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Background: Residual malaria transmissions in Africa may be associated with improved coverage of insecticide-treated nets, house features, and livestock husbandry. These human-land use activities may drive the ecology and behaviour of malaria vectors which sustain residual malaria transmission. This study was conducted to assess changes in the ecology and behaviour of Anopheles funestus and Anopheles arabiensis in villages with high coverage of insecticide-treated nets to guide the selection of complementary vector control strategies against residual malaria transmission., Methods: Mosquitoes were collected using a CDC-light trap, miniaturized double net trap, and Prokopack aspirator from 222 households in three villages (Ebuyu, Chirombora, and Mzelezi) within Kilombero Valley. Anopheles mosquitoes were morphologically identified to their physiological status and species-complex levels. A sub-sample of Anopheles mosquitoes was exposed to laboratory analyses of sibling species, host preference, and sporozoite rates. Additionally, the local houses were geo-referenced using Global Positioning Systems (GPS) devise, and house features were recorded and associated with vector abundance., Results: The population of An. funestus s.s was abundant with high Plasmodium sporozoite rates inside houses compared to An. arabiensis. However, these vector species equally blood-fed on humans inside houses, but they also flexibly mixed human and animal blood meal. Fewer An. funestus were caught in houses with metal- than grass roofs and houses with and without animals. Contrastingly, fewer An. arabiensis were caught from houses with screened eaves compared to houses with open eaves., Conclusions: This study confirms that An. funestus dominates residual malaria transmission over An. arabiensis. These vector species exhibit anthropophily and opportunistic blood-feeding behaviour in areas with high coverage of insecticide-treated nets, but they numerically respond differently to local house improvements. These results imply that integrating mosquito-proof houses, improved insecticide-treated nets, and livestock-based interventions could effectively reduce and eventually eliminate residual malaria transmission., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Limwagu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Attractive targeted sugar baits (ATSBs) are promising new interventions that can complement existing vector control tools. However, reproducible and quantitative information on the level of attractiveness of ATSBs under field conditions is needed. Therefore, we customized camera traps for close-up imaging. We integrated them into a rugged ATSB monitoring station for day and nighttime recording of mosquitoes landing on the bait., Methods: The camera traps were evaluated in a semifield system and then in the field in rural Tanzania. In semifield experiments, camera traps were set up in large cages (2 m × 5 m × 2 m) to record mosquitoes landing on an attractive sugar bait (ASB), a blank ASB, or 20% sucrose (w/v). Next, 198 mosquitoes (33 males and 33 females of Anopheles arabiensis, An. funestus and Aedes aegypti) were released into each large cage and allowed to seek a sugar meal for 72 h with a camera recording images of the mosquitoes present on the ASB at 1-min intervals. In the field, 16 camera traps were set in 16 households, 7 with ASB attractant, 7 with ASB blank, and 2 with 20% sucrose (w/v). Human landing catch (HLC) was performed on the same nights as the camera trap recordings., Results: Under semifield conditions, significantly more mosquitoes visited the ASBs than the blank baits, with An. funestus visiting more frequently than An. arabiensis. There were no significant differences between female and male An. arabiensis visits, but female An. funestus visited more than their conspecific males did. The duration of visits did not vary between the ASB and blank controls or between the mosquito species. Moreover, mosquitoes visited the ASB or sucrose equally, with An. arabiensis visiting the baits more than An. funestus. Compared with male mosquitoes, female mosquitoes visited the baits more often. There was no significant difference in visit duration between the species.  In the field study, a mean of 70 An. arabiensis were caught per person per night on HLC, while 1 individual was caught per night on ASBs. There were significantly more visits by mosquitoes to the ASB than to the ASB blanks or sucrose solution, with more An. arabiensis visiting the baits than An. funestus or Culex quinquefasciatus. Significantly more females than males visited the baits of all the species. Again, the duration of visits was similar among An. arabiensis, An. funestus and C. quinquefasciatus. Aedes aegypti very rarely visited ASBs in the semifield experiments, and none were observed on baits in the field., Conclusions: Using camera traps to record still images of mosquitoes on ASBs offers reliable, reproducible and quantitative information on their attractiveness in various environmental conditions. Thus, camera traps serve as effective tools for evaluating and improving ATSB technology., Competing Interests: Declarations. Ethical approval and consent to participate: Before starting the fieldwork, permission was granted by the Institutional Review Board of the Ifakara Health Institute (IHI/IRB/AMM/No: 24–2021) and Medical Research Coordination Committee of the National Institute for Medical Research in Tanzania (NIMR/HQ/R.8a/Vol.IX/3777). Consent for publication: Volunteers provided written informed consent to participate in the study and for the publication of this report and any accompanying images. Approval for publication was also obtained from the National Institute of Medical Research in Tanzania (BD.242/437/01C/57). Competing interests: Fredros Okumu is a Subject Editor for Parasites & Vectors and was not involved in the peer review of this paper. The authors declare that they have no other competing interests., (© 2024. The Author(s).)

Background: Larval source management (LSM) is re-emerging as a critical malaria intervention to address challenges associated with core vector control tools, such as insecticide-treated nets (ITNs), and to accelerate progress towards elimination. Presently, LSM is not widely used in rural settings and is instead more commonly applied in urban and arid settings. A systematic entomological assessment was conducted in rural communities of southeastern Tanzania, where insecticide-treated nets (ITNs) are widely used, to explore opportunities for deploying LSM to improve malaria control., Methods: Aquatic habitat surveys were conducted in 2022 and 2023 to understand habitat usage by different mosquito vectors, covering five villages during the rainy season and seven villages during the dry season. Additionally, samples of adult mosquitoes were collected to assess the role of various Anopheles species in malaria transmission in the area, and to explore opportunities for species sanitation using targeted LSM., Results: Adult mosquito surveys showed that in this area, the total entomological inoculation rates (EIR) for indoor collections were 20.1 and 6.5 infectious bites per person per year for outdoors. Anopheles funestus and Anopheles arabiensis were the only Anopheles vectors identified. Anopheles funestus was responsible for over 97.6% of the malaria transmission indoors and 95.4% outdoors. The concurrent larval surveys found that habitats with late instar An. arabiensis and An. funestus comprised only a small subset of 11.2%-16.5% of all water bodies in the rainy season, and 9.7%-15.2% in the dry season. In terms of size, these habitats covered 66.4%-68.2% of the total habitat areas in the wet season, reducing to 33.9%-40.6% in the dry season. From the rainy season to the dry season, the surface area of habitats occupied by An. arabiensis and An. funestus decreased by 92.0% to 97.5%, while the number of habitats occupied by An. arabiensis and An. funestus decreased by 38.0% to 57.3%. Anopheles funestus preferred large, permanent habitats with clear water and vegetation year-round, while An. arabiensis showed contrasting seasonal preferences, favouring sunlit still waters in the rainy season and larger, opaque habitats in the dry season., Conclusion: These findings suggest that An. funestus, which is the dominant malaria vector in the area, mediating over 95% of malaria transmission, preferentially occupies only a small subset of uniquely identifiable aquatic habitats in both wet and dry seasons. This presents an opportunity to expand LSM in rural settings by carefully targeting An. funestus habitats, which might be effective and logistically feasible as a complementary approach alongside existing interventions. Further research should assess the impact of targeted LSM for species sanitation compared to blanket LSM., Competing Interests: Declarations Ethics approval and consent to participate Ethics approvals for this study were obtained from the Institutional Review Board of Ifakara Health Institute (Ref no: IHI/IRB/No: 32-2021) and the Medical Research Coordinating Committee (MRCC) at the National Institute for Medical Research (Ref no: NIMR/HQ/R.8a/Vol. IX/3761). Consent for publication Permission to publish was granted by The National Institute for Medical Research (NIMR), Tanzania, Ref No. BD.242/437/01C/11. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Larval source management (LSM) effectively reduces mosquito populations at their breeding sites, addressing issues like insecticide resistance that limit the effectiveness of primary interventions such as insecticide-treated nets (ITNs). Although traditionally used in urban and dry areas, recent research suggests it might also be effective in rural settings in eastern and southern Africa, where Anopheles funestus thrives in permanent water bodies that sustain year-round transmission. Targeting these habitats could enhance LSM, but it requires understanding local community practices, as mosquito breeding sites often overlap with community water resources. This study examined how communities use aquatic habitats and how these practices may impact LSM strategies, with a focus on habitats used by An. funestus., Methods: This study was conducted in three villages in the Ulanga and Malinyi districts of southeastern Tanzania using a mixed-methods approach. Quantitative data were collected through cross-sectional surveillance, and qualitative data through unstructured interviews, focus group discussions, and field observations. Data analysis integrated both quantitative and qualitative findings to develop a comprehensive understanding of community perspectives., Results: A survey of 931 aquatic habitats found mosquito larvae in 73% of them, with late instar An. funestus present in 23%. River streams made up 41% of the habitats, while ground pools accounted for 4%. Most habitats (90%) were used by communities, including 95% of those with An. funestus larvae, for activities such as domestic chores, agriculture, livestock rearing, brickmaking, and fishing. Focus group discussions revealed a willingness to adopt LSM, with a preference for larviciding and habitat modification over habitat removal, as the water sources were vital for daily use. Community concerns centered on the safety of larvicides for humans and animals, the environmental impact, and the need for better awareness of how LSM affects health and livelihoods., Conclusion: This study highlights community perspectives on LSM, focusing on the dual function of aquatic habitats as mosquito breeding sites and essential community water sources. This dual role presents both challenges and opportunities, suggesting that LSM strategies must balance public health needs with socio-economic realities. There was a clear preference for larviciding and habitat modification over removal, with a strong emphasis on health and environmental safety. The study emphasizes the importance of educating communities on the safety and effectiveness of LSM, and tailoring LSM strategies to fit the needs and preferences of local communities., (© 2024. The Author(s).)

