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Abstract Background Attractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the insect alimentary canal, with potential to deliver a variety of compounds new to medical entomology and malaria control. Its potential to control mosquitoes was recently demonstrated in major field trials in Africa. The pyrrole chlorfenapyr is an insecticide new to malaria vector control, and through its unique mode of action—disruption of ATP mediated energy transfer in mitochondria—it may have direct action on energy transfer in the flight muscle cells of mosquitoes. It may also have potential to disrupt mitochondrial function in malarial parasites co-existing within the infected mosquito. However, little is known about the impact of such compounds on vector competence in mosquitoes responsible for malaria transmission. Methods In this study, ATSBs containing chlorfenapyr insecticide and, as a positive control, the anti-malarial drugs artemether/lumefantrine (A/L) were compared for their effect on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae sensu stricto (s.s.) and for their capacity to reduce vector competence. Female mosquitoes were exposed to ATSB containing either sublethal dose of chlorfenapyr (CFP: 0.025%) or concentrations of A/L ranging from 0.4/2.4 mg/ml to 2.4/14.4 mg/ml, either shortly before or after taking infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments. Results Both the prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or chlorfenapyr, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg/ml) totally erased P. falciparum parasites: 0% prevalence of infection in female mosquitoes exposed compared to 62% of infection in negative controls (df = 1, χ2 = 31.23 p

Abstract Background Eave tube technology is a novel method of insecticide application that uses an electrostatic coating system to boost insecticide efficacy against resistant mosquitoes. A series of previous experiments showed encouraging insecticidal effects against malaria vectors. This study was undertaken to assess the effects of the eave tube approach on other Culicidae, in particular Culex quinquefasciatus, under laboratory and semi-field conditions. Methods Larvae of Cx. quinquefasciatus from Bouaké were collected and reared to adult stage, and World Health Organization (WHO) cylinder tests were performed to determine their resistance status. WHO standard 3-min cone bioassays were conducted using PermaNet 2.0 netting versus eave tube-treated inserts. To assess the transient exposure effect on Cx. quinquefasciatus, eave tube assay utilizing smelly socks as attractant was performed with exposure time of 30 s, 1 min, and 2 min on 10% beta-cyfluthrin-treated inserts. Residual activity of these treated inserts was then monitored over 9 months. Field tests involving release–recapture of Cx. quinquefasciatus within enclosures around experimental huts fitted with windows and untreated or insecticide-treated eave tubes were conducted to determine house entry preference and the impact of tubes on the survival of this species. Results Bouaké Cx. quinquefasciatus displayed high resistance to three out of four classes of insecticides currently used in public health. After 3 min of exposure in cone tests, 10% beta-cyfluthrin-treated inserts induced 100% mortality in Cx. quinquefasciatus, whereas the long-lasting insecticidal net (LLIN) only killed 4.5%. With reduced exposure time on the eave tube insert, mortality was still 100% after 2 min, 88% after 1 min, and 44% after 30 s. Mortality following 1 h exposure on 10% beta-cyfluthrin-treated insert was > 80% continuously up to 7 months post-treatment. Data suggest that Cx. quinquefasciatus have a stronger preference for entering a house through the eaves than through windows. Beta-cyfluthrin-treated inserts were able to kill 51% of resistant Cx. quinquefasciatus released within the enclosure. Conclusions Eave tubes are a novel method for delivery of insecticide to the house. They attract nuisance host-seeking Cx. quinquefasciatus mosquitoes and are as effective in controlling them as they are against pyrethroid-resistant Anopheles gambiae, despite the high level of resistance Cx. quinquefasciatus have developed. Graphical Abstract

