Impacts of dual active-ingredient bed nets on the behavioural responses of pyrethroid resistant Anopheles gambiae determined by room-scale infrared video tracking.

BACKGROUND: The success of insecticide treated bed nets (ITNs) for malaria vector control in Africa relies on the behaviour of various species of Anopheles. Previous research has described mosquito behavioural alterations resulting from widespread ITN coverage, which could result in a decrease in net efficacy. Here, behaviours were compared including timings of net contact, willingness to refeed and longevity post-exposure to two next-generation nets, PermaNet® 3.0 (P3 net) and Interceptor® G2 (IG2 net) in comparison with a standard pyrethroid-only net (Olyset Net™ (OL net)) and an untreated net. METHODS: Susceptible and resistant Anopheles gambiae mosquitoes were exposed to the nets with a human volunteer host in a room-scale assay. Mosquito movements were tracked for 2 h using an infrared video system, collecting flight trajectory, spatial position and net contact data. Post-assay, mosquitoes were monitored for a range of sublethal insecticide effects. RESULTS: Mosquito net contact was focused predominantly on the roof for all four bed nets. A steep decay in activity was observed for both susceptible strains when P3 net and OL net were present and with IG2 net for one of the two susceptible strains. Total mosquito activity was higher around untreated nets than ITNs. There was no difference in total activity, the number, or duration, of net contact, between any mosquito strain, with similar behaviours recorded in susceptible and resistant strains at all ITNs. OL net, P3 net and IG2 net all killed over 90% of susceptible mosquitoes 24 h after exposure, but this effect was not seen with resistant mosquitoes where mortality ranged from 16 to 72%. All treated nets reduced the willingness of resistant strains to re-feed when offered blood 1-h post-exposure, with a more pronounced effect seen with P3 net and OL net than IG2 net. CONCLUSION: These are the first results to provide an in-depth description of the behaviour of susceptible and resistant Anopheles gambiae strains around next-generation bed nets using a room-scale tracking system to capture multiple behaviours. These results indicate that there is no major difference in behavioural responses between mosquito strains of differing pyrethroid susceptibility when exposed to these new ITNs under the experimental conditions used.

Quantifying late-stage host-seeking behaviour of Anopheles gambiae at the insecticidal net interface using a baited-box bioassay.

BACKGROUND: Insecticide-treated nets (ITNs) are losing efficacy against pyrethroid-resistant malaria vector populations throughout Africa. Safeguarding bed net efficacy, vital for effective malaria control, requires greater knowledge of mosquito-ITN interactions and how this impacts on the mosquito. METHODS: A purpose-built benchtop apparatus with a closed 10 cm cubic chamber (the 'Baited-box') was used to video record behaviour of individual free-flying female Anopheles gambiae during approach and blood-feeding on a human hand through untreated nets and ITNs at close range. Time and duration of defined behavioural events, and knockdown and mortality at 1- and 24-h post-exposure respectively, were recorded for pyrethroid susceptible and resistant mosquitoes. RESULTS: Using three human volunteers differing in relative attractiveness to mosquitoes, 328 mosquitoes were individually tested. There were no significant differences between response rates to ITNs and untreated nets (P > 0.1) or between resistant (Tiassalé) and susceptible (Kisumu) mosquito strains, at untreated nets (P = 0.39) or PermaNet 2.0 (P = 1). The sequence of behavioural events from host-seeking to completion of blood-feeding was consistent in all tests but duration and start time of events involving net contact were reduced or delayed respectively with ITNs. Blood-feeding durations at untreated nets (means from 4.25 to 8.47 min (95% confidence interval (CI) = 3.39-9.89) at 3 human volunteers) were reduced by 37-50% at PermaNet 2.0, in susceptible (mean 2.59-4.72 min, 95% CI = 1.54-5.5, P = < 0.05) and resistant (mean 4.20 min, 95% CI = 3.42-4.97, P = 0.01) strains. Total accumulated net contact was approximately 50% lower at PermaNet and Olyset ITNs (P < 0.0001) in susceptible (two of the three volunteers) and resistant mosquitoes. Times prior to first net contact were similar at untreated nets and ITNs (P > 0.2), and neither ITN type showed detectable spatial repellency. After initial contact, blood-feeding commenced later at Olyset (mean 2.76 min, 95% CI = 1.74-3.76, P = 0.0009) and PermaNet (mean 2.4 min, 95% CI = 1.52-3.33, P = 0.0058) than untreated netting (mean 0.68 min, 95% CI = 0.42-0.94). CONCLUSIONS: The baited box offers a simple method for detailed characterization of mosquito behavioural responses to insecticidal nets, for comparing entomological modes of action between nets and for defining the behavioural responses of particular mosquito strains or populations. The device has potential as a screening assay in the search for novel net treatments and for investigations into behavioural resistance mechanisms.

Improving the efficiency of aerosolized insecticide testing against mosquitoes.

Developing robust and standardised approaches for testing mosquito populations against insecticides is vital for understanding the effectiveness of new active ingredients or formulations. Methods for testing mosquito susceptibility against contact insecticides or products, such as those delivered through public health programmes, are well-established and standardised. Nevertheless, approaches for testing volatile or aerosolized insecticides used in household products can be challenging to implement efficiently. We adapted WHO guidelines for household insecticides to develop a standardised and higher-throughput methodology for testing aerosolized products in a Peet Grady test chamber (PG-chamber) using caged mosquitoes and an efficient decontamination method. The new approach was validated using insecticide resistant and susceptible Aedes and Anopheles mosquito colonies. An added feature is the inclusion of cage-facing cameras to allow real-time quantification of knockdown following insecticide exposure. The wipe-based decontamination method was highly effective for removing pyrethroids' aerosolized oil-based residues from chamber surfaces, with < 2% mortality recorded for susceptible mosquitoes tested directly on the surfaces. There was no spatial heterogeneity for knockdown or mortality of caged mosquitoes within the PG chamber. The dual-cage approach we implement yields eight-times the throughput compared to a free-flight protocol, allows simultaneous testing of different mosquito strains and effectively discriminates susceptible and resistant mosquito colonies tested side-by-side.

Diffuse retro-reflective imaging for improved video tracking of mosquitoes at human baited bednets.

Robust imaging techniques for tracking insects have been essential tools in numerous laboratory and field studies on pests, beneficial insects and model systems. Recent innovations in optical imaging systems and associated signal processing have enabled detailed characterization of nocturnal mosquito behaviour around bednets and improvements in bednet design, a global essential for protecting populations against malaria. Nonetheless, there remain challenges around ease of use for large-scale in situ recordings and extracting data reliably in the critical areas of the bednet where the optical signal is attenuated. Here, we introduce a retro-reflective screen at the back of the measurement volume, which can simultaneously provide diffuse illumination, and remove optical alignment issues while requiring only one-sided access to the measurement space. The illumination becomes significantly more uniform, although noise removal algorithms are needed to reduce the effects of shot noise, particularly across low-intensity bednet regions. By systematically introducing mosquitoes in front of and behind the bednet in laboratory experiments, we are able to demonstrate robust tracking in these challenging areas. Overall, the retro-reflective imaging set-up delivers mosquito segmentation rates in excess of 90% compared to less than 70% with backlit systems.

A highly stable blood meal alternative for rearing Aedes and Anopheles mosquitoes.

We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts.