Interventions that effectively target Anopheles funestus mosquitoes could significantly improve control of persistent malaria transmission in south–eastern Tanzania

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

Effects of a new outdoor mosquito control device, the mosquito landing box, on densities and survival of the malaria vector, Anopheles arabiensis, inside controlled semi-field settings

Background The significance of malaria transmission occurring outdoors has risen even in areas where indoor interventions such as long-lasting insecticidal nets and indoor residual spraying are common. The actual contamination rates and effectiveness of recently developed outdoor mosquito control device, the mosquito landing box (MLB), on densities and daily survival of host-seeking laboratory Anopheles arabiensis, which readily bites humans outdoors was demonstrated. Methods Experiments were conducted in large semi-field systems (SFS) with human volunteers inside, to mimic natural ecosystems, and using MLBs baited with natural or synthetic human odours and carbon dioxide. The MLBs were dusted with 10 % pyriproxyfen (PPF) or entomopathogenic fungi (Metarhizium anisopliae) spores to mark mosquitoes physically contacting the devices. Each night, 400 laboratory-reared An. arabiensis females were released in one SFS chamber with two MLBs, and another chamber without MLBs (control). Mosquitoes were individually recaptured while attempting to bite volunteers inside SFS or by aspiration from SFS walls. Mosquitoes from chambers with PPF-treated MLBs and respective controls were individually dipped in water-filled cups containing ten conspecific third-instar larvae, whose subsequent development was monitored. Mosquitoes recaptured from chambers with fungi-treated MLBs were observed for fungal hyphal growth on their cadavers. Separately, effects on daily survival were determined by exposing An. arabiensis in chambers having MLBs treated with 5 % pirimiphos methyl compared to chambers without MLBs (control), after which the mosquitoes were recaptured and monitored individually until they died. Results Up to 63 % (152/240) and 43 % (92/210) of mosquitoes recaptured inside treatment chambers were contaminated with pyriproxyfen and M. anisopliae, respectively, compared to 8 % (19/240) and 0 % (0/164) in controls. The mean number of larvae emerging from cups in which adults from chambers with PPF-treated MLBs were dipped was significantly lower [0.75 (0.50–1.01)], than in controls [28.79 (28.32–29.26)], P < 0.001). Daily survival of mosquitoes exposed to 5 % pirimiphos methyl was nearly two-fold lower than controls [hazard ratio (HR) = 1.748 (1.551–1.920), P < 0.001]. Conclusion High contamination rates in exposed mosquitoes even in presence of humans, demonstrates potential of MLBs for controlling outdoor-biting malaria vectors, either by reducing their survival or directly killing host-seeking mosquitoes. The MLBs also have potential for dispensing filial infanticides, such as PPF, which mosquitoes can transmit to their aquatic habitats for mosquito population control. Keywords: Mosquito landing box; Malaria; Elimination; Anopheles arabiensis ; Pirimiphos methyl; Outdoor biting; Pyriproxyfen; Metarhizium anisopliae ; Semi-field syste

Evaluation of an ultraviolet LED trap for catching Anopheles and Culex mosquitoes in south-eastern Tanzania

