A unique visual pigment expressed in green, red and deep-red receptors in the eye of the small white butterfly, Pieris rapae crucivora
1 Graduate School of Integrated Science, Yokohama City University, Yokohama
236-0027, Japan
2 Department of Neurobiophysics, University of Groningen, Groningen, The
Netherlands
* Author for correspondence (e-mail: arikawa{at}yokohama-cu.ac.jp)
Accepted 12 May 2004
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Summary |
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Key words: compound eye, colour vision, spectral filter, rhodopsin, spectral sensitivity
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Introduction |
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The sensitivity wavelength range of a photoreceptor cell is principally
determined by the absorption spectrum of its visual pigment. A visual pigment
molecule consists of an opsin protein with an 11-cis retinal
chromophore. Absorption of light by the visual pigment molecule converts the
11-cis retinal to all-trans retinal, which then triggers the
signal transduction cascade, resulting in a change of the membrane potential
of the photoreceptor cell. The wavelength range where light effectively
isomerizes the chromophore depends on the interaction of certain amino acids
of the opsin with the chromophore; i.e. the opsin's amino acids together with
the chromophore determine the spectral sensitivity of a photoreceptor. A
distinct spectral sensitivity correlates with a unique amino acid sequence, as
has been established for honeybees
(Mardulyn and Cameron, 1999;
Townson et al., 1998
). This
also holds for the Japanese yellow swallowtail butterfly, Papilio
xuthus, where green and red receptors express different mRNAs encoding
different visual pigment opsins (Kitamoto
et al., 1998
).
Optical factors often play a modulatory role in the photoreceptor spectral
sensitivity. For example, the spectral sensitivity of the red receptors of
Papilio xuthus peaks at 600 nm, but the spectrum is considerably
narrower than predicted for a visual pigment with peak absorbance at 600 nm.
The red receptors of Papilio are located in the proximal tier of
those ommatidia where the rhabdom is surrounded by red pigmentation, which
acts as a red transmittant spectral filter. While the absorption spectrum of
the visual pigment peaks at 575 nm, the filter shifts the spectral sensitivity
such that it peaks at 600 nm (Arikawa et
al., 1999b).
We here report an extreme case of spectral filtering in the eye of the
small white butterfly, Pieris rapae crucivora. Pieris has three types
of long wavelength photoreceptors, peaking at 560 nm (green), 620 nm (red),
and 640 nm (deep-red), accordingly called L560, L620 and L640 receptors,
respectively (Qiu and Arikawa,
2003a,b
).
The Pieris eye consists of three distinct types of ommatidia, which
are characterized by the perirhabdomeral pigmentation: a pale-red pigment in
type I and III ommatidia and a deep-red pigment in type II ommatidia
(Qiu et al., 2002
). In all
ommatidial types, two of the four distal photoreceptors, R3 and R4, are L560
receptors. The proximal photoreceptors of type I and type III ommatidia are
L620 receptors, whereas the proximal photoreceptors of type II ommatidia are
L640 receptors (see Table
1).
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Materials and methods |
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Molecular cloning
The method of molecular cloning of Pieris opsins was as described
previously (Kitamoto et al.,
1998). Briefly, retinal mRNA was extracted using the QuickPrep
Micro mRNA purification kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ,
USA) from eyes rapidly frozen in liquid nitrogen. For amplifying the cDNAs of
long-wavelength absorbing visual pigments by RT-PCR, we designed two sets of
degenerate primers based on amino acid sequences conserved in long-wavelength
absorbing visual pigments of the swallowtail butterflies Papilio
xuthus (Kitamoto et al.,
1998
) and Papilio glaucus
(Briscoe, 1998
) and the
hawkmoth Manduca sexta (Chase et
al., 1997
); for the sequence of the primers, see the legend of
Fig. 1. RT-PCR using all of
these primers identified a single DNA fragment with an opsin-like sequence. To
obtain the full-length cDNA, we carried out 3' and 5'-RACE.
|
To compare the amino acid sequence deduced from the cloned cDNA with opsins of other insects so far identified, the sequences were aligned using an alignment program (CLUSTAL W 1.6), and then a phylogenetic analysis was performed by the neighbour joining method (PHYLIP 3.572), with octopus opsin as an outgroup.
