Investigating the potential role of metabolic resistance genes in conferring cross-resistance to pyrethroids and polycyclic aromatic hydrocarbon pollutants in the major malaria vector Anopheles coluzzii

Investigating the potential role of metabolic resistance genes in conferring cross-resistance to pyrethroids and polycyclic aromatic hydrocarbon pollutants in the major malaria vector Anopheles coluzzii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigating the potential role of metabolic resistance genes in conferring cross-resistance to pyrethroids and polycyclic aromatic hydrocarbon pollutants in the major malaria vector Anopheles coluzzii Abdullahi Muhammad, Sulaiman S. Ibrahim, Hanafy M. Ismail, Helen Irving, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6079555/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous and recalcitrant environmental pollutants generated from petroleum activities and/or biological conversion of organic materials. Environmental exposure of mosquito to these pollutants can potentially select resistance to insecticides used in public health for vector control. To understand the cross-resistance potentials between PAHs and pyrethroid insecticides, microsomal fractions prepared from Anopheles coluzzii mosquitoes obtained from agricultural sites and a laboratory susceptible strain, Ngousso, were tested with three major PAHs - fluorene, fluoranthene and naphthalene. Recombinant P450s previously associated with pyrethroids resistance in Anopheles gambiae ( CYPs 6M2, 6Z2, 6Z3, 9J5, 6P3, 6P4, 6P5 CYP9K1) and Anopheles funestus CYP6P9a were also used to investigate metabolism of the above PAHs alongside the microsome. Results: Microsomes prepared from pyrethroid resistant Anopheles coluzzii significantly (p = 0.001) depleted fluorene and fluoranthene with percentage depletions of 73%±0.5 and 43%.0±2.2, respectively. Steady state kinetic study demonstrated the microsome having a high affinity for the fluorene with a Km and turnover, respectively of 58.69µM±20.47 and 37.016 min- 1 ±3.67. On the other hand, significant metabolism of fluorene up to 47.9%±2.3 and 52.8%±0.8 depletions were observed with recombinant CYP6P3 and CYP6Z3, respectively. Other P450s showed little to no metabolism with fluorene. CYP6P3 and CYP6Z3 metabolised fluoranthene with percentage depletions of 50.4%±4.9 and 60.3% ±5.3, respectively. However, there was no observed metabolism of naphthalene with all the recombinant P450s used in this study. Conclusion : This study demonstrates that P450 monooxygenases from the malaria vectors can metabolise PAHs, highlighting the potential possibility of this environmental pollutants selecting the P450s, driving insecticide resistance in field populations of major malaria vectors. Cytochrome P450 microsome Anopheles coluzzii Polycyclic aromatic hydrocarbons cross-resistance insecticide malaria Figures Figure 1 Figure 2 Background Insecticide resistance is a strong threat to the gains made in malaria control using vector control tools ( 1 – 3 ). Insecticide-based interventions such as the insectcicide treated nets (ITNs) and indoor residual spraying (IRS) are being challenged by the evolution/escalation of resitance even to newer chemistries ( 4 ). Resistance to at least one insecticide has been reported in virtually all the WHO African regions with the strong indication of cross-resistance to other insecticides in most parts ( 2 , 5 , 6 ). When a mosquito population becomes resistant to insecticides it has not been exposed to using the same mechanism of another insecticide is termed as the cross-resistance ( 7 ). Cross-resistance is most pronounced through the metabolic resistance mechanisms due to the substrate specificity and/or promiscuity ( 8 ) of the enzymes involved in the sequestration, metabolism and excretion of insecticides leading to the metabolism of wide range of compounds ( 9 , 10 ). The key enzymes involved in insecticide resistance are the cytochrome P450 monooxygenases (P50s), glutathione S-transferases (GSTs) and carboxylesterases ( 11 ). On the other hand, prior exposure to environmental pollutants and/or agrichemicals has been shown to increase selection pressure on mosquito vectors, leading to elevated levels of metabolic resistance genes, which is linked to cross-resistance to vector control insecticides. Several studies have demonstrated how survival of mosquito in polluted breeding sites led to the increase in their resistance and increased urban malaria transmission ( 12 – 14 ). Members of Anopheles gambiae complex are most implicated in this adaptation to breeding in polluted waters ( 15 – 17 ). Hence, it is crucial to understand the molecular mechanisms through which environmental pollutants contribute to the selection of insecticide resistance in malaria vectors, thereby unravelling the cross-resistance potentials of individual compounds ( 18 – 21 ). Even though many studies above have documented the potential link between exposure to pollutants, including polyaromatic hydrocarbons (PAHs) and insecticide resistance, little is known of the underlying molecular mechanisms driving the cross resistance in Anopheles mosquitoes. Microsomes are cytosolic subcellular fractions generated from the ultracentrifugation of homogenised tissues contain the membrane bound enzymes, including the cytochrome P450 monooxygenases ( 22 ). Microsomal fractions from higher organisms has been proven very useful in the studies of drug and other compounds metabolisms, as well as toxicity studies in drug discovery and related disciplines ( 23 – 25 ). Thus, they can potentially be explored in the study of insecticide cross-resistance in malaria vectors ( 24 ). Microsomes isolated from field resistant populations of malaria vectors can be a useful tool in determining the potential cross-resistance of newer chemistries (e.g., synthetic insecticides) and other environmental pollutants, e.g., the PAHs ( 26 – 28 ). Several studies have implicated overexpression and overactivity of key Anopheles cytochrome P450 monooxygenases in insecticide resistance. Some of these P450s have been functionally validated, in vitro using heterologous expression in E. coli , coupled with metabolism assays ( 9 , 29 , 30 ) and in vivo , using transgenic expression in Drosophila flies ( 31 ). For example, in An. funestus for example, the duplicated CYP6P9a/b have been found to be the major drivers of pyrethroid resistance and demonstrated to metabolize and confer resistance to even non-pyrethroid insecticides ( 8 , 32 – 34 ). This is in addition to other P450s, such as CYP9K1 ( 35 ) which was shown to metabolize type II pyrethroid (deltamethrin) but with no affinity for type I (permethrin), the highly polymorphic CYP6M7 which was shown to metabolise pyrethroids ( 36 ) and CYP6AA1 which was shown to metabolise stablished as pyrethroid insecticides and bendiocarb ( 37 ). In An. gambiae , several P450s have also been implicated in the metabolism of pyrethroid insecticides, these include CYP6M2 , a strong metaboliser of pyrethroids including permethrin and deltamethrin ( 38 ), CYP6P3 , found to significantly metabolize types I and II pyrethroids ( 39 ), CYP6P5 located on the pyrethroid resistance locus with appreciable copy number variations ( 40 ) in the CYP6 cluster ( 41 ). Other important pyrethroid associated P450s include the CYP6Z3 ( 42 ), CYP6Z2 , found to have broad range of substrates specificity suggesting its potential roles in survival at larval stage (McLaughlin et al. , 2008). CYP9K1 ( 45 ), and CYP9J5 (Nkya et al. , 2014). To study the cross-resistance between PAHs and pyrethroids, the above P450s panels from An. gambiae ( CYPs 6Z2, 6Z3, 6P5, 9K1, 6M2, 9J5, 6P3 and 6P4 )) and An. funestus CYP6P9a were investigated for metabolic activities toward the PAHs and pyrethroid insecticides. Isolated microsomes prepared from field population as well as from the fully susceptible An. coluzzii lab colony, Ngousso were utilised in metabolism assays to investigate cross-resistance between pyrethroid insecticides and the PAHs. In addition, the above P450s were heterologously expressed in E. coli and utilised to investigate their potential to confer the cross resistance to these two unrelated groups of compounds. Methods Mosquito collection and rearing Blood fed, indoor resting female Anopheles mosquitoes were caught using electric aspirators (John. W. Hock, Florida, USA) in Auyo town ( 47 ) in September 2019. The F 0 female mosquitoes were morphologically identified (Gilles and Coetzee, 1987) as members of Anopheles gambiae complex and kept in standard insectary conditions of 25 o C and 75% relative humidity and 12:12h light:dark cycles, and maintained on 10% sucrose solution until they were gravid. Gravid females were individually forced to lay eggs ( 49 ) and the eggs were transported to Liverpool School of Tropical Medicine for the downstream analysis. Using the genomic DNA extracted from the F 0 individuals, species were confirmed using SINE 200 PCR ( 50 ). Eggs were pooled into trays and allowed to hatch. The laboratory susceptible Anopheles coluzzii colony, Ngousso ( 