Allomonal effect of breath contributes to differential attractiveness of humans to the African malaria vector Anopheles gambiae

Background Removal of exhaled air from total body emanations or artificially standardising carbon dioxide (CO2) outputs has previously been shown to eliminate differential attractiveness of humans to certain blackfly (Simuliidae) and mosquito (Culicidae) species. Whether or not breath contributes to between-person differences in relative attractiveness to the highly anthropophilic malaria vector Anopheles gambiae sensu stricto remains unknown and was the focus of the present study. Methods The contribution to and possible interaction of breath (BR) and body odours (BO) in the attraction of An. gambiae s.s. to humans was investigated by conducting dual choice tests using a recently developed olfactometer. Either one or two human subjects were used as bait. The single person experiments compared the attractiveness of a person's BR versus that person's BO or a control (empty tent with no odour). His BO and total emanations (TE = BR+BO) were also compared with a control. The two-person experiments compared the relative attractiveness of their TE, BO or BR, and the TE of each person against the BO of the other. Results Experiments with one human subject (P1) as bait found that his BO and TE collected more mosquitoes than the control (P = 0.005 and P < 0.001, respectively), as did his BO and the control versus his BR (P < 0.001 and P = 0.034, respectively). The TE of P1 attracted more mosquitoes than that of another person designated P8 (P < 0.021), whereas the BR of P8 attracted more mosquitoes than the BR of P1 (P = 0.001). The attractiveness of the BO of P1 versus the BO of P8 did not differ (P = 0.346). The BO from either individual was consistently more attractive than the TE from the other (P < 0.001). Conclusions We demonstrated for the first time that human breath, although known to contain semiochemicals that elicit behavioural and/or electrophysiological responses (CO2, ammonia, fatty acids) in An. gambiae also contains one or more constituents with allomonal (~repellent) properties, which inhibit attraction and may serve as an important contributor to between-person differences in the relative attractiveness of humans to this important malaria vector.


Background
Mosquitoes in search of a blood meal integrate information from host-related visual, physical and chemical cues during the host-seeking process [1][2][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 intraand 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][9][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][12][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.

Mosquitoes
Experiments were conducted using the Ifakara strain of laboratory-reared Anopheles gambiae Giles sensu stricto, originally colonized from wild-caught gravid females in Njage, South-east Tanzania, in 1996. The mosquito larvae were reared under ambient temperature and light conditions in screenhouse insectaries at the Mbita Point Research and Training Centre of the International Centre of Insect Physiology and Ecology (00°25'S, 34°13'E). The larvae were reared using fresh water from Lake Victoria and were fed on Tetramin ® fish food three times per day (the total amount of food provided was 0.3 grams tetramin ® /100 larvae/day). Pupae were collected from rearing trays and transferred to an insectary where they were kept in mesh-covered 30 cm cubic cages in which rolls of filter-paper soaked in 6% glucose solution were provided. The colony was maintained by routinely offering a human arm to feed upon. Adult females with no prior access to blood were used for experiments when they were four to eight days old and were transferred from the holding cages into release cups six hours before the onset of experiments. Only water-wet cotton wool pads were provided as liquid source on the mesh-topped open ends of the release cups. For further details see [16].

Experimental set-up
Experiments were conducted using two arms of a previously described three-port olfactometer (Figure 1), situated within a large semi-field screenhouse where the ambient atmospheric conditions were not controlled (for details see [16]). Approximately 100 mosquitoes (the exact number was recorded for each experimental release), released into a choice chamber located ~1 metre away from the participants, were used for each experiment. Mosquitoes flying upwind in response to host stimuli were caught in traps, without a chance of entering the tents (for details see [16]).

Human subjects
Two healthy African males, designated person P 1 and person P 8 (numbers refer to the same individuals used in our previous study [16]), were recruited to participate in the experiments. P 1 was aged nineteen years (weight, 80 kg; height, 1.80 m), P 8 was aged 22 years (weight, 79 kg, height 1.85 m). The participants wore only shorts at the time of the experiment and bathed with non-perfumed soap one hour before starting the experiments. No attempt was made to control their daily diet except prohibiting them from ingesting alcohol, a factor that has recently been shown to influence the relative attractiveness of humans for Aedes albopictus [17]. Their malaria infection status was observed daily by microscopic examination of thin and thick smears of finger-prick blood stained with Giemsa. Previous work has demonstrated that P 1 is nearly three times (P < 0.05) more attractive to An. gambiae than P 8 with the mean number of mosquitoes caught during experiments being 20.14 ± 3.17 and 6.78 ± 1.01, respectively [16].
Attraction to total emanations, body odour and breath P 1 was recruited to assess the response of mosquitoes to his total body emanations (TE) or either his breath (BR) or body odour (BO) alone. In this context TE refers to BR plus all volatile discharges of the skin and BO refers to volatiles discharged solely from the skin. BR and BO were separated using a one-way breathing valve (Harvard-Douglas ® ). The test person wore the breathing valve as a mouthpiece and fitted a sprung nose clip so that he inhaled and exhaled air through the mouth only. Thus, he inhaled air from within the confines of the screen house via a polyvinyl chloride (PVC) pipe and exhaled it via a bendable, corrugated PVC pipe. The BR was discharged to a destination dictated by the needs of each experiment ( Figure 1). Separated BR was either recombined with BO to reconstitute the TE (Experiment 1), diverted to the other tent exhaust (Experiment 2), or vented from the apparatus completely through the main air exhaust (Experiment 3).
The first of these three alternative arrangements allowed the response of mosquitoes to TE to be compared with that to a control tent lacking a bait host or body emanations. The second and third of these three arrangements allowed the attractiveness of BO to be compared with BR and with a control tent lacking a bait host or body emanations. The attractiveness of BR, compared with an empty tent, was also assessed. In that case the test person sat out-side the tents but exhaled into one of the tents' exhausts (Experiment 4). Experiments were conducted over 30-min test periods, between 19.30-20.00 and 20.30-21.00 hours. After each experiment all mosquitoes were removed from the apparatus and the number trapped counted. Each of the four possible arrangements were repeated 16 (Experiments 1 and 2) or eight (Experiments 3 and 4) times with the human bait switching position so that he occupied each of the two tents for half of the replicates per experiment. Previous experiments found no effect of residual odours on the behavioural responses of An. gambiae [16].

