Genetic markers associated with the widespread insecticide resistance in malaria vector Anopheles funestus populations across Tanzania.

BACKGROUND: Anopheles funestus is a leading vector of malaria in most parts of East and Southern Africa, yet its ecology and responses to vector control remain poorly understood compared with other vectors such as Anopheles gambiae and Anopheles arabiensis. This study presents the first large-scale survey of the genetic and phenotypic expression of insecticide resistance in An. funestus populations in Tanzania. METHODS: We performed insecticide susceptibility bioassays on An. funestus mosquitoes in nine regions with moderate-to-high malaria prevalence in Tanzania, followed by genotyping for resistance-associated mutations (CYP6P9a, CYP6P9b, L119F-GSTe2) and structural variants (SV4.3 kb, SV6.5 kb). Generalized linear models were used to assess relationships between genetic markers and phenotypic resistance. An interactive R Shiny tool was created to visualize the data and support evidence-based interventions. RESULTS: Pyrethroid resistance was universal but reversible by piperonyl-butoxide (PBO). However, carbamate resistance was observed in only five of the nine districts, and dichloro-diphenyl-trichloroethane (DDT) resistance was found only in the Kilombero valley, south-eastern Tanzania. Conversely, there was universal susceptibility to the organophosphate pirimiphos-methyl in all sites. Genetic markers of resistance had distinct geographical patterns, with CYP6P9a-R and CYP6P9b-R alleles, and the SV6.5 kb structural variant absent or undetectable in the north-west but prevalent in all other sites, while SV4.3 kb was prevalent in the north-western and western regions but absent elsewhere. Emergent L119F-GSTe2, associated with deltamethrin resistance, was detected in heterozygous form in districts bordering Mozambique, Malawi and the Democratic Republic of Congo. The resistance landscape was most complex in western Tanzania, in Tanganyika district, where all five genetic markers were detected. There was a notable south-to-north spread of resistance genes, especially CYP6P9a-R, though this appears to be interrupted, possibly by the Rift Valley. CONCLUSIONS: This study underscores the need to expand resistance monitoring to include An. funestus alongside other vector species, and to screen for both the genetic and phenotypic signatures of resistance. The findings can be visualized online via an interactive user interface and could inform data-driven decision-making for resistance management and vector control. Since this was the first large-scale survey of resistance in Tanzania's An. funestus, we recommend regular updates with greater geographical and temporal coverage.

Changes in contributions of different Anopheles vector species to malaria transmission in east and southern Africa from 2000 to 2022.

BACKGROUND: Malaria transmission in Africa is facilitated by multiple species of Anopheles mosquitoes. These vectors have different behaviors and vectorial capacities and are affected differently by vector control interventions, such as insecticide-treated nets and indoor residual spraying. This review aimed to assess changes in the contribution of different vector species to malaria transmission in east and southern Africa over 20 years of widespread insecticide-based vector control. METHODS: We searched PubMed, Global Health, and Web of Science online databases for articles published between January 2000 and April 2023 that provided species-specific sporozoite rates for different malaria vectors in east and southern Africa. We extracted data on study characteristics, biting rates, sporozoite infection proportions, and entomological inoculation rates (EIR). Using EIR data, the proportional contribution of each species to malaria transmission was estimated. RESULTS: Studies conducted between 2000 and 2010 identified the Anopheles gambiae complex as the primary malaria vector, while studies conducted from 2011 to 2021 indicated the dominance of Anopheles funestus. From 2000 to 2010, in 57% of sites, An. gambiae demonstrated higher parasite infection prevalence than other Anopheles species. Anopheles gambiae also accounted for over 50% of EIR in 76% of the study sites. Conversely, from 2011 to 2021, An. funestus dominated with higher infection rates than other Anopheles in 58% of sites and a majority EIR contribution in 63% of sites. This trend coincided with a decline in overall EIR and the proportion of sporozoite-infected An. gambiae. The main vectors in the An. gambiae complex in the region were Anopheles arabiensis and An. gambiae sensu stricto (s.s.), while the important member of the An. funestus group was An. funestus s.s. CONCLUSION: The contribution of different vector species in malaria transmission has changed over the past 20 years. As the role of An. gambiae has declined, An. funestus now appears to be dominant in most settings in east and southern Africa. Other secondary vector species may play minor roles in specific localities. To improve malaria control in the region, vector control should be optimized to match these entomological trends, considering the different ecologies and behaviors of the dominant vector species.

Sterilized Anopheles funestus can autodisseminate sufficient pyriproxyfen to the breeding habitat under semi-field settings.

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. CONCLUSION: 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.

Effects of vegetation densities on the performance of attractive targeted sugar baits (ATSBs) for malaria vector control: a semi-field study.

