Background Adult mosquito survival is a critical factor in malaria transmission because Anopheles vectors must live at least 11–14 days before they can transmit. However, there is no direct method to assess age. Traditional methods of mosquito age-grading rely on indirect proxies of reproductive history derived from dissection and observation of ovarian features (Detinova and Polovodova methods). Both approaches are labour-intensive, time-consuming, and demand specialized expertise; with little known about the extent to which are used in malaria surveillance programmes, and why they may be deprioritised. This study assessed current practices, capacity, and constraints related to mosquito age-grading for malaria vector surveillance in Africa, including knowledge and use of existing dissection-based methods and emerging alternatives such as infrared spectroscopy. Methods We conducted an online survey of 285 stakeholders from across Africa, including researchers, entomologists, and National Malaria Programme (NMP) personnel, to assess the practices, priorities, and barriers to mosquito age-grading. This was complemented with a series of in-depth interviews and focus group discussions to collect insights and perspectives from stakeholders regarding their familiarity and use of age-grading methods. Findings: More than 70% of survey respondents reported that malaria vector surveillance was routinely conducted by their institutions or countries, with the highest priority given to vector density, species identification, human biting rate, and insecticide resistance. Familiarity (awareness as opposed to knowledge) of age-grading methods was highest for the Detinova (55%) and Polovodova (44%), and lower for newer approaches, including infrared-spectroscopy. Only 50% of respondents indicated that they regularly assessed mosquito age (mostly Detinova method); with > 1/3 considering age-grading to be a high-priority in vector surveillance. Reported barriers to conducting mosquito-age-grading included insufficient technical expertise, perceived impracticality of ovary dissections for large-scale surveillance, inadequate tools, and limited funding. Conclusions Despite its critical role in malaria transmission, mosquito survival and age are rarely assessed in African vector surveillance programs. There is need for greater acknowledgment of these measures and their implications for disease transmission and control, and investment in tools, training, and funding to overcome current operational barriers. Integrating more practical and scalable age-assessment methods could enhance targeting of interventions.

Introduction : Effective larval source management (LSM) requires detailed knowledge of the aquatic habitats supporting dominant malaria vector. This study investigated intra-annual variations in Anopheles funestus sensu lato habitats in south-eastern Tanzania, where this species drives malaria transmission, and evaluated pictorial and drone-based methods to complement standard entomological surveys. Methods Monthly entomological surveys (July-2022 to June-2023) quantified larval productivity in 184 An. funestus sensu lato habitats and characterized their environmental features (vegetation cover, shading, turbidity, water movement, size, and depth) and physicochemical properties (temperature, pH, dissolved oxygen, and total dissolved solids); temporal variation was analysed using generalised additive mixed models. Pictorial surveys of a habitat’s subset yielded 132 standardized monthly images, independently assessed by five trained raters and inter-rater reliability quantified. A 6 km² drone survey conducted in May 2023 was validated against concurrent ground-truth data. Key informant interviews assessed stakeholder perceptions of pictorial and drone approaches, analysed thematically. Results Large ground pools and rivers sustain An. funestus sensu lato larvae year-round, human-made habitats like ditches declined sharply during the dry season. Physicochemical and environmental characteristics varied significantly over time, but larval densities fluctuating primarily by environmental characteristics. Habitats with partial shading, moderate vegetation cover, and limited water disturbance, conditions most common after the rains and during the early dry season, consistently supported the highest An. funestus sensu lato larval densities. Pictorial surveys reliably tracked temporal habitat changes, showing good inter-rater reliability for water coverage (ICC = 0.809), vegetation quantity (ICC = 0.761), and water presence (κ = 0.839), and were viewed by stakeholders as a practical for documenting habitat conditions. Drone imagery underperformed, detecting only 35.7% of confirmed habitats with 60% classification accuracy (κ = 0.295), limited by vegetation canopy cover. Conclusion The main An. funestus sensu lato habitats in south-eastern Tanzania persisted year-round, though their characteristics varied significantly by month. Environmental, not physicochemical, factors drove intra-annual variations in larval density, suggesting scope for seasonally adapted larval source management. Ground-based pictorial monitoring offers a practical supplement to entomological surveys, while drone-based mapping needs validations.

