Chem. Senses 27: 453-459,
2002
© Oxford University Press 2002
A Cluster of Candidate Odorant Receptors from the Malaria Vector Mosquito, Anopheles gambiae
Department of Biological Sciences, Program in Developmental Biology and Center for Molecular Neuroscience, Vanderbilt University, Nashville, TN 37235 USA 1 These authors contributed equally to this work
Correspondence to be sent to: L.J. Zwiebel, Department of Biological Sciences, Program in Developmental Biology and Center for Molecular Neuroscience, Vanderbilt University, Nashville, TN 37235 USA. e-mail: l.zwiebel{at}vanderbilt.edu
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Abstract |
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Olfaction is critical to the host preference selection behavior of many disease-transmitting insects, including the mosquito Anopheles gambiae sensu stricto (hereafter A. gambiae), one of the major vectors for human malaria. In order to more fully understand the molecular biology of olfaction in this insect, we have previously identified several members member of a family of candidate odorant receptor proteins from A. gambiae (AgORs). Here we report the cloning and characterization of an additional AgOR gene, denoted as AgOr5, which shows significant similarity to putative odorant receptors in A. gambiae and Drosophila melanogaster and which is selectively expressed in olfactory organs. AgOr5 is tightly clustered within the A. gambiae genome to two other highly homologous candidate odorant receptors, suggesting that these genes are derived from a common ancestor. Analysis of the developmental expression within members of this AgOR gene cluster reveals considerable variation between these AgORs as compared to candidate odorant receptors from D. melanogaster.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chemoreception in general and olfaction in particular represent critical sensory inputs into many behaviors, including host preference selection, among insect agricultural pests and disease vectors (Takken, 1991
;
Hildebrand and Shepherd, 1997).
These economically and medically important insects include several species of
mosquitoes that transmit malaria, dengue, West Nile encephalitis, and yellow
fever. While all of these diseases pose significant threats to human health,
malaria, which is transmitted by several species of Anopheline mosquitoes,
remains of particular concern, as it is responsible for millions of deaths per
year in Africa alone (Collins and
Paskewitz, 1995). The overall ability of these mosquitoes to
transmit malaria (vectorial capacity) is, in part, determined by host
preference selection. Therefore a molecular analysis of mosquito olfaction may
provide opportunities for disrupting vector—host inter-actions, thereby
reducing incidence of disease transmission.
As a first step in this process, the cloning and characterization of
components of the olfactory signal transduction cascade from Anopheles
gambiae will facilitate molecular and biochemical studies of this
mosquito's olfactory processes. Olfactory signal transduction, which is
mediated by G-protein coupled receptors (GPCRs) and their downstream
effectors, is widely conserved across a broad spectrum of organisms, including
mammals, fish, crustaceans and nematodes [reviewed in Hildebrand and Shepherd
(Hildebrand and Shepherd,
1997)]. Indeed, the cloning and characterization of the GPCRs
involved in this cascade, known as odorant receptors (ORs)
(Buck and Axel, 1991;
Ngai et al., 1993),
has significantly accelerated the molecular analysis of olfaction across a
wide array of vertebrate systems [reviewed in Mombaerts
(Mombaerts, 1999)]. The first
invertebrate organism in which candidate ORs were identified was
Caenorhabditis elegans through the screening of a genome project for
potential signaling molecules (Troemel
et al., 1995). As is the case for vertebrate ORs, the
C. elegans ORs are seven transmembrane GPCRs, although the C.
elegans ORs bear almost no similarity to the vertebrate ORs. Within
C. elegans, there is only between 10 and 48% identity among the ORs,
indicating that they are much more divergent than the vertebrate ORs.
Moreover, genes encoding candidate ORs are found in characteristic clusters
throughout the mouse (Xie et al.,
2000) and C. elegans
(Troemel et al.,
1995) genomes.
