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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)
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
*Author for correspondence (e-mail: vogt{at}biol.sc.edu)
Accepted 10 December 2001
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Key words: Manduca sexta, Drosophila melanogaster, odorant binding protein, olfactory receptor, odor degrading enzyme, gene expression, olfactory sensilla, olfaction.
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; Krieger et al., 1997; Vogt et al., 1999; Hekmat-Scafe et al., 2000), and independently derived OBPs have also been identified in several vertebrate species (Pelosi et al., 1982; Pelosi, 1996; Pevsner et al., 1985, 1988a,b; Lee et al., 1987; Lobel et al., 1998; Tegoni, 2000). Large families of homologous but divergent ORs have been identified in the insect Drosophila melanogaster (Clyne et al., 1999; Gao and Chess, 1999; Scott et al., 2001; Vosshall et al., 1999, 2000), the nematode Caenorhabditis elegans (Troemel et al., 1995; Bargmann et al., 1998; Robertson et al., 2000, 2001), and several vertebrate species (e.g. Buck 1996; Freitag et al., 1998; Dryer, 2000; Rouquier et al., 2000). A variety of ODEs have been identified in insects (Vogt and Riddiford, 1981, 1986; Vogt et al., 1985; Rybczynski et al., 1989, 1990; Rogers et al., 1999), as well as lobster (Gleeson et al., 1992) and mammals (Ben-Arie et al., 1993). In insects, where olfactory neurons are compartmentalized within cuticular hairs (sensilla), unique combinations of ORs, OBPs and ODEs are thought to influence the odor specificities and sensitivities of the olfactory neurons (Rybczynski et al., 1990; Vogt et al., 1991a, 1999; Pelosi and Maida, 1995; Steinbrecht, 1999; Rogers et al., 1999).
Insect OBPs are small, globular, water-soluble proteins that are expressed in the support cells of olfactory sensilla and are secreted into the extracellular fluid occupying the lumen of the sensilla hairs and surrounding the ciliary dendrite projections of olfactory receptor neurons (Vogt and Riddiford, 1981; Steinbrecht et al., 1992, 1995; Leal et al., 1999; Sandler et al., 2000). OBPs are the first gene products in the biochemical pathway detecting diverse environmental odorants, and are thought to transport odor molecules from the inner openings of pores that penetrate the sensillum cuticle to receptor proteins (ORs) located in the membranes of the olfactory receptor neurons (Vogt et al., 1985, 1999; Krieger and Breer, 1999; Wojtasek and Leal, 1999; Kaissling, 2001). The insect behaviors associated with specific odor molecules have presumably subjected the OBP gene family to selective pressures that have driven the diversification of this family. OBP homologues have been identified in numerous species of holometabolous and hemipteran insects; if they are shown also to exist in orthopteroids they would arguably be represented throughout the Neoptera, or in more than 98 % of all insect species (Vogt et al., 1999). Seven OBP sequences have been published for Manduca sexta (Györgyi et al., 1988; Vogt et al., 1991b; Robertson et al., 1999). Previous studies identified six OBPs in D. melanogaster (McKenna et al., 1994; Pikielny et al., 1994; Kim et al., 1998), and as many as 32 have been suggested to be present in the fully sequenced D. melanogaster genome (Kim and Smith, 2001).
OBPs are differentially expressed among diverse classes of sensilla, which have unique odor specificities. This was first suggested by the identification of three distinct OBP classes in lepidopteran species, based on N-terminal sequence analysis: the pheromone binding proteins (PBPs) and the general odorant binding proteins GOBP1 and GOBP2 (Vogt et al., 1991a). PBPs were specific to or highly enriched in male antennae, while GOBP1 and GOBP2 proteins were more equivalently expressed in antennae of both sexes. These patterns suggest that PBPs are associated with sex-pheromone-specific trichoid sensilla and GOBPs are associated with plant volatile sensitive basiconic sensilla (Vogt et al., 1991a). Differential expression of OBPs was subsequently substantiated by a series of elegant electron microscopical (EM) immunocytochemical studies in the lepidoptera Antheraea polyphemus and Bombyx mori (Laue and Steinbrecht, 1997; Maida et al., 1997, 1999; Steinbrecht, 1996, 1999; Steinbrecht et al., 1992, 1995, 1996) and in the dipteran D. melanogaster (Hekmat-Scafe et al., 1997; Park et al., 2000). These EM studies demonstrated both unique and combinatorial expression of different OBPs in association with morphologically and functionally distinct classes of olfactory sensilla.