Abstract Background Evidence of natural infection with Wolbachia and its negative correlation with Plasmodium falciparum among wild malaria vectors has opened new avenues for utilization of Wolbachia in malaria vector control. However, the interaction between Wolbachia and Plasmodium parasites in mosquitoes tends to be species-specific and may show ecological variations. Among the primary malaria vectors in Tanzania, natural Wolbachia infection has only been observed in Anopheles arabiensis, while there is still limited information on Wolbachia natural infection in Anopheles funestus sensu lato, and its interaction with P. falciparum in the mosquito species. Therefore, this study investigated the prevalence of natural infection and co-infection of Wolbachia and P. falciparum in the An. funestus s.l. in southeastern Tanzania, and characterized the Wolbachia strains detected. Methods The study was conducted in five villages in southeastern Tanzania between March and June 2024. Mosquitoes were collected from 52 households using Centers for Disease Control and Prevention (CDC) light traps and Prokopack aspirators, followed by morphological identification. Detection of An. funestus sibling species and Wolbachia was performed using conventional polymerase chain reaction (PCR) and nested PCR (Wolbachia only). Sanger sequencing was performed as a confirmatory test followed by phylogenetic analysis of the detected Wolbachia strains. P. falciparum sporozoites were detected using enzyme-linked immunosorbent assay (ELISA). Results Wolbachia was detected in almost half of all wild An. funestus s.l. tested using the primary PCR (prevalence = 46.5%, N = 400); and more than half when nested PCR approach was used (prevalence = 70.8%, N = 400). Only three mosquitoes carried P. falciparum sporozoites (prevalence = 0.8%, N = 400) and only one showed co-infection with Wolbachia (prevalence = 0.3%, n = 400). Sequencing and phylogenetic analysis involving both the 16S rDNA, coxA, and wsp Wolbachia genes showed that the detected strains clustered with Wolbachia supergroup B, specific for Dipterans. Conclusions Unlike findings from the previous study, this study demonstrates that An. funestus s.l. in southeastern Tanzania are infected with Wolbachia, at a surprisingly high prevalence. This study also provides the first report on Wolbachia–P. falciparum co-infection status in An. funestus s.l. in Tanzania. Further studies with larger sample sizes are needed to confirm the association between native Wolbachia and P. falciparum in wild An. funestus s.l. in southeastern Tanzania. Graphical Abstract

BACKGROUND:Current malaria surveillance methods are considered too expensive to scale-up within limited-resource settings; hence, new technologies and approaches are necessary to maximize the collection of data and ultimately design new malaria control tools. Effective mosquito surveillance can be enhanced through the utilization of digital technologies and the engagement of citizens in real-time data collection. This study used the HumBug acoustic sensor with the MozzWear app to detect and identify host-seeking mosquitoes based on their flight sounds, with citizens receiving airtime incentives for recording and uploading sounds. METHODS:A randomized controlled trial was used to assess the role of incentives to encourage the local community to record and upload mosquito sounds using the MozzWear application. Participants were randomized into two groups: (1) a control group, in which no incentive was provided; and (2) an incentive group, in which airtime credit was provided to participants. Both groups were provided with HumBug smartphones running the MozzWear app plus adapted mosquito bed nets ('HumBug Nets') to hold the phones during recording and were asked to record and upload mosquito flight tone data once per week for a period of four months. The intervention group was rewarded with an airtime incentive every week after the data were uploaded. At the end of the study, an experience survey was administered to participants in both groups to assess their experience participating in this study. RESULTS:The overall results indicate that the control group performed well in terms of the number of nights spent recording and uploading data compared to the incentive group. The level of intrinsic and extrinsic motivation differs between demographic variables. Their feedback suggested that fighting against malaria was more important and was everyone's concern in rural Tanzania. In addition, the participants expressed their interest in being involved in future research related to mosquito surveillance and the fight against malaria. CONCLUSION:Citizens can play a valuable role in scientific research; even without giving them incentives, they can still participate in the study. By participating in mosquito surveillance and malaria prevention studies, community members can make significant contributions to addressing mosquito-borne diseases and improving health outcomes.

