The olfactory gating of visual preferences to human skin and visible spectra in mosquitoes

Abstract

Mosquitoes track odors, locate hosts, and find mates visually. The color of a food resource, such as a flower or warm-blooded host, can be dominated by long wavelengths of the visible light spectrum (green to red for humans) and is likely important for object recognition and localization. However, little is known about the hues that attract mosquitoes or how odor affects mosquito visual search behaviors. We use a real-time 3D tracking system and wind tunnel that allows careful control of the olfactory and visual environment to quantify the behavior of more than 1.3 million mosquito trajectories. We find that CO2 induces a strong attraction to specific spectral bands, including those that humans perceive as cyan, orange, and red. Sensitivity to orange and red correlates with mosquitoes’ strong attraction to the color spectrum of human skin, which is dominated by these wavelengths. The attraction is eliminated by filtering the orange and red bands from the skin color spectrum and by introducing mutations targeting specific long-wavelength opsins or CO2 detection. Collectively, our results show that odor is critical for mosquitoes’ wavelength preferences and that the mosquito visual system is a promising target for inhibiting their attraction to human hosts.

Full text

ARTICLE
The olfactory gating of visual preferences to human
skin and visible spectra in mosquitoes
Diego Alonso San Alberto1,4, Claire Rusch1,4, Yinpeng Zhan2, Andrew D. Straw3, Craig Montell2&
Jeffrey A. Riffell 1✉
Mosquitoes track odors, locate hosts, and find mates visually. The color of a food resource,
such as a flower or warm-blooded host, can be dominated by long wavelengths of the visible
light spectrum (green to red for humans) and is likely important for object recognition and
localization. However, little is known about the hues that attract mosquitoes or how odor
affects mosquito visual search behaviors. We use a real-time 3D tracking system and wind
tunnel that allows careful control of the olfactory and visual environment to quantify the
behavior of more than 1.3 million mosquito trajectories. We find that CO
2
induces a strong
attraction to specific spectral bands, including those that humans perceive as cyan, orange, and
red. Sensitivity to orange and red correlates with mosquitoes’strong attraction to the color
spectrum of human skin, which is dominated by these wavelengths. The attraction is eliminated
by filtering the orange and red bands from the skin color spectrum and by introducing
mutations targeting specific long-wavelength opsins or CO
2
detection. Collectively, our results
show that odor is critical for mosquitoes’wavelength preferences and that the mosquito visual
system is a promising target for inhibiting their attraction to human hosts.
https://doi.org/10.1038/s41467-022-28195-x OPEN
1Department of Biology, University of Washington, Seattle, WA 98195, USA. 2University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
3Institute of Biology I & Bernstein Center Freibug, Albert-Ludwigs-Univesität Freiburg, Freiburg im Breisgau, Germany.
4
These authors contributed equally:
Diego Alonso San Alberto, Claire Rusch. ✉email: jriffell@uw.edu
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The behavioral preference of insects for certain bands in the
visible light spectrum plays a profound role in structuring
ecological communities by mediating processes such as
plant-insect/predator-prey interactions and disease transmission1–3.
For biting insects, such as mosquitoes, tsetse flies, and kissing bugs,
vision plays an essential role in various behaviors, including flight
control, object tracking for host- or nectar-finding, and locating
oviposition sites4. The visual stimuli that mediate these behaviors
are integrally tied to other host-related cues, such as scent and heat.
For instance, when combined with an odor lure, tsetse flies are
highly attracted to what humans perceive as blue color5,6,and
kissing bugs prefer visual objects only when also associated with
odors7. Visually guided mosquito behaviors are also thought to play
a role in host attraction8–10.Ithaslongbeenknownthatmosqui-
toes are attracted to dark, high-contrast objects9,11, which has led to
the development of black traps10.Forhigh-contrastvisualstimuli,
recent work has shown that certain odors stimulate visual search
behaviors in Aedes aegypti mosquitoes. This species is not attracted
to black objects in the absence of CO
2
, but after encountering a CO
2
plume, they become highly attracted to such objects11. Other cues
(heat, water vapor, skin volatiles) mediate behaviors such as landing
and biting11–13.
Despite the potential importance of color in mediating mos-
quito biting behaviors, surprisingly, details regarding other
wavelengths that attract mosquitoes or how odors sensitize that
attraction remain unclear. The visual spectra of important
resources can be diverse and dominated by short and medium
wavelengths (e.g., flowers or oviposition sites) or long wave-
lengths (e.g., human skin) (Fig. 1a, b). Despite interest in devel-
oping traps and lures that exploit mosquito spectral preferences,
only a few studies have examined these preferences, and the
results of those studies have been contradictory. For instance,
studies of Ae. aegypti have shown no difference in spectral pre-
ference in the 450–600 nm wavelength range14,15. By contrast,
other studies have demonstrated specific preferences but for
different wavelength bands: Ae. aegypti mosquitoes were attracted
to blue in one study16 and only to green–yellow in other
studies17,18. Other studies have shown that mosquitoes some-
times prefer red14,17,19, although it is thought that mosquitoes
lack opsin receptors sensitive to these wavelengths. Because many
mosquito species are attracted to dark visual objects, responses to
long wavelengths (red to human observers) may represent
achromatic responses from visual channels that are sensitive to
medium-wavelengths and therefore are perceived as dark gray
or black when presented against a light-colored background.
Nevertheless, these prior studies did not characterize the actual
flight trajectories of the mosquitoes, nor control for the change in
behavioral state associated with the smell of a host. Accurate
control of both a visual object’sreflectance and its contrast with
the background is required to determine whether mosquitoes are
attracted to specific wavelengths.
Here in this study, we use a large wind tunnel and a computer
vision system to close knowledge gaps regarding mosquito visual
and olfactory responses by examining Ae. aegypti free-flight
responses to objects of different wavelengths, with and without
thepresenceofCO
2
.Aedes aegypti provides an excellent model for
studies aimed at elucidating spectral preferences and determining
how these preferences are modulated by odor. Aedes aegypti, which
are active during the dawn and dusk periods20, have 10 rhodopsins,
5 of which are expressed in the adult eye21. Little is known about
opsin tuning, although they are orthologs of medium-wavelength
sensitive opsins (green), and previous electroretinogram (ERG)
studies suggested that Ae. aegypti is sensitive to medium-long
wavelengths in the green–yellow spectrum22,23.Weshowthat
when encountering odor, mosquitoes become particularly attracted
to hues that are dominant in human skin. We also demonstrate
that knockout of either the olfactory channel that gates visual
attraction or the opsins that allow detection of objects that reflect
long wavelengths eliminates attraction to skin tones.
Results
Olfactory gating of spectral preferences of Ae. aegypti mos-
quitoes. Examining olfactory and visual search behaviors in
mosquitoes often requires simulating conditions in which the
statistics of the stimuli (e.g., intensity, duration) and resulting
mosquito behavior are as natural as possible. We therefore
examined Ae. aegypti behavior in a large wind tunnel spanning
450 mosquito body lengths and equipped with a 16-camera, real-
time tracking system for monitoring and quantifying mosquito
behaviors11,24. A checkerboard pattern was projected on the
bottom (floor) of the wind tunnel, and a low-contrast gray hor-
izon was projected on each side of the tunnel to provide optic
flow (Fig. 1c). Similar to our previous assays, we placed two
identically sized circles (3 cm diameter) on the floor of the tunnel
in the upwind area of the working section, 18 cm apart and 33 cm
from the odor source (Fig. 1c). In each experimental trial, 50
mated Ae. aegypti females were co-released into the tunnel, and
their trajectories were recorded over a 3 h period (1.3 million total
trajectories were recorded, with an average trajectory duration of
3 s). Simultaneous release of the mosquitoes provided an efficient
method to examine their olfactory-visual responses, and was not
statistically different from when the mosquitoes were released
singly (see Materials and Methods—Statistical analyses for
details). The tunnel was filled with filtered air for 1 h, after which
aCO
2
plume (95% filtered air, 5% CO
2
) located 33 cm away and
separate from the visual objects was introduced into the tunnel
and left for 1 h (Fig. 1d). Measurements of the plume in the
working section showed an exponential decay, typical of turbu-
lent diffusion, with a concentration of ~1500 ppm ~30 cm from
the odor source. In the last hour of the experiment, only filtered
air was released into the wind tunnel.
During exposure to filtered air, the mosquitoes exhibited
random behavior to the odor source and visual objects, and they
spent much of their time exploring the ceiling and walls of the
tunnel and rarely investigated the visual objects (Fig. 1e). By
contrast, upon exposure to the CO
2
plume, the number of flying
mosquitoes more than doubled (Fig. 1f, h; Wilcoxon signed-rank
test, number of trajectories in Air vs. CO
2
,P< 0.002). During this
time, the mosquitoes exhibited odor-tracking behavior, spending
most of the time in the working section’s central area with
significantly elevated flight velocities (Fig. 1f, s1; Kruskal–Wallis
test: df =2, Chi-square =597.23, P< 0.0001). The CO
2
also
triggered an attraction to visual objects. Here, we define attraction
as the amount of time a trajectory spends around an object
relative to the evenly reflecting control (white to the human
observer). The Ae. aegypti mosquitoes showed no interest in the
objects during the filtered air treatment (only 1–4% of mosquitoes
investigated), but during CO
2
release, the percentage and number
of mosquitoes investigating the visual objects increased signifi-
cantly (21%; paired Student’sttest: P=0.002) (Supplementary
Fig. S1). After the plume was stopped, the attraction to the visual
objects ceased (Fig. 1e, Supplementary Fig. S1e; Wilcoxon signed-
rank test, CO
2
vs. Post-CO
2
:P< 0.001).
