Influence of the agrochemicals used for rice and vegetable cultivation on insecticide resistance in malaria vectors in southern Côte d’Ivoire

Background Vector control can contribute to the development of resistance to insecticides in malaria vectors. As the swamps and wetlands used for some agricultural activities constitute productive breeding sites for many mosquito species, agricultural pest control may increase the selection pressure for insecticide resistance in mosquitoes. Understanding the use of agrochemicals by farmers is important to plan and initiate effective integrated pest and vector management interventions. Methods A knowledge–attitude–practice study, using questionnaires, was undertaken with 102 rice farmers in Tiassalé and 106 vegetable farmers in Dabou (South Côte d’Ivoire) in order to generate information on pesticide usage. In addition, insecticide susceptibility bioassays were conducted using adult mosquitoes obtained from larvae collected within farms, and the persistence of agricultural pesticides in the farming environment, including sediment and mosquito breeding site water, was investigated by HPLC. Results Herbicides and insecticides appeared to be the most frequently used pesticides for both crops. Amino phosphonates (mostly glyphosate) represented the most used herbicides (45 % for rice up to 89 % for vegetables). Pyrethroids appeared to be the most used insecticides (accounting for 90 % of all the insecticide use reported). Approximately 75 % of respondents had not been to school and do not understand product labels. Only about 45 % of farmers respect the recommended pesticide dosage and about 10–15 % of pesticides used for rice and vegetable, respectively, are not recommended for these crops. As per WHO criteria, the mosquito local populations from the two localities were resistant to three of the four insecticides tested, as mortalities were less than 35 % for deltamethrin, DDT and bendiocarb. Higher susceptibility was observed for malathion, although the population was considered resistant in Dabou (80 % mortality) and susceptible in Tiassalé (98 % mortality). With the exception of glyphosate, residues from each of six chemicals tested for were detected in each of the sites visited in the two localities. Conclusion The study describes the use of insecticides and herbicides on crops and highlights the importance of considering agriculture practices when attempting to manage resistance in malaria vectors. Inter-sectoral collaboration between agriculture and public health is required to develop efficient integrated pest and vector management interventions

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

Relationship between insecticide resistance profiles in Anopheles gambiae sensu lato and agricultural practices in Côte d’Ivoire

Abstract Background Insecticide-based malaria vector control is increasingly undermined due to the development of insecticide resistance in mosquitoes. Insecticide resistance may partially be related to the use of pesticides in agriculture, while the level and mechanisms of resistance might differ between agricultural practices. The current study aimed to assess whether phenotypic insecticide resistance and associated molecular resistance mechanisms in Anopheles gambiae sensu lato differ between agricultural practices. Methods We collected An. gambiae s.l. larvae in six sites with three different agricultural practices, including rice, vegetable and cocoa cultivation. We then exposed the emerging adult females to discriminating concentrations of bendiocarb (0.1%), deltamethrin (0.05%), DDT (4%) and malathion (5%) using the standard World Health Organization insecticide susceptibility test. To investigate underlying molecular mechanisms of resistance, we used multiplex TaqMan qPCR assays. We determined the frequency of target-site mutations, including Vgsc-L995F/S and Vgsc-N1570Y, and Ace1-G280S. In addition, we measured the expression levels of genes previously associated with insecticide resistance in An. gambiae s.l., including the cytochrome P450-dependent monooxygenases CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and the glutathione S-transferase GSTe2. Results The An. gambiae s.l. populations from all six agricultural sites were resistant to bendiocarb, deltamethrin and DDT, while the populations from the two vegetable cultivation sites were additionally resistant to malathion. Most tested mosquitoes carried at least one mutant Vgsc-L995F allele that is associated with pyrethroid and DDT resistance. In the cocoa cultivation sites, we observed the highest 995F frequencies (80–87%), including a majority of homozygous mutants and several in co-occurrence with the Vgsc-N1570Y mutation. We detected the Ace1 mutation most frequently in vegetable-growing sites (51–60%), at a moderate frequency in rice (20–22%) and rarely in cocoa-growing sites (3–4%). In contrast, CYP6M2, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, previously associated with metabolic insecticide resistance, showed the highest expression levels in the populations from rice-growing sites compared to the susceptible Kisumu reference strain. Conclusion In our study, we observed intriguing associations between the type of agricultural practices and certain insecticide resistance profiles in the malaria vector An. gambiae s.l. which might arise from the use of pesticides deployed for protecting crops. Graphical Abstrac

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

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

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