Human scent guides mosquito thermotaxis and host selection under naturalistic conditions

Abstract

The African malaria mosquito Anopheles gambiae exhibits a strong innate drive to seek out humans in its sensory environment, classically entering homes to land on human skin in the hours flanking midnight. To gain insight into the role that olfactory cues emanating from the human body play in generating this epidemiologically important behavior, we developed a large-scale multi-choice preference assay in Zambia with infrared motion vision under semi-field conditions. We determined that An. gambiae prefers to land on arrayed visual targets warmed to human skin temperature during the nighttime when they are baited with carbon dioxide (CO₂) emissions reflective of a large human over background air, body odor from one human over CO₂, and the scent of one sleeping human over another. Applying integrative whole body volatilomics to multiple humans tested simultaneously in competition in a six-choice assay, we reveal high attractiveness is associated with whole body odor profiles from humans with increased relative abundances of the volatile carboxylic acids butyric acid, isobutryic acid, and isovaleric acid, and the skin microbe-generated methyl ketone acetoin. Conversely, those least preferred had whole body odor that was depleted of carboxylic acids among other compounds and enriched with the monoterpenoid eucalyptol. Across expansive spatial scales, heated targets without CO₂ or whole body odor were minimally or not attractive at all to An. gambiae. These results indicate that human scent acts critically to guide thermotaxis and host selection by this prolific malaria vector as it navigates towards humans, yielding intrinsic heterogeneity in human biting risk.

Keywords: Anopheles gambiae; host preference; human scent; malaria; mosquito; olfaction; thermotaxis; volatile organic compound; volatilomics.

Figure 1.. OGTA for measuring Anopheles gambiae landing behavior at night

(A) Schematic of the odor-guided thermotaxis assay (OGTA) for use in laboratory conditions. An. gambiae females (n = 25 per trial) were introduced into a cage containing a heated landing platform surrounded by infrared LEDs. CO₂ was puffed into the cage at t = 0 min and t = 15 min adjacent to the landing platform (arrow, 1 min per pulse) and mosquito landings were recorded. (B) Example image frame of mosquitoes landing on the heated platform illuminated by the surrounding infrared light array. (C) Heatmaps of platform occupancy from a representative experiment for each stimulus combination. (D) Percentage of mosquitoes landing on the platform throughout the experiment for each stimulus combination. The arrows indicate the time points of stimulus onset. Bars represent the length of the heat stimulus duration. Mean ± SEM plotted. (E) Total number of landings on the platform for each stimulus combination. Mean ± SEM plotted. n = 5/stimulus combination. The letters indicate significant differences (p < 0.05): Fisher’s exact permutation tests with Benjamini-Hochberg correction. See also Data S1A.

Figure 2.. Semi-field system for multi-choice assays of mosquito olfactory preference during host-seeking

(A) Aerial view of the screened mosquito flight cage arena with roof (green) surrounded by 8 one-person tents. Tents (2 per side) are connected to deliver olfactory stimuli via a ducting fan assembly to the flight cage arena. (B) Side view of the mosquito flight cage arena. (C) Schematic of the multi-choice olfactory preference assay. n = 200 mosquitoes per nightly trial. (D) OGTA for use in the semi-field system. The ducting from each tent vents the target olfactory stimulus over a heated landing platform and mosquito landings are recorded. See also Figure S1.

Figure 3.. Anopheles gambiae prefers to land on heated targets baited with CO2 emissions reflective of a large human over background air

(A) Stimulus position and rotation during eight-choice trials with these stimuli. The CO₂ stimulus (position i), 400 ppm above atmospheric concentrations to mimic the presence of a large human, was shuffled every night. The background air control positions (ii–viii) were labeled in reference to the CO₂ stimulus position in a clockwise manner. All platforms were heated to 35°C throughout the experiment. (B) Heatmap of platform occupancy for an example night (night 1). (C) Number of mosquito landings on the OGTA platforms throughout the experiment. Mean ± SEM plotted. (D) Percentage of the total landings per platform per night. Mean ± SEM plotted. n = 6 trials. The letters indicate significant differences (p < 0.001): observed inter-individual mean landing percentage differences were compared (based on 5,000 permutation resampling simulations) against the null hypothesis assuming these arise only from positional bias, with Benjamini-Hochberg correction. See also Figures S1, S6, and Data S1B.

