Chemical Senses vol. 30 no. 2 © Oxford University Press 2005; all rights reserved.
Synergism between ammonia, lactic acid and carboxylic acids as kairomones in the host-seeking behaviour of the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae)
Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH, Wageningen, The Netherlands
Correspondence to be sent to: Willem Takken, Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands. e-mail: willem.takken{at}wur.nl
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Abstract |
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Host odours play a major role in the orientation and host location of blood-feeding mosquitoes. Anopheles gambiae Giles sensu stricto, which is the most important malaria vector in Africa, is a highly anthropophilic mosquito species, and the host-seeking behaviour of the females of this mosquito is guided by volatiles of human origin. Ammonia, lactic acid and several carboxylic acids are known to be present in the human odour blend. We investigated the effect of these compounds on naïve female mosquitoes using a dual-port olfactometer. Ammonia was an attractant on its own, whereas lactic acid was not attractive. Carboxylic acids, offered as a mixture of 12 compounds, were repellent at the concentration tested. The addition of ammonia to the carboxylic acid mixture overruled the repellent effect of the latter. Combining ammonia with either lactic acid or the carboxylic acids did not enhance the attractiveness of ammonia alone. However, a synergistic effect was found when ammonia, lactic acid and the carboxylic acids were applied as a blend. Our findings indicate that An. gambiae s.s. relies on the combination of ammonia, lactic acid and carboxylic acids in its orientation to human hosts. The role of lactic acid in this tripartite synergism differs from that reported for the yellow fever mosquito Aedes aegypti.
Key words: dual-port olfactometer, host-odours, human volatiles, olfaction, sweat
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Introduction |
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Females of the mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) (henceforth termed An. gambiae) are important vectors of malaria in Africa. The dominance of this mosquito as a malaria vector is largely due to its preference for human blood (White, 1974
; Coluzzi et al., 1979; Pates et al., 2001a). Because female mosquitoes use host odours to find their blood-hosts (Takken, 1991; Takken and Knols, 1999) and because An. gambiae females are highly anthropophilic, it is likely that the latter use human-specific odour compounds for orientation.
Field studies in Africa showed that carbon dioxide (CO2), a major component of breath, accounts for only a minor part of the attractiveness of a human host to An. gambiae females. Human body odour appeared to play a larger role (Costantini et al., 1996, 1998; Mboera et al., 1997). This is in accordance with the hypothesis mentioned above since all warm-blooded vertebrates exhale CO2 and it is unlikely that An. gambiae, which has a preference for humans, will be guided to its host by this compound alone.
Laboratory studies aimed at elucidating the compounds constituting human-produced odour blends that mosquitoes use for host location have yielded several active mixtures and individual substances. Human sweat was found to be attractive to An. gambiae (Braks et al., 1997; Braks and Takken, 1999; Healy and Copland, 2000). One of its components is ammonia and it was shown in a dual-port olfactometer that ammonia is a kairomone for An. gambiae (Braks et al., 2001). Carboxylic acids make up an important part of human sweat (Cork and Park, 1996). However, an artificial blend of 22 aliphatic carboxylic acids did not elicit landings of An. gambiae females whereas 2-oxopentanoic acid did, although the latter compound was not attractive from a distance (Healy and Copland, 2000). In contrast, Knols et al. (1997) found that a synthetic mixture of 12 aliphatic carboxylic acids was attractive to females of An. gambiae in a dual-port olfactometer. Human sweat also contains lactic acid (Cork and Park, 1996; Healy and Copland, 2000) and humans seem to have uniquely high levels of this compound on their skin compared to other animals (Dekker et al., 2002). Although Healy and Copland (2000) found that An. gambiae females land on filter papers impregnated with human sweat, the concentration of lactic acid that was present in sweat did not elicit landing responses. Braks et al. (2001) showed that this compound was slightly attractive to An. gambiae females in an olfactometer.
Lactic acid is known to play an important role in the host-seeking behaviour of another anthropophilic mosquito species, the yellow fever mosquito, Aedes aegypti L. (Acree et al., 1968; Smith et al., 1970; Geier et al., 1996). Ammonia was also identified as an attractant for Ae. aegypti; it is not attractive when tested alone, but it enhances the attractiveness of lactic acid (Geier et al., 1999). Fatty acids of chain length C1–C3, C5–C8 or C13–C18 had the same effect when mixed with lactic acid. The combination of lactic acid, ammonia and two fatty acids appeared to be almost as attractive as an extract of human skin residues (Bosch et al., 2000).
