Polymorphism of red receptors: sensitivity spectra of proximal photoreceptors in the small white butterfly Pieris rapae crucivora
Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan
* Author for correspondence (e-mail: arikawa{at}yokohama-cu.ac.jp)
Accepted 13 May 2003
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Summary |
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Key words: compound eye, retina, photoreceptor, rhabdom, color vision, butterfly, Pieris rapae crucivora
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Introduction |
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The white butterfly, Pieris, is a cosmopolitan genus that has long
been used for vision research. Early anatomical work revealed that the
ommatidia contain nine photoreceptor cells, numbered R19, and bear red
pigmentation in the ventral half of the compound eye
(Kolb, 1978;
Ribi, 1978b
). The
photoreceptors were also shown to be strongly diverse in terms of their
sensitivity spectra, with peak sensitivities ranging from the ultraviolet to
the red wavelengths (Shimohigashi and
Tominaga, 1991
). The different spectral receptor types participate
in various behavioral tasks such as egg laying and feeding
(Kolb and Scherer, 1982
).
Behavioral aspects related to color vision of Pieris have also been
studied (Goulson and Cory,
1993
; Kandori and Ohsaki,
1996
,
1998
), and some behavioral
experiments (Scherer and Kolb,
1987
) and associated model calculations
(Kelber, 2001
) have together
demonstrated that Pieris rapae has true color vision, as in
Papilionid species (Kinoshita et al.,
1999
; Kelber and Pfaff,
1999
).
Our research on the yellow swallowtail Papilio xuthus has revealed
that the eyes contain three types of ommatidia, each with a distinct set of
photoreceptors, with sensitivity spectra shaped by the rhodopsin absorption
spectra of both visual and filtering pigments
(Arikawa and Stavenga, 1997;
Arikawa et al.,
1999a
,b
).
The accumulated information about the retinal anatomy and the photoreceptor
spectra of the Pieris compound eye inspired us to investigate whether
this butterfly has a similar ommatidial heterogeneity. As expected, we
identified three types of anatomically distinct ommatidia in the compound eye
of Pieris rapae crucivora (Qiu et
al., 2002
). All ommatidia of a Pieris eye are tiered,
containing four distal (R14) and four proximal (R58)
photoreceptor cells, the rhabdomeres, of which together form the fused
rhabdom, a cylindrical structure along the central axis of the ommatidium. The
R9 cell contributes to the rhabdom at the very base (i.e. is most proximal)
(Fig. 1). Except for the dorsal
part of the eye, all ommatidia are prominently pigmented
(Ribi, 1978b
). The proximal
photoreceptors, R58, bear a dense pigmentation around the rhabdom,
which appears as four reddish spots in transverse sections. From the
arrangement of the pigment clusters three ommatidial types can be clearly
distinguished: in type I, the pigment clusters are arranged trapezoidally, in
type II, in a square, and in type III, in a rectangle
(Qiu et al., 2002
; see also,
for example, Fig. 2C). The red
pigmentation around the rhabdom functions as a red-transmittant spectral
filter, with the rhabdom acting as a waveguide, because its diameter in
Pieris is less than 2 µm (Qiu
et al., 2002
) and the refractive indices of the rhabdom and
surrounding medium only differ slightly. When light propagates along the
slender rhabdom, a significant proportion of the light flux travels outside
its boundary, and therefore the red pigmentation lining the rhabdom can absorb
light in the boundary wave.
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The photoreceptors in the three types of ommatidia appear to be spectrally
heterogeneous. Electrophysiological recordings of photoreceptors in the distal
tier revealed the existence of UV (U), blue (B), double-peaked blue (dB),
green (G) and green with depressed sensitivity at 420 nm (dG)receptors
(Qiu and Arikawa, 2003). Using
a combination of electrophysiology and histology, we demonstrate that these
spectral receptors occur in different combinations in the three ommatidial
types (see Table 1). In
addition to these spectral receptors, Shimohigashi and Tominaga
(1991
) reported finding red
receptors in the eye of Pieris rapae. By contrast, we did not
encounter red receptors in the distal tier in our previous study
(Qiu and Arikawa, 2003
). In
fact, in the eye of the Japanese yellow swallowtail Papilio xuthus,
red receptors are found exclusively in the proximal tier of the retina, so we
conjectured that the red receptors must be located in the proximal tier in the
eye of Pieris rapae as well.
