) 61–68www.elsevier.com/locate/gene

Gene 394 (2007

Differential expression of genes in pyrethroid resistant and susceptiblemosquitoes, Culex quinquefasciatus (S.)

Nannan Liu a,b,⁎, Huqi Liu a,1, Fang Zhu a, Lee Zhang c

a Department of Entomology and Plant Pathology, Auburn University, Auburn AL 36849, USAb Cellular and Molecular Biosciences Program, Auburn University, Auburn AL 36849, USA

c Genomics and Sequencing Laboratory, Auburn University, Auburn, Alabama 36849-5413, USA

Received 4 November 2006; received in revised form 24 January 2007; accepted 26 January 2007

Available online

Received by J.G. Zhang

20 February 2007

Abstract

The transcriptional regulation of gene expression is a primary means by which insects adapt to a changing environment. The evolution ofinsecticide resistance is conferred through mechanisms, typically requiring the interaction of multiple genes. Consequently, the characterizationof gene regulation and interactions in resistance is fundamental for achieving an understanding of the complex processes responsible forresistance. cDNA macroarray technology offers a promising new approach for investigating the complicated processes responsible for resistancedevelopment by revealing the interrelations of all of the elements in a resistant system simultaneously. In the current study, we compared thegene expression profiles of resistant and susceptible Culex quinquefasciatus mosquitoes, using a combination of subtractive PCR hybridizationand cDNA microarray technique. By screening of 1500 cDNA clones from a resistant-susceptible mosquito subtractive library, we identified aset of genes with up-regulated expression in insecticide resistant Culex mosquitoes through transcriptional profiling compared to those insusceptible mosquitoes. These genes are vital for cellular and molecular metabolism, signal transduction, vesicular and molecular transport,protein biosynthesis, ubiquitination, and neuronal survival, but most have not previously been implicated in insecticide resistance. Functionalstudies of resistance-associated up-regulated genes should shed new light on both the molecular basis of resistance and the regulatory pathwaysthat lead to it.© 2007 Elsevier B.V. All rights reserved.

Keywords: Subtractive hybridization; EST; Evolutionary selection; Gene expression

1. Introduction

Insecticide resistance is assumed to be a pre-adaptivephenomenon, in that prior to insecticide exposure rare individualsalready exist who carry an altered genome that results in one or

Abbreviations: EST, expressed sequence tag; WNV, West Nile virus; SLEV,Saint Louis encephalitis virus; SSH, suppression subtractive hybridization;ORF, open reading frame; Rho, Rhodopsin; GPCR, G-protein-coupled receptor;GRK, G-protein-coupled receptor kinase.⁎ Corresponding author. Department of Entomology and Plant Pathology, 301

Funchess Hall, Auburn University, Auburn, AL 36849-5413, USA. Tel.: +1 334844 2661; fax: +1 334 844 5005.

E-mail address: nliu@acesag.auburn.edu (N. Liu).1 Current address: Department of Biology, Northwest Sci-Tech University of

Agriculture and Forestry, Yangling 712100, Shaanxi, China.

0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2007.01.032

more possible mechanisms (genes) allowing them to survive theevolutionary selection pressure of insecticides (World HealthOrganization, 1957). Gene overexpression, amplification, andstructural mutations have frequently been linked to insecticideresistance (Dong, 1997; Raymond et al., 1998; Scott, 1999;Soderlund and Knipple, 2003; Hemingway et al., 2004;Nabeshima et al., 2004; Feyereisen, 1999, 2005) and a largenumber of studies have indicated that multiple resistancemechanisms or genes are involved in insecticide resistance ofindividual insect species (Raymond et al., 1989; Liu and Yue,2000, 2001; Hemingway et al., 2002; Ranson et al., 2002; Pedraet al., 2004; Liu et al., 2005; Xu et al., 2005; Vontas et al., 2006).Transcriptional overexpression of genes in resistant insectsappears to be a common determining event in the evolution ofresistance development in insects (Carino et al., 1994; Liu and

62 N. Liu et al. / Gene 394 (2007) 61–68

Scott, 1997; 1998; Vontas et al., 2006). Furthermore, trans and/orcis transcriptional factors are thought to form the linkages andarchitectures of regulatory networks controlling overexpressionlevels for genes that mediate physiological responses toinsecticides (Grant and Hammock, 1992; Carino et al., 1994;Liu and Scott, 1997, 1998; Hemingway et al., 2004). Takentogether, the above findings not only indicate a commonphenomenon of insecticide resistance conferred by multi-resistance mechanisms or genes, but also offer a tantalizinghint of a regulatory interaction among different mechanisms and/or genes in resistance. However, it remains unclear how manygenes are involved and how the products of these genes interactto with each other. Recently, Vontas et al. (2006) reportedmultiple genes that were differentially expressed betweensusceptible and DDT resistant Anopheles gambiae mosquitoes,including a cytochrome P450 gene, CYP314A1. However, mostof these genes have not been reported in insecticide resistancepreviously. While this result may be puzzling as to whether thesegenes are associated with resistance, it values more thecomplexity of an organism that in response to the evolution ofinsecticide selection.

The mosquito Culex quinquefasciatus is an importantdisease vector throughout the wet tropics. In the SoutheasternUSA, this species is moderately competent as a vector of WestNile virus (WNV) (Sardelis et al., 2001) and is a primaryvector of Saint Louis encephalitis virus (SLEV) in manyurban settings (Jones et al., 2002). The approach used tocontrol transmission of these pathogens and other mosquito-associated diseases has primarily relied on the application ofinsecticides (McCarroll and Hemingway, 2002). However,mosquito-borne diseases are now resurgent, largely becauseof the insecticide resistance that has developed in mosquitovectors (Phillips, 2001; Hemingway et al., 2002). Recently, amosquito strain of C. quinquefasciatus, HAmCqG0, fromHuntsville, Alabama, has been established in the laboratory.The HAmCqG0 mosquito strain exhibited high levels ofresistance to pyrethroids (Liu et al., 2004) and was furtherselected in the laboratory with permethrin for 6 generations,generate a strain of HAmCqG6 (Xu et al., 2006). In earlierstudies, we identified multiple resistance mechanisms, such asP450-, hydrolase-, and/or GST-mediated detoxication andtarget site insensitivity, that were involved in pyrethroidresistance in this Culex strain (Liu et al., 2005; Xu et al., 2005,2006). We hypothesize that gene expression patterns must bealtered in Culex mosquitoes in response to resistance as thosethat have been recently reported in other insect species (Pedraet al., 2004; Vontas et al., 2006). To test our hypothesis and toisolate the genes or factors governing resistance, in the currentstudy we monitored the genes that were differentiallyexpressed within the entire genome of Culex mosquitoes inresponse to resistance, using a combination of suppressionsubtractive hybridization (SSH) and cDNA macroarrays (dotblot macroarrays). Using this approach, we identified 24genes that are up-regulated in resistant mosquitoes comparedwith susceptible ones. These genes will provide useful toolswith which to examine the function of the genes in insecticideresistance.

2. Materials and methods

2.1. C. quinquefasciatus strains

Two strains of mosquito C. quinquefasciatus were used inthis study. HAmCqG6 were collected from Huntsville County,Alabama, USA (Liu et al., 2004) and further selected withpermethrin at larvae stages for 6 generations (Xu et al., 2006)after collection; and S-Lab, a insecticide-susceptible strain thatwas obtained from Dr. Laura Harrington at Cornell University.All mosquitoes were reared at 25±2 °C under a photoperiod of12:12 (L:D) h.

2.2. Construction of a subtractive HAmCqG6/S-Lab PCR cDNAlibrary

Total RNA of 3rd instar larvae (∼20 larvae) for eachmosquito strain was extracted by the acidic guanidinethiocyanate–phenol–chloroform method (Liu and Scott,1997). Poly(A)+RNA from each sample was isolated withOligotex-dT suspension (QIAGEN) as described by themanufacturer. Double-stranded cDNA was synthesized from2 μg poly(A)+RNA of each sample using the PCR-selectedcDNA Subtraction Kit (Clontech) as described by themanufacturer. The cDNAs that contained specific transcriptsare referred to here as “testers” (i.e. from HAmCqG6) and thereference cDNAs as “drivers” (i.e. from S-Lab). The double-stranded cDNAs of both testers and drivers were digested withRsa 1 to create smaller blunt-ended fragments to be used astesters or drivers according to the manufacturer's instructions(Clontech). The tester cDNAs were subdivided into twoportions, A and B, and modified by ligating with cDNAadaptors 1 and 2 (provided in the kit, Clontech), respectively, asdescribed by the manufacturer.

Two-step subtractive hybridizations were carried out. In thefirst step hybridization, two primary hybridization reactions (Aand B) were performed by adding an excess amount ofunmodified driver cDNA to separate portions A and B of testercDNA samples at a 10:1 ratio. Samples were denatured for2 min at 98 °C and allowed to anneal for 8 h at 68 °C. Theremaining single-stranded, adaptor-ligated tester cDNAs (i.e.,the differentially expressed tester sequences) were dramaticallyenriched in each of the hybridization reactions for differentiallyexpressed sequences because non-target cDNAs present in thetester and driver formed hybrids. For the second stephybridization, A and B primary hybridization reaction solutionswere mixed together without denaturing. Thus, the remainingsubtracted single-stranded tester cDNAs were allowed to re-associate and form new hybrids. These new hybrids weredouble-stranded tester molecules (i.e., the differentially ex-pressed tester sequences) with different 5′ ends correspondingto the sequences of two different adaptors. Fresh denatureddriver DNA was added to the reaction (again, without dena-turing the subtraction mix) to further enrich the new double-stranded tester molecules. After filling in the ends with DNApolymerase (Clontech), the differentially expressed sequences(tester cDNA) had different annealing sites on their 3′ and 5′

Table 1Analysis of ESTs with signal intensity differences greater than 2-fold (Pb0.01)in the HAmCqG6–S-Lab subtractive library

HamCqG6–S-Lab subtractive library

# of total sequences a 24# of high quality sequences 24 (100%)# of contigs 1# of sequences in contigs 3 (13% of the mRNA mass)# of singletons 21 (87% of the mRNA mass)# of unique sequences 22 (singletons+contigs)Redundancy 8% (24–22/24)

a EST sequences showed statistically significant differences across any two ofthree different tests.

