Molecular cloning, nucleotide sequence and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant mosquito, Culex quinquefasciatus Say

Insect Biochemistry and Molecular Biology 30 (2000) 163–171 www.elsevier.com/locate/ibmb

Molecular cloning, nucleotide sequence and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant mosquito, Culex quinquefasciatus Say Shinji Kasai a, Indira S. Weerashinghe a, Toshio Shono a, Minoru Yamakawa

b,*

a b

Laboratory of Applied Zoology, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Laboratory of Biological Defense, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan Received 5 March 1999; received in revised form 5 October 1999; accepted 6 October 1999

Abstract To analyze cytochrome P450s in the southern house mosquito, Culex quinquefasciatus, we quantified the content of P450s and b5 in larval microsomes of guts and carcasses. Results indicated that content was 30 times higher in guts than in carcasses. A conserved region in the alignment of insect P450 family 6 (CYP6) proteins served as a guide for the synthesis of degenerate oligonucleotide primers to clone P450 cDNAs. Primers were used in the reverse transcription–polymerase chain reaction (RT–PCR) of gut mRNA from 4th-instar larvae of the permethrin-susceptible or resistant C. quinquefasciatus. PCR products of ca. 250 base pairs (bp) were cloned, and nucleotide sequences of 35 clones from susceptible and 28 from resistant strains determined. Alignment of the deduced amino acid sequences from these clones showed them to be classifiable into six isoforms. We next screened a cDNA clone (CYP6F1) from a gut cDNA library and determined the nucleotide sequence. Northern blot analysis showed that the CYP6F1 gene in the permethrin-resistant strain appeared to be expressed more strongly than in the susceptible strain. The deduced amino acid of CYP6F1 showed that it has conserved domains of a membrane-anchoring signal, reductase binding sites, a heme-binding site, ETLR motif and substrate recognition sites in P450s. Phylogenetic analysis showed that CYP6F1 is strongly related to CYP6D1 involved in pyrethroid detoxification.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Insect cytochrome P450; CYP6F1; Gene expression; Insecticide resistance; Mosquito; Permethrin

1. Introduction Cytochrome P450 monooxygenases are one of the most important enzyme systems associated with insecticide detoxification or activation. P450 genes such as CYP6A1 (Feyereisen et al., 1989), CYP6A2 (Waters et al., 1992) and CYP6D1 (Tomita and Scott, 1995) isolated from insecticide resistant strains were analyzed for their involvement in insecticide resistance from the house fly, Musca domestica, and fruit fly, Drosophila melanogaster. All these P450 isoforms are constitutively overexpressed in insecticide resistant strains. CYP6A2 was over-expressed in malathion resistant strain (91–R) compared to a susceptible strain (Waters et al., 1992).

* Corresponding author. Tel.: +81-298-38-6154; fax: +81-298-386028. E-mail address: [email protected] (M. Yamakawa)

CYP6A1 gene expression was 10 times higher in the diazinon resistant “Rutgers” strain compared to the susceptible sbo strain (Feyereisen et al., 1995). Of the P450 isoforms reported, only CYP6D1 from the house fly was shown to be involved in insecticide resistance by direct evidence (Wheelock and Scott, 1992; Zhang and Scott, 1996; Scott, 1999). In the pyrethroid resistant LPR strain of house fly, the CYP6D1 gene was expressed 9-fold higher than in the susceptible strain (Liu and Scott, 1997). In the case of CYP6A1 and CYP6D1, overexpression in the resistant strain was due to enhanced gene transcription (Liu and Scott, 1998) but not gene amplification (Feyereisen et al., 1995; Tomita et al., 1995). Although resistant mechanisms mediated by P450s to insecticides in flies have been extensively analyzed, those in mosquitos remain totally obscure. To our knowledge, there are at present only two reports on P450 characterization in mosquitos (Scott et al., 1994; Kasai

0965-1748/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 9 9 ) 0 0 1 1 4 - 9