In situ hybridization
The compound eyes of Pieris were fixed in 4% paraformaldehyde in
0.1 mol l1 sodium phosphate buffer (pH 7.2) for 0.52
h at 25°C. After dehydration with an ethanol series, we embedded the eyes
in paraplast. The paraplastembedded eyes were sectioned at 8 µm
thickness with a rotary microtome.
Probes for in situ hybridization were designed to hybridize to
400 bases of the mRNA at the non-coding region downstream of the
C-terminal. The corresponding cDNA region was first subcloned into pGEM-3zf(+)
vector, and then digoxigenin (DIG)-labelled cRNA was generated using the
DIG-RNA labelling kit (Roche, Mannheim, Germany).
For labelling, sections were first de-paraffinized and treated with hybridization solution [300 mmol l1 NaCl, 2.5 mmol l1 EDTA, 200 mmol l1 Tris-HCl (pH 8.0), 50% formamide, 10% dextran sulphate, 1 mg ml1 yeast tRNA, 1x Denhardt's medium], containing 0.5 µg ml1 of the cRNA probe, at 45°C overnight. After a brief rinse, the sections were incubated in 50% formamide in 2x SSC (saline sodium citrate buffer) at 55°C for 2 h and then treated with RNase (10 µg ml1) at 37°C for 1 h. The probes were further visualized by anti-DIG immunocytochemistry.
Calculation of the absorbance spectra of the photoreceptor screening pigment
Four clusters of pigment surround the rhabdoms distally in all ommatidia of
the fronto-ventral eye of Pieris. The pigment clusters thus act as
absorption filters for the R58 proximal photoreceptors. The pigment is
pale-red in type I and III ommatidia and deep-red in type II ommatidia. The
R58 photoreceptors in the different ommatidial types have spectral
sensitivities depending on the wavelength, , peaking in the red and
deep-red, respectively (Qiu and Arikawa,
2003b
). The central hypothesis of the present paper is that the
two types of distal screening pigment create the two types of R58
spectral sensitivities by selective spectral filtering.
The spectral sensitivity of a photoreceptor, S(), is
experimentally determined by measuring at several wavelengths the number of
photons (Ic) necessary to elicit a chosen criterion
voltage response, assuming that this response is always the result of the same
number of photons absorbed by the visual pigment (Iabs).
The spectral sensitivity is then given by
S(
)=(Iabs/Ic)n,
where the index n indicates normalization. The stimulus light first passes the
dioptric apparatus and then enters the rhabdom. There, the light propagates in
distinct light patterns, the waveguide modes. The number of allowed modes
depends on the waveguide number:
![]() | (1) |
![]() | (2) |
![]() | (3) |
Hence:
![]() | (4) |
![]() | (5) |
The distal absorbance, Ad, as given by
equation 5,consists of three
terms: (1) the absorbance of the dioptrics; (2) the absorbance of the various
visual (and possibly other absorbing) pigments within the rhabdom and (3) the
absorbance of the red screening pigments near the rhabdom boundary. The first
term is probably, in very good approximation, a constant, i.e. independent of
wavelength (Stavenga, 2004).
The second term is described by
Adv=log(Tdv), where the
transmittance of the distal visual pigment, Tdv, in the
green to red wavelength range is given by an expression similar to
equation 2:
Tvd=exp(f3,4
max
Ld),
where f3,4=f3+f4
is the sum of the rhabdom volume fractions of photoreceptors R3 and R4, and
Ld is the length of the distal rhabdom tier. Anatomical
data show that f3,4=0.3 and Ld=250
µm (Qiu et al., 2002
).
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Results |
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R38 photoreceptors of Pieris rapae all contain the same PrL mRNA
Fig. 3 shows the results of
histological in situ hybridization, together with a diagrammatical
sketch of the ommatidium (Fig.
3A) and unstained histological sections of the Pieris
retina through the distal (Fig.
3B) and the proximal (Fig.
3C) tier of the retina. In the distal tier, the probe for PrL mRNA
hybridized the R3 and R4 photoreceptors in all ommatidia
(Fig. 3D). In the proximal
tier, the same probe labelled the R58 photoreceptors in all ommatidia
(Fig. 3E). We could not
identify any obvious labelling in the R9 basal photoreceptors.
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Filtering by photoreceptor screening pigment results in different photoreceptor classes
The spectral sensitivity of the distal R3,4 photoreceptors, determined by
intracellular recordings (Qiu and Arikawa,
2003a), well approximates the absorption spectrum of a visual
pigment peaking at 563 nm (Fig.
