Infrared video tracking of Anopheles gambiae at insecticide-treated bed nets reveals rapid decisive impact after brief localised net contact

Long-lasting insecticidal bed nets (LLINs) protect humans from malaria transmission and are fundamental to malaria control worldwide, but little is known of how mosquitoes interact with nets. Elucidating LLIN mode of action is essential to maintain or improve efficacy, an urgent need as emerging insecticide resistance threatens their future. Tracking multiple free-flying Anopheles gambiae responding to human-occupied bed nets in a novel large-scale system, we characterised key behaviours and events. Four behavioural modes with different levels of net contact were defined: swooping, visiting, bouncing and resting. Approximately 75% of all activity occurred at the bed net roof where multiple brief contacts were focussed above the occupant’s torso. Total flight and net contact times were lower at LLINs than untreated nets but the essential character of the response was unaltered. LLINs did not repel mosquitoes but impacted rapidly: LLIN contact of less than 1 minute per mosquito during the first ten minutes reduced subsequent activity; after thirty minutes, activity at LLINs was negligible. Velocity measurements showed that mosquitoes detected nets, including unbaited untreated nets, prior to contact. This is the most complete characterisation of mosquito-LLIN interactions to date, and reveals many aspects of LLIN mode of action, important for developing the next generation of LLINs

Finding a Husband: Using Explainable AI to Define Male Mosquito Flight Differences

Mosquito-borne diseases account for around one million deaths annually. There is a constant need for novel intervention mechanisms to mitigate transmission, especially as current insecticidal methods become less effective with the rise of insecticide resistance among mosquito populations. Previously, we used a near infra-red tracking system to describe the behaviour of mosquitoes at a human-occupied bed net, work that eventually led to an entirely novel bed net design. Advancing that approach, here we report on the use of trajectory analysis of a mosquito flight, using machine learning methods. This largely unexplored application has significant potential for providing useful insights into the behaviour of mosquitoes and other insects. In this work, a novel methodology applies anomaly detection to distinguish male mosquito tracks from females and couples. The proposed pipeline uses new feature engineering techniques and splits each track into segments such that detailed flight behaviour differences influence the classifier rather than the experimental constraints such as the field of view of the tracking system. Each segment is individually classified and the outcomes are combined to classify whole tracks. By interpreting the model using SHAP values, the features of flight that contribute to the differences between sexes are found and are explained by expert opinion. This methodology was tested using 3D tracks generated from mosquito mating swarms in the field and obtained a balanced accuracy of 64.5% and an ROC AUC score of 68.4%. Such a system can be used in a wide variety of trajectory domains to detect and analyse the behaviours of different classes, e.g., sex, strain, and species. The results of this study can support genetic mosquito control interventions for which mating represents a key event for their success

Keeping track of the enemy : Flight analyses of the host-seeking malaria mosquito Anopheles gambiae s.s

Female mosquitoes can transmit pathogens to their host during blood feeding and are an important vector of human diseases such as dengue, chikungunya, filariasis and malaria. After 15 years of decline in the number of fatal malaria cases, this decline came to a halt in 2016. Growing resistance against drugs and insecticides pose a serious threat for future human health. This thesis focuses on the behaviour of host-seeking malaria mosquitoes by analysing their flight paths during their approach to different host cues. Fundamental knowledge on the role of selected host cues was acquired. In addition, studies to support successful implementation of vector control interventions were performed in both wind-tunnel settings and the semi-field in Kenya. My research demonstrates that automated tracking systems can strengthen behavioural-ecological studies on disease vectors, in addition to conventional bio-assays such as olfactometers, by providing detailed information on the approach behaviour of mosquitoes to different targets. The attraction towards the host-sensory cue CO2 was investigated in an olfactometer bioassay. Trap catches of female Anopheles gambiae s.s. were enhanced by separation of the CO2 source from the source of human skin emanations. Close-range deterrent effects of CO2 were overcome by the simultaneous presence of skin emanations. Flight path analysis of mosquitoes in a wind tunnel, showed that exposure to human odour resulted in prolonged and highly convoluted flight tracks. The combination of odour with heat was crucial to induce landings of host-seeking mosquitoes. A semi-field study in Kenya revealed that house-entering mosquitoes approached the eave of a house in a wide angle to the house at eave level, where the proportion that entered uninterruptedly (23%) spent just a few seconds around the eave area. The presence of insecticide-treated nets inside a house did not repel mosquitoes as measured by the number of house entries. At close range, in a wind tunnel, free-flight exposure of mosquitoes to deltamethrin-treated nets in combination with human odour did not reveal any (excito-) repellent effect and resulted in lower mortality rates compared to standard bioassays where contact with the treated material is enforced. The knowledge obtained on the behavioural responses of mosquitoes to host cues has indirectly affected vector control tool implementations in the field. For example, in the development of an odour-baited trap, a CO2 release pipe was included that is separated from the attractive odour plume. The role of heat was exploited in the development of a repellent bioassay and a heat source was added to another trap model. Insights in house-entry behaviour and mosquito responses to bed nets support the successful implementation of push-pull systems, installation of eave tubes or implementing house improvement operations to reduce malaria transmission. An integrated vector management approach is required to further develop existing control tools by adding and improving alternative intervention techniques.</p