Abstract Background Increased global trade, while beneficial economically, can also increase the spread of vector-borne diseases, particularly those transmitted by Aedes mosquitoes spreading via trade routes. Given the heightened trade-induced activity at ports of entry, it is particularly crucial to assess the risk of mosquito-borne diseases in these settings. This study compared the risks of Aedes-borne disease in and around the eastern Tanzanian seaport of Tanga. Methods A 200 m × 200 m grid-based system was used to sample mosquitoes within the port area, and in surrounding areas at 2 km, 2.5 km, and 5 km away, between June and December 2023. We characterized mosquito breeding habitats, collected mosquito larvae using standard dippers and tested susceptibility of raised adult Aedes aegypti populations to different insecticides. Adult mosquitoes were collected using BG sentinel traps (daytime) and Centers for Disease Control (CDC) light traps (night-time). Additionally, more than 200 port users and neighboring residents were surveyed to assess their experiences with and perceptions of mosquito biting and disease risks. Results There were 2931 breeding sites, with (60.8%, n = 1782) positive for Aedes larvae. The percentage of water-holding containers infested with Aedes immatures, i.e., the container index (CI), was highest in the port area (66.2%), and lowest 5 km away (44.6%). The port area also had a greater proportion of temporary breeding sites (64.9%) than did the surrounding areas. The adult mosquito surveys revealed 20,449 mosquito species including: Culex quinquefasciatus (56.2%), Mansonia uniformis (38.6%), Ae. aegypti (5.1%), Anopheles gambiae (0.1%), and Anopheles funestus. Ae. aegypti were more abundant in the port area than in the surrounding areas (P < 0.001), whereas Culex sp., and Mansonia sp., were significantly outside (P < 0.001). Adult Anopheles sp., were found only in the port area, but Anopheles larvae were found both within and outside the port areas. Tests on Ae. aegypti sp., revealed susceptibility to bendiocarb and DDT, and resistance to permethrin. Awareness of mosquito-borne diseases among respondents was high for malaria (64.8%), but low for dengue (26.3%) and Chikungunya (1.7%). Most respondents reported being bothered by mosquitoes mostly at night (53.4%) or in the evening (40.7%). In addition to insecticidal bednets, which are used primarily against malaria, preventive measures for Aedes-borne diseases are limited. Conclusions This study identified significant potential risk of Aedes species, specifically Ae. aegypti sp., and associated diseases, but low perception of risk and inadequate personal protection measures in the study area. This low perception of risk highlights the need to improve public knowledge of the transmission and control of Aedes-borne diseases. Graphical Abstract

House improvement is associated with remarkable reductions in indoor mosquito bites and disease incidences, even in typical rural houses. However, its exploitation remains extremely poor in Tanzania and other endemic countries due to limited financial resources. Nevertheless, village community banks (VICOBA), practiced in Tanzania for nearly two decades, have proven to provide financial services to rural communities that would otherwise not be able to get them from formal financial institutions. This study explored the need, opinion, and willingness of VICOBA members to use VICOBA platforms as a source of finance for improving local houses and eventually controlling mosquito-borne diseases. A mixed-methods approach was used in this study, whereby a survey was administered to 150 participants and twelve focus group discussions were done in three villages in Ulanga district, rural Tanzania. The FGDs comprised eight participants each, with equal representation of males and females. The FGD guide was used to probe the opinions of study participants on malaria transmission, housing condition improvements, and financial resources. About 99% of all participants indicated the urgent need to improve their houses to prevent mosquito bites and were willing to utilize VICOBA for improving their houses. In the focus group discussion, the majority of people who participated were also in need of improving their houses. All participants confirmed that they were at the highest risk of getting mosquito-borne diseases, and they were willing to use money that was either saved or borrowed from their VICOBA for housing improvements and vector control. A self-sustaining financial system destined for house improvement and related interventions against malaria and other mosquito-borne diseases is crucial. The community members were willing to use VICOBA as a source of finance for house improvement and disease control; however, there was limited knowledge and sensitization on how they could utilize VICOBA for disease control.

