The effect of eave and window modifications on house entry behavior of Anopheles gambiae

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Lankheet, Remco P.M. Pieters, Miracle Gadamika, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5545048/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jul, 2025 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Background Anopheles gambiae mosquitoes transmit malaria parasites to humans mostly by biting them indoors at night. An. gambiae predominantly enter houses through ventilation openings such as open eaves and windows. Methods Here, we studied how flying An. gambiae approach and enter a house, and whether barriers to reduce mosquito house entry alter mosquito flight patterns. We used stereoscopic high-speed videography to reconstruct nearly 70,000 three-dimensional tracks of mosquitoes flying around a house during 30 experimental nights, with five combinations of closed or screened eaves and windows. Results We found that these eave and window treatments did not affect the number of mosquitoes attracted to the house. In all cases, mosquitoes were most active during the early evening, with lower but sustained activity throughout the night. Most An. gambiae approached the house by flying directly towards the eave in a straight, upward sloping path, and most flight activity near the house was in front of the eave. Due to the highly attractive nature of the eave area of the house, window treatments had limited to no effect on the number of house entries when eaves were left open, highlighting the importance of closing or screening eaves to prevent mosquito house entry. For the screened eave treatment, An. gambiae spent about 10× as much time near the eave over the course of the night compared to treatments with open or closed eaves. Moreover, these mosquitoes returned multiple times, persistently trying to enter the house. When the eave was fully closed, mosquitoes deferred from the eave area towards the screened window, but the initial approach flights remained towards the closed eave. Conclusions Taken together, these results demonstrate the tendency of An. gambiae to direct house entry toward the eaves, and to only divert to other house entry points as a secondary option. The persistent mosquito flight near screened eaves may provide guidance for the placement of outdoor vector control tools. Anopheles gambiae Malaria Mosquito control Housing Insect flight Videography Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 BACKGROUND Malaria remains a serious global health challenge, despite the progress made over the past two decades in reducing the malaria burden. From 2000 to 2015, the prevalence of Plasmodium falciparum infection in endemic regions of Africa was reduced by 50%, and an estimated 663 million clinical cases of malaria were averted [ 1 ]. Vector control, mostly by means of insecticide treated bed nets, contributed to more than 70% of this reduction [ 1 ], demonstrating the importance of interrupting mosquito-human contact. Still, the progress made until 2015 has stalled in recent years [ 2 ]. Anopheles gambiae s.s. (hereafter An. gambiae ) is one of the main vectors of malaria parasites in Africa and is typically nocturnal, anthropophilic and endophilic, i.e., preferring to feed from humans indoors at night [ 3 , 4 , 5 , 6 ]. Hence, a critical component of finding a bloodmeal host is for the mosquito to find and enter an inhabited house. Navigation towards inhabited houses is most likely triggered by carbon dioxide (CO 2 ) levels exceeding background levels [ 7 ]. Locating a house, or house entry point, is presumably based on the integration of perceived CO 2 levels, volatile host odors, and visual cues or contrasts between the house and the landscape [ 8 , 9 ]. The perception of volatile cues by mosquitoes is affected by odor plume structure, and this, in turn, is affected by habitat characteristics and house design [ 8 , 10 ]. As such, climate conditions and indoor micro-climate affect the approach and time spent indoors by mosquitoes [ 10 , 11 ]. Finally, it should be noted that the behavioral set of responses for house entry and indoor resting can be distinct from that for actual host finding [ 12 , 13 ]. Traditional house designs in many malaria-endemic regions of Africa include three types of openings potentially used by mosquitoes as entry points: doors, windows, and open eaves. Open eaves, i.e. open gaps usually running the length of the wall where the roof and wall meet but do not join, are particularly important house entry points for An. gambiae [ 12 , 14 ]. Screening eaves reduces the number of An. gambiae and other malaria vectors in houses [ 15 , 16 ], even when the windows and doors are left open [ 17 ]. Screening windows and doors can reduce the number of An. gambiae in houses with closed eaves [ 10 ], but not with open eaves [ 17 ]. Traditional house designs, including open eaves and unfinished materials for walls, floors and roofs, are generally associated with an increased risk of malaria parasite infection and malaria cases when compared to improved house designs (closed eaves and finished materials) [ 18 , 19 ]. In randomized trials, house modifications (some combination of screening or closing eaves, windows, doors and ceilings) have been associated with reduced malaria parasite prevalence and reduced anemia prevalence [ 20 ]. This has led to growing support from policy makers for structural house modifications as a strategy for malaria vector control [ 21 ]. Understanding mosquito flight towards window and eave openings, the time spent near these entry areas, and success rates of finding entry openings provides valuable information for designing mosquito-proof homes [ 14 , 16 ]. Filming and analyzing mosquito flight tracks around house entry points allows for the collection of detailed information about the flight behavior of mosquitoes when approaching a house, and how this leads to finding and entering houses (e.g. flight trajectories, time spent near windows and eaves, persistence and angle of approach). This fundamental knowledge on strategies used by mosquitoes to locate inhabited houses is essential for designing effective house modifications to prevent mosquito entry [ 22 , 23 ], and it is also key for the design and optimal placement of other vector control tools used on or near houses, such as eave tubes [ 24 ], spatial repellents, odor baited traps, or a combined push-pull strategy [ 25 , 26 , 27 ]. For the current study, we assessed how different modifications of eaves and windows affect the flight dynamics of female An. gambiae mosquitoes around the house, and consequently the house entry behavior and rates. Over the course of 240 hours of experimental recordings, we captured nearly seventy thousand mosquito flight tracks, amounting to approximately 76 hours of mosquito flight data. We used these data to quantify the spatial-temporal distribution of mosquitoes flying around the house, their typical house approach and entry behavior, and how the eave and window treatments affected these. METHODS Mosquito colony All mosquitoes used in this study were colony-reared An. gambiae (Kisumu strain). Eggs to establish the colony were initially obtained from the Malaria Alert Centre, Blantyre, Malawi, and the colony was maintained in the laboratory facilities at Majete Wildlife Park in Chikwawa District, Malawi. The colony rearing facility was not climate controlled. Temperature and relative humidity in the colony rearing facility ranged from 24°C to 36°C and from 62–85%, respectively. Mosquitoes were blood fed twice per week on a human arm, and eggs were distributed over larval rearing trays measuring 46 × 30 × 9 cm. Larval trays were filled with water from a well near the laboratory facility or from a tap at the nearby Kapichira Power Station. Three hundred to four hundred larvae per tray were fed on ground pellets of Marltons koi and pond fish food (Marltons Pet Care Pty Ltd, South Africa). Pupae were collected daily and placed in cages for emergence to adults. All cages with adult mosquitoes were provided a 10% sucrose solution via a piece of soaked cotton wool. Cages with experimental mosquitoes were not provided with a blood meal prior to the experiments. Experimental set-up Experiments were performed in a semi-field screened enclosure measuring 12.0 × 12.0 × 2.1–4.0 m (length, width, height) at the Majete Wildlife Park in Chikwawa District, Malawi. The walls of the screened enclosure were made from fiber glass, mosquito-proof screening (Phifer Inc, USA), and the roof was a waterproof tarpaulin. Within this enclosure, we built an experimental house measuring 5.0 × 3.0 × 2.2–2.7 m in length, width and height (Fig. 1 ). The walls of the experimental house were constructed from locally produced bricks and plastered with cement, and the roof was made with corrugated iron sheets, including a 20 cm overhang. The front side of the experimental house was fitted with a door (197 × 60 cm inner dimensions) in the middle, two windows (30 × 30 cm), and four removable eave frames with inner dimensions of 90 × 10 cm per frame. The back side of the house also contained two windows and the four eave frames, but no door. The wooden door, window frames and eave frames were painted with black, water-based chalkboard paint. The windows and eave frames could be left completely open, fitted with insect screens, or closed completely with wooden shutters. The screens were made of charcoal-colored fiber glass (Wire Weaving Co. Dinxperlo, The Netherlands), and the shutters were made of plywood painted black with water-based chalkboard paint. Using this system, we were able to systematically investigate the effect of window and eave closure and screening on mosquito house entry behavior. The door remained closed overnight for all experimental treatments. Two beds were positioned inside the house, one along each side wall, and each covered with an untreated bed net. During experimental nights, one adult man slept in each bed, under the bed nets, to act as a bait for mosquitoes. Three pairs of adult men volunteered to sleep in the house for 10 experimental nights each. Written informed consent was obtained from the volunteer sleepers. The College of Medicine Research and Ethics Committee (COMREC) in Malawi approved the study (Proposal Number P.02/19/2598). A CDC light trap (John W. Hock Ltd, USA) was placed near each bed to collect a sample of mosquitoes that entered the house [ 28 , 29 ]. Camera and real-time mosquito tracking set-up We used a multi-camera videography system to track the three-dimensional flight kinematics of An. gambiae mosquitoes around the experimental house. The videography system consisted of four synchronized machine-vision cameras (Basler acA2040-90umNIR, USB 3.0, Basler AG, Germany), equipped with 16 mm f1.4 wide-angle lenses (Kowa LM16HC, Kowa Optical Products Co., Ltd., Japan), with lens aperture set at f2.8. The cameras were operating at 50 frames per second (fps), and a 1 ms exposure time. To improve light sensitivity of the cameras, pixels within each 2×2-megapixel camera image were binned 2×2. Binning combines the charge from adjacent pixels (in this case, 2×2 pixel bins), resulting in increased light sensitivity but a reduced spatial resolution (in this case, reduced to 1×1 megapixel). Image capture on the cameras was synchronized by means of an external trigger pulse, generated by an Arduino Uno (Arduino, Italy) ( https://github.com/strawlab/triggerbox.git ). To protect the cameras and lenses from water, heat and dust, each set was placed in a camera housing (Transpac THP 4000, Basler AG Germany). These camera housings were mounted onto an aluminum frame (MayTec Aluminium Systemtechnik GmbH, Germany) that was fixed to the concrete floor on which the house was built (Fig. 1 b). The cameras were placed at an approximate distance of 2.5 m from the front wall of the house, at heights between 0.8 m and 1.3 m. As a result, the camera system imaged the front, right side of the experimental house, including half the door and one window. The cameras were oriented slightly upwards to film the volume below the roof near the eave area. The dimensions of the area in front of the house where mosquitoes could be tracked had a size of approximately 2.5 × 1.0 × 1.5 m (Fig. 1 d). The filming volume was illuminated with eight near-infrared light-emitting-diode (NIR-LED) lights (2× ABUS TVAC71000-60° and 6x ABUS TVAC71070-95°, ABUS Germany). The NIR-LED lights were mounted on a frame placed on the concrete slab directly below the area of interest (Fig. 1 b). The NIR-LED lights were directed upwards and arranged to uniformly light the filming volume near the eave and window, aiming for optimal contrast between the illuminated mosquitoes and the dark background of the house. We used an automated tracking software [ 30 ] to track in real-time the positions of multiple mosquitoes flying in the four camera views, and from these we reconstructed the three-dimensional flight tracks. The tracking software ran on a single laptop (Lenovo ThinkPad P51, Lenovo, China) with Intel Xeon E3-v6 processor and Ubuntu Linux operating system, which thus performed the real-time image analysis and object tracking for all four cameras, and the three-dimensional flight track reconstruction. Based on pilot recordings, sensor gain was set to 1.0 for all cameras, and the maximum number of simultaneously tracked mosquitoes was set to 10. Tracks were reconstructed only when the mosquito was visible in at least two of the four camera views. A dynamic background model was used with update intervals for each 100 frames and a 1% weight factor to compensate for slow changes in illumination conditions. Cameras were calibrated with the multi camera self-calibration routine [ 31 ] by tracking a single moving LED light by each of the four cameras (Cree SunBright 535 nm Green LED, CreeLED Inc, USA). This calibration was aligned to world reference points based on landmarks on the experimental house. The resulting coordinate system in the world reference frame was defined as {X,Y,Z}, with the X-axis oriented perpendicular to the house front wall, the Y-axis parallel to the house front wall along the ground, and the Z-axis vertically. We defined values within this coordinate system as { x,y,z }, with the origin { x,y,z }={0,0,0} located against the house front wall ( x = 0), on the ground in front of the house ( z = 0), and ( y = 0) at the right side of the door frame as observed from the cameras. The calibration procedure was repeated every experimental day, in case cameras would accidentally have changed position over the course of the experiment. A correction for lens distortions was generated for each camera at the start of the experiment, using a 6 × 10 checkerboard pattern with 90 mm squares. Distortion parameters were computed using openCV procedures ( https://docs.opencv.org ). Tracking results were corrected for lens distortions. Videography experiments were performed from 20:00h to 04:00h. If volunteers shortly left the experimental house during the night, a five-minute buffer period was marked prior to leaving and post re-entering the experimental house. Tracking data within those time slots were removed from further analyses. Eave and window modifications We evaluated five experimental house modifications (Fig. 2 ). For our control condition, both the windows and screens were fully open (eave open-window open; EO-WO). We used two treatments to test the effect of window modifications on mosquito house entry behavior. In the first treatment, we screened the window and left the eaves open (EO-WS), and in the second treatment we closed the window while leaving the eaves open (EO-WC). To test the effect of eave modifications on mosquito house entry behavior, we used two treatments in which we screened and closed the eaves, while keeping the window screened (ES-WS and EC-WS, respectively). Experimental procedure Before each experiment, the house was prepared by closing, screening, or leaving open the eaves and windows, as randomly assigned for each replicate night of the study (Fig. 2 ). Each treatment was in place for six replicate nights (see experimental treatment schedule in Table S1 ). On the day of each experimental replicate, 500 female mosquitoes (5–8 days old and not previously blood-fed), were selected before 12:00h and set aside in the insectary in a release bucket (Ø: 12.5 cm, height: 12.5 cm), covered with a mesh and provided with water-soaked cotton wool. Two volunteers slept inside the house under untreated bed nets, starting at 19:30h. The volunteers’ heads were positioned at the front (door) side of the house, and each pair shifted beds (left or right side of the house) after each replicate. At 19:30h the two CDC light traps at the end of each bed were turned on, with their lights switched off, and the bucket with mosquitoes was placed in the screened enclosure, 5.8 m in front of the experimental house. At 20:00h, mosquitoes were released from the bucket by lifting the mesh via a fishing line operated from outside the screened enclosure. Mosquito flight was tracked until 04:00h, after which the CDC light traps were turned off, and the volunteers could leave the house. Temporary absence of volunteers during the recording period was recorded in a logbook. A Prokopack aspirator (John W. Hock Company, USA) was used to collect mosquitoes from inside the experimental house at 04:00h. Together with these Prokopack catches, CDC light trap catches were shortly frozen and collected mosquitoes were then counted. Mosquitoes remaining in the release bucket were also counted, and the number of responding mosquitoes for each replicate night was defined by subtracting the number remaining in the release bucket from the initial 500 mosquitoes. Remaining mosquitoes found inside the screened enclosure later that day were removed with the Prokopack and discarded after freezing. Experimental replicates were carried out no more frequently than every other day to ensure proper preparation and allow any uncaught mosquitoes remaining in the screened enclosure to die before the next experimental replicate. Data analysis The real-time tracking algorithm used a Kalman predictor to reconstruct three-dimensional flight paths from stereoscopic videography data [ 30 ], and thus the output data consisted of Kalman-filtered flight paths defined by location, flight velocity, and the Kalman covariance error e( t ). In post-processing, we filtered the resulting database of flight tracks in two steps. To remove potential extrapolation errors from the Kalman predictor, we deleted the end of tracks if either the estimated flight speed was greater than 1.5 m/s or the Kalman covariance error was larger than 0.01. We then discarded all tracks that were shorter than 10 cm or less than 0.2 seconds (10 video frames at 50 fps). These settings were based on a sensitivity analysis and the assumption that flying Anopheles mosquitoes have a maximum flight speed of less than 1.5 m/s. The resulting flight paths consisted of the temporal dynamics of the three-dimensional location { x ( t ), y ( t ), z ( t )} and velocity { u ( t ), v ( t ), w ( t )} of each flying mosquito; these were used for our subsequent analyses. We used all combined flight tracks per treatment to calculate average mosquito density distributions and flight velocity distributions around the house. For this, we divided the filming volume into 40×40×40 voxels (spatial bins), resulting in an approximate voxel size of 5 cm in the X- and Z-direction, and 7.5 cm in the Y-direction. In each voxel we estimated the mosquito density as the relative proportion of time mosquitoes spent in that voxel, defined as T * = T i / T total , where T i is the time spent in voxel i , and T total is the total flight time. We visualized these density distributions as heat maps projected on three 2D planes (X-Y, X-Z, and Y-Z). We determined the flight velocity vector in each voxel as the mean flight velocity of all mosquitoes that passed through that voxel. We visualized the velocity distributions using streamline plots derived from these velocity fields, projected on the same set of 2D planes as for the density distributions (X-Y, X-Z, and Y-Z). For measuring and comparing flight activities near the eave and window area, we defined volumes-of-interest around the eave and window (Fig. 1 e and 1 f, respectively). These volumes had the same rectangular or square shape as the eave or window, respectively, but extended 10 cm on each side (in the Y- and Z-direction). The volumes started at the wall and extended 30 cm outward in the direction perpendicular to the wall (in the X-direction). We then identified all flight tracks that intersected these volumes around the eave and window. Based on these, we quantified flight activity around the window and eave using the time that mosquitoes spent in the corresponding volumes. We determined this time spent in each volume by summing all durations that flight tracks remained in the defined volume. We did this for each experimental night, and for an array of time bins with a temporal resolution of 10 minutes. Next, we used the flight tracks around the window and eave to study when and how mosquitoes visited the window and eave, and when and how they arrived, departed, remained in, and returned to these volumes. We defined ‘arrivals’ as flight tracks that started at least 10 cm outside the volume of interest and ended within the volume. ‘Departures’ started within the volume of interest and ended at least 10 cm outside the volume. ‘Visitors’ started outside the volume, entered the volume, left the volume, and then ended outside the volume. ‘Returnees’ started inside the volume, left the volume, re-entered the volume, and finally ended inside the volume. ‘Remainers’ started and ended inside the volume, without moving outside the volume. Note that if a flight track ended within the window or eave volume, the mosquito might have entered the house or have landed on the house, because the tracking algorithm only tracked mosquitoes flying outside the house. Based on these data, we determined the number of mosquitoes that showed each type of flight behavior (visiting, arriving, departing, remaining and returning). We then used the flight kinematics data to determine the behavior-specific flight dynamics around the eave and window. Specifically, we constructed streamline plots, both per treatment and across all 30 replicates, for all mosquitoes that arrived at the volumes around the eave and window. To focus on the approach kinematics only, we removed the parts of the tracks after arrival. We used ANOVA to test for differences among treatments in various flight kinematics and house entry parameters. The dependent parameters were the number of responding mosquitoes, the percentage of responding mosquitoes collected inside the experimental house, flight track duration (time spent), and the number of flight tracks. We used Tukey’s HSD for pairwise comparisons when the ANOVA test showed a significant difference between treatments. We also used ANOVA to test for differences in house entry rates among the three pairs of volunteer sleepers. RESULTS We performed experiments on 30 nights ( n = 6 replicates per treatment) in the period between 16 March to 20 June 2020. The average number of responding mosquitoes, i.e. those that left the release bucket overnight, was 491.1 ± 5.5 per night (98%). There was no effect of house treatment on the number of responding mosquitoes (Table S2, ANOVA, P = 0.459). Indoor mosquito collections House entry rates based on CDC light trap and indoor Prokopack collections were independent of the three volunteer pairs used to lure the mosquitoes into the experimental house (ANOVA, P = 0.885). The percentage of responding mosquitoes that entered the house was high for all treatments with open eaves, with a median of 47.3% (IQR: 12.1%), 28.9% (IQR: 24.0%), and 46.3% (IQR: 12.8%) for the EO-WO, EO-WC, and EO-WS treatments, respectively (Fig. 3 , Table S2). Among the three treatments with open eaves, we did not see a significant effect of window treatment (open, closed or screened) on indoor mosquito collections (Fig. 3 , TukeyHSD, P > 0.05). As expected, among the three treatments with screened windows, closing or screening the eaves drastically reduced the number of mosquitoes found indoors compared to open eaves (Fig. 3 , TukeyHSD, P < 0.001). The percentage of responding mosquitos caught indoors was reduced to a median of 1.9% (IQR: 1.4%) and 1.8% (IQR: 0.3%) when eaves were screened or closed, respectively (Fig. 3 , Table S2). Mosquito flight activity in space and time In total, we recorded and reconstructed 69,025 flight tracks, which resulted in a median of 3.6 (IQR: 4.4) flight tracks per responding mosquito per experimental night (Fig. 4 a), with a median cumulative track duration per responding mosquito per night of 11.5 (IQR: 17.5) seconds (Fig. 4 b), and a median track duration per flight of 3.7 (IQR: 1.2) seconds (Fig. 4 c). The number of simultaneously recorded tracks was well below our tracking algorithm limit of 10 simultaneous tracks (Fig. 4 d). The mean number of tracks per video frame varied from approximately one flight track in the first hour of the night (for treatments with eaves screened or closed), to values below 0.5 towards the end of the night, irrespective of treatment (Fig. 4 d). The number of flight tracks per responding mosquito per night (Fig. 4 a) was not statistically different between house treatments (ANOVA, Pr(F) = 0.383). The cumulative track duration per responding mosquito per night (Fig. 4 b) was marginally different between treatments (ANOVA, Pr(F) = 0.062); the greatest differences in pairwise comparisons were between the treatment with eaves and windows screened (ES-WS) and those with open eaves (EO-WO, EO-WC, and EO-WS; TukeyHSD, adjusted P = 0.126, 0.099, and 0.077, respectively). Mean duration per track was significantly longer for the house treatment with both eaves and windows screened (ES-WS), than for the treatments with open eaves (EO-WO, EO-WC, and EO-WS; Fig. 4 c, TukeyHSD, P < 0.05), but not for the treatment with eaves closed and windows screened (EC-WS; Fig. 4 c, TukeyHSD, P = 0.120). The spatial distribution of mosquito flight tracks in front of the experimental house, as measured by relative proportion of time spent in the 40×40×40 voxels, was generally concentrated near the eaves of the house (Fig. 5 ). Based on these spatial distributions of relative proportion of time spent, we estimated the total time spent flying in front of the house by all mosquitoes combined within each treatment (Fig. 6 a). The time spent flying in the full trackable area was marginally different between house treatments (Fig. 6 a, ANOVA, Pr(F) = 0.059). Based on the spatial distributions shown in Fig. 5 , the concentration of flight activity near the eaves appeared strongest on nights with eaves and windows screened (Fig. 5 e; ES-WS). Indeed, when we compared the time spent (total track duration) in the volume around the eave, mosquitoes spent significantly more time in the eave area on nights with eaves and windows screened (ES-WS), compared to any other treatment (Fig. 6 b, TukeyHSD, P < 0.05). Flight activity near the window of the house was generally lower (i.e. less relative proportion of time spent) than near the eaves (Fig. 5 ). Nights with eaves closed and windows screened (EC-WS) appeared to be an exception (Fig. 5 d), with roughly equal amounts of relative proportion of time spent near the windows and eaves on those nights. Indeed, time spent (total track duration) in the defined window area was marginally different among the five treatments (Fig. 6 c, ANOVA, Pr(F) = 0.056), with the eaves closed and windows screened (EC-WS) treatment appearing higher than the other treatments. Figure 7 shows the time spent over night in the full recording volume, and near the eave and window, separated in time bins of 10 minutes. Differences in flight activity among treatments were clearest in the first part of nightly trials, from 20:00h when measurements were started, to about 21:30h. During this period flight activity in front of the house was relatively high, and particularly so for treatments with the eave screened or closed (ES-WS and EC-WS, respectively; Fig. 7 a). For the case with the eave closed (EC-WS), this flight activity rapidly dropped within the first hour, but for the eave screened treatment (ES-WS), this flight activity remained relatively high throughout the night. Moreover, the location of this increased