4A). We therefore may conclude that PrL, expressed in R3,4, is an
R563 visual pigment. This conclusion immediately implies that all R58
photoreceptors have an R563, although their spectral sensitivities severely
deviate from an R563 absorption spectrum
(Fig. 4B). The R58
photoreceptors form two classes, L620 and L640, that correlate with the colour
of the pigment clusters in the ommatidium of the photoreceptor, being pale-red
(PR; in type I and III ommatidia) or deep-red (DR; in type II ommatidia),
respectively.
The two photoreceptor classes can be explained with a simple model, based on the assumption that the pale-red and deep-red pigments act as spectral filters, which are positioned fully distally, i.e. in front of the proximal rhabdom, formed by the rhabdomeres of photoreceptors R58. The absorbance spectra of the screening pigments can be estimated with the procedure outlined in the Materials and methods, as visualized in Fig. 4B,C. The first step is the calculation of the log absorptance of the proximal photoreceptors, log(1Tp), where Tp is the transmittance of the proximal tier of the rhabdom. Red pigments are assumed to be transparent at long wavelengths, and hence the log sensitivity curves should match the log absorptance spectrum at long wavelengths. This indeed occurs for both L620 and L640 receptors (Fig. 4B). The difference between the log absorptance and log sensitivity curves then yields the absorbance spectra for the retinal material situated distally (Fig. 4C).
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Discussion |
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Absorbance spectra of red screening pigments
The absorbance spectra of the screening pigments in the pale-red (PR)
ommatidia (type I and III) and deep-red (DR) ommatidia (type II) in the distal
retina of the Pieris eye were estimated by subtraction of the
experimental log spectral sensitivities from the calculated log absorptance
spectrum of the proximal rhabdom tier. The resulting spectra show a
substantial absorption of approximately 1 and 2 log units at wavelengths of
<600 nm, but moreover clearly show a difference in spectral cut-off for the
two types of screening pigments. Whereas the PR absorbance is low above
620 nm, the DR absorbance is only minor above 660 nm. This is in full
agreement with measurements of the eye shine, which showed that eye
reflectance sharply drops at wavelengths below 620 and 660 nm for the two
ommatidial types that can be distinguished with optical methods
(Qiu et al., 2002
). It should
be noted, however, that the derived absorbance spectra of
Fig. 4C are not exclusively due
to the screening pigments. The visual pigments in the distal rhabdom tier also
function as spectral filters. The absorbance, given by spectrum
Adv in Fig.
4B (see Materials and methods), is maximally
0.17, so the
effect of the distal visual pigment is minor. The absorbance spectra include
the possible spectral effects of the dioptric apparatus. Although the facet
lens and crystalline cone will be fully transparent, the channelling of light
into the rhabdom will not be spectrally flat. Presumably, the spectral
modulations are very minor (see the case of fly eyes: Stavenga,
2003a
,b
).
Furthermore, as explained in the Materials and methods, the light reflected by
the tapetal mirror slightly modulates the absorptance spectrum of the proximal
photoreceptors. The maximal contribution from the tapetum is given by
log(1+Tp), but it is probably much smaller. At any rate,
the absorbance spectra of Fig.
4C will not essentially change when the contributions of distal
visual pigment filtering and tapetal mirror are fully accounted for. A
detailed analysis cannot be given here because first the absorption
characteristics of the short-wavelength visual pigments and then the existing
short-wavelength spectral filters have to be analyzed in more detail
(Arikawa et al., 1999a
).
Variability of long-wavelength photoreceptors
Many lepidopterans appear to have a long-wavelength absorbing visual
pigment in six of the eight main photoreceptors. The long-wavelength visual
pigments characterized photochemically in the nymphalids Vanessa
cardui (Briscoe et al.,
2003) and Polygonia c-album
(Vanhoutte, 2003
) absorb
maximally at
530 nm, and the similarly dominant visual pigment of the
moth Manduca sexta peaks at 520 nm
(White et al., 2003
). This can
be compared with the honeybee, where six of the eight main photoreceptors have
a 530 nm visual pigment (M. Kurasawa, M. Giurta and K. Arikawa, manuscript in
preparation). (Note also that, in flies, six large photoreceptors have the
same visual pigment, peaking at 490 nm.) In Apis, Vanessa and
Manduca, the two additional photoreceptors are UV and/or blue
receptors, which are distributed heterogeneously in the ommatidial lattice.
