Background: Anopheles funestus, a major vector of malaria in Africa, has proven difficult to colonize in laboratory settings, impeding research on its biology and control. After several attempts, our team recently succeeded in colonizing a strain of An. funestus from Tanzania (FUTAZ). The objective of this study was to analyse the key fitness and genotypic characteristics of these mosquitoes during multiple filial generations of laboratory adaptation and compare them to wild An. funestus from Tanzania and a pre-existing colony of An. funestus from Mozambique (FUMOZ). Methods: Measures of mating success (percentage of female mosquitoes inseminated), body size (wing length), fecundity (number of eggs laid per female), and insecticide susceptibility (percentage of 24-hour mortality after exposure to insecticides) were compared between the newly established colonies of Tanzanian An. funestus (FUTAZ colonies), the long-established FUMOZ colonies, and a colony of Anopheles arabiensis maintained in the same laboratory. The maternal lineages of the An. funestus mosquitoes were investigated through a hydrolysis probe analysis of their mitochondrial DNA to identify distinct clades, I and II. Additionally, other intragenomic variations were examined through a PCR analysis of restriction fragment length polymorphisms (RFLP) on the third domain of 28S ribosomal DNA. These molecular markers were used to compare the FUTAZ colonies, FUMOZ colonies in Tanzania and South Africa, and the wild-collected An. funestus from Tanzania. Result: The mating success and body size of FUTAZ females declined significantly from filial generations F1 to F6 relative to the founder population (F0), but then increased from F7 onwards eventually matching FUMOZ by F9. Fecundity was similar across all colonies tested. However, it took significantly longer for 50% of the females in the FUTAZ and FUMOZ colonies (over 10 days) to mate compared to females in the An. arabiensis colony (approximately 5 days). Insecticide resistance appeared to be lost during colonization, but this varied with insecticide classes. Majority of mosquitoes in the FUTAZ colony, as well as the wild-caught Tanzanian An. funestus belonged to Clade I (80.4-89.4%) and RFLP type "Y" (90.5-91.4%), while the FUMOZ colonies were mostly Clade II (65.5-88.5%) and RFLP type "MW" (90.5-91.5%). Conclusion: This study suggests that the mating success and body size of An. funestus decreases significantly during the early stages of colonization, then increase as the mosquitoes adapt to laboratory conditions. It is therefore crucial to have a large enough founder population to persist through these early generations in order to achieve stable colonization of An. funestus. The Clade and RFLP genotyping demonstrated the genetic similarities between the FUTAZ mosquitoes and wild-caught Tanzanian An. funestus, but also showed that the new colony can be distinguished from the FUMOZ colony. ### Competing Interest Statement The authors have declared no competing interest.

Anopheles funestus, a major vector of malaria in Africa, has proven difficult to colonize in laboratory settings, impeding research on its biology and control. After several attempts, our team recently succeeded in colonizing a strain of An. funestus from Tanzania (FUTAZ). The objective of this study was to analyze the key fitness and genotypic characteristics of these mosquitoes during multiple filial generations of laboratory adaptation and compare them to wild An. funestus from Tanzania and a pre-existing colony of An. funestus from Mozambique (FUMOZ). Measures of mating success, body size, fecundity, and insecticide resistance were compared between the newly established FUTAZ colonies, the long-established FUMOZ colonies, and a colony of Anopheles arabiensis maintained in the same laboratory. The maternal lineages of the An. funestus mosquitoes were investigated through a hydrolysis probe analysis of their mitochondrial DNA to identify distinct clades. Other intragenomic variations were examined by D3-RFLP genotyping of 28S ribosomal DNA. These molecular markers were used to compare the FUTAZ colonies, FUMOZ colonies in Tanzania (TZ) and South African (SA) laboratories, and the wild-collected Tanzanian An. funestus. The mating success and body size of FUTAZ females declined significantly from filial generations F1 to F6 relative to the founder population (F0) then increased from F7 onwards, eventually matching FUMOZ by F9. Fecundity was similar across all colonies tested. However, it took significantly longer for 50

