Substrate promiscuity of key resistance P450s confers clothianidin resistance while increasing chlorfenapyr potency in malaria vectors.

Novel insecticides were recently introduced to counter pyrethroid resistance threats in African malaria vectors. To prolong their effectiveness, potential cross-resistance from promiscuous pyrethroid metabolic resistance mechanisms must be elucidated. Here, we demonstrate that the duplicated P450s CYP6P9a/-b, proficient pyrethroid metabolizers, reduce neonicotinoid efficacy in Anopheles funestus while enhancing the potency of chlorfenapyr. Transgenic expression of CYP6P9a/-b in Drosophila confirmed that flies expressing both genes were significantly more resistant to neonicotinoids than controls, whereas the contrasting pattern was observed for chlorfenapyr. This result was also confirmed by RNAi knockdown experiments. In vitro expression of recombinant CYP6P9a and metabolism assays established that it significantly depletes both clothianidin and chlorfenapyr, with metabolism of chlorfenapyr producing the insecticidally active intermediate metabolite tralopyril. This study highlights the risk of cross-resistance between pyrethroid and neonicotinoid and reveals that chlorfenapyr-based control interventions such as Interceptor G2 could remain efficient against some P450-based resistant mosquitoes.

Association of a rapidly selected 4.3kb transposon-containing structural variation with a P450-based resistance to pyrethroids in the African malaria vector Anopheles funestus.

Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance.

Indoor residual spraying of experimental huts in Cameroon highlights the potential of Fludora® Fusion to control wild pyrethroid-resistant malaria vectors.

Elevated resistance to pyrethroids in major malaria vectors has led to the introduction of novel insecticides including neonicotinoids. There is a fear that efficacy of these new insecticides could be impacted by cross-resistance mechanisms from metabolic resistance to pyrethroids. In this study, after evaluating the resistance to deltamethrin, clothianidin and mixture of clothianidin + deltamethrin in the lab using CDC bottle assays, the efficacy of the new IRS formulation Fludora® Fusion was tested in comparison to clothianidin and deltamethrin applied alone using experimental hut trials against wild free-flying pyrethroid-resistant Anopheles funestus from Elende and field An. gambiae collected from Nkolondom reared in the lab and released in the huts. Additionally, cone tests on the treated walls were performed each month for a period of twelve months to evaluate the residual efficacy of the sprayed products. Furthermore, the L1014F-kdr target-site mutation and the L119F-GSTe2 mediated metabolic resistance to pyrethroids were genotyped on a subset of mosquitoes from the EHT to assess the potential cross-resistance. All Anopheles species tested were fully susceptible to clothianidin and clothianidin + deltamethrin mixture in CDC bottle assay while resistance was noted to deltamethrin. Accordingly, Fludora® Fusion (62.83% vs 42.42%) and clothianidin (64.42% vs 42.42%) induced significantly higher mortality rates in EHT than deltamethrin (42.42%) against free flying An. funestus from Elende in month 1 (M1) and no significant difference in mortality was observed between the first (M1) and sixth (M6) months of the evaluation (P > 0.05). However, lower mortality rates were recorded against An. gambiae s.s from Nkolondom (mortality rates 50%, 45.56% and 26.68%). In-situ cone test on the wall showed a high residual efficacy of Fludora® Fusion and clothianidin on the susceptible strain KISUMU (> 12 months) and moderately on the highly pyrethroid-resistant An. gambiae strain from Nkolondom (6 months). Interestingly, no association was observed between the L119F-GSTe2 mutation and the ability of mosquitoes to survive exposure to Fludora® Fusion, whereas a trend was observed with the L1014F-kdr mutation. This study highlights that Fludora® Fusion, through its clothianidin component, has good potential of controlling pyrethroid-resistant mosquitoes with prolonged residual efficacy. This could be therefore an appropriate tool for vector control in several malaria endemic regions.

Entomological surveys and insecticide susceptibility profile of Aedes aegypti during the dengue outbreak in Sao Tome and Principe in 2022.