The current study examines the genomic organization and patterns of expression of a subset of OBP genes of M. sexta: pbp1Msex, gobp1Msex and gobp2Msex. Previous studies suggested that these three genes are differentially expressed among distinct classes of olfactory sensilla (Györgyi et al., 1988; Vogt et al., 1991b), and as such are suitable models for elucidating genetic regulatory mechanisms underlying the determination of diverse sensillum phenotypes. The characterization of these OBP genes establishes the necessary background for investigating regulatory elements that control their spatial and temporal expression. The study concludes with an examination of the genomic organization and relationships of 25 OBP homologues in D. melanogaster, utilizing the completely characterized genome of this species, and a comparison between these D. malanogaster OBPs and 14 M. sexta OBPs.
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For the experiment examining GOBP2 expression through a larval molt cycle (Fig. 8), larvae were staged from Dr Riddiford’s colony with his assistance, after the protocols of Curtis et al. (1984) and Langelan et al. (2000). Five individuals were taken and analyzed from each stage. Staging was based on morphological characteristics as follows. Spiracle apolysis (SA): an area of clear cuticle is visible surrounding the abdominal spiracles, indicating that epidermal retraction has begun (Langelan et al., 2000). Slipped head (SH): a zone of clear cuticle is visible just behind the fourth instar head capsule, revealing the underlying fifth instar head capsule. The head cap slips downward further to finally lie on top of the mandibles of the fifth instar larva. ‘SH+22’ and ‘SH+30’ were based on the appearance of the fifth instar mandibles viewed through the cuticle of the fourth instar head capsule. Approximately 22 h after SH, the head capsule is still fluid-filled and the mandibles have acquired a yellow appearance from the tanning process. Approximately 30 h after SH, the fluid within the fourth instar head capsule is reabsorbed, leaving them air-filled, and the mandibles appear dark brown.
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) and Rogers et al. (1997). PCR products were generated from purified plasmid (Qiagen) using the M13 forward or reverse primers and an appropriate insert gene-specific primer. RNA was synthesized from 1 µg of PCR product in the presence of 2 mmol l–1 each of ATP, CTP, GTP, 0.6 mmol l–1 UTP and 0.3 mmol l–1 digoxigenin-UTP using T7 or T3 RNA polymerase. The PBP1Msex probe encompassed base pairs (bp) 588–910 (GenBank accession number, GB-M21798) (Györgyi et al., 1988), bounded by the first internal EcoRI site and an EcoR1 site in the 3' untranslated region (UTR). GOBP1Msex (GM-M73797) and GOBP2Msex (GB-M73798) probes encompassed full-length coding regions (Vogt et al., 1991b), and were 501 bp and 483 bp, respectively. For in situ hybridization studies, probes were degraded to an average size of 160 bp (Byrd et al., 1996).
Southern blot analysis
Genomic DNA (SDS-proteinase K isolation from a single M. sexta larva) was digested with EcoRV, ClaI, HincII, ScaI, HaeII or BglII restriction enzymes. Digested DNAs were electrophoresed overnight on a 0.8 % agarose gel (10 µg per lane), and depurinated (0.25 mol l–1 HCl, 25 min), denatured (0.5 mol l–1 NaOH, 1.5 mol l–1 NaCl, 45 min) and neutralized (1.0 mol l–1 Tris-HCl, pH 8.0, 1.5 mol l–1 NaCl, 45 min) on soaked Whatman paper. Digested DNAs were then transferred onto nylon membrane (Amersham; Hybond-N). A lane containing molecular mass marker was excised and stained with Methylene Blue (0.02 % in 300 mmol l–1 sodium acetate). The nylon membrane was prehybridized for 2.5 h at 50°C (50°C in 5x SSC, 0.1 % N-lauroylsarcosine, 0.02 % SDS, 2x Denhardt’s solution, 100 µg l–1 herring sperm DNA) (1x SSC is 0.15 mol l–1 NaCl, 0.015 mol l–1 sodium citrate) and hybridized with a digoxigenin-labeled antisense RNA probe for 16 h (20 ng l–1; 50°C in prehybridization solution containing 50 % formamide) under the same conditions in a solution containing 50 % formamide, followed by washing at room temperature in 2x SSC, 0.1 % SDS (500 ml, 5 min wash) and twice at 60°C in 0.5x SSC, 0.1 % SDS (500 ml wash, 15 min first wash, 1 h second wash). The same membrane was hybridized three separate times with individual OBP probes (PBP1Msex, GOBP1Msex and GOBP2Msex) and visualized by luminous detection (Roche Biochemicals; Lumiphos-530) on X-ray film. Between hybridizations, the membrane was stripped of probe (0.2 mol l–1 NaOH, 0.1 % SDS, 37°C, 30 min), equilibrated in 2x SSC (5 min), and rehybridized with a different OBP probe following a prehybridization step.
