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The ovipositor cue indole inhibits animal host attraction in Aedes aegypti (Diptera: Culicidae) mosquitoes

Background Mosquitoes are responsible for disease transmission worldwide. They possess the ability to discriminate between different ecological resources, including nectar sources, animal hosts and oviposition sites, a feature mediated by their olfactory system. Insect repellents, such as N,N-diethyl-meta-toluamide (also called DEET), have been shown to activate and inhibit mosquito odorant receptors, resulting in behavioral modulation. This and other repellents currently available for personal protection against mosquitoes are topically applied to the skin and operate at a short range. In our search for potential long-range inhibitors of attractants to human hosts, we have hypothesized that the shared chemical similarities between indole and DEET may confer the former with the ability to block odorant receptor function and inhibit human host attraction in a similar way as DEET. Methods We used the two-electrode voltage clamp system to assay Xenopus laevis oocytes as a platform to compare the pharmacological effect of commercially available insect repellents and indole on the Aedes aegypti (R)-1-octen-3-ol receptor, OR8, a receptor involved in the decision-making of female mosquitoes to identify human hosts. We also conducted arm-in-a-cage and wind-tunnel bioassays to explore the effect of indole on human host-seeking female Aedes aegypti mosquitoes. Results Our results demonstrate that indole inhibited the Aedes aegypti (R)-1-octen-3-ol receptor OR8. In our arm-in-a-cage assay, 1 M of DEET reduced mosquito visits on average by 69.3% while the same indole concentration achieved 97.8% inhibition. This effect of indole on flight visits was dose-dependent and disappeared at 1 μM. In the flight tunnel, indole elicited on average 27.5% lower speed, 42.3% lower upwind velocity and 30.4% higher tortuosity compared to the control. Conclusions Indole significantly inhibits OR8 activation by (R)-1-octen-3-ol, mosquito visits to a human hand and long-range human host-seeking. The volatility of indole may be leveraged to develop a novel insect repellent in the context of personal mosquito protection. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-022-05545-8.


Introduction
Mosquitoes are a major vector of infectious diseases worldwide due to their ability to transmit pathogens and are also a major source of annoyance. A spatial repellent, such as N,N-diethyl-meta-toluamide (DEET), inhibits mosquito attraction at various distances from the host, ranging from a few centimeters to close to 1 m [1][2][3]. However, despite the protection such repellents provide, mosquitoes are still able to efficiently locate potential human hosts, hover around them and locate any unprotected area that can be targeted for biting. It is therefore desirable to find alternative solutions with an extended protection range, such as volatile odorant repellents [1]. Plant volatiles, such as geraniol, citral, citronellal, eugenol and anisaldehyde, have been shown to exhibit spatial repellency against mosquitoes [4]. In the future, these plant odorants may play a major role in the development of spatial repellent technology.
Noting that DEET and indole exhibit overlapping chemical features, including the presence of an aromatic ring and a neighboring nitrogen atom (Fig. 1a), we hypothesized that indole might interfere with OR8 function. In the present study we used the two-electrode voltage clamp method to assay Xenopus laevis oocytes expressing Ae. aegypti OR8 and showed that indole inhibited OR8-mediated current depolarizations elicited by (R)-1-octen-3-ol. We also showed that sustained exposures to indole and repellents generated an increase of the current baseline. We observed that co-administration of indole and (R)-1-octen-3-ol evoked significant hyperpolarizations that were concentration dependent. In light of these pharmacological results, we tested the potential behavioral effect of indole on animal host-seeking female Aedes aegypti using an arm-in-a-cage assay, providing evidence that indole acted as a repellent at various doses. Using a wind tunnel, we showed that indole decreased anemotactic flight elicited by a synthetic human host blend composed of CO 2 and 1-octen-3-ol. Our findings suggest that volatile compounds such as indole may be discovered based on pharmacological knowledge of ORs. The volatility of indole and the phylogenetic conservation of the CO 2 and octenol receptors in the Culicidae family may be leveraged to develop a novel insect repellent with a broad spectrum of action in the context of personal mosquito protection.