To ensure that Ae. aegypti mosquito visual preference
behaviors were in response to discrete wavelength bands rather
than object contrast, we employed visual stimuli in the range
430–660 nm (violet to red to a human observer), with each visual
stimulus having the same approximate contrast with the back-
ground (Fig. 1g). While investigating the visual objects, the
mosquitoes would fly upwind and hover immediately downwind
of a visual object, at ~3–5 cm, while exhibiting brief excursions
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before returning to the objects (Fig. 1e). During CO
2
release, the
same total number of mosquitoes was recruited to the visual
objects of differing dominant wavelengths (Supplementary
Fig. S1e). Relative to the evenly reflecting object (white to the
human observer) that served as a control, however, the
mosquitoes preferred certain dominant wavelengths, such as
496 nm and longer wavelengths ≥590 nm as demonstrated by
their focused clustering around the object (Fig. 1e). By contrast,
other dominant wavelengths (437 nm, 452 nm, 510 nm, and
520 nm; appearing violet, blue, green and green–yellow to the
human observer, respectively) elicited no attraction responses
compared to the evenly reflecting control (Fig. 1e, i). Across all
dominant wavelengths, CO
2
had a strong effect on flight velocity
and duration, but there were no significant differences between
treatments (Supplementary Fig. S1b, d; Kruskal–Wallis test: Chi-
square < 16.82, P> 0.11), demonstrating that the presence of CO
2
c
400 500 600 700
50
100
Reflectance (%)
b
puddle
host
flower
0
Wavelength (nm)
a
0.08
Occupancy (%)
0
2500
CO
2
(ppm)
400
Clean air
d
e
f
top view
side view
CO2
Clean air CO2
top view
side view
Preference Index
***
437 452 496 510 520 590 600 660 __
control:
test:
peak (nm):
Black Wavelengths
-pre CO2
-post
0
1
2
3
Flight Activity
400 500 600
0
Wavelength (nm)
50
100
Reflectance (%)
g
h
i
700
0.5
0
-0.1
***
***
***
*** ***
__ __
***
***
-pre CO2
-post
2 m
0.6 m
0.6 m
3D tracking
cameras
CO source
2
Visual
targets
top view
top
view
side
view
20 cm
white
590nm
600nm
660nm
437nm
520nm
510nm
452nm
496nm
black
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is necessary for attraction to specific bands of the visual spectrum,
and that object attraction did not result from higher flight velocity
increasing the probability of a mosquito randomly encountering a
visual object.
To further investigate Ae. aegypti mosquito spectral prefer-
ences, a preference index value (defined as the time spent
investigating a spectral object minus the time investigating the
evenly reflecting control, divided by the sum of the times spent
investigating both objects) was calculated for each mosquito that
investigated a visual object. The dominant wavelengths differed
significantly in terms of mosquito preference (Fig. 1i,
Kruskal–Wallis test: df =8, Chi-square =597.23, P< 0.0001);
several dominant wavelengths were more attractive to mosquitoes
than the evenly reflecting object that served as a non-attracting
control (Fig. 1i, one-sample t-test: P< 0.001). As the dominant
wavelengths transitioned from 510 to 660 nm, the attractiveness
of the object also increased. For instance, 600 nm and 660 nm
visual objects were strongly preferred by female mosquitoes
(multiple comparison Kruskal–Wallis test: P< 0.05), whereas
510 nm, 452 nm, and 437 nm objects (green, blue and violet to the
human observer, respectively) were not more attractive than the
control object (multiple comparisons Kruskal–Wallis test:
P> 0.05). However, mosquitoes were not strictly attracted to
the longest wavelengths, as they were also significantly attracted
to objects with a dominant wavelength at 496 nm (cyan to the
human observer). As a control to test for the effect of visual object
attraction relative to other regions of the working section, we
examined the preference between the evenly reflecting (control)
object and a randomly selected volume in the wind tunnel.
Compared with the randomly selected volume, female mosqui-
toes investigated the evenly reflecting control object significantly
more in presence of CO
2
(preference index =−0.53 ± 0.03
(mean ± sem), one-sample t-test: P< 0.001).
Behavioral preferences for orange-red wavelengths reflected
from human skin. Across all skin tones and differences in pig-
mentation, human skin is dominated in the long-wavelength
range (590–660 nm) (Fig. 2a)25, but it is unclear which bands in
the human skin spectrum are most attractive to Ae. aegypti
mosquitoes. To examine whether Ae. aegypti exhibit different
preferences for certain spectral bands reflected by human skin, we
first utilized color cards designed for cosmetics purposes to match
human skin tones (Pantone SkinTone Guide) (Fig. 2a). Beha-
vioral experiments were performed in the wind tunnel to indi-
vidually test various faux skin tones that ranged from light to dark
(Y02, Y10, R10, and an unpleasant orange shade typical of
individuals using cheap tanning lotion [which we designated “vile
45”]) using the evenly reflecting object (white to the human
observer) as a control. Similar to the previous experiments, only
during CO
2
release did mosquitoes become highly attracted to
skin tones (Fig. 2c, e; Kruskal–Wallis test: df =8, Chi-square =
184.37, P< 0.001), exhibiting no behavior characteristic of
attraction before exposure to CO
2
(paired t-test: P=0.09).
Moreover, the mosquitoes exhibited the same level of attraction
to each skin tone, with the skin tones being not significantly
different from one another (Fig. 2c, e; Kruskal–Wallis test with
multiple comparisons: P> 0.05).
To determine which region of the human skin visual spectrum
is most attractive to Ae. aegypti, we overlaid the R10 skin color
card with optical filters to attenuate discrete bands (Fig. 2b).
Whereas the 450 nm optical filter had no significant effect on
behavioral attraction to the skin tone compared with the positive
controls (Fig. 2f; Kruskal–Wallis test with multiple comparisons:
P> 0.58), filters blocking longer wavelengths (550–700 nm)
reduced the attractiveness of the visual object (P< 0.05). In
particular, application of the 600 nm filter was associated with a
300% reduction in attraction compared with the positive controls
(Fig. 2c–f). Importantly, results for controls consisting of an
overlaid infrared (IR) filter or a clear nylon coverslip did not
differ significantly from the unmanipulated skin tone
(Kruskal–Wallis test with multiple comparisons: P> 0.33). To
further examine the importance of visual and olfactory integra-
tion in controlling Ae. aegypti visual preferences, we examined
single and double mutants of the long-wavelength photoreceptors
opsin-1 and opsin-2 and a line with a mutation in the Gr3
receptor14, which transduces CO
2
signals (Fig. 2g). Whereas the
Gr3-heterozygote, opsin-1 and opsin-2 single mutants, and wild-
type control mosquitoes were significantly attracted to the skin
tone during CO
2
exposure (one-sample t-test: P< 0.001), neither
the opsin-1,opsin-2 double mutant nor Gr3 mutant were attracted
to the skin tones (Fig. 2h; one-sample t-test: P> 0.31).
The behavioral preference to the long wavelengths in the skin
color cards may not reflect mosquito behaviors to the hues reflected
fromhumanskin.Toexaminethisfurther,wetestedAe. aegypti
mosquitoes in a smaller opaque cage (45 cm × 30 cm × 30 cm)
wheretheywereexposedtotwosmallwindows(16cm
2)(Fig.3a, b).
The cage and its environment were constructed to prevent
uncontrolled contamination from thermal or olfactory cues from
the human volunteer, and the windows being made from clear, heat
absorptive glass to block the radiant heat from the skin
(Supplementary Fig. S2). Subsequent testing showed no contamina-
tion during the experimental trials. The back of a hand was
displayed in one window, and the back of a heat-protective white
Fig. 1 Olfactory gating of mosquito color preference. a The Ae. aegypti eye. Image courtesy of Alex Wild (with permission). bSpectral reflectance of
behaviorally important objects for Ae. aegypti females: human skin (brown line); flower (Platanthera obtusata; green line) and within a small puddle filled
with Ae. aegypti larvae (blue line). Lines are mean; shaded area around the mean is the ±sem (n=6–10). cWind tunnel system with real-time tracking
system, odor and visual stimulation. dHeat map of the CO
2
plume in the wind tunnel. eExample of individual trajectories (top: [x-,y-axes], bottom: side
view [x-,z-axes]). The arrows represent the start of a trajectory; circles are the visual objects. fHeat (occupancy) maps showing the distribution of female
mosquitoes without (left panels) and with CO
2
delivery (right panels) while in presence of a white and a red objects (top), or white and green objects
(bottom). Plots shows the top and side views of the tunnel working section. gReflectance of the visual stimuli used in the experiments, as quantified using
a spectrophotometer and calibrated with a white Spectralon standard. Different colored traces correspond to the different stimuli. hRelative flight activity
between the different phases of the experiments (pre-, CO
2
and post-CO
2
) for black and white circles (n=12) and color and white circles (n=51). There
were no significant differences when the tested visual object was black vs. objects of different wavelengths (Kruskal–Wallis test, df =1, Chi-sq =0.01,
P=0.92), although for both groups CO
2
significantly elevated the number of flying mosquitoes compared to the filtered Air treatment (P< 0.002). iMean
preference index for the test object vs. the control (white) object. There was a significant effect of color on the attraction to the tested object
(Kruskal–Wallis test, Preference index ~ pair of visual stimuli used: df =8, Chi-sq =597.23, P< 0.0001). Several hues were significantly more attractive
than the control, white object (one-sample two-tailed t-test: ***: P< 0.001). Boxplots are the mean (line) with 95% confidence interval (shaded area)
(n=25,529; 17,729; 53,786; 23,694; 34,343; 31,037; 32,257; 24,774; 42,595; 20,929; and 48,198 mosquito trajectories for the all-white, all-black, black-
and-white, Bv-T2, Bw-, Gw-T1, Gc-, YGc-, Yw-, O- and R-Hue treatments, respectively).
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glove was displayed in the other window (as an internal control).
Similartotheassaysinthewindtunnel,wefoundthatmosquitoes
were highly activated by CO
2
(Fig. 3c–e), and this increased their
visual attraction to visual stimuli, including skin (Fig. 3b, c, f;
Kruskal–Wallis test: df =2, Chi-square =84.04, P< 0.001). Mos-
quitoes showed no preference during control experiments with two
white gloves displayed in the window, but significantly preferred
skin (Fig. 3f; Kruskal–Wallis test with multiple comparisons:
P< 0.001). However, when optical filters were placed over the
window, blocking the longer wavelengths (550–700 nm), the
attraction was significantly reduced (Kruskal–Wallis test with
multiple comparisons: P< 0.001) and not significantly different
from the negative control (Fig. 3f; Kruskal–Wallis test with multiple
comparisons: P=0.34). Collectively, these results demonstrate that
the long-wavelength band of the visual spectrum plays an important
role in determining mosquito attraction to skin color. In addition,
knockout of either visual or olfactory detection receptors suppresses
mosquito visual attraction to long-wavelength host cues.
