Background
Mosquitoes in search of a blood meal integrate information from host-related visual, physical and chemical cues during the host-seeking process [
1‐
3]. Vision is considered more important among diurnally active mosquitoes [
3], whereas physical and olfactory cues are the dominant cues for nocturnal species [
1,
2]. Many species of blood-feeding insects display non-random host selection at the intra- and interspecific level and, although this has important epidemiological implications, the evolutionary basis for this selection remains poorly understood [
4].
Host odour is one of the components influencing host choice. For example, the sandfly
Lutzomyia longipalpis responds to hand odour from different humans at significantly different rates [
5], and attraction of
Simulium blackfly species to total human emanations varies depending on the individual person used as the source of kairomones [
6]. Attractiveness of a human arm and hand odour to
Anopheles stephensi [
7],
Aedes aegypti [
8‐
10] and
An. quadrimaculatus [
10] has been shown to vary substantially between individual human baits. The response of members of the
An. gambiae complex to individual humans also varies considerably [
11‐
13], and these intra-specific differences can be observed in traps baited with total body emanations, including those from which the body heat component has been excluded [
14,
15]. Recently it was shown that either removal of exhaled air from total emanations [
5] or artificially standardising CO
2 outputs [
15] eliminates differential attraction of humans to blackflies and mosquitoes, respectively.
We used a recently developed multi-choice olfactometer [
16] to investigate how breath and body odour contribute to and might possibly interact as components of the attractiveness of humans to
An. gambiae s.s., one of the most anthropophilic, abundant and efficient vectors of malaria in Africa. Previous work with this system enabled us to rank the attractiveness of nine male Kenyans [
16], two of whom were involved in the experiments reported here.
Discussion
The behavioural response of
An. gambiae, as assessed through choice experiments making all possible dual comparisons by total emanations, body odours, breath and a control originating from a single human subject, demonstrated an allomonal effect of breath and an overall kairomonal effect of body odours and total emanations. Whereas the allomonal effect of breath was not known previously, total emanations have been shown to be responsible for over 90% of the attractiveness of humans to
An. gambiae s.l. [
18].
Surprisingly, there was no difference in the number of mosquitoes attracted by the two persons, who were otherwise consistently different in their attractiveness (see [
16]), when breath was excluded from their total emanations. Since host seeking is modulated by olfactory cues [
1,
2], and
An. gambiae preferentially responds to human rather than other vertebrate-host cues/odours [
19,
20], regardless of a person's degree of attractiveness, our present results suggest that breath is a key factor responsible for variability in human attractiveness to
An. gambiae. Our data also show a clear interaction between components of breath and body odour as the attractiveness of person P
1, who was significantly more attractive than person P
8 based on responses to their total emanations, was reversed when mosquitoes were allowed to make choices between their breaths.
These findings corroborate findings for other blood-feeding insects: removal of breath from total emanations has been shown to eliminate individual differences in attractiveness of humans to
Simulium species [
6] and artificially standardising outputs of carbon dioxide, a major component of breath, has been shown to equalise human attractiveness to
An. gambiae s.l. and
An. funestus [
15].
Human breath has been reported to be attractive to
Anopheles mosquitoes [
21,
22] and
Aedes aegypti [
23]. Krotozynski et al. [
24] identified 102 organic compounds of endogenous and exogenous origin in human breath, obtained from a group of 28 carefully selected healthy individuals. Since then several hundred additional volatile organic compounds have been identified [
25]. Carbon dioxide is by far the most abundant compound, and 97% of the remaining chemicals have a mean concentration between 0.06 and 9.5 ng L
-1. Acetone, isoprene and acetonitrile, with concentrations of 120, 33 and 24 ng L
-1, respectively, account for 51% of the mean organic contents. Compounds of bacterial origin, such as dimethylsulphide or methanethiol have also been found [
26].
Several of the above compounds have been shown to influence the host-seeking process of
An. gambiae. CO
2, for example [
27], contributes to the overall attractiveness of humans in the range of 9–40% [
18,
28] and human equivalents (300–500 ml min
-1) yield significant responses both in the laboratory [
29,
30] as well as in the field [
31]. Acetone, present in concentrations ranging from 293–870 ppb [
32], has also been shown to affect
An. gambiae behaviour [
33], as does ammonia [
34,
35], for which concentrations of 422–2389 ppb have been recorded from breath samples [
32]. The precise nature of such behavioural responses remains largely unknown, and breath has only been shown to be directly responsible for influencing the selection of a landing/biting site by
An. atroparvus [
36] and
An. albimanus [
37].
Considering the importance of breath in affecting host selection by
An. gambiae, three interesting findings emerge. First, given the minute concentrations at which most of these compounds occur, one or more very potent repellent(s) may be isolated, microgrammes of which may suffice to inhibit the host-seeking response. Second, given the intense exchange of metabolic gases at the alveolar interface between the bloodstream and lung cavity, malarial disease may alter the composition of exhaled breath and as such affect the host-selection process [
38]. Third, our results show that mosquito host selection is not just a matter of 'attractiveness', but also determined by a person's repellency, the sum of which may affect the threshold level for a mosquito to initiate close-range and biting behaviour once near a host.
Conclusions
We have identified an allomonal effect of human breath for
An. gambiae s.s. and shown this to contribute to between-person differences in relative attractiveness. Unfortunately, whereas factors that might be responsible for the allomonal effects remain unknown, it is interesting to contrast our observations with those who have noted that CO
2, a major component of breath, is a potent activator for
An. gambiae [
27]. It is also interesting to note that the orientation behaviour of such a specialised human-feeding mosquito as
An. gambiae is inhibited by breath and that this species prefers to bite the ankles and feet of its chosen host [
36]. We hypothesize that the allomonal properties of human breath combined with the attraction to such extremities as the feet and ankles may represent a mechanism that facilitates successful, undetected feeding by
An. gambiae upon their favoured human hosts.
Authors' contributions
WRM designed the olfactometer, conducted all the experimental work, and drafted the original manuscript. GFK assisted with statistical data analysis and interpretation. BGJK conceived the study, obtained funding for it, supervised the experimental work and edited the final version of the paper in collaboration with WRM, GFK and WT.