flight activity differed between these two treatments (ES-WS and EC-WS). For the eave screened treatment, the increased flight activity throughout the night was concentrated in the volume near the eave (Figs. 5 and 7 b). In contrast, the increased flight activity in the early evening for the closed eave case was mostly concentrated in front of the window (Figs. 5 and 7 c). For all other treatments, flight activity during the night was much lower and less strongly concentrated in specific areas (Fig. 7 ), although in all cases the highest flight activity remained near the eave (Fig. 5 ). Overall, less activity was observed near the window area than the eave area throughout the night (Figs. 5 and 7 b-c). We categorized the mosquito tracks according to the flight behavior around the eave and window, for the various house treatments (Fig. 8 ). Here, we characterized all flights near both the eave and window as “arrivals”, “departures”, “returnees”, “remainers”, and “visitors”. The distinct similarities in these categories between eave and window region show that the flight behavior around the window and eave are strikingly similar. This is particularly apparent when comparing the behaviors that lead to increased activity near the eave and window for the treatments with the eave screened and closed, respectively (ES-WS and EC-WS in Figs. 5 , 6 and 7 ). Our behavioral classification shows that the resulting increased flight activity near the eave (for ES-WS) and window (for EC-WS) are both largely driven by an increased number of ‘departures’, ‘returnees’, and ‘remainers’ for those treatments, compared to the other treatments (Fig. 8 ). The number of ‘arrivals’ and ‘visitors’ have a smaller effect on the treatment-specific increased flight activity near the eave and window. This suggests that the increased flight activity near the eave and window for the screened and closed eave cases is caused primarily by mosquitoes remaining near, or departing and returning to, those house structures when they are unable to enter the house through the screening, and not by differences between treatments in the initial flight to arrive at these structures. Mosquito flight pattern on approaching a house Next, we zoom in on the approach flights of mosquitoes towards the eave and window (Figs. 9 and S1-S2). Treatment-specific streamline plots of approach flights towards the eave and window (Figures S1 -S2, respectively) show a consistent approach behavior between the different treatments. We therefore combined all recorded approach flight tracks for the different treatments to reconstruct the average approach flight kinematics for mosquitoes arriving at the eave or window (Figs. 9 a-c and 9 d-f, respectively). For both cases, we show the average flight pattern of mosquitoes approaching the eave and window using streamlines color-coded with relative track density, and streamline thicknesses defining the flight speed. The streamline data are projected on the three planes defined in Fig. 1 d, being the projections on the house front wall (Y-Z), house symmetry plane (X-Z), and ground surface (X-Y). The results for mosquitoes approaching the eave (Fig. 9 a-c) show that, on average, mosquitoes approached the eave using a steady ascending flight, starting at an approximate distance of 1 meter from the house front wall. Particularly the side view projection shows that the majority of flight tracks (highest density) followed this steady ascending flight straight towards the eave. A second smaller group of mosquitoes (lower density) approached the eave more from below by ascending more steeply once they get closer to the house front wall. This pattern seems independent of treatment (Figure S1 ). The streamlines projected on the ground surface (Fig. 9 c), reveal that, on average, the mosquitoes approach the house with minimal variation in flight speed parallel to the house (left-right deviations). These results combined show that mosquitoes approaching the eave seem to fly in a highly targeted manner by rapidly ascending in a straight line towards the eave. The mosquitoes approaching the window showed a similar highly consistent flight pattern (Fig. 9 d-f). The top view projection shows that most mosquitoes approached the window in a straight line, starting from relatively far from the house, and only few mosquitoes approached the window from the sides (Fig. 9 f). That said, the side view projection shows that the highest density of flight tracks was located near and directed towards the eave (Fig. 9 e), suggesting that many of the mosquitoes arriving at the window flew first towards the eave, after which they turned towards the window. This flight pattern is similar for all treatments (Figure S2). The combined density and streamline plots suggest that the majority of flying mosquitoes approached the house by ascending in a straight line towards the eave (Figs. 5 and 9 ). We tested this by estimating flight height (relative to the eave) at various distance from the house, for all mosquitoes that approached the eave (Figs. 10 and S3). This analysis is based on the same dataset as shown in Fig. 9 a-c and S1. On average, the mosquitoes approached the eave by ascending approximately 40 cm over a 1 m distance while flying towards the house, resulting in an average climbing flight angle of 22 degrees during this approach. The increase in flight height with decreasing distance from the house is similar between the five house treatment experiments (Figure S3). This suggests that the climbing approach flight towards the eave is highly characteristic, and does not change with house modifications, even when the eave is fully closed (EC-WS). DISCUSSION Here, we studied how host-seeking An. gambiae approach and enter a house, and how modifications to common house entry points change these flight behaviors, including persistent attempts to enter when blocked by screening. We tracked free-flying mosquitoes released in a semi-field enclosure, allowing us to capture the detailed dynamics of An. gambiae flight in a large region in front of the house throughout the night. Across all five combinations of eave and window modifications tested here, An. gambiae approached the house by flying directly towards the eave, in an upward sloping path. When the eaves were open, a large percentage of An. gambiae entered the house, regardless of whether the windows were open, closed, or screened. When the eaves and windows were screened, An. gambiae spent more time in the area near the eave, persistently attempting to enter via the eave throughout the night. In contrast, with eaves closed and windows screened, An. gambiae spent more time in the area near the window – generally after first approaching the closed eave. Taken together, our results highlight the tendency of An. gambiae to direct house entry toward the eaves, and to only divert to other house entry points as a secondary option. Using our real-time videography-based tracking algorithms, we recorded flight activity over an extended period each night (from 20:00h to 4:00h). While flight activity continued throughout the night for all house treatments, peak activity occurred near the start of the recording period, directly after release of the mosquitoes (20:00h to 21:00h). We released 500 female An. gambiae mosquitoes per experimental night, with about 98% leaving the release bucket, resulting in a median of about 1,700 flight tracks per night. Although some mosquito flights may have been recorded over multiple tracks (e.g. if the flight exited and then re-entered the tracking area in front of the house), our data set of nearly 70,000 flight tracks represents about 76 hours of mosquito flight in front of an occupied house. We consistently observed An. gambiae approaching the occupied house by flying directly towards the eave along upward sloping flight paths, irrespective of the eave and window modifications. This characteristic flight pattern was apparent across several methods of visualizing the tracked flights, including the density distribution of all flight tracks (Fig. 5 ) and the streamline plots of flight tracks categorized as approaching the eave (Fig. 9 a-c) and window (Fig. 9 d-f). These results align with previous studies of An. gambiae house entry suggesting an increase in flight altitude to eave level based on indirect observations [ 12 , 14 ]. Our tracking data show directly that this increase in altitude is initiated at least 1 m from the house for the majority of An. gambiae , at a climbing angle of approximately 20 degrees – however, we do not know the point at which these mosquitoes initiate this ascending path, as the mosquito release point was beyond our tracking area. It is likely that straight, upward sloping flights by An. gambiae are specific to approaching an occupied house, representing a unique stage of host seeking. Host seeking by An. gambiae and other mosquitoes at distances > 10 m from a host (beyond visual range) is thought to consist of zigzag, cast-and-surge flight patterns dependent on wind and habitat factors that determine host odor plume characteristics, based on studies of other insect taxa [ 32 ], and supported by wind tunnel experiments in mosquitoes [ 33 , 34 ]. As mosquitoes move closer to a host, they likely integrate additional sensory cues, including visual and thermal cues, with corresponding changes in flight patterns dependent on the specific mix of cues [ 35 , 36 , 37 ]. Our observations of An. gambiae approaching an occupied house are the first direct evidence of their flight patterns at this stage of host seeking, filling a critical knowledge gap considering this species is generally endophilic [ 3 , 4 ]. Our results confirm that the eave is the most attractive region of human-occupied houses, and that the open eave is the primary entry point for An. gambiae [ 17 , 38 ]. Additionally, the behavioral responses to eave modifications confirm that odor cues from the house occupants are important for attracting An. gambiae to the eave [ 10 , 39 ]. When the eave was screened and odor could still exit the eave, mosquitoes continued flying there throughout the night while trying to enter the eave. In contrast, when the eave was fully closed and thereby the odor dispersing airflow was blocked, mosquitoes moved away from the eave and towards the screened window. In this configuration, the window was most likely the primary source of human odor dispersal, causing the mosquitoes to continue flying there following the initial approach to the eave. This initial approach toward the eave, even when the eave is fully closed, is striking and suggests that other sensory cues apart from odor may be important for approaching the house and eave. Although we did not measure CO 2 or other host odors, and some odor cues may have been present in the eave area when the eaves were fully closed, the difference between closed and screened eave treatments in mosquito activity near the eaves, with mosquitoes either leaving or persisting in the eave area, respectively, suggests a meaningful difference in the way these treatments were perceived by the mosquitoes. Taken together, the consistent flight pattern of An. gambiae when initially approaching the house, and the divergent subsequent behaviors of these mosquitoes in response to either screened eaves or closed eaves, provides guidance for the optimum placement of vector control tools on or near houses, such as eave tubes [ 24 ], odor-baited traps [ 40 , 41 , 42 ], or push-pull strategies [ 25 , 26 , 27 ]. The low amount of flight activity at ground level near the house suggests that placing odor-baited traps or other attractant-based interventions here would be less effective than placing them closer to the eave or farther from the house. The persistence of An. gambiae to attempt house entry near screened, but not closed, eaves suggests that placing odor-baited traps near screened eaves would be more effective than near closed eaves. However, effectiveness of odor-baited traps would also depend on the attractiveness of the trap relative to competing attractants and the capture efficiency of the trap [ 43 ], warranting further studies with specific odor-baited traps to determine the optimal location for maximum catch rates. Screening and closing the window while leaving the eave open had a strikingly small effect on An. gambiae flight behavior and did not reduce house entries. When the eaves were completely closed, we detected an increased flight activity around the screened windows, but these were mostly secondary approaches after visiting the closed eave (Fig. 9 e). These results confirm that window modifications such as screens or shutters are ineffective vector control tools for houses with open eaves if not paired with eave modifications, as previously shown [ 17 ]. CONCLUSIONS Our study provides the first direct evidence that female An. gambiae approach a house using a characteristic flight pattern, flying directly towards the house eave along a climbing flight path. Preventing house entry with screened eaves resulted in prolonged flight activity near the eave as mosquitoes continued to attempt entry at this same point. When the eave was fully closed, presumably preventing host odors from accumulating in the eave area, mosquitoes were deflected to the screened window after the initial approach to the eave. These divergent behaviors of An. gambiae after approaching screened and closed eaves may provide guidance for effective positioning of odor-baited traps or other outdoor vector control tools to remove mosquitoes from the population. Further studies on how mosquitoes approach and enter houses could build on our findings by incorporating additional house designs, for example increasing ventilation or the presence of indoor lights [ 39 , 44 ]. Abbreviations EO-WO: eave open-window open; EO-WS eave open-window screened; EO-WC eave open-window closed; ES-WS eave screened-window screened; EC-WS eave closed-window screened. Declarations Acknowledgements We thank Themba Mzilahowa at the Malaria Alert Centre for providing mosquito eggs to establish the An. gambiae colony used in this study, Florence Ndalakwanji for her work maintaining the mosquito colony, and the team at the Training and Research Unit of Excellence (TRUE) for their support of the study team. We are grateful to the African Parks Network, and especially the team at Majete Wildlife Reserve, for access to the facilities at Majete and for their assistance with setting up the semi-field enclosure. We would also like to acknowledge the support of the Building Out Vector-borne diseases in sub-Saharan Africa (BOVA) network, which provided a Pump-priming Grant to KSP for this study. Funding Funding for this study was provided through a Pump-priming Grant to KSP from the BOVA network (primary grant from BBSRC, MRC and NERC, no. BB/R00532X/1 to Steve Lindsay). RSM received additional support from NIH award no. T32AI007524 and K01TW011770. Availability of data and materials Experimental data that supports the findings of this study are available in the online repository Dryad at [ repository link to be added to next version ]. Authors’ contributions Conceptualization: JS, MJL, IP, KSP, FTM, RSM. Data curation: JS, RPMP, MG. Formal analysis: MJL, RPMP, AC, RSM. Funding acquisition: JS, MJL, IP, JGL, CJMK, KSP, FTM, RSM. Investigation: JS, RPMP, MG. Project administration: JS, KSP, RSM. Resources: KSP. Software: RPMP, MJL. Supervision: JGL, CJMK, KSP, FTM, RSM. Visualization: JS, MJL, AC, FTM, RSM. Writing - original draft: JS, CJMK, FTM, RSM. Writing - review & editing: JS, MJL, RPMP, MG, IP, AC, JGL, CJMK, KSP, FTM, RSM. All authors read and approved the final manuscript. Ethics approval and consent to participate Written informed consent was obtained from the volunteer sleepers. The College of Medicine Research and Ethics Committee (COMREC) in Malawi approved the study (Proposal Number P.02/19/2598). Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. References Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015;526:207–11; doi: 10.1038/nature15535 . 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McCann","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBADOQSTHYh5GBgYGwhoMUYwmYnUkohQQEiL+bTDDz/83HEnfX57dwIzb45dvsFh5mcP3jDYyG44gF2LzO00Y8neM89yN5w5u4GZd1uy5YbDbOaGcxjSjHFpkZDOYWPgbTucu0EiF6SF2cDgMIOZNA/D4UR8Whj/th1Ol58B1lIP1ML+DajlP14tzEBbEhhugLUcBmrhAdlyAI+WNGNp2bbDhiC/HJy77biB5GGeMsk5BsnGM3FqSX748W3bYXn59t6ND95uqzbgO96+TeJNhZ1sHw4tKACsRgFMGhChHA7kG0hRPQpGwSgYBSMBAACWw1vsJguT8wAAAABJRU5ErkJggg==","orcid":"","institution":"University of Maryland, Baltimore","correspondingAuthor":true,"prefix":"","firstName":"Robert","middleName":"S.","lastName":"McCann","suffix":""}],"badges":[],"createdAt":"2024-11-28 21:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5545048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5545048/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06887-9","type":"published","date":"2025-07-03T15:58:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81507067,"identity":"b4ad105b-9b37-42d7-86ea-0cc4ac717bd0","added_by":"auto","created_at":"2025-04-28 05:33:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3670239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe experimental set-up for studying house-entry behavior of female malaria mosquitoes.\u003c/strong\u003e \u003cstrong\u003e(a) \u003c/strong\u003eSchematic top view of the screened enclosure (12 x 12 m) including the experimental house (brown rectangle 3 x 5 m), the four high-speed cameras (labeled C1a, C2a, C1b, C2b), the Infra-Red lights for camera illumination (IR), and the mosquito release point (R). \u003cstrong\u003e(b) \u003c/strong\u003ePicture of the experimental setup, showing the large screened enclosure with in it the experimental house with door, window and metal roof with eave, and the four high-speed cameras and Infra-Red lights. \u003cstrong\u003e(c) \u003c/strong\u003eExample showing an overlay of all mosquito flight tracks within one experimental night with eaves and windows screened. A blue line was drawn each time a single mosquito entered the view. Orange to red colors were used to indicate when more individuals were tracked at the same time, \u003cstrong\u003e(d) \u003c/strong\u003eThe 3D coordinate system and volume in front of the house in which the mosquitoes could be tracked using our videography system. The x-axis and y-axis are oriented normal and parallel to the front wall of the house, and the z-axis is pointing vertically up. The 3D trackable volume is highlighted in white and projected on the floor, house, and the house symmetry plane. The location of the eave and window are indicated in red with the eave height between 2.12 m to 2.31 m and window height between 1.48 m to 1.97 m, respectively. \u003cstrong\u003e(e,f)\u003c/strong\u003e To study the flight activity near the eave and window, we defined corresponding volumes-of-interest near these structures, as defined by the blue boxes.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/5f9bd6334b52412b65e55b1e.png"},{"id":81507056,"identity":"a675f381-1b0d-47dd-afef-665dd96adc6f","added_by":"auto","created_at":"2025-04-28 05:33:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1899595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the five different experimental treatments, in which we systematically closed or screened the window and eave.\u003c/strong\u003e In the overview, the three rows show the different window treatment conditions (from bottom to top: open, closed and screened), and the three columns show the eave treatments (from left to right: open, closed and screened). Each condition was defined using a four-letter code, where E, W, O, C, and S stand for Eave, Window, Open, Closed, and Screened, respectively. The door was closed during all experiments. Eave and window treatments were changed using removable frames, as shown in the inset image. The inset image shows the back of the experimental house, where the eave and window treatments were the same as the front.\u003c/p\u003e","description":"","filename":"Fig201.png","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/e50717d76a9bdf7e184f33bd.png"},{"id":81507078,"identity":"5f0cd528-448e-416c-b2f9-533077601947","added_by":"auto","created_at":"2025-04-28 05:33:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":430718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePercentage of responding mosquitoes that were collected indoors at the end of each experiment.\u003c/strong\u003e Mosquitoes that flew out of the release bucket were marked as “responding”, which ranged from 477 to 499 (out of 500) per replicate. Counts of mosquitoes collected indoors were based on the sum of CDC light trap and Prokopack aspiration. Boxplots show median, 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentiles and fences of collected mosquitoes per night, by treatment (\u003cem\u003en\u003c/em\u003e=6 replicate nights per treatment, indicated by open circles).\u003c/p\u003e","description":"","filename":"fig3Perc.resp.mosq.found.indoorbyTreatment7Sep2023.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/e5a0052dc79ae48d7fe3f4da.jpg"},{"id":81508935,"identity":"db1ab8b0-b3fd-4b04-a155-b207bc60b729","added_by":"auto","created_at":"2025-04-28 05:41:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":800690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean flight activity throughout the night per treatment (a-c), and the corresponding temporal dynamics of flight activity per treatment (d).\u003c/strong\u003e Data for different treatments are color-coded, as defined at the bottom right panel.\u003cstrong\u003e (a-c) \u003c/strong\u003eWe estimated mean flight activity per treatment using \u003cstrong\u003e(a)\u003c/strong\u003e the mean number of recorded flight tracks per responding mosquito per night, \u003cstrong\u003e(b)\u003c/strong\u003e the mean flight track duration per responding mosquito per night, and \u003cstrong\u003e(c)\u003c/strong\u003e the mean duration per flight track. \u003cstrong\u003e(d)\u003c/strong\u003e We estimated the temporal dynamics of flight activity per treatment using the mean number of observed flight tracks per video frame within 10-minute bins. Boxplots in \u003cstrong\u003e(a-c) \u003c/strong\u003eshow median, 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentiles and fences (\u003cem\u003en\u003c/em\u003e=6 replicate nights per treatment, indicated by open circles). Dots in \u003cstrong\u003e(d)\u003c/strong\u003e represent means, and bars show 95% confidence intervals.\u003c/p\u003e","description":"","filename":"fig4Totaltracksanddurationbytreatment5April2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/e328b6733387bbde57e251ef.jpg"},{"id":81507065,"identity":"111d1094-7e1f-41ba-b51b-6b37051e70dd","added_by":"auto","created_at":"2025-04-28 05:33:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1811909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe density distribution of mosquitoes flying in front of the experimental house, for the five house treatments.\u003c/strong\u003e The spatial distribution of relative proportion of time spent in front of the house was estimated in 3D. Here, the 3D spatial distribution is projected on to flattened 2D planes for visualization. The X-Y plane shows the flattened distribution as observed from above, looking down to the ground. The Y-Z plane shows the flattened distribution as observed from behind the cameras, looking towards the front surface of the house. The X-Z plane shows the distribution as observed from Y ≥ 2.5 m, looking towards Y = 0 m. The location of the eave and window are indicated using red rectangles on the Y-Z plane. The relative proportion of time spent is shown using the colors as indicated by the color bar, where yellow and blue show high and low activity in that area, respectively.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/eafbe3ee0cc344d6abd098f6.png"},{"id":81507076,"identity":"d2ceff90-8b42-4c3c-9b94-98becd974414","added_by":"auto","created_at":"2025-04-28 05:33:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":376052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlight activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAn. gambiae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein front of the house (a), and near the eave (b) or window (c), separated by treatment. \u003c/strong\u003eFlight activity was expressed as the time spent in the specified volume over the full night of tracking (20:00h to 04:00h). \u003cstrong\u003e(a)\u003c/strong\u003e All activity in the trackable area in front of the house (as depicted in Figure 1d). \u003cstrong\u003e(b,c)\u003c/strong\u003e Flight activity within the volumes near the eave and window (\u003cstrong\u003e(b)\u003c/strong\u003e and \u003cstrong\u003e(c)\u003c/strong\u003e, respectively), as indicated by the blue boxes in the house schematics. Results are color-separated by treatment as indicated at the bottom. Boxplots show the median, 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentiles and fences (\u003cem\u003en\u003c/em\u003e=6 replicate nights per treatment, indicated by open circles).\u003c/p\u003e","description":"","filename":"fig6TotaltimespentineaveandwindowareabyTreatment11Sep2023copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/72cf6275b26be7fb29a4f040.jpg"},{"id":81507075,"identity":"e1e1b5af-c32d-4000-a899-7c3df05c1e29","added_by":"auto","created_at":"2025-04-28 05:33:45","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":738238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemporal dynamics of flight activity throughout the night, in front of the house (a), and near the eave (b) or window (c), separated by treatment. \u003c/strong\u003eFlight activity was quantified as time spent in the specified volume in 10-minute bins, from 20:00h to 04:00h. \u003cstrong\u003e(a)\u003c/strong\u003e All flight activity in the trackable area in front of the house. The trackable area is depicted in Figure 1d. Data in (\u003cstrong\u003ea\u003c/strong\u003e) are the same as Figure 4d, but scaled differently for relevant comparisons. \u003cstrong\u003e(b,c)\u003c/strong\u003e Flight activity in the volumes near the eave and window (\u003cstrong\u003e(b)\u003c/strong\u003e and \u003cstrong\u003e(c)\u003c/strong\u003e, respectively), as indicated by the blue boxes in the house schematics. Results are color-separated by treatment as indicated on the right and show the mean (dots) and 95% confidence intervals (bars) per treatment (\u003cem\u003en\u003c/em\u003e=6 replicate nights per treatment).\u003c/p\u003e","description":"","filename":"fig7Timespentas10minbins11Sep2023.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/ed867e57dd5186f78050c155.jpg"},{"id":81507071,"identity":"5c491b56-cea3-40b9-8976-575e9c722d32","added_by":"auto","created_at":"2025-04-28 05:33:44","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":475727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumber of flight tracks that either arrived, departed, returned, remained, or visited the eave or window-specific volumes (top or bottom row, respectively)\u003c/strong\u003e. The eave-specific or window-specific volumes are defined as shown in Figure 1e. Results are color-separated by treatment as indicated at the bottom. Boxplots show the median, 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentiles and fences (\u003cem\u003en\u003c/em\u003e=6 replicate nights per treatment, indicated by open circles).\u003c/p\u003e","description":"","filename":"fig8Arrivaldeparturesetc2023.12.02.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/504940f85a4cec86ca8e1cd7.jpg"},{"id":81507084,"identity":"365c2e2d-6dbf-40f2-9b68-a8d6691e23ff","added_by":"auto","created_at":"2025-04-28 05:33:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3207365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe flight patterns of all mosquitoes approaching the eave (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ea-c\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) and window (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ed-f\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e).