Chem. Senses 24: 647-653,
1999
© Oxford University Press 1999
Ammonia as an Attractive Component of Host Odour for the Yellow Fever Mosquito, Aedes aegypti
Institut für Zoologie, Universität Regensburg, Universitätsstraße 31, D-93040 Regensburg, Germany
Correspondence to be sent to: Martin Geier, Institut für Zoologie, Universität Regensburg, Universitätsstraße 31, D-93040 Regensburg, Germany. e-mail:martin.geier{at}biologie.uni-regensburg.de
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Abstract |
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Behavioural responses of Aedes aegypti mosquitoes to ammonia were investigated in a modified Y-tube olfactometer. Ammonia was attractive in concentrations from 17 ppb to 17 ppm in air when presented together with lactic acid. Aqueous solutions of ammonia salts in concentrations comparable to those found in human sweat also increased the attractiveness of lactic acid. The role of lactic acid as an essential synergist for ammonia became further apparent by the fact that ammonia alone or in combination with carbon dioxide was not effective, even though the synergistic effect of carbon dioxide and lactic acid was corroborated. An extract from human skin residues, which attracts ~80% of the tested mosquitoes, contains both lactic acid and ammonia. The combination of these compounds, however, attracts no more than 45%, indicating that other components on human skin also play a role in host finding. Preparative liquid chromatography of the skin extract yielded three behaviourally active fractions which work together synergistically. Fraction III contains lactic acid as the effective principle; the compositions of the other two have not been clarified yet. The attractiveness of fraction I was augmented considerably when ammonia was added, whereas the effect of fraction II was not influenced by ammonia. These results suggests that ammonia is part of the effective principle of fraction II and contributes to the attractive effect of host odours.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Olfactory cues are widely used by bloodsucking insects to detect and to find their sources for blood meals. Since mosquitoes are one of the most important groups of vectors for human and animal disease, many attempts have been undertaken to explore the attractive blend of host odours. Different mosquito species develop different host preferences, and it is generally assumed that host selection and discrimination is mainly based on olfactory cues (Takken, 1991
). Until
today,
however, only a few attractive components of host odour have been identified and we know little
about
the role of these volatiles in the complex network of behavioural sequences which lead these
insects to
their warm blooded hosts. Almost all mosquito species use carbon dioxide, which is given off
from
hosts with breath, as an alerting and attractive signal. L-(+)-Lactic acid, a major
component in breath and on human skin, attracts Aedes aegypti in that it acts as an
essential
synergist when combined with carbon dioxide as well as with volatiles from the skin (Acree,
1968; Smith et al., 1970; Eiras and
Jepson,
1991; Geier et al., 1996). Recently, a synthetic
mixture
of 12 aliphatic fatty acids, identified in the head space of Limburger cheese, has been implicated
as
attractive for Anopheles gambiae (Knols et al., 1997).
In the
past, certain amines, oestrogens, amino acids and alcohols have also been reported to attract
mosquitoes, but many of these results were contradictory and synthetic odour blends never
matched the
effect of the natural host odour (Hocking, 1971; Takken, 1991). Previous studies in our laboratory with yellow fever mosquitoes, Ae. aegypti,
have revealed that other components besides lactic acid contribute to the high attractiveness of
human
skin residues (Geier et al., 1996). Interestingly, the components
were
only attractive in combination with lactic acid. These findings indicate that attractive effects of
certain
compounds can be discovered in a bioassay only in combination with lactic acid. A promising
candidate
of such an attractant is ammonia, since this compound has been identified in effluvia from
vertebrates (Larson et al., 1979; Norwood et al., 1992). Attractive effects of ammonia have already been reported for a range of
haematophagous arthropods (Taneja and Guerin, 1997; Hribar et al., 1992). Müller (Müller, 1968),
Brown
(Brown, 1952) and Rössler (Rössler, 1961) could not find any attractive effect of ammonia in behavioural experiments with Ae.
