Insecticides Resistance Status of An. gambiae in Areas of Varying Agrochemical Use in Côte D’Ivoire

Background. Insecticide resistance monitoring of the malaria vectors to different classes of insecticides is necessary for resistance management. Malaria vector control management approaches are essentially based on IRS and LLINs. However, insecticide resistance is caused by several sources of selection and in case the selection pressure is from agricultural practices, then measures need to be taken to avoid a failure of the control methods put in place. The current study was undertaken to monitor the susceptibility of vectors to different classes of insecticides in areas of varying agrochemical use patterns. Methods. A survey to determine the agricultural chemical use pattern was undertaken in ten localities across Côte d’Ivoire. In addition, WHO susceptibility tests were carried out on adults Anopheles gambiae s.l. mosquitoes emerging from collected larvae from the sites surveyed. Four insecticides from each class of the four classes of insecticides were evaluated using the standard susceptibility test methods. Furthermore, the target site mutations involved in resistance mechanisms were identified following the Taqman assay protocols and mosquito species were identified using SINE-PCR. Results. The mortalities of all the An. gambiae s.l populations were similar regardless of the pesticide use pattern. The vectors were resistant to DDT, deltamethrin, and bendiocarb in all localities. In contrast, mosquitoes showed high susceptibility to malathion. High frequency of the Kdr-West gene allele was observed (70-100%). A single Kdr-East mutation was identified in a mosquito that harboured both Ace-1 and Kdr-West genes. Conclusion. Cultivated marshlands representing good habitats for mosquito development may deeply contribute to the selection of resistance genes given the intensive use of agrochemical for crop protection. In view of these, special attention must be given to them to mitigate mosquito resistance to insecticides

The impact of agrochemical pollutant mixtures on the selection of insecticide resistance in the malaria vector Anopheles gambiae: insights from experimental evolution and transcriptomics

Abstract Background There are several indications that pesticides used in agriculture contribute to the emergence and spread of resistance of mosquitoes to vector control insecticides. However, the impact of such an indirect selection pressure has rarely been quantified and the molecular mechanisms involved are still poorly characterized. In this context, experimental selection with different agrochemical mixtures was conducted in Anopheles gambiae. The multi-generational impact of agrochemicals on insecticide resistance was evaluated by phenotypic and molecular approaches. Methods Mosquito larvae were selected for 30 generations with three different agrochemical mixtures containing (i) insecticides, (ii) non-insecticides compounds, and (iii) both insecticide and non-insecticide compounds. Every five generations, the resistance of adults to deltamethrin and bendiocarb was monitored using bioassays. The frequencies of the kdr (L995F) and ace1 (G119S) target-site mutations were monitored every 10 generations. RNAseq was performed on all lines at generation 30 in order to identify gene transcription level variations and polymorphisms associated with each selection regime. Results Larval selection with agrochemical mixtures did not affect bendiocarb resistance and did not select for ace1 mutation. Contrastingly, an increased deltamethrin resistance was observed in the three selected lines. Such increased resistance was not majorly associated with the presence of kdr L995F mutation in selected lines. RNA-seq identified 63 candidate resistance genes over-transcribed in at least one selected line. These include genes coding for detoxification enzymes or cuticular proteins previously associated with insecticide resistance, and other genes potentially associated with chemical stress response. Combining an allele frequency filtering with a Bayesian FST-based genome scan allowed to identify genes under selection across multiple genomic loci, supporting a multigenic adaptive response to agrochemical mixtures. Conclusion This study supports the role of agrochemical contaminants as a significant larval selection pressure favouring insecticide resistance in malaria vectors. Such selection pressures likely impact kdr mutations and detoxification enzymes, but also more generalist mechanisms such as cuticle resistance, which could potentially lead to cross-tolerance to unrelated insecticide compounds. Such indirect effect of global landscape pollution on mosquito resistance to public health insecticides deserves further attention since it can affect the nature and dynamics of resistance alleles circulating in malaria vectors and impact the efficacy of control vector strategies

Deltamethrin and transfluthrin select for distinct transcriptomic responses in the malaria vector Anopheles gambiae

Abstract Background The widespread use of pyrethroid insecticides in Africa has led to the development of strong resistance in Anopheles mosquitoes. Introducing new active ingredients can contribute to overcome this phenomenon and ensure the effectiveness of vector control strategies. Transfluthrin is a polyfluorinated pyrethroid whose structural conformation was thought to prevent its metabolism by cytochrome P450 monooxygenases in malaria vectors, thus representing a potential alternative for managing P450-mediated resistance occurring in the field. In this study, a controlled selection was used to compare the dynamics of resistance between transfluthrin and the widely used pyrethroid deltamethrin in the mosquito Anopheles gambiae. Then, the associated molecular mechanisms were investigated using target-site mutation genotyping and RNA-seq. Methods A field-derived line of An. gambiae carrying resistance alleles at low frequencies was used as starting material for a controlled selection experiment. Adult females were selected across 33 generations with deltamethrin or transfluthrin, resulting in three distinct lines: the Delta-R line (selected with deltamethrin), the Transflu-R line (selected with transfluthrin) and the Tiassale-S line (maintained without selection). Deltamethrin and transfluthrin resistance levels were monitored in each selected line throughout the selection process, as well as the frequency of the L1014F kdr mutation. At generation 17, cross-resistance to other public health insecticides was investigated and transcriptomes were sequenced to compare gene transcription variations and polymorphisms associated with adaptation to each insecticide. Results A rapid increase in resistance to deltamethrin and transfluthrin was observed throughout the selection process in each selected line in association with an increased frequency of the L1014F kdr mutation. Transcriptomic data support a broader response to transfluthrin selection as compared to deltamethrin selection. For instance, multiple detoxification enzymes and cuticle proteins were specifically over-transcribed in the Transflu-R line including the known pyrethroid metabolizers CYP6M2, CYP9K1 and CYP6AA1 together with other genes previously associated with resistance in An. gambiae. Conclusion This study confirms that recurrent exposure of adult mosquitoes to pyrethroids in a public health context can rapidly select for various resistance mechanisms. In particular, it indicates that in addition to target site mutations, the polyfluorinated pyrethroid transfluthrin can select for a broad metabolic response, which includes some P450s previously associated to resistance to classical pyrethroids. This unexpected finding highlights the need for an in-depth study on the adaptive response of mosquitoes to newly introduced active ingredients in order to effectively guide and support decision-making programmes in malaria control

Additional file 1 of The impact of agrochemical pollutant mixtures on the selection of insecticide resistance in the malaria vector Anopheles gambiae: insights from experimental evolution and transcriptomics

Additional file 1: Figure S1. Venn diagrams. Venn diagrams showing the numbers of genes significantly differentially transcribed in each selected line as compared to the control line (FC ≥ 1.5-fold in either direction and corrected P value ≤ 0.005). The number of resistance candidate genes are shown within brackets