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Insect Biochemistry and Molecular Biology 35 (2005) 1367–1377

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Pteropsin: A vertebrate-like non-visual opsin expressed inthe honey bee brain

Rodrigo A. Velardea, Colin D. Sauera, Kimberly K. O. Waldena,Susan E. Fahrbachb, Hugh M. Robertsona,�

aDepartment of Entomology, University of Illinois at Urbana-Champaign, 320 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, USAbDepartment of Biology, Wake Forest University, Winston-Salem, NC 27109, USA

Received 27 July 2005; received in revised form 7 September 2005; accepted 8 September 2005

Abstract

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are

members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive

chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of

wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and

invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated

pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to

invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies

of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound

eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes

(AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis,

and therefore represents the presence of this vertebrate-like light-detecting system in insects.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Apis mellifera; G-protein-coupled receptors; Honey bee; Opsin; Pteropsin

1. Introduction

Opsins, the protein components of animal visualpigments, are members of the rhodopsin family of thesuperfamily of G-protein-coupled receptors (GPCRs)(Brody and Cravchik, 2000; Hill et al., 2002). Withinphotoreceptor cells, opsin proteins are coupled to light-sensitive, Vitamin A-derived chromophores; interactionsbetween opsins and their chromophores are primarydeterminants of the sensitivity of the chromophore to lightof different wavelengths (Briscoe and Chittka, 2001).Diversification of opsins during animal evolution thereforeprovided the molecular basis for wavelength-specificbehavior and color vision (Pichaud et al., 1999).

e front matter r 2005 Elsevier Ltd. All rights reserved.

mb.2005.09.001

ing author. Tel.: +1217 333 0489; fax: +1 217 244 3499.

ess: hughrobe@life.uiuc.edu (H.M. Robertson).

Many arthropods have excellent trichromatic colorvision, and the photoreceptor opsins of insects in particularhave been well-characterized, both in terms of their cellularfunction and sequence (Briscoe and Chittka, 2001).Sequence analysis has revealed that the opsins found inthe photoreceptor cells of the insect compound eye aredistinct from the vertebrate visual opsins (Hill et al., 2002;Briscoe and Chittka, 2001). Their closest known relativesare found among the relatively poorly studied flatwormand molluscan opsins (Hoffman et al., 2001; Hall et al.,1991; Hara-Nishimura et al., 1993).The discovery that many vertebrate opsins are expressed

in cells that are not canonical photoreceptors opened a newera in opsin research (Kojima and Fukada, 1999). Forexample, the pineal and Vertebrate Ancient (VA) opsins ofnon-mammals are similar (440% identical in amino acidsequence) to the vertebrate visual opsins, but pineal andVA opsins are not expressed in rods and cones (Okano

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et al., 1994; Soni et al., 1998). Their functions are largelyunknown, but may include providing non-visual photicinputs to biological clocks, such as the pineal pacemakerthat controls the rhythmic production of melatonin(Kasahara et al., 2002).

Melanopsin is another non-visual opsin of vertebratesthat, like the pineal opsins, is implicated as a source ofinputs to endogenous circadian pacemakers (Provencioet al., 1998). But melanopsins are notable in that theyresemble invertebrate visual opsins more closely than thevertebrate visual, pineal, or VA opsins (Bellingham et al.,2002). This discovery suggested that there were at least twomajor lineages of opsins in the common ancestor ofvertebrates and invertebrates (deuterostomes and proto-stomes): diversification in one of these lineages led to theinvertebrate visual opsins and the vertebrate melanopsins,while the other led to the vertebrate visual, pineal, and VAopsins.

Until recently, an invertebrate branch of this secondopsin lineage has been missing. The delay in its discoveryresults from its apparent evolutionary loss from theDrosophila lineage. In documenting the GPCR repertoireof the mosquito Anopheles gambiae, it was noted that thegenome of this insect encodes two related opsins (Agop11and 12) that appear to constitute this missing invertebrateopsin lineage (Hill et al., 2002). We also recognized arepresentative of this missing opsin lineage among a set ofabout 100 expressed sequence tags (ESTs) from the honeybee brain that encode proteins with orthologs in verte-brates and/or nematodes, but which have no apparentDrosophila orthologs (Whitfield et al., 2002). Here, wedescribe in detail this new vertebrate-like opsin lineage ininsects, which we propose to name pteropsin (‘‘wing’’opsin). We also show that, based upon a detailed analysisof the tissue distribution of the mRNAs encoding thecomplete set of honey bee opsins, pteropsin is unlikely toperform a visual role in insects. Insect pteropsin may beorthologous with another novel opsin recently describedfrom an annelid worm, which is also expressed in its brainand hence has a non-visual function (Arendt et al., 2004).

