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Abstract The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with

Abstract Background The efficacy of vector control tools depends on the behavior of the vector species. Many studies have sought to determine the feeding behavior of Anopheles mosquitoes in different settings of Ethiopia. We have performed a systematic review aimed to generate pooled evidence on the overall and species-specific blood meal sources of Anopheles mosquitoes in Ethiopia. Methods A search for relevant articles was performed in two electronic databases (PubMed and Science Direct) and three search engines (Google Scholar, Research Gate and Google) between 11 March and 2 April 2024. Following the initial identification of articles, we used EndNote X8 software and removed duplicate articles and screened the remaining articles by careful reading of their titles and abstracts. The full text of articles that passed this screening phase was retrieved, read and evaluated against predetermined selection criteria. The final decision for inclusion in the systematic review was made after a methodological quality check using the JBI critical appraisal checklist. All relevant data were extracted from tables, figures and texts of the included articles using a premade template in Excel, and the data were analyzed using Stata version 14 software. Results Of the 2431 studies identified, 27 met the inclusion criteria; all were published between 1997 and 2024. At 215 data points (frequency of tests of each Anopheles species by location and method of mosquito collections), 18,771 Anopheles mosquitoes belonging to 23 species or species complexes were tested for blood meal sources. The commonest sources of blood meals for Anopheles mosquitoes were bovine (36.0%, n = 6758) and human (29.4%, n = 5520). Among the tested anophelines, Anopheles (An.) arabiensis accounted for 67.9% (n = 12,741), followed by An. pharoensis, An. demeilloni and An. stephensi at 10.0%, 5.6% and 4.4%, respectively. Overall, there was no difference in the mean proportion of An. arabiensis detected with domestic animal blood (33.4%, 95% confidence interval [CI] 32.4–34.4%) and those detected with human blood (31.8%, 95% CI 30.9–32.8%). However, a greater proportion of the outdoor collected An. arabiensis were found to feed on bovines (47.9%, 95% CI 35.3–60.6) compared to humans (12.9%, 95% CI 0.8–24.9, P

Abstract The two main Afrotropical malaria vectors - Anopheles coluzzii and An. gambiae – are genetically distinct and reproductively isolated across West Africa. However, populations at the western extreme of their range are assigned as “intermediate” between the two species by whole genome sequence (WGS) data, and as hybrid forms by conventional molecular diagnostics. By exploiting WGS data from 1190 specimens collected across west Africa via the Anopheles gambiae 1000 Genomes network, we identified a putative taxon in the far-west (provisionally named Bissau molecular form), which did not arise by admixture but rather may have originated at the same time as the split between An. coluzzii and An. gambiae. Intriguingly, this taxon lacks insecticide resistance mechanisms commonly observed in the two main species. These findings lead to a change of perspective on malaria vector species in the far-west region with potential for epidemiological implications, and a new challenge for genetic-based mosquito control approaches.

Abstract 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. Graphical Abstract

Abstract Resistance to insecticides and adaptation to a diverse range of environments present challenges to Anopheles gambiae s.l. mosquito control efforts in sub-Saharan Africa. Whole-genome-sequencing is often employed for identifying the genomic basis underlying adaptation in Anopheles, but remains expensive for large-scale surveys. Reduced coverage whole-genome-sequencing can identify regions of the genome involved in adaptation at a lower cost, but is currently untested in Anopheles mosquitoes. Here, we use reduced coverage WGS to investigate population genetic structure and identify signatures of local adaptation in Anopheles mosquitoes across southern Ghana. In contrast to previous analyses, we find no structuring by ecoregion, with Anopheles coluzzii and Anopheles gambiae populations largely displaying the hallmarks of large, unstructured populations. However, we find signatures of selection at insecticide resistance loci that appear ubiquitous across ecoregions in An. coluzzii, and strongest in forest ecoregions in An. gambiae. Our study highlights resistance candidate genes in this region, and validates reduced coverage WGS, potentially to very low coverage levels, for population genomics and exploratory surveys for adaptation in Anopheles taxa.

Abstract Background Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. Methods Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households’ exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. Results Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75–100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9–100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). Conclusions This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission. Graphical Abstract

Abstract Background Aedes aegypti is the main vector of arboviral diseases worldwide. The species invaded and became established in southern Iran in 2020. Insecticide-based interventions are primarily used for its control. With insecticide resistance widespread, knowledge of resistance mechanisms is vital for informed deployment of insecticidal interventions, but information from Iranian Ae. aegypti is lacking. Methods Fifty-six Ae. aegypti specimens were collected from the port city of Bandar Lengeh in Hormozgan Province in the South of Iran in 2020 and screened for kdr mutations. The most common kdr mutations in Latin America and Asia (V410L, S989P, V1016G/I and F1534C), especially when present in combinations, are highly predictive of DDT and pyrethroid resistance were detected. Phylogenetic analyses based on the diversity of S989P and V1016G/I mutations were undertaken to assess the phylogeography of these kdr mutations. Results Genotyping all four kdr positions of V410L, S989P, V1016G/I and F1534C revealed that only 16 out of the 56 (28.57%) specimens were homozygous wild type for all kdr mutation sites. Six haplotypes including VSVF (0.537), VSVC (0.107), LSVF (0.016), LSIF (0.071), VPGC (0.257) and LPGC (0.011) were detected in this study. For the first time, 11 specimens harbouring the V410L mutation, and 8 samples with V1016I mutation were found. V410L and V1016I were coincided in 8 specimens. Also, six specimens contained 1016G/I double mutation which was not reported before. Conclusions The relatively high frequency of these kdr mutations in Iranian Ae. aegypti indicates a population exhibiting substantial resistance to pyrethroid insecticides, which are used widely in control operations and household formulations. The detection of the 410L/1016I kdr mutant haplotype in Iranian Ae. aegypti suggests possible convergence of invasive populations from West Africa or Latin America. However, as Iran has very limited maritime/air connections with those African countries, a Latin American origin for the invasive Ae. aegypti in Iran is more plausible. Graphical abstract

Abstract Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods.

Abstract Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestus group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestus group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa.