A major insecticide resistance mechanism in insect pests is knock-down resistance (kdr) caused by mutations in the voltage-gated sodium channel (Vgsc) gene. Despite being common in most malaria Anopheles vector species, kdr mutations have never been observed in Anopheles funestus, the principal malaria vector in Eastern and Southern Africa, with resistance mainly being conferred by detoxification enzymes. In a parallel study, we monitored 10 populations of An. funestus in Tanzania for insecticide resistance unexpectedly identified resistance to a banned insecticide, DDT, in the Morogoro region. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found eight novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (equivalent to L995F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region and were accompanied by weak signatures of a selective sweep, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). The WHO prequalifies no DDT products for vector control, and the chemical is banned in Tanzania. Widespread DDT contamination and a legacy of extensive countrywide stockpiles may have selected for this mutation. Continued monitoring is necessary to understand the origin of kdr in An. funestus, and the threat posed to insecticide-based vector control in Africa., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Background: Afrotropical malaria vectors are generally believed to bite nocturnally, leading to the predominant use of insecticide-treated nets (ITNs), which target indoor, nighttime-biting mosquitoes. This focus is reinforced by biases in entomological surveys, which largely overlook daytime mosquito activity. However, recent evidence challenges this paradigm, showing that Anopheles biting can extend way into the daytime, coinciding with human activities at dawn, daytime and evenings, suggesting a broader risk spectrum and potential protection gaps. We have therefore investigated the diurnal and nocturnal biting patterns of the malaria vectors Anopheles arabiensis and Anopheles funestus in south-eastern Tanzania, to better understand the scope of residual transmission and inform strategies for improved control., Methods: Host-seeking mosquitoes were collected hourly using miniaturized double net traps, both indoors and outdoors over 24-h periods between June 2023 and February 2024. Concurrently, human activities indoors and outdoors were monitored half-hourly to correlate with mosquito collections. A structured questionnaire was used to assess household members' knowledge, perceptions and experiences regarding exposure to mosquito bites during both nighttime and daytime., Results: Nocturnal biting by An. arabiensis peaked between 7 p.m. and 11 p.m. while that of An. funestus peaked later, between 1 a.m. and 3 a.m. Daytime biting accounted for 15.03% of An. arabiensis catches, with peaks around 7-11 a.m. and after 4 p.m., and for 14.15% of An. funestus catches, peaking around mid-mornings, from 10 a.m. to 12 p.m. Nighttime exposure to An. arabiensis was greater outdoors (54.5%), while daytime exposure was greater indoors (80.4%). For An. funestus, higher exposure was observed indoors, both at nighttime (57.1%) and daytime (69%). Plasmodium falciparum sporozoites were detected in both day-biting and night-biting An. arabiensis. Common daytime activities potentially exposing residents during peak biting hours included household chores, eating, sleeping (including due to sickness), resting in the shade or under verandas and playing (children). From evenings onwards, exposures coincided with resting, socializing before bedtime and playtime (children). Nearly all survey respondents (95.6%) reported experiencing daytime mosquito bites, but only 28% believed malaria was transmissible diurnally., Conclusions: This study updates our understanding of malaria vector biting patterns in south-eastern Tanzania, revealing considerable additional risk in the mornings, daytime and evenings. Consequently, there may be more gaps in the protection provided by ITNs, which primarily target nocturnal mosquitoes, than previously thought. Complementary strategies are needed to holistically suppress vectors regardless of biting patterns (e.g. using larval source management) and to extend personal protection limits (e.g. using repellents). Additionally, community engagement and education on mosquito activity and protective measures could significantly reduce malaria transmission risk., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background: The degree to which Anopheles mosquitoes prefer biting humans over other vertebrate hosts, i.e. the human blood index (HBI), is a crucial parameter for assessing malaria transmission risk. However, existing techniques for identifying mosquito blood meals are demanding in terms of time and effort, involve costly reagents, and are prone to inaccuracies due to factors such as cross-reactivity with other antigens or partially digested blood meals in the mosquito gut. This study demonstrates the first field application of mid-infrared spectroscopy and machine learning (MIRS-ML), to rapidly assess the blood-feeding histories of malaria vectors, with direct comparison to PCR assays., Methods and Results: Female Anopheles funestus mosquitoes (N = 1854) were collected from rural Tanzania and desiccated then scanned with an attenuated total reflectance Fourier-transform Infrared (ATR-FTIR) spectrometer. Blood meals were confirmed by PCR, establishing the 'ground truth' for machine learning algorithms. Logistic regression and multi-layer perceptron classifiers were employed to identify blood meal sources, achieving accuracies of 88%-90%, respectively, as well as HBI estimates aligning well with the PCR-based standard HBI., Conclusions: This research provides evidence of MIRS-ML effectiveness in classifying blood meals in wild Anopheles funestus, as a potential complementary surveillance tool in settings where conventional molecular techniques are impractical. The cost-effectiveness, simplicity, and scalability of MIRS-ML, along with its generalizability, outweigh minor gaps in HBI estimation. Since this approach has already been demonstrated for measuring other entomological and parasitological indicators of malaria, the validation in this study broadens its range of use cases, positioning it as an integrated system for estimating pathogen transmission risk and evaluating the impact of interventions., (© 2024. The Author(s).)

Background: Accurately determining the age and survival probabilities of adult mosquitoes is crucial for understanding parasite transmission, evaluating the effectiveness of control interventions and assessing disease risk in communities. This study was aimed at demonstrating the rapid identification of epidemiologically relevant age categories of Anopheles funestus, a major Afro-tropical malaria vector, through the innovative combination of infrared spectroscopy and machine learning, instead of the cumbersome practice of dissecting mosquito ovaries to estimate age based on parity status., Methods: Anopheles funestus larvae were collected in rural south-eastern Tanzania and reared in an insectary. Emerging adult females were sorted by age (1-16 days old) and preserved using silica gel. Polymerase chain reaction (PCR) confirmation was conducted using DNA extracted from mosquito legs to verify the presence of An. funestus and to eliminate undesired mosquitoes. Mid-infrared spectra were obtained by scanning the heads and thoraces of the mosquitoes using an attenuated total reflection-Fourier transform infrared (ATR-FT-IR) spectrometer. The spectra (N = 2084) were divided into two epidemiologically relevant age groups: 1-9 days (young, non-infectious) and 10-16 days (old, potentially infectious). The dimensionality of the spectra was reduced using principal component analysis, and then a set of machine learning and multi-layer perceptron (MLP) models were trained using the spectra to predict the mosquito age categories., Results: The best-performing model, XGBoost, achieved overall accuracy of 87%, with classification accuracy of 89% for young and 84% for old An. funestus. When the most important spectral features influencing the model performance were selected to train a new model, the overall accuracy increased slightly to 89%. The MLP model, utilizing the significant spectral features, achieved higher classification accuracy of 95% and 94% for the young and old An. funestus, respectively. After dimensionality reduction, the MLP achieved 93% accuracy for both age categories., Conclusions: This study shows how machine learning can quickly classify epidemiologically relevant age groups of An. funestus based on their mid-infrared spectra. Having been previously applied to An. gambiae, An. arabiensis and An. coluzzii, this demonstration on An. funestus underscores the potential of this low-cost, reagent-free technique for widespread use on all the major Afro-tropical malaria vectors. Future research should demonstrate how such machine-derived age classifications in field-collected mosquitoes correlate with malaria in human populations., (© 2024. The Author(s).)

