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A comparative study of odorant binding protein genes: differential expression of the PBP1-GOBP2 gene cluster in Manduca sexta (Lepidoptera) and the organization of OBP genes in Drosophila melanogaster (Diptera)

Richard G. Vogt^*, Matthew E. Rogers, Marie-dominique Franco and Ming Sun

Department of Biological Sciences, University of South Carolina, Columbia, SC 29208 USA
Present address: Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Present address: Department of Biology, Regis College, 3333 Regis Boulevard, Denver, CO 80221, USA

View larger version (53K): [in a new window]   Fig. 1. Hybridization analysis of PBP1Msex, GOBP1Msex and GOBP2Msex. (A) Southern blot analysis using genomic DNA isolated from a single individual; a single blot was sequentially hybridized with each probe, following stripping of the previous probe. Numbers (1–4) mark DNA fragments that appeared to hybridize with multiple OBP probes. Size markers (kb) are from HindIII-digested DNA. Labelled bands are discussed in the text. (B) DNA hybridization analysis of isolated genomic clones, processed under the same conditions as the Southern blot but on separate filters. Arrays of 25 clones were analyzed, and the numbers of positive clones are indicated. Arrows indicate two colonies which hybridized to both PBP1Msex and GOBP2Msex probes. Colony 2 (M2-1S) was chosen for sequence analysis.

View larger version (55K): [in a new window]   Fig. 2. Genomic organization of lepidopteran OBPs. (A) Sequence map of clone M2-1S, containing both gobp2Msex and pbp1Msex. Numbers indicate the upstream and downstream bases demarking translational initiation sites (2393, 6626), termination codons (3832, 7795), polyadenylation signals (gobp2Msex, AGTAAA, bp 3885; pbp1Msex, AATAAA, bp 8373) and boundaries between exons (heavy bars) and introns. Restriction sites relevant to Fig. 1 are indicated. The full-length sequence of M2-1S is available from GenBank (accession number AF323972). (B) Size comparison of exons and introns of OBPs. Exon/intron organization within coding regions are compared between GOBP2Msex and PBP1Msex, PBPs of several other moth species, and six OBPs of Drosophila melanogaster. Exon/intron boundaries were determined by comparing derived amino acid sequences with translated genomic DNA sequences. Genomic sequences are represented by large filled boxes (exons), joined by thin lines (introns); lengths are proportional to the scale bar. The 5' ends correspond to the start ATGs and the 3' ends correspond to the termination codons. Genes, taxa and GenBank accession numbers are: MsexG2 (M. sexta GOBP2, AF323972), Msex PBP1 (M. sexta PBP1, AF323972); AperPBP (Antheraea pernyi PBP1, X57562); AvelPBP (Argyrotaenia velutinana AvelE PBP, AF177641); CmurPBP (Choristinoneura murinana Cmur4 PBP, AF177662); CpinPBP (Choristinoneura pinus Cpin4 PBP; AF177653); CrosPBP (Choristinoneura rosaceana CrosC PBP; AF177654); OnubPBP (Ostrinia nubilalis UZ4 PBP, AF133643). PgosPBP (Pectinophora gossypiella PBP, AF177656) DmelOSE, DmelOS-F, DmelPBRP1 DmelPBRP2, DmelPBRP5 DmelLUSH – Drosophila melanogaster OS-E (AE003601); OS-F (PBPRP3) (AE003601); PBPRP1 (AE003541); PBPRP2 (AE003571); PBPRP5 (AE003617); LUSH (AE003516). (Krieger et al., 1991; Pikielny et al., 1994; McKenna et al., 1994; Hekmat-Scafe et al., 1997; Willett and Harrison, 1999; Willett, 2000). (C) Comparison of exon boundaries in OBP proteins. Amino acid alignments of OBP proteins in Fig. 4 are shown. The alignment is limited to regions surrounding the lepidopteran exon boundaries. Sequences were aligned using Clustal X (Thompson et al., 1994). Three of six conserved cysteine residues are marked (X). Intron/exon boundaries of the PBPs and GOBP2 are indicated by numbers (1,2); boundaries in the Drosophila proteins (Dmel) are indicated by letters (A–D). The C-terminal amino acids of exon domains are enclosed by boxes.

