Oligonucleotides were synthesized by Sigma-Aldrich and enzymes for DNA manipulation were supplied by New England Biolabs. Isopropyl-ß-D-thio-galactopyranoside (IPTG), 5-aminolevulinic acid (ALA), and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) were supplied by Melford (UK). Insecticides were supplied by ChemService: 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT), (S)-α-cyano-3-phenoxybenzyl (1R,3R)-cis-2,2-dimethyl-3-(2,2-dibromovinyl)-cyclopropanecarboxylate (deltamethrin), N-[1-[(6-chloro-3-pyridyl)methyl]-4,5-dihydroimidazol-2-yl]nitramide (imidocloprid), 3-phenoxybenzyl (1R,S)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (permethrin, mixture of four isomers), and 2-isopropoxyphenyl N-methylcarbamate (propoxur). (4S)-4,5-dihydro-2-(6 ′-hydroxy-2 ′-benzothiazolyl)-4-thiazolecarboxylic acid (Beetle luciferin) and proluciferin P450 substrates were supplied by Promega. HPLC solvents and ethanol were supplied by Fisher Scientific. Other chemicals were obtained from Sigma-Aldrich unless indicated otherwise.

Total RNA was extracted with either the Arcturus PicoPure Kit (Applied Biosystems) or TRI reagent (Sigma-Aldrich) from ten adult Ae. aegypti mosquitoes, either “Isla Mujeres” [19] or Merida strains. Complementary DNA was prepared using Superscript III (Invitrogen) with an oligo(dT)₂₀ primer and used as a template for amplifying full-length genes with KOD DNA polymerase (Merk Chemicals). The gene-specific primers used in these high-fidelity PCRs were designed according to the Ae.aegypti genome sequence (Table 1). Amplified genes were ligated into pGEM T-easy (Promega) and sequenced. For expression, the ompA leader sequence (ompA), was engineered onto the amino-terminus to direct the P450 to the E. coli outer membrane during expression. The ompA-leader was inserted into the expression plasmid pCWmod1 and linked to the 5′-end of the P450 gene with codons for either: Ala-Pro by blunt ligation via an NaeI site, or Ala-Gly using cohesive ligation with the isoschizomer NgoMIV. Both linkers were tested with CYP6CB1 and no difference in expression quality or quantity was observed. All genes used cohesive ligation at the 3′-end and the restriction enzymes are indicated in Table 2. Another approach was used for CYP9J19 or CYP9J26: these genes were prepared with an ompA-Ala-Pro leader sequence by fusion PCR and cloned into pCW-ori+ using vector plasmid, pB13 [20] via NdeI and EcoRI sites as previously described [16].

Competent E. coli DH5α cells were co-transformed with the pCW- P450 plasmid and and pACYC-AgCPR [16]. Transformants were selected on Luria-Bertani (LB) agar plates with 50 mg/L ampicilin (pCW-P450 selection) and 34 mg/L chloramphenicol (pACYC-AgCPR selection). After overnight growth at 37°C, a single colony was used to inoculate 5 mL of LB with antibiotics (50 mg/L ampicilin and 34 mg/L chloramphenicol) for overnight growth at 37°C with shaking. Two mL of this starter culture was then used to inoculate 200 mL of Terrific Broth with antibiotics that was then incubated at 37°C with orbital shaking. Once the cultures had reached early log-phase growth (A595 = 0.8–1.0), the culture was cooled to 25°C before adding 1 mM IPTG and 0.5 mM ALA (final concentrations) and continuing incubation at 25°C with orbital shaking. Initially, cultures were monitored daily to find the incubation time for optimal P450 expression: 6CB1, 1 day; 9J19, 2 days; 9J24, 3 days; 9J26, 2 days; 9J28, 2 days; 9J32, 1 day. P450 expression was estimated by resuspending whole cells in Spectrum Buffer (100 mM Tris-HCl, pH 7.4, 10 mM CHAPS, 20% (v/v) glycerol, 1 mM EDTA) [20], adding about 1 mg/mL of sodium dithionite as a reducing agent and recording the absorption spectra (500-400 nm) change after exposing to CO for 1 min. The peak height at 450 nm was used to calculate the P450 concentration [21].

After the optimal incubation period at 25°C, the cells were harvested and membranes prepared based on the method by Pritchard et al. [20]. Immediately after membrane isolated by ultracentrifugation (1 hr at 180000 g) the sample was homogenized in TSE buffer (50 mM Tris-acetate, pH 7.6, 250 mM sucrose, 0.25 mM EDTA) using a Dounce homogenizer. Typically 1 mL of TSE was used per 200 mL of culture processed.