Background: Surveillance of malaria vectors is crucial for assessing the transmission risk and impact of control measures. Human landing catches (HLC) directly estimate the biting rates but raise ethical concerns due to the exposure of volunteers to mosquito-borne pathogens. A common alternative is the CDC-light trap, which is effective for catching host-seeking mosquitoes indoors but not outdoors. New, exposure-free methods are needed for sampling mosquitoes indoors and outdoors in ways that reflect their natural risk profiles. The aim of this study was therefore to evaluate the efficacy of the miniaturized double net trap (DN-Mini) for sampling host-seeking mosquitoes in south-eastern Tanzania, where malaria transmission is dominated by Anopheles funestus., Methods: Adult mosquitoes were collected from 222 randomly selected houses across three villages (74 per village) in Ulanga district, south-eastern Tanzania, using the DN-Mini traps, CDC-Light traps, and Prokopack aspirators. First, we compared CDC-light and DN-Mini traps for collecting indoor host-seeking mosquitoes, while Prokopack aspirators were used for indoor-resting mosquitoes. Second, we deployed the DN-Mini and Prokopack aspirators to collect host-seeking and resting mosquitoes indoors and outdoors. Generalized linear mixed models (GLMM) with a negative binomial distribution were used to compare the effectiveness of the traps for catching different mosquito species., Results: The DN-Mini was 1.53 times more efficient in collecting An. funestus indoors (RR = 1.53, 95% CI: 1.190-1.98) compared to the CDC-Light trap. However, for Anopheles arabiensis, the DN-Mini caught only 0.32 times as many mosquitoes indoors as the CDC-Light traps (RR = 0.32, 95% CI: 0.183-0.567). Both An. funestus and An. arabiensis were found to be more abundant indoors than outdoors when collected using the DN-Mini trap. Similarly, the Prokopack aspirator was greater indoors than outdoors for both An. funestus and An. arabiensis., Conclusion: The DN-Mini outperformed the CDC-light trap in sampling the dominant malaria vector, An. funestus species, but was less effective in capturing An. arabiensis, and for both vector species, the biting risk was greater indoors than outdoors when measured using the DN-Mini trap. These findings highlight the importance of selecting appropriate trapping methods based on mosquito species and behaviors., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Limwagu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Anopheles funestus is a major malaria vector in Eastern and Southern Africa and is currently the dominant malaria-transmitting vector in many parts of Tanzania. Previous research has identified its preference for specific aquatic habitats, especially those that persist in dry months. This observation suggests the potential for targeted control through precise habitat mapping and characterization. In this study, we investigated the influence of habitat characteristics, land cover and human population densities on An. funestus distribution during dry seasons. Based on the results, we developed a habitat suitability model for this vector species in south-eastern Tanzania., Methods: Eighteen villages in south-eastern Tanzania were surveyed during the dry season from September-December 2021. Water bodies were systematically inspected for mosquito larvae and characterized by their physico-chemical characteristics and surrounding environmental features. A generalized linear model was used to assess the presence of An. funestus larvae as a function of the physico-chemical characteristics, land use and human population densities. The results obtained from this model were used to generate spatially explicit predictions of habitat suitability in the study districts., Results: Of the 1466 aquatic habitats surveyed, 440 were positive for An. funestus, with river streams having the highest positivity (74%; n = 322) followed by ground pools (15%; n = 67). The final model had an 83% accuracy in predicting positive An. funestus habitats, with the most important characteristics being permanent waters, clear waters with or without vegetation or movement and shading over the habitats. There was also a positive association of An. funestus presence with forested areas and a negative association with built-up areas. Human population densities had no influence on An. funestus distribution., Conclusions: The results of this study underscore the crucial role of both the specific habitat characteristics and key environmental factors, notably land cover, in the distribution of An. funestus. In this study area, An. funestus predominantly inhabits river streams and ground pools, with a preference for clear, perennial waters with shading. The strong positive association with more pristine environments with tree covers and the negative association with built-up areas underscore the importance of ecological transitions in vector distribution and malaria transmission risk. Such spatially explicit predictions could enable more precise interventions, particularly larval source management, to accelerate malaria control., (© 2024. The Author(s).)

Effective larviciding for malaria control requires detailed studies of larvicide efficacies, aquatic habitat characteristics, and life history traits of target vectors. Mosquitoes with brief larval phases present narrower timeframes for biolarvicidal effects than mosquitoes with extended periods. We evaluated two biolarvicides, VectoBac (Bacillus thuringiensis israelensis (Bti)) and VectoMax (Bti and Bacillus sphaericus) against Anopheles funestus and Anopheles arabiensis in shaded and unshaded habitats; and explored how larval development might influence retreatment intervals. These tests were done in semi-natural habitats using field-collected larvae, with untreated habitats as controls. Additionally, larval development was assessed in semi-natural and natural habitats in rural Tanzania, by sampling daily and recording larval developmental stages. Both biolarvicides reduced larval densities of both species by >98% within 72 h. Efficacy lasted one week in sun-exposed habitats but remained >50% for two weeks in shaded habitats. An. funestus spent up to two weeks before pupating (13.2(10.4-16.0) days in semi-natural; 10.0(6.6-13.5) in natural habitats), while An. arabiensis required slightly over one week (8.2 (5.8-10.6) days in semi-natural; 8.3 (5.0-11.6) in natural habitats). The findings suggest that weekly larviciding, which is essential for An. arabiensis might be more effective for An. funestus whose prolonged aquatic growth allows for repeated exposures. Additionally, the longer residual effect of biolarvicides in shaded habitats indicates they may require less frequent treatments compared to sun-exposed areas., (© 2024. The Author(s).)

Background: Gene drive modified mosquitoes (GDMMs) have the potential to address Africa's persistent malaria problem, but are still in early stages of development and testing. Continuous engagement of African stakeholders is crucial for successful evaluation and implementation of these technologies. The aim of this multi-country study was, therefore, to explore the insights and recommendations of key stakeholders across Africa on the potential of GDMMs for malaria control and elimination in the continent., Methods: A concurrent mixed-methods study design was used, involving a structured survey administered to 180 stakeholders in 25 countries in sub-Saharan Africa, followed by 18 in-depth discussions with selected groups and individuals. Stakeholders were drawn from academia, research and regulatory institutions, government ministries of health and environment, media and advocacy groups. Thematic content analysis was used to identify key topics from the in-depth discussions, and descriptive analysis was done to summarize information from the survey data., Results: Despite high levels of awareness of GDMMs among the stakeholders (76.7%), there was a relatively low-level of understanding of their key attributes and potential for malaria control (28.3%). When more information about GDMMs was provided to the stakeholders, they readily discussed their insights and concerns, and offered several recommendations to ensure successful research and implementation of the technology. These included: (i) increasing relevant technical expertise within Africa, (ii) generating local evidence on safety, applicability, and effectiveness of GDMMs, and (iii) developing country-specific regulations for safe and effective governance of GDMMs. A majority of the respondents (92.9%) stated that they would support field trials or implementation of GDMMs in their respective countries. This study also identified significant misconceptions regarding the phase of GDMM testing in Africa, as several participants incorrectly asserted that GDMMs were already present in Africa, either within laboratories or released into the field., Conclusion: Incorporating views and recommendations of African stakeholders in the ongoing research and development of GDMMs is crucial for instilling stakeholder confidence on their potential application. These findings will enable improved planning for GDMMs in Africa as well as improved target product profiles for the technologies to maximize their potential for solving Africa's enduring malaria challenge., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestus group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestus group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© 2023. The Author(s).)

Background: Malaria disproportionately affects low-income households in rural communities where poor housing is common. Despite evidence that well-constructed and mosquito-proofed houses can reduce malaria risk, housing improvement is rarely included in malaria control toolboxes. This study assessed the need, magnitude, and opportunities for housing improvement to control malaria in rural Tanzania., Methods: A mixed-methods study was conducted in 19 villages across four district councils in southern Tanzania. A structured survey was administered to 1292 community members to assess need, perceptions, and opportunities for housing improvement for malaria control. Direct observations of 802 houses and surrounding environments were done to identify the actual needs and opportunities, and to validate the survey findings. A market survey was done to assess availability and cost of resources and services necessary for mosquito-proofing homes. Focus group discussions were conducted with key stakeholders to explore insights on the potential and challenges of housing improvement as a malaria intervention., Results: Compared to other methods for malaria control, housing improvement was among the best understood and most preferred by community members. Of the 735 survey respondents who needed housing improvements, a majority needed window screening (91.1%), repairs of holes in walls (79.4%), door covers (41.6%), closing of eave spaces (31.2%) and better roofs (19.0%). Community members invested significant efforts to improve their own homes against malaria and other dangers, but these efforts were often slow and delayed due to high costs and limited household incomes. Study participants suggested several mechanisms of support to improve their homes, including government loans and subsidies., Conclusion: Addressing the need for housing improvement is a critical component of malaria control efforts in southern Tanzania. In this study, a majority of the community members surveyed needed modest modifications and had plans to work on those modifications. Without additional support, their efforts were however generally slow; households would take years to sufficiently mosquito-proof their houses. It is, therefore, crucial to bring together the key players across sectors to reduce barriers in malaria-proofing housing in endemic settings. These may include government subsidies or partnerships with businesses to make housing improvement more accessible and affordable to residents., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Background: Old mosquitoes are more likely to transmit malaria than young ones. Therefore, accurate prediction of mosquito population age can drastically improve the evaluation of mosquito-targeted interventions. However, standard methods for age-grading mosquitoes are laborious and costly. We have shown that Mid-infrared spectroscopy (MIRS) can be used to detect age-specific patterns in mosquito cuticles and thus can be used to train age-grading machine learning models. However, these models tend to transfer poorly across populations. Here, we investigate whether applying dimensionality reduction and transfer learning to MIRS data can improve the transferability of MIRS-based predictions for mosquito ages., Methods: We reared adults of the malaria vector Anopheles arabiensis in two insectaries. The heads and thoraces of female mosquitoes were scanned using an attenuated total reflection-Fourier transform infrared spectrometer, which were grouped into two different age classes. The dimensionality of the spectra data was reduced using unsupervised principal component analysis or t-distributed stochastic neighbour embedding, and then used to train deep learning and standard machine learning classifiers. Transfer learning was also evaluated to improve transferability of the models when predicting mosquito age classes from new populations., Results: Model accuracies for predicting the age of mosquitoes from the same population as the training samples reached 99% for deep learning and 92% for standard machine learning. However, these models did not generalise to a different population, achieving only 46% and 48% accuracy for deep learning and standard machine learning, respectively. Dimensionality reduction did not improve model generalizability but reduced computational time. Transfer learning by updating pre-trained models with 2% of mosquitoes from the alternate population improved performance to ~ 98% accuracy for predicting mosquito age classes in the alternative population., Conclusion: Combining dimensionality reduction and transfer learning can reduce computational costs and improve the transferability of both deep learning and standard machine learning models for predicting the age of mosquitoes. Future studies should investigate the optimal quantities and diversity of training data necessary for transfer learning and the implications for broader generalisability to unseen datasets., (© 2023. The Author(s).)

