Eyeshine and spectral tuning of long wavelength-sensitive rhodopsins: no evidence for red-sensitive photoreceptors among five Nymphalini butterfly species
1 Comparative and Evolutionary Physiology Group, Department of Ecology and
Evolutionary Biology, University of California, Irvine, CA 92697,
USA
2 Department of Electrical Engineering, University of Washington, Seattle,
WA 98195-2500, USA
* Author for correspondence (e-mail: abriscoe{at}uci.edu)
Accepted 15 December 2004
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Summary |
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Key words: color vision, rhodopsin, photoreceptor, spectral tuning, Lepidoptera, Inachis io, Junonia coenia, Nymphalis antiopa, Siproeta stelenes, Vanessa cardui
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Introduction |
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There is great diversity among butterfly species, in the wavelength for
peak sensitivity (max) of rhodopsins found in
photoreceptors of the compound eyes. Typically, rhodopsins are categorized
according to the range of wavelengths within which their
max values fall: short wavelength (SW, 300-400 nm), middle
wavelength (MW, 400-500 nm) and long wavelength (LW, 500-600 nm). At the
molecular level, it is clear that these arbitrary physiological
classifications roughly correspond to three major clades of rhodopsin
apoproteins (opsins): UV, blue and long wavelength. Different butterfly
species express different numbers of opsins, depending upon the family. For
instance, papilionid butterflies have six opsins, one UV, one blue and four LW
rhodopsins (Briscoe, 1998
;
Briscoe, 2000
;
Kitamoto et al., 1998
), five
of which are expressed in the retina
(Kitamoto et al., 2000
). The
nymphalid butterfly, Vanessa cardui, by contrast, has only three
opsins expressed in the retina, one UV, one blue and one LW
(Briscoe et al., 2003
), two of
which (UV and LW) are also expressed in the remnants of the larval stemmata in
the adult optic lobe (Briscoe and White,
2005
). The butterfly Pieris rapae has apparently only one
major LW opsin transcript expressed in the R3-8 photoreceptor cells
(Wakakuwa et al., 2004
).
Phylogenetic analysis of the opsin gene family indicates that two duplications
of an ancestral LW opsin gene occurred along the papilionid lineage since
papilionids and nymphalids shared a common ancestor
(Briscoe, 2001
;
Wakakuwa et al., 2004
). This
is evident in the large differences in opsin expression pattern between
papilionid and nymphalid adult eyes. In V. cardui (and apparently the
pattern is the same in the moth Manduca sexta;
White et al., 2003
), the UV
and blue opsins are expressed in a non-overlapping fashion in the R1 and R2
cells, and the LW opsin is expressed in the R3-9 cells in all ommatidia of the
main retina.
The fact that the R3-9 cells of Vanessa cardui (Nymphalini) retina
express a single LW opsin and that nymphalid butterfly eyes have a mirrored
tapetum makes quantitative study of the relationship between Nymphalini LW
opsin genotype and phenotype especially attractive. The tapetum is a
specialized manifold of the tracheal system that subtends the photoreceptor
cells of each ommatidium. It is composed of alternating layers of air and
cytoplasm, spaced in a regular way, that function optically as an interference
filter, reflecting broad-band light (300-700 nm) as in V. cardui
(Briscoe et al., 2003), or
reflecting relatively narrow-band light (320-590 nm) as in Junonia
coenia (Bernard, unpublished). The ommatidial tapetum is the reason why
most butterfly eyes (excluding papilionids) exhibit colorful eyeshine
(Bernard and Miller, 1970
;
Stavenga,
2002a
,b
,
2001
), and the reason why
epi-microspectrophotometric measurements of rhodopsin absorption spectra can
be made (see below).
