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Olfactory responses of host-seeking Anopheles gambiae s.s. Giles (Diptera: Culicidae)
Acta Tropica, 59(1995)333-335 © 1995 Elsevier Science B.V. All rights reserved 0001-706X/95/$09.50
333
ACTROP 00473
Short Communication
Olfactory responses of host-seeking Anopheles gambiae s.s. Giles (Diptera: Culicidae) R u u r d de J o n g a n d B a r t G.J. K n o l s Department of Entomology, WageningenAgricultural University, The Netherlands (Received 25 December 1994 accepted 21 March 1995) Key words: Anopheles gambiae sensu stricto; host seeking; kairomone; breath; Limburger cheese odour; olfactometer
Anopheles gambiae s.s. Giles, a highly anthropophilic mosquito species, is Africa's most important malaria vector (White, 1974). It has been shown that this species is attracted to 4.5% carbon dioxide (Knols et al., 1994a), a dose thought to be equivalent to what is emitted by humans during breathing. Also, evidence exists for an activating and attracting effect of carbon dioxide in the field (Gillies, 1980; Snow, 1970). Carbon dioxide, however, is not a human-specific kairomone and the attraction of An. gambiae to this chemical alone cannot explain its anthropophilic behaviour. We therefore decided to investigate whether chemicals present in exhaled human breath, other than CO2, might play a role in the attraction of this mosquito species. Breath has been found attractive for other Anopheles spp. (Mer et al., 1947; Laarman, 1955; Knols et al., 1994b; De Jong and Knols, 1995) and for Aedes aegypti L. (Khan et al., 1972). Breath samples from three resting male Caucasian volunteers (aged 27, 38 and 50 years) were collected in Tedlar ® gas bags and analyzed for their carbon dioxide concentrations by using an infrared COz gas analyser. A similar bag was then filled with cleaned, moistened air and adjusted to a similar CO2 concentration as measured in the breath sample, by adding COz from a pressurized gas cylinder. 10 ml of distilled water were then added to both bags which were then stored for 24 h at 25°C in order to obtain equal, near-saturated, moisture levels. Both odours were then tested against each other by pumping them at 230 ml min -I into traps fitted in a windtunnel olfactometer (for a description of the apparatus and experimental procedures see Knols et al., 1994a). The results (Table 1A) show that breath-related chemicals do not enhance the attractiveness of CO2 alone. When breath was tested against clean air (Table 1B) we found a slightly significant attraction for only one of the subjects (PH). It seems therefore that the doses of carbon dioxide present in Correspondence address: R. de Jong, Department of Entomology, Wageningen Agricultural University, P.O. Box 8031, 6700 EH, Wageningen, The Netherlands. SSDI 0001-706X(95)00090-9
334 TABLE 1 Numbers of An. gambiae s.s. caught in traps baited with human breath against a similar concentration of CO(A), human breath against clean air (B) or CO2 against clean air (C). N: Total number of mosquitoes tested (not all of which reacted to either stimulus) Person (A) RJ BK PH (B) RJ BK PH
(c)
Treatments Breath
CO2
22 43 36
37 47 37
Breath
Clean Air
34 36 50
32 36 30
CO2
Clean Air
57
42
[CO21%
Y
)~2
3.30 3.65 3.49
194 196 191
3.81ns 0.18ns 0ns
3.80 3.28 3.54
191 191 186
0.06ns 0ns 5.00*
3.56
187
2.26ns
ns: not significant; *: P<0.05.
