All moving animals, including flies [], sharks [], and humans [], experience a dynamic sensory landscape that is a function of both their trajectory through space and the distribution of stimuli in the environment. This is particularly apparent for mosquitoes, which use a combination of olfactory, visual, and thermal cues to locate hosts []. Mosquitoes are thought to detect suitable hosts by the presence of a sparse COplume, which they track by surging upwind and casting crosswind []. Upon approach, local cues such as heat and skin volatiles help them identify a landing site []. Recent evidence suggests that thermal attraction is gated by the presence of CO], although this conclusion was based experiments in which the actual flight trajectories of the animals were unknown and visual cues were not studied. Using a three-dimensional tracking system, we show that rather than gating heat sensing, the detection of COactually activates a strong attraction to visual features. This visual reflex guides the mosquitoes to potential hosts where they are close enough to detect thermal cues. By experimentally decoupling the olfactory, visual, and thermal cues, we show that the motor reactions to these stimuli are independently controlled. Given that humans become visible to mosquitoes at a distance of 5–15 m [], visual cues play a critical intermediate role in host localization by coupling long-range plume tracking to behaviors that require short-range cues. Rather than direct neural coupling, the separate sensory-motor reflexes are linked as a result of the interaction between the animal’s reactions and the spatial structure of the stimuli in the environment.

Results

−1 laminar flow and recorded over 20,000 flight trajectories (mean length >6 s) using a three-dimensional real-time tracking system [ 17 Straw A.D.

Branson K.

Neumann T.R.

Dickinson M.H. Multi-camera real-time three-dimensional tracking of multiple flying animals. 2 level of 400 ppm, we introduced a CO 2 plume with a peak concentration of 2,500 ppm for 3 hr during the mosquitoes’ circadian activity peak. In separate experiments, we measured the CO 2 concentration at 65 points in the tunnel and constructed a spatial model of the plume ( 1 van Breugel F.

Dickinson M.H. Plume-tracking behavior of flying Drosophila emerges from a set of distinct sensory-motor reflexes. 2 , the female mosquitoes did not exhibit plume-tracking behavior ( 2 plume, the female mosquitoes showed stereotypical cast and surge behavior in response to CO 2 concentrations greater than 500–600 ppm ( 11 Dekker T.

Cardé R.T. Moment-to-moment flight manoeuvres of the female yellow fever mosquito (Aedes aegypti L.) in response to plumes of carbon dioxide and human skin odour. 18 Dekker T.

Geier M.

