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First published online June 6, 2005
Journal of Experimental Biology 208, 2347-2361 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01634

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Molecular characterization and expression of the UV opsin in bumblebees: three ommatidial subtypes in the retina and a new photoreceptor organ in the lamina

Johannes Spaethe^* and Adriana D. Briscoe

Comparative and Evolutionary Physiology Group, Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA

View larger version (69K): [in a new window]   Fig. 1. The nucleotide sequence, including the untranslated regions, and the deduced amino acids of the UV opsin cDNA. Unbolded numbers are for nucleotides, and bolded numbers are for amino acids. The nucleotide sequence to which the gene-specific reverse primer binds is boxed. The location of the degenerate primer used in 3' RACE is underlined. Grey boxes indicate the amino acid residues that are conserved between B. impatiens and P. glaucus within the peptide domain used to generate the anti-UV antibody (Lampel et al., in press). The first two boxes indicate candidate glycosylation sites (NXS/T) (O'Tousa, 1992), and the third box indicates the lysine (K) that confers UV sensitivity (Salcedo, 2001; Salcedo et al., 2003). A pair of conserved cysteines (C) that form an essential disulfide bridge in bovine rhodopsin (Karnik et al., 1988) are located at amino acid positions 120 and 197 (boxed). The site of chromophore attachment (amino acid 318; Bownds, 1967) is also boxed. A highly conserved motif (E/DR) involved in G-protein binding/activation (Baldwin, 1997) is present at amino acid positions 144 and 145. GenBank accession number: AY655163.

View larger version (31K): [in a new window]   Fig. 2. Phylogeny of insect UV opsins based on 1st and 2nd nucleotide positions and the Tajima-Nei model of evolution with complete deletion of gaps. The A. mellifera blue opsin sequence was used as an outgroup. Bootstrap values based on 500 replications are shown above nodes for 1st and 2nd positions and below nodes for amino acids (italics). Branch length indicates nucleotide substitutions per site. GenBank accession numbers for sequences used in our analysis are as follows: Anopheles gambiae GPRop8 (AAAB01008960 gi|19612143: 3070327-13075141), Apis mellifera (UV, AF004169; blue, AF004168), Bicyclus anynana (AF484248), Camponotus abdominalis (U32502), Cataglyphis bombycina (U32501), Drosophila melanogaster (Rh3, M17718; Rh4, M17719), Drosophila pseudoobscura (Rh3, X65879; Rh4, X65880), Manduca sexta (L78081), Megoura viciae (AF189715), Papilio glaucus (AF077191), Papilio xuthus (AB028218).

View larger version (26K): [in a new window]   Fig. 3. UV opsin immunoreactivity on a western blot of crude protein extracted from retinas of four B. impatiens female workers with and without peptide treatment. (A) Lane 1, MagicMark protein standard (Invitrogen). Lane 2, anti-UV opsin antibody. The primary antibody concentration was 1:1000 (0.00453 nmol ml–1) and the membrane was incubated at 4°C overnight. The specific opsin immunoreactive band with an approximate molecular mass of 41 kDa is indicated by an arrow. Two smaller bands, which might represent alternatively glycosylated states of the UV opsin protein and/or proteolytic breakdown products, are present at approximately 30 and 29 kDa (arrows). Lane 3, anti-UV opsin antibody + UV opsin peptide. The antibody (0.00453 nmol ml–1) was mixed with 0.453 nmol ml–1 peptide for 1 h before incubation with the membrane at 4°C overnight. (B) Same membranes as above except stained with Coomassie blue to visualize total protein present on blots.

