High insecticide resistance in the major malaria vector Anopheles coluzzii in Chad Republic

Background The Sahel region of Chad Republic is a prime candidate for malaria pre-elimination. To facilitate pre-elimination efforts in this region, two populations of Anopheles coluzzii from Central Chad Republic were characterized, their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated. Methods Bloodfed female Anopheles gambiae s.l. resting indoor, were collected at N’djamena and Massakory, Chad in 2018 and characterized for species composition, and infection rate was determined using the TaqMan assay. Susceptibility to various insecticides was assessed using WHO tube bioassays. Cone bioassays were conducted using various long-lasting insecticidal nets (LLINs). Results were analysed using Chi Square test. Knockdown resistance (kdr) and ace-1 markers were investigated by TaqMan genotyping. Results Anopheles coluzzii was the major vector found in N’djamena (100%) and Massakory (~ 94%). No Plasmodium was found in 147 bloodfed F0An. coluzzii (82 from N’djamena and 65 from Massakory). High intensity pyrethroid resistance was observed with mortalities of < 2% for permethrin, deltamethrin and etofenprox, and with < 50% and < 60% dead following exposure to 10× diagnostic doses of deltamethrin and permethrin, respectively. For both sites, < 10% mortalities were observed with DDT. Synergist bioassays with piperonylbutoxide significantly recovered pyrethroid susceptibility in Massakory populations, implicating CYP450s (mortality = 13.6% for permethrin, χ2 = 22.8, df = 1, P = 0.0006; mortality = 13.0% for deltamethrin, χ2 = 8.8, df = 1, P < 0.00031). Cone-bioassays established complete loss of efficacy of the pyrethroid-based LLINs; and a 100% recovery of susceptibility following exposure to the roof of PermaNet®3.0, containing piperonylbutoxide. Both populations were susceptible to malathion, but high bendiocarb resistance was observed in Massakory population. The absence of ace-1 mutation points to the role of metabolic resistance in the bendiocarb resistance. Both 1014F and 1014S mutations were found in both populations at around 60% and < 20% respectively. Sequencing of intron-1 of the voltage-gated sodium channel revealed a low genetic diversity suggesting reduced polymorphism. Conclusions Multiple resistance in An. coluzzii populations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in the Sahel of this country. The pyrethroid-synergists LLINs (e.g. PermaNet®3.0) and organophosphate-based IRS maybe the alternatives for malaria control in this region.

Transcriptomic analysis of insecticide resistance in the lymphatic filariasis vectorCulex quinquefasciatus

ABSTRACTCulex quinquefasciatusplays an important role in transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF), as well as many arboviral diseases. Currently, efforts to tackleC. quinquefasciatusvectored diseases are based on either mass drug administration (MDA) for LF, or insecticide-based interventions. Widespread and intensive insecticide usage has resulted in increased resistance in mosquito vectors, includingC. quinquefasciatus. Herein, the transcriptome profile of Ugandan bendiocarb-resistantC. quinquefasciatuswas explored to identify candidate genes associated with insecticide resistance. Resistance to bendiocarb in exposed mosquitoes was marked, with 2.04% mortality following 1h exposure and 58.02% after 4h. Genotyping of the G119SAce-1target site mutation detected a highly significant association (p<0.0001; OR=25) between resistance andAce1-119S. However, synergist assays using the P450 inhibitor PBO or the esterase inhibitor TPP resulted in markedly increased mortality (to ≈80%), suggesting a role of metabolic resistance in the resistance phenotype. Using a novel, custom 60K whole-transcriptome microarray 16 genes significantly overexpressed in resistant mosquitoes were detected, with the P450Cyp6z18showing the highest differential gene expression (>8-fold increase vs unexposed controls). These results provide evidence that bendiocarb-resistance in UgandanC. quinquefasciatusis mediated by both target-site mechanisms and over-expression of detoxification enzymes.

Fine-scale spatial and temporal heterogeneities in insecticide resistance profiles of the malaria vector, Anopheles arabiensis in rural south-eastern Tanzania

Background: Programmatic monitoring of insecticide resistance in disease vectors is mostly done on a large scale, often focusing on differences between districts, regions or countries. However, local heterogeneities in residual malaria transmission imply the need for finer-scale data. This study reports small-scale variations of insecticide susceptibility in Anopheles arabiensis between three neighbouring villages across two seasons in Tanzania, where insecticidal bed nets are extensively used, but malaria transmission persists. Methods: WHO insecticide susceptibility assays were conducted on female and male An. arabiensis from three proximal villages, Minepa, Lupiro, and Mavimba, during dry (June-December 2015) and wet (January-May 2016) seasons. Adults emerging from wild-collected larvae were exposed to 0.05% lambda-cyhalothrin, 0.05% deltamethrin, 0.75% permethrin, 4% DDT, 4% dieldrin, 0.1% bendiocarb, 0.1% propoxur, 0.25% pirimiphos-methyl and 5% malathion. A hydrolysis probe assay was used to screen for L1014F (kdr-w) and L1014S (kdr-e) mutations in specimens resistant to DDT or pyrethroids. Synergist assays using piperonly butoxide (PBO) and triphenol phosphate (TPP) were done to assess pyrethroid and bendiocarb resistance phenotypes. Results: There were clear seasonal and spatial fluctuations in phenotypic resistance status in An. arabiensis to pyrethroids, DDT and bendiocarb. Pre-exposure to PBO and TPP, resulted in lower knockdown rates and higher mortalities against pyrethroids and bendiocarb, compared to tests without the synergists. Neither L1014F nor L1014S mutations were detected. Conclusions: This study confirmed the presence of pyrethroid resistance in An. arabiensis and showed small-scale differences in resistance levels between the villages, and between seasons. Substantial, though incomplete, reversal of pyrethroid and bendiocarb resistance following pre-exposure to PBO and TPP, and absence of kdr alleles suggest involvement of P450 monooxygenases and esterases in the resistant phenotypes. We recommend, for effective resistance management, further bioassays to quantify the strength of resistance, and both biochemical and molecular analysis to elucidate specific enzymes responsible in resistance.

Temephos resistance and the associated cross-resistance spectrum in a strain of Culex quinquefasciatus Say (Diptera: Culicidae) from Peliyagoda, Sri Lanka

A heterogeneous population (Pel) of Culex quinquefasciatus Say was selected, by single family rearing, to give a strain, Pel SS, which had low levels of hydrolysis of the esterase substrates α and β-naphthyl acetate. The level of temephos resistance was three-fold lower in this strain than the original parental population, and the levels of resistance to malathion, fenitrothion, pirimiphos-methyl, chlorpyrifos and permethrin were also reduced. In contrast, temephos selection increased the temephos-resistance in the Pel-RR strain ten-fold compared to the original parental population and 29-fold compared to Pel SS. The level of temephos-resistance in Pel RR compared to Pel SS at the LC50 level was 37-fold. Larval cross-resistance, at different levels, was observed to the organophosphorus insecticides, malathion, fenitrothion, parathion, pirimiphos-methyl, chlorpyrifos, fenthion and phoxim, and to the carbamates, propoxur and bendiocarb, but the level of fenthion, propoxur and bendiocarb resistance was less than three-fold. Adults showed cross-resistance to malathion and fenitrothion, but not to propoxur. There was negative cross-resistance to permethrin in both the larvae and adults of Pel RR compared to the Pel and Pel SS strains.