Exposure of Anopheles gambiae larvae to a sub-lethal dose of an agrochemical mixture induces tolerance to adulticides used in vector control management
Introduction
In 2020, malaria caused 627,000 deaths. These mainly occurred in Africa (96%) and more than 80% were children less than 5 years old (WHO, 2021). Despite significant advances in malaria vaccines and treatments, control of Anopheles mosquitoes in Africa remains an important malaria control strategy that decreases malaria prevalence and mortality in endemic regions (Bhatt et al., 2015; Coleman et al., 2017; Lim et al., 2011; Tizifa et al., 2018; WHO, 2021). Vector control relies on the use of insecticide treated nets (ITN) or indoor residual insecticide sprays (IRS). Pyrethroid insecticides are most commonly used because of their strong effects on the target mosquitoes and their low toxicity to the environment and mammals (Zaim et al., 2000).
However, after decades of use, pyrethroid efficacy has been reduced by resistance mechanisms selected in Anopheles populations (Chouaibou et al., 2017; Fane et al., 2012; Nwane et al., 2013; Reid and McKenzie, 2016). Pyrethroid resistance is mainly associated with target site mutations affecting the voltage gated sodium channel (VGSC, Knock Down Resistance ‘kdr’ mutations) and increased insecticide metabolism mediated by detoxification enzymes (metabolic resistance). Kdr mutations are widely distributed in African Anopheles populations and encompass kdr West (L1014F) and kdr East (L1014S) mutations (Martinez-Torres et al., 1998; Ranson et al., 2000). Metabolic resistance occurs through increased activities of detoxification enzymes, resulting in increased insecticide metabolism (Li et al., 2007). The main detoxification enzyme families involved include cytochrome P450 monooxygenases (P450, CYP for genes), carboxyl/cholinesterases (CCE), glutathione-S-transferase (GST), UDP-glycosyl-transferases (UDPGT) and sulfotransferases (SULT) (David et al., 2013; Liu, 2015; Moyes et al., 2017; Riveron et al., 2018). In addition to target-site modifications and metabolic resistance, additional mechanisms involving cuticle modifications, altered insecticide transport and sequestration, sensory appendage protein (SAP) and chemosensory proteins (CSP) have been reported or suggested (Ingham et al., 2020, 2018; Liu, 2015; Yahouedo et al., 2017).
Introduction of new insecticides with different modes of action has been suggested as a component of insecticide resistance management strategies (Darriet and Chandre, 2013; WHO, 2012). Among the novel insecticides proposed for managing pyrethroid resistance, neonicotinoids have been presented as a good alternative because they target nicotinic acetylcholine receptors (nAChR) which represent a new biochemical target in public health insects (Agossa et al., 2018; Ngufor et al., 2017). A new insecticide formulation combining the neonicotinoid clothianidin and the pyrethroid deltamethrin (8:1 w/w) under the brand name Fludora® Fusion was developed by Bayer for indoor residual spraying (IRS) as a tool for insecticide resistance management (IRM). Field trials using Fludora® Fusion demonstrated its high efficacy against various Malaria vectors, including pyrethroid-resistant populations (Fongnikin et al., 2020; Kamaraju et al., 2021; Ngufor et al., 2017). Fludora® Fusion may also help slow the selection of pyrethroid and neonicotinoid resistance alleles (Zoh et al., 2021). However, environmental factors that may affect the sensitivity of mosquitoes to Fludora® Fusion remain to be investigated.
Agriculture yields in Africa are associated with the use of agrochemicals. Heavy use of agrochemicals, including herbicides and insecticides, in intensive agricultural areas often leads to contamination of nearby mosquito larvae breeding sites (Chouaibou et al., 2016). Exposure to complex agrochemical mixtures can lead to reduced sensitivity to insecticides due to ‘gene x environment’ interactions (Liu, 2015; Riveron et al., 2018). These interactions can affect the transient expression of a range of proteins though the induced expression of detoxification enzymes. Cuticle proteins and transporters may also play roles in the increased resistance phenotypes observed. For example, Riaz et al. (2009) showed that Aedes aegypti larvae exposed to a sublethal dose of the herbicide glyphosate or the PAH benzo[a]pyrene showed increased tolerance to the insecticides imidacloprid and permethrin associated with increased transcription of several P450 genes. Similarly, exposure of Ae. aegypti larvae to a sublethal dose of the fungicide copper sulfate led to increased tolerance to permethrin and was associated with over-transcription of several P450 genes (Poupardin et al., 2008, 2010). Transcriptomic data confirmed the association between this increased insecticide tolerance and the overexpression of genes encoding detoxification enzymes, cuticle proteins and xenobiotic transporters (David et al., 2010; Poupardin et al., 2010). Exposure of Anopheles gambiae larvae to phytochemical leaf extracts of the curry tree revealed significant induction of transcripts associated with cuticular proteins and detoxification enzymes (Mang'era et al., 2021).