BACKGROUND: Attractive targeted sugar baits (ATSBs) control sugar-feeding mosquitoes with oral toxicants, and may effectively complement core malaria interventions, such as insecticide-treated nets even where pyrethroid-resistance is widespread. The technology is particularly efficacious in arid and semi-arid areas. However, their performance remains poorly-understood in tropical areas with year-round malaria transmission, and where the abundant vegetation constitutes competitive sugar sources for mosquitoes. This study compared the efficacies of ATSBs (active ingredient: 2% boric acid) in controlled settings with different vegetation densities. METHODS: Potted mosquito-friendly plants were introduced inside semi-field chambers (9.6 m by 9.6 m) to simulate densely-vegetated, sparsely-vegetated, and bare sites without any vegetation (two chambers/category). All chambers had volunteer-occupied huts. Laboratory-reared Anopheles arabiensis were released nightly (200/chamber) and host-seeking females recaptured using human landing catches outdoors (8.00 p.m.-9.00 p.m.) and CDC-light traps indoors (9.00 p.m.-6.00 a.m.). Additionally, resting mosquitoes were collected indoors and outdoors each morning using Prokopack aspirators. The experiments included a "before-and-after" set-up (with pre-ATSBs, ATSBs and post-ATSBs phases per chamber), and a "treatment vs. control" set-up (where similar chambers had ATSBs or no ATSBs). The experiments lasted 84 trap-nights. RESULTS: In the initial tests when all chambers had no vegetation, the ATSBs reduced outdoor-biting by 69.7%, indoor-biting by 79.8% and resting mosquitoes by 92.8%. In tests evaluating impact of vegetation, the efficacy of ATSBs against host-seeking mosquitoes was high in bare chambers (outdoors: 64.1% reduction; indoors: 46.8%) but modest or low in sparsely-vegetated (outdoors: 34.5%; indoors: 26.2%) and densely-vegetated chambers (outdoors: 25.4%; indoors: 16.1%). Against resting mosquitoes, the ATSBs performed modestly across settings (non-vegetated chambers: 37.5% outdoors and 38.7% indoors; sparsely-vegetated: 42.9% outdoors and 37.5% indoors; densely-vegetated: 45.5% outdoors and 37.5% indoors). Vegetation significantly reduced the ATSBs efficacies against outdoor-biting and indoor-biting mosquitoes but not resting mosquitoes. CONCLUSION: While vegetation can influence the performance of ATSBs, the devices remain modestly efficacious in both sparsely-vegetated and densely-vegetated settings. Higher efficacies may occur in places with minimal or completely no vegetation, but such environments are naturally unlikely to sustain Anopheles populations or malaria transmission in the first place. Field studies therefore remain necessary to validate the efficacies of ATSBs in the tropics.

Common predators and factors influencing their abundance in Anopheles funestus aquatic habitats in rural south-eastern Tanzania.

BACKGROUND: The role of larval predators in regulating the Anopheles funestus population in various malaria-endemic countries remains relatively unknown. This study aimed to investigate the common predators that co-exist with Anopheles funestus group larvae and evaluate factors that influence their abundance in rural south-eastern Tanzania. METHODS: Mosquito larvae and predators were sampled concurrently using standard dipper (350 ml) or 10 L bucket in previously identified aquatic habitats in selected villages in southern Tanzania. Predators and mosquito larvae were identified using standard identification keys. All positive habitats were geo-located and their physical features characterized. Water physicochemical parameters such as dissolved oxygen (DO), pH, electrical conductivity (EC), total dissolved solids (TDS) and temperature were also recorded. RESULTS: A total of 85 previously identified An. funestus aquatic habitats in nine villages were sampled for larvae and potential predators. A total of 8,295 predators were sampled. Of these Coenagrionidae 57.7% (n = 4785), Corixidae 12.8% (n = 1,060), Notonectidae 9.9% (n = 822), Aeshnidae 4.9% (n = 405), Amphibian 4.5% (n = 370), Dytiscidae 3.8% (n = 313) were common. A total of 5,260 mosquito larvae were sampled, whereby Anopheles funestus group were 60.3% (n = 3,170), Culex spp. 24.3% (n = 1,279), An. gambie s.l. 8.3% (n = 438) and other anophelines 7.1% (n = 373). Permanent and aquatic habitats larger than 100m2 were positively associated with An. funestus group larvae (P<0.05) and predator abundance (P<0.05). Habitats with submerged vegetation were negatively associated with An. funestus group larvae (P<0.05). Only dissolved oxygen (DO) was positively and significantly affect the abundance of An. funestus group larvae (P<0.05). While predators' abundance was not impacted by all physicochemical parameters. CONCLUSION: Six potential predator families were common in aquatic habitats of An. funestus group larvae. Additional studies are needed to demonstrate the efficacy of different predators on larval density and adult fitness traits. Interventions leveraging the interaction between mosquitoes and predators can be established to disrupt the transmission potential and survival of the An. funestus mosquitoes.

Corrigendum to "Persistently high proportions of plasmodium-infected Anopheles funestus mosquitoes in two villages in the Kilombero valley, South-Eastern Tanzania" [Parasite Epidemiology and Control 18 (2022) e00264].

[This corrects the article DOI: 10.1016/j.parepi.2022.e00264.].

Small-scale field evaluation of transfluthrin-treated eave ribbons and sandals for the control of malaria vectors in rural Tanzania.