BackgroundEndemic African malaria vectors are poorly adapted to typical urban ecologies. However, Anopheles stephensi, an urban malaria vector formerly confined to South Asia and the Persian Gulf, was recently detected in Africa and may change the epidemiology of malaria across the continent. Little is known about the public health implications of An. stephensi in Africa. This study is designed to assess the relative importance of household exposure to An. stephensi and endemic malaria vectors for malaria risk in urban Sudan and Ethiopia.MethodsCase-control studies will be conducted in 3 urban settings (2 in Sudan, 1 in Ethiopia) to assess the association between presence of An. stephensi in and around households and malaria. Cases, defined as individuals positive for Plasmodium falciparum and/or P. vivax by microscopy/rapid diagnostic test (RDT), and controls, defined as age-matched individuals negative for P. falciparum and/or P. vivax by microscopy/RDT, will be recruited from public health facilities. Both household surveys and entomological surveillance for adult and immature mosquitoes will be conducted at participant homes within 48 hours of enrolment. Adult and immature mosquitoes will be identified by polymerase chain reaction (PCR). Conditional logistic regression will be used to estimate the association between presence of An. stephensi and malaria status, adjusted for co-occurrence of other malaria vectors and participant gender.ConclusionsFindings from this study will provide evidence of the relative importance of An. stephensi for malaria burden in urban African settings, shedding light on the need for future intervention planning and policy development.

Abstract Malaria control uses insecticides to kill Anopheles mosquitoes. Recent successes in malaria control are threatened by increasing levels of insecticide resistance (IR), requiring insecticide resistance management (IRM) strategies to mitigate this problem. Field trials of IRM strategies are usually prohibitively expensive with long timeframes, and mathematical modeling is often used to evaluate alternative options. Previous IRM models in the context of malaria control assumed IR to have a simple (monogenic) basis, whereas in natural populations, IR will often be a complex polygenic trait determined by multiple genetic variants. A quantitative genetics model was developed to model IR as a polygenic trait. The model allows insecticides to be deployed as sequences (continuous deployment until a defined withdrawal threshold, termed “insecticide lifespan”, as indicated by resistance diagnosis in bioassays), rotations (periodic switching of insecticides), or full‐dose mixtures (two insecticides in one formulation). These IRM strategies were compared based on their “strategy lifespan” (capped at 500 generations). Partial rank correlation and generalized linear modeling was used to identify and quantify parameters driving the evolution of resistance. Random forest models were used to identify parameters offering predictive value for decision‐making. Deploying single insecticides as sequences or rotations usually made little overall difference to their “strategy lifespan”, though rotations displayed lower mean and peak resistances. Deploying two insecticides in a full‐dose mixture formulation was found to extend the “strategy lifespan” when compared to deploying each in sequence or rotation. This pattern was observed regardless of the level of cross resistance between the insecticides or the starting level of resistance. Statistical analysis highlighted the importance of insecticide coverage, cross resistance, heritability, and fitness costs for selecting an appropriate IRM strategy. Full‐dose mixtures appear the most promising of the strategies evaluated, with the longest “strategy lifespans”. These conclusions broadly corroborate previous results from monogenic models.

Abstract Background Since 2000, Burkina Faso has experienced regular dengue cases and outbreaks, making dengue an increasingly important health concern for the country. Previous studies in Burkina Faso reported that resistance of Aedes aegypti to pyrethroid insecticides was associated with the F1534C and V1016I kdr mutations. The current study reports high resistance of Ae. aegypti populations to pyrethroid insecticides, likely supported by mutations in the voltage-gated sodium channel, here evidenced by genotyping the kdr SNPs V410L, V1016I and F1534C. We also describe a new multiplex PCR-based diagnostic of F1534C and V1016I kdr SNPs. Methods Larvae of Ae. aegypti were collected from three health districts of Ouagadougou in 2018. The resistance status of Ae. aegypti to permethrin (15 μg/ml) and deltamethrin (10 μg/ml) was tested using bottles and to malathion (5%) using WHO tube tests. All bioassays used 1-h exposure and mortality recorded 24 h post-exposure. Bioassay results were interpreted according to WHO thresholds for resistance diagnosis. The kdr mutations were screened using AS-PCR and TaqMan methods in exposed and non-exposed Aedes mosquitoes. Results Females from all health districts were resistant to permethrin and deltamethrin (

Abstract Background Outbreaks of Aedes-borne arboviral diseases are becoming rampant in Africa. In Ghana, there is no organized arboviral control programme with interventions restricted to mitigate outbreaks. Insecticide application is a crucial part of outbreak responses and future preventative control measures. Thus, knowledge of the resistance status and underlying mechanisms of Aedes populations is required to ensure optimal insecticide choices. The present study assessed the insecticide resistance status of Aedes aegypti populations from southern Ghana (Accra, Tema and Ada Foah) and northern Ghana (Navrongo) respectively. Methods Phenotypic resistance was determined with WHO susceptibility tests using Ae. aegypti collected as larvae and reared into adults. Knockdown resistance (kdr) mutations were detected using allele-specific PCR. Synergist assays were performed with piperonyl butoxide (PBO) to investigate the possible involvement of metabolic mechanisms in resistance phenotypes. Results Resistance to DDT was moderate to high across sites (11.3 to 75.8%) and, for the pyrethroids deltamethrin and permethrin, moderate resistance was detected (62.5 to 88.8%). The 1534C kdr and 1016I kdr alleles were common in all sites (0.65 to 1) and may be on a trajectory toward fixation. In addition, a third kdr mutant, V410L, was detected at lower frequencies (0.03 to 0.31). Pre-exposure to PBO significantly increased the susceptibility of Ae. aegypti to deltamethrin and permethrin (P

Abstract Developing robust and standardised approaches for testing mosquito populations against insecticides is vital for understanding the effectiveness of new active ingredients or formulations. Methods for testing mosquito susceptibility against contact insecticides or products, such as those delivered through public health programmes, are well-established and standardised. Nevertheless, approaches for testing volatile or aerosolized insecticides used in household products can be challenging to implement efficiently. We adapted WHO guidelines for household insecticides to develop a standardised and higher-throughput methodology for testing aerosolized products in a Peet Grady test chamber (PG-chamber) using caged mosquitoes and an efficient decontamination method. The new approach was validated using insecticide resistant and susceptible Aedes and Anopheles mosquito colonies. An added feature is the inclusion of cage-facing cameras to allow real-time quantification of knockdown following insecticide exposure. The wipe-based decontamination method was highly effective for removing pyrethroids' aerosolized oil-based residues from chamber surfaces, with

