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.

Keywords: Agriculture; Anopheles gambiae; Côte d’Ivoire; Gene expression; Insecticide resistance; Malaria vectors; P450 genes; Target-site mutation.

Fig. 1

Map of the mosquito larval collection sites in Côte d’Ivoire. In Agbovile and Tiassalé, larvae were collected in the rice fields, while they were collected in the vegetable fields in Azaguié and Dabou. In the western part, mosquito larvae were collected in the cocoa fields of Issia and Soubré. The map was created with QGIS (2022, QGIS Geographic Information System. QGIS.ORG Association; http://www.qgis.org). Basemap source: Sentinel-2 cloudless (https://s2maps.eu) by EOX IT Services GmbH (contains modified Copernicus Sentinel data 2020)

Fig. 2

Kaplan–Meier ‘survival’ curves showing the cumulative knockdown over the 60 min exposure to diagnostic concentrations of deltamethrin (0.05%) and DDT (4%) in the WHO insecticide susceptibility assay. The log-rank test used for Kaplan–Meier test showed P-values < 0.0001 between Kisumu and rice, vegetable and cocoa sites, respectively

Fig. 3

24 h mortality rates against bendiocarb (0.1%), DDT (4%), deltamethrin (0.05%) and malathion (5%) diagnostic concentrations. Mortality rates above 98% (red line) indicate susceptibility according to WHO criteria of phenotypic resistance. Error bars indicate 95% confidence intervals

Fig. 4

Allelic frequency of target-site resistance mutations Vgsc-L995F, Vgsc-N1570Y and Ace1-G280S. The percentages indicate the frequency of the resistance alleles while the bars show the actual numbers

Fig. 5

Gene expression levels of detoxification enzymes in Anopheles gambiae s.l. from the six field populations with three different agricultural practices and in the Kisumu susceptible strain. The boxes indicate the 25–75% quartiles. The whiskers show the 5–95% range and the dots represent outliers

Fig. 6

Differential expression levels for measured putative detoxifying genes across field populations. The fold changes give the change in expression level of a population against the susceptible reference strain Kisumu on the log₂ scale. The fold changes were estimated using generalised linear regression models for each gene. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001