Molecular characterization and expression of the UV opsin in bumblebees: three ommatidial subtypes in the retina and a new photoreceptor organ in the lamina
Comparative and Evolutionary Physiology Group, Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA
Author for correspondence (e-mail:
abriscoe{at}uci.edu)
Accepted 6 April 2005
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Summary |
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Key words: circadian rhythm, visual pigment, photoreceptor, color vision, ultraviolet
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Introduction |
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One of the most intensively studied groups of arthropods with respect to UV
vision are the bees, since they were first found to be sensitive to UV light
more than 80 years ago(Kühn,
1924). In a bee's life, UV perception plays an essential role in
flower detection and discrimination (Dyer
and Chittka, 2004
; Spaethe et
al., 2001
), polarization vision
(Brines and Gould, 1979
;
Von Frisch, 1949
) and
orientation (Edrich et al.,
1979
; Moller, 2002). It has also been shown that UV information is
utilized in both real color vision and wavelength-specific behavior.
UV perception in general is mediated by visual pigments, which are composed
of a chromophore and an opsin protein. Opsins belong to the large group of
G-protein-coupled receptors and are subdivided into distinct classes according
to the part of the light to which they tune the absorbance properties of the
chromophore. Physiological and behavioral studies have shown that all bees
investigated so far have one receptor that is most sensitive in UV light
(Briscoe and Chittka, 2001;
Peitsch et al., 1992
).
However, despite the huge amount of data showing the significance of UV
perception in Apidae, molecular data for the UV opsin are rare. Among the
estimated 20 000 bee species (Michener,
2000
), the UV opsin from only a single species, the honeybee
Apis mellifera, is molecularly characterized
(Townson et al., 1998
), and
opsin spatial expression data are completely missing. The goal of the present
study, therefore, was to characterize molecularly the UV opsin-encoding gene
and determine its protein expression pattern in order to obtain a better
understanding of possible functions of the UV visual pigment in bees. We used
the bumblebee Bombus impatiens as our study system. This species is
one of the most common North American bumblebee species and is intensively
used in agriculture for pollination of cucumbers, peppers, tomatoes,
strawberries, melons and squash (Meisels
and Chiasson, 1997
; Stubbs and
Drummond, 2001
; van Ravestijn
and van der Sande, 1991
). The spectral sensitivity of the B.
impatiens retina for short-wavelength light has been characterized and
shows a single peak most sensitive to 350 nm
(Bernard and Stavenga,
1978
).
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Materials and methods |
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Phylogenetic analysis
Compared to the insect long-wavelength (LW)-sensitive opsin gene family,
for which more than 240 complete or partial sequence data are published
(Ascher et al., 2001;
Briscoe, 2001
;
Kawakita et al., 2003
;
Ortiz-Rivas et al., 2004
;
Spaethe and Briscoe, 2004
),
only a few UV-sensitive opsin gene sequences are available in GenBank
(Briscoe, 2000
;
Chase et al., 1997
;
Gao et al., 2000
;
Kitamoto et al., 2000
;
Smith et al., 1997
;
Vanhoutte et al., 2002
). We
downloaded 13 full-length UV opsin cDNA sequences from four insect orders and
the blue-sensitive opsin sequence from Apis mellifera as an outgroup
into the Alignment Explorer in MEGA 3.0
(Kumar et al., 2004
). We
aligned the translated amino acid sequences using the ClustalW algorithm
(Thompson et al., 1994
).
Heterogeneous patterns of nucleotide or amino acid substitution between
sequences can produce erroneous branching patterns (see literature cited in
Kumar and Gadagkar, 2001). We
therefore tested 1st + 2nd nucleotide positions, 3rd nucleotide positions and
amino acid sequences for composition homogeneity among lineages using the
disparity index test (Kumar and Gadagkar,
2001
) as implemented in MEGA 3.0
(Kumar et al., 2004
). Our goal
was to only use molecular characters in our phylogenetic reconstruction that
appear to be evolving with a similar pattern of substitution and/or to remove
any sequences that violated the homogeneity assumption. Third nucleotide
positions appeared to have evolved in a significantly non-homogeneous fashion
(in 71% of all comparisons after Bonferroni correction). First + 2nd
nucleotide positions and the protein sequences were found to be homogeneous
except for 1st + 2nd nucleotide positions in Camponotus abdominalis
and for amino acids in Drosophila Rh4, which were significantly
different from one or more of the other species sequences
(P<0.002; all P-values were adjusted for multiple
comparisons using Bonferroni correction; data not shown). We therefore decided
to use 1st + 2nd nucleotide positions and amino acid sequences in the
phylogenetic analysis. In addition, we looked for possible effects of the
non-homogeneously evolving sequences on the tree topology by running
phylogenetic analyses both with and without them. We applied the
neighbor-joining (NJ) algorithm under the Tajima-Nei model for 1st + 2nd
nucleotide positions with complete deletion of gaps, and Poisson correction
for the amino acid sequences to construct phylogenetic trees, including a
total of 1176 aligned nucleotide and 392 aligned amino acid sites. Robustness
of the NJ trees was tested using the bootstrap method with 500 replicates. All
phylogenetic analyses were conducted using MEGA 3.0
(Kumar et al., 2004
).