breath (in all three samples lower than 4.5%) were not attractive to An. gambiae, nor was CO2 alone at 3.56% when tested against clean air (Table 1C). Biting of An. gambiae on a naked motionless human host preferentially occurs on feet and ankles. Washing of this region with a bactericidal soap diverts An. gambiae from biting this region to biting other parts of the body, suggesting that foot odour plays a role in the host location of this species (De Jong and Knols, 1995). We tested in our experimental set-up the responses of East and West African An. gambiae to the odour of Limburger cheese, an odour which, to the human nose, TABLE 2 Numbers of West or East African An. gambiae s.s caught in traps baited with either headspace of (i.e. air blown over) Limburger cheese (30 or 500 mg, for Tanzanian and Liberian mosquitoes respectively) or odourless air (A); headspace of 220 lal of water or odourless air (B); headspace of Limburger cheese or headspace of 220 gl of water (C) Origin (A)
Treatments
N
Z2
Cheese
Control
Liberia Tanzania
181 100 Water
75 31 Control
392 233
43.9*** 36.3***
Liberia
92 Cheese
104 Water
369
0.7ns
Liberia
142
85
383
14.3"**
(B) (c)
ns: not significant; ***: P<0.0001; N: total number of mosquitoes tested (not all of which reacted to either stimulus).
335
resembles foot odour. The results (Table 2A) show that traps baited with odour emanating from Limburger cheese catch significantly higher numbers of mosquitoes than control traps. Since this cheese contains 44% water (EI-Erian, 1969), and moisture is reported to influence mosquito behaviour (Wright and Kellogg, 1962; Clements, 1963), we incorporated tests to assess its importance by testing the headspace of (i.e. the air pumped over) the equivalent of water present in the larger amount of cheese (Table 2B,C). No evidence was found for any influence of moisture on the catches, thus confirming the attractiveness of cheese odour. Our results indicate that, in human breath, volatiles other than carbon dioxide do not play an important role in the host-seeking behaviour of An. gambiae s.s.. However, we do not know whether an interaction between breath odours and other host-related odours, a phenomenon found for other haematophagous insects (Willemse and Takken, 1994), exists. Nevertheless, we have shown that host-seeking females of this highly anthropophilic mosquito species are attracted by Limburger cheese odour, which, like foot odour, is of bacterial origin (Noble, 1982). This suggests that the kairomones used by An. gambiae to locate its human host are skinrelated and of microbial origin.
Acknowledgement The present work is supported by the European Community as part of the project 'Behavioural studies on malaria vectors' under contract numbers TS3-CT92-0101 and TS3-CT91-0032. References Clements, A.N. (1963) The Physiology of Mosquitoes. pp. 267-291 Pergamon Press, Oxford. De Jong, R. and Knols, B.G,J. (1995) Selection of biting sites on man by two malaria mosquito species. Experientia 51, 80-84. E1-Erian, A.F.M. (1969) Bacteriological studies on Limburger cheese. PhD thesis, Wageningen Agricultural University, The Netherlands. Gillies, M.T. (1980) The role of carbon dioxide in host-finding by mosquitoes (Diptera: Culicidae): a review. Bull. ent. Res. 70, 525-532. Khan, A.A. and Maibach, H.I. (1972) Effect of human breath on mosquito attraction to man. Mosq. News 32, 11-15. Knols, B.G.J., De Jong, R. and Takken, W. (1994a) Trapping system for testing olfactory responses of the malaria mosquito Anopheles gambiae in a wind tunnel. Med. Vet. Entomol. 8, 386-388. Knols, B.G.J., Takken, W. and De Jong, R. (1994b) Influence of human breath on selection of biting sites by Anopheles albimanus. J. Am. Mosq. Contr. Assoc. 10, 423426. Laarman, J.J. (1955) The host-seeking behaviour of the malaria mosquito Anopheles atroparvus. Acta Leid. 25, 1-144. Mer, G., Birnbaum, D., and Aioub, A. (1947) The attraction of mosquitoes by human beings. Parasitol. 38, 1-9. Noble, W.C. (1982) Microbiology of human skin. Lloyd Luke, London. Snow, W.F. (1970) The effect of a reduction in expired carbon dioxide on the attractiveness of human subjects to mosquitoes. Bull. ent. Res. 60, 43-48. White, G.B. (1974) Anopheles gambiae complex and disease transmission in Attica. Trans. Roy. Soc. Trop. Med. Hyg. 68, 278-299. Willemse, L. and Takken, W. (1994) Odor-induced host location in tsetse flies (Diptera: Glossinidae). J. Med. Entomol. 31,775-794. Wright, R.H. and Kellogg, F.E. (1962) Response of Aedes aegypti to moist convection currents. Nature 194, 402-403.
