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. 2026 May 28;6(4):1081–1090. doi: 10.1021/acsmeasuresciau.6c00054

Metabolomic Changes Underpinning Permethrin Resistance in the Anopheles gambiae Vector from Cameroon

Steve V Djova 1,2,3, Michael W Christopher 4, Sulaiman S Ibrahim 3,7, Mersimine Kouamo 3, Rhoel R Dinglasan 5, Timothy J Garrett 1,*, Charles S Wondji 3,6,*
PMCID: PMC13495491  PMID: 42631101

Abstract

Insecticide resistance reduces the effectiveness of current malaria-vector-control interventions. To understand the mechanisms underlying permethrin resistance in field-derived Anopheles gambiae vectors collected from Cameroon, we used ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) to comprehensively analyze metabolic changes in resistant and susceptible samples to gain insights into mechanisms driving permethrin resistance. Resistant mosquitoes exhibited upregulated levels of inosine, nicotinic acid, dipeptides, amino acids, fumarate, uracil, and aldopentoses. These data suggest that altered metabolic-based detoxification, as well as target site and metabolic shifts, and enhanced energy production contribute to permethrin resistance. Conversely, susceptible mosquitoes showed increased levels of N-acetyl-aspartic acid, xanthurenic acid, 2-hydroxyglutarate, 3-hydroxykynurenine, propanoylcarnitine, and l-pipecolic acid. These metabolites are associated with neurotoxicity, energy disruption, as well as tryptophan and lysine catabolism, leading to susceptible phenotypes. These findings provide deeper metabolic insight into An. gambiae permethrin resistance and compel further investigations into how these metabolic pathways contribute to the emergence of pyrethroid cross-resistance.

Keywords: An. gambiae, permethrin resistance, metabolome, UHPLC/HRMS/MS, resistance biomarkers, susceptibility biomarkers

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Introduction

Malaria remains a significant global health concern, with 597,000 deaths reported in 2023 alone. Sub-Saharan African countries continue to carry the heaviest burden of mortality, with 95% of the estimated malaria deaths worldwide, and 76% of those deaths among children under the age of 5 are caused primarily by Plasmodium falciparum. Malaria prevention still relies heavily on the use of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) for vector control. However, the effectiveness of these tools is currently challenged by the spread of pyrethroid resistance across major malaria mosquito vectors, such as Anopheles gambiae , and Anopheles funestus. − Mosquitoes have evolved a variety of ways to detoxify pyrethroid insecticides, with the two main mechanisms being (i) target-site resistance, known as knockdown resistance (kdr), a mutation in the voltage-gated sodium channel that reduces pyrethroid binding and subsequent toxicity in mosquitoes, − and (ii) metabolic resistance through increased gene expression/amplification or allelic variation of cytochrome P450s, carboxylesterases (COEs), glutathione S-transferases (GSTs), and other oxidation/reduction genes. , While extensive ITN distribution and coverage are primary drivers of pyrethroid resistance in Cameroon, , evidence suggests the involvement of other contributing factors. Notably, the application of pesticides in agriculture may increase pyrethroid tolerance in mosquitoes. A case in point is Nkolondom, a peri-urban agricultural site in Yaoundé, Cameroon, where routine pesticide application by farmers has increased pyrethroid resistance in mosquitoes.