BACKGROUND:To detect and identify mosquitoes using their characteristic high-pitched sound, we have developed a smartphone application, known as the 'HumBug sensor', that records the acoustic signature of this sound, along with the time and location. This data is then sent remotely to a server where algorithms identify the species according to their distinctive acoustic signature. Whilst this system works well, a key question that remains is what mechanisms will lead to effective uptake and use of this mosquito survey tool? We addressed this question by working with local communities in rural Tanzania and providing three alternative incentives: money only, short message service (SMS) reminders and money, and SMS reminders only. We also had a control group with no incentive. METHODS:A multi-site, quantitative empirical study was conducted in four villages in Tanzania from April to August 2021. Consenting participants (n = 148) were recruited and placed into one of the three intervention arms: monetary incentives only; SMS reminders with monetary incentives; and SMS reminders only. There was also a control group (no intervention). To test effectiveness of the mechanisms, the number of audio uploads to the server of the four trial groups on their specific dates were compared. Qualitative focus group discussions and feedback surveys were also conducted to explore participants' perspectives on their participation in the study and to capture their experiences of using the HumBug sensor. RESULTS:Qualitative data analysis revealed that for many participants (37 out of 81), the main motivation expressed was to learn more about the types of mosquitoes present in their houses. Results from the quantitative empirical study indicate that the participants in the 'control' group switched on their HumBug sensors more over the 14-week period (8 out of 14 weeks) when compared to those belonging to the 'SMS reminders and monetary incentives' trial group. These findings are statistically significant (p < 0.05 or p > 0.95 under a two-sided z-test), revealing that the provision of monetary incentives and sending SMS reminders did not appear to encourage greater number of audio uploads when compared to the control. CONCLUSIONS:Knowledge on the presence of harmful mosquitoes was the strongest motive for local communities to collect and upload mosquito sound data via the HumBug sensor in rural Tanzania. This finding suggests that most efforts should be made to improve flow of real-time information back to the communities on types and risks associated with mosquitoes present in their houses.

A large-scale multi-species dataset of acoustic recordings Dataset compatible with two papers: The Computational Paralinguistics ChallengE (ComParE): Mosquito Event Detection Task https://github.com/EIHW/ComParE2022/tree/MOS-C An update to: HumBugDB: a large-scale acoustic mosquito dataset: NeurIPS 2021 Paper https://github.com/HumBug-Mosquito/HumBugDB. A large-scale multi-species dataset containing recordings of mosquitoes collected from multiple locations globally, as well as via different collection methods. In total, we present 20 hours of labelled mosquito data with 15 hours of corresponding background noise, recorded at the sites of 8 experiments. Of these, 64,843 seconds contain species metadata, consisting of 36 species (or species complexes). This repository contains: Audio files to be extracted into audio/data/train and audio/data/dev/{a/b} respectively Metadata in csv format: neurips_2021_zenodo_0_0_2.csv

Abstract 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.

Abstract Background: Malaria transmission can be highly heterogeneous between and within villages, and is influenced by factors such as survival and biting frequencies of Anopheles mosquitoes. This study investigated the correlations between biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes in south-eastern Tanzania; and how these correlations may inform improved vector control strategies.Methods: Female An. funestus were collected in houses located 50-100m, 150-200m or over 200m from the nearest known aquatic habitats. The mosquitoes were exposed to 1×, 5× and 10× the diagnostic doses of either deltamethrin or permethrin, or to the synergist, piperonyl butoxide (PBO) followed by the pyrethroids, then monitored for 24hr-mortality. Ovaries of exposed and non-exposed mosquitoes were dissected to assess parity status as a proxy for biological age. Adults emerging from larval collections in the same villages were exposed to the same insecticides at 3-5, 8-11 or 17-20 days old to compare resistance. Findings: Mosquitoes collected nearest to the aquatic habitats (50-100m) had the lowest mortality compared to other distances, with a maximum of 51% mortality at 10× permethrin. For the age-synchronized mosquitoes collected as larvae, the percentage 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 restored 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 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 females. Further studies should investigate whether fine-scale targeting of key interventions (e.g. preferentially spraying homes near the centres of villages) would be more impactful.

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.

This paper presents the first large-scale multi-species dataset of acoustic recordings of mosquitoes tracked continuously in free flight. We present 20 hours of audio recordings that we have expertly labelled and tagged precisely in time. Significantly, 18 hours of recordings contain annotations from 36 different species. Mosquitoes are well-known carriers of diseases such as malaria, dengue and yellow fever. Collecting this dataset is motivated by the need to assist applications which utilise mosquito acoustics to conduct surveys to help predict outbreaks and inform intervention policy. The task of detecting mosquitoes from the sound of their wingbeats is challenging due to the difficulty in collecting recordings from realistic scenarios. To address this, as part of the HumBug project, we conducted global experiments to record mosquitoes ranging from those bred in culture cages to mosquitoes captured in the wild. Consequently, the audio recordings vary in signal-to-noise ratio and contain a broad range of indoor and outdoor background environments from Tanzania, Thailand, Kenya, the USA and the UK. In this paper we describe in detail how we collected, labelled and curated the data. The data is provided from a PostgreSQL database, which contains important metadata such as the capture method, age, feeding status and gender of the mosquitoes. Additionally, we provide code to extract features and train Bayesian convolutional neural networks for two key tasks: the identification of mosquitoes from their corresponding background environments, and the classification of detected mosquitoes into species. Our extensive dataset is both challenging to machine learning researchers focusing on acoustic identification, and critical to entomologists, geo-spatial modellers and other domain experts to understand mosquito behaviour, model their distribution, and manage the threat they pose to humans.

