Exploitation of adult Anopheles arabiensis behaviour and ecology for the dissemination of pyriproxyfen, a novel technique for malaria vector control in Tanzania

Effective larviciding to manage mosquito aquatic habitats offers an additional strategy for malaria vector control by complementing benefits already achieved by long lasting insecticidetreated nets (LLINs) and indoor residual spraying (IRS). Sustainable implementation of larviciding requires comprehensive understanding of the ecology of disease vectors and robust monitoring of factors governing local disease transmission. Treatment of aquatic habitats with the juvenile hormone analogue Pyriproxyfen (PPF), inhibits adult mosquito emergence at extremely low concentrations that are potentially deliverable by PPF-contaminated gravid adult females, a phenomenon termed „autodissemination‟. The primary aim of this thesis was to investigate a range of adult mosquito behaviours that might be exploited to disseminate PPF. The effectiveness of PPF to sterilize adult mosquitoes for malaria vector control was also assessed in a controlled system. Vector dynamics, malaria transmission intensity and risk factors were evaluated at the field site where the PPF autodissemination strategy would be evaluated in field trials and potentially implemented. Field monitoring of indoor malaria transmission risk factors revealed that even in the communities with high coverage of bednets, LLINs did not reduce the indoor densities of An. gambiae s.l (RR= 0.74 (0.50 - 1.11, p > 0.05) but reduced An. funestus indoor densities by 56% (RR= 0.44 (0.23 - 0.87, p < 0.05)). Houses with eave gaps had 3.3 and 5.5 times more An. gambiae s.l. (RR= 3.3 (2.39 - 4.56, p < 0.05)) and An. funestus ((RR = 5.55 (3.25 - 9.46, p < 0.05)) respectively. Intact screening over windows reduced up to 66% (RR = 0.34 (0.17 - 0.69)) and 83% (RR = 0.17 (0.08 - 0.39)) indoor entry of An. gambiae s.l. and An. funestus respectively. Furthermore, surveillance of wild malaria vectors populations and susceptibility to insecticide resistance demonstrated significant increase in An. funestus densities in 2012 (RR=1.56 (1.33-1.69)) compared to An.gambiae s.l. (p <0.0001). In 2014, the proportion of An. gambiae s.l. catches (67%; 4373/6373) was higher than An. funestus (33%; 2100/6373). PCR results revealed change in relative proportion between the two sibling species of An.gambaie s.l. with a significant decrease in An. gambiae s.s. from approximately 14% (414/2,924) in 2008 to 0% (0/435) in 2014. Insecticide susceptibility tests indicated high resistance in An. funestus against deltamethrin (mortality rate in discriminating dose assay = 87%), lambda cyhalothrin (74%), permethrin (65%), bendiocarb (65%), and DDT (66%). Similarly, An. arabiensis showed insecticide resistance to permethrin (77%), deltamethrin (64%) and lambda cyhalothrin (42%) in 2014. In large screened cages it was demonstrated that adult An. arabiensis can disseminate PPF from clay pots treated with PPF to the aquatic habitats, resulting in 76.5% reduction in adult emergence, with higher mean proportion of adult emerging from untreated chamber, 0.95 (0.56 -1.34) compared to the treated chamber, 0.21 (0.09 - 0.51, p 95% (89.3 - 102.9%) reduction in adult An. arabiensis production. This research provides evidence on the need of better housing and larviciding to complement LLINs in controlling the remaining malaria transmission transmitted by An. funestus and An. arabiensis. It also demonstrated for the first time that the PPF autodissemination strategy and sterilization of adult females present a promising malaria vector control option for field trial. PPF-autodissemination can be integrated into a vector management toolbox to control outdoor malaria transmission and also target multiple disease-carrying mosquitoes that share aquatic habitats with malaria vectors. These findings highlight the potential of PPF for controlling outdoor and indoor malaria vectors and call for further testing in the field

A Geographical Location Model for Targeted Implementation of Lure-and-Kill Strategies Against Disease-Transmitting Mosquitoes in Rural Areas