The same long-wavelength visual pigment appears to exist in all
long-wavelength receptors, however, indicating that these animals have a
homogeneous retina, when considering the long-wavelength range. This agrees
with optical observations on common nymphalids, which show a homogeneous eye
shine (Stavenga, 2002a
).
The heterogeneous eye shine observable in many butterflies, including
Pieris, demonstrates that many butterflies have diversified their
long-wavelength receptors. As a first example, the dorsal parts of the eyes of
the satyrine Bicyclus anynana have ommatidia, which all have the same
greenorange eye shine. Ventrally, red-reflecting ommatidia intersperse
the yelloworange shining ones. Spectral measurements strongly suggest
that in the red-reflecting ommatidia red spectral filters occur, presumably
shifting the spectral sensitivity of sets of long-wavelength receptors
(Stavenga, 2002b).
Pieris applies one of two types of red spectral filters in the
ommatidia in the fronto-ventral eye area. Papilio xuthus also uses
two types of screening pigment, i.e. either a red or a yellow pigment, but
moreover combines these pigments with different types of
long-wavelength-sensitive visual pigments. Papilio xuthus exercises
further extravagance by expressing two different long-wavelength visual
pigments in certain photoreceptors. Presumably, these differences increase the
potential for colour vision. At least, Papilio xuthus can boast
extreme colour discrimination capacities
(Kinoshita and Arikawa, 2000
;
Kinoshita et al., 1999
).
Eye shine studies suggest that the diversification of spectral properties
strongly depends on species and eye region
(Stavenga, 2002a). For
example, whereas the ommatidia in the main part of the eye of Pieris
rapae reflect red or deep-red light, the ommatidia in the dorsal part of
the eye reflect in the yellow wavelength range, in agreement with the absence
of red screening pigment dorsally (Ribi,
1979
). The red spectral filters in the fronto-ventral area create
extremely long-wavelength shifted photoreceptors, presumably to improve the
capacity to discriminate food plants
(Kelber, 1999
), which are more
often seen with the ventral than the dorsal eye area.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arikawa, K. (2003). Spectral organization of the eye of a butterfly Papilio. J. Comp. Physiol. A 189,791 -800.
Arikawa, K., Inokuma, K. and Eguchi, E. (1987). Pentachromatic visual system in a butterfly. Naturwissenschaften 74,297 -298.
Arikawa, K., Mizuno, S., Scholten, D. G. W., Kinoshita, M., Seki, T., Kitamoto, J. and Stavenga, D. G. (1999a). An ultraviolet absorbing pigment causes a narrow-band violet receptor and a single-peaked green receptor in the eye of the butterfly Papilio.Vision Res. 39,1 -8.[CrossRef][Medline]
Arikawa, K., Scholten, D. G. W., Kinoshita, M. and Stavenga, D. G. (1999b). Tuning of photoreceptor spectral sensitivities by red and yellow pigments in the butterfly Papilio xuthus. Zool. Sci. 16,17 -24.
Bernard, G. D. (1979). Red-absorbing visual pigment of butterflies. Science 203,1125 -1127.
Briscoe, A. D. (1998). Molecular diversity of visual pigments in the butterfly Papilio glaucus.Naturwissenschaften 85,33 -35.[CrossRef][Medline]
Briscoe, A. D., Bernard, G. D., Szeto, A. S., Nagy, L. M. and White, R. H. (2003). Not all butterfly eyes are created equal: rhodopsin absorption spectra, molecular identification and localization of UV-, blue- and greensensitive rhodopsin encoding mRNA in the retina of Vanessa cardui. J. Comp. Neurol. 458,334 -349.[CrossRef][Medline]
Chase, M. R., Bennett, R. R. and White, R. H.
(1997). Three opsin-encoding cDNAS from the compound eye of
Manduca sexta. J. Exp. Biol.
200,2469
-2478.
Govardovskii, V. I., Fyhrquist, N., Reuter, T., Kuzmin, D. G. and Donner, K. (2000). In search of the visual pigment template. Vis. Neurosci. 17,509 -528.[CrossRef][Medline]
Kelber, A. (1999). Ovipositing butterflies use
a red receptor to see green. J. Exp. Biol.