BACKGROUND:In rural south-eastern Tanzania, Anopheles funestus is a major malaria vector, and has been implicated in nearly 90% of all infective bites. Unfortunately, little is known about the natural ecological requirements and survival strategies of this mosquito species. METHODS:Potential mosquito aquatic habitats were systematically searched along 1000 m transects from the centres of six villages in south-eastern Tanzania. All water bodies were geo-referenced, characterized and examined for presence of Anopheles larvae using standard 350 mLs dippers or 10 L buckets. Larvae were collected for rearing, and the emergent adults identified to confirm habitats containing An. funestus. RESULTS:One hundred and eleven habitats were identified and assessed from the first five villages (all < 300 m altitude). Of these, 36 (32.4%) had An. funestus co-occurring with other mosquito species. Another 47 (42.3%) had other Anopheles species and/or culicines, but not An. funestus, and 28 (25.2%) had no mosquitoes. There were three main habitat types occupied by An. funestus, namely: (a) small spring-fed pools with well-defined perimeters (36.1%), (b) medium-sized natural ponds retaining water most of the year (16.7%), and (c) slow-moving waters along river tributaries (47.2%). The habitats generally had clear waters with emergent surface vegetation, depths > 0.5 m and distances < 100 m from human dwellings. They were permanent or semi-permanent, retaining water most of the year. Water temperatures ranged from 25.2 to 28.8 °C, pH from 6.5 to 6.7, turbidity from 26.6 to 54.8 NTU and total dissolved solids from 60.5 to 80.3 mg/L. In the sixth village (altitude > 400 m), very high densities of An. funestus were found along rivers with slow-moving clear waters and emergent vegetation. CONCLUSION:This study has documented the diversity and key characteristics of aquatic habitats of An. funestus across villages in south-eastern Tanzania, and will form an important basis for further studies to improve malaria control. The observations suggest that An. funestus habitats in the area can indeed be described as fixed, few and findable based on their unique characteristics. Future studies should investigate the potential of targeting these habitats with larviciding or larval source management to complement malaria control efforts in areas dominated by this vector species.

Malaria control in Africa relies extensively on indoor residual spraying (IRS) and insecticidetreated nets (ITNs). Indoor residual spraying typically targets mosquitoes resting on walls, and in few cases, roofs and ceilings, using contact insecticides. Unfortunately, little attention is paid to where malaria vectors actually rest indoors, and how such knowledge could be used to improve IRS. This study investigated preferred resting surfaces of two major malaria vectors, Anopheles funestus and Anopheles arabiensis, inside four common house types in rural south-eastern Tanzania. The assessment was done inside 80 houses in south-eastern Tanzania across four villages. In each house, resting mosquitoes were captured using Prokopack aspirators from walls, undersides of roofs, floors, furniture, utensils, clothes and bed-nets. Overall, only 26% of An. funestus and 18% of An. arabiensis were found on walls. In grass-thatched houses, 33-55% of An. funestus and 43-50% of An. arabiensis rested under roofs, while in metal-roofed houses, only 1620% of An. funestus and 8-30% of An. arabiensis rested under roofs. Considering all data together, approximately 40% of mosquitoes rested on surfaces not typically targeted by IRS, i.e. floors, furniture, utensils, clothes and bed-nets. These proportions were particularly high in metal-roofed houses (47-53% of An. funestus; 60-66% of An. arabiensis). While IRS uses contact insecticides to target adult mosquitoes on walls, and occasionally roofs and ceilings, significant proportions of vectors rest on surfaces not usually sprayed. This gap exceeds one-third of malaria mosquitoes in grass-thatched houses, and can reach two-thirds in metal-roofed houses. Where field operations exclude roofs during IRS, the gaps can be much greater. In conclusion, there is need for locallyobtained data on mosquito resting behaviours and how these influence the overall impact and costs of IRS.