Isolation of M. sexta OBP genomic clones
A M. sexta genomic library in EMBL3 (generously provided by Dr F. Horodyski, University of Ohio) was plated at a density of 6.3x104 plaque-forming units (p.f.u.) per 150 mm Petri dish on a layer of Escherichia coli LE392 (Promega). DNA was transferred to nylon membrane (ICN), denatured (5 min) and neutralized (5 min) as above, and UV-crosslinked (in 10x SSC) on soaked Whatman paper. Membranes were prehybridized for 2.5 h at 68°C (5x SSC, 0.1 % N-lauroylsarcosine, 2x Denhardt’s solution, 0.02 % SDS, 100 µg l–1 herring sperm DNA) and hybridized with a mixture of digoxigenin-labeled PBP1, GOBP2 and GOBP1 antisense RNA probes (25 ng ml–1 probe–1) under the same conditions in a solution containing 50 % formamide. Following washes (twice at 60°C in 0.5x SSC, 0.1 % SDS), hybridized probe was visualized by luminous detection (Roche Biochemicals; Lumiphos 530) on X-ray film (Kodak, X-OMAT). Positive plaques were isolated and rescreened at low density under identical conditions. DNA from select positive clones was isolated using the Wizard Lambda Prep Kit (Promega) following recommended protocols.
Clone identities were determined by dot blot hybridization. 1 µl of each DNA sample was spotted onto dry nylon membrane (ICN) and consecutively hybridized with individual PBP1, GOBP1 and GOBP2 RNA probes following the same procedure outlined for the genomic DNA library screen (see above). After each hybridization, the membrane was stripped of probe (0.2 mol l–1 NaOH, 0.1 % SDS, 37°C, 30 min), equilibrated in 2x SSC (5 min), and rehybridized with a different OBP probe following the prehybridization step. A clone that was positive for both PBP1 and GOBP2, designated M2-1S, was chosen for further analysis.
Subcloning M. sexta genomic clone M2-1S by polymerase chain reaction
The polymerase chain reaction (PCR) was used to generate four subclones of the M2-1S insert. Several primers were designed from published cDNA sequences for PBP1 (Györgyi et al., 1988) and GOBP2 (Vogt et al., 1991b), and the left and right arm sequences of the EMBL3 cloning vector (Stratagene). All PCR reactions were performed using the Expand Long Template PCR System (Roche Biochemicals). Each reaction (4x50 µl) used the supplied enzyme mix (1.75 U; mixture of Taq and Pwo DNA polymerases) and buffer no. 3, with 350 µmol l–1 dNTP, 300 nmol l–1 of each primer, and 20 ng M2-1S DNA. PCR was performed on a Cetus Thermocycler under oil overlay: the sequence was 3 min at 94°C followed by 30 cycles at 94°C (25 s), 60°C (40 s), 68°C (12 min for 10 cycles + a 20 s extension for each remaining cycle), and 1 cycle at 68°C (7 min). Pooled samples were purified by phenol–chloroform extraction and precipitation (Maniatis et al., 1982). Resuspended PCR products were reamplified by PCR using primers containing either EcoRI or BamHI sites at the 5' end of the same gene-specific sequence. The resulting products were purified as above, digested with the appropriate restriction enzyme, and cloned into pBluescript (SK+; Stratagene).
Sequencing M2-1S subclones
All clones were fully sequenced in both directions using vector primers or primers designed to internal sequence. Sequencing was done at the University of Florida DNA Sequencing Core Laboratory (Gainesville, FL, USA) using ABI Prism Dye Terminator cycle sequencing protocols (part number 402078) developed by Applied Biosystems (Perkin Elmer Corp., Foster City, CA, USA). The fluorescently labeled extension products were analyzed on an Applied Biosystems Model 373 Stretch DNA Sequencer (Perkin Elmer Corp.). Oligo primers were designed using OLIGO 4.0 (National BioSciences, Inc., Plymouth, MN, USA) and synthesized at the DNA Synthesis Core Laboratory (University of Florida, Gainesville, FL, USA). Nucleotide sequences were aligned and assembled using programs in the Sequencer 3.0 package (Gene Codes Corp., Ann Arbor, MI, USA).