63N. Liu et al. / Gene 394 (2007) 61–68

ends. The molecules were then subjected to suppression sub-traction PCR as described by the manufacturer (Clontech) usingthe Advantage 2 polymerase (Clontech) and primers that cor-responded to sequences of the adaptors. In the entire populationof molecules, only those double-stranded tester molecules withtwo different adaptors at the ends were amplified exponentially.Subsequently, the subtractive HAmCqG6/S-Lab (tester versesdriver) library was constructed by directly cloning PCR-cDNAproducts that were recovered after subtraction into PCR™ 2.1vectors with a PCR™ 2.1 Original TA Cloning Kit (Invitrogen),as described by the manufacturer.

2.3. cDNA macroarray and data analysis

cDNA fragments (inserts) from these plasmid DNAs wereamplified by PCR across the multiple cloning site of the TAvector using M13 reverse and forward primers. Equal amountsof each PCR product were printed in triplicate on each Nytranmembrane (Schleicher and Schuell). In each experiment, fouridentical parallel membrane sets with arrayed PCR productswere hybridized by α-32P-dCTP labeled subtracted (HAmCqG6/S-Lab and S-Lab/HAmCqG6) and unsubtracted (HAmCqG6 andS-Lab) cDNA probes synthesized using PCR-Select DifferentialScreening Kit (Clontech) according to the manufacturer'sinstruction. Each cDNA macroarray experiment was repeatedthree times, each time with newly prepared membrane sets andprobes from different samples. As a result, the macroarrayexperimental design for the HAmCqG6/S-Lab library was: 4identical membrane sets with cDNA arrays (for 4 differentialscreening probes)×3 replicates (newly prepared probes fromdifferent preparations of samples). In total, there were 12parallel sets of array membranes for the HAmCqG6/S-Lablibrary. Our macroarray design with technical and biologicalreplicates would permit us to explore not only variations in thetechnology we are using but also the differences in theunderlying biological system we are interested in. A Student'st-test was applied for the comparisons, using the SAS v9.1software package (SAS Institute Inc.) to determine significantdifference in the gene expression. A value of P≤0.01 wasconsidered statistically significant (Patel et al., 2005). Up-regulation was considered when clones showed ≥2-fold(Pb0.01) difference in the signal intensity in no less than tworepeated membranes.

2.4. Northern blotting analysis

Total RNAs of 3rd instar larvae (∼20 larvae) and adults (20males and 20 females) for each mosquito strain were extracted.Ten micrograms of total RNA from each sample (larvae andadults of HAmCqG6 and S-Lab) was separated by formaldehydedenaturing gel electrophoresis. The cDNAs, based on macro-array results, were derived from differentially expressedcandidate colons, labeled with [α-32P] dCTP using a Primer-ItII Random Primer Labeling kit (Stratagene) according to themanufacturer's instruction, and hybridized with RNA blotsusing QuickHyb solution (Stratagene). The amount of RNAloaded in each lane was standardized by comparing the density

of the 18S ribosomal RNA (rRNA) band on agarose gel underUV before transfer (Liu and Zhang, 2004). All northernanalyses were repeated three times with different preparationsof RNA samples. The radiographic signal intensity wasquantitatively analyzed by QuantiScan v3.0 (Biosoft) as ourprevious analyses (Liu and Zhang, 2004). The statisticalanalysis of the gene up-regulation was performed usingANOVA for multiple comparisons. A value of P≤0.01 wasconsidered statistically significant.

3. Results

3.1. Identification of genes overexpressed in resistant C.quinquefasciatus mosquitoes

In order to obtain a molecular profile of the differential geneexpressions associated with insecticide resistant mosquito C.quinquefasciatus, a combination of subtractive hybridizationand cDNA macroarray techniques was used to detect differ-ences in the gene expression between resistant and susceptibleCulex strains. A cDNA subtractive library, HAmCqG6/S-Lab,was constructed using cDNA from HAmCqG6 as the tester,which was enriched for the cDNAs expressed from resistantHAmCqG6 mosquitoes. A total of 1500 ESTs from the HAmCq-G6/S-Lab subtractive cDNA library were screened. Of the ESTstested, 24 showed up-regulation with ≥2-fold (Pb0.01)difference in the signal intensity in no less than two repeatedtests. These ESTs were selected for further study. Sequenceanalysis revealed that these clones contained partial fragmentsof genes. In these 24 high quality up-regulated ESTs, 3 wereassembled into 1 contig. The remaining 21 ESTs weresingletons. The combination of the contig and singletons was22 in the subtractive cDNA library, representing uniquetranscripts and suggesting an 8% redundancy (Table 1). Thelow redundancy in our subtractive library indicates theadvantages of using SSH in the equalization (normalization)of transcript abundance in the target population. Further, the useof SSH makes it possible to combine normalization, whichequalizes transcript abundance in the target population, withsubtraction, which excludes the common sequences shared byboth the target and driver populations, in the same procedure.Thus, using of SSH allows to dramatically increase theprobability of obtaining differentially expressed rare transcripts(Diatchenko et al., 1996; Gurskaya et al., 1996).