The objective of this study was to determine whether agrochemicals found in Anopheline larval breeding sites can affect the tolerance of adult mosquitoes to commonly used insecticides such as Fludora® Fusion. In this study, An. gambiae larvae were exposed to a sub-lethal dose of a mixture of agrochemicals representative of those used in an agriculture region of the Ivory Coast. Comparative bioassays were conducted between adult mosquitoes exposed, or nonexposed, to the pesticide mixture at the larval stage. A transcriptomic analysis using RNA sequencing (RNA-seq) was conducted to study the molecular mechanisms underlying the observed insecticide sensitivity changes.
Section snippets
Mosquitoes
An. gambiae sensu stricto larvae were collected near Tiassalé (south of Ivory Coast). This is a rice field area with classic vector control activities (mainly mosquito nets impregnated with pyrethroids) and heavy use of pesticides for crop protection, including herbicides, fungicides and insecticides from the pyrethroid, organophosphate, carbamate and neonicotinoid families (Chouaibou et al., 2016). Malaria vectors in this area are resistant to multiple insecticides, including pyrethroids, DDT,
Bioassays
Exposure of Tiassalé-S larvae to a sublethal dose of the agrochemical mixture differentially affected the tolerance of adult females to the Fludora Fusion mixture and its two insecticide components (Fig. 1). Comparative bioassays on adult females showed a 2.5-fold and a 2.2-fold increased tolerance to clothianidin and the Fludora Fusion mixture respectively compared to the nonexposed control (Fisher p-value <0.001). Though a slight increased tolerance to deltamethrin was observed in exposed
Discussion
Mosquito larval development often occurs in areas exposed to natural xenobiotics and anthropogenic pollutants. Xenobiotics, including residual agrochemicals, can affect subsequent larval tolerance to chemical insecticides used for mosquito control (Poupardin et al., 2010; Riaz et al., 2009; Suwanchaichinda and Brattsten, 2001). The xenobiotics most affective on larval insecticide sensitivity often act as detoxification enzyme inducers, supporting the key role of the detoxification system in
Conclusion
The WHO has encouraged development of novel vector control products that can be used for managing pyrethroid resistance (Turner et al., 2016; WHO, 2012). Among them, pyrethroid-neonicotinoid combinations such as the IRS formulation Fludora® Fusion show good potential for use against African malaria vectors (Fongnikin et al., 2020; Fuseini et al., 2019; Oxborough et al., 2019). Such combinations also show a good potential for delaying the selection of insecticide resistance alleles (Zoh et al.,
Data availability statement
The RNA-seq sequence data from this study have been deposited at NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/info/ submission.html) under the accession number GSE189345.
CRediT authorship contribution statement
Marius Gonse Zoh: Methodology, Investigation, Data curation. Jordan Tutagata: . Behi K. Fodjo: Methodology, Investigation. Chouaïbou S. Mouhamadou: Methodology. Christabelle G. Sadia: Methodology, Investigation. Justin McBeath: Conceptualization. Frederic Schmitt: Conceptualization. Sebastian Horstmann: Conceptualization. Jean-Philippe David: Conceptualization, Methodology, Investigation, Data curation, Writing – original draft, Writing – review & editing, Supervision. Stéphane Reynaud:
Declaration of Competing Interest
The funding organization (Bayer) developed and commercialized Fludora® Fusion used in this study and proposed the original test hypothesis. The funder had no role in the study design, data collection, analysis and interpretation of results. JMB, FS and SH were employed by Bayer. MGZ PhD was funded by Bayer and hosted at the Laboratoire d'Ecologie Alpine (LECA). The position of JT was funded by Bayer and hosted at LECA. All other authors declare no conflict of interest.
Acknowledgements
Editorial services, including language editing and correction, were provided by XpertScientific Editing and Consulting Services.
References (72)
Evidence of multiple pyrethroid resistance mechanisms in the malaria vector Anopheles gambiae sensu stricto from Nigeria
Trans. R. Soc. Trop. Med. Hyg.