\u003c/strong\u003e Results are shown as average streamlines color-coded with relative track density, as defined at the top of each column. Streamline thicknesses show variations in mean flight speed, where thicker lines indicate higher flight speeds. Data are projected on the three planes defined in figure 1d: \u003cstrong\u003e(a,d)\u003c/strong\u003e the house front wall (Y-Z), \u003cstrong\u003e(b,e)\u003c/strong\u003e the house symmetry plane (X-Z), and \u003cstrong\u003e(c,f)\u003c/strong\u003e the ground surface (X-Y). The flight patterns are based on all flight tracks of mosquitoes arriving at the eave-specific volume \u003cstrong\u003e(a-c)\u003c/strong\u003e and the window-specific volume \u003cstrong\u003e(d-f)\u003c/strong\u003e, as defined in figure 1e and shown here in blue. The other house structures, including the house wall, door outline, and non-used eave-specific and window-specific volume are shown in grey.\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/3d93567b1c2e0a2dd3e1aaf4.png"},{"id":81507086,"identity":"6ffa9b94-9cf7-469d-9ffe-ebe16165092e","added_by":"auto","created_at":"2025-04-28 05:33:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":444499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe flight height relative to the height of the eave versus distance from the house, for all mosquitoes approaching the eave.\u003c/strong\u003e The results are shown as boxplots per distance from the house, at a range of distances from 0.1 m to 1.0 m from the house with increments of 0.1 m. Each boxplot shows the median height, the 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentiles and fences (\u003cem\u003en\u003c/em\u003e=30 replicate nights for all treatments combined, indicated by open circles).\u003c/p\u003e","description":"","filename":"fig10Meandzacrossalltreatmentsat0.1mintervals13Jul2023.png","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/6f05766a4b64db2821c94a60.png"},{"id":86179896,"identity":"fa3f0b00-0f80-4f80-825b-edc836ff9475","added_by":"auto","created_at":"2025-07-07 16:20:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15155116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/62869aca-e5e5-4604-af5a-761e429d6b89.pdf"},{"id":81507058,"identity":"3e0a6763-b0ae-473c-9f96-5b16dcabc28c","added_by":"auto","created_at":"2025-04-28 05:33:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3213611,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5545048/v1/3cb71660e9240e5a7027229a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of eave and window modifications on house entry behavior of Anopheles gambiae","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eMalaria remains a serious global health challenge, despite the progress made over the past two decades in reducing the malaria burden. From 2000 to 2015, the prevalence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e infection in endemic regions of Africa was reduced by 50%, and an estimated 663\u0026nbsp;million clinical cases of malaria were averted [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Vector control, mostly by means of insecticide treated bed nets, contributed to more than 70% of this reduction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], demonstrating the importance of interrupting mosquito-human contact. Still, the progress made until 2015 has stalled in recent years [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnopheles gambiae s.s.\u003c/em\u003e (hereafter \u003cem\u003eAn. gambiae\u003c/em\u003e) is one of the main vectors of malaria parasites in Africa and is typically nocturnal, anthropophilic and endophilic, i.e., preferring to feed from humans indoors at night [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hence, a critical component of finding a bloodmeal host is for the mosquito to find and enter an inhabited house. Navigation towards inhabited houses is most likely triggered by carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) levels exceeding background levels [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Locating a house, or house entry point, is presumably based on the integration of perceived CO\u003csub\u003e2\u003c/sub\u003e levels, volatile host odors, and visual cues or contrasts between the house and the landscape [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The perception of volatile cues by mosquitoes is affected by odor plume structure, and this, in turn, is affected by habitat characteristics and house design [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As such, climate conditions and indoor micro-climate affect the approach and time spent indoors by mosquitoes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Finally, it should be noted that the behavioral set of responses for house entry and indoor resting can be distinct from that for actual host finding [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditional house designs in many malaria-endemic regions of Africa include three types of openings potentially used by mosquitoes as entry points: doors, windows, and open eaves. Open eaves, i.e. open gaps usually running the length of the wall where the roof and wall meet but do not join, are particularly important house entry points for \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Screening eaves reduces the number of \u003cem\u003eAn. gambiae\u003c/em\u003e and other malaria vectors in houses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], even when the windows and doors are left open [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Screening windows and doors can reduce the number of \u003cem\u003eAn. gambiae\u003c/em\u003e in houses with closed eaves [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], but not with open eaves [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditional house designs, including open eaves and unfinished materials for walls, floors and roofs, are generally associated with an increased risk of malaria parasite infection and malaria cases when compared to improved house designs (closed eaves and finished materials) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In randomized trials, house modifications (some combination of screening or closing eaves, windows, doors and ceilings) have been associated with reduced malaria parasite prevalence and reduced anemia prevalence [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This has led to growing support from policy makers for structural house modifications as a strategy for malaria vector control [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnderstanding mosquito flight towards window and eave openings, the time spent near these entry areas, and success rates of finding entry openings provides valuable information for designing mosquito-proof homes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Filming and analyzing mosquito flight tracks around house entry points allows for the collection of detailed information about the flight behavior of mosquitoes when approaching a house, and how this leads to finding and entering houses (e.g. flight trajectories, time spent near windows and eaves, persistence and angle of approach). This fundamental knowledge on strategies used by mosquitoes to locate inhabited houses is essential for designing effective house modifications to prevent mosquito entry [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and it is also key for the design and optimal placement of other vector control tools used on or near houses, such as eave tubes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], spatial repellents, odor baited traps, or a combined push-pull strategy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the current study, we assessed how different modifications of eaves and windows affect the flight dynamics of female \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes around the house, and consequently the house entry behavior and rates. Over the course of 240 hours of experimental recordings, we captured nearly seventy thousand mosquito flight tracks, amounting to approximately 76 hours of mosquito flight data. We used these data to quantify the spatial-temporal distribution of mosquitoes flying around the house, their typical house approach and entry behavior, and how the eave and window treatments affected these.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMosquito colony\u003c/h2\u003e \u003cp\u003eAll mosquitoes used in this study were colony-reared \u003cem\u003eAn. gambiae\u003c/em\u003e (Kisumu strain). Eggs to establish the colony were initially obtained from the Malaria Alert Centre, Blantyre, Malawi, and the colony was maintained in the laboratory facilities at Majete Wildlife Park in Chikwawa District, Malawi. The colony rearing facility was not climate controlled. Temperature and relative humidity in the colony rearing facility ranged from 24\u0026deg;C to 36\u0026deg;C and from 62\u0026ndash;85%, respectively. Mosquitoes were blood fed twice per week on a human arm, and eggs were distributed over larval rearing trays measuring 46 \u0026times; 30 \u0026times; 9 cm. Larval trays were filled with water from a well near the laboratory facility or from a tap at the nearby Kapichira Power Station. Three hundred to four hundred larvae per tray were fed on ground pellets of Marltons koi and pond fish food (Marltons Pet Care Pty Ltd, South Africa). Pupae were collected daily and placed in cages for emergence to adults. All cages with adult mosquitoes were provided a 10% sucrose solution via a piece of soaked cotton wool. Cages with experimental mosquitoes were not provided with a blood meal prior to the experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental set-up\u003c/h3\u003e\n\u003cp\u003eExperiments were performed in a semi-field screened enclosure measuring 12.0 \u0026times; 12.0 \u0026times; 2.1\u0026ndash;4.0 m (length, width, height) at the Majete Wildlife Park in Chikwawa District, Malawi. The walls of the screened enclosure were made from fiber glass, mosquito-proof screening (Phifer Inc, USA), and the roof was a waterproof tarpaulin. Within this enclosure, we built an experimental house measuring 5.0 \u0026times; 3.0 \u0026times; 2.2\u0026ndash;2.7 m in length, width and height (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The walls of the experimental house were constructed from locally produced bricks and plastered with cement, and the roof was made with corrugated iron sheets, including a 20 cm overhang. The front side of the experimental house was fitted with a door (197 \u0026times; 60 cm inner dimensions) in the middle, two windows (30 \u0026times; 30 cm), and four removable eave frames with inner dimensions of 90 \u0026times; 10 cm per frame. The back side of the house also contained two windows and the four eave frames, but no door. The wooden door, window frames and eave frames were painted with black, water-based chalkboard paint.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe windows and eave frames could be left completely open, fitted with insect screens, or closed completely with wooden shutters. The screens were made of charcoal-colored fiber glass (Wire Weaving Co. Dinxperlo, The Netherlands), and the shutters were made of plywood painted black with water-based chalkboard paint. Using this system, we were able to systematically investigate the effect of window and eave closure and screening on mosquito house entry behavior. The door remained closed overnight for all experimental treatments.\u003c/p\u003e \u003cp\u003eTwo beds were positioned inside the house, one along each side wall, and each covered with an untreated bed net. During experimental nights, one adult man slept in each bed, under the bed nets, to act as a bait for mosquitoes. Three pairs of adult men volunteered to sleep in the house for 10 experimental nights each. Written informed consent was obtained from the volunteer sleepers. The College of Medicine Research and Ethics Committee (COMREC) in Malawi approved the study (Proposal Number P.02/19/2598). A CDC light trap (John W. Hock Ltd, USA) was placed near each bed to collect a sample of mosquitoes that entered the house [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCamera and real-time mosquito tracking set-up\u003c/h3\u003e\n\u003cp\u003eWe used a multi-camera videography system to track the three-dimensional flight kinematics of \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes around the experimental house. The videography system consisted of four synchronized machine-vision cameras (Basler acA2040-90umNIR, USB 3.0, Basler AG, Germany), equipped with 16 mm f1.4 wide-angle lenses (Kowa LM16HC, Kowa Optical Products Co., Ltd., Japan), with lens aperture set at f2.8. The cameras were operating at 50 frames per second (fps), and a 1 ms exposure time. To improve light sensitivity of the cameras, pixels within each 2\u0026times;2-megapixel camera image were binned 2\u0026times;2. Binning combines the charge from adjacent pixels (in this case, 2\u0026times;2 pixel bins), resulting in increased light sensitivity but a reduced spatial resolution (in this case, reduced to 1\u0026times;1 megapixel). Image capture on the cameras was synchronized by means of an external trigger pulse, generated by an Arduino Uno (Arduino, Italy) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/strawlab/triggerbox.git\u003c/span\u003e\u003cspan address=\"https://github.com/strawlab/triggerbox.git\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo protect the cameras and lenses from water, heat and dust, each set was placed in a camera housing (Transpac THP 4000, Basler AG Germany). These camera housings were mounted onto an aluminum frame (MayTec Aluminium Systemtechnik GmbH, Germany) that was fixed to the concrete floor on which the house was built (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The cameras were placed at an approximate distance of 2.5 m from the front wall of the house, at heights between 0.8 m and 1.3 m. As a result, the camera system imaged the front, right side of the experimental house, including half the door and one window. The cameras were oriented slightly upwards to film the volume below the roof near the eave area. The dimensions of the area in front of the house where mosquitoes could be tracked had a size of approximately 2.5 \u0026times; 1.0 \u0026times; 1.5 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eThe filming volume was illuminated with eight near-infrared light-emitting-diode (NIR-LED) lights (2\u0026times; ABUS TVAC71000-60\u0026deg; and 6x ABUS TVAC71070-95\u0026deg;, ABUS Germany). The NIR-LED lights were mounted on a frame placed on the concrete slab directly below the area of interest (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The NIR-LED lights were directed upwards and arranged to uniformly light the filming volume near the eave and window, aiming for optimal contrast between the illuminated mosquitoes and the dark background of the house.