Molecular surveillance of resistance is an increasingly important part of vector borne disease control programmes that utilise insecticides. The visceral leishmaniasis (VL) elimination programme in India uses indoor residual spraying (IRS) with the pyrethroid, alpha-cypermethrin to control Phlebotomus argentipes the vector of Leishmania donovani, the causative agent of VL. Prior long-term use of DDT may have selected for knockdown resistance (kdr) mutants (1014F and S) at the shared DDT and pyrethroid target site, which are common in India and can also cause pyrethroid cross-resistance. We monitored the frequency of these marker mutations over five years from 2017-2021 in sentinel sites in eight districts of north-eastern India covered by IRS. Frequencies varied markedly among the districts, though finer scale variation, among villages within districts, was limited. A pronounced and highly significant increase in resistance-associated genotypes occurred between 2017 and 2018, but with relative stability thereafter, and some reversion toward more susceptible genotypes in 2021. Analyses linked IRS with mutant frequencies suggesting an advantage to more resistant genotypes, especially when pyrethroid was under-sprayed in IRS. However, this advantage did not translate into sustained allele frequency changes over the study period, potentially because of a relatively greater net advantage under field conditions for a wild-type/mutant genotype than projected from laboratory studies and/or high costs of the most resistant genotype. Further work is required to improve calibration of each 1014 genotype with resistance, preferably using operationally relevant measures. The lack of change in resistance mechanism over the span of the study period, coupled with available bioassay data suggesting susceptibility, suggests that resistance has yet to emerge despite intensive IRS. Nevertheless, the advantage of resistance-associated genotypes with IRS and under spraying, suggest that measures to continue monitoring and improvement of spray quality are vital, and consideration of future alternatives to pyrethroids for IRS would be advisable.

Abstract Background Declines in global malaria cases and deaths since the millennium are currently challenged by multiple factors including funding limitations, limits of, and resistance to vector control tools, and also recent spread of the invasive vector species, Anopheles stephensi—especially into novel urban settings where malaria rates are typically low. Coupled with general increases in urbanization and escalations in the number of conflicts creating rapid and unplanned population displacement into temporary shelter camps within host urban areas, particularly in the Middle East and sub-Saharan Africa, increased urban malaria is a major threat to control and elimination. Methods Entomological monitoring surveys (targeting Aedes aegypti) of water containers across urban areas hosting internally displaced people (IDP) communities in Aden city, Yemen, were performed by The MENTOR Initiative, a non-governmental organisation. As part of these surveys in 2021 23 larvae collected and raised to adults were morphologically identified as An. stephensi. Twelve of the samples were sent to Liverpool School of Tropical Medicine for independent morphological assessment and genetic analysis by sequencing the ribosomal ITS2 region and the mitochondrial COI gene. Results All twelve samples were confirmed morphologically and by sequence comparison of the single ITS2 and COI haplotype detected to the NCBI BLAST database as An. stephensi. Phylogenetic analysis with comparable COI sequences indicated close relationship to haplotypes found in Djibouti and Ethiopia. Conclusion The study results confirm the presence of An. stephensi in Yemen. Confirmation of the species in multiple urban communities hosting thousands of IDPs living in temporary shelters with widescale dependency on open water containers is of particular concern due to the vulnerability of the population and abundance of favourable breeding sites for the vector. Proactive monitoring and targeted integrated vector management are required to limit impacts in this area of typically low malaria transmission, and to prevent further the spread of An. stephensi within the region.

Abstract Background Outbreaks of dengue fever caused by viruses transmitted by Aedes aegypti mosquitoes are repeated occurrences in West Africa. In recent years, Burkina Faso has experienced major dengue outbreaks, most notably in 2016 and 2017 when 80% of cases were recorded in Ouagadougou City (Central Health Region). In order to better understand the ecology of this vector and to provide information for use in developing control measures, a study on the characteristics of Aedes container breeding sites and the productivity of such sites, as measured by the abundance of immature stages and resultant adult body size, was undertaken in three health districts (Baskuy, Bogodogo and Nongremassom) of Ouagadougou. Methods Adult mosquitoes were collected indoors and outdoors in 643 households during the rainy season from August to October 2018. The presence of water containers was systematically recorded and the containers examined for the presence or absence of larvae. Characteristics of the container breeding sites, including size of the container and temperature, pH and conductivity of the water contained within, were recorded as well as the volume of water. Traditional Stegomyia indices were calculated as quantitative indicators of the risk of dengue outbreaks; generalised mixed models were fitted to larval and pupal densities, and the contribution of each covariate to the model was evaluated by the Z-value and associated P-value. Results A total of 1061 container breeding sites were inspected, of which 760 contained immature stages of Ae. aegypti (‘positive’ containers). The most frequent container breeding sites found in each health district were tyres and both medium (buckets/cans/pots) and large (bins/barrels/drums) containers; these containers were also the most productive larval habitats and the types that most frequently tested positive. Of the Stegomyia indices, the Breteau, House and Container indices exceeded WHO dengue risk thresholds. Generalised linear mixed models showed that larval and pupal abundances were associated with container type, physicochemical characteristics of the water and collection month, but there were significant differences among container types and among health districts. Aedes aegypti body size was positively associated with type and diameter of the container, as well as with electrical conductivity of the water, and negatively associated with pH and temperature of the water and with the level of exposure of the container to sunlight. Conclusion This study provides data on putative determinants of the productivity of habitats regarding Ae. aegypti immature stages. These data are useful to better understand Ae. aegypti proliferation. The results suggest that identifying and targeting the most productive container breeding sites could contribute to dengue vector control strategies in Burkina Faso. Graphical Abstract

Understanding the contribution of asymptomatic Plasmodium carriers in malaria transmission might be helpful to design and implement new control measures. The present study explored the prevalence of asymptomatic and symptomatic Plasmodium infections (asexual and sexual stages) and the contribution of asymptomatic P. falciparum carriers to Anopheles-mediated malaria transmission in Ouidah (Benin). Thick and thin blood smears were examined from finger-prick blood specimens using light microscopy, and the density of both asexual and sexual stages of Plasmodium species was calculated. Infectivity of gametocyte-infected blood samples to Anopheles gambiae was assessed through direct membrane feeding assays. The prevalence of asymptomatic Plasmodium infections was 28.73% (289/1006). All the asymptomatic gametocyte-carriers (19/19), with gametocytaemia ranging from 10 ̶ 1200 gametocytes/μL of blood, were infectious to An. gambiae mosquitoes. The mean oocyst prevalences varied significantly (χ2 = 16.42, df = 7, p = 0.02) among laboratory mosquito strains (6.9 ̶ 39.4%) and near-field mosquitoes (4.9 ̶ 27.2%). Likewise, significant variation (χ2 = 56.85, df = 7, p = 6.39 × 10−10) was observed in oocyst intensity. Our findings indicate that asymptomatic Plasmodium carriers could significantly contribute to malaria transmission. Overall, this study highlights the importance of diagnosing and treating asymptomatic and symptomatic infection carriers during malaria control programmes.

