Download RESEARCH ARTICLES Characterization of the Long

RESEARCH ARTICLES
Characterization of the Long-Wavelength Opsin from Mecoptera and
Siphonaptera: Does a Flea See?
Sean D. Taylor,* Katharina Dittmar de la Cruz,* Megan L. Porter, and Michael F. Whiting*
*Brigham Young University, Department of Integrative Biology; and Brigham Young University, Department of Microbiology and
Molecular Biology
Mecoptera and Siphonaptera represent two insect orders that have largely been overlooked in the study of insect vision.
Recent phylogenetic evidence demonstrates that Mecoptera (scorpionflies) is paraphyletic, with the order Siphonaptera
(fleas) nesting as sister to the family Boreidae (snow fleas), showing an evolutionary trend towards reduction in gross eye
morphology within fleas. We provide the first molecular characterization of long-wavelength opsins from these three
lineages (opsin gene from fleas [FL-Opsin], the Boreidae [B-Opsin], and a mecopteran family [M-Opsin]) and assess
the effects of loss of visual acuity on the structure and function of the opsin gene. Phylogenetic analysis implies a physiological sensitivity in the red-green spectrum for these opsins. Analysis of intron splice sites reveals a high degree of
similarity between FL-Opsin and B-Opsin as well as conserved splice sites across insect blue-green and long-wavelength
opsins. Calculated rates of evolution and tests for destabilizing selection indicate that FL-Opsin, B-Opsin, and M-Opsin are
evolving at similar rates with no radical selective pressures, implying conservative evolution and functional constraint
across all three lineages.
Introduction
Insects represent a group of organisms in which visual
perception and the associated structural components are
highly developed (Briscoe and Chittka 2001). The class
Insecta is one of the largest and most diverse classes of
organisms on the planet, accounting for over 70% of
all known species (Resh and Carde 2003). Insects have
adapted to nearly every ecological niche available, from
arctic to tropic and mountaintop to river bottom. However,
despite this wide diversity of habitats and lifestyles, finding
links in the adaptation of visual systems to visual ecology
has proven to be difficult (Briscoe and Chittka 2001). It is
important, therefore, to continue to characterize the visual
system for a diversity of insect groups in order to gain a
better understanding of the selective pressures and constraints that guide visual evolution in insects.
The photoreceptive cells in the insect compound eye
contain light-sensitive visual pigments, which consist of an
opsin apoprotein, bound by a protonated Schiff’s base linkage to a photosensitive chromophore molecule, usually
11-cis-retinal. The visual cascade is initiated when the chromophore absorbs a photon of light, causing it to isomerize
to the trans form. A corresponding conformational change
in the opsin protein, a member of the G protein–coupled
receptors, initiates a transducin-mediated response, which
ultimately sends a neural signal to the brain. For a review
of opsin physiology, see Nathans (1987). A single individual may possess several opsin variants, each sensitive to
different wavelengths of light, thus conferring a specific
range of spectral sensitivity to that organism (Kochendoerfer
et al. 1999). Typically, insects possess trichromatic vision,
conferred by at least three opsin classes: UV-, blue-, and
green-sensitive variants. All insects studied have green
receptors (;530 nm), most express UV (;330 nm) and
Key words: opsin, evolution, Mecoptera, Siphonaptera, insect vision.
E-mail: [email protected].
Mol. Biol. Evol. 22(5):1165–1174. 2005
doi:10.1093/molbev/msi110
Advance Access publication February 9, 2005
Published by Oxford University Press 2005.
blue (;440 nm), and only a few have been shown to contain red (.565 nm) (Briscoe and Chittka 2001). Molecular
characterizations and spectral tuning studies have been performed on opsin proteins found in many insect groups,
including bees (Bellingham et al. 1997), moths and butterflies (Kitamoto et al. 1998; Briscoe 2000), mantids (Towner
and Gartner 1994), fireflies (Cronin et al. 2000), and fruit
flies (Neufeld, Carthew, and Rubin 1991; Carulli et al.
1994).
Mecoptera and Siphonaptera represent two insect
groups that have largely been overlooked in the study of
vision. Mecoptera is a relatively small, holometabolous
insect order with approximately 600 described species
(Penny and Byers 1979). For purposes of this study, two
families are of particular interest. Panorpidae (true scorpionflies) is the most speciose family of mecopterans. They
have a scavenging lifestyle, feeding primarily on dead, softbodied arthropods and often robbing spiders’ webs of the
encased insects (Byers and Thornhill 1983). They have
well-developed eyes and are highly visually oriented, with
a maximum spectral sensitivity peak at 490–520 nm and a
minor peak at 360 nm (Burkhardt and De LaMotte 1972).
Boreidae (snow fleas) are wingless, alpine insects that live
and feed exclusively on mosses and are most often collected
on winter snow (Penny 1977). The eyes of boreids are
smaller and appear slightly less well developed than those
of panorpids. We have observed that boreids will often
escape collection by jumping when they are too closely
approached, indicating that vision plays an important role
in sensing the environment. Little is known, however, of
boreid visual ecology.
