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( E)-Caryophyllene and α-Humulene: Aedes aegypti Oviposition Deterrents Elucidated by Gas Chromatography-Electrophysiological Assay of Commiphora leptophloeos Leaf Oil
research-article
Author(s):
Rayane Cristine Santos da Silva
1
,
2 ,
Paulo Milet-Pinheiro
1
,
3 ,
Patrícia Cristina Bezerra da Silva
1 ,
Alexandre Gomes da Silva
2 ,
Marcia Vanusa da Silva
2 ,
Daniela Maria do Amaral Ferraz Navarro
1
,
* ,
Nicácio Henrique da Silva
2
Publication date (Electronic):
9 December 2015
Journal:
PLoS ONE
Publisher:
Public Library of Science
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Abstract
Aedes aegypti is responsible for the transmission of dengue, a disease that infects millions of people each year. Although essential oils are well recognized as sources of compounds with repellent and larvicidal activities against the dengue mosquito, much less is known about their oviposition deterrent effects. Commiphora leptophloeos, a tree native to South America, has important pharmacological properties, but the chemical profile and applicability of its essential oil in controlling the spread of the dengue mosquito have not been investigated. The aim of this study was to determine the composition of C. leptophloeos leaf oil and to evaluate its larvicidal and oviposition deterrent effects against A. aegypti. Fifty-five components of the essential oil were detected by gas chromatography (GC)—mass spectrometry, with α-phellandrene (26.3%), ( E)-caryophyllene (18.0%) and β-phellandrene (12.9%) identified as the major constituents. Bioassays showed that the oil exhibited strong oviposition deterrent effects against A. aegypti at concentrations between 25 and 100 ppm, and possessed good larvicidal activity (LC 50 = 99.4 ppm). Analysis of the oil by GC coupled with electroantennographic detection established that seven constituents could trigger antennal depolarization in A. aegypti gravid females. Two of these components, namely ( E)-caryophyllene and α-humulene, were present in substantial proportions in the oil, and oviposition deterrence assays confirmed that both were significantly active at concentrations equivalent to those present in the oil. It is concluded that these sesquiterpenes are responsible, at least in part, for the deterrent effect of the oil. The oviposition deterrent activity of the leaf oil of C. leptophloeos is one of the most potent reported so far, suggesting that it could represent an interesting alternative to synthetic insecticides. The results of this study highlight the importance of integrating chemical and electrophysiological methods for screening natural compounds for their potential in combating vectors of insect-borne diseases.
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Most cited references33
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Medicinal plants of the caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach.
The caatinga (semi-arid vegetation) is a Brazilian biome with a significant but poorly studied biodiversity closely associated with a diverse cultural heritage. The present work focused on analyzing published information available concerning medicinal plants used by traditional communities. We sought to contribute to future phytochemical and pharmacological investigations by documenting the therapeutic uses of native caatinga plants within the aims of modern ethnopharmacological research. Twenty-one published works cited a total of 389 plant species used by indigenous and rural communities in northeastern Brazil for medicinal purposes. The relative importance index (RI) of each species in these inventories was calculated, and information concerning the plant's local status (spontaneous or cultivated), distribution, and habit was recorded. Of the 275 spontaneous (non-cultivated) species cited, 15.3% were endemic to the caatinga. A statistical relationship was verified between the relative importance of the species and their endemic status (p<0.05). Herbaceous plants were more numerous (169) than trees (90) or shrubs and sub-shrubs (130) at a statistically significant level (p<0.05). A survey of published information on the phytochemical and pharmacological status of the plants demonstrating the highest RI supported the veracity of their attributed folk uses.
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Chemical ecology and behavioral aspects of mosquito oviposition.
Christy Bentley, Tyler Day (1988)
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Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism
Shinji Kasai, Osamu Komagata, Kentaro Itokawa … (2014)
Introduction The yellow fever mosquito Aedes aegypti inhabits tropical and subtropical regions worldwide and is the major vector of dengue fever (DF) and yellow fever. DF is a rapidly growing health issue, with the average annual number of cases being approximately 100 million and a 30-fold increase in the past 50 years [1], [2]. The disease is endemic in at least 112 countries, especially in south and Southeast Asia, and an estimated 2.5 billion people are currently living in risk areas [1]. At present, DF causes more illness and death than any other arbovirus disease in humans [3]-[5]. Successful population control of vector insects is the key to prevent transmission and epidemics of infectious diseases. This strategy relies heavily on insecticides, particularly pyrethroid, a popular class of insecticides with high and rapid toxic activity toward insects and low toxicity to mammals [6]. Pyrethroids are used to control and/or prevent adult mosquitoes by ultra-low volume sprays, thermal fogging, pyrethroid-impregnated nets, etc. However, many dengue endemic areas are now facing the problem of pyrethroid resistance due to frequent and intensive use of these chemicals [7]. Resistance of A. aegypti to pyrethroids has been reported from various countries [8]. Understanding the level and mechanism of resistance to insecticides is essential for developing appropriate vector control measures. A. aegypti is present in most residential areas of Singapore. Despite a well-established national vector control program that includes community engagement, law enforcement, and inter-sectional coordination, Singapore continues to face the risk of DF resurgence [9]–[11]. During an outbreak in 2005, more than 14,000 DF cases were reported [12]. Since 2005, surveillance for dengue control has been based on four pillars: (1) case surveillance through mandatory notification of dengue cases to the Ministry of Health, by all medical practitioners; (2) vector surveillance through premises checks by vector control officers from National Environment Agency (NEA); (3) virus genotype surveillance at Environmental Health Institute of NEA; and (4) monitoring of other environmental parameters such as weather factors and population density. Clustering of cases and development of risk maps using the surveillance data allows