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et al., 1998a). One report indicated that partial sequences of 17 reverse transcription polymerase chain reaction (RT–PCR) products of the CYP4 P450 family in a mosquito, Anopheles albimanus, were classified into eight subfamily members in pyrethroid-susceptible and seven subfamily members in resistant strains, of which four were commonly detected in both strains (Scott et al., 1994). We previously studied the mechanism of pyrethroid resistance in the permethrin resistant strain of C. quinquefasciatus (JPal-per strain). This strain was originally collected from Saudi Arabia (Amin and Hemingway, 1989) and showed a high level of resistance to permethrin; the resistance ratio was 2000-fold. Our studies showed that the cytochrome P450 monooxygenase system was a major mechanism of resistance in the JPal–per strain; piperonyl butoxide (PBO) synergized permethrin toxicity in vivo and inhibited P450 mediated metabolism of permethrin in vitro (Kasai et al., 1998b). A cDNA encoding P450 from this mosquito was recently cloned in our laboratory and the nucleotide sequence determined (Kasai et al., 1998a). The amino acid sequence deduced from the nucleotide sequence of this clone indicated that this P450 species belongs to the CYP6 family (Kasai et al., 1998a). Thus, the P450 clone was designated CYP6E1 as assigned by the P450 nomenclature commitee and became the first P450 species from mosquitos whose full-length sequence was determined. Analysis of CYP6E1 gene expression in C. quinquefasciatus, however, showed that this gene is very weakly expressed in both permethrin-resistant and susceptible strains (Kasai et al., 1998a). Our previous study indicated that cytochrome P450 and b5 content in an entire body was about 2.4 times higher in the permethrin-resistant JPal–per strain than in the susceptible strain (Kasai et al., 1998b). We therefore analyzed P450 gene transcripts other than CYP6E1 in this mosquito to understand the mechanisms resistant to permethrin in larvae of the JPal– per strain. In the present study, we cloned cDNAs encoding cytochrome P450s by RT-PCR using degenerate primers designed from CYP6 families. The nucleotide sequence of a clone was determined and this clone was designated CYP6F1, as assigned by the nomenclature comittee. Northern blotting showed that the CYP6F1 gene is expressed at a higher level in the permethrin-resistant strain than in the susceptible strain. Phylogenetic analysis of CYP6F1 indicated that it is closely related to CYP6D1 involved in pyrethroide insecticide resistance. 2. Materials and methods 2.1. Biological materials The pyrethroid-resistant strain (JPal-per) of C. quinquefasciatus was kindly provided by Dr. J. Hemingway

(College of Cardiff, University of Wales) collected in Saudi Arabia and selected by permethrin for 20 consecutive generations at a mortality level of 60–70% (Amin and Hemingway, 1989). The susceptible strain was collected in Chichijima, Ogasawara Islands, Japan, in 1968 and cultured without exposure to insecticides. Mosquitos were reared at 27±1°C and a photoperiod of 16:8 (L:D)h. 2.2. Quantitative analysis of cytochrome P450 and b5 Cytochrome P450 and b5 content in the larval microsomes from guts and carcasses was quantitatively analyzed at least three times according to the methods of Omura and Sato (1964) as described previously (Kasai et al., 1998b). Larvae were dissected and, guts and carcasses separated on ice. Five hundred guts and carcasses were used for each measurement. 2.3. Primer peparation and RT-PCR Guts from 100 4th instar larvae of permethrin-resistant JPal–per or susceptible strains were excised and their content removed. Gut samples were homogenized and mRNAs purified using a QuickPrep Micro mRNA Purification Kit (Pharmacia, Stockholm, Sweden). cDNA was then synthesized using a cDNA Synthesis Kit (Pharmacia, Stockholm, Sweden). The following degenerate primers containing EcoRI sites for PCR were designed based on the homologous region of a cytochrome P450 CYP6 family (ET(T/L)RKYP for forward primer and PFG(A/D/E)GP for reverse primer): 5⬘CGGAATTCGA(A/G)AC(A/G/C/T)(A/C/T)(C/T) (A/G/C/T)(A/C)G(A/G/C/T)CC(A/G/C/T)(G/T)C-3⬘ for a forward primer and 5⬘-CGGAATTCGG(A/G/C/T)CC (A/G/C/T)(G/T)C(A/G/C/T)CC(A/G)AA (A/G/C/T)GG3⬘ for a reverse primer. RT-PCR was done under the following conditions: The reaction mixture was first kept at 94°C for 1 min, then 30 cycles of PCR (94°C for 30 s, 45°C for 30 s, 72°C for 1 min) were done, and the sample finally kept at 72°C for 5 min. PCR products of ca. 250 bp were cloned into a pUC118 vector. Resultant PCR clones (35 clones from permethrin-susceptible and 28 from resistant strains) were sequenced with dye terminator cycle sequencing using a DNA sequencer (ABI 373A, Foster City, CA). 2.4. Construction of cDNA library and CYP6F1 cloning A gut cDNA library was constructed with the purified mRNA from 4th instar larvae of the permethrin-resistant strain in the λgt10 vector (Stratagene, La Jolla, CA) using a cDNA Synthesis Kit (Pharmacia, Stockholm, Sweden) and GigaPack II Packaging Extract (Stratagene, La Jolla, CA). The titer of this library was 3.9×106 pfu/ml. One PCR fragment designated CYP6F1 was lab-

