In vitro labelling and gel-based analysis of PM targets in An. gambiae homogenates

Mosquito homogenate preparation and probe treatment

Twenty individuals of 3–5-day-old female An. gambiae (Kisumu strain) were homogenized in 500 µl of 1× DPBS (Invitrogen, UK) buffer and centrifuged at 21130 × g (Eppendorf 5425 R Centrifuge) for 5 min at 4 °C. The supernatant protein concentration was determined using the Bradford assay⁶⁹ and normalized to 1 mg/ml using 1× DPBS. Aliquots (48 µl) of the normalized homogenate were then incubated with 1 µl of inhibitors pirimiphos-methyl (10 or 100 µM final concentration), pirimiphos-methyl oxon (10 or 100 µM final concentration), desthiobiotin-fluorophosphonate probe (FP) probe (2 or 10 µM final concentration), or dimethyl sulfoxide (DMSO, control) for 1 h at 30 °C with shaking at 1200 rpm. Subsequently, 1 µl of T-FP probe (100 µM) was added to each sample to give 2 µM final concentration, except for the negative control, which received 1 µl of DMSO⁷⁰. Heat-denatured homogenates treated with 2 µM final concentration of T-FP served as negative controls to confirm activity-dependent labelling of SHs. Incubation continued for an additional 1 h, then 50 µl of 1x-SDS page Gel loading buffer was added to each sample, mixed gently, and heated at 85 °C for 2–3 min, followed by a brief centrifugation.

Gel electrophoresis and analysis

Samples (20 µL) and 5 µL of protein marker were resolved on NuPAGE™ Bis-Tris Mini Gels (Thermo Fisher Scientific, 4–12% gradient) using NuPAGE MOPS SDS Running Buffer for medium-to-large proteins. Electrophoresis was performed at 200 V for 50 min. Gels were imaged using the iBright FL1500 Imaging System (ThermoFisher Scientific, UK) with a tetramethylrhodamine (TRITC) filter, followed by destaining in acid-methanol for 1 h. Gels were then stained with colloidal stain, destained with water, and re-imaged.

Proteomic analysis of pirimiphos-methyl oxon targets in An. gambiae using FP-ABPP

FP Probe treatment

Homogenates from insecticide-susceptible female An. gambiae mosquitoes (Kisumu strain) were used to study protein interactions with the active organophosphate metabolite pirimiphos-methyl oxon (Sigma-Aldrich, UK). Each 500 µL homogenate sample (2 mg/mL protein) was pre-incubated with 10 µL of 5 mM pirimiphos-methyl oxon for 1 h at 30 °C with shaking. Subsequently, 10 µL of 100 µM ActivX™ Desthiobiotin-FP Serine Hydrolase Probe (Thermo-Fisher Scientific) was added, resulting in final concentrations of 100 µM PMO and 2 µM FP probe. Probe concentration was initially optimized for binding saturation, with 2 µM selected in line with ABPP published protocol⁷⁰, and inhibitor dose–response confirmed near-saturating competition and robust target detection (Supplementary Fig. 1). The samples were incubated for an additional hour at 30 °C with shaking. Control samples, including both active and heat-denatured homogenates treated with the FP probe, served as positive and negative controls, respectively. All treatments, including the PMO-treated groups and controls, were performed in triplicate to ensure reliability and reproducibility. After incubation, samples were chilled on dry ice and stored at −80 °C for further analysis.

Removal of excess reagents and protein precipitation

Frozen samples were thawed on ice, then centrifuged to pellet the proteins at 21130 × g (Eppendorf 5425R Centrifuge, UK) for 10 min at 4 °C. The supernatant was discarded, and pellets were washed thrice with cold methanol, using sonication for resuspension between centrifugations. Next, 0.65 mL of 2.5% SDS in Ca- and Mg-free D-PBS was added to each pellet, followed by sonication and heating steps to solubilize proteins. Samples were centrifuged again, and the supernatant discarded. Additional heating and sonication were applied when precipitate still visible to ensure complete protein solubilization. Finally, the samples were adjusted to 3.5 mL with D-PBS (reaching 0.5% SDS) and frozen overnight.