Background Aedes -borne diseases such as dengue and chikungunya constitute constant threats globally. In Tanzania, these diseases are transmitted by Aedes aegypti, which is widely distributed in urban areas, but whose ecology remains poorly understood in small towns and rural settings. Methods A survey of Ae. aegypti aquatic habitats was conducted in and around Ifakara, a fast-growing town in south-eastern Tanzania. The study area was divided into 200 × 200 m search grids, and habitats containing immature Aedes were characterized. Field-collected Ae. aegypti were tested for susceptibility to common public health insecticides (deltamethrin, permethrin, bendiocarb and pirimiphos-methyl) in the dry and rainy seasons. Results Of 1515 and 1933 aquatic habitats examined in the dry and rainy seasons, 286 and 283 contained Aedes immatures, respectively (container index, CI: 18.9–14.6%). In the 2315 and 2832 houses visited in the dry and rainy seasons, 114 and 186 houses had at least one Aedes -positive habitat, respectively (house index, HI: 4.9–6.6%). The main habitat types included: (i) used vehicle tires and discarded containers; (ii) flowerpots and clay pots; and (iii) holes made by residents on trunks of coconut trees when harvesting the coconuts. Used tires had highest overall abundance of Ae. aegypti immatures, while coconut tree-holes had highest densities per habitat. Aedes aegypti adults were susceptible to all tested insecticides in both seasons, except bendiocarb, against which resistance was observed in the rainy season. Conclusions To our knowledge, this is the first study on ecology and insecticide susceptibility of Ae. aegypti in Ifakara area, and will provide a basis for future studies on its pathogen transmission activities and control. The high infestation levels observed indicate significant risk of Aedes -borne diseases, requiring immediate action to prevent potential outbreaks in the area. While used tires, discarded containers and flowerpots are key habitats for Ae. aegypti , this study also identified coconut harvesting as an important risk factor, and the associated tree-holes as potential targets for Aedes control. Since Ae. aegypti mosquitoes in the area are still susceptible to most insecticides, effective control could be achieved by combining environmental management, preferably involving communities, habitat removal and insecticide spraying.

Background: Long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have greatly reduced malaria transmission in sub-Saharan Africa, but are threatened by insecticide resistance. In south-eastern Tanzania, pyrethroid-resistant Anopheles funestus are now implicated in > 80% of malaria infections, even in villages where the species occurs at lower densities than the other vector species, Anopheles arabiensis . This study compared the intensities of resistance between the two malaria vectors, so as to improve options for control . Methods: The study used WHO assays with 1×, 5× and 10× insecticide doses to assess levels of resistance, followed by synergist bioassays to understand possible mechanisms of the observed resistance phenotypes. The tests involved adult mosquitoes collected from villages across two districts in south-eastern Tanzania and identified using morphological and molecular approaches. Findings: At baseline doses (1×), both species were resistant to the two pyrethroids (permethrin and deltamethrin) but susceptible to the organophosphate (pirimiphos-methyl). An. funestus , but not An. arabiensis was also resistant to the carbamate (bendiocarb) at baseline doses. Both species were generally resistant to DDT, except An.arabiensis from one village. An. funestus showed strong resistance to pyrethroids, surviving the 5× and 10× doses except in one village. Pre-exposure to the synergist, piperonyl butoxide (PBO), enhanced the potency of pyrethroid in both An. arabiensis and An. funestus achieving mortalities >98%, except for An. funestus from two villages for which permethrin-associated mortalities exceeded 90% but not 98%. Conclusions : In these communities where An. funestus dominates malaria transmission, this study may suggest that the species also have much stronger resistance to pyrethroids than its counterpart, An. arabiensis and can survive more classes of insecticides, including carbamates. The pyrethroid resistance in both species appears to be mostly metabolic and may be temporarily addressed using synergists, e.g. PBO. These findings may explain the continued persistence and dominance of An. funestus despite widespread use of pyrethroid-treated LLINs, and inform new choices of interventions to tackle malaria transmission in such settings. These may include PBO-based LLINs or improved IRS with compounds to which the vectors are susceptible. Additional field validation of these indications will be necessary using age-synchronized mosquitoes.

