The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14 C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae. malaria | insecticide resistance | hydrocarbons | mosquito cuticle | cytochrome P450

Background Anopheles funestus is one of the primary vectors of human malaria, which causes a million deaths each year in sub-Saharan Africa. Few scientific resources are available to facilitate studies of this mosquito species and relatively little is known about its basic biology and evolution, making development and implementation of novel disease control efforts more difficult. The An. funestus genome has not been sequenced, so in order to facilitate genome-scale experimental biology, we have sequenced the adult female transcriptome of An. funestus from a newly founded colony in Burkina Faso, West Africa, using the Illumina GAIIx next generation sequencing platform.Methodology/Principal FindingsWe assembled short Illumina reads de novo using a novel approach involving iterative de novo assemblies and “target-based” contig clustering. We then selected a conservative set of 15,527 contigs through comparisons to four Dipteran transcriptomes as well as multiple functional and conserved protein domain databases. Comparison to the Anopheles gambiae immune system identified 339 contigs as putative immune genes, thus identifying a large portion of the immune system that can form the basis for subsequent studies of this important malaria vector. We identified 5,434 1∶1 orthologues between An. funestus and An. gambiae and found that among these 1∶1 orthologues, the protein sequence of those with putative immune function were significantly more diverged than the transcriptome as a whole. Short read alignments to the contig set revealed almost 367,000 genetic polymorphisms segregating in the An. funestus colony and demonstrated the utility of the assembled transcriptome for use in RNA-seq based measurements of gene expression.Conclusions/SignificanceWe developed a pipeline that makes de novo transcriptome sequencing possible in virtually any organism at a very reasonable cost ($6,300 in sequencing costs in our case). We anticipate that our approach could be used to develop genomic resources in a diversity of systems for which full genome sequence is currently unavailable. Our An. funestus contig set and analytical results provide a valuable resource for future studies in this non-model, but epidemiologically critical, vector insect.

Background and methodsA longitudinal Anopheles gambiae s.l. insecticide-resistance monitoring programme was established in four sentinel sites in Burkina Faso. For three years, between 2008 and 2010, WHO diagnostic dose assays were used to measure the prevalence of resistance to all the major classes of insecticides at the beginning and end of the malaria transmission season. Species identification and genotyping for target site mutations was also performed and the sporozoite rate in adults determined.ResultsAt the onset of the study, resistance to DDT and pyrethroids was already prevalent in An. gambiae s.l. from the south-west of the country but mosquitoes from the two sites in central Burkina Faso were largely susceptible. Within three years, DDT and permethrin resistance was established in all four sites. Carbamate and organophosphate resistance remains relatively rare and largely confined to the south-western areas although a small number of bendiocarb survivors were found in all sites by the final round of monitoring. The ace-1R target site resistance allele was present in all localities and its frequency exceeded 20% in 2010 in two of the sites. The frequency of the 1014F kdr mutation increased throughout the three years and by 2010, the frequency of 1014F in all sites combined was 0.02 in Anopheles arabiensis, 0.56 in An. gambiae M form and 0.96 in An. gambiae S form. This frequency did not differ significantly between the sites. The 1014S kdr allele was only found in An. arabiensis but its frequency increased significantly throughout the study (P = 0.0003) and in 2010 the 1014S allele frequency was 0.08 in An. arabiensis. Maximum sporozoite rates (12%) were observed in Soumousso in 2009 and the difference between sites is significant for each year.ConclusionPyrethroid and DDT resistance is now established in An. gambiae s.l. throughout Burkina Faso. Results from diagnostic dose assays are highly variable within and between rounds of testing, and hence it is important that resistance monitoring is carried out on more than one occasion before decisions on insecticide procurement for vector control are made. The presence of 1014S in An. gambiae s.l., in addition to 1014F, is not unexpected given the recent report of 1014S in Benin but highlights the importance of monitoring for both mutations throughout the continent. Future research must now focus on the impact that this resistance is having on malaria control in Burkina Faso.

In the city of Bobo-Dioulasso in Burkina Faso, Anopheles arabiensis has superseded Anopheles gambiae s.s. as the major malaria vector and the larvae are found in highly polluted habitats normally considered unsuitable for Anopheles mosquitoes. Here we show that An. gambiae s.l. adults emerging from a highly polluted site in the city centre (Dioulassoba) have a high prevalence of DDT resistance (percentage mortality after exposure to diagnostic dose = 65.8% in the dry season and 70.4% in the rainy season, respectively). An investigation into the mechanisms responsible found an unexpectedly high frequency of the 1014S kdr mutation (allele frequency = 0.4), which is found at very low frequencies in An. arabiensis in the surrounding rural areas, and an increase in transcript levels of several detoxification genes, notably from the glutathione transferase and cytochrome P450 gene families. A number of ABC transporter genes were also expressed at elevated levels in the DDT resistant An. arabiensis. Unplanned urbanisation provides numerous breeding grounds for mosquitoes. The finding that Anopheles mosquitoes adapted to these urban breeding sites have a high prevalence of insecticide resistance has important implications for our understanding of the selective forces responsible for the rapid spread of insecticide resistant populations of malaria vectors in Africa.

BackgroundMeasuring human exposure to mosquito bites is a crucial component of vector-borne disease surveillance. For malaria vectors, the human landing catch (HLC) remains the gold standard for direct estimation of exposure. This method, however, is controversial since participants risk exposure to potentially infected mosquito bites. Recently an exposure-free mosquito electrocuting trap (MET) was developed to provide a safer alternative to the HLC. Early prototypes of the MET performed well in Tanzania but have yet to be tested in West Africa, where malaria vector species composition, ecology and behaviour are different. The performance of the MET relative to HLC for characterizing mosquito vector population dynamics and biting behaviour in Burkina Faso was evaluated.MethodsA longitudinal study was initiated within 12 villages in Burkina Faso in October 2016. Host-seeking mosquitoes were sampled monthly using HLC and MET collections over 14 months. Collections were made at 4 households on each night, with METs deployed inside and outside at 2 houses, and HLC inside and outside at another two. Malaria vector abundance, species composition, sporozoite rate and location of biting (indoor versus outdoor) were recorded.ResultsIn total, 41,800 mosquitoes were collected over 324 sampling nights, with the major malaria vector being Anopheles gambiae sensu lato (s.l.) complex. Overall the MET caught fewer An. gambiae s.l. than the HLC (mean predicted number of 0.78 versus 1.82 indoors, and 1.05 versus 2.04 outdoors). However, MET collections gave a consistent representation of seasonal dynamics in vector populations, species composition, biting behaviour (location and time) and malaria infection rates relative to HLC. As the relative performance of the MET was somewhat higher in outdoor versus indoor settings, this trapping method slightly underestimated the proportion of bites preventable by LLINs compared to the HLC (MET = 82.08%; HLC = 87.19%).ConclusionsThe MET collected proportionately fewer mosquitoes than the HLC. However, estimates of An. gambiae s.l. density in METs were highly correlated with HLC. Thus, although less sensitive, the MET is a safer alternative than the HLC. Its use is recommended particularly for sampling vectors in outdoor environments where it is most sensitive.