UNASSIGNED: The resurgence of Anopheles funestus, a dominant vector of human malaria in western Kenya was partly attributed to insecticide resistance. However, evidence on the molecular basis of pyrethroid resistance in western Kenya is limited. Noncoding RNAs (ncRNAs) form a vast class of RNAs that do not code for proteins and are ubiquitous in the insect genome. Here, we demonstrated that multiple ncRNAs could play a potential role in An. funestusresistance to pyrethroid in western Kenya.
UNASSIGNED: Anopheles funestus mosquitoes were sampled by aspiration methods in Bungoma, Teso, Siaya, Port Victoria and Kombewa in western Kenya. The F1 progenies were exposed to deltamethrin (0.05%), permethrin (0.75%), DDT (4%) and pirimiphos-methyl (0.25%) following WHO test guidelines. A synergist assay using piperonyl butoxide (PBO) (4%) was conducted to determine cytochrome P450s\' role in pyrethroid resistance. RNA-seq was conducted on a combined pool of specimens that were resistant and unexposed, and the results were compared with those of the FANG susceptible strain. This approach aimed to uncover the molecular mechanisms underlying pyrethroid resistance.
UNASSIGNED: Pyrethroid resistance was observed in all the sites with an average mortality rate of 57.6%. Port Victoria had the highest level of resistance to permethrin (MR=53%) and deltamethrin (MR=11%) pyrethroids. Teso had the lowest level of resistance to permethrin (MR=70%) and deltamethrin (MR=87%). Resistance to DDT was observed only in Kombewa (MR=89%) and Port Victoria (MR=85%). A full susceptibility to P-methyl (0.25%) was observed in all the sites. PBO synergist assay revealed high susceptibility (>98%) to the pyrethroids in all the sites except for Port Victoria (MR=96%, n=100). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by imipenemase (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP-binding cassettes (2%) and carboxylesterases(1%).
UNASSIGNED: This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention development could help in insecticide resistance management.

Ecosystem change can profoundly affect human well-being and health, including through changes in exposure to vector-borne diseases. Deforestation has increased human exposure to mosquito vectors and malaria risk in Africa, but there is little understanding of how socioeconomic and ecological factors influence the relationship between deforestation and malaria risk. We examined these interrelationships in six sub-Saharan African countries using demographic and health survey data linked to remotely sensed environmental variables for 11,746 children under 5 years old. We found that the relationship between deforestation and malaria prevalence varies by wealth levels. Deforestation is associated with increased malaria prevalence in the poorest households, but there was not significantly increased malaria prevalence in the richest households, suggesting that deforestation has disproportionate negative health impacts on the poor. In poorer households, malaria prevalence was 27%-33% larger for one standard deviation increase in deforestation across urban and rural populations. Deforestation is also associated with increased malaria prevalence in regions where Anopheles gambiae and Anopheles funestus are dominant vectors, but not in areas of Anopheles arabiensis. These findings indicate that deforestation is an important driver of malaria risk among the world\'s most vulnerable children, and its impact depends critically on often-overlooked social and biological factors. An in-depth understanding of the links between ecosystems and human health is crucial in designing conservation policies that benefit people and the environment.

BACKGROUND: Spatial repellents (SR) may complement current vector control tools and provide additional coverage when people are not under their bednets or are outdoors. Here we assessed the efficacy of a metofluthrin-based SR in reducing exposure to pyrethroid-resistant Anopheles funestus in Siaya County, western Kenya.
METHODS: Metofluthrin was vaporized using an emanator configured to a liquid petroleum gas (LPG) canister, placed inside experimental huts (phase 1) or outdoors (phase 2), and evaluated for reductions in human landing rate, density, knockdown and mortality rates of An. funestus, which are present in high density in the area. To demonstrate the mosquito recruiting effect of LPG, a hut with only an LPG cooker but no metofluthrin was added as a comparator and compared with an LPG cooker burning alongside the emanator and a third hut with no LPG cooker as control. Phase 2 evaluated the protective range of the SR product while emanating from the centre of a team of mosquito collectors sitting outdoors in north, south, east and west directions at 5, 10 and 20 feet from the emanating device.
RESULTS: Combustion of LPG with a cook stove increased the density of An. funestus indoors by 51% over controls with no cook stove. In contrast, huts with metofluthrin vaporized with LPG combustion had lower indoor density of An. funestus (99.3% less than controls), with knockdown and mortality rates of 95.5 and 87.7%, respectively, in the mosquitoes collected in the treated huts. In the outdoor study (phase 2), the outdoor landing rate was significantly lower at 5 and 10 feet than at 20 feet from the emanator.
CONCLUSIONS: Vaporized metofluthrin almost completely prevented An. funestus landing indoors and led to 10 times lower landing rates within 10 feet of the emanator outdoors, the first product to demonstrate such potential. Cooking with LPG inside the house could increase exposure to Anopheles mosquito bites, but the use of the metofluthrin canister eliminates this risk.