aegypti, but they never tested this compound in combination with lactic acid. Taking the
synergistic
effects of lactic acid and also of carbon dioxide into account, we reinvestigated whether ammonia
could
be an attractant also for Ae. aegypti. In a modified Y-tube olfactometer we tested the
attractiveness of different sources of ammonia over a wide range of concentrations alone and in
different combinations with lactic acid, carbon dioxide and behaviourally active fractions of an
extract
from human skin residues. Such an extract has been shown to attract Ae. aegypti and
was
separated by means of liquid chromatography in three behaviourally active parts: fractions I and
II do
not contain lactic acid and have no attractive effect on their own, but they act synergistically
together
with fraction III, which contains lactic acid as the effective principle (Geier et al., 1996).
We
tested whether ammonia enhances the effect of one of these fractions. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Insects
Female Ae. aegypti, 5–15 days old, from cultures of the centre for Plant Research at Bayer AG in Monheim, were used in our experiments. The larvae were fed with Tetramin® fish food. Some 300–500 adults were kept in containers (50 x 40 x 25 cm) at 26–28°C and 60–70% relative humidity, with a 12 h:12 h L:D photoperiod. They had access to a 10% glucose solution on filter paper. Since male and females were kept together, we presume that all females had been mated before they were used in the experiments. Shortly before the experiments, we lured the mosquitoes out of their containers by means of the human hand as bait. This ensured that the tested insects were able to respond to the host.
Olfactometer
A modified Y-tube olfactometer (Geier, 1995) was used to measure
the
attractiveness of odours (Figure 1). The branch of the Y-tube consisted of
a
rectangular Plexiglas chamber, in which the two arms run into one side and the stem runs into the
opposite side (Figure 1). Each of both arms fitted into a PVC® stimulus chamber where the odours were mixed with the air flushing the olfactometer.
The
release chamber with the mosquitoes was attached to the downwind end of the stem. Rotating
screens
in the release chamber as well as in both arms at the downwind end permit the release or the
entrapment of the mosquitoes respectively. A constant airstream (flow rate: 80 l/min) from the
institute's pressurized air system was purified by a filter of activated charcoal, heated and
humidified before passing through the olfactometer. Further details of this experimental
arrangement are
described elsewhere (Geier, 1995). The temperature was 28 ±
1°C, the relative humidity 70 ± 5% and the wind speed 0.2 m/s in the arms and 0.4
m/s in
the stem respectively. The olfactometer was placed on a white table with white cardboard shields
(height: 20 cm) on both sides to prevent visual stimulation by the experimenter. The room was
illuminated by two 40 W light bulbs.
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Odour stimuli and stimulus delivery
Three different sources of ammonia stimuli were tested. To measure the
dose–response
curve (experiment 1) different amounts of ammonia were produced according to the procedure of
Ough and Stone (Ough and Stone, 1961). Charcoal-filtered air at flow
rates of
0.03–300 ml/min was passed through an Erlenmeyer flask filled with 50 ml of an aqueous
solution of 0.13 mmol/l NH3 (p.A., Merck, Darmstadt, Germany) in distilled water.
Charcoal-filtered air at a flow rate of 300 ml/min passed through an Erlenmeyer flask filled with
50 ml
distilled water served as a control. The air was passed over the surface of the solutions. To
determine
the output of NH3, the air of four different flow rates, 0.3, 3, 30 and 300 ml/min, was
trapped in gas wash flasks with a solution of 0.01 N HCl for 23 h, 1 h, 30 min and 12 min
respectively.
At the highest flow rate the trapping flasks were filled with 50 ml of the HCl solution; at the
lower flow
rates they were filled with 10 ml. We connected up to four trapping flasks in series to monitor
any
breakthrough from a previous one. The amount of ammonia was determined by titration with
0.01 N
NaOH solution (Poethke, 1987). Figure 2 shows
the
relationship between the flow rate through the stimulus source and the total amount of NH3
trapped per minute. In experiments 2, 3 and 5 the ammonia was delivered in the same way with a
flow
rate of 3 ml/min, which resulted in an output of 5 µg/min and a concentration of 7 nmol/l air
(170
ppb) in the test chamber of the olfactometer.
