Journal of Insect Physiology 57 (2011) 915–929

Characterization of olfactory genes in the antennae of the Southern housemosquito, Culex quinquefasciatus

Julien Pelletier, Walter S. Leal *

Honorary Maeda-Duffey Laboratory, Department of Entomology, University of California Davis, Davis, CA, USA

A R T I C L E I N F O

Article history:

Received 11 January 2011

Received in revised form 25 March 2011

Accepted 1 April 2011

Keywords:

Olfaction

Culex quinquefasciatus

Antennae

Olfactory genes

A B S T R A C T

Odorant reception in insects is mediated by different families of olfactory proteins. Here we focus on the

characterization of odorant-binding proteins (OBPs), ‘‘plus-C’’ odorant-binding proteins (‘‘plus-C’’ OBPs),

chemosensory proteins (CSPs) and sensory neuron membrane proteins (SNMPs) families from the

Southern house mosquito, Culex quinquefasciatus, a vector of pathogens implicated in multiple human

diseases. Using bioinformatics and molecular approaches, we have identified a diversity of genes in the

genome of Culex quinquefasciatus and examined their expression profiles by RT-PCR and real-time

quantitative PCR. Based on their high transcript enrichment in female antennae compared to non-

olfactory tissues, we have identified twelve OBPs, two ‘‘plus-C’’ OBPs and two SNMPs that likely play

important roles in odorant reception. Transcripts of two genes were clearly enriched in female antennae

compared to male antennae, whereas other genes displayed relatively equivalent transcript levels in

antennae of both sexes. Additionally, eight genes were found to be transcribed at very high levels in

female antennae compared to CquiOR7, suggesting they might encode highly abundant olfactory

proteins. Comparative analysis across different mosquito species revealed that olfactory genes of Culex

quinquefasciatus are related to putative orthologs in other species, indicating that they might perform

similar functions. Understanding how mosquitoes are able to detect ecologically relevant odorant cues

might help designing better control strategies. We have identified olfactory genes from different families

which are likely important in Culex quinquefasciatus behaviors, thus paving the way towards a better

understanding of the diversity of proteins involved in the reception of semiochemicals in this species.

� 2011 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Journal of Insect Physiology

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate / j in sp h ys

1. Introduction

Mosquitoes and other insects rely on their olfactory system tolocate hosts, oviposition sites, food sources and mates viaspecialized sensory structures, the olfactory sensilla. Thesefunctional units are present on different olfactory organs suchas antennae, maxillary palps and proboscis. A recent detailedmapping of the olfactory sensilla on the antennae and maxillarypalps of the mosquito Culex quinquefasciatus Say (Diptera,Culicidae) has highlighted a diversity of functional types ofsensilla with their own specificities and sensitivities (Hill et al.,2009; Syed and Leal, 2007, 2009). Large multigenic families ofmosquito odorant receptors (ORs) have been identified inAnopheles gambiae (An. gambiae) (Hill et al., 2002), Aedes aegypti

(Ae. aegypti) (Bohbot et al., 2007) and Culex quinquefasciatus (Cx.

quinquefasciatus) (Arensburger et al., 2010; Pelletier et al., 2010b)and a large set of ORs from these species have been functionally

* Corresponding author. Tel.: +1 530 752 7755; fax: +1 530 752 1537.

E-mail addresses: jppellet@ucdavis.edu (J. Pelletier), wsleal@ucdavis.edu

(W.S. Leal).

0022-1910/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2011.04.003

characterized (Bohbot and Dickens, 2009; Bohbot et al., 2010;Carey et al., 2010; Hallem et al., 2004; Hughes et al., 2010; Pelletieret al., 2010b; Wang et al., 2010), providing valuable insights onhow mosquitoes can detect and discriminate behaviorally relevantodorants (semiochemicals). However, the complete molecularmechanisms which confer the response profile to a given sensillaare still elusive. Indeed, we showed previously that the selectivityof a heterologously expressed OR from Cx. quinquefasciatus

(CquiOR2) towards indole and methylindoles was different fromthe selectivity of the native CquiOR2-expressing olfactory receptorneuron (ORN) in antennae, suggesting that other proteins, absentin heterologous system, are necessary to confer the native responseprofile of antennal ORNs (Pelletier et al., 2010b).

In mosquitoes, it has been clearly demonstrated that odorant-binding proteins (OBPs) are involved in the peripheral events ofodorant reception (Biessmann et al., 2010; Pelletier et al., 2010a).Insect OBPs, first identified in moths (Vogt and Riddiford, 1981), aresoluble proteins displaying a characteristic pattern of six conservedcysteine residues (‘‘classic’’ motif) and possess an N-terminal signalpeptide sequence. It has been suggested that OBPs ensure theliaison between the port of entry on the sensilla (the pore tubules)and the ORNs, thus contributing to the delivery of odorants to the

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929916

ORs (Vogt, 2005). The first mosquito OBP identified, Cx. quinque-

fasciatus OBP1 (CquiOBP1) (Ishida et al., 2002b), has since beenshown to bind to a mosquito oviposition pheromone (MOP) in a pH-dependent manner and to be expressed in a subset of olfactorysensilla, including one type responding to this pheromone (Lealet al., 2008). Gene knockdown significantly affected the antennalsensitivity to MOP, indole and 3-methylindole but not to nonanal,suggesting that CquiOBP1 is important for the reception of specificodorants (Pelletier et al., 2010a). Additionally, knockdown of aputative OBP1 ortholog in An. gambiae, AgamOBP1, reduced theantennal sensitivity to indole and 3-methylindole (Biessmann et al.,2010), indicating that both proteins might be involved in thereception of common semiochemicals in different species. Struc-tural analysis of orthologous OBP1 proteins in different mosquitospecies revealed highly similar three dimensional structures,suggesting that these related proteins share a common mechanismof odorants recognition (Leite et al., 2009; Mao et al., 2010; Woguliset al., 2006). Recently, AgamOBP1 was shown to be expressed insupport cells of olfactory sensilla across most antennal segments offemale and male antennae by fluorescent in situ hybridization,which clearly supports a role in olfaction (Schymura et al., 2010).Genome analysis allowed the identification of large multigenicfamilies of OBPs in An. gambiae (Li et al., 2005; Xu et al., 2003), Ae.

aegypti (Zhou et al., 2008) and Cx. quinquefasciatus (Pelletier andLeal, 2009), indicating that multiple OBPs might be important inodorant reception in mosquito antennae.

Contrary to soluble OBPs, sensory neuron membrane protein(SNMP) genes encode transmembrane proteins representing a sub-clade of the insect CD36 gene family (Nichols and Vogt, 2008).Different studies support a role of insect SNMPs in pheromonalcommunication. A Drosophila melanogaster SNMP1 has beenshown to be essential in the detection of the sex pheromone(Z)-11-vaccenyl acetate in T1 sensilla (Benton et al., 2007; Jin et al.,2008) whereas in moths, SNMP1s have been shown to beassociated with sex pheromone detecting neurons in antennae(Forstner et al., 2008; Rogers et al., 1997, 2001a,b). The SNMPfamily has been recently studied in insects, including in Cx.

quinquefasciatus with three SNMP1 paralogs and one SNMP2 genealready identified (Vogt et al., 2009). However in mosquitoes,where no sex pheromone communication has been characterized,the function of SNMPs remains intriguing.

In addition to OBPs and SNMPs, other proteins might beinvolved in odorant reception in mosquitoes. Classified into theOBP family but different from ‘‘classic’’ OBPs, ‘‘plus-C’’ odorant-binding proteins (‘‘plus-C’’ OBPs) possess at least three additionalconserved cysteine residues (‘‘plus-C’’ motif), but have never beenclearly demonstrated to play a role in olfaction. First reported in D.

melanogaster (Hekmat-Scafe et al., 2002), ‘‘plus-C’’ OBP genes havebeen identified in An. gambiae (Li et al., 2005; Xu et al., 2003; Zhouet al., 2004) and Ae. aegypti (Zhou et al., 2008). In An. gambiae, asemi-quantitative RT-PCR approach revealed that five ‘‘plus-C’’OBPs showed no evidence of being enriched in olfactory tissues (Liet al., 2005), whereas another study using real-time quantitativePCR (qPCR) showed that the transcripts of four ‘‘plus-C’’ OBP geneswere highly enriched in antennae, suggesting that they mightencode olfactory proteins (Biessmann et al., 2005).