Abstract Background Vector control is the main intervention used to control arboviral diseases transmitted by Aedes mosquitoes because there are no effective vaccines or treatments for most of them. Control of Aedes mosquitoes relies heavily on the use of insecticides, the effectiveness of which may be impacted by resistance. In addition, rational insecticide application requires detailed knowledge of vector distribution, dynamics, resting, and feeding behaviours, which are poorly understood for Aedes mosquitoes in Africa. This study investigated the spatiotemporal distribution and insecticide resistance status of Aedes aegypti across ecological extremes of Ghana. Methods Immature mosquitoes were sampled from containers in and around human dwellings at seven study sites in urban, suburban, and rural areas of Ghana. Adult Aedes mosquitoes were sampled indoors and outdoors using Biogents BG-Sentinel 2 mosquito traps, human landing catches, and Prokopack aspiration. Distributions of immature and adult Aedes mosquitoes were determined indoors and outdoors during dry and rainy seasons at all sites. The phenotypic resistance status of Aedes mosquitoes to insecticides was determined using World Health Organization susceptibility bioassays. The host blood meal source was determined by polymerase chain reaction. Results A total of 16,711 immature Aedes were sampled, with over 70% found in car tyres. Significantly more breeding containers had Aedes immatures during the rainy season (11,856; 70.95%) compared to the dry season (4855; 29.05%). A total of 1895 adult Aedes mosquitos were collected, including Aedes aegypti (97.8%), Aedes africanus (2.1%) and Aedes luteocephalus (0.1%). Indoor sampling of adult Aedes yielded a total of 381 (20.1%) and outdoor sampling a total of 1514 (79.9%) mosquitoes (z = − 5.427, P = 0.0000) over the entire sampling period. Aedes aegypti populations were resistant to dichlorodiphenyltrichloroethane at all study sites. Vectors showed suspected resistance to bendiocarb (96–97%), permethrin (90–96%) and deltamethrin (91–96%), and were susceptible to the organophosphate for all study sites. Blood meal analysis showed that the Aedes mosquitoes were mostly anthropophilic, with a human blood index of 0.9 (i.e. humans, 90%; human and dog, 5%; dog and cow, 5%). Conclusions Aedes mosquitoes were found at high densities in all ecological zones of Ghana. Resistance of Aedes spp. to pyrethroids and carbamates may limit the efficacy of vector control programmes and thus requires careful monitoring. Graphical abstract

Abstract Background Resistance in malaria vectors to pyrethroids, the most widely used class of insecticides for malaria vector control, threatens the continued efficacy of vector control tools. Target-site resistance is an important genetic resistance mechanism caused by mutations in the voltage-gated sodium channel (Vgsc) gene that encodes the pyrethroid target-site. Understanding the geographic distribution of target-site resistance, and temporal trends across different vector species, can inform strategic deployment of vector control tools. Results We develop a Bayesian statistical spatiotemporal model to interpret species-specific trends in the frequency of the most common resistance mutations, Vgsc-995S and Vgsc-995F, in three major malaria vector species Anopheles gambiae, An. coluzzii, and An. arabiensis over the period 2005–2017. The models are informed by 2418 observations of the frequency of each mutation in field sampled mosquitoes collected from 27 countries spanning western and eastern regions of Africa. For nine selected countries, we develop annual predictive maps which reveal geographically structured patterns of spread of each mutation at regional and continental scales. The results show associations, as well as stark differences, in spread dynamics of the two mutations across the three vector species. The coverage of ITNs was an influential predictor of Vgsc allele frequencies, with modelled relationships between ITN coverage and allele frequencies varying across species and geographic regions. We found that our mapped Vgsc allele frequencies are a significant partial predictor of phenotypic resistance to the pyrethroid deltamethrin in An. gambiae complex populations. Conclusions Our predictive maps show how spatiotemporal trends in insecticide target-site resistance mechanisms in African An. gambiae vary across individual vector species and geographic regions. Molecular surveillance of resistance mechanisms will help to predict resistance phenotypes and track their spread.

Monitoring insecticide resistance is crucial in disease-transmitting mosquitoes to allow assessment of viable candidate insecticides to use for control and to provide indication of changes in resistance. Insecticide resistance bioassays are typically performed on young female mosquitoes, yet disease is transmitted by older females, which may also have encountered insecticide multiple times during their adult life. If insecticide mortality rates increase with age directly, or indirectly via cumulative toxicity from repeated exposure, the strategy of testing young mosquitoes as the least susceptible cohort would be supported. We tested three hypotheses via examination of how age and cumulative exposure impact mortality rates to the pyrethroid deltamethrin in strains of Aedes aegypti from Jeddah, Saudi Arabia and the Cayman Islands, which show differences in resistance mechanisms. Females of different ages (5, 7, 10 and 14 days-old) were exposed using WHO tube assays to either a single dose of insecticide, or in a second experiment females (initially 5 days-old) were exposed daily over 10 days. Age only increased mortality in the Jeddah strain at 14 days-old and had no impact on the Cayman strain. This is consistent with greater impact linked to metabolic resistance in the Jeddah strain, though results from qPCR of four candidate genes, failed to provide evidence for a candidate underpinning an age-dependent change in resistance. With repeated exposure, mortality rates of surviving females decreased to very low levels, suggesting that surviving older cohorts of females may exhibit substantially lower susceptibility than young females in single exposure assays. Our results indicate that testing young females with a single insecticide exposure should capture minimum susceptibility for the majority of the population, but a small fraction of older females may prove particularly unresponsive to pyrethroid-based control measures.

Insecticide resistance threatens recent progress on malaria control in Africa. To characterize pyrethroid resistance in Uganda, Anopheles gambiae (s.s.) and Anopheles arabiensis were analyzed from 11 sites with varied vector control strategies. Mosquito larvae were collected between May 2018 and December 2020. Sites were categorized as receiving no indoor-residual spraying (‘no IRS’, n ​= ​3); where IRS was delivered from 2009 to 2014 and in 2017 and then discontinued (‘IRS stopped’, n ​= ​4); and where IRS had been sustained since 2014 (‘IRS active’, n ​= ​4). IRS included bendiocarb, pirimiphos methyl and clothianidin. All sites received long-lasting insecticidal nets (LLINs) in 2017. Adult mosquitoes were exposed to pyrethroids; with or without piperonyl butoxide (PBO). Anopheles gambiae (s.s.) and An. arabiensis were identified using PCR. Anopheles gambiae (s.s.) were genotyped for Vgsc-995S/F, Cyp6aa1, Cyp6p4-I236M, ZZB-TE, Cyp4j5-L43F and Coeae1d, while An. arabiensis were examined for Vgsc-1014S/F. Overall, 2753 An. gambiae (s.l.), including 1105 An. gambiae (s.s.) and 1648 An. arabiensis were evaluated. Species composition varied by site; only nine An. gambiae (s.s.) were collected from ‘IRS active’ sites, precluding species-specific comparisons. Overall, mortality following exposure to permethrin and deltamethrin was 18.8% (148/788) in An. gambiae (s.s.) and 74.6% (912/1222) in An. arabiensis. Mortality was significantly lower in An. gambiae (s.s.) than in An. arabiensis in ‘no IRS’ sites (permethrin: 16.1 vs 67.7%, P ​