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In experiment 4 we tested an aqueous solution of 10 mmol/l ammonium chloride (p.A., Merck, Darmstadt, Germany) in distilled water. The pH value of this solution, determined by means of a pH indicator paper (Merck), was found to be 6.5. Small open glass vials (height: 30 mm; inner diam.: 16 mm) were filled with 2 ml of this solution and placed into the stimulus chamber. A 10 mmol/l NaCl solution served as control. In this experiment a L-(+)-lactic acid/ ammonia buffer which simulates the composition of human sweet was also tested. For this, 400 mg of L-(+)-lactic acid (p.A., Merck) and 270 mg of 25% ammonia were dissolved in 50 ml of distilled water. This resulted in a lactate/ammonium salt solution of 89 mmol/l lactic acid and 80 mmol/l ammonia with a pH value of 5 and a surplus of free lactic acid; 0.5 ml of this solution was applied to filter paper discs (7 cm diam.) and the wet discs were put into the stimulus chamber. A lactic acid/sodium lactate buffer with 450 mg of L-(+)-sodium lactate (p.A. Merck) and 40 mg of L-(+)-lactic acid dissolved in 50 ml of distilled water served as a control. The pH value of this solution (80 mmol/l sodium lactate and 9 mmol/l lactic acid) was also 5, indicating the same amount of free lactic acid as in the lactate/ammonium salt solution.
Lactic acid stimuli were generated using a set-up similar to one based on the design of Ough
and
Stone (Ough and Stone, 1961), described above. Charcoal-filtered
compressed air at a flow rate of 15 ml/min was passed through a 250 ml Erlenmeyer flask filled
with 10
ml of L-(+)-lactic acid solution (90% in aq. sol.; Merck, Darmstadt, Germany).
According to the calibration of Geier et al. (Geier et al., 1999), at this flow rate an output of 3 µg/min lactic acid was generated and led into
the
stimulus chamber. This dose is in the range of the lactic acid given off from human hands
(0.4–2.22 µg/min) after data from Smith et al. (Smith et al.,
1970).
The carbon dioxide used in experiment 3 was taken from a gas cylinder having the trade-standard purity of 99.9% (Linde, Nürnberg, Germany). The gas was injected into the stimulus chamber at a flow rate of 1600 ml/min and homogeneously mixed with the olfactometer air, yielding a concentration of 4% in the test arm of the olfactometer.
A skin extract was obtained according to a method described in detail by Geier et al.
(Geier et al., 1996). Hands, forearms, feet and calves were
rubbed for
5 min with pads, which were then extracted with methanol (p.A. Fluka, Germany). The extracts
from
50 pads sampled within a period of 2 months were combined, concentrated to 30 ml by
evaporation in
a rotary evaporator, and then centrifuged at –20°C (950 g; 2 h) to yield a
clear
yellow supernatant extract with a concentration of free L-(+)-lactic acid of 6 mmol/l
(Geier et al., 1996). The NH3 concentration was 7.4 mmol/l, measured
using a gas-sensitive NH3 electrode after the method of standard addition in the
research laboratory of the Bayer AG (Camman, 1979).
The skin extract was fractionated on a preparative silicagel column with acetonitrile and
ethanol,
yielding three separate fractions—fractions I, II and III (Geier et al.,
1996). A blank extract made from 50 cotton pads served as a control. For stimulus
delivery a
volume of 0.01 ml of skin extract or fractions, respectively, were applied to the inner side of a
glass
cartridge (inner diam.: 3.3 mm; length: 5 cm). After the solvent had evaporated, the glass
cartridge was
placed into a heating element on top of the stimulus chamber and air was blown through it at a
rate of
2.8 l/min to deliver the odours from the surface of the glass cartridge as described elsewhere (Geier and Boeckh, 1999). The flow rate of the airstream was regulated
and
controlled by flow meters (Rota GmbH, Germany).