Another family of soluble proteins, variously called chemosen-sory proteins (CSPs) (Angeli et al., 1999), sensory appendage proteins(SAPs) (Robertson et al., 1999), olfactory-specific-D proteins (OS-D)(McKenna et al., 1994) or A-10 proteins (Pikielny et al., 1994), hasbeen identified across multiple insect orders and possess a typicalconserved cysteine pattern (‘‘CSP’’ motif) (Wanner et al., 2004; Xuet al., 2009; Zhou et al., 2006). In ants, CSPs have been shown to behighly expressed in antennae (Gonzalez et al., 2009; Ishida et al.,2002a; Ozaki et al., 2005) where they might be involved in nest materecognition signals (Gonzalez et al., 2009; Ozaki et al., 2005). Seven

CSP genes have been identified in the mosquito An. gambiae and thetranscripts of those genes were detected in antennae by microarrayhybridization, indicating that they might encode olfactory proteins(Biessmann et al., 2005). In Cx. quinquefasciatus, ‘‘plus-C’’ OBP andCSP families have never been characterized.

The availability of the genome sequence from Cx. quinquefas-

ciatus (Arensburger et al., 2010), an important vector of pathogens(Nasci and Miller, 1996), offers the opportunity to identify largesets of putative olfactory genes belonging to different families.However, little effort has been dedicated to ‘‘plus-C’’ OBP, CSP andSNMP families compared to OBPs and ORs and little is known abouttheir functions in mosquitoes. Thus it becomes important tocharacterize members of these families in order to assess theirpotential involvement in odorant reception. As a part of ourongoing effort to understand the olfactory mechanisms in Cx.

quinquefasciatus, the present study focuses on four main objec-tives: (1) identify members of the ‘‘plus-C’’ OBP and CSP families byusing the genome sequence of Cx. quinquefasciatus (Arensburgeret al., 2010) and carry out comparative analysis with othermosquito species, in order to obtain a better understanding of thediversity within these families, (2) clone the full-length sequencesof SNMP genes to confirm the diversity of this family inCx. quinquefasciatus, (3) characterize candidate ‘‘plus-C’’ OBPs,CSPs and SNMPs enriched in the antennae of Cx. quinquefasciatus byusing a semi-quantitative RT-PCR screening approach, (4) quantifythe transcripts levels of candidate genes identified in aim 3, as wellas candidate OBPs identified previously (Pelletier and Leal, 2009),by qPCR in female and male antennae as well as in non-olfactorytissues, in order to get insights into the expression profiles ofpotentially important olfactory genes.

In the present study, we have identified 13 members of the ‘‘plus-C’’ OBP family and twenty seven members of the CSP family andreconstructed the full-length sequences of four SNMP genes in Cx.

quinquefasciatus. Comparative analysis with other mosquito specieshas allowed the identification of putative orthologs as well asspecies-specific expansions within these families, including a largeexpansion of the CSP family in Culicinae mosquitoes. By usingmolecular approaches, we have identified 16 genes (twelve OBPs,two ‘‘plus-C’’ OBPs, and two SNMPs) which likely encode olfactoryproteins. Among those, eight genes (seven OBPs and one ‘‘plus-C’’OBP) were found to be transcribed at very high levels in femaleantennae compared to CquiOR7 (Xia and Zwiebel, 2006), suggestingthey might encode highly abundant olfactory proteins. Two genes(CquiOBP12 and CquiOBP+C2) displayed clear transcript enrichmentin female antennae compared to male antennae, indicating that theymight be more specifically involved in the reception of semiochem-icals important for females (e.g. selection of oviposition sites or hostsfor blood-feeding). Furthermore, comparative analysis revealed thatthe olfactory genes of Cx. quinquefasciatus are related to putativeorthologs in other mosquitoes, indicating that they might performsimilar functions across different species. A better understanding ofthe role of these proteins in the specificity and sensitivity of thedifferent types of sensilla might provide a better understanding ofhow mosquitoes are able to detect ecologically relevant odorantcues. Additionally, comparison of chemosensory adaptations indifferent mosquitoes might help to understand how differentspecies can occupy different ecological niches, which in turn isessential for species-specific control strategies.

2. Materials and methods

2.1. Identification of ‘‘plus-C’’ OBP and CSP sequences in Cx.

quinquefasciatus

The predicted protein database of Cx. quinquefasciatus

(cquinquefasciatus.PEPTIDES-CpipJ1.2) was downloaded from

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929 917

VectorBase (http://cpipiens.vectorbase.org/index.php) to performhomology searches using Blastp algorithm (Altschul et al., 1997).Amino acid sequences of ‘‘plus-C’’ OBPs and CSPs from D.

melanogaster (twelve ‘‘plus-C’’ OBPs and four CSPs) An. gambiae

(19 ‘‘plus-C’’ OBPs and seven CSPs) and Ae. aegypti (17 ‘‘plus-C’’OBPs) were used as queries in Blast searches. Similar searches wereperformed to identify CSP genes in the Ae. aegypti predicted proteindatabase (aaegypti.PEPTIDES-AaegL1.2). Multiple protein align-ments and sequence identities were computed using GeneDocsoftware (http://www.nrbsc.org/gfx/genedoc/ebinet.htm). N-ter-minal signal peptides were predicted using SignalP v3.0 (http://www.cbs.dtu.dk/services/SignalP) (Bendtsen et al., 2004). Molec-ular weights and isoelectric points of mature proteins werecomputed using ExPASy proteomics server (http://www.expasy.ch/tools/pi_tool.html). Blast in NCBI conserved domainsdatabase was performed to identify ‘‘pheromone-binding family,A10/OS-D’’ (pfam03392) CSP motif.

Gene structures and relative positions of ‘‘plus-C’’ OBP and CSPgenes on genomic supercontigs were studied using VectorBaseannotations. A few gene annotations have been reconstructedmanually, verified by cDNA cloning and sequencing and submittedto GenBank database. The other sequences identified hereoriginate from VectorBase gene annotations and were notconfirmed by cDNA cloning. Mosquito OBP nomenclature makesno distinction between ‘‘classical’’ and ‘‘plus-C’’ OBPs, but for thesake of clarity we decided to adopt a ‘‘plus-C’’ specific nomencla-ture for Cx. quinquefasciatus ‘‘plus-C’’ OBPs (CquiOBP+C1 toCquiOBP+C13). Gene names for ‘‘plus-C’’ OBPs and CSPs of Cx.

quinquefasciatus (and CSPs of Ae. aegypti) were given according tothe relative positions of genes on supercontigs.

2.2. Phylogenetic analysis of mosquito ‘‘plus-C’’ OBPs and CSPs

Amino acid sequences of ‘‘plus-C’’ OBPs identified in mosqui-toes were combined to create an entry file for phylogeneticanalysis in MEGA 4.0.2 (Tamura et al., 2007). An unrootedconsensus neighbor joining tree (Saitou and Nei, 1987) wascalculated at default settings with pairwise gap deletions. Branchsupport was assessed by bootstrap analysis based on 1000replicates. Mosquito ‘‘plus-C’’ OBPs used in phylogenetic analysisfollow the nomenclature established in An. gambiae (Xu et al.,2003) and Ae. aegypti (Zhou et al., 2008). A similar approach wasused to build the tree for mosquito CSPs. An. gambiae CSPs followthe nomenclature we retrieved from NCBI database. Two geneswhich encode identical proteins, AgamCSP1 (CAG26923) andAgamCSP2 (CAG26924) are referred here as AgamCSP2.

2.3. The Cx. quinquefasciatus SNMP gene family annotation

Genomic sequences of supercontig 3.529 containing previouslyidentified and partially annotated SNMP genes were analyzedmanually to reconstruct full-length coding sequences of four SNMPgenes (CquiSNMP1a, b, c and CquiSNMP2). N-terminus and C-terminus regions were predicted based on multiple alignmentswith other dipteran SNMPs. Full-length reconstructed SNMP geneswere confirmed by cDNA cloning and sequencing. Prediction oftransmembrane domains was performed using TMHMM 2.0 server(http://www.cbs.dtu.dk/services/TMHMM/).

2.4. Expression patterns by non-quantitative RT-PCR

Tissue dissection of female Cx. quinquefasciatus mosquitoes(one-to-seven-day-old sugar-fed adults), RNA extraction, cDNAsynthesis and PCR amplifications were performed as described in(Pelletier and Leal, 2009) with minor modifications. Templates forRT-PCR were prepared from females tissues (antennae, maxillary

palps, proboscis, legs and bodies devoid of heads and appendages).Gene-specific primers for 13 ‘‘plus-C’’ OBP, five CSP and four SNMPgenes were designed manually. PCR reactions were performed ontwo independent cDNA samples. All reactions were carried out in afinal volume of 25 ml with 2 units GoTaq1 DNA polymerase(Promega, Madison, WI). Primers used in RT-PCR experiments,including ribosomal protein L8 control gene (CquiRpL8) (Vector-Base CPIJ000162), are listed in Supplemental Table 4.