BackgroundDengue's emergence in West Africa was typified by the Burkina Faso outbreaks in 2016 and 2017, the nation's largest to date. In both years, we undertook three-month surveys of Aedes populations in or near the capital city Ouagadougou, where the outbreaks were centered.MethodologyIn 1200LG (urban), Tabtenga (peri-urban) and Goundry (rural) localities, we collected indoor and outdoor resting mosquito adults, characterized larval habitats and containers producing pupae and reared immature stages to adulthood in the laboratory for identification. All mosquito adults were identified morphologically. Host species (from which bloodmeals were taken) were identified by PCR. Generalized mixed models were used to investigate relationships between adult or larval densities and multiple explanatory variables.ResultsFrom samples in 1,780 houses, adult Ae. aegypti were significantly more abundant in the two urban localities (Tabtenga and 1200 LG) in both years than in the rural site (Goundry), where Anopheles spp. were far more common. Results from adult collections indicated a highly exophilic and anthropophilic (>90% bloodmeals of human origin) vector population, but with a relatively high proportion of bloodfed females caught inside houses. Habitats producing most pupae were waste tires (37% of total pupae), animal troughs (44%) and large water barrels (30%). While Stegomyia indices were not reliable indicators of adult mosquito abundance, shared influences on adult and immature stage densities included rainfall and container water level, collection month and container type/purpose. Spatial analysis showed autocorrelation of densities, with a partial overlap in adult and immature stage hotspots.ConclusionResults provide an evidence base for the selection of appropriate vector control methods to minimize the risk, frequency and magnitude of future outbreaks in Ouagadougou. An integrated strategy combining community-driven practices, waste disposal and insecticide-based interventions is proposed. The prospects for developing a regional approach to arbovirus control in West Africa or across Africa are discussed.

Anopheles stephensi mosquitoes are urban malaria vectors in Asia that have recently invaded the Horn of Africa. We detected emergence of An. stephensi mosquitoes in 2 noncontiguous states of eastern Sudan. Results of mitochondrial DNA sequencing suggest the possibility of distinct invasions, potentially from a neighboring country.

Abstract Background With widespread insecticide resistance in mosquito vectors, there is a pressing need to evaluate alternatives with different modes of action. Blood containing the antihelminthic drug ivermectin has been shown to have lethal and sub-lethal effects on mosquitoes. Almost all work to date has been on Anopheles spp., but impacts on other anthropophagic vectors could provide new options for their control, or additional value to anti-malarial ivermectin programmes. Methods Using dose-response assays, we evaluated the effects of ivermectin delivered by membrane feeding on daily mortality (up to 14 days post-blood feed) and fecundity of an Indian strain of Aedes aegypti. Results The 7-day lethal concentration of ivermectin required to kill 50% of adult mosquitoes was calculated to be 178.6 ng/ml (95% confidence intervals 142.3–218.4) for Ae. aegypti, which is much higher than that recorded for Anopheles spp. in any previous study. In addition, significant effects on fecundity and egg hatch rates were only recorded at high ivermectin concentrations (≥ 250 ng/ul). Conclusion Our results suggest that levels of ivermectin present in human blood at current dosing regimes in mass drug administration campaigns, or even those in a recent higher-dose anti-malaria trial, are unlikely to have a substantial impact on Ae. aegypti. Moreover, owing to the strong anthropophagy of Ae. aegypti, delivery of higher levels of ivermectin in livestock blood is also unlikely to be an effective option for its control. However, other potential toxic impacts of ivermectin metabolites, accumulation in tissues, sublethal effects on behaviour, or antiviral action might increase the efficacy of ivermectin against Ae. aegypti and the arboviral diseases it transmits, and require further investigation.

Long lasting insecticidal nets (LLINs) are a proven tool to reduce malaria transmission, but in Africa efficacy is being reduced by pyrethroid resistance in the major vectors. A previous study that was conducted in Muleba district, Tanzania indicated possible involvement of cytochrome P450 monooxygenases in a pyrethroid resistance in An. gambiae population where pre-exposure to piperonyl butoxide (PBO) followed by permethrin exposure in CDC bottle bioassays led to partial restoration of susceptibility. PBO is a synergist that can block pyrethroid-metabolizing enzymes in a mosquito. Insecticide resistance profiles and underlying mechanisms were investigated in Anopheles gambiae and An. funestus from Muleba during a cluster randomized trial. Diagnostic dose bioassays using permethrin, together with intensity assays, suggest pyrethroid resistance that is both strong and very common, but not extreme. Transcriptomic analysis found multiple P450 genes over expressed including CYP6M2, CYP6Z3, CYP6P3, CYP6P4, CYP6AA1 and CYP9K1 in An. gambiae and CYP6N1, CYP6M7, CYP6M1 and CYP6Z1 in An. funestus. Indeed, very similar suites of P450 enzymes commonly associated with resistant populations elsewhere in Africa were detected as over expressed suggesting a convergence of mechanisms across Sub-Saharan African malaria vectors. The findings give insight into factors that may correlate with pyrethroid PBO LLIN success, broadly supporting model predictions, but revision to guidelines previously issued by the World Health Organization is warranted.