2. Methods

2.1. Sequence and phylogenetic analysis

A total of 60 opsin sequences representing the diversityknown from invertebrates and vertebrates were alignedusing CLUSTALX after removal of any long N- or C-terminal tails beyond the central conserved seven trans-membrane (TM) domains (Jeanmougin et al., 1998; Hall,2001; Chenna et al., 2003). The input FASTA and outputCLUSTALX files are available as supplementary files tothis paper, as is the analyzed PAUP* file. Phylogeneticanalysis was performed using only the central conservedseven TM domains with the minimum distance heuristicalgorithm in PAUP* v4.0b10 (Swofford, 2001), withdistances corrected for multiple changes in the past using

the maximum likelihood model in TREE-PUZZLE v5(Schmidt et al., 2002) and the BLOSUM62 amino acidexchange matrix (Henikoff and Henikoff, 1992).

2.2. Bees

Bee colonies were maintained in the field according tostandard commercial techniques at the University ofIllinois Bee Research Facility (Urbana, IL). Colonies inthis region are derived from a mixture of European races ofApis mellifera. Foragers were collected at the hive entrance.Newly eclosed bees were collected from brood framestransferred to a laboratory incubator (33 1C, 95% RF) 1 or2 days prior to completion of metamorphosis. Drones werecollected directly from the upper frames of a typicalcolony.

2.3. In situ hybridization

Probes for in situ hybridization were prepared for eachbee opsin from PCR products using specific primers withT3 and T7 promoters attached to the 50 and 30 primers,respectively (primers are listed in a supplementary file tothis paper). Synthesis of riboprobes and digoxigenin-labeling were performed by means of in vitro transcriptionusing Roche RNA Labeling Mix (Roche 1277073). Probeswere 400–700 bases in length. Brains to be used inhybridization studies were dissected from the head capsuleof cold-anesthetized bees in a small drop of bee saline(Huang et al., 1991). Dissected brains were immediatelytransferred to Bright Cryo-M-Bed embedding compound,frozen onto cryostat chucks using powdered dry ice,sectioned at 10 mm, and thaw-mounted onto FisherPlusslides. After overnight air-drying, sections were fixed in 4%paraformaldehyde, deproteinized with proteinase K, andtreated with acetic anhydride prior to hybridization with adigoxigenin-labeled riboprobe (1000 ng/ml) at 50 1C over-night in 50% formamide. Following posthybridizationrinses, sections were incubated with a sheep anti-digox-igenin-alkaline phosphatase antibody (Roche 1093274),treated with levamisole to block endogenous alkalinephosphatase activity, and developed in NBT/BCIP (VectorLaboratories). Developed slides were coverslipped withCrystalMount (Biomeda) or glycerol. Sense strand probeswere used as controls. All solutions used prior tohybridization were RNase free.

2.4. Data deposition

The following accession numbers in the Third PartyAnnotation database at GenBank have been assigned tothe novel opsins described in this study: conceptual cDNAof A. mellifera (Am) pteropsin BK005510, based on ESTsand our RT/PCR fragment AAT68000.1; Am pteropsingene, BK005511; AmBLop, BK005512; AmUVop,BK005513; AmLop1, BK005514; AmLop2, BK005515;BmLop1, BK005516.

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3. Results

3.1. Identification of novel genes encoding insect opsins

The complete sequence of the bee brain cDNA cloneBB170024A10F03 (Whitfield et al., 2002) from which theoriginal 50 EST was derived revealed that, althoughpotentially full-length with 234 bp of 50 untranslated region(UTR), it was from a mis-spliced mRNA from which alarge middle section was removed. This led to a shortenedand frameshifted ORF followed by a 128bp 30UTR.Amplification and sequencing of a 500 bp fragment acrossthis gap from reverse transcribed bee head RNA providedthe missing sequence (GenBank Accession # AAT68000.1).Comparison of this full-length cDNA sequence (TPAAccession # BK005510) with the draft honey bee genomeassembly v3.0 from the Baylor Human Genome Sequen-cing Center (http://www.hgsc.bcm.tmc.edu/projects/honeybee/) revealed a 9 kbp gene with five introns in themiddle of 546 kbp scaffold Group 9.2 (supplementary files,TPA Accession #BK005511). The original mis-splicedmRNA/cDNA was missing exons 4 and 5. We subse-quently identified a pteropsin gene in the Japanese 3X andChinese 6X draft assemblies of the silkmoth Bombyx mori