2.5. Expression analysis by real-time quantitative PCR (qPCR)

Twenty three genes, plus the endogenous control ribosomalprotein S7 encoding gene (CquiRpS7) (VectorBase CPIJ006763)(Iatrou and Biessmann, 2008), were selected to perform qPCRanalysis. Gene-specific primers were designed using OligoPerfectTM

Designer (http://tools.invitrogen.com/content.cfm?pageid= 9716)according to several criteria: an annealing temperature between58 8C and 60 8C, an amplicon size between 80 and 120 base pairs,a G/C content around 50% and, if possible, an amplification productspanning a predicted intron. Primer sequences were also designed toprevent non specific amplifications, especially in the case of highsequence conservation within gene families (OS-E/OS-F, OBP19a‘‘classical’’ OBPs, ‘‘plus-C’’ OBP paralogs, SNMP1 paralogs and CSPparalogs). The specificity of each pair of primers was confirmed byvisualization of a single band amplicon at the expected size.Template cDNAs from female antennae, male antennae, female legsand female bodies were synthesized from 1 mg total RNA usingSuperScript1 VILO cDNA synthesis kit (Invitrogen, Carlsbad, CA),following manufacturer’s instructions. Integrity of cDNAs wasconfirmed by amplification of CquiRpL8 control gene and 10 timesdilutions were prepared prior to qPCR experiments. Reactions werecarried out using EXPRESS SYBR1 GreenERTM qPCR SuperMixUniversal (Invitrogen) in a final volume of 20 ml. Each reactioncontained 10 ml of qPCR SuperMix, 4 ml of diluted cDNA template,200 nM of each primer and 500 nM of ROX reference dye. Mastermixes were prepared to ensure better homogeneity. Reactions weredistributed into MicroAmp1 optical 96-well plates (AppliedBiosystems, Foster City, CA). A standard cycling program, 50 8C for2 min, 95 8C for 2 min, 40 cycles of 95 8C for 15 s and 60 8C for 1 min,was run on AB7300 real-time PCR system (Applied Biosystems). Notemplate control reactions were included for each set of primers.Two replicates were performed on two independent biologicalsamples (RNA and cDNA). Primers and cDNAs were tested inconventional PCR reactions and no bands corresponding to genomicproducts were observed after 40 cycles of amplification. Primersused in qPCR experiments are listed in Supplemental Table 4.

Analysis of qPCR data was performed with SDS software(Applied Biosystems) using the comparative Ct method (DDCt)(Livak and Schmittgen, 2001). A threshold cycle (Ct) was calculatedfor each sample based on experimental amplification plots.Relative DCt values have been obtained by subtracting the Ct

value of a target gene with the Ct value of the endogenous control(CquiRpS7) in each cDNA sample. Additionally, CquiOR7 was usedas an endogenous control to normalize the expression of targetgenes in olfactory tissues (female antennae and male antennaecomparison). Female antennae have been used as a calibrator tocalculate DDCt values between tissues (DCt male antennae orfemale legs or female bodies – DCt female antennae). Finally,�2

DDCt values have been calculated to determine the relativequantity of transcripts between tissues.

To determine the relative transcript levels of different genes infemale antennae, the threshold cycle was manually set to aconstant value of CquiRpS7 fluorescence signal. Then, correspond-ing Ct values for each target gene were used to calculate DCt values(Ct target gene – Ct RpS7). We used CquiOR7 as a calibrator todetermine DDCt values (DCt target gene – DCt OR7). Finally,

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929918

�2DDCt values were calculated to determine the relative amount of

transcript for each gene compared to CquiOR7.

2.6. Cloning and sequencing

Full-length coding sequences of CquiOBP+C1, CquiOBP+C2,CquiCSP1, CquiSNMP1a, CquiSNMP1b, CquiSNMP1c, CquiSNMP2,CquiOBP10 and CquiOBP29 have been manually reconstructed.Full-length PCR products have been amplified from antennal cDNAusing Pfu UltraTM II Fusion HS DNA polymerase (Stratagene, LaJolla, CA) with specific primers designed in the N-terminus and C-terminus regions of reconstructed gene sequences (see below).Purified PCR products have been cloned into pBlueScript SK+(Stratagene, Santa Clara, CA) and sequenced (Davis Sequencing Inc,Davis, CA). Sequences have been deposited into GenBank database.Accession numbers are indicated in Supplemental Table 1 forCquiOBP+C1, CquiOBP+C2, CquiCSP1, CquiSNMP1a, CquiSNMP1b,CquiSNMP1c and CquiSNMP2. Other accession numbers are asfollows: CquiOBP10 (HQ845076), CquiOBP29 (HQ845077).

fl-CquiOBP+C1 forward: 50-ATGAAGCTAGTGCTCACAGTGTCTG-T-30

fl-CquiOBP+C1 reverse: 50-TTAAATAACTGGGCACATTTTGTTGT-30

fl-CquiOBP+C2 forward: 50-ATGCTCCGTAACAGGATCGCCGT-30

fl-CquiOBP+C2 reverse: 50-TCAATTCAGTGGACACTTGGCGTG-30

fl-CquiCSP1 forward: 50-ATGGCGGTCGGGGTCGCGCTGGC-30

fl-CquiCSP1 reverse: 50-TTAGACGGCACCGTACTTGCGTATC-30

fl-CquiSNMP1a forward: 50-ATGAATTTGGCGGACGTGAATTTC-A-30

fl-CquiSNMP1a reverse: 50-CTAGAATCTTTCACGCTGATCCGAC-30

fl-CquiSNMP1b forward: 50-ATGAAGCTGGAAGAGTTAAACTTT-A-30

fl-CquiSNMP1b reverse: 50-TCAGTAACGCTGCTCAACCGGTGCC-30

fl-CquiSNMP1c forward: 50-ATGAAGCTCGATGAGTTGAATTTC-A-30

fl-CquiSNMP1c reverse: 50-TCAGTATCGTTCCTTTTGCGCCGGA-30

fl-CquiSNMP2forward:50-ATGGTGCAATGTACGCTGGTGCTGG-30

fl-CquiSNMP2 reverse: 50- TCATTTGTCGGGATTCCCGCCAAGA-30

fl-CquiOBP10 forward: 50-ATGAGCTGGCGAAGTATCATGCT-30

fl-CquiOBP10 reverse: 50-TTAGGGGAAGAAAAATTTCGGAT-30

fl-CquiOBP19 forward: 50-ATGAAGTGTGCAATGTTGCTAGT-30

fl-CquiOBP19 reverse: 50-CTAAAACAAACCGGCTTTGTTCT-30

fl-CquiOBP29 forward: 50-ATGTGTCGCCTGCAGGTTGTAGTT-30

fl-CquiOBP29 reverse: 50-TTACAGCTGTATTCCTCTCTTCCGC-30

3. Results and discussion

3.1. The ‘‘plus-C’’ OBP family in Cx. quinquefasciatus

We have explored the diversity of the ‘‘plus-C’’ OBP family in thepredicted peptide database of Cx. quinquefasciatus by Blast search(Altschul et al., 1997) using previously identified sequences fromD. melanogaster (Hekmat-Scafe et al., 2002), An. gambiae (Xu et al.,2003; Zhou et al., 2004) and Ae. aegypti (Zhou et al., 2008) asqueries. Candidate sequences were manually screened to retrievethe characteristic features of the family: the presence of apredicted signal peptide sequence and a characteristic eightcysteines/one proline conserved spacing pattern (‘‘plus-C’’ motif),C1-X8-41-C2-X3-C3-X39-47-C4-X17-29-C4a-X9-C5-X8-C6-P-X9–11-C6a (Zhou et al., 2008). Database search allowed the identificationof 13 ‘‘plus-C’’ OBP genes in Cx. quinquefasciatus, a rangecomparable with the diversity found in D. melanogaster (12 genes)(Hekmat-Scafe et al., 2002), and slightly lower than in An. gambiae

(19 genes) (Xu et al., 2003; Zhou et al., 2004) and Ae. aegypti (17

genes) (Zhou et al., 2008). Structural features and accessionnumbers of ‘‘plus-C’’ OBPs from Cx. quinquefasciatus are compiledin Supplemental Table 1. We have carried out cloning andsequencing of two ‘‘plus-C’’ OBP genes, CquiOBP+C1 andCquiOBP+C2. Like in other mosquito species, Cx. quinquefasciatus

‘‘plus-C’’ OBPs share very low average identity as only cysteineresidues are completely conserved in all proteins. The conservedproline residue after C6 is missing in CquiOBP+C5 andCquiOBP+C13. These two genes might represent incompleteannotations, both lacking full-length N-terminus and the firstalso lacking a small portion of C-terminus. Surprisingly, despite apresumably correct full-length annotation, CquiOBP+C4 lacks apredicted signal peptide sequence, indicating it might not be asecreted protein.