Whole-cell currents were monitored and recorded using the two-microelectrode voltage-clamp technique. Holding potential was maintained at − 80 mV using an OC-725C oocyte clamp (Warner Instruments, LLC, Hamden, CT, USA). Oocytes were placed in a RC-3Z oocyte recording chamber (Warner Instruments, LLC) and To determine the sensitivity of OR8 towards the different indole treatments, we interpolated the half-maximal effective concentration (EC 50 ) values, which correspond to the ligand concentrations eliciting 50% of the maximal current response.

Arm-in-cage assay for human host-seeking behavior
To investigate the potential role of indole in human hostseeking behavior, we developed an arm-in-cage bioassay in which the experimenter's hand is presented to fifteen 5-10-day-old post emergence adult females. The bioassay comprises custom-designed three-dimensionalprinted interlocking ring that creates a 55-mm-diameter opening in a powder-free latex glove worn by the experimenter. A plastic screen is wedged between the two ring components to protect against mosquito bites. The ring supports an odorant delivery platform located at the center of the ring, which receives a 10-mm-diameter cover glass and two 5-mm-diameter filter discs (WHA10016508; Merck) (Additional file 1: Figure S1, Additional file 2, Additional file 3). Mosquitoes are introduced in a 20.3-cm 3 metal cage located inside an incubator for at least 10 min before each experiment (26 °C, 80% RH) for acclimatation. This cage was chosen based on an earlier study reporting the effects of repellents on flight approach in Ae. aegypti [2]. The cage is then placed at room temperature (23-25 °C) under a video camera (EOS 70D, lance: MACRO 0.25/0.8ft; Canon Inc., Tokyo, Japan) to monitor and quantify the numbers of mosquito visits and their duration on the plastic screen. We used the solvent diethyl ether (DEE) as a vehicle. On a blank filter disc, 25 μl of DEE, indole or DEET was deposited on the filter paper and allowed to evaporate for 2 min prior to mosquito exposure. We used tenfold dilutions of indole ranging from 10 -6 to 10 -1 M, and 10 -1 M DEET. The experimenter rubbed the ring-mounted glove against the shirt and skin. All experiments were conducted during the first 3 h of the diurnal period and lasted 10 min. This schedule was chosen for practical reasons and also because mosquitoes consistently exhibited attraction to the human hand. To determine the repellency of indole against human host-seeking mosquitoes, we recorded the number of females landing per minute and counted the duration of their stay on the screen surface. Mosquito landing count was normalized ( x max(x)−min(x) * 100) . Mosquitoes returning to the region of interest were counted as a new visit. Statistical comparisons of mosquito landing were carried out with a Kruskal-Wallis H-test and post-hoc pairwise comparisons were carried out using the Wilcoxon rank sum exact test (P-value adjustment method: Benjamini-Hochberg [BH]). A oneway analysis of variance (ANOVA) analysis was used to analyze mosquito duration.

Wind tunnel bioassay
A wind tunnel system was used to measure the response of female Ae. aegypti to CO 2 alone, 1-octen-3-ol and indole stimuli. The wind tunnel is a rectangularly shaped chamber (2200 × 500 × 500 mm) composed of three compartments: a release, flight and odor delivery chamber, respectively. The release chamber holds the mosquitoes prior to the experiment, while the odorant chamber releases an odor from a clean air delivery system (Sigma Scientific LLC., Micanopy, FL, USA). Ten females (same age as previously described) were transferred to a release box (200 × 160 × 200 mm) inside the release chamber (500 × 200 × 500 mm) and allowed to acclimate for 1 h in darkness at 27 ± 0.7 °C. Experiments were conducted between 10:00 and 13:00 local time. Three experiments were conducted for each treatment. The flight tunnel (1500 × 500 × 500 mm) was illuminated with five infrared lights (RT VAR2-i2-1; Raytec Ltd., Ashington, UK), and a laminar air flow was applied at a speed of 0.1 m/s. After acclimatization, the injection air flow was manually opened for 15 min (duration of each experiment) with compressed air (4 l/m), which delivered the stimulus. Immediately afterwards, flight coordinates were recorded using TrackIt system (SciTrackS GmbH, Pfaffhausen, Switzerland) and the release box door opened. The time interval between each experiment was at least 30 min under constant room ventilation with the wind tunnel top cover removed. Manual maintenance of the wind tunnel was carried out using nitril latex-free gloves to prevent odor contamination.