Progress in understanding pyrethroid resistance mechanisms in An. gambiae sensu lato (s.l.) has revealed both target-site resistance, through the kdr (L1014F) alleles, ,, and metabolic resistance, associated with the upregulation of detoxification genes including P450s (cyp6P3, cyp6p4, cyp6z3, and cyp9k1) and glutathione S-transferases genes (gste2, gsttd1–6, and gstd1–4). − However, the metabolic changes accompanying the upregulation of these resistance genes and key biomarkers of resistance (such as key metabolites) remain unknown. In vitro metabolism assays with recombinant enzymes have confirmed the role of many cytochrome P450 enzymes in pyrethroid resistance in An. gambiae. Stevenson et al. used recombinant An. gambiae cytochrome P450 CYP6M2 to perform an in vitro analysis of deltamethrin metabolism. Their finding revealed a sequential breakdown of deltamethrin with 4′-hydroxylation identified as a major metabolism route. The study of Kengne-Ouafo et al. found that a recombinant Cyp6p3-E205D from An. gambiae has a 2.5-fold increase in its ability to metabolize α-cypermethrin and permethrin insecticides. While in vitro functional assays make identifying primary pyrethroid metabolites straightforward, the detoxification process in mosquitoes is more complex. Previous research has identified new features of pyrethroid detoxification in mosquito vectors. Permethrin resistance in Drosophila melanogaster involves insecticide detoxification and multiple cellular pathways, with metabolomic alterations in tryptophan catabolism as a secondary target. Building on these findings, we used UHPLC-HRMS/MS to conduct untargeted metabolite profiling to measure metabolome-associated changes in permethrin-resistant field-derived An. gambiae sensu stricto (s.s.) from Cameroon.

Results

Metabolic Response of An. gambiae s.s. from Nkolondom Exposed to Permethrin 0.75%

All mosquitoes from Nkolondom were confirmed by PCR as An. gambiae s.s., and WHO bioassay test tubes revealed permethrin resistance for these mosquitoes with 76.3% mortality after 1 h and 25.8% mortality after 30 min (0.5 h) exposure to permethrin 0.75% (Figure A). Our untargeted analysis identified a large number of features and adducts in positive and negative ionization (Tables S1 and S2). From ANOVA, we identified 2767 significant features, including adducts in negative ion mode and 2524 in positive ion mode (Tables S3 and S4,respectively). Principal component analysis (PCA) indicated that the metabolome resulted in three clusters: alive at 0.5 h, dead at 0.5 h, and unexposed clustering together; alive at 1 h; and dead at 1 h (Figure B). Sparse partial least-squares-discriminant analysis (sPLS-DA; performance shown in Figure SI) successfully resolved four distinct clusters corresponding to mosquitoes that were alive at 1 h, dead at 1 h, and dead at 0.5 h and a combined group of unexposed (controls) and alive at 0.5 h mosquitoes (Figure C). These sPLS-DA models demonstrate the distinct metabolic responses associated with permethrin (0.75%) exposure over time.

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Metabolic response ofAnopheles gambiae s.s.to permethrin exposure. (a) Susceptibility profile of An. gambiae s.s. collected in Nkolondom and exposed during 1 and 0.5 h to permethrin 0.75%. The data shown are mean ± SD, N = 100 at 1 and 0.5 h; ANOVA, post hoc Fisher’s tests ***p < 0.0001. (b) Principal component analysis (PCA) of metabolomes resulted in three clusters (unexposed, alive at 0.5 h, and dead at 0.5 h clustered together; alive at 1 h; and dead at 1 h). (c) Sparse partial least squares-discriminant analysis (sPLS-DA) provided greater discrimination, identifying four distinct clusters: unexposed and alive at 0.5 h clustered together, dead at 0.5 h, dead at 1 h, and alive at 1 h.

UHPLC-HRMS/MS analysis revealed distinct metabolite profiles when comparing permethrin-resistant to susceptible mosquitoes. In the first column cluster, resistant and unexposed mosquitoes exhibited elevated levels of l-phenylalanine, l-isoleucine, l-methionine, and the dipeptides alanyl-l-methionine and glycyl-l-methionine, while the levels were low in susceptible phenotypes. Conversely, in the second column cluster, dead, susceptible mosquitoes showed increased levels of xanthurenic acid (XA), 2-hydroxyglutarate, propanoylcarnitine, l-pipecolic acid, N-acetyl-aspartic acid (NAA), hexanoylcarnitine, N-acetyl-glutamate, N-acetyltyramine, butanoylcarnitine, acetyl-l-carnitine, 3-hyhydroxykynurenine, and orotate, while their concentration was low in resistant and unexposed phenotypes (Figure a). Negative ion mode also confirmed, in the first column cluster, higher levels of 5-hydroxy-l-tryptophan, fumarate, aldopentose, nicotinate, inosine, l-threonine, l-tryptophan, l-methionine, l-isoleucine, l-valine, and uracil in resistant and unexposed mosquitoes. In the second column cluster, N-acetyl-aspartate, mannitol, xanthurenic acid, orotate, and 2-methylglutarate revealed elevated levels in dead susceptible mosquitoes compared to resistant and unexposed groups (Figure b).