View larger version (102K): [in a new window]   Fig. 3. Morphology of antennae and male expression of three OBPs. (A–D) Scanning electron micrographs of adult male (A,C) and female (B,D) antennae. Arrows in A point to sensilla of the peripheral (1) and mid-annular sensory (2) regions in A and C, and to a female sensillum in D. Insert diagrams in A and B indicate the structural organization of the male or female annulus; asterisks indicate scale (non-sensory) regions and hatching or solid black, sensory regions. (C) The boundary between the peripheral and mid-annular sensory regions of a male annulus; arrows identify the long trichoid sensilla (1) and the short sensilla of the mid-annular region (2). (D) A comparable region of a female antenna; the arrow points to one of many slender hair-like sensilla. Short protrusions underlying the sensilla can be seen in both C and D; these are non-sensory protrusions in the antennal cuticle. (E) Side view diagram of a male annulus, showing the distributions of olfactory sensilla (arrows) in the peripheral (left annulus) and mid-annular (right annulus) sensory regions) (Lee and Strausfeld, 1990). (F) Diagram of three male and female annuli. Sensory and scale (non-sensory, asterisk) regions are noted, as are the peripheral (black) and mid-annular (hatched) sensory regions of male and the more-or-less single homogeneous sensory region (hatched) of female. Size bar, 278 µm (A,B), 114 µm (C,D).

View larger version (124K): [in a new window]   Fig. 4. Expression of PBP1 and GOBP2 in male and female antennae, in whole mount. (A–F) Bisected antennae of male (m; A–D) and female (f; E,F) adult M. sexta are shown probed with antisense RNA encoding PBP1 (P) or GOBP2 (G2). Insert diagrams indicate the orientation of the bisection. (C) shows details of cells of the mid-annular region expressing PBP1 (D, arrows). (G,H) Control in situ hybridizations (Con). Arrows in (G) indicate holes through the cuticle belonging to the long trichoid sensilla and through which olfactory dendrites pass to enter sensillum hairs. Tissue was from pharate adult animals. Size bar, 150 µm (A,B,E,F); 411 µm (C); 26 µm (D); 125 µm (G,H).

View larger version (118K): [in a new window]   Fig. 5. Expression of PBP1 and GOBP2 in sections of male and female antennae. (A–F) Male (m; A,B) and female (f; C,F) antennae were sectioned and probed with antisense RNA encoding PBP1Msex (P) or GOBP2Msex (G2). Insert diagrams indicate the positions and orientations of sections. (G–H) Comparison of the vertical distribution of PBP1 mRNA (G, in situ hybridization using GOBP2Msex probe) and PBP1 protein (H, immunocytochemistry using PBP1Msex antiserum) in the female antenna. (I) Control in situ hybridization. Tissue was from pharate adult animals. Size bar in I, 100 µm (A–F,I); in G, 25 µm (G,H).

View larger version (132K): [in a new window]   Fig. 6. Expression of GOBP1 in adult male and female antennae. (A–D) Male (m) antennae of adult M. sexta are shown probed in whole mount with antisense RNA encoding GOBP2 (G2), or three different clones of GOBP1 (G1). G1 probe (B) encoded the entire coding cDNA region, G1-15 probe (C) encoded the 5' third of the coding region, and G1-28 probe (D) encoded the middle third of the coding region. G1-15 and G1-28 probes were contiguous but non-overlapping. Asterisks mark the peripheral annular regions occupied by the long trichoid sensilla. (E,F) Female (f) antennae of adult M. sexta are shown probed in section with antisense RNA encoding GOBP2 (G2) or GOBP1 (G1). Arrows in F indicate positive staining cells. Insert diagrams indicate the positions and orientations of cuts. Tissue was from pharate adult animals. Size bar, 188 µm (A–D) or 50 µm (E,F).

