3D video tracking analysis reveals that mosquitoes pass more likely through holes in permethrin-treated than in untreated nets.

In addition to killing, mosquito nets treated with permethrin have been claimed to repel mosquitoes, reducing their success in passing through a holed net. We have tested this hypothesis by tracking mosquitoes in a modified World Health Organization tunnel test. In the original assay, mosquitoes are released at one end of the tunnel and have to pass through a holed piece of net to reach the bait at the other end. The mosquitoes are left in the tunnel overnight, while mortality and feeding rates are scored the following morning. Since the original test does not reveal how mosquitoes move within the tunnel, we combined the tunnel with a 3D video camera system. We tracked susceptible and permethrin-resistant Anopheles gambiae s.s. as they moved in the tunnel and interacted with an untreated or a permethrin-treated net (Olyset Net®). Surprisingly, while permethrin increased the mortality and reduced blood-feeding rates, twice as many mosquitoes passed through the holes of the permethrin-treated net. The flight trajectories reveal that upon exposure to the permethrin-treated net, both mosquito colonies showed increased 'excitation', thereby augmenting their chance of getting through the holes in the net. The study underlines the complexity of behavioural modes of action of insecticides.

Repurposing Know-how for Drug Development: Case Studies from the Swiss Tropical and Public Health Institute.

In pursuing novel therapeutic solutions, drug discovery and development rely on efficiently utilising existing knowledge and resources. Repurposing know-how, a strategy that capitalises on previously acquired information and expertise, has emerged as a powerful approach to accelerate drug discovery and development processes, often at a fraction of the costs of de novo developments. For 80 years, collaborating within a network of partnerships, the Swiss Tropical and Public Health Institute (Swiss TPH) has been working along a value chain from innovation to validation and application to combat poverty-related diseases. This article presents an overview of selected know-how repurposing initiatives conducted at Swiss TPH with a particular emphasis on the exploration of drug development pathways in the context of neglected tropical diseases and other infectious diseases of poverty, such as schistosomiasis, malaria and human African trypanosomiasis.

Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes.

BACKGROUND: Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor® G2 (IG2), a 'first-in-class' dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr, was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insect's mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2's public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. METHODS: We tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with L1014F and L1014S knockdown resistance alleles and expression levels of pyrethroid metabolisers CYP6P3, CYP6M2, CYP4G16 and CYP6P1 confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) prior to conducting experiments were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 h overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. RESULTS: Both prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI 0.23-0.46) and 0.43 (95% CI 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI 0.22-0.41). CONCLUSION: We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes.

Relationship between insecticide resistance profiles in Anopheles gambiae sensu lato and agricultural practices in Côte d'Ivoire.

BACKGROUND: Insecticide-based malaria vector control is increasingly undermined due to the development of insecticide resistance in mosquitoes. Insecticide resistance may partially be related to the use of pesticides in agriculture, while the level and mechanisms of resistance might differ between agricultural practices. The current study aimed to assess whether phenotypic insecticide resistance and associated molecular resistance mechanisms in Anopheles gambiae sensu lato differ between agricultural practices. METHODS: We collected An. gambiae s.l. larvae in six sites with three different agricultural practices, including rice, vegetable and cocoa cultivation. We then exposed the emerging adult females to discriminating concentrations of bendiocarb (0.1%), deltamethrin (0.05%), DDT (4%) and malathion (5%) using the standard World Health Organization insecticide susceptibility test. To investigate underlying molecular mechanisms of resistance, we used multiplex TaqMan qPCR assays. We determined the frequency of target-site mutations, including Vgsc-L995F/S and Vgsc-N1570Y, and Ace1-G280S. In addition, we measured the expression levels of genes previously associated with insecticide resistance in An. gambiae s.l., including the cytochrome P450-dependent monooxygenases CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and the glutathione S-transferase GSTe2. RESULTS: The An. gambiae s.l. populations from all six agricultural sites were resistant to bendiocarb, deltamethrin and DDT, while the populations from the two vegetable cultivation sites were additionally resistant to malathion. Most tested mosquitoes carried at least one mutant Vgsc-L995F allele that is associated with pyrethroid and DDT resistance. In the cocoa cultivation sites, we observed the highest 995F frequencies (80-87%), including a majority of homozygous mutants and several in co-occurrence with the Vgsc-N1570Y mutation. We detected the Ace1 mutation most frequently in vegetable-growing sites (51-60%), at a moderate frequency in rice (20-22%) and rarely in cocoa-growing sites (3-4%). In contrast, CYP6M2, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, previously associated with metabolic insecticide resistance, showed the highest expression levels in the populations from rice-growing sites compared to the susceptible Kisumu reference strain. CONCLUSION: In our study, we observed intriguing associations between the type of agricultural practices and certain insecticide resistance profiles in the malaria vector An. gambiae s.l. which might arise from the use of pesticides deployed for protecting crops.