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Heatmaps illustrate the most significant differences in the mosquito metabolomic profile across five biological replicates for each phenotype. Positive mode (a) and negative mode (b).

Metabolite Markers of Permethrin Detoxification in Permethrin-Resistant An. gambiae

While metabolome profiling for permethrin detoxification presents inherent challenges, variations in the critical metabolite abundance provide valuable insights into the specific enzymatic pathways involved. Notably, resistant mosquitoes exhibited elevated levels of nicotinate (Figure a and Figure S2), a precursor for NAD+ and NADP coenzymes, both indispensable for cytochrome P450 activation, regeneration, and metabolic energy generation. We also observed higher levels of uracil (Figure b and Figure S3) in resistant mosquitoes. Additionally, the upregulation of inosine in resistant mosquitoes is revealed (Figure d and Figure S4). Conversely, susceptible mosquitoes exhibited elevated orotate levels (Figure c and Figure S5).

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Confirmation of metabolic and target-site resistance, as well as purine and pyrimidine metabolism pathways associated withAn. gambiae permethrin-resistance phenotypes. ANOVA, post hoc Fisher with p value threshold = 0.05; statistical annotation: ns, nonsignificant. (a) Nicotinate: *p value = 0.0146, **p value = 0.0082, ***p value = 0.0005, ****p value < 0.0001. (b) Uracil: **p value = 0.0094, ***p value = 0.0008, ****p value < 0.0001. (c) Orotate: **p value = 0.0029, ***p value = 0.0004. (d) Inosine: *p value = 0.0131, **p value = 0.0018.

Amino Acid Profiles Differentiate An. gambiae Permethrin-Resistance Phenotypes

This study associated elevated levels of the dipeptides alanyl-l-methionine and glycyl-l-methionine, along with the free amino acids l-methionine, l-isoleucine, l-leucine, l-phenylalanine, l-tryptophan, l-valine, and l-threonine, with permethrin resistance in An. gambiae mosquitoes (Figures S6–S15).

Energy Metabolism Is Swift in Resistant Phenotypes

Several key metabolites that are involved in energy reserve catabolism were identified. Resistant and unexposed mosquitoes maintained higher aldopentose and fumarate levels than did susceptible mosquitoes, indicating unaffected energy production. In contrast, all mosquitoes that died following 0.5 or 1 h exposure showed significantly elevated mannitol (Figure a–c and Figures S16–S18).

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Energy metabolism responses associated withAn. gambiae permethrin-resistance phenotypes. ANOVA, post hoc Fisher with p value threshold = 0.05; statistical annotation: ns, nonsignificant. (a) Fumarate: **p = 0.0015, ****p value < 0.0001. (b) Aldopentose: ***p value = 0.0003. (c) Mannitol: *p value = 0.0154, **p value = 0.0021.

Hypoxia Markers Associated with Permethrin Neurotoxic Effects

l-2-Hydroxyglutaric acid and 2-methylglutaric acid were significantly elevated in susceptible mosquitoes compared to resistant and unexposed groups, as shown in Figures S19–21.

Amino Acid N-Acetylation Pathway Associated with Susceptible Phenotypes

N-Acetyl-l-glutamic acid, N-acetyl-tyramine, and N-acetyl-l-aspartic acid (NAA) were significantly elevated in susceptible mosquitoes (Figure a–c and Figures S22–S24). We used an NAA standard to confirm the chromatographic retention time of 2.09 min and MS/MS spectra to validate its exclusive presence among dead mosquito samples (Figure S25).

