Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae

Abstract

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of (14)C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An gambiae.

Keywords: cytochrome P450; hydrocarbons; insecticide resistance; malaria; mosquito cuticle.

Fig. 1.

TEM analysis of cuticle thickness. (A, Left) A representative image from a cross-section of the apical region of femur leg segment cuticle. The epicuticle (Epi) is the outermost gray zone. (Right) A higher-magnification image depicts numerous lipids or lipoprotein droplets deposited at the base of epicuticle (44). (B) Box-and-whisker plots of cuticle (Left) and epicuticle (Right) thickness. The boxes represent the 25% and 75% percentiles of five independent measurements for resistant (left box in each panel) and susceptible (right box in each panel) mosquitoes. The horizontal black line within the box indicates the mean.

Fig. 2.

Analysis of adult An. gambiae CHCs. (A) Representative total ion current (TIC) profile of An. gambiae HCs. Numbers indicating major HC peaks correspond to peak numbers from Dataset S1. (Inset) TLC for the separation of lipid species. Cuticular lipids from male (M) and female (F) mosquitoes were extracted by hexane and subsequently separated on 2D TLC. HCs are the major lipid species, compared with waxes (W), sterols (St), diglycerides (DGs), or triglycerides (TGs). First dimension of TLC: hexane; second dimension: hexane/diethyl ether/acetic acid. Visualization of lipids was performed after spraying the plates with 5% sulfuric acid in 95% ethanol, and charring at 180–200 °C for 20 min. (B) Comparison of CHCs in resistant (Res) and susceptible (Sus) females. The CHCs derived from resistant and susceptible adult (12- to 14-d-old) female mosquitoes were quantified by GC-MS/FID (20 insects pet vial) and were found to be significantly higher in resistant than in susceptible mosquitoes (980.5 ± 18.6 ng CHCs/mg of mosquito and 757.5 ± 72.5 ng CHCs/mg of mosquito, respectively). The box plots show the 25th and 75th percentile; the mean is shown as a black line within the box; error bars correspond to the 10th and 90th percentiles.

Fig. S1.

Analysis of cyp4g16 transcripts. (A) Alignment of the predicted amino acid sequences from the extreme C terminus of CYP4G16PA (PA) and CYP4G16PD (PD) in VectorBase, together with CYP4G16 PD1 (PD1) characterized in this study. An alternative splice event (in intron 4) is predicted to produce CYP4G16PD, but the actual sequence of CYP4G16 PD1 contains intron 4 (bold letters). (B) Relative expression levels of cyp4g16RA(BC) (ABC) and cyp4g16RD1 (D1) on cDNA from dissected abdominal walls.

Fig. 3.

Immunohistochemical localization of An. gambiae 4G P450 cytochromes. (A, Left column) Longitudinal sections from mosquito specimens were immunostained with α-CYP4G17–, α-CYP4G16–, or α-CPR–specific antibodies (green), respectively. (Center column) Cell nuclei are stained red with TOPRO. (Right column) Merged immunohistochemical images of P450 and nuclei staining. (Scale bars, 50 μm.) Malp. tub., Malpighian tubules; oe, oenocytes. (B) Merged immunohistochemical images as in A focusing on oenocytes, showing the subcellular localization of CYP4G17 in the cytoplasm (presumably bound to ER) and CYP4G16 associated with PM. (Scale bars, 10 μm.)

Fig. S2.

Confirmation, orientation, and colocalization of CYP4G16 with CPR. (A) Expression of CYP4G16 upon RNAi-based silencing of the corresponding gene. Immunolocalization of CYP4G16 to longitudinal cryosections from mosquito specimens that were injected with dsGFP (Upper) or ds4g16 (Lower). (Scale bars, 50 μm.) (B) Whole-mount staining using α-CYP4G16 and α-CPR, in the presence or absence of Triton X-100. (Upper) Triton X-100 was used in parallel with primary antibodies, and green fluorescence was detected. (Lower) Triton X-100 was not included, and the signal was lost for both proteins (CPR and CYP4G16), indicating the absence of protein epitopes outside the cells and the intracellular orientation of the CYP4G16. (Scale bars, 50 μm.) oe, oenocytes. (C) Double staining on abdominal walls with α-CPR and α-CYP4G16. Colocalization of CPR (green) and CYP4G16 (red) appears yellow. TOPRO stains the nucleus blue.

Fig. 4.

Decarbonylase activity of microsomal CYP4G16-CPR fused protein. (A) Conversion of [9,10]³H-octadecanal to n-heptacosane after 1-h incubation. CYP4G2-CPR is the positive control (21). Values are shown as means ± SE; n = 3. (B) In vitro expression and decarbonylase activity of microsomal CYP4G16-CPR fused protein: effect of time on the conversion of tritiated octadecanal to n-heptadecane. Filled squares, CYP4G16-CPR; inverted triangle, CPR control. Values are shown as means ± SE; n = 3. cpm, Counts per minute.