Abstract Background Malaria vector control and research rely heavily on monitoring mosquito populations for the development of resistance to public health insecticides. One standard method for determining resistance in adult mosquito populations is the World Health Organization test (WHO bioassay). The WHO bioassay kit consists of several acrylic pieces that are assembled into a unit. Parts of the kit commonly break, reducing the capacity of insectaries to carry out resistance profiling. Since there is at present only a single supplier for the test kits, replacement parts can be hard to procure in a timely fashion. Methods Using computer-aided design software and widely available polylactic acid (PLA) filament as a printing material, we 3D designed and printed replacement parts for the WHO bioassay system. We conducted a comparison experiment between original WHO bioassay kits and 3D printed kits to assess congruence between results. The comparison experiment was performed on two Kenyan laboratory strains of Anopheles gambiae (s.s.), Kilifi and Mbita. Studentʼs t-tests were used to assess significant differences between tube types. Finally, we exposed the PLA filament to common solutions used with the bioassay kit. Results We were able to design and print functional replacements for each piece of the WHO bioassay kit. Replacement parts are functionally identical to and interchangeable with original WHO bioassay parts. We note no significant difference in mortality results obtained from PLA printed tubes and WHO acrylic tubes. Additionally, we observed no degradation of PLA in response to prolonged exposure times of commonly used cleaning solutions. Conclusions Our designs can be used to produce replacement parts for the WHO bioassay kit in any facility with a 3D printer, which are becoming increasingly widespread. 3D printing technologies can affordably and rapidly address equipment shortages and be used to develop bespoke equipment in laboratories.

Abstract Background Strategies for combatting residual malaria by targeting vectors outdoors are gaining importance as the limitations of primary indoor interventions are reached. Strategies to target ovipositing females or her offspring are broadly applicable because all mosquitoes require aquatic habitats for immature development irrespective of their biting or resting preferences. Oviposition site selection by gravid females is frequently studied by counting early instar larvae in habitats; an approach which is valid only if the number of larvae correlates with the number of females laying eggs. This hypothesis was tested against the alternative, that a higher abundance of larvae results from improved survival of a similar or fewer number of families. Methods In a controlled experiment, 20 outdoor artificial ponds were left uncovered for 4 days to allow oviposition by wild mosquitoes, then covered with netting and first and second instar larvae sampled daily. Natural Anopheles habitats of two different types were also identified, and all visible larvae sampled. All larvae were identified to species, and most samples of the predominant species, Anopheles arabiensis, were genotyped using microsatellites for sibling group reconstructions using two contrasting softwares, BAPS and COLONY. Results In the ponds, the number of families reconstructed by each software significantly predicted larval abundance (BAPS R2 = 0.318, p = 0.01; COLONY R2 = 0.476, p = 0.001), and suggested that around 50% of females spread larvae across multiple ponds (skip oviposition). From natural habitats, the mean family size again predicted larval abundance using BAPS (R2 = 0.829, p = 0.017) though not using COLONY (R2 = 0.218, p = 0.68), but both softwares once more suggested high rates of skip oviposition (in excess of 50%). Conclusion This study shows that, whether in closely-located artificial habitats or natural breeding sites, higher early instar larval densities result from more females laying eggs in these sites. These results provide empirical support for use of early instar larval abundance as an index for oviposition site preference. Furthermore, the sharing of habitats by multiple females and the high skip-oviposition rate in An. arabiensis suggest that larviciding by auto-dissemination of insecticide may be successful.

Abstract The past 40 years have seen a dramatic emergence of epidemic arboviral diseases transmitted primarily by mosquitoes. The frequency and magnitude of the epidemics, especially those transmitted by urban Aedes species, have progressively increased over time, accelerating in the past 10 years. To reduce the burden and threat of vector-borne diseases, the World Health Organization (WHO) has recently adopted the Global Vector Control Response (GVCR) in order to support countries in implementing effective sustainable vector control. The evidence-base to support vector control is however limited for arboviral diseases which make prioritization difficult. Knowledge gaps in the distribution, mechanisms and impact of insecticide resistance on vector control impedes the implementation of locally tailored Aedes control measures. This report summarizes the main outputs of the second international conference of the Worldwide Insecticide resistance Network (WIN) on “Integrated approaches and innovative tools for combating insecticide resistance in arbovirus vectors” held in Singapore, 1–3 October 2018. The aims of the conference were to review progress and achievements made in insecticide resistance surveillance worldwide, and to discuss the potential of integrated vector management and innovative technologies for efficiently controlling arboviral diseases. The conference brought together 150 participants from 26 countries.

Abstract Background Culex quinquefasciatus has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. In C. quinquefasciatus two non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of the Vgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda. Results This new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches, pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles at Vgsc-L1014F, with genotyping results strongly correlated with pyrosequencing and TaqMan results (98.64% and 100% respectively). Conclusions Our results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

Abstract Background Resistance to pyrethroid insecticides involving kdr mutations is widespread in Aedes aegypti (L.) (Diptera: Culicidae) and potentially could impact control efforts in endemic countries. Dengue cases had been sporadic in Burkina Faso for over a decade prior to the 2016–2017 outbreak that resulted in 15,074 suspected cases and 36 deaths, mainly in Ouagadougou. These outbreaks highlighted the lack of information on numerous aspects of the biology, behaviour and insecticide status of local dengue vector populations that are fundamental to vector control. Results We investigated the insecticide resistance profiles and the kdr mutations involved in pyrethroid resistance of Ae. aegypti from Somgandé, a district of Ouagadougou. WHO bioassays revealed that the local Ae. aegypti populations were highly resistant to pyrethroids with mortalities of 15% for permethrin and 37% for deltamethrin. Resistance to carbamates was also detected with mortalities of 55% for propoxur and 90% for bendiocarb, but high mortalities (> 97%) to organophosphates (malathion and fenitrothion) indicated susceptibility. Allele-specific PCR and voltage-gated sodium channel gene sequencing showed a very high frequency (97%) of the F1534C kdr allele whilst the V1016I kdr mutation frequency was 46%. Association of dual-locus kdr mutations was detected for permethrin resistance. Conclusion We conclude that in this locality of Burkina Faso, Ae. aegypti is resistant to pyrethroid and carbamate insecticides but remains susceptible to organophosphates, providing useful information for possible future control.

[This corrects the article DOI: 10.1371/journal.pntd.0005625.].

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d'Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions.

Durability monitoring of insecticide-treated nets (ITNs) containing a pyrethroid in combination with a second active ingredient (AI) must be adapted so that the insecticidal bioefficacy of each AI can be monitored independently. An effective way to do this is to measure rapid knock down of a pyrethroid-susceptible strain of mosquitoes to assess the bioefficacy of the pyrethroid component and to use a pyrethroid-resistant strain to measure the bioefficacy of the second ingredient. To allow robust comparison of results across tests within and between test facilities, and over time, protocols for bioefficacy testing must include either characterisation of the resistant strain, standardisation of the mosquitoes used for bioassays, or a combination of the two. Through a series of virtual meetings, key stakeholders and practitioners explored different approaches to achieving these goals. Via an iterative process we decided on the preferred approach and produced a protocol consisting of characterising mosquitoes used for bioefficacy testing before and after a round of bioassays, for example at each time point in a durability monitoring study. We present the final protocol and justify our approach to establishing a standard methodology for durability monitoring of ITNs containing pyrethroid and a second AI.