Tissue preparation
Bumblebee workers were immobilized with CO2, decapitated and the
heads cut sagittally in two halves and fixed in phosphate-buffered
paraformaldehyde (4%) for 13 h atroom temperature or at 4°C
overnight. In some bees, for a better overview of the entire brain, we cut a
small window in the head capsule and pre-fixed the brain for 30 min. The brain
with the entire retina and cornea was then removed from the head capsule and
fixed for a total of 13 h at room temperature. After fixation, the
tissue was stepped through a 10% and 20% phosphate-buffered sucrose solution
and finally infiltrated for at least 12 h in a 30% sucrose solution. It was
then embedded and frozen in Tissue Tek O.C.T. freezing compound (Sakura
Finetek, Inc., Torrance, CA, USA) and 1214 µm sections were cut on a
cryostat (MICROM HM 500 OM, Walldorf, Germany).
Recent investigations have shown that, in the honeybee, period
mRNA expression level is highest during the first hours of the dark phase when
bees were entrained to a 12 h:12 h L:D cycle
(Bloch et al., 2003;
Toma et al., 2000
). Based on
this finding, we expected the highest level of period protein (PER) during the
first hours of the dark phase. To obtain the strongest immunoreactivity when
applying anti-PER antibody, we kept groups of bumblebees for at least three
days in small boxes (15x15x20 cm) entrained to a 12 h:12 h L:D
cycle before dissecting the retina and brain two hours after the commencement
of the dark phase under red light conditions as described above.
UV opsin western blot analysis
The immunoaffinity-purified polyclonal rabbit anti-PglRh5 (UV) antibody we
used was made against a 15 amino acid peptide domain within the C-terminus of
the butterfly Papilio glaucus Rh5 opsin
(Briscoe and Nagy, 1999;
Lampel et al., in press
).
Twelve of the 15 amino acid sites were conserved between the butterfly opsin
and the predicted B. impatiens UV opsin sequence
(Fig. 1), so we assumed that
cross-reactivity of the Papilio Rh5 antibody with the B.
impatiens UV opsin was very likely. To test for the likelihood that the
antibody might also cross-react with other peptides, we performed a tBLASTn
search of GenBank, including against the whole translated honeybee genome,
using both the butterfly peptide sequence and the bee sequence, which varies
by three amino acids. We then tested the antibody on a western blot of eye
extract subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). Eight retinas of four worker bees were dissected and homogenized
for 10 min in 25 µl ice-cold homogenization buffer, pH 7.5 (50 mmol
l1 Mops, 1 mmol l1 EDTA, 1 mmol
l1 EGTA, 120 mmol l1 KCl, 5 mmol
l1 MgCl2, 250 mmol l1 sucrose
and 1x Halt protease inhibitor cocktail mix; Pierce, Rockford, IL, USA).
After centrifuging for 5 min at 3000 g and 4°C, the
supernatant was transferred into a new tube and centrifuged for 15 min at 18
000 g and 4°C (Jouan BR4i; ThermoElectron Corporation,
Milford, MA, USA). The supernatant was diluted in 2x NuPAGE LDS sample
buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 1x NuPAGE
Reducing agent and 1x Halt protease inhibitor, to a final concentration
of 0.15 retina eq. µl1. Proteins from 3.75 retina
supernatant equivalents per lane were separated on NuPAGE 412% gradient
Bis-Tris SDS-PAGE minigel and transferred onto a PVDF membrane. The blot was
blocked for 1 h in 2.5% normal horse serum (Vectastain ABC kit; Vector
Laboratories, Burlingame, MA, USA) and then either probed with the primary
antibody (0.00453 nmol ml1) diluted in 1x TTBS (25
mmol l1 Tris, pH 7.5, 150 mmol l1 NaCl,
0.1% Tween 20) overnight at 4°C in the presence or absence of 100x
molar excess of peptide (0.453 nmol ml1) mixed with antibody
(0.00453 nmol ml1) for 1 h at room temperature prior to
being applied to the blot. Then the membrane was incubated for 30 min with a
secondary antibody conjugated with biotinylated horseradish peroxidase
(Vectastain ABC kit) and visualized with DAB (Pierce). As a control, each
membrane was subsequently stained with Coomassie blue to ensure that
comparable amounts of protein were loaded in each lane.