Background: Insecticidal mosquito-proof netting screens could combine the best features of insecticide-treated nets (ITNs) and indoor residual spraying (IRS), the two most important front line vector control interventions in Africa today, and also overcome the most important limitations of these methods. This study engaged members of a rural Tanzanian community in developing and evaluating simple, affordable and scalable procedures for installing readily available screening materials on eave gaps and windows of their own houses, and then treating those screens with a widely used IRS formulation of the organophosphate insecticide pirimiphos-methyl (PM)., Methods: A cohort of 54 households recruited upon consent, following which the structural features and occupant demographics of their houses were surveyed. Indoor mosquito densities were surveyed longitudinally, for approximately 3 months before and over 5 months after participatory house modification and screening using locally available materials. Each house was randomly assigned to one of three study arms: (1) No screens installed until the end of the study (negative control), (2) untreated screens installed, and (3) screened installed and then treated with PM, the insecticidal activity of which was subsequently assessed using standard cone assays., Results: Almost all (52) recruited households participated until the end, at which point all houses had been successfully screened. In most cases, screening was only installed after making enabling structural modifications that were accepted by the enrolled households. Compared to unscreened houses, houses with either treated or untreated screens both almost entirely excluded Anopheles arabiensis (Relative reduction (RR) ≥ 98%, P < < 0.0001), the most abundant local malaria vector. However, screens were far less effective against Culex quinquefasciatus (RR ≤ 46%, P < < 0.0001), a non-malaria vector causing considerable biting nuisance, regardless of their treatment status. While PM did not augment household level protection by screens against either mosquito species (P = 0.676 and 0.831, respectively), 8 months after treatment it still caused 73% and 89% mortality among susceptible insectary-reared Anopheles gambiae following exposures of 3 and 30 min, respectively., Conclusions: Participatory approaches to mosquito proofing houses may be acceptable and effective, and installed screens may be suitable targets for residual insecticide treatments., (© 2022. The Author(s).)

Background: Monitoring the biological attributes of mosquitoes is critical for understanding pathogen transmission and estimating the impacts of vector control interventions on the survival of vector species. Infrared spectroscopy and machine learning techniques are increasingly being tested for this purpose and have been proven to accurately predict the age, species, blood-meal sources, and pathogen infections in Anopheles and Aedes mosquitoes. However, as these techniques are still in early-stage implementation, there are no standardized procedures for handling samples prior to the infrared scanning. This study investigated the effects of different preservation methods and storage duration on the performance of mid-infrared spectroscopy for age-grading females of the malaria vector, Anopheles arabiensis., Methods: Laboratory-reared An. arabiensis (N = 3681) were collected at 5 and 17 days post-emergence, killed with ethanol, and then preserved using silica desiccant at 5 °C, freezing at - 20 °C, or absolute ethanol at room temperature. For each preservation method, the mosquitoes were divided into three groups, stored for 1, 4, or 8 weeks, and then scanned using a mid-infrared spectrometer. Supervised machine learning classifiers were trained with the infrared spectra, and the support vector machine (SVM) emerged as the best model for predicting the mosquito ages., Results: The model trained using silica-preserved mosquitoes achieved 95% accuracy when predicting the ages of other silica-preserved mosquitoes, but declined to 72% and 66% when age-classifying mosquitoes preserved using ethanol and freezing, respectively. Prediction accuracies of models trained on samples preserved in ethanol and freezing also reduced when these models were applied to samples preserved by other methods. Similarly, models trained on 1-week stored samples had declining accuracies of 97%, 83%, and 72% when predicting the ages of mosquitoes stored for 1, 4, or 8 weeks respectively., Conclusions: When using mid-infrared spectroscopy and supervised machine learning to age-grade mosquitoes, the highest accuracies are achieved when the training and test samples are preserved in the same way and stored for similar durations. However, when the test and training samples were handled differently, the classification accuracies declined significantly. Protocols for infrared-based entomological studies should therefore emphasize standardized sample-handling procedures and possibly additional statistical procedures such as transfer learning for greater accuracy., (© 2022. The Author(s).)

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., Competing Interests: The authors declared that they have no competing interest.

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

The malaria parasite, which is transmitted by several Anopheles mosquito species, requires more time to reach its human-transmissible stage than the average lifespan of mosquito vectors. Monitoring the species-specific age structure of mosquito populations is critical to evaluating the impact of vector control interventions on malaria risk. We present a rapid, cost-effective surveillance method based on deep learning of mid-infrared spectra of mosquito cuticle that simultaneously identifies the species and age class of three main malaria vectors in natural populations. Using spectra from over 40, 000 ecologically and genetically diverse An. gambiae, An. arabiensis, and An. coluzzii females, we develop a deep transfer learning model that learns and predicts the age of new wild populations in Tanzania and Burkina Faso with minimal sampling effort. Additionally, the model is able to detect the impact of simulated control interventions on mosquito populations, measured as a shift in their age structures. In the future, we anticipate our method can be applied to other arthropod vector-borne diseases., (© 2022. The Author(s).)

Background: In sub-Saharan Africa, house design and ventilation affects the number of malaria mosquito vectors entering houses. This study hypothesized that indoor light from a CDC-light trap, visible from outside a hut, would increase entry of Anopheles arabiensis, an important malaria vector, and examined whether ventilation modifies this effect., Methods: Four inhabited experimental huts, each situated within a large chamber, were used to assess how light and ventilation affect the number of hut-entering mosquitoes in Tanzania. Each night, 300 female laboratory-reared An. arabiensis were released inside each chamber for 72 nights. Nightly mosquito collections were made using light traps placed indoors. Temperature and carbon dioxide concentrations were measured using data loggers. Treatments and sleepers were rotated between huts using a randomized block design., Results: When indoor light was visible outside the huts, there was an 84% increase in the odds of collecting mosquitoes indoors (Odds ratio, OR = 1.84, 95% confidence intervals, 95% CI 1.74-1.95, p < 0.001) compared with when it was not. Although the odds of collecting mosquitoes in huts with closed eaves (OR = 0.54, 95% CI 0.41-0.72, p < 0.001) was less than those with open eaves, few mosquitoes entered either type of well-ventilated hut. The odds of collecting mosquitoes was 99% less in well-ventilated huts, compared with poorly-ventilated traditional huts (OR = 0.01, 95% CI 0.01-0.03, p < 0.001). In well-ventilated huts, indoor temperatures were 1.3 °C (95% CI 0.9-1.7, p < 0.001) cooler, with lower carbon dioxide (CO 2 ) levels (mean difference = 97 ppm, 77.8-116.2, p < 0.001) than in poorly-ventilated huts., Conclusion: Although light visible from outside a hut increased mosquito house entry, good natural ventilation reduces indoor carbon dioxide concentrations, a major mosquito attractant, thereby reducing mosquito-hut entry., (© 2022. The Author(s).)