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Protein N-acetylation and degradation associated withAn. gambiae permethrin-resistance phenotypes. ANOVA, post hoc Fisher p value threshold = 0.05; statistical annotation: ns, nonsignificant. (a) N-Acetyl-l-aspartic acid: ****p value < 0.0001. (b) N-Acetyl-l-glutamic acid: ***p value = 0.0002, ****p value < 0.0001. (c) N-Acetyl-tyramine: **p value = 0.002, **p value = 0.0002.

Tryptophan and Lysine Metabolism Pathways Associated with Susceptible Phenotypes

Levels of xanthurenic acid (XA) and 3-hydroxykynurenine, neurotoxic biomarkers and tryptophan catabolites (Figure a,b and Figures S26 and S27), were elevated in susceptible mosquitoes compared to those resistant at 0.5 and 1 h. The upregulation of 5-hydroxy-l-tryptophan, a precursor of serotonin neurotransmitter, in the resistant 1 h group may indicate a potential action in permethrin resistance (Figure c and Figure S28). l-Pipecolic acid (L-PA), a catabolite of lysine, was also elevated in the susceptible group (Figure d and Figure S29).

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Tryptophan and lysine catabolism pathway associated with An. gambiae permethrin-resistance phenotypes. ANOVA, post hoc Fisher p value threshold = 0.05; statistical annotation: ns, nonsignificant. (a) XA: *p value = 0.0257, **p value = 0.0072, ***p value = 0.0001, ****p value < 0.0001. (b) 3-Hydroxykynurenine: **p value = 0.0018, ***p value = 0.0001. (c) 5-Hydroxy-l-tryptophan: *p value = 0.018, ***p value = 0.0001. (d) L-PA: ****p value < 0.0001.

Carnitine/Acylcarnitine Shuttle Alteration

A mosquito’s capacity to survive permethrin exposure is directly linked to its ability to generate supplemental energy. The findings showed increased levels of hexanoylcarnitine, propanoylcarnitine, butanoylcarnitine, and N-acetyl-l-carnitine exclusively in susceptible mosquitoes (compared with resistant and unexposed groups). This pattern suggests energy depletion and a subsequent alteration of the acylcarnitine shuttle, leading to the accumulation of these short-chain fatty acids in susceptible mosquitoes (Figure a–d and Figures S30–S33).

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Carnitine/acylcarnitine shuttle associated with An. gambiae permethrin-resistance phenotype. ANOVA, post hoc Fisher p value threshold = 0.05; statistical annotation: ns, nonsignificant. (a) Acetyl-l-carnitine: *p value = 0.0113, **p value = 0.0028, ***p value = 0.0001, ****p value < 0.0001. (b) Hexanoylcarnitine: **p = 0.0034, ***p = 0.0004, ****p value < 0.0001. (c) Butanoylcarnitine: **p = 0.0019, ****p value < 0.0001. (d) Propanoylcarnitine: ****p value < 0.0001.

Discussion

Permethrin metabolism is a known multistep process. Carboxylesterases and cytochromes sequentially act on permethrin, producing intermediates such as 3-phenoxybenzyl alcohol. Cytochrome orthologs then convert this alcohol to 3-phenoxybenzaldehyde followed by aldehyde dehydrogenase transformation into a nontoxic phenoxybenzoic acid. − These nontoxic metabolites are subsequently excreted from cells and tissues via efflux pumps or the mosquito’s Malpighian tubules. In this study, none of the multiple intermediates of permethrin have been identified as a significant metabolite in a resistant phenotype. This result may reflect the role of ABCG transporters predicted to act as efflux pumps that clear pyrethroids or toxic intermediates from cells or tissue in An. gambiae mosquitoes. Nevertheless, the presence of metabolite signals like cis-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (m/z 206.9926) and phenoxybenzoic acid (3-PBA) (m/z 213.0557) indicates that permethrin detoxification is likely mediated by carboxylesterases and monooxygenase cytochrome P450 enzymes.