51 ), which is fully susceptible to all insecticides was used for comparison to the field populations. Microsome preparation The two strains of Anopheles coluzzii Auyo and Ngousso were reared in the insectary under standard insectary conditions to generate substantial amount of 4th instar larvae for the microsome preparations. Microsome preparation was conducted according the method reported earlier with some modifications ( 52 ). About 600 of 4th instar larvae were homogenised in 20 ml of ice-cold potassium phosphate buffer, pH 7.4 that has been supplemented with 1x protease inhibitor (Roche complete, ultra EDTA free, Sigma Aldrich, MA, USA). The larvae were homogenized in a 40 ml Dounce homogenizer equipped with a loose B pestle (Wheaton Science, Millville, NJ, USA). Filtration was conducted with layers of nylon filters to remove the residual debris. The filtrate was initially centrifuged at 10,000 xg for 10 min at 4 ˚C to separate the cytosolic fractions (supernatant) from other heavier cellular components. The supernatant was then centrifuged at 200,000 xg for 45 min at 4 ˚C to collect the pellet and discard the new supernatant. The pellet was reconstituted in 0.1M potassium phosphate buffer, pH 7.4, containing 20% glycerol. Total protein content of the microsomes were determined using the Bradford method ( 53 ). Cytochrome P450 content (Cary WinUV Software, Agilent Technologies) and microsomal P450 activity were also determined using spectral activity assay ( 54 , 55 ). Heterologous expression of recombinant P450s The recombinant P450s used in this study were acquired from the enzyme characterization group (ECG) lead, Dr. Mark J.I. Paine of the Vector Biology Department, Liverpool School of Tropical Medicine. All the P450s were expressed from field-resistant populations of An. gambiae except for CYP6P9a was expressed from An. funestus. The heterologous expression of the P450s was conducted according to the previously described approaches ( 29 , 38 , 44 ). Briefly, P450s were expressed by using ompA and pelB signal sequences to direct P450 and An. gambiae CPR (AgCPR) respectively to the inner membrane of E. coli a functional monooxygenase. The P450 sequences were amplified from cDNA and fused to the ompA signal peptides in simple PCR reactions. This is followed by ligation of the digested fragments into linearized expression vector pCWori + thereby creating a construct pB13:: ompA + 2 -P450. Competent JM109 cells were co-transformed with plasmids pB13:: ompA + 2 -P450 and pACYC-AgCPR for the expression of functional P450 and An. gambiae CPR respectively. Colonies carrying the two plasmids as confirmed by the colony PCR are used for a 12–14 h starter cultures overnight. Typically, 0.2 L cultures were supplemented with 2 mL from the starter cultures and incubated at 37 o C and 200 rpm shaking while the absorbance at 600 nm was being monitored. When the OD reached 0.6–0.8, the cultures were cooled down to 25 o C for 30 minutes with shaking at 150 rpm. Induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 0.5 mM 5-aminolaevulinic acid (ALA) was conducted. Determination of the P450 content and CPR activity were conducted using the procedure of ( 55 ) and (Guengerich 2009), respectively. Measurement of microsomal P450 activity using model probe substrate P450 activity was determined in the prepared microsomes from both Auyo and Ngousso strains using diethoxyfluorescein (DEF) as the model fluorogenic substrate using the methods earlier described ( 10 ). Briefly, the enzyme buffer mix consisted of 0.05 µM of cytochrome P450 (microsomes) 50mM potassium phosphate buffer, pH 7.4, 0.5 µM cytochrome b5 and 10 µM DEF. To initiate the reactions in a 96-well plates (Thermo Labsystems, Basingstoke, UK), an NADPH (nicotinamide adenine dinucleotide) regeneration mix (consisting of 1mM glucose-6-phosphate, 0.1M NADP+, 0.25mM magnesium chloride, IU/ml glucose-6-phosphate dehydrogenase and 50Mm potassium phosphate buffer pH 7.4) was added in the positive replicates whereas the same buffer with no NADPH was added in the negative wells. Using the excitation (482 nm) and emission (520 nm) wavelengths of DEF, the absorbance was read for 15 min and the relative fluorescence unit per min per picomole of the P450 (RFU/min/pmol of P450) was determined. In vitro metabolism assay of PAHs with heterologously expressed P450s. To compare the metabolic activity of the microsomes and the recombinant cytochrome P450s towards PAH metabolism assays were conducted side by side as described earlier ( 32 , 37 ). The NADPH regeneration mixtures (consisting of 0.1M NADP+, 50mM potassium phosphate buffer, pH 7.4, 1mM glucose-6-phosphate, 0.25mM magnesium chloride and 1U/ml glucose-6-phosphate dehydrogenase) was used in the positive replicates. For the negatives, the same buffer with no added NADP + was used. Enzyme-buffer mix comprised of 0.05 µM recombinant P450 or microsome, 0.4 µM cytochrome b5, 50mM potassium phosphate buffer pH 7.4 and 20 µM of the PAH substrates. To initiate the reactions, regeneration mixture is added to the enzyme-buffer mix in a 1:1 ratio (total of 200 µl) following activation for 5 min at 30 o C and 1200 rpm shaking. The reaction was run for 2 h at 30 o C with 200 rpm shaking. To stop the reaction, 200 µl of ice-cold HPLC grade acetonitrile was added followed by additional shaking for 5 min to precipitate the proteins. The mixture was kept on ice for 10 min prior to centrifuging at 16,000xg for 20 minutes. The supernatants were filtered through 0.45 µm PTFE filters (ThermoFisher Scientific, MA USA) and filtrate transferred to HPLC vials for onward analysis Using the Agilent HPLC 1200 infinity series, analysis of the PAHs metabolism was conducted. The HPLC conditions consisted of mobile phases of HPLC grade acetonitrile and water in the ratio of 80/20 and detected at a wavelength of 254 nm. 50 µl of the supernatant was injected on the HPLC and peaks were separated on a 250 × 4.6 mm (5 µm) Supelcosil LC-18-DB column (Supelco, Sigma-Aldrich, Gillingham, U.K.) in a 20 min run. Fluorene turnover assay using microsomes Because of the higher percentage of depletion observed in the Auyo microsomal metabolism of fluorene, it was chosen to be analysed further for its turnover and kinetic studies. To understand the turnover rates of the fluorene depletions, reaction run time was varied between 15 to 150 min. Specific points used were 15, 30, 45, 60, 90, 120 and 150 mins while the fluorene concentration was maintained at 30 µM in all these time points. Determination of steady-state kinetics parameters for the microsomal metabolism of fluorene For the determination of steady-state kinetic parameters, assays were conducted for 30 min with 0.05 µM microsomal P450 while varying the concentrations of fluorene (0-600 µM). Kinetics plot of velocity against the substrate concentrations was made using the least squares non-linear regression in GraphPad Prism 6.03 Software (GraphPad Inc. la Jolla CA, USA) that fits into the canonical Michaelis-Menten model as previously described for pyrethroid insecticides ( 37 ). Results P450 content and activity in microsomal fractions Microsomal preparations from both the field resistant strain (Auyo) and laboratory susceptible strain (Ngousso) of An. coluzzii produced P450 contents of 0.33 nmol P450/mg and 0.226 nmol P450/ mg m, respectively. The microsomes were screened for P450 activities using the model fluorogenic substrate diethoxyfluorescein (DEF). DEF activities of 4.9 RFU/min /pmole P450 and 4.4 RFU/min/ pmole P450 respectively (Fig. 1 A), were established for the Auyo and Ngousso microsomes, confirming the presence of functional cytochrome P450s. A total protein content of 2.0 mg/ml/100 larvae and 2.3 mg/ml/100 larvae were also determined for the Auyo and Ngousso microsomal preparations, respectively (Fig. 1 A) Metabolism of the PAHs by microsomal fractions Microsomal P450 demonstrated metabolic activity towards PAHs (fluorene, fluoranthene and naphthalene) with higher percentage depletions observed in the Auyo microsomes. For example, significant depletion of fluorine (73.3 ± 0.44%, P value = 0.0001) was seen with the Auyo microsome compared to the Ngousso microsome (Fig. 1 B). Similar profile was obtained with fluoranthene with 22.8 ± 5.2% significantly depleted by Auyo microsome (P value = 0.001), compared with the Ngousso microsome. Similar pattern was observed with naphthalene. Steady state kinetic parameters were investigated with fluorene, the most significantly depleted PAH by the Auyo microsomes. Turnover of 37.02 min − 1 ± 3.67 was recorded. The microsome demonstrated moderate affinity towards fluorene (Fig. 1 C), with Km value of 58.69 µΜ ± 20.47. Catalytic rate of (K cat ) of 4.196 min − 1 ± 0.436 with a corresponding high catalytic efficiency of 0.0715 ± 0.026 min − 1 µM − 1 (Fig. 1 D). The metabolic activity of recombinant pyrethroid associated P450s towards PAHs With the aim of understanding the potential cross-resistance liabilities between the recalcitrant environmental pollutant PAHs and pyrethroids, cytochrome P450s previously implicated in pyrethroid