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

The oviposition cue indole inhibits animal-host attraction in Aedes aegypti (Diptera: Culicidae) mosquitoes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The oviposition cue indole inhibits animal-host attraction in Aedes aegypti (Diptera: Culicidae) mosquitoes Amir Dekel, Evyatar Sar-Shalom, Yuri Vainer, Esther Yakir, Jonathan D. Bohbot This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1135352/v2 This work is licensed under a CC BY 4.0 License Status: Under Review Version 2 posted 8 You are reading this latest preprint version Show more versions Abstract Background Mosquitoes represent a major source of disease transmission worldwide. They possess the uncanny ability to discriminate between different ecological resources, including nectar sources, animal-hosts, and oviposition sites, a feature mediated by their exquisite 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 available for personal protection against mosquitoes are topically applied on the skin and operate at a short range. In our search for potential long-range inhibitors of human-host attractants, we have hypothesized that the shared chemical similarities between indole and DEET may confer the former the ability to block odorant receptor function and inhibit human-host attraction. Methods We used the two-electrode voltage clamp of Xenopus laevis oocytes as a pharmacological 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 of female mosquitoes to identify human hosts. We conducted an arm-in-a-cage and a wind-tunnel bioassays to explore the effect of indole on human-host seeking female Aedes aegypti mosquitoes. Results We provide evidence that indole inhibits the Aedes aegypti ( R )-1-octen-3-ol receptor OR8, a receptor involved in the decision of female mosquitoes to identify human hosts. In our arm-in-a-cage assay, one molar 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 one micromolar. In our long-range bioassay, indole elicited on average 27.5% lower speed, 42.3% lower upwind velocity and 30.4% higher tortuosity compared to our synthetic blend. 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. Aedes aegypti indole DEET IR3535 (R)-1-octen-3-ol OR8 repellent Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mosquitoes are a major source of infectious diseases worldwide due to their ability to transmit pathogens and are also a major source of annoyance. While N,N-Diethyl-meta-toluamide (DEET) and other topical repellents provide sufficient personal protection to ward off these insects, these compounds act at a short range [ 1 – 3 ]. Despite the protection 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 extended protection range such as volatile odorant repellents [ 1 ]. Unlike topical repellents, odorants such as geraniol, citral, citronellal, eugenol, and anisaldehyde have been shown to exhibit various degrees of spatial repellency against mosquitoes (Hao et al. 2008). In the future, these plant volatiles may play a major role in the development of spatial repellent technology. Mosquito repellents such as DEET or Insect Repellent 3535 (IR3535) exhibit low volatility and act at a short range. These compounds exhibit complex interactions with odorant receptors (ORs), activating some ORs while inhibiting others [ 4 – 6 ]. Notwithstanding its odor masking properties [ 7 – 9 ], DEET alone activates the Ae. aegypti indole receptor AaegOR2 and also inhibits ( R )-1-octen-3-ol-activated AaegOR8 [ 4 ]. The OR8 gene is expressed in basiconic sensilla on the maxillary palps of mosquitoes belonging to species of the Anopheline and Culicine families [ 10 , 11 ]. This phylogenetically-conserved receptor specifically responds to the ( R )-1-octen-3-ol enantiomer in the nanomolar range [ 12 , 13 ] and is believed to synergize the effect of CO 2 in the attraction to human-hosts [ 14 – 16 ]. Indole alone does not mediate oviposition in Anopheles gambiae (Diptera: Culicidae) [ 17 ] and Ae. aegypti [ 18 ] unless combined with 3-methyl-1-butanol [ 17 ] in An. gambiae , and nonanal, p-cresol, phenol and dimethyltrisulfide in Culex spp. (Diptera: Culicidae) [ 19 ]. High indole dosages have been demonstrated to repel the oviposition of mosquitoes such as Toxorhynchites spp. (Diptera: Culicidae) [ 20 ], Cx. quinquefasciatus [ 21 ], Aedes spp. [ 18 ] and An. gambiae [ 22 ]. Noting that DEET