BACKGROUND: Early-evening and outdoor-biting mosquitoes may compromise the effectiveness of frontline malaria interventions, notably insecticide-treated nets (ITNs). This study aimed to evaluate the efficacy of low-cost insecticide-treated eave ribbons and sandals as supplementary interventions against indoor-biting and outdoor-biting mosquitoes in south-eastern Tanzania, where ITNs are already widely used. METHODS: This study was conducted in three villages, with 72 households participating (24 households per village). The households were divided into four study arms and assigned: transfluthrin-treated sandals (TS), transfluthrin-treated eave ribbons (TER), a combination of TER and TS, or experimental controls. Each arm had 18 households, and all households received new ITNs. Mosquitoes were collected using double net traps (to assess outdoor biting), CDC light traps (to assess indoor biting), and Prokopack aspirators (to assess indoor resting). Protection provided by the interventions was evaluated by comparing mosquito densities between the treatment and control arms. Additional tests were done in experimental huts to assess the mortality of wild mosquitoes exposed to the treatments or controls. RESULTS: TERs reduced indoor-biting, indoor-resting and outdoor-biting Anopheles arabiensis by 60%, 73% and 41%, respectively, while TS reduced the densities by 18%, 40% and 42%, respectively. When used together, TER & TS reduced indoor-biting, indoor-resting and outdoor-biting An. arabiensis by 53%, 67% and 57%, respectively. Protection against Anopheles funestus ranged from 42 to 69% with TER and from 57 to 74% with TER & TS combined. Mortality of field-collected mosquitoes exposed to TER, TS or both interventions was 56-78% for An. arabiensis and 47-74% for An. funestus. CONCLUSION: Transfluthrin-treated eave ribbons and sandals or their combination can offer significant household-level protection against malaria vectors. Their efficacy is magnified by the transfluthrin-induced mortality, which was observed despite the prevailing pyrethroid resistance in the study area. These results suggest that TER and TS could be useful supplementary tools against residual malaria transmission in areas where ITN coverage is high but additional protection is needed against early-evening and outdoor-biting mosquitoes. Further research is needed to validate the performance of these tools in different settings, and assess their long-term effectiveness and feasibility for malaria control.

Relationships between biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes in south-eastern Tanzania.

BACKGROUND: Malaria transmission can be highly heterogeneous between and within localities, and is influenced by factors such as survival and biting frequencies of Anopheles mosquitoes. This study investigated the relationships between the biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes, which currently dominate malaria transmission in south-east Tanzania. The study also examined how such relationships may influence malaria transmission and control. METHODS: Female An. funestus were collected in houses located 50-100 m, 150-200 m or over 200 m from the nearest known aquatic habitats. The mosquitoes were exposed to 1×, 5× and 10× the diagnostic doses of deltamethrin or permethrin, or to the synergist, piperonyl butoxide (PBO) followed by the pyrethroids, then monitored for 24 h-mortality. Ovaries of exposed and non-exposed mosquitoes were dissected to assess parity as a proxy for biological age. Adults emerging from larval collections in the same villages were tested against the same insecticides at 3-5, 8-11 or 17-20 days old. FINDINGS: Mosquitoes collected nearest to the aquatic habitats (50-100 m) had the lowest mortalities compared to other distances, with a maximum of 51% mortality at 10× permethrin. For the age-synchronized mosquitoes collected as larvae, the insecticide-induced mortality assessed at both the diagnostic and multiplicative doses (1×, 5× and 10×) increased with mosquito age. The highest mortalities at 1× doses were observed among the oldest mosquitoes (17-20 days). At 10× doses, mortalities were 99% (permethrin) and 76% (deltamethrin) among 8-11 day-olds compared to 80% (permethrin) and 58% (deltamethrin) among 3-5 day-olds. Pre-exposure to PBO increased the potency of both pyrethroids. The proportion of parous females was highest among mosquitoes collected farthest from the habitats. CONCLUSION: In this specific setting, older An. funestus and those collected farthest from the aquatic habitats (near the centre of the village) were more susceptible to pyrethroids than the younger ones and those caught nearest to the habitats. These findings suggest that pyrethroid-based interventions may remain at least moderately effective despite widespread pyrethroid-resistance, by killing the older, less-resistant and potentially-infective mosquitoes. Further studies should investigate how and whether these observations could be exploited to optimize malaria control in different settings.

Persistently high proportions of plasmodium-infected Anopheles funestus mosquitoes in two villages in the Kilombero valley, South-Eastern Tanzania.

