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

Outdoor Mosquito Control Using Odour-Baited Devices: Development and Evaluation of a Potential New Strategy to Complement Indoor Malaria Prevention Methods

A considerable effort is currently underway to develop a malaria vaccine based on live Plasmodium falciparum sporozoites. The first requisite of a sporozoite vaccine is the guarantee of parasite arrest prior to the onset of the pathogenic blood stage. Immunisation with genetically attenuated parasites (GAP) that arrest in the liver forms a promising approach. Work in this thesis describes the development and characterisation of a P. berghei Δb9Δslarp GAP that fully arrests in the liver. Immunisation of multiple mouse strains with low numbers of Δb9Δslarp GAP resulted in sterile protection. The Δb9Δslarp GAP is there- fore the leading GAP vaccine candidate. Work in this the- sis further describes the effect of varying the parameters of sporozoite inoculation on parasite liver load. These findings provide a rationale for the design of clinical trials aimed at the administration of live attenuated P. falciparum sporozoites

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

The Automatic Classification of Pyriproxyfen-Affected Mosquito Ovaries.

Pyriproxyfen (PPF) may become an alternative insecticide for areas where pyrethroid-resistant vectors are prevalent. The efficacy of PPF can be assessed through the dissection and assessment of vector ovaries. However, this reliance on expertise is subject to limitations. We show here that these limitations can be overcome using a convolutional neural network (CNN) to automate the classification of egg development and thus fertility status. Using TensorFlow, a resnet-50 CNN was pretrained with the ImageNet dataset. This CNN architecture was then retrained using a novel dataset of 524 dissected ovary images from An. gambiae s.l. An. gambiae Akron, and An. funestus s.l., whose fertility status and PPF exposure were known. Data augmentation increased the training set to 6973 images. A test set of 157 images was used to measure accuracy. This CNN model achieved an accuracy score of 94%, and application took a mean time of 38.5 s. Such a CNN can achieve an acceptable level of precision in a quick, robust format and can be distributed in a practical, accessible, and free manner. Furthermore, this approach is useful for measuring the efficacy and durability of PPF treated bednets, and it is applicable to any PPF-treated tool or similarly acting insecticide

Dramatic decreases of malaria transmission intensities in Ifakara, south-eastern Tanzania since early 2000s.

BACKGROUND: Ongoing epidemiological transitions across Africa are particularly evident in fast-growing towns, such as Ifakara in the Kilombero valley, south-eastern Tanzania. This town and its environs (population ~ 70,000) historically experienced moderate to high malaria transmission, mediated mostly by Anopheles gambiae and Anopheles funestus. In early 2000s, malaria transmission [Plasmodium falciparum entomological inoculation rate (PfEIR)] was estimated at ~ 30 infectious bites/person/year (ib/p/yr). This study assessed the PfEIR after 15 years, during which there had been rapid urbanization and expanded use of insecticide-treated nets (ITNs). METHODS: Randomly-selected 110 households were sampled across Ifakara town and four adjacent wards. Mosquitoes were trapped nightly or monthly (June.2015-May.2016) using CDC-light-traps indoors, Suna® traps outdoors and human landing catches (HLC) indoors and outdoors. All Anopheles mosquitoes were morphologically identified and analysed by ELISA for Plasmodium circumsporozoite proteins. Mosquito blood meals were identified using ELISA, and sub-samples of An. gambiae and An. funestus examined by PCR to distinguish morphologically-similar siblings. Insecticide resistance was assessed using WHO-susceptibility assays, and some Anopheles were dissected to examine ovariole tracheoles for parity. RESULTS: After 3572 trap-nights, one Plasmodium-infected Anopheles was found (an An. funestus caught outdoors in Katindiuka-ward by HLC), resulting in overall PfEIR of 0.102 ib/p/yr. Nearly 80% of malaria vectors were from Katindiuka and Mlabani wards. Anopheles gambiae densities were higher outdoors (64%) than indoors (36%), but no such difference was observed for An. funestus. All An. funestus and 75% of An. gambiae dissected were parous. Anopheles gambiae complex consisted entirely of Anopheles arabiensis, while An. funestus included 84.2% An. funestus s.s., 4.5% Anopheles rivulorum, 1.4% Anopheles leesoni and 9.9% with unamplified-DNA. Anopheles gambiae were susceptible to bendiocarb and malathion, but resistant to pyrethroids, DDT and pirimiphos-methyl. Most houses had brick walls and/or iron roofs (> 90%), and 52% had screened windows. CONCLUSION: Malaria transmission in Ifakara has decreased by > 99% since early-2000s, reaching levels nearly undetectable with current entomological methods. These declines are likely associated with ITNs use, urbanization and improved housing. Remaining risk is now mostly in peri-urban wards, but concerted efforts could further decrease local transmission. Parasitological surveys are required to assess actual prevalence, incidence and importation rates

Patterns of pesticide usage in agriculture in rural Tanzania call for integrating agricultural and public health practices in managing insecticide-resistance in malaria vectors.

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, where Anopheles mosquitoes 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

Sub-lethal aquatic doses of pyriproxyfen may increase pyrethroid resistance in malaria mosquitoes.

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

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