64 N. Liu et al. / Gene 394 (2007) 61–68

3.2. Quantitative analysis of up-regulated genes in resistant C.quinquefasciatus mosquitoes

The cDNAs of 22 unique transcripts that showed up-regulation with ≥2-fold (Pb0.01) difference were chosen forfurther analyses by Northern blotting analysis to investigatetheir expression level in the larva and adult stages of bothresistant and susceptible mosquitoes. Northern blot analysisindicated that 20 of the 22 transcripts showed consistent up-regulation with ≥2-fold (P≤0.01) in the expression in both thelarva and adult stages of the resistant HAmCqG6 straincompared with the susceptible S-Lab strain (Fig. 1). Two ESTsequences (#14 (P13b7) and #21 (P19d4)) showed differences≥1.5-fold (P≤0.01) in the expression between these twostrains. The Northern blotting results confirmed the reliability ofour subtractive PCR hybridization and cDNA macroarraysstudy. The low false positive rate identified here may be largelydue to the macroarray experiment design (with three indepen-dent replications) and because the positive clone was chosenfrom at least two independent tests. The probability of the samefalse positives occurring by chance in two separate tests is quitelow, reflecting the product of the P-value probabilities found onboth tests (for example, 0.01×0.01=0.0001), and thereforeproviding enhanced statistical confidence in the scientificinferences (Blalock et al., 2005). It should be noted, however,that failure to repeat a significant expression change does notnecessarily imply that the gene alteration observed in one studywas a false positive. In addition, the reliability (reproducibility)of the findings for the statistically significant clones across anytwo of the three different tests but not necessarily all three testswas designed to eliminate increasing the rate of false negativeerrors. The replication of macroarray experiments and the

Fig. 1. Northern blot analysis of differentially expressed gene patterns from the cDNAof HAmCqG6 or S-Lab) was separated by formaldehyde denaturing gel electrophoresimacroarray results. The ethidium bromide stain of 18s ribosomal RNA in agarose g

statistical analysis of the up-regulated clones across all thereplications are important factors in reducing the false positiverate. The high percentage for the genes that were notdifferentially expressed in our resistant vs. susceptible mosquitosubtractive library is likely to be due primarily to the lowefficiency of the subtraction, which has a lower ratio (10:1) forthe driver vs. tester compared to the commonly used highstringency ratio of 100:1. This relatively low stringency ratiowas used in order to obtain most, if not all, of the differentiallyexpressed genes since if the ratio of driver vs. tester is too high,some of the differentially expressed cDNAs will be lost duringthe subtraction process (Konietzko and Kuhl, 1998).

3.3. Sequencing analysis of up-regulated genes in resistantC. quinquefasciatus mosquitoes

The deduced amino acid sequences of 22 unique clones werecompared to known proteins using a BCBI BLAST search. Sixof the 22 cDNAs had no database match to proteins in theGenBank protein database with known functions and were thusdefined as unknown genes, while the remaining 16 clonesexhibited protein homologies with the GenBank database(Table 2). These results indicate that this approach of usingSSH and cDNA macroarrays to identify differentially expressedgenes between resistant and susceptible mosquitoes is highlysuccessful, uncovering a number of up-regulated genes inresistant mosquitoes. At first glance, the number of non-matched sequences (6 of 22) is somewhat striking, indicatingthat 27% of the cDNAs failed to be matched with specifictranscripts. The short 3′ sequences of the clones, which mighthave failed to reveal specific transcript information, could beone explanation for the unknown sequences. However, an open

macroarrays. Ten micrograms of total RNA from each sample (larvae or adultss. Blots were hybridized with the cDNA probes derived from clones based on theel is shown at the bottom.

Table 2Up-regulated genes identified in resistant mosquitoes, HAmCqG6, compared with susceptible S-Lab mosquitoes

Function and clone name Accession number Signal intensity (Pb0.01) Homologous protein % similarity, organisms

Cellular and molecular metabolism#6 (P16g10) EC093821 18±5.0 HAD-superfamily hydrolase subfamily IIB 25%, Clostridium beijerincki#10 (P24g9) EC093825 2.5±0.4 Fatty acid synthase 49%, Canis familiaris#18 (P15a8) EC093833 17.4±5.5 Cytochrome P450 CYP9K1 45%, Anopheles gambiae#24 (P49a11) EC093837 15.2±4.2 Cytochrome P450 CYP9F2 48%, Tribolium castaneum

Signal transudation and regulation#4 (P8f9), 20, 22 EC093819 5.0±2.0 Rhodopsin 77%, Papilio glaucus#23 (P47c2) EC093836 10±3.1 Arrestin 67%, Apis mellifera