Supplementary tools are required to address the limitations of insecticide-treated nets (ITNs) and indoor residual spraying (IRS), which are currently the core vector control methods against malaria in Africa. The eave ribbons technology exploits the natural house-entry behaviours of major malaria vectors to deliver mosquitocidal or repellent actives around eave spaces through which the Anopheles mosquitoes usually enter human dwellings. They confer protection by preventing biting indoors and in the peri-domestic outdoor spaces, and also killing a significant proportion of the mosquitoes. Current versions of eave ribbons are made of low-cost hessian fabric infused with candidate insecticides and can be easily fitted onto multiple house types without any additional modifications. This article reviews the evidence for efficacy of the technology, and discusses its potential as affordable and versatile supplementary approach for targeted and efficient control of mosquito-borne diseases, particularly malaria. Given their simplicity and demonstrated potential in previous studies, future research should investigate ways to optimize scalability and effectiveness of the ribbons. It is also important to assess whether the ribbons may constitute a less-cumbersome, but more affordable substitute for other interventions, such as IRS, by judiciously using lower quantities of selected insecticides targeted around eave spaces to deliver equivalent or greater suppression of malaria transmission., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

Background: Different forms of mosquito modifications are being considered as potential high-impact and low-cost tools for future malaria control in Africa. Although still under evaluation, the eventual success of these technologies will require high-level public acceptance. Understanding prevailing community perceptions of mosquito modification is, therefore, crucial for effective design and implementation of these interventions. This study investigated community perceptions regarding genetically-modified mosquitoes (GMMs) and their potential for malaria control in Tanzanian villages where no research or campaign for such technologies has yet been undertaken., Methods: A mixed-methods design was used, involving: (i) focus group discussions (FGD) with community leaders to get insights on how they frame and would respond to GMMs, and (ii) structured questionnaires administered to 490 community members to assess awareness, perceptions and support for GMMs for malaria control. Descriptive statistics were used to summarize the findings and thematic content analysis was used to identify key concepts and interpret the findings., Results: Nearly all survey respondents were unaware of mosquito modification technologies for malaria control (94.3%), and reported no knowledge of their specific characteristics (97.3%). However, community leaders participating in FGDs offered a set of distinctive interpretive frames to conceptualize interventions relying on GMMs for malaria control. The participants commonly referenced their experiences of cross-breeding for selecting preferred traits in domestic plants and animals. Preferred GMMs attributes included the expected reductions in insecticide use and human labour. Population suppression approaches, requiring as few releases as possible, were favoured. Common concerns included whether the GMMs would look or behave differently than wild mosquitoes, and how the technology would be integrated into current malaria control policies. The participants emphasised the importance and the challenge of educating and engaging communities during the technology development., Conclusions: Understanding how communities perceive and interpret novel technologies is crucial to the design and effective implementation of new vector control programmes. This study offers vital clues on how communities with no prior experience of modified mosquitoes might conceptualize or respond to such technologies when deployed in the context of malaria control programmes. Drawing upon existing interpretive frames and locally-resonant analogies when deploying such technologies may provide a basis for more durable public support in the future.

Background: Larval source management was historically one of the most effective malaria control methods but is now widely deprioritized in Africa, where insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are preferred. However, in Tanzania, following initial successes in urban Dar-es-Salaam starting early-2000s, the government now encourages larviciding in both rural and urban councils nationwide to complement other efforts; and a biolarvicide production-plant has been established outside the commercial capital. This study investigated key obstacles and opportunities relevant to effective rollout of larviciding for malaria control, with a focus on the meso-endemic region of Morogoro, southern Tanzania., Methods: Key-informants were interviewed to assess awareness and perceptions regarding larviciding among designated health officials (malaria focal persons, vector surveillance officers and ward health officers) in nine administrative councils (n = 27). Interviewer-administered questionnaires were used to assess awareness and perceptions of community members in selected areas regarding larviciding (n = 490). Thematic content analysis was done and descriptive statistics used to summarize the findings., Results: A majority of malaria control officials had participated in larviciding at least once over the previous three years. A majority of community members had neutral perceptions towards positive aspects of larviciding, but overall support for larviciding was high, although several challenges were expressed, notably: (i) insufficient knowledge for identifying relevant aquatic habitats of malaria vectors and applying larvicides, (ii) inadequate monitoring of programme effectiveness, (iii) limited financing, and (iv) lack of personal protective equipment. Although the key-informants reported sensitizing local communities, most community members were still unaware of larviciding and its potential., Conclusions: The larviciding programme was widely supported by both communities and malaria control officials, but there were gaps in technical knowledge, implementation and public engagement. To improve overall impact, it is important to: (i) intensify training efforts, particularly for identifying habitats of important vectors, (ii) adopt standard technical principles for applying larvicides or larval source management, (iii) improve financing for local implementation and (iv) improve public engagement to boost community awareness and participation. These lessons could also be valuable for other malaria endemic areas wishing to deploy larviciding for malaria control or elimination.

Background: Malaria infection during pregnancy has negative health consequences for both mothers and offspring. Sub-microscopic malaria infection during pregnancy is common in most African countries. We sought to identify factors associated with sub-microscopic placental malaria, and its association with adverse pregnancy outcomes among HIV-negative pregnant women in Dar es Salaam, Tanzania., Methods: We recruited a cohort of pregnant women during their first trimester and assessed for the occurrence of placental malaria and pregnancy outcomes. The follow-up was done monthly from recruitment until delivery. Histopathology placental malaria positive results were defined as the presence of malaria pigment or parasitized erythrocytes on the slide (histology-positive (HP)), and the sub-microscopic placental infection was defined as positive Plasmodium falciparum DNA by polymerase chain reaction (DNA PCR) amplification in a negative histopathology test. Adverse pregnancy outcomes investigated included low birth weight (birth weight below 2.5 kg), prematurity (live birth below 37 weeks), and small-for-gestational-age (SGA) (live born with a birth weight below 10th percentile for gestational age and sex). Weighted baseline category logit, log-binomial, and log-Poisson models were used to assess factors associated with placental malaria, and its association with adverse pregnancy outcomes., Results: Among 1115 women who had histopathology and DNA PCR performed, 93 (8%) had HP placental infection, and 136 (12%) had the sub-microscopic placental infection. The risk of sub-microscopic placental malaria was greater in women who did not use mosquito prevention methods such as bed nets, fumigation, or mosquito coils (odds ratio (OR) = 1.75; 95% confidence interval (CI): 1.05-2.92; P = 0.03) and in women who were anemic (OR = 1.59; 95% CI: 1.20-2.11; P = 0.001). Women who were underweight had reduced odds of sub-microscopic placental malaria infection (OR = 0.33; 95% CI: 0.17-0.62; P = 0.001). Women who were overweight/obese had 1.48 times higher the odds of HP placental malaria compared to normal weight (OR = 1.48; 95% CI: 1.03-2.11; P = 0.03). HP placental malaria infection was associated with an increased risk of SGA births (RR = 1.30, 95% CI: 0.98-1.72, P = 0.07). In contrast, the sub-microscopic infection was associated with a reduced risk of SGA births (RR = 0.61, 95% CI: 0.43-0.88, P = 0.01). Placental malaria was not associated with low birth weight or prematurity., Conclusion: Malaria prevention methods and maternal nutrition status during early pregnancy were important predictors of sub-microscopic placental malaria. More research is needed to understand sub-microscopic placental malaria and the possible mechanisms mediating the association between placental malaria and SGA.

Background: Malaria control in Tanzania currently relies primarily on long-lasting insecticidal nets and indoor residual spraying, alongside effective case management and behaviour change communication. This study explored opinions of key stakeholders on the national progress towards malaria elimination, the potential of currently available vector control interventions in helping achieve elimination by 2030, and the need for alternative interventions that could be used to supplement malaria elimination efforts in Tanzania., Methods: In this exploratory qualitative study, Focus group discussions were held with policy-makers, regulators, research scientists and community members. Malaria control interventions discussed were: (a) improved housing, (b) larval source management, (c) mass drug administration (MDA) with ivermectin to reduce vector densities, (d) release of modified mosquitoes, including genetically modified or irradiated mosquitoes, (e) targeted spraying of mosquito swarms, and (f) spatial repellents., Results: Larval source management and spatial repellents were widely supported across all stakeholder groups, while insecticide-spraying of mosquito swarms was the least preferred. Support for MDA with ivermectin was high among policy makers, regulators and research scientists, but encountered opposition among community members, who instead expressed strong support for programmes to improve housing for poor people in high transmission areas. Policy makers, however, challenged the idea of government-supported housing improvement due to its perceived high costs. Techniques of mosquito modification, specifically those involving gene drives, were viewed positively by community members, policy makers and regulators, but encountered a high degree of scepticism among scientists. Overall, policy-makers, regulators and community members trusted scientists to provide appropriate advice for decision-making., Conclusion: Stakeholder opinions regarding alternative malaria interventions were divergent except for larval source management and spatial repellents, for which there was universal support. MDA with ivermectin, housing improvement and modified mosquitoes were also widely supported, though each faced concerns from at least one stakeholder group. While policy-makers, regulators and community members all noted their reliance on scientists to make informed decisions, their reasoning on the benefits and disadvantages of specific interventions included factors beyond technical efficiency. This study suggests the need to encourage and strengthen dialogue between research scientists, policy makers, regulators and communities regarding new interventions.

Background: Malaria control in Africa relies extensively on indoor residual spraying (IRS) and insecticide-treated nets (ITNs). IRS 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., Methods: The assessment was done inside 80 houses including: 20 with thatched roofs and mud walls, 20 with thatched roofs and un-plastered brick walls, 20 with metal roofs and un-plastered brick walls, and 20 with metal roofs and plastered brick walls, across four villages. In each house, resting mosquitoes were sampled in mornings (6 a.m.-8 a.m.), evenings (6 p.m.-8 p.m.) and at night (11 p.m.-12.00 a.m.) using Prokopack aspirators from multiple surfaces (walls, undersides of roofs, floors, furniture, utensils, clothing, curtains and bed nets)., Results: 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 16-20% 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, clothing and bed nets. These proportions were particularly high in metal-roofed houses (47-53% of An. funestus; 60-66% of An. arabiensis)., Conclusion: While IRS typically 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 locally-obtained data on mosquito resting behaviours and how these influence the overall impact and costs of IRS. This study also emphasizes the need for alternative approaches, e.g. house screening, which broadly tackle mosquitoes beyond areas reachable by IRS and ITNs.