Pharmacokinetics of ivermectin metabolites and their activity against Anopheles stephensi mosquitoes.

BACKGROUND: Ivermectin (22,23-dihydroavermectin B1a: H2B1a) is an endectocide used to treat worm infections and ectoparasites including lice and scabies mites. Furthermore, survival of malaria transmitting Anopheles mosquitoes is strongly decreased after feeding on humans recently treated with ivermectin. Currently, mass drug administration of ivermectin is under investigation as a potential novel malaria vector control tool to reduce Plasmodium transmission by mosquitoes. A "post-ivermectin effect" has also been reported, in which the survival of mosquitoes remains reduced even after ivermectin is no longer detectable in blood meals. In the present study, existing material from human clinical trials was analysed to understand the pharmacokinetics of ivermectin metabolites and feeding experiments were performed in Anopheles stephensi mosquitoes to assess whether ivermectin metabolites contribute to the mosquitocidal action of ivermectin and whether they may be responsible for the post-ivermectin effect. METHODS: Ivermectin was incubated in the presence of recombinant human cytochrome P450 3A4/5 (CYP 3A4/5) to produce ivermectin metabolites. In total, nine metabolites were purified by semi-preparative high-pressure liquid chromatography. The pharmacokinetics of the metabolites were assessed over three days in twelve healthy volunteers who received a single oral dose of 12 mg ivermectin. Blank whole blood was spiked with the isolated metabolites at levels matching the maximal blood concentration (Cmax) observed in pharmacokinetics study samples. These samples were fed to An. stephensi mosquitoes, and their survival and vitality was recorded daily over 3 days. RESULTS: Human CYP3A4 metabolised ivermectin more rapidly than CYP3A5. Ivermectin metabolites M1-M8 were predominantly formed by CYP3A4, whereas metabolite M9 (hydroxy-H2B1a) was mainly produced by CYP3A5. Both desmethyl-H2B1a (M1) and hydroxy-H2B1a (M2) killed all mosquitoes within three days post-feeding, while administration of desmethyl, hydroxy-H2B1a (M4) reduced survival to 35% over an observation period of 3 days. Ivermectin metabolites that underwent deglycosylation or hydroxylation at spiroketal moiety were not active against An. stephensi at Cmax levels. Interestingly, half-lives of M1 (54.2 ± 4.7 h) and M4 (57.5 ± 13.2 h) were considerably longer than that of the parent compound ivermectin (38.9 ± 20.8 h). CONCLUSION: In conclusion, the ivermectin metabolites M1 and M2 contribute to the activity of ivermectin against An. stephensi mosquitoes and could be responsible for the "post-ivermectin effect".

Species composition and insecticide resistance in malaria vectors in Ellibou, southern Côte d'Ivoire and first finding of Anopheles arabiensis in Côte d'Ivoire.