Histological analyses
Adult tissue was prepared as described above (Animals); tissue for analysis was selected from 70 % methanol stocks. For larval tissues, heads were rehydrated to PBS, and the majority of tissue cut away from the larval antenna and maxillary palps, leaving enough head tissue for handling and orientation. For whole-mount analysis, sensory appendages (antenna, palp, galea) were cut open longitudinally by a single passage of a micro-scalpel (blade breaker, George Tiemann, Hauppauge, NY, USA) to allow probe access.
Whole-mount in situ hybridizations (for adult and larval tissues) were done as described by Byrd et al. (1996) and Rogers et al. (1999). Tissue was prehybridized overnight at 55°C (in 0.6 mol l–1 NaCl, 10 mmol l–1 Tris, pH 7.5, 2 mmol l–1 EDTA, 1x Denhardt’s, 50 µg ml–1 herring sperm DNA and 50 µg ml–1 tRNA) and hybridized for at least 24 h at 60°C with 100 ng ml–1 digoxigenin-labeled probes in the pre-hybridization solution containing 50 % formamide. After washing, tissue was incubated in blocking solution alone (5 % non-fat dry milk in PBS-Tw, 2 h, 20°C) followed by blocking solution containing alkaline phosphatase-coupled anti-digoxigenin antibody (Roche–Boehringer Mannheim; dilution 1:5000, overnight, 4°C). Hybridized probe was visualized using Nitroblue Tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) at 20°C following the recommended protocol (Roche–Mannheim). Tissue was photographed in whole mount under dark field illumination.
Sectioned in situ hybridizations were done as described by Byrd et al. (1996) and Rogers et al. (1997). Tissue was dehydrated through a graded series of ethanol and toluene (tissue stored in 70 % methanol was transferred to 70 % ethanol and carried forward), and incubated in melted paraffin (Periplast +) for 2–4 h before being embedded in plastic molds. Paraffin was additionally hardened on dry ice after trimming; sections (10 µm) were taken using razor blades mounted on top of a microtome blade, and transferred to water drops on electrostatically charged microscope slides (SuperFrost II, Fisher). After drying, slides were dewaxed by immersion in xylene, and sections were treated with Proteinase K [5 µg ml–1 in PBS-Tw, 15 min, room temperature (RT)]. Tissue was then treated with fix and acetic anhydride as described above. Slides were washed twice with glycine/PBS-Tw (2 mg glycine ml–1, 5 min per wash) between treatments. Sections were prehybridized overnight at 42°C (0.6 mol l–1 NaCl, 10 mmol l–1 Tris, pH 7.5, 2 mmol l–1 EDTA, 1x Denhardt’s solution, 50 µg ml–1 herring sperm DNA and 50 µg ml–1 tRNA; 1 ml per slide) and hybridized with 100 ng ml–1 digoxigenin-labeled probes under the same conditions but in the presence of 50 % formamide. Following hybridization, sections were washed as described above. Tissue sections were then blocked, treated with alkaline phosphatase-coupled anti-digoxigenin antibody and stained as described above. Coverslips were placed on slides with Aquamount mounting medium (Lerner Laboratories) and samples photographed with differential interference contrast (DIC) optics. For pre-hybridization and hybridizations, slides were placed on parallel glass rods mounted on the floor of plastic Petri dish (four slides per dish) containing wet tissue and sealed with parafilm to maintain humidity; temperature-controlled incubations and washes were performed in a bacterial incubator.
Immunocytochemistry of whole-mount and sectioned material was done as described in Rogers et al. (1997) and Callahan et al. (2000). Tissues were prepared as described above for in situ analysis. Whole-mount tissue or dewaxed sections were blocked in 3 % non-fat dry milk (NFDM), incubated with primary antiserum (diluted 1:500, overnight, 4°C) followed by goat IgG–horseradish peroxidase conjugate (ICN; diluted 1:100, 2 h, RT) and stained with VIP substrate (Vector) following the recommended protocols. For a negative control, sections were incubated with pre-immune serum under identical conditions. All washes and antibody treatments included 3 % NFDM in PBS-Tx (PBS containing 0.1 % Triton X-100). Permount (Fisher) was used to place coverslips on slides, which were photographed using brightfield or DIC optics. Antisera were immunohistochemically active at dilutions to 1:10,000. Primary antisera were anti-PBPMsexta (Györgyi et al., 1988) or anti-rGOBP2Msexta. rGOBP2Msexta was expressed from cDNA (Vogt et al., 1991b; Feng and Prestwich, 1997) and antiserum was generated in a rabbit using rGOBP2Msexta dissolved in 50 % Freund’s Complete Adjuvant (University of South Carolina Institute for Biological Research Technology Antibody Facility).