Population genetic analysis of mosquitoes is important for understanding the distribution of insecticide resistance alleles, devising sustainable control approaches, and understanding how vector populations are structured in space. Anopheles funestus is the dominant malaria vector in most parts of East and Southern Africa. To better understand its population genetic structure in Tanzania, we sequenced the genomes of 334 individual An. funestus mosquitoes from 11 regions across the country. Signs of reduced migration between western and eastern cohorts across the semi-arid central region containing the Rift Valley suggest a partial barrier to gene flow between these populations. This was evidenced by population structure between the eastern and western cohorts, as well as asynchronous selective sweeps and copy number variant profiles at the Cyp9k1 gene and Cyp6p gene cluster. Eastern cohorts, despite having less diversity and greater inbreeding, also share genetic histories characterized by low genome-wide Fst values with those in the west. This suggests that the barrier to gene flow is porous and likely represents continuous spatial structure rather than a complete barrier to migration. The observed population disconnectedness should be considered for insecticide deployment, resistance management, and the rollout of novel genetic-based vector control approaches. These findings provide the most detailed study of Tanzanian An. funestus population structure and resistance genetics to date. Future research should examine the epidemiological relevance of this partial discontinuity in gene flow and whether these populations have different malaria transmission abilities.

Population genetic analysis of mosquitoes is becoming increasingly important for understanding the distribution of insecticide resistance alleles, devising sustainable insecticide-based vector control approaches, and how malaria vector populations are structured in space. Anopheles funestus is the dominant malaria vector in Tanzania and most parts of East and Southern Africa. To better understand its population genomic structure in Tanzania, we sequenced the genomes of 334 individual An. funestus mosquitoes from 11 administrative regions. We found two genetically differentiated populations; one inland and at high altitude (found in Katavi, Kagera, Kigoma, and Mwanza) and a second coastal, at low altitude (found in Pwani, Morogoro, Tanga, Ruvuma, Mtwara, Dodoma, and Lindi), with differences in genetic diversity and inbreeding. We found asynchronous selective sweeps, associated with insecticide resistance phenotypes, at the Cyp9k1 gene, and Cyp6p gene cluster, with distinct copy number-variant profiles between the coastal and inland populations. These results suggest that inland and coastal An. funestus populations have divergent histories, with the arid, central region of Tanzania, which also contains the Rift Valley being a possible barrier to gene flow. Such population disconnectedness should be considered for insecticide deployment, resistance management, and the rollout of novel genetic-based vector control approaches. These findings provide the most detailed study of Tanzanian An. funestus population structure and resistance genetics to date. Future research should examine the epidemiological relevance of this discontinuity in gene flow and whether these populations have different malaria transmission abilities.

Introduction:Larval source management, particularly larviciding, is mainly implemented in urban settings to control malaria and other mosquito-borne diseases. In Tanzania, the government has recently expanded larviciding to rural settings across the country, but implementation faces multiple challenges, notably inadequate resources and limited know-how by technical staff. This study evaluated the potential of training community members to identify, characterize and target larval habitats of Anopheles funestus mosquitoes, the dominant vector of malaria transmission in south-eastern Tanzania. Methods:A mixed-methods study was used. First, interviewer-administered questionnaires were employed to assess knowledge, awareness, and perceptions of community members towards larviciding (N = 300). Secondly community-based volunteers were trained to identify and characterize aquatic habitats of dominant malaria vector species, after which they treated the most productive habitats with a locally-manufactured formulation of the biolarvicide, Bacillus thuringiensis var. israelensis. Longitudinal surveys of mosquito adults and larvae were used to assess impacts of the community-led larviciding programme in two villages in rural south-eastern Tanzania. Results:At the beginning of the program, the majority of village residents were unaware of larviciding as a potential malaria prevention method, and about 20 % thought that larvicides could be harmful to the environment and other insects. The trained community volunteers identified and characterized 360 aquatic habitats, of which 45.6 % had Anopheles funestus, the dominant malaria vector in the area. The preferred larval habitats for An. funestus were deep and had either slow- or fast-moving waters. Application of biolarvicides reduced the abundance of adult An. funestus and Culex spp. species inside human houses in the same villages, by 46.3 % and 35.4 % respectively. Abundance of late-stage instar larvae of the same taxa was also reduced by 74 % and 42 %, respectively. Conclusion:This study demonstrates that training community members to identify, characterize, and target larval habitats of the dominant malaria vectors can be effective for larval source management in rural Tanzania. Community-led larviciding reduced the densities of adult and late-stage instar larvae of An. funestus and Culex spp. inside houses, suggesting that this approach may have potential for malaria control in rural settings. However, efforts are still needed to increase awareness of larviciding in the relevant communities.