BACKGROUND: Knowing the species composition and insecticide resistance status of the target vector population is important to guide malaria vector control. The aim of this study was to characterize the malaria vector population in terms of species composition, insecticide susceptibility status and potential underlying resistance mechanisms in Ellibou, southern Côte d'Ivoire. METHODS: A 1-year longitudinal entomological survey was conducted using light traps and pyrethroid spray catches to sample adult mosquitoes in combination with larval sampling. The susceptibility status of Anopheles gambiae sensu lato (s.l.) to bendiocarb, deltamethrin, DDT and malathion was assessed using the World Health Organization insecticide susceptibility test. Additionally, An. gambiae specimens were screened for knockdown (kdr) and acetylcholineesterase (ace1) target site resistance alleles, and the expression levels of eight metabolic resistance genes, including seven cytochrome P450 monooxygenases (P450s) and one glutathione S-transferase (GST), measured with reverse transcription quantitative real-time polymerase chain reaction (qPCR). RESULTS: Overall, 2383 adult mosquitoes from 12 different taxa were collected with Culex quinquefasciatus and An. gambiae being the predominant taxa. Molecular identification of An. gambiae s.l. revealed the presence of Anopheles arabiensis, Anopheles coluzzii, An. gambiae sensu stricto (s.s.) and Anopheles coluzzii/An. gambiae s.s. hybrids. Anopheles gambiae mosquitoes were resistant to all insecticides except malathion. PCR diagnostics revealed the presence of ace1-G280S and the kdr L995F, L995S and N1570Y target-site mutations. Additionally, several genes were upregulated, including five P450s (i.e., CYP6P3, CYP6M2, CYP9K1, CYP6Z1, CYP6P1) and GSTE2. CONCLUSION: This is the first documented presence of An. arabiensis in Côte d'Ivoire. Its detection - together with a recent finding further north of the country - confirms its existence in the country, which is an early warning sign, as An. arabiensis shows a different biology than the currently documented malaria vectors. Because the local An. gambiae population was still susceptible to malathion, upregulation of P450s, conferring insecticide resistance to pyrethroids, together with the presence of ace1, suggest negative cross-resistance. Therefore, organophosphates could be an alternative insecticide class for indoor residual spraying in the Ellibou area, while additional tools against the outdoor biting An. arabiensis will have to be considered.

A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

Multiple introductions and overwintering shape the progressive invasion of Aedes albopictus beyond the Alps.

Aedes albopictus originates from Southeast Asia and is considered one of the most invasive species globally. This mosquito is a nuisance and a disease vector of significant public health relevance. In Europe, Ae. albopictus is firmly established and widespread south of the Alps, a mountain range that forms a formidable biogeographic barrier to many organisms. Recent reports of Ae. albopictus north of the Alps raise questions of (1) the origins of its recent invasion, and (2) if this mosquito has established overwintering populations north of the Alps. To answer these questions, we analyzed population genomic data from >4000 genome-wide SNPs obtained through double-digest restriction site-associated DNA sequencing. We collected SNP data from specimens from six sites in Switzerland, north and south of the Alps, and analyzed them together with specimens from other 33 European sites, five from the Americas, and five from its Asian native range. At a global level, we detected four genetic clusters with specimens from Indonesia, Brazil, and Japan as the most differentiated, whereas specimens from Europe, Hong Kong, and USA largely overlapped. Across the Alps, we detected a weak genetic structure and high levels of genetic admixture, supporting a scenario of rapid and human-aided dispersal along transportation routes. While the genetic pattern suggests frequent re-introductions into Switzerland from Italian sources, the recovery of a pair of full siblings in two consecutive years in Strasbourg, France, suggests the presence of an overwintering population north of the Alps. The suggestion of overwintering populations of Ae. albopictus north of the Alps and the expansion patterns identified points to an increased risk of further northward expansion and the need for increased surveillance of mosquito populations in Northern Europe.

Correction: Integrated Aedes management for the control of Aedes-borne diseases.

[This corrects the article DOI: 10.1371/journal.pntd.0006845.].

An evidence synthesis approach for combining different data sources illustrated using entomological efficacy of insecticides for indoor residual spraying.