Tracking software and data analysis
Flight trajectories were recorded using two acA2000-165um infrared-sensitive cameras (Basler AG, Germany) and processed by the 3D software TrackIt (SciTrackS GmbH, Switzerland). Output data was analyzed through R version 4.0.2 programing language [24]. A description of the functions to quantify parameters of flight trajectories can be found in Additional file 4: Table S1. In total, the combined treatments consisted of 90m614 raw X, Y, Z coordinates. Coordinates located outside the flight tunnel (reflection) were removed. The visual field was cropped to define a 800 × 500 × 500 region of interest (ROI; Fig. 5a). Coordinates within the first 3 min of recording were retained and analyzed. We randomly sampled 25% of the remaining coordinates (5192 coordinates) for statistical analysis. Statistical comparisons between treatments were carried out with a Kruskal-Wallis H-test, and post-hoc pairwise comparisons were carried out using the Wilcoxon rank sum exact test (P value adjustment method: Bonferroni).

Odorant stimuli
The injected stimuli included: (i) CO 2 (600 ± 20 ppm) + 25 µl of DEE; (ii) CO 2 + 25 µl of DEE + 2 µl of 1-octen-3-ol (168 mg); and (iii) CO 2 + 25 µl of DEE + 2 µl of 1-octen-3-ol (168 mg) with indole 5 M (14.625 mg). Each stimulus was deposited on a double Whatman filter paper (WHA10016508; Merck & Co., Inc.) as described above in the arm-in-cage bioassay. To allow DEE to evaporate, the holder was placed for 2 min in a separate room, following which the chemical holder was placed inside an OSI-4550 inline volatile collection chamber (Sigma Scientific LLC) connected between the clean air delivery system and the injector. CO 2 was delivered by a steel compressed gas cylinder, and its content was monitored at the injector site using a TES-1370 CO 2 analyser (TES Electrical Electronic Corp., Taipei, Taiwan). During all these procedures, the person conducting the experiment wore laboratory gloves and a mask.
To determine the nature of this inhibitory effect, we established a series of concentration-response curves using OR8-Orco-injected oocytes exposed to increasing concentrations of (R)-1-octen-3-ol alone or combined with 10 -4 M, 10 -3 M or 10 -2 M indole (Fig. 1c). Two types of currents were observed, including the expected agonist-induced depolarization currents and unusual indoledependent hyperpolarization or reduction in baseline currents.
The interpolated EC 50 values for octenol alone versus 10 -4 M and 10 -3 M indole were not statistically significant ( Fig. 1d; Additional file 5: Table S2). The EC 50 value elicited by 10 -2 M indole was significantly different from that elicited by (R)-1-octen-3-ol alone, but the response was moderate. These findings suggest that indole does not have an important effect on the sensitivity of this receptor for (R)-1-octen-3-ol. Contrary to our initial results (Fig. 1b), we did not observe any significant inhibition of the amplitude response with increasing indole concentration (Fig. 1e). However, current amplitudes are contingent on oocyte inherent variability. To address this limitation, we determined the systematic effect of indole on depolarization current amplitudes by normalizing all of the current responses to the initial 10 -7 M (R)-1octen-3-ol exposure (Fig. 1c). We noted that 10 -2 M indole consistently reduced the current amplitude across the concentration of (R)-1-octen-3-ol, excluding 10 -10 M and confirmed that 10 -3 M indole significantly reduced the response amplitude of AaegOR8 to 10 -7 M (R)-1octen-3-ol (Fig. 1f ).