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Sections were first post-fixed in 100% ice-cold acetone, washed three times for 10 min in 1x PBS, once for 5 min in 0.5% SDS in 1x PBS, and then blocked for 30 min with 4% normal goat or normal donkey serum in PBST (1x PBS, 0.3% Triton X-100). Either affinity-purified rabbit anti-UV antibody (1:200 to 1:1000) or anti-PER serum (1:100 to 1:500) was then added to sections and incubated for 23 h at room temperature or overnight at 4°C. After washing for 30 min in 1x PBS, sections were incubated with a secondary antibody [Cy3-conjugated goat anti-rabbit (Jackson Immuno Research Laboratories, Inc., West Corore, PA, USA) or Alexa Fluor 488-conjugated donkey anti-rabbit (Molecular Probes, Eugene, OR, USA); 1:1000 in PBS] for 1 h, washed for 30 min and counterstained for 15 min with 0.1% 4,5-diamidino-2-phenylidone (DAPI; Molecular Probes). Slides were then washed in PBS again and mounted with Aqua Poly Mount (Polysciences, Inc., Warrington, PA, USA). As a control for specificity of the antibody, we also performed a peptide competition experiment in which a 0.023 nmol ml1 dilution of the anti-UV opsin antibody in blocking solution was mixed for 1 h with 2.3 nmol ml1 peptide (Biosource, Hopkinton, MA, USA), and then applied to slides. The slides were then processed as described above in parallel with adjacent sections to which a 0.023 nmol ml1 dilution of the anti-UV opsin antibody had been applied (peptide omitted). Sections were examined and photographed using an Axioskop 2 plus microscope connected to an AxioCam HRc (Zeiss, Goettingen, Germany), and images were processed using Adobe Photoshop (version 7.0).
In situ hybridization
As an additional control for the specificity of our anti-UV opsin antibody,
we also performed in situ hybridizations to examine the distribution
of the UV opsin mRNA transcript in the compound eye and optic lobe. Sense and
antisense RNA probes (riboprobes) of the UV opsin cDNA were synthesized with
digoxigenin-labeled UTPs using a DIG RNA labeling kit (Roche Diagnostics,
Indianapolis, IN, USA). Sections were incubated in hybridization buffer in a
humid chamber for 30 min at 60°C as previously described
(Briscoe et al., 2003). The
labeled probe was diluted in the hybridization buffer, corresponding to
approximately 45 ng of probe per slide. The sections were incubated overnight
at 5560°C in a humid chamber and then washed for 10 min with, in
turn, 2x, 1x and 0.1x standard saline citrate (SSC) and 0.1%
Tween to increase accessibility of the probe. The probes were localized by
incubation with an anti-digoxigenin alkaline phosphatase-conjugated antibody
(1:1000), diluted in 1x PBS plus 0.1% Tween for two hours. After three
10 min washes with 1x PBS, the slides were washed in alkaline
phosphatase developing solution with levamisol for 5 min before detection. The
probes were detected by a colorimetric reaction produced by nitro blue
tetrazolium, 5-bromo-4-chloro-3-indolylphosphate and 10% Tween in alkaline
phosphatase developing solution.
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Results |
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UV opsin gene phylogeny
The phylogenetic analysis using 1st and 2nd nucleotide positions showed
that the B. impatiens sequence is most closely related to the A.
mellifera opsin (100% bootstrap support) and that both sequences form,
together with the other two hymenopteran sequences, a well-supported group
(100% bootstrap support) (Fig.
2). Removing the Camponotus sequence from the analysis
did not affect the tree structure, thus we retained the sequence in our
subsequent analyses. All lepidopteran and dipteran sequences formed two
strongly supported groups (100% and 84%, respectively). Using amino acid
sequences instead of 1st and 2nd nucleotide positions revealed a very similar
tree structure except that Anopheles forms, with the hymenopteran
sequences, a sister group to all Drosophila opsins. However, when we
removed the Drosophila Rh4 amino acid sequences, which have evolved
under a significantly different rate from Rh3
(Carulli and Hartl, 1992), the
identical tree structure to that obtained with 1st and 2nd nucleotide
positions was recovered (data not shown).