Streptavidin enrichment of FP-labelled proteins

The SDS-solubilized proteins were diluted with calcium- and magnesium-free Dulbecco’s Phosphate-Buffered Saline (D-PBS, Thermo Fisher Scientific, UK) to achieve a final SDS concentration of 0.2%. Pre-washed streptavidin beads (Invitrogen, UK) were added to the samples, which were rotated to enrich for FP-labelled proteins. After centrifugation, most of the supernatant was removed, and the remaining beads were transferred to a new tube. The beads were sequentially washed with 1% SDS (Sigma-Aldrich), 6 M urea (Sigma-Aldrich), and Ca- and Mg-free D-PBS, with centrifugation and supernatant removal between each wash.

On-bead reduction, alkylation, and digestion

The washed beads were resuspended in 500 µL of 6 M urea in Ca- and Mg-free D-PBS. Next, 25 µL of 200 mM dithiothreitol (DTT, Sigma-Aldrich, UK) was added to achieve a final concentration of 10 mM, and the samples were heated at 65 °C for 15 min. Following reduction, 25 µL of 500 mM iodoacetamide (IAA, Sigma-Aldrich, UK) was added, reaching a final concentration of 25 mM, and the samples were incubated at room temperature in the dark for 30 min. The beads were centrifuged at 1400 × g for 2 min, and the supernatant was removed. The beads were washed once with 1 mL of Ca- and Mg-free D-PBS. For trypsin digestion, the beads were resuspended in 200 µL of 2 M urea in Ca- and Mg-free D-PBS, 2 µL of 100 mM calcium chloride (CaCl₂, Sigma-Aldrich, UK) to achieve a final concentration of 1 mM, and 4 µL of 0.5 mg/mL trypsin (2 µg total; Promega, UK). The samples were incubated overnight at 37 °C with gentle agitation.

Elution of tryptic peptides

After digestion, the samples were centrifuged at 1400 × g for 2 min, and the supernatant containing the tryptic peptides was transferred to a clean microcentrifuge tube (Fisherbrand™ Premium Microcentrifuge Tubes, Thermo Fisher Scientific). To recover any remaining peptides, 100 µL of Ca- and Mg-free D-PBS was added to the beads, and the resulting solution was combined with the initial supernatant for a final volume of 300 µL. The peptide solution was acidified with 17 µL of 90% formic acid (Fisher Chemical, UK) to reach a final concentration of 5%. The samples were either prepared for immediate mass spectrometry (MS) analysis or stored at −80 °C for future use.

LC-MS/MS analysis for protein identification and quantification

Tryptic peptides generated from on-bead digestion were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the FingerPrints Proteomics Facility, College of Life Sciences, University of Dundee. Peptides were resuspended in 1% formic acid (FA, Thermo Fisher Scientific) and purified using Empore Solid Phase Extraction Cartridges, C18 (#41155D, 3 M). Samples were dried using a SpeedVac (Thermo Scientific) and resuspended in 50 µl of 1% formic acid. The resuspended peptides were centrifuged, and the supernatant was transferred to HPLC vials, with 8 µl of the sample analyzed directly on a chromatography-mass spectrometry system. Peptides were separated and analyzed using an Ultimate 3000 RSLCnano system (Thermo Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific). Samples were injected and washed on an Acclaim PepMap 100 C18 trap column (100 µm × 2 cm, Thermo Scientific) with 2% Buffer B for 5 min before applying a gradient using Buffer A (0.1% formic acid) and Buffer B (80% acetonitrile in 0.1% formic acid). The separation was performed on an Easy-Spray PepMap RSLC C18 analytical column (75 µm × 50 cm, Thermo Scientific) under gradient conditions starting with 2% Buffer B, held for 5 min, followed by a linear increase to 35% Buffer B over 125 min, reaching 98% Buffer B after a further 2 min, and maintained for 10 min, before re-equilibration to 35% Buffer B after 1 min. The flow rate was maintained at 0.3 µl/min throughout the run, and the sample was transferred to the mass spectrometer via an Easy-Spray source set to 50 °C with a source voltage of 3.0 kV and capillary temperature of 250 °C. A scan cycle comprised a MS1 scan (m/z range 335–1800, ion injection time of 25 ms, resolution 60,000 and automatic gain control of 3.0 × 106 acquired in profile mode, followed by 20 sequential dependent MS2 scans (resolution, 15,000) of the most intense ions fulfilling predefined selection criteria (AGC 5 × 103, maximum ion injection time 40 ms, isolation window of 1.4 m/z). The HCD collision energy was set to 27%. Mass accuracy was checked before the samples were run.