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eled with digoxigenin-conjugated dUTP (Boehringer Mannheim, Mannheim, Germany) and used as a probe. Filter lifts (Gene Screen Plus membrane, NEN, Boston, MA) of 6.2×104 plaques were screened using the probe under the following conditions: Prehybridization at 40°C for 1 h in the presence of 50% formamide, hybridization with the probe at 40°C overnight, washing once with 2×SSC containing 0.1% sodium dodecyl sulfate (SDS) for 15 min and twice with 0.2×SSC containing 0.1% SDS at 65°C for 20 min. Two of the positive plaques were screened four times and purified. Plaques contained a 1.6 kbp cDNA as an insert. cDNA inserts were excised and subcloned into a pUC 118 vector. Sequencing was done using the following primers for walking designed based on determined sequences: 5⬘-TGTAAAACGACGGCCATT-3⬘ (M13-21), 5⬘-CAGGAAACAGC TATGAC-3⬘ (M1 3RV), 5⬘-CAATGCTGGTGG TCAAC-3⬘ (Q1F), 5⬘-CAATGCTGGTGGTCA AC-3⬘ (Q2F), 5⬘-CCTTAGAACACTCACGG-3⬘ (Q3R), 5⬘CCCCAAACGGAAGATAC-3⬘(Q5R), 5⬘-CATGGGACATCGAAGTG-3⬘(Q6F), 5⬘-CACTTCGATGTC CC ATC-3⬘(Q6R), 5⬘-AAATCACGCACAAGCAC-3⬘(Q7R), 5⬘-ACGTTTCGCC ACGTGATC-3⬘(Q9R), 5⬘-CGGTT GGATTCGGAATC-3⬘ (Q10F) and 5⬘-ATTC CAGTCCTCGGACTA-3 (Q11F). 2.5. Phylogenetic analysis Computer-aided phylogenetic analysis was done using the DNA star provided by DNA Star Inc. (Madison, WI, USA) via Clustal method of alignment. 2.6. Northern blot analysis Northern blotting was done with RNA samples from guts of permethrin-susceptible and resistant strains (Sambrook et al., 1989). To determine overall P450 gene expression, we used mixed probes, which were 250 bp PCR products synthesized using degenerate primers as described above. In CYP6F1 gene expression, a CYP6F1 cDNA fragment (position 1121–1317, Fig. 2) was used as a probe. Probes were labeled with digoxigenine-conjugated dUTP. Prehybridization and hybridization were done at 42°C in the presence of 50% formaldehyde (Sambrook et al., 1989). The membrane was washed twice with 2×SSC plus 0.1% SDS for 15 min at room temperature followed by two washings with 0.2×SSC plus 0.1% SDS for 60 min at 68°C. As an internal marker, 28S rRNA was visualized on the agarose gel by staining with ethidium bromide. For another internal marker, the following primers designated based on the homologous region of actin genes from insects such as Bombyx mori (Mounier et al., 1987), D. melanogaster (accession no. K00668 and K00669) and Anopheles gambiae (Salazar et al., 1994), 5⬘-AGCAGGAGATGGCCACC-3⬘ (forward primer) and 5⬘-TCCA-

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CATCTGCTGGAAGG-3⬘ (reverse primer), were synthesized to obtain a PCR product, which was then used as a probe. The quantity of signals was determined using NIH image software (National Institute of Health, Maryland, USA) after film scanning.