genome (Mita et al., 2004; Xia et al., 2004), where the sevenexons are spread across 6 and 3 contigs, respectively. (TheN-terminal is missing from the Chinese assembly; this geneannotation and those of the visual opsins from B. mori

cannot be submitted to the TPA database at GenBankbecause they are not based on contiguous sequences, butare available in our supplementary FASTA file). Wehave also annotated the pteropsin gene from theinitial draft genome assembly contigs for the red flourbeetle Tribolium castaneum (http://www.hgsc.bcm.tmc.edu/projects/tribolium/). No orthologous gene sequences couldbe identified using TBLASTN searches of the Drosophila

melanogaster euchromatic genome sequence, or among the7274,000 ESTs available at NCBI, or the available D.

melanogaster heterochromatic sequences at FlyBase, or thedraft assembly of the Drosophila pseudoobscura genome(Richards et al., 2005). The pteropsin gene thereforeappears to have been lost from the Drosophila lineagesometime between the split of mosquitoes from other fliesroughly 250Myr ago and the melanogaster/pseudoobscura

split roughly 25–50Myr ago (Gaunt and Miles, 2002;Richards et al., 2005).

Bee pteropsin is a 328aa protein with only short N- andC-terminal extensions beyond the central conserved 7 TMcore of approximately 300aa, and the T. castaneum proteinis similarly compact at 350aa. In contrast, the mosquitoAgop11 and 12 proteins have extended C-terminal tails,making them 460 and 433aa, respectively, as does the B.

mori protein at 560aa. Bee pteropsin has 45% amino acididentity in the 7 TM region to these two Anopheles

relatives, and 31–35% identity to the vertebrate encepha-lopsins, Takifugu Teleost multiple tissue (TMT), andAmphioxus opsins 4 and 5, its closest relatives among the

other opsins. In the central conserved 7 TM core, the insectpteropsins retain all the major signature features of animalopsins, including an ER(F/Y/W) motif near the cytoplas-mic side of TM3 involved in G-protein linkage. Thepresence of a glutamic acid in this motif rather than anaspartic acid allies insect pteropsin with the vertebrateopsins. Pteropsin has the two expected conserved cysteinesin extracellular loops 1 and 2 involved in a disulfide bond.Pteropsin has a relatively short intracellular loop 3, unlikethe melanopsins and invertebrate visual opsins, but like thevertebrate opsins. Pteropsin has the conserved lysine inTM7 implicated as the chromophore binding site distinc-tive to opsins. Finally, after the highly conserved NPIIYdomain in TM7, the immediate cytoplasmic region ofMNTQFR is more similar to the vertebrate opsins than tomelanopsin or the invertebrate opsins.For comparative purposes, we also examined the visual

opsin genes from bee, silkmoth, and flour beetle. Three beeopsins were previously described: a blue-sensitive opsinAmBLop (TPA Accession #BK005512), a UV-sensitiveopsin AmUVop (TPA Accession #BK005513), and a long-wavelength-sensitive opsin AmLop (TPA Accession#BK005514) (Townson et al., 1998; Chang et al., 1996).We propose to rename the latter AmLop1 because we haveidentified a second opsin of this kind that we nameAmLop2 (TPA Accession #BK005515): independent iden-tification of the AmLop2 lineage in other Hymenoptera hasrecently been reported (Spaethe and Briscoe, 2004). In thesilkmoth genome, we identified homologs of each of thesethree insect visual opsins (BmBLop, BmUVop, BmLop1,and BmLop2) as well as a homolog of the Drosophila Rh7(Papatsenko et al., 2001) and Anopheles Op10 (Hill et al.,2002) opsins of unknown wavelength sensitivity (BmU-nop). Only BmLop2 is intact, full-length, and encoded by asingle contig: TPA Accession # BK005516. Each of theothers is encoded by multiple contigs (and the N-terminalexons could not be confidently identified for the BmUVopand BmUnOp in the draft genome assemblies), and theytherefore cannot be submitted to the Third Party Annota-tion database at GenBank. They are available in thesupplemental FASTA and alignment files that accompanythis paper. Only two visual opsins are apparent in the firstdraft assembly of the T. castaneum genome, a single longwavelength opsin and the UV opsin (TcLop and TcUVop).An ortholog of the DmRh7/AgOp10 lineage could not beidentified in the bee-assembled genome sequence or theunassembled reads, and has therefore most likely been lostfrom the bee genome, although it remains formally possiblethat it is encoded by an unclonable region of the beegenome.

