Entomological survey of sibling species in the Anopheles funestus group in Tanzania confirms the role of Anopheles parensis as a secondary malaria vector.

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.

Discovery of knock-down resistance in the major African malaria vector Anopheles funestus.

A major mechanism of insecticide resistance 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. While monitoring 10 populations of An. funestus in Tanzania, we unexpectedly found resistance to DDT, a banned insecticide, in one location. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found 8 novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (L1014F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to the exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). Further study is necessary to identify the origin and spread of kdr in An. funestus, and the potential threat to current insecticide-based vector control in Africa.

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

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

Advances in the genetic characterization of the malaria vector, Anopheles funestus, and implications for improved surveillance and control.

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.

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

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

Evaluation of different deployment strategies for larviciding to control malaria: a simulation study.

BACKGROUND: Larviciding against malaria vectors in Africa has been limited to indoor residual spraying and insecticide-treated nets, but is increasingly being considered by some countries as a complementary strategy. However, despite progress towards improved larvicides and new tools for mapping or treating mosquito-breeding sites, little is known about the optimal deployment strategies for larviciding in different transmission and seasonality settings. METHODS: A malaria transmission model, OpenMalaria, was used to simulate varying larviciding strategies and their impact on host-seeking mosquito densities, entomological inoculation rate (EIR) and malaria prevalence. Variations in coverage, duration, frequency, and timing of larviciding were simulated for three transmission intensities and four transmission seasonality profiles. Malaria transmission was assumed to follow rainfall with a lag of one month. Theoretical sub-Saharan African settings with Anopheles gambiae as the dominant vector were chosen to explore impact. Relative reduction compared to no larviciding was predicted for each indicator during the simulated larviciding period. RESULTS: Larviciding immediately reduced the predicted host-seeking mosquito densities and EIRs to a maximum that approached or exceeded the simulated coverage. Reduction in prevalence was delayed by approximately one month. The relative reduction in prevalence was up to four times higher at low than high transmission. Reducing larviciding frequency (i.e., from every 5 to 10 days) resulted in substantial loss in effectiveness (54, 45 and 53% loss of impact for host-seeking mosquito densities, EIR and prevalence, respectively). In seasonal settings the most effective timing of larviciding was during or at the beginning of the rainy season and least impactful during the dry season, assuming larviciding deployment for four months. CONCLUSION: The results highlight the critical role of deployment strategies on the impact of larviciding. Overall, larviciding would be more effective in settings with low and seasonal transmission, and at the beginning and during the peak densities of the target species populations. For maximum impact, implementers should consider the practical ranges of coverage, duration, frequency, and timing of larviciding in their respective contexts. More operational data and improved calibration would enable models to become a practical tool to support malaria control programmes in developing larviciding strategies that account for the diversity of contexts.

Addressing key gaps in implementation of mosquito larviciding to accelerate malaria vector control in southern Tanzania: results of a stakeholder engagement process in local district councils.

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.

Aquatic habitats of the malaria vector Anopheles funestus in rural south-eastern Tanzania.

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.