Background: In south-eastern Tanzania where insecticide-treated nets have been widely used for >20 years, malaria transmission has greatly reduced but remains highly heterogenous over small distances. This study investigated the seasonal prevalence of Plasmodium sporozoite infections in the two main malaria vector species, Anopheles funestus and Anopheles arabiensis for 34 months, starting January 2018 to November 2020. Methods: Adult mosquitoes were collected using CDC-light traps and Prokopack aspirators inside local houses in Igumbiro and Sululu villages, where earlier surveys had found very high densities of An. funestus. Collected females were sorted by taxa, and the samples examined using ELISA assays for detecting Plasmodium circumsporozoite protein in their salivary glands. Results: Of 7859 An. funestus tested, 4.6% (n = 365) were positive for Pf sporozoites in the salivary glands. On the contrary, only 0.4% (n = 9) of the 2382 An. arabiensis tested were positive. The sporozoite prevalence did not vary significantly between the villages or seasons. Similarly, the proportions of parous females of either species were not significantly different between the two villages (p > 0.05) but was slightly higher in An. funestus (0.50) than in An. arabiensis (0.42). Analysis of the 2020 data determined that An. funestus contributed 97.7% of all malaria transmitted in households in these two villages. Conclusions: In contexts where individual vector species mediate most of the pathogen transmission, it may be most appropriate to pursue a species-focused approach to better understand the ecology of the dominant vectors and target them with effective interventions to suppress transmission. Despite the ongoing efforts on tackling malaria in the two study villages, there is still persistently high Plasmodium infection prevalence in local populations of An. funestus, which now carry ~97% of all malaria infections and mediates intense year-round transmission. Further reduction in malaria burden in these or other similar settings requires effective targeting of An. funestus.

A statistical calibration tool for methods used to sample outdoor-biting mosquitoes.

BACKGROUND: Improved methods for sampling outdoor-biting mosquitoes are urgently needed to improve surveillance of vector-borne diseases. Such tools could potentially replace the human landing catch (HLC), which, despite being the most direct option for measuring human exposures, raises significant ethical and logistical concerns. Several alternatives are under development, but detailed evaluation still requires common frameworks for calibration relative to HLC. The aim of this study was to develop and validate a statistical framework for predicting human-biting rates from different exposure-free alternatives. METHODS: We obtained mosquito abundance data (Anopheles arabiensis, Anopheles funestus and Culex spp.) from a year-long Tanzanian study comparing six outdoor traps [Suna Trap (SUN), BG Sentinel (BGS), M-Trap (MTR), M-Trap + CDC (MTRC), Ifakara Tent Trap-C (ITT-C) and Mosquito Magnet-X Trap (MMX)] and HLC. Generalised linear models were developed within a Bayesian framework to investigate associations between the traps and HLC, taking intra- and inter-specific density dependence into account. The best model was used to create a calibration tool for predicting HLC-equivalents. RESULTS: For An. arabiensis, SUN catches had the strongest correlation with HLC (R2 = 19.4), followed by BGS (R2 = 17.2) and MTRC (R2 = 13.1) catches. The least correlated catch was MMX (R2 = 2.5). For An. funestus, BGS had the strongest correlation with the HLC (R2 = 53.4), followed by MTRC (R2 = 37.4) and MTR (R2 = 37.4). For Culex mosquitoes, the traps most highly correlated with the HLC were MTR (R2 = 45.4) and MTRC (R2 = 44.2). Density dependence, both between and within species, influenced the performance of only BGS traps. An interactive Shiny App calibration tool was developed for this and similar applications. CONCLUSION: We successfully developed a calibration tool to assess the performance of different traps for assessing outdoor-biting risk, and established a valuable framework for estimating human exposures based on the trap catches. The performance of candidate traps varied between mosquito taxa; thus, there was no single optimum. Although all the traps tested underestimated the HLC-derived exposures, it was possible to mathematically define their representativeness of the true biting risk, with or without density dependence. The results of this study emphasise the need to aim for a consistent and representative sampling approach, as opposed to simply seeking traps that catch the most mosquitoes.

Susceptibility status of major malaria vectors to novaluron, an insect growth regulator South-Eastern Tanzania.

Introduction: application of Insect Growth Regulator (IGR) such as pyriproxyfen has shown a promising result in controlling malaria transmitting mosquitoes through autodissemination technique. Novaluron that inhibits the chitin development at mosquito larval stage present a promising candidate IGR for rotation with pyriproxyfen to prevent a chance of resistance development. This study assessed the susceptibility of immature stages of Anopheles arabiensis, Anopheles gambiae and Anopheles funestus to novaluron. Methods: susceptibility bioassays using technical grade novaluron (98% active ingredient) were performed inside the semi-field system using first instar larvae of Anopheles species. For each tested species, a total of 1500 larvae were used in the bioassay. Concentration range of 0.01 mg/l to 2 mg/l of novaluron were tested to establish Lethal Concentration (LC) sufficient to kills 50%, 90% and 99% of the exposed larvae by using log-dose response analysis. Results: of the tested mosquitoes, Anopheles gambiae were highly susceptible to novaluron followed by An. Arabiensis and then An. funestus. Lethal concentrations, LC50, LC90 and LC99 (95%CI) in mg/l for An. gambiae were 0.018, 0.332 and 2.001 respectively. For An. arabiensis were 0.026, 0.546 and 2.013; and for An. funestus were 0.032, 1.00 and 5.580. High larval mortality was recorded at high concentration (2mg/L), with 80% mortality within 3 days post exposure. Conclusion: the study demonstrates the efficacy of novaluron in controlling Anopheles mosquito species at immature stages via larval mortality. These findings warrant further testing of novaluron for autodissemination by different vector species for its inclusion in rotation to prevent development of resistance.