Relative position of genes on genomic supercontigs revealedthat most ‘‘plus-C’’ OBP genes are located on three differentgenomic clusters (Supplemental Table 2). A first cluster encom-passes six genes (CquiOBP+C1 to CquiOBP+C6) within 14.7 kb onsupercontig 3.21 (cluster 1) whereas two other clusters containsthree genes (CquiOBP+C7 to CquiOBP+C9) within 25.2 kb onsupercontig 3.70 (cluster 2) and two genes (CquiOBP+C10 andCquiOBP+C11) within 3.9 kb on supercontig 3.219 (cluster 3).Similar organizations on genomic clusters have also been shownfor other mosquitoes An. gambiae (Xu et al., 2003) and Ae. aegypti

(Zhou et al., 2008) ‘‘plus-C’’ OBP genes.To understand the relationships among mosquito ‘‘plus-C’’

OBPs, we have combined peptide sequences of three species (An.

gambiae, Ae. aegypti and Cx. quinquefasciatus) for phylogeneticanalysis. A sequence comparison tree constructed by the neighborjoining method is shown in Fig. 1. The consensus tree highlights atleast four main subdivisions in the mosquito ‘‘plus-C’’ OBP family,with each group supported by high bootstrap values (based on1000 replicates). A large group (group A, 99% bootstrap support)encompasses 28 proteins and includes different subgroups ofrelated proteins among the three species. A second group (group B,100% bootstrap support) encompasses nine proteins separated intotwo different subgroups, each containing related members of thethree species. The last two groups (group C and group D,respectively 96% and 98% bootstrap support) enclose specificlineages of An. gambiae ‘‘plus-C’’ OBPs probably originating from anexpansion event in this species. Four proteins (AgamOBP54,AgamOBP55, CquiOBP+C10, CquiOBP+C11) do not belong to anyof these groups. In Cx. quinquefasciatus, six ‘‘plus-C’’ OBPs fromcluster 1 belong to phylogenetic group A whereas three ‘‘plus-C’’OBPs from cluster 2 belong to phylogenetic group B, indicating thatmembers of each cluster share more identity between them thanwith other proteins. Gene clustering and sequence conservationwithin clusters suggests that the Cx. quinquefasciatus ‘‘plus-C’’ OBPgene family has evolved by gene duplication events, as previouslysuggested for An. gambiae (Xu et al., 2003) and Ae. aegypti (Zhouet al., 2008). Most ‘‘plus-C’’ OBPs are highly divergent betweenmosquito species, but several ‘‘plus-C’’ OBPs belonging to groups Aand B share more than 40% amino acids identity across differentspecies (Supplemental Table 2). The highest conservation levels areobserved within subdivisions of group A (subgroups A1, A2 and A3)and within subdivisions of group B (subgroups B1 and B2).

By using microsynteny analysis we could identify putativeorthologs among mosquito ‘‘plus-C’’ OBP genes belonging tosubgroups A1, A2 and A3 (Fig. 2). Indeed, members of A1(CquiOBP+C1/AgamOBP48), A2 (CquiOBP+C2/AaegOBP47/Aga-

mOBP47) and A3 subgroups (CquiOBP+C3/AaegOBP48/AgamOBP46)share conserved positions on genomic contigs, display the sameorientations (except for AgamOBP47), possess identical genesstructures (except for AgamOBP46) and share a common neigh-boring gene, which itself displays high conservation amongspecies. In an evolutionary context, these findings clearly suggest

Fig. 1. Phylogenetic relationships of mosquito ‘‘plus-C’’ OBPs. The sequence comparison tree was generated by the neighbor joining method. Cx. quinquefasciatus ‘‘plus-C’’

OBPs are in black, An. gambiae ‘‘plus-C’’ OBPs are in blue and Ae. aegypti ‘‘plus-C’’ OBPs are in red. Robust groupings characterized by high bootstrap values are indicated in

bold. Only bootstrap values above 60% are indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929 919

that a subset of orthologous ‘‘plus-C’’ OBP genes has beenconserved in different mosquito species. Other genes located onthe same contig in Cx. quinquefasciatus (CquiOBP+C4, 5 and 6) andAe. aegypti (AaegOBP49, 50, 51, 52, 53 and 54) do not display clearorthology relationships and share low identity between species,suggesting they might have originated from independent species-specific expansion events in both Culex and Aedes mosquitoes.Interestingly, we could not find an ortholog of CquiOBP+C1/AgamOBP48 in Ae. aegypti at the expected genomic location(upstream AaegOBP47) but we found two pairs of clustered genes(AaegOBP42-43 and AaegOBP62-63) related to CquiOBP+C1 and

AgamOBP48 (Fig. 1) (Supplemental Table 2). Within these clusters,AaegOBP42 shares 98% identity with AaegOBP63 whereas Aae-gOBP43 shares 100% identity with AaegOBP62, and both pairsshare a conserved intergenic region, indicating that both clustersmight have originated from a common ancestor gene. Takentogether, our data suggest that mosquito ‘‘plus-C’’ OBP genefamilies expanded by multiple species-specific duplication events,with only a few genes still sharing clear orthology relationships. Itis still unclear how these diversified repertoires of ‘‘plus-C’’ OBPgenes might contribute to the differences in the ecology of thedifferent mosquito species.

Fig. 2. Microsynteny analysis within a subgroup of the ‘‘plus-C’’ OBP family. Colored blocks represent ‘‘plus-C’’ OBP genes on Cx. quinquefasciatus contig 3.21 (in black), Ae.

aegypti contig 1.584 (in red) and An. gambiae chromosome 2L (in blue). Empty blocks represent highly conserved genes with a putative ‘‘hotdog’’ domain. Direction of genes is

indicated by arrowheads. Distance between genes and gene structures are also indicated. Putative orthologous ‘‘plus-C’’ OBP genes are indicated by double-headed arrows.

Red box highlights an Ae. aegypti expansion leading to two pairs of genes related to CquiOBP+C1/AgamOBP48 on contig 1.495 and 1.3337. Genes on Cx. quinquefasciatus contig

3.21: CquiOBP+C1 (CPIJ002105), CquiOBP+C2 (CPIJ002106), ‘‘hotdog’’ gene (CPIJ002107), CquiOBP+C3 (CPIJ002108), CquiOBP+C4 (CPIJ002109), CquiOBP+C5 (CPIJ002110) and

CquiOBP+C6 (CPIJ002111); Ae. aegypti contig 1.584: AaegOBP47 (EAT36414), ‘‘hotdog’’ gene (EAT36415), AaegOBP48 (EAT36416), AaegOBP49 (EAT36419), AaegOBP50

(EAT36420), AaegOBP51 (EAT36423), AaegOBP52 (EAT36425), AaegOBP53 (EAT36426) and AaegOBP54 (EAT36427); An. gambiae chromosome 2L: AgamOBP48 (AGAP007286),

AgamOBP47 (AGAP007287), ‘‘hotdog’’ gene (AGAP007288) and AgamOBP46 (AGAP007289). (For interpretation of the references to colour in this figure legend, the reader is

referred to the web version of the article.)

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929920

3.2. The CSP family in Cx. quinquefasciatus

Using the same approach described above for ‘‘plus-C’’ OBPs, wehave explored the diversity of the CSP family in Cx. quinquefasciatus

and Ae. aegypti. Candidate sequences were manually screened toretrieve the characteristic features of the CSP family: the presenceof a predicted signal peptide sequence, a conserved four cysteinesspacing pattern (‘‘CSP’’ motif), C1-X6–8-C2-X16–21-C3-X2-C4 (Zhouet al., 2006), and the presence of a ‘‘pheromone-binding family,A10/OS-D’’ (pfam03392) motif characteristic of the CSP family. Wehave identified respectively 27 and 43 CSP genes in Cx.

quinquefasciatus and Ae. aegypti, a higher number than in D.

melanogaster (four genes) (Wanner et al., 2004) and An. gambiae

(seven genes) (Zhou et al., 2006), indicating that large expansionsof the family occurred in both Culex and Aedes mosquitoes. Wefound another CSP gene in the genome of An. gambiae, (VectorBaseAGAP001303), which likely encodes a partial CSP lacking a full-length N-terminus, bringing to eight the repertoire of CSP genes inthis species. We refer here to this new CSP as AgamCSP6. Structuralfeatures and accession numbers of CSPs from Cx. quinquefasciatus

are compiled in Supplemental Table 1, whereas accession numbersof CSPs from Ae. aegypti and An. gambiae are compiled inSupplemental Table 3. All putative CSPs share a conservedcharacteristic motif (pfam03392) indicating that they likely belongto the CSP family. In Cx. quinquefasciatus, one CSP gene (CquiCSP27)likely encodes an incomplete protein lacking a full-length N-terminus whereas another (CquiCSP1) has been reconstructed fromtwo partially annotated genes. Reconstruction of CquiCSP1 wasconfirmed by cloning and sequencing.