The primary reason for the failure of malaria vector control across endemic regions is the widespread insecticide resistance observed in Anopheles vectors. The most dominant African vectors of malaria parasites are Anopheles gambiae and Anopheles funestus mosquitoes. These species often exhibit divergent behaviours and adaptive changes underscoring the importance of deploying active and effective measures in their control. Unlike An. gambiae, An. funestus mosquitoes are poorly studied in Benin Republic. However, recent reports indicated that An. funestus can adapt and colonize various ecological niches owing to its resistance against insecticides and adaptation to changing breeding habitats. Unfortunately, scientific investigations on the contribution of An. funestus to malaria transmission, their susceptibility to insecticide and resistance mechanism developed are currently insufficient for the design of better control strategies. In an attempt to gather valuable information on An. funestus, the present review examines the progress made on this malaria vector species in Benin Republic and highlights future research perspectives on insecticide resistance profiles and related mechanisms, as well as new potential control strategies against An. funestus. Literature analysis revealed that An. funestus is distributed all over the country, although present in low density compared to other dominant malaria vectors. Interestingly, An. funestus is being found in abundance during the dry seasons, suggesting an adaptation to desiccation. Among the An. funestus group, only An. funestus sensu stricto (s.s.) and Anopheles leesoni were found in the country with An. funestus s.s. being the most abundant species. Furthermore, An. funestus s.s. is the only one species in the group contributing to malaria transmission and have adapted biting times that allow them to bite at dawn. In addition, across the country, An. funestus were found resistant to pyrethroid insecticides used for bed nets impregnation and also resistant to bendiocarb which is currently being introduced in indoor residual spraying formulation in malaria endemic regions. All these findings highlight the challenges faced in controlling this malaria vector. Therefore, advancing the knowledge of vectorial competence of An. funestus, understanding the dynamics of insecticide resistance in this malaria vector, and exploring alternative vector control measures, are critical for sustainable malaria control efforts in Benin Republic.

BACKGROUND: Children in Kenya spend a substantial amount of time at school, including at dawn and dusk when mosquitoes are active. With changing vector behaviour towards early morning biting, it is important to determine whether there is an additional risk of transmission in schools. This study sought to understand whether late morning biting by Anopheles funestus, previously documented in households in western Kenya, was replicated in schools.
METHODS: From the 4th to the 6th of August 2023, human landing collections were conducted hourly in four schools in Alego Usonga sub-County, Siaya County. The collections were conducted in and outside five classrooms in each school and ran for 17 h, starting at 18:00 until 11:00 h the next morning.
RESULTS: Anopheles funestus was the predominant species collected, forming 93.2% (N = 727) of the entire collection, with peak landing between 06:00 and 07:00 h and continuing until 11:00 h. More than half of the collected An. funestus were either fed or gravid, potentially indicative of multiple bloodmeals within each gonotrophic cycle, and had a sporozoite rate of 2.05%.
CONCLUSIONS: School children spend up to 10 h of their daytime in schools, reporting between 06:00 and 07:00 h and staying in school until as late as 17:00 h, meaning that they receive potentially infectious mosquito bites during the morning hours in these settings. There is a need to consider vector control approaches targeting schools and other peridomestic spaces in the morning hours when An. funestus is active.