(2009)- et al.
The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance
Insect Biochem Mol Biol
(2014) Anopheles gambiae resistance to pyrethroid-treated nets in cotton versus rice areas in Mali
Acta Trop.
(2012)Evaluation of the residual effectiveness of Fludora fusion WP-SB, a combination of clothianidin and deltamethrin, for the control of pyrethroid-resistant malaria vectors on Bioko Island, Equatorial Guinea
Acta Trop.
(2019)Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides
Insect Biochem. Mol. Biol.
(2008)Do pollutants affect insecticide-driven gene selection in mosquitoes? Experimental evidence from transcriptomics
Aquat Toxicol
(2012)Molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in the dengue vector Aedes aegypti
Aquat Toxicol
(2013)Impact of glyphosate and benzo[a]pyrene on the tolerance of mosquito larvae to chemical insecticides. Role of detoxification genes in response to xenobiotics
Aquat. Toxicol.
(2009)- et al.
Effects of Exposure to Pesticides on Carbaryl Toxicity and Cytochrome P450 Activities in Aedes albopictus Larvae (Diptera: culicidae)
Pestic Biochem Physiol
(2001) Small-scale field evaluation of the efficacy and residual effect of Fludora((R)) Fusion (mixture of clothianidin and deltamethrin) against susceptible and resistant Anopheles gambiae populations from Benin, West Africa
Malar. J.
(2018)
Investigation of mechanisms of bendiocarb resistance in Anopheles gambiae populations from the city of Yaounde, Cameroon
Malar. J.
Investigating the molecular basis of multiple insecticide resistance in a major malaria vector Anopheles funestus (sensu stricto) from Akaka-Remo, Ogun State, Nigeria
Parasit Vectors
Controlling the false discovery rate: a practical and powerful approach to multiple testing
Journal of the Royal Statistical Society: Series B (Methodological)
The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015
Nature
Trimmomatic: a flexible trimmer for Illumina sequence data
Bioinformatics
Evolution and function of the insect hexamerins
Eur. J. Entomol.
Mapping insecticide resistance in Anopheles gambiae (s.l.) from Cote d'Ivoire
Parasit Vectors
Evolution of phenotypic plasticity in extreme environments
Philos. Trans. R. Soc. Lond., B, Biol. Sci.
Influence of the agrochemicals used for rice and vegetable cultivation on insecticide resistance in malaria vectors in southern Cote d'Ivoire
Malar. J.
A reduction in malaria transmission intensity in Northern Ghana after 7 years of indoor residual spraying
Malar. J.
Efficacy of six neonicotinoid insecticides alone and in combination with deltamethrin and piperonyl butoxide against pyrethroid-resistant Aedes aegypti and Anopheles gambiae (Diptera: culicidae)
Pest Manag. Sci.
Transcriptome response to pollutants and insecticides in the dengue vector Aedes aegypti using next-generation sequencing technology
BMC Genomics
Role of cytochrome P450s in insecticide resistance: impact on the control of mosquito-borne diseases and use of insecticides on Earth
Philos. Trans. R. Soc. Lond., B, Biol. Sci.
The Anopheles gambiae Detoxification chip: A highly Specific Microarray to Study Metabolic-Based Insecticide Resistance in Malaria Vectors
The choreography of the chemical defensome response to insecticide stress: insights into the Anopheles stephensi transcriptome using RNA-Seq
Sci Rep
Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s. from Southern Benin and Nigeria
BMC Genomics
Multiple-insecticide resistance in Anopheles gambiae mosquitoes, Southern Cote d'Ivoire
Emerging Infect. Dis.
Efficacy of Fludora(R) Fusion (a mixture of deltamethrin and clothianidin) for indoor residual spraying against pyrethroid-resistant malaria vectors: laboratory and experimental hut evaluation
Parasit Vectors
Resistance of Anopheles gambiae to the new insecticide clothianidin associated with unrestricted use of agricultural neonicotinoids in Yaoundé, Cameroon
BioRxiv
Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments
Fonctional ecology
The flexible stem hypothesis: evidence from genetic data
Dev. Genes Evol.
The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America
Drug Metab. Dispos.
Binding of insecticides to lipophorin and arylphorin, 2 hemolymph-proteins of Heliothis-zea
Arch. Insect Biochem. Physiol.
Changes in both trans- and cis-regulatory elements mediate insecticide resistance in a lepidopteron pest, Spodoptera exigua
PLoS Genet
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