Cloning and characterization of NYD-OP7 , a novel deltamethrin resistance associated gene from Culex pipiens pallens (cid:2)

One mosquito opsin gene, NYD-OP7 , has been cloned from Culex pipiens pallens . An open reading frame (ORF) of 1116bp was found to encode a putative 371 amino acids protein which exhibits high identity with opsins from Aedes and Anopheles mosquitoes. Transcript expression of NYD-OP7 was determined by real-time PCR in all life stages of deltamethrin-susceptible and -resistant strains of the Culex mosquito. The results demonstrated that this gene is expressed at all developmental stages, and it is expressed predominantly at the pupae and adult stages. Meanwhile, in pupae and adults, NYD-OP7 is overexpressed in deltamethrin-resistant strain than in -susceptible strain. Importantly, stable expression of NYD-OP7 in the mosquito C6/36 cells can confer moderate deltamethrin resistantce. Our study pro-vides the W rst direct evidence that increased expression of an opsin gene may play some role in the development of deltamethrin resistantce in Cx. pipiens pallens . © 2006 Elsevier Inc. All rights reserved.


Introduction
Opsins, the visual pigments of animal photoreceptors, belong to the G-protein coupled receptor family which is characterized by a seven-helix transmembrane topology and by the ability to activate heterotrimeric G-proteins [1][2][3].
Since the Wrst sequence of an opsin, bovine rhodopsin, was determined by conventional protein sequencing and cDNA sequencing, more than 1000 opsins have been identi-Wed [4,5]. In invertebrates, some photoreceptor opsins have been well-characterized, both in terms of their sequences and cellular functions [1]. When sequence alignments are made of the invertebrate and vertebrate opsins, it is evident that individual sequence may be quite diVerent, but the tertiary structure appears conserved [5,6].
Insecticide resistance is a major obstacle to the control of vector-borne diseases [7][8][9]. Many studies have suggested that the insecticide resistance phenotype evolves rapidly based on the selection of major eVect genes [10,11]. However, recent genome-wide transcription proWling indicated that a broader range of genes may be involved and that insecticide resistance may be more complex than previously considered [12][13][14].
As part of an ongoing study of insecticide resistance in Cx. pipiens pallens, we have employed suppression subtractive hybridization (SSH) and cDNA microarray to identify diVerentially expressed genes between deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens. Opsin is one of only ten genes that showed over-expression in deltamethrin-resistant strain [15]. In the present study, we isolated and sequenced the complete sequence of this gene and termed it NYD-OP7 2 . We then characterized the expression pattern of NYD-OP7 gene in diVerent developmental stages of Cx. pipiens pallens. In addition, we investigated the role of NYD-OP7 in deltamethrin resistance by transfecting this gene into the mosquito C6/36 cells and tried to determine if over-expression of NYD-OP7 can change the sensitivity of the cells to deltamethrin.

Mosquito strains
The deltamethrin-susceptible strain of Cx. pipiens pallens was obtained from the Shanghai Insect Institute of the Chinese Academy of Sciences and was maintained in our laboratory. The mosquitoes had never been exposed to any insecticides and the median lethal concentrations (LC 50 ) of deltamethrin (Roussel Uclaf, France) was 0.0008 mg/L. The strain was reared and bred at 25-27°C in a 16 h light/8 h dark photoperiod. The deltamethrin-resistant strain was derived from the susceptible early fourth instar larvae by selection with deltamethrin for 10 generations until the resistant ratio (LC 50 of Wlial generation/LC 50 of parental generation) reached 400 [16].

Construction of cDNA library
Total RNA were extracted from 300 adult females of resistant strain using the RNeasy maxi kit (Qiagen, Germany) according to the manufacture's instructions. The integrity of total RNA was determined by denaturing agarose gel electrophoresis and the yield and purity of total RNA was estimated by spectrophotometry. The poly (A+) mRNA was puriWed from total RNA with the PolyAtract mRNA isolation system (Promega, USA), and 2 g polyA mRNA was used for cDNA library construction with Excell/NotI/EcoRI/CIP vector system (Pharmacia, USA) following the manufacturer's protocols. The cDNA library contained approximately 5 £ 10 5 clones and was ampliWed.