BACKGROUND: Over a decade of vector control by indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) distribution on the mainland, and only LLINs on islands had a minimal impact on disease burden in Nchelenge district, northern Zambia. Anopheles funestus and Anopheles gambiae are vectors known only from the mainland. Understanding vector bionomics in the district is necessary for planning and targeting effective vector control. This study aimed to provide information on abundance, seasonality, and Plasmodium falciparum sporozoite infectivity of malaria vectors in Nchelenge, including islands.
METHODS: Mosquitoes were collected in 192 CDC indoor light traps set in 56 households between January 2015 and January 2016. Morphological and molecular species identifications and P. falciparum circumsporoites by ELISA were performed. Mosquito counts and relative abundances from the islands and mainland were compared, and household factors associated with vector counts were determined.
RESULTS: A total of 5888 anophelines were collected during the study. Of these, 5,704 were female Anopheles funestus sensu lato (s.l.) and 248 female An. gambiae s.l. The highest proportion of An. funestus (n = 4090) was from Chisenga Island and An. gambiae (n = 174) was from Kilwa Island. The highest estimated counts per trap for An. funestus s.l. were from Chisenga island, (89.9, p < 0.001) and from the dry season (78.6, p < 001). For An. gambiae the highest counts per trap were from Kilwa island (3.1, p < 0.001) and the rainy season (2.5, p = 0.007). The highest estimated annual entomological inoculation rate was from Chisenga Island with 91.62 ib/p/y followed by Kilwa Island with 29.77 ib/p/yr, and then Mainland with 19.97 ib/p/yr.
CONCLUSIONS: There was varied species abundance and malaria transmission risk across sites and seasons. The risk of malaria transmission was perennial and higher on the islands. The minimal impact of vector control efforts on the mainland was evident, but limited overall. Vector control intervention coverage with effective tools needs to be extended to the islands to effectively control malaria transmission in Nchelenge district.

BACKGROUND: Anopheles funestus, the main malaria vector, prefer to oviposit in permanent and/or semi-permanent breeding habitats located far from human dwellings. Difficulties in identifying and accessing these habitats jeopardize the feasibility of conventional larviciding. In this way, a semi-field study was conducted to assess the potential of autodissemination of pyriproxyfen (PPF) by An. funestus for its control.
METHODS: The study was conducted inside a semi-field system (SFS). Therein, two identical separate chambers, the treatment chamber with a PPF-treated clay pot (0.25 g AI), and the control chamber with an untreated clay pot. In both chambers, one artificial breeding habitat made of a plastic basin with one litre of water was provided. Three hundred blood-fed female An. funestus aged 5-9 days were held inside untreated and treated clay pots for 30 min and 48 h before being released for oviposition. The impact of PPF on adult emergence, fecundity, and fertility through autodissemination and sterilization effects were assessed by comparing the treatment with its appropriate control group.
RESULTS: Mean (95% CI) percentage of adult emergence was 15.5% (14.9-16.1%) and 70.3% (69-71%) in the PPF and control chamber for females exposed for 30 min (p < 0.001); and 19% (12-28%) and 95% (88-98%) in the PPF and control chamber for females exposed for 48 h (p < 0.001) respectively. Eggs laid by exposed mosquitoes and their hatch rate were significantly reduced compared to unexposed mosquitoes (p < 0.001). Approximately, 90% of females exposed for 48 h retained abnormal ovarian follicles and only 42% in females exposed for 30 min.
CONCLUSIONS: The study demonstrated sterilization and adult emergence inhibition via autodissemination of PPF by An. funestus. Also, it offers proof that sterilized An. funestus can transfer PPF to prevent adult emergence at breeding habitats. These findings warrant further assessment of the autodissemination of PPF in controlling wild population of An. funestus, and highlights its potential for complementing long-lasting insecticidal nets.