In addition to the membrane-bound rhodopsins, photoreceptor cells of
butterflies may contain intracellular red, orange or yellow photostable
pigment granules that function optically as lateral filters, bleeding
short-wavelength light from the rhabdom and thereby red-shifting both the
spectral sensitivity of photoreceptors and the reflectance spectrum of
eyeshine. Red intracellular pigment granules, which are responsible for
saturated red eyeshine, were first described in photoreceptor cells of
Pieridae (Ribi, 1979), as
intracellular granules packed densely around rhabdomeres of proximal retinular
cells. The substantial red-shifts caused by similar red and yellow photostable
filtering pigments on spectral sensitivity of Papilio xuthus
photoreceptor cells were measured electrophysiologically
(Arikawa et al., 1999
). Effects
of deep-red and pale-red filtering pigments on Pieris photoreceptor
cells (Qiu et al., 2002
) were
described more recently.
In the present study, we examined the LW opsin genes from a group of
closely related butterflies in the Nymphalini subfamily in order to further
our understanding of the molecular basis of spectral tuning. The long
wavelength class of rhodopsin is particularly notable in butterflies because
it is so very diverse (compared to the heavily studied hymenopterans; reviewed
by Briscoe and Chittka, 2001).
Because of the recent work on the spatial expression patterns of opsins
described above, the LW opsin genotype can be correlated unambiguously to its
max phenotype. Quantitative epi-microspectrophotometry of
butterfly eyeshine can determine the number of LW rhodopsins and their
absorbance spectra, and can detect the presence or document the absence of
colored photostable filtering pigments.
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Materials and methods |
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Procedures for using eyeshine to make photochemical measurements from
butterfly eyes have been described previously (Bernard,
1983a,b
;
Briscoe et al., 2003
).
Briefly, a completely intact butterfly was mounted in a slotted plastic tube
fixed to the goniometric stage, oriented to select an eye region of
approximately 20 ommatidia for study, then the microscope objective was
focused on the deep pseudopupil for optimal collection of eyeshine and
reduction of stray light. After at least 1 h in the dark, the reflectance
spectrum of eyeshine was measured with a series of dim monochromatic flashes
(see Fig. 3). This was
used as a reference spectrum against which difference spectra were computed
following treatment with photo-isomerizing flashes.
|
Our experimental goal was to measure the difference spectrum for a partial
bleach of the rhodopsin that is most sensitive at long wavelengths, then
characterize it by max, the wavelength of maximal
absorbance. This was achieved using the epi-microspectrophotometric apparatus
(Fig. 1) with the adapting beam
equipped with one of a series of 3 mm Schott (Duryea, PA, USA) cutoff filters
in position Fc, and illuminating at full intensity for 5 s, and
measuring the reflectance spectrum several minutes after the flash. This was
started with filter RG665 but if the flash caused no measurable photochemical
change, the filter was changed to RG645, then RG630, RG610, RG590, RG570
successively, until the procedure of flashing and spectral measuring created a
difference spectrum that revealed modest photo-conversion of rhodopsin to its
blue-shifted photoproduct (
max approximately 490 nm). Then
that filter was used to deliver multiple flashes separated by dark periods as
described by Bernard (1983b
),
to partially bleach the eye of its LW-sensitive rhodopsin. Bleaching of the
rhabdom is possible because metarhodopsin photoproducts decay from the rhabdom
more rapidly than rhodopsin content regenerates. The photoproduct-free
difference spectrum for this partial bleach yields directly a two-way
absorbance spectrum. Least-squares fitting to template absorbance spectra
(Stavenga et al., 1993
) yields
an estimate of
max.
Photographs of butterfly eyeshine
The apparatus depicted in Fig.
1 was modified for photomicrography by exchanging the MPV
photometer with a Leitz 543-040 microphotographic attachment, Leica MDa camera
body, and GF16X eyepiece. The film used was Kodak ASA160 Daylight-Ektachrome.
The illuminator slide was replaced with a Leitz Mecablitz-III microflash. The
microscope objective was 8x/0.18P, the back focal plane of which was
filled by the Leitz Opak epi-illuminator. An intact butterfly was mounted in a
slotted plastic tube fixed to the goniometric stage, then oriented to set the
eye's direction of view. The microscope was adjusted to center the eyeshine
spot in the field of view, focused on the cornea. After at least several
minutes of dark-adaptation, the shutter of the camera was opened long enough
for the eye to be flashed at full intensity by the Mecablitz strobe light.