Cloning and sequencing of NYD-OP7 cDNA
To isolate the full-length of culex opsin gene, we used the standard SP6 and T7 library vector primers and the primers designed based on the sequence of the SSH fragment we reported previously (GenbBank Accession No. BE247812). The sequence of the oligonucleotide primers was: forward 5Ј-GTCGCTCTGGTGACCATTTC-3Ј; reverse 5Ј-CACG ATCGGGTTGTAGACAG-3Ј. PCR conditions were: initial denaturation at 94°C for 5 min, followed by 32 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 90 s with Wnal 10 min extension at 72°C. The PCR products were separated by 1% agarose gel electrophoresis and puriWed using a QIA quick Gel extraction kit (Qiagen, Germany). Products were then cloned into the pGEM-T easy vector (Promega, USA) and sequenced at Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). Then the sequences of above three fragments were assembled to generate a putative full-length cDNA of opsin. A pair of PCR primers: forward 5Ј-TTAAGTCTGAAGTAGTTC-3Ј and reverse 5Ј-CTAGCAAAGATCAGCAATC-3Ј were designed for the ampliWcation of full-length cDNA according to the assembled sequence of opsin. PCR was carried out using LA Taq polymerase (TaKaRa, Japan) using the following protocol: 94°C for 5 min, followed by 32 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 90 s with Wnal 10 min extension at 72°C. Then the PCR product was puriWed, cloned into the pGEM-T easy vector and sequenced.

Sequence alignment and phylogenetic tree
The standard protein-/protein BLAST sequence comparison (blastp; www.ncbi.nlm.nih.gov / BLAST/) and PSI-BLAST programs were used to search for sequences in the GenBank and SWISS-PROT databases with similarities to the translated sequences of NYD-OP7 [17]. Sequence alignment and phylogenetic tree analysis using the neighbour-joining method was carried out by the CLUSTAL W program [18]. The opsins included in our analysis were: twelve sequences from An. gambiae genome [2], six sequences from Drosophila melanogaster, three sequences from Manduca sexta, six sequences from Papilio glaucus and two sequences from Aedes aegypti [6].

RNA extraction and cDNA synthesis
Total RNA were extracted from embryos, Wrst, second, third and fourth instar larvae, pupae, and adults of both susceptible and resistant strains using the RNeasy mini kit (Qiagen, Germany) according to the manufacturer's protocol, and contaminant genomic DNA was removed by DNase I treatment. cDNA was synthesized from 2 g of total RNA with M-MLV reverse transcriptase (Promega, USA) and random oligonucleotide primers according to the manufacturer's protocol.

Real-time PCR of NYD-OP7 in mosquito developmental stages
Aliquots of cDNA were ampliWed on an ABI PRISM 7300 (Applied Biosystems) using the SYBR green PCR kit (PE Applied Biosystems) with 25 l reaction mixtures. The PCR mixture contained 1£ SYBR PCR buVer, 2.0 mM MgCl 2 , 0.2 mM (each) dNTP, 0.2 M primers, and 0.3 l of Taq polymerase (2.5 U/ l). The primers used were: NYD-OP7: forward 5Ј-GTGGGCTTTCGCACTCTT-3Ј, reverse 5Ј-GTTCACGCATCTGTTCCTC-3Ј; -actin: forward 5Ј-C GCTTCCTCGTCTACACTGG-3Ј, reverse 5Ј-GTGTTGG CGAACAGATCCTT-3Ј. For ampliWcation, the following program was employed: 40 cycles of 95°C for 15 s, 60°C for 1 min. A melting curve program was run immediately after the PCR program. Control measurement was done in the absence of reverse transcriptase to make sure that the level of genomic DNA contaminants was negligible.
Real-time PCR data were analyzed with a 7300 System SDS Software v1.2.1 (Applied Biosystems) to estimate transcript copy numbers for each sample. The raw threshold cycle (Ct) values were normalized against -actin standard to obtain normalized Ct values, which were then used to calculate relative expression levels in the samples. The expression level of NYD-OP7 in susceptible strain egg was considered as background level, or 1. To verify reproducibility, the real-time PCR analysis was repeated twice in independent experiments, with three replicates for each sample.