Odour distribution
Since we know that the spatial distribution of odours influences the attractiveness of odour
sources
(Geier et al., 1999), we visualized the distribution of the
odorants by
means of TiCL4 smoke. In the arms of the olfactometer smoke was equally
distributed
similar to the homogeneous plume type outlined by Geier et al. (Geier et
al., 1999). More turbulent odour eddies, i.e. odour clouds and filaments,
emerged in the
rectangular Plexiglas chamber and the stem of the Y-tube respectively, where the two airstreams
of the
arms come together.
Bioassay
Bioassays were conducted as described in detail elsewhere (Geier et al.,
1999). Groups of 18–22 female mosquitoes were used for the tests. Before
stimulation, the mosquitoes were given 20 min to acclimatize. Between the tests a constant flow
of fresh
air flushed the olfactometer; the bioassays ran from 9:00 a.m. to 6:00 p.m. The odour stimuli
were
tested in five blocks of tests, in which the stimuli were tested repeatedly in random order. For
each
block of experiments a different mosquito population was used.
Evaluation of activation and attraction
In each test we distinguished two behavioural categories of responses: (i) the percentage of
mosquitoes found outside the release chamber after 30 s was taken as a measure for activation,
which
included taking flight and short upwind progress. (ii) The percentages of mosquitoes trapped at
the
upwind end of the testand control chambers, respectively, were taken as measures for
attractiveness of
the testand control odours. For each stimulus the means (± SE) of activation and
attractiveness
were calculated. Since the data are percentage values, they were transformed using angle
transformation (Sokal and Rohlf, 1981) for further statistical analysis.
The
transformed means were analysed independently by a one-way ANOVA using the LSD method
as a post hoc test for comparison of the treatments. All calculations and statistics were
performed
with the statistics program SPSS 8.0 for Windows.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Lactic acid at the dose of 3 µg/min, which is in the range of evaporation rates from human hands (Smith et al., 1970
), attracted ~20% of the mosquitoes (Figure 3). The attractiveness of this stimulus was significantly increased
by
addition of ammonia over a wide range of concentrations (Figure 3). The
effective concentration range of ammonia was between 0.7 and 700 nmol/l air (17 ppb–17
ppm). The lowest ammonia concentration of 0.07 nmol/l (1.7 ppb) did not affect the response to
the
lactic acid stimulus, indicating a threshold between 0.07 and 0.7 nmol/l air (1.7–17 ppb).
Addition of 7 nmol/l air (170 ppb) ammonia doubled the percentage of mosquitoes attracted to
lactic
acid alone. Higher concentrations did not further raise the attractiveness. From 0.7 to 70 nmol/l
air
(17–1700 ppb) ammonia alone was not attractive compared with controls.
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In a direct choice situation, when ammonia and lactic acid were tested simultaneously as separate stimuli, all mosquitoes preferred significantly the lactic acid source (experiment 2, Table 1). A combination of lactic acid and ammonia, however, was significantly more attractive than lactic acid alone. When the stimulus in both chambers was lactic acid, both chambers were equally attractive.
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In contrast to the synergism found between ammonia and lactic acid, no such effect was observed when ammonia was added to carbon dioxide (experiment 3,Table 1). A concentration of 4% carbon dioxide had a strong activating as well as a slight attractive effect upon the mosquitoes. By adding ammonia at a concentration of 7 nmol/l air (170 ppb) neither the percentage of activated mosquitoes nor the percentage of attracted mosquitoes was significantly higher than with carbon dioxide alone. In the same experiment, however, a combination of carbon dioxide and lactic acid attracted nearly 80% of the mosquitoes, indicating a strong synergistic effect between these stimuli.
