Exploring the molecular mechanisms of increased intensity of pyrethroid resistance in Central African population of a major malaria vector Anopheles coluzzii.

Molecular mechanisms driving the escalation of pyrethroid resistance in the major malaria mosquitoes of Central Africa remain largely uncharacterized, hindering effective management strategies. Here, resistance intensity and the molecular mechanisms driving it were investigated in a population of Anopheles coluzzii from northern Cameroon. High levels of pyrethroid and organochloride resistance were observed in An. coluzzii population, with no mortality for 1× permethrin; only 11% and 33% mortalities for 5× and 10× permethrin diagnostic concentrations, and <2% mortalities for deltamethrin and DDT, respectively. Moderate bendiocarb resistance (88% mortality) and full susceptibility to malathion were observed. Synergist bioassays with piperonyl butoxide recovered permethrin susceptibility, with mortalities increasing to 53.39%, and 87.30% for 5× and 10× permethrin, respectively, implicating P450 monooxygenases. Synergist bioassays with diethyl maleate (DEM) recovered permethrin and DDT susceptibilities (mortalities increasing to 34.75% and 14.88%, respectively), implicating glutathione S-transferases. RNA-seq-based genome-wide transcriptional analyses supported by quantitative PCR identified glutathione S-transferase, GSTe2 (RNA-seqFC = 2.93 and qRT-PCRFC = 8.4, p < 0.0043) and CYP450, CYP6Z2 (RNA-seqFC = 2.39 and qRT-PCRFC = 11.7, p < 0.0177) as the most overexpressed detoxification genes in the pyrethroid-resistant mosquitoes, compared to mosquitoes of the susceptible Ngousso colony. Other overexpressed genes include P450s, CYP6M2 (FC = 1.68, p < 0.0114), CYP4G16 (FC = 2.02, p < 0.0005), and CYP4G17 (FC = 1.86, p < 0.0276). While high frequency of the 1014F kdr mutation (50%) and low frequencies of 1014S (6.61%) and 1575Y (10.29%) were observed, no ace-1 mutation was detected in bendiocarb-resistant populations, suggesting the preeminent role of metabolic mechanism. Overexpression of metabolic resistance genes (including GSTe2 and CYP6Z2 known to confer resistance to multiple insecticides) in An. coluzzii from the Sudan Savannah of Cameroon highlights the need for alternative management strategies to reduce malaria burden in northern Cameroon.

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon.

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a village located 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adult progeny. Bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having a similar sporozoite rate. Both species exhibited high levels of resistance to pyrethroids (<40% mortality). An. gambiae s.s. was also resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively . Furthermore, the high pyrethroid/DDT resistances in An. gambiae s.s. corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the high Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon.

BACKGROUND: The scale-up in the distribution of long-lasting insecticidal nets (LLINs) and indoor residual spraying has significantly reduced malaria burden and mortality. However, insecticide resistance, among other factors, is responsible for a recent rebound in malaria transmission in 2015-2016, threatening the progress so far made. As a contribution towards understanding patterns of resistance and its mechanism in the field we characterized a population of Anopheles gambiae (s.l.) from Gounougou, a Guinea savanna of north/central Cameroon. RESULTS: Indoor collection conducted in September 2017 identified Anopheles coluzzii and Anopheles arabiensis as the unique Anopheles vector species, with abundances of 83 and 17%, respectively. Analysis of infection with TaqMan assays using heads/thoraces of indoor collected females of An. coluzzii revealed a low Plasmodium falciparum parasite rate of 4.7%. Bioassays conducted with female An. coluzzii revealed extreme resistance, with low mortalities of only 3.75 ± 1.25%, 3.03 ± 1.59% and 1.45 ± 1.45%, respectively, for permethrin, deltamethrin and DDT. In contrast, high susceptibility was obtained with the organophosphates and carbamates, with mortalities in the range of 98-100%. Synergist assays with piperonyl butoxide (PBO) recovered some susceptibility with increased mortality for permethrin to 14.88 ± 8.74%, and for deltamethrin to 32.50 ± 10.51% (~27-fold increase compared to mortalities with deltamethrin alone, χ2 = 29, df = 1, P < 0.0001). These correlated with the results of cone bioassays which revealed complete loss of efficacy of Olyset®Net (0% mortality) and PermaNet®2.0 (0% mortality), and the considerable loss of efficacy of Olyset®Plus (mortality of 2 ± 2%), PermaNet®3.0 side panel (mortality of 2 ± 2%) and PermaNet3.0® roof (mortality of 16 ± 5.1%). Time-course bioassays conducted with deltamethrin established a high intensity of resistance, with LT50 of 309.09 (95% CI 253.07-393.71, Fiducial), and a resistance ratio of 93.09 compared with the fully susceptible Ngoussou laboratory colony. TaqMan genotyping revealed a high frequency of the 1014F allele (65.25%) in the An. coluzzii populations. Sequencing of a fragment of the voltage-gated sodium channel identified a single An. arabiensis female harbouring the 1014S kdr mutation. CONCLUSIONS: This finding of high pyrethroid and DDT resistance in An. coluzzii from north-central Cameroon is a major obstacle to malaria control using pyrethroid bednets and indoor residual spraying with DDT.

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 F0 An. 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.