Recombinant insect expression vector construction
The 1116 bp NYD-OP7 coding sequence, from the putative translational ATG to the end of the ORF, was ampli-Wed by PCR using the cDNA library as the template. Forward and reverse primers, containing at their ends, respectively, EcoRI and NotI restriction sites, were: forward 5Ј-CGGAATTCTGGCAGCTTTCGTTGAGCC-3Ј; reverse 5Ј-ACTAGCGGCCGCTGTACTTAAGCCTTCTCG-3Ј. The PCR product was ligated into the pGEM-T easy vector. Then the recombinant plasmid was digested with SacII and NotI and the puriWed fragment was ligated into the SacII-NotI linearized insect expression vector pIE1-3 (Novagen, USA), resulting in the pIE1-3/OP7 construct. The integrity of the construct was veriWed by restriction enzyme digestion and sequencing. The pIE1-3 vector is designed for constitutive expression of recombinant protein from the baculovirus ie1 promoter in stably transfected insect cells [19]. To obtain stably transfected cells, a pIE1-3 recombinant should be cotransfected with pIE1-neo vector (Novagen, USA), which contains the Neo selectable marker under ie1 control.

Cell culture and transfection
Mosquito C6/36 cells were obtained from China Center for Type Culture Collection (CCTCC, China). The cells were maintained in Eagle's minimum essential media (EMEM) (Invitrogen, USA) supplemented with non-essential amino acids, fetal bovine serum (10%) (Sijiqing, China), and antibiotics. The cells were grown in a 5% CO 2 humidi-Wed incubator at 28°C. Transfection was performed using Cellfectin reagent (Invitrogen, USA) according to the manufacturer's protocol. BrieXy, 4 £ 10 5 cells/well were plated into a 6-well plate in growth medium without antibiotics and cultured overnight. Then the pIE1-3/OP7construct and the pIE1-3 vector only, were each separately cotransfected with pIE1-neo construct at a ratio 4:1 into C6/36 cells according to the manufacturer's protocol. After 24 h, the cells were selected with 300 g/ml G418 (Calbiochem, Germany) for 2 weeks and the medium was changed every 4 days. Following selection, G418-resistant colonies were isolated and expanded as individual clonal cell lines for subsequent analysis.

RT-PCR analysis
Total RNA was extracted from cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol and the reverse transcript reaction was performed as above. The resulting cDNA preparation was subjected to PCR ampliWcation using the forward primer speciWc to NYD-OP7 (5Ј-GTGGGCTTTCGCACTCTT-3Ј) and the pIE1-3 vector-speciWc HRAS reverse primer (5Ј-AAGATTAGCGAC GCTGCT-3Ј). Reaction conditions were as follows: 94°C for 5 min; 30 cycles (25 cycles for -actin) of 94°C for 30 s, 58°C for 30 s, 72°C for 40 s; Wnal extension at 72°C for 10 min. To conWrm equal loading, PCR ampliWcation of the -actin gene was also done in parallel. The primers used for -actin PCR ampliWcation were: forward 5Ј-CACCAGGGT GTGATG GTCGG-3Ј, reverse 5Ј-CCACCGATCCAGAC GGAGT-3Ј. The PCR products were analyzed by electrophoresis on 1% agarose gel containing ethidium bromide, and photographed under UV light.

Statistics
Data were analyzed by Student's t test. The level of sig-niWcance was set at p < 0.05.

Isolation full-length of the Cx. pipiens pallens NYD-OP7 gene
To isolate opsin gene from the Cx. pipiens pallens, we performed a RT-PCR screen of cDNA library using the standard SP6 and T7 library vector primers and primers designed based on the sequences we reported previously (GenBank Accession No. BE247812). The sequences we got were assembled to generate a putative full-length cDNA of opsin. Then a pair of PCR primers was designed for the ampliWcation of full-length cDNA. Sequencing of the ampliWed transcript led to the identiWcation of a novel opsin gene which we termed NYD-OP7 (GenBank Accession No. AY749413). The NYD-OP7 cDNA has an open reading frame of 1116 bp, encoding 371 amino acids (Fig. 1).