prioritization of vector control operations. Though the program is largely based on source reduction and large scale fogging has not been conducted by the authorities since 2005, insecticides continue to be used by the authority for indoor misting in areas with dengue transmission, by private arrangement and household use of aerosol cans. Regular monitoring for insecticide has revealed that Singapore's A. aegypti larvae have developed resistance to synthetic pyrethroids, with resistance ratio to permethrin in the range of 29–47 times [13]. Generally, insecticide resistance in insects is caused by three major mechanisms: (1) reduced sensitivity of the target site, (2) reduced penetration of the insecticide due to altered cuticles, and (3) increased activity or level of detoxification enzyme(s). Insects develop resistance to insecticides by obtaining one or more of these mechanisms. Amino acid substitution in the voltage-sensitive sodium channel (Vssc), the target site of DDT and pyrethroids, is the most well-understood mechanism conferring resistance against pyrethroid insecticides [14], [15]. This mechanism is called “knockdown resistance,” with its inherited trait, “kdr,” being first identified in the housefly as a leucine–phenylalanine substitution at position 1014 (L1014F) of Vssc. Recently, many other mutations that cause reduced sensitivity to Vssc have been identified from many arthropod species and are called either “kdr-like factor” or “knockdown resistance.” Knockdown resistance is unaffected by synergists that inhibit the activity of metabolic enzymes such as carboxyl esterases or cytochrome P450 monooxygenases (P450s). Several amino acid substitutions have been detected in A. aegypti Vssc: S989P, I1011M, I1011V, V1016G, V1016I, F1534C, and D1763Y [16]–[22]. Of these, only substitutions V1016G, V1016I, and F1534C have been shown to strongly correlate with pyrethroid resistance [17], [23]–[27]. The dominance of these three mutations on pyrethroid sensitivity, however, is not well understood. Metabolism-mediated insecticide resistance is now considered a key mechanism in insects [7], [28], [29]. Three families of metabolic enzymes have been implicated in the metabolism of insecticides: esterases, glutathione transferases (GSTs), and P450s. The genome project of A. aegypti identified 26 esterases, 49 GSTs, and 160 P450s [30]. Identification of the specific enzymes involved in insecticide resistance, however, has proven challenging. P450s has been shown to be the metabolic enzyme most strongly linked to the development of pyrethroid resistance [31]–[33]. However, due to the large number of P450 genes and the structural similarity among different isoforms, identification of the isoform(s) associated with resistance has been difficult, with only few exceptions [34], [35]. Recently, microarray analysis that compares gene expression between susceptible- and resistant-strains unearthed candidate genes that confer insecticide resistance in different insect species [36]–[38]. Although molecular diagnosis of insecticide resistance that targets Vssc mutations is widely reported, such a system has not been reported for P450 genes in Aedes mosquitoes. Since insecticide resistance in a mosquito population could be concurrently affected by more than one mechanism, an accurate molecular diagnosis thus needs to consider the various possible mechanisms. Metabolism-mediated pyrethroid resistance of A. aegypti requires further study especially in the populations collected from Southeast Asia, the largest endemic area of DF. Reduced cuticle penetration is the least understood mechanism among the three. Though it may have a primary role in resistance [39]–[41], it more often acts in combination with the other mechanism(s). This study examined the mechanisms conferring pyrethroid resistance in an A. aegypti strain collected from Singapore and investigated three major mechanisms of resistance. The results are expected to lead to the development of more accurate resistance monitoring systems and contribute to the discovery of new insecticide target sites to overcome the challenge of insecticide resistant mosquitoes. Methods Ethics statement Mice were used as the blood source for mosquitoes. This study complies with the guidelines for animal experiments performed at National Institute of Infectious Diseases, Japan. The protocol for the utility of mice was approved by the Animal Use Committee of National Institute of Infectious Diseases (registration numbers: 209052, 210044, 211031 and 112029). A. aegypti population and strains A laboratory-susceptible reference strain of A. aegypti, SMK, was obtained from Sumitomo Chemical Co., Ltd. in 2009. This strain was originally from USA and has been maintained in the laboratory for at least 20 years without exposure to insecticides. The pyrethroid-resistant population, SPS0, was collected from Singapore in 2009. The SPS0 population was selected by exposure to permethrin for 10 generations as described below and the SP strain ( = SPS10) was established. Permethrin doses used for the selections are listed in Table 1. Larvae were fed a ground diet of insect food (Oriental Yeast Co., Ltd., Tokyo, Japan), and the adults were maintained on 10% sucrose. Females were given blood meals from mice. Both larvae and adults were reared at 27 ± 1°C with a photoperiod of 16∶8 (L∶D) h. 10.1371/journal.pntd.0002948.t001 Table 1 Selection of the SP strain of Aedes aegypti in the laboratory by permethrin. Selection No. Selected population Gender Permethrin dose (ng/mosquito) No. mosquitoes treated Mortality (%) 1 SPS0G3 ♂ 10 600 52.0 ♀ 60 600 62.8 2 SPS1G1 ♂ 27 480 60.6 ♀ 177 480 76.9 3 SPS2G1 ♂ 44 480 57.1 ♀ 333 490 85.3 4 SPS3G1 ♂ 61 513 31.8 ♀ 444 551 69.1 5 SPS4G1 ♂ 111 515 74.0 ♀ 444 524 50.2 6 SPS5G2 ♂ 167 629 89.2 ♀ 821 482 79.0 7 SPS6G1 ♂ 167 527 71.3 ♀ 821 510 78.4 8 SPS7G1 ♂ 167 583 82.7 ♀ 821 613 86.8 9 SPS8G1 ♂ 167 542 65.1 ♀ 821 512 73.6 10 SPS9G1 ♂ 167 529 46.3 ♀ 821 547 45.2 The subscript numbers in the population name indicate the permethrin selections in the population. For example, SPS5G2 indicates the 2nd generation of SPS5 which was selected by permethrin for five generations. Chemicals Three technical grade insecticides were used: permethrin (94.1%, Sumitomo Chemical Co., Ltd., Osaka, Japan), temephos (94.9%, Wako Chemical Pure Industries, Ltd., Osaka, Japan), and pirimiphos methyl (98.0%, Wako Chemical Pure Industries, Ltd., Osaka, Japan). An inhibitor of P450s, piperonyl butoxide (PBO, 98.0%, Wako Chemical Pure Industries, Ltd., Osaka, Japan), was used for the adult and larval bioassays. [14C]-(1RS)-trans-permethrin (specific activity, 4.44 GBq/m mol, purity >99%) labeled at the phenoxy benzyl group and 4′-hydroxy-3-phenoxy-benzyl 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-carboxylate (4′-HO-permethrin) were generously provided by Sumitomo Chemical Co., Ltd. (Osaka, Japan). [14C]-(1RS)-trans-permethrin was purified by thin layer