Indole concentrations of 10 -3 M and 10 -2 M elicited hyperpolarization currents (reductions in current baseline) in the presence of (R)-1-octen-3-ol concentrations ranging from 10 -10 M to 10 -7 M (Fig. 1c). These hyperpolarization currents were concentration dependent and were surmounted at higher (R)-1-octen-3-ol concentrations (Fig. 1g). To understand the contribution of indole alone to these currents, we exposed OR8 to increasing concentrations of indole without any other ligand present (Fig. 2a) and observed that 10 -6 to 10 -4 M indole evoked small depolarization currents. The two highest indole concentrations elicited either depolarization or hyperpolarization currents that were a fraction of those elicited by the initial 10 -7 M (R)-1-octen-3-ol stimulation. To better characterize these small yet inconsistent effects, we focused on the currents elicited by 10 -2 M indole and consistently observed these small hyperpolarization and Depol.
Indole modifies the OR8-mediated current baseline of the oocyte membrane.
The cause of the observed hyperpolarization currents caused by indole in the presence of (R)-1-octen-3-ol required further investigation. It mirrored a phenomenon previously documented with AaegOR8, AaegOR2 and AaegOR10 [5,7] and more recently with additional ORs from Cx. quinquefasciatus, Ae. aegypti, and An. gambiae [25]. To investigate whether the hyperpolarization current was a transient response or a durable modification of the current baseline, we exposed OR8-injected oocytes to a change of perfusion buffer by switching from the ND96 buffer solution to a 5.10 -3 M indole perfusion. We also administered increasing 10-fold dilutions of (R)-1-octen-3-ol before reversing the perfusion solution back to ND96 (Fig. 3a). Prior to and after the exchange of the two perfusion buffers, the oocyte was exposed to a transient stimulation of 10 -7 M (R)-1-octen-3-ol for control purposes. The switch from ND96 buffer to indole elicited a stable decrease in the baseline current not observed in water-injected oocytes (Fig. 3a). (R)-1-Octen-3-ol produced very little depolarization current at all tested concentrations. By comparison, DEET and IR3535 evoked larger currents. The switch in buffers in the opposite direction exhibited a stable decrease in baseline current that was most pronounced in the case of DEET and IR3535 (Fig. 3a). In these experiments, we treated the oocytes with a lower indole concentration, as compared to DEET and IR3535, because 10 -2 M indole consistently killed the perfused oocytes. All observed currents elicited by (R)-1-octen-3-ol, indole, a mixture of these two ligands and buffer switch are summarized in Fig. 3b.

Indole inhibits close-range human-host attraction.
To explore the behavioral role of indole, we exposed a human hand to female mosquitoes using an arm-in-acage assay (Fig. 4a insert). The hand was covered with a protective glove which allowed mosquitoes to detect human skin odor through a window created by an open area on the dorsal side of the hand (Additional file 6: Video S1 ). This open area was protected by a screen and was equipped with an odor delivery system (Additional file 1: Figure S1, Additional file 2, Additional file 3). Increasing doses of indole ranging from 10 -6 to 10 -1 M were deposited on this delivery system, and repellency was measured in terms of number of mosquito visits and duration of visits. The repellency effect of DEET was significantly higher than that of the DEE vehicle and indole 10 -6 M, respectively (Fig. 4a) (Kruskal-Wallis H-test, H = 106.11, df = 8, P < 0.0001). All treatments with indole, except for 10 -6 M, were significantly different those with the vehicle (Fig. 4a). Increasing indole doses reduced the number of mosquito visits from 40.6% to 93.8%. We observed a 3.6% inhibition with a 10 -6 M indole, but this effect was not statistically significant. Looking at the accumulated landing numbers, DEE vehicle and 10 -6 M indole elicited overlapping temporal dynamics (Fig. 4b).