Building upon the results of Geier et al. (1999) and Bosch et al. (2000) with Ae. aegypti and with the knowledge that the human sweat compounds ammonia, L-lactic acid and several carboxylic acids were attractive to An. gambiae, we examined the effects of blends of these compounds on the behaviour of this mosquito. Initially, because the amount released by humans is to our knowledge unknown, the concentration range within which ammonia is attractive to An. gambiae females was examined. Subsequently experiments were done to examine whether ammonia might cause synergism when combined with lactic acid and/or a mixture of carboxylic acids.
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Materials and Methods |
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Mosquitoes
The Anopheles gambiae Giles sensu stricto colony at Wageningen University, The Netherlands, originated from Suakoko, Liberia. The mosquitoes have been cultured in the laboratory since 1988 with blood meals from a human arm twice a week. The adult mosquitoes were maintained in 30 x 30 x 30 cm gauze cages at 27 ± 1°C, 80 ± 5% relative humidity, and a photo-scotophase of 12:12 light:dark. They had access to a 6% glucose solution on filter paper. The larvae were reared in tap water in plastic trays and fed daily with Tetramin® baby fish food. Pupae were collected daily and placed in adult cages for emergence.
Olfactometer
A dual-port olfactometer (see Pates et al., 2001b), consisting of a Perspex flight chamber of 1.60 x 0.66 x 0.43 m, was used to study the behavioural responses of female mosquitoes to different odour stimuli. Pressurized air was charcoal filtered, humidified and led through two Perspex mosquito trapping devices, which were linked to two ports (diameter 4 cm, 28 cm apart), into the flight chamber with a speed of 0.22 ± 0.02 m/s. The light of one tungsten light bulb (75 W) was filtered and scattered through a screen of yellow cloth hanging 
1 m above the flight chamber. This resulted in dim light of 1 lux in the olfactometer. The experimental room was maintained at a temperature of 28 ± 1.5°C and a relative humidity of 60 ± 6%. The temperature inside the flight chamber was equal to that of the room and the relative humidity was maintained at 65 ± 6%. The relative humidity of the air flowing out of the ports was maintained above 80% and the temperature was 28 ± 1.5°C.
Odour stimuli
Compounds were tested singly and in combinations with each other. They were tested either against clean air or against one or two of the test stimuli. The bioassay methods described by Braks et al. (2001) were used for the preparation of ammonia and lactic acid; the mixture of carboxylic acids was made following the method of Knols et al. (1997).
Ammonia
One day before the experiments, a specified amount of an aqueous ammonia solution (Stock: Merck, min. 25%, 1 l = 0.91 kg) was injected in a 80 l dual stainless steel fitted Tedlar air sample bag (SKC Inc., USA). Subsequently, the bag was filled with 60 l of humidified and filtered warm, pressurized air at least 17 h prior to the experiments to allow the ammonia to evaporate. This procedure resulted in ammonia concentrations of 13.6, 136, 1,364, 13,637, 27,274 or 136,371 p.p.m. in different bags (Table 3). Another 80 l air sample bag filled with corresponding volumes of distilled water and 60 l air was prepared in a similar way to be used as the control stimulus. During the experiments, the air was pumped at 230 ml/min (MG-4 Ametek air pump, Ametek, USA) from the air sample bags through silicon tubes (diameter 7 mm; Rubber BV, The Netherlands) into one or both trapping devices where it mixed with the main airstream (Braks et al., 2001). Based on the response of the mosquitoes to these six concentrations of ammonia (Figure 1), an ammonia concentration of 13.7 p.p.m. was used in subsequent experiments.
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Lactic acid
L-(+)-Lactic acid sodium salt (98%, L-7022, Sigma, St Louis, MO, USA) was dissolved in ethanol (Merck, absolute ethanol, pro analysis) following the method used by Braks et al. (2001)
and Geier et al. (1999) who found certain concentrations of this compound to be attractive to An. gambiae and Ae. aegypti. For each experiment 100 µl of a lactic acid solution was applied on filter paper (5 x 2 cm; Whatman 2 or Schleicher & Schuell 595) which was placed in an iron clip. The ethanol was allowed to evaporate before the clip with the filter paper was put in a trapping device. For the control stimulus an equivalent amount of ethanol was applied in a similar way and placed in the other trapping device (Braks et al., 2001).