chromatography (TLC) according to the modified method described by Shono et al. [42]. We used a solvent system of toluene/carbon tetrachloride (1∶1) instead of benzene/carbon tetrachloride (1∶1) in order to separate trans-permethrin from other degraded compounds. Permethrin selections and adult bioassay Three- to seven-day-old adult male and virgin females were used for the permethrin selections. In order to avoid measurement error caused by insect locomotive activity, we used a topical application method, instead of filter paper method, for pyrethroid selections and adult bioassays. The mosquitoes were anaesthetized with CO2 and placed in a 200-ml Erlenmeyer glass flask. They were then re-anaesthetized for 3 min with 100 µl diethyl ether absorbed onto cotton. A volume of 0.22 µl permethrin in acetone solution was dropped onto the thoracic notum of each mosquito with a Hamilton repeating dispenser PB-600 equipped with 10 µl syringe 701SNR (Hamilton, Reno, NV, USA). The insects were placed in groups of 40 in paper cups sealed with a nylon mesh. Water-absorbed cotton was placed on the top of the mesh. Approximately 1000 mosquitoes were selected from each generation (Table 1). The doses of permethrin used for the selections were determined by a preliminary experiment for each gender. Twenty-four hours after treatment, all the mosquitoes were put into a cage, provided with sucrose water, and maintained for the next generation. A bioassay to monitor permethrin susceptibility was conducted for each selected generation as described below. Adult bioassays were conducted by topical application using 3- to 7-day-old female mosquitoes and consisted of at least five doses of permethrin causing >0% and 15,000 A. aegypti Liverpool transcripts identified in the genome project (https://www.vectorbase.org/content/aedes-aegypti-liverpooltranscriptsaaegl13fagz). Each probe was spotted at least two times at different positions on each array. A probe of each P450 gene was spotted six times. Microarrays in a 4 × 44 k format were constructed using contract manufacturing carried out by Agilent Technologies. The entire design of the 44 k array used in this study is available from the NCBI Gene Expression Omnibus (GEO) site as accession # GPL17604. The strains were reared in parallel in order to minimize variation resulting from breeding conditions. For each life stage (larvae, adult males and females), four RNA sources were prepared from each of the SP and SMK strains reared in different trays and cages ( = four biological replicates). Total RNA was extracted from 10 fourth instar larvae or 10 three-day-old adults using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan). Genomic DNA was removed by digesting the total RNA samples with DNase I using TURBO DNase (Life Technologies Co., Carlsbad, CA, USA). The quality and quantity of total RNA were assessed by spectrophotometry using Nanodrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and a bioanalyzer MultiNA (Shimadzu Co., Kyoto, Japan). The purified RNA was mixed with the internal control RNA supplied in a RNA Spike-In Kit (One-color, Agilent Technologies). Fluorescein-labeled cRNA were synthesized via a double-stranded cDNA intermediate using a Low Input Quick Amp Labeling Kit (Agilent Technologies). The cRNA derived from the SP and SMK strains were differentially labeled with cyanine-3 (Cy-3) dye-conjugating CTP (PerkinElmer Inc., Waltham, MA, USA). cRNA was then purified with Qiagen RNeasy Mini Kit (Qiagen, Venlo, Netherlands), and the overall efficacy of cRNA synthesis and fluorescein-labeling was measured using a Nanodrop ND-1000 spectrophotometer. The labeled cRNA were pooled and hybridized to microarray probes in a hybridization oven at 65°C for 17 h under rotation at 10 rpm using a Gene Expression Hybridization Kit (Agilent Technologies, Santa Clara, CA, USA). After hybridization washing, the fluorescent dyes were stabilized against ozone oxidization with the Gene Expression Wash Buffer (Agilent Technologies) and the microarray plate was then dried in nitrogen gas. The fluorescence of Cy-3 on each spot was scanned using a DNA Microarray Scanner G565BA (Agilent Technologies, Santa Clara, CA, USA). Spot identification and quantification were performed using Feature Extraction Software v. 7.5 (Agilent Technologies, Santa Clara, CA, USA) in the default setting. A linear and LOWESS algorithm (a combination of the linear method and traditional LOWESS method) was used for dye normalization. Flagged spots (such as saturated, low intensity, and statistical outlier) were ignored in the final data analysis. The ratio of transcription levels for each gene in the microarray experiment for the SP and SMK strains was called the “microarray ratio.” A representative microarray ratio in each hybridization experiment was expressed as the average of all spots for a gene on an array, where the spot number for a gene is calculated as “unique probe number” x “replicating spot number (ideally n = 4).” The P-value for every spot was calculated by Agilent's Universal Error Model. The raw results of the 10 microarray hybridizations are available from GEO (series accession# GSE50069). The genes over expressed in the SP strain were selected using a cut-off of >3-fold relative change in expression and a P 5) and in males (>3) in SP relative to SMK in microarray analysis. CYP9M7 was also analyzed as it forms a gene cluster with CYP9M4, CYP9M5, and CYP9M6 within 65 kbp on the same supercontig (1.29) and is structurally related to these genes. The primer sequences and their regions are shown in Table S1 and Figure S1. Genomic DNA was extracted from individual male mosquitoes as described above. The PCR was conducted using high fidelity polymerase (PrimeSTAR GXL DNA Polymerase; Takara Bio Inc., Shiga, Japan), treated with illustra ExoStar (GE Healthcare UK Ltd., Little Chalfont, England) to remove unincorporated primers and dNTPs, followed by direct sequencing with the primers listed in Table S1. Because two haplotypes of CYP9M6, namely CYP9M6v1 and CYP9M6v2, were identified from the SP strain, the primer sets, 9M6F31/9M6R21 and 9M6F23/9M6R35 for CYP9M6v1, and 9M6F30/9M6R22 and 9M6F18/9M6R35 for CYP9M6v2 were used to amplify each variant, followed by direct sequencing of the PCR products (Figure S2 and Table S1). Quantitative real-time PCR cDNA was synthesized using total RNA isolated for microarray analysis, and the QT' primer (Table S1) and reverse transcriptase ReverTra Ace (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Real time quantitative PCR was performed using a PikoReal Real Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). The PCR primers were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). Each PCR reaction of 10 µl final volume contained 5 µl SYBR Premix Ex Taq II (Takara Bio Inc., Shiga, Japan), 1 µl cDNA (equivalent to 10 ng total RNA), 0.4 µl of each forward and reverse primer (10 µM, Table S1), and 3.2 µl ddH2O. The