Siphonaptera (fleas) is a highly specialized holometabolous insect order with approximately 2,380 described species (R. E. Lewis and J. H. Lewis 1985). Fleas are strict
vertebrate ectoparasites and are of tremendous economic
importance as vectors of pathogens, transmitting diseases
such as plague, murine typhus, and tularemia (Dunnet
and Mardon 1991). Little is known about flea vision,
due mainly to the radical divergence in flea eye structure
1166 Taylor et al.
Table 1
Primers Used to Amplify LW-Opsin
Primer
LF1 (fw)
Barney (fw)
Rh6 5.1F (fw)
Mike (fw)
Rh6 5.1R (rv)
Google (rv)
Scylla (rv)
FIG. 1.—Condensed phylogeny of Mecoptera and Siphonaptera presented by Whiting (2002) and eye morphology of Panorpidae, Boreidae,
and Siphonaptera.
from the typical insect bauplan (Crum, Knapp, and Wite
1974). Fleas show a transformation of the multifaceted
eyes and ommatidia of most insects, replaced instead with
heavily sclerotized, atypical ocelli, or ‘‘eyespots,’’ or in
some cases, a complete absence of any eye at all (Dunnet
and Mardon 1991). The species used in our analysis exhibit
a diversity of eyespot morphologies, ranging from large
eyespots in Pulex irritans, to small eyespots in Ctenophthalmus agyrtes and Jellisonia sp., to no discernable eyespot in Uropsylla tasmanica. Despite the drastic reduction
in eye structure, Crum, Knapp, and Wite (1974) demonstrated that cat fleas (Ctenophalides felis) and oriental rat fleas
(Xenopsylla cheopis) have peak light sensitivities at 330
and 530 nm, respectively, although the light elicited different responses in the two species. Osbrink and Rust (1985)
further demonstrated that visual cues seem to be among the
primary factors influencing host selection in cat fleas. However, this eyespot probably cannot form an acute image but
merely acts as a light sensor (Osbrink and Rust 1985; Rust
and Dryden 1997; Land 2003).
Whiting (2002) recently demonstrated that Mecoptera
is paraphyletic, with Siphonaptera nesting within Mecoptera as sister group to Boreidae, and the obscure family
Nannochoristidae placed as sister to Boreidae 1 Siphonaptera (fig. 1). This phylogenetic hypothesis provides an evolutionary framework in which to evaluate shifts in visual
ecology and search for possible correlates in morphological
and molecular changes associated with vision in these
insects. Given that nannochoristids are visual mecopterans,
optimization of vision on this phylogeny implies that there
was a reduction of visual acuity in the flea lineage. We compare opsin structure and function in the ectoparasitic fleas,
which have a single-lens light sensor and only general light-
Sequence (5# / 3#)
CAYTGGTAYCARTWYCCICCIATG
GAYMGITAYAAYGTIATIGTIAARGG
GGMTGGAAYMGRTATGTWCCTGARG
ATGMGIGAICARGCIAARAARATG
CYTCAGGWACATAYCKRTTCCAKCC
CATYTTYTTIGCYTGITCICKCAT
TTRTAIACIGCRTTIGCYTTIGCRAA
dark sensitivity, with that of their boreid sister group, which
are detritus feeders and visually oriented insects. These in
turn are compared with their highly visual panorpid relatives, which have a fully developed, image-forming eye.
Because nannochoristids are rare insects, we have not
included them in this study.
This study focuses on the effects of the shift to parasitism in fleas, the associated loss of visual acuity, and the
molecular processes underlying the evolution of the opsin
protein in these insects. Given the demonstrated photosensitivity of the fleas and scorpionflies to green light, we isolated the long-wavelength (LW) opsin gene from fleas (FLOpsin), their close sister group, the Boreidae (B-Opsin),
and the Panorpidae, a highly visual mecopteran family
(M-Opsin). We provide the first molecular characterization
of insect opsins from these three groups. In addition, we
compare the gene structure and evolutionary history of
these three opsin groups in order to assess the effects of loss
of visual acuity on the structure and function of the opsin
gene. We specifically test for relaxation of functional constraint on the FL-Opsin protein due to the gross reduction in
eye structure and visual sensitivity.
Materials and Methods
DNA Analysis: Polymerase Chain Reaction and
Sequencing
LW opsin sequences were obtained from four flea species representing four families, six panorpid species representing the single diverse genus Panorpa, and one boreid
species. For a detailed list of all taxa included in the analysis, see Appendix 1 (Supplementary Material online).
Genomic DNA was extracted from specimens using
the Qiagen DNeasy extraction kit using manufacturer’s
guidelines. Because of the low yield of DNA expected from
fleas, the DNA was eluted in only 100 ll of solution.
Genomic DNA vouchers and specimen vouchers are deposited in the Insect Genomics Collection, M. L. Bean
Museum, Brigham Young University.
Polymerase chain reaction (PCR) primers were developed from conserved regions of arthropod LW opsins and,
once sufficient sequences were obtained, redesigned for use
in Mecoptera and Siphonaptera. The amplified region spans
the first six of the transmembrane helices and ;60% of the
seventh. Primer sequences are given in table 1. PCR amplification used the following three-step cycling protocol with
Amplitaq Gold polymerase (ABI, Foster City, Calif.): 35
cycles of denaturation at 94°C for 1 min, annealing at
55°C for 1 min, and extension at 72°C for 3 min, followed
Flea and Mecoptera Opsin 1167
FIG. 2.—Comparison of FL-Opsin, M-Opsin, and B-Opsin intron splice sites to other insect opsins, based on Briscoe (1999). Numbers above hatch
marks indicate position of splice site relative to Drosophila melanogaster Rh1 amino acid sequence. Numbers in parentheses indicate observed size (bp) of
introns (shown only for LW opsins). Regions in black were not amplified in this analysis. Intron positions and lengths determined from alignment of our
novel opsin sequences with the following: Drosophila melanogaster Rh1 (X65877), Papilio glaucus Rh3 (AF098283), Bee LW1 (Bombus impatiens:
AY485302, Bombus terrestris: AY485301, Drosophila afflicta: AY485303, Drosophila rinconis: AY485304, Osmia rufa: AY572828), Bee LW2 (B.
impatiens: AY485306, B. terrestris: AY485305, D. afflicta: AY485308, D. rinconis: AY485307, O. rufa: AY572829, Apis mellifera: XM_397398.1),
Anopheles LW (GPRop5, GPRop6, GPRop7; see Appendix 1, available as Supplementary Material online).
by an additional 7 min of elongation at 72°C. PCR products
were visualized by gel electrophoresis and purified using
the Millipore Montage purification system. Initial amplifications were attempted on all taxa using LF1 and Scylla
primers in order to obtain the entire target region in a single
amplification. If unsuccessful, smaller overlapping segments were amplified with internal primers.
Because opsin variants arose through a series of gene
duplication events (Deininger, Fuhrman, and Hegemann
2000), the possibility exists that paralogous opsin variants
may coamplify. In order to separate potential paralogous
copies, the PCR products were cloned using the pCR
2.1-TOPO cloning kit (Invitrogen) following the manufacturer’s protocol. Ten clones from each PCR product were
picked and then reamplified and sequenced using the provided M13 vector primers (Invitrogen, Carlsbad, Calif.) and
BigDye Terminator chemistry (ABI). Sequence data were
collected using the ABI Prism 3730 capillary autosequencer
in the BYU DNA Sequencing Center.
The vector ends were trimmed from all cloned products and the resulting sequence blasted in GenBank to
insure that an opsin product was indeed amplified. Clones
and overlapping PCR regions from the same taxa were initially included in the phylogenetic analysis as separate terminals to verify that the sequences did not represent
paralogous gene copies. If they were shown to group
together, the sequences were then combined, with any discrepancies coded as ambiguous or missing data, and
included as a single terminal in the final analysis.
Phylogenetic Analysis
Sequences from crustaceans and the LW, blue-green
(BG), blue (BL), and UV insect opsin variants were downloaded from GenBank and used to determine the phylogenetic placement of FL-Opsin, M-Opsin, and B-Opsin (see
Appendix 1). The sequence of bovine rhodopsin, as the
only opsin to have its tertiary structure characterized,
was included as an out-group. Initial alignments were performed manually in Sequencher 4.2 (GeneCodes 2003).
Introns were identified through comparison to GenBank
insect cDNA sequences (fig. 2). The resulting coding
sequences were translated using MacClade (W. P. Maddison
and D. R. Maddison 2003) and the amino acid sequences
aligned via ClustalX (Thompson et al. 1997). The alignment was further refined by performing a homology alignment of the Jellisonia sp. amino acid sequence (the most
complete opsin isolated in this study) to high-resolution
bovine opsin crystal structure (Palczewski et al. 2000) in
the Swiss-Pdb Viewer and submitted to the SwissModel
Server for homology modeling (Guex and Peitsh 1997).
This allowed us to optimize our overall nucleotide alignment based on both amino acid sequence homology and
structural homology.
Phylogenetic analyses were performed using the
nucleotide sequences after intron removal. The phylogeny
was reconstructed using Bayesian methods coupled with
Markov chain Monte Carlo (BMCMC) techniques as
implemented in MrBayes v3.0b4, which allows for mixed
1168 Taylor et al.
model analyses (Huelsenbeck and Ronquist 2001; Ronquist
and Huelsenbeck 2003). Because different codon positions
have different structural constraints, the data set was partitioned into first, second, and third codon positions. Models
for each partition were selected using the procedure implemented in Modeltest v.3.6 (Posada and Crandall 1998).
Mixed models were used with unlinked parameters between
partitions, treating model parameters as unknown variables
with uniform default priors. Three independent BMCMC
analyses were run, each consisting of 10 independent
Markov chains started from a random tree and run for
3 3 106 generations, with every 1,000th generation
sampled. To confirm that each separate Bayesian analysis
converged and mixed well, the fluctuating value of the likelihood and all phylogenetic parameters were examined
graphically. To check for congruence among independent
runs, the mean likelihood score and parameter values and
the posterior probabilities (pP) for individual clades were
compared. All sample points prior to reaching stationary
values were discarded as burn-in, and the remaining trees
from each independent analysis were combined to calculate
the maximum a posteriori (MAP) tree (Huelsenbeck and
Imennov 2002; Huelsenbeck et al. 2002).
Opsin Evolution
The ratio of nonsynonymous versus synonymous
changes (dN/dS) for the insect LW portion of the tree was
estimated with maximum likelihood using the codon-based
substitution model of the CODEML software package in
PAML v3.14 (Yang 1998). Several different site-specific
models in which selective pressure varies among different
sites but the site-specific pattern is identical across all lineages were implemented (Yang et al. 2000): model M0
(null model with no variation among sites), M1 (‘‘neutral’’
model, with two categories of site with fixed dN/dS ratios of
0 and 1), M2 (‘‘selection’’ model with three categories of
site, two with fixed dN/dS ratios of 0 and 1, and a third estimated dN/dS ratio), M3 (‘‘discrete’’ model with three categories of site, where the dN/dS ratio is free to vary for each
site), M7 (‘‘beta’’ model—10 categories of site, with 10 dN/
dS ratios in the range 0–1 taken from a discrete approximation of the beta distribution), and M8 (‘‘beta plus omega’’
model—10 categories of site from a beta distribution as in
model M7 plus an additional category of site with a dN/dS
ratio that is free to vary from 0 to greater than 1). PAML
estimates the dN/dS ratios that are free to vary under these
models, as well as the proportion of sites with each ratio. All
models were run twice with starting omega values of less
than and greater than 1 as suggested by the PAML manual
to test for entrapment in local optima (Yang 1997). Likelihood ratio tests, to determine whether more complex models provided a significantly better fit to the data than more
simple models, were performed by comparing the likelihood ratio test statistic (ÿ2[ln L1 ÿ ln L2]) to critical values
of the chi-square distribution with the appropriate degrees
of freedom (Yang 1998).
Although dN/dS ratios are useful for detecting selection,
they do not indicate how the identified selection affects the
overall structure and function of the protein. Amino acid substitutions can have a wide range of effects on a protein
depending on the difference in physicochemical properties
and location in the protein structure; in order to evaluate these
effects we used the insect LW portion of our Bayesian tree in
TreeSAAP v3.0 (Woolley et al. 2003) to test for evidence of
selection among 31 amino acid properties in the three lineages of interest. TreeSAAP allows us to identify these property changes and classify them into categories on a gradient
from conservative to radical change. Based on data set–
specific nucleotide substitution patterns, a neutral model
of expected change for each category is generated. This null
model is then compared to the observed numbers of amino
acid replacements in the data set, and a z-score is calculated
for each category. Categories where the observed number of
amino acid replacements is significantly different than neutral expectations are considered as potentially being affected
by selective pressures. In this study, we specified a scale of 20
categories of change. Greater than expected numbers of
replacements (relative to the neutral model) in categories
1–3 indicate significant stabilizing selection, whereas the
same situation in categories 18–20 indicate significant destabilizing selection (table 3). By mapping the identified amino
acid replacements back onto the protein structure, we are able
to make inferences about the influence of potential selective
pressures on protein function and on the evolutionary history
of particular lineages (Woolley et al. 2003).
Results and Discussion
Opsin Gene Isolation and Phylogenetic Analysis
PCR amplification of the LW opsin gene for the Mecoptera and Siphonaptera yielded ;1,030- and ;1,200-bp fragments, respectively. We were unable to obtain ;460 bp from
the 5# end of Panorpa gracilis and Boreus coloradensis. The
translated sequences used in the analysis ranged between 203
and 278 amino acids. Interestingly, Siphonaptera have a
unique four–amino acid–long expansion segment in transmembrane helix 6, at amino acid positions 119–222 in our
alignment (fig. 3).
The MAP tree (ln L 5 ÿ24,065.002 and pP 5 0.002)
used in PAML and TreeSAAP analyses is shown in figure 4.
Although not entirely congruent with the species phylogeny
presented by Whiting (2002), there are nonetheless several
significant features about this topology. Briscoe (2000)
demonstrated that opsins will cluster together primarily
by physiological similarity, i.e., similar spectral sensitivities, and secondarily by species relationships. Our data
strongly support these results, with opsins of physiological
similarity clustering together across the topology. FLOpsins and M-Opsins are well supported as monophyletic
groups within the insect LW clade, suggesting sensitivity in
the red-green spectrum. We note, however, that although
support for the ordinal grouping of the FL-Opsins, MOpsins, and B-Opsin sequences is strong, the opsin gene
generally provides little or no support for the resolution
of these ordinal relationships to each other.
Furthermore, a recent study of the Anopheles gambiae
genome revealed the presence of a higher number of opsin
genes than any other characterized insect, with at least half of
the identified copies being duplicates of LW-sensitive genes
(Hill et al. 2002). Phylogenetic analysis of this LW gene
complement indicated at least one early gene duplication
Flea and Mecoptera Opsin 1169
FIG. 3.—Two-dimensional representation of the homology model of FL-Opsin showing preservation of the seven-helix structure. Numbering begins
with the first amino acid included in this analysis. Black circles represent amino acids not obtained. Boxed residues show an expansion region found only
in FL-Opsin. Circles with a cross in them show key conserved residues described by Briscoe (1999). The results of the TreeSAAP analysis are mapped
onto the model. Blue-colored circles show sites of conservative amino acid (AA) replacement, and red-colored circles show sites of radical AA replacement found along the branches leading to FL-Opsin, M-Opsin, or B-Opsin clades. Circles shaded in light gray indicate sites strictly conserved across the
three lineages. White circles represent AA residue variation among the three lineages.
within insects before the emergence of the orders Orthoptera,
Mantodea, Hymenoptera, Lepidoptera, and Diptera
(Spaethe and Briscoe 2004). In our analysis, the B-Opsin,
FL-opsin, and M-opsin sequences do not cluster with either
the A. gambiae or the bee LW2 genes (fig. 4). Analysis of our
alignment using the methods described by Spaethe and Briscoe (2004) produced a tree similar to their results, with the
newly characterized B-Opsin, FL-Opsin, and M-Opsin
sequences grouping within the insect LW1 clade (results
not shown). Although the weak support in the LW clade
of our MAP tree makes inferences about opsin gene duplication and evolution difficult, the phylogenetic relationships
of the opsin gene copies in A. gambiae and Hymenoptera
indicate that there may be many more yet unidentified gene
duplication events in the insect LW clade.
Opsin Gene Structure
Figure 2 shows comparative intron splice site positions
of FL-Opsin, B-Opsin, and M-Opsin relative to other insect
LW opsins. Within the regions amplified, the putative
Siphonaptera opsin gene contains five introns, two Panorpidae, and four Boreidae. Interestingly, the B-opsin and FLopsins (putative LW1) and bee LW2 share a unique intron
at position 156, although they are not a monophyletic clade
in our phylogeny. Manual mapping of this character onto
the MAP tree (fig. 4) indicates a single gain of this intron
site, with secondary loss in the Bee LW1 clade.
Briscoe (1999) reported three splice sites conserved
across insect BG and LW opsin groups (sites 190, 239,
and 332, fig. 2). Our data support this observation for both
sites 190 and 239, although secondary intron deletions are
observed in the bee LW1 (position 239) and Anopheles and
Panorpidae (position 190) lineages. Site 332 is outside the
region amplified in this study and cannot be further evaluated. Additionally, our data indicate a unique splice site
at 119 that, with the exception of bee LW1 and Anopheles,
appears to be characteristic of LW opsins, and we predict it
will also be present in boreids.
Of the remaining intron insertions, sites 79 in Papilio
and 108 in bee LW1 appear to be autapomorphies (fig. 2).
Several sites (277, Papilio and bee LW1 and LW2; 296,
Boreidae and Siphonaptera) appear in only a few taxa and
may indicate their presence before the divergence of those
lineages. All of the Anopheles LW opsin gene copies investigated (GPRop5, GPRop6, GPRop7) have lost all intron
splice sites except 239, implying that patterns of intron
insertion-deletion can change considerably within lineages.