High nicotinic acid levels in resistant mosquitoes, a precursor to NAD+ and NADP+, support the role of monooxygenase cytochrome P450 enzymes in permethrin detoxification, consistent with findings from Hannemann et al. on NAD­(P)­H-dependent ferredoxin and the fact that monooxygenases are not electron-transfer proteins. The increased nicotinamide levels observed by Brinzer et al. in Drosophila melanogaster exposed to permethrin suggest that this is a conserved metabolic response in Diptera. The elevated uracil levels detected in permethrin-resistant and unexposed mosquitoes, in contrast to susceptible groups, likely fuel RNA synthesis, a process that correlates with P450-mediated permethrin detoxification and could explain the necessity for high uracil levels to support the increased production of detoxifying enzymes. The accumulation of orotate, an intermediate of UMP synthesis, in susceptible mosquitoes following permethrin exposure suggests a potential disruption of the RNA synthesis. This observation aligns with previous research demonstrating permethrin’s impact on pyrimidine metabolism.

Inosine is an intermediate in purine metabolism. Increased inosine levels in resistant mosquitoes may suggest IMP metabolism disruption, xanthine metabolism inhibition, or post-transcriptional modification (A-to-I editing) of the pre-mRNA encoding the voltage-gated sodium channel. While two data sets dispute the genotype–phenotype link, our findings align with previous studies showing that A-to-I editing of para transcripts in An. gambiae, Aedes aegypti styl, and Drosophila affects invariant amino acids to varying degrees in mRNA, thereby modulating the sodium channel function. This A-to-I editing of kdr could explain resistance in mosquitoes and its connection to the genotype-resistance phenotype observed in Musca domestica, Blattella germanica, Culex quinquefasciatus, and Aedes albopictus. ,

A significant change in energy metabolism was observed in resistant, unexposed mosquitoes, evidenced by increased aldopentose levels compared with those of susceptible mosquitoes. This finding suggests the mobilization and utilization of stored glycogen, consistent with the depletion of glycogen energy reserves previously reported upon permethrin exposure.

Permethrin exposure to mosquitoes may disrupt the Krebs cycle, as the observed upregulation of fumarate, a key intermediate in both resistant and unexposed mosquitoes compared to susceptible ones, strongly suggests a perturbation in energy metabolism.

Multiple complex resistance mechanisms result in greater energy consumption and are upregulated when mosquitoes are exposed to permethrin. Stress-induced protein hydrolysis and release of free amino acids are one source of energy production through the tricarboxylic acid cycle in insects. ,− Previous studies using targeted and untargeted GC/MS metabolomic approaches have shown increased levels of free amino acids in An. gambiae mosquitoes and specific to pyrethroid insecticides. , Compared to unexposed and susceptible mosquitoes, resistant mosquitoes showed increased levels of dipeptides and free amino acids. These elevated amino acid levels may indicate upregulated pathways associated with insecticide resistance, as previously demonstrated by Forman et al. Altered amino acid profiles, including these changes, are established markers of resistance.

In mosquitoes,, the carnitine/acylcarnitine shuttle facilitates the transport of short-chain fatty acids from the cytoplasm into the mitochondrial matrix for β-oxidation and ATP production. , Similarly, acylcarnitine is a major intermediate in Aedes aegypti, mediating fatty acid-CoA transport for mitochondrial β-oxidation and ATP generation.

In permethrin-resistant An. gambiae, increased energy demands during detoxification may drive an elevated fatty acid influx via the carnitine/acylcarnitine shuttle. Notably, susceptible mosquitoes exhibited altered carnitine/acylcarnitine shuttle activity and elevated levels of hexanoylcarnitine, propanoylcarnitine, butanoylcarnitine, and N-acetylcarnitine compared to resistant and unexposed mosquitoes. The observed alterations in the carnitine/acylcarnitine shuttle point to potential disruptions in energy production and a possible role in modulating permethrin resistance in An. gambiae.