PCR reactions were performed under the following conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. The 2−ΔΔCt method [50] was used to quantify the relative expression level of P450s, with RPS3 gene acting as the internal control. For each gene analyzed, serial dilutions of cDNA showed that the efficacy of amplification for all P450 genes and the RPS3 gene were >0.998. Three replicates of the PCR reactions were performed for each sample (technical replications) and each experiment was repeated four times using an independent RNA source (biological replications). Identification of gene copy number Gene copy number was determined by quantitative PCR using the same primer sets described above. Genomic DNA was individually extracted from eight virgin females of each strain using Get Pure DNA Kit-Cell, Tissue (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's instructions. The quantity and quality of the DNA was assessed using a spectrophotometer Nanodrop ND-1000 (Thermo Fisher Scientific Inc., MA, USA). The DNA samples were diluted to 100 ng/µl and 1 µl used as a template. Data were normalized using RPS3 gene and the exact single gene copy number reported by the genome project. Three replicates of the PCR reactions were performed for each sample (technical replications) and each experiment was conducted using 8 individual DNA source (biological replications). Association between permethrin excretion and CYP9M6 and CYP6BB2 genotypes One hundred virgin males and females of the SP and SMK strains were cross-mated. The offspring were then interbred to obtain F2 mosquitoes. Three-day-old F2 females were dosed with a sub-lethal amount of [14C]-permethrin (ca 600 dpm, 0.88 ng) as described above. Each treated mosquito was isolated in a scintillation vial and the amount of radioisotope excreted was quantified 24 h later. Genomic DNA was isolated from six legs of each mosquito as described above using a REDExtract-N-Amp Tissue PCR Kit. Both CYP9M6 and CYP6BB2 were genotyped. Genotyping of CYP9M6 was carried by real time quantitative PCR using three sets of primer: 9M6F88/9M6R89 (common), 9M6F95/9M6R97 (specific to SP), and 9M6F96/9M6R97 (specific to SMK). The primer sequences are listed in Table S1. Genotyping of CYP6BB2 was carried out by analysis of the melting curve of different peaks in the post-PCR assay. PCR was performed using a TaKaRa Ex Taq Hot Start Version (Takara Bio, Shiga, Japan), 6BB2F21/6BB2R22 primers, 0.5 µl EvaGreen (Biotium, Inc., Hayward, CA, USA), and 1 µl genomic DNA in 10 µl of reaction mixture. The cycling conditions for PCR were as follows: initial denaturation of 95°C for 1 min, followed by 50 cycles of 95°C for 10 s and 60°C for 5 s. This reaction produced 43 bp PCR products (Figure S3). The PCR and dissociation analysis were performed using a PikoReal Real Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). Total RNA was isolated from the remaining bodies of the mosquitoes and cDNA was synthesized as described above. Real time quantitative PCR was performed for CYP9M6 and CYP6BB2 to quantify the levels of transcription of these genes, as described above. Forty-two females (24 females for each crossing) were used for the analysis. Expression of P450s, reductase, and b5 in Sf9 cells Full-length cDNAs encoding CYP9M6 and CYP6BB2 from the SP strain were cloned into the pPSC8 protein expression vector (Wako Chemical Pure Industries, Ltd., Osaka, Japan) using unique restriction sites positioned in the vector regions (XbaI and PstI for CYP9M6 and XbaI and KpnI for CYP6BB2). Because we identified two variants for CYP9M6 in the SP strain (CYP9M6v1 and CYP9m6v2), these two genes were amplified using primers 9M6F85/9M6R86 for CYP9M6v1 and 9M6F85/9M6R87 for CYP9M6v2. For amplification of CYP6BB2 cDNA, 6BB2F19 and 6BB2R20 primers were used and cloned into the pPSC8 vector (Table S1 and Figure S1). A 200-µl aliquot of Sf900II medium (Life Technologies Co., CA, USA) containing 2 µg of the vectors (pPSC8/CYP9M6 or pPSC8/CYP6BB2), 85 ng Linear AcNPV (the baculovirus Autographa californica nuclear Polyhedrosis Virus), 5 µl Insect GeneJuice Transfection Reagent (Merck KGaA, Damstadt, Germany) was transfected to expressSF+ cells (1 × 106 cells in 25 cm2 flask, Wako Chemical Pure Industries, Ltd., Osaka, Japan). The infected cells were incubated at 28°C for five days, centrifuged at 3000 xg for 30 min at 4°C, and the supernatant used for protein expression. Full-length cDNA encoding NADPH cytochrome P450 reductase (PRE) and cytochrome b5 cDNAs was also amplified with the aegREDF9/aegREDR5 and aegb5F1/aegb5R2 primer sets, respectively, and then cloned into the pFastBac1 vector (Life Technologies Co., Carlsbad, CA, USA) using multiple cloning sites (StuI and XbaI for PRE and EcoRI and XbaI for b5). Recombinant constructs (pFastBac1/PRE and pFastBac1/b5) were used to transform MAX Efficiency DH10Bac competent cells (Life Technologies Co., Carlsbad, CA, USA). Recombinant bacmid DNA was isolated according to the manufacturer's instructions. The Sf9 cells were infected with recombinant baculovirus in Grace's Insect Medium with Cellfectin II (Life Technologies Co., Carlsbad, CA, USA), according to the manufacturer's instructions. For preparation of cell lysates expressing P450, 50 ml of confluent cells were infected in a 125-ml flask, and incubated at 28°C with shaking at 130 rpm. Twenty-four hours after infection, hemin (in 50% ethanol and 0.1 N NaOH) was added to the media to a final concentration of 2 µg/ml, followed by further incubation for 48 hours. The cells were then centrifuged at 3000 xg at 4°C for 30 min and the pellets were used for the in vitro permethrin metabolism study. Metabolism of permethrin by heterologously expressed CYP9M6 and CYP6BB2 The cell pellets prepared as described above were resuspended in 5 ml of homogenization buffer [49] and sonicated with an ultrasonic processor Sonics Vibra-Cell VCX130 (Sonics & Materials, Inc., Newtown, CT, USA) for 2 min on ice water (pulse on 10 s, pulse off 10 s, with 20% amplitude). The homogenates were centrifuged at 10,000 xg for 15 min and the supernatant was then centrifuged at 100,000 xg for 1 h as described above for the preparation of microsomes. The microsomal pellets were resuspended by homogenizing in 2 ml of resuspension buffer [49], and the protein concentration was determined using Bradford's reagent (Coomassie Plus Protein Assay; Thermo Fisher Scientific Inc., Waltham, MA, USA). The in vitro permethrin metabolism study was conducted as described above using 7 mg of microsomal proteins. The experiment was replicated for tree times. Results Establishment of a permethrin-resistant strain A field population of A. aegypti collected from Singapore was selected for permethrin resistance by subjecting a wild type population (SPS0) to the chemical for 10 generations (Table 1). Initially, the resistance ratio (RR) of SPS0 was 35-fold. The RR rose rapidly over the generations and the established SP strain ( = SPS10) developed 1650-fold resistance after 10 generations of permethrin selection (Figure 1A). The RR for each generation is summarized in Table 2. To determine the degree of dominance of the resistance [51], the SP strain was crossed with a susceptible reference strain (SMK) and bioassays were performed on the progenies. The degrees of dominance for F1 (SP♀ × SMK♂) and F1 (SMK♀ × SP♂) were −0.337 and −0.383, respectively (Figure 1B). 