Carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours
1 Department of Entomology, University of California, Riverside, CA 92521,
USA
2 Institut fur Zoologie, Universität Regensburg,
Universitätsstrasse 31, Regensburg 93040, Germany
* Author for correspondence at present address: Division of Chemical Ecology, Department of Crop Science, SLU, Alnarp, SE-23053, Sweden (e-mail: teun.dekker{at}vv.slu.se)
Accepted 6 June 2005
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Summary |
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Key words: Aedes aegypti, mosquito, orientation, sensitisation, host odour, carbon dioxide, human skin odour
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Introduction |
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In mosquitoes, however, many different classes of sensory neurons are
involved in host odour detection, and the odour mix that emanates from a host
is very complex, consisting of several hundred compounds
(Bowen, 1996;
Meijerink and Van Loon, 1999
;
Pappenberger et al., 1996
).
The cues picked up by the antenna are ambiguous, as many odours are not
specific to a vertebrate host, remain present in the absence of a live host
(such as inside houses), and vary among individuals of the same host species
and from day to day (Bernier et al.,
2001
). Filtering and integration of odour mixtures should be
exceedingly important for host recognition by mosquitoes.
In contrast, exhaled CO2, a nearly universal mosquito activator
and attractant (Rudolfs, 1922;
Reeves, 1953
;
Gillies, 1980
), is diluted
against atmospheric CO2 levels (background around 0.035%,
vs 4% for exhaled CO2). Fluctuating levels of
CO2 therefore invariably signify a nearby living vertebrate. We
tested the sensitivity of mosquitoes to CO2 and skin odours, and
whether, besides being a strong activator and attractant, CO2 can
influence the mosquito's olfactory response to skin odours, thereby acting as
a `releasing stimulus'. We used the synanthropic subtropical mosquito
Aedes aegypti L., an important vector of dengue and yellow fever.
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Methods |
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Experimental setup and testing procedures
We recorded the flight behaviour of mosquitoes in a wind tunnel
(Fig. 1) with a transparent
Plexiglas flight chamber of 150 cm (length)x50 cm (width)x50 cm
(height), the sides of which were covered with Medium Red light filter
(Roscolux, Rosco Laboratories, Stamford, CT, USA, which blocks light <600
nm) to prevent orientation of the mosquitoes to visual cues outside of the
tunnel. Transparent red dots (6 cm diameter, approx. 100 m2,
Medium Red, Roscolux, Stamford, CT, USA) randomly arranged on the floor of the
wind tunnel provided `non-directional' optomotor cues. Outside air from 8 m
above ground was pushed by a centrifugal in-line duct fan through the wind
tunnel; air flowing through the tunnel was free of human odours. The air was
filtered by an activated charcoal filter, humidified to 70±5% RH, and
adjusted to 27±2°C. A turbulence-free airflow of 30 cm
s1 was created by passing the air through an activated
carbon air filter, an aluminum honeycomb laminizer (consisting of cells of 1.5
cm diameter and 15 cm long), and two stainless steel screens (150 mesh).
Mosquitoes were illuminated against the background by four infrared lights
(UFL 694, Rainbow, Irvine, CA, USA) at the tunnel's downwind end. Each had 60
LEDs (940 nm), equipped with cut-off filters permeable to light of >950 nm.
During the experiments the light level in the human visual spectrum from
diffuse fluorescent lights was approximately 15 lux.
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Odours
We tested CO2 and human skin odour at various concentrations.
CO2 was obtained from 100% (99.9% purity) and 4% pressurized
cylinders. Skin odour was obtained by inserting a human arm (belonging to
T.D.) in a 10 cm diameter glass tube and pushing 30 l min1
clean air from a pressurized cylinder through the tube
(Fig. 2). A stainless steel fan
was mounted at one end of the tube to create a strong air movement over the
hand, ensuring a high uptake of skin odour and thorough mixing of the
odour-laden air. We used Teflon® tubing (0.3 cm i.d.) from the skin-odour
tube to the plume generators. The tubes were 15 cm long, thereby minimizing
odour adsorption in the tubes. Except for the hand used in the skin-odour
tube, we used Fisherbrand® (Pittsburgh, PA, USA) latex examination gloves
during the experiments to avoid contamination with any experimental device. 3
h before the experiments the arm was washed with tapwater for 1 min. The
laminizing screens of the wind tunnel were replaced whenever the concentration
of skin odour of a new experiment was lower than used in the previous
experiment. Screens were washed thoroughly with water and soap.
Plumes
Two kinds of plumes were used.