Background: Long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have greatly reduced malaria transmission in sub-Saharan Africa, but are threatened by insecticide resistance in dominant malaria vectors. In south-eastern Tanzania, pyrethroid-resistant Anopheles funestus now transmit more than 80% of malaria infections even in villages where the species occurs at far lower densities than other vectors such as Anopheles arabiensis.Methods: To better understand the dominance of An. funestus in these settings and improve options for its control, this study compared intensities of resistance between females of this species and those of An. arabiensis , using WHO assays with 1×, 5× and 10× insecticide doses. Additional tests were done to assess the reversibility of such resistance using synergists. The mosquitoes were collected from villages across two districts in south-eastern Tanzania.Findings: Both species were resistant to the two pyrethroids (permethrin and deltamethrin) and the organochloride (DDT) but susceptible to the organophosphate (pirimiphos-methyl) at standard baseline doses (1×). However, An. funestus as opposed to An. arabiensis was also resistant to the carbamate (bendiocarb) at standard doses (1×). An. funestus showed strong resistance to pyrethroids, surviving the 5× doses and 10× doses except in one village. Pre-exposure to the synergist, piperonyl butoxide (PBO), reversed the pyrethroid-resistance in both An. arabiensis and An. funestus achieving mortalities >98%, except for An. funestus from two villages for which permethrin-associated mortalities exceeded 90% but not 98%.Conclusions : In these communities where An. funestus now dominates malaria transmission, the species also displays much stronger resistance to pyrethroids than its counterpart, An. arabiensis, and can readily survive more classes of insecticides, including carbamates. The resistance to pyrethroids in both mosquito species appears to be mostly metabolic and can be reversed significantly using synergists such as PBO. These findings may explain the continued persistence and dominance of An. funestus despite widespread use of pyrethroid-treated LLINs, and will also inform future choices of interventions to tackle malaria transmission in this area and other similar settings. Such interventions may include PBO-based LLINs or improved IRS with compounds such as organophosphates against which the vectors are still susceptible.

Background Long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have greatly reduced malaria transmission in sub-Saharan Africa, but are threatened by insecticide resistance. In south-eastern Tanzania, pyrethroid-resistant Anopheles funestus are now implicated in > 80% of malaria infections, even in villages where the species occurs at lower densities than the other vector, Anopheles arabiensis . This study compared the insecticide resistance phenotypes between the two malaria vectors in an area where pyrethroid-LLINs are widely used . Methods The study used the World Health Organization (WHO) assays with 1×, 5× and 10× insecticide doses to assess levels of resistance, followed by synergist bioassays to understand possible mechanisms of the observed resistance phenotypes. The tests involved adult mosquitoes collected from three villages across two districts in south-eastern Tanzania and included four insecticide classes. Findings At baseline doses (1×), both species were resistant to the two candidate pyrethroids (permethrin and deltamethrin), but susceptible to the organophosphate (pirimiphos-methyl). Anopheles funestus , but not An. arabiensis was also resistant to the carbamate (bendiocarb). Both species were resistant to DDT in all villages except in one village where An. arabiensis was susceptible. Anopheles funestus showed strong resistance to pyrethroids, surviving the 5× and 10× doses, while An. arabiensis reverted to susceptibility at the 5× dose. Pre-exposure to the synergist, piperonyl butoxide (PBO), enhanced the potency of the pyrethroids against both species and resulted in full susceptibility of An. arabiensis (> 98% mortality). However, for An. funestus from two villages, permethrin-associated mortalities after pre-exposure to PBO only exceeded 90% but not 98%. Conclusions In south-eastern Tanzania, where An. funestus dominates malaria transmission, the species also has much stronger resistance to pyrethroids than its counterpart, An. arabiensis, and can survive more classes of insecticides. The pyrethroid resistance in both species appears to be mostly metabolic and may be partially addressed using synergists, e.g. PBO. These findings may explain the continued persistence and dominance of An. funestus despite widespread use of pyrethroid-treated LLINs, and inform new intervention choices for such settings. In short and medium-term, these may include PBO-based LLINs or improved IRS with compounds to which the vectors are still susceptible.