The membrane samples were analysed for P450 quality and content by 100-fold dilution in Spectrum Buffer and CO-difference spectroscopy [21]. Cytochrome c reductase activity was used to measure CPR content [22], and protein content was estimated by Bradford assay. Samples were stored in aliquots at −70°C.

Anopheles gambiae cytochrome b5 was prepared as described previously [18] .

5 mM stock concentrations of insecticides were prepared in ethanol and diluted to 10× the assay concentration in 20% (v/v) ethanol immediately before each experiment to minimise precipitation of insecticide. Standard reactions contained a final ethanol concentration of 2% (v/v) with 10 µM insecticide, 0.1 µM P450, 0.8 µM cyt b5 in 200 mM Tris-HCl for pH 7.4, and NADPH regeneration components (1 mM glucose-6-phosphate (G6P), 0.25 mM MgCl₂, 0.1 mM NADP⁺, and 1 U/mL glucose-6-phosphate dehydrogenase (G6PDH)). Reactions were started, after pre-incubation at 30°C for 5 min, by adding enzyme samples for a final reaction volume of 0.1 mL. These were incubated for a specified time at 30°C with 1200 rpm orbital shaking and quenched by adding 0.1 mL of acetonitrile. Samples were then incubated with shaking as before for an additional 10 min before centrifuging at 20000 g for 5 min. 0.15 mL of the supernatant was then transferred to HPLC vials, stored at room temperature and analyzed within 24 hrs. Reactions were performed in triplicate and a paired T-test of sample reactions (+NADPH) vs negative control (−NADPH) used for statistical measurements of substrate depletion.

For enzyme kinetic measurements the reaction rate in response to insecticide concentration was used to estimate Michaelis-Menton parameters. Reactions were performed, as described above, for 10 min with insecticide concentrations of: 0.25, 0.5, 1, 2, 4, 8, or 16 µM. The reactions were performed in parallel against a negative control (−NADPH) at 16 µM. Non-linear regression of results of three independent experiments were used for estimations of K_M and k_cat (SigmaPlot 11, Systat Software, Inc.).

100 µl of acetonitrile-quenched reaction supernatant was analyzed by reverse-phase HPLC with a 250 mm C18 column (Acclaim 120, Dionex) and a mobile phase consisting of 90% acetonitrile and 10% water. The system was run at 23°C with 1 mL/min flow rate. Reactions with with permethrin, deltamethrin or DDT, were monitored by absorbance at 232 nm, whereas imidacloprid or propoxur were monitored at 270 nm. The insecticide was quantified by peak integration (Chromeleon software, Dionex). Elution times were 9.2 min for deltamethrin and 10.1 min for cis- and 11.9 min for trans-permethrin stereoisomers. There was no significant difference in the change of area for the two permethrin peaks in these reactions, therefore permethrin concentration was measured as the total area under the two peaks.

Four resorufin ethers were tested as fluorogenic substrates and six luciferin-based substrates (P450-Glo, Promega) were tested against each of the Ae.aegypti P450 membrane preps. Three An. gambiae P450/CYP membrane preps were included for additional comparisons: CYP6Z2 [16], CYP6P3 [17], and CYP6M2 [18].

Each of these probe substrate were tested in reactions at 25°C buffered with 0.1 M KPi at pH 7.4 and included: 1 mM G6P, 0.25 mM MgCl₂, 5 µM substrate, 0.1 or 0.2 µM P450, and cyt b5 at twice the P450 concentration. Positive reactions included 0.1 mM NADP and 1 U/mL GADPH whereas negative reactions had neither of these components and could not generate NADPH. Reactions were run in opaque white 96-well (flat-based) plates in triplicate. Luciferin (Promega) or resorufin standard curves were analyzed with each 96-well plate and used to calculate product formation rate.

Resorufin-producing reactions were monitored in a fluorescence plate-reader (Ex = 565 nm, Em = 585 nm) with 200 ms measurements recorded 24 s apart. The rate of resorufin molecules produced per P450 molecule per min (turnover) was determined by linear regression of the measurements between 2 min and 8 min after the reactions began. Luciferin reactions were run for 30 min before quenching as described by the P450-Glo kit . The endpoint signal was then measured by a luminescence plate-reader and the turnover calculated. Three replicates of positive and negative control reactions were run for each P450/substrate combination. Significant differences were determined by one-tailed T-tests (assuming equal variances).