\u003c/p\u003e \u003cp\u003eWe used an automated tracking software [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] to track in real-time the positions of multiple mosquitoes flying in the four camera views, and from these we reconstructed the three-dimensional flight tracks. The tracking software ran on a single laptop (Lenovo ThinkPad P51, Lenovo, China) with Intel Xeon E3-v6 processor and Ubuntu Linux operating system, which thus performed the real-time image analysis and object tracking for all four cameras, and the three-dimensional flight track reconstruction. Based on pilot recordings, sensor gain was set to 1.0 for all cameras, and the maximum number of simultaneously tracked mosquitoes was set to 10. Tracks were reconstructed only when the mosquito was visible in at least two of the four camera views. A dynamic background model was used with update intervals for each 100 frames and a 1% weight factor to compensate for slow changes in illumination conditions.\u003c/p\u003e \u003cp\u003eCameras were calibrated with the multi camera self-calibration routine [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] by tracking a single moving LED light by each of the four cameras (Cree SunBright 535 nm Green LED, CreeLED Inc, USA). This calibration was aligned to world reference points based on landmarks on the experimental house. The resulting coordinate system in the world reference frame was defined as {X,Y,Z}, with the X-axis oriented perpendicular to the house front wall, the Y-axis parallel to the house front wall along the ground, and the Z-axis vertically. We defined values within this coordinate system as {\u003cem\u003ex,y,z\u003c/em\u003e}, with the origin {\u003cem\u003ex,y,z\u003c/em\u003e}={0,0,0} located against the house front wall (\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0), on the ground in front of the house (\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0), and (\u003cem\u003ey\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0) at the right side of the door frame as observed from the cameras.\u003c/p\u003e \u003cp\u003eThe calibration procedure was repeated every experimental day, in case cameras would accidentally have changed position over the course of the experiment. A correction for lens distortions was generated for each camera at the start of the experiment, using a 6 \u0026times; 10 checkerboard pattern with 90 mm squares. Distortion parameters were computed using openCV procedures (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://docs.opencv.org\u003c/span\u003e\u003cspan address=\"https://docs.opencv.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Tracking results were corrected for lens distortions.\u003c/p\u003e \u003cp\u003eVideography experiments were performed from 20:00h to 04:00h. If volunteers shortly left the experimental house during the night, a five-minute buffer period was marked prior to leaving and post re-entering the experimental house. Tracking data within those time slots were removed from further analyses.\u003c/p\u003e\n\u003ch3\u003eEave and window modifications\u003c/h3\u003e\n\u003cp\u003eWe evaluated five experimental house modifications (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For our control condition, both the windows and screens were fully open (eave open-window open; EO-WO). We used two treatments to test the effect of window modifications on mosquito house entry behavior. In the first treatment, we screened the window and left the eaves open (EO-WS), and in the second treatment we closed the window while leaving the eaves open (EO-WC). To test the effect of eave modifications on mosquito house entry behavior, we used two treatments in which we screened and closed the eaves, while keeping the window screened (ES-WS and EC-WS, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eExperimental procedure\u003c/h3\u003e\n\u003cp\u003eBefore each experiment, the house was prepared by closing, screening, or leaving open the eaves and windows, as randomly assigned for each replicate night of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Each treatment was in place for six replicate nights (see experimental treatment schedule in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the day of each experimental replicate, 500 female mosquitoes (5\u0026ndash;8 days old and not previously blood-fed), were selected before 12:00h and set aside in the insectary in a release bucket (\u0026Oslash;: 12.5 cm, height: 12.5 cm), covered with a mesh and provided with water-soaked cotton wool. Two volunteers slept inside the house under untreated bed nets, starting at 19:30h. The volunteers\u0026rsquo; heads were positioned at the front (door) side of the house, and each pair shifted beds (left or right side of the house) after each replicate. At 19:30h the two CDC light traps at the end of each bed were turned on, with their lights switched off, and the bucket with mosquitoes was placed in the screened enclosure, 5.8 m in front of the experimental house.\u003c/p\u003e \u003cp\u003eAt 20:00h, mosquitoes were released from the bucket by lifting the mesh via a fishing line operated from outside the screened enclosure. Mosquito flight was tracked until 04:00h, after which the CDC light traps were turned off, and the volunteers could leave the house. Temporary absence of volunteers during the recording period was recorded in a logbook. A Prokopack aspirator (John W. Hock Company, USA) was used to collect mosquitoes from inside the experimental house at 04:00h. Together with these Prokopack catches, CDC light trap catches were shortly frozen and collected mosquitoes were then counted. Mosquitoes remaining in the release bucket were also counted, and the number of responding mosquitoes for each replicate night was defined by subtracting the number remaining in the release bucket from the initial 500 mosquitoes. Remaining mosquitoes found inside the screened enclosure later that day were removed with the Prokopack and discarded after freezing. Experimental replicates were carried out no more frequently than every other day to ensure proper preparation and allow any uncaught mosquitoes remaining in the screened enclosure to die before the next experimental replicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe real-time tracking algorithm used a Kalman predictor to reconstruct three-dimensional flight paths from stereoscopic videography data [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and thus the output data consisted of Kalman-filtered flight paths defined by location, flight velocity, and the Kalman covariance error e(\u003cem\u003et\u003c/em\u003e). In post-processing, we filtered the resulting database of flight tracks in two steps. To remove potential extrapolation errors from the Kalman predictor, we deleted the end of tracks if either the estimated flight speed was greater than 1.5 m/s or the Kalman covariance error was larger than 0.01. We then discarded all tracks that were shorter than 10 cm or less than 0.2 seconds (10 video frames at 50 fps). These settings were based on a sensitivity analysis and the assumption that flying \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes have a maximum flight speed of less than 1.5 m/s. The resulting flight paths consisted of the temporal dynamics of the three-dimensional location {\u003cem\u003ex\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e),\u003cem\u003ey\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e),\u003cem\u003ez\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e)} and velocity {\u003cem\u003eu\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e),\u003cem\u003ev\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e),\u003cem\u003ew\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e)} of each flying mosquito; these were used for our subsequent analyses.\u003c/p\u003e \u003cp\u003eWe used all combined flight tracks per treatment to calculate average mosquito density distributions and flight velocity distributions around the house. For this, we divided the filming volume into 40\u0026times;40\u0026times;40 voxels (spatial bins), resulting in an approximate voxel size of 5 cm in the X- and Z-direction, and 7.5 cm in the Y-direction. In each voxel we estimated the mosquito density as the relative proportion of time mosquitoes spent in that voxel, defined as \u003cem\u003eT\u003c/em\u003e* = \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e/\u003cem\u003eT\u003c/em\u003e\u003csub\u003etotal\u003c/sub\u003e, where \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the time spent in voxel \u003cem\u003ei\u003c/em\u003e, and \u003cem\u003eT\u003c/em\u003e\u003csub\u003etotal\u003c/sub\u003e is the total flight time. We visualized these density distributions as heat maps projected on three 2D planes (X-Y, X-Z, and Y-Z). We determined the flight velocity vector in each voxel as the mean flight velocity of all mosquitoes that passed through that voxel. We visualized the velocity distributions using streamline plots derived from these velocity fields, projected on the same set of 2D planes as for the density distributions (X-Y, X-Z, and Y-Z).\u003c/p\u003e \u003cp\u003eFor measuring and comparing flight activities near the eave and window area, we defined volumes-of-interest around the eave and window (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, respectively). These volumes had the same rectangular or square shape as the eave or window, respectively, but extended 10 cm on each side (in the Y- and Z-direction). The volumes started at the wall and extended 30 cm outward in the direction perpendicular to the wall (in the X-direction). We then identified all flight tracks that intersected these volumes around the eave and window. Based on these, we quantified flight activity around the window and eave using the time that mosquitoes spent in the corresponding volumes. We determined this time spent in each volume by summing all durations that flight tracks remained in the defined volume. We did this for each experimental night, and for an array of time bins with a temporal resolution of 10 minutes.\u003c/p\u003e \u003cp\u003eNext, we used the flight tracks around the window and eave to study when and how mosquitoes visited the window and eave, and when and how they arrived, departed, remained in, and returned to these volumes. We defined \u0026lsquo;arrivals\u0026rsquo; as flight tracks that started at least 10 cm outside the volume of interest and ended within the volume. \u0026lsquo;Departures\u0026rsquo; started within the volume of interest and ended at least 10 cm outside the volume. \u0026lsquo;Visitors\u0026rsquo; started outside the volume, entered the volume, left the volume, and then ended outside the volume. \u0026lsquo;Returnees\u0026rsquo; started inside the volume, left the volume, re-entered the volume, and finally ended inside the volume. \u0026lsquo;Remainers\u0026rsquo; started and ended inside the volume, without moving outside the volume. Note that if a flight track ended within the window or eave volume, the mosquito might have entered the house or have landed on the house, because the tracking algorithm only tracked mosquitoes flying outside the house.\u003c/p\u003e \u003cp\u003eBased on these data, we determined the number of mosquitoes that showed each type of flight behavior (visiting, arriving, departing, remaining and returning). We then used the flight kinematics data to determine the behavior-specific flight dynamics around the eave and window. Specifically, we constructed streamline plots, both per treatment and across all 30 replicates, for all mosquitoes that arrived at the volumes around the eave and window. To focus on the approach kinematics only, we removed the parts of the tracks after arrival.\u003c/p\u003e \u003cp\u003eWe used ANOVA to test for differences among treatments in various flight kinematics and house entry parameters. The dependent parameters were the number of responding mosquitoes, the percentage of responding mosquitoes collected inside the experimental house, flight track duration (time spent), and the number of flight tracks. We used Tukey\u0026rsquo;s HSD for pairwise comparisons when the ANOVA test showed a significant difference between treatments. We also used ANOVA to test for differences in house entry rates among the three pairs of volunteer sleepers.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eWe performed experiments on 30 nights (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 replicates per treatment) in the period between 16 March to 20 June 2020. The average number of responding mosquitoes, i.e. those that left the release bucket overnight, was 491.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 per night (98%). There was no effect of house treatment on the number of responding mosquitoes (Table S2, ANOVA, P\u0026thinsp;=\u0026thinsp;0.459).