Abstract Background Mutations in the voltage-gated sodium channel at codon 1014 confer knock-down resistance (kdr) to pyrethroids in a wide range of insects. Anopheles gambiae exhibits two mutant alleles at codon 1014, serine and phenylalanine; and both are now widespread across Africa. Existing screening methods only allow for one resistant allele to be detected per assay. A new locked nucleic acid (LNA) qPCR assay was developed for the simultaneous detection of both mutant alleles and the wild type allele in a single assay. This tri-allelic detection assay was assessed as part of a study of the insecticide resistance in An. gambiae sensu stricto (s.s.) in the previously un-sampled area of Nord Ubangi, Democratic Republic of the Congo. Methods Samples from three sites were tested for insecticide susceptibility using WHO bioassays, with and without the synergist PBO preceding pyrethroid exposures, and were subsequently analysed for frequency and resistance-association of the Vgsc-1014 and Vgsc-N1575Y mutations. Results from the LNA-kdr 1014 assay were compared to results from standard TaqMan-kdr assays. Results Anopheles gambiae sensu lato (s.l.) was by far the predominant vector captured (84%), with only low frequencies of Anopheles funestus s.l. (9%) detected in Nord Ubangi. Molecular identification found An. gambiae s.s. to be the principal vector (99%) although Anopheles coluzzii was detected at very low frequency. Anopheles gambiae were susceptible to the carbamate insecticide bendiocarb, but resistant to DDT and to the pyrethroids permethrin and deltamethrin. Susceptibility to both pyrethroids was partially restored with prior exposure to PBO suggesting likely involvement of metabolic resistance. Anopheles gambiae s.s. was homozygous for kdr resistant alleles with both the L1014F and L1014S mutations present, and the N1575Y polymorphism was present at low frequency. The LNA-kdr assay simultaneously detected both resistant alleles and gave results entirely consistent with those from the two TaqMan-kdr assays. Conclusion This study provides rare data on insecticide resistance and mechanisms in Anopheles from the centre of Africa, with the first detection of N1575Y. Nord Ubangi populations of An. gambiae s.s. show insecticide resistance mediated by both metabolic mechanisms and Vgsc mutations. The LNA-kdr assay is particularly suitable for use in populations in which both 1014S and 1014F kdr alleles co-occur and provides robust results, with higher throughput and at a quarter of the cost of TaqMan assays.

Abstract Background The spread of insecticide resistance (IR) is a major threat to vector control programmes for mosquito-borne diseases. Early detection of IR using diagnostic markers could help inform these programmes, especially in remote locations where gathering reliable bioassay data is challenging. Most current molecular tests for genetic IR markers are only suitable for use in well-equipped laboratory settings. There is an unmet need for field-applicable diagnostics. Methods A single-cartridge test was designed to detect key IR mutations in the major African vector of malaria, Anopheles gambiae. Developed on the portable, rapid, point-of-care compatible PCR platform - Genedrive® (genedrive® plc), the test comprises two assays which target single nucleotide polymorphisms (SNPs) in the voltage gated sodium channel (VGSC) gene that exert interactive effects on knockdown resistance (kdr): L1014F, L1014S and N1575Y. Results Distinct melt peaks were observed for each allele at each locus. Preliminary validation of these assays using a test panel of 70 An. gambiae samples showed complete agreement of our assays with the widely-used TaqMan assays, achieving a sensitivity and specificity of 100%. Conclusion Here we show the development of an insecticide resistance detection assay for use on the Genedrive® platform that has the potential to be the first field-applicable diagnostic for kdr.

Pyrethroid resistance is widespread in malaria vectors. However, differential mortality in discriminating dose assays to different pyrethroids is often observed in wild populations. When this occurs, it is unclear if this differential mortality should be interpreted as an indication of differential levels of susceptibility within the pyrethroid class, and if so, if countries should consider selecting one specific pyrethroid for programmatic use over another. A review of evidence from molecular studies, resistance testing with laboratory colonies and wild populations, and mosquito behavioural assays were conducted to answer these questions. Evidence suggested that in areas where pyrethroid resistance exists, different results in insecticide susceptibility assays with specific pyrethroids currently in common use (deltamethrin, permethrin, α-cypermethrin, and λ-cyhalothrin) are not necessarily indicative of an operationally relevant difference in potential performance. Consequently, it is not advisable to use rotation between these pyrethroids as an insecticide-resistance management strategy. Less commonly used pyrethroids (bifenthrin and etofenprox) may have sufficiently different modes of action, though further work is needed to examine how this may apply to insecticide resistance management.

Abstract Vector-borne diseases transmitted by insect vectors such as mosquitoes occur in over 100 countries and affect almost half of the world’s population. Dengue is currently the most prevalent arboviral disease but chikungunya, Zika and yellow fever show increasing prevalence and severity. Vector control, mainly by the use of insecticides, play a key role in disease prevention but the use of the same chemicals for more than 40 years, together with the dissemination of mosquitoes by trade and environmental changes, resulted in the global spread of insecticide resistance. In this context, innovative tools and strategies for vector control, including the management of resistance, are urgently needed. This report summarizes the main outputs of the first international workshop on Insecticide resistance in vectors of arboviruses held in Rio de Janeiro, Brazil, 5–8 December 2016. The primary aims of this workshop were to identify strategies for the development and implementation of standardized insecticide resistance management, also to allow comparisons across nations and across time, and to define research priorities for control of vectors of arboviruses. The workshop brought together 163 participants from 28 nationalities and was accessible, live, through the web (> 70,000 web-accesses over 3 days).