Observing the distribution of mosquito bites on humans to inform personal protection measures against malaria and dengue vectors.

BACKGROUND: Understanding mosquito biting behaviours is important for designing and evaluating protection methods against nuisance biting and mosquito-borne diseases (e.g. dengue, malaria and zika). We investigated the preferred biting sites by Aedes aegypti and Anopheles arabiensis on adult volunteers in standing or sleeping positions; and estimated the theoretical protection limits affordable from protective clothing or repellent-treated footwear. METHODS: Adult volunteers dressed in shorts and t-shirts were exposed to infection-free laboratory-reared mosquitoes inside screened chambers from 6am to noon (for day-biting Ae. aegypti) or 6pm to midnight (night-biting An. arabiensis). Attempted bites on different body parts were recorded. Comparative observations were made on same volunteers while wearing sandals treated with transfluthrin, a vapour-phase pyrethroid that kills and repels mosquitoes. RESULTS: An. arabiensis bites were mainly on the lower limbs of standing volunteers (95.9% of bites below the knees) but evenly-distributed over all exposed body surfaces when the volunteers were on sleeping positions (only 28.8% bites below knees). Ae. aegypti bites were slightly concentrated on lower limbs of standing volunteers (47.7% below knees), but evenly-distributed on sleeping volunteers (23.3% below knees). Wearing protective clothing that leave only hands and head uncovered (e.g. socks + trousers + long-sleeved shirts) could theoretically prevent 78-83% of bites during sleeping, and at least 90% of bites during non-sleeping hours. If the feet are also exposed, protection declines to as low as 36.3% against Anopheles. The experiments showed that transfluthrin-treated sandals reduced An. arabiensis by 54-86% and Ae. aegypti by 32-39%, but did not change overall distributions of bites. CONCLUSION: Biting by An. arabiensis and Ae. aegypti occur mainly on the lower limbs, though this proclivity is less pronounced in the Aedes species. However, when hosts are on sleeping positions, biting by both species is more evenly-distributed over the exposed body surfaces. High personal protection might be achieved by simply wearing long-sleeved clothing, though protection against Anopheles particularly requires covering of feet and lower legs. The transfluthrin-treated footwear can reduce biting risk, especially by An. arabiensis. These findings could inform the design and use of personal protection tools (both insecticidal and non-insecticidal) against mosquitoes and mosquito-borne diseases.

Using ecological observations to improve malaria control in areas where Anopheles funestus is the dominant vector.

The most important malaria vectors in sub-Saharan Africa are Anopheles gambiae, Anopheles arabiensis, Anopheles funestus, and Anopheles coluzzii. Of these, An. funestus presently dominates in many settings in east and southern Africa. While research on this vector species has been impeded by difficulties in creating laboratory colonies, available evidence suggests it has certain ecological vulnerabilities that could be strategically exploited to greatly reduce malaria transmission in areas where it dominates. This paper examines the major life-history traits of An. funestus, its aquatic and adult ecologies, and its responsiveness to key interventions. It then outlines a plausible strategy for reducing malaria transmission by the vector and sustaining the gains over the medium to long term. To illustrate the propositions, the article uses data from south-eastern Tanzania where An. funestus mediates over 85% of malaria transmission events and is highly resistant to key public health insecticides, notably pyrethroids. Both male and female An. funestus rest indoors and the females frequently feed on humans indoors, although moderate to high degrees of zoophagy can occur in areas with large livestock populations. There are also a few reports of outdoor-biting by the species, highlighting a broader range of behavioural phenotypes that can be considered when designing new interventions to improve vector control. In comparison to other African malaria vectors, An. funestus distinctively prefers permanent and semi-permanent aquatic habitats, including river streams, ponds, swamps, and spring-fed pools. The species is therefore well-adapted to sustain its populations even during dry months and can support year-round malaria transmission. These ecological features suggest that highly effective control of An. funestus could be achieved primarily through strategic combinations of species-targeted larval source management and high quality insecticide-based methods targeting adult mosquitoes in shelters. If done consistently, such an integrated strategy has the potential to drastically reduce local populations of An. funestus and significantly reduce malaria transmission in areas where this vector species dominates. To sustain the gains, the programmes should be complemented with gradual environmental improvements such as house modification to maintain biting exposure at a bare minimum, as well as continuous engagements of the resident communities and other stakeholders.

Outdoor biting and pyrethroid resistance as potential drivers of persistent malaria transmission in Zanzibar.