BACKGROUND: The first dengue outbreak in Sao Tome and Principe was reported in 2022. Entomological investigations were undertaken to establish the typology of Aedes larval habitats, the distribution of Ae. aegypti and Ae. albopictus, the related entomological risk and the susceptibility profile of Ae. aegypti to insecticides, to provide evidence to inform the outbreak response. METHODOLOGY/PRINCIPAL FINDINGS: Entomological surveys were performed in all seven health districts of Sao Tome and Principe during the dry and rainy seasons in 2022. WHO tube and synergist assays using piperonyl butoxide (PBO) and diethyl maleate (DEM) were carried out, together with genotyping of F1534C/V1016I/V410L mutations in Ae. aegypti. Aedes aegypti and Ae. albopictus were found in all seven health districts of the country with high abundance of Ae. aegypti in the most urbanised district, Agua Grande. Both Aedes species bred mainly in used tyres, discarded tanks and water storage containers. In both survey periods, the Breteau (BI > 50), house (HI > 35%) and container (CI > 20%) indices were higher than the thresholds established by WHO to indicate high potential risk of dengue transmission. The Ae. aegypti sampled were susceptible to all insecticides tested except dichlorodiphenyltrichloroethane (DDT) (9.2% mortality, resistant), bendiocarb (61.4% mortality, resistant) and alpha-cypermethrin (97% mortality, probable resistant). A full recovery was observed in Ae. aegypti resistant to bendiocarb after pre-exposure to synergist PBO. Only one Ae. aegypti specimen was found carrying F1534C mutation. CONCLUSIONS/SIGNIFICANCE: These findings revealed a high potential risk for dengue transmission throughout the year, with the bulk of larval breeding occurring in used tyres, water storage and discarded containers. Most of the insecticides tested remain effective to control Aedes vectors in Sao Tome, except DDT and bendiocarb. These data underline the importance of raising community awareness and implementing routine dengue vector control strategies to prevent further outbreaks in Sao Tome and Principe, and elsewhere in the subregion.

Genetic markers associated with the widespread insecticide resistance in malaria vector Anopheles funestus populations across Tanzania.

BACKGROUND: Anopheles funestus is a leading vector of malaria in most parts of East and Southern Africa, yet its ecology and responses to vector control remain poorly understood compared with other vectors such as Anopheles gambiae and Anopheles arabiensis. This study presents the first large-scale survey of the genetic and phenotypic expression of insecticide resistance in An. funestus populations in Tanzania. METHODS: We performed insecticide susceptibility bioassays on An. funestus mosquitoes in nine regions with moderate-to-high malaria prevalence in Tanzania, followed by genotyping for resistance-associated mutations (CYP6P9a, CYP6P9b, L119F-GSTe2) and structural variants (SV4.3 kb, SV6.5 kb). Generalized linear models were used to assess relationships between genetic markers and phenotypic resistance. An interactive R Shiny tool was created to visualize the data and support evidence-based interventions. RESULTS: Pyrethroid resistance was universal but reversible by piperonyl-butoxide (PBO). However, carbamate resistance was observed in only five of the nine districts, and dichloro-diphenyl-trichloroethane (DDT) resistance was found only in the Kilombero valley, south-eastern Tanzania. Conversely, there was universal susceptibility to the organophosphate pirimiphos-methyl in all sites. Genetic markers of resistance had distinct geographical patterns, with CYP6P9a-R and CYP6P9b-R alleles, and the SV6.5 kb structural variant absent or undetectable in the north-west but prevalent in all other sites, while SV4.3 kb was prevalent in the north-western and western regions but absent elsewhere. Emergent L119F-GSTe2, associated with deltamethrin resistance, was detected in heterozygous form in districts bordering Mozambique, Malawi and the Democratic Republic of Congo. The resistance landscape was most complex in western Tanzania, in Tanganyika district, where all five genetic markers were detected. There was a notable south-to-north spread of resistance genes, especially CYP6P9a-R, though this appears to be interrupted, possibly by the Rift Valley. CONCLUSIONS: This study underscores the need to expand resistance monitoring to include An. funestus alongside other vector species, and to screen for both the genetic and phenotypic signatures of resistance. The findings can be visualized online via an interactive user interface and could inform data-driven decision-making for resistance management and vector control. Since this was the first large-scale survey of resistance in Tanzania's An. funestus, we recommend regular updates with greater geographical and temporal coverage.

The internal transcribed spacer 1 sequence polymorphism brings updates to tsetse species distribution in the northern Cameroon: Importance in planning efficient vector control.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning.

Contribution of Anopheles gambiae sensu lato mosquitoes to malaria transmission during the dry season in Djoumouna and Ntoula villages in the Republic of the Congo.