View larger version (118K): [in a new window]   Fig. 4. UV opsin immunoreactivity (-ir) in the retina, visualized with a secondary antibody attached to Alexa Fluor 488 (green) or Cy3 (red). Cell nuclei were stained with DAPI (blue). (A) Frontal section showing the distally twisted rhabdomeres with UV opsin-ir (arrow). Note the strong autofluorescence of the crystalline cones (cc). (B) Tangential section of the central part of the eye showing three types of ommatidia, those with zero (double arrowhead), one (single arrowhead) and two (arrows) UV opsin-ir rhabdomeres. An electron microscopy study of the bumblebee fused rhabdom indicates that the rhabdomeres of R1, 2 and 5,6 are physically separated by the rhabdomeres of the other photoreceptor cells (Meyer-Rochow, 1981). (C) Semi-tangential section showing the heterogeneous UV opsin-ir in the rhabdomeres of different ommatidia. Ommatidium with one (arrowhead), two (arrows) and no (double arrowhead) UV opsin-ir retinula cells. Note the UV opsin-ir vesicles (flecks) in the cell bodies adjacent to the UV opsin-ir rhabdoms. (D) Tangential section showing UV opsin mRNA expression in the cell bodies of R1 and R5 photoreceptor cells, visualized with antisense digoxigenin-labeled riboprobes and an alkaline phosphatase-conjugated anti-digoxigenin antibody. Dotted lines indicate the boundaries of individual ommatidia. Three ommatidial types are shown, those with zero, one or two UV opsin mRNA-expressing photoreceptor cells. (E) Diagram of a longitudinal view of a single bee ommatidium showing the cornea (c), crystalline cone (cc), primary pigment cells (darkly speckled), secondary pigment cells (lightly speckled) and retinular cells (white) surrounding the fused rhabdom (hatched). Tangential views of the three ommatidial types observed in the Bombus retina with respect to UV opsin-ir (purple) are shown. Numbers indicate retinula cells R1–8. The R1 and R5 cells are UV opsin-ir, following the convention of Menzel and Blakers (1976). Note that in ommatidia in which only one rhabdomere is UV opsin-ir, that cell can be either the R1 or R5 cell (only one shown). (F) Longitudinal section of the retina showing the more densely packed UV opsin-ir dorsal rim area (Dra) (arrow). La, lamina; Re, retina. Scale bars, 20 µm (A,C); 10 µm (B); 25 µm (D); 50 µm (F).

View larger version (62K): [in a new window]   Fig. 5. UV opsin-ir in the dorsal ocelli of a worker bee (Alexa Fluor 488; green). Nuclei were stained with DAPI (blue). (A) Tissue was fixed for 45 min at 4°C and then 20 min at room temperature in 4% paraformaldehyde before being sectioned on a cryostat. Sections (12 µm) were incubated with a 0.023 nmol ml–1 dilution of the anti-UV opsin antibody. Arrows indicate UV opsin-ir rhabdomeres in some but not all photoreceptor cells in both ocelli. The median ocellus is on the left and one of the lateral ocelli is on the right. Electron microscopy studies of the dorsal ocelli in the worker bee, Apis mellifera, have previously identified both planar and non-planar rhabdoms (Toh and Kuwabara, 1974). The arrow on the right points to a planar-like rhabdom, and on the left to a non-planar rhabdom. The rhabdoms lie directly beneath a layer of corneagenous cell nuclei (cn). The cell bodies of the retinular cells subtend the rhabdomeres and are filled with pigment granules (pg) (not shown). Arrowheads indicate strongly autofluorescing cuticle. (B) Adjacent section incubated with 0.023 nmol ml–1 dilution of the anti-UV opsin antibody mixed for 30 min with 2.3 nmol ml–1 peptide prior to application to the slides and parallel processing with the section shown in A. Arrows indicate the location of the rhabdomere layer, where the UV opsin protein immunoreactivity has been abolished. Arrowheads indicate the strongly autofluorescing cuticle. The corneagenous cells of the ocelli, like that of the compound eye, are themselves also autofluorescing. cn, nucleus of the corneagenous cell; pg, pigment granule layer of retinular cells. Scale bar, 50 µm.