Data processing and analysis

The LC-MS/MS RAW data were processed with MaxQuant version 2.1.1.0. and peptides were identified from the MS/MS spectra searched against the Anopheles gambiae reference proteome (UniProtKB, proteome ID UP000007062, downloaded from https://www.uniprot.org/proteomes/UP000007062 on 23 February 2022). Cysteine carbamidomethylating was used as a fixed modification, and methionine oxidation, asparagine/glutamine deamidation, methionine/tryptophan dioxidation, glutamine to pyro-Glu and N-term acetylation. The false discovery rate was set to 0.01 at the protein level & peptide-spectrum match (PSM) level. The match between runs option was enabled, and protein grouping was achieved by selecting a unique and razor peptides mode that calculates ratios from unique and razor peptides (razor peptides were uniquely assigned to protein groups). Other parameters were used as pre-set in the software. The built-in label-free quantification algorithm (MaxLFQ) was used to perform the LFQ experiments in MaxQuant software⁷¹. MaxLFQ allows comparative analysis of high-resolution MS data generated from two biological samples by comparing peptide peak intensities. For statistical analysis and bioinformatics, Protein Groups file processed using LFQ-Analyst, an interactive web-platform designed for analyzing and visualizing proteomics data pre-processed with MaxQuant⁷². LFQ data were log₂-transformed, and missing values imputed using the “Missing Not At Random” (MNAR) method, drawing from a left-shifted Gaussian distribution (mean shift: 1.8 standard deviations; width: 0.3). Significantly differentiated proteins were identified based on an adjusted p-value ≤ 0.05 (Benjamini-Hochberg correction) and an absolute log₂ fold change ≥ 1. Pearson correlation coefficients compared protein labelling intensities across replicates and treatment conditions, visualized in a correlation matrix. Hierarchical clustering was performed on Z-score normalized; log₂-transformed LFQ intensities.

Processed data, including the complete dataset and quantitative proteomic results, are provided in the Supplementary Data Files (S1–S6) as detailed in the Data Availability section.

Crude sub-cellular fractionation from mosquito thoraxes

Mitochondria were isolated from the other cell compartments, i.e., nuclei and cytoplasm, as described in Njiru et al. 2022⁷³, using approximately 40 mg thoraxes (~ 80 thoraxes, after removal of the wings and the legs) of 3–5 days old female mosquitoes of the docking line A11, homogenized in 1 ml of freshly-prepared, iced-cold Phosphate- free sucrose MOPS buffer pH 8.0, containing 440 mM sucrose, 20 mM MOPS, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF and 1 mM protease inhibitors (SIGMAFAST tablets, Sigma), using Dounce homogenizer. The suspension was centrifuged twice at 1000 × g for 10 min, at 4 °C, to separate carcass and nuclei (pellet) from mitochondria and cytosol (supernatant). Afterwards, the supernatant was centrifuged at 15,000 × g for 30 min, at 4 °C, to sediment mitochondria and leave cytoplasm in the supernatant. A small amount of each fraction was kept and stored at −80 °C, for subsequent Western blot analysis. Approximately 30 μg of total protein were loaded from each fraction, in two separate 10% SDS acrylamide gels, and immuno-blotted against three protein targets: Coeae6g (rabbit anti-Coeae6g, Davids Biotechnologie, dilution 1/1.000 in 2% milk/1× TBST; Supplementary Methods), alpha-tubulin (serving as cytosolic marker; mouse anti-alpha-tubulin, DSHB #12G10, dilution 1/2.000 in 2% milk/1× TBST) and ATP5A (serving as mitochondrial marker; mouse anti-ATP5A, Abcam 15H4C4, dilution 1/1.000 in 2% milk/1× TBST). Antibody binding was detected using goat anti-rabbit IgG (Cell Signaling; #7074) and goat anti-mouse IgG (Merck; #12-349), both horse-radish peroxidase (HRP)-linked, at dilutions 1/5.000 in 2% milk/1× TBST, and ECL substrates (Thermo Scientific; SuperSignal West Pico Plus).

