Trapping of the malaria vector Anopheles gambiae with odour-baited MM-X traps in semi-field conditions in western Kenya

Development of odour-baited trapping devices for biting insects remains a challenge for many important species, including African malaria vectors [1, 2]. Such traps may find application in mosquito surveillance [3], risk assessment and forecasting [4], and/or be used en masse for population suppression and disease transmission reduction similar to trap-bait systems developed for tsetse flies [5‐7]. There are three important components of trap development, namely the 'attractant', the physical trap design, and trapping mechanism used. A fourth set of essentials follows, namely the cost, applicability and acceptance of such devices by end-users in anticipated market sectors.

Anopheles gambiae s.s. is a highly anthropophilic mosquito, with a tendency to blood feed and rest inside houses [8]. Studies on semiochemicals affecting its host-seeking behaviour have intensified since the late 1980s [1], with the main aim to replace the Human Biting Catch (HBC). Some traps use whole human odour, like the CDC light trap placed next to an occupied bednet [9], the OBET [10, 11] and Mbita traps [12‐14]. However, humans vary substantially in their innate attractiveness towards An. gambiae s.s. [15, 16], which has been attributed to the presence of allomonal effects of breath [17], variability in skin microfloral composition [18, 19], or both. Identification of specific kairomones with key involvement in governing the host-seeking process has therefore been advocated [20].

Of the several hundreds of volatiles produced by humans [21, 22], a fair number have been reported to elicit behavioural responses by An. gambiae s.s. These comprise commonly known kairomones like carbon dioxide [23‐25], but also carboxylic fatty acids [26, 27], oxo-carboxylic acids [28], ketones [29], phenols [29, 30], L-lactic acid [31], and ammonia [32].

It remains speculative why attempts to reproduce laboratory studies under field conditions have been unsuccessful to date, although the substantial differences between olfactometer-based studies [26‐31] and field-based trapping methods may be a prime cause for this. Alternatively, prolonged maintenance of mosquito strains under artificial laboratory conditions may result in distorted behaviour and responses to 'attractants' that would not be similar in nature. A third reason relates to the highly endophagic behaviour of An. gambiae s.s. that results in strong 'competition' between odour-baited traps and human hosts present in the indoor environment, arguably always in favour of humans expressing the full range of physical and chemical cues. Finally, and in contrast with laboratory studies, ambient climatic conditions may vary over space and time, and cause highly variable trap catches in the field.

The development and commercialization of traps based on counterflow geometry technology in the late 1990s by the American Biophysics Corporation has resulted in efficacy evaluation studies for a range of mosquito species in various geographical settings, including Africa [33‐35]. Many of these evaluations inferred superiority of this technology over conventional traps such as the CDC light trap [36], which supports the view that trap design rather than the stimuli used affect the trapping efficiency.

In the present study, it was aimed to reproduce some previous laboratory findings under semi-natural conditions in western Kenya. In large outdoor cages (for a description see [37]) experimental counterflow geometry traps were deployed (see below), baited with human foot odour, CO₂, NH₃, 1-octen-3-ol, or various combinations thereof, as a first step to develop more appropriate research tools for anticipated open field studies.

When traps were baited with worn socks only, average (± SD) catches over eight test nights were 47 ± 21 for BNN and 73 ± 20 for BGJK (Table 1). Three out of four completed Latin square blocks yielded highly significant attraction to foot odour and in spite of high catches in the first block of BGJK, the conservative discriminatory levels needed when applying a double replicate of 2× 2 Latin square cross-over designs caused insignificance because of the control trap yielding 21 specimens in the first night. The overall effect of fabric (nylon or cotton) was not significant.

When evaluating the attractiveness of foot odours collected on socks worn for 12 hours only, unexpectedly high levels of residual activity were observed (Table 2). This effect was stronger for BGJK than for BNN. Nevertheless, two days after collection of foot odour of BNN, 39% (78/200) of released mosquitoes were caught, whereas BGJK's residual odours still trapped nearly 27.5% (55/200) of the females released as long as seven days after initial odour collection.