One of the key findings was the upregulation of XA and 3-hydroxykynurenine, a bypass product of tryptophan in susceptible mosquitoes compared to resistant and unexposed groups. Xanthurenic acid is also a byproduct of the ommochrome pathway involved in mosquito eye pigmentation and is associated as a signaling molecule to Plasmodium gametocytes to undergo gametogenesis inside the mosquito midgut. Brinzer et al. attested that permethrin susceptibility in Drosophila melanogaster is mediated by the production of XA. Furthermore, they found that the knockdown of the CG6950 gene, which is involved in tryptophan catabolism, increased the resistance to permethrin. Our findings confirm that the production of XA and a neurotoxic 3-hydroxykynurenine through tryptophan catabolism makes An. gambiae more sensitive to permethrin.

We found a high concentration of NAA, N-acetyl-l-glutamic acid, and N-acetyl-tyramine in susceptible mosquitoes. NAA is present in flies and plays a crucial role in cellular energy production, particularly during stress or nutrient deprivation, by influencing the overall brain energy state of the Drosophila nervous system. This finding suggests a potential role of NAA in disrupting the state of brain energy in An. gambiae when exposed to permethrin.

On the other hand, NAA is a precursor of N-acetyl-l-aspartylglutamate (NAAG), a prominent peptide neurotransmitter in the mammalian nervous system. While NAAG has been detected in the Drosophila nervous system, with concentrations depending on the fly’s condition, , its presence and function in the An. gambiae nervous system remain unknown. Although NAAG itself has not been detected in Drosophila, the related tripeptide NAAG2 has been identified using LC/MS by Kozik et al. Furthermore, enzymes capable of N-acetylation reactions are present in Drosophila. , This finding warrants further investigation into the potential role of NAA in modulating permethrin sensitivity in this key malaria vector.

Permethrin exposure disrupts mosquito nerve signaling, leading to paralysis and death. , However, it is unclear what other intrinsic and extrinsic factors, including stressors, can potentiate the effect of permethrin in mosquitoes. Increased levels of l-2-hydroxyglutarate and 2-methylglutarate in susceptible mosquitoes compared with those that survived exposure (resistant) and unexposed mosquitoes may be underappreciated factors that increase permethrin toxicity. A previous study showed that l-2-hydroxyglutarate accumulates under hypoxic stress and contributes to mitochondrial dysfunction and lethality in Drosophila melanogaster. While the D. melanogaster study suggests an association with hypoxic stress, under controlled O2 conditions, it is unclear if insecticide-induced chemical hypoxia or other environmental stressors in the field-collected mosquitoes are responsible for the observed differential responses in susceptible vs resistant phenotypes. Additional research exploring, more broadly, the inducers of l-2-hydroxyglutarate and 2-methylglutarate upregulation may provide further insight into the interactions among abiotic stressors with permethrin toxicity in An. gambiae.

L-PA is a cyclic amino acid derived from lysine catabolism and known to act as an osmo-protectant among bacteria and a key metabolite of immune defense in plants. , L-PA was upregulated in susceptible mosquitoes compared to resistant and unexposed ones. To the best of our knowledge, this is the first instance of L-PA being identified and associated with permethrin susceptibility in An. gambiae. Furthermore, the biochemical pathways allowing resistant mosquitoes to suppress L-PA production or the mechanisms by which L-PA increases insecticide toxicity are currently unknown. It has also been suggested that L-PA from the lysine catabolic derivative is an important metabolite that is used by bacteria for growth and adaptation to stress. Lower L-PA levels in the resistant and unexposed mosquitoes compared with the susceptible phenotype may reflect differences in lysine metabolism and usage of L-PA within the mosquitoes’ microbiome and strengthen the hypothesis that microbiome actively mediates in permethrin detoxification. These results highlight a new, bipartite metabolic axis with respect to An. gambiae permethrin resistance that warrants further investigation.