Sequence and phylogenetic analysis
Alignment of the deduced NYD-OP7 amino acid sequence with Ae. aegypti and An. gambiae opsins revealed sequence identities of 87% with Ops1, 86% with GPRop1-4, 83% with Ops2, 79% with GPRop6, and 63% with GPRop5 (Fig. 2). The newly isolated opsin displays typical hallmarks of rhodopsins, such as a highly conserved lysine residue (Lys-317) in the seventh transmembrane region, to which the retinal chromophore is attached; putative phosphorylation sites (Ser, Thr) near the C-terminus; putative N-glycosylation sites (Asn-Xaa-Ser/Thr) near the N-terminus, and two cysteine residues (Cys-121 and Cys-198) in the Wrst and second extracellular loop, which may form a disulphide bond. NYD-OP7 contains a DRY sequence at the junction of helix III and the second intracellular loop that is very similar to the ERY sequence found in bovine rhodopsin. This sequence contains a charged pair that is required for transducin activation [20,21]. Although dissimilar from the vertebrate opsins, the sequence QAKKMNV found in the N-terminal half of cytoplasmic loop 3 is very highly conserved among the invertebrate opsins and is located within a region of the protein that is also required for transducin activation [22]. Most insect rhodopsins have a His-Glu-Lys (HEK) motif at the beginning of a conserved region in the third cytoplasmatic loop, and it is implicated in rhodopsin-G-protein interaction. Interestingly, the HEK motif is mod-iWed to His-Glu-Glu in NYD-OP7. Other insect rhodopsins in which the lysine residue of the HEK motif is substituted include two M. sexta rhodopins [23], two An. gambiae rhodopsins [2], one calliphora rhodopsin [24] and one Ae. aegypti opsin [6]. Fig. 3 shows the molecular phylogenetic relationship between NYD-OP7 and some other insect opsins. Results showed that the NYD-OP7 sequence is most closely related to the Ae. aegypti Ops1, and it clearly belongs to a group comprising the known arthropod opsins.

Expression proWle of the NYD-OP7 gene
The expression of the NYD-OP7 gene was analyzed by real-time PCR ampliWcation of the corresponding mRNA from diVerent Cx. pipiens pallens developmental stages. As shown in Fig. 4, NYD-OP7 is expressed at signiWcantly high levels in pupae and adults, whereas expression is very low in the egg, Wrst, second, third and fourth instar larvae stages. Interestingly, in pupae and adults, the expression of NYD-OP7 is signiWcantly higher in resistant strain than in susceptible strain.

