2001) and the M opsin is not sensitive to light of 620 nm
(Fig. 2).
Different kind of opsins and their expression in the retina
In order to see if the capability of H. erato to discriminate
between stimuli of long wavelengths is based on the expression of more than
one L opsin we screened a cDNA library synthesized from adult eyes. More than
200 clones were screened and only one L opsin-encoding mRNA was found in the
compound eyes of H. erato, along with blue and ultraviolet
opsin-encoding mRNA transcripts. The GenBank accession numbers for these genes
are as follows: UVRh, AY918904; BlueRh, AY918906;
LWRh, AY918907. To see how these three opsins are expressed, at least
50 eyes were analyzed after performing in situ hybridization in the
frontal and fronto-lateral part of the compound eye.
Fig. 5 shows an example of the
hybridization pattern obtained after using UVRh, BlueRh and
LWRh digoxigenin-labeled antisense riboprobes.
Fig. 5A shows the pattern
produced by the UVRh riboprobe. Some ommatidia show two cells
stained, others show only one cell stained and some no staining. The same
configuration can be observed when the BlueRh riboprobe was used
(Fig. 5B). Since the
sections in Fig. 5A,B are
consecutive, the position of the cells in both pictures can be followed (black
circles in the insets mark the same ommatidia). We observed three different
types of ommatidia. The ommatidia that express UVRh in two cells do
not express BlueRh. Reciprocally, the ommatidia that express
BlueRh in two cells do not express UVRh. The third type of
ommatidia expresses each opsin mRNA in one cell. This is the same situation as
in V. cardui (Briscoe et al.,
2003), P. xuthus
(Arikawa, 2003), Pieris
rapae (Arikawa et al.,
2005; Wakakuwa et al.,
2004), Manduca sexta
(White et al., 2003), and in
Danaus plexippus (Sauman et al.,
2005). Fig 5C,D
show the LWRh pattern, in which six cells are stained in all
ommatidia across the whole section. Each cell expresses only one of the three
opsins, and we found no evidence of co-expression. This pattern was seen in
all the eyes examined. We, therefore, conclude that the difference between
H. erato and V. atalanta in their ability to discriminate
light in the long wavelength part of the spectrum cannot be attributed to the
presence of a second L opsin.
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Eye glow and histology: Lateral filtering pigment heterogeneity in H. erato
Since H. erato can extend their color vision range into the long
wavelengths using only one L opsin which is homogeneously distributed, we next
studied whether the ommatidia differ in aspects other than the S and M opsin
expression. We first examined the light that is reflected by the tapetum and
emerges from the compound eye (eye glow) of H. erato, using an
ophthalmoscope, and found two classes of ommatidia
(Fig. 6A). One class of
ommatidia reflects yellow light and the other reflects red. By contrast, all
ommatidia of V. atalanta reflect an homogeneous orange
(Fig. 6D) in the same fashion
as in V. cardui, a close relative
(Briscoe and Bernard,
2005).
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), the
different degrees of closure are a result of different sensitivities of the
receptors in both ommatidial classes to these wavelengths. Considering the
wavelengths of the test lights, we conclude that the L receptors must differ
in their spectral sensitivity between ommatidial classes, and this could
provide the basis for color vision in the L range. In V. atalanta all
ommatidia close completely and at the same speed when illuminated with 620 nm
and 590 nm. When illuminated with 650 nm a partial closure was observed
indicating that the photoreceptors are slightly sensitive to this
wavelength.
We hypothesized that, as has been shown in Pieris rapae
(Wakakuwa et al., 2004) the
occurrence of the two physiologically distinct H. erato ommatidial
classes might result from different populations of lateral filter pigments.
Because of the waveguide properties of the narrow butterfly rhabdom
(Nilsson et al., 1988),
lateral filter pigments can affect the wavelengths of light to which the
receptors are sensitive (Miller and
Bernard, 1968; Ribi,
1979; Stavenga,
2002a; Stavenga,
2002b).
We tested this hypothesis by inspecting serial sections through the compound eye, and found that the difference between the heterogeneous eye glow of H. erato and the homogeneous eye glow of V. atalanta may be due to differences in the presence or absence, and distribution of lateral filter pigments. In a tangential section of the fronto-lateral region of the compound eye of H. erato (190 µm from the cornea), the presence of pupillary pigments can be observed (Fig. 6B). In dark-adapted eyes these pigments are further away from the rhabdoms than in light adapted eyes (picture not shown). These pigments disappear completely around 220 µm and after a gap, a second type of red pigmentation appears around 320 µm from the cornea. The pigments at this depth (Fig. 6C, 370 µm from the cornea) are closer to the rhabdom than the pupillary pigments (distance between opposite pigment spots 0.68±0.20 µm, mean ± s.d.; N=10) and do not move as a function of light adaptation. Their coating of the rhabdom means that they can filter the short wavelength light traveling in the waveguide, and therefore change the spectral sensitivity. The color of this pigment is heterogeneous among different ommatidia (see circles marked a and b, in Fig. 6C). This pigment heterogeneity is also evident at sections taken more proximal in the eye (480 µm, picture not shown). We propose that this heterogeneity of pigmentation may be the cause of the heterogeneous eye glow and the difference in speed of the pupil closure (i.e. different sensitivities).
In the eye of V. atalanta (Fig.