BACKGROUND: Mosquitoes belonging to the Anopheles gambiae sensu lato complex play a major role in malaria transmission across Africa. This study assessed the relative importance of members of An. gambiae s.l. in malaria transmission in two rural villages in the Republic of the Congo. METHODS: Adult mosquitoes were collected using electric aspirators from June to September 2022 in Djoumouna and Ntoula villages and were sorted by taxa based on their morphological features. Anopheles gambiae s.l. females were also molecularly identified. A TaqMan-based assay and a nested polymerase chain reaction (PCR) were performed to determine Plasmodium spp. in the mosquitoes. Entomological indexes were estimated, including man-biting rate, entomological inoculation rate (EIR), and diversity index. RESULTS: Among 176 mosquitoes collected, An. gambiae s.l. was predominant (85.8%), followed by Culex spp. (13.6%) and Aedes spp. (0.6%). Three members of the An. gambiae s.l. complex were collected in both villages, namely An. gambiae sensu stricto (74.3%), Anopheles coluzzii (22.9%) and Anopheles arabiensis (2.8%). Three Plasmodium species were detected in An. gambiae s.s. and An. coluzzii (Plasmodium falciparum, P. malariae and P. ovale), while only P. falciparum and P. malariae were found in An. arabiensis. In general, the Plasmodium infection rate was 35.1% (53/151) using the TaqMan-based assay, and nested PCR confirmed 77.4% (41/53) of those infections. The nightly EIR of An. gambiae s.l. was 0.125 infectious bites per person per night (ib/p/n) in Djoumouna and 0.08 ib/p/n in Ntoula. The EIR of An. gambiae s.s. in Djoumouna (0.11 ib/p/n) and Ntoula (0.04 ib/p/n) was higher than that of An. coluzzii (0.01 and 0.03 ib/p/n) and An. arabiensis (0.005 and 0.0 ib/p/n). CONCLUSIONS: This study provides baseline information on the dominant vectors and dynamics of malaria transmission in the rural areas of the Republic of the Congo during the dry season. In the two sampled villages, An. gambiae s.s. appears to play a predominant role in Plasmodium spp.

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.

Entomological indicators of Plasmodium species transmission in Goma Tsé-Tsé and Madibou districts, in the Republic of Congo.

BACKGROUND: Malaria remains a major public health problem in the Republic of Congo, with Plasmodium falciparum being the deadliest species of Plasmodium in humans. Vector transmission of malaria is poorly studied in the country and no previous report compared rural and urban data. This study aimed to determine the Anopheles fauna and the entomological indices of malaria transmission in the rural and urban areas in the south of Brazzaville, and beyond. METHODS: Indoor household mosquitoes capture using electric aspirator was performed in rural and urban areas during raining and dry seasons in 2021. The identification of Anopheles species was done using binocular magnifier and nested-PCR. TaqMan and nested-PCR were used to detect the Plasmodium species in the head/thorax and abdomens of Anopheles. Some entomological indices including the sporozoite infection rate, the entomological inoculation rate and the man biting rate were estimated. RESULTS: A total of 699 Anopheles mosquitoes were collected: Anopheles gambiae sensu lato (s.l.) (90.7%), Anopheles funestus s.l. (6.9%), and Anopheles moucheti (2.4%). Three species of An. gambiae s.l. were identified including Anopheles gambiae sensu stricto (78.9%), Anopheles coluzzii (15.4%) and Anopheles arabiensis (5.7%). The overall sporozoite infection rate was 22.3% with a predominance of Plasmodium falciparum, followed by Plasmodium malariae and Plasmodium ovale. Anopheles aggressiveness rate was higher in households from rural area (1.1 bites/night) compared to that from urban area (0.8 ib/p/n). The overall entomological inoculation rate was 0.13 ib/p/n. This index was 0.17 ib/p/n and 0.092 ib/p/n in rural and in urban area, respectively, and was similar during the dry (0.18 ib/p/n) and rainy (0.14 ib/p/n) seasons. CONCLUSION: These findings highlight that malaria transmission remains high in rural and urban area in the south of Republic of Congo despite the ongoing control efforts, thereby indicating the need for more robust interventions.

Clothianidin-resistant Anopheles gambiae adult mosquitoes from Yaoundé, Cameroon, display reduced susceptibility to SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying.