Background: Improved surveillance techniques are required to accelerate efforts against major arthropod-borne diseases such as malaria, dengue, filariasis, Zika and yellow-fever. Light-emitting diodes (LEDs) are increasingly used in mosquito traps because they improve energy efficiency and battery longevity relative to incandescent bulbs. This study evaluated the efficacy of a new ultraviolet LED trap (Mosclean) against standard mosquito collection methods. Methods: The study was conducted in controlled semi-field settings and in field conditions in rural south-eastern Tanzania. The Mosclean trap was compared to commonly used techniques, namely CDC-light traps, human landing catches (HLCs), BG-Sentinel traps and Suna traps. Results: When simultaneously placed inside the same semi-field chamber, the Mosclean trap caught twice as many Anopheles arabiensis as the CDC-light trap, and equal numbers to HLCs. Similar results were obtained when traps were tested individually in the chambers. Under field settings, Mosclean traps caught equal numbers of An. arabiensis and twice as many Culex mosquitoes as CDC-light traps. It was also better at trapping malaria vectors compared to both Suna and BG-Sentinel traps, and was more efficient in collecting mosquitoes indoors than outdoors. The majority of An. arabiensis females caught by Mosclean traps were parous (63.6%) and inseminated (89.8%). In comparison, the females caught by CDC-light traps were 43.9% parous and 92.8% inseminated. Conclusions: The UV LED trap (Mosclean trap) was efficacious for sampling Anopheles and Culex mosquitoes. Its efficacy was comparable to and in some instances better than traps commonly used for vector surveillance. The Mosclean trap was more productive in sampling mosquitoes indoors compared to outdoors. The trap can be used indoors near human-occupied nets, or outdoors, in which case additional CO2 improves catches. We conclude that this trap may have potential for mosquito surveillance. However, we recommend additional field tests to validate these findings in multiple settings and to assess the potential of LEDs to attract non-target organisms, especially outdoors

Observing the distribution of mosquito bites on humans to inform personal protection measures against malaria and dengue vectors

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

Protecting migratory farmers in rural Tanzania using eave ribbons treated with the spatial mosquito repellent, transfluthrin.

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 CO2-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

Fine-scale distribution of malaria mosquitoes biting or resting outside human dwellings in three low-altitude Tanzanian villages.

BACKGROUND: While malaria transmission in Africa still happens primarily inside houses, there is a substantial proportion of Anopheles mosquitoes that bite or rest outdoors. This situation may compromise the performance of indoor insecticidal interventions such as insecticide-treated nets (ITNs). This study investigated the distribution of malaria mosquitoes biting or resting outside dwellings in three low-altitude villages in south-eastern Tanzania. The likelihood of malaria infections outdoors was also assessed. METHODS: Nightly trapping was done outdoors for 12 months to collect resting mosquitoes (using resting bucket traps) and host-seeking mosquitoes (using odour-baited Suna® traps). The mosquitoes were sorted by species and physiological states. Pooled samples of Anopheles were tested to estimate proportions infected with Plasmodium falciparum parasites, estimate proportions carrying human blood as opposed to other vertebrate blood and identify sibling species in the Anopheles gambiae complex and An. funestus group. Environmental and anthropogenic factors were observed and recorded within 100 meters from each trapping positions. Generalised additive models were used to investigate relationships between these variables and vector densities, produce predictive maps of expected abundance and compare outcomes within and between villages. RESULTS: A high degree of fine-scale heterogeneity in Anopheles densities was observed between and within villages. Water bodies covered with vegetation were associated with 22% higher densities of An. arabiensis and 51% lower densities of An. funestus. Increasing densities of houses and people outdoors were both associated with reduced densities of An. arabiensis and An. funestus. Vector densities were highest around the end of the rainy season and beginning of the dry seasons. More than half (14) 58.3% of blood-fed An. arabiensis had bovine blood, (6) 25% had human blood. None of the Anopheles mosquitoes caught outdoors was found infected with malaria parasites. CONCLUSION: Outdoor densities of both host-seeking and resting Anopheles mosquitoes had significant heterogeneities between and within villages, and were influenced by multiple environmental and anthropogenic factors. Despite the high Anopheles densities outside dwellings, the substantial proportion of non-human blood-meals and absence of malaria-infected mosquitoes after 12 months of nightly trapping suggests very low-levels of outdoor malaria transmission in these villages

Eave ribbons treated with transfluthrin can protect both users and non-users against malaria vectors

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.; 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.; 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.; 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

Evaluation of a push–pull system consisting of transfluthrin-treated eave ribbons and odour-baited traps for control of indoor- and outdoor-biting malaria vectors