10.1371/journal.pntd.0002948.g001 Figure 1 Log dose-probit mortality lines of Aedes aegypti. (A) The development of permethrin resistance in a field-collected population of adult A. aegypti under laboratory selection conditions. SPS0 is a field-collected population prior to permethrin selection. SPS10 is the strain established by permethrin selection for 10 generations and is designated as SP. SMK is a susceptible reference strain. (B) Dose–mortality lines of adult F1 (SP♀ × SMK♂ or SMK♀ × SP♂) and BC1 (F1♀ × SMK♂ or SMK♀ × F1♂) showing the inheritance of SP resistance. (C) Synergistic effects of PBO on permethrin toxicity in adults. (D) Susceptibility of larvae to permethrin and the synergistic effects of PBO. 10.1371/journal.pntd.0002948.t002 Table 2 Toxicity of permethrin and pirimiphos methyl by topical application to adult Aedes aegypti and the synergistic effects of PBO. Insecticide Population or strain na Slope ± SE LD50 (ng/♀) 95% CL SRb RRc Permethrin SMK 354 7.0 ± 0.64 1.5 1.4–1.6 1.0 SPS0 480 2.0 ± 0.14 52 37–74 35 SPS1 440 2.2 ± 0.18 106 92–123 71 SPS2 199 3.9 ± 0.41 212 157–278 141 SPS3 200 4.7 ± 0.51 318 212–443 212 SPS4 280 4.2 ± 0.37 509 461–562 339 SPS5 400 3.2 ± 0.26 685 610–769 457 SPS6 280 7.4 ± 0.75 757 684–838 505 SPS7 360 5.2 ± 0.50 1090 959–1240 727 SPS8 360 4.7 ± 0.46 1650 1440–1890 1100 SPS9 280 6.4 ± 0.65 1900 1690–2130 1270 SPS10 ( = SP) 280 5.0 ± 0.59 2470 2160–2840 1650 F1(SP♀ × SMK♂) 200 11.8 ± 1.5 14 13–15 9.3 F1(SMK♀ × SP♂) 240 7.8 ± 0.84 12 11–14 8.0 BC1(SMK♀x F1(SP♂ × SMK♀)♂) 320 5.8 ± 0.62 3.6 3.2–4.0 2.4 BC1(F1(SP♀ × SMK♂)♀ × SMK♂) 320 5.9 ± 0.62 4.9 4.4–5.5 3.3 Permethrin +PBOd SMK 300 6.0 ± 0.55 0.89 0.83–0.95 1.7 1.0 SPS0 420 2.8 ± 0.22 9.8 7.3–13 5.3 11 SPS10 ( = SP) 360 5.8 ± 0.57 30 26–34 82 34 SP♀ × SMK♂ 200 9.7 ± 1.4 3.3 3.0–3.6 4.2 3.7 SMK♀ × SP♂ 240 10.5 ± 1.2 2.3 2.1–2.5 5.2 2.6 Pirimiphos methyl SMK 360 10.5 ± 0.94 7.7 7.0–8.4 1.0 SPS0 359 6.5 ± 0.56 20 19–21 2.6 SPS10 ( = SP) 200 16.2 ± 2.2 38 35–41 4.9 a n, number of females tested. b SR, synergistic ratio = LD50 (without PBO)/LD50 (with PBO). c RR, resistance ratio = LD50 (each population)/LD50 (SMK). d PBO (5 µg/mosquito) was administered 1 h prior to permethrin treatment. The toxicity of permethrin was markedly increased in the SP strain by pretreatment with the P450 inhibitor piperonyl butoxide (PBO), with the RR decreasing from 1650- to 33-fold. The synergistic ratio (SR) of the SP and SMK strains was 1.7 and 83, respectively, indicating a significant role of P450s in permethrin resistance of SP (Figure 1C and Table 2). The low SR of SPS0, at only 5.3, suggested that the P450-mediated resistance mechanism was initiated or enhanced during the process of permethrin selection. We observed an approximately three-fold difference in the LD50s between SPS0 (9.8 ng/female) and SP (30 ng/female) in the presence of PBO. This suggested that a mechanism, other than P450s also contributed to the development of high level of permethrin resistance in SP (Table 2). The SP strain exhibited a 4.9-fold cross-resistance to pirimiphos methyl after permethrin selection. Although the laboratory selections were conducted during the adult stage, larvae of the SP strain also developed a high level of resistance to permethrin (Figure 1D and Table 3), from RR of 160-fold for SPS0 to 8790-fold for SP. PBO also increased the toxicity of permethrin in the larval stage. The SRs of SMK, SPS0, and SP were 3.5, 8.7, and 126, respectively. The SP strain showed a low level of resistance to organophosphates, with the RRs of larvae to temephos and pirimiphos methyl being 1.5- and 4.2-fold, respectively (Table 3). 10.1371/journal.pntd.0002948.t003 Table 3 Toxicity of permethrin, temephos, and pirimiphos methyl to larval Aedes aegypti and the synergistic effects of PBO. Insecticide Population or strain na Slope±SE LC50 (µg/ml) 95% CL SRb RRc Permethrin SMK 372 4.0 ± 0.32 0.0028 0.0021–0.0038 1.0 SPS0 655 1.2 ± 0.08 0.45 0.31–0.72 160 SPS10 ( = SP) 472 1.2 ± 0.15 24.6 15.6–45.3 8790 Permethrin + PBOd SMK 539 3.5 ± 0.27 0.00079 0.00073–0.00087 3.5 1.0 SPS0 421 2.2 ± 0.18 0.039 0.033–0.044 11.5 49 SPS10 ( = SP) 398 1.9 ± 0.20 0.20 0.14–0.26 123 253 Temephos SMK 313 10 ± 0.85 0.016 0.015–0.017 1.0 SPS0 316 7.0 ± 0.71 0.019 0.016–0.023 1.2 SPS10 ( = SP) 320 7.7 ± 0.94 0.024 0.022–0.027 1.5 Pirimiphos methyl SMK 506 4.0 ± 0.39 0.031 0.026–0.043 1.0 SPS0 312 5.2 ± 0.50 0.067 0.063–0.073 2.2 SPS10 ( = SP) 300 10 ± 1.1 0.13 0.12–0.14 4.2 a n, number of larvae tested. b SR, synergistic ratio = LD50 (without PBO)/LD50 (with PBO). c RR, resistance ratio = LD50 (each population)/LD50 (SMK). d PBO (5 µg/ml) was administered 1 h prior to permethrin treatment. Frequencies of knockdown resistance type alleles We genotyped five positions of Vssc, which potentially cause decreased sensitivity to pyrethroid insecticides. Sequencing of the partial DNA of Vssc identified three amino acid substitutions in the SPS0 population compared with the SMK strains: S989P, V1016G, and F1534C. Of these substitutions, P989 and G1016 always appeared together, indicating that these polymorphisms are located on the same haplotype. All mosquitoes in the SPS0 population possessed either the P989+G1016 or C1534 haplotypes, with a frequency of 44% and 56%, respectively. After the first selection, the frequency of P989+G1016 increased to 79%, and following the second selection, the C1534 haplotypes were no longer detected (Table 4). In SMK, all individuals were homozygous for wild-type at all five amino acid positions. The typical kdr mutation, L1014F, was not detected in any of the Singapore populations and would not be expected in this species because kdr in Aedes would require a 2 nucleotides change. 10.1371/journal.pntd.0002948.t004 Table 4 Transition of the frequency of the Vssc genotype during permethrin selection in the SP strain of Aedes aegypti. Amino acid location Percentage of mosquitoes (number of mosquitoes) 989 1011 1014 1016 1534 1763 SMK (n = 24) SPS0 (n = 48) SPS1 (n = 48) SPS2 (n = 24) SPS10 (n = 24) S/S I/I L/L V/V F/F D/D 100 (24) 0 0 0 0 S/S I/I L/L V/V C/C D/D 0 29.2 (14) 2.0 (1) 0 0 S/P I/I L/L V/G C/F D/D 0 54.2 (26) 37.5 (18) 0 0 P/P I/I L/L G/G F/F D/D 0 16.7 (8) 60.4 (29) 100 (24) 100 (24) Frequencies of P989+G1016 0% 43.8% 79.2% 100% 100% The subscript numbers in the population name indicate the number of generations exposed to permethrin. For example, SPS2 was the population which was selected by permethrin for two generations. The location of amino acids are numbered according to the sequence of Vssc from the house fly, Musca domestica (accession nos. AAB47604 and AAB47605). Rate of permethrin reduction from cuticle We examined the rate of permethrin reduction from cuticle by measuring the disappearance of insecticide from the external surface of the mosquitoes (Figures 2A and B). There was no difference in the rate of permethrin reduction from cuticle between the SP and SMK strains, rather slightly higher in SP strain, with penetration rate constants of 0.358 h−1 and 0.252 h−1, respectively. 