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Here we have investigated the sensitivity spectra of the proximal receptors, R58, in the Pieris ommatidia. Using a combination of electrophysiology and histology, we found that the proximal receptors in all three types of ommatidia are specifically sensitive in the long wavelength region, i.e. red. Interestingly, the sensitivity spectra of these receptors differ between the ommatidial types, probably reflecting their differences in pigmentation, tapeta and/or autofluorescence.
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Materials and methods |
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Electrophysiology
Electrophysiological methods were as described before
(Qiu et al., 2002). Briefly, a
butterfly was mounted on a plastic stage set in a Faraday cage. A silver wire
inserted in the stump of an antenna served as the indifferent electrode. To
insert a glass micropipette into the eye, a hole covering about 1020
facets was made in the dorsal region of the cornea with a razor blade. The eye
was then positioned at the center of a Cardan arm perimeter device. The
dorsoventral axis of the compound eye was adjusted to a vertical
orientation.
Monochromatic stimuli were provided by a 500 W Xenon arc lamp through a series of narrow-band interference filters. The light was focused on the tip of an optical fiber that was attached to the perimeter device, where it provided a point source of light (1° in diameter). The quantum flux of each monochromatic stimulus was measured by a radiometer (Model-470D, Sanso, Tokyo, Japan), and the maximum quantum flux of each monochromatic stimulus at the corneal surface was adjusted to 5.0x1011 photons cm-2 s-1.
A glass microelectrode filled with a fluorescing dye, Alexafluor 568
(excitation at 576 nm, emission at 599 nm), 1% in 50 mmol l-1
potassium phosphate, pH 7, resistance approximately 100 M, was inserted
into the eye through the hole made in the cornea. After impalement of the
electrode into a photoreceptor, the optical axis of the photoreceptor was
located by moving the tip of the optical fiber to maximize the response.
Light flashes were of 30 ms duration. First, the sensitivity spectrum of the photoreceptor was determined by stimulating the cell with a series of monochromatic flashes. The stimulus intensityresponse function was measured at the peak wavelength of the given receptor over an intensity range of 5 log units. The photoreceptor was subjected to further analyses only when the maximal response amplitude exceeded 30 mV.
After recording, photoreceptors were marked by injecting the Alexafluor 568 through the recording electrode into the photoreceptor by applying a 4 nA hyperpolarizing current for approximately 4 min. The eyes were then fixed in 4% paraformaldehyde in 0.1 mol l-1 sodium cacodylate buffer, pH 7.4, at room temperature for 30 min, and embedded in Spurr's resin. Unstained transverse sections of 10 µm thickness were observed and photographed with regular transmission microscopy and with fluorescence microscopy using green excitation light (dichroic cube U-WIG: excitation band-pass filter 550 nm, emission cut-off filter 580 nm), to determine the anatomical identity of the recorded photoreceptor and the type of ommatidium to which it belongs.
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Results |
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The type of receptors encountered most frequently has a peak sensitivity at 620 nm (Fig. 2). Epi-fluorescence microscopy revealed that the particular receptor which yielded the spectrum of Fig. 2A was a proximal photoreceptor R8 (Fig. 2B). Regular transmission microscopy showed that the photoreceptor was located in an ommatidium whose pigment clusters are arranged trapezoidally, meaning that the ommatidium was of type I. We thus identified six cells peaking at 620 nm, which belonged to the R58 set of photoreceptors in type I ommatidia. Photoreceptors peaking at 620 nm were also found in type III ommatidia. The particular example of Fig. 3 was identified as an R6 in a type III ommatidium. We identified three such receptors (Fig. 3B,C).
