N Falé Sagnon

Pyrethroid resistance in the Anopheles vectors of malaria is driving an urgent search for new insecticides that can be used in proven vector control tools such as insecticide treated nets (ITNs). Screening for potential new insecticides requires access to stable colonies of the predominant vector species that contain the major pyrethroid resistance mechanisms circulating in wild populations. Southwest Burkina Faso is an apparent hotspot for the emergence of pyrethroid resistance in species of the Anopheles gambiae complex. We established stable colonies from larval collections across this region and characterised the resistance phenotype and underpinning genetic mechanisms. Three additional colonies were successfully established (1 An. coluzzii, 1 An. gambiae and 1 An. arabiensis) to add to the 2 An. coluzzii colonies already established from this region; all 5 strains are highly resistant to pyrethroids. Synergism assays found that piperonyl butoxide (PBO) exposure was unable to fully restore susceptibility although exposure to a commercial ITN containing PBO resulted in 100% mortality. All colonies contained resistant alleles of the voltage gated sodium channel but with differing proportions of alternative resistant haplotypes. RNAseq data confirmed the role of P450s, with CYP6P3 and CYP6Z2 elevated in all 5 strains, and identified many other resistance mechanisms, some found across strains, others unique to a particular species. These strains represent an important resource for insecticide discovery and provide further insights into the complex genetic changes driving pyrethroid resistance.

The Anopheles gambiae complex consists of multiple morphologically indistinguishable mosquito species including the most important vectors of the malaria parasite Plasmodium falciparum in sub-Saharan Africa. Several lineages have only recently been described as distinct, including the cryptic taxon GOUNDRY in central Burkina Faso. The ecological, immunological, and reproductive differences among these taxa will critically impact population responses to disease control strategies and environmental changes. Here we examine whole-genome sequencing data from a longitudinal study of putative A. coluzzii in western Burkina Faso. Surprisingly, many samples are genetically divergent from A. coluzzii and all other Anopheles species and represent a new taxon, here designated Anopheles TENGRELA (AT). Population genetic analysis suggests that GOUNDRY sensu stricto represents an admixed population descended from both A. coluzzii and AT. AT harbors low nucleotide diversity except for the 2La inversion polymorphism which is maintained by overdominance. It shows numerous fixed differences with A. coluzzii concentrated in several regions reflecting selective sweeps, but the two taxa are identical at standard diagnostic loci used for taxon identification and thus AT may often go unnoticed. We present an amplicon-based genotyping assay for identifying AT which could be usefully applied to numerous existing samples. Misidentified cryptic taxa could seriously confound ongoing studies of Anopheles ecology and evolution in western Africa, including phenotypic and genotypic surveys of insecticide resistance. Reproductive barriers between cryptic species may also complicate novel vector control efforts, for example gene drives, and hinder predictions about evolutionary dynamics of Anopheles and Plasmodium .

Enhancers are cis-regulatory elements that control most of the developmental and spatial gene expression in eukaryotes. Genetic variation of enhancer sequences is known to influence phenotypes, but the effect of enhancer variation upon enhancer functional activity and downstream phenotypes has barely been examined in any species. In the African malaria vector, Anopheles coluzzii , we identified a pilot set of candidate enhancers in the proximity of genes relevant for immunity, insecticide resistance, and development. The candidate enhancers were functionally validated using luciferase reporter assays, and their activity was found to be essentially independent of their physical orientation, a typical property of enhancers. All of the enhancer intervals segregated genetically polymorphic alleles, which displayed significantly different levels of functional activity, and inactive null alleles were also observed. Deletion mutagenesis and functional testing revealed a modular structure of positive and negative regulatory elements within the tested enhancers. The enhancer alleles carry genetic polymorphisms that also segregate in wild A. coluzzii populations in West Africa, indicating that enhancer variants with likely phenotypic consequences are frequent in nature. These results demonstrate the feasibility of screening for naturally polymorphic A. coluzzii enhancers that underlie important aspects of malaria transmission and vector biology.