View larger version (135K): [in a new window]   Fig. 6. (A–E) UV opsin-ir in the retina and optic lobe (Cy3, red). Nuclei were stained with DAPI (blue). (A) UV opsin-ir of the most proximal part of the lamina (C-layer), adjacent to the first optic chiasm (arrow). (B) Adjacent section to A processed with the primary antibody omitted. Strong autofluorescence of the cuticle and trachea are indicated here and in C by arrowheads. (C) UV opsin-ir in the retina and the lamina. The width of the labeled neuropil increases towards the lateral edges of the lamina (bottom left). (D) Perikarya cluster at the ventral edge of the lamina (arrow). The maximum number of labeled cells we could detect in a single section was 15. (E) The column-like structure of the UV opsin-ir neuropil in the proximal part of the lamina. Two layers of nuclei border the labeled neuropil (blue). The structure of UV opsin-ir columns found in our study shows high similarity with the optical cartridges described at the same location in the honeybee by Sinakevitch et al. (2003) using reduced silver staining. Arrow indicates cell layer comprising glial nuclei or nuclei of the monopolar cells that communicate with the long photoreceptor cell fibers. (F) UV opsin mRNA detected by in situ hybridization using an antisense riboprobe. Hybridization signal was found in the perikarya of the two layers bordering the lamina columns (see also arrow in E) in between which the UV opsin-ir was detected and in perikarya at the distal rim of the medulla (arrowhead), adjacent to the first optic chiasm. (Inset) PER-like-ir (green) was found in the perikarya layer that is located adjacent to the distal rim of the medulla in the first optic chiasm and in the 3rd layer of the lamina (arrow). Nuclei are stained with DAPI (blue). Arrows in F and in the inset indicate similar location. D, dorsal; 1. Oc, first optic chiasm; La, lamina; Me, medulla; Re, retina; V, ventral. Scale bars, 50 µm (A,B,C,F); 20 µm (D,E).

View larger version (113K): [in a new window]   Fig. 7. UV opsin-ir (Alexa Fluor 488, green or Cy3, red) in perikarya along the edge of the medulla (Me) and the protocerebrum (Pr), within the central body (CB) and antennal lobes (AL), and PER-ir in the AL. Nuclei are stained with DAPI (blue). (A) UV opsin-like ir was found in a cluster of approximately 10 perikarya located at the ventral edge of the medulla (arrow). Arrowhead indicates autofluorescing cuticle. (B) An adjacent section to A except that the antibody was incubated prior to application with the peptide against which it was raised for 1 h at room temperature (see Materials and methods). Arrow indicates the location of perikarya in which the UV opsin-ir is undetectable. (C) Sketch of a posterior section through the optic lobe of a worker bee showing the location of the UV opsin-ir perikarya (red asterisk) shown in A. (D,E) UV opsin-ir (Cy3, red) of a cell cluster between the dorsal-median rim of the lobula (Lo) and the lateral calyces of the mushroom bodies. These cells are in a similar location to pigment dispersing hormone (PDH)-ir cells in the honeybee (Bloch et al., 2003). The arrow in D is pointing to the cell cluster and in E to an individual cell. (F) Sketch of an anterior section of a worker bee brain showing the location of the UV opsin-ir cell cluster (red asterisk) shown in D and E, which is located between the anterior lateral protocerebrum and the optic lobe. (G) Frontal section of the antennal lobe. Strongest UV opsin-ir was found in the core region of each glomeruli. (H) UV opsin-ir (Alexa Fluor 488, green) was found in the upper part of the central body. (I) Adjacent section to H, processed with the primary antibody omitted. (J) PER-ir in the deuterocerebrum. Strongest labeling was found in the lateral cell cluster (arrow). These perikarya belong to local interneurons and projection neurons (Rybak and Menzel, 1993). Dorsal and ventral clusters exhibit weaker labeling. (K) Same section as in J, showing nuclei of all antennal lobe cell bodies labeled by DAPI (blue). CB(u), central body, upper region; CB(l), central body, lower region; Lo, lobula; Me, medulla; Pr, protocerebrum; OL, optic lobe; lCa, lateral calyx; mCa, median calyx; D, dorsal; V, ventral. Scale bars, 20 µm (A,B,E, G,H,I); 50 µm (D,J,K).

View larger version (39K): [in a new window]   Fig. 8. Schematic frontal view of a bumblebee brain showing the location of UV opsin-ir (left) and PER-ir (right), in grey, found in this study. AL, antennal lobe; CB, central body; LA, lamina; lMB, mushroom body – lateral calyx; LO, lobula; mMB, mushroom body – median calyx; ME, medulla; PER, period protein; PR, protocerebrum; RE, retina.