Abstract Background The Anopheles funestus group includes at least 11 sibling species, with Anopheles funestus Giles being the most studied and significant malaria vector. Other species, like Anopheles parensis, are understudied despite their potential role in transmission. This article provides insights into the biology and insecticide susceptibility of An. parensis, with observations of its densities in northwestern Tanzania. Methods Mosquitoes were collected in three villages in Misungwi district, northwestern Tanzania, using CDC light traps and battery-powered aspirators indoors and human-baited double net traps outdoors. Female Anopheles adults were morphologically sorted and identified by PCR, and a subset was tested by ELISA for vertebrate blood meal sources and Plasmodium sporozoite infections. Insecticide susceptibility was assessed using the WHO protocol (2nd edition, 2018). Unfed females were dissected to assess parity, gonotrophic status and insemination status, while blood-fed females were monitored for oviposition to estimate egg counts. The prevalence of An. parensis was generally < 24% across all sites, except in Ngaya village, where it unexpectedly constituted 84% of PCR-amplified An. funestus sensu lato. This species was present in both indoor and outdoor collections, yet the females exclusively fed on non-human vertebrates, with no human blood meals detected. Parity rates were approximately 49% for resting and 46% for host-seeking females, with slightly higher percentages of both parous and inseminated females in the dry season compared to the wet season. Most parous females had oviposited once or twice, with those in the dry season ovipositing significantly more eggs. The average wing length of female An. parensis was 2.93 mm, and there was no significant impact of body size on parity, fecundity or insemination. The An. parensis mosquitoes were fully susceptible to pyrethroids, carbamates, organophosphates and organochlorides. Results The prevalence of An. parensis was generally < 24% across all sites, except in Ngaya village, where it unexpectedly constituted 84% of PCR-amplified An. funestus sensu lato. This species was present in both indoor and outdoor collections, yet the females exclusively fed on non-human vertebrates, with no human blood meals detected. Parity rates were approximately 49% for resting and 46% for host-seeking females, with slightly higher percentages of both parous and inseminated females in the dry season compared to the wet season. Most parous females had oviposited once or twice, with those in the dry season ovipositing significantly more eggs. The average wing length of female An. parensis was 2.93 mm, and there was no significant impact of body size on parity, fecundity or insemination. The An. parensis mosquitoes were fully susceptible to pyrethroids, carbamates, organophosphates and organochlorides.. Conclusion This study offers insights into the behaviours and insecticide susceptibility of An. parensis. Primarily feeding on non-human hosts, An. parensis is less significant in malaria transmission than more anthropophilic vectors. Unlike the pyrethroid-resistant An. funestus sensu stricto, An. parensis remains fully susceptible to public health insecticides despite the use of insecticidal bed nets. These findings provide a foundation for future research and may inform control strategies targeting residual malaria transmission involving An. parensis. Graphical Abstract