3.2. Phylogenetic analyses

Phylogenetic analysis reveals that the Bombyx, Apis, andTribolium visual opsins cluster confidently with theirrespective Drosophila, Anopheles, and other insect relatives(Fig. 1). Certain visual opsin lineages have expanded in

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Fig. 1. Phylogenetic tree of the animal opsins showing clustering of the insect pteropsin lineage with the vertebrate encephalopsin, TMT, visual, pineal,

and Very Ancient (VA) opsins, as well as the annelid Platynereis ciliary opsin. The opsins added in this manuscript are highlighted with bold underlining,

using Am for Apis mellifera, Bm for Bombyx mori, and Tc for Tribolium castaneum. The different opsin lineages are indicated on the right. Insect and

related invertebrate opsins are differentiated in red to green shades, while vertebrate and related opsins are differentiated in blue shades. Support for the

major branches is indicated as percentage of 1000 bootstrap replicates of neighbor joining using uncorrected distances, 1000 bootstrap replicates of

heuristic parsimony analysis, and 25,000 maximum likelihood quartet puzzling steps in TREE-PUZZLE v5. The human and Takifugu proteins were

chosen to represent the vertebrate melanopsin, encephalopsin, RGRopsin, peropsin, and neuropsin lineages, while representative sequences were used for

the vertebrate visual, pineal, parapineal, and VA opsins. Many additional insect visual opsins are known, and in particular the various lepidopteran opsins

cluster confidently with the appropriate new Bombyx mori opsins in this tree, but are not included because of space limitations.

R.A. Velarde et al. / Insect Biochemistry and Molecular Biology 35 (2005) 1367–13771370

particular insects. Examples are the duplication of the UV-sensitive opsins in Drosophila (DmRh3 and DmRh4), andespecially the apparently independent and often multipleduplications of the long-wavelength-sensitive opsins inlepidopterans (Briscoe, 2001; Spaethe and Briscoe, 2004).

Strikingly, the pteropsins do not cluster with the insectvisual opsin lineages. Therefore, phylogenetic analysis of

all major available opsin lineages was performed todetermine the relationships of the insect pteropsin lineage,using corrected distance methods (Fig. 1). The tree wasrooted with the vertebrate RGRopsin (Shen et al., 1994),peropsin (Sun et al., 1997), and neuropsin (Tarttelin et al.,2003), plus three of the six opsins described from thecephalochordate Amphioxus (Koyanagi et al., 2002). In this

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tree, the insect pteropsin lineage clusters confidently withthe vertebrate visual, pineal, VA, and TMT opsins andencephalopsin (Blackshaw and Snyder, 1999), as well astwo of the Amphioxus opsins. The level of support for thisclustering is comparable to that for the clustering of thevertebrate melanopsin with the invertebrate visual opsins.In particular, the insect pteropsin lineage appears to bemost closely related to the TMT opsin of the pufferfishTakifugu rubripes (Moutsaki et al., 2003), encephalopsin,and the Amphioxus opsins 4 and 5, all of which haveunknown functions.

An opsin from the annelid worm Platynereis has beenproposed as an ortholog of the insect mosquito AgOp11and 12 pteropsins (Arendt et al., 2004). We agree with thisassessment that the annelid opsin is a similarly vertebrate-like opsin in an invertebrate; Arendt and colleaguesconsider this to indicate the presence of a vertebrate-likeciliary-type eye in the brains of invertebrates. However, ourphylogenetic analysis does not confidently identify thisannelid opsin and the insect pteropsin as orthologouslineages. As shown in Fig. 1, the corrected distance analysisyields a trifurcation between the annelid opsin, the insectpteropsins, and the vertebrate encephalopsin, fish TMT,and two Amphioxus opsins. Maximum parsimony andlikelihood analyses place this annelid opsin even morebasally, equivalent to these and the vertebrate visual/pineal/VA lineages. Thus it is possible that, like verte-brates, invertebrates contained more than one ciliary-opsinlineage, with different lineages persisting in annelids andinsects.