BACKGROUND: Prospective malaria public health interventions are initially tested for entomological impact using standardised experimental hut trials. In some cases, data are collated as aggregated counts of potential outcomes from mosquito feeding attempts given the presence of an insecticidal intervention. Comprehensive data i.e. full breakdowns of probable outcomes of mosquito feeding attempts, are more rarely available. Bayesian evidence synthesis is a framework that explicitly combines data sources to enable the joint estimation of parameters and their uncertainties. The aggregated and comprehensive data can be combined using an evidence synthesis approach to enhance our inference about the potential impact of vector control products across different settings over time. METHODS: Aggregated and comprehensive data from a meta-analysis of the impact of Pirimiphos-methyl, an indoor residual spray (IRS) product active ingredient, used on wall surfaces to kill mosquitoes and reduce malaria transmission, were analysed using a series of statistical models to understand the benefits and limitations of each. RESULTS: Many more data are available in aggregated format (N = 23 datasets, 4 studies) relative to comprehensive format (N = 2 datasets, 1 study). The evidence synthesis model had the smallest uncertainty at predicting the probability of mosquitoes dying or surviving and blood-feeding. Generating odds ratios from the correlated Bernoulli random sample indicates that when mortality and blood-feeding are positively correlated, as exhibited in our data, the number of successfully fed mosquitoes will be under-estimated. Analysis of either dataset alone is problematic because aggregated data require an assumption of independence and there are few and variable data in the comprehensive format. CONCLUSIONS: We developed an approach to combine sources from trials to maximise the inference that can be made from such data and that is applicable to other systems. Bayesian evidence synthesis enables inference from multiple datasets simultaneously to give a more informative result and highlight conflicts between sources. Advantages and limitations of these models are discussed.

Multi-insecticide resistant malaria vectors in the field remain susceptible to malathion, despite the presence of Ace1 point mutations.

Insecticide resistance in Anopheles mosquitoes is seriously threatening the success of insecticide-based malaria vector control. Surveillance of insecticide resistance in mosquito populations and identifying the underlying mechanisms enables optimisation of vector control strategies. Here, we investigated the molecular mechanisms of insecticide resistance in three Anopheles coluzzii field populations from southern Côte d'Ivoire, including Agboville, Dabou and Tiassalé. All three populations were resistant to bendiocarb, deltamethrin and DDT, but not or only very weakly resistant to malathion. The absence of malathion resistance is an unexpected result because we found the acetylcholinesterase mutation Ace1-G280S at high frequencies, which would typically confer cross-resistance to carbamates and organophosphates, including malathion. Notably, Tiassalé was the most susceptible population to malathion while being the most resistant one to the pyrethroid deltamethrin. The resistance ratio to deltamethrin between Tiassalé and the laboratory reference colony was 1,800 fold. By sequencing the transcriptome of individual mosquitoes, we found numerous cytochrome P450-dependent monooxygenases - including CYP6M2, CYP6P2, CYP6P3, CYP6P4 and CYP6P5 - overexpressed in all three field populations. This could be an indication for negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate pro-insecticides, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathione S-transferases and other candidates putatively involved in insecticide resistance.

AIMSurv: First pan-European harmonized surveillance of Aedes invasive mosquito species of relevance for human vector-borne diseases.

Human and animal vector-borne diseases, particularly mosquito-borne diseases, are emerging or re-emerging worldwide. Six Aedes invasive mosquito (AIM) species were introduced to Europe since the 1970s: Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus. Here, we report the results of AIMSurv2020, the first pan-European surveillance effort for AIMs. Implemented by 42 volunteer teams from 24 countries. And presented in the form of a dataset named "AIMSurv Aedes Invasive Mosquito species harmonized surveillance in Europe. AIM-COST Action. Project ID: CA17108". AIMSurv2020 harmonizes field surveillance methodologies for sampling different AIMs life stages, frequency and minimum length of sampling period, and data reporting. Data include minimum requirements for sample types and recommended requirements for those teams with more resources. Data are published as a Darwin Core archive in the Global Biodiversity Information Facility- Spain, comprising a core file with 19,130 records (EventID) and an occurrences file with 19,743 records (OccurrenceID). AIM species recorded in AIMSurv2020 were Ae. albopictus, Ae. japonicus and Ae. koreicus, as well as native mosquito species.

Radio-frequency exposure of the yellow fever mosquito (A. aegypti) from 2 to 240 GHz.