Mannitol is a six-carbon sugar produced mainly through microbial fermentation, including members of the mosquito microbiome. An adult mosquito’s susceptibility to insecticides is strongly shaped by the microbial community it encounters during its larval stage. This underscores a vital link between environmental factors and chemical resistance. Nkolondom is a complex ecological environment, and the widespread use of permethrin-based pesticides in agriculture has likely driven the microbiota evolution and selection of permethrin resistance in An. gambiae populations. Specifically, Djondji Kamga et al. showed that the microbiome contributes to pyrethroid resistance escalation in the main malaria vectors, An. gambiae collected from Nkolondom. Within field populations of An. gambiae, two bacterial strains, Pseudomonas and Bosea, were consistently more abundant in survivors’ permethrin 1X mosquitoes than in unexposed mosquitoes and in the laboratory-susceptible Kisumu strains. Moreover, Silva et al. reported a link between Pseudomonas fluorescens and increased pyrethroid resistance in An. gambiae. P. fluorescens produces mannitol-2-dehydrogenase, which converts mannitol into fructose, providing energy for permethrin detoxification. We therefore hypothesize that P. fluorescens or other similar bacteria species within the mosquito microbiota may enhance permethrin resistance in An. gambiae by converting stored mannitol into energy for detoxification. This microbial activity helps mosquitoes defend against permethrin more effectively, resulting in lower mannitol levels in resistant mosquitoes compared with susceptible phenotypes, which lack this metabolic pathway. Functional validation assays are needed to confirm the mannitol metabolic pathway implicated in permethrin toxicity.

Materials and Methods

Mosquito Sample Collections

The mosquito larvae were collected from breeding sites in Nkolondom (3°57′05″ N and 11°29′24″ E), a peri-urban area located 10 km from Yaoundé, Cameroon, where the intensive use of pesticides by farmers for their daily activity and strong pyrethroid resistance have been documented for An. gambiae. The collection was conducted in July 2023; the larvae were brought to the Centre of Research in Infectious Diseases (CRID), where they were reared to adult F0 mosquitoes. Species ID was confirmed by PCR using the PCR molecular identification described by Bass et al.

Insecticide Susceptibility Bioassay

Two to five day old female adult F0 mosquitoes were transferred to WHO bioassay test tubes and exposed to permethrin following an established method. Four groups of 25 mosquitoes were exposed to 0.75% of permethrin-impregnated papers for 30 min and 1 h. After the bioassays, the mosquitoes that were alive at 30 min and 1 h, those that were dead at 30 min and 1 h, and unexposed controls were preserved at −80 °C to avoid metabolome degradation and quench metabolic activity.

Metabolome Extraction

Metabolite extraction was performed using a method developed by Safari Yazd et al. Five mosquitoes from each group were individually homogenized using twenty 0.7 mm zirconia beads. Metabolites were then extracted from each sample using an ice-cold mixture of 800 μL ACN/MeOH/acetone (8:1:1, v/v/v), and the supernatant was removed, dried with nitrogen through the OA-HEAT heating system, and reconstituted in water containing 0.1% formic acid (FA).

