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

Abstract

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.

Figure 1. Flight activity of Anopheles gambiae at unbaited, baited and insecticide-treated bed nets.

(A) The experimental insectary, showing the bed and fitted bed net, with two pairs of Fresnel lenses visible on the left and right; illumination at 850 nm was from two LEDs (not in image) located behind the right Fresnel lenses with the light from each forming an approximated parallel beam across the bed, through the left Fresnel lenses and focused into the camera beyond (not in photograph). The LEDs and camera were positioned 1.2 m behind the lenses, aligned to an optical axis through the centre of the Fresnel lenses. Mosquitoes were released on the wall behind the photographer at a height of 2 m. (B–D) Examples of a full 60-minute record of a test showing flight tracks of Anopheles gambiae at: (B) an unbaited untreated bed net; (C) a human-baited untreated bed net; (D) a human-baited insecticide-treated bednet (LLIN; Permanet 2^®; Vestergaard-Frandsen, Lausanne, Switzerland). Twenty-five mosquitoes were released in all tests and activity was recorded for 60 minutes. Each coloured track is the path of a single mosquito flight event. Tracks are colour-coded according to time they first appeared in the field of view as shown in the key: blue tracks at the start through to red at the end of the 60-minute test. (E–H) Images showing representative tracks for Anopheles gambiae flight in each of the four behaviour modes as defined in the text. See also Supplementary Video. (I) The proportion of time spent in each behaviour mode for each bed net type: Unbaited = unbaited untreated bed net; Untreated = human-baited untreated bed net; LLIN = human-baited insecticide-treated bed net (LLIN).

Figure 2. Distribution of Anopheles gambiae flight activity, behaviour modes and net contact at different regions on and around a bed net

(A) Distribution map key showing region codes to which each mosquito track was assigned. Bed net surfaces 1–6 were on the horizontal roof, 7 and 10 the vertical head and foot end walls, respectively, 8 and 9 the vertical side walls. Portions of flight tracks visible beyond the net surfaces were assigned to the spatial regions 11–14 as shown. Regions 15 and 16 record flight activity without net contact (i.e. swooping) that occurred in front of net, on the left (15) and right (16) portions of the field of view, respectively. (B) Distribution of initial net contacts by region, showing the first point of net contact for those tracks occurring in the first ten minutes of testing. (C) The total duration (seconds) of all contacts (includes mid-flight brief contacts made during visiting and bouncing, and resting behaviour) by all mosquitoes on each region of the bed net surface over the 60 minute test (means of 3,10 and 10 replicate tests for unbaited, baited and LLIN, respectively). Charts D–H show the density of activity (s/m²) around and on the bed net surface: (D) All activity combined; (E) Swooping; (F) Visiting; (G) Bouncing; (H) Resting. Although tests controlled for the orientation of the human bait in relation to the mosquito release point, all figures show the volunteer (when present) with the head on the left.

Figure 3. Velocity of Anopheles gambiae during landing at bednets.

Mean velocity of mosquitoes during final approach, contact and departure from the bed net surface. The figure represents a 1.3 s track segment, with the bed net contact point at 0 mm; positive x-axis values indicate position before contact, negative values after contact. The grey region either side of the contact point represents the ± 3 mm region where tarsal contact with the bed net was possible. The average points at which deceleration started for each net type are marked with ‘X’. Note that the graph presents the averages of multiple repeat test values and hence the position of the point of deceleration does not correspond perfectly with the average approach track as illustrated. Coloured bars show standard deviation at each track point.

Figure 4. Rates of Anopheles gambiae activity throughout the 60 minute test period.

(A) Total activity at untreated baited nets and LLINs.(B,C) Mosquito activity as in Fig. 3A separated by behavioural mode, at untreated baited nets (B) and LLINs (C). X-axis units are mean (±SD) activity per 5-minute inclusive interval, i.e. 5 (0–4 min 59 s), 10 (5 min–9 min 59 s), 15 (10–14 min 59 s), etc.