Cardé R.T. Carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours. Figure 1 Wind Tunnel, CO 2 Plume, and Example Flight Trajectories Show full caption (A) Wind tunnel used in our experiments. Color borders indicate top, side, and upwind views used in subsequent panels. 2 plume based on 65 measurements in the wind tunnel; see the (B) Heatmap of a turbulent-flow, particle-diffusion model of the COplume based on 65 measurements in the wind tunnel; see the Experimental Procedures and Figure S1 for details. The white dot indicates a mosquito, drawn to scale. (C) Example flight trajectory in clean air. The two colored arrowheads show synchronized points across side and top views. The spacing between the points (33-Hz intervals) indicates the animal’s speed. (D) Example flight trajectory in the presence of a CO 2 plume, showing the mosquitoes’ stereotypical behavior exploring the high-contrast object after sensing CO 2 . 2 (or control) experience is plotted below and color-coded within the trajectories using the scale in (B). See also In both (C) and (D), an estimate of the animal’s instantaneous CO(or control) experience is plotted below and color-coded within the trajectories using the scale in (B). See also Figure S1 for additional trajectories and Movie S1 for animations. Figure 2 CO 2 Triggers Mosquitoes to Explore High-Contrast Dark Objects Show full caption (A) Heatmap showing where female mosquitoes spent their time over a 3-hr period. The top panel shows a side view of the data. The bottom panel shows a top down view of the data over the altitude range indicated by the vertical pink line in the top panel. The right panel shows a photograph of the wind tunnel. (B) Same as (A), but in the presence of a CO 2 plume. (C) Same as (A), but in the presence of a CO 2 plume and with a black and a white visual object on the floor of the tunnel. (D) Same as (A), but with male mosquitoes in clean air. We did not find any qualitative differences in male mosquitoes’ behavior in the presence of a CO 2 plume (not shown). (E) Relative flight activity, measured as the ratio of time mosquitoes spent flying in the presence of a CO 2 or clean air plume compared to their prior activity. (F) The ratio of the total time mosquitoes spent near the object divided by the total time they spent elsewhere for CO 2 and clean air conditions. Shading shows bootstrapped 95% confidence intervals of the mean. (G) Time elapsed between when mosquitoes left the plume (conservatively defined here as 401 ppm) and when they approached to within 3 cm of the object. (H) Example trajectories (top row, side view; bottom row, top-down view) that contributed to the histogram shown in (G), demonstrating the circuitous path many mosquitoes took from the plume to the object. Only the trajectory segments between plume exit (pink arrow) and object approach are shown. See also Figure S2 for a description of plume-tracking behavior and Figure S3 To study host-seeking behavior in Aedes aegypti, we released mated females in a wind tunnel with 40 cm slaminar flow and recorded over 20,000 flight trajectories (mean length >6 s) using a three-dimensional real-time tracking system [] ( Figure 1 A). We projected a low-contrast checkerboard pattern on the entire floor of the tunnel and placed one high-contrast spot 20 cm from the upwind end ( Figure S1 A). After allowing the mosquitoes to acclimatize for 1 hr in clean air, which contained a background COlevel of 400 ppm, we introduced a COplume with a peak concentration of 2,500 ppm for 3 hr during the mosquitoes’ circadian activity peak. In separate experiments, we measured the COconcentration at 65 points in the tunnel and constructed a spatial model of the plume ( Figures 1 B, S1 B, and S1C). When combined with our three-dimensional tracking, the plume model made it possible to reconstruct the olfactory experiences of the mosquitoes along each individual trajectory [] (see the Experimental Procedures for additional details). In control experiments, in which we injected clean air instead of CO, the female mosquitoes did not exhibit plume-tracking behavior ( Figures 1 C, 2 A , and S1 ). In the presence of the COplume, the female mosquitoes showed stereotypical cast and surge behavior in response to COconcentrations greater than 500–600 ppm ( Figure S2 ), as has been reported previously [].

2 plume, they spent much of their time exploring the dark visual feature on the floor of the wind tunnel, despite its location approximately 10 cm below the CO 2 plume ( 2 presentation ( 10 Bidlingmayer W.L. How mosquitoes see traps: role of visual responses. 19 Gibson G.

Torr S.J. Visual and olfactory responses of haematophagous Diptera to host stimuli. 20 Muir L.E.

Kay B.H.

Thorne M.J. Aedes aegypti (Diptera: Culicidae) vision: response to stimuli from the optical environment. 21 Browne S.M.

Bennett G.F. Response to mosquitoes (Diptera: Culicidae) to visual stimuli. 2 , the males did not show any qualitative behavioral changes compared to their clean air responses and did not exhibit any evidence of plume-tracking behaviors. Thus, the influence of CO 2 on the reaction to visual features appears to be a sex-specific behavior associated with blood foraging. In addition to plume tracking and object attraction, CO 2 also elicited a significant increase in the general flight activity of females (3 versus 0.88%/mm3). They also spent much of their time in the vicinity of the tube through which CO 2 was introduced into the tunnel. Although we tried to minimize the visual signature of this tube, it is likely that the mosquitoes could see it to some degree. The most salient result of our experiments, however, was the influence of odor on the attractiveness of the visual object. When the female mosquitoes were exposed to clean air, they explored the ceiling and walls of the wind tunnel but rarely approached the visual feature. By contrast, when mosquitoes were exposed to the COplume, they spent much of their time exploring the dark visual feature on the floor of the wind tunnel, despite its location approximately 10 cm below the COplume ( Figures 1 D, 2 B, and S1 and Movie S1 ). The attraction to this visual feature persisted through the entire length of the 3-hr COpresentation ( Figure S3 ). During these exploratory bouts, the mosquitoes hovered near the visual object at a distance of approximately 3 cm. These results are, to our knowledge, the first direct observation of odor-gated visual attraction in mosquitoes. In previous unpublished experiments, however, Richard Dow came to similar conclusions using trap assays without access to detailed knowledge of either the animals’ trajectories or the structure of the odor plume []. In experiments in which we provided both a bright-white and a dark-black object on a gray background, the mosquitoes only explored the dark object ( Figure 2 C), consistent with trap assays using wild mosquitoes []. In contrast to the female mosquitoes, males vigorously explored the visual feature in clean air ( Figure 2 D). In the presence of CO, the males did not show any qualitative behavioral changes compared to their clean air responses and did not exhibit any evidence of plume-tracking behaviors. Thus, the influence of COon the reaction to visual features appears to be a sex-specific behavior associated with blood foraging. In addition to plume tracking and object attraction, COalso elicited a significant increase in the general flight activity of females ( Figure 2 E). Averaged across all of the trajectories, the female mosquitoes spent close to 5% of their time near the visual object compared to all other parts of the wind tunnel ( Figure 2 F). Normalized for volume, the mosquitoes spent ten times more time near the object than in the rest of the tunnel (8.8%/mmversus 0.88%/mm). They also spent much of their time in the vicinity of the tube through which COwas introduced into the tunnel. Although we tried to minimize the visual signature of this tube, it is likely that the mosquitoes could see it to some degree.