Repeated photos from the same spot required several minutes of dark-adaptation
between flashes to ensure full recovery from pupillary responses prior to each
photo.
PCR, cloning and sequencing
Genomic DNA templates of Inachis io and Siproeta stelenes
were gifts from Dr Andrew Brower. The locality where the S. stelenes
specimen was collected is given in Brower and DeSalle
(1998). Wild-collected eggs of
Nymphalis antiopa were kindly provided by Dr Peter Bryant and the
hatched larvae were fed on willow leaves (Salix exigua) collected in
the San Joaquin Freshwater Marsh Reserve maintained by the University of
California Natural Reserve System. Two sets of primers were used in PCRs to
obtain fragments of the long wavelength opsin, 80 (5'-GAA CAR GCW AAR
AAR ATG A-3') and OPSRD (5'-CCR TAN ACR ATN GGR TTR TA-3'),
which amplifies a short fragment of the gene. Once the gene-specific fragment
had been cloned, species-specific reverse primers were designed to amplify a
longer fragment (Inachis RD 5'-CAG ATA GTG GCA AGA GGA GTG
AT-3; Siproeta RD 5'-TCG AAG ATA CCG GAA TAG TTG AT-3'),
in combination with the forward primer LWFD (5'-CAY YTN ATH GAY CCN CAY
TGG-3'). For Nymphalis antiopa, total RNA was extracted from
adult head tissue using TRIZOL and a cDNA library was synthesized using the
Marathon cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, CA, USA).
3'RACE products were obtained by pairing primer 80 with an adaptor
primer and 5'RACE products were obtained by use of a gene-specific
reverse primer (Nymphalis RD 5'-GCA GTT TCG AAG ATA CCA GCA
TAG-3'). In all cases, the following PCR conditions were used: 94°C
for 1 min then 35 cycles of 94°C for 30 s, 50°C for 1 min, 68°C
for 1 min. In all PCR reactions, overlapping pieces of the gene were amplified
and cloned independently. PCR products were purified with the GeneClean Kit,
and then cloned using the pGEM T-easy Vector System II (Promega, Madison, WI,
USA). For each cloned PCR product, two to six clones were purified (Eppendorf)
and sequenced using the ABI Big-Dye Terminator Reaction Kit v. 3.1 in forward
and reverse directions and run on an ABI Automated Sequencer (Foster City, CA,
USA).
Phylogenetic analysis
Translated amino acid sequences were aligned by eye in MacClade 4.0
(Maddison and Maddison, 2000).
We tested first plus second nucleotide positions, third nucleotide positions,
and the translated protein sequences for composition homogeneity among
lineages using the disparity index test
(Kumar and Gadagkar, 2001
). We
found that third nucleotide positions appear to have evolved in a
significantly non-homogeneous fashion in all nymphalid opsins and all other
papilionid and moth sequences while in nymphalids, third nucleotide positions
have evolved homogeneously (data not shown). Therefore, because the protein
sequences and first plus second positions were homogeneous, first plus second
nucleotide positions or amino acids were used in the phylogenetic analyses.
Both maximum parsimony (MP) and neighbor-joining (NJ) analyses were conducted
(PAUP*, MEGA 3.0) (Kumar et
al., 2004
). A total of 145 parsimony-informative amino acid sites
were included, and a stepmatrix of amino acid changes derived from a larger
data set of G protein-coupled receptors
(Rice, 1994
; see supplementary
material in Spaethe and Briscoe,
2004
) was employed as a weighting scheme to account for unequal
probabilities of amino acid change in the MP analysis. The reliability of the
MP trees was tested by bootstrap analysis in PAUP*
(Swofford, 1998
). For the NJ
analysis, a total of 532 aligned nucleotide sites (first plus second
positions) were used with Tamura-Nei correction (which takes into account both
transition/transversion and GC content biases;
Nei and Kumar, 2000
) and
complete deletion of gaps.