Indole at 10 -1 M had a significantly higher temporal repellency than all other treatments, including DEET at the same concentration. Other indole treatments exhibited intermediate temporal repellency between these two extremes. However, the only significant differences were observed between the vehicle and 1 M indole (Additional

Indole reduces 1-octen-3-ol-mediated attraction.
The results of the present study up to this point provided circumstantial pharmacological evidence that indole may in part affect OR8-mediated detection, but they did not give any direct indication that indole affects 1-octen-3-ol-mediated attraction in the context of human hostseeking. To explore this possibility, we used a flight tunnel (Fig. 5a) to expose human-host seeking female mosquitoes to a synthetic blend composed of CO 2 and 1-octen-3-ol. We used three odor treatments, including CO 2 alone or in combination with 1-octen-3-ol and indole, respectively. The ROI was divided into three sections (ROI-1, 2, 3; Fig. 5a) to explore possible differences in terms of trajectory speed, velocity and tortuosity. Kernel density estimations of mosquito locations along the X-axis were statistically different between the three treatments (Fig. 5b). A bird's-eye view (X-Y plane) of flight trajectories representing flight speed suggested differences between the treatments ( Fig. 5c; see example of individual trajectories in Additional file 8: Fig. S3). 1-Octen-3-ol seemed to increase the number of trajectories and coverage of the ROI while indole appeared to reduce flight speed across that same area. Among all three sections of the ROI, speed was higher and more consistent in response to CO 2 in ROI-1 and -2 than in ROI-3 (Fig. 5d) (Kruskal-Wallis H-test, H = 86.8, df = 2, P < 0.0001). As a result, we focused on ROI-1 and -2 for further analyses. As reflected in Fig. 5c, higher speeds were elicited with the addition of 1-octen-3-ol than with CO 2 alone (Fig. 5e) , with increasing concentrations of (R)-1-octen-3-ol, ND96 buffer perfusion and 100 nM (R)-1-octen-3-ol. The same protocol was also applied to water-injected oocytes. In AaegOR8-Orco-injected oocytes, perfusion buffer transitions from ND96 buffer to indole, DEET or IR3535 are shown as solid red arrows, while the transitions in the reverse direction are shown as a solid black arrow. In water-injected oocytes, the corresponding buffer transitions are indicated by empty red and black arrows. The baseline currents of OR8-Orco-injected oocytes exhibited significant increases and decreases compared to those of water-injected oocytes. Statistical significance was determined using the Wilcoxon-Mann-Whitney test (n = 5-9). b Under saline perfusion (ND96), AaegOR8-Orco elicitd a stable current baseline. A stimulus pulse of (R)-1-octen-3-ol activated AaegOR8-Orco, which translated into a transient whole-cell depolarization current. A pulse of (R)-1-octen-3-ol (low concentration) and indole (high concentration) mixture transiently induced a hyperpolarization current. Increasing (R)-1-octen-3-ol concentrations reversed indole blockade. High indole concentrations elicited small depolarization and hyperpolarization currents. Under indole perfusion, the current baseline was increased. IR3535, Ethyl butylacetylaminopropionate elicited significant decreases in speed compared to those observed with CO 2 alone or those in combination with 1-octen-3-ol (Kruskal-Wallis H-test, H = 301.2, df = 2, P < 0.0001). While upwind velocity did not show any statistical differences between CO 2 and 1-octen-3-ol, indole elicited lower upwind velocities (Fig. 5f ) (Kruskal-Wallis H-test, H = 35.5, df = 2, P < 0.0001). Finally, indoleinduced tortuosity was significantly higher than the tortuosity elicited by CO 2 alone or in combination with 1-octen-3-ol (Fig. 5g) (Kruskal-Wallis H-test, H = 20.9, df = 2, P < 0.0001).