Background Zanzibar provides a good case study for malaria elimination. The islands have experienced a dramatic reduction in malaria burden since the introduction of effective vector control interventions and case management. Malaria prevalence has now been maintained below 1% for the past decade and the islands can feasibly aim for elimination. Methods To better understand factors that may contribute to remaining low-level malaria transmission in Zanzibar, layered human behavioural and entomological research was conducted between December 2016 and December 2017 in 135 randomly selected households across six administrative wards. The study included: (1) household surveys, (2) structured household observations of nighttime activity and sleeping patterns, and (3) paired indoor and outdoor mosquito collections. Entomological and human behavioural data were integrated to provide weighted estimates of exposure to vector bites, accounting for proportions of people indoors or outdoors, and protected by insecticide-treated nets (ITNs) each hour of the night. Results Overall, 92% of female Anopheles mosquitoes were caught in the rainy season compared to 8% in the dry season and 72% were caught outdoors compared to 28% indoors. For individual ITN users, ITNs prevented an estimated two-thirds (66%) of exposure to vector bites and nearly three quarters (73%) of residual exposure was estimated to occur outdoors. Based on observed levels of ITN use in the study sites, the population-wide mean personal protection provided by ITNs was 42%. Discussion/conclusions This study identified gaps in malaria prevention in Zanzibar with results directly applicable for improving ongoing programme activities. While overall biting risk was low, the most notable finding was that current levels of ITN use are estimated to prevent less than half of exposure to malaria vector bites. Variation in ITN use across sites and seasons suggests that additional gains could be made through targeted social and behaviour change interventions. However, even for ITN users, gaps in protection remain, with a majority of exposure to vector bites occurring outdoors before going to sleep. Supplemental interventions targeting outdoor exposure to malaria vectors, and groups that may be at increased risk of exposure to malaria vectors, should be explored.

Aedes -borne infections such as dengue and chikungunya constitute constant threats globally. In Tanzania, the most important vector of these diseases is Aedes aegypti , which is widely distributed in urban areas, but whose ecology remains poorly-understood in small towns and rural settings. We surveyed the aquatic habitats of Ae. aegypti mosquitoes in and around Ifakara, a fast-growing town in south-eastern Tanzania. The area was divided into 200m × 200m search grids, and habitats containing Ae. aegypti larvae or pupae characterized physically. Additionally, using WHO guidelines, we examined susceptibility of field-collected Aedes to common public health insecticides, i.e. pyrethroids (permethrin, 0.75% and deltamethrin, 0.05%), organophosphate (pirimiphos methyl, 0.25%), carbamate (bendiocarb, 0.1%) and organochloride (dieldrin, 4%), in both dry and rainy seasons (Nov-2018 to May-2019). A total of 1,515 and 1,933 larval habitats were visited and characterized in the dry and rainy season. Of these only 286 (18.87%) and 283 (14.64%) contained Aedes immatures. A total of 2,315 and 2,832 houses were visited in the dry and rainy season surveys, out of which, 114 (4.9%) and 186 (6.6%) houses had at least one positive breeding site respectively. Majority of Ae. aegypti habitats were associated with improper garbage disposal (used tires and discarded containers), peri-domestic aesthetics and water storage (flower pots and clay pots) or coconut harvesting (holes made by residents on trunks of coconut trees to support climbing harvesters). Used tires had highest overall abundance of Aedes immatures, while coconut tree holes had highest densities per habitat. Habitats containing Ae. aegypti had clear stagnant water, mostly in shaded areas. The mosquitoes were susceptible to all insecticides in both seasons, except bendiocarb, which had reduced susceptibility during rainy season tests. This is the first study on ecology and insecticide susceptibility of Aedes mosquitoes in this area, and will provide a basis for future evaluation of its role in pathogen transmission, as well as options for its control. Infestation levels observed indicate that immediate actions should be taken to prevent outbreaks. The larval indices (container index, house index and breteaux index) are high enough to signal significant risk of Aedes -borne diseases in the area. We also recommend that similar assessments should be done in other towns and their surrounding areas. Addition to that, such research should involve community to help eradicate the breeding sites through an integrated environmental management program leveraging on any existing initiatives such as the monthly clean-up campaigns in Tanzania. Fortunately, the Ae. aegypti in the area are still susceptible to majority of insecticides used in public health, indicating available opportunities to include insecticides in the control programs. While used tires, discarded containers and flower pots are key habitats for Aedes in the area, this study also identified coconut harvesting as an important risk factor, and the associated tree-holes as vital targets for Aedes control.