Fifth generation networks (5G) will be associated with a partial shift to higher carrier frequencies, including wavelengths comparable in size to insects. This may lead to higher absorption of radio frequency (RF) electromagnetic fields (EMF) by insects and could cause dielectric heating. The yellow fever mosquito (Aedes aegypti), a vector for diseases such as yellow and dengue fever, favors warm climates. Being exposed to higher frequency RF EMFs causing possible dielectric heating, could have an influence on behavior, physiology and morphology, and could be a possible factor for introduction of the species in regions where the yellow fever mosquito normally does not appear. In this study, the influence of far field RF exposure on A. aegypti was examined between 2 and 240 GHz. Using Finite Difference Time Domain (FDTD) simulations, the distribution of the electric field in and around the insect and the absorbed RF power were found for six different mosquito models (three male, three female). The 3D models were created from micro-CT scans of real mosquitoes. The dielectric properties used in the simulation were measured from a mixture of homogenized A. aegypti. For a given incident RF power, the absorption increases with increasing frequency between 2 and 90 GHz with a maximum between 90 and 240 GHz. The absorption was maximal in the region where the wavelength matches the size of the mosquito. For a same incident field strength, the power absorption by the mosquito is 16 times higher at 60 GHz than at 6 GHz. The higher absorption of RF power by future technologies can result in dielectric heating and potentially influence the biology of this mosquito.

Pyrethroid and Etofenprox Resistance in Anopheles gambiae and Anopheles coluzzii from Vegetable Farms in Yaoundé, Cameroon: Dynamics, Intensity and Molecular Basis.

Previous studies have indicated widespread insecticide resistance in malaria vector populations from Cameroon. However, the intensity of this resistance and underlying mechanisms are poorly known. Therefore, we conducted three cross-sectional resistance surveys between April 2018 and October 2019, using the revised World Health Organization protocol, which includes resistance incidences and intensity assessments. Field-collected Anopheles gambiae s.l. populations from Nkolondom, Nkolbisson and Ekié vegetable farms in the city of Yaoundé were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1× insecticide diagnostic concentrations for resistance incidence, then 5× and 10× concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan® qPCR assays. In Nkolbisson, both An. coluzzii (96%) and An. gambiae s.s. (4%) were found together, whereas only An. gambiae s.s. was present in Nkolondom, and only An. coluzzii was present in Ekié. All three populations were resistant to the four insecticides (<75% mortality rates-MR1×), with intensity generally fluctuating over the time between mod-erate (<98%-MR5×; ≥98%-MR10×) and high (76-97%-MR10×). The kdr L995F, L995S, and N1570Y, and the Ace-1 G280S-resistant alleles were found in An. gambiae from Nkolondom, at 73%, 1%, 16% and 13% frequencies, respectively, whereas only the kdr L995F was found in An. gambiae s.s. from Nkolbisson at a 50% frequency. In An. coluzzii from Nkolbisson and Ekié, we detected only the kdr L995F allele at 65% and 60% frequencies, respectively. Furthermore, expression levels of Cyp6m2, Cyp9k1, and Gste2 metabolic genes were highly upregulated (over fivefold) in Nkolondom and Nkolbisson. Pyrethroid and etofenprox-based vector control interventions may be jeopardized in the prospected areas, due to high resistance intensity, with multiple mechanisms in An. gambiae s.s. and An. coluzzii.

The pharmacokinetics and drug-drug interactions of ivermectin in Aedes aegypti mosquitoes.