NYD-OP7 expression and deltamethrin sensitivity analysis of pIE1-3/OP7 transfected C6/36 cells
To elucidate the contribution of the NYD-OP7 gene product to deltamethrin resistance, the mosquito C6/36 cells were transfected with the pIE1-3/OP7 insect expression vector. After G418 selection, one stably transfected NYD-OP7 subclone was obtained. Analysis of this transfectant demonstrates a higher expression of NYD-OP7 gene as compared with the empty vector transfectant control (Fig. 5). These subclones did not show any growth morphological changes and then they were examined for deltamethrin sensitivity by [ 3 H]-TdR assay. As shown in Fig. 6, NYD-OP7 transfected C6/36 cells are relatively resistant to deltamethrin at the concentrations of 15, 20 and 25 M (p < 0.05). Meanwhile, a slight increase of cell viability was also observed at 30 M deltamethrin but without statistical signiWcance (p D 0.0744).
In this experiment, we studied a novel cDNA encoding opsin (NYD-OP7) isolated from Cx. pipiens pallens. Considering the amino acid sequence of this gene, the translated protein is expected to have a seven-transmembrane structure, and it has all the conserved residues required for a functional opsin protein. This feature and the amino acid identity of NYD-OP7 with known opsins suggest that it encodes a functional opsin protein. We have compared the amino acid sequence of the novel opsin with those of other insect opsins. The phylogenetic analysis indicates that NYD-OP7 is a member of the group including Ae. aegypti Ops1 and An. gambiae GPRop1-3. Real-time PCR analysis revealed the expression of NYD-OP7 at all developmental stages both in deltamethrin-susceptible and -resistant strains and it appeared that NYD-OP7 mRNA levels are signiWcantly lower in Cx. pipiens pallens embryos and larvae than in older developmental stages. Whether the expression pattern reXects a function for NYD-OP7 in eye development remains to be seen.
In previous study, we identiWed 16 genes that were diVerentially transcribed among the deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens and as shown by microarray, one opsin gene is 3.06-fold overexpressed in the resistant strain [15]. In this paper, we also found that in pupae and adults, NYD-OP7 is overexpressed in resistant strain than in susceptible strain, which shows that NYD-OP7 may play a role in the deltamethrin resistance phenotype. However, it remains possible that NYD-OP7 over-expression is an indirect consequence of acquired deltamethrin resistance. To determine whether NYD-OP7 directly participates in the establishment of the deltamethrin resistance phenotype, we transfected this gene into the mosquito C6/36 cells and to determine if over-expression of NYD-OP7 can change the deltamethrin sensitivity in C6/36 cells. The results showed that NYD-OP7 transfected cells are relatively resistant to deltamethrin at low drug concen-trations which suggests that NYD-OP7 is directly involved in the development of deltamethrin resistance.
Previously, by using a small-scale microarray representing all Drosophila P450 genes, Daborn et al. and Le GoV et al. suggested that resistance to DDT in the Weld is monogenic and is due to the over-expression of a single P450 gene, Cyp6g1 [10,32]. However, by using a detox chip and a genome-wide microarray of An. gambiae, David et al. and Vontas et al. found that many genes that belong to families not usually associated with insecticide resistance (such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism) were over-transcribed in the resistant strain [13,14]. Meanwhile, by using genomewide microarray, Pedra et al. demonstrated that DDTmetabolic resistance in Drosophila is associated with over-expression of detoxiWcation related genes but also overexpression of ion transport, signal transduction, RNA transcription, and lipid and sugar metabolism pathways [12]. Interestingly, they also reported that Drosophila UV-sensitive opsin (Rh3 and Rh4) and Blue-sensitive opsin (NinaE) were overexpressed in the DDT-resistant strain, suggesting that opsin gene may play some role in DDT-resistance [12]. Fig. 2 (continued) In the case of NYD-OP7, we hypothesized that as one member of G-protein coupled receptor family, NYD-OP7 may contribute to the phenotype of insecticide resistance by activating downstream G-protein and signal transduction pathway. It is also possible that the deltamethrin-resistant phenotype requires the coordinated expression of many diVerent genes so that over-expression of individual gene is necessary, but maybe not suYcient, to generate a high resistance phenotype. It should also be mentioned that because of lack of a proper antibody, we cannot do western blot to conWrm the over-expression of NYD-OP7 in stably transfected cells at the protein level. So there is a possibility that the expression of NYD-OP7 at the protein level is not as high as the mRNA level which may be the reason for why high NYD-OP7 transcriptional level expression only causes a modest increase in resistance. In a recent study, we have proved that the cotransfection of two genes (NYD-Tr and NYD-Ch) into C6/36 cells can induce deltamethrin resistance [33]. All these data suggest that insecticide resistance is more complex than we considered and we believe that further investigation should be done to fully elucidate the role that NYD-OP7 has in insecticide resistance.
In summary, we have reported the cloning and identiWcation of a novel opsin gene (NYD-OP7) from Cx. pipiens pallens. Based on the expression proWles and previous SSH and microarray results, this gene appears to have some role in deltamethrin resistance. Over-expression of NYD-OP7 in mosquito C6/36 cells can confer moderate resistance to   deltamethrin, suggesting that NYD-OP7 directly participates in the development of deltamethrin resistance and the precise deWnition of its role awaits further investigation.