Malaria vectors have developed resistance to the four families of insecticides available for public health purposes. For example, the kdr mutation is associated with organochlorines and pyrethroids resistance. It is of particular concern that organophosphate and carbamate resistance associated with the G119S ace-1 R mutation has recently increased in West Africa in extent and frequency, and is now spreading through the Anopheles gambiae malaria vector population. There is an urgent need to improve resistance management using existing insecticides and new tools to quickly assess resistance level for rapid decision-making. DNA extracted from field-collected mosquitoes was used to develop the method. Specific primers were designed manually to match the mutation region and an additional mismatched nucleotide in the penultimate position to increase specificity. Other primers used are common to both wild and mutant types. The allele specific (AS)-LAMP method was compared to the PCR restriction fragment length polymorphism (PCR-RFLP) and real-time PCR (RT-PCR) methods using the genomic DNA of 104 field-collected mosquitoes. The primers designed for LAMP were able to distinguish between the wild type (ace-1 S ) and mutated type allele (ace-1 R ). Detection time was 50 min for the wild type homozygous and 64 min for the heterozygous. No amplification of the resistant allele took place within the 75-min test period when using the wild type primers. For the ace-1 R resistant type, detection time was 51 min for the resistant homozygous and 55 min for the heterozygous. No amplification of the wild type allele took place within the 75-min test period when using the resistant type primers. Gel electrophoresis of LAMP products confirmed that amplification was primer-DNA specific, i.e., primers could only amplify their target specific DNA. AS-LAMP, PCR-RFLP, and RT-PCR showed no significant difference in the sensitivity and specificity of their ace-1 R detection ability. The AS-LAMP method could detect the ace-1 R mutation within 60 min, which is faster than conventional PCR-RFLP. This method may be used to quickly detect the ace-1 R mutation for rapid decision-making, even in less well-equipped laboratories.

The genome-wide association study (GWAS) techniques that have been used for genetic mapping in other organisms have not been successfully applied to mosquitoes, which have genetic characteristics of high nucleotide diversity, low linkage disequilibrium, and complex population stratification that render population-based GWAS essentially unfeasible at realistic sample size and marker density. We designed a novel mapping strategy for the mosquito system that combines the power of linkage mapping with the resolution afforded by genetic association. We established founder colonies from West Africa, controlled for diversity, linkage disequilibrium and population stratification. Colonies were challenged by feeding on the infectious stage of the human malaria parasite, Plasmodium falciparum, mosquitoes were phenotyped for parasite load, and DNA pools for phenotypically similar mosquitoes were Illumina sequenced. Phenotype-genotype mapping was carried out in two stages, coarse and fine. In the first mapping stage, pooled sequences were analysed genome-wide for intervals displaying relativereduction in diversity between phenotype pools, and candidate genomic loci were identified for influence upon parasite infection levels. In the second mapping stage, focused genotyping of SNPs from the first mapping stage was carried out in unpooled individual mosquitoes and replicates. The second stage confirmed significant SNPs in a locus encoding two Toll-family proteins. RNAi-mediated gene silencing and infection challenge revealed that TOLL 11 protects mosquitoes against P. falciparum infection. We present an efficient and cost-effective method for genetic mapping using natural variation segregating in defined recent Anopheles founder colonies, and demonstrate its applicability for mapping in a complex non-model genome. This approach is a practical and preferred alternative to population-based GWAS for first-pass mapping of phenotypes in Anopheles. This design should facilitate mapping of other traits involved in physiology, epidemiology, and behaviour.

Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. It is known that a large haplotype shared between An. gambiae and Anopheles coluzzii by introgression carries point mutations of the voltage-gated sodium channel gene para, including the L1014F kdr mutation associated with insensitivity to pyrethroid insecticides. Carriage of L1014F kdr is also correlated with higher susceptibility to infection with Plasmodium falciparum. However, the genetic mechanism and causative gene(s) underlying the parasite susceptibility phenotype are not known. Mosquitoes from the wild Burkina Faso population were challenged by feeding on natural P. falciparum gametocytes. Oocyst infection phenotypes were determined and were tested for association with SNP genotypes. Candidate genes in the detected locus were prioritized and RNAi-mediated gene silencing was used to functionally test for gene effects on P. falciparum susceptibility. A genetic locus, Pfin6, was identified that influences infection levels of P. falciparum in mosquitoes. The locus segregates as a ~3 Mb haplotype carrying 65 predicted genes including the para gene. The haplotype carrying the kdr allele of para is linked to increased parasite infection prevalence, but many single nucleotide polymorphisms on the haplotype are also equally linked to the infection phenotype. Candidate genes in the haplotype were prioritized and functionally tested. Silencing of para did not influence P. falciparum infection, while silencing of a predicted immune gene, serine protease ClipC9, allowed development of significantly increased parasite numbers. Genetic variation influencing Plasmodium infection in wild Anopheles is linked to a natural ~3 megabase haplotype on chromosome 2L that carries the kdr allele of the para gene. Evidence suggests that para gene function does not directly influence parasite susceptibility, and the association of kdr with infection may be due to tight linkage of kdr with other gene(s) on the haplotype. Further work will be required to determine if ClipC9 influences the outcome of P. falciparum infection in nature, as well as to confirm the absence of a direct influence by para.