Vesicular and molecular transport#2 (P6h12) EC093817 4.8±1.2 Rab-protein 5 87%, Drosophila melanogaster#17 (P14g2) EC093832 5.3±2.0 Mitochondrial phosphate carrier protein 93%, Aedes aegypti

Cytoskeletal network#3 (P7f1) EC093818 6.7±1.4 Actin 100%, Culex pipiens pipiens#9 (P21g8) EC093824 7.2±1.7 Muscle-specific actin 3 95%, Aedes aegypti

Protein biosynthesis and ubiquitination#1 (P6c4) EC093816 8.8±1.2 F-box 74%, Danio rerio#8 (P18g9) EC093823 7.4±1.4 Ribosomal protein L38 74%, Spodoptera frugiperda#16 (P14d9) EC093831 3.7±0.9 Ribosomal protein L19 92%, Aedes aegypti#19 (P15g8) EC093834 2.7±1.1 Ribosomal protein L2/L8 100%, Aedes aegypti#21 (P19d4) EC093835 2.2±0.5 Elongation factor 1-alpha 70%, Drosophila melanogaster

Neuronal survival#12 (P38a7) EC093827 7.4±2.9 Enolase 85%, Drosophila subobscura

Unknown#5 (P12a11) EC093820 4.3±0.8 Unknown#7 (P18a9) EC093822 15±4.8 Unknown#11 (P26c9) EC093826 4.2±0.6 Unknown#13 (P2g3) EC093828 5.5±1.1 Unknown#14 (P13b7) EC093829 3.4±0.3 Unknown#15 (P13e11) EC093830 8.8±1.9 Unknown

65N. Liu et al. / Gene 394 (2007) 61–68

reading frame (ORF) analysis revealed that 5 of the 6 (83%) ofthese non-matched sequences actually contain identifiableprotein coding regions, suggesting that these function-unknowngenes may encode completely new classes of proteins in Culexmosquitoes (Hinton, 1997). Annotation of the 16 up-regulatedcDNAs that were functionally interpretable based on theirsimilarities to genes of known function showed an enrichmentof gene groups associated with cellular and molecularmetabolism (clones #6, #10, #18, and #24), signal transductionand regulation (clones #4 and #23), vesicular and moleculartransport (clones #2 and #17), cytoskeletal network (clones #3and #9), protein biosynthesis and ubiquitination (clones #1,#8, #16, #19, and #21), and neuronal survival (clone #12)(Table 2).

4. Discussion

Transcriptional overexpression of genes in resistant insectsappears to be a common determining event in the developmentof resistance in insects, and transcriptional factors are known toform the linkages and architectures of the regulatory networksthat control overexpression levels for genes. Developing the

novel strategies that are urgently needed to circumvent and/ordelay resistance development and control resistant mosquitoeswill require building a fundamental understanding of the genesand regulatory networks governing resistance development. Toaddress this issue, this study serves as the first attempt todecipher the genes that are up-regulated in resistant mosquitoes,C. quinquefasciatus, using a combination of subtractive PCRhybridization and cDNA macroarray techniques. Our resultsrevealed a cluster of co-regulated genes with increasedexpression in resistant Culex mosquitoes. Twenty two uniquetranscripts were found to be up-regulated in the resistantmosquito strain. Yet, besides the role of cytochrome P450s thathas been clearly demonstrated in insecticide resistance(Feyereisen, 1999, 2005; Scott, 1999), the function of most ofthese genes in insecticide resistance has not yet been reported,which agrees with the previous study on A. gambiae (Vontaset al., 2006). The high degree of similarity to known proteinsequences may strongly suggest possible functions althoughthese putative proteins may diverge into different or new classesof proteins, especially when partial sequences of clones arecompared (Evans and Wheeler, 1999). Therefore, the highdegree of similarity shown by these clones (Table 1) indicates

66 N. Liu et al. / Gene 394 (2007) 61–68

that the functions of their homologous proteins merit seriousconsideration in insecticide resistance.

Genes that play a role in the metabolic function that underliesthe detoxification of both endogenous and exogenous (includ-ing insecticides) compounds were among those that increasedmost significantly in the resistant mosquitoes (Table 2, Fig. 1).Two cytochrome P450s showed significantly increased expres-sion in the resistant mosquitoes. P450s have been known to becentrally involved in metabolic detoxification of xenobiotics,including insecticides. Previous studies have indicated that anumber of resistance mechanisms are present in HAmCqG6 andthat P450-mediated detoxication is one of the major resistancemechanisms involved based on a synergistic study usingpiperonyl butoxide, an inhibitor of cytochrome P450s (Xu etal., 2005). Nevertheless, no individual P450 genes have yetbeen identified in this strain as being responsible for resistance.Although our previous study indicated that hydrolase-mediateddetoxification was one of the factors in pyrethroid resistance inCulex mosquitoes (Xu et al., 2005), HAD-superfamily hydro-lase subfamily IIB identified in the study has not beenpreviously reported in resistance. Characterizing the functionof the overexpressed putative cytochrome P450 and hydrolasesubfamily IIB genes in HAmCqG6 will shed new light on themechanisms of resistance in HAmCqG6 and provide funda-mental information to increase our understanding of metabolic-mediated resistance in mosquitoes.