Analysis of Drosophila OBP genes
Twenty five OBP homologues were identified from the D. melanogaster genome data base using the Blast network servers at National Center for Biotechnology Information (NCBI) and Berkeley Drosophila Genome Project (BDGP, http://www.fruitfly.org/blast/) (see Table 1). The database was initially screened using six previously identified OBP sequences: OS-E, OS-F(PBPRP3), PBPRP1, PBPRP2, PBPRP5 and LUSH (McKenna et al., 1994; Pikielny et al., 1994; Kim et al., 1998), and rescreened using newly identified sequences. Criteria for selecting candidate OBPs were based on Blast e-values <0.05, a cutoff considered to be statistically significant (Karlin and Altschul, 1990). Data associated with the gene product accession number (AAF#) include the gene product sequence as well as a locus accession number (AE#) referencing a gene scaffold, with annotations describing the coding regions and their orientation within the scaffold sequence. Gene loci were determined using the NCBI Entrez Genome Web Server for D. melanogaster (www.ncbi.nlm.nih.gov/PMGifs/Genomes/7227.html) and using the gene product identifier (CG# or specific name) noted in the sequence reference file or scaffold annotation. Introns and exons of D. melanogaster genes were identified by comparing translations of genomic nucleotide sequences with predicted amino acid sequences, both obtained from the gene scaffold data entries for the respective genes.
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); the alignment included several non-Drosophila proteins identified during the Blast search as having significant similarity to the D. melanogaster proteins. Phylogenetic analysis was performed on the ClustalX alignment matrix using Paup (Version 4.0b8 for Macintosh) (Swofford, 2000). Three analyses were performed and results displayed on a single tree (see Fig. 10A): Neighbor Joining (Saitou and Nei, 1987) (default settings, 5000 replicates) and Maximum Likelihood (Quartet Puzzling) (Strimmer and von Haeseler, 1996) (default settings, 50,000 puzzling steps), both based on Paup’s mean character differences, and Maximum Parsimony (heuristic search; default settings, 5000 replicates). Algorithms ignored missing data resulting from alignment gapping.
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AAF50909 (gene scaffold AE003571)
Annotation suggested three exons. A truncation of the first exon was required to permit alignment using a start ATG situated mid-exon 1 (scaffold nucleotide modification: <97221..97368, 97434..97656, 98027..98227>).
AAF51918 (gene scaffold AE003600)
Annotation suggested three exons, but Blast analysis indicated that only exons 1 and 2 are OBP-related. The stop codon used was seven codons downstream from the annotated end of exon 2, adding six amino acid residues to the exon 2 domain (scaffold nucleotide modification: <70038..70376, 70440..70499>). Searching with AAF51918 identified AAF51919 as a significant homologue, but significance was only in the rejected exon 3 and AAF51519 was thus rejected as an OBP-related homologue.
AAF57521 (gene scaffold AE003795)
Annotation suggested four exons, but Blast analysis indicated that only exons 3 and 4 are OBP-related. The start ATG used was from the middle of exon 3 (scaffold nucleotide modification: complement <250658..251011, 251074..251133>).
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Isolation and characterization PBP1 and GOBP2 genes
A genomic DNA library (8x105 plaques) was screened with a single mixture of digoxigenin-labeled PBP1Msex, GOBP1Msex and GOBP2Msex antisense RNA probes. 19 positive clones were subjected to dot blot hybridization with individual probes to determine their identity (Fig. 1B): PBP1Msex probe hybridized to five clones; GOBP1Msex probe hybridized to eight clones; GOBP2Msex probe hybridized to five clones. Two clones were positive to both PBP1Msex and GOBP2Msex (arrows). One of these clones (no. 2, Fig. 1B) was designated M2-1S and sequenced.
