DEET (N,N-Diethyl-m-toluamide) is one of the most widely used mosquito repellents. Although DEET has been shown to be extremely effective, recent studies have revealed that certain individual insects are unaffected by its presence. A genetic basis for this has been shown in Aedes aegypti mosquitoes and the fruit fly Drosophila melanogaster, but, for the triatomine bug, Rhodnius prolixus, a decrease in response to DEET occurred shortly after previous exposure, indicating that non-genetic factors may also be involved in DEET “insensitivity”. In this study, we examined host-seeking behaviour and electrophysiological responses of A. aegypti after pre-exposure to DEET. We found that three hours after pre-exposure the mosquitoes showed behavioural insensitivity, and electroantennography revealed this correlated with the olfactory receptor neurons responding less to DEET. The change in behaviour as a result of pre-exposure to DEET has implications for the use of repellents and the ability of mosquitoes to overcome them.

It has been shown that changes in behavioural responses by insects to compounds can occur through forms of ‘conditioning’ or ‘learned behaviours’ that are not genetically determined. Conditioning has been shown in R. prolixus [10] , [11] , D. melanogaster [12] , and the parasitic wasp Microplitis croceipes [13] . For mosquitoes, some studies have found no evidence for behavioral adaptation [14] , [15] , while more recent work has demonstrated conditioning to odours [16] – [18] . Other investigations have shown that mosquitoes have a preference for returning to hosts they have successfully fed on previously [19] . These preferences are not passed on to their offspring and are therefore likely to be due to learned behaviour [20] . It has also been shown that mosquitoes return to sites where they have previously oviposited [21] , [22] , demonstrating that they can adapt their behaviour based on previously successful events. Interestingly, mosquitoes which emerge from eggs in sites where repellents are present have been shown to return to the same oviposition site, unaffected by the presence of the repellent [23] , [24] , suggesting that they can overcome repellents when accustomed to them or when they are associated with a reward.

The insect repellent N,N-diethyl-m-toluamide (DEET) is one of the most commonly used repellents worldwide [1] . However, despite its common use over the last 60 years, and evidence that it can repel 100% of mosquitoes in the laboratory, semi-field and field tests [2] – [4] , there are several studies suggesting that certain individual insects are not repelled by DEET. For example, a small proportion of individuals in populations of Aedes aegypti mosquitoes and Drosophila melanogaster fruit flies will move towards an attractant despite the presence of DEET, a genetic “insensitivity” which can be selected for in the population [5] – [8] , and which corresponds to changes in the function of the peripheral olfactory system [8] . However, in a recent study, the triatomine bug, Rhodnius prolixus, showed a decrease in behavioural repellency after continuous stimulation with DEET [9] , indicating that other, non-genetic, factors may play a role in preventing insects from responding to DEET.

To determine whether the antennal olfactory system was involved in the altered behavioural responses to DEET, we looked at EAG responses following the behavioural tests. This showed that there were no significant differences in EAG responses to DEET between any of the DEET-insensitive mosquitoes tested from the groups which had been treated at 3 hrs with DEET after pre-exposure to either a control arm (CA/DAi) or an arm with DEET on it (DA/DAi), or those with only an initial exposure to DEET (−/DAi) ( Fig. 2 ). However, the DEET-sensitive mosquitoes, collected after a second exposure to DEET (DA/DAs), had a significantly greater response to DEET than the three groups of DEET-insensitive mosquitoes (p = 0.001, p = 0.019, p<0.001 respectively). The response to DEET of the control group (−/CA) was not significantly different from the DEET-sensitive mosquitoes or the DEET-insensitive mosquitoes collected during initial exposure to DEET (−/DAi+s), but was significantly greater than the response of the DEET-insensitive mosquitoes exposed to DEET after a control arm (CA/DAi) (p = 0.01) or a DEET arm (DA/DAi) (p = 0.006).

To eliminate the possibility of an interaction between host volatiles and DEET being involved in the observed changes in behavioural responses, we tested the mosquitoes with an artificial heating device in place of the arm ( Fig. 1B ). In this experiment, mosquitoes also probed significantly more in response to DEET upon second exposure (HD/HD) than when exposed to DEET for the first time (−/HD) (p = 0.016), although the proportion probing was still lower than the responses to the artificial heat source control (−/H) (p<0.001). There was no significant difference in response between mosquitoes at first and second exposure to the artificial heat source control (H/H). However, mosquitoes exposed to DEET on the heating device 3 h after pre-exposure to the artificial heat source control (H/HD) probed significantly less than in response to all other treatments (p = 0.001).