BACKGROUND: Chronic exposure of mosquito larvae to pesticide residues and cross-resistance mechanisms are major drivers of tolerance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid prequalified for Indoor Residual Spraying (IRS). METHODS: Using standard bioassays, we tested if reduced susceptibility to clothianidin can affect the efficacy of SumiShield® 50WG, one of four new IRS formulations containing clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp sampled from urban, suburban and agricultural areas of Yaoundé, Cameroon. RESULTS: We found that in this geographic area, the level of susceptibility to the active ingredient predicted the efficacy of SumiShield 50WG. This formulation was very potent against populations that reached 100% mortality within 72 h of exposure to a discriminating concentration of clothianidin. By contrast, mortality leveled off at 75.4 ± 3.5% within 7 days of exposure to SumiShield 50WG in An. gambiae adults collected from a farm where the spraying of the two neonicotinoids acetamiprid and imidacloprid for crop protection is likely driving resistance to clothianidin. CONCLUSIONS: Despite the relatively small geographic extend of the study, the findings suggest that cross-resistance may impact the efficacy of some new IRS formulations and that alternative compounds could be prioritized in areas where neonicotinoid resistance is emerging.

Overexpression and nonsynonymous mutations of UDP-glycosyltransferases are potentially associated with pyrethroid resistance in Anopheles funestus.

UDP-glycosyltransferases (UGTs) enzymes are pivotal in insecticide resistance by transforming hydrophobic substrates into more hydrophilic forms for efficient cell elimination. This study provides the first comprehensive investigation of Anopheles funestus UGT genes, their evolution, and their association with pyrethroid resistance. We employed a genome-wide association study using pooled sequencing (GWAS-PoolSeq) and transcriptomics on pyrethroid-resistant An. funestus, along with deep-targeted sequencing of UGTs in 80 mosquitoes Africa-wide. UGT310B2 was consistently overexpressed Africa-wide and significant gene-wise Fst differentiation was observed between resistant and susceptible populations: UGT301C2 and UGT302A3 in Malawi, and UGT306C2 in Uganda. Additionally, nonsynonymous mutations in UGT genes were identified. Gene-wise Tajima's D density curves provide insights into population structures within populations across these countries, supporting previous observations. These findings have important implications for current An. funestus control strategies facilitating the prediction of cross-resistance to other UGT-metabolised polar insecticides, thereby guiding more effective and targeted insecticide resistance management efforts.

Geographical emergence of sulfadoxine-pyrimethamine drug resistance-associated P. falciparum and P. malariae alleles in co-existing Anopheles mosquito and asymptomatic human populations across Cameroon.

Malaria molecular surveillance remains critical in detecting and tracking emerging parasite resistance to anti-malarial drugs. The current study employed molecular techniques to determine Plasmodium species prevalence and characterize the genetic diversity of Plasmodium falciparum and Plasmodium malariae molecular markers of sulfadoxine-pyrimethamine resistance in humans and wild Anopheles mosquito populations in Cameroon. Anopheles mosquito collections and parasitological survey were conducted in villages to determine Plasmodium species infection, and genomic phenotyping of anti-folate resistance was accomplished by sequencing the dihydrofolate-reductase (dhfr) and dihydropteroate-synthase (dhps) genes of naturally circulating P. falciparum and P. malariae isolates. The malaria prevalence in Elende was 73.5% with the 5-15 years age group harboring significant P. falciparum (27%) and P. falciparum + P. malariae (19%) infections. The polymorphism breadth of the pyrimethamine-associated Pfdhfr marker revealed a near fixation (94%) of the triple-mutant -A16I51R59N108I164. The Pfdhps backbone mediating sulfadoxine resistance reveals a high frequency of the V431A436G437K540A581A613 alleles (20.8%). Similarly, the Pmdhfr N50K55L57R58S59S114F168I170 haplotype (78.4%) was predominantly detected in the asexual blood stage. In contrast, the Pmdhps- S436A437occured at 37.2% frequency. The combined quadruple N50K55L57R58S59S114F168I170_ S436G437K540A581A613 (31.9%) was the major circulating haplotype with similar frequency in humans and mosquitoes. This study highlights the increasing frequency of the P. malariae parasite mostly common in asymptomatic individuals with apparent P. falciparum infection. Interventions directed at reducing malaria transmission such as the scaling-up of SP are favoring the emergence and spread of multiple drug-resistant alleles between the human and mosquito host systems.

Impact of a small-scale tsetse fly control operation with deltamethrin impregnated "Tiny Targets" on tsetse density and trypanosomes' circulation in the Campo sleeping sickness focus of South Cameroon.