Using a variety of approaches, a large family of candidate ORs was recently
identified in D. melanogaster (Gao
and Chess, 1999; Clyne et
al., 1999; Vosshall
et al., 1999). These genes are members of a highly
divergent family of receptors, displaying between 10% and 75% identity and
bearing no significant homology to any other GPCR family
(Smith, 1999). Like ORs from
mouse and C. elegans, a subset of Drosophila ORs have been
mapped to several gene clusters within the D. melanogaster genome.
Furthermore, several studies have used a variety of methods to begin to
examine OR—odorant interactions
(Zhang et al., 1997;
Zhao et al., 1998;
Wetzel et al., 1999;
Storkuhl and Kettler, 2001;
Wetzel et al.,
2001).
Recently, our group used genomics and molecular-based approaches to
identify and characterize four A. gambiae odorant receptor (AgOR)
genes, AgOr1, AgOr2, AgOr3 and AgOr4, that encode candidate odorant receptor
proteins from A. gambiae (Fox
et al., 2001). We have demonstrated that these AgORs
display several of the characteristics expected of OR family members. They are
all predicted to encode seven transmembrane domains, show significant homology
to D. melanogaster ORs (DORs), and are selectively expressed in
olfactory tissues. Here, we report the cloning and characterization of a fifth
OR gene, AgOr5, which is tightly clustered within the A. gambiae
genome with AgOr3 and AgOr4. Analysis of the developmental profiles of these
linked AgORs reveals that, unlike Drosophila ORs, AgOrs 3, 4 and 5
display novel and diverse patterns of pre-adult expression.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Sequencing of A. gambiae BAC clones
BAC clone 08K09 was generously provided by Dr Frank Collins (Notre Dame University, South Bend, IN) and directly sequenced or subcloned into pBluescript II KS (+) (Stratagene, La Jolla, CA) prior to sequencing, which was performed with an ABI 377 auto- mated sequencer using Big-Dye chemistry (PE Biosystems, Foster City, CA) using custom primers.
Phylogenetic analysis
Deduced amino acid sequences of AgORs 2-5 were aligned with four
representative D. melanogaster odorant receptors and one
representative D. melanogaster gustatory receptor to serve as an
outgroup using ClustalX v1.6 (Thompson
et al., 1997). Phylogenetic analysis was performed using
the exhaustive method by PAUP* v4.0b4
(Swofford, 2001), and
optimality criterion set at maximum parsimony. Bootstrap analysis was used to
assess statistical support for relationships via branch and bound analysis of
1000 pseudo-replicated data sets. Similar trees were obtained using
neighbor-joining and heuristic searches (data not shown).
Reverse-transcriptase polymerase chain reaction (RT-PCR)
One hundred mosquitoes were dissected and RNA was extracted (RNeasy, Qiagen, Valencia, CA) and resuspended in 30 µl. Total RNA was reverse transcribed using oligo-dT primers (Roche Molecular Biochemical, Indianapolis, IN) and SuperScriptII reverse transcriptase (Gibco BRL, Rockville, MD). A 10 µl volume of the RNA was used to synthesize cDNA, and 1 µl of each cDNA was used in each PCR reaction. PCR amplifications were carried out with the following forward (f) and reverse (r) primer pairs:
- AgOr3, f5'-GGAAAAGGAGCTGAACGAGA-3' and
r5'-CTAAAACTGCTCCTTCAGTA-3' (product size: 309 base pairs (bp)
cDNA, 367 bp genomic DNA);
- AgOr4, f5'-ATTTACGGCGGCAGTATCTT-3' and
r5'-TCACTGTACATCCATCTTTA-3' (product size: 450 bp cDNA, 610 bp
genomic DNA);
- AgOr5, f5'-TATGTGGTACGCATCAATCA-3' and
r5'-AAACAGTACACCCACGTNTGC-3' (product size: 561 bp cDNA, 687 bp
genomic DNA);
- rps7, f5'-GGCGATCATCATCTACGTGC-3' and
r5'-GTAGCTGCTGCAAACTTCGG-3' (product size: 458 bp cDNA, 610 bp
genomic DNA). Optimal annealing temperature, as tested empirically, was
58°C for all AgOR primer pairs and rps7.