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Results |
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Log-reflectance spectra
Eyeshine log-reflectance spectra were measured from a medio-ventral region
of the dark-adapted eyes of nymphalid butterflies Inachis io, Junonia
coenia, Nymphalis antiopa, and Siproeta stelenes as well as the
pierid butterfly Pieris rapae. These data are presented in
Fig. 3 in addition to
previously published (Bernard,
1983b; Briscoe et al.,
2003
) spectra for Vanessa cardui.
Absorbance spectra
The max value of the LW rhodopsin expressed in the
retina was estimated to be 522 nm in Siproeta stelenes, 530 nm in
Inachis io, 534 nm in Nymphalis antiopa, and 510 nm in
Junonia coenia. One-way absorbance spectra of these rhodopsins,
determined from partial bleaches, are shown in
Fig. 4.
|
All five species were subjected to the photochemical series described in
Materials and methods. In all cases the first measurable photochemical
difference spectra were well fitted by photo-conversion of the visual pigment
reported in the preceding paragraph. There is no evidence at all for visual
pigments of greater max in eyes of these five species.
Long wavelength opsin sequences
The primer combination 80-OPSRD yielded one band for both Inachis
io and Siproeta stelenes that was 316 and 320 bp in length,
respectively. The gene-specific reverse primers designed from these sequences
in combination with LWFD primer also yielded a single PCR band for each
template. In total, we obtained a 1512 bp genomic fragment of the long
wavelength-sensitive opsin gene from Siproeta stelenes (GenBank
accession no. AY740908) and a 1437 bp genomic fragment from Inachis
io (AY740906). Each of the genomic sequences contained six exons and
introns (ranging from 69-158 bp in length), and encompassed seven
transmembrane domains. From Nymphalis antiopa cDNA we also sequenced
overlapping 3' and 5'RACE products, which when combined formed a
1612 bp full-length opsin cDNA (AY740907). This sequence included both a start
and a stop codon and encodes a protein that contains 378 amino acids. The
translated protein sequences are shown in
Fig. 5 aligned with those of
other nymphalid species.
|
Opsin phylogeny and evolution of eyeshine in nymphalids
Phylogenetic analysis of the opsin-coding region using the Neighbor-Joining
(NJ) algorithm yielded a tree with good (50-100%) bootstrap support in all but
one node (Fig. 6). The
branching pattern of the NJ tree was identical in all respects to the 50%
majority-rule consensus MP tree except for the placement of the two
Pieris opsins; in the MP tree they form a sister clade to the
Papilio Rh1-Rh3 clade. For instance, all included nymphalid opsin
sequences were recovered with 92% bootstrap support, providing further
evidence for the previous observation of Briscoe
(2001), that two novel gene
duplication events have occurred within papilionids since papilionid and
nymphalid butterflies shared a common ancestor. The opsin sequences obtained
from Inachis io and Siproeta stelenes genomic DNA cluster
with the opsin sequences obtained from retina-derived cDNAs of other
Nymphalini tribe members, i.e. Vanessa and Junonia
(bootstrap support 98%), indicating the homology of the genomic sequences with
the majority LW opsin expressed in the retina of related butterflies. We also
repeated the phylogenetic analysis including the Bicyclus anynana LW
opsin fragment (Vanhoutte et al.,
2002
) that had been cloned from RNA extracted from eye tissue and
found similar results with this shorter alignment.
|
Mapping of eyeshine characteristics onto the opsin phylogeny indicates that
the orange eyeshine observed in both Vanessa cardui and Inachis
io (Stavenga, 2002a) and
`blue-orange' eyeshine observed in Siproeta stelenes and
Nymphalis antiopa, is probably a result of their common ancestry
(Fig. 6), while the blue
eyeshine observed in Junonia coenia is a derived trait among the
sampled Nymphalini.