Mosquitoes are vectors of major diseases such as dengue fever and malaria. Mass drug administration of endectocides to humans and livestock is a promising complementary approach to current insecticide-based vector control measures. The aim of this study was to establish an insect model for pharmacokinetic and drug-drug interaction studies to develop sustainable endectocides for vector control. Female Aedes aegypti mosquitoes were fed with human blood containing either ivermectin alone or ivermectin in combination with ketoconazole, rifampicin, ritonavir, or piperonyl butoxide. Drug concentrations were quantified by LC-MS/MS at selected time points post-feeding. Primary pharmacokinetic parameters and extent of drug-drug interactions were calculated by pharmacometric modelling. Lastly, the drug effect of the treatments was examined. The mosquitoes could be dosed with a high precision (%CV: ≤13.4%) over a range of 0.01-1 µg/ml ivermectin without showing saturation (R2: 0.99). The kinetics of ivermectin were characterised by an initial lag phase of 18.5 h (CI90%: 17.0-19.8 h) followed by a slow zero-order elimination rate of 5.5 pg/h (CI90%: 5.1-5.9 pg/h). By contrast, ketoconazole, ritonavir, and piperonyl butoxide were immediately excreted following first order elimination, whereas rifampicin accumulated over days in the mosquitoes. Ritonavir increased the lag phase of ivermectin by 11.4 h (CI90%: 8.7-14.2 h) resulting in an increased exposure (+29%) and an enhanced mosquitocidal effect. In summary, this study shows that the pharmacokinetics of drugs can be investigated and modulated in an Ae. aegypti animal model. This may help in the development of novel vector-control interventions and further our understanding of toxicology in arthropods.

Surveillance of invasive Aedes mosquitoes along Swiss traffic axes reveals different dispersal modes for Aedes albopictus and Ae. japonicus.

Over the past three decades, Europe has witnessed an increased spread of invasive aedine mosquito species, most notably Aedes albopictus, a key vector of chikungunya, dengue and Zika virus. While its distribution in southern Europe is well documented, its dispersal modes across the Alps remain poorly investigated, preventing a projection of future scenarios beyond its current range in order to target mosquito control. To monitor the presence and frequency of invasive Aedes mosquitoes across and beyond the Alps we set oviposition and BG-Sentinel traps at potential points of entry with a focus on motorway service areas across Switzerland. We placed the traps from June to September and controlled them for the presence of mosquitoes every other week between 2013 and 2018. Over the six years of surveillance we identified three invasive Aedes species, including Ae. albopictus, Ae. japonicus and Ae. koreicus. Based on the frequency and distribution patterns we conclude that Ae. albopictus and Ae. koreicus are being passively spread primarily along the European route E35 from Italy to Germany, crossing the Alps, while Ae. japonicus has been expanding its range from northern Switzerland across the country most likely through active dispersal.

Use of Insecticides in Agriculture and the Prevention of Vector-Borne Diseases: Population Knowledge, Attitudes, Practices and Beliefs in Elibou, South Côte d'Ivoire.

People's knowledge, attitudes, practices and beliefs (KAPB) pertaining to malaria are generally well described. However, little is known about population knowledge and awareness of insecticide resistance in malaria vectors. The aim of this study was to investigate KAPB related to insecticide resistance in malaria vectors due to the use of insecticides in agriculture and the prevention against mosquitoes. In mid-2017, we carried out a cross-sectional survey in Elibou, South Côte d'Ivoire, employing a mixed methods approach. Quantitative data were obtained with a questionnaire addressed to household heads. Interviews were conducted with key opinion leaders, including village chiefs, traditional healers, heads of health centres and pesticide sellers. Focus group discussions were conducted with youth and elders. A total of 203 individuals participated in the questionnaire survey (132 males, 65%). We found that people had good knowledge about malaria and mosquitoes transmitting the disease, while they felt that preventing measures were ineffective. Pesticides were intensively used by farmers, mainly during the rainy season. Among the pesticides used, insecticides and herbicides were most commonly used. While there was poor knowledge about resistance, the interviewees stated that insecticides were not killing the mosquitoes anymore. The main reason given was that insecticides were diluted by the manufacturers as a marketing strategy to sell larger quantities. More than a third of the farmers used agricultural pesticides for domestic purposes to kill weeds or mosquitoes. We observed a misuse of pesticides among farmers, explained by the lack of specific training. In the community, long-lasting insecticidal nets were the most common preventive measure against malaria, followed by mosquito coils and insecticide sprays. The interviewees felt that the most effective way of dealing with insecticide resistance was to combine at least two preventive measures. In conclusion, population attitudes and practices related to insecticides used in agriculture and the prevention against mosquitoes could lead to resistance in malaria vectors, while people's knowledge about insecticide resistance was limited. There is a need to raise awareness in communities about the presence of resistance in malaria vectors and to involve them in resistance management.