Relative positions on genomic supercontigs revealed that mostmosquito CSP genes are located at a single genomic location(Supplemental Tables 2 and 3). In Cx. quinquefasciatus, 24 CSPgenes (CquiCSP1 to CquiCSP24) are located within 314.262 kb onsupercontig 3.42. We observed the same trend in A. aegypti, with aset of 36 CSP genes (AaegCSP1 to AaegCSP36) located within963.088 kb on supercontig 1.47, and in An. gambiae with seven CSPgenes located within 105.755 kb on chromosome 3R.

Using the same approach described above for ‘‘plus-C’’ OBPs, wehave built a sequence comparison tree combining CSPs from three

mosquito species (An. gambiae, Ae. aegypti and Cx. quinquefascia-

tus). The consensus tree highlights two main subdivisions withinthe mosquito CSP family (Fig. 3). The upper part has been named‘‘Culicinae expansion group’’ because it contains several subgroupsof Culex or Aedes CSPs but no Anopheles CSPs. In contrast, the lowerpart contains eight subgroups of putative orthologous CSPs highlyconserved among the three mosquito species (Supplemental Table2). These subgroups were named based on the An. gambiae familyannotation in NCBI database, except for newly identifiedAgamCSP6 (Supplemental Table 3). The SAP orthologous group(88% bootstrap support) consists of three subgroups, SAP1, SAP2and SAP3, whereas five independent CSP groups could beidentified, CSP2, CSP3, CSP4, CSP5 and CSP6. Contrary to othersubgroups, CSP6 subgroup encompasses putative orthologs whichare not located within the large clusters of mosquito CSPs. Incontrast to ‘‘plus-C’’ OBPs, mosquitoes CSPs display much moreconservation between species (Supplemental Table 2).

By using microsynteny analysis we could identify putativeorthologs among mosquito CSP genes belonging to SAP and CSPsubgroups (Fig. 4). Indeed, members of CSP4 (CquiCSP1/AaegCSP1/AgamCSP4), CSP2 (CquiCSP2/AaegCSP2/AgamCSP2), CSP5 (CquiCSP3/AaegCSP3/AgamCSP5), CSP3 (CquiCSP4/AaegCSP4/AgamCSP3), SAP3(CquiCSP5/AaegCSP5/AgamSAP3), SAP2 (CquiCSP22/AaegCSP35/AgamSAP2) and SAP1 (CquiCSP23-CquiCSP24/AaegCSP36/Agam-

SAP1) share high identity between them (Supplemental Table 2),are located at conserved locations on genomic contigs, display thesame orientations (except for AgamCSP3) and possess identicalgene structures (except for members of CSP5 subgroup). Moreover,microsynteny analysis revealed the presence of neighboring geneshighly conserved between mosquitoes. In SAP1 subgroup, we haveidentified two genes in Cx. quinquefasciatus (CquiCSP23 andCquiCSP24) related to AgamSAP1 and AaegCSP36. These two geneswhich share 88% amino acids identity might have originated from aputative ancestor ortholog by gene duplication.

Interestingly, genes originating from expansion events aremostly located at identical genomic locations in Cx. quinquefascia-

tus (16 out of 19 expanded genes) and Ae. aegypti (29 expandedgenes), respectively between CquiCSP5 and CquiCSP22 and betweenAaegCSP5 and AaegCSP35 genes (Fig. 4). Additionally, another group

Fig. 3. Phylogenetic relationships of mosquito CSPs. The sequence comparison tree was generated by the neighbor joining method. Cx. quinquefasciatus CSPs are in black, An.

gambiae CSPs are in blue and Ae. aegypti CSPs are in red. Robust groupings characterized by high bootstrap values are indicated in bold. Only bootstrap values above 60% are

indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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of two CSPs from Culex expansion (CquiCSP26 and CquiCSP27) andsix CSPs from Aedes expansion (AaegCSP37 to AaegCSP42) areclustered on another genomic location. Comparison of CSPsoriginating from expansion events revealed that they share high

sequence conservation within and between species (typicallyabove 50% amino acids identity). Moreover, CSPs from expansionevents are more related to SAP than CSP orthologs (Fig. 3)(Supplemental Table 2), suggesting that expansions in Culex and

Fig. 4. Microsynteny analysis within a large genomic cluster of CSPs. Colored blocks represent CSP genes on Cx. quinquefasciatus contig 3.42 (in black), Ae. aegypti contig 1.47

(in red) and An. gambiae chromosome 3R (in blue). Empty blocks represent three triplets of highly conserved genes. Direction of genes is indicated by arrowheads. Distance

between genes and gene structures are also indicated. Putative orthologous CSP genes are indicated by double-headed arrows. Genes on Cx. quinquefasciatus contig 3.42:

CquiCSP1 (CPIJ002600-CPIJ002601); ‘‘chitotriosidase-1’’ (CPIJ002602); ‘‘imaginal disc growth factor’’ (CPIJ002604); CquiCSP2 (CPIJ002605); CquiCSP3 (CPIJ002607);

CquiCSP4 (CPIJ002608); CquiCSP5 (CPIJ002609); CquiCSP22 (CPIJ002627); CquiCSP23 (CPIJ002628); CquiCSP24 (CPIJ002629); ‘‘membrane traffic protein’’ (CPIJ002634). Ae.

aegypti contig 1.47: AaegCSP1 (EAT46822); ‘‘imaginal disc growth factor’’ (EAT46823); ‘‘imaginal disc growth factor’’ (EAT46826); AaegCSP2 (EAT46827); AaegCSP3

(EAT46830); AaegCSP4 (EAT46831); AaegCSP5 (EAT46832); AaegCSP35 (EAT46863); AaegCSP36 (EAT46864); ‘‘membrane traffic protein’’ (EAT46869); An. gambiae

chromosome 3R: AgamCSP4 (AGAP008062); ‘‘protein coding gene’’ (AGAP008061); ‘‘protein coding gene’’ (AGAP008060); AgamCSP2 (AGAP008059); AgamCSP5

(AGAP008058); AgamCSP3 (AGAP008055); AgamSAP3 (AGAP008054); AgamSAP2 (AGAP008052); AgamSAP1 (AGAP008051); ‘‘protein coding gene’’ (AGAP008046). (For

interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Aedes species might have a common origin. To support thishypothesis, we found putative orthology relationship betweenCquiCSP21 and AaegCSP34, both genes sharing conserved location,orientation, structure and high identity between them (84%amino acids conservation). The high identity within subsets of CSPparalogs in Culex and Aedes (sometimes above 90% amino acidsidentity) also suggests recent species-specific duplication events.Thus, the large expansion of the CSP family in both Culex and Aedes

species likely occurred from multiple duplication events after thesplit with An. gambiae, considerably increasing the repertoire ofSAP-like genes in Culicinae mosquitoes. The physiologicalsignificance of such expansions in the CSP family of Culex andAedes mosquitoes represents an interesting topic for furtherstudies.

Gene duplication events leading to expansions of multigenicfamilies are proposed as the main explanation for the increase inthe total number of predicted genes in Cx. quinquefasciatus andto a lesser extent in Ae. aegypti when compared to An. gambiae

(Arensburger et al., 2010). The expansion of the CSP family inCulicinae offers another illustration of this phenomenon.Previously, expansions of different families of olfactory genessuch as the OR family (Arensburger et al., 2010), the ionotropicreceptor (IR) family (Croset et al., 2010) and the ‘‘classical’’ OBPfamily (Pelletier and Leal, 2009) have been documented in Cx.

quinquefasciatus and Ae. aegypti. However, it is still unclearwhether the increase of the number of paralogs within thesefamilies together with gene diversification events, are correlatedwith an increased chemosensory capability to occupy newecological niches.