\u003c/p\u003e\n\u003ch3\u003eIndoor mosquito collections\u003c/h3\u003e\n\u003cp\u003eHouse entry rates based on CDC light trap and indoor Prokopack collections were independent of the three volunteer pairs used to lure the mosquitoes into the experimental house (ANOVA, P\u0026thinsp;=\u0026thinsp;0.885). The percentage of responding mosquitoes that entered the house was high for all treatments with open eaves, with a median of 47.3% (IQR: 12.1%), 28.9% (IQR: 24.0%), and 46.3% (IQR: 12.8%) for the EO-WO, EO-WC, and EO-WS treatments, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table S2). Among the three treatments with open eaves, we did not see a significant effect of window treatment (open, closed or screened) on indoor mosquito collections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, TukeyHSD, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). As expected, among the three treatments with screened windows, closing or screening the eaves drastically reduced the number of mosquitoes found indoors compared to open eaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, TukeyHSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The percentage of responding mosquitos caught indoors was reduced to a median of 1.9% (IQR: 1.4%) and 1.8% (IQR: 0.3%) when eaves were screened or closed, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMosquito flight activity in space and time\u003c/h3\u003e\n\u003cp\u003eIn total, we recorded and reconstructed 69,025 flight tracks, which resulted in a median of 3.6 (IQR: 4.4) flight tracks per responding mosquito per experimental night (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), with a median cumulative track duration per responding mosquito per night of 11.5 (IQR: 17.5) seconds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), and a median track duration per flight of 3.7 (IQR: 1.2) seconds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The number of simultaneously recorded tracks was well below our tracking algorithm limit of 10 simultaneous tracks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The mean number of tracks per video frame varied from approximately one flight track in the first hour of the night (for treatments with eaves screened or closed), to values below 0.5 towards the end of the night, irrespective of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe number of flight tracks per responding mosquito per night (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) was not statistically different between house treatments (ANOVA, Pr(F)\u0026thinsp;=\u0026thinsp;0.383). The cumulative track duration per responding mosquito per night (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) was marginally different between treatments (ANOVA, Pr(F)\u0026thinsp;=\u0026thinsp;0.062); the greatest differences in pairwise comparisons were between the treatment with eaves and windows screened (ES-WS) and those with open eaves (EO-WO, EO-WC, and EO-WS; TukeyHSD, adjusted P\u0026thinsp;=\u0026thinsp;0.126, 0.099, and 0.077, respectively). Mean duration per track was significantly longer for the house treatment with both eaves and windows screened (ES-WS), than for the treatments with open eaves (EO-WO, EO-WC, and EO-WS; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, TukeyHSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not for the treatment with eaves closed and windows screened (EC-WS; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, TukeyHSD, P\u0026thinsp;=\u0026thinsp;0.120).\u003c/p\u003e \u003cp\u003eThe spatial distribution of mosquito flight tracks in front of the experimental house, as measured by relative proportion of time spent in the 40\u0026times;40\u0026times;40 voxels, was generally concentrated near the eaves of the house (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Based on these spatial distributions of relative proportion of time spent, we estimated the total time spent flying in front of the house by all mosquitoes combined within each treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The time spent flying in the full trackable area was marginally different between house treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, ANOVA, Pr(F)\u0026thinsp;=\u0026thinsp;0.059). Based on the spatial distributions shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the concentration of flight activity near the eaves appeared strongest on nights with eaves and windows screened (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee; ES-WS). Indeed, when we compared the time spent (total track duration) in the volume around the eave, mosquitoes spent significantly more time in the eave area on nights with eaves and windows screened (ES-WS), compared to any other treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, TukeyHSD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Flight activity near the window of the house was generally lower (i.e. less relative proportion of time spent) than near the eaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Nights with eaves closed and windows screened (EC-WS) appeared to be an exception (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), with roughly equal amounts of relative proportion of time spent near the windows and eaves on those nights. Indeed, time spent (total track duration) in the defined window area was marginally different among the five treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, ANOVA, Pr(F)\u0026thinsp;=\u0026thinsp;0.056), with the eaves closed and windows screened (EC-WS) treatment appearing higher than the other treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the time spent over night in the full recording volume, and near the eave and window, separated in time bins of 10 minutes. Differences in flight activity among treatments were clearest in the first part of nightly trials, from 20:00h when measurements were started, to about 21:30h. During this period flight activity in front of the house was relatively high, and particularly so for treatments with the eave screened or closed (ES-WS and EC-WS, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). For the case with the eave closed (EC-WS), this flight activity rapidly dropped within the first hour, but for the eave screened treatment (ES-WS), this flight activity remained relatively high throughout the night.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the location of this increased flight activity differed between these two treatments (ES-WS and EC-WS). For the eave screened treatment, the increased flight activity throughout the night was concentrated in the volume near the eave (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). In contrast, the increased flight activity in the early evening for the closed eave case was mostly concentrated in front of the window (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). For all other treatments, flight activity during the night was much lower and less strongly concentrated in specific areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), although in all cases the highest flight activity remained near the eave (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Overall, less activity was observed near the window area than the eave area throughout the night (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-c).\u003c/p\u003e \u003cp\u003eWe categorized the mosquito tracks according to the flight behavior around the eave and window, for the various house treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Here, we characterized all flights near both the eave and window as \u0026ldquo;arrivals\u0026rdquo;, \u0026ldquo;departures\u0026rdquo;, \u0026ldquo;returnees\u0026rdquo;, \u0026ldquo;remainers\u0026rdquo;, and \u0026ldquo;visitors\u0026rdquo;. The distinct similarities in these categories between eave and window region show that the flight behavior around the window and eave are strikingly similar. This is particularly apparent when comparing the behaviors that lead to increased activity near the eave and window for the treatments with the eave screened and closed, respectively (ES-WS and EC-WS in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our behavioral classification shows that the resulting increased flight activity near the eave (for ES-WS) and window (for EC-WS) are both largely driven by an increased number of \u0026lsquo;departures\u0026rsquo;, \u0026lsquo;returnees\u0026rsquo;, and \u0026lsquo;remainers\u0026rsquo; for those treatments, compared to the other treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The number of \u0026lsquo;arrivals\u0026rsquo; and \u0026lsquo;visitors\u0026rsquo; have a smaller effect on the treatment-specific increased flight activity near the eave and window. This suggests that the increased flight activity near the eave and window for the screened and closed eave cases is caused primarily by mosquitoes remaining near, or departing and returning to, those house structures when they are unable to enter the house through the screening, and not by differences between treatments in the initial flight to arrive at these structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMosquito flight pattern on approaching a house\u003c/h2\u003e \u003cp\u003eNext, we zoom in on the approach flights of mosquitoes towards the eave and window (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and S1-S2). Treatment-specific streamline plots of approach flights towards the eave and window (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S2, respectively) show a consistent approach behavior between the different treatments. We therefore combined all recorded approach flight tracks for the different treatments to reconstruct the average approach flight kinematics for mosquitoes arriving at the eave or window (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-c and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed-f, respectively). For both cases, we show the average flight pattern of mosquitoes approaching the eave and window using streamlines color-coded with relative track density, and streamline thicknesses defining the flight speed. The streamline data are projected on the three planes defined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, being the projections on the house front wall (Y-Z), house symmetry plane (X-Z), and ground surface (X-Y).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results for mosquitoes approaching the eave (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-c) show that, on average, mosquitoes approached the eave using a steady ascending flight, starting at an approximate distance of 1 meter from the house front wall. Particularly the side view projection shows that the majority of flight tracks (highest density) followed this steady ascending flight straight towards the eave. A second smaller group of mosquitoes (lower density) approached the eave more from below by ascending more steeply once they get closer to the house front wall. This pattern seems independent of treatment (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The streamlines projected on the ground surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec), reveal that, on average, the mosquitoes approach the house with minimal variation in flight speed parallel to the house (left-right deviations). These results combined show that mosquitoes approaching the eave seem to fly in a highly targeted manner by rapidly ascending in a straight line towards the eave.\u003c/p\u003e \u003cp\u003eThe mosquitoes approaching the window showed a similar highly consistent flight pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed-f). The top view projection shows that most mosquitoes approached the window in a straight line, starting from relatively far from the house, and only few mosquitoes approached the window from the sides (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef). That said, the side view projection shows that the highest density of flight tracks was located near and directed towards the eave (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee), suggesting that many of the mosquitoes arriving at the window flew first towards the eave, after which they turned towards the window. This flight pattern is similar for all treatments (Figure S2).\u003c/p\u003e \u003cp\u003eThe combined density and streamline plots suggest that the majority of flying mosquitoes approached the house by ascending in a straight line towards the eave (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). We tested this by estimating flight height (relative to the eave) at various distance from the house, for all mosquitoes that approached the eave (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e and S3). This analysis is based on the same dataset as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-c and S1. On average, the mosquitoes approached the eave by ascending approximately 40 cm over a 1 m distance while flying towards the house, resulting in an average climbing flight angle of 22 degrees during this approach. The increase in flight height with decreasing distance from the house is similar between the five house treatment experiments (Figure S3). This suggests that the climbing approach flight towards the eave is highly characteristic, and does not change with house modifications, even when the eave is fully closed (EC-WS).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we studied how host-seeking \u003cem\u003eAn. gambiae\u003c/em\u003e approach and enter a house, and how modifications to common house entry points change these flight behaviors, including persistent attempts to enter when blocked by screening. We tracked free-flying mosquitoes released in a semi-field enclosure, allowing us to capture the detailed dynamics of \u003cem\u003eAn. gambiae\u003c/em\u003e flight in a large region in front of the house throughout the night. Across all five combinations of eave and window modifications tested here, \u003cem\u003eAn. gambiae\u003c/em\u003e approached the house by flying directly towards the eave, in an upward sloping path. When the eaves were open, a large percentage of \u003cem\u003eAn. gambiae\u003c/em\u003e entered the house, regardless of whether the windows were open, closed, or screened. When the eaves and windows were screened, \u003cem\u003eAn. gambiae\u003c/em\u003e spent more time in the area near the eave, persistently attempting to enter via the eave throughout the night. In contrast, with eaves closed and windows screened, \u003cem\u003eAn. gambiae\u003c/em\u003e spent more time in the area near the window \u0026ndash; generally after first approaching the closed eave. Taken together, our results highlight the tendency of \u003cem\u003eAn. gambiae\u003c/em\u003e to direct house entry toward the eaves, and to only divert to other house entry points as a secondary option.\u003c/p\u003e \u003cp\u003eUsing our real-time videography-based tracking algorithms, we recorded flight activity over an extended period each night (from 20:00h to 4:00h). While flight activity continued throughout the night for all house treatments, peak activity occurred near the start of the recording period, directly after release of the mosquitoes (20:00h to 21:00h). We released 500 female \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes per experimental night, with about 98% leaving the release bucket, resulting in a median of about 1,700 flight tracks per night. Although some mosquito flights may have been recorded over multiple tracks (e.g. if the flight exited and then re-entered the tracking area in front of the house), our data set of nearly 70,000 flight tracks represents about 76 hours of mosquito flight in front of an occupied house.