Background: Many subsistence farmers in rural southeastern Tanzania regularly relocate to distant farms in river valleys to tend to crops for several weeks or months each year. While there, they live in makeshift semi-open structures, usually far from organized health systems and where insecticide-treated nets (ITNs) do not provide adequate protection. This study evaluated the potential of a recently developed technology, eave ribbons treated with the spatial repellent transfluthrin, for protecting migratory rice farmers in rural southeastern Tanzania against indoor-biting and outdoor-biting mosquitoes., Methods: In the first test, eave ribbons (0.1 m × 24 m each) treated with 1.5% transfluthrin solution were compared to untreated ribbons in 24 randomly selected huts in three migratory communities over 48 nights. Host-seeking mosquitoes indoors and outdoors were monitored nightly (18.00-07.00 h) using CDC light traps and CO 2 -baited BG malaria traps, respectively. The second test compared efficacies of eave ribbons treated with 1.5% or 2.5% transfluthrin in 12 huts over 21 nights. Finally, 286 farmers were interviewed to assess perceptions about eave ribbons, and their willingness to pay for them., Results: In the two experiments, when treated eave ribbons were applied, the reduction in indoor densities ranged from 56 to 77% for Anopheles arabiensis, 36 to 60% for Anopheles funestus, 72 to 84% for Culex, and 80 to 98% for Mansonia compared to untreated ribbons. Reduction in outdoor densities was 38 to 77% against An. arabiensis, 36 to 64% against An. funestus, 63 to 88% against Culex, and 47 to 98% against Mansonia. There was no difference in protection between the two transfluthrin doses. In the survey, 58% of participants perceived the ribbons to be effective in reducing mosquito bites. Ninety per cent were willing to pay for the ribbons, the majority of whom were willing to pay but less than US$2.17 (5000 TZS), one-third of the current prototype cost., Conclusions: Transfluthrin-treated eave ribbons can protect migratory rice farmers, living in semi-open makeshift houses in remote farms, against indoor-biting and outdoor-biting mosquitoes. The technology is acceptable to users and could potentially complement ITNs. Further studies should investigate durability and epidemiological impact of eave ribbons, and the opportunities for improving affordability to users.

Background: Eave ribbons treated with spatial repellents effectively prevent human exposure to outdoor-biting and indoor-biting malaria mosquitoes, and could constitute a scalable and low-cost supplement to current interventions, such as insecticide-treated nets (ITNs). This study measured protection afforded by transfluthrin-treated eave ribbons to users (personal and communal protection) and non-users (only communal protection), and whether introducing mosquito traps as additional intervention influenced these benefits., Methods: Five experimental huts were constructed inside a 110 m long, screened tunnel, in which 1000 Anopheles arabiensis were released nightly. Eave ribbons treated with 0.25 g/m 2 transfluthrin were fitted to 0, 1, 2, 3, 4 or 5 huts, achieving 0, 20, 40, 60, 80 and 100% coverage, respectively. Volunteers sat near each hut and collected mosquitoes attempting to bite them from 6 to 10 p.m. (outdoor-biting), then went indoors to sleep under untreated bed nets, beside which CDC-light traps collected mosquitoes from 10 p.m. to 6 a.m. (indoor-biting). Caged mosquitoes kept inside the huts were monitored for 24 h-mortality. Separately, eave ribbons, UV-LED mosquito traps (Mosclean) or both the ribbons and traps were fitted, each time leaving the central hut unfitted to represent non-user households and assess communal protection. Biting risk was measured concurrently in all huts, before and after introducing interventions., Results: Transfluthrin-treated eave ribbons provided 83% and 62% protection indoors and outdoors respectively to users, plus 57% and 48% protection indoors and outdoors to the non-user. Protection for users remained constant, but protection for non-users increased with eave ribbons coverage, peaking once 80% of huts were fitted. Mortality of mosquitoes caged inside huts with eave ribbons was 100%. The UV-LED traps increased indoor exposure to users and non-users, but marginally reduced outdoor-biting. Combining the traps and eave ribbons did not improve user protection relative to eave ribbons alone., Conclusion: Transfluthrin-treated eave ribbons protect both users and non-users against malaria mosquitoes indoors and outdoors. The mosquito-killing property of transfluthrin can magnify the communal benefits by limiting unwanted diversion to non-users, but should be validated in field trials against pyrethroid-resistant vectors. Benefits of the UV-LED traps as an intervention alone or alongside eave ribbons were however undetectable in this study. These findings extend the evidence that transfluthrin-treated eave ribbons could complement ITNs.

The BG-Malaria trap (BGM) is an adaptation of the well-known BG-Sentinel trap (BGS) with greater trapping efficiencies for anopheline and culicine mosquitoes. Its continued optimization requires greater understanding of mosquito flight behaviors near it. We used three high-resolution infrared cameras (68 frames/second) to track flight behaviors of laboratory-reared Anopheles arabiensis females in vicinity of the BGM in comparison with BGS. Additional comparisons were done for BGM at 20, 40 and 80cm heights, and for BGMs baited with Ifakara blend plus CO2, CO2 alone, or no bait. More mosquitoes were observed near BGM than BGS. Both BGMs installed 20cm above the floor and baited with CO2 received more visits by host-seeking mosquitoes than the other BGMs evaluated in their respective experiments. Trap designs, height and attractants all influence mosquito activity in vicinity of the traps which can be readily visualized using infrared cameras to accelerate trap development and testing. The greater activity of host-seeking mosquitoes near BGM than BGS supports the proven superiority of BGM traps in field and semi-field settings., Competing Interests: The authors have declared that no competing interests exist.

To accelerate malaria elimination in areas where core interventions such as insecticide-treated nets (ITNs) are already widely used, it is crucial to consider additional factors associated with persistent transmission. Qualitative data on human behaviours and perceptions regarding malaria risk was triangulated with quantitative data on Anopheles mosquito bites occurring indoors and outdoors in south-eastern Tanzania communities where ITNS are already used but lower level malaria transmission persists. Each night (18:00h-07:00h), trained residents recorded human activities indoors, in peri-domestic outdoor areas, and in communal gatherings. Host-seeking mosquitoes were repeatedly collected indoors and outdoors hourly, using miniaturized exposure-free double net traps (DN-Mini) occupied by volunteers. In-depth interviews were conducted with household representatives to explore perceptions on persistent malaria and its control. Higher proportions of people stayed outdoors than indoors in early-evening and early-morning hours, resulting in higher exposures outdoors than indoors during these times. However, exposure during late-night hours (22:00h-05:00h) occurred mostly indoors. Some of the popular activities that kept people outdoors included cooking, eating, relaxing and playing. All households had at least one bed net, and 83.9% of people had access to ITNs. Average ITN use was 96.3%, preventing most indoor exposure. Participants recorgnized the importance of ITNs but also noted that the nets were not perfect. No complementary interventions were reported being used widely. Most people believed transmission happens after midnight. We conclude that insecticide-treated nets, where properly used, can still prevent most indoor exposures, but significant risk continues unabated before bedtime, outdoors and at communal gatherings. Such exposure is greatest for rural and low-income households. There is therefore an urgent need for complementary interventions, particularly those targeting outdoor-biting and are applicable for all people including the marginalised populations such as migratory farmers and fishermen. Besides, the differences in community understanding of ongoing transmission, and feedback on imperfections of ITNs should be considered when updating malaria-related communication and interventions., Competing Interests: The authors have declared that no competing interests exist.

Background: Natural infections of the endosymbiont bacteria Wolbachia have recently been discovered in populations of the malaria mosquito Anopheles gambiae (s.l.) in Burkina Faso and Mali, West Africa. This Anopheles specific strain wAnga limits the malaria parasite Plasmodium falciparum infections in the mosquito, thus it offers novel opportunities for malaria control., Results: We investigated Wolbachia presence in Anopheles arabiensis and Anopheles funestus, which are the two main malaria vectors in the Kilombero Valley, a malaria endemic region in south-eastern Tanzania. We found 3.1% (n = 65) and 7.5% (n = 147) wAnga infection prevalence in An. arabiensis in mosquitoes collected in 2014 and 2016, respectively, while no infection was detected in An. funestus (n = 41). Phylogenetic analysis suggests that at least two distinct strains of wAnga were detected, both belonging to Wolbachia supergroup A and B., Conclusions: To our knowledge, this is the first confirmation of natural Wolbachia in malaria vectors in Tanzania, which opens novel questions on the ecological and genetic basis of its persistence and pathogen transmission in the vector hosts. Understanding the basis of interactions between Wolbachia, Anopheles mosquitoes and malaria parasites is crucial for investigation of its potential application as a biocontrol strategy to reduce malaria transmission, and assessment of how natural wAnga infections influence pathogen transmission in different ecological settings.