and indole exhibit overlapping chemical features, including the presence of an aromatic ring and a neighboring nitrogen atom (Fig. 1 A), we surmised that indole might interfere with OR8 function. Using the two-electrode voltage clamp of Xenopus laevis oocytes expressing Ae. aegypti OR8 , we show that indole inhibits OR8-mediated current depolarizations elicited by ( R )-1-octen-3-ol. We also show that sustained exposures to indole and repellents generate an increase of the current baseline. We observed that co-administration of indole and ( R )-1-octen-3-ol evokes significant hyperpolarizations that are 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, and provide evidence that indole acts as a repellent at various doses. Using a wind tunnel, we show that indole decreases 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 the 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. Methods Aedes aegypti mosquitoes The mosquito laboratory-reared colony developed by Prof. Joel Margalit is maintained in an air-controlled insect chamber with a temperature of 26 o C, 80% relative humidity, and 12:12h light/dark photoperiod. Mosquito larvae were kept in plastic pans containing one liter of water. Larvae were fed with a ground mixture of Novocrabs feed (JBL GmbH & Co, Germany), mice feed and dry yeast until adult emergence. Pupae were transferred into plastic cages and eclosed adults were allowed to mate and feed ad libitum with a 10% sucrose solution. Females were fed with cow blood using a membrane feeding system. AaegOR8-Orco mRNA expression in Xenopus laevis oocytes In vitro transcription and two-microelectrode voltage-clamp electrophysiological recordings were carried out as previously described (Dekel et al., 2016). Both AaegOR8 and AaegOrco were synthesized using the mMESSAGE mMACHINE® SP6 kit (ThermoFisher Scientific) from the linearized pSP64tRFA expression vector. X. laevis oocytes were manually collected, separated, and treated for 30 min with a 8mg/mL collagenase solution at 18°C to remove the follicular layer. Stage V-VI oocytes were rinsed in Ringer solution (96 mM NaCl, 2mM KCl, 5mM MgCl 2 and 5mM HEPES, pH 7.6) and microinjected with a mixture of 1 µL AaegOR2 (3 µg/µL), 1 µL AaegOrco (3 µg/µL) and 1 µL of double-distilled water. Injected oocytes were maintained at 18°C for 3 days in Ringer’s solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl 2 , 0.8 mM CaCl 2 and 5 mM HEPES, pH 7.6) supplemented with 5% dialyzed horse serum, 50 µg/mL tetracycline, 100 µg/mL streptomycin and 550 µg/mL sodium pyruvate. 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, Hamden, CT, USA) and exposed for 8 seconds to different concentrations of indole (CAS 120-72-9, Sigma-Aldrich), ( R )-1-octen-3-ol (CAS 3687-48-7, 98.2%, Bedoukian Research Inc.), N,N-Diethyl-m-toluamide (DEET; CAS number 134-62-3, Sigma-Aldrich), and IR3535 (CAS 52304-36-6, Merck). All compounds were solubilized in 200 µL of dimethyl sulfoxide (DMSO) prior to dilutions in Ringer’s buffer. Orco alone was expressed in oocytes and exposed to N -(4-Ethylphenyl)-2-{[4-ethyl-5-(pyridin-3-yl)-4 H -1,2,4-triazol-3-yl]sulfanyl}acetamide (VUAA1, from Innovapharm Ltd) as a positive control. Currents were allowed to return to baseline between odorant applications. Data acquisition were carried out with a Digidata 1550A and pCLAMP10 (Molecular Devices, Sunnyvale, CA, USA). Plotting and statistical analyses of the data were performed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). Human-host seeking arm-in-cage bioassay To investigate the potential role of indole in human host-seeking 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 is comprised of a custom-designed 3D-printed 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: Fig. S1.; Additional files 2&3). Mosquitoes are introduced in a 20.3 cm 3 metal cage located inside an incubator for at least 10 minutes before each experiment (26°C, 80% RH) for acclimatation. The cage is then placed at room temperature of (23–25 °C) under a video camera (Canon, EOS 70D, lance: MACRO 0.25/0.8ft) to monitor and quantify the numbers of mosquito visits and their duration. On a blank filter discs, 25 µL of the solvent diethyl ether (vehicle), indole or DEET were deposited on the filter paper and allowed to evaporate for 2 minutes prior to mosquito exposure. We used 10-fold dilutions of indole ranging from 10 − 6 -10 − 1 M, and 10 − 1 M DEET. The experimenter rubbed the ring-mounted glove against the shirt and skin. All experiments were conducted between 10:00 and 13:00 and lasted 10 minutes. 