Albeit at lower levels, NH₃ when offered in various concentrations, raised trap catches significantly (at 0.1, 1 and 10%; Table 3A) except when offered undiluted. No clear dose-response effect was observed. As expected, CO₂, a well-known kairomone, elicited strong behavioural responses, with an average (± SD) of 110 ± 26 females collected (~55% of the number released) per night. In contrast, 1-octen-3-ol at 0.5 mg/hr did not raise catches significantly over those collected by the control trap (Table 3C). When foot odour was combined with NH₃ (10% v/v) catches remained significantly different from the control trap, though on average similar to catches with foot odour only (Table 3D). However, when CO₂ was added to this blend, a sharp and significant increase in catches was observed, with an average (± SD) catch of 181 ± 43 females (~91% of the number released; Table 3E). In fact, it was noticed that, even in the absence of sugar sources in the greenhouse, mosquitoes managed to survive between test periods (12 hrs), as catches in this series twice exceeded the 200 insects released during the experimental night.

When four traps were tested simultaneously, and three of these contained odour baits, trap catches with NH₃ were similar to those of the control trap and significantly reduced catches when combined with foot odour over foot odour alone (Table 4A). In a further series (Table 4B), NH₃ again did not increase trap catches significantly, nor did it affect catches when combined with carbon dioxide. As observed in previous experiments (Table 3E), a strong increase in catches when foot odours were combined with CO₂ was expected. Table 4C shows not only that foot odour and CO₂ alone caused significant increases in catches over control traps, but also that the combination of the two led to a significant and synergistic increase in catch levels (i.e. the combination of the two yielded significantly higher catches than either or the sum of the two baits alone).

An overview of the experimental baits and catch levels is shown in Figure 2. Overall, catch levels of both strains of An. gambiae s.s. when responding to foot odour, appeared similar.

In the early 1990s, foot odour was first incriminated as influencing the selection of biting sites by An. gambiae s.s. on humans [40]. Subsequently it was found that this mosquito also responds strongly to Limburger cheese volatiles, reminiscent of human foot odour, in laboratory bioassays [26, 41, 42]. Repeated efforts to show similar effects in the field, when baiting various trap models and electric nets [43] with these complex odours were disappointing [44] and not reported (Knols and Mboera, unpublished data). However, in the present study it was clearly demonstrated that foot odours, when collected on either cotton or nylon sock fabric, can significantly increase trap catches and thus confirm earlier laboratory findings [40]. It is noteworthy that the residual activity of these emanations spanned several days, corroborating earlier studies claiming that previously occupied houses or worn clothing attracted mosquitoes several days after having been vacated or worn, respectively [45]. These findings clearly indicate that as yet unknown kairomones with low volatility are present in foot odour. Progression in the identification of active fractions of foot odour is currently underway (A. Hassanali, personal communication).

Ammonia has been demonstrated to elicit behavioural and sensory physiological responses of many haematophagous arthropods including Afrotropical Anopheles [46‐48]. Either on its own, but particularly when combined with lactic acid and a blend of carboxylic fatty acids, laboratory assays confirmed the kairomonal effect of this chemical [48]. The current findings show a mildly attractive effect of ammonia alone, but did not indicate an additive or synergistic effect when combined with either foot odour or CO₂. It should be noted though that the high volatility of this compound in an aqueous solution could have caused only short-term effects. Alternatively, with ammonia already naturally present in foot odour additional ammonia did not cause any measurable behavioural effect. A slow-release system, analogous to the continuous production of ammonia on the human skin through microbial breakdown of urea and amino acids [46], might improve trap catches.

Carbon dioxide, in all experiments, dramatically increased trap catches. Regretfully, the use of this kairomone either in gaseous form or as dry ice remains a major hurdle for its inclusion in trapping devices in the tropics. Although alternative delivery systems have been developed (such as the catalytic combustion of propane), these still depend on costly gas tanks not widely available. Although it has been argued that anthropophilic vectors cannot depend solely on this chemical to locate humans [1], CO₂ clearly synergises responses to other human-specific substances. Although mosquito surveillance programmes may use CO₂-baited devices, widespread use of traps at household level will ultimately require identification of potent bait without the need for CO₂. At present, kairomones present in foot odour seem to offer the best promise for this. It should be noted though, that semi-field systems cannot replace field studies and that verification of findings should always take place. Similarly, although this trapping system has proven useful for field sampling of mosquitoes (Yu Tong Qiu et al., unpublished data from field studies in the Gambia), it should be considered primarily as an experimental tool to evaluate candidate kairomones and not directly as a replacement for existing sampling tools for adult anophelines.