Genes that play a role in signal transduction systems wereresistance up-regulated, notably the G-protein-coupled signaltransduction system, with increased expression of bothRhodopsin (Rho) and Arrestin (Table 2, Fig. 1). Rho is thearchetypal class A G-protein-coupled receptor (GPCR) (Filipeket al., 2003). GPCRs act as transducers for a range of differentsensory, chemotactic, hormonal, and neuronal signals, and areinvolved in many essential physiological functions of organ-isms. Arrestins are a gene family of regulatory proteins that,along with G-protein-coupled receptor kinases (GRKs) andother co-factors, regulate signaling and trafficking of G-protein-coupled receptors by virtue of their preferential binding to thephosphorylated active form of the receptor (Pierce et al., 2002).Up-regulation of these two genes in resistant mosquitoes maysuggest that the neuronal signaling is affected in the resistantmosquitoes. G-protein coupled receptors are widely expressedin the body and play a fundamental role in physiology andpathophysiology and have been used as potential targets fortherapeutic intervention in many human diseases (Wise et al.,2002). Nevertheless, the role of GPCR-mediated signaltransduction system in insecticide resistance had not beenexamined prior to this study. The resistance-specific over-expression of Arrestins and Rhodopsin in resistant mosquitoesthat was identified in this study highlights the functionalimportance of the signal transduction system in the regulation ofinsecticide resistance.

The genes involved in vesicular and molecular transportshowed increased expression in resistant mosquitoes, includingRab GTPase and mitochondrial phosphate carrier protein. Rabproteins are members of the family of small GTPases, whichincludes proteins that are involved in a variety of biological

processes including signal transduction (Ras), regulation of thecytoskeleton (Rac and Rho), nuclear transport (Ran), andintracellular traffic (Rab) (Amillet et al., 2006). Rab proteinshave been directly implicated in the regulation of vesiculartransport and membrane traffic and localized to variouscompartments of both the biosynthetic/secretory and endocyticpathways of eukaryotic cells (Martinez and Goud, 1998).Moreover, genes that are involved in protein biosynthesis,ubiquitination, and/or degradation, such as ribosomal proteinsand F-box, a motif for ubiquitin dependent protein lysis in cellregulation and signal transduction, are overexpressed in theresistant mosquitoes. This suggests that the rapid regulation andturnover of proteins in the resistant mosquitoes are involved inresponse to resistance. Therefore, it is not surprising that genesinvolved in vesicular and molecular transport are also over-expressed in the resistant mosquitoes.

The neuron-specific marker, enolase, increased significantlyin the resistant mosquitoes. Neuron-specific enolase has beenshown to promote the development, growth, and survival ofneurons, increasing neuron numbers and modifying their energymetabolism (Yang et al., 2006). The significant increase in theexpression of the enolase gene observed in the resistantmosquitoes suggests that the resistance may be associatedwith major changes in the number of neuronal cells, promotingtheir survival and modifying their metabolism. Since the insectnervous system is the primary target for most insecticides,including pyrethroids, promoting neuron survival and prevent-ing neuronal dysfunction in resistant insects is not unexpected.

Two up-regulated ESTs were highly homologous to actingenes that are involved in cytoskeletal networks. Cytoskeletalproteins serve important cell biologic functions, includingmaintaining cellular mechanical integrity, stress responses,vesicle transport, cell division, and motility, among otherfunctions (Pollard, 2003). In addition, actin overexpression mayserve either protective or damage-promoting roles, as has beensuggested in human studies (Zhong and Omary, 2004). One ofthe actin EST (clone #9) showed 95% similarity to the codingregion of muscle-specific actin 3, AeAct-3, which has beenreported as a male-specific actin gene in Aedes aegypti(Vyazunova and Lan, 2004). Whether this actin EST is male-specific gene in Culex mosquitoes as its homologous one in A.aegypti or is involved in mosquito resistance needs furtherinvestigation. However, it will not be surprising if genes that arein response to insecticide resistance or physiological processesare sex dependent. Recently, Goff et al. (2006) reported thatseveral genes (such as P450s and GSTs) that were associatedwith insecticide resistance and xenobiotic induction were sex-dependently expressed. Cyp312a1, a cytochrome P450 geneinvolved in ecdysteroid biosynthesis, was male-specificallyexpressed (Kasai and Tomita, 2003).

In conclusion, our study strongly supported the hypothesisthat biological and physiological pathways and geneticsignatures are significantly altered in response to insecticideselection. Identifying all the genes in the resistance system of anorganism is like listing all the parts of a vehicle. By itself it is notsufficient to reveal the complexity underlying the engineeredobject (Kitano, 2003). However, to understand how a particular

67N. Liu et al. / Gene 394 (2007) 61–68

system functions, we must first examine the individual genes inthe resistance system. The genomic resources and knowledgedeveloped by this study will contribute to better understandingmolecular mechanisms that govern development of insecticideresistance. Based on this study, our next step must be tocharacterize the functions of these genes, map out the networkof gene interactions, and examine the biochemical pathways bywhich such interactions modulate the physiological propertiesin insecticide resistant systems. The new knowledge andunderstanding of genes in resistance and regulatory networkwill provide us the important information for designing novelstrategies to control the mosquito vectors, circumventing and/ordelaying resistance development, and reducing the prevalenceof mosquito-borne diseases.