2 was not detectable using a sensitive meter (LI-6262 CO 2 /H 2 O analyzer, LI-COR), and our spatial model based on measurements within the plume estimated a concentration at the floor that was no different from background levels. Thus, when the mosquitoes approached and explored the visual object, they were not experiencing CO 2 above the background. We calculated the time elapsed between leaving the plume and approaching the visual object for the 126 trajectories that contained continuous, un-fragmented data between these two events ( 2 plume, whereas others took circuitous paths in which more than 10 s elapsed before reaching the object ( The visual object was placed 10 cm below the plume, at which distance the COwas not detectable using a sensitive meter (LI-6262 CO/HO analyzer, LI-COR), and our spatial model based on measurements within the plume estimated a concentration at the floor that was no different from background levels. Thus, when the mosquitoes approached and explored the visual object, they were not experiencing COabove the background. We calculated the time elapsed between leaving the plume and approaching the visual object for the 126 trajectories that contained continuous, un-fragmented data between these two events ( Figure 2 G). Some mosquitoes approached the object immediately after leaving the COplume, whereas others took circuitous paths in which more than 10 s elapsed before reaching the object ( Figure 2 H). Many of the mosquitoes that approached the visual feature continued to explore the area for another 10 s or more without reencountering the plume (see Movie S1 ). These results indicate that attraction to visual features can be triggered by a brief prior exposure to odor and does not require simultaneous experience of the two cues. Our estimates are necessarily conservative because our tracking system cannot reliably maintain the identities of individual mosquitoes over periods longer than 10–20 s.

2 -gated thermal attraction described in a previous study [ 6 McMeniman C.J.

Corfas R.A.

Matthews B.J.

Ritchie S.A.