3.3. Intron locations

This relationship of the insect pteropsin lineage withthese vertebrate opsin lineages is further supported byanalysis of their gene structures. Intron locations and

Fig. 2. Gene structure for the insect pteropsins compared with the vertebr

melanopsin and invertebrate visual opsins; and the vertebrate RGRopsin, pero

domain 300 amino acid core are shown as numbers indicating their phases. Co

precisely shared position and phase. The Foster # line indicates the numbers giv

the insect visual opsins, multiple novel intron locations are present in the diff

phases with respect to encoded amino acids have com-monly been used to illuminate opsin and other gene familyevolution (Tarttelin et al., 2003; Bellingham et al., 2003). Inparticular, intron analysis has been used to support theconclusion that vertebrate melanopsin is most closelyrelated to the invertebrate opsin lineages because theyshare three homologous intron locations. Fig. 2 shows thatthe insect pteropsin lineage, specifically the Anopheles,Bombyx, and Tribolium genes, shares at least three intronlocations with the vertebrate visual, pineal, and VA opsinsand encephalopsin, out of four highly conserved intronlocations in the vertebrate opsins commonly numbered 1–4and referred to here as Foster #s (Bellingham et al., 2003).It is also possible that the central phase 2 intron in theAnopheles, Bombyx, and Tribolium pteropsin genes ishomologous with the phase 2 second intron in thevertebrate opsins. This intron is not in precisely the sameconserved codon, so intron slippage would have to beinvoked to consider this to be a homologous intronlocation. In contrast, the insect pteropsin lineage sharesno homologous intron locations with either melanopsin orthe insect visual opsins, nor does it share intron locationswith the outgroup of peropsin, RGRopsin, and neuropsin,whose clustering is supported by their sharing of at leastthree or four unique intron locations (Tarttelin et al.,2003). The Anopheles, Bombyx, and Tribolium pteropsingenes share another two homologous introns in phase 0and 2, while Bombyx has an additional unique phase 0intron location. Remarkably, the Apis pteropsin gene hasfive unique intron locations that are not shared with anyother known opsin gene, implying that it has lost fiveancestral introns and acquired five new ones. The annelidciliary-opsin cannot be included in this analysis because itis only known from a cDNA sequence, but we predict thatits gene will contain at least the three intron locationsshared by the insect pteropsins and the vertebrate opsins.

ate encephalopsin, TMT, visual, pineal, and VA opsins; the vertebrate

psin, and neuropsin genes. Intron locations relative to the conserved 7 TM

lored lines connect introns that are considered to be homologous based on

en to conserved intron locations by Bellingham et al. (2003). In the case of

erent lineages, and all are conflated in the one schematic shown.

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Fig. 3. Distribution of mRNAs encoding AmLop1, AmLop2, AmBLop,

and AmUVop in the retina of the compound eye and in the

photoreceptors of the dorsal ocelli. Left panels show the presence or

absence of in situ hybridization signal in the retina (a, c, e, g); right panels

show the presence or absence of signal in the ocelli (b, d, f, h). Non-visual,

non-opsin screening pigments are visible at the distal margin of the retina

in the compound eye in all sections; these non-opsin pigments are visible in

freshly dissected tissue and remain visible throughout the hybridization

process. AmLop1 and AmLop2 do not overlap in distribution, while

AmUVop is expressed in both the compound eyes and the ocelli. (a, b)

Probe for AmLop1. (c, d) Probe for AmLop2. (e, f) Probe for AmBLop.

(g, h) Probe for AmUvop. Scale bar ¼ 200mm (a, b, c) or 100mm (d, e, f, g,

h). For orientation, see Fig. 6a.

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3.4. In situ hybridization

As a first step towards understanding the function ofpteropsin, we undertook extensive in situ hybridizationstudies of the expression pattern of pteropsin mRNAin the heads of honey bees using digoxigenin-labeledriboprobes. The visual system of the honey bee is well-characterized in terms of both structure and function. Thehoney bee is a UV-blue-green trichromat, which is typical ofmany insects that are generalist flower visitors (Briscoe andChittka, 2001). The compound eye of the worker honey beeis composed of about 5500 facets, or ommatidia (Lehrer,1998). Each ommatidium contains nine receptor cells. Thehoney bee head also has three dorsal photoreceptive organs,the ocelli. Each of the three ocelli has a single flattenedspheroidal lens and about 800 receptor cells (Toh andKuwabara, 1974). Electrophysiological studies have revealedthat there are three uniformly distributed receptor types inthe compound eye, representing three different spectralsensitivities: green (544nm), blue (436nm), and ultraviolet(344nm) (Peitsch et al., 1992). Each ommatidium has beenreported to contain four long-wavelength (green) receptorcells, two medium-wavelength (blue) receptor cells, and threeshort-wavelength (uv) receptor cells (Peitsch et al., 1992). Thehoney bee ocellus contains two types of photoreceptor,reported to be maximally sensitive at 499nm and335–340nm (Goldsmith and Ruck, 1958; Mizunami, 1994).Honey bee workers are all female; male bees, or drones, alsohave trichromatic vision, but their compound eye iscompartmentalized into distinct dorsal and ventral regionswith different spectral sensitivities (Menzel et al., 1991).

For purposes of comparison with pteropsin, we firstexamined the tissue distribution of the mRNAs encodingthe four visual opsins identified in bees, including the newlydiscovered AmLop2 (Fig. 3). The previously clonedAmLop1 is abundantly expressed in all ommatidia of theworker compound eyes (Fig. 4), verifying that this opsin islikely the major long wavelength receptor of the honey bee(with a minimum of 4/9 cells expressing the transcript ineach ommatidium). AmBLop and AmUVop were alsoexpressed throughout the worker retina, but more sparselythan AmLop1, consistent with reports from other insects(White et al., 2003; Arikawa, 2003; Spaethe and Briscoe,2005). A more detailed analysis would be required toaddress the question of possible heterogeneity of ommati-dial composition in the worker retina.

AmLop2 was not expressed in any receptor cells withinthe compound eye. Examination of the ocelli, however,revealed that the likely functional role of AmLop2 is as thelong wavelength opsin of these dorsal photoreceptivestructures (Fig. 3). There was no overlap in the distributionof AmLop1 and AmLop2, leading us to designate AmLop2as ocellus-specific. The uv-sensitive opsin of the honey bee,AmUVop, however, is expressed by photoreceptor cells inboth the compound eyes and the ocelli. There was noexpression of AmLop1, AmLop2, AmUVop, or AmBLopin any region of the brain (Fig. 5).

The pattern for the four visual opsins of the honey bee isin sharp contrast to the pattern for Apis pteropsin. Noexpression of pteropsin was detected in the compound eye

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Fig. 4. In situ hybridizations of transverse and sagittal sections through the compound eye of the honey bee. In all panels, the top edge is dorsal and the

distal retina is to the right. (a) Transverse section hybridized with the probe for AmLop 1. Compare with lower magnification section presented in Fig. 3.

(b) Sagittal section hybridized with same probe as in (a) Circled region indicates a cross-section through a single ommatidium, revealing a hybridization

signal in four retinula cells (R2, R3, R6, and R7). Note that the compound eye of the honey bee is a curved structure, so that the entire complement of

AmLop1-positive retinula cells is not visible for most ommatidia shown in this section. (c) Transverse section hybridized with the probe for AmBLop. (d)

Sagittal section hybridized with same probe as in (c) Circled region indicates a cross-section through a single ommatidium, revealing hybridization in two

retinula cells (R4 and R8). These figures suggest that a minimum of 4/9 and 2/9 retinula cells in many ommatidia of the worker bee compound eye express

Lop1 and BLop, respectively, with spatial heterogeneity of ommatidial types within the compound eye of the honey bee a possibility. Scale bars ¼ 40mm.

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or ocelli of more than 50 bee brains processed for in situhybridization with anti-sense probes. Instead, a smallcluster of cells located in the brain at the boundarybetween the lateral protocerebrum and the medulla of theoptic lobes was consistently labeled in our in situhybridization studies (Fig. 6). Approximately 12 suchsmall cells were labeled on each side of the brain. We alsodetected hybridization signal in 3–4 large cells at the dorsalmidline of the protocerebrum. No signal was produced inany case on adjacent control sections hybridized to sensestrand riboprobes.

3.5. Sex difference in honey bee opsin expression

The compound eye of the drone honey bee can bedivided into distinct dorsal and ventral areas on the basisof differences in facet diameter, interommatidial angles,rhabdom length, and distribution of non-visual screeningpigment granules (Menzel et al., 1991). Previous reportshave indicated that drones have predominantly longwavelength receptors in the ventral area, and only UVand blue receptors in the dorsal area. A male-specificdistribution of the visual opsins was confirmed in thepresent study (Fig. 7), but the distribution of pteropsinmRNA was identical in workers (females) and drones (datanot shown).