Active dispersal of Aedes albopictus: a mark-release-recapture study using self-marking units.

BACKGROUND: Understanding the dispersal dynamics of invasive mosquito species is fundamental to improve vector surveillance and to target control efforts. Aedes albopictus has been deemed a poor flyer as its range of active dispersal is generally assumed to be limited to a few hundred metres, while laboratory studies suggest this mosquito could actually fly several kilometres. The discrepancy may be due to differences in the local environment or to the methodological approach. In Switzerland, Ae. albopictus has been present since 2003 and has since then expanded its range. While passive dispersal is a key driver, it remains unclear how far this mosquito spreads through active flight and what the age structure and size of the local population are, all important parameters for vector surveillance and control. METHOD: We investigated the active dispersal, daily survival rate and population size of Ae. albopictus in mark-release-recapture studies in Coldrerio and Lugano, two areas of intensive control in Switzerland. To mark mosquitoes emerging from breeding sites, we used self-marking units with fluorescent pigment that have minimal impact on mosquito survival and behaviour. We recaptured the adult mosquitoes with BG-Sentinel traps within a radius of 1 km from the marking units over 22 consecutive days. RESULTS: We found that 77.5% of the recaptured Ae. albopictus individuals flew further than 250 m, the limit that is usually deemed sufficient for vector containment. The average age of females and males was 8.6 days and 7.8 days in Coldrerio and Lugano, respectively, while the estimated mosquito population densities were 134 mosquitoes/ha in Coldrerio and 767 mosquitoes/ha in Lugano. CONCLUSIONS: Self-marking units are an effective tool to mark wild mosquitoes. Using this approach, we found that mosquitoes survive long enough to potentially transmit arboviral disease in our study area and that host-seeking Ae. albopictus females may travel further than previously assumed for European mosquito populations. This finding has direct implications for vector control as emergency treatments around positive cases, as well as surveillance and control around detections of new infestations, might need to be extended beyond the usual recommended range of just a few hundred metres.

Evaluation of honey-baited FTA cards in combination with different mosquito traps in an area of low arbovirus prevalence.

BACKGROUND: The threat of mosquito-borne diseases is increasing in continental Europe as demonstrated by several autochthonous chikungunya, dengue and West Nile virus outbreaks. In Switzerland, despite the presence of competent vectors, routine surveillance of arboviruses in mosquitoes is not being carried out, mainly due to the high costs associated with the need of a constant cold chain and laborious processing of thousands of mosquitoes. An alternative approach is using honey-baited nucleic acid preserving cards (FTA cards) to collect mosquito saliva that may be analysed for arboviruses. Here, we evaluate whether FTA cards could be used to detect potentially emerging viruses in an area of low virus prevalence in combination with an effective mosquito trap. METHODS: In a field trial in southern Switzerland we measured side-by-side the efficacy of the BG-Sentinel 2, the BG-GAT and the Box gravid trap to catch Aedes and Culex mosquitoes in combination with honey-baited FTA cards during 80 trapping sessions of 48 hours. We then screened both the mosquitoes and the FTA cards for the presence of arboviruses using reverse-transcription PCR. The efficacy of the compared trap types was evaluated using generalized linear mixed models. RESULTS: The Box gravid trap collected over 11 times more mosquitoes than the BG-GAT and BG-Sentinel 2 trap. On average 75.9% of the specimens fed on the honey-bait with no significant difference in feeding rates between the three trap types. From the total of 1401 collected mosquitoes, we screened 507 Aedes and 500 Culex females for the presence of arboviruses. A pool of six Cx. pipiens/Cx. torrentium mosquitoes and also the FTA card from the same Box gravid trap were positive for Usutu virus. Remarkably, only two of the six Culex mosquitoes fed on the honey-bait, emphasising the high sensitivity of the method. In addition, two Ae. albopictus collections but no FTA cards were positive for mosquito-only flaviviruses. CONCLUSIONS: Based on our results we conclude that honey-baited FTA cards, in combination with the Box gravid trap, are an effective method for arbovirus surveillance in areas of low prevalence, particularly where resources are limited for preservation and screening of individual mosquitoes.