A less frequent class of photoreceptors peaks at 640 nm. Fig. 4 shows an example of a recording from such a photoreceptor in a type II ommatidium. We labeled four cells peaking at 640 nm, all of which were proximal photoreceptors of the R58 set in type II ommatidia (Fig. 4B,C).
Fig. 5 shows the mean
sensitivity spectra of the two classes, peaking at 620 nm and 640 nm,
respectively. Both sensitivity profiles are rather narrow, with half-bandwidth
of approximately 40 nm. This value is much lower than that of a normal
rhodopsin absorption spectrum (Stavenga et
al., 1993). Clear differences can be seen in a few points. For
example, in the 640 nm peaking receptors, the sensitivity at 620 nm is less
than 50%, and a sensitivity depression exists in the short wavelength region,
always with the lowest sensitivity at 420 nm.
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Discussion |
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The monochromatic stimuli used in the present study had 20 nm periodic
intervals. Therefore, one might argue that the difference between the two
spectra could be attributed to some experimental error. It is true that the
precise profiles of the sensitivity spectra may be slightly distorted by the
low sampling rate, and therefore the peak values, 620 and 640 nm, are only
approximate. Nevertheless, clear differences exist between them, which
strongly indicate that the physiological properties of LW620 and LW640
receptors are in fact different. Most importantly, the two receptor classes
exist in different types of ommatidia. As described by Qiu et al.
(2002), the pigmentation
around the rhabdom of types I and III ommatidia has the same, pale-red color.
The pigmentation of type II ommatidia is deep-red. Pigments accumulated near
the rhabdom act as spectral filters, which thus can considerably alter the
sensitivity spectrum of the photoreceptors
(Arikawa et al., 1999b
;
Stavenga, 1989
,
2002a
,b
).
Deep-red pigment will shift the sensitivity spectrum of the proximal
photoreceptors to longer wavelengths more strongly than pale-red pigment
(Qiu et al., 2002
;
Stavenga 2002a
), in agreement
with the finding that the ommatidia with pale-red pigment and deep-red pigment
have LW620 and LW640 receptors, respectively.
This view is further supported by the difference in the properties of the
tapetum. The tapetum is a tracheole folded into a stack of layers, alternately
consisting of air and cytoplasm, thus creating an interference reflection
filter. Incident light that has propagated along the rhabdom is reflected at
the tapetum, travels back through the rhabdom and leaves the eye again,
visible as the so-called eyeshine. The eyeshine reflectance spectra of all
ommatidia have a more-or-less gaussian shape, with a half-bandwidth of
approximately 50 nm, but the spectra nevertheless distinctly differ between
the types of ommatidia: the reflectance spectra of types I and III ommatidia
are approximately the same and peak at 635 nm, whereas the spectrum of type II
peaks at 675 nm (Table 1). The
eyeshine is due to light reflected by the tapetum and filtered by the clusters
of screening pigment located adjacent to the rhabdom
(Qiu et al., 2002;
Stavenga, 2002a
). The slope of
the screening pigment's absorption spectrum at the long-wavelength side
determines the left-hand (short-wavelength) slope of the eyeshine reflectance
spectrum, and the long-wavelength tail of the tapetal reflectance spectrum
determines the right-hand (long-wavelength) slope of the eyeshine reflectance
spectrum. The types I and III ommatidia thus have a pale-red screening pigment
coupled with a tapetum reflecting up to approximately 670 nm, and the type II
ommatidia have a deep-red screening pigment coupled with a tapetum reflecting
up to approximately 710 nm (Qiu et al.,
2002
). The wider spectral range of the latter tapetum will be
beneficial for an extended red sensitivity.
A point of specific interest is the depressed sensitivity of the LW640
receptors in the short wavelength region. LW640 receptors exist in type II
ommatidia, which fluoresce under epi-illumination of 420 nm light
(Qiu et al., 2002). In the
distal tier of type II ommatidia, we encountered dB receptors and dG
receptors, both having a depressed sensitivity at 420 nm
(Qiu and Arikawa, 2003
). The
depressed sensitivity is presumably due to the filtering effect of the
fluorescing material, absorbing strongly at 420 nm, functioning as a
blueviolet-absorbing filter, similar to the 3-hydroxyretinol in
Papilio type II ommatidia, which functions as a UV-absorbing filter
(Arikawa et al., 1999a
). If
this is the case, the blueviolet filter must also act on the proximal
photoreceptors. Interestingly, the LW640 receptor has a depressed sensitivity
in the short wavelength region, with the minimum sensitivity always at 420
nm.