Spectral sensitivity of the Aedes eye. The preference of Ae.
aegypti for long wavelengths in the orange-red band motivated us
to examine the sensitivity of the retina by recording ERGs that
extracellularly measure the summed responses of retinal cells to
visual stimuli (Fig. 4a). In the first series of experiments, a moving
bar of differing dominant wavelengths (blue [peak 451 nm], green
[537 nm], or red [640 nm], all at the same intensity; 18° wide at
30°/s clockwise) was projected on a black background while
conducting the ERG recordings (Fig. 4b). When the moving bar
reached the mosquito’s visual field, the ERG exhibited a negative
response that quickly returned to baseline after the bar moved
past the mosquito’sfield of view (Fig. 4b). A significant difference
in wavelength-evoked responses was observed (Kruskal–Wallis
test: df =3, 40.03, P< 0.001), with the 451 nm and 537 nm bars
eliciting the strongest responses (Fig. 4b). Although responses to
the 640 nm bar were significantly weaker, this dominant wave-
length still elicited ERG responses that were significantly higher
than the baseline and those of the no-stimulus controls (Wil-
coxon signed-rank test: P< 0.001).
To further characterize Ae. aegypti spectral sensitivity, we used
a scanning monochromator to examine ERG responses across the
near ultraviolet (UV) to far-red wavelength range (350–750 nm).
Ae. aegypti exhibited the highest sensitivity to short (410 nm,
3.2 mV) and medium wavelengths (520 nm, 2.8 mV) (Fig. 4c, d).
Strong ERG responses (0.47–1.27 mV) were still noted in the
medium-long wavelengths (>590 nm), although at >700 nm, the
responses decreased to approximately 0.25 mV, which was still
significantly higher than the baseline control (Wilcoxon signed-
rank test, P< 0.03).
The role of visual contrast in determining Ae. aegypti pre-
ferences. Mosquitoes are very sensitive to detecting dark objects
that contrast highly with the background8,11. In the above
experiments, we kept the total object contrast (400–700 nm) with
the background approximately the same, but the Ae. aegypti
preference for long-wavelength objects and skin tones motivated
0
20
40
60
80
400 500 600 700 400 500 600 700
Preference Index
0.5
0
Y02
+450nm filter
+600nm filter
+700nm filter
Reflectance (%)
450 nm
filter
600 nm
filter
700 nm
filter
Host skin
Faux skin hue
a,b
ab
c
Wavelength (nm)
a
c
b,c
+IR filter
Vile 45
Y10
R10
ns
top view
side view
Occupancy (%)
CO | R10
2 CO | R10+600nm filter
2
d
ef
a
+coverslip
X
CO2
XGr3
mutant
op-1,2
mutant
g
Preference Index
0.5
(-|-)
Gr3
(-|+)
Gr3
RG
op-1 ,op-2
0
-0.2
R
op-1
G
op-2
wt
h
0.08
0
Fig. 2 The contribution of orange-red wavelengths in attraction to faux
human skin. a Spectral reflectance of human skin and faux skin used in
behavioral experiments. Lines are the mean and shaded area is the ±sem
(n=6). bUltra-thin optical filters (450nm, 600nm, and700nm)attenuated
discrete bands in the object’sreflected spectrum. c,dOccupancy maps of the
mosquito’s distribution around the visual objects during exposure to CO
2
.
During CO
2
, mosquitoes were significantly attracted to the faux skin color
compared to the white (control) object (c). However, the optical filter
attenuating the 550–630 nm band reduced the number of mosquitoes
investigating the faux skin color (d). e,fMosquitoes significantly preferred the
faux human skin colors (e), although optical filters in the yellow to red
wavelengths significantly decreased the attractiveness of the visual object (f).
Boxplots are the mean (line) with 95% confidence interval (shaded area);
letters denote statistically significant differences between groups. gMosquito
lines deficient in long-wavelength opsins (opsin1 and opsin2), or unable to
detect CO
2
(Gr3 mutant), were tested in their attraction of human skin color.
hMean preference indices for the Gr3 mutants (blue) and opsin mutants
(orange). All mosquito lines showed similar preferences to the white and skin
color visual objects during exposure to filtered air (Kruskal–Wallis test: df =3,
Chi-sq =1.68, P=0.64). However, during CO
2
the lines were significantly
different from one another in their visual preferences (Kruskal–Wallis test with
multiple comparisons: df =3, Chi-sq =96.01, P<0.001): only the
heterozygote (Gr3−/+) and wild-type (LVP) lines showed significant attraction
to the skin color (one-sample t-test: P< 0.001), whereas the opsin double
mutant line (op-1/op-2)andtheGr 3−/−mutants showed no attraction (one-
sample two-tailed t-test: P> 0.31). Boxplots are the mean (line) with 95%
confidence interval (shaded area) (n=13,999; 15,035; 14,016; 48,624; 19,284;
20,713;40,690;26,135;39,649;16,208;3,550;9,277;13,799;10,948;and
5,679 mosquito trajectories for the Y02, Vile 45, Y10, R10, IR filter, coverslip,
450 nm filter, 600 nm filter, 700 nm filter, wt, Gr3−/−
,Gr3
−/+,op1-R,op-2G,
and op1-R,op2-G treatments, respectively).
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us to evaluate whether these responses were due to contrast alone
(calculated as the Weber contrast, or the difference in spectral
energy reflected by an object and the background, divided by the
sum of the two) or whether mosquitoes can discriminate long-
wavelength objects >590 nm independently of intensity. As the
first step in determining how features of a visual stimulus impact
mosquito visual preference, gray objects that contrasted differ-
ently with the background were tested against the evenly
reflecting control (Fig. 5, Weber Contrasts: −0.28–0.02). Similar
to the above results and across all tested stimuli, the presence of
CO
2
increased mosquito flight activity and the number of visits to
the visual objects (Fig. 5c, Wilcoxon signed-rank test, Air vs. CO
2
:
P< 0.001). Female mosquitoes exhibited significantly greater
attraction to the majority of the gray visual objects than the
control object (Fig. 5d, one-sample t-test: P< 0.001). However,
when exposed to the lightest gray object, which closely approxi-
mated the background and the evenly reflecting control, mos-
quitoes showed no preference for either object (Fig. 5d; Student’s
ttest: P=0.33). Overall, object darkness and contrast with the
lighter background was significantly related to mosquito pre-
ference, with mosquitoes investigating and preferring darker
objects (Fig. 5d; Kruskal–Wallis test: df =5, Chi-square =634.16,
P< 0.001). Although Ae. aegypti showed a distinct preference for
darker objects, mosquito flight velocity and duration did not
significantly differ across treatments (Kruskal–Wallis test: Chi-
square > 7.40, df =5, P> 0.055).
Effects of contrast and spectral discrimination on Aedes pre-
ferences. Given the difference between high retinal sensitivity to
medium-long (green) wavelengths, lack of attraction to those
objects, and the relatively low ERG sensitivity to long (orange/
red) wavelengths but strong behavioral attraction to those objects,
we asked the following question: what role does the darkness of
the object (i.e., its contrast) have on behavioral preferences vs. the
object’s dominant wavelength? Although we lacked the photo-
receptor tunings that would allow us to normalize the inputs
between spectral channels, we experimentally manipulated object
contrast to equalize the perceptual contrast of the objects to the
mosquitoes and then examined the mosquito’s ability to dis-
criminate between different bands of the visual spectrum.
We first performed behavioral experiments in which we altered
the darkness of the gray objects to determine the contrast that
matched the attraction shown to the 660 nm object (red to human
Skin Skin+filters
-pre CO2
-post
0
2
4
Flight Activity
***
***
-pre CO2
-post 0 6 12 18 24
0
50
100
Time (min.)
0
0.35
0.70 a
b
b
Mosquito Visits (%)
Preference Index
test:
control:
white glove skin skin+filters
3D tracking
cameras
windows
CO | Skin
2
front view
CO | Skin + 600nm filters
2
0.08
Occupancy (%)
0
ac
def
front view
b
skin
filters
control
control
filters
skin
control
Fig. 3 The importance of long wavelengths in attraction to human skin. a Cage assay with real-time tracking system, odor, and visual stimulation
through two windows on the front of the cage. bExample of individual trajectories (top: skin and control (white glove), bottom: skin+filters
(550–700 nm), and control). cOccupancy maps showing the distribution of female mosquitoes during CO
2
stimulation while in presence of the skin
and control (top), and the skin+filters (550–700 nm) and control (bottom). dRelative flight activity between the different phases of the experiments
(pre-, CO
2,
and post-CO
2
) for the skin and skin+filters treatments (n=6 trials/treatment). There was no significant difference in the relative activity
during the CO
2
phase between the skin and skin+600-nm filter treatments (Kruskal–Wallis test, df =1, Chi-sq =0.004, P=0.96). eThe percentage of
mosquitoes visiting the windows over the duration of the experiment. Few mosquitoes investigated the windows before the CO
2
exposure. However,
exposure to CO
2
significantly increased the numbers of mosquitoes visiting the windows relative to the pre-CO
2
period (Kruskal–Wallis test with
multiple comparisons: df =5, Chi-sq. =277.85, P< 0.0001), although during CO
2
there were no significant differences in the total number of
mosquitoes investigating the windows between treatment groups (Kruskal–Wallis test with multiple comparisons: P> 0.57). Lines are the means and
shaded areas the ±sem. fMean preference index for the different treatment groups (white glove vs. white glove, skin vs. white glove, and skin +filter
(550–700 nm) vs. white globe). Boxplots area the mean (line) with 95% confidence interval (shaded area). Different letters denote statistically
significant differences between groups (Kruskal–Wallis test with multiple comparisons, P<0.01).(n=13,597 for the skin treatment group; n=9502 for
the for the skin +filters treatment group; and n=9368 for the control group).
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observer)(Fig. 6a, b). Across a range of gray contrast levels, the
660 nm object was significantly more attractive to Ae. aegypti
than the gray objects (Fig. 6a, c, e; Kruskal–Wallis test: Chi-
square =149.75, df =6, P< 0.0001). As the gray objects became
darker, however, they became more attractive to the mosquitoes,
and the strong preference for the 660 nm object decreased
(Fig. 6e). The attractiveness of the 660 nm object equaled that of
the darkest gray and black objects (Fig. 6e; one-sample t-test:
P=0.07 and P=0.08 for the darkest gray [gray 1.5] and black
objects, respectively). We then determined which contrast of a
510 nm object (green to human observer) matched the attraction
to the 660 nm object. For this purpose, we used the darkest gray
object that was equally attractive as the 660 nm object and tested
it against objects with dominant wavelengths of 510 nm but
differing in their background contrast (Fig. 6b). The dark gray
object was significantly more attractive than most 510 nm objects,
but the darkest 510 nm object elicited the same level of attraction
(Fig. 6f; preference index =−0.01; one-sample t-test: P=0.66).