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 ( \({x}^{\text{'}}= \frac{x-\text{m}\text{i}\text{n}\left(x\right)}{\text{max}\left(x\right)-\text{m}\text{i}\text{n}\left(x\right)}*100)\) . 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 using Wilcoxon rank sum exact test ( P value adjustment method: Benjamini-Hochberg (BH)). A one-way 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 x 500 x 500 mm), which is composed of three compartments, including release, flight, and odor delivery chambers. The release chamber holds the mosquitoes prior to the experiment, while the odorant chamber releases odor from a clean air delivery system (Sigma Scientific LLC., USA). Ten females (same age as previously described) were transferred to a release box (200 x 160 x 200 mm) inside the release chamber (500 mm x 200 mm x 500 mm) and allowed to acclimate for 1 hour in darkness with 27 ± 0.7 o C. Experiments were conducted between 10:00 and 13:00 local time. Three experiments were conducted for each treatment. The flight tunnel (1500 x 500 x 500 mm) was illuminated with 5 infra-red lights (RT VAR2-i2-1, Raytec Ltd., United Kingdom) 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 the experiments) with compressed air (4 L/m), which delivered the stimulus. Immediately afterwards, flight coordinates were recorded using TrackIt (SciTrackS GmbH, 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 [ 23 ]. 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 90614 raw X,Y,Z coordinates. Coordinates located outside the flight tunnel (reflection) were removed. The visual field was cropped to define a 800 x 500 x 500 region of interest (ROI, Fig. 5 A). Coordinates within the first three minutes 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 using 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 (29 mg). Each stimulus was deposited on a double Whatman filter paper (WHA10016508, Merck) as described above in the arm-in-cage bioassay. To allow DEE to evaporate, the holder was placed for 2 minutes in a separate room. Afterwards, the chemical holder was placed inside an OSI-4550 inline volatile collection chamber (Sigma Scientific LLC., USA) connected between the clean air delivery system and the injector. Carbon dioxide was delivered by a steel compressed gas cylinder. The CO 2 content was monitored at the injector site using a TES-1370 CO 2 analyser (TES electrical electronic Corp., Taiwan). During all these procedures, the experimenter wore laboratory gloves and a mask. Results Indole inhibits AaegOR8 activation by ( R )-1-octen-3-ol. Based on the chemical similarities between indole and DEET (Fig. 1A), we surmised that indole exerts an inhibitory effect on AaegOR8. A 10 -3 M indole concentration reduced by approximately 30% the AaegOR8 current amplitude elicited by 10 -7 M ( R )-1-octen-3-ol (Fig. 1B). This effect was not observed in Orco -injected oocytes. 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 indole-dependent 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 ( R )-1-octen-3-ol alone but 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 as the indole concentration increased (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 the current responses to the initial 10 -7 M ( R )-1-octen-3-ol exposure (Fig. 1C). 10 -2 M indole consistently reduced the current amplitude across the concentration of ( R )-1-octen-3-ol, excluding 10 -10 M. We confirmed that 10 -3 M indole significantly reduced the response amplitude of AaegOR8 to 10 -7 M ( R )-1-octen-3-ol (Fig. 1F). Indole concentration 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 (Fig. 2A). 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 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 hyperpolarization currents (Fig. 2B). By comparison, this same concentration of indole, in the presence of ( R )-1-octen-3-ol at concentrations as low as 10 -10 M, elicited currents reaching the initial response to 10 -7 M ( R )-1-octen-3-ol, suggesting that the significant indole-induced hyperpolarization currents require the presence of ( R )-1-octen-3-ol (Fig. 2C). Indole modifies the OR8-mediated current baseline of the oocyte membrane. The cause for the observed hyperpolarization currents caused by indole in the presence of ( R )-1-octen-3-ol was intriguing. It mirrored a phenomenon previously documented with AaegOR8, AaegOR2 and AaegOR10 [4,6] and more recently with additional ORs from Culex quinquefasciatus , Aedes aegypti , and Anopheles gambiae [24]. 