Background: Behaviour changes in mosquitoes from indoor to outdoor biting result in continuing risk of malaria from outdoor activities, including routine household activities and occasional social and cultural practices and gatherings. This study aimed to identify the range of social and cultural gatherings conducted outdoors and their associated risks for mosquito bites., Methods: A cross-sectional study was conducted in four villages in the Kilombero Valley from November 2015 to March 2016. Observations, focus group discussions, and key informant interviews were conducted. The recorded data were transcribed and translated from Swahili to English. Thematic content analysis was used to identify perspectives on the importance of various social and cultural gatherings that incidentally expose people to mosquito bites and malaria infection., Results: Religious, cultural and social gatherings involving the wider community are conducted outdoors at night till dawn. Celebrations include life course events, religious and cultural ceremonies, such as Holy Communion, weddings, gatherings at Easter and Christmas, male circumcision, and rituals conducted to please the gods and to remember the dead. These celebrations, at which there is minimal use of interventions to prevent bites, contribute to individual satisfaction and social capital, helping to maintain a cohesive society. Bed net use while sleeping outdoors during mourning is unacceptable, and there is minimal use of other interventions, such as topical repellents. Long sleeve clothes are used for protection from mosquito bites but provide less protection., Conclusion: Gatherings and celebrations expose people to mosquito bites. Approaches to prevent risks of mosquito bites and disease management need to take into account social, cultural and environmental factors. Area specific interventions may be expensive, yet may be the best approach to reduce risk of infection as endemic countries work towards elimination. Focusing on single interventions will not yield the best outcomes for malaria prevention as social contexts and vector behaviour vary.

Background: Long-lasting insecticide-treated nets and indoor residual spraying protect against indoor-biting and indoor-resting mosquitoes but are largely ineffective for early-biting and outdoor-biting malaria vectors. Complementary tools are, therefore, needed to accelerate control efforts. This paper describes simple hessian ribbons treated with spatial repellents and wrapped around eaves of houses to prevent outdoor-biting and indoor-biting mosquitoes over long periods of time., Methods: The eave ribbons are 15 cm-wide triple-layered hessian fabrics, in lengths starting 1 m. They can be fitted onto houses using nails, adhesives or Velcro, without completely closing eave-spaces. In 75 experimental nights, untreated ribbons and ribbons treated with 0.02%, 0.2%, 1.5% or 5% transfluthrin emulsion (spatial repellent) were evaluated against blank controls using two experimental huts inside a 202 m 2 semi-field chamber where 500 laboratory-reared Anopheles arabiensis were released nightly. Two volunteers sat outdoors (one/hut) and collected mosquitoes attempting to bite them from 6 p.m. to 10 p.m. (outdoor-biting), then went indoors and slept under bed nets, beside which CDC-light traps collected mosquitoes from 10 p.m. to 6.30 a.m. (indoor-biting). To assess survival, 200 caged mosquitoes were suspended near the huts nightly and monitored for 24 h thereafter. Additionally, field tests were done in experimental huts in a rural Tanzanian village to evaluate treated ribbons (1.5% transfluthrin). Here, indoor-biting was assessed using window traps and Prokopack ® aspirators, and outdoor-biting assessed using volunteer-occupied double-net traps., Results: Indoor-biting and outdoor-biting decreased > 99% in huts fitted with eave ribbons having ≥ 0.2% transfluthrin. Even 0.02% transfluthrin-treated ribbons provided 79% protection indoors and 60% outdoors. Untreated ribbons however reduced indoor-biting by only 27% and increased outdoor-biting by 18%, though these were non-significant (P > 0.05). Of all caged mosquitoes exposed near treated huts, 99.5% died within 24 h. In field tests, the ribbons provided 96% protection indoors and 84% outdoors against An. arabiensis, plus 42% protection indoors and 40% outdoors against Anopheles funestus. Current prototypes cost ~ 7USD/hut, are made of widely-available hessian and require no specialized expertise., Conclusion: Transfluthrin-treated eave ribbons significantly prevented outdoor-biting and indoor-biting malaria vectors and could potentially complement current tools. The technique is simple, low-cost, highly-scalable and easy-to-use; making it suitable even for poorly-constructed houses and low-income groups.

BG-Malaria (BGM) trap is a simple adaptation of the widely-used BG-Sentinel trap (BGS). It is proven to be highly effective for trapping the Brazilian malaria vector, Anopheles darlingi, in field conditions, and the African vector, Anopheles arabiensis, under controlled semi-field environments, but has not been field-tested in Africa. Here, we validated the BGM for field sampling of malaria vectors in south-eastern Tanzania. Using a series of Latin-Square experiments conducted nightly (6pm-7am) in rural villages, we compared mosquito catches between BGM, BGS and human landing catches (HLC). We also compared BGMs baited with different attractants (Ifakara-blend, Mbita-blend, BG-Lure and CO2). Lastly, we tested BGMs baited with Ifakara-blend from three odour-dispensing methods (BG-Cartridge, BG-Sachet and Nylon strips). One-tenth of the field-collected female Anopheles gambiae s.l. and Anopheles funestus were dissected to assess parity. BGM captured more An. gambiae s.l. than BGS (p < 0.001), but HLC caught more than either trap (p < 0.001). However, BGM captured more An. funestus than HLC. Proportions of parous An. gambiae s.l. and An. funestus consistently exceeded 50%, with no significant difference between methods. While the dominant species caught by HLC was An. gambiae s.l. (56.0%), followed by Culex spp. (33.1%) and Mansonia spp. (6.0%), the BGM caught mostly Culex (81.6%), followed by An. gambiae s.l. (10.6%) and Mansonia (5.8%). The attractant-baited BGMs were all significantly superior to un-baited controls (p < 0.001), although no difference was found between the specific attractants. The BG-Sachet was the most efficient dispenser for capturing An. gambiae s.l. (14.5(2.75-42.50) mosquitoes/trap/night), followed by BG-Cartridge (7.5(1.75-26.25)). The BGM caught more mosquitoes than BGS in field-settings, but sampled similar species diversity and physiological states as BGS. The physiological states of malaria vectors caught in BGM and BGS were similar to those naturally attempting to bite humans (HLC). The BGM was most efficient when baited with Ifakara blend, dispensed from BG-Sachet. We conclude that though BGM traps have potential for field-sampling of host-seeking African malaria vectors with representative physiological states, both BGM and BGS predominantly caught more culicines than Anopheles, compared to HLC, which caught mostly An. gambiae s.l., Competing Interests: The authors have declared that no competing interests exist.

Background: House improvement and environmental management can significantly improve malaria transmission control in endemic communities. This study assessed the influence of physical characteristics of houses and surrounding environments on mosquito biting risk in rural Tanzanian villages, and examined knowledge and perceptions of residents on relationships between these factors and malaria transmission. The study further assessed whether people worried about these risks and how they coped., Methods: Entomological surveys of indoor mosquito densities were conducted across four villages in Ulanga district, south-eastern Tanzania. The survey involved 48 sentinel houses sampled monthly and other sets of 48 houses randomly recruited each month for one-off sampling over 12 months. Physical characteristics of the houses and surrounding environments were recorded. Questionnaire surveys were administered to 200 household heads to assess their knowledge and concerns regarding the observed housing and environmental features, and whether they considered these features when constructing houses. Focus group discussions, were conducted to clarify emergent themes on people's perceptions on relationships between housing or environmental factors and malaria transmission., Results: The entomological surveys showed statistically higher indoor densities of the malaria vectors (Anopheles arabiensis and Anopheles funestus) in houses with mud walls compared to plastered or brick walls, open eaves compared to closed eaves and unscreened windows compared to screened windows. Most respondents reported that their houses allowed mosquito entry, at least partially. Participants were aware that house structure and environmental characteristics influenced indoor mosquito densities and consequently malaria transmission. They were concerned about living in poorly-constructed houses with gaps on eaves, walls, windows and doors but were constrained by low income., Conclusion: In rural south-eastern Tanzania, significant proportions of people still live in houses with open eaves, unscreened windows and gaps on doors. Though they are fully aware of associated mosquito biting and pathogen transmission risks, they are constrained by low-income levels. The study proposes that community-based house improvement initiatives combined with targeted subsidies could lower the financial barriers, improve access to essential construction materials or designs, and significantly accelerate malaria transmission control in these communities.