Mosquito rearing and blood feeding
Anopheles gambiae (G3 strain) embryos were either kindly provided by Dr Mark Benedict (Centers for Disease Control and Prevention, Atlanta, GA) or generated in-house and disinfected with 0.05% sodium hypochlorite prior to hatching in flat plastic pans with distilled water. Larvea were reared on a diet of ground Whiskas Original Recipe cat food (KalKan Inc., Vernon, CA) that was applied to the surface of the water. Pupae were transferred to plastic cups in 1 gallon plastic containers, where newly emerged adults were collected the following morning. Adult mosquitoes were maintained in 1 gallon plastic containers at 27°C with 75% relative humidity under a 12:12 h photoperiod and fed daily with a 10% dextrose solution.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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An alignment of the deduced amino acid sequences of three candidate odorant receptors from A. gambiae, AgOr3, AgOr4 and AgOr5 is shown in Figure 1A, where a significant degree of sequence conservation is evident among them. Specifically, the strongest pair-wise identity (59%) and similarity (80%) are observed between AgOr3 and AgOr5. AgOr4 and AgOr5 share 29% identity and 64% similarity, while AgOr3 and AgOr4 share 19% identity and 62% similarity, respectively. The relative positions of a subset of introns, as well as the overall length of the deduced proteins (averaging 400 amino acids), are conserved among all five AgORs and also between putative AgORs and DORs. Here we show that AgOr3 and AgOr5 maintain complete conservation with regard to predicted exon/intron positions (Figure 1B), while the positions of the first and second introns of AgOr4 correspond with the first and third introns of AgOr3 and AgOr5, respectively. AgOr4 lacks the last two introns of AgOr3 and AgOr5 and instead maintains overall amino acid homology by combining exons 4, 5 and 6 of AgOr3 and AgOr5 into a single exon 3. In addition to primary sequence similarity between AgORs and DORs, an analysis of AgOr3, AgOr4 and AgOr5 reveals multiple hydrophobic regions that indicate seven possible transmembrane domains (Figure 2) that are characteristic of this family of GPCRs. Four separate analyses were performed on the predicted protein sequences to estimate the positions of the transmembrane domains [Kyte—Doolittle (Kyte and Doolittle, 1982
),
Hopp Woods (Hopp and Woods,
1981), and Eisenberg et al.
(Eisenberg et al.,
1984)] using DNA Strider, Version 1.2
(Marck, 1988) and TMPRED
(www.ch.embnet.org/software/TMPRED_form.html
). For all three protein sequences, the majority of the transmembrane domains
were predicted by all four algorithms. Even though these types of analyses
remain fundamental predictions, the fact that four independent algorithms
predicted many of the transmembrane domains significantly increases their
reliability.
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It is especially interesting to note that AgOr3, AgOr4 and AgOr5 are
tightly clustered together within the A. gambiae genome
(Figure 1B). AgOr5 and AgOr4
are separated by 310 bp while AgOr4 and AgOr3 are separated by 747 bp. It is
interesting to note that if 
100 AgORs (see Discussion for rationale for
this approximation) were equally spaced along the A. gambiae genome
(270 Mb), the average distance between each AgOR would be 2.7 Mb, much more
distance than separates these clustered genes. Close chromosomal linkage is
characteristic of odorant and taste receptor genes from D.
melanogaster (Clyne et al.,
1999,
2000;
Gao and Chess, 1999; Vosshall
et al., 1999,
2000), as well as OR genes
from C. elegans (Troemel et
al., 1995) and mouse (Xie
et al., 2000). Taken together, these data are consistent
with the classification of these genes as candidate olfactory receptors from
A. gambiae.
In order to more fully assess their relationships, four AgOR sequences were
aligned with four representative DORs and one gustatory receptor from D.
melanogaster to serve as an outgroup. From this alignment, phylogenetic
trees were generated and bootstrap analysis was used to assess statistical
support for the relationships observed.