Even though two groups may share a particular intron
splice site, the length of the insert was observed to vary. For
1170 Taylor et al.
FIG. 4.—Maximum a posteriori estimate of phylogeny (MAP) tree used in PAML and TreeSAAP analyses, ln L 5 ÿ24,065.002 and pP 5 0.002.
Posterior probabilities .0.95 are indicated by a gray circle on the corresponding branch. A detailed list of all taxa names and their respective accession
numbers can be found in Appendix 1. The shared insertion at site 156 between Boreidae, Siphonaptera, and bee LW2 is mapped onto the respective
branches, indicating a gain (red rectangle) and a loss (white rectangle).
example, at position 296 the boreid has a long intron (537
bp) and the fleas have relatively short introns (58–84 bp).
Intron lengths vary even among taxa within the same group,
e.g., sequence lengths for position 119 in Jellisonia sp., C.
agyrtes, and P. irritans are 99, 119, and 66 bp, respectively.
These results indicate that the splice sites appear to be more
phylogenetically conserved than the actual makeup of the
intron.
Removal of introns reveals a preserved open reading
frame in the boreid-, panorpid-, and flea-lineages. Homology modeling of the inferred amino acid sequence of the
Jellisonia sp. FL-Opsin suggests that the seven transmembrane helices characteristic of G protein–coupled receptors
and all known insect opsins are preserved (fig. 3). Additionally, Briscoe (1999) described seven amino acid motifs that
are necessary for a functional opsin protein and that are conserved across all known insect opsins. Four of these motifs
are located within the region amplified in this study, all four
of which are conserved in FL-Opsin, B-Opsin, and
M-Opsin (fig. 3). These residues include (1) Leu36 and
(2) Asn41 found in all G protein–coupled receptors (numbered according to the first residue obtained in FL-Opsin);
(3) the G protein–coupled receptor motif, E/D R, in the third
transmembrane region involved in transducin activation
(Asp102 and Arg103); and (4) a disulfide bridge connecting
transmembrane region 3 with the second cytostolic loop at
Flea and Mecoptera Opsin 1171
Table 2
Results from PAML Analysis of FL-Opsin, B-Opsin, and
M-Opsin Lineages
Model Code
M0
M1
M2
M3
M7
M8
(one-ratio)
(neutral)
(selection)
(discrete)
(beta)
(beta&w)
ln L
ÿ3,175.547
ÿ3,137.736
ÿ3,137.736
ÿ3,092.321
ÿ3,099.321
ÿ3,099.321
Different
dN/dSb
from
FL-Opsin B-Opsin M-Opsin
Neutrala
—
—
N
Y
—
N
0.0424
0.0651
0.0651
0.0437
0.0610
0.0610
0.0424
0.0649
0.0649
0.0437
0.0611
0.0611
0.0424
0.0649
0.0649
0.0437
0.0610
0.0610
a
Results from likelihood ratio test (P 5 0.01).
b
Ratios calculated from branches leading to FL-Opsin, B-Opsin, and M-Opsin
clades, respectively, and compared to the average ratio across the tree for each model
analyzed. In each model, rates are not significantly different (P 5 0.05) from the
average rate across the tree.
Cys78/155. As expected, many of these key residues occur in
regions for which the amino acid sequence is conserved
across all insect LW opsins.
Opsin Evolution
The results of the PAML analysis are summarized in
table 2. For the majority of the comparisons made, we failed
to reject the null hypothesis that models that allow for positive selection fit the data significantly better than models
that do not. However, model M3 showed a significantly better score (P 5 0.001) than the neutral model M1, indicating
that selective pressure is present. Although model M3 fit the
data better, no substitutions were found in categories where
x . 1, rejecting the hypothesis of diversifying selection.
This result is confirmed by examining the individual rates
of evolution found in M-Opsin, B-Opsin, and FL-Opsin and
comparing them to the other lineages in the topology. If, for
instance, in the flea lineage there was either strong selection
away from the ancestral protein or a loss of functional constraint of the opsin protein due to the reduction in eye structure, we expect to see a greater number of nucleotide
substitutions as well as a larger proportion of nonsynonymous to synonymous substitutions (Yokoyama et al. 1995).
The dN/dS ratios on the branch leading to the FL-Opsin
clade are not significantly different (P 5 0.05) from those
leading to the B-Opsin and M-Opsin clades (see table 2).
These ratios are also nearly identical to the average rates
of evolution across the entire topology (P 5 0.05). In addition, all ratios are observed to be less than 1. As stated earlier, this indicates that any selective pressure on opsin
evolution is purifying across all the insects observed such
that the structural and functional properties of the protein
are being conserved (Yang et al. 2000).
Analysis in TreeSAAP also confirmed that a low rate
of radical diversifying selection (destabilizing selection)
was occurring with respect to amino acid properties. Only
9.6% of all sites showed destabilizing selection among 7 of
the 31 properties tested. On the other hand, 21.4% of all
sites are under strongly stabilizing selection for 22 of 31
amino acid properties tested, and 60% of the amino acid
residues in FL-Opsin, B-Opsin, and M-Opsin were identical. Although several of the properties showing destabilizing selection (e.g., turn tendencies, coil tendencies, or
Table 3
Conservative (Stabilizing) and Radical (Destabilizing)
Amino Acid Properties Under Selection Identified from 31
Tested Amino Acid Properties in TreeSAAP Analysisa
Conservative
Selection
Categories
Amino Acid Property
1
Alpha-helica tendencies
Average number of surrounding
residues
Beta-structure tendencies
Bulkiness
Buriedness
Chromatographic index
Coil tendencies
Composition
Compressibility
Equilibrium constant
Helical contact area
Hydropathy
Isoelectric point
Long-range non-bonded
energy
Mean root square mean fluctuation
displacement
Molecular volume
Molecular weight
Normalized consensus
hydrophobicity
Partial specific volume
Polar requirement
Polarity
Power to be at the C-terminal
Power to be at the middle of
alpha helix
Power to be at N-terminal
Refractive index
Short/-medium range non-bonded
energy
Solvent accessible reduction ratio
Surrounding hydrophobicity
Thermodynamic transfer
hydrophobicity
Total non-bonded energy
Turn tendencies
X
X
X
X
X
X
X
X
X
X
X
X
X
X
2
3
X
X
X
X
X
X
Radical
Selection
Categories
18
19
20
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
a
Twenty-two properties show selection at least once in the first three categories
(conservative selection). Only seven properties show selection in the last three categories (radical selection).