Acknowledgements

This study was supported by the U.S. Department ofAgriculture National Research Initiative (USDA-NRI) Com-petitive Grants Program Grants 2001-35302-10956 to N.L. andL.Z.; AAES Foundation Grants to N.L.; and the Hatch ProjectALA08-029. We thank Jan Szechi for the editorial assistance.

References

Amillet, J.M., et al., 2006. Characterization of human Rab20 overexpressed inexocrine pancreatic carcinoma. Human Pathol. 37, 256–263.

Blalock, E.M., et al., 2005. Harnessing the power of gene microarrays for thestudy of brain aging and Alzheimer's disease: statistical reliability andfunctional correlation. Ageing Res. Rev. 4, 481–512.

Carino, F.A., Koener, J.F., Plapp Jr., F.W., Feyereisen, R., 1994. Constitutiveoverexpression of the cytochromeP450 geneCYP6A1 in a house fly strainwithmetabolic resistance to insecticides. Insect Biochem. Mol. Biol. 24, 411–418.

Diatchenko, L., et al., 1996. Suppression subtractive hybridization: a method forgenerating differentially regulated or tissue-specific cDNA probes andlibraries. Proc. Natl. Acad. Sci. U. S. A. 93, 6025–6030.

Dong, K., 1997. A single amino acid change in the para sodium channel proteinis associated with knockdown-resistance (kdr) to pyrethroid insecticides inGerman cockroach. Insect Biochem. Mol. Biol. 27, 93–100.

Evans, J.D., Wheeler, D.E., 1999. Differential gene expression betweendeveloping queens and workers in the honey bee, Apis mellifera. Proc.Natl. Acad. Sci. U. S. A. 96, 5575–5580.

Feyereisen, R., 1999. Insect P450 enzymes. Annu. Rev. Entomol. 44, 507–533.Feyereisen, R., 2005. Insect cytochrome P450. In: Gilbert, L.I., Iatrou, K., Gill,

S. (Eds.), Comprehensive Molecular Insect Science, vol. 4. Elsevier, Oxford,pp. 1–77.

Filipek, S., Stenkamp, R.E., Teller, D.C., Palczewski, K., 2003. G protein-coupled receptor rhodopsin: a prospectus. Annu. Rev. Physiol. 65, 851–879.

Grant, D.F., Hammock, B.D., 1992. Genetic and molecular evidence for a trans-acting regulatory locus controlling glutathione S-tranferase-2 expression inAedes aegypti. Mol. Gen. Genet. 234, 169–176.

Goff, G.L., et al., 2006. Xenobiotic response in Drosophila melanogaster: sexdependence of P450 and GST gene induction. Insect Biochem. Mol. Biol.36, 674–682.

Gurskaya, N.G., et al., 1996. Equalizing cDNA subtraction based on selectivesuppression of polymerase chain reaction: cloning of jurkat cell transcriptsinduced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal.Biochem. 240, 90–97.

Hemingway, J., Field, L., Vontas, J., 2002. An overview of insecticideresistance. Science 298, 96–97.

Hemingway, J., Hawkes, N.J., McCarroll, L., Ranson, H., 2004. The molecularbasis of insecticide resistance in mosquitoes. Insect Biochem. Mol. Biol. 34,653–665.

Hinton, J.C., 1997. The Escherichia coli genome sequence: the end of an era orthe start of the FUN? Mol. Microbiol. 26, 417–422.

Jones, S.C., Morris, J., Hill, G., Alderman, M., Ratard, R.C., 2002. St. Louisencephalitis outbreak in Louisiana in 2001. J. La. State Med. Soc. 154,303–306.

Kasai, S., Tomita, T., 2003. Male specific expression of a cytochrome P450(Cyp312a1) in Drosophila melanogaster. Biochem. Biophys. Res. Com-mun. 300, 894–900.

Kitano, H., 2003. Systems biology: a brief overview. Science 295, 1662–1664.Konietzko, U., Kuhl, D., 1998. A subtractive hybridization method for the

enrichment of moderately induced sequences. Nucleic Acids Res. 26,1359–1361.

Liu, N., Scott, J.G., 1997. Phenobarbital induction of CYP6D1 is due to a transacting factor on autosome 2 in house flies, Musca domestica. Insect Mol.Biol. 6, 77–81.

Liu, N., Scott, J.G., 1998. Increased transcription of CYP6D1 causescytochrome P450-mediated insecticide resistance in house fly. InsectBiochem. Mol. Biol. 28, 531–535.

Liu, N., Yue, X., 2000. Insecticide resistance and cross-resistance in the housefly (Diptera: Muscidae). J. Econ. Entomol. 93, 1269–1275.

Liu, N., Yue, X., 2001. Genetics of pyrethroid resistance in a strain (ALHF) ofhouse flies (Diptera: Muscidae). Pestic. Biochem. Physiol. 70, 151–158.