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Retina and ocelli
Bee ommatidia are composed of one small proximal and eight elongate distal
photoreceptor cells. UV opsin immunoreactivity (-ir) was found in most but not
all ommatidia across the retina (Fig.
4AC,F). The strongest staining was found in the rhabdoms of
the ommatidia, where most of the opsin protein is localized at high
concentration in the microvillous membranes. In some cases, vesicles in the
cytoplasm of some photoreceptor cells also showed UV opsin-ir
(Fig. 4C). We found three
different ommatidial types with respect to UV opsin-ir: those containing two,
one and no UV opsin-ir photoreceptor cells
(Fig. 4B,C). A diagram of a
single ommatidium in longitudinal view and of the three ommatidial types in
cross section found in the main retina is shown in
Fig. 4E. Because of its small
size, we did not investigate the presence or absence of the UV opsin in the
tiny ninth cell. In the median-central part of the retina of a worker bee
(N=3), we estimate that 25% of ommatidia (63 out of 252) contain two
UV opsin-ir photoreceptor cells, 48% (121 out of 252) contain one UV-ir
photoreceptor cell, and 27% (68 out of 252) contain no UV opsin-ir
photoreceptor cells. UV opsin-ir receptor cells were most densely packed in
the dorsal rim area (Dra) of the eye compared with the rest of the retina
(Fig. 4F). We could not tell
whether there were more than two photoreceptor cells per ommatidium in the Dra
that had UV opsin expression, as has been suggested by some investigations
(Meyer, 1984).
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At the top of the bee head, three additional photoreceptor organs are located: the median and two lateral ocelli. We detected UV opsin expression in some of the retinula cell rhabdoms in all three ocelli of the worker bee (Fig. 5A). Incubation of adjacent sections with the anti-UV opsin antibody and peptide for 1 h abolished the specific staining in the rhabdoms of both the retina (Fig. S2A,B in supplementary material) and ocelli (Fig. 5B).
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Optic lobe
UV opsin-ir was found in the first optical layer, the lamina, as well as in
at least 10 perikarya located ventrally of the lamina
(Fig. 6). Immunoreactivity
within the lamina was restricted to the 3rd layer (C-layer;
Mobbs, 1985), which is formed
by the lamina cartridges (Ribi,
1979
) and is embedded between two layers of cell bodies
(Fig. 6AE).
Immunoreactivity in this layer was found throughout the entire lamina and
formed a disc-like shape with an increasing thickness towards the laminar
margin (Fig. 6C). Sections
incubated with the secondary antibody but without the primary anti-UV antibody
showed no labeling (Fig. 6B), and sections labeled with the primary antibody mixed with the peptide also
showed no labeling (compare Fig. S2C,D in supplementary material). In most
immunolabelings, we also observed a weak UV opsin-ir in the medulla [where the
long fibers of the UV opsin-expressing retinula cells terminate
(Menzel and Blakers, 1976
)],
which was not found in any of the controls (Figs S1, S2C,D in supplementary
material). In situ hybridization using a DIG-labeled UV opsin
antisense riboprobe revealed mRNA expression in the retina
(Fig. 4D), in the cell bodies
that form part of the 3rd layer of the lamina and in the cell bodies along the
distal margin of the medulla (Fig.
6F).
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Spatial characterization of the period protein
The period protein (PER) plays a central role in the maintenance of the
circadian rhythms and in photoperiodic timing of annual rhythms that are
entrained by light (Hall,
1995), and thus we were interested if partial overlap of PER and
visual pigment expression occurs. The anti-PER serum we applied was raised
against the full length of the Drosophila melanogaster PER protein
(Liu et al., 1992
) and was
shown to be cross-reactive in the honeybee
(Bloch et al., 2003
). PER-ir
was obtained in the 1st and 2nd layer of the optic lobe and in the
deuterocerebrum. We found PER-ir in the perikarya layer, which is located
adjacent to the distal rim of the medulla in the outer chiasm
(Fig. 6F, inset). A second
nuclear staining was found in the 3rd layer of the lamina. A similar pattern
of PER expression in the optic lobe was previously reported in the honeybee
(Bloch et al., 2003
). Double
staining of the sections with DAPI confirmed that PER-ir perikarya in the
lamina form the double layer of cell bodies that borders the lamina
cartridges, i.e. the lamina region in which we detected UV opsin-ir
(Fig. 6F, inset) as well as UV
opsin mRNA expression (Fig.
6F). None of the other perikarya within the lamina exhibit PER-ir
(data not shown).