Knowing when and where infected mosquitoes bite is required for estimating accurate measures of malaria risk, assessing outdoor exposure, and designing intervention strategies. This study combines secondary analyses of a human behaviour survey and an entomological survey carried out in the same area to estimate human exposure to malaria-infected Anopheles mosquitoes throughout the night in rural villages in south-eastern Tanzania. Mosquitoes were collected hourly from 6PM to 6AM indoors and outdoors by human landing catches in 2019, and tested for Plasmodium falciparum sporozoite infections using ELISA. In nearby villages, a trained member in each selected household recorded the whereabouts and activities of the household members from 6PM to 6AM in 2016 and 2017. Vector control use was high: 99% of individuals were reported to use insecticide-treated nets and a recent trial of indoor residual spraying had achieved 80% coverage. The risk of being bitten by infected mosquitoes outdoors, indoors in bed, and indoors but not in bed, and use of mosquito nets was estimated for each hour of the night. Individuals were mainly outdoors before 9PM, and mainly indoors between 10PM and 5AM. The main malaria vectors caught were Anopheles funestus sensu stricto and An. arabiensis. Biting rates were higher in the night compared to the evening or early morning. Due to the high use of ITNs, an estimated 85% (95% CI 81%, 88%) of all exposure in children below school age and 76% (71%, 81%) in older household members could potentially be averted by ITNs under current use patterns. Outdoor exposure accounted for an estimated 11% (8%, 15%) of infective bites in children below school age and 17% (13%, 22%) in older individuals. Maintaining high levels of ITN access, use and effectiveness remains important for reducing malaria transmission in this area. Interventions against outdoor exposure would provide additional protection.

Abstract 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. Graphical Abstract

Abstract 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.

Background Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an anthropophilic vector, which is now strongly resistant to public health insecticides, and several sibling species, which remain largely understudied despite their potential as secondary vectors. This paper provides the initial results of a cross-country study of the species composition, distribution and malaria transmission potential of members of the Anopheles funestus group in Tanzania. Methods Mosquitoes were collected inside homes in 12 regions across Tanzania between 2018 and 2022 using Centres for Disease Control and Prevention (CDC) light traps and Prokopack aspirators. Polymerase chain reaction (PCR) assays targeting the noncoding internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA (18S rDNA) were used to identify sibling species in the An. funestus group and presence of Plasmodium infections, respectively. Where DNA fragments failed to amplify during PCR, we sequenced the ITS2 region to identify any polymorphisms. Results The following sibling species of the An. funestus group were found across Tanzania: An. funestus s.s. (50.3%), An. parensis (11.4%), An. rivulorum (1.1%), An. leesoni (0.3%). Sequencing of the ITS2 region in the nonamplified samples showed that polymorphisms at the priming sites of standard species-specific primers obstructed PCR amplification, although the ITS2 sequences closely matched those of An. funestus s.s., barring these polymorphisms. Of the 914 samples tested for Plasmodium infections, 11 An. funestus s.s. (1.2%), and 2 An. parensis (0.2%) individuals were confirmed positive for P. falciparum. The highest malaria transmission intensities [entomological inoculation rate (EIR)] contributed by the Funestus group were in the north-western region [108.3 infectious bites/person/year (ib/p/y)] and the south-eastern region (72.2 ib/p/y). Conclusions Whereas An. funestus s.s. is the dominant malaria vector in the Funestus group in Tanzania, this survey confirms the occurrence of Plasmodium-infected An. parensis, an observation previously made in at least two other occasions in the country. The findings indicate the need to better understand the ecology and vectorial capacity of this and other secondary malaria vectors in the region to improve malaria control.