We examined whether female A. aegypti mosquitoes would change their behaviour when tested twice with a DEET treatment on a human arm. Mosquito responses were determined using an arm-on-cage repellency assay [8] , during which mosquitoes which attempted to probe despite the presence of DEET were considered insensitive. Mosquitoes probing in response to DEET on an arm when first exposed were removed from the experiment, thus, mosquitoes probing on second exposure to DEET were all initially sensitive to DEET and had altered their behaviour. We found that previously DEET-sensitive females, which were exposed again to an arm treated with DEET (DA/DA), landed and probed significantly more on the second DEET exposure than mosquitoes tested for the first time with DEET (−/DA), or tested with DEET following exposure to a control arm (CA/DA) (p<0.001) ( Fig. 1A ). However, the proportion of mosquitoes probing on second exposure to DEET was still lower than the response to the untreated arm (−/CA) (p<0.001). Mosquitoes did not change their behaviour to the untreated control arm if pre-exposed to it (CA/CA). There was also no significant difference between the number of mosquitoes probing in response to DEET upon first exposure (−/DA) and to DEET tested 3 h after pre-exposure to the untreated control arm (CA/DA).

Discussion

The genetic insensitivity to DEET found in previous studies [5]–[8] cannot be the cause of the change in behaviour of A. aegypti which occurred over a short, three hour, period in the experiments reported here. Our observed increase in insensitivity to DEET on a second exposure, by previously DEET-sensitive mosquitoes, initially suggested they may have adapted to DEET, possibly by associating it with the presence of a host arm, and were able to ‘overcome’ the natural repellent effect. This would be consistent with other studies showing that mosquitoes can learn to respond differently to odours to maximise feeding success [19], [20]. Both Culex quinquefasciatus mosquitoes [17], the parasitic wasp M. croceipes [13], and the triatomine bug R. Prolixus [31]–[33] can learn to associate a neutral odour with a food source through Pavlovian conditioning, and adapt their host-seeking preferences accordingly. In C. quinquefasciatus this conditioning could last for up to 24 hours in colony mosquitoes, though fewer mosquitoes responded over time [18]. However, in our study, altered behaviour towards DEET did not result in a reward (i.e. the mosquitoes were not given a blood meal) other than the ability to move towards a human arm/heat source, and this behaviour occurred even when there was no host-related stimulus present. Interestingly, mosquitoes showed increased repellency by DEET on the artificial heat source when pre-exposed to the heat, which was not seen towards DEET on an arm after pre-exposure to the control arm (Fig. 1A, B). The presence of human volatiles with the DEET stimulus may have been a greater incentive for the mosquitoes to persist in host seeking when re-exposed, compared to the weaker attraction of heat alone. Overall, the increased response to a second treatment with DEET on an attractive stimulus, after pre-exposure to DEET with no attractant present, indicates that the learned behaviour is not by association with an attractant as was found in other studies with host-seeking insects [11], [18], [33]. It is instead a direct response to a single exposure to the DEET, Such habituation to DEET has been shown in R. prolixus, where continuous stimulation led to 10–20 minutes of reduced repellency [9]. Thus it seems likely that in our experiments increased DEET-insensitivity results from sensory adaptation or habituation, whereby there is an decrease in response to a stimulus (in this case, DEET) after repeated exposure [34], [35].

The phenomenon of insects changing their response to a compound after pre-exposure or conditioning has been investigated with EAG in D. melanogaster, M. croceipes, Apis mellifera and Protophormia terraenovae [25]–[27], [36], [37]. In some cases no changes in EAG were found, even though the insects were exhibiting changed behavioural responses [36], [37]. In D. melanogaster, the behavioural change was suggested to be caused by a reduction in the volume of glomeruli, and corresponding synapse loss, over a week's exposure to the chemical. In contrast, other trials with D. melanogaster, A. mellifera and P. terraenovae, all using pre-exposures of less than 60 seconds, a decrease in EAG responses to the compounds was observed [25]–[27]. For D. melanogaster, the insects were no longer behaviourally repelled by a repellent, and the EAG decrease only lasted for a brief time, with responses returning to half the normal level in four minutes [25]. In our study, the mosquitoes that had become behaviourally insensitive to DEET also showed a lower EAG response to the repellent (Fig. 2), in contrast to the D. melanogaster study where no change in EAG responses was seen [36]. This supports the possibility that habituation is occurring in our study, as exposure to a chemical for a week, as in the D. melanogaster study, would give time for different changes causing behavioural alteration to occur, such as the loss of synapses, compared to the changes induced in the peripheral olfactory system after brief exposure [25]–[27].