In experiment 4 we tested two other ammonia sources, which mimic the composition of human sweat. The results summarized inTable 1 show that an aqueous solution of ammonium chloride increased the attractiveness of the lactic acid standard stimulus. In addition, the aqueous buffer system of lactic acid and ammonia attracted a significant higher percentage of mosquitoes than a buffer of lactate/lactic acid at the same pH.
Both fractions I and II, obtained by means of preparative liquid chromatography of a highly
attractive skin extract, have been shown to increase the attractiveness of lactic acid, but they had
no
effect on their own (Geier et al., 1996). When ammonia was
added
to a combination of either one with lactic acid, a significant increase of attractiveness was
observed only
with fraction I, but not with fraction II (experiment 5,Table 1). The
combination of fraction I, lactic acid and ammonia, however, was less attractive than the
combination of
fractions I and II and lactic acid.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The data presented here clearly demonstrate an attractive effect of ammonia on Ae. aegypti in concentration ranges which exist around or downwind from human hosts.
The concentration of this compound in human breath has been found by several investigators
to be
between 120 and 3170 ppb (Larson et al., 1979; Norwood et al., 1992). The lowest concentration which caused a significant
behavioural response in the olfactometer was found to be 0.7 nmol/l air (17 ppb), which is clearly
below the concentration of ammonia in breath. Another major source of ammonia is the human
skin.
Sweat produced by the eccrine sweat glands contains 0.7–25 mmol/l (12–425
mg/l)
ammonia and 3.9–67.7 mmol/l (235–4000 mg/l) urea, which is quickly
decomposed to
ammonia by the bacterial microflora on the skin surface (Fiedler, 1968; Ciba-Geigy, 1977). The high lactic acid concentration (27–37
mmol/l =
2.5–3.4 g/l) of human sweat sets the pH value of human skin between 5 and 6.8 (Fiedler, 1968). At this pH value most of the ammonia is bound as salts
and composes a
buffer system together with lactate/lactic acid. Since we do not know the evaporation rate of
gaseous
ammonia above the human skin surface, we tested an aqueous solution of ammonium chloride
(pH 6.5)
and an aqueous lactic acid/ammonia buffer (pH 5) in concentration ranges similar to that found in
human
sweat. Both stimuli significantly enhanced the responses to lactic acid, indicating a considerable
evaporation of ammonia from these sources. These results and also the dose–response
characteristics (Figure 3) indicate that Ae. aegypti is
sensitive
to ammonia at the levels which are given off by humans with their breath as well as from their
skin.
From our data, we assume a sensory threshold to ammonia in a concentration range between
2 and
17 ppb, which is similar to the one found in the haematophagous bug Triatoma infestans
(Taneja and Guerin, 1997). Nymphs of these bugs were attracted to
concentrations of 3 and 17 ppb on a servosphere, whereas no significant response was found at
0.3
ppb. Other examples of attraction or aggregation to ammonia sources have been documented for
a
variety of both haematophageous and non-haematophagous arthropods, such as the horse-fly Hybomitra lasiophtalma (Hribar et al., 1992), the human
body
louse Pediculus humanus (Mumcuoglu et al., 1986),
the
cockroach Blatella germanica (Sakuma and Fukami, 1991) and
the
Mediterranean fruit fly (Mazor et al., 1987). Female fruit flies
use
ammonia as an attractive odour cue in a similar context as female Aedes mosquitoes:
they are
attracted towards ammonia-releasing proteinaceous sources in order to retrieve protein for egg
maturation. In contrast to yellow fever mosquitoes, which respond to ammonia only in
combination with
lactic acid, fruit flies and Triatoma bugs are attracted by ammonia alone (Taneja
and Guerin, 1997). This might reflect the different behavioural contexts in which
ammonia
is used by mosquitoes with their narrow host range on the one hand and by more opportunistic
insects
on the other. Only in combination with a specific human skin component such as lactic acid
might
ammonia contribute to the host recognition of the anthropophilic mosquito Ae. aegypti.