3. Results 3.1. Quantitative analysis of P450 and b5 To determine resistant mechanisms in the southern house mosquito, C. quinquefaciatus, to pyrethroids, we focused on P450s, since our previous results had suggested that P450s play an important role in the resistance of this insect to permethrin (Kasai et al., 1998b). Guts and carcasses from mosquito larvae were separated and the content of P450 and b5 quantified. P450 and b5 content per proteins in guts is 30 times higher than in carcasses, indicating that more than 80% of cytochrome P450 in larvae is present in the gut (Table 1). This result differed from that of the house fly, which contains most P450s in the fat body (Scott and Lee, 1993). P450 content in guts was 2.7 times higher in the JPal-per strain than in the susceptible strain and the relative value was 2.8 in carcasses. These values well reflect previous quantitative results using entire larvae (2.5 times). 3.2. Characterization of RT-PCR poducts Ten P450s belonging to the CYP6 family have been identified in four different insect species (Feyereisen et al., 1989; Waters et al., 1992; Tomita and Scott, 1995; Kasai et al., 1998a; Wang and Hobbs, 1995; Cohen et al., 1992). We took advantage of the four conserved regions of this family to prepare primers for RT-PCR. Of 15 possible candidate primers, we selected and synthesized two based on annealing temperature and GC content. As a source of mRNAs from permethrin-susceptible and resistant strains, guts were chosen because this tissue was shown to contain a higher level of P450 activity than other tissues (Table 1). RT-PCR indicated clear bands of ca. 250 bp. PCR products were subcloned and colonies containing objective plasmids selected at random, i.e., 35 clones from permethrin-susceptible and 28 from resistant strains. Plasmids from these clones were purified and the nucleotide sequences of inserted PCR products determined. Deduced amino acid sequences of these clones showed that PCR products from both strains can be classified into six isoforms containing one clone, previously analyzed (CYP6E1) and the nucleotide sequence determined (Kasai et al., 1998a). The permethrin-susceptible strain contained four groups and the resistant strain six (Table 2). Two clones, CYP6E1 and CYP6F1, appeared to have a one nucleotide difference between permethrin-susceptible and resistant

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Table 1 Contents of cytochrome P450s and b5 in the gut and the carcass of susceptible and JPal-per larvae of Culex quinquefasciatus Cytochrome P450 Gut

Strain

0.53 (0.026)a 1.44 (0.17) 2.72

Susceptible JPal-per Ratio a

Cytochrome b5 Gut

Carcass 0.015 (0.004) 0.042 (0.007) 2.80

Carcass

0.31 (0.003) 0.52 (0.093) 1.68

0.015 (0.004) 0.019 (0.009) 1.27

Results are expressed as nmol/mg protein. All values are mean of at least three replicates (±SE in parentheses).

Table 2 Occurrence of Culex P450 clones in permethrin-susceptible and resistant JPal-per larvae of C. quinquefasciatus Strain Genes

Susceptible na

%

JPal-per na

%

CYP6F1 PCul-3 PCul-4 CYP6E1 PCul-5 PCul-6 Total

20 12 1 2 0 0 35

57.1 34.3 2.9 5.7 0 0 100

17 5 3 1 1 1 28

60.7 17.9 10.7 3.6 3.6 3.6 100

a

Number of cDNA clones found.

Fig. 1. Alignment of amino acid sequences deduced from the nucleotide sequences of cloned PCR products. RT-PCR was performed with primers designed based on the conserved region of the CYP6 family. Resulting PCR products (ca. 250 bp) were cloned and sequenced. Amino acid sequences were deduced from the nucleotide sequences of clones, designated CYP6E1, CYP6F1, PCul-3, PClu-4, PClu-5 and PClu-6. Computeraided alignment was done using Genetyx-Mac software.

Table 3 Percent identity of the partial nucleotide sequence of Culex P450 cDNA and the deduced amino acid sequencesa

CYP6E1 CYP6F1 PCul-3 PCul-4 PCul-5 PCul-6 a

CYP6E1

CYP6F1

PCul-3

PCul-4

PCul-5

PCul-6

60.9 58.1 52.0 59.1 53.7

47.8 60.2 54.7 59.3 56.2

32.8 37.3 51.7 55.0 52.5

43.3 40.3 25.0 57.9 63.6

44.8 41.8 34.3 43.3 55.5

41.8 44.8 37.3 50.8 46.3 -

Figures in upper right (bold) and lower left are % identity of deduced amino acid and at lower left % identity of nucleotide sequences.

strains in the ca. 250 bp PCR products but no difference was observed at the amino acid level. Fig. 1 shows the amino acid sequences of six P450 isomers. A comparison of nucleotide sequences among the six groups showed the percentage identity from 51.2 to 63.6% (Table 3). Likewise, the percentage identity at the amino acid level was from 25.0 to 50.8% (Table 3).