6E,F), the only pigments detected were the pupillary pigments (see
also Stavenga, 1979). We found
no histological evidence of a lateral filtering pigment or pigment
heterogeneity between ommatidia, which is consistent with the homogeneous eye
glow, the pupil closure, experimental reflectance spectra (G. Bernard,
personal communication), and with the data from the other species of this
genus that was recently examined, V. cardui
(Briscoe et al., 2003).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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We have compared the color vision abilities of two butterfly species that
possess three opsin genes coding for opsins in the S, M and L range. One of
the species, V. atalanta, was able to discriminate between blue (440
nm) and yellow (590 nm) with high accuracy but not between yellow (590 nm) and
red (620 nm; Fig. 4A,B). This
indicates that only one receptor type is sensitive to both long wavelengths.
The blue-sensitive receptor is insensitive to these long wavelengths making a
comparison of different receptor signals (and thus color vision) impossible.
In the absence of a color difference, V. atalanta used the intensity
of the stimulus as a choice criterion. This result is similar to that obtained
with a sphingid that also possesses three spectral types of receptors leaving
it unable to discriminate yellow from red
(Kelber and Hénique,
1999).
In contrast, H. erato is able to discriminate 620 nm not only from
590 nm but also from 640 nm (Fig.
4C,D). Two spectral types of receptors must therefore be sensitive
to light of 620 nm wavelength. In P. aegeus, the ability to
discriminate 590 nm from 630 nm is mediated by a separate red receptor
containing a separate red opsin (Kelber
and Pfaff, 1999; Matic et al., 1983). This second L opsin evolved
as a result of a gene duplication event, which occurred after the divergence
of the nymphalid and papilionid lineages
(Briscoe, 2001). In H.
erato, our extensive search for an additional L opsin gene was
unsuccessful. Six of eight proximal photoreceptors in each ommatidium, and
probably the ninth basal receptor express the same known green-sensitive opsin
(Fig. 5C,D). The remaining two
receptors are two UV receptors, two blue receptors or one of each
(Fig. 5A,B). This situation is
the same in V. cardui, a close relative of V. atalanta
(Briscoe et al., 2003). The
blue receptors of both species have a peak sensitivity at longer wavelengths
than other insect blue receptors (470 nm)
(Struwe, 1972) [for comparison
see Briscoe and Chittka (Briscoe and
Chittka, 2001)] but this cannot explain our results
(Fig. 2). We conclude that
H. erato has color vision in the red range, without having two opsins
sensitive in this spectral range.
Do lateral filtering pigments create a second long-wavelength receptor?
The ophthalmoscope studies of H. erato revealed two classes of
ommatidia differing in eye glow color (Fig.
6A). This is similar to many butterfly eyes studied by Stavenga
(Stavenga, 2002a;
Stavenga, 2002b), including
another species of the large genus Heliconius, H. melpomene. By
contrast, a uniform eye glow was seen in V. atalanta
(Fig. 6D), similar to that of
V. cardui and other nymphalids including Nymphalis antiopa,
Siproeta steneles, Inachis io and Polygonia c-album
(Briscoe and Bernard, 2005;
Stavenga, 2002a). Possible
mechanisms underlying the different eye glow colors include lateral filtering
pigments, different opsin densities and tapetal reflection. Different opsin
densities are hard to prove; nonetheless, we cannot exclude them as a possible
cause for the difference in the ommatidial reflection seen in H.
erato. Differences in tapetal reflection have little relevance since they
only affect light absorbed on the way out of the receptor. This is a very
small amount as most light is absorbed on the way into the eye. The most
likely candidate causing differences between receptor spectra are the lateral
filtering pigments found in H. erato but not in V. atalanta
(Fig. 6). These pigments are
close enough to the rhabdom to act as lateral filters. Different pigments in
different ommatidia (Fig. 6C)
can result in two kinds of ommatidia with different spectral sensitivities as
has been shown for P. rapae
(Wakakuwa et al., 2004).
Further studies including electrophysiological measurements are needed to
reveal the exact receptor sensitivities.
Color vision with filter pigments?
To our knowledge, H. erato is the first animal shown to use two
photoreceptors containing the same opsin for color vision. We hypothesize that
lateral filtering pigments may be the basis of the difference between the two
receptor types in Heliconius as well. Filtering pigments can extend
the range of color vision by shifting the L receptor sensitivity in one class
of ommatidia. The comparison between different receptors created in this way
allows the animal to discriminate colors at longer wavelengths than those
expected by the opsins alone. The total range of vision, which is set by the
sensitivity of the opsins, is unaltered. The fact that Pieris and
Papilio also have lateral filtering pigments
(Arikawa, 2003;
Wakakuwa et al., 2004) makes
it probable that this mechanism is older than the evolution of a separate red
opsin in Papilio, and common in butterflies. There is a high
probability that butterflies such as Sasakia charonda, Polygonium
c-aureum (Kinoshita et al.,
1997) and Pieris rapae are also able to discriminate
colors in the red range. Given the receptor curves measured
(Wakakuwa et al., 2004), the
color discrimination ability of P. rapae might extend even further
into the red range than color vision of H. erato.
Red filtering pigments that may change receptor sensitivity have earlier
been found in a hymenopteran insect (Ribi,
1978). We expect that careful studies will reveal more such cases
and conclude that the study of opsin genes and their expression is not
sufficient to understand the evolution of color vision systems in animals.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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