333
ACTROP 00473
Short Communication
Olfactory responses of host-seeking Anopheles gambiae s.s. Giles (Diptera: Culicidae) R u u r d de J o n g a n d B a r t G.J. K n o l s Department of Entomology, WageningenAgricultural University, The Netherlands (Received 25 December 1994 accepted 21 March 1995) Key words: Anopheles gambiae sensu stricto; host seeking; kairomone; breath; Limburger cheese odour; olfactometer
Anopheles gambiae s.s. Giles, a highly anthropophilic mosquito species, is Africa's most important malaria vector (White, 1974). It has been shown that this species is attracted to 4.5% carbon dioxide (Knols et al., 1994a), a dose thought to be equivalent to what is emitted by humans during breathing. Also, evidence exists for an activating and attracting effect of carbon dioxide in the field (Gillies, 1980; Snow, 1970). Carbon dioxide, however, is not a human-specific kairomone and the attraction of An. gambiae to this chemical alone cannot explain its anthropophilic behaviour. We therefore decided to investigate whether chemicals present in exhaled human breath, other than CO2, might play a role in the attraction of this mosquito species. Breath has been found attractive for other Anopheles spp. (Mer et al., 1947; Laarman, 1955; Knols et al., 1994b; De Jong and Knols, 1995) and for Aedes aegypti L. (Khan et al., 1972). Breath samples from three resting male Caucasian volunteers (aged 27, 38 and 50 years) were collected in Tedlar ® gas bags and analyzed for their carbon dioxide concentrations by using an infrared COz gas analyser. A similar bag was then filled with cleaned, moistened air and adjusted to a similar CO2 concentration as measured in the breath sample, by adding COz from a pressurized gas cylinder. 10 ml of distilled water were then added to both bags which were then stored for 24 h at 25°C in order to obtain equal, near-saturated, moisture levels. Both odours were then tested against each other by pumping them at 230 ml min -I into traps fitted in a windtunnel olfactometer (for a description of the apparatus and experimental procedures see Knols et al., 1994a). The results (Table 1A) show that breath-related chemicals do not enhance the attractiveness of CO2 alone. When breath was tested against clean air (Table 1B) we found a slightly significant attraction for only one of the subjects (PH). It seems therefore that the doses of carbon dioxide present in Correspondence address: R. de Jong, Department of Entomology, Wageningen Agricultural University, P.O. Box 8031, 6700 EH, Wageningen, The Netherlands. SSDI 0001-706X(95)00090-9
334 TABLE 1 Numbers of An. gambiae s.s. caught in traps baited with human breath against a similar concentration of CO(A), human breath against clean air (B) or CO2 against clean air (C). N: Total number of mosquitoes tested (not all of which reacted to either stimulus) Person (A) RJ BK PH (B) RJ BK PH
(c)
Treatments Breath
CO2
22 43 36
37 47 37
Breath
Clean Air
34 36 50
32 36 30
CO2
Clean Air
57
42
[CO21%
Y
)~2
3.30 3.65 3.49
194 196 191
3.81ns 0.18ns 0ns
3.80 3.28 3.54
191 191 186
0.06ns 0ns 5.00*
3.56
187
2.26ns
ns: not significant; *: P<0.05.
breath (in all three samples lower than 4.5%) were not attractive to An. gambiae, nor was CO2 alone at 3.56% when tested against clean air (Table 1C). Biting of An. gambiae on a naked motionless human host preferentially occurs on feet and ankles. Washing of this region with a bactericidal soap diverts An. gambiae from biting this region to biting other parts of the body, suggesting that foot odour plays a role in the host location of this species (De Jong and Knols, 1995). We tested in our experimental set-up the responses of East and West African An. gambiae to the odour of Limburger cheese, an odour which, to the human nose, TABLE 2 Numbers of West or East African An. gambiae s.s caught in traps baited with either headspace of (i.e. air blown over) Limburger cheese (30 or 500 mg, for Tanzanian and Liberian mosquitoes respectively) or odourless air (A); headspace of 220 lal of water or odourless air (B); headspace of Limburger cheese or headspace of 220 gl of water (C) Origin (A)
Treatments
N
Z2
Cheese
Control
Liberia Tanzania
181 100 Water
75 31 Control
392 233
43.9*** 36.3***
Liberia
92 Cheese
104 Water
369
0.7ns
Liberia
142
85
383
14.3"**
(B) (c)
ns: not significant; ***: P<0.0001; N: total number of mosquitoes tested (not all of which reacted to either stimulus).