\u003c/p\u003e \u003cp\u003eWe consistently observed \u003cem\u003eAn. gambiae\u003c/em\u003e approaching the occupied house by flying directly towards the eave along upward sloping flight paths, irrespective of the eave and window modifications. This characteristic flight pattern was apparent across several methods of visualizing the tracked flights, including the density distribution of all flight tracks (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and the streamline plots of flight tracks categorized as approaching the eave (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-c) and window (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed-f). These results align with previous studies of \u003cem\u003eAn. gambiae\u003c/em\u003e house entry suggesting an increase in flight altitude to eave level based on indirect observations [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our tracking data show directly that this increase in altitude is initiated at least 1 m from the house for the majority of \u003cem\u003eAn. gambiae\u003c/em\u003e, at a climbing angle of approximately 20 degrees \u0026ndash; however, we do not know the point at which these mosquitoes initiate this ascending path, as the mosquito release point was beyond our tracking area. It is likely that straight, upward sloping flights by \u003cem\u003eAn. gambiae\u003c/em\u003e are specific to approaching an occupied house, representing a unique stage of host seeking. Host seeking by \u003cem\u003eAn. gambiae\u003c/em\u003e and other mosquitoes at distances\u0026thinsp;\u0026gt;\u0026thinsp;10 m from a host (beyond visual range) is thought to consist of zigzag, cast-and-surge flight patterns dependent on wind and habitat factors that determine host odor plume characteristics, based on studies of other insect taxa [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and supported by wind tunnel experiments in mosquitoes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. As mosquitoes move closer to a host, they likely integrate additional sensory cues, including visual and thermal cues, with corresponding changes in flight patterns dependent on the specific mix of cues [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our observations of \u003cem\u003eAn. gambiae\u003c/em\u003e approaching an occupied house are the first direct evidence of their flight patterns at this stage of host seeking, filling a critical knowledge gap considering this species is generally endophilic [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results confirm that the eave is the most attractive region of human-occupied houses, and that the open eave is the primary entry point for \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additionally, the behavioral responses to eave modifications confirm that odor cues from the house occupants are important for attracting \u003cem\u003eAn. gambiae\u003c/em\u003e to the eave [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. When the eave was screened and odor could still exit the eave, mosquitoes continued flying there throughout the night while trying to enter the eave. In contrast, when the eave was fully closed and thereby the odor dispersing airflow was blocked, mosquitoes moved away from the eave and towards the screened window. In this configuration, the window was most likely the primary source of human odor dispersal, causing the mosquitoes to continue flying there following the initial approach to the eave. This initial approach toward the eave, even when the eave is fully closed, is striking and suggests that other sensory cues apart from odor may be important for approaching the house and eave. Although we did not measure CO\u003csub\u003e2\u003c/sub\u003e or other host odors, and some odor cues may have been present in the eave area when the eaves were fully closed, the difference between closed and screened eave treatments in mosquito activity near the eaves, with mosquitoes either leaving or persisting in the eave area, respectively, suggests a meaningful difference in the way these treatments were perceived by the mosquitoes.\u003c/p\u003e \u003cp\u003eTaken together, the consistent flight pattern of \u003cem\u003eAn. gambiae\u003c/em\u003e when initially approaching the house, and the divergent subsequent behaviors of these mosquitoes in response to either screened eaves or closed eaves, provides guidance for the optimum placement of vector control tools on or near houses, such as eave tubes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], odor-baited traps [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], or push-pull strategies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The low amount of flight activity at ground level near the house suggests that placing odor-baited traps or other attractant-based interventions here would be less effective than placing them closer to the eave or farther from the house. The persistence of \u003cem\u003eAn. gambiae\u003c/em\u003e to attempt house entry near screened, but not closed, eaves suggests that placing odor-baited traps near screened eaves would be more effective than near closed eaves. However, effectiveness of odor-baited traps would also depend on the attractiveness of the trap relative to competing attractants and the capture efficiency of the trap [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], warranting further studies with specific odor-baited traps to determine the optimal location for maximum catch rates.\u003c/p\u003e \u003cp\u003eScreening and closing the window while leaving the eave open had a strikingly small effect on \u003cem\u003eAn. gambiae\u003c/em\u003e flight behavior and did not reduce house entries. When the eaves were completely closed, we detected an increased flight activity around the screened windows, but these were mostly secondary approaches after visiting the closed eave (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee). These results confirm that window modifications such as screens or shutters are ineffective vector control tools for houses with open eaves if not paired with eave modifications, as previously shown [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eOur study provides the first direct evidence that female \u003cem\u003eAn. gambiae\u003c/em\u003e approach a house using a characteristic flight pattern, flying directly towards the house eave along a climbing flight path. Preventing house entry with screened eaves resulted in prolonged flight activity near the eave as mosquitoes continued to attempt entry at this same point. When the eave was fully closed, presumably preventing host odors from accumulating in the eave area, mosquitoes were deflected to the screened window after the initial approach to the eave. These divergent behaviors of \u003cem\u003eAn. gambiae\u003c/em\u003e after approaching screened and closed eaves may provide guidance for effective positioning of odor-baited traps or other outdoor vector control tools to remove mosquitoes from the population. Further studies on how mosquitoes approach and enter houses could build on our findings by incorporating additional house designs, for example increasing ventilation or the presence of indoor lights [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEO-WO: eave open-window open; EO-WS eave open-window screened; EO-WC eave open-window closed; ES-WS eave screened-window screened; EC-WS eave closed-window screened.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Themba Mzilahowa at the Malaria Alert Centre for providing mosquito eggs to establish the \u003cem\u003eAn. gambiae\u0026nbsp;\u003c/em\u003ecolony used in this study, Florence Ndalakwanji for her work maintaining the mosquito colony, and the team at the Training and Research Unit of Excellence (TRUE) for their support of the study team. We are grateful to the African Parks Network, and especially the team at Majete Wildlife Reserve, for access to the facilities at Majete and for their assistance with setting up the semi-field enclosure. We would also like to acknowledge the support of the Building Out Vector-borne diseases in sub-Saharan Africa (BOVA) network, which provided a Pump-priming Grant to KSP for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding for this study was provided through a Pump-priming Grant to KSP from the BOVA network (primary grant from BBSRC, MRC and NERC, no. BB/R00532X/1 to Steve Lindsay). RSM received additional support from NIH award no. T32AI007524 and K01TW011770.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental data that supports the findings of this study are available in the online repository Dryad at [\u003cem\u003erepository link to be added to next version\u003c/em\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: JS, MJL, IP, KSP, FTM, RSM. Data curation: JS, RPMP, MG. Formal analysis: MJL, RPMP, AC, RSM. Funding acquisition: JS, MJL, IP, JGL, CJMK, KSP, FTM, RSM. Investigation: JS, RPMP, MG. Project administration: JS, KSP, RSM. Resources: KSP. Software: RPMP, MJL. Supervision: JGL, CJMK, KSP, FTM, RSM. Visualization: JS, MJL, AC, FTM, RSM. Writing - original draft: JS, CJMK, FTM, RSM. Writing - review \u0026amp; editing: JS, MJL, RPMP, MG, IP, AC, JGL, CJMK, KSP, FTM, RSM. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the volunteer sleepers. The College of Medicine Research and Ethics Committee (COMREC) in Malawi approved the study (Proposal Number P.02/19/2598).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. 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Malaria journal. 2022;21 1:36; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12936-022-04063-3\u003c/span\u003e\u003cspan address=\"10.1186/s12936-022-04063-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org/10.1186/s12936-022-04063-3.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Anopheles gambiae, Malaria, Mosquito control, Housing, Insect flight, Videography","lastPublishedDoi":"10.21203/rs.3.rs-5545048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5545048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAnopheles gambiae\u003c/em\u003e mosquitoes transmit malaria parasites to humans mostly by biting them indoors at night. \u003cem\u003eAn. gambiae\u003c/em\u003e predominantly enter houses through ventilation openings such as open eaves and windows.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHere, we studied how flying \u003cem\u003eAn. gambiae\u003c/em\u003e approach and enter a house, and whether barriers to reduce mosquito house entry alter mosquito flight patterns. We used stereoscopic high-speed videography to reconstruct nearly 70,000 three-dimensional tracks of mosquitoes flying around a house during 30 experimental nights, with five combinations of closed or screened eaves and windows.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that these eave and window treatments did not affect the number of mosquitoes attracted to the house. In all cases, mosquitoes were most active during the early evening, with lower but sustained activity throughout the night. Most \u003cem\u003eAn. gambiae\u003c/em\u003e approached the house by flying directly towards the eave in a straight, upward sloping path, and most flight activity near the house was in front of the eave. Due to the highly attractive nature of the eave area of the house, window treatments had limited to no effect on the number of house entries when eaves were left open, highlighting the importance of closing or screening eaves to prevent mosquito house entry. For the screened eave treatment, \u003cem\u003eAn. gambiae\u003c/em\u003e spent about 10\u0026times; as much time near the eave over the course of the night compared to treatments with open or closed eaves. Moreover, these mosquitoes returned multiple times, persistently trying to enter the house. When the eave was fully closed, mosquitoes deferred from the eave area towards the screened window, but the initial approach flights remained towards the closed eave.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTaken together, these results demonstrate the tendency of \u003cem\u003eAn. gambiae\u003c/em\u003e to direct house entry toward the eaves, and to only divert to other house entry points as a secondary option. The persistent mosquito flight near screened eaves may provide guidance for the placement of outdoor vector control tools.\u003c/p\u003e","manuscriptTitle":"The effect of eave and window modifications on house entry behavior of Anopheles gambiae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 05:33:38","doi":"10.21203/rs.3.rs-5545048/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-23T14:49:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T09:32:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T09:32:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T18:29:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77788549414243111790774740623633306048","date":"2025-04-22T18:52:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18196773826910432403861019778558776439","date":"2025-04-18T13:25:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T18:13:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-17T09:29:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-04-15T15:55:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6fc9d730-1206-438c-b8ad-05dbe0623d3d","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:12:32+00:00","versionOfRecord":{"articleIdentity":"rs-5545048","link":"https://doi.org/10.1186/s13071-025-06887-9","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-07-03 15:58:24","publishedOnDateReadable":"July 3rd, 2025"},"versionCreatedAt":"2025-04-28 05:33:38","video":"","vorDoi":"10.1186/s13071-025-06887-9","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06887-9","workflowStages":[]},"version":"v1","identity":"rs-5545048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5545048","identity":"rs-5545048","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}