3.3. Screening and sequencing of a CYP6F1 cDNA clone from a gut cDNA library To determine the complete amino acid sequence of a P450 clone, a gut cDNA library of the permethrin-resistant strain was screened with a CYP6F1 PCR product as a probe for hybridization. Eleven positive clones were

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Fig. 2. Nucleotide and deduced amino acid sequences of a CYP6F1 cDNA clone. A cDNA encoding CYP6F1 was screened from the gut cDNA library of the permethrin-resistant JPal-per strain of C. quinquefasciatus using the CYP6F1 PCR product as a probe. Nucleotide sequences expressed in uppercasing indicate the open reading frame. Deduced amino acids are shown by single uppercased letters under nucleotide sequences. The poly A addition signal is indicated in bold and the translation stop codon indicated by a star. Underlining denotes the positions of primers used for RTPCR. Figures at left and right margins are nucleotide numbers.

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obtained from 6.2×104 plaques. Two plaques from these positive clones were subjected to nucleotide sequencing and found to be the same. Sequencing of this cDNA clone showed that it contains an open reading frame that encodes 508 amino acids and confirmed that the probe region is present in a typical P450 sequence (Fig. 2). The sequence flanking the PCR product corresponded exactly to conserved amino acid sequences used to design degenerate PCR primers. A poly A addition signal was localized in a typical position of the 3⬘-region. 3.4. Expression of P450 genes in permethrinsusceptible and resistant strains We first studied overall gene expression of P450s by Northern blotting using degenerate mixed primers as probes in permethrin-susceptible and resistant strains. A RNA sample from guts of the permethrin-resistant strain gave a signal stronger than that of the susceptible strain (Fig. 3, Panel A). We then conducted Northern blotting using a CYP6F1 cDNA fragment as a probe. Results showed a significantly stronger signal in the resistant strain than in the susceptible strain (Fig. 3, Panel B). The signal of CYP6F1 gene transcripts was not detected from carcasses, although three times more RNA was analyzed, suggesting that the CYP6F1 gene is mainly expressed in the gut. To determine if the enhanced CYP6F1 mRNA expression is due to gene amplification or not, we probed genomic DNA from the JPal-per strain with a CYP6F1 specific cDNA (Southern blotting). In this procedure, we digested the genomic DNA with EcoRI and EcoRV as restriction enzymes. However, the resultant signals identified DNA fragments of different sizes in the susceptible and permethrin-resistant strains (data not shown) making us unable to determine if gene amplification caused the overexpression of CYP6F1 gene.

4. Discussion We previously showed that microsomal fractions of guts and carcasses of JPal-per larvae of the southern house mosquito, C. quinquefasciatus, distinctly degrade permethrin in the presence of NADPH (Kasai et al., 1998b), strongly suggesting that P450 monooxygenase works as a mechanism of resistance in the JPal-per strain in this insect. We therefore studied the content of P450 and b5 in an entire body and found that the permethrinresistant JPal-per strain contains about 2.4 times more of these enzymes than the susceptible strain (Kasai et al., 1998b). Interestingly, the P450 monooxygenase of the JPal-per strain metabolized five times more permethrin in the gut and about 20 times more in carcasses of the JPal-per strain than in the susceptible strain, although microsomal fractions of JPal-per larvae contained only about 2.4 times more cytochrome P450 (Kasai et al., 1998b). Thus, isozymes that metabolize permethrin may increase their quantity ratio in total P450 and bring greater permethrin metabolism in the JPal-per strain, suggesting isoform overexpression. Specific inhibitors for P450 strongly suppressed the degradation of permethrin by microsomal P450 enzymes in the JPalper strain, suggesting the involvement of P450 in resistance to the pyrethroid. We therefore focused on the P450 and tried to clone cDNAs encoding P450s as a first step toward understanding the mechanisms resistant to permethrin in C. quinquefasciatus. Before cloning, we determined P450 and b5 content in guts and carcasses and confirmed that guts are a suitable tissue for preparing cDNA library to screen P450 cDNAs (Table 1). We cloned CYP6-related cDNAs from permethrinsusceptible and resistant strains by RT-PCR. Nucleotide sequences of 63 total clones from both strains showed them to be classifiable into six isoforms. Four common clones found in both strains, whereas two clones were

Fig. 3. Northern blot analysis of RNA samples from guts and carcasses of permethrin-susceptible and resistant JPal-per strains of C. quinquefasciatus. In panel A, mixtures of 250 bp probes were used and 3 µg of RNA samples were electrophoresed. In panel B, a CYP6F1 probe was used, and 3 µg RNA samples from guts and 10 µg from carcasses were loaded. Actin gene transcript or 28S rRNA is used as an internal marker. S and R denote permethrin-susceptible and resistant strains. For details on experimental conditions, see Materials and methods.