In work on P. terraenovae, the authors concluded that non-associative learning processes, such as habituation, occurred with repeated doses of a repellent [27]. When re-tested with the repellent, approximately 50% of flies no longer responded, which is similar to the level of behavioural DEET-insensitivity found in our study on a second exposure. This might suggest that the same mechanism may be responsible for the behavioural changes. However, in D. melanogaster, P. terraenovae, and indeed in vertebrates, habituation causing a change in response only lasts for a few minutes to half an hour [25], [27], [38], after which the responses return to normal (dishabituation, which is a key characteristic of habituation) [35], [39]. This is in contrast to the altered behavioural and EAG responses to DEET seen in our study with A. aegypti, where the effect lasted for at least 3 hours. It is possible that sensory adaptation and habituation vary between species, and last longer in A. aegypti than in R. prolixus, D. melanogaster or P. terraenovae, and experiments carried out over longer time periods would ascertain if A. aegypti responses did return to normal. However, there is evidence in the nematode Caenorhabditis elegans for two separate causes of decreased response to an odour, with low concentrations resulting in habituation, where the responses return to normal, and high concentrations resulting in sensory adaptation, with responses not returning to normal [39]. In the nematodes the cause of the adaptation was thought to be sensory or receptor fatigue. The same could be true for the mosquitoes in our study, if the ORs are desensitized to DEET after first exposure. This would, however, have to occur differentially between the mosquitoes which altered after first exposure to show behavioural insensitivity to DEET, and those which did not.

If, as shown here, mosquitoes can change their response to a repellent after pre-exposure, then caution should be taken when testing insects multiple times in behavioural repellency bioassays. Methods which retest the same mosquitoes are commonly used, and could be affected by the adaptive behaviour shown in our study [4], [40]–[43]. It should also be determined whether the adaptive behaviour occurs in an arm-in-cage experiment to find out if mosquitoes should not be re-used in repellency tests. DEET has been shown to be 100% effective for up to 5 hours in arm-in cage tests [3], possibly due to the higher concentrations used (28%) or different behaviour triggered on more frequent, every 10–15 min, exposure. When tested at similar concentrations to those in our study, complete repellency lasted for under 4 hours, and insensitivity to the repellent could have occurred after this time. The time for the receptors to return to normal should also be investigated to determine whether dishabituation occurs, or if the change in olfaction is due to receptor fatigue or indeed any other cause. It would be interesting to discover other compounds which may have this effect on mosquito olfaction, and if it could be artificially induced to lower responses to attractants. Investigating the ORNs involved and finding the mechanism responsible may lead to improved control methods. Perhaps the most urgent need is to examine whether the insensitivity also occurs in ‘semi-field’ or ‘field’ situations to determine whether mosquitoes might be less sensitive to repellents if they encounter them for the second time. As research in the field suggests repellents are an important part of transmission prevention strategies in communities [44]–[45], and that some insecticides have repellent properties [46], the likelihood of a mosquito encountering a repellent multiple times is increased. A. aegypti, while primarily dawn and dusk feeders, will continue to feed throughout the day, particularly in shaded or forested areas. Thus the behavioural insensitivity seen here after 3 hours is relevant to their host-seeking period. The effect of pre-exposure, and the relevance to the cycle of feeding activity, may differ for different mosquitoes depending how long the insensitivity lasts. In the field, the concentrations of DEET applied as personal protection would also diminish over time, which could increase the proportion of mosquitoes altering their behaviour. After three hours DEET is still 100% effective, but over longer time periods decreasing effectiveness might have a greater impact. It is therefore important to study this phenomenon over longer times, as this would have clear implications for use of repellents for personal protection, and the use of repellents has been shown to have a direct impact on disease transmission [47].