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

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.

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.

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

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.

Phenotypic resistance to pyrethroid associated to metabolic mechanism in Vgsc-L995F-resistant Anopheles gambiae malaria mosquitoes.

Background: The indiscriminate use of insecticides in agriculture and public health lead to a selection of resistance mechanisms in malaria vectors compromising vector control tools and strategies. This study investigated the metabolic response in the Vgsc-L995F Anopheles gambiae Tiassalé resistance strain after long-term exposure of larvae and adults to deltamethrin insecticide. Methods:  Vgsc-L995F An. gambiae Tiassalé strain larvae were exposed over 20 generations to deltamethrin (LS) and adults to PermaNet 2.0 (AS) and combining exposure at larvae and adult stages (LAS) and compared to unexposed (NS) group. All four groups were subjected to the standard World Health Organization (WHO) susceptibility tube tests using deltamethrin (0.05%), bendiocarb (0.1%) and malathion (5%). Vgsc-L995F/S knockdown-resistance ( kdr) mutation frequency was screened using multiplex assays based on Taqman real-time polymerase chain reaction (PCR) method. Additionally, expression levels of detoxification enzymes associated to pyrethroid resistance, including CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and glutathione S-transferase GSTe2 were measured. Results: Our results indicated that deltamethrin resistance was a response to insecticide selection pressure in LS, AS and LAS groups, while susceptibility was observed in NS group. The vectors showed varied mortality rates with bendiocarb and full susceptibility to malathion throughout the selection with LS, AS and LAS groups. Vgsc-L995F mutation stayed at high allelic frequency level in all groups with a frequency between 87% and 100%. Among the overexpressed genes, CYP6P4 gene was the most overexpressed in LS, AS and LAS groups. Conclusion: Long-term exposure of larvae and adults of Vgsc-L995F resistant- An. gambiae Tiassalé strain to deltamethrin and PermaNet 2.0 net induced resistance to deltamethrin under a significant effect of cytochromes P450 detoxification enzymes. These outcomes highlight the necessity of investigating metabolic resistance mechanisms in the target population and not solely kdr resistance mechanisms prior the implementation of vector control strategies for a better impact.

Trapping and killing performance of a PermaNet 2.0 hybrid mosquito trapping bednet: an experimental hut evaluation.

Background: Despite the huge global effort , there has been an increase in malaria morbidity and mortality in sub-Saharan Africa since 2015, from 212 million cases and 429,000 deaths in 2015 to 241 million cases and 627,000 deaths in 2020 mainly because of resistance to insecticide. Therefore, advancing innovative approaches is the only sustainable way to fight malaria. Methods: Taking advantage of the behavior of mosquitoes around the net, which is almost 70-90% concentrated on the roof, we have developed a two-compartment mosquito bednet, the so-called T-Net for mass mosquito trapping and killing. In the current study, we investigated in an experimental hut trial, the efficacy of trapping-long-lasting insecticide-treated nets (T-LLINs) against Anopheles gambiae s.l. in an insecticide resistance context. Five different arms have been considered in this study including three positive control arms e.g. PermaNet 2.0 LLIN, Tsara boost LLIN and Interceptor generation 2 (IG2) LLIN), one negative control arm using insecticide-free bednet, and one candidate arm using a hybrid-treated trapping bednet made with PermaNet 2.0 LLIN mounted with an insecticide-free compartment (T-LLIN). Results: The highest average daily mortality was recorded with the T-LLIN. In total, 678 mosquitoes were killed by T-LLIN among the 760 collected, i.e. 89.2%. Out of these, 317 were found in the trap compartment, representing 46.75% of mortality directly attributable to the mechanical effect of this net. This added value made it possible to quantify the increased in the killing effect that this net would have over the positive control arms: this would be 52% higher than the killing effect of PN2.0, 25.2% higher than that of Tsara boost and 23% higher than that of IG2. Conclusion: The current study shows potential to maximize the efficiency of the WHO-recommended LLINs by an addition of an insecticide-free trap compartment on top of the net.

Exposure of Anopheles gambiae larvae to a sub-lethal dose of an agrochemical mixture induces tolerance to adulticides used in vector control management.

The heavy use of pesticides in agricultural areas often leads to the contamination of nearby mosquito larvae breeding sites. Exposure to complex mixtures of agrochemicals can affect the insecticide sensitivity of mosquito larvae. Our study objective was to determine whether agrochemical residues in Anopheline larval breeding sites can affect the tolerance of adults to commonly used adulticides. We focussed on Fludora® Fusion, a vector control insecticide formulation combining two insecticides (deltamethrin and clothianidin) with different modes of action. An. gambiae larvae were exposed to a sub-lethal dose of a mixture of agrochemical pesticides used in a highly active agricultural area on the Ivory Coast. Comparative bioassays with Fludora Fusion mixture and its two insecticide components (deltamethrin and clothianidin) were carried out between adult mosquitoes exposed or not to the agrochemicals at the larval stage. A transcriptomic analysis using RNA sequencing was then performed on larvae and adults to study the molecular mechanisms underlying the phenotypic changes observed. Bioassays revealed a significantly increased tolerance of adult females to clothianidin (2.5-fold) and Fludora Fusion mixture (2.2-fold) following larval exposure to agrochemicals. Significantly increased tolerance to deltamethrin was not observed suggesting that insecticide exposure affects the adult efficacy of the Fludora Fusion mixture mainly through mechanisms acting on clothianidin. Transcriptomic analysis revealed the potential of agrochemicals to induce various resistance mechanisms including cuticle proteins, detoxification action and altered insecticide sequestration. These results suggest that although the Fludora Fusion mixture is effective for adult vector control, its efficacy may be locally affected by the ecological context. The present study also suggests that, although the complex interactions between the use of agrochemicals and vector control insecticides are difficult to decipher in the field, they still must be considered in the context of insecticide resistance management programmes.

Multi-insecticide resistant malaria vectors in the field remain susceptible to malathion, despite the presence of Ace1 point mutations.

Insecticide resistance in Anopheles mosquitoes is seriously threatening the success of insecticide-based malaria vector control. Surveillance of insecticide resistance in mosquito populations and identifying the underlying mechanisms enables optimisation of vector control strategies. Here, we investigated the molecular mechanisms of insecticide resistance in three Anopheles coluzzii field populations from southern Côte d'Ivoire, including Agboville, Dabou and Tiassalé. All three populations were resistant to bendiocarb, deltamethrin and DDT, but not or only very weakly resistant to malathion. The absence of malathion resistance is an unexpected result because we found the acetylcholinesterase mutation Ace1-G280S at high frequencies, which would typically confer cross-resistance to carbamates and organophosphates, including malathion. Notably, Tiassalé was the most susceptible population to malathion while being the most resistant one to the pyrethroid deltamethrin. The resistance ratio to deltamethrin between Tiassalé and the laboratory reference colony was 1,800 fold. By sequencing the transcriptome of individual mosquitoes, we found numerous cytochrome P450-dependent monooxygenases - including CYP6M2, CYP6P2, CYP6P3, CYP6P4 and CYP6P5 - overexpressed in all three field populations. This could be an indication for negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate pro-insecticides, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathione S-transferases and other candidates putatively involved in insecticide resistance.