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Fig. 4. Comparison of the complete amino acid sequences of CYP6F1 to those of other insect P450s. CYP6E1, CYP6A1, CYP6B2 and CYP6D1 are derived from C. quinquefasciatus (Kasai et al., 1998a), M. domestica (Feyereisen et al., 1989) and H. armigera (Salazar et al., 1994) and M. domestica (Tomita and Scott, 1995). Conserved amino acids are highlighted. Alignment is as described in Fig. 1.

detected only in the JPal-per strain under our experimental conditions (Table 2). The complete amino acid sequence deduced from the CYP6F1 nucleotide sequence showed 40.0% identity to CYP6E1 from C. quinquesfasciatus (Kasai et al., 1998a), 35.5% identity to CYP6A1 and 39.2% to CYP6D1 from a house fly, M. domestica (Feyereisen et al., 1989; Tomita and Scott, 1995), and 35.1% identity to CYP6B2 from a cotton bollworm, H. armigera (Wang and Hobbs, 1995) (Fig.

4). A comparison of these P450s indicated that CYP6F1 contains important conserved domains (Gotoh, 1992) such as a putative membrane-anchoring signal (positions 3 to 20), putative reductase binding sites (positions 362 to 371 and 416 to 427), a typical heme-binding site (positions 441 to 450), ETLR motif (positions 368-371) and substrate recognition sites (positions 97 to 117, 115 to 154 and 311 to 314). Computer-aided phylogenetic analysis of CYP6F1 showed it to be closely correlated

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Fig. 5. Dendrogram of insect P450s. The phylogenetic relationship of CYP6F1 from C. quinquefasciatus to 20 insect cytochrome P450s was analyzed by computer-aided methods via Clustal method of alignment. The scale under the tree indicates the distance between sequences (the number of substitution events). Accession numbers of amino acid sequences of insect cytochrome P450s are as follows: CYP6A1 (P13527), CYP6A2 (P33270), CYP6A3 (U09231), CYP6A4 (U09232), CYP6A8 (L46859), CYP6B1 (M80828), CYP6B2 (U18085), CYP6B3 (U25819), CYP6B4 (U47059), CYP6B6 (Q95031), CYP6B7 (061387) CYP6C1 (U09233), CYP6C2 (U09345), CYP6D1 (U15168), CYP6E1 (AB001323), CYP6F1 (AB001324), CYP4C1 (P29981), CYP4C1 (P29981), CYP4D1 (P33269), CYP4E2 (JC5236), CYP9A1 (Y23506), CYP12A1 (U86618), CYP28A1 (U866187), CYP28A2 (U89746) and CYP28A2 (U89747).

with CYP6D1 and previously analyzed CYP6E1 (Kasai et al., 1998a) (Fig. 5). These results strongly suggest that CYP6F1 is a member of the CYP6P450 family. Some of the other RT-PCR products are also speculated to be members of the CYP6 family because of their close structural similarities (Fig. 1), although complete sequences have not yet been determined. Our phylogenetic analysis showed that CYP6F1 is closely related to the house fly CYP6D1. CYP6D1 is the only P450 proven to detoxify pyrethroids (Wheelock and Scott, 1992; Zhang and Scott, 1996). It would be interesting to determine whether CYP6F1 also contributes to resistance in C. quinquefasciatus. Our Northern blot analysis indicated that the CYP6F1 gene is more strongly expressed in a permethrin-resistant strain than in a susceptible strain. Similar overexpression in insecticide resistant-populations have also been seen in Helicoverpa armigera CYP6B7 (Ranasinghe and Hobbs, 1998), Drosophila CYP6A2 (Waters et al., 1992), M. domestica CYP6A1 (Carino et al., 1994) and CYP6D1 (Tomita and Scott, 1995). Although we have no direct evidence at present to show that CYP6F1 is involved in permethrin resistance, our results suggest that CYP6F1 is a good candidate. An analysis of the effects of CYP6F1 antibodies on permethrin degradation in a JPal-per strain should help demonstrate the involvement of CYP6F1 in permethrin resistance. P450 families other than CYP6 need to be examined in permethrinresistant mosquitos because insect P450 family 12 was

also recently found to be involved in the metabolism of xenobiotics (Guzov et al., 1998).

Acknowledgements We thank Dr. D. Taylor for a critical reading of this manuscript. This work was supported by Enhancement of Center of Excellence, Special Coordination Funds for Promoting Science and Technology Agency, Japan. Nucleotide sequence data reported here has been submitted to the DDBJ nucleotide sequence databases with accession number AB001324.

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