A physical map of the fully sequenced M2-1S insert (9186 bp, GenBank accession number AF323972) is presented in Fig. 2A. The translational initiation and termination codons and the exon/intron boundaries of each gene were determined by alignment with published cDNA sequences for GOBP2Msex (Vogt et al., 1991b) and for PBP1Msex (Györgyi et al., 1988). Gobp2Msex spans 1492 bp from start codon to polyadenylation signal and pbp1Msex spans 1747 bp from start codon to polyadenylation signal. Both genes are oriented in the same direction, with gobp2 upstream (5') of pbp1Msex; 2741 bp separate the polyadenylation signal of gobp2Msex and the initiation codon of pbp1Msex. The coding region of each gene contains three exons, the first encoding at least part of the 5' UTRs and the amino acid signal peptides (Vogt et al., 1991a). TATA box motifs reside 292 bp and 508 bp upstream from the respective gobp2Msex and pbp1Msex initiation codons. Also, the octamer PyCATTTPuPy, which may represent an enhancer motif (Hekmat-Scafe et al., 1997), was found 318 bp and 439 bp upstream from the respective gobp2Msex and pbp1Msex initiation codons.
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Expression of PBP1Msex, GOBP1Msex and GOBP2Msex in adult male and female antennae
In a previous study, PBP1Msex, GOBP1Msex and GOBP2Msex proteins were partially sequenced directly from both male and female antennae (Vogt et al., 1991a). PBP1Msex was more abundant in male antennae than female antennae, and was shown to associate with pheromone-sensitive long trichoid sensilla of male antennae. In females, it was not determined whether the expression of PBP1Msex was restricted to a subset of sensilla or occurred at low levels in the general population of sensilla. Both GOBP1Msex and GOBP2Msex were present at similar levels in male and female antennae but neither associated with pheromone-sensitive trichoid sensilla isolated from male antennae, suggesting that both GOBPs associated with sensilla involved in the detection of plant volatiles. To clarify these general observations, in situ hybridization and immunocytochemical studies were performed on male and female antennae.
The anatomy of male and female adult antennae is reviewed in Fig. 3. Both male and female M. sexta adults have flagellum-shaped antennae, which are subdivided into approximately 80 segment-like annuli (Sanes and Hildebrand, 1976; Keil, 1989; Lee and Strausfeld, 1990; Shields and Hildebrand, 1999a,b). Fig. 3A–D shows male (A,C) and female (B,D) antennae; single annuli are represented in the inserts. Each annulus is divided into a sensory region rich in olfactory sensilla (arrows 1 and 2 in Fig. 3A) and a largely non-sensory region (marked by asterisks) containing scales and very few sensory structures (Fig. 3F). In male antennae, the sensory region of an annulus is divided into two zones. A peripheral sensory zone (Fig. 3E, left) contains the single class of long trichoid sensilla (type I); these sensilla appear to form a horseshoe pattern when the antenna is viewed from the side as in Fig. 3E. A mid-annular sensory zone (Fig. 3E, right) contains several types of short sensilla, intermixed, including many short trichoid (type II) and basiconic (type I and II) sensilla, and a few coeloconic and styliform sensilla (Fig. 3E, right). In general, a sensillum contains 1–3 sensory neurons plus three supporting cells (thecogen, trichogen and tormogen cells). Each male antenna contains about 100,000 sensilla and 250,000 sensory neurons (Sanes and Hildebrand, 1976; Lee and Strausfeld, 1990); the long type I trichoid sensilla contain neurons that respond specifically to sex pheromone, while the mid-annular mixture of sensilla contain neurons thought to respond to plant volatiles. In female antennae, the sensory region is constructed of a single sensory zone of intermixed sensilla types, which include all those of the male antenna except for the long trichoid sensilla (Fig. 3B,D,F); a recent study identified two classes of trichoid sensilla on female antennae, suggesting that one of these classes (type A) is the equivalent of the male type I trichoid sensilla, though much shorter (Shields and Hildebrand, 2001). Several publications suggest that the total number of sensilla on female and male is similar (Sanes and Hildebrand, 1976; Lee and Strausfeld, 1990; Shields and Hildebrand, 1999a,b); Oland and Tolbert (1988) estimated that a female antenna contained 300,000–340,000 neurons.
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). PBP1 mRNA appears to be restricted to the basal region of the epithelium, suggesting that it is primarily expressed in the trichogen cells; a single layer of negatively stained, round nuclei is clearly visible. Expression of GOBP2Msex in males was restricted to the mid-annular regions (Fig. 5B). Staining appears to be associated with single elongate cells (nuclei are visible by negative staining) and restricted to the basal region of the epithelium. The location of these nuclei and their appearance in a single layer suggest that GOBP2 expression may also occur predominantly in the trichogen cells of these sensilla. Unstained areas between expressing cells suggest the locations of sensilla cell clusters not expressing GOBP2.