Figure 3 shows that AgOr2
confidently groups with DORs 30a, 49b and 43a. In some analyses, AgOr2 forms a
monophyletic group with 43a [data not shown and Fox et al.
(Fox et al., 2001)],
while in the tree presented in Figure
2 the monophyletic group of AgOr2, 30a and 49b are branched with
respect to 43a with low bootstrap support. AgOr3, AgOr4 and AgOr5 form a
monophyletic group with very strong support in all analyses, with AgOr3 and
AgOr5 grouping together in every bootstrap replicate
(Figure 3 and data not shown).
DOR56a is paraphyletic to the AgOr3-5 clade in this analysis with 63%
bootstrap support (Figure 3),
but it can be placed paraphyletically to the AgOr2 clade in other analyses
[data not shown and Fox et al.
(Fox et al.,
2001)].
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In order to determine whether AgOr5 expression is restricted to olfactory
tissues, a characteristic that has been observed for all candidate AgORs to
date (Fox et al.,
2001), RT-PCR-based studies were performed. In these assays,
4-day-old adult mosquitoes were hand dissected into antennae/maxillary palps
(olfactory tissues), head (from which olfactory tissue has been removed, but
with proboscis attached), body and legs. These tissues were used to generate
RNA and, subsequently, cDNA template pools for PCR. Furthermore, as an
additional control, all reactions were carried out using oligonucleotide
primers that were designed to span predicted introns in order to distinguish
between genomic DNA and cDNA templates, as well as oligonucleotide primers
against the A. gambiae ribosomal protein S7 (rps7)
(Salazar et al.,
1993). The rps7 gene is constitutively expressed at high
levels in all tissues of the mosquito and, therefore, provides a control for
the integrity of the cDNA templates.
Consistent with its phylogenetic groupings with the other AgORs, olfactory-specific expression of AgOr5 is observed (Figure 4). In these studies, RT-PCR products of the predicted size are seen exclusively in reactions using antennae/maxillary palp cDNA templates. Importantly, no AgOr5 cDNA products are observed with head/proboscis, body or leg cDNA templates. It is noteworthy that the rps7 amplifications are more robust for the head, body and leg templates, reflecting the higher template amounts used in these parallel reactions, further demonstrating there is no detectable expression of AgOr5 in non-olfactory tissues. Genomic DNA contamination of cDNA templates prepared from olfactory and head tissue is detectable and, as a result of primer design, is clearly distinguishable from cDNA products. To further verify the specificity of these reactions, the AgOr5 RT-PCR product was subcloned and sequenced, revealing that an AgOr5-specific product had indeed been obtained (data not shown). Lastly, to additionally assure that AgOr5 is not expressed in any tissues other than antennae/maxillary palp, an additional 15 cycles of PCR were added to the control reactions containing head, body and leg cDNA templates. Even under these extremely sensitive conditions, AgOr5 cDNA RT-PCR products are undetectable in non-olfactory tissues (data not shown).
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We have made repeated attempts to detect the olfactory-specific expression
of AgOr3, AgOr4 and AgOr5 by means of in situ hybridization (ISH)
but, in each case, obtained inconclusive staining patterns. This is not
entirely surprising, given the low expression levels of DORs, of which a
sizable subset (30%) are undetectable using ISH methods
(Vosshall et al.,
2000). Furthermore, in two studies involving ISH of candidate
Drosophila taste receptor genes, only a small fraction was detected
(Clyne et al., 2000;
Scott et al., 2001).
In light of these studies, it is likely that the expression levels of the
three AgOR genes reported here are also beneath the detection threshold for
ISH.