Introduction: Larval source management (LSM) can effectively suppress mosquito populations at source and provides an opportunity to address major challenges such as insecticide resistance that undermine primary interventions like insecticide-treated nets (ITNs). While mostly implemented in urban and arid settings, emerging research indicates its potential in some rural settings in east and southern Africa, where the main malaria vector, Anopheles funestus, prefers permanent and semi-permanent water bodies that support year-round transmission. Targeting these unique habitats could amplify effectiveness of LSM but requires careful considerations of local societal practices and expectations - particularly since mosquito breeding sites often also serve as community water resources. The aim of this study was therefore to explore how the societal uses of aquatic habitats by local communities in rural south-eastern Tanzania might influence LSM strategies, focusing on habitats frequented 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 a cross-sectional surveillance of all aquatic habitats, while qualitative data were gathered via a combination of individual unstructured interviews, focus group discussions with various community groups and field observations of community practices and activities. Data analysis employed weaving and inferencing techniques to integrate findings from both quantitative and qualitative components, thereby developing a comprehensive understanding from the respondents' perspectives. Results: A survey of 931 aquatic habitats revealed that 73% contained mosquito larvae, with late instar An. funestus identified in 23% of these habitats. River streams segments were the most common habitat type, accounting for 41%, followed by ground pools at 4%; other types included pits, rice fields, ditches, and puddles. Community use was noted for 90% of these habitats, including 95% of those with An. funestus larvae, for activities such as domestic chores such as cooking, washing utensils, washing clothes and bathing, agriculture, livestock rearing, brickmaking, and fishing. Focus group discussions indicated community readiness to implement LSM, favoring larviciding and habitat manipulation over habitat removal. Community concerns regarding LSM centered on the safety of larvicides for animal and human health and their environmental impact. The discussions proved the need for LSM interventions to integrate seamlessly with daily activities; and for community education on LSM safety and efficacy. Conclusion: This study offers valuable insights into community perspectives on LSM for malaria control in rural settings, emphasizing the dual role of aquatic habitats as both mosquito breeding sites and community water sources. This presents a set of unique challenges and opportunities – suggesting that LSM strategies must address both the biological aspects of mosquito control and the socio-economic realities of local communities. Notably, there was a marked preference for larviciding and habitat manipulation over habitat removal, with a strong emphasis on health and environmental safety. Overall, the study highlights the critical importance of educating communities, adopting culturally sensitive approaches to LSM, and aligning LSM strategies with the needs, perspectives, and daily lives of local communities.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis work was supported in whole by the Bill & Melinda Gates Foundation [Grant No. INV-002138 to Ifakara Health Institute]. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethical approval for the study was obtained from the Ifakara Health Institute Institutional Review Board (Ref: IHI/ IRB/No: 26-2020) and the Medical Research Coordinating Committee (MRCC) at the National Institute for Medical Research-NIMR (Ref: NIMR/HQ/R.8a/Vol. IX/3495)I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesUpon reasonable request

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

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

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.

An amendment to this paper has been published and can be accessed via the original article.

Simulation datasets for: "Evaluation of different deployment strategies for larviciding to control malaria – a simulation study". The folders include a description folder with relevant input files for simulations in OpenMalaria and the processedExpResults includes the Rdataframes, generated from the raw simulation outputs, used for analysis.

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

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

Background Unrestricted use of pesticides in agriculture is likely to increase insecticide resistance in mosquito vectors. Unfortunately, strategies for managing insecticide resistance in agriculture and public health sectors lack integration. This study explored the types and usage of agricultural pesticides, and awareness and management practices among retailers and farmers in Ulanga and Kilombero districts in south-eastern Tanzania, whereAnophelesmosquitoes are resistant to pyrethroids. Methods An exploratory sequential mixed-methods approach was employed. First, a survey to characterize pesticide stocks was conducted in agricultural and veterinary (agrovet) retail stores. Interviews to assess general knowledge and practices regarding agricultural pesticides were performed with 17 retailers and 30 farmers, followed by a survey involving 427 farmers. Concurrently, field observations were done to validate the results. Results Lambda-cyhalothrin, cypermethrin (both pyrethroids) and imidacloprids (neonicotinoids) were the most common agricultural insecticides sold to farmers. The herbicide glyphosate (amino-phosphonates) (59.0%), and the fungicides dithiocarbamate and acylalanine (54.5%), and organochlorine (27.3%) were also readily available in the agrovet shops and widely used by farmers. Although both retailers and farmers had at least primary-level education and recognized pesticides by their trade names, they lacked knowledge on pest control or proper usage of these pesticides. Most of the farmers (54.4%, n = 316) relied on instructions from pesticides dealers. Overall, 93.7% (400) farmers practised pesticides mixing in their farms, often in close proximity to water sources. One-third of the farmers disposed of their pesticide leftovers (30.0%, n = 128) and most farmers discarded empty pesticide containers into rivers or nearby bushes (55.7%, n = 238). Conclusion Similarities of active ingredients used in agriculture and malaria vector control, poor pesticide management practices and low-levels of awareness among farmers and pesticides retailers might enhance the selection of insecticide resistance in malaria vectors. This study emphasizes the need for improving awareness among retailers and farmers on proper usage and management of pesticides. The study also highlights the need for an integrated approach, including coordinated education on pesticide use, to improve the overall management of insecticide resistance in both agricultural and public health sectors.

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.