10.1371/journal.pntd.0002948.g002 Figure 2 In vivo distribution of [14C]-permethrin topically applied to SP and SMK strains of Aedes aegypti. (A) Schematic of the procedure of in vivo assay. (B) Percentage of the applied radiolabel recovered in the external rinse. [14C]-Permethrin (600 dpm; 0.88 ng) was applied to the thoracic notum. PBO (5 µg) was also applied to the thoracic notum or thoracic sternum 1 h prior to permethrin application to observe its effect on permethrin penetration. Acetone was also applied 1 h prior to permethrin application to confirm whether the solvent had any side effects. Values are expressed as the means (±SE) for four mosquitoes. (C) Percentage of radioactivity detected in the mosquitoes. (D) Percentage of radioactivity excreted in the vials. (E) Thin-layer chromatogram of the compounds excreted by females of SP strain. Developing solvents: toluene/ethyl acetate (6∶1). The presence of 4′-HO-permethrin was determined by co-chromatography with an authenticated chemical. (F) Percentage of permethrin and its metabolites excreted by females of SP strain. Values represent the average and bars represent the SE of the mean (n = 3). (G) Re-chromatography of the high-polar compounds stuck around origin. Developing solvents: chloroform/methanol/water (65∶25∶4). Effects of PBO on permethrin penetration Some previous studies suggested that synergistic effects of synergists are not due to inhibition of enzymes but due to enhancement of penetration rate of insecticides by synergists [52]–[54]. Therefore, we investigated the effects of PBO on permethrin penetration (Figure 2A). Pretreatment with PBO in the SP strain did not enhance permethrin penetration but resulted in significantly delayed its penetration (Figure 2B). Twelve hours after treatment, less than 20% of the insecticide had penetrated the cuticle in insects pretreated with PBO compared with 90% in insects without PBO treatment. A reduced rate of permethrin penetration was also observed even when we administered PBO to the thoracic sternum of mosquitoes. As shown in Figure 2B, pretreatment of the SP strain with acetone did not affect permethrin penetration. In vivo metabolism The marked synergistic effects of PBO on permethrin toxicity implicated P450s being involved in resistance. Therefore we further investigated trends of permethrin after penetration through the cuticle using isotopic tracer tests. It was observed that in the SMK strain, internal radioactivity reached a peak at approximately 12 h after permethrin treatment, and then gradually decreased (Figure 2C). In contrast, in the SP strain, internal radioactivity reached a much earlier peak and was then rapidly eliminated. The percentage of the initial radioactivity that remained after 24 h in the SMK and SP strains was 59.5% and 14.8%, respectively. Unlike the groups without PBO pretreatment, the internal dose of radioactivity gradually increased in the groups that received PBO pretreatment and did not decrease until about 48 h after treatment. The internal radioactivity at 48 h with or without PBO in SP strain was 42.7% and 4.9%, respectively (Figure 2C). Consistent with the above findings, we found that the SP strain excreted radioactivity more rapidly than the SMK strain (Figure 2D). The percentage of the radioactivity that was excreted 24 h after treatment in the SMK and SP strains was 32.7% and 82.8%, respectively. This suggested that the SP strain can eliminate permethrin more efficiently. The rate constants of excretion (hr−1) for SMK and SP were 0.022 ± 0.002 and 0.101 ± 0.008, respectively. PBO significantly reduced permethrin excretion, suggesting that permethrin is excreted after being metabolized by P450s. The high performance thin layer chromatography (HPTLC) analysis of excrete from SP strain revealed that more than 85% of the excreted radioisotope consisted of high polar compounds and approximately 10% of the permethrin was excreted without being metabolized (Figures 2E and F). Further investigation using another solvent specific for water-soluble metabolites showed that the compounds consisted of a number of metabolites (Figure 2G). In vitro metabolism We carried out in vitro metabolism studies to determine whether the changes observed in vivo were related to the activity of P450s. Microsomes of the SP strain metabolized permethrin at a much higher rate than those of the SMK strain (Figures 3A and B). Permethrin metabolism by microsomes was apparently due to P450 as it was NADPH-dependent and was inhibited by PBO (Figure 3A). Only a very small amount of permethrin was metabolized by enzymes in the 100,000 xg supernatant, suggesting that carboxyl esterase have only minimal involvement in the development of resistance. The rate constant for metabolism (min−1) of SMK and SP by microsomal enzymes was 0.0007 and 0.0042, respectively. At the start of incubation, 4′-HO-permethrin was the major metabolite in SP, although its percentage did not increase. In contrast, the percentage of high polar metabolites increased over time (Figure 3B, Table S2). The metabolites located around the origin of the HPTLC plates were collected and further developed with chloroform-based solvent (Figure 3C). The high-polar compounds consisted of a number of metabolites and were consistent with those in the in vivo study. 10.1371/journal.pntd.0002948.g003 Figure 3 In vitro [14C]-permethrin metabolism studies. (A) Thin-layer chromatogram of in vitro permethrin metabolism in adult Aedes aegypti with or without NADPH or PBO. Microsomes (100,000 × g pellet) and the soluble fraction (100,000 × g supernatant) were used as the enzyme source. The presence of 4′-HO-permethrin was determined by co-chromatography with an authenticated chemical. (B) Metabolites of permethrin in adult A. aegypti at various incubation times. For detailed data, see Table S2. (C) Thin-layer chromatogram of high-polar compounds (SP) located at the origin of the first HPTLC. Developing solvents: chloroform/methanol/water (65∶25∶4). (D) Inhibition of permethrin metabolism by 4′-HO-permethrin. Microsomes of the SP strain were incubated for 30 min with [14C]-permethrin, NADPH, and ×7-, ×70-, or ×140-fold higher doses of 4′-HO-permethrin than permethrin. In order to assess the effect of 4′-HO-permethrin on permethrin metabolism, we performed an inhibition assay by incubating different doses of unlabeled 4′-HO-permethrin with [14C]-permethrin. This showed that