resistance/metabolism were recombinantly expressed and used for metabolism assays with PAHs. The pyrethroid metabolism associated P450s showed varied levels of metabolism towards the three select PAHs, naphthalene, fluorene and fluoranthene. For example, none of the P450s used in this study depleted up to 10% of naphthalene, suggesting no cross-resistance towards this PAH and pyrethroid insecticides through the P450 metabolism route (Fig. 1 D). On the other hand, fluoranthene was significantly depleted by the recombinant CYP6Z2 and CYP6Z3, with percentage depletions of 60% ± 4.9 and 50.4% ± 5.3 respectively. In the case of fluorene, highest depletions were seen with the recombinant CYP6Z3 (52.8 ± 0.8%) and CYP6P3 (47.9 ± 2.3%) (Fig. 1 B), with much lower depletions obtained from CYP9K1 (8.5% ± 2.5), CYP6P4 (9.3% ± 4.3), and CYP9J5 (7.7% ± 2.5). Discussion Chemical insecticides used in vector control tools and agrichemicals used in agriculture are considered the major drivers of insecticide resistance in addition to the environmental pollutants. ( 46 , 56 – 58 ). Microsomal fractions isolated from pyrethroid resistant and susceptible strains of An. coluzzii were used to study the cross-resistance between pyrethroids and PAHs as a class of ubiquitous environmental pollutant. Microsomes are the cytoplasmic fractions of the cells containing the membrane-bound enzymes including the cytochrome P450s and their usage in drug discovery and toxicity studies have been well documented ( 23 , 25 ) compared to their use in vector biology studies. They can thus be explored for the studies of insecticide resistance liabilities without the need for expressing individual P450s recombinantly. Successful isolation of microsomes from different insect species have been reported including Aedes aegypti mosquitoes ( 27 , 28 , 59 – 61 ). In the present studies, microsomes with substantial P450 activity and content were successfully isolated from the larvae of pyrethroid resistant and susceptible strains of An. coluzzii. The P450 yield and activity were higher (0.33 nmol P450/mg protein for the resistant and 0.226 nmol P450/mg for the susceptible) than those seen in a recent study ( 59 ) but lower than what was obtained from southern armyworm ( Spodeptera eridania) microsomal preparations ( 62 ). The microsomal fractions from pyrethroid resistant strains metabolised all the three PAHs significantly more than the pyrethroid susceptible ones (P values = 0.0001, 0.001 and 0.001 in the cases of fluorene, naphthalene and fluoranthene metabolism, respectively). Suggesting the cross-resistance potentials between the pyrethroids and the environmental pollutants. In a related study ( 28 ) on Aedes aegypti , the microsomes from pyrethroids resistant strains metabolised more permethrin than their susceptible counterpart. So basically, these findings are suggesting prior exposure to pyrethroids can lead to resistance to other pollutants/insecticides and vice-versa. The highest percentage depletions in both studies with microsomes and recombinant P450s was observed in the Auyo microsomal metabolism of fluorene (73.3 ± 0.4%) and was thus further characterized to understand the kinetics and turnover of this reaction. The Km (58.69 ± 20.47 µM) Kcat (4.20 ± 0.44 min − 1 ) were higher than those previously obtained with recombinant P450s metabolism of insecticides, this might probably be because microsomal fractions contain a mixture many P450s which may all have varied affinity towards the substrate thereby compounding the whole catalytic effects. Examples include the case of metabolism of ecdysone by microsomes of African migratory locust ( 63 ), metabolism of permethrin and deltamethrin by the duplicated An. funestus CYP6P9a/b ( 36 ) permethrin and deltamethrin metabolism by CYP6AA1 of An. funestus ( 37 ) and the deltamethrin metabolism by An. minimus CYP6AA3. Cytochrome P450 monooxygenases as the important Phase I enzymes ( 64 ) in the metabolism of xenobiotics have been implicated as the major players in conferring resistance to insecticides in disease vectors. A good number of them have been extensively characterized and found to confer resistance to certain insecticides through metabolism, sequestration and excretion of the soluble metabolites. Some of these notable ones associated with pyrethroids resistance in An. gambiae and An. funestus were deployed to study their potentials in metabolising PAHs thereby understanding their cross-resistance abilities. CYP6P3 is one of the most extensively characterized P450 in An. gambiae ( 65 , 66 ) and it is very promiscuous in its ability to metabolise a wide range of compounds including types I and II pyrethroids ( 39 ). In this study, it metabolised fluorene and fluoranthene with more than 40% depletions, suggesting its potential role in cross-resistance between PAHs and pyrethroids. The An. funestus CYP6P9a is an orthologue of the An. gambiae CYP6P3 and it is equally very promiscuous with catalytic activity towards wide range of substrates including pyrethroids and non-pyrethroids insecticides but for some reason did not metabolise the PAHs ( 31 , 32 , 34 ). Furthermore, despite the major role of CYP6M2 in pyrethroid resistance and its metabolism ( 38 ), there was no activity observed with all the three select PAHs. CYP6Z2 serves an important marker pyrethroid resistance even though it was found to not metabolise the parent pyrethroid insecticides ( 44 ) but able to metabolize the carboxylesterase metabolites of pyrethroid metabolism (3-phenoxybenzoic alcohol and 3-phenoxybenzaldehyde) ( 43 ), thus solidifying its role in pyrethroid resistance. Both CYP6Z2 and CYP6Z3 demonstrated strong metabolic activity towards fluorene and fluoranthene, indicating their potential role in the survival of An. gambiae in polluted breeding sites. Earlier studies ( 67 ) have suggested that the CYP6Z family of cytochrome P450s in An. gambiae are most notably expressed at the early life stages of larvae and pupae. This means they inherently help mosquito’s timely survival in polluted breeding sites due to their characteristic broad range substrate specificity capable of metabolizing wide range of compounds including plants’ secondary metabolites ( 68 ). This similar trend was also reported in Aedes aegypti mosquito’s CYP6Z8, an orthologue of the An. gambiae CYP6Z2 showing catalytic activity on broad range of substrates including α-naphthoflavone, resveratrol, and diethylstilbestrol ( 43 ). Conclusion Differential level of microsomal metabolism between pyrethroid resistant and susceptible strains also indicate the potential relationship between PAHs and pyrethroids. Cytochrome P450s associated with pyrethroids resistance were found to metabolise PAHs in metabolic assays confirming the cross-resistance potentials between these pollutants and insecticides of public health importance. These findings therefore stress the importance of cross-resistance studies especially for newer chemistries before they are rolled out in vector control tools. Declarations Ethics approval and consent to participate Not Applicable Consent for publication Not Applicable Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. Competing interests The authors declare that they have no competing interests Funding The work was supported from the petroleum technology development fund (PTDF) overseas scholarship awarded to AM. The funders had no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of this manuscript. Authors' contributions AM, CSW, SSI and HMI conceptualized the ideas while AM carried out the investigations in the laboratory. SSI and HI contributed in data analysis while MJIP and CSW provided the necessary supervision and data validations. AM wrote the first draft of the manuscript. All authors have read and approved the final version of the manuscript. Acknowledgements The authors would like to appreciate the contributions of the ECG group members at LSTM for their contributions in the provision of the P40 clones for expression References Ranson H, Lissenden N. Insecticide Resistance in African Anopheles Mosquitoes: A Worsening Situation that Needs Urgent Action to Maintain Malaria Control. Trends Parasitol [Internet]. 2016;32(3):187–96. Available from: http://dx.doi.org/10.1016/j.pt.2015.11.010 WHO. World Malaria Report 2021. World Malaria report Geneva: World Health Organization. (2021). Licence: CC. 2021. 2013–2015 p. 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Knights KM, Stresser DM, Miners JO, Crespi CL. In vitro drug metabolism using liver microsomes. Curr Protoc Pharmacol. 2016;2016(September):7.8.1–7.8.24. Zhang L, Kasai S, Shono T. In Vitro Metabolism of Pyriproxyfen by Microsomes from Susceptible and Resistant Housefly Larvae. Arch Insect Biochem Physiol. 1998;37(3):215–24. Suwanchaichinda C, Brattsten LB. Induction of microsomal cytochrome P450s by tire-leachate compounds, habitat components of Aedes albopictus mosquito larvae. Arch Insect Biochem Physiol. 2002;49(2):71–9. Kasai S, Komagata O, Itokawa K, Shono T, Ng LC, Kobayashi M et al. Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism. PLoS Negl Trop Dis. 2014;8(6). Pritchard MP, Ossetian R, Li DN, Henderson CJ, Burchell B, Wolf CR, et al. A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: Expression of CYP3A4, CYP2A6, and CYP2E1. Arch Biochem Biophys. 1997;345(2):342–54. Lees RS, Ismail HM, Logan RAE, Malone D, Davies R, Anthousi A et al. New insecticide screening platforms indicate that Mitochondrial Complex I inhibitors are susceptible to cross-resistance by mosquito P450s that metabolise pyrethroids. Sci Rep [Internet]. 2020;10(1):1–10. Available from: https://doi.org/10.1038/s41598-020-73267-x Riveron JM, Irving H, Ndula M, Barnes KG, Ibrahim SS, Paine MJI, et al. Directionally selected cytochrome P450 alleles are driving the spread of pyrethroid resistance in the major malaria vector Anopheles funestus. Proc Natl Acad Sci U S A. 2013;110(1):252–7. Ibrahim SS, Riveron JM, Bibby J, Irving H, Yunta C, Paine MJI, et al. Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector. PLoS Genet. 2015;11(10):1–25. Mugenzi LMJ, Tekoh A, Ibrahim TS, Muhammad S, Kouamo A, Wondji M et al. MJ,. The duplicated P450s CYP6P9a/b drive carbamates and pyrethroids cross-resistance in the major African malaria vector Anopheles funestus. PLoS Genet [Internet]. 2023;19(3):e1010678. Available from: http://dx.doi.org/10.1371/journal.pgen.1010678 Ibrahim SS, Kouamo MFM, Muhammad A, Irving H, Riveron JM, Tchouakui M et al. Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a / - b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus. Int J Mol Sci. 2024;25(8092). Hearn J, Djoko Tagne CS, Ibrahim SS, Tene-Fossog B, Mugenzi LMJ, Irving H, et al. Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa. Mol Ecol. 2022;31(13):3642–57. Riveron JM, Ibrahim SS, Chanda E, Mzilahowa T, Cuamba N, Irving H, et al. The highly polymorphic CYP6M7 cytochrome P450 gene partners with the directionally selected CYP6P9a and CYP6P9b genes to expand the pyrethroid resistance front in the malaria vector Anopheles funestus in Africa. BMC Genomics. 2014;15(1):1–19. Ibrahim SS, Amvongo-Adjia N, Wondji MJ, Irving H, Riveron JM, Wondji CS. Pyrethroid resistance in the major malaria vector anopheles funestus is exacerbated by overexpression and overactivity of the P450 CYP6AA1 across Africa. Genes (Basel). 2018;9(3):1–17. Stevenson BJ, Bibby J, Pignatelli P, Muangnoicharoen S, O’Neill PM, Lian LY et al. Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed. Insect Biochem Mol Biol [Internet]. 2011;41(7):492–502. Available from: http://dx.doi.org/10.1016/j.ibmb.2011.02.003 Muller P, Warr E, Stevenson BJ, Pignatelli PM, Morgan JC, Yawson AE et al. Field-Caught Permethrin-Resistant Anopheles gambiae Overexpress CYP6P3, a P450 That Metabolises Pyrethroids. PLoS Genet. 2008;4(11). Lucas ER, Nagi SC, Egyir-Yawson A, Essandoh J, Dadzie S, Chabi J, et al. Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae and Anopheles coluzzii. Nat Commun. 2023;14(1):1–23. Irving H, Riveron JM, Ibrahim SS, Lobo NF, Wondji CS. Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus. Heredity (Edinb) [Internet]. 2012;109(6):383–92. Available from: http://dx.doi.org/10.1038/hdy.2012.53 Moyes CL, Lees RS, Yunta C, Walker KJ, Hemmings K, Oladepo F et al. Assessing cross-resistance within the pyrethroids in terms of their interactions with key cytochrome P450 enzymes and resistance in vector populations. Parasites and Vectors [Internet]. 2021;14(1):1–13. Available from: https://doi.org/10.1186/s13071-021-04609-5 Chandor-Proust A, Bibby J, Régent-Kloeckner M, Roux J, Guittard-Crilat E, Poupardin R, et al. The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural mode. Biochem J. 2013;455(1):75–85. McLaughlin LA, Niazi U, Bibby J, David JP, Vontas J, Hemingway J, et al. Characterization of inhibitors and substrates of Anopheles gambiae CYP6Z2. Insect Mol Biol. 2008;17(2):125–35. Vontas J, Grigoraki L, Morgan J, Tsakireli D, Fuseini G, Segura L, et al. Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities. Proc Natl Acad Sci U S A. 2018;115(18):4619–24. Nkya TE, Akhouayri I, Poupardin R, Batengana B, Mosha F, Magesa S et al. Insecticide resistance mechanisms associated with different environments in the malaria vector Anopheles gambiae: A case study in Tanzania. Malar J. 2014;13(1). Ibrahim SS, Manu YA, Tukur Z, Irving H, Wondji CS. High frequency of kdr L1014F is associated with pyrethroid resistance in Anopheles coluzzii in Sudan savannah of northern Nigeria. BMC Infect Dis. 2014;14(1):1–9. Gilles M, Coetzee M. A supplement to anophelinae of Africa south of Sahara (Afro-tropical region). In Soth Africa; 1987. p. 55:1–143. Morgan JC, Irving H, Okedi LM, Steven A, Wondji CS. Pyrethroid resistance in an anopheles funestus population from uganda. PLoS ONE. 2010;5(7):1–8. Santolamazza F, Mancini E, Simard F, Qi Y, Tu Z, Della Torre A. Insertion polymorphisms of SINE200 retrotransposons within speciation islands of Anopheles gambiae molecular forms. Malar J. 2008;7:1–10. Harris C, Lambrechts L, Rousset F, Abate L, Nsango SE, Fontenille D et al. Polymorphisms in Anopheles gambiae immune genes associated with natural resistance to plasmodium falciparum. PLoS Pathog. 2010;6(9). Lee SS, Scott JG. An improved method for preparation, stabilization, and storage of house fly (Diptera: Muscidae) microsomes. J Econ Entomol. 1989;82(6):1559–63. Bradford MA, Rapid. and Snsitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem. 1976;(72):248–54. Guengerich FP, Martin MV, Sohl CD, Cheng Q. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nat Protoc. 2009;4(9):1245–51. Sato RYO, Omura T. The Carbon Monoxide-binding Pigment of Liver Microsomes. J Biol Chem [Internet]. 1964;239(7):2370–8. Available from: http://dx.doi.org/10.1016/S0021-9258(20)82244-3 Nkya TE, Akhouayri I, Kisinza W, David JP. Impact of environment on mosquito response to pyrethroid insecticides: Facts, evidences and prospects. Insect Biochem Mol Biol [Internet]. 2013;43(4):407–16. Available from: http://dx.doi.org/10.1016/j.ibmb.2012.10.006 Kamdem C, Fouet C, Gamez S, White BJ. Pollutants and Insecticides Drive Local Adaptation in African Malaria Mosquitoes. Mol Biol Evol. 2017;34(5):1261–75. Awolola TS, Oduola AO, Obansa JB, Chukwurar NJ, Unyimadu JP. Anopheles gambiae s.s. breeding in polluted water bodies in urban Lagos, southwestern Nigeria. J Vector Borne Dis. 2007;44(December):241–4. Sterkel M, Haines LR, Casas-Sánchez A, Adung’a VO, Vionette-Amaral RJ, Quek S et al. Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis. PLoS Biol. 2021;19(1). Wilkinson CF, Brattsten LB. Microsomal drug metabolizing enzymes in insects. Drug Metab Rev. 1972;1(1):153–227. Nolden M, Brockmann A, Ebbinghaus-Kintscher U, Brueggen K-U, Horstmann S, Paine MJI et al. Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification. Curr Res Parasitol Vector-Borne Dis [Internet]. 2021;1(June):100041. Available from: https://doi.org/10.1016/j.crpvbd.2021.100041 Brattsten LB, Gunderson CA. Isolation of insect microsomal oxidases by rapid centrifugation. Pestic Biochem Physiol. 1981;16(3):187–98. Feyreisen R, Durst F. Ecdysterone Biosynthesis: A Microsomal Cytochrome-P‐450‐Linked Ecdysone 20‐Monooxygenase from Tissues of the African Migratory Locust. Eur J Biochem. 1978;88(1):37–47. Iyanagi T. Molecular Mechanism of Phase I and Phase II Drug-Metabolizing Enzymes: Implications for Detoxification. Int Rev Cytol. 2007;260:35–112. Adolfi A, Poulton B, Anthousi A, Macilwee S, Ranson H, Lycett GJ. Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, Anopheles gambiae. Proc Natl Acad Sci U S A. 2019;116(51):25764–72. Edi CV, Djogbénou L, Jenkins AM, Regna K, Muskavitch MAT, Poupardin R et al. CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae. PLoS Genet. 2014;10(3). Nikou D, Ranson H, Hemingway J. An adult-specific CYP6 P450 gene is overexpressed in a pyrethroid- resistant strain of the malaria vector, Anopheles gambiae. Gene. 2003;318(1–2):91–102. Vontas J, Hemingway J, Ranson H, Sutcliffe MJ, Paine MJI. Characterization of inhibitors and substrates of Anopheles gambiae CYP6Z2. Insect Mol Biol. 2008;17(February):125–35. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6079555","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":421885397,"identity":"c8f0c965-9915-4b87-9279-824dc688bc40","order_by":0,"name":"Abdullahi Muhammad","email":"data:image/png;base64,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","orcid":"","institution":"Vector Biology Department, Liverpool School of Tropical Medicine","correspondingAuthor":true,"prefix":"","firstName":"Abdullahi","middleName":"","lastName":"Muhammad","suffix":""},{"id":421885398,"identity":"b194a9b8-687f-4f01-8f2b-b9a4d7388583","order_by":1,"name":"Sulaiman S. Ibrahim","email":"","orcid":"","institution":"Department of Biochemistry, Bayero University, PMB 3011, Kano Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Sulaiman","middleName":"S.","lastName":"Ibrahim","suffix":""},{"id":421885399,"identity":"baf4f16d-8f14-44b5-806e-a8e2929568e0","order_by":2,"name":"Hanafy M. Ismail","email":"","orcid":"","institution":"Vector Biology Department, Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hanafy","middleName":"M.","lastName":"Ismail","suffix":""},{"id":421885400,"identity":"219da6f5-da05-475c-99fc-150c513501bd","order_by":3,"name":"Helen Irving","email":"","orcid":"","institution":"Vector Biology Department, Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Helen","middleName":"","lastName":"Irving","suffix":""},{"id":421885401,"identity":"be0a409a-7252-47e8-b1bf-e464c2f06d5e","order_by":4,"name":"Mark J.I. Paine","email":"","orcid":"","institution":"Vector Biology Department, Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"J.I.","lastName":"Paine","suffix":""},{"id":421885402,"identity":"0f8704de-abe0-45f9-967b-85aba18a8489","order_by":5,"name":"Charles S. Wondji","email":"","orcid":"","institution":"Vector Biology Department, Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"S.","lastName":"Wondji","suffix":""}],"badges":[],"createdAt":"2025-02-21 12:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6079555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6079555/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77940129,"identity":"7bf9a444-9086-402d-824a-c39005349303","added_by":"auto","created_at":"2025-03-07 05:10:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47905,"visible":true,"origin":"","legend":"\u003cp\u003eCatalytic activity of the Auyo and Ngousso microsomes towards PAHs and the enzyme kinetics studies of the Auyo microsomes with fluorene as substrate. A) Def activity and the protein content of the isolated microsomes. B) Percentage depletions of the metabolism of the select PAHs by the microsomes isolated from the pyrethroid resistant and susceptible strains of \u003cem\u003eAnopheles coluzzii.\u003c/em\u003e Results are presented as mean±SD of three replicates of the positives (+NADPH) compared to the negatives (-NADPH). *** Significant difference between the Ngousso and Auyo percentage depletions for each substrate. C) Time course of fluorene turnover, substrate concentration 20 µM. C) Michaelis-Menten plot of fluorene metabolism by microsomes isolated from the field resistant strain (Auyo).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6079555/v1/618bf37adfcdcc290b877690.png"},{"id":77940130,"identity":"1f197dfb-aec7-424a-93d5-53ab0447cdcc","added_by":"auto","created_at":"2025-03-07 05:10:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59909,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolism of PAHs by pyrethroid associated recombinant cytochrome P450 monooxygenases. Details of the P450 membranes used in the study including the P450 content and CPR activity (\u003cstrong\u003eA).\u003c/strong\u003ePercentage depletions of fluorene (B), fluoranthene (C) and naphthalene (D) for the various recombinant cytochrome P450s. Values are presented as mean±S.D. of three technical replicates in each case.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6079555/v1/957f82a5b4fd2427760158f3.png"},{"id":77941141,"identity":"2931b881-b8ea-45ab-b9bf-7b8f7ca72af5","added_by":"auto","created_at":"2025-03-07 05:34:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":909589,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6079555/v1/16b2a8af-1d7a-417c-9a9e-e0e46ab38841.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating the potential role of metabolic resistance genes in conferring cross-resistance to pyrethroids and polycyclic aromatic hydrocarbon pollutants in the major malaria vector Anopheles coluzzii","fulltext":[{"header":"Background","content":"\u003cp\u003eInsecticide resistance is a strong threat to the gains made in malaria control using vector control tools (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Insecticide-based interventions such as the insectcicide treated nets (ITNs) and indoor residual spraying (IRS) are being challenged by the evolution/escalation of resitance even to newer chemistries (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Resistance to at least one insecticide has been reported in virtually all the WHO African regions with the strong indication of cross-resistance to other insecticides in most parts (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). When a mosquito population becomes resistant to insecticides it has not been exposed to using the same mechanism of another insecticide is termed as the cross-resistance (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Cross-resistance is most pronounced through the metabolic resistance mechanisms due to the substrate specificity and/or promiscuity (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) of the enzymes involved in the sequestration, metabolism and excretion of insecticides leading to the metabolism of wide range of compounds (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The key enzymes involved in insecticide resistance are the cytochrome P450 monooxygenases (P50s), glutathione S-transferases (GSTs) and carboxylesterases (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, prior exposure to environmental pollutants and/or agrichemicals has been shown to increase selection pressure on mosquito vectors, leading to elevated levels of metabolic resistance genes, which is linked to cross-resistance to vector control insecticides. Several studies have demonstrated how survival of mosquito in polluted breeding sites led to the increase in their resistance and increased urban malaria transmission (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Members of \u003cem\u003eAnopheles gambiae\u003c/em\u003e complex are most implicated in this adaptation to breeding in polluted waters (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Hence, it is crucial to understand the molecular mechanisms through which environmental pollutants contribute to the selection of insecticide resistance in malaria vectors, thereby unravelling the cross-resistance potentials of individual compounds (\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Even though many studies above have documented the potential link between exposure to pollutants, including polyaromatic hydrocarbons (PAHs) and insecticide resistance, little is known of the underlying molecular mechanisms driving the cross resistance in \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes.\u003c/p\u003e \u003cp\u003eMicrosomes are cytosolic subcellular fractions generated from the ultracentrifugation of homogenised tissues contain the membrane bound enzymes, including the cytochrome P450 monooxygenases (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Microsomal fractions from higher organisms has been proven very useful in the studies of drug and other compounds metabolisms, as well as toxicity studies in drug discovery and related disciplines (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Thus, they can potentially be explored in the study of insecticide cross-resistance in malaria vectors (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Microsomes isolated from field resistant populations of malaria vectors can be a useful tool in determining the potential cross-resistance of newer chemistries (e.g., synthetic insecticides) and other environmental pollutants, e.g., the PAHs (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have implicated overexpression and overactivity of key Anopheles cytochrome P450 monooxygenases in insecticide resistance. Some of these P450s have been functionally validated, \u003cem\u003ein vitro\u003c/em\u003e using heterologous expression in \u003cem\u003eE. coli\u003c/em\u003e, coupled with metabolism assays (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) and \u003cem\u003ein vivo\u003c/em\u003e, using transgenic expression in Drosophila flies (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). For example, in \u003cem\u003eAn. funestus\u003c/em\u003e for example, the duplicated \u003cem\u003eCYP6P9a/b\u003c/em\u003e have been found to be the major drivers of pyrethroid resistance and demonstrated to metabolize and confer resistance to even non-pyrethroid insecticides (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). This is in addition to other P450s, such as \u003cem\u003eCYP9K1\u003c/em\u003e (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) which was shown to metabolize type II pyrethroid (deltamethrin) but with no affinity for type I (permethrin), the highly polymorphic \u003cem\u003eCYP6M7\u003c/em\u003e which was shown to metabolise pyrethroids (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and \u003cem\u003eCYP6AA1\u003c/em\u003e which was shown to metabolise stablished as pyrethroid insecticides and bendiocarb (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eAn. gambiae\u003c/em\u003e, several P450s have also been implicated in the metabolism of pyrethroid insecticides, these include \u003cem\u003eCYP6M2\u003c/em\u003e, a strong metaboliser of pyrethroids including permethrin and deltamethrin (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), \u003cem\u003eCYP6P3\u003c/em\u003e, found to significantly metabolize types I and II pyrethroids (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), \u003cem\u003eCYP6P5\u003c/em\u003e located on the pyrethroid resistance locus with appreciable copy number variations (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) in the CYP6 cluster (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Other important pyrethroid associated P450s include the \u003cem\u003eCYP6Z3\u003c/em\u003e (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), \u003cem\u003eCYP6Z2\u003c/em\u003e, found to have broad range of substrates specificity suggesting its potential roles in survival at larval stage (McLaughlin \u003cem\u003eet al.