To determine whether Ae. aegypti mosquitoes can discriminate
between objects of different dominant wavelengths but similar
levels of apparent contrast, we tested attraction to the 660 nm vs.
the darkest 510 nm object (Fig. 6g, h). During exposure to CO
2
,
mosquitoes were strongly attracted to 660 nm but not the dark
510 nm object (Fig. 6g, h; one-tailed t-test: P< 0.0001). We also
determined whether mosquitoes could discriminate between
objects that overlapped in their spectral bands. For this purpose,
we matched the apparent contrast of the non-attractive 452 nm
object (blue to human observer) to an attractive gray. Next, we
tested mosquito responses to the 497 nm object (another
attractive spectral band) and the dark 452 nm object, as well as
the 497 nm object vs. the dark 510 nm object. Similar to the
results observed with the 660 nm object, mosquitoes significantly
preferred the 497 nm object over the dark 452 and 510 nm objects
(Fig. 6h; one-tailed t-test: P< 0.0001). Thus, mosquitoes easily
discriminated between objects with overlapping and distinct
spectral bands, even when the object contrasts matched.
By incorporating object contrasts, reflectance values, and peak
wavelengths as independent variables into a series of linear
models, the results of the behavioral tests offered a means to
examine the relative contributions of these variables toward
mosquito preferences. In these models, all possible combinations
were tested, and the best model was selected based on its Akaike
Information Criterion (AIC) score, where the AIC estimates the
value of each model and lower scores reflect the quality of the
statistical model. Using combinations of the independent
variables, we found the best model (and hence, lowest AIC
score) relied on object peak wavelength and contrast, and
excluded reflectance (Supplementary Fig. S3a, b). But which
dominant wavelengths might be critical for mediating these
behaviors? To further explore the relationship between the object
350 400 450 500 550 600 650 700 750
0
20
40
60
80
100
2 sec.
1 mV
350 nm 410 nm 450 nm 520 nm 600 nm 650 nm 700 nm
Wavelength (nm)
Sensitivity (% ± SE)
noise
a
c
projector
3 sec.
1 mV
d
b
ERG
Fig. 4 Retinal sensitivity to visual stimuli. a Experimental setup for the
electroretinogram (ERG) experiments. bERG responses to a blue (410 nm),
green (520 nm) or red (>590 nm) moving bar (mean ± sem; n=7
mosquitoes). Responses to the moving object were significantly higher than
the baseline for all tested dominant wavelengths (410 nm, 520 nm or
590 nm), although 410 nm and 520 nm bars elicited stronger responses
(Kruskal–Wallis test, Amplitude responses~wavelength moving bar, df =2,
Chi-sq. =40.03, P< 0.001, n=7 mosquitoes). cERG responses to pulses
of light (350–750 nm). Traces are the mean responses (shaded area is the
±sem; n=8 mosquitoes) to discrete wavelengths showing the elevated
responses to 410 nm (violet to human observer) and 520 nm bands. d
Retinal sensitivity curve across the tested wavelengths from 350 to
750 nm. Two maxima occurred at the short (420 nm) and medium
(~530 nm) wavelengths, although responses were still significantly elevated
above the noise at wavelengths more than 650 nm (two-sided t-test, ERG
vs. noise: P< 0.01).
Occupancy (%)
0
Black Greys
**
***
***
***
***
***
*** ***
control:
test:
0.5
0
-0.2
Preference Index
Flight Activity
4
2
0
cd
-pre CO2
-post
-pre CO2
-post
contrast:
b
a
top view
side view
top view
side view
0.02 -0.30
CO | Grey9.5
2 CO | Grey4.0
2 0.08
Fig. 5 The effect of achromatic contrast on mosquito attraction to visual
objects. a Occupancy map of the distribution of female mosquitoes in the
wind tunnel (top and side views) during CO
2
delivery in presence of a white
and a light gray object (Weber Contrast: −0.05). bAs in a, except the gray
object has a higher contrast (−0.23). cRelative flight activity between the
different phases of the experiments (pre-, CO
2
and post-CO
2
). Mosquitoes
exhibited similar flight activities across all tested visual objects (Kruskal–Wallis
test, df =1, Chi-sq =3.24, P=0.07). dMean preference indices for the test
(gray, or black) vs. control object (white) with 95% confidence interval
(n=12,764; 27,537; 37,085; 28,644; 36,050; 25,896; and 21,514 mosquito
trajectories for the white, grey9.5, grey6.5, grey4.5, grey4.0, grey2.5, and black
treatments, respectively). Object contrast had a significant effect on the
attraction to the tested object (Kruskal–Wallis test: Preference Index~contrast
tested object, df =5, Chi-sq =634.16, P< 0.001). All gray objects were
significantly more attractive than the control, white object (one-sample tw-
tailed t-test, **: P< 0.01, ***: P<0.001) except for the lightest gray object
(−0.05), which was not more attractive (one-sample t-test: P=0.33).
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wavelengths and its attractiveness, a series of linear models were
run using a multivariate analysis (Principal Components
Analysis, PCA) of each object’s visual spectrum. The PCA
analysis allowed reduction of highly collinear and dimensional
spectral data into reduced components, which can then be used as
independent variables in the model. The best model explained
~17% of the variance in object attractiveness (Supplementary
Fig. S3c) and indicated that preference is negatively correlated
with the content of the medium wavelength band (500–575 nm)
in the object’s visual spectrum (Supplementary Fig. S3d, e).
Comparison of wavelength preference between mosquito species.
The strong and specific responses of Ae. aegypti to specificbandsin
the visual spectrum and the similarity in long-wavelength opsin
gene expansion in other mosquito species21 motivated us to
examine the wavelength preferences in Anopheles (An.) stephensi
and Culex (Cx.) quinquefasciatus mosquitoes. To examine the visual
preferences in An. stephensi and Cx. quinquefasciatus,wefirst
conducted ERG recordings of 7-day-old females and examined their
retinal responses to discrete wavelengths from 350 to 750 nm
(Fig. 7a). Both mosquito species exhibited the strongest response to
short wavelengths (350–420 nm; UV-visible violet to the human
observer), and the second strongest response was observed in
the medium wavelengths (500–520 nm; cyan-green). The strong
response of An. stephensi and Cx. quinquefasciatus to the short
wavelengths of 370 nm contrasted with that of Ae. aegypti (dashed
line), which exhibited the strongest response at ~400 nm (Figs. 3d
and 7b, c). All three species exhibited similar responses to the long
wavelengths in the orange to red band (620–750 nm).
Are mosquito species’behavioral preferences for visual objects
correlated with their ERG responses, and are their color preferences
similar? To answer these questions, we tested the responses of An.
stephensi and Cx. quinquefasciatus mosquitoes to objects that
dominate in wavelengths at 452 nm, 510 nm, 660 nm, and black
objects in the wind tunnel using a methodology similar to that used
for Ae. aegypti, except at lower light levels (1.28 µW/cm2). As with
the results for Ae. aegypti,encounteringtheCO
2
plume caused a
doubling in the number of flying mosquitoes and increased
the percentage of mosquitoes that investigated the visual objects
5.58- to 9.15-fold relative to air-only treatment for An. stephensi and
Cx. quinquefasciatus, respectively (Fig. 7d; Kruskal–Wallis test:
df =2, Chi-square > 7, P< 0.01). However, in contrast to the tight
clustering around attractive visual objects by Ae. aegypti (Fig. 1f),
occupancy maps showed that the responses of An. stephensi
and Cx. quinquefasciatus mosquitoes were much more diffuse
(Fig. 7e, f). Nonetheless, Cx. quinquefasciatus formed a clustering
hotspot around the outlet of the odor plume nozzle that was much
stronger than the responses of the other two species (Figs. 1f, 7e, f).
Although we tried to minimize the odor nozzle’s visual signature by
using clear acrylic and tubing, Cx. quinquefasciatus mosquitoes
might have located the plume source based on the high CO
2
concentration or by seeing the nozzle to some degree.
We next examined the preferences of the mosquito species for
different spectral objects (black, blue, green, or red) relative to the
evenly reflecting white object (the non-attractive control). Similar
to Ae. aegypti, when both An. stephensi and Cx. quinquefasciatus
mosquitoes were subjected to filtered air treatment, they showed
no preference for the black object or any of the spectral objects
relative to the white control object (preference indices of -0.04
and 0.08 for An. stephensi and Cx. quinquefasciatus, respectively;
Kruskal–Wallis test: df =3, Chi-square < 3.66, P> 0.30). How-
ever, their spectral preferences changed when exposed to CO
2
.
After encountering the CO
2
plume, An. stephensi preferred the
ab
cd
ef
Reflectance (%)
0
50
100
400 500 600 700
Reflectance (%)
0
50
100
400 500 600 700
0.08
Occupancy (%)
0
top view
side view
top view
side view
-0.6
0
0.2
-0.6
0
0.2
control:
test:
h
-0.1
0
0.5
control:
test:
*** *** ***
*** ***
***
***
*** ***
***
***
***
Preference Index
Preference Index
Preference Index
g
top view
CO | Red & Grey2.5
2 CO | Grey1.5 & Green-S2
2
CO | Red & Green-S2
2 0.08
Occupancy (%)
0
Fig. 6 The role of dominant wavelength vs. contrast in mosquito visual
attraction. a Spectral reflectance of 660 nm (R-Hue), black, and gray
objects used in the experiments: Gray 9.5, 6.5, 4.5, 4.0, 2.5, and 1.5, with
Weber contrast values of −0.17, −0.30, −0.05, −0.10, −0.20, −0.24,
−0.27, and −0.28, respectively. bSpectral reflectance of 496 nm and
510 nm objects used in the experiments: Gw-T1, Gw-T3, Gc-T1, Gc-Hue,
G-S1, and G-S2, with Weber contrast values of −0.18, −0.11, −0.17,
−0.20, −0.25, and −0.27, respectively. c,dOccupancy maps showing
the distribution of female mosquitoes in the wind tunnel (top and side
views) during CO
2
delivery in presence of the 660 nm (R-Hue) and
Grey2.5 objects (c), or 510 nm (G-S2) and Grey1.5 objects (d); both the
510 nm and Grey1.5 objects have the same levels of contrast with the
background (Weber Contrasts of −0.27). eMean preference indices for
the 660 nm vs. gray objects with different levels of contrast with the
background. The Grey1.5 object (Weber Contrast value of −0.28) was
not significantly different from 0 (one-sample two-tailed t-test: P=0.07)
and was subsequently used in experiments in (f) (blue arrow). fThe
Grey1.5 object was significantly more attractive than most of the 510 nm
objects (one-sample two-tailed t-test: P< 0.001, ***), although the
darkest 510 nm object (G-S2; Weber Contrast =−0.27) was not
significantly different from 0 (one-sample two-tailed t-test: P=0.66))
(n=10,098–15,578 trajectories for each tested object). gAs in (c),
occupancy maps showing the distribution of trajectories around the
660 nm and dark 510 nm objects. hMean preference indices for 660 nm
vs. dark 510 nm objects, and 496 nm vs. dark 452 nm or dark 510 nm
objects. Mosquitoes significantly preferred the 660 nm and 496 nm
objects over the dark chromatic objects (510 nm and 452 nm)(one-
sample two-tailed t-test: P< 0.0001). For (e,fand h), boxplots area the
mean (line) with 95% confidence interval (shaded area), and asterisks
denote P< 0.001 (one-sample t-test) (n=7191–27,717 mosquito
trajectories for eachtestedobject).