Vosshall L.B. Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans. 2 plume (p < 0.01), indicating that CO 2 does not appear to directly gate the attraction to warm objects. Significantly more mosquitoes approached the warm high-contrast object than the warm nearly invisible object. This result shows that attraction to visual features increases the probability of localizing a warm object. However, more mosquitoes found the warm nearly invisible object than a room-temperature nearly invisible object, indicating that thermal signals can provide an independent source of information about the location of potential hosts. Figure 3 Visual Stimuli Provide an Intermediate Cue, Linking Long-Range Olfactory Cues and Short-Range Heat Sensing Show full caption (A) Photograph of the ITO-coated glass pad. (B) Measurements of the thermal plume created by the heated glass pads at altitudes of 0.5, 2.5, and 6.5 cm, colored orange, purple, and black, respectively. (C) Photographs and thermal images of the stimuli in the wind tunnel. (D) Mean fraction of trajectories that entered an 8 × 8 × 4 cm3 volume above and downwind of either the left or right object (see F). Shading indicates 95% confidence intervals. The letters at the top indicate significantly different groups (Mann-Whitney U test with Bonferroni correction, p = 0.01). (E) Mean preference index for the test object versus control object with 95% confidence intervals. Statistics were calculated as in (D). (F) Sample trajectory entering one of the test volumes (green) used in (D) and (E). The trajectory is color-coded red for the 2 s prior to when it entered the volume. (G) Spatial representation of preference index prior to when mosquitoes entered either test volume shown in (F). For each trajectory, we selected the segments 2 s prior to when they entered either volume, in addition to the portions spent inside the volumes (red region of the trajectory shown in F). We then calculated the preference index for each 2 × 2 cm2 rectangular region as the amount of time spent on the side of the wind tunnel of the test object compared to the control object, divided by their sum. We then calculated the mean preference index for each 2 × 2 cm2 region across all trajectories and its 95% confidence interval. Colors indicate preference index for regions where the 95% confidence interval was smaller than 0.5 (out of the total range of −1 to +1); the regions with higher uncertainty are shown in black. Blue or pink colors that are more saturated than the arrows on the scale bar represent regions where the mosquitoes showed a statistically significant preference for one side or the other. The average approach trajectory for all the mosquitoes in each trial is shown as a magenta line. Because the average approach trajectories to the two objects were indistinguishable, this line shows the average approach of all trajectories for simplicity. The light-green box shows a side view of the volumes shown in (F). The colored arrows indicate the altitudes at which the temperature of the air was measured in (B). The number of trajectories that approached the test (orange), control (blue), or both (black) objects is indicated in the top right of each panel. Odor-induced visual attraction could be one mechanism by which mosquitoes first navigate toward and localize potential hosts, but other cues, such as warmth, might also aid in the final stages of foraging. To investigate the potential interaction between vision and CO-gated thermal attraction described in a previous study [], we constructed two transparent objects from indium tin oxide (ITO)-coated glass, which could be heated to a desired temperature ( Figure 3 A). We could independently manipulate the thermal and visual features of the stimulus by placing a long-pass gel filter over the glass. Such a filter appears dark to the mosquitoes but transparent to our cameras ( Figures 3 B and 3C). In each experiment, we presented the mosquitoes with two objects: a dark room-temperature control object and a test object that was either dark or nearly invisible and either room temperature or heated to 37°C ( Figures 3 D and 3E). When presented with two dark objects, one of which was warm, the female mosquitoes showed a significant preference for the warm object (p < 0.01). Although fewer mosquitoes approached either object in clean air, those that did showed a preference for the warm object that was not different than in the presence of a COplume (p < 0.01), indicating that COdoes not appear to directly gate the attraction to warm objects. Significantly more mosquitoes approached the warm high-contrast object than the warm nearly invisible object. This result shows that attraction to visual features increases the probability of localizing a warm object. However, more mosquitoes found the warm nearly invisible object than a room-temperature nearly invisible object, indicating that thermal signals can provide an independent source of information about the location of potential hosts.

The data shown in Figure 3 E provide only a simplified view of the behavioral algorithm used by the mosquitoes. To indicate how the visual and thermal cues influence behavior on a finer spatial scale, we constructed a preference index as a function of tunnel position for the 2 s before the mosquitoes approached either object ( Figures 3 F and 3G). On average, mosquitoes initially approached the objects without encountering the heat plume, which was only detectable 2–3 cm above the floor of the tunnel ( Figure 3 B). The mosquitoes that approached the object from just above the floor did, however, show a preference for the warm object from as far away as 20 cm.

12 Eiras A.E.

Jepson P.C. Responses of female Aedes aegypti (Diptera: Culicidae) to host odours and convection currents using an olfactometer bioassay. 2 O (p < 0.01) ( 12 Eiras A.E.

Jepson P.C. Responses of female Aedes aegypti (Diptera: Culicidae) to host odours and convection currents using an olfactometer bioassay. Water vapor from rapidly evaporated perspiration has been attributed as another cue mediating host-seeking behavior in mosquitoes []. To investigate the possible role of water vapor on host localization in combination with visual and thermal cues, we placed a small petri dish containing a moist KimWipe over each glass pad in combination with the infrared pass filter that provides the visual cue. In this case, mosquitoes showed a significantly stronger response to the warm object at altitudes of 6–8 cm, rather than the narrow 2-cm region above the floor in which mosquitoes responded to the heat plume without HO (p < 0.01) ( Figure 3 G). These results suggest that the secondary effect of increased humidity over a warm object may be a more important cue than the temperature of the object itself. This behavior would help mosquitoes differentiate warm radiant objects, such as dark rocks heated by the sun, from animals, which increase the humidity around them when perspiring [].