unlabeled 4′-HO-permethrin inhibited effective permethrin metabolism, possibly by acting as a feedback inhibitor (Figure 3D). Microarrays A microarray that contained 60- mer probes for all genes identified by the genome project [55] was constructed. We then compared the levels of transcription in the SP and SMK strains of the adult males and females and the fourth instar larvae. In this study, we focused on P450s and their related genes because our investigations including bioassays and in vivo and in vitro metabolism studies showed that this metabolic enzyme is the key factor in the development of resistance. The genes over expressed in the SP strain were selected using a cut-off of >3-fold relative change in expression and a P-value 20-fold transcription level with a significantly low ( 3-fold, P 3-fold and a P-value 3-fold and a P-value (negative log10 scale) >2. The symbol (↑) indicates over expression of the gene reported in the resistant population or the following strains: A, Cayman (females) [57]; B, Cuba (females) [57]; C, Vauclin (larvae) [28]; D, Vauclin (males+females) [28]; E, PMDR (females) [30]; F, IM (males+females) [30]; G, IM (larvae) [30]; H, Iquitos (males+females) [56]; I, Calderitas (males+females) [56]; J, Lazaro C. (males+females) [56]; K, Merida (males+females) [56]; and L, Noexp-Perm (larvae) [58]. Sequencing analysis of P450s In order to design primers for real time quantitative PCR, we used eight mosquitoes individually to obtain the sequence of partial or full length DNA for seven P450 genes (CYP6BB2, CYP6Z7, CYP6Z8, CYP9M4, CYP9M5, CYP9M6 and CYP9M7) and an internal control gene, ribosomal protein S3 (RPS3). We focused on these P450 genes according to the following criteria: overexpressed in females (>5) and in males (>3) in SP relative to SMK in microarray analysis. CYP9M7 was also analyzed as it forms a gene cluster with CYP9M4, CYP9M5, and CYP9M6 within 65 kbp on the same supercontig (1.29) and is structurally related to these genes (Figure 5F). The full length cDNA and deduced amino acid sequences of CYP6BB2 were identical between the SP and SMK strains. However, on the ninth nucleotide after the stop codon, we observed a C1533T replacement in the SP strain (Figure S3). All eight SP mosquitoes possessed two CYP9M6 genes, designated as CYP9M6v1 (accession number AB840269) and CYP9M6v2 (accession number AB840270) (Figures S3 and S4). The nucleotide and amino acid identities of these alleles were 97.6% (1563/1602) and 98.9% (527/533), respectively. In the SMK strain, we found three CYP9M6 variants; two mosquitoes were homozygous for CYP9M6v3 (accession number AB846835) or CYP9M6v4 (accession number AB846836), two were homozygous for CYP9M6v5 (accession number AB846837), and four were heterozygous CYP9M6 variants. The CYP9M6v5 protein appeared to be incomplete as an active enzyme because there was a stop codon at amino acid position 473 (Figure S4). 10.1371/journal.pntd.0002948.g005 Figure 5 Real time quantitative PCR analysis of selected genes from the microarray experiments. Transcription levels of seven P450 genes were individually validated in adult female (A), adult male (B), and fourth instar larvae (C) in SP and SMK strains of Aedes aegypti. Ribosomal protein S3 gene (RPS3) was used to normalize the data. Expression ratios (SP/SMK) are expressed in parentheses. (D) Correlation between the microarray and real time quantitative PCR (R2 = 0.824). (E) Gene copy number of 7 P450s and RPS3 quantified by real time PCR. The ratios of relative copy number (SP/SMK) are expressed in parentheses. (F) Gene clusters of cytochrome P450 and the CYP9M subfamily on the A. aegypti supercontig 1.29 on the 2nd chromosome. The error bars represent standard errors of three (A, B, and C) and eight (E) biological replicates. Quantitative real-time PCR We performed real-time quantitative PCR (qPCR) for seven P450 genes to verify the results of the microarray analyses. We focused these genes according to the criteria described above. Primers for quantitative real time PCR were designed from sequences that were consensus among eight SP and SMK mosquitoes. Five of these genes, CYP6BB2, CYP6Z7, CYP9M4, CYP9M5, and CYP9M6 were over expressed during the larval stage and in adult males and females (Figures 5A–C) in SP relative to SMK. As shown in Figure 5D there was a good correlation between the results of the microarrays and qPCR (R2 = 0.824). This confirmed that the results of the microarray were accurate. CYP9M6 showed the largest relative change in the seven genes and was over expressed 22.9-fold in males, 38.9-fold in females, and 28.2-fold in larvae of the SP strain relative to SMK. The relative expression level of CYP6BB2 to RPS3 was also higher (close to CYP9M6), especially in adult females (Figure 5A), with the gene reported to be over expressed in five other pyrethroid-resistant strains (Table 5). Identification of the gene copy number The gene copy numbers of the seven P450s in the SP and SMK strains were compared by qPCR (Figure 5E). RPS3 gene was used to normalize the data. While the majority of the P450 genes in the SMK strain were likely to be a single copy, some genes in SP were amplified significantly. The average copy number of CYP6Z7, CYP9M4, CYP9M5, and CYP9M6 in SP were 3.1-, 4.3-, 4.8-, and 4.6-fold more than SMK, respectively. This clearly shows that the over expression of these P450 genes, at least in part, was due to gene amplification. Association between permethrin excretion and CYP9M6 and CYP6BB2 In order to determine whether CYP9M6 and CYP6BB2 have a role in permethrin metabolism, F2 progeny (interbred population of F1 (SMK × SP)) were treated with a sub-lethal dose of [14C]-permethrin, and the excretion rate was measured 24 h after treatment in individuals. The genotypes and transcription levels of CYP9M6 and CYP6BB2 were determined for each mosquito to examine the association between these parameters and the rate of excretion (Figures 6A and D). The transcription levels for CYP9M6 between RR (homozygote of SP allele) and SS (homozygote of SMK allele) were significantly different (P 3-fold in all samples (males, females, or larvae) of the resistant SP strain compared with the susceptible SMK strain (Table 5). It is noteworthy that 15 of these genes have been reported previously to be overexpressed in one or more resistant populations or strains collected from different regions of the world [28], [30], [56], [57]. In this study, we found that CYP9M6 and CYP6BB2 were responsible for resistance. In addition, CYP9J26 and CYP9J28, which have the ability to metabolize permethrin [59], [76], were also over expressed in the SP strain. It remains to be determined as to how many P450s contribute to the resistance in SP mosquitoes. However, it is not likely that all genes over expressed in the SP strain are involved in the development of resistance, as it is generally known that transcription of genes forming clusters are occasionally