power to be at the N-terminal) seem to be related to structural aspects of the protein, none are properties previously
identified to affect spectral tuning (i.e., polarity). Additionally, all of the conserved residues as described by Briscoe
(1999) also show conservation in our data set. These results
are shown in figure 3 mapped onto the two-dimensional
projection of FL-Opsin. See table 3 for a complete list of
all properties tested. As a whole, these data not only imply
functional constraint but also similar functional properties
across FL-Opsin, B-Opsin, and M-Opsin.
Sensitivity to Topology
Because of the weak support of the ordinal relationships found in our analysis, the PAML and TreeSAAP analyses were also calculated after constraining the data to the
Whiting (2002) species tree. The results in all cases were
identical, suggesting that the data are relatively insensitive
1172 Taylor et al.
to organization of the ordinal relationships, as predicted by
Yang et al. (2000). We observe that despite the overlying
reduction in eye morphology and loss of visual sensitivity
across these three lineages, there remains strong conservative, purifying selection on the opsin protein.
Functional Constraint of FL-Opsin, B-Opsin,
and M-Opsin
Despite the considerable variation in lifestyle, habitat,
and visual structure, the LW opsin gene appears to be
remarkably well preserved across panorpids, boreids, and
fleas. This is not entirely surprising, given that all three lineages demonstrate sensitivity and responsiveness to visual
cues at some level. Perhaps with gross reduction in flea eye
morphology and the incapability to perceive images, we
might have expected to see some corresponding change
in the mechanism of photoreception itself. However, as discussed above, not only does FL-Opsin appear to have
retained functional constraint but also no evidence is found
for unusual selective pressure on the amino acid sequence
and structural properties. On the contrary, we observed a
high degree of conservative selection, preserving amino
acid properties and structure similar to other functional
opsins. These results provide evidence that although vast
differences in perceptual abilities exist between Panorpidae,
Boreidae, and Siphonaptera, e.g., ability to form images,
the underlying physiology involved in the mechanisms
of photoreception appears to be preserved.
In addition, the preservation of opsin structure in
Siphonaptera, combined with the apparent reduction of
macroscopic eye structure may also indicate the presence
of an extraretinal photoreceptor system. Indeed, preliminary transmission electron studies on flea eyespots indicate
no preservation of typical microscopic adult insect eye
structures (e.g., rhabdom, crystalline cone, corneal lens)
as well as a heavily sclerotized layer of chitin covering
the flea ‘‘eyes’’ (Dittmar de la Cruz, unpublished data). This
structural degradation implies that photoreception may no
longer occur within the eye. Extraretinal, nonvisual photoreceptors are common among insects and have been found
at sites located in the central nervous system (Briscoe and
Nagy 1999; Shimizu et al. 2001), the posterior margin of the
compound eye (Yasuyama and Meinertzhagen 1999), and
the peripheral abdominal segments (Arikawa 1997; see G.
Fleissner and G. Fleissner 2003 for a review of nonvisual
photoreceptors). A number of LW opsin copies have been
implicated as extraretinal (Briscoe and Nagy 1999; Spaethe
and Briscoe 2004), and Spaethe and Briscoe (2004) speculate that these extraretinal opsins evolved early in invertebrate evolution. Interestingly, similar to the peripheral
photoreceptors found on the last abdominal segments
(genitalia) of Papilio xuthus (Arikawa et al. 1980), fleas
have a sensillial plate (or pygidium) on the last abdominal
segment, which is packed with receptor-like structures.
Unfortunately, the function of these ‘‘receptors’’ is still a
source of speculation.
Furthermore, extraretinal photoreceptors appear to be
involved in the photic entrainment of the circadian clock
(Shimizu et al. 2001; Malpel, Klarsfeld, and Rouyer
2002). Koehler, Leppla, and Patterson (1989) demonstrated
the presence of circadian rhythms in fleas, which could be
mediated by these types of nonvisual receptors.
Other studies have also shown examples of organisms
that do not have functional eye structures, yet seem to retain
functional opsin proteins. R. Yokoyama and S. Yokoyama
(1990), for example, presented a case where putative functional opsin genes were isolated from Astyanax fasciatus,
a blind cave fish. This phenomenon was confirmed by
Crandall and Hillis (1997), who demonstrated that some
species of cave crayfish still possess a functional opsin with
no significant differences from opsins isolated from surface
species. As there is no light available, there would be little
reason to maintain a traditional photoreceptor. They concluded that opsin must be involved in other pathways
besides vision, although this remains highly speculative.
Conclusions
(1) We have isolated and characterized the first LW opsin
genes from two orders: Mecoptera and Siphonaptera.
(2) Phylogenetic analysis implies a physiological sensitivity in the red-green spectrum for these opsins, consistent
with previously identified spectral sensitivities.
(3) Analysis of intron splice sites reveals the presence of
two introns (190 and 239) conserved across the BG
and LW groups. In addition, one intron (119) seems
to be unique to LW opsins.