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black and 660 nm objects (Fig. 7h; Kruskal–Wallis test: df =3,
Chi-square =38.6, P< 0.001), but they were not significantly
attracted to the 452 nm or 510 nm objects (Fig. 7h; multiple
comparison Kruskal–Wallis test: P> 0.05). By contrast, Cx.
quinquefasciatus mosquitoes preferred the 452 nm and 660 nm
objects (Kruskal–Wallis test: df =3, Chi-square =13.6, P< 0.01;
with multiple comparisons: P< 0.05) but were not significantly
attracted to the 510 nm or black objects (Fig. 7i; multiple
comparison Kruskal–Wallis test: P> 0.05). Collectively, these
results show that odor strongly sensitizes attraction to visual
objects across mosquito species; however, spectral preferences can
be species-specific.
Discussion
Free-flight behavioral experiments with Ae. aegypti mosquitoes
have shown that these insects integrate olfactory, visual, skin
volatiles, and thermal cues to function efficiently and robustly in
complex environments11,13,26. However, we know very little
about mosquito visual-guided behaviors or how vision is involved
in host selection. In this study, we utilized real-time tracking of
mosquito behaviors in a large wind tunnel. The wind tunnel
system enables control of aerodynamic conditions to structure the
odor plume and allow the plume and visual objects to be
decoupled in time and space. Both are important considerations
when testing olfactory-visual integration. In this study, and
similar to our previous work using achromatic objects11,26, the
presence of CO
2
increased mosquito responses to visual objects in
a wavelength-specific manner. Both chromaticity and contrast
were important components in visual object attraction and could
partly explain Ae. aegypti mosquito preferences for objects that
appear orange and red to human observers. These results were
qualitatively similar to those reported by Smart and Brown
(1957), who examined the landing responses of mosquitoes on
colored cloth in a field16. The results of their study showed that
CO | An. stephensi
2CO | Cx. quinquefasciatus
2
350 450 550 650 750
0
50
100
350 450 550 650 750
0
50
100
ERG
Ae. aegypti
An. stephensi
Cx. quinque.
An. stephensi Cx. quinque.
1 mV
2 mV
4 mV
1 s
350 nm 400 nm 700 nm Wavelength (nm) Wavelength (nm)
abc
Retinal sensitivity (%)
Retinal sensitivity (%)
def
0
Preference Index
0.5
-0.3
control:
test:
Ae. aegypti An. stephensi Cx. quinquefas.
b
b
a
aa
b
c
c
c
a
b,c
a,b
0
Preference Index
0.5
-0.3
0
Preference Index
0.5
-0.3
ghi
top
view
side
view
top
view
side
view
top view
top view
top view
top view
An. stephensi
Cx. quinquefasciatus 0.08
Occupancy (%)
0
Fig. 7 Species-specific responses to dominant wavelengths. a Electroretinogram (ERG) recordings from Ae. aegypti,An. stephensi and Cx. quinquefasciatus
mosquitoes. Each trace is the mean ± sem of 7–9 mosquitoes/species. b,cRetinal sensitivity to discrete wavelengths show that both An. stephensi (b) and
Cx. quinquefasctiatus (c) have the strongest responses in the UV (360 nm) and green (520 nm) bands. The dashed line is the retinal sensitivity of Ae.
aegypti.dRepresentative flight trajectories [(x,y) and (x,z)] of Cx. quinquefasciatus (left) and An. stephensi (right) mosquitoes. Cx. quinquefasciatus showed a
mild attraction to the blue object (top), whereas An. stephensi showed an attraction to the red object (bottom). e,fOccupancy maps (x,y) of An. stephensi
(e) and Cx. quinquefasciatus (f) distribution around the 452 nm (top) and 660 nm (bottom) objects during CO
2
exposure. g–iThe preference indices for Ae.
aegypti (g), An. stephensi (h) and Cx. quinquefasciatus (i) in response to the black, 660 nm (R-Hue), 510 nm (Gc-T1), and 452 nm (Bw-T1) objects. Lines are
the means and shaded bars are the 95% confidence intervals, and letters above bars denote statistical comparisons (Kruskal–Wallis test with multiple
comparisons: P< 0.05) (for Ae. aegypti,n=29,254; 13,580; 18,190; and 12,086 trajectories; for An. stephensi,n=5817; 9134; 2153; and 5627 trajectories;
and for Cx. quinquefasciatus,n=3746; 13,553; 4238; and 2835 for black, 660 nm, 510 nm, and 452 nm treatments, respectively).
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mosquitoes (Aedes sp.) are attracted to red and black cloths, with
darker colors being more attractive than lighter shades16. Our
ERG results and wind tunnel assay data suggest that mosquitoes
can detect and are attracted to the long-wavelength bands in the
orange and red portions of the human visual spectrum, although
if objects contrast highly with the background and/or are darker,
then they become more attractive. The demonstrated spectral
preferences of mosquitoes and their lack of attraction to spectral
bands in the 450 nm region, and 510–550 nm regions (blue and
green to human observer), impacted the fashion industry in the
early twentieth century. For instance, Nuttall and Shipley
(1902)27 suggested wearing khaki pants would be appropriate for
ensembles in a tropical environment, and the US military chan-
ged its dress shirts from dark blue to light blue in part to mitigate
mosquito biting28. Nonetheless, a key question is what drives
these responses in mosquitoes, particularly the attraction to
darker and higher-contrast colors? An important component
could be the visual OFF responses to the visual background29.In
our wind tunnel, we projected a light gray checkerboard pattern
on the bottom of the tunnel to provide optic flow and contrast
with the visual objects. The lack of opsins for the long wave-
lengths could cause an OFF response in downstream neurons that
receive input from the photoreceptors. One complication with
this hypothesis is that mosquitoes still preferred the long wave-
lengths when tested against a dark 510 nm object that matched it
in apparent contrast, suggesting they have an ability to detect
these long wavelengths. This was further demonstrated by the
ERG responses to long wavelengths. Recent work in Drosophila
has shown that photopigments provide a mechanism for flies to
long wavelengths >600 nm30, and it is possible that similar pro-
cesses play a role in mosquitoes.
Although the contrast with a lighter background did impact the
attraction responses of Ae. aegypti mosquitoes, the preferences for
the dominant wavelengths that appear red and cyan to humans
were greater than the apparent contrasts of the competing visual
cues (objects with dominant wavelengths at 510 nm, or 452 nm).
The opsin-2 gene, which is tuned to the green to orange band of
the visual spectrum, is highly expressed in the mosquito retina.
The results of this study and those reported elsewhere24 suggest
that opsin-1 and opsin-2 play important roles in determining
object preference and visual attraction to human skin. Additional
research will be needed to identify the opsins and neural circuits
involved in small-field object detection and color preference and
determine how odor modulates those responses.
Compared with other insects, such as honeybees or the tsetse
fly, we know little about mosquito visual ecology or how visual
cues are integrated with other senses in these insects. Whereas
shallow pools of water can be rich in medium-long wavelengths
and flowers dominant in short-medium wavelengths, hosts’skin
is dominated by long wavelengths in the >600 nm region of the
visual spectrum (Fig. 1b). Abundant work by the cosmetics
industry has shown that human skin—irrespective of skin tone or
pigmentation—has a lower peak in the green wavelength
(530 nm, ~20%) and a dominant reflectance in the long wave-
lengths (>600 nm, 20–60%). The diurnal Ae. aegypti and noc-
turnal and crepuscular An. stephensi and Cx. quinquefasciatus
mosquitoes are all active during periods in which these longer
wavelengths are dominant. For example, Ae. aegypti exhibit peak
activity in the mornings and late afternoons, and An. stephensi
and Cx. quinquefasciatus mosquitoes are especially active during
moonlit nights—both environments are long-wavelength
shifted20,31,32. In this study, we show that mosquitoes are espe-
cially sensitive to long wavelengths (590–660 nm) for host
detection; blocking these wavelengths can suppress object
attraction. Moreover, we found that mosquitoes can distinguish
between overlapping and discrete spectral bands (Fig. 6h), even
when their apparent contrasts match. Insect photoreceptors
within an ommatidial cartridge transduce light intensity and
spectral information. At the photoreceptor terminals, they also
provide antagonistic inputs to downstream neuron targets, thus
allowing discrimination of spectral inputs (termed ‘color oppo-
nency’). In Drosophila melanogaster, color opponency at the
photoreceptor terminals plays an important role in their color
discrimination and preference29, and it could be that similar
processes are at play in mosquitoes. In a variety of insects,
including flies, bees, and butterflies, spectrally sensitive photo-
receptors form connections with transmedulla neurons that
project into the lobula, where additional spectral and motion
processing—including color opponency—occurs33–35.