BACKGROUND: Low-level of malaria transmission persist in Zanzibar despite high coverage of core vector control interventions. This study was carried out in hot-spot sites to better understand entomological factors that may contribute to residual malaria transmission in Zanzibar. METHODS: A total of 135 households were randomly selected from six sites and consented to participate with 20-25 households per site. Mosquito vector surveillance was carried out indoors and outdoors from 6:00 pm-7:00 am using miniaturized double net trap (DN-Mini™). Additional collections were done indoors using mouth aspirators to retrieve resting mosquitoes from wall and ceiling surfaces, and outdoors using resting bucket and pit traps. All collected mosquitoes were morphologically and genetically (PCR) analysed in the laboratory. All collected anopheline and blood-fed mosquitoes were analysed for sporozoite infection and blood meal host preferences by Circumsporozoite Protein ELISA and blood meal ELISA, respectively. The differences between indoor and outdoor mosquito biting rates were analysed using generalized linear mixed models. Levels of resistance to commonly used insecticides were quantified by WHO susceptibility tests. RESULTS: Out of 704 malaria vectors collected across 135 households, PCR analysis shows that 98.60% were Anopheles arabiensis, 0.6% Anopheles merus and 0.6% Anopheles gambiae sensu stricto. Sporozoite ELISA analysis indicates that all mosquitoes were negative for the malaria parasite. The results show that more An. arabiensis were collected outdoor (~ 85%) compared to indoor (~ 15%). Furthermore, large numbers of An. arabiensis were caught in outdoor resting sites, where the pit trap (67.2%) collected more mosquitoes compared to the outdoor DN-Mini trap (32.8%). Nearly two-thirds (60.7%) of blood-fed mosquitoes had obtained blood meals from non-human hosts. Mosquitoes displayed non-uniform susceptibility status and resistance intensity among the tested insecticides across the study sites to all WHO recommended insecticides across the study sites. CONCLUSION: This study suggests that in contexts such as Zanzibar, testing of novel techniques to complement indoor protection and targeting outdoor biting and/or resting mosquitoes, may be warranted to complement existing interventions and contribute to malaria elimination efforts. The study highlights the need to implement novel interventions and/or adaptations of strategies that can target outdoors biting mosquitoes.

Using Bayesian state-space models to understand the population dynamics of the dominant malaria vector, Anopheles funestus in rural Tanzania.

BACKGROUND: It is often assumed that the population dynamics of the malaria vector Anopheles funestus, its role in malaria transmission and the way it responds to interventions are similar to the more elaborately characterized Anopheles gambiae. However, An. funestus has several unique ecological features that could generate distinct transmission dynamics and responsiveness to interventions. The objectives of this work were to develop a model which will: (1) reconstruct the population dynamics, survival, and fecundity of wild An. funestus populations in southern Tanzania, (2) quantify impacts of density dependence on the dynamics, and (3) assess seasonal fluctuations in An. funestus demography. Through quantifying the population dynamics of An. funestus, this model will enable analysis of how their stability and response to interventions may differ from that of An. gambiae sensu lato. METHODS: A Bayesian State Space Model (SSM) based on mosquito life history was fit to time series data on the abundance of female An. funestus sensu stricto collected over 2 years in southern Tanzania. Prior values of fitness and demography were incorporated from empirical data on larval development, adult survival and fecundity from laboratory-reared first generation progeny of wild caught An. funestus. The model was structured to allow larval and adult fitness traits to vary seasonally in response to environmental covariates (i.e. temperature and rainfall), and for density dependency in larvae. The effects of density dependence and seasonality were measured through counterfactual examination of model fit with or without these covariates. RESULTS: The model accurately reconstructed the seasonal population dynamics of An. funestus and generated biologically-plausible values of their survival larval, development and fecundity in the wild. This model suggests that An. funestus survival and fecundity annual pattern was highly variable across the year, but did not show consistent seasonal trends either rainfall or temperature. While the model fit was somewhat improved by inclusion of density dependence, this was a relatively minor effect and suggests that this process is not as important for An. funestus as it is for An. gambiae populations. CONCLUSION: The model's ability to accurately reconstruct the dynamics and demography of An. funestus could potentially be useful in simulating the response of these populations to vector control techniques deployed separately or in combination. The observed and simulated dynamics also suggests that An. funestus could be playing a role in year-round malaria transmission, with any apparent seasonality attributed to other vector species.

Effects of agricultural pesticides on the susceptibility and fitness of malaria vectors in rural south-eastern Tanzania.

BACKGROUND: Agricultural pesticides may exert strong selection pressures on malaria vectors during the aquatic life stages and may contribute to resistance in adult mosquitoes. This could reduce the performance of key vector control interventions such as indoor-residual spraying and insecticide-treated nets. The aim of this study was to investigate effects of agrochemicals on susceptibility and fitness of the malaria vectors across farming areas in Tanzania. METHODS: An exploratory mixed-methods study was conducted to assess pesticide use in four villages (V1-V4) in south-eastern Tanzania. Anopheles gambiae (s.l.) larvae were collected from agricultural fields in the same villages and their emergent adults examined for insecticide susceptibility, egg-laying and wing lengths (as proxy for body size). These tests were repeated using two groups of laboratory-reared An. arabiensis, one of which was pre-exposed for 48 h to sub-lethal aquatic doses of agricultural pesticides found in the villages. RESULTS: Farmers lacked awareness about the linkages between the public health and agriculture sectors but were interested in being more informed. Agrochemical usage was reported as extensive in V1, V2 and V3 but minimal in V4. Similarly, mosquitoes from V1 to V3 but not V4 were resistant to pyrethroids and either pirimiphos-methyl or bendiocarb, or both. Adding the synergist piperonyl butoxide restored potency of the pyrethroids. Pre-exposure of laboratory-reared mosquitoes to pesticides during aquatic stages did not affect insecticide susceptibility in emergent adults of the same filial generation. There was also no effect on fecundity, except after pre-exposure to organophosphates, which were associated with fewer eggs and smaller mosquitoes. Wild mosquitoes were smaller than laboratory-reared ones, but fecundity was similar. CONCLUSIONS: Safeguarding the potential of insecticide-based interventions requires improved understanding of how agricultural pesticides influence important life cycle processes and transmission potential of mosquito vectors. In this study, susceptibility of mosquitoes to public health insecticides was lower in villages reporting frequent use of pesticides compared to villages with little or no pesticide use. Variations in the fitness parameters, fecundity and wing length marginally reflected the differences in exposure to agrochemicals and should be investigated further. Pesticide use may exert additional life cycle constraints on mosquito vectors, but this likely occurs after multi-generational exposures.