(4) Flea opsin sequences showed a unique, four–amino
acid–long expansion segment.
(5) Calculated rates of evolution indicate that FL-Opsin,
B-Opsin, and M-Opsin are evolving at similar rates
with no radical selective pressures, implying conservative evolution and functional constraint.
The amino acid composition of FL-Opsin, B-Opsin,
and M-Opsin is remarkably well preserved. Over 60% of
the amino acid residues are identical across the three lineages,
with an additional 22% that are under stabilizing, conservative selection. Among these are several key amino acid motifs
that are necessary for proper opsin function. Panorpidae, Boreidae, and Siphonaptera exhibit vast differences in lifestyle
and ecology that are reflected in the adaptations of the visual
systems of each group. These lines of evidence indicate that
despite the reduction in eye structure and loss of visual acuity,
fleas have retained a remarkable similarity in LW opsin structure and amino acid properties. Although it is highly unlikely
that fleas perceive visual images, our data support the possibility of fleas being sensitive to LW light. That the underlying
mechanisms of photoreception could be preserved across visual systems of such immensely different qualities, speaks
much of the flexibility, ease of adaptation, and potential of
our biological world.
Supplementary Material
Supplementary data are available at Molecular Biology
and Evolution online (www.mbe.oupjournals.org).
Acknowledgments
We gratefully acknowledge K. Miller, S. Cameron, and
G. Svenson for their support and comments on this work. We
Flea and Mecoptera Opsin 1173
are alsograteful to D. McClellan for his invaluable insights and
help with the TreeSAAP analysis, as well as to M. PérezLosadaforhishelpwithPAML.ThankstoJ.Pricefortheinitial
design of opsin PCR primers, to M. Hastriter for his indefatigable knowledge of fleas and the use of his collection, and to
R. Trowbridge for his photography. This research was funded
by NSF grants DEB-9983195 (M.F.W.) and DEB-0120718
(M.F.W.), the Karl-Enigk-Foundation for Experimental Parasitology (Hannover, Germany), and the Brigham Young University Office of Research and Creative Activities.
Literature Cited
Arikawa, K., E. Eguchi, A. Yoshida, and K. Aoki. 1980. Multiple
extraocular photoreceptive areas on genitalia of butterfly,
Papilio xuthus. Nature 288:700–702.
Arikawa, K. 1997. Hindsight by genitalia: photo-guided copulation in butterflies. J. Comp. Physiol. A. 180:295–299.
Bellingham, J., S. E. Wilkie, A. G. Morris, J. K. Bowmaker, and
D. M. Hunt. 1997. Characterization of the ultraviolet-sensitive
opsin gene in the honey bee, Apis mellifera. Eur. J. Biochem.
243:775–781.
Briscoe, A. D. 1999. Intron splice sites of Papilio glaucus PglRh3
corroborate insect opsin phylogeny. Gene 230:101–109.
———. 2000. Six opsins from the butterfly Papilio glaucus:
molecular phylogenetic evidence for paralogous origins of
red-sensitive visual pigments in insects. J. Mol. Evol.
51:110–121.
Briscoe, A. D., and L. Chittka. 2001. The evolution of color vision
in insects. Annu. Rev. Entomol. 46:471–510.
Briscoe, A. D., and L. Nagy. 1999. Spatial expression of opsins
in the retina and brain of the tiger swallotail Papilio glaucus.
Am. Zool. 39:254B.
Burkhardt, D., and I. De LaMotte. 1972. Electrophysiological
studies on the eyes of Diptera, Mecoptera, and Hymenoptera.
Pp. 137–145 in R. Wehner, ed. Information processing in
the visual systems of arthropods. Springer-Verlag, Berlin,
Germany.
Byers, G. W., and R. Thornhill. 1983. Biology of the Mecoptera.
Annu. Rev. Entomol. 28:203–228.
Carulli, J. P., D.-M. Chen, W. S. Stark, and D. L. Hartl. 1994.
Phylogeny and physiology of Drosophila opsins. J. Mol. Evol.
38:250–262.
Crandall, K. A., and D. M. Hillis. 1997. Rhodopsin evolution in
the dark. Nature (Lond.) 387:667–668.
Cronin, T. W., M. Jarvilehto, M. Weckstrom, and A. B. Lall. 2000.
Tuning of photoreceptor spectral sensitivity in fireflies
(Coleoptera: Lampyridae). J. Comp. Physiol. 186:1–12.
Crum, G. E., F. W. Knapp, and G. M. Wite. 1974. Response of the
cat flea, Ctenocephalides felis (Bouché), and the oriental rat
flea, Xenopsylla cheopis (Rothschild), to electromagnetic radiation in the 300–700 nanometer range. J. Med. Entomol.
11:88–94.
Deininger, W., M. Fuhrman, and P. Hegemann. 2000. Opsin evolution: out of the wild green yonder? Trends Genet. 16:158–159.
Dunnet, G. M., and D. K. Mardon. 1991. Siphonaptera. Pp. 705–716
in CSIRO, ed. The insects of Australia: a textbook for students
and research workers. CSIRO and Cornell University Press,
Melbourne, Australia.
GeneCodes. 2003. Sequencher. v4.2. Ann Arbor, Mich.
Guex, N., and M. C. Peitsh. 1997. SWISS-MODEL and the SwissPdb viewer: an environment for comparative protein modeling.
Electrophoresis 18:2714–2723.
Fleissner G., and G. Fleissner. 2003. Nonvisual photoreceptors
with emphasis on their putative role as receptors of natural
Zeitgeber stimuli. Chronobiology 20(4):593–616.
Hill, C. A., A. N. Fox, R. J. Pitts, L. B. Kent, P. L. Tan, M. A.
Chrystal, A. Cravchik, F. H. Collings, H. M. Robertson, and
L. F. Zwiebel. 2002. G protein-coupled receptors in Anopheles
gambiae. Science 298:176–178.