Initially, five different concentrations of lactic acid (0.1, 0.01, 0.001, 0.0001 and 0.00001 g/ml) were tested in combination with gaseous ammonia (136 p.p.m.) and against gaseous ammonia only (136 p.p.m.) to obtain the highest attractiveness of the mixture of these two compounds together. Based on the results of this experiment and the results previously obtained by Braks et al. (2001), the concentration of lactic acid used in the subsequent experiments was 0.001 g/ml.
Mixture of carboxylic acids
A mixture of carboxylic acids was dissolved in diethyl ether (Merck, min. 98%) based on the relative amounts of each acid in the acid extracts of Limburger cheese samples, which were shown to be attractive to An. gambiae (De Jong and Knols, 1995; Knols et al., 1997). A sandblasted glass slide (5 x 2 cm) with 100 µl of the 108 times diluted synthetic mixture of the 12 carboxylic acids (Sigma, min. 99%) was placed in a trapping device. A control glass slide with an equivalent amount of diethyl ether was placed in the opposite trapping device after the diethyl ether had evaporated.
Experimental procedure
Thirty female mosquitoes, 5–8 days old, which had not received a blood meal, were randomly collected from their cage 14–18 h before the start of the experiments and placed in a cylindrical release cage (diameter 8 cm, height 10 cm) with access to tap water from damp cotton wool placed on top of the cage.
The experiments were performed during the last 4 h of the dark period, when An. gambiae is normally active. In each trial test odours were released in the airstream before a group of mosquitoes was set free from a cage which was placed at the downwind end of the flight chamber, 1.60 m from the two ports. Mosquitoes were left in the flight chamber for 15 min. The female mosquitoes that had entered either trapping device were counted at the end of the experiments, after anaesthetization with 100% CO2. Mosquitoes remaining in the flight chamber were removed with a vacuum cleaner.
Each trial started with new mosquitoes, clean trapping devices and new stimuli. Experiments were repeated at least six times on different days. The sequence of test odours was randomized on the same day and between days. Test stimuli were alternated between right and left ports in different replicates to rule out any positional effects. Experiments with clean air only in either port were done to test the symmetry of the trapping system. Surgical gloves were worn by the experimenter to avoid contamination of the equipment with human volatiles.
Statistical analysis
For each two-choice test a 
2 test was used to analyse whether the total (i.e. sum of all replicates) number of mosquitoes that was trapped in the treatment trapping device and the total number that was trapped in the control trapping device differed from a 1:1 distribution. A generalized linear model (GLM; binomial, linked in logit; Genstat, release 4.2) was used to investigate the effect of the different odour stimuli on the total response (i.e. total number of mosquitoes that entered both the treatment and control trapping device as fraction of the total number of mosquitoes that left the release cage). Two-sided t-probabilities were calculated to test pairwise differences between means. Effects were considered to be significant at P < 0.05 (Oude Voshaar, 1994; Sokal and Rohlf, 1998).
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Results |
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Symmetry of trapping system
An. gambiae females responded to a humidified odourless airstream as was shown previously by Knols et al. (1994). In all our experiments in which we tested clean air coming from both ports, equal numbers of mosquitoes were caught in both trapping devices (2 test; P 
0.50), which demonstrated that the olfactometer was symmetrical (Figure 1).
Dose–response effects of ammonia
Figure 1 shows the dose-dependent response of An. gambiae to gaseous ammonia. Odorous air from sample bags containing 13.6, 136, 1,364 or 13,637 p.p.m. ammonia attracted significantly more mosquitoes than clean air (2 test; P < 0.05). Catches with air containing 27,274 p.p.m. ammonia did not differ from the catches in the trapping device with clean air (2 test; P 0.05). The mosquitoes preferred the clean air to the highest concentration of ammonia tested, 136,371 p.p.m. (2 test; P = 0.02), indicating that this concentration is repellent for An. gambiae.
