Combining larviciding with insecticide treated nets (ITNs) can reduce malaria transmission. However, most modelling analyses use generalized scenarios rather than incorporating local epidemiological and ecological contexts. In Tanzania and other countries, larviciding is increasingly being prioritized in national strategies, with growing advocacy for its broader implementation, to achieving sustained malaria reduction. District-specific modelling is therefore essential to capture variation in transmission ecology, seasonality, and varying coverage levels, providing evidence that is both rigorous and actionable for malaria control programs. The Vector Control Optimization Model (VCOM) was adapted and extended to incorporate local seasonality, simulating the impact of larviciding across a range of coverage levels combined with ITNs. The model was parameterized using district-level field-data on mosquito mortality collected before (2016-2017) and after (2019-2021) larviciding implementation. Mosquito mortality rates were estimated using Bayesian inference. Outcomes were evaluated specifically for Anopheles gambiae s.l. including annual entomological inoculation rates (EIR) and mosquito density. Sensitivity analysis explored the influence of key parameters driving transmission in this scenario study. The immature mosquito mortality rate due to larviciding was 61% based on field data. VCOM simulation showed that, at 80%, ITNs coverage, larviciding substantially reduced mosquito densities and EIR. Specifically, combining ITNs at 80% and larviciding coverage ≥ 60% lowered EIR below 1 ib/p/year, the threshold required to interrupt malaria transmission. Sensitivity analyses highlighted the high impact of targeting immature mosquitoes, suggesting larviciding can effectively complement ITNs to control vectors, including invasive species like An. stephensi, regardless of feeding preference, resting, and biting behaviors, which hinder the effectiveness of most vector control tools. This study provides local evidence that larviciding is an effective complement to ITNs for interrupting malaria transmission. Implementation should leverage innovative approaches, such as drones for precise mapping and targeted application of biological larvicides, to maximize coverage, and scalability for district-level malaria control and elimination.

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.

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.

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

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

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.

Background Despite significant progress against malaria, the use of two core interventions, namely insecticide-treated nets (ITNs) and indoor residual spraying (IRS), are becoming increasingly vulnerable due to insecticide resistance and outdoor biting by mosquitoes. One representative example is Kilombero Valley in Tanzania where transmission continues despite over 80% of the population using ITNs. Further progress in this and similar African settings will require identifying which supplementary tools combine most effectively with ITNs to reduce mosquito exposure. Methods Using the Kilombero Valley as a case study, the Vector Control Optimization Model (VCOM) was adapted and extended to simulate the impact of adding eave ribbons treated with spatial repellent (ER) as supplementary intervention in an area of high ITN coverage. Simulation was conducted to assess the impact of varying coverage of this supplementary intervention on the entomological inoculation rates (EIR) generated by two common vectors in Kilombero, Anopheles arabiensis and Anopheles funestus . Finally, the impact of introducing this intervention on the combined EIR from An. arabiensis and An. funestus was assessed to identify scenarios in which values fell below 1; the likely threshold required for malaria interruption. Results ER was predicted to substantially reduce the EIR in Kilombero valley when combined with 80% ITN coverage. However, the nature of the impact varied notably between vector species. ER was predicted to have a much larger effect on transmission mediated by An. funestus than An. arabiensis . Additionally, in the situation where EIR from both An. arabiensis and An. funestus was combined, substantial coverage of this supplementary intervention was predicted to lower EIR to below one. Conclusion Despite the significant impact of ER in combination with ITNs on one of the two vectors ( An. funestus or An. arabiensis ), this intervention is insufficient when combined with ITNs to reduce the EIR to below one in settings like Kilombero Valley where both species contribute to malaria transmission.

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.

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.