Butterfly spectral tuning sites are shared with vertebrate cone opsins
We were interested in identifying candidate spectral tuning sites
responsible for the observed variation in max values among
the Nymphalini rhodopsins. These species have been chosen in part because
their short evolutionary history would narrow the number of variable sites. We
also limited our comparisons to those rhodopsins for which
max values had been measured on our experimental apparatus,
in order to reduce one source of experimental uncertainty - a difference in
experimental paradigm.
The maximum parsimony-reconstructed amino acid substitutions that occurred
along the blue-shifted Junonia-Siproeta branch are shown in
Table 1. Amino acid residues
that on the basis of homology modeling
(Briscoe, 2002) are found
within the binding pocket facing the chromophore are also indicated. Notably,
we found two amino acid sites that have known spectral tuning effects in the
vertebrate rhodopsins. The first occurs in transmembrane domain I, is part of
the region immediately surrounding the Schiff base, and corresponds to amino
acid 44 in bovine rhodopsin (Palczewski et
al., 2000
). Mutagenesis of this site causes a 3 nm blue-shift in
rhodopsin absorption spectrum (Andres et
al., 2003
). The second site is found in transmembrane domain IV
and corresponds to amino acid 180 in human red cone opsin (or 164 in bovine
rhodopsin). Mutagenesis of the serine residue at this site to an alanine
causes a 5 nm blue-shift (Asenjo et al.,
1994
). Both blue-shifted rhodopsins in our data set, those of
Junonia coenia (R510) and Siproeta stelenes (R522) have
alanine at this site. Therefore, we suggest that the 8 nm difference between
R522 of Siproeta stelenes and the ancestral R530 rhodopsin is largely
accounted for by the substitution at this site and that of the former site.
Six other amino acid sites have also changed along the blue-shifted opsin
branch (Table 1). While most of
these are likely to be neutral mutations with respect to spectral tuning, it
is interesting to note that specific mutations at two of these sites, (amino
acids 45 and 46 in bovine rhodopsin) along with the two sites mentioned above
(amino acids 44 and 164), are responsible for the human eye disease, autosomal
dominant retinitis pigmentosa (Andres et
al., 2003
; Briscoe et al.,
2004
; Rodriguez et al.,
1993
; Sung et al.,
1991
).
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Discussion |
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The eyeshine in Vanessa cardui arises from a tapetum that has a
broad, flat reflectance across the visible spectrum, decreasing above 680 nm,
and ommatidia that express ultraviolet (UV), blue (B) and green (G) absorbing
rhodopsins. This combination of tapetal reflection and rhodopsin absorption
creates orange eyeshine (Bernard,
1983b; Briscoe et al.,
2003
), the dark-adapted log-reflectance spectrum of which is shown
in Fig. 3A (filled green
circles). The filled red circles in the same figure show a log-reflectance
spectrum of Pieris rapae (ventral) cherry-red eyeshine. Note that the
reflectance of Pieris is very low for wavelengths less than 600 nm.
Its spectrum does not exhibit a large secondary maximum in the blue, as does
Vanessa. The optical basis for these striking spectral differences is
that Pieris retinular cells contain short-wavelength absorbing,
lateral-filtering granules and Vanessa retinular cells do not.
The spectrum shown by the open green circles in
Fig. 3A is an example of a
photochemical change in the Vanessa reflectance spectrum caused by
photo-conversion of R530 to its photoproduct M490 (data from fig. 1c/A of
Bernard, 1983b). A similar
photo-conversion of the LW rhodopsin R563 of Pieris
(Wakakuwa et al., 2004
)
exhibits quite a different reflectance spectrum, shown by the open red circles
in Fig. 3A. Here, the expected
decrease in reflectance in the neighborhood of 500 nm, associated with
accumulation of metarhodopsin, is not observable because the lateral-filtering
granules of Pieris are so effective in absorbing short-wavelength
light that enters the rhabdom. None is reflected.