BACKGROUND: Significant progress has been made towards African sleeping sickness elimination in the last decade. Indeed, the World Health Organization (WHO) global goal of eliminating the chronic form of the disease as a public health problem was achieved in 2020 (i.e., < 2,000 new cases per year). Vector control has played an important role in achieving this goal. In this study, we evaluated the impact of the insecticide impregnated Tiny Targets on tsetse fly densities and their infection rates with Trypanosoma spp in the Campo sleeping sickness focus of South Cameroon. METHODS: The study site was divided into two areas: (i) the south-west experimental area, which included vector control, and (ii) the eastern part as the non-intervention area. After compiling the baseline entomological data (tsetse densities and trypanosome infection rates), around 2000 Tiny Targets were deployed in the South-West area and replaced every six months for two years. Post-intervention surveys were conducted every six months to determine tsetse densities and levels of trypanosome infections with PCR-based methods. RESULTS: Following the intervention, tsetse mean catches decreased by 61% after six months, and up to 73% after twelve months (pre-intervention: 2.48 flies/trap/day, 95%CI [1.92-3.14]; 12-months post-intervention: 0.66 tsetse/trap/day, 95%CI [0.42-0.94]). This decrease was not sustained after 18 months, and the mean catch doubled compared to that after 12 months. After 24 months, the mean catches still increased by 17% (18 months: 1.45 tsetse/trap/day, 95%CI [1.07-1.90] and 24 months: 1.71 tsetse/trap/day, 95%CI [1.27-2.24]). In the non-intervention area, a variation in tsetse catches was observed during the two years, with a general increase from 2.43 [0.73-5.77] to 3.64 [1.47-7.70] tsetse/trap/day. In addition, trypanosome infection rates dropped by 75% in both areas (P-value < 0.001) from 21.20% to 5.06% and from 13.14% to 3.45% in intervention and control areas respectively. CONCLUSION: Tiny targets have proven useful in reducing tsetse population densities and trypanosome infection rates, providing evidence for the integration of this tool in current strategies towards trypanosomiasis elimination in Campo. The non-sustained decrease of tsetse densities after one year may indicate reinvasions from neighbouring breeding sites or that the intervention area was not large enough. Our results show the need to scale up by accessing difficult breeding sites and extend the tiny targets to the whole transborder focus.

Community evaluation of the physical and insecticidal durability of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide incorporated mosquito net: protocol for a multi-country study in West, Central and East Africa.

BACKGROUND: Pyrethroid-PBO nets have demonstrated improved impact against clinical malaria transmitted by pyrethroid resistant mosquito vectors and are being scaled up across Africa. However very little is known about their physical and insecticidal durability under operational conditions. This study will investigate the attrition, fabric integrity, insecticide content and bioefficacy of DuraNet® Plus, a new WHO prequalified alphacypermethrin and PBO incorporated net developed by Shobikaa Impex Private Limited over 3 years of field use in communities in Benin, Cameroon and Tanzania. METHODS: The study will be conducted in parallel in selected villages in Zakpota District in Benin, Mbalmayo, District in Cameroon and Muheza District in Tanzania. In each country, ~ 1800 households will be recruited and randomised to receive DuraNet® Plus or DuraNet® (a WHO prequalified alphacypermethrin-only ITN). Follow up surveys will be performed at 1 month post distribution to investigate adverse events and subsequently every 6-12 months to assess ITN attrition and fabric integrity following standard WHO procedures. A second cohort of nets will be withdrawn every 6-12 months and assessed for alpha-cypermethrin and PBO content and for entomological activity in laboratory bioassays (cone bioassays and tunnel tests). Alpha-cypermethrin bioefficacy will be monitored using the susceptible Anopheles gambiae Kisumu strain in cone bioassays while PBO bioefficacy will be monitored using pyrethroid resistant strains with overexpressed P450 enzymes in tunnel tests to determine the proportion of efficacious nets (≥ 95% knockdown, ≥ 80% mortality or ≥ 90% blood feeding inhibition in tunnels) at each time point. Nets withdrawn at 12, 24 and 36 months from each country will also be tested in experimental hut trials against wild free-flying pyrethroid resistant Anopheles gambiae sl in Côvè Benin to investigate the superiority of DuraNet® Plus over DuraNet® at each time point under semi field conditions. CONCLUSION: This large-scale multi country trial will provide useful information on the durability of a pyrethroid-PBO net (DuraNet® Plus) in 3 different regions in sub-Saharan Africa. The methods proposed for bioefficacy testing could also contribute towards the development of new standardised guidelines for monitoring the insecticidal efficacy of pyrethroid-PBO nets under operational conditions.