Baculovirus-mediated expression of recombinant Coeae6g in Sf9 cells

The An. coluzzii Coeae6g coding region was cloned into the pFastBac/ CT-TOPO vector (Bac-to-Bac TOPO Cloning Kit; Gibco), using primers Coeae6g Forw and Rev (Supplementary Data S7). DH10Bac competent E. coli cells were transformed to generate bacmid DNA, using the Bac-to-Bac Baculovirus Expression System (Gibco). Colonies with recombinant bacmids were selected on kanamycin/tetracycline/gentamycin plates by blue-white selection, and the presence of the Coeae6g coding sequence was verified with PCR (pUC/M13 Forward -M13 Reverse primer pair) and via Sanger sequencing (GENEWIZ, Azenta Life Sciences, Germany), using primers pUC/M13 Forward and M13 Reverse, and Coeae6g Fin and Rin (Supplementary Data S7). Recombinant baculovirus expressing YFP was also generated and used as a negative control in all experiments henceforth.

To obtain esterase expression, fresh Sf9 cells (Gibco, Cat. Number 11496015) at 10⁶ cells/mL were infected for 72 h\ with baculovirus stock at a multiplicity of infection of 5 (virus stocks were titrated using the baculoQUANT ALL-IN-ONE Kit, Oxford Expression Technologies Ltd) in SF-900 II SFM (Gibco) medium, supplemented with 10% FBS and 10% Penicillin-Streptomycin (Gibco). Uninfected Sf9 cells were included as control. Harvested and washed with 1× ice-cold Phosphate Buffered Saline (PBS)- cell pellets were resuspended in 0.02 M Sodium Phosphate Buffer pH 7.2, disrupted by mild sonication, and then centrifuged at 1000 × g for 5 min, at 4 °C, to remove cell debris. Supernatants were subjected to Western blot analysis, to verify successful Coeae6g expression (Supplementary methods; Supplementary Fig. 6), prior biochemical assays.

Biochemical assays and inhibition kinetics in Sf9 cells expressing Coeae6g

Recombinant An. coluzzii Coeae6g esterase activity was assessed against the model substrates, α-naphthyl acetate (α-ΝΑ), β-naphthyl acetate (β-NA) (both diluted in methanol) and p-nitrophenyl acetate (p-NPA) (diluted in acetonitrile) using a SpectraMax-M2 multimode microplate reader (Molecular Devices, Berkshire, UK). The reaction mixture for α-ΝΑ and β-NA consisted of 10 μl of cell homogenate, containing approximately 10 μg of protein, and 200 μl of 300 μΜ substrate in 0.02 M SPB pH 7.2. Reactions were incubated for 20 min, at room temperature upon which 50 μl of 3 mg/ ml Fast Blue dye were added per well. Formation of the naphthol—Fast Blue RR dye complex was measured endpoint at 570 nm. Standard curves of α-naphthol and β-naphthol were used to convert initial slopes into specific activities. For p-NPA, the substrate was diluted in 50 mM SPB and the rate of p-nitrophenol formation (kinetics) was monitored at 405 nm, at 20 s intervals during a 2 min period. P- nitrophenol concentration was determined based on the absorbance and using a molecular extinction coefficient of 18,000 M⁻¹ cm^−1,74. Control reactions using protein homogenates from Sf9 expressing YFP, as well as uninfected Sf9, were included for all substrates.

Recombinant Coeae6g kinetics against α-ΝΑ were determined by using a range of final substrate’s concentrations (1 μM to 1 mM). K_m and V_max values were defined using non-linear regression in GraphPad Prism 8.0.2. Eight different insecticide compounds were tested for their potential to inhibit recombinant Coeae6g activity against α-ΝΑ, including pirimiphos-methyl (Dr. Ehrenstorfer), malathion (PESTANAL Sigma-Aldrich) and their toxic oxon analogs, pirimiphos-methyl oxon (ChemService) and malaoxon (PESTANAL Sigma-Aldrich), respectively, bendiocarb (PESTANAL Sigma-Aldrich), propoxur (ChemService), permethrin (PESTANAL Sigma-Aldrich), and deltamethrin (PESTANAL Sigma-Aldrich). Insecticide stock solutions were prepared in 5% acetonitrile. 10 μg of Sf9 expressing Coeae6g homogenates were incubated with 10 μl of each compound concentration for 10 min, prior the 20 min incubation with α-ΝΑ, at a concentration equal to the K_m value. Half-maximal inhibitory concentration values (IC₅₀), corresponding to the concentration of each insecticide required to inhibit Coeae6g activity against α-ΝΑ by 50%, were calculated using GraphPad Prism 8.0.2.