Log-reflectance spectra for all five Nymphalini species we examined showed prominent secondary maxima in the blue. Although not shown, substantial measurable decreases in reflectance, associated with accumulation of metarhodopsin following photo-conversion of LW rhodopsin, was measured in all five species. We conclude that these five species have no lateral-filtering granules in their photoreceptor cells.
Most of the Nymphalini species we examined visually had eyeshine that was similar to that of Vanessa (Fig. 2). Tapetal reflectance for Nymphalis, Inachis and Vanessa is high in the red, out to at least 680 nm (Fig. 3B). An exception is Junonia, which has blue eyeshine owing to a modification of the tapetal structure in which wavelengths of light above 600 nm arepoorly reflected. Siproeta is an intermediate case in which tapetal reflectance is low for wavelengths greater than 620 nm.
Orange eyeshine has been reported in Inachis io and Polygonia
c-album (Stavenga,
2002a), which are both members of the Nymphalini subfamily. Our
phylogenetic analysis of the LW opsin gene as well as phylogenetic studies of
the EF1alpha and wingless genes
(Wahlberg et al., 2003
)
indicates that Nymphalis antiopa is most closely related to
Vanessa cardui. We also find that V. cardui shares orange
eyeshine with Nymphalis antiopa and Inachis io. This
observation, in combination with previous studies
(Stavenga, 2002b
) that found
that most Nymphalini butterflies have yellow-orange eyeshine, tentatively
suggests that the orange eyeshine may be the ancestral Nymphalini eyeshine
state, whereas the distinctive blue eyeshine we find in Junonia
coenia is a trait of recent origin that likely represents a change in
tapetal development. We also note that we did not observe any dramatic
dorsal-ventral differences in the eyeshine of any of the included species, as
has been reported for other nymphalids, e.g. Bicyclus anynana, Pararge
aegeria, Hypolimnas anthedon
(Stavenga, 2002b
;
Stavenga, 2002a
). With an
expanded collection of butterfly eyeshine data mapped onto a robust phylogeny
of the nymphalids (e.g. Wahlberg et al.,
2003
), it would be interesting to estimate how frequently
dorsal-ventral eyeshine patterning differences have evolved within butterflies
in order to begin to identify ecological factors correlated with their
evolution.
From the notable lack of saturated red ommatidial eyeshine, and measured
reflectance spectra that have high reflectivity in the blue, we conclude that
there is no evidence for the presence of photostable filtering pigments within
the surveyed Nymphalini. From our photochemical experiments with multiple
cutoff filters we conclude that there is only a single long
wavelength-sensitive rhodopsin present. Indeed an extensive PCR-based screen
of the eye-specific cDNA of Nymphalis antiopa indicates the presence
of only three opsins, UV, blue and LW (M. P. Sison-Mangus and A.D.B.,
unpublished data). This result suggests that the color vision system of
Junonia, Nymphalis, Siproeta, Polygonia, Inachis and Vanessa
(the Nymphalini `eye ground plan') is probably trichromatic in its most
fundamental aspects, like that of the hawkmoth, Manduca sexta
(White et al., 2003), a
prediction that can be tested behaviorally using the paradigms of Kelber et
al. (2003
) and Kinoshita et
al. (1999
). [The dorsal
retina, which in V. cardui, expresses almost entirely UV and
green-sensitive opsins, may be employed for brightness or contrast
discrimination, while the ventral retina, which expresses all three opsins
(Briscoe et al., 2003
), is
probably functionally trichromatic.]
Spectral tuning of the long wavelength sensitive rhodopsins of Nymphalini
Since we found no evidence of either a second LW rhodopsin in any of the
investigated species, or of red lateral-filtering pigments that would produce
two or more receptor types from one rhodopsin, our observations have permitted
the exploration of the relationship between the peak absorbance of the
rhodopsin and its genotype.
Intriguingly, we found evidence that the amino acid substitution homologous to S180A in visual pigments of human cones causes a similar blue-shift of absorption spectrum in the visual pigments of butterflies. This suggests that some of the same polymorphic sites that are involved in generating red-green color vision in New World primates may be segregating in butterflies.
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Acknowledgments |
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