Outdoor devices for luring and killing disease-transmitting mosquitoes have been proposed as potential com- plementary interventions alongside existing intra-domiciliary methods namely insecticide treated nets and house spraying with residual insecticides. To enhance effectiveness of such outdoor interventions, it is essential to optimally locate them in such a way that they target most of the outdoor mosquitoes. Using odour-baited lure and kill stations (OBS) as an example, we describe a map model derived from: 1) com-munity participatory mapping conducted to identify mosquito breeding habitats, 2) entomological field studies conducted to estimate outdoor mosquito densities and to determine safe distances of the OBS from human dwellings, and 3) field surveys conducted to map households, roads, outdoor human aggregations and landmarks. The resulting data were combined in a Ge- ographical Information Systems (GIS) environment and analysed to determine optimal locations for the OBS. Separately, a GIS-interpolated map produced by asking community members to rank different zones of the study area and show where they expected to find most mosquitoes, was visually compared to another map interpolated from the entomological survey of outdoor mosquito densities. An easy-to-interpret suitability map showing optimal sites for placing OBS was produced, which clearly depicted areas least suitable and areas most suitable for locating the devices. Comparative visual interpretation of maps derived from interpolating the community knowledge and entomological data revealed major similarities between the two maps. Using distribution patterns of human and mosquito populations as well as characteristics of candidate outdoor interventions, it is possible to readily determine suitable areas for targeted positioning of the interventions, thus improve effectiveness. This study also highlights possibilities of relying on community knowledge to approximate areas where mosquitoes are most abundant and where to locate outdoor complementary interventions such as odour-baited lure and kill stations for controlling disease-transmitting mosquitoes.\u

A Simple and Efficient Tool for Trapping Gravid Anopheles at Breeding Sites.

No effective tool currently exists for trapping ovipositing malaria vectors. This creates a gap in our ability to investigate the behavior and ecology of gravid Anopheles.\ud Here we describe a simple trap that collects ovipositing Anopheline and Culicine mosquitoes. It consists of an acetate sheet coated in glue that floats on the water surface. Ten breeding sites were selected in rural Tanzania and 10 sticky traps set in each. These caught a total of 74 gravid Anopheles (54 An. arabiensis, 1 An. gambiae s.s. and 16 unamplified) and 1333 gravid Culicines, in just two trap nights. This simple sampling tool provides an opportunity to further our understanding of the behavior and ecology of gravid female Anophelines. It strongly implies that at least two of the major vectors of malaria in Africa land on the water surface during the oviposition process, and demonstrates that Anophelines and Culicines often share the same breeding sites. This simple and efficient trap has clear potential for the study of oviposition site choice and productivity, gravid dispersal, and vector control techniques which use oviposition behavior as a means of disseminating larvicides

First report of Metarhizium anisopliae IP 46 pathogenicity in adult Anopheles gambiae s.s. and An. arabiensis (Diptera; Culicidae).

The entomopathogenic fungus Metarhizium anisopliae isolate IP 46, originating from a soil sample collected in 2001 in the Cerrado of Central Brazil, was tested for its ability to reduce the survival of adult male and female Anopheles gambiae s.s. and An. arabiensis mosquitoes. A 6-h exposure to the fungus coated on test paper at a concentration of 3.3 x 106 conidia cm-2 reduced the daily survival of both mosquito species (HR = 3.14, p < 0.001), with higher risk of dying in An. gambiae s.s relative to An. arabiensis (HR = 1.38, p < 0.001). Fungal sporulation was observed in >95% of mosquito cadavers in the treatment groups. The results indicate that M. anisopliae IP 46 has the potential to be a bio-control agent for African malaria vector species, and is a suitable candidate for further research and development

Most outdoor malaria transmission by behaviourally-resistant Anopheles arabiensis is mediated by mosquitoes that have previously been inside houses