In addition, we have examined the expression of the AgOr3, AgOr4 and AgOr5 gene cluster during A. gambiae development. As shown in Figure 5, AgOr3 is first detectable in fourth instar larvae and thereafter is maintained into adult stages while AgOr4 is not expressed until reaching sexual maturity in 4-day-old adults. Lastly, AgOr5 is detectable in all stages from first instar larvae through sexually mature adulthood. Moreover, all three AgORs are expressed in both female and male adult mosquitoes.
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It is important to note that non-quantitative RT-PCR was performed in these experiments. Therefore, any fluctuations in product amount may not be due to changes in gene expression, and conclusions to relative gene expression levels should not be drawn from these data. These experiments simply examine qualitative aspects of AgOR expression in the various developmental stages.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study we have identified and characterized an additional gene, AgOr5, encoding a candidate OR from the malaria vector mosquito, A. gambiae. AgOr5 is highly similar to two previously identified (Fox et al., 2001
)
candidate OR genes, AgOr3 and AgOr4. Importantly, in keeping with the paradigm
established for the other AgORs previously described
(Fox et al., 2001),
AgOr5 is similar to several DORs, displays approximately seven transmembrane
domains, and is selectively expressed in olfactory tissues of the mosquito. In
addition to these OR characteristics, AgOr5 contains a subset of introns whose
positions are conserved in DORs and AgORs, and maintains the general
conservation of protein length of 400 amino acids that has been observed
for AgORs and DORs. The fact that AgOr3, AgOr4 and AgOr5 are clustered within
the A. gambiae genome is in keeping with the characterization of many
ORs from mouse (Xie et al.,
2000), C. elegans
(Troemel et al.,
1995) and D. melanogaster
(Clyne et al., 1999;
Vosshall et al.,
1999). It is intriguing to speculate as to the underlying
principle behind this apparent conservation of linkage among some ORs. In this
regard, the ability (see below) to generate a large gene family such as the
ORs through duplication events (that would tend to favor close chromosomal
linkage) is an especially appealing rationale. Phylogenetic analyses of several AgORs confidently groups AgORs 3, 4 and 5 together as a monophyletic lineage on the resulting tree. The monophyly of these chromosomally adjacent genes is consistent with their origin through two intra-chromosomal duplication events: the first resulting in AgOr4 and the ancestral gene copy of AgOr3 and AgOr5, and the second yielding the latter gene copies. The sequences and exon/intron structures of AgORs 3 and 5 further supports this scenario and suggest these duplications to be relatively recent events. The positions of two introns are absolutely conserved among all three AgORs. The positions of the remaining three introns of AgOr3 and AgOr5 are also exactly conserved and their sequences are less divergent (35-50% identity; data not shown) than the two absolutely conserved introns (>25% identity; data not shown). The placement of AgOr4 between the more highly homologous AgOr3 and AgOr5 copies is suggestive of some role for recombination during or following the second duplication event, although the precise mechanism is not of central concern for the present investigation. It is also possible that additional AgORs will be found in this cluster pending the completion of the A. gambiae genome sequencing project.
AgOr3, AgOr4 and AgOr5 do not have readily apparent orthologs within the family of DORs (Figure 3). These three AgOR genes might therefore represent a class of receptors associated with behaviors that are unique to insects such as A. gambiae. It is intriguing to note that these behaviors include complex activities such as responses to ovipositional and host preference cues. Host preference cues for an anthropophilic mosquito such as A. gambiae are of critical importance for establishing the insect's vectorial capacity and might be expected to consist largely of human-specific odorants.
While our data show that AgOr3, AgOr4 and AgOr5 are detectable in both male
and female olfactory tissues (Figure
5), there is little reason to preclude the possibility that these
genes could play a role in sex-specific olfactory behaviors such as blood meal
host preference selection that are present in hematophagous insects such as
A. gambiae. In support of this, male mosquitoes have been shown to
respond to vertebrate host-specific odorants in the vicinity of the host
(Takken and Knols, 1999),
probably to facilitate mating in proximity to host. Further study is required
to determine the precise behaviors these AgORs underlie.