Mosquito rearing

All mosquitoes were maintained under standard insectary conditions, at temperature 26 °C ± 2 °C and relative humidity 70% ± 10%, under L12:D12 hour photoperiod. All larval stages were fed on ground fish food (Tetramin tropical flakes, Tetra, Blacksburg, VA, USA), and adults were provided with 10% sucrose solution. Details of mosquito strains, including colony origin, generation history, and rearing conditions, are provided in Supplementary Data S8.

Construction of UAS-Coeae6g responder plasmids and creation of UAS-Coeae6g An. gambiae transgenic lines by ΦC31- mediated cassette exchange

The Coeae6g coding regions were amplified from cDNA of the An. gambiae Kisumu and An. coluzzii NGousso laboratory colonies using Phusion HF DNA polymerase (NEB) with primer pairs, Ang006727forRI: Ang006727revNheI and Coeae6gForwNheI: Coeae6gRevXhoI (Supplementary Data S7), respectively. These fragments were then either subcloned and sequence verified (AngCoeae6g) in pJET before cloning into a YFP-marked UAS plasmid (pSL* attB:3 × P3-eYFP:gyp-UAS14i-gyp:attB, previously described in Lynd et al.⁴²), or directly cloned (AncCoeae6g) into the same UAS plasmid and then sequenced using primers Coeae6g NheI For, Coeae6g Fin, Coeae6g XhoI Rev, and Coeae6g Rin (Supplementary Data S7), as well as the universal primers Hsp70 and SV40 polyA.

Embryos of the Ubi-GAL4 (for AncCoeae6g) or A11 (for AngCoeae6g) docking line (previously described in Adolfi et al. and Lynd et al., respectively)^42,75, bearing attP sites and marked with 3 × P3-eCFP (Supplementary Fig. 7), were microinjected with a mix of 350 ng/μl of responder UAS- Coeae6g plasmid and 150 ng/μl of an integrase helper plasmid (pKC40) encoding the phiC31 integrase⁷⁶ following standard procedures (Poulton et al.)⁷⁷. Emerging F₀ larvae, with transient YFP expression, were selected using fluorescent stereomicroscopy and outcrossed with wild type G3 individuals of the opposite sex. The G1 larvae expressing only eYFP and no eCFP, in their nerve cord and eyes (denoting successful cassette exchange), were identified by fluorescence microscopy using YFP and CFP filters (Leica) and inter-crossed. The colony was maintained as a mixed stock of homozygotes (UAS- Coeae6g / UAS- Coeae6g) and heterozygotes (UAS- Coeae6g/+) but enriched with fluorescent progeny in each generation.

Driver × Responder line crosses

To promote the expression of Coeae6g, individuals of both Ang and Anc UAS-Coeae6g responder lines (marked eYFP) were crossed with opposite sex individuals from Ubi-GAL4 driver line (generated in Adolfi et al., 2018⁴¹, that carries the GAL4 factor under the control of the An. gambiae polyubiquitin promoter and is marked with eCFP) (Supplementary Fig. 7). Transheterozygous Ubi-GAL4/UAS- Coeae6g progeny were selected by screening pupae positive for both eYFP and eCFP.

Insecticide resistance profile of the GAL4/UAS progeny

Susceptibility to insecticides in the progeny of the cross Ubi-GAL4 × UAS- Coeae6g, as well as the parental docking lines, were assessed using WHO tube bioassays and insecticide discriminating doses⁴³. These doses are fixed at twice the lethal concentration that kills 99% of the susceptible mosquitoes 24 h after 1 h of exposure, and mortality of less than 90% is the threshold to define diagnostic resistance (98–100% mortality shows susceptibility, while 90–97% indicates the possibility of upcoming resistance)⁴³. A minimum of three replicates of 20–25 female mosquitoes, 3–5 days old, were conducted for each insecticide. Statistical significance between the mortality percentages of the GAL4/UAS and the parental lines was assessed using Welch’s t-test. The LD₅₀ (lethal dose 50; exposure dose resulting in 50% mortality) was performed as described by Lees et al. 2020 in at least two replicates for a range of PM dilutions in acetone covering 0.0001%–0.01%. The LT₅₀ (lethal time 50; exposure time resulting in 50% mortality) was determined using WHO insecticide-impregnated papers and varying the exposure time. Test values were calculated using Polo Plus 2.0.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.