Background Anopheles arabiensis is stereotypical of diverse vectors that mediate residual malaria transmission globally, because it can feed outdoors upon humans or cattle, or enter but then rapidly exit houses without fatal exposure to insecticidal nets or sprays. Methods Life histories of a well-characterized An. arabiensis population were simulated with a simple but process-explicit deterministic model and relevance to other vectors examined through sensitivity analysis. Results Where most humans use bed nets, two thirds of An. arabiensis blood feeds and half of malaria transmission events were estimated to occur outdoors. However, it was also estimated that most successful feeds and almost all (>98 %) transmission events are preceded by unsuccessful attempts to attack humans indoors. The estimated proportion of vector blood meals ultimately obtained from humans indoors is dramatically attenuated by availability of alternative hosts, or partial ability to attack humans outdoors. However, the estimated proportion of mosquitoes old enough to transmit malaria, and which have previously entered a house at least once, is far less sensitive to both variables. For vectors with similarly modest preference for cattle over humans and similar ability to evade fatal indoor insecticide exposure once indoors, >80 % of predicted feeding events by mosquitoes old enough to transmit malaria are preceded by at least one house entry event, so long as ≥40 % of attempts to attack humans occur indoors and humans outnumber cattle ≥4-fold. Conclusions While the exact numerical results predicted by such a simple deterministic model should be considered only approximate and illustrative, the derived conclusions are remarkably insensitive to substantive deviations from the input parameter values measured for this particular An. arabiensis population. This life-history analysis, therefore, identifies a clear, broadly-important opportunity for more effective suppression of residual malaria transmission by An. arabiensis in Africa and other important vectors of residual transmission across the tropics. Improved control of predominantly outdoor residual transmission by An. arabiensis, and other modestly zoophagic vectors like Anopheles darlingi, which frequently enter but then rapidly exit from houses, may be readily achieved by improving existing technology for killing mosquitoes indoors

Predicting Scenarios for Successful Autodissemination of Pyriproxyfen by Malaria Vectors from Their Resting Sites to Aquatic Habitats; Description and Simulation Analysis of a Field-Parameterizable Model

Background Large-cage experiments indicate pyriproxifen (PPF) can be transferred from resting sites to aquatic habitats by Anopheles arabiensis - malaria vector mosquitoes to inhibit emergence of their own offspring. PPF coverage is amplified twice: (1) partial coverage of resting sites with PPF contamination results in far higher contamination coverage of adult mosquitoes because they are mobile and use numerous resting sites per gonotrophic cycle, and (2) even greater contamination coverage of aquatic habitats results from accumulation of PPF from multiple oviposition events. Methods and Findings Deterministic mathematical models are described that use only field-measurable input parameters and capture the biological processes that mediate PPF autodissemination. Recent successes in large cages can be rationalized, and the plausibility of success under full field conditions can be evaluated a priori. The model also defines measurable properties of PPF delivery prototypes that may be optimized under controlled experimental conditions to maximize chances of success in full field trials. The most obvious flaw in this model is the endogenous relationship that inevitably occurs between the larval habitat coverage and the measured rate of oviposition into those habitats if the target mosquito species is used to mediate PPF transfer. However, this inconsistency also illustrates the potential advantages of using a different, non-target mosquito species for contamination at selected resting sites that shares the same aquatic habitats as the primary target. For autodissemination interventions to eliminate malaria transmission or vector populations during the dry season window of opportunity will require comprehensive contamination of the most challenging subset of aquatic habitats that persist or retain PPF activity (Ux) for only one week , where Ux = 7 days). To achieve >99% contamination coverage of these habitats will necessitate values for the product of the proportional coverage of the ovipositing mosquito population with PPF contamination (CM) by the ovitrap-detectable rates of oviposition by wild mosquitoes into this subset of habitats , divided by the titre of contaminated mosquitoes required to render them unproductive , that approximately approach unity . Conclusions The simple multiplicative relationship between CM and , and the simple exponential decay effect they have upon uncontaminated aquatic habitats, allows application of this model by theoreticians and field biologists alike

Effective autodissemination of pyriproxyfen to breeding sites by the exophilic malaria vector Anopheles arabiensis in semi-field settings in Tanzania