It is important to note that our current experiments did not
incorporate close-range cues from a host, such as heat, water
vapor, or skin volatiles. These cues play critical roles in control-
ling landing and biting behaviors, and future work could deter-
mine how visual spectra are processed in tandem with these other
stimuli. Nonetheless, previous work has shown that visual cues
can promote mosquito orientation and search behaviors in
combination with odor, heat, or water vapor11,13. The integration
of multimodal stimuli in driving behavioral responses raises
questions regarding how the sensory systems are linked in the
brain. Odor stimulation increases visual responses in the object-
detecting neuropil in the Ae. aegypti lobula26. Neuropil in this
brain region is responsive to moving objects but not wide-field
motion. Interestingly, whereas olfactory stimulation increases
visual responses in the lobula, visual stimulation does not mod-
ulate glomerular responses in the antennal lobe, the primary site
for processing olfactory information in the mosquito brain. Why
might this occur? Mosquitoes have a relatively poor visual reso-
lution (~10°); thus, vision may not provide fine-scale information
about the identity of an object. Instead, an object’s odor may
provide information about its identity, whereas vision can provide
details regarding the location of the object.
Despite the potential importance, few studies have examined
retinal responses to long wavelengths in mosquitoes22,23, and how
peripheral and downstream visual circuits, such as those in the
optic lobes, process this information remains unknown. Evolu-
tionary analyses of long-wavelength opsins in diverse mosquito
species have suggested these genes are functionally important. In
Ae. aegypti,Anopheles coluzzii, and Cx. quinquefasciatus, these
genes have undergone duplication events and may be under
positive selection, explaining the commonalities in ERG respon-
ses. Aedes aegypti has 10 putative opsins, five of which are
potential medium- to long-wavelength opsins in the adult
(>500 nm)21. One of these genes, opsin-1, is expressed in the
largest group of photoreceptor cells (R1–R6) in the Ae. aegypti
eye22. The R1-R6 photoreceptors form a trapezoid-like structure
in the ommatidial cartridge, with the R7 and R8 cells in the
middle. Perhaps similar to D. melanogaster with rhodopsin-1
(Rh1), the Ae. aegypti opsin-1 is likely involved in motion- and
dim-light sensitivity36. The inner R7 and R8 photoreceptors are
involved in color vision, and the Ae. aegypti opsin-2 is expressed
in the R7 photoreceptors located in distinct bands on the dorsal
and ventral surfaces of the eye, and possibly used for navigation
and biting behaviors. For mosquitoes, opsins that differ in spec-
tral tuning can be co-expressed in the R7 photoreceptor in the
female retina (e.g., the long-wavelength opsin-2 and short-
wavelength opsin-9), thereby increasing their range in wave-
length sensitivity37. This may contrast the opsin co-expression in
D. melanogaster R7 cells in the dorsal region of the eye, where two
short (UV)-sensitive opsins are co-expressed38, presumably to
increase the spectral contrast with the green-absorbing opsins in
the R8 layer. The opsin co-expression and broadening of spectral
input in mosquitoes could be advantageous under low-light
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conditions, as photon capture would be maximized, thus allowing
for detection of suitable hosts or perhaps sources of nectar37,39,
but this advantage would come at the cost of detection of parti-
cular wavelength bands. In our study, however, mosquitoes were
able to discriminate between distinct and overlapping spectral
bands (510 nm and 660 nm; 496 nm and 510 nm; and 496 and
452 nm), all at the same apparent contrasts, suggesting that other
opsins, perhaps in the R8 cells, or downstream circuits, play a role
in increasing the separability of those hues.
Preferences for certain dominant wavelengths plays a critical
role in a diverse array of insect vectors, and it is integrally tied to
olfaction. For instance, traps that incorporate visual and olfactory
cues have proven transformative as a low-cost method for con-
trolling tsetse flies in parts of Africa40. Like Ae. aegypti, the
majority of tsetse flies (e.g., Glossina morsitans morsitans and G.
pallidipes) locate hosts based on smell, and once they are within
close range (<10 m), visual cues cause the insects to investigate
and potentially bite if the object is a host. Beginning with Vale
(1974)41 and continuing into the 1990s, researchers found that
flies are most attracted to a specific blue (~460 nm), followed by
red and black, but they are not attracted to green and white5,6.
Researchers subsequently found that incorporating blue and black
hues in traps was particularly effective at inducing flies to come
into contact with insecticide-treated screens. Triatoma infestans
(i.e., the kissing bug) is another example of an insect that inte-
grates odor and visual cues to mediate attraction to targets.
Exposure to an aggregation pheromone modified the responses of
T. infestans to colored objects, including the red, blue, and black
hues, although the bugs always reject green (~525 nm) and white
hues7. In a similar manner, our results obtained from tests of
different mosquito species demonstrate the importance of olfac-
tion in mediating mosquito spectral preferences. In the absence of
CO
2
, mosquitoes did not demonstrate any preference between
evenly reflecting (white to humans) and dominant wavelengths,
but they became attracted to certain dominant wavelengths in the
presence of CO
2
. However, there were species-specific differences
in their wavelength preferences. Whereas Ae. aegypti was equally
attracted to both 660 nm and black objects, An. stephensi was
most attracted to black, followed by 660 nm objects. By contrast,
Cx. quinquefasciatus was attracted to 452 nm, followed by 660 nm
objects; surprisingly, however, this species was not attracted to
small black objects. Collectively, the results of our current study
and those of other studies show that the visual systems of insect
disease vectors and their behaviors constitute attractive targets for
the development of traps incorporating visual features that can be
species-specific in terms of attraction, thus providing incentive to
identify molecular targets that compromise mosquito olfactory-
visual responses.
Methods
Mosquitoes, odor delivery, and wind tunnel. Mosquitoes (Aedes aegypti: Rock-
efeller, Liverpool, Gr3[ECFP]13 and opsin-1,opsin-2 mutant lines; Anopheles ste-
phensi (Indian strain) and Culex quinquefasciatus) were raised at the University of
Washington campus. Mosquito lines were provided from BEI Resources (Mana-
ssas, VA, USA) (Ae. aegypti: Rockefeller, Liverpool, Gr3[ECFP]13;An. stephensi
and Cx. quinquefasciatus). In the case of the Ae. aegypti opsin-1,opsin-2, and opsin-
1,opsin-2 lines, we used existing mosquito lines that were generated as previously
described24. Briefly, the op1 and op2 alleles were generated by selecting short-guide
RNAs that targeted the GPROp1 (LOC5568060) and GPROp2 (LOC5567680)
loci24. Lines were homogenized and verified by PCR before testing24 (Supple-
mentary Tables 1–3). Mosquitoes were raised in groups of 100 individuals and
anesthetized with cold to sort males from females after cohabitating for 7 days. At
this time, more than 90% of the females have been mated, as indicated by their
developing embryos. For each experimental trial in the wind tunnel, we released 50
females into the wind tunnel working section 3 h prior to the mosquito’s subjective
sunset (the time period of peak activity for Ae. aegypti). Each visual stimulus was
tested in 4–12 experimental trials (on average, ~5575 trajectories were quantified
per trial) and 12,300 Ae. aegypti were flown in total across all experiments (99.4%
of all mosquitoes flew in the working section, and thus their behaviors were
captured by the real-time tracking system), for a total of 1,305,695 trajectories.
After one hour, the 5% CO
2
plume (or filtered air in control experiments), was
automatically released from a point source at the immediate upwind section of the
tunnel and at a height of 20 cm and in the centerline of the tunnel. The CO
2
remained on for 1 h, before switching off for another hour of filtered air (post
CO
2
). The 1 h time periods were chosen because mosquitoes did not adapt or
habituate to the CO
2
plume due to the diameter of the plume (~1.5 cm) and the
size of the tunnel lowered the encounter probability of the mosquitoes to the
plume. In addition, the 1 h periods provided a baseline of behavior before CO
2
release, and after encountering the CO
2
, mosquitoes would remain activated and
visually sensitized for 5–10 min—thus, the 1 h post-CO
2
period allowed sufficient
time for mosquitoes to return to baseline.
The CO
2
and filtered air were automatically delivered using two mass flow
controllers (MC-200SCCM-D, Alicat Scientific, Tucson, AZ) that were controlled
by a Python script that allowed synchronizing odor and filtered air delivery with
the trajectory behaviors. The CO
2
plume was quantified using a Li-Cor LI-6262
CO2/H2O analyzer (Li-Cor, Lincoln, NE) for a total of 500 locations throughout
the tunnel (Fig. 1). Data yielded an exponential decay similar to a model of
turbulent diffusion at the airflow (40 cm/s) and turbulent intensities (5%) of this
tunnel, such that 20 cm from the source and parallel to the wind flow the plume
was ~1700 ppm (Fig. 1c, d), which is in the range of the plume of human breath.
All behavioral experiments took place in a low-speed wind tunnel (ELD Inc.,
Lake City, MN), with a working section of 224 cm long, 61 cm wide, by 61 cm high
with a constant laminar flow of 40 cm/sec (Fig. 1). We used three short-throw
projectors (LG PH450U, Englewood Cliffs, NJ) and rear projection screens
(SpyeDark, Spye, LLC, Minneapolis, MN) to provide a low contrast checkerboard
on the floor of the tunnel and gray horizons on each side of the tunnel. The
intensity of ambient light from the projectors was 96 lux across the 420–670 nm
range. A 3D real-time tracking system (the open-source Braid system)11,42 was
used to track the mosquitoes’trajectories. Sixteen cameras (Basler AC640gm,
Exton, PA) were mounted on top of the wind tunnel and recorded mosquito
trajectories at 60 frames/sec. All cameras had an opaque IR Optical Wratten Filter
(Kodak 89B, Kodak, Rochester, NY) to mitigate the effect of light in the tracking.
IR backlights (HK-F3528IR30-X, LedLightsWorld, Bellevue, WA) were installed
below and the sides of the wind tunnel to provide constant illumination beyond the
visual sensitivity of the mosquitoes. The temperature within the wind tunnel,
measured using ibuttons and FLIR cameras (FLIR One Pro, FLIR Systems Inc.,
Goleta, CA USA), was 22.5 °C and did not show any variability within the working
section11,24. Ambient CO
2
was constantly measured outside of the tunnel and was
~400 ppm.
Visual stimuli and experimental series in the wind tunnel. To determine the
role of odor in the innate spectral preferences of mosquitoes, and identify the role
of achromatic contrast and wavelength discrimination, a series of different
experiments were conducted. In each experiment, two visual stimuli, separated by
18 cm, were presented to the mosquitoes on the floor in the upwind area of the
tunnel and perpendicular to the direction of airflow. Visual stimuli consisted of
paper circles that were 3 cm diameter (Color-aid Corp., Hudson Fall, NY, USA).