\u003c/em\u003e, 2008). \u003cem\u003eCYP9K1\u003c/em\u003e (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), and \u003cem\u003eCYP9J5\u003c/em\u003e (Nkya \u003cem\u003eet al.\u003c/em\u003e, 2014).\u003c/p\u003e \u003cp\u003eTo study the cross-resistance between PAHs and pyrethroids, the above P450s panels from \u003cem\u003eAn. gambiae\u003c/em\u003e (\u003cem\u003eCYPs 6Z2, 6Z3, 6P5, 9K1, 6M2, 9J5, 6P3 and 6P4\u003c/em\u003e)) and \u003cem\u003eAn. funestus CYP6P9a\u003c/em\u003e were investigated for metabolic activities toward the PAHs and pyrethroid insecticides.\u003c/p\u003e \u003cp\u003eIsolated microsomes prepared from field population as well as from the fully susceptible \u003cem\u003eAn. coluzzii\u003c/em\u003e lab colony, Ngousso were utilised in metabolism assays to investigate cross-resistance between pyrethroid insecticides and the PAHs. In addition, the above P450s were heterologously expressed in \u003cem\u003eE. coli\u003c/em\u003e and utilised to investigate their potential to confer the cross resistance to these two unrelated groups of compounds.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMosquito collection and rearing\u003c/h2\u003e \u003cp\u003eBlood fed, indoor resting female Anopheles mosquitoes were caught using electric aspirators (John. W. Hock, Florida, USA) in Auyo town (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) in September 2019. The F\u003csub\u003e0\u003c/sub\u003e female mosquitoes were morphologically identified (Gilles and Coetzee, 1987) as members of \u003cem\u003eAnopheles gambiae\u003c/em\u003e complex and kept in standard insectary conditions of 25\u003csup\u003eo\u003c/sup\u003eC and 75% relative humidity and 12:12h light:dark cycles, and maintained on 10% sucrose solution until they were gravid. Gravid females were individually forced to lay eggs (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) and the eggs were transported to Liverpool School of Tropical Medicine for the downstream analysis. Using the genomic DNA extracted from the F\u003csub\u003e0\u003c/sub\u003e individuals, species were confirmed using SINE 200 PCR (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Eggs were pooled into trays and allowed to hatch. The laboratory susceptible \u003cem\u003eAnopheles coluzzii\u003c/em\u003e colony, Ngousso (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), which is fully susceptible to all insecticides was used for comparison to the field populations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicrosome preparation\u003c/h3\u003e\n\u003cp\u003eThe two strains of \u003cem\u003eAnopheles coluzzii\u003c/em\u003e Auyo and Ngousso were reared in the insectary under standard insectary conditions to generate substantial amount of 4th instar larvae for the microsome preparations. Microsome preparation was conducted according the method reported earlier with some modifications (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). About 600 of 4th instar larvae were homogenised in 20 ml of ice-cold potassium phosphate buffer, pH 7.4 that has been supplemented with 1x protease inhibitor (Roche complete, ultra EDTA free, Sigma Aldrich, MA, USA). The larvae were homogenized in a 40 ml Dounce homogenizer equipped with a loose B pestle (Wheaton Science, Millville, NJ, USA). Filtration was conducted with layers of nylon filters to remove the residual debris. The filtrate was initially centrifuged at 10,000 xg for 10 min at 4 ˚C to separate the cytosolic fractions (supernatant) from other heavier cellular components. The supernatant was then centrifuged at 200,000 xg for 45 min at 4 ˚C to collect the pellet and discard the new supernatant. The pellet was reconstituted in 0.1M potassium phosphate buffer, pH 7.4, containing 20% glycerol. Total protein content of the microsomes were determined using the Bradford method (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Cytochrome P450 content (Cary WinUV Software, Agilent Technologies) and microsomal P450 activity were also determined using spectral activity assay (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eHeterologous expression of recombinant P450s\u003c/h3\u003e\n\u003cp\u003eThe recombinant P450s used in this study were acquired from the enzyme characterization group (ECG) lead, Dr. Mark J.I. Paine of the Vector Biology Department, Liverpool School of Tropical Medicine. All the P450s were expressed from field-resistant populations of \u003cem\u003eAn. gambiae\u003c/em\u003e except for CYP6P9a was expressed from \u003cem\u003eAn. funestus.\u003c/em\u003e The heterologous expression of the P450s was conducted according to the previously described approaches (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Briefly, P450s were expressed by using \u003cem\u003eompA\u003c/em\u003e and \u003cem\u003epelB\u003c/em\u003e signal sequences to direct P450 and \u003cem\u003eAn. gambiae\u003c/em\u003e CPR (AgCPR) respectively to the inner membrane of \u003cem\u003eE. coli\u003c/em\u003e a functional monooxygenase. The P450 sequences were amplified from cDNA and fused to the ompA signal peptides in simple PCR reactions. This is followed by ligation of the digested fragments into linearized expression vector pCWori\u0026thinsp;+\u0026thinsp;thereby creating a construct pB13::\u003cem\u003eompA\u0026thinsp;+\u0026thinsp;2\u003c/em\u003e-P450. Competent JM109 cells were co-transformed with plasmids pB13::\u003cem\u003eompA\u0026thinsp;+\u0026thinsp;2\u003c/em\u003e-P450 and pACYC-AgCPR for the expression of functional P450 and \u003cem\u003eAn. gambiae\u003c/em\u003e CPR respectively. Colonies carrying the two plasmids as confirmed by the colony PCR are used for a 12\u0026ndash;14 h starter cultures overnight. Typically, 0.2 L cultures were supplemented with 2 mL from the starter cultures and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC and 200 rpm shaking while the absorbance at 600 nm was being monitored. When the OD reached 0.6\u0026ndash;0.8, the cultures were cooled down to 25 \u003csup\u003eo\u003c/sup\u003eC for 30 minutes with shaking at 150 rpm. Induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 0.5 mM 5-aminolaevulinic acid (ALA) was conducted. Determination of the P450 content and CPR activity were conducted using the procedure of (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) and (Guengerich 2009), respectively.\u003c/p\u003e\n\u003ch3\u003eMeasurement of microsomal P450 activity using model probe substrate\u003c/h3\u003e\n\u003cp\u003eP450 activity was determined in the prepared microsomes from both Auyo and Ngousso strains using diethoxyfluorescein (DEF) as the model fluorogenic substrate using the methods earlier described (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Briefly, the enzyme buffer mix consisted of 0.05 \u0026micro;M of cytochrome P450 (microsomes) 50mM potassium phosphate buffer, pH 7.4, 0.5 \u0026micro;M cytochrome b5 and 10 \u0026micro;M DEF. To initiate the reactions in a 96-well plates (Thermo Labsystems, Basingstoke, UK), an NADPH (nicotinamide adenine dinucleotide) regeneration mix (consisting of 1mM glucose-6-phosphate, 0.1M NADP+, 0.25mM magnesium chloride, IU/ml glucose-6-phosphate dehydrogenase and 50Mm potassium phosphate buffer pH 7.4) was added in the positive replicates whereas the same buffer with no NADPH was added in the negative wells. Using the excitation (482 nm) and emission (520 nm) wavelengths of DEF, the absorbance was read for 15 min and the relative fluorescence unit per min per picomole of the P450 (RFU/min/pmol of P450) was determined.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003emetabolism assay of PAHs with heterologously expressed P450s.