Guidelines from the World Health Organization for monitoring insecticide resistance in disease vectors recommend exposing insects to a predetermined discriminating dose of insecticide and recording the percentage mortality in the population. This standardized methodology has been widely adopted for malaria vectors and has provided valuable data on the spread and prevalence of resistance. However, understanding the potential impact of this resistance on malaria control requires a more quantitative measure of the strength or intensity of this resistance. Bioassays were adapted to quantify the level of resistance to permethrin in laboratory colonies and field populations of Anopheles gambiae sensu lato. WHO susceptibility tube assays were used to produce data on mortality versus exposure time and CDC bottle bioassays were used to generate dose response data sets. A modified version of the CDC bottle bioassay, known as the Resistance Intensity Rapid Diagnostic Test (I-RDT), was also used to measure the knockdown and mortality after exposure to different multipliers of the diagnostic dose. Finally cone bioassays were used to assess mortality after exposure to insecticide treated nets. The time response assays were simple to perform but not suitable for highly resistant populations. After initial problems with stability of insecticide and bottle washing were resolved, the CDC bottle bioassay provided a reproducible, quantitative measure of resistance but there were challenges performing this under field conditions. The I-RDT was simple to perform and interpret although the end point selected (immediate knockdown versus 24 h mortality) could dramatically affect the interpretation of the data. The utility of the cone bioassays was dependent on net type and thus appropriate controls are needed to interpret the operational significance of these data sets. Incorporating quantitative measures of resistance strength, and utilizing bioassays with field doses of insecticides, will help interpret the possible impact of resistance on vector control activities. Each method tested had different benefits and challenges and agreement on a common methodology would be beneficial so that data are generated in a standardized format. This type of quantitative data are an important prerequisite to linking resistance strength to epidemiological outcomes.

The effectiveness of pyrethroid-treated bednets for malaria control in sub-Saharan Africa is under threat because of high levels of resistance to pyrethroid insecticides in the vectors. Here we assess the durability of polyethylene nets with a novel combination of permethrin, a pyrethroid, with pyriproxyfen, an insect juvenile mimic (PPF-LLIN), in comparison with a typical permethrin-treated long-lasting insecticidal net (LLIN). This is a cluster randomised controlled trial of net durability in Burkina Faso, with clustering at the level of the compound and includes entomological outcome measurements. Half the compounds in each village will be randomly allocated PPF-LLIN and half the LLIN. All sleeping places in a compound will be provided with one type of net. We will distribute the nets at the start of the first transmission season and follow net use at the start and end of each transmission season for 3 years. In one village, bio-efficacy and chemical content will be recorded immediately after net distribution and then at 6, 12, 18, 24, 30 and 36 months. In the other village net survivorship and fabric integrity will be recorded immediately after distribution, and then at 6, 12, 18, 24, 30 and 36 months. Routine measurements of indoor temperature and relative humidity will be made in both villages during the study. Residents will be followed for possible side effects of the PPF-LLIN by surveillance of known asthmatic subjects during the first month post-distribution and pregnancy outcomes will be monitored from antenatal clinic records. The protocol is novel on two accounts. Firstly, it is the first to describe the procedure for measuring net durability following recent World Health Organisation (WHO) guidelines. Meeting the minimum requirements set in the guidelines is essential before a new type of net can be recommended by WHO’s Pesticide Evaluation Scheme (WHOPES). Secondly, it describes methods to monitor the persistence of an active ingredient that reduces vector fertility and fecundity. If the PPF-LLIN is both effective and persistent it will provide an alternative vector control strategy where pyrethroid-resistant vectors are present. ISRCTN30634670 assigned 13 August 2014.