UHPLC-HRMS/MS Conditions for Untargeted Metabolome

Untargeted metabolomic profiling was performed using a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer coupled to an UltiMate 3000 UHPLC system. Chromatographic separation was achieved on an Avantor ACE Excel C18-pfp (100 × 2.1 mm, 2 μm) column. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. Briefly, the conditions begin at 100% A for the first 3 min followed by a gradient up to 80% B at 13 min, where it was held for 3 min before returning to starting conditions. The flow rate was 0.350 mL/min, and the column temperature was 25 °C. The total run time per sample was 17 min. Heated electrospray ionization (HESI) with injection volumes of 4 and 2 μL was used, respectively, in both negative and positive ion modes. HESI parameters were as follows: a sheath gas flow rate of 55 arb, an auxiliary gas flow rate of 10 arb, a sweeping gas flow rate of 1 arb, and a capillary temperature of 325 °C. The spray voltage was 3.00 kV for the negative mode and 3.50 kV for the positive mode. The S-lens RF level was set to 30. Full scan data (m/z 70–1000) were acquired at a resolving power of 35,000. For tandem mass spectrometry (MS/MS) with biological samples, an isolation window of 2.0 m/z was used. The automatic gain control (AGC) target was 5 × 105, with a maximum injection time (IT) of 75 ms. The normalized collision energy (NCE) was 40, and data were acquired at a resolving power of 17,500. For analysis of chemical standards, the same parameters were used.

Data Processing and Statistical Analysis

Raw UHPLC-HRMS/MS data were converted to mzXML using Raw Converter and subsequently processed with MZmine 2.53. , Statistical analysis was performed using MetaboAnalyst 6.0 and GraphPad Prism 10. Data filtering was done with variance filter by standard deviation and abundance filter by mean intensity value with RDS greater than 25%. Normalization was grouped into three categories: normalization by sum, data log transformation (base 10), and data autoscaling. ANOVA was performed using post hoc Fisher’s tests with false discovery rate (FDR)-corrected p value threshold = 0.05.

Conclusions

Through comprehensive untargeted metabolomic profiling, we have characterized the metabolomic profile of permethrin resistance of field-derived An. gambiae from Cameroon. We observed metabolite signals consistent with P450 monooxygenase-mediated detoxification and target-site resistance mechanisms and identified metabolic shifts indicative of enhanced energy production in resistant mosquitoes. We further revealed that NAA may influence neuronal energy homeostasis, which may modulate the toxicity of permethrin. Both XA and 3-hydroxykynurenine, derived from tryptophan catabolism, as well as l-pipecolic acid, a catabolite of lysine, are associated with permethrin toxicity. In addition to the well-known permethrin resistance mechanisms based on enzymatic metabolic detoxification and target-site insensitivity, this work shows that permethrin resistance in An. gambiae results from a complex coordinated biological process, potentially influenced by the mosquito microbiome. This process is driven by a shift in energy production, inhibition of NAA synthesis, and suppression of tryptophan and lysine catabolism, creating a robust metabolic cross talk and defense against permethrin. These findings improve our understanding of the role of metabolism in An. gambiae permethrin resistance and compel further interrogation of the role of these pathways in permethrin cross-resistance.

Supplementary Material

tg6c00054_si_001.csv (15.7MB, csv)
tg6c00054_si_002.csv (17.2MB, csv)
tg6c00054_si_003.csv (285.8KB, csv)
tg6c00054_si_004.csv (302.4KB, csv)
tg6c00054_si_005.pdf (1.7MB, pdf)

Acknowledgments

This study was supported, in part, by U19AI181594 from the US National Institutes of Health to R.R.D., C.S.W., and T.J.G. and a Bill and Melinda Gates Foundation project INV-006003 grant to C.S.W. The table of contents image was generated in Biorender.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.6c00054.

  • Positive features An. gambie (CSV)

  • Negative features An. gambie (CSV)

  • ANOVA post hoc negative features An. gambie (CSV)

  • ANOVA post hoc positive features An. gambie (CSV)

  • Performance of sparse partial least-squares-discriminant (sPLS-DA) analysis; details of chromatograms, full-scan mass spectrum, and tandem mass spectrum of the identified features in positive or negative mode; summary of features detected in both positive and negative modes; and summary of the significant features in both positive and negative modes (PDF)

The authors declare no competing financial interest.

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Supplementary Materials

tg6c00054_si_001.csv (15.7MB, csv)
tg6c00054_si_002.csv (17.2MB, csv)
tg6c00054_si_003.csv (285.8KB, csv)
tg6c00054_si_004.csv (302.4KB, csv)
tg6c00054_si_005.pdf (1.7MB, pdf)

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