Survival and gonotrophic cycle duration are important determinants of the vectorial capacity of malaria vectors but there are a limited number of approaches to estimate these quantities from field data. Time-series of observations of mosquitoes at different stages in the life-cycle are under-used.
Anopheles funestus mosquitoes were caught using various methods over a 7.6-year period in Furvela, Mozambique. Survival and oviposition cycle duration were estimated using (i) an existing time-series approach for analysing dissections of mosquitoes caught in light-traps, extended to allow for variability in the duration of the cycle; (ii) an established approach for estimating cycle duration from resting collection data; (iii) a novel time-series approach fitted to numbers and categories of mosquitoes caught in exit-traps.
Data were available from 7,396, 6,041 and 1,527 trap-nights for exit-traps, light-traps and resting collections respectively. Estimates of cycle duration varied considerably between the different methods. The estimated proportion of female mosquitoes surviving each day of 0.740 (95% credible interval [0.650-0.815]) derived from light-trap data was much lower than the estimated daily survival of male mosquitoes from the model fitted to exit-trap data (0.881, 95% credible interval [0.747-0.987]). There was no tendency for the oviposition cycle to become shorter at higher temperature while the odds of survival of females through the cycle was estimated to be multiplied by 1.021 for every degree of mean weekly temperature increase (95% credible interval [0.991-1.051]). There was negligible temperature dependence and little inter-annual variation in male survival.
The time-series approach fitted to the exit-traps suggests that male An. funestus have higher survival than do females, and that male survival was temperature independent and unaffected by the introduction of long-lasting insecticidal nets (LLINs). The patterns of temperature dependence in females are at variance with results of laboratory studies. Time series approaches have the advantage for estimating survival that they do not depend on representative sampling of mosquitoes over the whole year. However, the estimates of oviposition cycle duration were associated with considerable uncertainty, which appears to be due to variability between insects in the duration of the resting period, and the estimates based on exit-trap data are sensitive to assumptions about relative trapping efficiencies.

Evaluating the susceptibility of malaria vectors to the new WHO-recommended products is a key step before large-scale deployment. We mapped the susceptibility profile of Anopheles funestus to neonicotinoids across Africa and established the diagnostic doses of acetamiprid and imidacloprid with acetone + MERO as solvent. Indoor resting An. funestus were collected in 2021 in Cameroon, Malawi, Ghana and Uganda. Susceptibility to clothianidin, imidacloprid and acetamiprid was evaluated using CDC bottle assays and offsprings of the field-caught adults. The L119F-GSTe2 marker was genotyped to assess the potential cross-resistance between clothianidin and this DDT/pyrethroid-resistant marker. Mosquitoes were susceptible to the three neonicotinoids diluted in acetone + MERO, whereas low mortality was noticed with ethanol or acetone alone. The doses of 6 µg/mL and 4 µg/mL were established as diagnostic concentrations of imidacloprid and acetamiprid, respectively, with acetone + MERO. Pre-exposure to synergists significantly restored the susceptibility to clothianidin. A positive correlation was observed between L119F-GSTe2 mutation and clothianidin resistance with the homozygote resistant mosquitoes being more able to survive than heterozygote or susceptible. This study revealed that An. funestus populations across Africa are susceptible to neonicotinoids, and as such, this insecticide class could be effectively implemented to control this species using IRS. However, potential cross-resistance conferred by GSTe2 calls for regular resistance monitoring in the field.

Microbiome composition has been associated with insecticide resistance in malaria vectors. However, the contribution of major symbionts to the increasingly reported resistance escalation remains unclear. This study explores the possible association of a specific endosymbiont, Asaia spp., with elevated levels of pyrethroid resistance driven by cytochrome P450s enzymes and voltage-gated sodium channel mutations in Anopheles funestus and Anopheles gambiae. Molecular assays were used to detect the symbiont and resistance markers (CYP6P9a/b, 6.5 kb, L1014F, and N1575Y). Overall, genotyping of key mutations revealed an association with the resistance phenotype. The prevalence of Asaia spp. in the FUMOZ_X_FANG strain was associated with the resistance phenotype at a 5X dose of deltamethrin (OR = 25.7; p = 0.002). Mosquitoes with the resistant allele for the markers tested were significantly more infected with Asaia compared to those possessing the susceptible allele. Furthermore, the abundance correlated with the resistance phenotype at 1X concentration of deltamethrin (p = 0.02, Mann-Whitney test). However, for the MANGOUM_X_KISUMU strain, findings rather revealed an association between Asaia load and the susceptible phenotype (p = 0.04, Mann-Whitney test), demonstrating a negative link between the symbiont and permethrin resistance. These bacteria should be further investigated to establish its interactions with other resistance mechanisms and cross-resistance with other insecticide classes.