In a dramatic departure from Drosophila ORs where expression is restricted to pupal and adult stages, AgOr3 and AgOr5 display robust expression as early as the first larval instars. While the limited number of AgORs examined in this study makes it difficult to put this difference in developmental expression in precise biological context, it is nevertheless worthwhile noting the radically different life-cycles these two dipterans undergo. For example, in contrast to D. melanogaster where the pre-adult stages are entirely terrestrial, the equivalent stages of the A. gambiae life-cycle are aquatic. It is reasonable to speculate that such differences in environmental constraints might very well result in the utilization of AgORs in chemosensory-based behaviors at unique times relative to the academic model insect D. melanogaster.
Our studies indicate that AgOr3, AgOr4 and AgOr5 exhibit different developmental expression profiles during the mosquito's life cycle. This may be indicative of the presence of several unique regulatory sequences capable of directing distinct temporal expression within or close to the borders of this locus. Furthermore, the ability to detect distinct AgOR expression patterns during development is consistent with the hypothesis in which the presence of a particular OR or novel combinations of OR genes might be correlated with a unique set of behavioral objectives. For example, AgOr4 is only expressed in sexually mature (4 day old) adult mosquitoes, but not in immature (1 day old) mosquitoes. It is intriguing to speculate that AgOr4 may be involved in behaviors restricted to adults capable of mating, perhaps playing a role in mating itself. In addition to indicating the roles these AgORs might play in mosquito behavior, investigating the developmental expression of AgORs may lend insight into what types of odorants these putative ORs interact with, i.e. odorants that are specific for larvae, adults or both. Information regarding developmental expression may also indicate at which life stage potential antagonists or other novel treatments directed against particular AgORs might be applied within the context of olfaction-based mosquito control programs.
With the identification of five members of a family of candidate OR genes
in A. gambiae, biochemical, behavioral and transgenic studies may now
begin to determine the specific classes of odorant ligands that activate these
receptors. Approximately 5% of the A. gambiae genome has been
screened to date for the presence of AgORs. This leads to an estimate of
100 AgORs, a number on the order of current estimates for DORs
(Drosophila Receptor Nomenclature Committee, 2000). Identification
and characterization of the whole family of AgORs may indicate potential
mosquito attractants and/or repellants. Furthermore, comparative studies of
putative ORs from hematophagous and non-hematophagous insects, as well as
between anthropophilic and zoophilic species of Anopheline mosquitoes, may
provide information concerning the molecular basis for host preference
selection among these insects. This information could lead to novel
disease-control strategies targeting vector—host interactions.
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Acknowledgments |
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We acknowledge the work of Dr Hugh M. Robertson (University of Illinois at Urbana—Champaign) for his collaborative efforts to elucidate and characterize the AgOR gene family. We would like to thank Patricia Russell for mosquito care and C. Elaine Merrill for helpful discussions. Special thanks to Dr Daniel J. Funk (Vanderbilt University) for help with phylogenetic analysis and discussion. BAC clones were generously provided by Dr Frank Collins. This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR V30/181/208 to L.J.Z.) and by grants from the Division of Integrative Biology & Neuroscience of the National Science Foundation (IBN 0075338 to L.J.Z.) and the National Institute of Deafness and Other Communication Disorders (1 R01 DC04692-01 to L.J.Z.).
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Accepted February 25, 2002
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J. Krieger, E. Grosse-Wilde, T. Gohl, Y. M. E. Dewer, K. Raming, and H. Breer Genes encoding candidate pheromone receptors in a moth (Heliothis virescens) PNAS, August 10, 2004; 101(32): 11845 - 11850. [Abstract] [Full Text] [PDF]
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R. J. Pitts, A. N. Fox, and L. J. Zwiebel A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector Anopheles gambiae PNAS, April 6, 2004; 101(14): 5058 - 5063. [Abstract] [Full Text] [PDF]
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