Personal protection with insect repellents is a popular method for preventing contact with arthropod disease vectors. While their modes of action may vary, repellents generally prevent host seeking vectors from landing on or biting the user. The vectors, which under normal circumstances would be attracted to the person, are either diverted away or disoriented in such a way that they fail to bite the host [1]. These are the most important determinants of effectiveness of any repellent, though there are certainly other essential factors such as costs, availability and user acceptability [2].

The efficacy of repellent compounds aimed for public health use must be rigorously ascertained through laboratory and field trials. Standard test procedures involve controlled experiments in which repellent-treated limbs of human volunteers are exposed to either laboratory reared or wild species of the target insects [3]. Other than the risks of mosquito-borne infections and toxicological concerns [4], the heterogeneity among individual human attraction to blood seeking mosquitoes introduces bias into the data generated by in vivo screening methods [5]. Many haematophagous insects have preferences for different host emanations and some are more likely to bite certain parts of the body than others [6, 7].

High throughput screening of chemical databases to identify new repellents may, therefore, be better accomplished using proxies of humans such as synthetic odour blends with proven attractiveness to a particular vector species. The challenge is to formulate synthetic attractants with fairly consistent attractiveness, when compared to actual humans, and which consist of the important kairomones isolated from human emanations. In addition, factors such as temperature and humidity which also affect insect responses must be held at levels comparable to humans and their environments [8].

The objective of this study was to determine whether synthetic odour blends that are attractive to the malaria vector Anopheles gambiae s s can be used instead of human volunteers to screen for the repellent properties of compounds. To achieve this objective, responses of laboratory-reared An. gambiae s.s to a synthetic attractant blend [9] were evaluated after adding different concentrations of two known mosquito repellents, N, N diethyl-3-methylbenzamide (deet) and para-methane-3, 8, diol (PMD) onto the blend. Deet is the gold-standard insect repellent, with proven high efficacy against a number of disease-carrying and nuisance biting arthropods [10, 11]. Having been used for the past fifty years, it is the active ingredient of several commercially marketed repellents and is recommended for topical skin applications on humans at doses up to 100% [12, 13].

PMD is the active ingredient of waste distillates of lemon-eucalyptus oil (Corymbia citriodora), originally isolated in China as a byproduct of bio-oil distillation [14]. Unlike deet, PMD has only been recently registered for public health use [15]. Nonetheless, recent studies have shown that it has repellency effects that are comparable to or greater than those exhibited by deet [15]. Unlike deet which works mainly as a close range olfactory inhibitor [16], the mode of action of PMD has not been clearly established, though it is likely to be diverting mosquitoes over longer distances from potential blood sources.

Mosquito repellents are normally evaluated on the basis of several different parameters including but not limited to the relative numbers of landing and biting mosquitoes, the duration of protection achieved and user acceptability [4, 15]. Of these, the basic indicator is the decrease in number of host seeking mosquitoes that land or bite the host when the repellents are applied. The experiments reported here were limited to measuring changes in the attractiveness of the synthetic odour blend to the target vector, An. gambiae s.s., whenever the selected repellents were added, and then using these changes an indicator of repellency.

This study demonstrates that when repellent compounds are added onto a blend of attractive compounds, the attractiveness of the blend is not necessarily reduced, but may occasionally be increased depending on which repellent is used. Deet, the most commonly used repellent elicited responses that are comparable to those recorded when human volunteers are the test subjects [20–22]. However, there was no dose response effect seen, and the duration of protection was not evaluated on an hour-by-hour basis.

On the other hand, PMD which has proven high repellency at concentrations between 20% and 50% [15] unexpectedly increased the attractiveness of the synthetic blend. Though this outcome could not be readily explained, it is hypothesized that PMD possibly interacted with one or more of the blend constituents in such away that it resulted in a more attractive blend. Such a phenomenon would, however, not have been possible to directly observe using behavioural assays. Scientists working on development of synthetic lures recognize that the attractiveness of such lures depend not only on attributes of the individual constituents but also on the combinations of the constituents [23]. In fact, host-seeking females do not recognize humans based on only the individual components of their body emanations, but also on the basis of the combinations of different chemicals that they produce [5, 9]. Adding any behaviourally active volatile (attractant or repellent), to an attractive blend may, therefore, shift the equilibrium of olfactory responses in either direction, i.e. attraction or repulsion as has been well-documented in oviposition semiochemicals of mosquitoes [24, 25].

In addition, PMD may have been volatilized by the air current of MMX^® trap used in these experiments, effectively reducing its concentration to low levels that could induce attraction [26]. It is known that certain compounds such as aliphatic carboxylic acids and deet may have both repellent and attractant properties and can express these properties at different concentrations and distances from source [9, 16]. Deet was described by Dogan et al as an inhibitor in the presence of a host but an attractant in the absence of host [16]. The authors further elucidated that a component of human emanations, L-lactic acid was the target of this effect. Indeed some scientists believe that repellents work by interacting with volatile compounds emanating from the host so as to modify their perception by the insect vector [16, 27]. It can, therefore, be expected that the test repellents would elicit different mosquito responses depending on the dose and also on which constituents of the synthetic lure they interacted with. One obvious implication of this limitation of synthetic odours as a test medium is that, if for example the method had been originally used to evaluate PMD, the active ingredient may not have been considered a commercially viable repellent against the mosquitoes.

Whether this or any other synthetic odour blend can be used for repellent screening remains unanswered, although the results obtained from the experiments with deet are comparable to those obtainable with human tests [20–22]. However, it is encouraging that trap catches were less variable than achievable with in vivo trials involving human volunteers whose attractiveness to mosquitoes may show greater heterogeneity [5]. It is proposed that though synthetic odour blends could be used for repellent testing, they would have to be tested with a wide range of known repellents and with multiple delivery methods.

It is concluded that different repellents may elicit different modes of action when added onto synthetic human odour blends, the resulting combinations being either attractive or repellent to the same vector species. Interactions between the repellent and the constituents of the synthetic lure, or changes in repellent concentration are the likely causes of such disparities. In effect, these results revealed a potential limitation of using synthetic human odours to screen for mosquito repellents. Thus, the best method to determine if chemicals provide protection for humans against biting vectors still remains human volunteers. Finally, the synthetic odour blend tested here is not yet sufficiently refined to serve as replacement for humans in repellent testing, but is nonetheless a suitable and consistent lure that can be developed further and evaluated in different formats for exposure-free repellent testing purposes in future.

Ethical approval for the study was granted by Ifakara Health Institute Ethical Review Board, approval number IHRDC/EC4/CL.N96/2004 and Tanzania National Institute of Medical Research, approval number NIMR/HQ/R.8a/Vol.IX/345