Discussion
The overlapping chemical structures between DEET and indole provided the initial impetus to test the potential blocking effect of the latter on AaegOR8. We observed a significant reduction in the current baseline of OR8 at high indole concentrations, with indole generating (R)-1-octen-3-ol-dependent hyperpolarization currents (Fig. 1c). In comparison to DEET and IR3535, these increases in current baseline were robust and exhibited different degrees of reversibility, with indole showing the highest level of insurmountability (i.e. 1-octen-3-ol failed to elicit significant responses and the switch to ND96 biffer did not restore the baseline current). This finding indicates that the affinity of indole for OR8 is higher than that of the other two tested insect repellents. We also did not observe that indole affected whole-cell currents of oocytes expressing Orco, suggesting that indole acts as an allosteric modulator of OR8. We had previously reported on these currents, elicited by the highest concentrations of DEET, IR3535 and picaridin, without providing a molecular mechanism underlying this phenomenon [5,7]. Recently, similar observations were reported with Cx. quinquefasciatus and An. gambiae ORs, suggesting that these hyperpolarization currents may be mediated by chloride influx [25,26]. The reversible decrease of the baseline current in OR8-injected oocytes suggests that these hyperpolarization currents may not be generated by the activation and subsequent opening of the OR8 ion channel but, rather, may reflect a reduction of its constitutive activity. This interpretation is consistent with the observed moderate yet significant increase in baseline current.
What is the ecological role of (R)-1-octen-3-ol? OR8 is expressed in the maxillary palps of adult mosquitoes [11,12]. It is selectively activated by nanomolar concentrations of (R)-1-octen-3-ol when expressed in Xenopus oocytes [13]. The evidence presented in this study  Fig. 4 Indole repels human-seeking female Aedes aegypti mosquitoes. a Diagram of the arm-in-cage assay (inset). The gloved had of the person participating in the experiment is introduced inside a cage containing 15 Ae. aegypti female mosquitoes. The exposed skin area was monitored by a recording camera (see Additional file 6: Video S1 for a representative video). The number of mosquito visits were counted and normalized as a function of treatments, including treatments with the vehicle (diethyl ether [DEE]), DEET 1 M and 7 indole concentrations. All treatment groups were analyzed using a Wilcoxon rank-sum test (n = 3, see Methods section for more details). The percentage of inhibition is indicated below the graph in the gray shaded area. Different lowercase letters above bars indicate significant difference at P ≤ 0.05). b Hyperbola line fitting of the cumulative number of mosquito landings over time. Points represented are mean ± standard deviation of the mean (n = 3). See Additional file 7: Fig. S2. c Histogram of the normalized average time duration mosquitoes spent on the exposed skin area. A one-way ANOVA analysis was used to analyze all treatment groups. The Shapiro-Wilk normality test (W = 0.95056, P = 0.2212) was performed on the square root of the dependent variable (n = 3). Vertical lines indicate SEM suggests a correlative relationship between the observed repellency and one possible molecular mechanism for eliciting this behavior. We recognize that indole repellency may be generated by other molecular targets, such as the indolergic or other receptors. Indeed, indoleinduced repellency may have no direct relevance to 1-octen-3-ol/OR8-mediated behavior. The high concentrations used in the present study (up to 1 M) are unlikely to be found in nature. However, at the lower used concentrations, we also observed significant repellency. Whether the same or other detection mechanisms are involved is not known. It is possible that different indole concentrations target different molecular mechanisms, as has been observed for DEET [5,6,[27][28][29][30][31].