Background: Push–pull strategies have been proposed as options to complement primary malaria prevention tools, indoor residual spraying (IRS) and long-lasting insecticide-treated nets (LLINs), by targeting particularly early-night biting and outdoor-biting mosquitoes. This study evaluated different configurations of a push–pull system consisting of spatial repellents [transfluthrin-treated eave ribbons (0.25 g/m2 ai)] and odour-baited traps (CO2-baited BG-Malaria traps), against indoor-biting and outdoor-biting malaria vectors inside large semi-field systems. Methods: Two experimental huts were used to evaluate protective efficacy of the spatial repellents (push-only), traps (pull-only) or their combinations (push–pull), relative to controls. Adult volunteers sat outdoors (1830 h–2200 h) catching mosquitoes attempting to bite them (outdoor-biting risk), and then went indoors (2200 h–0630 h) to sleep under bed nets beside which CDC-light traps caught host-seeking mosquitoes (indoor-biting risk). Number of traps and their distance from huts were varied to optimize protection, and 500 laboratory-reared Anopheles arabiensis released nightly inside the semi-field chambers over 122 experimentation nights. Results: Push-pull offered higher protection than traps alone against indoor-biting (83.4% vs. 35.0%) and outdoor-biting (79% vs. 31%), but its advantage over repellents alone was non-existent against indoor-biting (83.4% vs. 81%) and modest for outdoor-biting (79% vs. 63%). Using two traps (1 per hut) offered higher protection than either one trap (0.5 per hut) or four traps (2 per hut). Compared to original distance (5 m from huts), efficacy of push–pull against indoor-biting peaked when traps were 15 m away, while efficacy against outdoor-biting peaked when traps were 30 m away. Conclusion: The best configuration of push–pull comprised transfluthrin-treated eave ribbons plus two traps, each at least 15 m from huts. Efficacy of push–pull was mainly due to the spatial repellent component. Adding odour-baited traps slightly improved personal protection indoors, but excessive trap densities increased exposure near users outdoors. Given the marginal efficacy gains over spatial repellents alone and complexity of push–pull, it may be prudent to promote just spatial repellents alongside existing interventions, e.g. LLINs or non-pyrethroid IRS. However, since both transfluthrin and traps also kill mosquitoes, and because transfluthrin can inhibit blood-feeding, field studies should be done to assess potential community-level benefits that push–pull or its components may offer to users and non-users

Semi-field evaluation of electrocuting eave tubes for the control of endophagic mosquitoes in south-east Tanzania

Background: Eave spaces are major entry points through which malaria vectors enter houses. Interventions that target mosquitoes at the eaves have recently been developed. However, most of these interventions are based on insecticides for which resistance has been reported. Here we evaluated the efficacy of mosquito electrocuting eave tubes (MEETs) against Anopheles gambiae sensu stricto (An. gambiae s.s.) and Anopheles funestus s.s. under semi-field conditions. Methods: Experiments were conducted in two semi-field chambers, each containing one experimental hut. Six electrocuting eave tubes were installed in each hut to assess their impact on laboratory-reared An. gambiae s.s. and An. funestus s.s.. Each species was assessed separately over 10 nights by releasing 200 unfed females per night into each chamber. One volunteer slept in each hut from 7 p.m. to 5 a.m. Mosquitoes were collected indoors and outdoors using mouth and Prokopack aspirators. Results: The placement of MEETs significantly reduced the nightly An. gambiae s.s. indoor and outdoor biting, by 21.1% and 37.4%, respectively. Indoor-biting An. funestus s.s. were reduced by 87.5% while outdoor-biting numbers of An. funestus s.s. declined by 10.4%. Conclusions: MEETs represent a promising tool for controlling mosquitoes at the point of house entry. Further validation of their potential under natural field conditions is necessary. Several advantages over insecticide-based eave tubes are indicated and discussed in this article. Graphical Abstract

Small-scale field evaluation of transfluthrin-treated eave ribbons and sandals for the control of malaria vectors in rural Tanzania

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 &amp; 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 &amp; 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