Broad plume
We created a broad continuous plume by pushing the odour-laden air into a
14 cm diameter plume generator placed upwind from the two laminizing screens
(Fig. 1B,C). The resulting flow
was isokinetic to the main flow through the wind tunnel. To ensure vigorous
mixing, clean air was blown at 4 l min1 into the plume
generator, immediately downwind of the point where the odour entered (see
Fig. 1B). To obtain the desired
concentration of CO2, we adjusted the flow rate from a cylinder of
100% or 4% CO2, with a background CO2 concentration of
350 p.p.m. and a flow of 360 l min1 through the plume
generator. Lower concentrations of skin odour were obtained by taking a
fraction of the skin-odour-laden (see above) air (verified `offline' using
bubble flow meters with a negligible resistance). As a control in the
broad-continuous skin-odour experiments, we also created a homogeneous
skin-odour plume by directly inserting a hand (belonging to T.D.) in the plume
generator upwind from the laminising screens
(Fig. 1C).
The plume structure was analyzed for homogeneity by simulating the plume
structure with air containing 1000 p.p.m. propylene as a chemical tracer gas
(Justus et al., 2002). We
analyzed the plume structure at 50 cm from the source, at various points along
the lateral and vertical axes using a mini-photoionization detector (mini-PID,
Aurora Scientific, Aurora, ONT, Canada) at a sampling rate of 100 Hz. This
demonstrated that the plume was fairly homogeneously distributed
(Fig. 3).
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The laminar flow through the wind tunnel allowed for accurate estimation of the plume's diameter and the position. The plume had a diameter of approximately 15 cm. The centre of the plume was 25 cm from the side and 17 cm above the wind tunnel floor. By turning the release cage at the start of the experiment, the cage was centred in the plume. Mosquitoes exiting the cage once it was turned were in contact with the broad plume.
Superimposed ribbon CO2 plume
We created a ribbon plume of 4% CO2 from a pipette 100 cm
downwind and 30 cm upwind of the release cage
(Fig. 1A). The ribbon plume had
a diameter of 0.5 cm. The plume passed through the centre of the release cage
and was superimposed within a broad homogeneous skin odour plume. Most
mosquitoes leaving the release cage contacted the ribbon plume.
Experiments
(1) Effect of odour concentration on activation, upwind flight and source finding
The following series were performed with broad continuous plumes:
(2) Effect of exposure to CO2 filaments on sensitivity to skin odour
In this experimental series we presented the mosquitoes with broad
continuous skin-odour plumes of various concentrations, with or without a
ribbon plume of 4% CO2 superimposed within the downwind end of the
skin odour plume (see Fig. 1A).
We tested 100% (i.e. the full skin-odour-laden air) skin odour and fivefold
steps dilutions thereof. The following treatments were tested:
We scored source finding for all treatments, and reconstructed the flight tracks for treatments a, b, c and h. We analyzed the flight tracks upwind from the ribbon CO2 plume. These tracks therefore represent flight tracks in the absence of a fluctuating CO2 signal. Treatments were randomized within each experimental day.
Data analysis
A Weibull distribution was used to characterise the activation rate of
Ae. aegypti (Crawley,
1993). The shape parameter
allows the activation rate
(`hazard') to increase (
>1) or decrease (
<1) over
experimental time, starting with a constant rate (exponential distribution,
=1). Mosquitoes that left the release cage before the cage was turned
into the plume were excluded from further analysis. We used a censoring
indicator for mosquitoes that did not take off within the experiment, allowing
for `non-responders' to contribute to the survivorship function. Differences
between the survivorship curves were assessed with
2-values
(Aitkin et al., 1989
;
Crawley, 1993
).
The percentage of mosquitoes that left the release cage and reached the source (the section of the upwind screen from where the plume entered the wind tunnel) was arcsine square-root transformed and analyzed by a two-way analysis of variance (ANOVA), followed by an LSD post hoc test to assess the significance of differences between the means.
Mosquitoes flying along the side of the wind tunnel were excluded from
track analysis. In the sensitization experiments, those parts of the flight
tracks downwind from the ribbon CO2 plume were excluded from
analysis. The subsequent tracks represented tracks in response to skin odour
only. The data were smoothed with the cubic spline algorithm, a method that is
particularly well suited for data that are parabolic in nature
(Jackson, 1979).