Abstract Background A better understanding of vector distribution and malaria transmission dynamics at a local scale is essential for implementing and evaluating effectiveness of vector control strategies. Through the data gathered in the framework of a cluster randomized controlled trial (CRT) evaluating the In2Care (Wageningen, Netherlands) Eave Tubes strategy, the distribution of the Anopheles vector, their biting behaviour and malaria transmission dynamics were investigated in Gbêkê region, central Côte d’Ivoire. Methods From May 2017 to April 2019, adult mosquitoes were collected monthly using human landing catches (HLC) in twenty villages in Gbêkê region. Mosquito species wereidentified morphologically. Monthly entomological inoculation rates (EIR) were estimated by combining the HLC data with mosquito sporozoite infection rates measured in a subset of Anopheles vectors using PCR. Finally, biting rate and EIR fluctuations were fit to local rainfall data to investigate the seasonal determinants of mosquito abundance and malaria transmission in this region. Results Overall, Anopheles gambiae, Anopheles funestus, and Anopheles nili were the three vector complexes found infected in the Gbêkê region, but there was a variation in Anopheles vector composition between villages. Anopheles gambiae was the predominant malaria vector responsible for 84.8% of Plasmodium parasite transmission in the area. An unprotected individual living in Gbêkê region received an average of 260 [222–298], 43.5 [35.8–51.29] and 3.02 [1.96–4] infected bites per year from An. gambiae, An. funestus and An. nili, respectively. Vector abundance and malaria transmission dynamics varied significantly between seasons and the highest biting rate and EIRs occurred in the months of heavy rainfall. However, mosquitoes infected with malaria parasites remained present in the dry season, despite the low density of mosquito populations. Conclusion These results demonstrate that the intensity of malaria transmission is extremely high in Gbêkê region, especially during the rainy season. The study highlights the risk factors of transmission that could negatively impact current interventions that target indoor control, as well as the urgent need for additional vector control tools to target the population of malaria vectors in Gbêkê region and reduce the burden of the disease.

Abstract Background A study was conducted prior to implementing a cluster-randomized controlled trial (CRT) of a lethal house lure strategy in central Côte d’Ivoire to provide baseline information on malaria indicators in 40 villages across five health districts. Methods Human landing catches (HLC) were performed between November and December 2016, capturing mosquitoes indoors and outdoors between 18.00 and 08.00 h. Mosquitoes were processed for entomological indicators of malaria transmission (human biting, parity, sporozoite, and entomological inoculation rates (EIR)). Species composition and allelic frequencies of kdr-w and ace-1 R mutations were also investigated within the Anopheles gambiae complex. Results Overall, 15,632 mosquitoes were captured. Anopheles gambiae sensu lato (s.l.) and Anopheles funestus were the two malaria vectors found during the survey period, with predominance for An. gambiae (66.2%) compared to An. funestus (10.3%). The mean biting rate for An. gambiae was almost five times higher than that for An. funestus (19.8 bites per person per night for An. gambiae vs 4.3 bites per person per night for An. funestus) and this was evident indoors and outdoors. Anopheles funestus was more competent to transmit malaria parasites in the study area, despite relatively lower number tested for sporozoite index (4.14% (63/1521) for An. gambiae vs 8.01% (59/736) for An. funestus; χ2 = 12.216; P 85%), coupled with high malaria transmission pattern, which could guide the use of Eave tubes in the study areas.

Abstract Background There is evidence that the knockdown resistance gene (Kdr) L1014F and acetylcholinesterase-1 gene (Ace-1 R ) G119S mutations involved in pyrethroid and carbamate resistance in Anopheles gambiae influence malaria transmission in sub-Saharan Africa. This is likely due to changes in the behaviour, life history and vector competence and capacity of An. gambiae. In the present study, performed as part of a two-arm cluster randomized controlled trial evaluating the impact of household screening plus a novel insecticide delivery system (In2Care Eave Tubes), we investigated the distribution of insecticide target site mutations and their association with infection status in wild An. gambiae sensu lato (s.l.) populations. Methods Mosquitoes were captured in 40 villages around Bouaké by human landing catch from May 2017 to April 2019. Randomly selected samples of An. gambiae s.l. that were infected or not infected with Plasmodium sp. were identified to species and then genotyped for Kdr L1014F and Ace-1 R G119S mutations using quantitative polymerase chain reaction assays. The frequencies of the two alleles were compared between Anopheles coluzzii and Anopheles gambiae and then between infected and uninfected groups for each species. Results The presence of An. gambiae (49%) and An. coluzzii (51%) was confirmed in Bouaké. Individuals of both species infected with Plasmodium parasites were found. Over the study period, the average frequency of the Kdr L1014F and Ace-1 R G119S mutations did not vary significantly between study arms. However, the frequencies of the Kdr L1014F and Ace-1 R G119S resistance alleles were significantly higher in An. gambiae than in An. coluzzii [odds ratio (95% confidence interval): 59.64 (30.81–131.63) for Kdr, and 2.79 (2.17–3.60) for Ace-1 R ]. For both species, there were no significant differences in Kdr L1014F or Ace-1 R G119S genotypic and allelic frequency distributions between infected and uninfected specimens (P > 0.05). Conclusions Either alone or in combination, Kdr L1014F and Ace-1 R G119S showed no significant association with Plasmodium infection in wild An. gambiae and An. coluzzii, demonstrating the similar competence of these species for Plasmodium transmission in Bouaké. Additional factors including behavioural and environmental ones that influence vector competence in natural populations, and those other than allele measurements (metabolic resistance factors) that contribute to resistance, should be considered when establishing the existence of a link between insecticide resistance and vector competence. Graphical Abstract