View larger version (100K): [in a new window]   Fig. 7. GOBP2 expression in larval antennae and maxillary palps. (A–C) Mouth parts. Frontal (A), side (B) and ventral (C) views of the oral region, illustrating the position of larval antennae (Ant) and maxilla with associated palps and galea. OC, ocelli. (A,B) Fifth and (C) fourth instar larvae. (D–I) Antenna. Single larval antennae shown diagrammatically (D), in whole mount (E) and in section (F–I). (E) A whole-mount in situ hybridization using the antisense GOBP2Msex RNA probe (G2is). (F–I) Immunoreactions using GOBP2Msex antiserum (G2ab). (D) is modified from Kent and Hildebrand (1987), with segments I, II and III indicated. Small arrows, three basiconic sensilla on the end of the second segment; large arrowhead, three basiconic sensilla on the tip of the third segment; asterisks, mechanosensory spines. (J–N) Maxilla. Larval maxilla shown diagrammatically (J), in whole mount (K,L) and in section (M,N). (K,L) Whole-mount in situ hybridizations using the antisense GOBP2Msex RNA probe (G2is). (M,N) Immunoreactions using GOBP2Msex antiserum (G2ab). (J) is modified from Hanson and Dethier (1973), showing palp and galea. S, styloconic sensilla; B, basiconic-like sensilla on the tip of the maxillary palp. Arrowhead points to region on third segment of maxillary palp containing several pore-plate sensilla. Asterisks note positions of extirpation in the experiments of Hanson and Dethier (1973), which suggested differential roles of these structures in feeding decisions. (O,P) Controls. Control antennae (O) and maxilla (P) probed with PBP1Msex antiserum (Pab). All tissues (E-I, K-P) are from day-3 fifth instar larvae (actively feeding), except for L, which is from a fourth instar larva. Arrows over histology indicate positive staining. Arrowheads indicate third antennal segment (E,I) and point of cuticular contact of stain in third palp segment (L,M). Size bar (lower right), 542 µm (A,B), 104 µm (E), 58 µm (F), 65 µm (G), 72 µm (H), 48 µm (I), 148 µm (K), 100 µm (L), 116 µm (M,N), 66 µm (O), 116 µm (P).

View larger version (102K): [in a new window]   Fig. 8. GOBP2 expression in larval antenna through a fourth-fifth instar molt cycle. (A,B) The relationship between the developmental stage, levels of ecdysteroid and juvenile hormone (JH) and gene expression are indicated from fourth instar to adult, modified from Riddiford (1995). Lettered asterisks (a-k) in (A) indicate developmental stages represented by tissues shown in (C). Significant developmental events are indicated in all-capitalized text above graph. Ecdysteroid titers are from Bollenbacher et al. (1981) and Warren and Gilbert (1986); JH titers are from Fain and Riddiford (1975) and Baker et al. (1987). Expression profiles of four ecdysteroid sensitive genes are shown in black, reviewed in Riddiford (1995): INS, insecticyanin; LCP 14, larval cuticle protein; LCP 16.6, larval endocuticle protein; DDC dopa decarboxylase. Temporal expression of GOBP2Msex in larva is indicated in (A), from this study; expression of GOBP2Msex and PBP1Msex in pupa is indicated in (B), from Vogt et al. (1993). (C) Whole-mount in situ hybridizations of developmentally staged tissue, each representative of five individuals. Arrows indicate positively stained cell clusters; asterisks indicate staining entering the third antennal segment. (a-k) Tissue correlated with the time points indicated by lettered asterisks in A. 4th, feeding fourth instar larva day 1; SA, spiracle apolysis; SH, slipped head, with hours after indicated (e.g. SH+3=SH+3 h). Ecd 5th, animal within 2 h of molting; Day-1 5th is 24 h after molting. ‘Wandering’ is an animal on the first day of wandering (W1, in A), a non-feeding pre-pupal stage that initiates about 5 days after molting. The time between SA 15-16 and SH was approximately 8 h, and between SH+30 and Ecd 5th, approximately 5 h. Apolysis, or the detachment of the epidermis from the cuticle, occurred following stage SA15-16, and is indicated by the loss of antennal form observed at SH (d). The formation of new fifth instar larval cuticle is indicated by the structural form the antenna has reacquired by stage SH+22 (f). The fourth instar larval cuticle is not shed until ecdysis (Ecd 5th, h), after which the cuticle becomes tanned, as indicated by the brown coloration in the Day-1 5th and ‘Wandering’ tissues (i,k). Size bar, 150 µm.