Custom-made programs in Visual Basic® for Applications were employed to analyze the flight tracks with respect to the mosquito's position relative to the plume. We verified the plume's position visually using TiCl4 `smoke,' and chemically using surrogate odour propylene in conjunction with the photoionization detector (mini-PID; for details, see above). We averaged flight parameters over 100 ms (3 frames) and scored how the flight parameters changed over time after flying into or out of the plume, first within each flight track followed by between flight tracks. Here we present only two flight parameters, the track angle and the flight speed. The track angle is defined as the angle of the insect with respect to wind, with upwind at 0°. The flight speed (in mm s1) is the velocity of the insect in 3-D.
The data were log transformed, checked for normality and day effects, and analyzed in Statistica (StatSoft, Inc., Tulsa, OK, USA) using a repeated-measure ANOVA, followed by an LSD post hoc test. Contrasts were used to test for significant changes in a parameter within a treatment after plume contact (repeated measures).
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Results |
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Track angle
Mosquitoes headed more due upwind within 300 ms after entering a 100% skin
odour or `hand' plume (Fig.
5A); mosquitoes leaving the plume headed more crosswind within 300
ms (skin odour and `hand'). No significant change in track angle was observed
after mosquitoes had entered or exited a plume of 20% skin odour, 4% skin
odour or clean air.
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(B) CO2 series
Activation
Fig. 4B shows the activation
rate for the treatments within the CO2 series. Activation was lower
for 1% CO2 and `hand' compared to the other treatments
(P<0.02 and <0.005, respectively). Activation rates with 1%
CO2 and `hand' were comparable.
Flight velocities
Flight velocities were similar for all treatments
(Table 1).
Track angle
Upon entering the plume, the track angle
(Fig. 5B) changed to more due
upwind within 200 ms (all concentrations of CO2) and 300 ms (skin
odour). Mosquitoes exiting the plume projected their tracks more crosswind
within 200 ms (skin odour, 0.05% and 0.1% CO2), 300 ms (0.3%), or
600 ms (1% CO2) of exiting the plume.
Source finding
Source finding was comparable for all treatments
(Table 1). The lower source
finding rate with `hand' was not significant (P=0.06).
(C) Sensitization series
Fig. 6 shows representative
tracks in response 20% skin odour, 20% skin odourCO2,
100% skin odour, and to CO2 only (ribbon plume). In the analysis,
the first part of the tracks downwind from the CO2 source (i.e. 100
cm and further downwind) were excluded, such that the data represent responses
to skin odour alone.
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Track angle
After entering a plume of 100% skin odour, mosquitoes aimed their thrust
more due upwind within 200 ms, and more crosswind within 200 ms after exiting
the plume (Fig. 5C). No changes
were observed with CO2 only and 20% skin odour. Mosquitoes that
intercepted one or more CO2 filaments, however, changed their
flight paths upon entering and exiting the 20% skin odour plume in a similar
fashion to mosquitoes responding to 100% skin odour.
Source finding
Table 1 shows that the
percentage of mosquitoes that reached the source was lower with 20% skin odour
than with 100% skin odour (P<0.001). However, the percentage of
mosquitoes that reached the source of 20% skin odourCO2
was comparable to the 100% skin-odour plume. After CO2 filament
encounters, mosquitoes showed increased orientation (i.e. upwind turning when
entering the plume) to 20% skin odour for a period of over 10 s. Source
finding was comparable for 20% skin odour and 4% skin
odourCO2, and higher than for 0.8% skin odour, 0.8% skin
odourCO2 or CO2 only.
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Discussion |
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Activation
CO2 is a very potent activator. Ae. aegypti was
activated by highly diluted, close to background concentrations of
CO2 (0.05%, background 0.035%). High activation rates by
CO2 have been frequently reported (e.g.
Gillies, 1980;
Geier and Boeckh, 1999
). After
the onset of the experiment, activation rates with 1% CO2 declined
over time, which may have been caused by sensory adaptation. CO2
receptor cells adapt rapidly under continuous stimulation
(Grant and O'Connell, 1996
;
Grant et al., 1995
). In
contrast to CO2, our data suggest that skin odour is only
activating at concentrations that occur near a human host. Although we scored
an initially higher activation rate at the start of the experiment (the
Weibull shape parameter, or `hazard,'
<1 for all treatments),
probably through a slight mechanical stimulation during turning of the cage
(e.g. vibration and reversal of the wind flow), this should have influenced
all treatments equally. Historically, activation and orientation have been
considered separate steps in the host-seeking process. We found a similarly
strong effect of CO2 on parameters of mosquito flight, including
speed and kinetic responses.
Flight speed
The flight speed increased with high concentrations of skin odour and with
all concentrations of CO2. Such orthokinetic responses have been
reported for moths upon repetitive interception of pheromone filaments (e.g.