Huelsenbeck, J. P., and N. S. Imennov. 2002. Geographic origin of
human mitochondrial DNA: accommodating phylogentic
uncertainty and model comparison. Syst. Biol. 51:155–165.
Huelsenbeck, J. P., B. Larget, R. E. Miller, and F. Ronquist. 2002.
Potential applications and pitfalls of Bayesian inference phylogeny. Syst. Biol. 51:673–688.
Huelsenbeck, J. P., and F. Ronquist. 2001. MrBayes: Bayesian
inference of phylogeny. Bioinformatics 17:754–755.
Kitamoto, J., K. Sakamoto, K. Ozaki, Y. Mishina, and K. Arikawa.
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 Papilip
xuthus. J. Exp. Biol. 201:1255–1261.
Kochendoerfer, G. G., S. W. Lin, T. P. Sakmar, and R. A. Mathies.
1999. How color visual pigments are tuned. Trends Biochem.
Sci. 8:300–305.
Koehler, P. G., N. C. Leppla, and R. S. Patterson. 1989. Circadian
rhythm of cat flea (Siphonaptera: Pulicidae) locomotion unaffected by ultrasound. J. Econ. Entomol. 82:516–518.
Land, M. F. 2003. Eyes and vision. Pp. 393–406 in V. H. Resh and
R. T. Carde, eds. Encyclopedia of insects. Academic Press,
New York.
Lewis, R. E., and J. H. Lewis. 1985. Notes on the geographical
distribution and host preferences in the order Siphonaptera.
J. Med. Entomol. 22:134–152.
Maddison, W. P., and D. R. Maddison. 2003. MacClade: analysis
of phylogeny and character evolution. Sinauer Associates,
Sunderland, Mass.
Malpel, S., A. Klarsfeld, and F. Rouyer. 2002. Larval optic nerve
and adult extra-retinal photoreceptors sequentially associate
with clock neurons during Drosophila brain development.
Development 129:1443–1453.
Nathans, J. 1987. Molecular biology of visual pigments. Annu.
Rev. Neurosci. 10:163–194.
Neufeld, T. P., R. W. Carthew, and G. M. Rubin. 1991. Evolution
of gene position: chromosomal arrangement and sequence
comparison of the Drosophila melanogaster and Drosophila
virilis sina and Rh4 genes. Proc. Natl. Acad. Sci. USA
88:10203–10207.
Osbrink, W. L., and M. K. Rust. 1985. Cat flea (Siphonaptera:
Pulicidae): factors influencing host-finding behavior in the laboratory. Ann. Entomol. Soc. Am. 78:29–34.
Palczewski, K., T. Kumasaka, T. Hori et al. (12 co-authors). 2000.
Crystal structure of rhodopsin: a G protein-coupled receptor.
Science 289:739–745.
Penny, N. D. 1977. A systematic study of the family Boreidae
(Mecoptera). Kans. Univ. Sci. Bull. 51:141–217.
Penny, N. D., and G. W. Byers. 1979. A check-list of the Mecoptera of the world. Acta Amazon. 9:365–388.
Posada, D., and K. A. Crandall. 1998. Modeltest: testing the model
of DNA substitution. Bioinformatics 14:817–818.
Resh, V. H., and R. T. Carde. 2003. Encyclopedia of insects. Academic Press, New York.
Ronquist, F., and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics
19:1572–1574.
Rust, M. K., and M. W. Dryden. 1997. The biology, ecology, and
management of the cat flea. Annu. Rev. Entomol. 42:451–473.
Shimizu, I., Y. Yamakawa, Y. Shimazaki, and T. Iwasa. 2001.
Molecular cloning of Bombyx cerebral ospin (Boceropsin)
1174 Taylor et al.
and cellular localization of its expression in the silkworm brain.
Biochem. Biophys. Res. Commun. 287:27–34.
Spaethe, J., and A. D. Briscoe. 2004. Early duplication and functional diversification of the opsin gene family in insects. Mol.
Biol. Evol. 21:1583–1594.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and
D. G. Higgins. 1997. The Clustal X windows interface: flexible
strategies for multiple sequence alignment aided by quality
analysis tools. Nucleic Acids Res. 24:4876–4882.
Towner, P., and W. Gartner. 1994. The primary structure of mantid opsin. Gene 143:227–231.
Whiting, M. F. 2002. Mecoptera is paraphyletic: multiple genes
and phylogeny of Mecoptera and Siphonaptera. Zool. Scr.
31:93–104.
Woolley, S., J. Johnson, M. J. Smith, K. A. Crandall, and D. A.
McClellan. 2003. TreeSAAP: selection on amino acid properties using phylogenetic trees. Bioinformatics 19:671–672.
Yang, Z. 1997. PAML: a program package for phylogenetic
analysis by maximum likelihood. Comput. Appl. BioSci.
13:555–556.
Yang, Z., R. Nielsen, N. Goldman, and A.-M. K. Pedersen. 2000.
Codon-substitution model for heterogeneous selection pressure at amino acid sites. Genetics 155:431–449.
Yasuyama, K., and I. Meinertzhagen. 1999. Extraretinal photoreceptors at the compound eye’s posterior margin in Drosophila
melanogaster. J. Comp. Neurol. 412:193–202.
Yokoyama, R., and S. Yokoyama. 1990. Convergent evolution
of the red- and green-like visual pigment genes in fish,
Astyanax facsiatus, and human. Proc. Natl. Acad. Sci. USA
87:9315–9318.
Yokoyama, S., A. Meany, H. Wilkens, and R. Yokoyama. 1995.
Initial mutational steps toward loss of opsin gene function in
cavefish. Mol. Biol. Evol. 12:527–532.
Claudia Schmidt-Dannert, Associate Editor
Accepted January 17, 2005