The lateral cell cluster of the ALs, which comprises predominantly
perikarya from local AL and projection neurons
(Rybak and Menzel, 1993;
Witthoeft, 1967
), also
exhibited strong PER-ir (Fig.
7J). In addition, many cell populations across the entire brain
were found to display a weak PER-ir that was not present in the controls (data
not shown). The high frequency of perikarya PER-ir found in our study is in
accordance with PER expression data from the honeybee and other insects
(Bloch et al., 2003
;
Wise et al., 2002
). A
schematic representation of UV opsin and PER protein distribution is shown in
Fig. 8.
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Discussion |
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The molecular basis of invertebrate ultraviolet vision has recently been
identified, through a series of site-directed mutagenesis experiments
(Salcedo et al., 2003), to be
due primarily to the presence of a lysine residue at a site that is homologous
to glycine 90 (G90) in bovine rhodopsin. We therefore inspected the translated
B. impatiens amino acid sequence for the presence of this residue,
which is important for conferring the short-wavelength spectral sensitivity of
the putative rhodopsin. We indeed found a lysine at this site in the B.
impatiens sequence and, on the basis of the presence of this amino acid,
and the robust results of our phylogenetic analysis, we conclude that the
encoded opsin protein is likely to produce the 350 nm UV-sensitive
photopigment previously characterized by Bernard and Stavenga
(1978
).
In addition to these inferences regarding the putative function of this protein from the sequence analysis, three major conclusions can be drawn from our anatomical results. (1) The UV-sensitive photoreceptor cells are heterogeneously distributed among the ommatidia in the retina. We found three different types: ommatidia with two, one and no UV opsin-ir receptor cells. (2) UV opsin-ir cells are not only present in the retina but were also found in the ocelli and the optic lobe, as well as in the protocerebrum and the ALs. (3) Both UV opsin-ir and PER-ir were found in the most proximal (internal plexiform) layer of the lamina and the AL. Collectively, these results (especially the latter) suggest that both the UV opsin and PER might be part of a light-sensitive extra-retinal input channel of the circadian system.
Retina
The honeybee, Apis mellifera, is the only hymenopteran species
where the distribution of different photoreceptor types within an ommatidium
has been investigated. Combined intracellular recordings with morphological
characterizations suggested the presence of only one ommatidial type within
the entire retina of the worker honeybee
(Menzel and Blakers, 1976).
Each ommatidium was thought to contain two UV-, two blue- and four
green-sensitive photoreceptor cells plus a small proximal ninth cell, which
was thought to be UV-sensitive. [Intracellular recordings from photoreceptor
cells in the Bombus hortorum compound eye confirm the presence of
three types of photoreceptor with a peak sensitivity in the UV (353 nm), the
blue (430 nm) and the green (548 nm);
Meyer-Rochow, 1981
.] However,
reliable morphological characterization of the different bee receptor types is
difficult due to the twist of the ommatidium around its long axis and possible
(but, in most cases, neglected) morphological receptor variation in different
parts of the eye (see discussion in Menzel
and Blakers, 1976
). Also, it has not been clear whether the
correlation between spectral sensitivity and morphology found in the honeybee
and two ant species (Menzel,
1972
; Menzel and Blakers,
1975
) is extendible to all hymenopterans.
In contrast to these early studies, our results have revealed three
different ommatidial types in the worker bumblebee retina with respect to both
UV opsin protein and mRNA expression (Fig.
4). Opsin immunocytochemistry and in situ hybridization
studies in dipterans (Chou et al.,
1996; Huber et al.,
1997
; Montell et al.,
1987
; Papatsenko et al.,
1997
; Zuker et al.,
1987
) and lepidopterans
(Briscoe et al., 2003
;
Kitamoto et al., 2000
;
Wakakuwa et al., 2004
;
White et al., 2003
) have
shown that the presence of different ommatidial types within the retina is a
common feature of all investigated insects. For instance, the major ommatidial
types in the butterfly Vanessa cardui are similar to those in the
bumblebee and comprise seven green-sensitive cells and either two UV-, one UV-
and one blue-, or two blue-sensitive cells (in Vanessa, the small
ninth cells express a green-sensitive opsin in contrast to the supposed
UV-sensitive opsin in honeybees) (Briscoe
et al., 2003
). Our data, together with results from other insects,
suggest that the honeybee retina is presumably also comprised of different
ommatidial types, which were not detected with the more classical techniques.
Opsin-expression studies on the honeybee retina are, of course, necessary to
verify this hypothesis.