Indole is synthesized by bacteria [32,33], fungi and plants [34] and released by animals [35] and may attract female mosquitoes in the contexts of animal host- [36] and oviposition-seeking [37]. In Culex mosquitoes, f Violin plot of upwind velocity in ROI-1 and -as a function of the odor treatments. g Violin plot of tortuosity in ROI-1 and -2 as a function of the odor treatments. In all violin graphs (e-g), the CO 2 treatment is colored green, the CO 2 + 1-often-3-ol treatment is colored blue and the CO 2 + 1-octen-3-ol + indole treatment is colored maroon. CO 2 concentration: 600 ppm; 1-octen-3-ol concentration: 168 mg; indole concentration: 14.625 mg. ROI, Region Of Interest. Different lowercase letters above all violin graphs indicate significant difference at P ≤ 0.05). Median values (speed, velocity and tortuosity) are provided below each violin graph indole has been associated with fermented Bermuda grass infusions, which attract gravid female mosquitoes [38][39][40]. However, indole alone does not appear to act as an oviposition attractant in Ae. aegypti [19] or in An. gambiae, while it is slightly repellent to ovipositing Aedes albopictus [19]. Indole may play a role in human host-seeking behavior as well [37]. Microbiota on the skin [41] and in sweat [35] release indole and may contribute to its attractiveness. We tested the olfactory-mediated effect of multiple indole doses in the context of human host-seeking behavior and provide observational evidence that indole repels female mosquitoes (Fig. 4). Whether these doses are ecologically relevant is not known, but this observation could provide one explanation as to why different individuals exhibit different levels of attractiveness towards mosquitoes.
To test whether indole-mediated OR8 inhibition would elicit repellency, we tested its effect against blood-seeking female Ae. aegypti. Our results suggest that indole acts as an olfactory repellent in a broad range of doses (1 M-10 -5 M) and loses its repellent activity at low doses (10 -6 M). We used DEET as a positive control since it is a recognized and effective insect repellent. DEET probably operates through different modes of action, including interacting with both smell and taste receptors [5,6,[27][28][29][30][31] and by reducing the volatility of odorants [9]. In our arm-in-a-cage assay, indole was not applied to the skin but rather onto a physically separate chemical holder, suggesting that the inhibitory effect of indole is not that of chemically decreasing odorant volatility ('masking'), as has been suggested in An. gambiae [8][9][10]. The number of mosquito visits was the most significant measure of repellency. By contrast, the durations of the visits were highly variable, with DEET standing out as compared to the vehicle. However, all of the tested treatments did not show any significant differences. Taken together, these results suggest that mosquitoes were repelled before making contact with the screen, which is located immediately below the chemical source. This would be consistent with an olfactory-mediated effect, whereas once landed on the screen, olfactory information may be downplayed by the brain while other senses, such as taste or close-range chemosensation, take precedence.
The highest indole concentrations used in our behavioral experiments are unpleasant (moth ball) to the human nose and may elicit rejection from consumers. However, we have shown that indole concentrations as low as 10 -5 M elicit significant repellency at short range. These concentrations are consistent with those available in the fragrance industry, which uses indoles in dilutions of ≤ 0.1% to create a floral effect in perfumes. Overall, indole demonstrates minimal toxicity at low concentrations [42]. When ingested, indole exhibits an LD 50 of 1000 mg [43]. The quantity we used in our flight tunnel was 14.625 mg, while the minimal dose to repel mosquitoes in our cage assay was 146 µg (10 −5 M). These quantities are well below the toxicity levels, hence the use of indole in perfumery. In addition, indole may be mixed with other volatiles to enhance this floral effect. While it is not known that perfumes containing indoles repel mosquitoes, other personal care products with plant odorants as part of their formulations have been shown to repel mosquitoes [44,45]. Future studies on indolic perfumes might also reveal an ability to create a repelling effect.

Conclusions
The results of our study provide strong support that indole, a mosquito kairomone of unclear ecological significance, inhibits human host-seeking Ae. aegypti females. We also provided pharmacological evidence that the OR8/1-octen-3-ol detection pathway is a potential molecular mode of action for this inhibition. Since indole activates multiple mosquito ORs, including OR2 and OR10, the indole-mediated reduction in anemotactic flight may be caused by distinct olfactory pathways. Our observations nevertheless raise the need for additional studies on the efficacy of indole as a potential mosquito spatial repellent.