Gene drive technology offers the promise for a high-impact, cost-effective, and durable method to control malaria transmission that would make a significant contribution to elimination. Gene drive systems, such as those based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein, have the potential to spread beneficial traits through interbreeding populations of malaria mosquitoes. However, the characteristics of this technology have raised concerns that necessitate careful consideration of the product development pathway. A multidisciplinary working group considered the implications of low-threshold gene drive systems on the development pathway described in the World Health Organization Guidance Framework for testing genetically modified (GM) mosquitoes , focusing on reduction of malaria transmission by Anopheles gambiae s.l. mosquitoes in Africa as a case study. The group developed recommendations for the safe and ethical testing of gene drive mosquitoes, drawing on prior experience with other vector control tools, GM organisms, and biocontrol agents. These recommendations are organized according to a testing plan that seeks to maximize safety by incrementally increasing the degree of human and environmental exposure to the investigational product. As with biocontrol agents, emphasis is placed on safety evaluation at the end of physically confined laboratory testing as a major decision point for whether to enter field testing. Progression through the testing pathway is based on fulfillment of safety and efficacy criteria, and is subject to regulatory and ethical approvals, as well as social acceptance. The working group identified several resources that were considered important to support responsible field testing of gene drive mosquitoes.

Background: Additional vector control tools (VCTs) are needed to supplement insecticide-treated nets (ITNs) and indoor residual spraying (IRS) to achieve malaria elimination in many settings. To identify options for expanding the malaria vector control toolbox, we conducted a systematic review of the availability and quality of the evidence for 21 malaria VCTs, excluding ITNs and IRS., Methods: Six electronic databases and grey literature sources were searched from January 1, 1980 to September 28, 2015 to identify systematic reviews, Phase I-IV studies, and observational studies that measured the effect of malaria VCTs on epidemiological or entomological outcomes across any age groups in all malaria-endemic settings. Eligible studies were summarized qualitatively, with quality and risk of bias assessments undertaken where possible. Of 17,912 studies screened, 155 were eligible for inclusion and were included in a qualitative synthesis., Results: Across the 21 VCTs, we found considerable heterogeneity in the volume and quality of evidence, with 7 VCTs currently supported by at least one Phase III community-level evaluation measuring parasitologically confirmed malaria incidence or infection prevalence (insecticide-treated clothing and blankets, insecticide-treated hammocks, insecticide-treated livestock, larval source management (LSM), mosquito-proofed housing, spatial repellents, and topical repellents). The remaining VCTs were supported by one or more Phase II (n=13) or Phase I evaluation (n=1). Overall the quality of the evidence base remains greatest for LSM and topical repellents, relative to the other VCTs evaluated, although existing evidence indicates that topical repellents are unlikely to provide effective population-level protection against malaria., Conclusions: Despite substantial gaps in the supporting evidence, several VCTs may be promising supplements to ITNs and IRS in appropriate settings. Strengthening operational capacity and research to implement underutilized VCTs, such as LSM and mosquito-proofed housing, using an adaptive, learning-by-doing approach, while expanding the evidence base for promising supplementary VCTs that are locally tailored, should be considered central to global malaria elimination efforts., (© 2018 Elsevier Ltd All rights reserved.)

Odour-baited technologies are increasingly considered for effective monitoring of mosquito populations and for the evaluation of vector control interventions. The BG-Malaria trap (BGM), which is an upside-down variant of the widely used BG-Sentinel trap (BGS), has been demonstrated to be effective to sample the Brazilian malaria vector, Anopheles darlingi. We evaluated the BGM as an improved method for sampling the African malaria vectors, Anopheles arabiensis. Experiments were conducted inside a large semi-field cage to compare trapping efficiencies of BGM and BGS traps, both baited with the synthetic attractant, Ifakara blend, supplemented with CO2. We then compared BGMs baited with either of four synthetic mosquito lures, Ifakara blend, Mbita blend, BG-lure or CO2, and an unbaited BGM. Lastly, we compared BGMs baited with the Ifakara blend dispensed via either nylon strips, BG cartridges (attractant-infused microcapsules encased in cylindrical plastic cartridge) or BG sachets (attractant-infused microcapsules encased in plastic sachets). All tests were conducted between 6P.M. and 7A.M., with 200-600 laboratory-reared An. arabiensis released nightly in the test chamber. The median number of An. arabiensis caught by the BGM per night was 83, IQR:(73.5-97.75), demonstrating clear superiority over BGS (median catch = 32.5 (25.25-37.5)). Compared to unbaited controls, BGMs baited with Mbita blend caught most mosquitoes (45 (29.5-70.25)), followed by BGMs baited with CO2 (42.5 (27.5-64)), Ifakara blend (31 (9.25-41.25)) and BG lure (16 (4-22)). BGM caught 51 (29.5-72.25) mosquitoes/night, when the attractants were dispensed using BG-Cartridges, compared to BG-Sachet (29.5 (24.75-40.5)), and nylon strips (27 (19.25-38.25)), in all cases being significantly superior to unbaited controls (p < 000.1). The findings demonstrate potential of the BGM as a sampling tool for African malaria vectors over the standard BGS trap. Its efficacy can be optimized by selecting appropriate odour baits and odour-dispensing systems.

Background: The extensive use of indoor residual spraying (IRS) and insecticide-treated nets (ITNs) in Africa has contributed to a significant reduction in malaria transmission. Even so, residual malaria transmission persists in many regions, partly driven by mosquitoes that bite people outdoors. In areas where Anopheles gambiae s.s. is a dominant vector, most interventions target the reduction of indoor transmission. The increased use of ITNs/LLINs and IRS has led to the decline of this species. As a result, less dominant vectors such as Anopheles funestus and Anopheles arabiensis, both also originally indoor vectors but are increasingly biting outdoors, contribute more to residual malaria transmission. The study reports the investigated community perceptions on malaria and their implications of this for ongoing outdoor malaria transmission and malaria control efforts., Methods: This was a qualitative study conducted in two rural villages and two peri-urban areas located in Kilombero Valley in south-eastern Tanzania. 40 semi-structured in-depth interviews and 8 focus group discussions were conducted with men and women who had children under the age of five. The Interviews and discussions focused on (1) community knowledge of malaria transmission, and (2) the role of such knowledge on outdoor malaria transmission as a contributing factor to residual malaria transmission., Results: The use of bed nets for malaria prevention has been stressed in a number of campaigns and malaria prevention programmes. Most people interviewed believe that there is outdoor malaria transmission since they use interventions while indoors, but they are unaware of changing mosquito host-seeking behaviour. Participants pointed out that they were frequently bitten by mosquitoes during the evening when outdoors, compared to when they were indoors. Most participants stay outdoors in the early evening to undertake domestic tasks that cannot be conducted indoors. House structure, poor ventilation and warm weather conditions were reported to be the main reasons for staying outdoors during the evening. Participants reported wearing long sleeved clothes, fanning and slapping themselves, using repellents, and burning cow dung and neem tree leaves to chase away mosquitoes., Conclusions: Community understanding of multiple prevention strategies is crucial given changes in mosquito host seeking behaviour and the increased incidence of outdoor biting. The current low use of outdoor control measures is attributed largely to limited awareness of outdoor transmission. Improved community understanding of outdoor malaria transmission is critical: efforts to reduce or eliminate malaria transmission will not be successful if the control of outdoor transmission is not emphasized.

Malaria is transmitted by many Anopheles species whose proportionate contributions vary across settings. We re-assessed the roles of Anopheles arabiensis and Anopheles funestus, and examined potential benefits of species-specific interventions in an area in south-eastern Tanzania, where malaria transmission persists, four years after mass distribution of long-lasting insecticide-treated nets (LLINs). Monthly mosquito sampling was done in randomly selected households in three villages using CDC light traps and back-pack aspirators, between January-2015 and January-2016, four years after the last mass distribution of LLINs in 2011. Multiplex polymerase chain reaction (PCR) was used to identify members of An. funestus and Anopheles gambiae complexes. Enzyme-linked immunosorbent assay (ELISA) was used to detect Plasmodium sporozoites in mosquito salivary glands, and to identify sources of mosquito blood meals. WHO susceptibility assays were done on wild caught female An. funestus s.l, and physiological ages approximated by examining mosquito ovaries for parity. A total of 20,135 An. arabiensis and 4,759 An. funestus were collected. The An. funestus group consisted of 76.6% An. funestus s.s, 2.9% An. rivulorum, 7.1% An. leesoni, and 13.4% unamplified samples. Of all mosquitoes positive for Plasmodium, 82.6% were An. funestus s.s, 14.0% were An. arabiensis and 3.4% were An. rivulorum. An. funestus and An. arabiensis contributed 86.21% and 13.79% respectively, of annual entomological inoculation rate (EIR). An. arabiensis fed on humans (73.4%), cattle (22.0%), dogs (3.1%) and chicken (1.5%), but An. funestus fed exclusively on humans. The An. funestus populations were 100% susceptible to organophosphates, pirimiphos methyl and malathion, but resistant to permethrin (10.5% mortality), deltamethrin (18.7%), lambda-cyhalothrin (18.7%) and DDT (26.2%), and had reduced susceptibility to bendiocarb (95%) and propoxur (90.1%). Parity rate was higher in An. funestus (65.8%) than An. arabiensis (44.1%). Though An. arabiensis is still the most abundant vector species here, the remaining malaria transmission is predominantly mediated by An. funestus, possibly due to high insecticide resistance and high survival probabilities. Interventions that effectively target An. funestus mosquitoes could therefore significantly improve control of persistent malaria transmission in south-eastern Tanzania.