202,2619
-2630.
Kelber, A. and Pfaff, M. (1999). True colour vision in the orchard butterfly, Papilio aegeus.Naturwissenschaften 86,221 -224.[CrossRef]
Kinoshita, M. and Arikawa, K. (2000). Colour
constancy of the swallowtail butterfly, Papilio xuthus. J. Exp.
Biol. 203,3521
-3530.
Kinoshita, M., Shimada, N. and Arikawa, K.
(1999). Colour vision of the foraging swallowtail butterfly
Papilio xuthus. J. Exp. Biol.
202,95
-102.
Kitamoto, J., Sakamoto, K., Ozaki, K., Mishina, Y. and Arikawa,
K. (1998). Two visual pigments in a single photoreceptor
cell: Identification and histological localization of three mRNAs encoding
visual pigment opsins in the retina of the butterfly Papilio xuthus.J. Exp. Biol. 201,1255
-1261.
Mardulyn, P. and Cameron, S. A. (1999). The major opsin in bees (Insecta: Hymenoptera): a promising nuclear gene for higher level phylogenetics. Mol. Phylogenet. Evol. 12,168 -176.[CrossRef][Medline]
Matic, T. (1983). Electrical inhibition in the retina of the butterfly Papilio. I. Four spectral types of photoreceptors. J. Comp. Physiol. A 152,169 -182.
Qiu, X. and Arikawa, K. (2003a). The photoreceptor localization confirms the spectral heterogeneity of ommatidia in the male small white butterfly, Pieris rapae crucivora. J. Comp. Physiol. A 189,81 -88.
Qiu, X. and Arikawa, K. (2003b). Polymorphism
of red receptors: sensitivity spectra of proximal photoreceptors in the small
white butterfly, Pieris rapae crucivora. J. Exp. Biol.
206,2787
-2793.
Qiu, X., Vanhoutte, K. A. J., Stavenga, D. G. and Arikawa, K. (2002). Ommatidial heterogeneity in the compound eye of the male small white butterfly, Pieris rapae crucivora. Cell Tissue Res. 307,371 -379.[CrossRef][Medline]
Ribi, W. A. (1979). Coloured screening pigments cause red eye glow hue in Pierid butterflies. J. Comp. Physiol. A 132,1 -9.
Shimohigashi, M. and Tominaga, Y. (1991). Identification of UV, green and red receptors, and their projection to lamina in the cabbage butterfly, Pieris rapae. Cell Tissue Res. 263,49 -59.
Stavenga, D. G. (2002a). Colour in the eyes of insects. J. Comp. Physiol. A 188,337 -348.
Stavenga, D. G. (2002b). Reflections on
colourful ommatidia of butterfly eyes. J. Exp. Biol.
205,1077
-1085.
Stavenga, D. G. (2003a). Angular and spectral sensitivity of fly photoreceptors. I. Integrated facet lens and rhabdomere optics. J. Comp. Physiol. A 189, 1-17.
Stavenga, D. G. (2003b). Angular and spectral sensitivity of fly photoreceptors. II. Dependence on facet lens F-number and rhabdomere type in Drosophila. J. Comp. Physiol. A 189,189 -202.
Stavenga, D. G. (2004). Angular and spectral sensitivity of fly photoreceptors. III. Dependence on the pupil mechanism in the blowfly Calliphora. J. Comp. Physiol. A 190,115 -129.
Townson, S. M., Chang, B. S. W., Salcedo, E., Chadwell, L. V.,
Pierce, N. E. and Britt, S. G. (1998). Honeybee blue- and
ultraviolet-sensitive opsins: cloning, heterologous expression in
Drosophila, and physiological characterization. J.
Neurosci. 18,2412
-2422.
Vanhoutte, K. A. J. (2003). Butterfly visual pigments: molecular cloning and optical reflections. Thesis, University of Groningen, The Netherlands.
Warrant, E. J. and Nilsson, D. E. (1998). Absorption of white light in photoreceptors. Vision Res. 38,195 -207.[CrossRef][Medline]
White, R. H., Xu, H., Munch, T., Bennett, R. R. and Grable, E.
A. (2003). The retina of Manduca sexta:
rhodopsin-expression, the mosaic of greenblueand UV-sensitive photoreceptors
and regional specialization. J. Exp. Biol.
206,3337
-3348.
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