4. Discussion

Identification of pteropsin in non-drosophilid insectssignificantly improves our understanding of the evolutionof the animal opsins. Our analyses strongly support theconclusion of Hill et al. (2002) and Arendt et al. (2004) thatthere were at least two lineages of opsins in the commonancestor of insects and vertebrates. One led to the visualopsins of insects and molluscs and melanopsin in verte-brates, while the other led to the visual opsins, the pinealopsins, the VA and TMT opsins, and encephalopsin ofvertebrates, and the pteropsin lineage in insects and theciliary-opsin protein in annelids. The RGRopsin, peropsin,and neuropsin lineages (along with three Amphioxus

opsins) appear to represent an equally old third lineageof animal opsins, based largely on their unique set of intronlocations (Fig. 2), but we have not been able to identifyhomologs of these chordate opsin lineages in the availableinsect genome sequences.The four visual opsins encoded by the bee genome

appear to account in full for the well-documented spectralsensitivity of honey bees to UV light, blue wavelengths, andlong wavelengths. As is the case in all other insectsexamined to date, the long-wavelength opsin genes haveduplicated, yielding two opsins (AmLop1 and AmLop2)that likely have overlapping spectral sensitivities. That

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Fig. 6. Location of mRNA encoding Apis pteropsin in the worker bee brain. (a) Diagram of the honey bee brain, indicating the location of the compound

eyes (blue), the ocelli (yellow), and the cells expressing pteropsin mRNA (purple). Magenta boxes correspond to panels b, c, and d of this figure, and are

labeled accordingly. (b) A cluster of approximately 12 cells located between the lateral protocerebrum and the medulla of the optic lobes labeled with the

anti-sense probe for Apis pteropsin. The optic lobe is to the right and the lateral protocerebrum is to the left; the mushroom body lateral calyx is dorsal. (c)

Four cells at midline of the dorsal protocerebrum expressed pteropsin mRNA. (d) No cells in the compound eye hybridized with the anti-sense probe for

Apis pteropsin Scale bar ¼ 100mm.

Fig. 5. Absence of hybridization signal for visual opsins in the brain of the honey bee. (a) Transverse section hybridized with probe for AmLop1. (b)

Transverse section hybridized with probe for AmLop2. (c) Transverse section hybridized with probe for AmBLop. (d) Transverse section hybridized with

probe for AmUVop. Scale bars ¼ 400mm.

R.A. Velarde et al. / Insect Biochemistry and Molecular Biology 35 (2005) 1367–13771374

these two related opsins have different functions is revealedby their non-overlapping distribution: AmLop1 is ex-pressed solely in the compound eye and therefore isresponsible for long wavelength-mediated behaviors ofthe bee that are mediated by the compound eyes alone,specifically the use of color vision at flowers and recogni-tion of the immediate surroundings of the hive entrance(Menzel and Muller, 1996). AmLop2 is expressed solely in

the ocelli, and therefore is only involved in the ocelli-dependent timing of the initiation and termination offoraging for the day (Mizunami, 1994). We predict that arecently described novel long wavelength opsin in a bumblebee (Bombus impatiens) is likely also to be an ocellus-specific opsin, comparable to AmLop2 (Spaethe andBriscoe, 2004). These hymenopteran ocellar opsins arenot orthologous to the Drosophila ocellus-specific opsin,

ARTICLE IN PRESS

Fig. 7. Localization of mRNA encoding AmLop1, AmBLop, and AmUVop in the drone compound eye. The drone retina is compartmentalized into

distinct dorsal and ventral regions with different spectral sensitivities. (a) Expression of AmLop1 mRNA in the ventral region of the retina only. (b)

Expression of AmBLop mRNA abundantly in the dorsal region and sparsely in the ventral region of the retina. (c) Expression of AmUVop primarily in

the dorsal region of the retina. Scale bar ¼ 100mm.

R.A. Velarde et al. / Insect Biochemistry and Molecular Biology 35 (2005) 1367–1377 1375

Dmrh2. Instead, Dmrh2 is a member of a pair ofduplicated long wavelength opsin genes that have noknown orthologs in other insects (Pollock and Benzer,1988; Mizunami, 1994). Unlike the lepidopteran B. mori,bees do not appear to have an ortholog of the DmRh7/Agop10 opsin of unknown wavelength sensitivity, so weare unable to provide any insight into the role of thisapparently visual opsin.