Entomological longitudinal surveys in two contrasted eco-climatic settings in Cameroon reveal a high malaria transmission from Anopheles funestus associated with GSTe2 metabolic resistance.

BACKGROUND: The impact of metabolic resistance to insecticides on malaria transmission remains poorly characterised notably through application of entomological parameters. The lack of resistance markers has been one of the limiting factors preventing a robust assessment of such impact. To this end, the present study sought to investigate how the L119F-Gste2 metabolic gene influences entomological parameters underpinning mosquitos' propensity to transmit Plasmodium spp. METHODS: Longitudinal studies were carried out in Mibellon and Elende, two different eco-climatic settings in Cameroon and mosquitoes were collected using Human Landing Catch (HLC), Centre for Disease Control Light Trap (CDC-LT) and Pyrethrum Spray Catch (PSC) technics. Plasmodium sporozoite parasites were detected by TaqMan and Nested PCR, and blood meal origin by ELISA. The allele-specific PCR (AS-PCR) method was used to genotype the L119F-GSTe2 marker and association with malaria transmission was established by comparing key transmission parameters such as the Entomological Inoculation Rate (EIR) between individuals with different L119F-GSTe2 genotypes. RESULTS: An. funestus s.l was the predominant malaria vector collected during the entomological survey in both sites (86.6% and 96.4% in Elende and Mibellon, respectively) followed by An. gambiae s.l (7.5% and 2.4%, respectively). Sporozoite infection rates were very high in both collection sites (8.7% and 11% in Elende and Mibellon, respectively). An. funestus s.s exhibited a very high entomological inoculation rate (EIR) (66 ib/h/month and 792 ib/h/year) and was responsible for 98.6% of all malaria transmission events occurring in both sites. The Human Blood Index was also high in both locations (HBI = 94%). An. funestus s.s. mosquitoes with both 119 F/F (RR) and L119F (RS) genotypes had a significantly higher transmission intensity than their susceptible L/L119 (SS) counterparts (IRR = 2.2, 95%CI (1.1-5.2), p = 0.03; IRR = 2.5, 95% CI (1.2-5.8), p = 0.01 respectively). CONCLUSION: This study highlights the major role that An. funestus s.s plays in malaria transmission in Cameroon with an aggravation from GSTe2-based metabolic resistance.

Can the use of larviciding with biological compounds contribute in increasing Anopheles gambiae s.l. susceptibility to pyrethroid in a population expressing high resistance intensity?

BACKGROUND: Larviciding using non-insecticide compounds is considered appropriate for controlling outdoor biting mosquitoes and for managing insecticide resistance. However, there is still not enough information on the influence of larviciding in managing pyrethroid resistance. In the present study, we checked whether the introduction of larviciding using the biolarvicide VectoMax G in the city of Yaoundé is contributing in restoring the susceptibility of An. coluzzii populations to pyrethroids. METHODOLOGY: The susceptibility status of field An. coluzzii population was evaluated at different time points before and during larviciding treatments. In addition, An. coluzzii larvae collected in the city of Yaoundé, were split into four groups and exposed to different selection regimes for many generations as follow; (i): deltamethrin 0.05%_only, (ii): Vectomax_only, (iii): Vectomax+deltamethrin 0.05%, (iv): VectoMax+deltamethrin 0.05% + susceptible. Life traits parameters were measured in the progeny and compared between colonies. The control was the susceptible laboratory strain "Ngousso". Kdr allele frequency and the profile of expression of different detoxification genes and oxidative stress genes was checked using qPCR analysis. Gene's expression was compared between the first and the last generation of each colony and in field populations collected before and during larviciding. RESULTS: An increase in mosquito susceptibility to deltamethrin and permethrin was recorded for the field populations after larviciding implementation. Resistance intensity to deltamethrin was found to decrease from high to low in field populations. Only the colony vectomax+deltamethrin+susceptible presented a high susceptibility to deltamethrin after 21 generations. The kdr gene frequency was found to be unchanged in the field population and laboratory colonies. A significant decrease in the overexpression profile of Gste2 was detected in field population after larviciding implementation. Other genes showing a similar pattern though not significant were Cyp6z1, Cyp6p1 and Cyp6g16. Concerning fitness only the colony vectomax+deltamethrin+susceptible was found to display a fitness profile similar to the susceptible colony with high fecundity, high hatching rate, short development time and long adult survival rate. CONCLUSION: The profile of the field population supported reversal of phenotypic resistance to pyrethroids however no reduction in the frequency of the kdr allele was recorded. Some detoxification genes were detected less overexpressed. The study suggest that reversal may take longer to achieve in a population expressing a very high resistance profile and under continuous insecticide selection pressure.

Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo.

Assessing patterns and evolution of insecticide resistance in malaria vectors is a prerequisite to design suitable control strategies. Here, we characterised resistance profile in Anopheles gambiae and Anopheles funestus in Kinshasa and assess the level of aggravation by comparing to previous 2015 estimates. Both species collected in July 2021 were highly resistant to pyrethroids at 1×, 5× and 10× concentrations (mortality < 90%) and remain fully susceptible to bendiocarb and pirimiphos methyl. Compared to 2015, Partial recovery of susceptibility was observed in A. gambiae after PBO synergist assays for both permethrin and α-cypermethrin and total recovery of susceptibility was observed for deltamethrin in 2021. In addition, the efficacy of most bednets decreased significantly in 2021. Genotyping of resistance markers revealed a near fixation of the L1014-Kdr mutation (98.3%) in A. gambiae in 2021. The frequency of the 119F-GSTe2 resistant significantly increased between 2015 and 2021 (19.6% vs 33.3%; P = 0.02) in A. funestus. Transcriptomic analysis also revealed a significant increased expression (P < 0.001) of key cytochrome P450s in A. funestus notably CYP6P9a. The escalation of pyrethroid resistance observed in Anopheles populations from Kinshasa coupled with increased frequency/expression level of resistance genes highlights an urgent need to implement tools to improve malaria vector control.

Characterization of the swarming behavior of Anopheles coluzzii and Anopheles gambiae (Diptera: Culicidae) populations in a hybrid zone of Senegal.

Anopheles gambiae and Anopheles coluzzii, often found in sympatry and synchronous, have undergone a premating reproductive isolation across their distribution range. However, in the Western coast of Africa, unexpected hybridization zones have been observed, and little is known about swarming behavior of these cryptic taxa. Here, we characterized the swarming behavior of An. coluzzii and An. gambiae to investigate its role in the high hybridization level in Senegal. The study was conducted in the south and central Senegal during the 2018 rainy season. Mating swarms of malaria vectors were surveyed at sunset and collected using an insect net. Meanwhile, indoor resting populations of malaria vectors were collected by pyrethrum spray catches. Upon collection, specimens were identified morphologically, and then members of the An. gambiae complex were identified at the species level by polymerase chain reaction (PCR). An. gambiae swarmed mainly over bare ground, whereas An. coluzzii were found swarming above various objects creating a dark-light contrast with the bare ground. The swarms height varied from 0.5 to 2.5 m. Swarming starting time was correlated with sunset whatever the months for both species, and generally lasted about 10 min. No mixed swarm of An. gambiae and An. coluzzii was found even in the high hybridization area. These results indicated a premating isolation between An. coluzzii and An. gambiae. However, the high hybridization rate in the sympatric area suggests that heterogamous mating is occurring, thus stressing the need for further extensive studies.

Larviciding intervention targeting malaria vectors also affects Culex mosquito distribution in the city of Yaoundé, Cameroon.

Although Culex species are considered to be equally affected by control measures targeting malaria vectors, there is still not enough evidence of the impact of interventions such as larviciding on the distribution of these mosquito species. The present study assessed the impact of a larviciding trial targeting malaria vectors on Culex mosquito species in the city of Yaoundé, Cameroon. A cluster randomized trial comparing 13 treated clusters and 13 untreated clusters was implemented. Data were collected at baseline and during the larviciding intervention, from March 2017 to November 2020. The microbial larvicide VectoMax G was applied once every 2 weeks in the intervention areas. Adult mosquitoes were collected using CDC light traps in both intervention and non-intervention areas and compared between arms. Globally, larviciding intervention was associated with 69% reduction in aquatic habitats with Culex larvae and 36.65% reduction of adult Culex densities in houses. Adult Culex densities were reduced both indoors (35.26%) and outdoors (42.37%). No change in the composition of Culex species was recorded. The study suggests a high impact of larviciding on Culex mosquito species distribution. The impact of the intervention can be improved if typical Culex breeding habitats including pit latrines are targeted.