View larger version (31K): [in a new window]   Fig. 9. D. melanogaster OBP homologues. (A) Physical positions of OBP (L1-L12) and DOR loci on chromosomes 1-3 (C1-C3). Numbers are in megabase units (MB); circles mark positions of centromeres. DORs are those identified by the Drosophila Odorant Receptor Nomenclature Committee (2000) and Leslie Voshall (67d; personal communication). Mid-point nucleotide positions of genes were determined using the NCBI Entrez genome server. (B) Spatial organization of genes at loci 2, 6, 10 and 12; based on gene scaffold annotations. Positions of all annotated genes within these regions are shown. Tall boxes are OBP genes (CG numbers) and short boxes intervening genes; exons are indicated only for OBPs, and arrows indicate orientations of OBP genes. Numbers indicate the nucleotide range of each diagram; diagrams of loci 6 and 12 are scaled (1:20, 1:10) relative to those of loci 2 and 10. Locus 7 is not illustrated; two OBP genes are separated by about 44 kbp with six unrelated annotated genes situated between. mtr, mitochondrial thioredoxin (CG8517); OR56a, olfactory receptor.

View larger version (115K): [in a new window]   Fig. 10. Alignment (ClustalX) of D. melanogaster OBP amino acid sequences. Drosophila sequences are those identified in Table 1; locus 6 genes were excluded because of significant divergence from the other OBPs. Other included proteins showed a significant relationship to the Drosophila proteins by Blast analysis: CAB64650, CAB64649 and CAB64645 are serum proteins of the medfly Ceratitis capitata, (Christophides et al., 2000), LAP (AF091118) is an OBP from the hemipteran Lygus lineolaris (Vogt et al., 1999) and ABPXMsex (AF117577) is an OBP from M. sexta (Robertson et al., 1999). This alignment preserved the relationships between six conserved cysteines, noted by ‘X’. Exon domains are alternately in bold and underlined.

View larger version (56K): [in a new window]   Fig. 11. Comparisons of OBP sequences. (A) Amino acid sequence comparisons of 23 D. melanogaster OBPs. A Neighbor Joining Distance tree is shown, derived from the alignment matrix shown in Fig. 9C. Branch lengths are proportional to percentage sequence difference (scale bar represents 10 % mean difference). Three methods were used for this analysis; numbers by nodes are triplets and refer in order to neighbor-joining bootstrap values (5000 replicates), maximum likelihood quartet puzzling support values (in parentheses, 50,000 puzzling steps), and maximum parsimony support values (5000 replicates). Numbers with asterisks indicate gene locus numbers identified in Table 1 and Fig. 9A. Branches are collapsed to 40 % support for at least one method of analysis; all three methods yielded identical topologies at this level of support. Non-drosopohilid taxa are indicated (<>); these were identified when searching the D. melanogaster homologues using Blast. (B) Comparisons of exon domains of 23 D. melanogaster OBPs. Graphical representation of aligned amino acid sequences shown in Fig. 9C, focusing on the alignment of exon domain boundaries within the proteins, including 23 D. melanogaster OBPs plus PBP1Msex and GOBP2Msex (transferred from Fig. 4). Alternate exon domains are shown as filled and unfilled boxes; C-terminal amino acid numbers of exon domain boundaries are indicated, referencing their character positions in the alignment (Fig. 9C). (C) Amino acid sequence comparisons of dipteran versus M. sexta OBPs. A Neighbor Joining Distance tree is shown (Paup 4.0b8), derived from a ClustalX alignment (not shown). Branch lengths are proportional to percentage sequence difference (scale bar represents 10 % mean difference); numbers by nodes are bootstrap values (1000 replicates). Sources of dipteran sequences are described above, except for the mosquito sequences; two sequences from Anopheles gambiae are from L. Zwiebel, and one sequence from Aedes aegypti is from J. Bohbot and R. Vogt. M. sexta sequences PBP1, PBP2, PBP3, GOBP1, GOBP2, ABPX and ABP1 were previously published (Györgyi et al., 1988; Vogt et al., 1991b; Robertson et al., 1999). The remaining M. sexta sequences were identified from ESTs submitted by H. Robertson (GenBank); this data set was downloaded and searched locally by Blast protocols using software obtained from the NCBI FTP site, and subsequently translated for alignment. Representative EST accession numbers are indicated. One sequence, ABP4, was provided by Hugh Robertson and is as yet unpublished. The broken bar at the bottom identifies major similarity groups in this analysis.