Abstract Background The widespread emergence of insecticide resistance in African malaria vectors remains one of the main challenges facing control programmes. Electrostatic coating that uses polarity to bind insecticide particles is a new way of delivering insecticides to mosquitoes. Although previous tests demonstrated the resistance breaking potential of this application method, studies screening and investigating the residual efficacy of a broader range of insecticides are necessary. Methods Eleven insecticide powder formulations belonging to six insecticide classes (pyrethroid, carbamate, organophosphate, neonicotinoid, entomopathogenic fungus and boric acid) were initially screened for residual activity over 4 weeks against pyrethroid resistant Anopheles gambiae sensu lato (s.l.) from the M’bé valley, central Côte d’Ivoire. Tests were performed using the eave tube assay that simulates the behavioural interaction between mosquitoes and insecticide-treated inserts. With the best performing insecticide, persistence was monitored over 12 months and the actual contact time lethal to mosquitoes was explored, using a range of transient exposure time (5 s, 30 s, 1 min up to 2 min) in the tube assays in laboratory. The mortality data were calibrated against overnight release-recapture data from enclosure around experimental huts incorporating treated inserts at the M’bé site. The natural recruitment rate of mosquitoes to the tube without insecticide treatment was assessed using fluorescent dust particles. Results Although most insecticides assayed during the initial screening induced significant mortality (45–100%) of pyrethroid resistant An. gambiae during the first 2 weeks, only 10% beta-cyfluthrin retained high residual efficacy, killing 100% of An. gambiae during the first month and > 80% over 8 subsequent months. Transient exposure for 5 s of mosquitoes to 10% beta-cyfluthrin produced 56% mortality, with an increase to 98% when contact time was extended to 2 min (P = 0.001). In the experimental hut enclosures, mortality of An. gambiae with 10% beta-cyfluthrin treated inserts was 55% compared to similar rate (44%) of mosquitoes that contacted the inserts treated with fluorescent dusts. This suggests that all host-seeking female mosquitoes that contacted beta-cyfluthrin treated inserts during host-seeking were killed. Conclusion The eave tube technology is a novel malaria control approach which combines house proofing and targeted control of anopheline mosquitoes using insecticide treated inserts. Beta-cyfluthrin showed great promise for providing prolonged control of pyrethroid resistant An. gambiae and has potential to be deployed year-round in areas where malaria parasites are transmitted by highly pyrethroid resistant An. gambiae across sub-Saharan Africa.

Abstract Background Eave tubes are a type of housing modification that provide a novel way of delivering insecticides to mosquitoes as they attempt to enter the house. The current study reports on a series of semi-field studies aimed at improving the understanding of how eave tubes might impact mosquito mortality and behaviour. Methods Experiments were conducted using West African style experimental huts at a field site in M’be, Côte d’Ivoire. Huts were modified in various ways to determine: (i) whether mosquitoes in this field setting naturally recruit to eave tubes; (ii) whether eave tubes can reduce house entry even in the absence of screening; (iii) whether mosquitoes suffer mortality if they attempt to exit a house via treated eave tubes; and, (iv) whether screening and eave tubes might deflect mosquitoes into neighbouring houses without the intervention. Results Ninety percent more mosquitoes (Anopheles gambiae sensu lato, and other species) entered huts through open eaves tubes compared to window slits. The addition of insecticide-treated eave tubes reduced mosquito entry by 60%, even when windows remained open. Those mosquitoes that managed to enter the huts exhibited a 64% reduction in blood feeding and a tendency for increased mortality, suggesting contact with insecticide-treated inserts prior to hut entry. When An. gambiae mosquitoes were deliberately introduced into huts with treated eave tubes, there was evidence of six times increase in overnight mortality, suggesting mosquitoes can contact treated eave tube inserts when trying to exit the hut. There was no evidence for deflection of mosquitoes from huts with screening, or screening plus eave tubes, to adjacent unmodified huts. Conclusions Eave tubes are a potentially effective way to target Anopheles mosquitoes with insecticides. That treated eave tubes can reduce mosquito entry even when windows are open is a potentially important result as it suggests that eave tubes might not need to be combined with household screening to have an impact on malaria transmission. The absence of deflection is also a potentially important result as coverage of eave tubes and/or screening is unlikely to be 100% and it is important that households that do not have the technology are not disadvantaged by those that do.