3.3. The SNMP gene family in Cx. quinquefasciatus

Members of the SNMP family have been previously identified inCx. quinquefasciatus, including three SNMP1 paralogs (CquiSNMP1a,CquiSNMP1b and CquiSNMP1c) and one SNMP2 (CquiSNMP2) (Vogtet al., 2009). Using genomic data and comparison with SNMPs fromother mosquito species (AgamSNMP1, AgamSNMP2, AaegSNMP1

and AaegSNMP2) (Vogt et al., 2009), we have reconstituted the full-length coding sequences of CquiSNMP1a, b and c as well asCquiSNMP2. Reconstructed sequences were further confirmed bycloning and sequencing. Structural features of Cx. quinquefasciatus

SNMPs and their accession numbers are available in SupplementalTable 1. All SNMPs were predicted to contain two transmembranedomains as well as a large extracellular loop, a characteristicfeature of insect SNMPs. The presence of three paralogous SNMP1genes in Cx. quinquefasciatus is unique in mosquitoes as both An.

gambiae and Ae. aegypti possess a single SNMP1 encoding gene(Vogt et al., 2009). Based on conserved gene structures (sevenexons/six introns, including conserved exon boundaries) and closerange localization, it is likely that Cx. quinquefasciatus SNMP1expansion arose from duplication events, as suggested previously(Vogt et al., 2009). Sequence comparison between paralogsrevealed that CquiSNMP1a shares 61% amino acids identity withboth CquiSNMP1b and CquiSNMP1c which share 77% amino acidsidentity between them, indicating that gene diversificationsignificantly affected the primary sequence of SNMP1 genes.Interestingly, we found that diversification between paralogsoccurred mostly in the C-terminus region located immediatelyafter the second transmembrane domain. Moreover, CquiSNMP1b

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and CquiSNMP1c are closer to other mosquito SNMP1s thanCquiSNMP1a (Supplemental Table 2) but it is unclear howdiversification of SNMP1 genes in Cx. quinquefasciatus has affectedtheir functional properties, especially regarding their putativeinvolvement in hypothetical sex pheromone reception.

3.4. Screening for transcripts enriched in antennae by RT-PCR

We have used a semi-quantitative RT-PCR approach to identifycandidate genes, among ‘‘plus-C’’ OBP, CSP and SNMP families,which present expression patterns compatible with a role inolfaction. Indeed, all genes previously shown to ensure olfactoryfunctions have been demonstrated to be preferentially transcribedin chemosensory tissues. We have previously carried out similarexperiments to characterize olfactory candidates in a largemultigenic family of 53 OBP genes (Pelletier and Leal, 2009). Thisapproach led to the identification of the transcripts of 13 genes thatwere detected exclusively in olfactory tissues, whereas transcriptsof 25 genes were detected in both olfactory and non-olfactorytissues indicating that they might encode encapsulins, notnecessarily involved exclusively in olfaction (Leal, 2005). In thepresent study, we have studied the expression patterns of 13 ‘‘plus-C’’ OBP, five CSP and four SNMP genes in female olfactory tissues(antennae, maxillary palps and proboscis) and non-olfactorytissues (legs and bodies devoid of heads and appendages) in orderto identify which transcripts are enriched in the main olfactoryorgan, the antennae (Fig. 5). Female tissues were chosen to perform

Fig. 5. Expression profiles of Cx. quinquefasciatus ‘‘plus-C’’ OBP, CSP and SNMP genes by

maxillary palps (FMp), proboscis (FPr), legs (FL) and bodies devoid of heads and appendag

have been tested. Amplification of CquiRpL8 was used as control of cDNA integrity.

this initial screening in order to identify genes involved in female-specific behaviors such as detection of host and oviposition sites.

Tested on female tissues, transcripts of ‘‘plus-C’’ OBP genesdisplayed heterogeneous patterns of expression (Fig. 5A). Twogenes, CquiOBP+C1 and CquiOBP+C2, consistently exhibited thestrongest bands in antennae compared to other genes. Moreover,CquiOBP+C1 was detected exclusively in olfactory tissues (anten-nae, maxillary palps and proboscis), whereas CquiOBP + C2 wasalso detected weakly in non-olfactory tissues (legs and bodies).Transcripts from other ‘‘plus-C’’ OBP genes were detectedubiquitously (CquiOBP+C8, 9, 10, 11 and 13), in all tissues minusantennae (CquiOBP+C5), in all tissues minus antennae and bodies(CquiOBP+C7), only in legs (CquiOBP+C12) or were not detected atall (CquiOBP+C3). Interestingly, CquiOBP+C4 and CquiOBP+C6

consistently exhibited a stronger band in maxillary palpscompared to other tissues, indicating that these transcripts mightbe enriched in this accessory olfactory organ. The expressionpatterns of 13 members of the ‘‘plus-C’’ OBP family in Cx.

quinquefasciatus resemble those observed in another mosquito,An. gambiae. In this species, a first study using a semi-quantitativeRT-PCR approach showed a wide distribution for five ‘‘plus-C’’ OBPgenes (AgamOBP46, 48, 51, 54 and 57) (Li et al., 2005). The authorsconcluded that none of these transcripts showed evidence of beingenriched in olfactory tissues. In another study, the expressionpatterns of several ‘‘plus-C’’ OBP genes were examined by using RT-PCR and qPCR (Biessmann et al., 2005). The authors showed thatfour genes were transcribed at high levels in female antennae

RT-PCR. Template cDNAs were prepared from adult female tissues: antennae (FA),

es (FB). Thirteen ‘‘plus-C’’ OBP genes (A), five CSP genes (B) and four SNMP genes (C)

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(AgamOBP47 (6th rank), 48 (2nd), 54 (11th) and 57 (18th)),comparable with those of ‘‘classical’’ OBPs, whereas two tran-scripts were not detected at all in antennae (AgamOBP50 and 51).Additionally, they showed that all genes were transcribed infemale maxillary palps and that three genes were also transcribedin female bodies (AgamOBP50, 51 and 54). Interestingly AgamOBP47

and AgamOBP48, two genes with highly enriched transcripts infemale antennae are putative orthologs of CquiOBP+C1 andCquiOBP+C2 (Fig. 2), two genes that display a strong transcriptionin Cx. quinquefasciatus female antennae (Fig. 5A). Taken together,these studies suggest that this subset of orthologous ‘‘plus-C’’ OBPgenes in Anopheles and Culex mosquitoes share high expression inchemosensory tissues, especially in the antennae. On the otherhand, most other ‘‘plus-C’’ OBP genes are broadly distributed in Cx.

quinquefasciatus and might be involved in a more general function,not specifically olfactory. No expression studies are available on Ae.

aegypti ‘‘plus-C’’ OBP genes.We have selected five CSP genes for tissue-specificity study,

focusing on the members of SAP1, SAP2, SAP3 (CquiCSP5, 22, 23 and24) and CSP2 (CquiCSP2) subgroups (Fig. 4). Indeed, in D.

melanogaster transcript of an SAP1-related gene, DmelOS-D, wasdetected exclusively in antennal tissues by northern blot(McKenna et al., 1994) whereas in An. gambiae, microarrayhybridization experiments revealed that transcripts of four genes(AgamSAP1, AgamSAP2, AgamSAP3 and AgamCSP3) were detected inantennae with only one of them (AgamSAP2) also detected inheadless bodies (Biessmann et al., 2005). When tested on Cx.

quinquefasciatus female tissues, five CSP genes clearly displayedubiquitous expression profiles, indicating they might not beinvolved specifically in olfaction (Fig. 5B). In many insects, CSPshave been suggested to ensure a general role in the transport ofhydrophobic molecules, based on their broad distribution patternsand broad binding specificity (Wanner et al., 2004; Zhou et al.,2006). Our data indicate that it might also be the case inmosquitoes, at least for a subset of Cx. quinquefasciatus CSPs.

Transcripts of paralogous SNMP1 genes have been detected inall female tissues (Fig. 5C). However, clear variations in bandintensities suggest that SNMP1a, SNMP1b and to a lesser extentSNMP1c might be enriched in antennae. On the other hand,CquiSNMP2 expression profile revealed clearly similar transcriptlevels in olfactory tissues and in legs, indicating that this genemight not be involved specifically in olfaction but may still beinvolved in the transduction of peripheral chemosensory signals,possibly in contact chemosensation. The expression patterns offour members of the SNMP family in Cx. quinquefasciatus resemblethose of SNMP genes in Ae. aegypti. Indeed, AaegSNMP1 andAaegSNMP2 were detected by RT-PCR in antennae, legs and wingsbut with a more intense band in antennae for AaegSNMP1 and innon-olfactory tissues for AaegSNMP2 (Vogt et al., 2009), whereas inAn. gambiae, AgamSNMP1 was detected by RT-PCR exclusively inantennae but not in body (Benton et al., 2007).

3.5. Selection of candidate genes for quantitative analysis

Odorant detection mediates important behaviors critical for thesurvival of mosquitoes. As these mechanisms are specificallylocated within olfactory tissues, it is reasonable to speculate thatolfactory mechanisms are achieved by abundant proteins prefer-entially enriched in these tissues. By using qPCR, our objective wasto determine accurately the expression profiles of 23 candidategenes (see below) in different tissues of Cx. quinquefasciatus, inorder to identify highly enriched transcripts in the main olfactoryorgan, the antennae.