Mafra-Neto and Cardé,
1994,
1995
;
Vickers and Baker, 1994
) and
for tsetse and stable flies in response to CO2, acetone and octenol
(Paynter and Brady, 1993
;
Schofield and Brady, 1997
).
However, mosquito flight speed remained fairly constant over time,
irrespective of flying in or outside of the plume. In contrast, male moths
increased their flight speed by twofold or more after contacting pheromone
filaments (Mafra-Neto and Cardé,
1994
,
1998
). The relatively constant
flight speed in mosquitoes may be a requirement when flight stability depends
on haltere input (Chan et al.,
1998
). Alternatively, mosquito flight may be characterized by
close to maximum flight muscle output, such that mosquitoes cannot increase
flight speed much without compromising flight stability
(Lehmann and Dickinson,
2001
).
Track angle
Within about 200 ms of encountering a single filament of pheromone, moths
aim their tracks more due upwind (Baker and
Vickers, 1997; Cardé
and Mafra-Neto, 1997
; Quero et
al., 2001
). Upwind turning in odour plumes also has been recorded
for tsetse and stable flies (Colvin et
al., 1989
; Paynter and Brady,
1993
; Schofield and Brady,
1997
) in response to CO2, octenol and acetone odours.
In a different bioassay setup using vertical, odour-laden convection currents,
Daykin et al. (1965
) reported
increased turning of Ae. aegypti when leaving but not when entering a
skin-odour plume. Our averaged flight tracks revealed a robust response of
Ae. aegypti to entering and to exiting a plume of CO2 and
high concentrations of skin odour. Entering such a plume resulted in a rapid
(i.e. within 200 ms) change of the track to more due upwind. Conversely,
exiting the plume resulted in a similarly rapid increase to more due
crosswind. At 1% CO2, mosquitoes exiting the plume increased their
angle more slowly than at lower CO2 concentrations. This may be
caused by sensory adaptation of CO2 olfactory sensory neurons, of
which the phasic response portion quickly adapts upon stimulation
(Grant and O'Connell, 1996
).
In contrast, with a high skin-odour concentration, a fivefold dilution of skin
odour evoked only a weak and irregular average upwind turning upon entering
the plume, and no change upon leaving the plume. Often this resulted in
mosquitoes not regaining contact with a lost skin-odour plume.
Source finding
Source finding is the most commonly used measure of `attractiveness.' Our
source-finding data parallel the activation and track analysis data by showing
a high source-finding rate at all CO2 concentrations and at 100%
skin odour, and a rapid loss of sensitivity at dilutions of skin odour. Our
source-finding results also suggest that Ae. aegypti mosquitoes are
more sensitive to CO2 than to skin odour. Although mosquitoes may
respond to highly diluted filaments of CO2, other environmental
factors, such as the constancy of wind direction, atmospheric stability and
habitat (Brady et al., 1990;
Murlis et al., 1992
), may
limit the range over which a CO2 plume can be followed to a
potential host.
In summary, our flight track analysis shows that upwind flight and source
finding of Ae. aegypti in response to skin odour wanes quickly with
dilution. Because undiluted skin odour was in the range of naturally
encountered concentrations, we conclude that Ae. aegypti may be less
sensitive to skin odours than previously supposed (e.g.
Gillies, 1980;
Takken and Knols, 1999
). In
contrast, our study shows that the sensitivity and the range of attraction of
CO2 may be greater than assumed. The previous conjecture of a
limited range of attraction for CO2 may partly have been caused by
the assumption that as CO2 is transported downwind in a plume, it
rapidly decreases to background concentrations
(Gillies, 1980
). In reality,
CO2 follows an asymptotic rather than linear dilution curve, i.e. a
100x dilution of 4% CO2 against a background of 0.035% yields
a concentration of 0.075% (asymptotic dilution) instead of 0.04% (linear
dilution). Moreover, filaments of odour can be transported in an odour plume
many meters downwind without appreciable dilution
(Murlis et al., 1992
). Because
a concentration difference of only 0.005% (i.e. an 800x dilution from
4%) changes the firing rate of CO2-sensitive cells for several
mosquito species (Grant and O'Connell,
1996
; Grant et al.,
1995
), and the mosquito shows behavioural sensitivity to slight
elevations in CO2 levels (this study), the range over which
CO2 can potentially attract mosquitoes may have been
underestimated. Zöllner et al.