In many insects, it has been shown that the dorsal-most rows of ommatidia
(Dra) contain an increased number of UV-sensitive receptor cells, which are
also sensitive to polarized light (Sauman
et al., 2005; Labhart and
Meyer, 1999
). In Drosophila, for example, it has been
shown that both the R7 and R8 cells of the Dra express the UV-sensitive RH3
opsin whereas, in the main retina, RH3 is expressed in only a subset of R7
cells and never in R8 (Wernet et al.,
2003
; Zuker et al.,
1987
). In this region in the bumblebee retina, we found the
highest density of UV opsin-ir ommatidia
(Fig. 4E). We were, however,
unable to determine the number of UV opsin-labeled cells per ommatidium in
this area.
Ocelli
Bumblebees have been shown to use their ocelli alone or in conjunction with
the dorsal portion of the compound eye to navigate by polarized light
(Wellington, 1974). While the
compound eye seems particularly critical for form and color perception and
works well during the day, when the environment is brightly illuminated,
ocelli are especially important at dusk, when the surroundings are too dim for
the compound eye to distinguish landmarks. By making use of polarized light
cues, bumblebees are thereby able to prolong their foraging. Physiological
investigations indicated that honeybee and bumblebee ocelli have a peak
sensitivity in the blue-green as well as in the UV part of the light
(Meyer-Rochow, 1981
;
Ruck and Goldsmith, 1958
).
Meyer-Rochow (1981
) estimated
the UV peak sensitivity of the bumblebee median ocellus to be 353 nm, which is
indistinguishable from the peak sensitivity of the UV visual pigment of the
compound eye (Bernard and Stavenga,
1978
). It is therefore not entirely surprising that we found UV
opsin-ir rhabdomeres in all three ocelli. The physiological similarity between
the UV visual pigment in the compound eye and ocelli, in combination with the
finding of only one UV opsin-encoding gene in the recently released Apis
mellifera genome and our immunohistochemical results, suggests that the
same opsin is expressed in both the compound eye and ocelli.
The honeybee ocelli show an additional peak sensitivity at around 490 nm
(Ruck and Goldsmith, 1958),
which is
50 nm shifted towards shorter wavelengths compared with the
long-wavelength photoreceptor cells found in the retina
(Menzel and Blakers, 1976
),
and in Bombus hortorum, the ocelli have a secondary peak around 519
nm (as estimated by ERG) (Meyer-Rochow,
1980
). In Drosophila, the Rh2 opsin is exclusively expressed in
the ocelli (Pollock and Benzer,
1988
) and shows a 60 nm blue-shift compared to the paralogous main
retinular opsin Rh1 (Britt et al.,
1993
; Zuker et al.,
1988
). We recently found a novel LW-sensitive opsin gene (LW
Rh2) that is paralogous to the already known LW opsin gene (LW
Rh1) in hymenopterans (Spaethe and
Briscoe, 2004
). The mRNA expression level of LW Rh2 is
much lower compared with the already known LW Rh1 opsin gene (J.S.
and A.D.B., unpublished observation) and, although its cellular location is
currently unclear, it is possible that it is expressed only in the ocelli,
like Rh2 in Drosophila.
Extra-retinal opsin expression
So far, extra-retinal opsin expression has been studied in only a few
insects and crustaceans (Sandeman et al.,
1990). As one example, the expression of green-, blue- and
UV-sensitive opsins in lepidopteran adult stemmata, which correspond to the
former larval photoreceptors, has only recently been examined
(Briscoe and White, 2005
;
Lampel et al., in press
). In
addition, even within the same insect order there appears to be heterogeneity
in terms of which opsins are expressed. In the butterfly Vanessa, for
instance, only UV and green opsin transcripts were detected in the stemmata,
while in hawkmoths, all three were found. In the Drosophila
HofbauerBuchner eyelet, the homologue of the adult lepidopteran
stemmata, the green-sensitive Rh5 and the blue-sensitive Rh6 rhodopsins were
found to be expressed but, in contrast to lepidopterans, no UV-sensitive (Rh3
or Rh4) rhodopsin was detected (Malpel et
al., 2002
; Yasuyama and
Meinertzhagen, 1999
). In both instances, all opsins that are found
in the stemmata are also expressed in the retina. Whether the UV opsin-ir
perikarya at the ventral rim of the lamina that we found in the bumblebee
(Fig. 6D) are homologous to the
butterfly stemmata is unknown; earlier studies suggested that stemmata are
probably absent in aculeate hymenopterans
(Gilbert, 1994
; but see
Felisberti and Ventura, 1996
).
Also, we found no associated crystalline cones with these perikarya, as were
found in lepidopterans (Briscoe and White,
2005
).