Based on RT-PCR screening experiments from the present studyand from a previous study of the OBP family (Pelletier and Leal,2009), we have selected a subset of candidate genes displaying

transcript enrichment in antennae for qPCR analysis: two ‘‘plus-C’’OBPs (CquiOBP+C1 and CquiOBP+C2), three SNMPs (CquiSNMP1a,CquiSNMP1b and CquiSNMP1c) and 13 OBPs (CquiOBP1 to 9,CquiOBP11 to 14). Additionally, three ubiquitous OBPs (CquiOBP19,29 and 50) and an OBP detected in olfactory tissues as well as inlegs (CquiOBP10) (Pelletier and Leal, 2009) were also selected foranalysis. The co-receptor CquiOR7 (newly named CquiOrco), anolfactory gene expressed in most types of olfactory sensilla (Xiaand Zwiebel, 2006), was also included.

3.5.1. Transcripts of 16 genes are highly enriched in female antennae

We have first determined the relative transcript levels ofcandidate genes in female adult tissues (antennae, legs and bodies)to identify which transcripts are enriched in antennae versus twodifferent non-olfactory tissues. The relative transcript ratios foreach gene between female antennae and female legs (FA/FL) andbetween female antennae and female bodies devoid of heads andappendages (FA/FB) were calculated after normalization byCquiRpS7 (Fig. 6).

We found the transcripts of 17 genes (including CquiOR7) highlyenriched in FA compared to FL (CquiOBP1 to CquiOBP4, FA/FLranging from 76667 to 213 times) (Fig. 6A). Transcripts ofremaining genes were slightly enriched in FA (CquiOBP9 toCquiSNMP1c, ranging from 50 to 7.8 times), equivalently expressed(CquiOBP50, 1.21 times) or enriched in FL (CquiOBP19, 0.53 times).Most transcripts enriched in FA (CquiOBP6 to CquiOBP4) displayed aFA/FL ratio comparable to CquiOR7 (652 times), whereas sixtranscripts (CquiOBP1, CquiOBP7, CquiOBP11, CquiOBP+C1,CquiOBP12, and CquiOBP2) displayed higher ratios (FA/FL rangingfrom 76667 to 2415). We observed the same trend when wecalculated the relative transcript ratios between FA and FB(Fig. 6B). We found the transcripts of 18 genes (including CquiOR7)highly enriched in FA compared to FB (CquiOBP1 to CquiSNMP1b,FA/FB ranging from 716667 to 1232 times). Transcripts ofremaining genes were moderately enriched in FA (CquiOBP29 toCquiOBP50, ranging from 455 to 3.3 times). Most transcriptsenriched in FA (CquiOBP3 to CquiSNMP1b) displayed a FA/FB ratiocomparable to CquiOR7 (4053 times), whereas seven transcripts(CquiOBP1, CquiOBP+C1, CquiOBP11, CquiOBP7, CquiOBP12,CquiOBP2 and CquiOBP6) displayed higher ratios (FA/FB rangingfrom 716,667 to 11,066).

Comparison of transcript ratios in antennae versus non-olfactory tissues (legs or bodies) confirmed that transcripts of16 genes (CquiOBP1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13 and 14; CquiOBP+C1

and 2; CquiSNMP1a and b) are indeed highly enriched in femaleantennae compared to female non-olfactory tissues and likelyencode important olfactory proteins of Cx. quinquefasciatus. Amongthose, six genes (CquiOBP1, 2, 6, 7, 11, 12 and CquiOBP+C1)displayed the highest transcript enrichment in FA in bothexperiments. Similar qPCR analyses showed that putative ortho-logs of CquiOBP1 and CquiOBP7 in Anopheles stephensi (respectivelyAsteOBP1 and AsteOBP7) and Ae. aegypti (respectively AaegOBP1 andAaegOBP2) also displayed high transcript enrichment in FAcompared to non-olfactory tissues (Sengul and Tu, 2008, 2009,2010), suggesting a common role for these orthologous genes inthe olfactory mechanisms of several mosquito species.

The present study also confirmed that most but not all OBPs areenriched in olfactory tissues. Indeed, transcripts of CquiOBP19 andCquiOBP50, two genes previously shown as ubiquitous based onRT-PCR profiles (Pelletier and Leal, 2009), were detected at similarlevels in antennae and in non-olfactory tissues by qPCR, suggestingthat they might be involved in broader functions and are notnecessarily dedicated to odorant reception. However, it is stillunclear whether four genes, which display only moderateenrichment in FA, can be considered as candidate olfactory genes.Indeed, CquiOBP9 (FA/FL = 50; FA/FB = 243), CquiOBP10 (FA/

Fig. 6. Transcript ratios of 23 candidate genes in female tissues by qPCR. Transcripts ratios have been calculated in female antennae versus female legs (FA/FL) (A), and in

female antennae versus female bodies devoid of heads and appendages (FA/FB) (B). Normalization of cDNA templates was achieved by using CquiRpS7 as an endogenous

control. A logarithmic scale (base 10) was used for y-axis. Genes order is based on decreasing ratio values.

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FL = 8.1; FA/FB = 4856), CquiOBP29 (FA/FL = 21.8; FA/FB = 455) andCquiSNMP1c (FA/FL = 7.8; FA/FB = 30.2) are all enriched in FAcompared to non-olfactory tissues but much less than other 16genes highly enriched in FA. Interestingly, we noticed a generalhigher FA/FB ratio compared to FA/FL ratio for most genes, typicallyaround one order of magnitude, indicating slight transcriptenrichment in FL compared to FB. We found one transcript,CquiOBP10, much more abundant in FL than in FB, suggesting thatthis gene might play a role in both odorant and contactchemoreception.

3.5.2. Female antennae versus male antennae

In order to identify genes potentially involved in sex-specificolfactory behaviors, we have calculated the transcript ratiosbetween female and male antennae (FA/MA) by using twoindependent endogenous controls, CquiRpS7 and CquiOR7 genes(Fig. 7). We noticed that FA/MA ratios for all genes wereconsistently higher (around 10 times) when CquiRpS7 was usedas a control (Fig. 7A) than when CquiOR7 was used as a control(Fig. 7B), confirming a trend previously observed by semi-quantitative RT-PCR analysis of Cx. quinquefasciatus OBPs (Pelletierand Leal, 2009) and by qPCR analysis of An. funestus OBPs (Xu et al.,2010). As previously discussed (Pelletier and Leal, 2009), thisdiscrepancy likely reflects the sexual dimorphism in antennal

structures, female antennae harboring olfactory sensilla through-out 13 segments whereas olfactory sensilla are restricted to the lasttwo distal segments of male antennae. In An. gambiae, qPCRexperiments showed that AgamOR7 transcript was around twelvetimes more abundant in FA than in MA when RpS7 was used ascontrol (Iatrou and Biessmann, 2008), which is comparable to whatwe obtained in Cx. quinquefasciatus for CquiOR7 (FA/MA = 9.5) byusing the same control. Due to differences in antennal structures,CquiOR7 might represent a better control to compare the transcriptlevels of olfactory genes between FA and MA, by providing a moreaccurate ‘‘per sensilla’’ normalization.

Based on CquiOR7 normalization we could identify twotranscripts, CquiOBP+C2 (FA/MA = 3.5) and CquiOBP12 (FA/MA = 2.13), enriched in FA (Fig. 7B). Most other genes (CquiOBP1

to CquiOBP9) displayed comparable ratios (FA/MA ranging from1.53 to 0.64), five were enriched in MA (CquiOBP14 toCquiOBP10) (FA/MA ranging from 0.54 to 0.39) and two werehighly enriched in MA (CquiOBP50 and CquiOBP19). Our datasuggest that most candidate olfactory genes are unlikelyinvolved in sex-specific olfactory behaviors of Cx. quinquefas-

ciatus and may rather be involved in the reception of commonsemiochemicals in the antennae of both sexes. Only a few geneswere found to be preferentially transcribed in female or maleantennae but no dramatic differences could be unveiled among

Fig. 7. Transcript ratios of 23 candidate genes in female and male antennae by qPCR. Transcripts ratios have been calculated in female antennae versus male antennae (FA/

MA). Normalization of cDNA templates was achieved by using two independent endogenous controls, CquiRpS7 (A) and CquiOR7 (B). Genes order is based on decreasing ratio

values.