The
opportunistic bug T. infestans, however, might make more versatile use of the
same
stimulus,
e.g. for finding their refuges, which are marked with ammonia-releasing faeces, and also for host
finding
(Taneja and Guerin, 1997). The finding that ammonia is attractive to
yellow
fever mosquitoes only in combination with lactic acid explains the results of Brown (Brown,
1952),
Rössler (Rössler, 1961) and Müller (Müller, 1968), who could not find behavioural responses to ammonia stimuli
because they did not test this compound together with lactic acid.
Previous studies on Ae. aegypti in our laboratory showed that enzymatic
decomposition of
lactic acid abolished the attractive effect of human skin residues (Geier et al., 1996). An attractive effect was regained by combining synthetic lactic acid
with the lactic
acid-deprived residues. This implies that all components which contribute to the attractiveness of
skin
odour are only effective when lactic acid is present concurrently. The observed behavioural
responses
to ammonia correspond with these findings. Lactic acid seems to play the key role in
odour-mediated
host finding of yellow fever mosquitoes. It acts synergistically together with carbon dioxide, as
well as
with ammonia and other unidentified compounds on human skin. The fact that no synergistic
effects
were found between ammonia and carbon dioxide shows that neither ammonia nor carbon
dioxide can
substitute lactic acid as a synergist in attracting Ae. aegypti. Therefore we
suggest an
olfactory
host recognition pattern in which different compounds of the attractive odour might act together
at
distinct levels of synergism. The highly attractive skin extract contained, beside many other
compounds,
considerable amounts of free lactic acid (6mmol/l) and ammonia (7.4 mmol/l). While the
complete skin
extract attracted 80–90% of the mosquitoes, a mixture of lactic acid and ammonia
attracted at
most 45%. Since this attractiveness did not increase even with higher doses of ammonia, it is
obvious
that additional components of the extract play a role. This is further confirmed by the results
from the
combinations of two behaviourally active fractions of the skin extract with ammonia. Both
fractions and
lactic acid combined are as effective as an equivalent amount of skin extract. Ammonia increased
the
attractiveness of fraction I plus lactic acid whereas no increase was observed with fraction II plus
lactic
acid. The combination of fraction I, lactic acid and ammonia, however, was less attractive than
the
combination fractions I and II and lactic acid. This suggests that ammonia is an effective
principal in
fraction II, but it is obviously not the only one.
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Acknowledgments |
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We thank the Bayer AG (Leverkusen, Germany) for supply of the mosquito eggs as well as for the chemical analysis of the skin extract.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Accepted June 14, 1999
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R. B. Barrozo and C. R. Lazzari Orientation Behaviour of the Blood-sucking Bug Triatoma infestans to Short-chain Fatty Acids: Synergistic Effect of L-Lactic Acid and Carbon Dioxide Chem Senses, November 1, 2004; 29(9): 833 - 841. [Abstract] [Full Text] [PDF]
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R. B. Barrozo and C. R. Lazzari The Response of the Blood-sucking Bug Triatoma infestans to Carbon Dioxide and other Host Odours Chem Senses, May 1, 2004; 29(4): 319 - 329. [Abstract] [Full Text] [PDF]
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B. M. Steib, M. Geier, and J. Boeckh The Effect of Lactic Acid on Odour-Related Host Preference of Yellow Fever Mosquitoes Chem Senses, June 1, 2001; 26(5): 523 - 528. [Abstract] [Full Text] [PDF]
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O. J. Bosch, M. Geier, and J. Boeckh Contribution of Fatty Acids to Olfactory Host Finding of Female Aedes aegypti Chem Senses, June 1, 2000; 25(3): 323 - 330. [Abstract] [Full Text] [PDF]
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