Lepidopterans and aphids are the only insect groups so far in which opsins
have also been reported in other parts of the brain. Shimizu et al.
(2001) cloned a
long-wavelength sensitive opsin (boceropsin) from a larval brain cDNA
of the silkworm, Bombyx mori, and showed its protein expression in
various neuronal clusters in the larval brain. Gao et al.
(1999
) tested several
antibodies directed against invertebrate (Drosophila Rh1) and
vertebrate (chicken cone, mammalian blue cone, and rod) opsins in the aphid
Megoura viciae, and an anterior ventral neuropil region in the brain
was labeled. However, in situ hybridization experiments with two
M. viciae opsin-encoding antisense riboprobes found expression only
in the retina but not in other parts of the brain, indicating that an
additional opsin might be expressed in the aphid brain
(Gao et al., 2000
). In four
different hawkmoth species, Lampel et al.
(in press
) found widespread
expression of a new LW-sensitive opsin (`brain opsin') in various parts of the
optic lobe, namely the lamina, lobula, lobula plate, medulla, accessory
medulla and adjacent neurons innervating the accessory medulla. In contrast to
the opsins found in both the retina and stemmata, the brain opsin in the
hawkmoths was exclusively expressed outside the retina, including in perikarya
along the ventral rim of the medulla, as we found in bees.
Our data show that in bumblebees, UV opsin that is expressed in the retina
and supposed to be used primarily for vision is broadly expressed in different
parts of the brain as well. In contrast to previous studies in lepidopterans
and dipterans, where expression of retinal opsins outside the retina was shown
to be restricted to stemmata, opsin-ir in Bombus was also found in
larger neuropils like the proximal rim of the lamina
(Fig. 6), the AL and central
body (Fig. 7). This situation
in Bombus is not unlike that of the crayfish Cherax
destructor, where the major rhodopsin of the retinula cells of the
compound eye was also found to be expressed in cell sommata along the anterior
margin of the cerebral ganglion (Sandeman
et al., 1990), and highlights the flexibility of opsin expression
patterns in different invertebrate lineages. It will be interesting to see
whether the blue and LW eye opsins of bumblebees are also expressed
extraretinally.
With all antibody studies, however, it is important to note that there is
always the possibility of cross-reactivity with multiple epitopes
(Marchalonis et al., 2001).
The results of our western blot indicate the presence of a major band at
41 kDa, which is the predicted size for the UV opsin protein. We also
note the presence of two smaller bands in the same blot, at 30 and 29 kDa.
Multiple bands have been observed in opsin western blots in
Drosophila (Bentrop et al.,
1997
), in hawkmoths (White et
al., 2003
) and in Limulus
(Battelle et al., 2001
), and
therefore the observation of multiple bands in our western blot is not
entirely surprising. In Drosophila, this is due to the fact that
nascent Rh1 opsin protein is glycosylated and then deglycosylated as it
matures. On a western blot, this is observed as multiple bands, at 40 and 35
kDa (Ozaki et al., 1993
). We
inspected the Bombus UV opsin amino acid sequence for the presence of
putative glycosylation sites, indicated by the consensus sequence NXS/T
(O'Tousa, 1992
), and found two
in the N-terminal domain (see Fig.
1), one more than is present in the Drosophila Rh1 opsin
(Katanosaka et al., 1998
).
Therefore, one possible explanation for these additional bands is that the UV
opsin protein in bees exists in several different states of glycosylation.
Another possibility is that these bands also represent proteolytic breakdown
products of the opsin protein. Lastly, the results of our tBLASTn search for
peptide sequences similar to that part of the protein to which we directed our
antibodies, including against the whole Apis mellifera genome,
yielded only UV-sensitive insect opsin proteins, so it would seem that this
peptide motif is, at least, not common among the known genomes in GenBank.
Several lines of evidence in fact suggest that the opsin expression
patterns we observed in the retina and brain may be specific. First, staining
in the ocelli (Fig. 5B),
perikarya at the edge of the medulla (Fig.
7B), in the retina, lamina organ, cluster of cells between the
lobula and the mushroom bodies (Fig. S2 in supplementary material), within the
antennal lobes and central body (not shown) were all abolished when the
antibody was first incubated with the peptide. Second, we confirmed the
pattern of opsin protein expression in the compound eye by performing in
situ hybridizations. Third, the cluster of UV opsin-ir cells that we
observed between the lobula plate and the calyces of the mushroom bodies is
similar in position to a cluster of pigment-dispersing hormone (PDH)-ir cells
in Apis mellifera (Bloch et al.,
2003). Neurons that are immunoreactive for PDH appear to be
significant components of the optic lobe pacemakers in a variety of insects
(Helfrich-Forster et al.,
1998
). Finally, in intracellular recordings from neurons in the
central body of the locust, several were found to be light sensitive
(Vitzthum et al., 2002
). Our
finding of UV opsin-like staining in the central body is consistent with this
and may in part explain some of these findings. Clearly, much additional work
needs to be performed to corroborate our results. As one example, it would be
interesting to examine the distribution of visual arrestin and PDH in relation
to the UV opsin expression described above.