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929926

our panel of OBP, ‘‘plus-C’’ OBP and SNMP genes. Indeed, wefound that the highest enrichment (based on CquiOR7 normali-zation) was 3.5 times in FA (CquiOBP+C2) and 2.6 times inMA (CquiOBP10), if we exclude CquiOBP19 and CquiOBP50 basedon their ubiquitous expression profiles. As a comparison, in amoth species, pheromone-binding proteins which are involvedin the reception of female sex pheromones in male antennaedisplayed much higher male-specific enrichments, from 14 to372 times (Allen and Wanner, 2011). Even if we cannot rule outthat other OBPs and ‘‘plus-C’’ OBPs not tested here might beinvolved, it is reasonable to speculate that sex-specific olfactorybehaviors in Cx. quinquefasciatus might be driven by thedifferential expression of a subset of ORs and/or IRs (or possiblyother unidentified genes). The recent identification of largefamilies of 181 ORs (Arensburger et al., 2010) and 69 IRs (Crosetet al., 2010) in this mosquito offers interesting perspectives todecipher gender-specific differences in the olfactory system ofCx. quinquefasciatus.

Interestingly, among all olfactory candidates, the gene with themost important antennal sex bias, CquiOBP+C2, is a putativeortholog to AgamOBP47 which was previously shown to be one ofthe most enriched transcripts in FA compared to MA (Biessmannet al., 2005), suggesting that these genes might be involved more

specifically in the reception of semiochemicals important forfemales across different mosquito species. Among SNMPs,CquiSNMP1a and CquiSNMP1b did not display any enrichmentbetween sexes (FA/MA = 0.92 and 1, respectively) whereasCquiSNMP1c was enriched in MA (FA/MA = 0.43), but it is difficultto speculate about the role of SNMP1 paralogs in antennae withoutevidence for a clear sex pheromone communication in Cx.

quinquefasciatus.

3.5.3. Transcripts abundance in female antennae

Based on the assumption that the genes with the mostabundant transcripts in antennae should ensure importantolfactory functions in this tissue, we have determined the relativetranscript amounts of each candidate gene in FA by using the co-receptor CquiOR7 as a calibrator (Fig. 8). From our panel ofcandidates, 13 genes displayed higher transcripts levels thanCquiOR7, the most abundant OR gene. We found that eight genes(CquiOBP1 to CquiOBP6) exhibit considerably higher transcriptlevels than CquiOR7 (35.1–4.2 times CquiOR7 level), six genes(CquiOBP5 to CquiOBP+C2) display slightly higher or similartranscript levels (1.56–0.92 times CquiOR7 level), five genes(CquiSNMP1b to OBP19) display lower transcript levels (0.61–0.16 times CquiOR7 level) and three genes (CquiSNMP1c to

Fig. 8. Transcript abundance of 23 candidate genes in female antennae by qPCR. Relative transcripts amounts were calculated in CquiRpS7 normalized cDNA by using CquiOR7

as a calibrator. Genes order is based on decreasing transcript amount values.

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929 927

CquiOBP50) display much lower transcript levels (0.06–0.007 timesCquiOR7 level).

This experiment revealed a high heterogeneity of transcriptabundance in FA among olfactory genes. Indeed, the mostabundant transcript, CquiOBP1, is around 92 times more abundantthan the less abundant transcript, CquiOBP4. We have identifiedeight genes with very high transcript levels in FA (CquiOBP1, 2, 6, 7,8, 11, and 12; CquiOBP+C1), suggesting that they encode highlyabundant olfactory proteins. We confirmed here that most OBPgenes that belong to the OS-E/OS-F, PBPRP1, LUSH, OBP19a andPBPRP4 mosquito OBP groups are indeed highly transcribed andenriched in FA, as we previously suggested (Pelletier and Leal,2009). On the other hand, genes with lower transcript enrichmentin FA compared to non-olfactory tissues (CquiOBP9, 10, 19, 29, 50

and CquiSNMP1c) also displayed lower transcript level in FA withthe exception of CquiOBP29, which represents the 10th mostabundant transcript (1.53 times CquiOR7 level), comparable witholfactory genes CquiOBP5 and CquiOBP13.

Among all candidates, CquiOBP+C1 represents the 4th mostabundant transcript in FA whereas CquiOBP+C2 ranks at the 15thplace. Interestingly, putative CquiOBP+C1 ortholog, AgamOBP48,was determined as the 2nd most abundant transcript in An.

gambiae FA (Biessmann et al., 2005), suggesting that these genesare transcribed at very high levels in FA across different mosquitospecies. The physiological significance of ‘‘plus-C’’ OBPs inolfactory tissues remains intriguing as little is known about theirfunction. It has been suggested that AgamOBP48 is capable ofinteracting with specific ‘‘classical’’ OBPs in vitro, leading to theformation of heterodimers and higher order complexes (Andro-nopoulou et al., 2006). However, it is still unclear how theseinteractions between ‘‘plus-C’’ and ‘‘classical’’ OBPs affect therecognition of odorants in vivo.

Two SNMP genes, CquiSNMP1a and CquiSNMP1b present similartranscription profiles, indicating that they might be co-regulated.Indeed, they share similar transcript amounts in FA (0.61 and 0.57times CquiOR7, rank 16th and 17th, respectively) as well as similarFA/FL, FA/FB (Fig. 6) and FA/MA ratios (Fig. 7). Based on theirphysical proximity in the genome, these genes might sharecommon regulatory regions. Moreover, the transcript levels of

CquiSNMP1a and CquiSNMP1b in FA are relatively high fortransmembrane protein encoding genes, suggesting that theymight be transcribed in a rather large population of sensilla on theantennae. Both transcripts are around 10 times more abundant inFA than CquiSNMP1c transcript (0.06 times CquiOR7, rank 21st)which also displays much lower enrichment in FA (Fig. 6) and isslightly enriched in MA (Fig. 7). Based on qPCR experiments, it isclear that CquiSNMP1a and CquiSNMP1b are differentiallyexpressed compared to CquiSNMP1c. These differences in expres-sion profiles indicate clear modifications in the regulatory regionsthat govern the transcription of SNMP1 genes. Taken together,expression profiles and gene diversification suggest an expandedfunctional role of SNMP1 paralogs in Cx. quinquefasciatus, asalready suggested (Vogt et al., 2009). Furthermore, it is reasonableto speculate that contrary to CquiSNMP1a and b, the role ofCquiSNMP1c might be more general and not exclusively restrictedto olfaction. The expansion of SNMP genes in Cx. quinquefasciatus

which likely occurred by gene duplication events was accompa-nied by both sequence diversification and modification of theregulatory regions (in the case of CquiSNMP1c). Considering theclose proximity of these genes in the genome, analysis of theirdistribution in different subtypes of sensilla, together with thecharacterization of their regulatory regions might provideinteresting insights about how evolution can modify the expres-sion patterns of evolutionary related genes.

4. Conclusions

To design new strategies for vector control, like the develop-ment of new attractants or repellents, it is critical to understandhow mosquitoes can detect ecologically relevant odorant cues. It islikely that the response profiles of different functional types ofsensilla rely on the differential expression of a diversity of genesbelonging to different families. Our work contributes to a betterunderstanding of the diversity of olfactory proteins in Cx.

quinquefasciatus. We demonstrate that transcripts of a subset ofOBPs, ‘‘plus-C’’ OBPs and SNMPs are highly enriched in the mainolfactory tissue, the antennae, indicating that these genes are likelyinvolved specifically in the reception of odorants. We showed

J. Pelletier, W.S. Leal / Journal of Insect Physiology 57 (2011) 915–929928

previously that an OBP, CquiOBP1, is important for the reception ofoviposition attractants in female antennae. By identifying a largeset of new olfactory genes, we provide new insights into themolecular mechanisms of odorant reception in Culex mosquitoes.Functional approaches will help to dissect the role of these proteinsand determine how they contribute to the acute sensitivity andspecificity of the different functional types of sensilla in Cx.

quinquefasciatus. Along with the characterization of responseprofiles from ORs and IRs, future experiments could provide abetter understanding of how Culex mosquitoes are able to detectand discriminate semiochemicals critical for their survival.

Acknowledgements

The authors would like to thank Professor Anthony Cornell forproviding Cx. quinquefasciatus mosquitoes for our laboratorycolony, Dr. Zainulabeuddin Syed for his critique of an early draftof the manuscript and Dr. Wei Hu for assistance in qPCRexperiments. This work was supported in part by the NationalScience Foundation grant 0918177, the USDA NIFA-AFRI grant2010-65105-20582, a Cooperative Agreement with BedoukianResearch, Inc., and the UC Davis Agricultural Experiment Station.Funding sources did not play any role in the design of the study,data collection, analysis and interpretation, writing of themanuscript and in the decision to submit the manuscript forpublication.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.jinsphys.2011.04.003.

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