In conclusion, we propose here that there exist at least two subclasses of
red sensitive photoreceptor cells in the eye of Pieris rapae
crucivora. To the best of our knowledge, this is the first example of
polymorphism of red receptors in insect compound eyes. In crustaceans, the
amazingly complex eye of the mantis shrimp contains at least two types of long
wavelength receptors in the wavelength region between 600 and 700 nm
(Marshall et al., 1991). The
possible benefit of having polymorphic red receptors is to increase the color
discrimination ability in this region of the spectrum. This needs to be tested
by appropriate behavioral experiments, however.
In the present study we focused on the sensitivity spectra of four proximal
photoreceptors R58, and did not try to record from the small basal
photoreceptor R9. Shimohigashi and Tominaga
(1991) successfully identified
one R9 as a red receptor peaking at 620 nm, which is similar to the LW620
receptor described in this study. At that time, however, ommatidial
heterogeneity of the Pieris retina had not yet been discovered, so
the ommatidial type of the recorded R9 was not characterized. The R9
photoreceptors occupy the most proximal location of the ommatidium
(Arikawa et al., 1999b
) and
therefore should be even more severely affected by the spectral filtering. It
is therefore likely that the red-sensitive R9 recorded by Shimohigashi and
Tominaga (1991
) was a member
of either a type I or type III ommatidium. If the R9 receptors of type II are
also red sensitive, their sensitivity spectrum would probably be similar to
that of the LW640.
As stated above, the precise profiles of the sensitivity spectra of LW620
and LW640 receptors could be slightly distorted due to our low wavelength
sampling rate. Electrophysiological measurements using a higher sampling rate
will be necessary for a more detailed characterization of the sensitivity
spectra. Interestingly, our preliminary studies on the molecular biology of
visual pigment opsins expressed in the proximal receptors indicated that all
proximal receptors contain identical mRNA, encoding a long
wavelength-absorbing visual pigment, so the LW620 and LW640 receptors may be
identical at the molecular biological level. However, we have also found in
the Papilio retina that some photoreceptors coexpress multiple types
of visual pigments (Kitamoto et al.,
1998,
2000
), resulting in distinct
sensitivity spectra (Arikawa et al., in press). Therefore, more complete
knowledge of the visual pigment opsins expressed in the proximal
photoreceptors in the Pieris eye is needed to substantiate our view
that Pieris has two different classes of red photoreceptors. Such
molecular biological information would also provide insight into how the
polymorphism of red receptors is established.
The existence of three different types of ommatidia in a single compound
eye has also been demonstrated in Papilio xuthus
(Kitamoto et al., 2000),
Vanessa cardui (Briscoe et al.,
2003
) and Manduca sexta (R. H. White, H. Xu, T. A. Munch,
R. R. Bennett and E. A. Grable, manuscript submitted), and seems to be a basic
design for the eyes of lepidopteran insects. One type of ommatidia commonly
contains two different short-wavelength receptors, namely UV and blue
receptors. This is probably important for discriminating colors of very small
targets in the short wavelength region of the spectrum. The difference is
obvious in the proximal tier. The sensitivity spectra of the proximal
photoreceptors are either the green, red or broad-band types in Papilio
xuthus (Arikawa et al.,
1999b
, in press), whereas there are two types of red-sensitive
receptors in Pieris. In M. sexta and V. cardui,
these photoreceptors are probably all green-sensitive
(Briscoe et al., 2003
; R. H.
White, H. Xu, T. A. Munch, R. R. Bennett and E. A. Grable, manuscript
submitted). The difference may reflect the difference in the color vision
properties, which should be demonstrated by careful behavioral analyses.
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Acknowledgments |
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References |
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