Putative non-visual extra-retinal photoreceptors
Besides vision, a primary function of photoreceptors in insects is the
entrainment of circadian and seasonal photoperiodic rhythms. Biological
rhythms are associated with organismal changes in physiology and behavior and
are controlled by clock genes at the cellular level
(Devlin and Kay, 2001;
Jackson et al., 2001
). The
`circa'-dian changes of clock gene expression levels are entrained by external
photic stimuli, for instance by changes in the surrounding light intensity or
spectral composition (Zordan et al.,
2001
). However, the contribution of the various non-visual photic
input channels is complex and, even in Drosophila, not fully
understood. Behavioral experiments with different fly mutants showed that,
besides the compound eyes and the ocelli, at least three different non-visual
channels are additionally involved in synchronizing the circadian clock: the
HofbauerBuchner eyelet (discussed above), the blue-light photopigment
cryptochrome, and unknown photopigments in clock-gene-expressing dorsal
neurons (Helfrich-Forster et al.,
2001
; Rieger et al.,
2003
). Other putative extra-retinal photoreceptors described in
different hemi- and holometabolous insect orders are the lamina and lobula
organs, located in the optic lobe
(Fleissner and Fleissner,
2003
). The laminar organs have been found on the proximal
dorso-frontal rim of the lamina, the part of the optic lobe where we found
strong UV opsin- and PER-immunoreactivity in the bumblebee
(Fig. 6). This photoreceptive
organ was detected by means of immunocytochemistry using antibodies against
proteins of the phototransduction cascade and cryptochrome in combination with
ultrastructural investigations. However, neither the presence of opsins nor
PER was tested. We speculate that the proximal part of the lamina in
bumblebees serves a similar function to the laminar organ in other insects.
However, to demonstrate that the proximal part of the lamina in bumblebees is
homologous to the previously described lamina organ in other insects, we need
to check for the presence of cryptochrome in the bumblebee lamina, for opsins
in the laminar organ of other insect orders, and for similar morphological and
developmental origins.
We also found UV opsin and PER expression in AL neurons. No photoreceptive
system has been described in the bee AL so far. However, the extraretinal LW
brain opsin in adult hawkmoths, mentioned above, is also expressed in the core
region of hawkmoth antennal lobe glomeruli
(Lampel et al., 2002; J.
Lampel, A.D.B. and L. T. Wasserthal, unpublished). Therefore, these neurons
might form independent photoreceptive clock neurons in the bee AL, as in
Drosophila, where autonomous circadian oscillators are found in
different organs and tissues all over the body
(Plautz et al., 1997
). Also,
electroantennograms in Drosophila have shown a robust circadian
rhythm of electrophysiological responses of the antenna that was abolished in
a mutant fly where PER expression was restricted to the optic lobe but was not
expressed in peripheral circadian oscillators
(Krishnan et al., 1999
). An
alternative explanation is that the UV opsin serves a different, yet unknown,
function in the AL than photoreception. For instance, arrestins play an
important role in visual signaling processes by interacting with the rhodopsin
and terminating visual stimulation. In recent studies in Anopheles
and Drosophila, it has been shown that visual arrestins are also
expressed in olfactory neurons and are required for normal olfactory
physiology, demonstrating a function in both visual and olfactory signal
transduction systems (Merrill et al.,
2002
).
To summarize, UV opsin was found in different parts of the bumblebee brain, the proximal rim of the lamina, antennal lobe, central complex and different cell clusters in the protocerebrum. Also, PER, an important component of the circadian clock, was found in the proximal rim of the lamina and the antennal lobe, indicating a putative function of these brain regions as putative extra-retinal photoreceptors in the entrainment of circadian rhythms. Further studies are needed, however, to clarify whether UV opsin and PER are co-expressed in the same cells and whether PER protein levels in the lamina and in other UV-expressing areas described here oscillate over a 24 h light:dark cycle.
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Acknowledgments |
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![]() |
Footnotes |
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* Present address: Institute for Zoology, Department of Evolutionary Biology,
University of Vienna, 1090 Vienna, Austria
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References |
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