Figure 4.. Anopheles gambiae prefers to land on heated targets baited with the whole body odor of one human over CO2 emissions reflective of a large human

(A) Stimulus position and rotation during eight-choice trials with these stimuli. The human stimulus (position i) was shuffled every night. The remaining positions (ii–viii, CO₂) were labeled in reference to the human position in a clockwise manner. All platforms were heated to 35°C throughout the experiment. (B) Heatmap of platform occupancy for an example night (night 2). (C) Number of mosquito landings on the OGTA platforms throughout the experiment. Mean ± SEM plotted. (D) Percentage of the total landings per platform per night. Mean ± SEM plotted. n = 6 trials. The letters indicate significant differences (p < 0.05): observed inter-individual mean landing percentage differences were compared (based on 5,000 permutation resampling simulations) against the null hypothesis assuming these arise only from positional bias, with Benjamini-Hochberg correction. See also Figures S2, S6, and Data S1C.

Figure 5.. Anopheles gambiae prefers heated targets baited with the body odor of one human over another

(A) Stimulus position and rotation during eight-choice trials with whole body odor from two individual humans versus background air. The position of Human 1 (blue) and the position of Human 2 (green) relative to Human 1 were shuffled every night. The control positions (air) and the position of Human 2 were labeled in reference to Human 1. Human 2 could take any of the positions ii–viii. (B) Heatmap of platform occupancy for an example night (night 1) where Human 2 took position viii. (C) Number of mosquito landings on the OGTA platforms. Mean ± SEM plotted. (D) Percentage of the total landings per platform per night. Mean ± SEM plotted. n = 7 trials (control positions have 6 data points since one of the positions was occupied by Human 2 every night). The letters indicate significant differences (p < 0.05): observed inter-individual mean landing percentage differences were compared (based on 5,000 permutation resampling simulations) against the null hypothesis assuming these arise only from positional bias, with Benjamini-Hochberg correction. (E) Preference index calculated from total landings on Human 1 and 2. One sample t test for significant difference to 0: **p = 0.0054. (F) Effect of distance between Human 1 and 2 on the preference index. Coefficient of determination R² = 0.009. See also Figures S3, S6, and Data S1D.

Figure 6.. Anopheles gambiae differentially prefers body odor from certain individuals in a six-choice context

(A) Stimulus position and rotation during six-choice trials with six humans. Humans were shuffled randomly every night and could occupy any of the six positions (i–vi) numbered relative to their position in the cage. (B) Heatmaps of platform occupancy for an example night (night 3). (C) Number of mosquito landings on the platforms. Mean ± SEM plotted. (D) Percentage of the total landings per platform per night. Mean ± SEM plotted. n = 6 trials. The letters indicate significant differences (p < 0.05): observed inter-individual mean landing percentage differences were compared (based on 5,000 permutation resampling simulations) against the null hypothesis assuming these arise only from positional bias, with Benjamini-Hochberg correction. See also Figures S4, S5, S6, Table S1, and Data S1E.

Figure 7.. Whole body volatilomics reveals candidate compounds modulating Anopheles gambiae olfactory preferences for human scent

(A) Heatmap of 15 identified volatile organic compounds in human whole body odor demonstrating substantial variation between participants. Scale bar represents the amounts of analytes detected normalized to the internal standard, with red indicating a higher concentration and blue indicating lower concentration. Normalized across all participants. Heatmap constructed using MetaboAnalyst 5.0. (B) PLS-DA score plot containing all chemical features detected. Ellipses indicate 95% confidence intervals. Plot constructed using MetaboAnalyst 5.0. (C) Variation in 4/15 compounds of interest: acetoin, isobutyric acid, isovaleric acid, and eucalyptol detected in whole body odor from these humans. The line indicates the median, the box marks the lower and upper quartile, and the whiskers the 1.5 interquartile distance; outliers are indicated by black crosses. n = 6 trials. The letters indicate significant differences (p < 0.05) in compound abundance between humans: Fisher’s exact permutation tests with Benjamini-Hochberg correction. See also Figure S7, Data S1F and S2.