Wild populations of malaria vectors can mate both inside and outside human dwellings.

BACKGROUND: Wild populations of Anopheles mosquitoes are generally thought to mate outdoors in swarms, although once colonized, they also mate readily inside laboratory cages. This study investigated whether the malaria vectors Anopheles funestus and Anopheles arabiensis can also naturally mate inside human dwellings. METHOD: Mosquitoes were sampled from three volunteer-occupied experimental huts in a rural Tanzanian village at 6:00 p.m. each evening, after which the huts were completely sealed and sampling was repeated at 11:00 p.m and 6 a.m. the next morning to compare the proportions of inseminated females. Similarly timed collections were done inside local unsealed village houses. Lastly, wild-caught larvae and pupae were introduced inside or outside experimental huts constructed inside two semi-field screened chambers. The huts were then sealed and fitted with exit traps, allowing mosquito egress but not entry. Mating was assessed in subsequent days by sampling and dissecting emergent adults caught indoors, outdoors and in exit traps. RESULTS: Proportions of inseminated females inside the experimental huts in the village increased from approximately 60% at 6 p.m. to approximately 90% the following morning despite no new mosquitoes entering the huts after 6 p.m. Insemination in the local homes increased from approximately 78% to approximately 93% over the same time points. In the semi-field observations of wild-caught captive mosquitoes, the proportions of inseminated An. funestus were 20.9% (95% confidence interval [CI]: ± 2.8) outdoors, 25.2% (95% CI: ± 3.4) indoors and 16.8% (± 8.3) in exit traps, while the proportions of inseminated An. arabiensis were 42.3% (95% CI: ± 5.5) outdoors, 47.4% (95% CI: ± 4.7) indoors and 37.1% (CI: ± 6.8) in exit traps. CONCLUSION: Wild populations of An. funestus and An. arabiensis in these study villages can mate both inside and outside human dwellings. Most of the mating clearly happens before the mosquitoes enter houses, but additional mating happens indoors. The ecological significance of such indoor mating remains to be determined. The observed insemination inside the experimental huts fitted with exit traps and in the unsealed village houses suggests that the indoor mating happens voluntarily even under unrestricted egress. These findings may inspire improved vector control, such as by targeting males indoors, and potentially inform alternative methods for colonizing strongly eurygamic Anopheles species (e.g. An. funestus) inside laboratories or semi-field chambers.

Fitness characteristics of the malaria vector Anopheles funestus during an attempted laboratory colonization.

BACKGROUND: The malaria vector Anopheles funestus is increasingly recognized as a dominant vector of residual transmission in many African settings. Efforts to better understand its biology and control are significantly impeded by the difficulties of colonizing it under laboratory conditions. To identify key bottlenecks in colonization, this study compared the development and fitness characteristics of wild An. funestus from Tanzania (FUTAZ) and their F1 offspring during colonization attempts. The demography and reproductive success of wild FUTAZ offspring were compared to that of individuals from one of the only An. funestus strains that has been successfully colonized (FUMOZ, from Mozambique) under similar laboratory conditions. METHODS: Wild An. funestus (FUTAZ) were collected from three Tanzanian villages and maintained inside an insectary at 70-85% RH, 25-27 °C and 12 h:12 h photoperiod. Eggs from these females were used to establish three replicate F1 laboratory generations. Larval development, survival, fecundity, mating success, percentage pupation and wing length were measured in the F1 -FUTAZ offspring and compared with wild FUTAZ and FUMOZ mosquitoes. RESULTS: Wild FUTAZ laid fewer eggs (64.1; 95% CI [63.2, 65.0]) than FUMOZ females (76.1; 95% CI [73.3, 79.1]). Survival of F1-FUTAZ larvae under laboratory conditions was low, with an egg-to-pupae conversion rate of only 5.9% compared to 27.4% in FUMOZ. The median lifespan of F1-FUTAZ females (32 days) and males (33 days) was lower than FUMOZ (52 and 49 for females and males respectively). The proportion of female F1-FUTAZ inseminated under laboratory conditions (9%) was considerably lower than either FUMOZ (72%) or wild-caught FUTAZ females (92%). This resulted in nearly zero viable F2-FUTAZ eggs produced. Wild FUTAZ wings appear to be larger compared to the lab reared F1-FUTAZ and FUMOZ. CONCLUSIONS: This study indicates that poor larval survival, mating success, low fecundity and shorter survival under laboratory conditions all contribute to difficulties in colonizing of An. funestus. Future studies should focus on enhancing these aspects of An. funestus fitness in the laboratory, with the biggest barrier likely to be poor mating.