Abstract Background Pyrethroid resistance is a threat to effective vector control of Aedes aegypti, the vector of dengue, Zika and other arboviruses, but there are many major knowledge gaps on the mechanisms of resistance. In Jeddah and Makkah, the principal dengue-endemic areas of Saudi Arabia, pyrethroids are used widely for Ae. aegypti control but information about resistance remains sparse, and the underlying genetic basis is unknown. Findings from an ongoing study in this internationally significant area are reported here. Methods Aedes aegypti collected from each city were raised to adults and assayed for resistance to permethrin, deltamethrin (with and without the synergist piperonyl butoxide, PBO), fenitrothion, and bendiocarb. Two fragments of the voltage-gated sodium channel (Vgsc), encompassing four previously identified mutation sites, were sequenced and subsequently genotyped to determine associations with resistance. Expression of five candidate genes (CYP9J10, CYP9J28, CYP9J32, CYP9M6, ABCB4) previously associated with pyrethroid resistance was compared between assay survivors and controls. Results Jeddah and Makkah populations exhibited resistance to multiple insecticides and a similarly high prevalence of resistance to deltamethrin compared to a resistant Cayman strain, with a significant influence of age and exposure duration on survival. PBO pre-exposure increased pyrethroid mortality significantly in the Jeddah, but not the Makkah strain. Three potentially interacting Vgsc mutations were detected: V1016G and S989P were in perfect linkage disequilibrium in each strain and strongly predicted survival, especially in the Makkah strain, but were in negative linkage disequilibrium with 1534C, though some females with the Vgsc triple mutation were detected. The candidate gene CYP9J28 was significantly over-expressed in Jeddah compared to two susceptible reference strains, but none of the candidate genes was consistently up-regulated to a significant level in the Makkah strain. Conclusions Despite their proximity, Makkah and Jeddah exhibit significant differences in pyrethroid resistance phenotypes, with some evidence to suggest a different balance of mechanisms, for example with more impact associated with CYP450s in the Jeddah strain, and the dual kdr mutations 989P and 1016G in the more resistant Makkah strain. The results overall demonstrate a major role for paired target site mutations in pyrethroid resistance and highlight their utility for diagnostic monitoring.

BackgroundThe landscape of mosquito-borne disease risk has changed dramatically in recent decades, due to the emergence and reemergence of urban transmission cycles driven by invasive Aedes aegypti and Ae. albopictus. Insecticide resistance is already widespread in the yellow fever mosquito, Ae. Aegypti; is emerging in the Asian tiger mosquito Ae. Albopictus; and is now threatening the global fight against human arboviral diseases such as dengue, yellow fever, chikungunya, and Zika. Because the panel of insecticides available for public health is limited, it is of primary importance to preserve the efficacy of existing and upcoming active ingredients. Timely implementation of insecticide resistance management (IRM) is crucial to maintain the arsenal of effective public health insecticides and sustain arbovirus vector control.Methodology and principal findingsThis Review is one of a series being generated by the Worldwide Insecticide resistance Network (WIN) and aims at defining the principles and concepts underlying IRM, identifying the main factors affecting the evolution of resistance, and evaluating the value of existing tools for resistance monitoring. Based on the lessons taken from resistance strategies used for other vector species and agricultural pests, we propose a framework for the implementation of IRM strategies for Aedes mosquito vectors.Conclusions and significanceAlthough IRM should be a fixture of all vector control programs, it is currently often absent from the strategic plans to control mosquito-borne diseases, especially arboviruses. Experiences from other public health disease vectors and agricultural pests underscore the need for urgent action in implementing IRM for invasive Aedes mosquitoes. Based on a plan developed for malaria vectors, here we propose some key activities to establish a global plan for IRM in Aedes spp.

BACKGROUND:Recent outbreaks of dengue and other Aedes aegypti-borne arboviruses highlight the importance of a rapid response for effective vector control. Data on insecticide resistance and underlying mechanisms are essential for outbreak preparedness, but are sparse in much of Africa. We investigated the levels and heterogeneity of insecticide resistance and mechanisms of Ae. aegypti from contrasting settings within and around Ouagadougou, Burkina Faso. METHODOLOGY/PRINCIPAL FINDINGS:Bioassays were performed on larvae and adults to diagnose prevalence of resistance, and to assess levels where resistance was detected. Investigation of resistance mechanisms was performed using synergist bioassays, knockdown resistance (kdr) target site mutation genotyping and quantitative PCR expression analysis of candidate P450 genes. Larval dose-response assays indicated susceptibility to the organophosphates tested. Adult females were also susceptible to organophosphates, but resistance to carbamates was suspected in urban and semi-urban localities. Females from all localities showed resistance to pyrethroids but resistance prevalence and level were higher in urban and especially in semi-urban areas, compared to the rural population. Environment was also associated with susceptibility: adults reared from larvae collected in tires from the semi-urban site were significantly less resistant to pyrethroids than those collected from large outdoor drinking water containers ('drums'). Susceptibility to both pyrethroids tested was largely restored by pre-exposure to Piperonyl Butoxide (PBO), suggesting a strong metabolic basis to resistance. The 1534C kdr mutation was nearly fixed in semi-urban and urban areas but was far less common in the rural area, where the 1016I kdr mutation frequency was also significantly lower. P450 gene analysis detected limited over-expression of single candidates but significantly elevated average expression in the semi-urban site compared to both a susceptible laboratory colony, and females from the other collection sites. CONCLUSIONS/SIGNIFICANCE:Our results reveal pyrethroid resistance and paired kdr mutations in both urban and semi-urban sites at levels that are unprecedented for mainland Africa. The combination of target site and metabolic mechanisms is common in Ae. aegypti populations from other continents but is a worrying finding for African populations. However, organophosphate insecticides are still active against both larvae and adults of Ae. aegypti, providing useful insecticidal options for control and resistance management.

Background: Growing insecticide resistance and changes in biting and resting behavior of malaria vectors threaten efficacy of insecticide treated nets and indoor residual spraying. Larval source management (LSM) is a promising approach that can target mosquitoes irrespective of their behavior as adults. However, the use of traditional monitoring methods for immature stages of Anopheles mosquitoes is a major challenge to LSM due to the variability in their breeding habitats. We evaluate the use of an environmental DNA (eDNA) analysis technique in monitoring Anopheles gambiae sensu lato larvae in experimental aquatic habitats. Methods: eDNA was simultaneously sampled and extracted from different volumes of water, number of larvae, and occupation time. Larval presence was detected using PCR and eDNA concentration in samples from 1 L habitats quantified using an IGS and cyt b TaqMan assays. The limit of detection of the two assays was tested and larval density correlated with eDNA positivity. Results: 74% of replicates in the 50 mL habitats were PCR positive with at least 6h required to get a signal from a single larva (0.02 larvae/mL). All 12 replicates where 1 L of water was used were positive with stronger PCR bands than replicates with the same larval density in 50 mL for 24 h. There was a correlation between larval densities and eDNA detection in both assays: IGS, r = 0.503, p = 0.047; and cyt b, r = 0.558, p = 0.025. There was stochasticity in eDNA detection rates, using both PCR and qPCR across all the dilutions. Conclusion: This study has demonstrated the potential use of eDNA analysis for detection and quantification of An. gambiae s.s. mosquito larvae in aquatic habitats. The stochasticity observed in eDNA detection suggest that this technique is best for monitoring aquatic habitats with many larvae at low densities.