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo compare the metabolic activity of the microsomes and the recombinant cytochrome P450s towards PAH metabolism assays were conducted side by side as described earlier (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The NADPH regeneration mixtures (consisting of 0.1M NADP+, 50mM potassium phosphate buffer, pH 7.4, 1mM glucose-6-phosphate, 0.25mM magnesium chloride and 1U/ml glucose-6-phosphate dehydrogenase) was used in the positive replicates. For the negatives, the same buffer with no added NADP\u0026thinsp;+\u0026thinsp;was used. Enzyme-buffer mix comprised of 0.05 \u0026micro;M recombinant P450 or microsome, 0.4 \u0026micro;M cytochrome b5, 50mM potassium phosphate buffer pH 7.4 and 20 \u0026micro;M of the PAH substrates. To initiate the reactions, regeneration mixture is added to the enzyme-buffer mix in a 1:1 ratio (total of 200 \u0026micro;l) following activation for 5 min at 30 \u003csup\u003eo\u003c/sup\u003eC and 1200 rpm shaking. The reaction was run for 2 h at 30 \u003csup\u003eo\u003c/sup\u003eC with 200 rpm shaking. To stop the reaction, 200 \u0026micro;l of ice-cold HPLC grade acetonitrile was added followed by additional shaking for 5 min to precipitate the proteins. The mixture was kept on ice for 10 min prior to centrifuging at 16,000xg for 20 minutes. The supernatants were filtered through 0.45 \u0026micro;m PTFE filters (ThermoFisher Scientific, MA USA) and filtrate transferred to HPLC vials for onward analysis\u003c/p\u003e \u003cp\u003eUsing the Agilent HPLC 1200 infinity series, analysis of the PAHs metabolism was conducted. The HPLC conditions consisted of mobile phases of HPLC grade acetonitrile and water in the ratio of 80/20 and detected at a wavelength of 254 nm. 50 \u0026micro;l of the supernatant was injected on the HPLC and peaks were separated on a 250 \u0026times; 4.6 mm (5 \u0026micro;m) Supelcosil LC-18-DB column (Supelco, Sigma-Aldrich, Gillingham, U.K.) in a 20 min run.\u003c/p\u003e\n\u003ch3\u003eFluorene turnover assay using microsomes\u003c/h3\u003e\n\u003cp\u003eBecause of the higher percentage of depletion observed in the Auyo microsomal metabolism of fluorene, it was chosen to be analysed further for its turnover and kinetic studies. To understand the turnover rates of the fluorene depletions, reaction run time was varied between 15 to 150 min. Specific points used were 15, 30, 45, 60, 90, 120 and 150 mins while the fluorene concentration was maintained at 30 \u0026micro;M in all these time points.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of steady-state kinetics parameters for the microsomal metabolism of fluorene\u003c/h2\u003e \u003cp\u003eFor the determination of steady-state kinetic parameters, assays were conducted for 30 min with 0.05 \u0026micro;M microsomal P450 while varying the concentrations of fluorene (0-600 \u0026micro;M). Kinetics plot of velocity against the substrate concentrations was made using the least squares non-linear regression in GraphPad Prism 6.03 Software (GraphPad Inc. la Jolla CA, USA) that fits into the canonical Michaelis-Menten model as previously described for pyrethroid insecticides (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eP450 content and activity in microsomal fractions\u003c/h2\u003e \u003cp\u003eMicrosomal preparations from both the field resistant strain (Auyo) and laboratory susceptible strain (Ngousso) of \u003cem\u003eAn. coluzzii\u003c/em\u003e produced P450 contents of 0.33 nmol P450/mg and 0.226 nmol P450/ mg m, respectively. The microsomes were screened for P450 activities using the model fluorogenic substrate diethoxyfluorescein (DEF). DEF activities of 4.9 RFU/min /pmole P450 and 4.4 RFU/min/ pmole P450 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), were established for the Auyo and Ngousso microsomes, confirming the presence of functional cytochrome P450s. A total protein content of 2.0 mg/ml/100 larvae and 2.3 mg/ml/100 larvae were also determined for the Auyo and Ngousso microsomal preparations, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMetabolism of the PAHs by microsomal fractions\u003c/h2\u003e \u003cp\u003eMicrosomal P450 demonstrated metabolic activity towards PAHs (fluorene, fluoranthene and naphthalene) with higher percentage depletions observed in the Auyo microsomes. For example, significant depletion of fluorine (73.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44%, P value\u0026thinsp;=\u0026thinsp;0.0001) was seen with the Auyo microsome compared to the Ngousso microsome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similar profile was obtained with fluoranthene with 22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2% significantly depleted by Auyo microsome (P value\u0026thinsp;=\u0026thinsp;0.001), compared with the Ngousso microsome. Similar pattern was observed with naphthalene.\u003c/p\u003e \u003cp\u003eSteady state kinetic parameters were investigated with fluorene, the most significantly depleted PAH by the Auyo microsomes. Turnover of 37.02 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026plusmn; 3.67 was recorded. The microsome demonstrated moderate affinity towards fluorene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), with Km value of 58.69 \u0026micro;Μ\u0026thinsp;\u0026plusmn;\u0026thinsp;20.47. Catalytic rate of (K\u003csub\u003ecat\u003c/sub\u003e) of 4.196 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026plusmn; 0.436 with a corresponding high catalytic efficiency of 0.0715\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026micro;M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe metabolic activity of recombinant pyrethroid associated P450s towards PAHs\u003c/h2\u003e \u003cp\u003eWith the aim of understanding the potential cross-resistance liabilities between the recalcitrant environmental pollutant PAHs and pyrethroids, cytochrome P450s previously implicated in pyrethroid resistance/metabolism were recombinantly expressed and used for metabolism assays with PAHs. The pyrethroid metabolism associated P450s showed varied levels of metabolism towards the three select PAHs, naphthalene, fluorene and fluoranthene. For example, none of the P450s used in this study depleted up to 10% of naphthalene, suggesting no cross-resistance towards this PAH and pyrethroid insecticides through the P450 metabolism route (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). On the other hand, fluoranthene was significantly depleted by the recombinant CYP6Z2 and CYP6Z3, with percentage depletions of 60% \u0026plusmn; 4.9 and 50.4% \u0026plusmn; 5.3 respectively. In the case of fluorene, highest depletions were seen with the recombinant CYP6Z3 (52.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%) and CYP6P3 (47.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), with much lower depletions obtained from CYP9K1 (8.5% \u0026plusmn; 2.5), CYP6P4 (9.3% \u0026plusmn; 4.3), and CYP9J5 (7.7% \u0026plusmn; 2.5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChemical insecticides used in vector control tools and agrichemicals used in agriculture are considered the major drivers of insecticide resistance in addition to the environmental pollutants. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Microsomal fractions isolated from pyrethroid resistant and susceptible strains of \u003cem\u003eAn. coluzzii\u003c/em\u003e were used to study the cross-resistance between pyrethroids and PAHs as a class of ubiquitous environmental pollutant. Microsomes are the cytoplasmic fractions of the cells containing the membrane-bound enzymes including the cytochrome P450s and their usage in drug discovery and toxicity studies have been well documented (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) compared to their use in vector biology studies. They can thus be explored for the studies of insecticide resistance liabilities without the need for expressing individual P450s recombinantly. Successful isolation of microsomes from different insect species have been reported including \u003cem\u003eAedes aegypti\u003c/em\u003e mosquitoes (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). In the present studies, microsomes with substantial P450 activity and content were successfully isolated from the larvae of pyrethroid resistant and susceptible strains of \u003cem\u003eAn. coluzzii.\u003c/em\u003e The P450 yield and activity were higher (0.33 nmol P450/mg protein for the resistant and 0.226 nmol P450/mg for the susceptible) than those seen in a recent study (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) but lower than what was obtained from southern armyworm (\u003cem\u003eSpodeptera eridania)\u003c/em\u003e microsomal preparations (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe microsomal fractions from pyrethroid resistant strains metabolised all the three PAHs significantly more than the pyrethroid susceptible ones (P values\u0026thinsp;=\u0026thinsp;0.0001, 0.001 and 0.001 in the cases of fluorene, naphthalene and fluoranthene metabolism, respectively). Suggesting the cross-resistance potentials between the pyrethroids and the environmental pollutants. In a related study (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) on \u003cem\u003eAedes aegypti\u003c/em\u003e, the microsomes from pyrethroids resistant strains metabolised more permethrin than their susceptible counterpart. So basically, these findings are suggesting prior exposure to pyrethroids can lead to resistance to other pollutants/insecticides and vice-versa.\u003c/p\u003e \u003cp\u003eThe highest percentage depletions in both studies with microsomes and recombinant P450s was observed in the Auyo microsomal metabolism of fluorene (73.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%) and was thus further characterized to understand the kinetics and turnover of this reaction. The Km (58.69\u0026thinsp;\u0026plusmn;\u0026thinsp;20.47 \u0026micro;M) Kcat (4.