To produce a catalytically active monooxygenase complex CYP's 6CB1, 9J10, 9J19, 9J24, 9J26, 9J28 and 9J32 were co-expressed with An. gambiae CPR in E. coli membranes [16]. As previously, An. gambiae b5 was also used to enhance P450 catalytic activity [23]; An. gambiae CPR and b5 are extremely similar to their Ae. aegypti homologues sharing 87% and 84% amino acid identity respectively, thus capable of reconstituting Aedes P450 activity. Apart from CYP9J10, which repeatedly failed to express functional enzyme in E. coli, all P450s produced the characteristic CO-reduced spectra indicative of active P450 (Figure 1). Apart from CYP6CB1 all P450s contained very low quantities of inactive P420 and high levels of P450 up to ∼200 nmol/L for CYP9J19 (Table 3). CYP6CB1 produced relatively low quantities of P450 (∼10 nmol/L; Table 3) and large amounts of P420, suggestive of poor enzyme quality.

In order to determine if the P450s were capable of pyrethroid metabolism, they were tested against permethrin and deltamethrin, representative Type I (non-cyano) and Type II (cyano) pyrethroids respectively. Catalytic activity was assessed by measuring substrate turnover (disappearance of substrate with time)(Figure 2). CYP's 9J24, 9J26, 9J28 produced low, but reproducible deltamethrin and permethrin turnover, whereas CYP9J32 demonstrated strong activity for deltamethrin and weak activity for permethrin (Figure 2). CYP's 9J19 and 6CB1 did not metabolise permethrin or deltamethrin. The ability of CYP's 9J24, 9J26, 9J28 and 9J32 to metabolise pyrethroids was further characterised by measuring substrate dependent reaction rates. The reactions followed Michaelis-Menten kinetics and the kinetic constants were estimated for each P450-pyrethroid combination (Table 4). The apparent K_M measurements for permethrin ranged from 2.3±1.1 µM (CYP9J32) to 4.2±0.9 µM (CYP9J26) and for deltamethrin from 1.2±0.2 µM for (CYP9J26) to 5.2±2.1 µM (CYP9J32). The turnover rates (k_cat) for permethrin ranged from 0.16±0.03 min⁻¹ (CYP9J24) to 0.8±0.1 min⁻¹ (CYP9J32) and from 0.22±0.02 min⁻¹ (CYP9J26 or CYP9J28) to 3.0±0.5 min⁻¹ (CYP9J32) for deltamethrin. Overall, the K_M values for all P450s were within the normal range, 1–50 µM, associated with substrate binding and P450 metabolism [18], [24]. In comparison with the normal broad range of k_cat values for P450s of 1–20 min⁻¹, k_cat values were low i.e. <1 min⁻¹. The exception was CYP9J32, which had a high k_cat value of ∼3 min⁻¹ for deltamethrin. Interestingly the k_cat for permethrin was ∼3 fold lower (0.8 min⁻¹). Finally, as for the pyrethroids, all P450s where tested against DDT (organochlorine), propoxur (carbamate), and imidacloprid (neonicotinoid), but there was no obvious substrate turnover detected.

Fluorescent and chemiluminescent substrates are routinely used in the pharmaceutical industry for monitoring P450 activity [25]. In the context of vector control these probes could be extremely useful for diagnostic monitoring of P450 levels for insecticide resistance [26]. We therefore screened the Ae. aegypti P450s for their ability to metabolise the four fluorogenic resorufin ethers; methyl (RME), ethyl (REE), pentyl (RPE), or benzyl ethers (RBE), as well as six P450 Glo™ proluciferin substrates, L-H, L-ME, L-CEE, L-BE, L-PFBE, L-PPXE. However, with the exception of CYP9J32, which produced a high rate of metabolism with the bulkiest luciferin compound L-PPXE, the Aedes P450s were largely unreactive against these substrates (Figure 3).

We extended the screen to include three An. gambiae P450s namely, CYP6P3 [17] and CYP6M2 [18], which metabolise pyrethroids and CYP6Z2, which does not metabolise pyrethroids [16]. Like CYP9J32, CYP6P3 and CYP6M2 showed a clear preference for the luciferin substrate L-PPXE. In contrast, CYP6Z2 showed a marked preference for the smaller probe substrates with the greatest activity towards RME for the resorufin ethers and L-ME for the proluciferins (Figure 3).