Sub-lethal aquatic doses of pyriproxyfen may increase pyrethroid resistance in malaria mosquitoes.

BACKGROUND: Pyriproxyfen (PPF), an insect growth hormone mimic is widely used as a larvicide and in some second-generation bed nets, where it is combined with pyrethroids to improve impact. It has also been evaluated as a candidate for auto-dissemination by adult mosquitoes to control Aedes and Anopheles species. We examined whether PPF added to larval habitats of pyrethroid-resistant malaria vectors can modulate levels of resistance among emergent adult mosquitoes. METHODOLOGY: Third-instar larvae of pyrethroid-resistant Anopheles arabiensis (both laboratory-reared and field-collected) were reared in different PPF concentrations, between 1×10-9 milligrams active ingredient per litre of water (mgAI/L) and 1×10-4 mgAI/L, or no PPF at all. Emergent adults escaping these sub-lethal exposures were tested using WHO-standard susceptibility assays on pyrethroids (0.75% permethrin and 0.05% deltamethrin), carbamates (0.1% bendiocarb) and organochlorides (4% DDT). Biochemical basis of pyrethroid resistance was investigated by pre-exposure to 4% PBO. Bio-efficacies of long-lasting insecticide-treated nets, Olyset® and PermaNet 2.0 were also examined against adult mosquitoes with or without previous aquatic exposure to PPF. RESULTS: Addition of sub-lethal doses of PPF to larval habitats of pyrethroid-resistant An. arabiensis, consistently resulted in significantly reduced mortalities of emergent adults when exposed to pyrethroids, but not to bendiocarb or DDT. Mortality rates after exposure to Olyset® nets, but not PermaNet 2.0 were also reduced following aquatic exposures to PPF. Pre-exposure to PBO followed by permethrin or deltamethrin resulted in significant increases in mortality, compared to either insecticide alone. CONCLUSIONS: Partially-resistant mosquitoes exposed to sub-lethal aquatic concentrations of PPF may become more resistant to pyrethroids than they already are without such pre-exposures. Studies should be conducted to examine whether field applications of PPF, either by larviciding or other means actually exacerbates pyrethroid-resistance in areas where signs of such resistance already exist in wild the vector populations. The studies should also investigate mechanisms underlying such magnification of resistance, and how this may impact the potential of PPF-based interventions in areas with pyrethroid resistance.

Fine-scale distribution of malaria mosquitoes biting or resting outside human dwellings in three low-altitude Tanzanian villages.

BACKGROUND: While malaria transmission in Africa still happens primarily inside houses, there is a substantial proportion of Anopheles mosquitoes that bite or rest outdoors. This situation may compromise the performance of indoor insecticidal interventions such as insecticide-treated nets (ITNs). This study investigated the distribution of malaria mosquitoes biting or resting outside dwellings in three low-altitude villages in south-eastern Tanzania. The likelihood of malaria infections outdoors was also assessed. METHODS: Nightly trapping was done outdoors for 12 months to collect resting mosquitoes (using resting bucket traps) and host-seeking mosquitoes (using odour-baited Suna® traps). The mosquitoes were sorted by species and physiological states. Pooled samples of Anopheles were tested to estimate proportions infected with Plasmodium falciparum parasites, estimate proportions carrying human blood as opposed to other vertebrate blood and identify sibling species in the Anopheles gambiae complex and An. funestus group. Environmental and anthropogenic factors were observed and recorded within 100 meters from each trapping positions. Generalised additive models were used to investigate relationships between these variables and vector densities, produce predictive maps of expected abundance and compare outcomes within and between villages. RESULTS: A high degree of fine-scale heterogeneity in Anopheles densities was observed between and within villages. Water bodies covered with vegetation were associated with 22% higher densities of An. arabiensis and 51% lower densities of An. funestus. Increasing densities of houses and people outdoors were both associated with reduced densities of An. arabiensis and An. funestus. Vector densities were highest around the end of the rainy season and beginning of the dry seasons. More than half (14) 58.3% of blood-fed An. arabiensis had bovine blood, (6) 25% had human blood. None of the Anopheles mosquitoes caught outdoors was found infected with malaria parasites. CONCLUSION: Outdoor densities of both host-seeking and resting Anopheles mosquitoes had significant heterogeneities between and within villages, and were influenced by multiple environmental and anthropogenic factors. Despite the high Anopheles densities outside dwellings, the substantial proportion of non-human blood-meals and absence of malaria-infected mosquitoes after 12 months of nightly trapping suggests very low-levels of outdoor malaria transmission in these villages.