The localization of Apis pteropsin mRNA to brain leadsus to speculate that pteropsin is involved in the regulationof circadian rhythms in non-drosophilid insects. Invertebrates, it is now accepted that regulatory input fromlight is mediated by multiple receptor proteins, includingvisual and pineal opsins and melanopsin (Beaule et al.,2003; Jenkins et al., 2003; Hattar et al., 2003; Panda et al.,2003) as well as the two mammalian cryptochromes (vanGelder, 2003). In Drosophila, the major regulatory inputfrom light is via the Drosophila cryptochrome protein,which is a paralog of the vertebrate cryptochromes (Emeryet al., 1998, 2000), although other pathways also contribute(Helfrich-Forster et al., 2001). Interestingly, we have beenunable to identify an ortholog of the Drosophila crypto-chrome in the draft bee genome, and its partner, timeless,also appears to be missing from the honey bee. Bees insteadencode an ortholog of the two mammalian cryptochromesand the so-called mammalian timeless gene, which isactually an ortholog of the Drosophila timeout or Tim2protein (Bloch G., SF, and HMR unpublished results).Bees therefore appear to have molecular machinery forregulation of circadian rhythms by light that is moresimilar to that of vertebrates than that of other insects, andpteropsin may play an important role in this. Thelocalization of mRNA encoding pteropsin to the lateralmargin of the protocerebrum is highly suggestive of a rolein the brain clock of insects. Cells expressing the period-and pigment dispersing hormone-immunoreactivity arefound in this region of the honey bee brain (Bloch et al.,2003), and numerous studies in a variety of insects haveindicated the importance of this brain region for regulationof circadian rhythms of locomotor activity (Helfrich-Forster, 1995; Petri and Stengl, 1997; Renn et al., 1999).

Our finding that Apis pteropsin is restricted in itsdistribution to cells in the brain is somewhat similar toreports that BmLop2 (boceropsin) of the silkworm, B.

mori, is expressed in a small number of cells in the larvalsilkworm brain (Shimizu et al., 2001) and a report that aUV opsin is expressed in protocerebral neurons in thebumblebee brain (Spaethe and Briscoe, 2005). Note,however, that both of these opsins are visual opsins, notpteropsin (Fig. 1). Expression of pteropsin in the brain isalso consistent with demonstration that the annelid ciliaryopsin is expressed in the brain, in a structure considered tobe homologous to the ciliary eyes of vertebrates (Arendtet al., 2004). Non-drosophilid insects can therefore also beconsidered to have such a light-sensitive structure, albeitwithout obvious morphological correlates, in their brains.Pteropsin is one of about 100 examples of bee brain

ESTs that encode proteins with vertebrate and/or nema-tode orthologs, but which appear to be missing from thegenome of D. melanogaster (Whitfield et al., 2002; HMRunpublished). These proteins have clearly been importantfor much of animal evolution and often are more ancientthan animals, yet drosophilid flies have lost these genes inthe past 7300Myr. This may have occurred either becauseflies no longer have use for the functions of the missinggenes, or because they have substituted for them with otherproteins (e.g. Orian et al., 2003). Examination of theDrosophila genome has previously revealed many suchexamples in comparison with yeast, nematodes, andmammals (Adams et al., 2000; Rubin et al., 2000) includingperhaps most famously the replacement of telomeres andtelomerase by regular insertion of HET-A and TARTfamily retrotransposons (Biessmann and Mason, 2003;Pardue and DeBaryshe, 2003). The 7100 examplesobtained from comparison of the bee brain ESTs withthe Drosophila genome are particularly striking becausethey represent cases where orthologs are already known toexist in other insects. This means that these 7100 genes/proteins, including pteropsin, cannot be studied in thetractable molecular genetic model animal system providedby Drosophila. Their presence in other insects providesstrong impetus for the continuing development of genomic

ARTICLE IN PRESSR.A. Velarde et al. / Insect Biochemistry and Molecular Biology 35 (2005) 1367–13771376

resources for other insects, including not only the honeybee but also the flour beetle T. castaneum and other insects(Robertson, 2005). Knockdown of pteropsin expression inthis and other insects using RNA interference, along withadditional studies of its expression patterns during devel-opment and over circadian time, will allow elucidation ofits role in insect behavior and evolution.

Acknowledgements

We thank Gene E. Robinson for access to the Universityof Illinois Bee Research Facility, Karen Pruiett forbeekeeping, and Takeuchi Hideaki for helpful commentson in situ hybridization on bee brains. This research wassupported by NIH Grant AI56081 (to HMR), NationalScience Foundation Grant IBN-0315552 (to SEF), and aUniversity of Illinois University Scholar Award (to SEF).

Appendix A. Supplementary Material

The online version of this article contains additional supple-mentary data. Please visit doi:10.1016/j.ibmb.2005.09.001.

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