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; Laue and Steinbrechet, 1997; Steinbrecht, 1999). Expression of GOBP1Msex is shown in Fig. 6. GOBP1Msex expression occurred in the same region as GOBP2Msex in both adult male (Fig. 6A–D) and female (Fig. 6D–F) antennae. In male whole mounts, cells expressing gobp1Msex appear to be somewhat smaller than those expressing gobp2Msex, suggesting that these two genes are differentially expressed within a common region. Double-labeling experiments would be necessary to confirm differential expression. GOBP1Msex probes consistently produced high background staining relative to the GOBP2Msex or PBP1Msex probes. This difference is evident in Fig. 6A and B; while full-length GOBP1Msex probe stained discrete cells in the mid-annular region (Fig. 6B), a more diffuse staining was also observed in the peripheral regions (asterisks, Fig. 6B) at a notably higher level than observed for the GOBP2Msex probe (asterisks, Fig. 6A). To improve specificity, probes were generated to specific subregions of the GOBP1Msex cDNA. A probe encoding the 5' third of the coding region (G1–15, Fig. 6C) displayed reduced cross-reactivity with cells of the periphery (asterisks). In contrast, a probe encoding the middle third of the coding region (G1–28, Fig. 6D) displayed increased cross-reactivity with the periphery (asterisks).
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). To test this hypothesis, expression of both GOBP2Msex and PBP1Msex was investigated in larval tissue. The larval mouth of M. sexta, typical of any lepidopteran, is surrounded by sensory detectors that are presumably designed to assess the quality of potential food (Hanson and Dethier, 1973; Kent and Hildebrand, 1987; Glendinning et al., 1999). A pair of relatively simple antennae are situated on either side of the mandibles (Fig. 7A,B), and a pair of bilobed maxilla are situated just below the mandibles (Fig. 7A–C) (Kent and Hildebrand, 1987; Keil, 1996; Laue, 2000). At least some sensilla of the antennae and maxillary palps are presumed to detect volatile odors (Hanson and Dethier, 1973; Kent and Hildebrand, 1987). M. sexta larval antennae and maxillary palps contribute in the discrimination of different solanaceous plants (Hanson and Dethier, 1973). Gustatory styloconic sensilla on the maxillary galea play a commanding role in larval discrimination between solanaceous (host) and non-solanaceous (non-host) plants (del Campo et al., 2001).
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), who used antisera against PBP and GOBP2 of A. polyphemus to immunodetect OBPs in sections of larval Bombyx mori and Helicoverpa armigera antennae at the EM level. Laue identified three basiconic sensilla (two large and one small), two chaetica and two campaniform sensilla on segment II, three basiconic sensilla (one large and two small) and one styloconic sensillum on segment III. GOBP2 antigenicity associated with the three large basiconic sensilla; the small basiconic sensilla were not immunoreactive and no anti-PBP antigenicity was observed (Laue, 2000).
The identity of GOBP2Msex in antenna was confirmed using the polymerase chain reaction (RT–PCR) (data not shown). Antennal mRNA was isolated, converted to cDNA, amplified with GOBP2-specific primers and the resulting product cloned and sequenced (Rogers et al., 1999). The resulting sequence exactly matched that of the adult antennal-derived GOBP2Msex sequence (Vogt et al., 1991b). Similar efforts using PBP1 specific primers yielded no product, supporting the negative histology which suggests that PBP1 is not expressed in larval antennae.
Cells in the maxillary palp express GOBP2 but not PBP1 (Fig. 7K–N,P). Each maxilla consists of two lobes, the galea and palp; the palp consists of three segments with candidate volatile-sensitive sensilla on segment III (Fig. 7C,J). Whole-mount in situ hybridizations of fifth and fourth instar maxillary palps revealed a single cluster of at least two cells expressing GOBP2 mRNA (Fig. 7K,L). Immunocytochemical detection in sectioned tissue suggests that this cluster is located within segment II of the palp (arrows) but makes contact with the cuticle near the base of the segment III (arrowheads) (Fig. 7M,N). GOBP2Msex was not detected in the galea, and PBP1Msex expression was not detected in the maxilla (Fig. 7P).