Liu, N., Zhang, L., 2004. CYP4AB1, CYP4AS1 and Gp-9 overexpression inworkers of the red imported fire ant, Solenopsis invicta. Gene 327, 81–87.

Liu, H., Cupp, E.W., Micher, K.M., Guo, A., Liu, N., 2004. Insecticideresistance and cross-resistance in Alabama and Florida strains of Culexquinquefasciatus. J. Med. Entomol. 41, 408–413.

Liu, H., Xu, Q., Zhang, L., Liu, N., 2005. Chlorpyrifos resistance in themosquito, Culex quinquefasciatus (S.). J. Med. Entomol. 42, 815–820.

Martinez, O., Goud, B., 1998. Rab proteins. Biochim. Biophys. Acta 1404,101–112.

McCarroll, L., Hemingway, J., 2002. Can insecticide resistance status affectparasite transmission in mosquitoes? Insect Biochem. Mol. Biol. 32,345–1351.

Nabeshima, T., et al., 2004. An amino acid substitution attributable toinsecticide-insensitivity of acetylcholinesterase in a Japanese encephalitisvector mosquito, Culex tritaeniorhynchus. Biochem. Biophys. Res. Com-mun. 313, 794–801.

Patel, O.V., et al., 2005. Validation and application of a high fidelity mRNAlinear amplification procedure for profiling gene expression. Vet. Immunol.Immunopathol. 105, 331–342.

Pedra, J.H., Mclntyre, L.M., Scharf, M.E., Pittendrigh, B.R., 2004. Genome-wide transcription profile of field- and laboratory-selected dichlorodiphenyl-trichloroethane (DDT)-resistant Drosophila. Proc. Natl. Acad. Sci. U. S. A.101, 7034–7039.

Phillips, R.S., 2001. Current status of malaria and potential for control. Clin.Microbiol. Rev. 14, 208–226.

Pierce, K.L., Premont, R.T., Lefkowitz, R.J., 2002. Seven-transmembranereceptors. Nat. Rev., Mol. Cell Biol. 3, 639–650.

Pollard, T.D., 2003. The cytoskeleton, cellular motility and the reductionistagenda. Nature 422, 741–745.

Ranson, H., et al., 2002. Evolution of supergene families associated withinsecticide resistance. Science 298, 179–181.

Raymond, M., Arnaouty, V.B., Sivasubramanian, N., Mouches, C., Georghiou,G.P., Pasteur, N., 1989. Amplification of various esterase B's responsible fororganophosphate resistance in Culex mosquitoes. Biochem. Genet. 27,417–423.

Raymond, M., Chevillon, C., Guillemaud, T., Lenormand, T., Pasteur, N., 1998.An overview of the evolution of overproduced esterases in the mosquitoCulex pipiens. Philos. Trans. R. Soc. Lond., B Biol. Sci. 353, 1707–1711.

Sardelis, M.R., Turell, M.J., Dohm, D.J., O'Guinn, M.L., 2001. Vectorcompetence of selected North American Culex and Coquillettidiamosquitoes for West Nile virus. Emerg. Infect. Dis. 7, 1018–1022.

Scott, J.G., 1999. Cytochromes P450 and insecticide resistance. Insect Biochem.Mol. Biol. 29, 757–777.

Soderlund, D.M., Knipple, D.C., 2003. The molecular biology of knockdownresistance to pyrethroid insecticides. Insect Biochem. Mol. Biol. 33,563–577.

68 N. Liu et al. / Gene 394 (2007) 61–68

Vontas, J., et al., 2006. Gene expression in insecticide resistant and susceptibleAnopheles gambiae strains constitutively or after insecticide exposure.Insect Mol. Biol. 14, 509–521.

Vyazunova, I., Lan, Q., 2004. Stage-specific expression of two actin genes in theyellow fever mosquito, Aedes aegypti. Insect Mol. Biol. 13, 241–249.

Wise, A., Gearing, K., Rees, S., 2002. Target validation of G-protein coupledreceptors. Drug Discov. Today 7, 235–246.

World Health Organization, 1957. WHO Expert Committee on Insecticides.WHO Tech Rp Ser 7th Rpt.

Xu, Q., Liu, H., Zhang, L., Liu, N., 2005. Mechanisms of permethrin resistance inmosquitoes, Culex quinquefasciatus (S.). Pest Manag. Sci. 61, 1096–1102.

Xu, Q., Wang, H., Zhang, L., Liu, N., 2006. Kdr allelic expression of the sodiumchannel gene in insecticide resistance of Culex quinquefasciatus. Biochem.Biophys. Res. Commun. 345, 774–780.

Yang, Y.L., Chen, H.F., Kuo, T.J., Lin, C.Y., 2006. Mutations on CaENO1 inCandida albicans inhibit cell growth in the presence of glucose. J. Biomed.Sci. 13, 313–321.

Zhong, B., Omary, M.B., 2004. Actin overexpression parallels severity ofpancreatic injury. Exp. Cell Res. 299, 404–414.