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

Entomological Surveillance as a Cornerstone of Malaria Elimination: A Critical Appraisal

Global capacity for developing new insecticides and vector control products, as well as mathematical models to evaluate their likely impact upon malaria transmission has greatly improved in recent years. Given that a range of new vector control products are now emerging that target a greater diversity of adult mosquito behaviours, it should soon be feasible to effectively tackle a broader range of mosquito species and settings. However, the primary obstacles to further progress towards more effective malaria vector control are now paucities of routine programmatic entomological surveillance, and capacity for data processing, analysis and interpretation in endemic countries. Well-established entomological methods need to be more widely utilized for routine programmatic surveillance of vector behaviours and insecticide susceptibility, the effectiveness of vector control products and processes, and their impacts on mosquito populations. Such programmatic data may also be useful for simulation analyses of mosquito life histories, to identify opportunities for pre-emptively intervening early in the life cycle of mosquitoes, rather than targeting transmission events occurring when they are older. Current obstacles to more effective utilization, archiving and sharing of entomological data largely centre around global inequities of analytical capacity. These prohibitive and unfair imbalances can be addressed by reorienting funding schemes to emphasize south-centred collaborations focused on malaria-endemic countries

The sub-lethal effects of pyrethroid exposure on Anopheles gambiae s.l. life-history traits, behaviour, and the efficacy of insecticidal bednets

Malaria control progress in Africa has stalled. Though the reasons for this will be multifaceted, increasing and intense resistance to pyrethroids in Anopheles gambiae s.l. is almost certainly a contributing factor. Standard methods to monitor insecticide resistance and evaluate vector control tools primarily focus on the immediate and lethal effects on the mosquito. These methods disregard other important delayed and sub-lethal effects, despite their implications for malaria transmission. In response to growing concerns over the sustained effectiveness of current control tools, next-generation products are being developed and evaluated. These aim to target insecticide-resistant mosquitoes or mosquitoes that contribute to residual malaria transmission. Adaptations to current standard efficacy tests are needed to evaluate the novel modes of action of such products. The effect of insecticide exposure on the longevity, reproductive output and blood-feeding behaviour of a wild highly pyrethroid-resistant Anopheles gambiae s.l. population was evaluated. Mosquitoes were exposed to a range of insecticides and insecticidal bednets using laboratory tests and semi-field experimental hut trials. Benchtop video tests were evaluated for their feasibly in measuring the effectiveness of standard and next-generation nets. Subsequently, these tests were used to investigate the behaviour of field-populations of An. gambiae s.l. at the bednet interface in response to a human host. Following exposure to both pyrethroid-only and next-generation nets, evidence of sub-lethal impacts were limited or non-existent. The mosquitoes exposed to insecticidal nets did not suffer from reduced lifespan or altered reproductive output. Evidence of delayed mortality was only recorded when mosquitoes were exposed to extremely high levels of pyrethroids in WHO tube bioassays. Some mosquitoes were inhibited from blood-feeding in experimental hut trials, however, lab tests suggest this effect is absent by 8-hour post net-exposure. The efficacy of next-generation nets on the field population was dependant on the product. Brief contact with PermaNet 3.0 roof (pyrethroid + PBO) caused rapid knock-down and 100% mortality in all tests. Exposure to all other insecticidal nets, including Interceptor G2 (pyrethroid + chlorfenapyr), resulted in low 24-hour mortality in both lab and semi-field experiments. Following adaptations for the field, video tests were able to collect behavioural data on mosquito responses to insecticidal nets such as flying, resting, and probing behaviour. Responses were similar between untreated and pyrethroid- only netting. Extreme reductions in activity were observed following exposure to PermaNet 3.0, and Interceptor G2 showed signs of repellence. The results suggest community protection offered by first-generation LLINs is extremely low in this setting, however, pyrethroid-PBO nets appear to be effective at controlling the highly pyrethroid-resistant population. This work highlights the need for additional studies of sub-lethal effects in other field populations, with lower insecticide resistance levels or differing mechanisms, to establish if such measurements should be incorporated into the evaluation of novel vector control tools