BACKGROUND Malaria vector control strategies that target adult female mosquitoes are challenged by the emergence of insecticide resistance and behavioural resilience. Conventional larviciding is restricted by high operational costs and inadequate knowledge of mosquito-breeding habitats in rural settings that might be overcome by the juvenile hormone analogue, Pyriproxyfen (PPF). This study assessed the potential for Anopheles arabiensis to pick up and transfer lethal doses of PPF from contamination sites to their breeding habitats (i.e. autodissemination of PPF). METHODS A semi-field system (SFS) with four identical separate chambers was used to evaluate PPF-treated clay pots for delivering PPF to resting adult female mosquitoes for subsequent autodissemination to artificial breeding habitats within the chambers. In each chamber, a tethered cow provided blood meals to laboratory-reared, unfed female An. arabiensis released in the SFS. In PPF-treated chambers, clay pot linings were dusted with 0.2 - 0.3 g AI PPF per pot. Pupae were removed from the artificial habitats daily, and emergence rates calculated. Impact of PPF on emergence was determined by comparing treatment with an appropriate control group. RESULTS Mean (95%CI) adult emergence rates were (0.21 +/- 0.299) and (0.95 +/- 0.39) from PPF-treated and controls respectively (p < 0.0001). Laboratory bioassay of water samples from artificial habitats in these experiments resulted in significantly lower emergence rates in treated chambers (0.16 +/- 0.23) compared to controls 0.97 +/- 0.05) (p < 0.0001). In experiments where no mosquitoes introduced, there were no significant differences between control and treatment, indicating that transfer of PPF to breeding sites only occurred when mosquitoes were present; i.e. that autodissemination had occurred. Treatment of a single clay pot reduced adult emergence in six habitats to (0.34 +/- 0.13) compared to (0.98 +/- 0.02) in the controls (p < 0.0001), showing a high level of habitats coverage amplification of the autodissemination event. CONCLUSION The study provides proof of principle for the autodissemination of PPF to breeding habitats by malaria vectors. These findings highlight the potential for this technique for outdoor control of malaria vectors and call for the testing of this technique in field trials

Attracting, trapping and killing disease-transmitting mosquitoes using odor-baited stations - The Ifakara Odor-Baited Stations

BACKGROUND: To accelerate efforts towards control and possibly elimination of mosquito-borne diseases such as malaria and lymphatic filariasis, optimally located outdoor interventions could be used to complement existing intradomicilliary vector control methods such as house spraying with insecticides and insecticidal bednets. METHODS: We describe a new odor-baited station for trapping, contaminating and killing disease-transmitting mosquitoes. This device, named the 'Ifakara Odor-baited Station' (Ifakara OBS), is a 4 m3 hut-shaped canvas box with seven openings, two of which may be fitted with interception traps to catch exiting mosquitoes. It is baited with synthetic human odors and may be augmented with contaminants including toxic insecticides or biological agents. RESULTS: In field trials where panels of fabric were soaked in 1% pirimiphos-methyl solution and suspended inside the Ifakara OBS, at least 73.6% of Anopheles arabiensis, 78.7% of Culex and 60% of Mansonia mosquitoes sampled while exiting the OBS, died within 24 hours. When used simply as a trap and evaluated against two existing outdoor traps, Ifakara Tent trap and Mosquito Magnet-X(R), the OBS proved more efficacious than the Ifakara Tent trap in catching all mosquito species found (P < 0.001). Compared to the Mosquito Magnet-X(R), it was equally efficacious in catching An. arabiensis (P = 0.969), but was less efficacious against Culex (P < 0.001) or Mansonia species (P < 0.001). CONCLUSION: The Ifakara OBS is efficacious against disease-carrying mosquitoes including the malaria vector, An. arabiensis and Culicine vectors of filarial worms and arboviruses. It can be used simultaneously as a trap and as a contamination or killing station, meaning most mosquitoes which escape trapping would leave when already contaminated and die shortly afterwards. This technique has potential to complement current vector control methods, by targeting mosquitoes in places other than human dwellings, but its effectiveness in the field will require cheap, long-lasting and easy-to-use mosquito lures

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