(2004
) released CO2
from a point source into two types of woodland habitats at a rate equivalent
to that emitted by a typical bovid host and measured CO2 levels at
various distances downwind. Fluctuations in CO2 levels could be
readily detected over background at distances up to 64 m (the maximum distance
sampled) in a riverine habitat. This indicates that intermittent bursts of
CO2 from a vertebrate host could be used as an orientation cue over
distances much greater than previously assumed.
Although CO2 does not signify a host-specific cue for mosquito
species exhibiting a preference to bite certain vertebrate species
(Mboera et al., 1998;
Takken and Knols, 1999
;
Pates et al., 2001
), it does
invariably indicate a live potential host. The behavioural sensitivity to
CO2 and innate olfactory sensitivity for certain odours may be
different for other mosquito species. The fact, however, that CO2
is the only odour that increases capture rates of many
(Mboera and Takken, 1997
)
mosquito species in the field, and the fact that CO2-sensitive
cells exhibit a similar sensitivity across various species
(Grant and O'Connell, 1996
),
suggest that CO2 is key in the host orientation of most mosquito
species.
Sensitization
Our results show that CO2 also modulates the female Ae.
Aegypti's threshold of sensitivity to skin odours. A brief encounter
with a CO2 filament instantaneously increased their sensitivity by
at least fivefold. Such sensitization persisted for at least 10 s (the time
between the last CO2 encounter and the tracks recorded). To our
knowledge, this is the first example of an instantaneous behavioural
sensitization of the olfactory circuitry. It differs from classic synergistic
responses (Acree et al., 1968;
Eiras and Jepson, 1994
;
Geier and Boeckh, 1999
) in
that the stimuli are separated in time. Because we analyzed flight tracks
upwind of the CO2 source, our results demonstrate that the
increased response to diluted skin odour is caused by true sensitization, i.e.
increased sensitivity of the olfactory system to skin odour.
CO2-induced sensitization may be more pronounced after deprivation
of fluctuating CO2 signals, such as in our study (for 12 h).
CO2-sensitive sensory cells of mosquitoes are located on the
maxillary palps, whereas skin odours are perceived by sensory cells on the
antennae (Kellogg, 1970;
Meijerink and Van Loon, 1999
).
This implies that sensitization takes place either at the level of the
olfactory lobe or at higher brain centres. In honeybees, a slow-acting
modulation of sensitivity of the antennal lobe via a protocerebral
feedback loop has been implied in olfactory learning
(Iwama and Shibuya, 1998
;
Faber et al., 1999
;
Sachse and Galizia, 2002
). In
locusts, repeated stimulation with the same odour enhanced oscillatory
synchronization of projection neurons
(Stopfer and Laurent, 1999
),
and induced a higher sensitivity of individual projection neurons
(Bäcker, 2002
). Other
potential underlying neuronal mechanisms of the observed behavioural
sensitization may involve a general neuromodulatory network in the antennal
lobe (e.g. Sachse and Galizia,
2002
), or feedback neurons from the antennal lobe to the OSNs, a
unique neuromodulatory pathway in mosquitoes
(Meola et al., 2000
;
Meola and Sittertz-Bhatkar,
2002
; Bäcker,
2002
).
Although the proximate cause of sensitization for skin odour by
CO2 remains to be established, such rapid sensitization may be
adaptive, because in a miasma of potential host odours, a fluctuating
CO2 signal reliably signifies a warm-blooded vertebrate. However,
as CO2 is not a host-specific cue, the question arises whether such
a high sensitivity to CO2 is indeed an adaptation by mosquitoes
that prefer to bite humans. This raises the issue of what portion of host
preference is determined by odours, and what by other factors, such as resting
behaviour. Host preference in Ae. aegypti is dependent in part on one
or a few genes that determine its propensity to either rest inside or outside
houses (Trpis and Hauserman,
1978). Similarly, blood meal analysis shows that the host
preference of Ae. aegypti is strongly influenced by the relative
availability of host species inside dwellings
(Tandon and Ray, 2000
).
Furthermore, Ae. aegypti oviposits predominantly close to or inside
dwellings, which implies that adults may not have to orient from far away to
their preferred host. In such habitats a high sensitivity to fluctuations in
CO2 levels would indeed help to alert the mosquito to the presence
of a host. The question of how these and other ecologically important cues
mediate host finding in the field necessitates further study.
Finally, the observed CO2-induced activation, sensitization and orientation to a potential host may be more important for day-active mosquitoes such as Ae. aegypti, which bite when the host may be moving. Whether other mosquito species and genera with other feeding habits, such as the nocturnal anopheline mosquitoes, which typically feed when the host is stationary, have a similar organisation of response to host odours remains to be determined.
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Acknowledgments |
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References |
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