335
resembles foot odour. The results (Table 2A) show that traps baited with odour emanating from Limburger cheese catch significantly higher numbers of mosquitoes than control traps. Since this cheese contains 44% water (EI-Erian, 1969), and moisture is reported to influence mosquito behaviour (Wright and Kellogg, 1962; Clements, 1963), we incorporated tests to assess its importance by testing the headspace of (i.e. the air pumped over) the equivalent of water present in the larger amount of cheese (Table 2B,C). No evidence was found for any influence of moisture on the catches, thus confirming the attractiveness of cheese odour. Our results indicate that, in human breath, volatiles other than carbon dioxide do not play an important role in the host-seeking behaviour of An. gambiae s.s.. However, we do not know whether an interaction between breath odours and other host-related odours, a phenomenon found for other haematophagous insects (Willemse and Takken, 1994), exists. Nevertheless, we have shown that host-seeking females of this highly anthropophilic mosquito species are attracted by Limburger cheese odour, which, like foot odour, is of bacterial origin (Noble, 1982). This suggests that the kairomones used by An. gambiae to locate its human host are skinrelated and of microbial origin.
Acknowledgement The present work is supported by the European Community as part of the project 'Behavioural studies on malaria vectors' under contract numbers TS3-CT92-0101 and TS3-CT91-0032. References Clements, A.N. (1963) The Physiology of Mosquitoes. pp. 267-291 Pergamon Press, Oxford. De Jong, R. and Knols, B.G,J. (1995) Selection of biting sites on man by two malaria mosquito species. Experientia 51, 80-84. E1-Erian, A.F.M. (1969) Bacteriological studies on Limburger cheese. PhD thesis, Wageningen Agricultural University, The Netherlands. Gillies, M.T. (1980) The role of carbon dioxide in host-finding by mosquitoes (Diptera: Culicidae): a review. Bull. ent. Res. 70, 525-532. Khan, A.A. and Maibach, H.I. (1972) Effect of human breath on mosquito attraction to man. Mosq. News 32, 11-15. Knols, B.G.J., De Jong, R. and Takken, W. (1994a) Trapping system for testing olfactory responses of the malaria mosquito Anopheles gambiae in a wind tunnel. Med. Vet. Entomol. 8, 386-388. Knols, B.G.J., Takken, W. and De Jong, R. (1994b) Influence of human breath on selection of biting sites by Anopheles albimanus. J. Am. Mosq. Contr. Assoc. 10, 423426. Laarman, J.J. (1955) The host-seeking behaviour of the malaria mosquito Anopheles atroparvus. Acta Leid. 25, 1-144. Mer, G., Birnbaum, D., and Aioub, A. (1947) The attraction of mosquitoes by human beings. Parasitol. 38, 1-9. Noble, W.C. (1982) Microbiology of human skin. Lloyd Luke, London. Snow, W.F. (1970) The effect of a reduction in expired carbon dioxide on the attractiveness of human subjects to mosquitoes. Bull. ent. Res. 60, 43-48. White, G.B. (1974) Anopheles gambiae complex and disease transmission in Attica. Trans. Roy. Soc. Trop. Med. Hyg. 68, 278-299. Willemse, L. and Takken, W. (1994) Odor-induced host location in tsetse flies (Diptera: Glossinidae). J. Med. Entomol. 31,775-794. Wright, R.H. and Kellogg, F.E. (1962) Response of Aedes aegypti to moist convection currents. Nature 194, 402-403.