The percentage of mosquitoes responding in the experiments with 13,637 (24%) and 27,274 (28.6%) p.p.m. ammonia was not significantly different from the control (clean air against clean air) (26.6%), whereas the response (varying from 33.2 to 57.3%) was significantly higher when the other concentrations were tested (GLM; Figure 1).
Different concentrations of lactic acid in combination with ammonia
None of the concentrations of lactic acid that were tested against ammonia alone (136 p.p.m.) increased the attractiveness of ammonia (2-test) (Table 2). The concentration of 0.0001 g/ml of lactic acid combined with ammonia was repellent.
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Responses to blends of ammonia, lactic acid and a synthetic mixture of 12 carboxylic acids
Gaseous ammonia at a dose of 136 p.p.m. alone was attractive when tested against clean air (2 test; P = 0.02), whereas lactic acid alone was not (2-test; P = 0.10). The combination of ammonia and lactic acid attracted significantly more mosquitoes than clean air (2 test; P = 0.0003; Table 3A).
The synthetic mixture of the 12 carboxylic acids was repellent when tested alone and in combination with lactic acid (against the solvents only) (2-test; P = 0.0002 and P = 0.047 respectively). However, the combination of the carboxylic acid mixture with ammonia and with both ammonia and lactic acid was significantly more attractive than clean air (2 test; P = 0.03 and P = 0.00005 respectively; Table 3A).
The attractiveness of ammonia was not increased by the addition of lactic acid or by the carboxylic acid mixture (2 test; P = 0.41 and P = 0.59 respectively; Table 3B). However, the combination of ammonia, lactic acid and carboxylic acids was significantly more attractive than ammonia alone (2 test; P = 0.02).
A mixture of carboxylic acids and lactic acid or a mixture of ammonia and lactic acid did not attract more mosquitoes than lactic acid alone (2 test; P = 1.000 and P = 0.26 respectively). But as with the previous series of experiments (Table 3B), the combination of all chemicals together attracted more mosquitoes than only one of the compounds, lactic acid (2-test; P = 0.004; Table 3C).
Lactic acid combined with the carboxylic acids was not more attractive than the latter alone (2 test; P = 0.78). Adding ammonia only or ammonia together with lactic acid overruled the repellency of the carboxylic acid mixture (2 test; P = 0.03 and P = 0.0006 respectively; Table 3D).
No significant differences were found between the number of mosquitoes attracted to the combination of ammonia, lactic acid and the carboxylic acid mixture and the numbers attracted to the mixture of ammonia and lactic acid or the combination of ammonia and the carboxylic acid mixture (2 test; P = 0.66 and P = 0.35 respectively; Table 3E). The combination of lactic acid and the carboxylic acid mixture was significantly less attractive than the mixture of all chemicals together (2 test; P = 0.002).
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Discussion |
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Ammonia, lactic acid and several carboxylic acids have been detected in human sweat and in human skin emanations (e.g. Eiras and Jepson, 1991
; Cork and Park, 1996; Bernier et al., 1999, 2000; Braks et al., 2001). Our experiments clearly show that the combination of these components affects the host-seeking behaviour of the highly anthropophilic females of the malaria mosquito An. gambiae s.s..