The role of breath and body odour in between-person differences in relative attractiveness
Behavioural responses of mosquitoes as a result of simultaneous exposure to emanations originating from two human subjects were assessed to determine their relative attractiveness when BR was included or excluded from their TE. BR was removed from the apparatus using a one-Apparatus used to study the response of An. gambiae s.s. mosquitoes to human breath, body odours and a combination thereof Figure 1 Apparatus used to study the response of An. gambiae s.s. mosquitoes to human breath, body odours and a combination thereof. Breath was separated from body odours using a one-way breathing valve and diverted to the exhaust of the same (1) or other tent (2), or vented out through the main air exhaust (3). Alternatively, the test person sat outside the tent and his breath was diverted to one of the tent exhausts (4). a: test person, b: tent, c,d: tent exhaust, e: mosquito release cup, f: choice chamber, g: trapping chamber, h: main air exhaust. Dimensions shown are in cm. Broken arrows depict the direction of movement of air currents. Further descriptive details of the experimental set up see [16].  Figure 1). Inclusion of BR did not involve re-direction as shown in path 1; the human subject occupied the tent without using the breathing valve. The following choice tests were carried out: (i) TE of person P 1 versus TE of person P 8 (Experiment 1), (ii) BR of person P 1 versus BR of person P 8 (Experiment 2), (iii) BO of person P 1 versus BO of person P 8 (Experiment 3), (iv) BO of person P 1 versus TE of person P 8 (Experiment 4) or (v) BO of person P 8 versus TE of person P 1 (Experiment 5). Experiment 2 was conducted with both participants outside the tents, each of them using a breathing valve in order to direct his BR to a separate tent exhaust. The number of mosquitoes trapped in all comparisons as a result of responses to stimuli originating from person P 1 or P 8 were counted and noted. Experiments were conducted thrice per night between 19.30-20.00, 20.30-21.00 and 21.30-22.00 hours. Each of the five experiments was repeated 12 or 18 times with the two human subjects switching between the two tents so that half of the tests were conducted with each subject in the two alternative tents. The number of replicates conducted for each experiment is shown in Figure 3.

Statistical analysis
Relative attractiveness was calculated as the number of mosquitoes trapped by the emanations of P 1 divided by the sum of the number trapped by P 1 and P 8 , thus representing the relative attractiveness of person P 1 . A relative attractiveness of greater than 0.5 indicates greater attractiveness of person P 1 , whereas values smaller than 0.5 indicate greater attractiveness of P 8 . Non-parametric statistical methods were used for analysis because of their robustness and flexibility. The significance of differences in attractiveness between the baits in the two traps in each experiment was assessed by Kendall's W test for related samples, comparing the catches of person P 1 with those of person P 8 in the same experiment. The significance of changes in relative attractiveness between experiments was assessed by the Kruskal-Wallis H test for independent samples, comparing the relative attractiveness estimates from repetitions of the same experiments with those of another. In all cases the number of repetitions or releases for each experiment is denoted by N, whereas the total number of mosquitoes which were actually trapped by either human bait is denoted by n. Analysis of the attractiveness of BO of person P 1 versus his BR or the control, as well as the attractiveness of his TE or BR versus the control followed the same procedures. Data were analysed using the Statistical Products and Service Solutions (SPSS, version 10.0).

Results
Experiments were carried out over a total of forty-eight nights, 24 nights for experiments involving the two human subjects and 24 nights for experiments concerning the single participant. None of the participants presented with malaria parasites over the duration of the study.

Response to total emanations, body odour and exhaled air
The attractiveness of the BO of person P 1 versus his BR or a control, as well as the attractiveness of his TE or BR versus a control, are shown in Figure 2. His BO (P = 0.005) and TE (P < 0.001) were significantly more attractive than a control (empty tent) as was his BO (P < 0.001) and the control (P = 0.034) over his BR.

The role of breath and body odour in between-person differences in relative attractiveness
The between-person differences in relative attractiveness, which was measured with respect to person P 1 (i.e. the number of mosquitoes trapped by emanations of person P 1 divided by the sum of the number of mosquitoes trapped by emanations of person P 1 and person P 8 ) following inclusion or exclusion of BR or BO from their TE, are shown in Figure 3. Person P 1 was more attractive than person P 8 based on mosquito responses to their TE (P = 0.021) whereas person P 8 was more attractive than person P 1 based on responses to their BR (P < 0.001). There was no significant difference in the attractiveness of the two persons based on responses to their BO (P = 0.346). The BO of person P 1 was more attractive than the TE of person P 8 (P = 0.001) and the BO of person P 8 was more attractive than the TE of person P 1 (P = 0.001). Thus TE without BR (=BO), from either individual, was consistently more attractive than TE from the other. Comparisons of the relative attractiveness of the study subjects between experiments (lower section of Figure 3) were all significant except between experiment 1 and experiment 3 (P = 0.253).

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 alve-olar 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 humanfeeding 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.