Reflectance spectra for all the visual stimuli were characterized using an Ocean
Optics USB2000 spectrophotometer with a deuterium tungsten halogen light
source (DH2000) calibrated with a white Spectralon standard (Labsphere, North
Sutton, NH, USA). The projector and working section light intensities were
measured using a cosine-corrected spectrophotometer (HR- +2000, Ocean Optics,
Dunedin, FL, USA) 5 cm from the projector source (63 µW/cm2). The achromatic
contrasts of the visual objects relative to the background were measured using the
calibrated spectrophotometer and the Weber contrasts were calculated by the
intensity (µW/cm2) of the object (I
object
) and background (I
backgroun
), where (I
object
-
I
background
)/I
background
. In the first experimental series, using wt (ROCK) Ae. aegypti,
we examined the innate preference of individual colors relative to the non-
attractive white object. Tested objects had distinct and peak wavelengths of 437 nm,
452 nm, 496 nm, 510 nm, 520 nm, 590 nm, 600 nm, and 660 nm (Bv-T2, Bw-, Gw-
T1, Gc-, YGc-, Yw-, O- and R-Hue; Color-aid Corp.). These stimuli all had similar
achromatic contrasts (−0.12 to −0.18) and peak reflectance values, but had distinct
peak wavelengths (Fig. 1g). The position of the respective hue and white object in
each replicate trial was randomized.
In the second experimental series, experiments were performed to examine
which spectral bands of the human skin might attract mosquitoes. To ensure the
replicability of the experiments, and enable the control of visual object humidity,
temperature, and odor, we first elected to first use faux skin mimics (Pantone
SkinTone Guide; Pantone LLC, Carlstadt, NJ 07072 USA). Using wt (ROCK) Ae.
aegypti, we tested four different skin tones (R10, Y10, and Y02) and a skin tone that
we named “vile 45”, which matched the putrid orange tone from individuals who
use cheap tanning lotion (PANTONE 16-1449X, Gold Flame). Similar to our
previous wind tunnel experiments, each individual skin tone was paired with an
evenly reflective object (white to human observer) that served as a control. To
attenuate different spectral bands reflected from the skin tone, we used ultra-thin
(~200 um thick) plastic filters that were placed over the object. Filters were selected
to attenuate the 450–530 nm band, the 550–630 nm band, or the 650–730 nm band
(36–333 [notch filter], 35–894 [long-pass filter], and 35–896 [long-pass filter],
respectively; Edmund Optics Inc., Barrington, NJ USA). Reflectance measurements
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of the object with the filters showed the transmission loss was <5% for bands
outside of the filtered wavelengths (Fig. 2B). As a control to determine if
attenuating a spectral band outside of the visual spectrum effects mosquito
behavior, we used an IR filter that allows transmission of 350–750 nm band of the
visual spectrum (14-547 [KG2], Edmund Optics Inc., Barrington, NJ USA). To
control for the physical effect that placing the plastic filter over the object may have
had on mosquito behavior, we used plastic coverslips with the same refractive index
as glass (72261-22; Electron Microscopy Services, Hatfield, PA USA). Experiments
were also conducted with Gr3−/−mutants13 and the opsin-1−/−,opsin-2−/−double
mutants24 to examine how the loss of olfactory or visual detection, respectively,
impacted mosquito attraction to the skin color. As controls, we used heterozygote
(Gr3−/+), single mutants (opsin-1−/−, opsin-2−/−), and the wild-type
(Liverpool) lines.
In the third series, the role of achromatic contrast, and not wavelength, on
mosquito visual preferences were examined. Gray circles (3 cm diameter), differing
in their Weber Contrast (−0.28 to −0.05) and ranging from near-black to very
light gray (Grays 1.5, 2.5, 4.0, 4.5, 6.5, and 9.5; Color-aid Corp., Greyset), were run
in combination with a white circle (Weber Contrast of 0.02). Similar to the above
experiments, wt (ROCK) Ae. aegypti were used in the experiments.
In the fourth experimental series, we examined whether the spectral preferences
of the mosquito (Ae. aegypti [ROCK]) while controlling for the apparent contrast
of the different hues. We normalized the perceptual contrasts of the visual objects
by testing a range of grays of different contrasts with the background (Grays 1.5,
2.5, 4.0, 4.5, 6.5, and 9.5 [Weber Contrasts of −0.28 to −0.05]; Color-aid Corp.,
Greyset) in combination with, and against, the 660 nm object (R-Hue; Weber
Contrast =−0.17). Once we identified the gray object that was as attractive as the
660 nm object, identified by the PI that not significantly from 0 (t-test: P> 0.05), we
then tested that gray (Gray 1.5; Weber Contrast =−0.28) against a range of
different objects that had the same peak wavelength (510 nm) but different Weber
Contrasts, from light to dark (−0.11 to −0.27). Based on the 510 nm object that
elicited the same level of attraction as the dark gray (PI =0), we then tested the
dark 510 nm object vs. the red object. Similar to experiments with the 510 nm
objects, we tested the dark gray (Gray 1.5; Weber Contrast =−0.28) against a
range of different objects that had the same peak wavelength (452 nm) but different
Weber Contrasts, from light to dark (−0.13 to −0.28), followed by testing the
660 nm object vs. the dark 452 object, and the attractive cyan (495 nm) object vs.
the dark 452 nm or dark 510 nm objects with the same apparent contrast.
Visual attraction to skin in a Cage-assay. To assay the visual attraction to the
skin, acrylic cages (45 × 30 × 30cm; McMaster-Carr; cat. # 8560K171) were con-
structed to allow for video recording and tracking from above. Thermal insulation
and white sheeting were wrapped around the cage’s exterior to prevent any heat
cues from outside of the cage while providing a uniform visual environment to the
interior. The sides and windows of the cage were sealed to prevent odor con-
tamination from the experimenter from leaking into the cage. Using thermal
imaging (FLIR One Pro, FLIR Systems Inc., Goleta, CA USA), solid-phase
microextraction fibers (75 um CAR/PD MS SPME fiber, 57344-U; Supelco, Belle-
fonte PA USA) for VOC collection and subsequent analysis using Gas Chroma-
tography with Mass Spectrometric Detection (Agilent Technologies, Palo Alto, CA,
USA), and CO
2
measurements both inside and outside of the cage, allowed for the
testing of odor and heat contamination within the cage. Results showed no odor,
CO
2
or heat contamination (Fig. S2). A small 4 cm vent on the cage side facing
away from the experimenter and underneath the hood ventilation system allowed
filtered air or 5% CO
2
input to the cage. In contrast to the wind tunnel assays,
ambient airflow was small (<10 cm/s) within the cage. To conduct the visual
preference assays, two 4 × 4 cm windows, spaced 18 cm apart, were cut into the
acrylic. Windows were sealed with heat absorptive glass (Schott KG2, Edmund
Optics) to prevent both thermal and odor contamination into the cage. Similar to
the visual stimuli used in the wind tunnel experiments, mosquitoes were tested
with a uniform reflective “control”in one window (white-colored glove to human
observer), and the other window displaying either human skin, or human skin
through long-wavelength optical filters (550–730 nm band; 36–333 and 35–894
filters; Edmund Optics Inc., Barrington, NJ USA). Positions of the visual stimuli
displayed in the windows were randomized between experimental replicates. To
test for any side preference or contamination in the cage, control experiments were
also conducted using two white gloves as the visual stimuli in the windows.
Experiments were performed in a chamber held at ~20–22.5 °C, and the cage was
situated underneath a hood ventilation system allowing air exchange. A CO
2
Flypad (Genesee Scientific; cat. # 59-119) was placed immediately adjacent to the
vent on the side of the cage, and similar to the wind tunnel experiments, CO
2
was
controlled by two mass flow controllers (MC-200SCCM-D, Alicat Scientific,
Tucson, AZ) via a Python script that allowed synchronizing odor and filtered air
delivery with the trajectory behaviors. Filtered air was released for the first 8 min of
each experiment, followed by the release of 5% CO
2
for 8 min, before switching off
for another 8 min (post-CO
2
). Two cameras (Basler AC640gm, Exton, PA) were
mounted above the cage and recorded mosquito trajectories at 100 frames/sec. IR
backlights (HK-F3528IR30-X, LedLightsWorld, Bellevue, WA) were installed above
the cage. Human skin reflectance measurements and assays were from three males
and three female individuals on the University of Washington (Seattle, WA USA)
campus (ages 25–46 years old), and volunteers were from various ethnic groups.
An ergonomic armstand set was used to position and keep the arms steady over the
24 min. period. Because CO
2
causes behavioral changes in the mosquitoes that can
last 5–10 min after CO
2
exposure is stopped, the 24 min. experiment was the
minimum time that allowed us to examine the mosquito responses at the different
time periods (Air, CO
2
, post-CO
2
). Protocols were reviewed and approved by the
University of Washington Institutional Review Board, and all human volunteers
gave their informed consent to participate in the research. Similar to the wind
tunnel experiments, we used 6–8 day-old, non-blood-fed, mated females who were
sucrose deprived for 24 h but had access to water. We released 50 Ae. aegypti
(ROCK) females into the cage for each experiment, and the assays were initiated
3 h before lights off (ZT12). Ambient CO
2
was constantly measured both inside
and outside of the cage.
Visual preferences in Cx. quinquefasciatus and An. stephensi.Anopheles ste-
phensi (Indian strain) and Culex quinquefasciatus mosquitoes were separately
raised in groups of 100 individuals and anesthetized with CO
2
to sort males from
females after cohabitating for 7 days. At the time of their subjective sunset, groups
of 50 female mosquitoes were released into the working section of the wind tunnel.
After 1 h of filtered air, 5% CO
2
was released from a point source at the upwind
section of the tunnel (height of 20 cm, and in the center of the tunnel) for 1 h, after
which filtered air was released from the point source. The intensity of ambient light
from the projectors was ~1.3 µW/cm2. The low-light intensity, relative to that used
with Ae. aegypti, was necessary to recruit females to the visual objects. At higher
light intensities An. stephensi and Cx. quinquefasciatus mosquitoes responded to
the CO
2
plume but they did not respond to the visual objects. We found that these
two species began to investigate the visual objects only at light intensities <5 lux.
Like our experiments with Ae. aegypti, the temperature within the wind tunnel was
~22.5 °C.