coordinately controlled by the same regulatory factor [77]. In association analysis using F2 progeny, the expression level of CYP9M5 and CYP9M6 showed a high correlation coefficient (R2 = 0.90), whereas no correlation was observed between the expression levels of CYP9M6 and CYP6BB2 (R2 = 0.10, data not shown). This suggested that CYP9M5 and CYP9M6 are controlled partially by a common mechanism. A further example of this mechanism is the finding that administration of phenobarbital to insects induces a multiple number of P450 genes [78], [79]. Therefore, over expressed genes do not always provide an advantage for insect survival. Heterologous expression and confirmation of the metabolic activity of each P450 isoform is necessary to gain a greater understanding of these processes. To the best of our knowledge, this is the first report showing a correlation between the expression level of each P450 and the rate of insecticide metabolism of individual insects. This analysis may provide a good tool for evaluating the rate of contribution of each P450 isoform to the development of resistance. In this analysis, both CYP9M6 and CYP6BB2 showed moderate correlation between the levels of transcription and the individual rate of permethrin metabolism. Furthermore, although no correlation was observed between the expression level of CYP9M6 and CYP6BB2 (R2 = 0.10), there was a prominent relationship between the rate of permethrin metabolism and standardized-combined expression level of CYP9M6 and CYP6BB2 (Figure 7G). These results strongly suggest that these two P450s are auxiliary parts of effective permethrin excretion. Although CYP9M6 showed the greatest relative change in gene expression in the comparison between the SP and susceptible strains, we still hesitate to conclude that this enzyme plays a major role in permethrin metabolism. First, homozygotes of CYP6BB2 had a rate of permethrin excretion as high as that observed in homozygotes of CYP9M6 in Figures 6C and F. Second, in the heterologous expression study, under our experimental conditions, CYP6BB2 showed a much higher activity of permethrin metabolism than CYP9M6. It is therefore possible that CYP9M6 may compensate for its low metabolic activity by its high mRNA level. Although both CYP9M4 and CYP9M5 were also over expressed in the SP strain, we did not investigate the permethrin metabolic activity of these P450s because their relative expression level was below 1/10th of that of CYP9M6. However, it is possible they may confer resistance depending on their unit activity of permethrin metabolism. Further work will be needed to clarify if these P450 isoforms are involved in the resistance. One of the goals of the study was to establish a more accurate molecular diagnosis method for monitoring field populations of A. aegypti by identifying metabolic enzymes that confer resistance. Findings of CYP9M6 and CYP6BB2 are good progress for this purpose, however, we also found that the high level of resistance in the SP strain was not due to one major mechanism but was actually the consequence of multiple P450 isozymes (at least 4) and reduced sensitivity of Vssc. This implies that we may need to recognize the reality that the development of molecular diagnoses targeting metabolic enzymes will be more complicated and challenging than that of targeting only Vssc [80]. We are currently focusing on elucidating the contribution degree of these P450s on pyrethroid resistance in the field populations of A. aegypti collected from different regions. Supporting Information Figure S1 Diagram depicting primer positions. (PDF) Click here for additional data file. Figure S2 Multiple alignments of CYP9M6 cDNA sequences identified from SP and SMK strains of Aedes aegypti. Hyphens indicate identical nucleotides to the sequence of CYP9M6v1 (SP). CYP9M6v1 and v2 are identified from SP strain. CYP9M6v3, v4, and v5 were identified from SMK strain. Primers used for genotyping were also indicated. For the deduced amino acid sequences, see Figure S4. (PDF) Click here for additional data file. Figure S3 Full length cDNA and deduced amino acid sequence of CYP6BB2 from SP and SMK strains of Aedes aegypti. The red letter indicates the only nucleotide differentiate between SP (cytosine) and SMK (thymidine) in this region. The 6BB2F21 and 6BB2R22 primers were used for genotyping. The poly(A) addition signal (aataaa) is also underlined. (PDF) Click here for additional data file. Figure S4 Multiple alignments of CYP9M6 amino acid sequences identified from Liverpool, SP, and SMK strains of Aedes aegypti. Hyphens indicate identical amino acids to the sequence of Liverpool strain which was used for the genome project. Asterisks indicate stop codons. (PDF) Click here for additional data file. Table S1 Primer lists. (XLSX) Click here for additional data file. Table S2 In vitro metabolism of [14C]-permethrin by microsome of SP and SMK strains of Aedes aegypti. (PDF) Click here for additional data file. Table S3 Metabolism of [14C]-permethrin by CYP9M6v1, CYP9M6v2, and CYP6BB2 expressed in Sf9 cells. (PDF) Click here for additional data file.
Author and article information
Contributors
Dmitri Boudko: Role: Editor
Journal
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Title:
PLoS ONE
Publisher:
Public Library of Science
(San Francisco, CA USA
)
ISSN
(Electronic):
1932-6203
Publication date
(Electronic):
9
December
2015
Publication date Collection: 2015
Volume: 10
Issue: 12
Electronic Location Identifier: e0144586
Affiliations
[1
]Laboratory of Chemical Ecology, Department of Fundamental Chemistry, Federal University
of Pernambuco (UFPE), Recife, Brazil
Author notes
Conceived and designed the experiments: DMAFN NHS RCSS. Performed the experiments: RCSS PMP PCBS AGS. Analyzed the data: DMAFN PMP PCBS RCSS. Contributed reagents/materials/analysis tools: DMAFN MVS. Wrote the paper: DMAFN PMP RCSS PCBS AGS.
* E-mail:
dmafn@
123456ufpe.br
Article
Publisher ID: PONE-D-15-21051DOI: 10.1371/journal.pone.0144586
PMC ID: 4674132
PubMed ID: 26650757
SO-VID: cc56b2b5-353c-4016-aa68-9d5f3f74b7e5
Copyright © © 2015 da Silva et al
License:
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
History
Date
received
: 21
May
2015
Date
accepted
: 21
November
2015
Page count
Figures: 3,
Tables: 1,
Pages: 14
Funding
The study was supported financially by Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) and Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE)/Programa
de Apoio à Núcleos de Excelência (PRONEX) and the following funding sources were not
involved in the study design, in the collection, analysis, and interpretation of data:
FACEPE/PRONEX (APQ-0981-1.06/08); Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq)CNPq/Universal (Processo: 482866/2011-0); CNPq/INCT-Dengue (15204).
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Subject:
Research Article
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Data availability:
ScienceOpen disciplines: Uncategorized