As was shown before by Braks et al. (2001), ammonia alone is attractive to An. gambiae females. Concentrations of 13.6–13,637 p.p.m. of ammonia in the sampling bag attracted 25–57% of the mosquitoes. Higher concentrations produced repellency (Figure 1). These data confirm that ammonia is an important kairomone derived from incubated sweat, an attractive complex odour source containing higher amounts of ammonia (49.4 mM) than fresh sweat (6.3 mM), which is not or only slightly attractive (Braks and Takken, 1999; Meijerink et al., 2000; Braks et al., 2001). By our calculations the dilution of ammonia from the air sample bags in the surrounding airstream was 100-fold, but due to the turbulence of the plume, causing heterogeneous mixing of odour components, it is likely that instantaneous peak concentrations of ammonia may have been present, similar to the ammonia concentration in the sample bags (Murlis et al., 1992), to which the mosquitoes were exposed, starting with the undiluted concentrations as released from the bags (Table 1). Assuming a 100-fold dilution, the lowest concentration for which we found attractiveness (136 ppb) is within the range found in human breath (between 40 and 3170 ppb; Larson et al., 1979). This concentration is above the threshold for attractiveness reported with Ae. aegypti (between 2 and 17 ppb; Geier et al., 1999). Another crucial difference between the two species is that in Ae. aegypti ammonia was only active in combination with lactic acid as an essential synergist. Conversely, lactic acid alone attracts Ae. aegypti (Geier et al., 1996, 1999), but not An. gambiae. In An. gambiae the mixture of ammonia and lactic acid does not attract more mosquitoes than ammonia alone (Tables 2 and 3B). The synthetic mixture of the 12 carboxylic acids did not increase the attractiveness of ammonia either (Table 3B). However, only the combination of ammonia, lactic acid and carboxylic acids was significantly more attractive than any of the three components alone (Table 3B–D). Therefore, enhanced attractiveness of the combined components by definition implies a tripartite synergistic effect between these stimuli. This tripartite synergism was not revealed for Ae. aegypti as the carboxylic acids have only been tested in combination with lactic acid (Bosch et al., 2000). These data also demonstrate the important role of ammonia in the blend, as the addition of lactic acid or carboxylic acids to binary blends of ammonia and carboxylic acids or ammonia and lactic acid, respectively, did not result in enhanced attractiveness, whereas addition of ammonia to a blend of lactic acid and carboxylic acids clearly caused significantly enhanced effects (Table 3E).
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Although in our experiments lactic acid alone was not significantly more attractive than clean air (Table 3A), contrary to what was found by Braks et al. (2001)
and Smallegange et al. (2002), but corresponding to Dekker et al. (2002), the data presented here show that lactic acid is necessary to evoke the synergistic effect with ammonia and carboxylic acids that we observed (Table 3).
It was remarkable that ammonia suppressed the repellent effect of the carboxylic acid mixture (Table 3A, B). When tested alone, the carboxylic acid mixture was repellent (Table 3A). This result appears in contrast with the first report of olfactory activity of carboxylic acids to An. gambiae from our laboratory, where a similar mixture of carboxylic acids at the same concentration (diluted by a factor of 108) was reported attractive (Knols et al., 1997). Impurities in the (commercially) obtained carboxylic acids may be one explanation for the differences in results between those of Knols et al. and the present study. The repellent effect of carboxylic acids alone is also illustrated by the very low response (2.4%) of mosquitoes when the acids were released from both odour ports of the olfactometer (Table 3D). Whether only one or several carboxylic acids caused the repellency of the 12-component mixture needs to be investigated. For Ae. aegypti, Bosch et al. (2000) found that especially C1–C3 and C5–C8 carboxylic acids enhanced attractiveness of lactic acid, whereas C9 and C11 reduced attractiveness. We already know from electroantennographic (EAG) studies that An. gambiae females can detect saturated carboxylic acids. These studies showed that short-chain carboxylic acids elicit higher EAG responses than less volatile, long-chain, acids (Cork and Park, 1996; Knols et al., 1997). Experiments with An. gambiae females that were done in Y-tube olfactometers showed that the synergistic effect could also be achieved when combining ammonia and lactic acid with only one of the short-chain carboxylic acids that was present in our synthetic mixture: hexanoic acid (Smallegange et al., 2002). In addition, Costantini et al. (2001) reported attractiveness of an unsaturated carboxylic acid, 7-octenoic acid, which is a human-specific component secreted from the apocrine sweat glands in the axillary regions (Zeng et al., 1991).
Our results show that a synthetic blend of volatiles, all naturally present in human odour (Bernier et al., 2000; Healy and Copland, 2000; Braks et al., 2001), provides a synergistic effect on olfaction-based trap-entry responses of An. gambiae mosquitoes. It is clear from field studies that An. gambiae is strongly attracted to natural human odours (Haddow, 1942; Ribbands, 1950; Costantini et al., 1996; Mboera et al., 1997), and the development of traps baited with highly attractive synthetic blends simulating human odours appears realistic.
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Acknowledgements |
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We thank F. van Aggelen, A. Gidding, and L. Koopman for rearing the mosquitoes. The research was financed by the Technology Foundation of the Netherlands Organisation for Scientific Research (STW-NWO) under project WBI.4834.
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References |
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Accepted December 14, 2004
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