Trajectories analysis. As described above, for each experimental trial we release a
group of 50 mosquitoes because the working section of the wind tunnel was large
enough to minimize any interactions between individuals, while allowing for the
efficient capturing of behaviors to the CO
2
plume and visual objects. Our tracking
system is unable to maintain mosquito identities for extended periods of time, but
we considered individual trajectories as independent for the sake of statistical
analysis. To ensure that the release of the 50 mosquitoes reflected behaviors of
single mosquitoes in the working section, experiments were performed as described
in Material and Methods—Visual Stimuli and Experimental Series in the Wind
Tunnel but using single mosquitoes. A 660 nm and evenly reflective objects (red
and white to the human observer) were used as the visual stimuli in these
experiments. Fifty Ae. aegypti mosquitoes were individually tested and released into
the working section of the tunnel and their behaviors were compared to mosqui-
toes that were co-released. Results of these experiments showed that all 50/50
individual mosquitoes flew in the tunnel, which compares with the 99.4% of
mosquitoes that flew in the tunnel in all the co-released experiments. We also
found that flight velocities, durations, and PIs of the individual mosquitoes to red
and white circles were not statistically different from co-released mosquitoes
(unpaired t-tests, P=0.75, 0.93, and 0.44 for flight duration, flight velocity, and PI,
respectively). During the CO
2
exposure, on average and for those mosquitoes that
investigated the visual objects (38% of the 50 mosquitoes individually-released into
the tunnel), one mosquito produced nine flight trajectories during the 1 h period
(range from 1 to 37 trajectories) and investigated the visual object 3.70 times
(±2.33). This was similar to the estimated number of trajectories per mosquito that
investigated the objects in co-released experiment s (4.76 ± 3.64). Since 38% of the
mosquitoes investigated the visual objects, this percentage provided evidence that
our trials were not biased from insufficient sampling. Therefore, co-release of the
mosquitoes allowed efficient testing of mosquito behavior to the visual objects and
did not differ from individually released mosquitoes.
Analyses were restricted to trajectories that were at least 90 frames (1.5 s) long.
Only trajectories that lasted for more than 1.5 s were analyzed (average length
trajectory: 3.1 s, longest trajectory: 96.4 s, total number of 1,305,695). The flight
activity in the different phases of an experimental trial (pre-, CO
2
and post-CO
2
)
where the mosquitoes encountered filtered air or CO
2
were quantified by the
number of trajectories recorded during one phase divided by the number of
trajectories recorded in the previous phase. To examine the mosquito behaviors
and preferences to the two visual stimuli in the tunnel, a fictive volume was created
around the visual cues (area: 14 × 14 cm, height: 4 cm). The volume was centered
over the object in the crosswind direction, and shifted slightly downwind in the
wind line direction. This volume was chosen as it captures the area of primary
activity of the mosquitoes. A sensitivity analysis was performed by adjusting the
volume size and demonstrated that this volume best captured the mosquitoes
investigating the visual objects while excluding mosquitoes transiting to other areas
of the working section.
Occupancy maps were calculated by dividing the wind tunnel into 0.3 cm2
squares. For each replicate experiment, the number of mosquito occurrences within
each square was summed and divided by the total number of occurrences in all
squares to yield a percentage of residency. We did not quantify landings on the
spots due to limitations of the camera angles needed to identify landings. During
the filtered “air”treatment, mosquitoes often investigated certain areas of the
working section, such as the top or corners of the working section, causing hot
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spots in the occupancy maps. This is typical for mosquito activity without a
stimulus. By contrast, when CO
2
is released, these hot spots are no longer apparent,
and instead, the mosquitoes investigate the visual objects or navigate to the odor
source, as demonstrated by a hotspot in the central area of the working section or
near the odor source. For each mosquito line, the replicate trials were pooled to
create an occupancy heat map for the tested visual stimulus.
To calculate the fractions of trajectories that approached either visual object, for
each trajectory we calculated a preference index by determining the amount of time
a trajectory spent in each volume divided by the total time it spent in both volumes.
If the trajectory spent all of its time in only one volume then it was assigned a
preference index of 1 (test object) or −1 (white neutral object). Approximately
25–50% of the trajectories approached either object. From these preference indices,
we calculated the global mean, and bootstrapped the 95% confidence interval of the
mean through random resampling of the individual trajectories 500 times. To
determine whether the mosquitoes preferred the visual objects compared to
elsewhere in the tunnel, we calculated the preference index for each trajectory at
each time point as the amount of time the mosquito spent in a particular
4 × 14 × 14 cm volume that was randomly selected in the tunnel and compared
them to the volumes containing the visual objects. Mean flight velocities were
calculated from the 3D tracks of each individual trajectory. To further examine
whether mosquito responses to the visual objects changed throughout the
experiment, the percent of time (per each minute interval) the mosquitoes
investigated the visual objects was calculated. Statistically significant groups were
estimated using a Kruskal–Wallis, Mann–Whitney U-test with Bonferroni
correction at a P=0.01 level, or the one-sample t-test. All recorded data were
analyzed using Matlab (Mathworks, 2019a release). To explore the potential impact
of pseudoreplication on the statistical results, we used a multilevel analysis using
the restricted maximum likelihood method and setting the experimental trial as a
random, nested effect. We found that, for the visual stimuli used in the
experiments, there was a significant impact of the visual spectra of the object on the
preference index (P< 0.001) but the experimental trial did not contribute to the
model results. Thus, potential for pseudoreplication due to biased sampling did not
contribute to our results. The model was created using R 4.0.3 and the lmer
function from the lme4 package43.
Electroretinogram (ERG) recordings. ERG recordings were performed by fixing
6 day-old, non-blood-fed female mosquitoes to a coverslip using Bondic glue. Mos-
quitoes were dark-adapted for 1h prior to stimulation. The recording glass electrode
(thin-wall glass capillaries; OD, 1.0mm; length, 76 mm; World Precision Instruments,
cat. # TW100F-3) was pulled using a micropipette puller (Sutter Instrument, p-2000),
and filled with Ringer’s solution (3 mM CaCl
2
, 182 mM K Cl, 46 mM Na Cl, 10 mM
Tris pH 7.2). The reference electrode, a sharpened tungsten wire, was placed into one
compound eye in a small drop of electrode gel (Parker, cat. # 17-05), and the
recording electrode was placed immediately on the surface of the contralateral eye.
Two different types of visual stimuli were presented to the mosquitoes. In the first
series, the mosquitoes were placed at the center of a semi-cylindrical visual arena
(frosted mylar, 10 cm diameter, 10 cm high); a video projector (Acer K132 WXGA
DLP LED Projector, 600 Lumens) positioned in front of the arena projected the visual
stimuli. To test the response to moving objects, similar to what the mosquito might
encounter in flight, we tested responses to a 19° wide bar moving from left to right
(Clockwise) (Fig. 4). The mosquitowas randomlytested with blue, green, and red bars
(distinct peaks at 455, 547 nm, and 633 nm, 18 lux), and each colored bar was tested
10–30 times per mosquito (n=7 mosquitoes).
The second stimulation method used a digital monochromator to examine
responses to different wavelengths across the mosquito visual spectra
(350–750 nm). Mosquitoes were exposed to a 1 s pulses of light (10 lux) from a
light source (35-watt Halogen; ThorLabs) and a fiber optic scanning
monochromator (MonoScan 2000, Mikropak GmbH, Ostfildern, Germany) that
provided control of the transmitted wavelengths (±2 nm). Light was transmitted via
optical fibers (QP600-1-SR-B X, Ocean Optics, FL 32792, USA) and through a
neutral density filter (fused silica, Thorlabs Inc., 0-1 OD). Each mosquito
preparation was tested to wavelengths of 350–750 nm in 10 nm increments (n=8
mosquitoes/species). The visual stimuli were calibrated using a cosine-corrected
spectrophotometer (HR- +2000, Ocean Optics, Dunedin, FL, USA) that was
placed immediate to the recording preparation, allowing us to scale the irradiance
of the tested stimuli. The light-induced responses were amplified by using an A-M
Systems amplifier (10-100x; A-M Systems, 1800) and digitized using a Digidata
data acquisition system (Digidata 1550B, Molecular Devices, San Jose, CA 95134).
Data were visualized and analyzed using Matlab software (Mathworks).
Linear models. Linear models were created using R 4.0.3 and the lm function with
the default option. Comparison between models were performed using the AIC
function that calculates the Aikaike’s Information Criterion (AIC) for each model.
For the first series of models, the dataset consisted of the mean preference index
per experiment, the contrast value, peak wavelength and brightness value for the
tested object. For the second series of models, the dataset contained the mean
preference index per experiment and the area under the curve (AUC) of the
reflectance measurement for the tested object calculated with bins of 25 nm from
350 to 675 nm. A Principal Component Analysis (PCA) was applied to the AUC
vector to remove collinearity in the object’s spectrum.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The source data underlying Figs. 1h, 3d, and 5c are provided as a Source Data file. The
wind tunnel data generated in this study have been deposited in the Dryad Data
Repository at https://doi.org/10.5061/dryad.d51c5b04d. Source data are provided with
this paper.
Code availability
Software is available on https://github.com/riffelllab (https://doi.org/10.5281/
zenodo.5579784) and https://github.com/strawlab/strand-braid.
Received: 27 July 2021; Accepted: 5 January 2022;
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Acknowledgements
We are grateful for the advice and discussions with C.E. Reisenman and F. Van Breugel.
Support for this project was funded by Air Force Office of Scientific Research under
grants FA9550-20-1-0422 (J.A.R.); the National Institutes of Health under grants R01-
AI148300 (J.A.R.), R21-AI137947 (J.A.R.); and an Endowed Professorship for Excellence
in Biology (J.A.R.). C.M. was supported by EY008117 (C.M.) from the NEI, AI165575
(C.M.) from NIAID, DC016278 (C.M.) from NIDCD, and from the U.S. Army Research
Office and accomplished under cooperative agreement W911NF-19-2-0026 (C.M.) for
the Institute for Collaborative Biotechnologies. A.D.S. was supported by the Momentum
program of the Volkswagen Foundation.
Author contributions
D.A.S.A., C.R., and J.A.R. designed research; D.A.S.A., C.R., and J.A.R. performed wind
tunnel experiments; C.R. and J.A.R. conducted electroretinogram experiments; A.D.S.
assisted in visual stimulus, wind tunnel software, and experimental designs; Y.Z. and
C.M. generated the opsin mutant lines. All authors wrote and edited the paper.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41467-022-28195-x.
Correspondence and requests for materials should be addressed to Jeffrey A. Riffell.
Peer review information Nature Communications thanks Claudio Lazzari, Marcelo
Lorenzo and Zainulabeuddin Syed for their contribution to the peer review of this work.
Peer reviewer reports are available.
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