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were higher than those previously obtained with recombinant P450s metabolism of insecticides, this might probably be because microsomal fractions contain a mixture many P450s which may all have varied affinity towards the substrate thereby compounding the whole catalytic effects. Examples include the case of metabolism of ecdysone by microsomes of African migratory locust (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e), metabolism of permethrin and deltamethrin by the duplicated \u003cem\u003eAn. funestus\u003c/em\u003e CYP6P9a/b (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) permethrin and deltamethrin metabolism by CYP6AA1 of \u003cem\u003eAn. funestus\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and the deltamethrin metabolism by \u003cem\u003eAn. minimus\u003c/em\u003e CYP6AA3.\u003c/p\u003e \u003cp\u003eCytochrome P450 monooxygenases as the important Phase I enzymes (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e) in the metabolism of xenobiotics have been implicated as the major players in conferring resistance to insecticides in disease vectors. A good number of them have been extensively characterized and found to confer resistance to certain insecticides through metabolism, sequestration and excretion of the soluble metabolites. Some of these notable ones associated with pyrethroids resistance in \u003cem\u003eAn. gambiae\u003c/em\u003e and \u003cem\u003eAn. funestus\u003c/em\u003e were deployed to study their potentials in metabolising PAHs thereby understanding their cross-resistance abilities. CYP6P3 is one of the most extensively characterized P450 in \u003cem\u003eAn. gambiae\u003c/em\u003e (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) and it is very promiscuous in its ability to metabolise a wide range of compounds including types I and II pyrethroids (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In this study, it metabolised fluorene and fluoranthene with more than 40% depletions, suggesting its potential role in cross-resistance between PAHs and pyrethroids. The \u003cem\u003eAn. funestus\u003c/em\u003e CYP6P9a is an orthologue of the \u003cem\u003eAn. gambiae\u003c/em\u003e CYP6P3 and it is equally very promiscuous with catalytic activity towards wide range of substrates including pyrethroids and non-pyrethroids insecticides but for some reason did not metabolise the PAHs (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Furthermore, despite the major role of CYP6M2 in pyrethroid resistance and its metabolism (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), there was no activity observed with all the three select PAHs.\u003c/p\u003e \u003cp\u003eCYP6Z2 serves an important marker pyrethroid resistance even though it was found to not metabolise the parent pyrethroid insecticides (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) but able to metabolize the carboxylesterase metabolites of pyrethroid metabolism (3-phenoxybenzoic alcohol and 3-phenoxybenzaldehyde) (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), thus solidifying its role in pyrethroid resistance. Both CYP6Z2 and CYP6Z3 demonstrated strong metabolic activity towards fluorene and fluoranthene, indicating their potential role in the survival of \u003cem\u003eAn. gambiae\u003c/em\u003e in polluted breeding sites. Earlier studies (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e) have suggested that the CYP6Z family of cytochrome P450s in \u003cem\u003eAn. gambiae\u003c/em\u003e are most notably expressed at the early life stages of larvae and pupae. This means they inherently help mosquito\u0026rsquo;s timely survival in polluted breeding sites due to their characteristic broad range substrate specificity capable of metabolizing wide range of compounds including plants\u0026rsquo; secondary metabolites (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). This similar trend was also reported in \u003cem\u003eAedes aegypti\u003c/em\u003e mosquito\u0026rsquo;s CYP6Z8, an orthologue of the \u003cem\u003eAn. gambiae\u003c/em\u003e CYP6Z2 showing catalytic activity on broad range of substrates including α-naphthoflavone, resveratrol, and diethylstilbestrol (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDifferential level of microsomal metabolism between pyrethroid resistant and susceptible strains also indicate the potential relationship between PAHs and pyrethroids. Cytochrome P450s associated with pyrethroids resistance were found to metabolise PAHs in metabolic assays confirming the cross-resistance potentials between these pollutants and insecticides of public health importance. These findings therefore stress the importance of cross-resistance studies especially for newer chemistries before they are rolled out in vector control tools.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to this article as no datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported from the petroleum technology development fund (PTDF) overseas scholarship awarded to AM. The funders had no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAM, CSW, SSI and HMI conceptualized the ideas while AM carried out the investigations in the laboratory. SSI and HI contributed in data analysis while MJIP and CSW provided the necessary supervision and data validations. AM wrote the first draft of the manuscript. All authors have read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to appreciate the contributions of the ECG group members at LSTM for their contributions in the provision of the P40 clones for expression\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRanson H, Lissenden N. 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Insect Mol Biol. 2008;17(February):125\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cytochrome P450, microsome, Anopheles coluzzii, Polycyclic aromatic hydrocarbons, cross-resistance, insecticide, malaria","lastPublishedDoi":"10.21203/rs.3.rs-6079555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6079555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous and recalcitrant environmental pollutants generated from petroleum activities and/or biological conversion of organic materials. Environmental exposure of mosquito to these pollutants can potentially select resistance to insecticides used in public health for vector control. To understand the cross-resistance potentials between PAHs and pyrethroid insecticides, microsomal fractions prepared from \u003cem\u003eAnopheles coluzzii\u003c/em\u003e mosquitoes obtained from agricultural sites and a laboratory susceptible strain, Ngousso, were tested with three major PAHs - fluorene, fluoranthene and naphthalene. Recombinant P450s previously associated with pyrethroids resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e (\u003cem\u003eCYPs 6M2, 6Z2, 6Z3, 9J5, 6P3, 6P4, 6P5 CYP9K1) \u003c/em\u003eand\u003cem\u003e Anopheles funestus\u003c/em\u003e \u003cem\u003eCYP6P9a\u003c/em\u003e were also used to investigate metabolism of the above PAHs alongside the microsome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Microsomes prepared from pyrethroid resistant \u003cem\u003eAnopheles coluzzii\u003c/em\u003e significantly (p = 0.001) depleted fluorene and fluoranthene with percentage depletions of 73%±0.5 and 43%.0±2.2, respectively. Steady state kinetic study demonstrated the microsome having a high affinity for the fluorene with a Km and turnover, respectively of 58.69µM±20.47\u003cbr\u003e\nand 37.016 min-\u003csup\u003e1\u003c/sup\u003e±3.67. On the other hand, significant metabolism of fluorene up to 47.9%±2.3 and 52.8%±0.8 depletions were observed with recombinant \u003cem\u003eCYP6P3 \u003c/em\u003eand \u003cem\u003eCYP6Z3, \u003c/em\u003erespectively. Other P450s showed little to no metabolism with fluorene. \u003cem\u003eCYP6P3 \u003c/em\u003eand \u003cem\u003eCYP6Z3\u003c/em\u003e metabolised fluoranthene with percentage depletions of 50.4%±4.9 and 60.3% ±5.3, respectively. However, there was no observed metabolism of naphthalene with all the recombinant P450s used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: This study demonstrates that P450 monooxygenases from the malaria vectors can metabolise PAHs, highlighting the potential possibility of this environmental pollutants selecting the P450s, driving insecticide resistance in field populations of major malaria vectors.\u003c/p\u003e","manuscriptTitle":"Investigating the potential role of metabolic resistance genes in conferring cross-resistance to pyrethroids and polycyclic aromatic hydrocarbon pollutants in the major malaria vector Anopheles coluzzii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-07 05:02:39","doi":"10.21203/rs.3.rs-6079555/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27510f32-33ac-428f-83d0-d05b0ec912c9","owner":[],"postedDate":"March 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-07T05:02:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-07 05:02:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6079555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6079555","identity":"rs-6079555","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}