GOBP2Msex expression in the maxillary palp may associate with pore plate sensilla in the side of segment III. Several reports have characterized sensory structures on the maxillary palps. Schoonhoven and Dethier (1966) described eight peg-shaped sensilla at the tip of segment III and four campaniform sensilla on the side of segment III in the region where GOBP2 immunoreactivity was observed to make cuticle contact (Fig. 7M,N). Several (2–4) of the tip sensilla were thought to be olfactory, on the basis of electrophysiological responses to plant volatiles, and the remainder were identified as gustatory or contact chemoreceptors (Schoonhoven and Dethier, 1966). Keil (1989) presented an ultrastructural analysis of the maxillary palp sensory structures in the moth Helicoverpa armigera and suggested that none of the tip sensilla were olfactory. Of the eight tip sensilla, five had single tip pores and three had both tip and side-wall pores; however, only the tip pores appeared not to penetrate the cuticle, suggesting that all eight sensilla were contact chemoreceptors. On the side of the palp (third segment), Keil (1989) described one singly innervated campaniform sensillum (proprioceptive), a large singly innervated digitiform organ, and two multiply innervated pore-plate sensilla. Based on structure and innervation, the digitiform organ is a candidate CO2 detector, and the pore-plate sensilla might be olfactory detectors (Keil, 1989). The location of expressed GOBP2Msex suggests that it associates with one of these side-wall sensilla, possibly one or both of the multiply innervated pore-plate sensilla described by Keil. These observations further suggest that a re-evaluation of the function and identity of M. sexta maxillary palp sensory structures is in order.
Downregulation of GOBP2Msex expression during the larval molt
During a larval molt, the outer cuticle, including the sensillum cuticle ensheathing chemosensory neurons, is lost. OBPs are secreted into the extracellular lumen of the sensillum and are thus subject to loss during the molt; continued secretion of OBPs in the absence of sensilla cuticle would result in an energetic loss. Also, the support cells that express OBPs alter their program during a molt to extend a protrusion, which molds the new sensillum hair, and to express and secrete the cuticular proteins, which form the sensillum hair. Coexpression of OBPs at this time might strain this hair-forming process. We therefore hypothesized that OBP expression would be downregulated during the molting process. Because larval molts are regulated by ecdysteroids (Fig. 8A), and because the M. sexta OBPs were previously shown to be regulated by ecdysteroids in the developing adult antenna (Fig. 8B) (Vogt et al., 1993), we further hypothesized that the downregulation of larval OBP expression would correspond temporally to changes in larval ecdysteroid levels. To explore these possibilities, the larval expression of GOBP2 was examined through the molt from fourth to fifth instar, selecting animals staged relative to known ecdysteroid levels.
Expression of GOBP2Msex was observed to be downregulated during the larval molt, corresponding temporally to the rise and fall of larval ecdysteroids (Fig. 8A,C). Fig. 8C shows a developmental series of larval antennae, subjected to in situ hybridization with antisense GOBP2Msex probe in whole mount; the relative age of these tissues is indicated graphically in Fig. 8A. The presence of GOBP2Msex mRNA was detected strongly at SA 3–5 (Fig. 8Cb), weakly at SA 15-16 (Fig. 8Cc), but not detected at stages SH or SH+3 (Fig. 8Cd,e). GOBP2Msex mRNA was clearly visible again at SH+30 (Fig. 8Cg); under direct observation, staining was faintly apparent at SH+22 (Fig. 8Cf). This study indicates that GOBP2Msex expression is downregulated during a molt, turned off by SH but reinitiated by SH+22 (summarized in Fig. 8A). The temporal expression of GOBP2Msex correlates with the rise and fall of ecdysteroid levels as well as with expression of several other genes, which are known to be regulated by ecdysteroid levels and juvenile hormone (JH) (Fig. 8A).
Analysis of OBP gene loci in Drosophila
The full characterization of the Drosophila genome (Adams et al., 2000) affords the opportunity to assess the genomic organization of a large set of OBP genes within a single species. To that end, we analysed 19 potential homologues of the six previously identified Drosophila OBPs. Note that only the six previously identified OBPs are known from cDNAs; the coding regions of the additional OBP homologues were identified by the algorithms used by Celera Genomics (Adams et al., 2000) to characterize coding regions and intron/exon boundaries and are thus subject to the errors that may be inherent within this approach. Several of these entries were modified, as indicated in Materials and methods.
All 25 Drosophila OBP homologues are listed in Table 1. These genes distribute among 12 loci on the three euchromatic chromosomes (Fig. 9A). Five of the 12 loci include multiple OBP genes, ranging from 2 to 6 (Fig. 9B). Many of the genes from a given multi-OBP locus are sequentially arranged; gene orientation within a multi-OBP locus appears to be arbitrary (Fig. 9B). Members of a locus tend to share significant similarity with each other based on Blast e-values; only the members of locus 2 shared no significant sequence similarity with other members of that locus. Further analysis might identify additional OBP homologues within the Drosophila genome but, for those identified here, multi-OBP loci are not the rule, but are not uncommon.
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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.