Feasibility and costs of monitoring efforts aimed to monitor mosquito species are strictly dependent on the presence of skilled entomologists directly in the field. However, in several contexts this is not possible or easy to organize, thus limiting the possibility to obtain crucial information on presence/abundance of potential disease vectors and of new invasive species. Digital imaging approaches could be extremely useful in the frame of medical entomology to overcome this limit. This work describes a surveillance approach to collect and morphologically identify host-seeking malaria vectors based on remote transmission of digital images of specimens collected with ad hoc modified traps. CDC light trap (CDC) and the BG-Sentinel trap (BG), both baited with BG-lure and CO2, were modified in order to have collected mosquitoes immobilized on a bi-dimensional surface. The performance of the two traps in the field was comparatively tested by Latin-square experiments in two villages of Burkina Faso under low and high mosquito densities. The efficiency of identifications based the inspection of digital images versus microscopic identifications of collected specimens was compared. A total of 1,519 mosquitoes belonging to 16 species were collected, of which 88.5% were microscopically identified as Anopheles gambiae s.l. (mainly Anopheles coluzzii, 85.7%). During dry season BG collected 15 times more females than CDC outdoors, whereas indoors the BG collected 0.4 times less than CDC. During rainy season the ratio BG/CDC was 6.4 and 0.7 outdoors and indoors, respectively. The efficiency of digital images versus microscopic identifications of collected specimens was 97.9%, 95.6% and 81.5% for Culicidae, Anophelinae and An. gambiae s.l., respectively. Results strongly encourage the use of BG-trap for collecting host-seeking An. gambiae particularly in the outdoor environment, providing new perspectives to the challenge of collecting this fraction of the biting population, whose epidemiological relevance is increasing due to the success of large-scale implementation of indoor malaria vector control strategies. Moreover, results show that the transmission of digital images of specimens collected by the ad hoc modified host-seeking traps efficiently allows identification of malaria vectors, thus opening the perspective to easily carry out mosquito monitoring also in the absence of entomologists directly in the field.

Recent reductions in malaria in sub-Saharan Africa have been associated with increased coverage with long-lasting insecticidal nets (LLINs). Pyrethroids are currently the only insecticide class used for treating nets, and the rapid increase in resistance to pyrethroids in vector mosquitoes may jeopardise future vector control. Nets containing a novel combination of permethrin, a pyrethroid, and pyriproxyfen, an insect juvenile hormone mimic, (PPF-LLIN) may enhance malaria control, as well as reducing the spread of pyrethroid-resistant mosquitoes. This trial will determine whether PPF-LLINs provide incremental protection against malaria over current best practice of LLINs and prompt treatment in an area with pyrethroid-resistant vectors. A 2 armed cluster-randomised controlled trial will be conducted in Burkina Faso to assess whether PPF-LLIN (containing 2% permethrin and 1% pyriproxyfen w/w) provide better protection against clinical malaria in children than 2% permethrin-treated LLINs. Study subjects will be recruited and provided with LLINs at the start of the study. The LLINs will be exchanged for PPF-LLIN by cluster in a step-wedge fashion so 3 months before the end of the 2 year trial all participants will have a PPF-LLIN. The primary endpoint will be clinical malaria incidence measured by passive case detection in a cohort of children, aged 6 months to 5 years. Anaemia and parasite prevalence will also be measured in children during cross-sectional surveys. Exposure to malaria parasites will be assessed using light traps followed by identification of common vector species and their sporozoite infection rates. Safety evaluation will include recording of adverse events and pregnancy outcomes. The main endpoint analysis will include adjusting for distance between village clusters with different types of nets, as the impact of PPF-LLIN is likely to increase as larger areas are dominated by PPF-LLIN, reducing the spill over of mosquitoes from villages with LLINs. The step-wedge design is to measure the impact of an incrementally delivered environmental intervention where we expect the degree of control to be improved as more people use PPF-LLIN over a larger area. Trial findings will help inform policy makers on the effectiveness of PPF-LLIN nets for malaria control in areas of pyrethroid resistance. ISRCTN21853394 – AvecNet, registered on 3 April 2013.