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 solution to a 5.10 -3 M indole perfusion. We also administered increasing ten-fold dilutions of ( R )-1-octen-3-ol before reversing the perfusion solution back to ND96 (Fig. 3A). Prior and after the two perfusion buffer exchanges, the oocyte was exposed with a transient stimulation of 10 -7 M ( R )-1-octen-3-ol for control purposes. The switch from ND96 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 currents at all tested concentrations. By comparison, DEET and IR3535 evoked larger currents. The opposite buffer switch exhibited a stable decrease in baseline current most pronounced in the case of DEET and IR3535 as well (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-a-cage assay (Fig. 4A insert). The hand was covered with a protective glove allowing 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: Fig. S1., Additional files 2&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 different from vehicle and indole 10 -6 M (Fig. 4A) (Kruskal-Wallis H test, H = 106.11, df = 8, p-value < 0.0001). All indole treatments, except 10 -6 M, were significantly different from 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 concentration but this effect was not statistically significant. Looking at the accumulated landing numbers, vehicle and 10 -6 M indole elicited overlapping temporal dynamic (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 1M indole (Additional file 7: Fig. S2). In terms of visit durations, mosquitoes spent on average the same amount of time on the open area when landing occurred (Fig. 4C) (ANOVA, F (8,18) = 1.219, P = 0.343). Indole reduces 1-octen-3-ol-mediated attraction. While we have circumstantial pharmacological evidence that indole may in part affect OR8-mediated detection, we do not have any direct indication that indole affects 1-octen-3-ol-mediated attraction in the context of human host-seeking. 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. We divided the ROI 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 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 ROIs, speed was higher and more consistent in response to CO 2 in ROI-1-2 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&2 for further analyses. As reflected in Fig. 5C, the addition of 1-octen-3-ol elicited higher speeds than with CO 2 alone (Fig. 5E). The addition of indole elicited significant decreases in speed compared to those observed with CO 2 alone or 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, indole-induced tortuosity was significantly higher than those 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, and indole generated ( R )-1-octen-3-ol-dependent hyperpolarization currents (Fig. 1 C). 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. This finding indicates that the affinity of indole for OR8 is higher than for 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 [ 4 , 6 ]. Recently, similar observations were observed with Cx. quinquefasciatus and An. gambiae ORs suggesting that these hyperpolarization currents may be mediated by chloride influx [ 24 , 25 ]. The reversible decrease of the baseline current in OR8-injected oocyte suggests that these hyperpolarization currents may not be generated by the activation and subsequent opening of the OR8 ion channel but may reflect a reduction of its constitutive activity. This interpretation is consistent with the 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 [ 10 , 11 ]. It is selectively activated by nanomolar concentrations of ( R )-1-octen-3-ol when expressed in Xenopus oocytes [ 12 ]. The evidence presented in this study suggest 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, indole-induced repellency may have no direct relevance to 1-octen-3-ol/OR8-mediated behavior. The high concentrations used here (up to 1 M) are unlikely to be found in nature. However, at the lower used concentrations, we also overserved 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 observed with DEET [ 4 , 5 , 26 – 30 ]. Indole is synthesized by bacteria [ 31 , 32 ], fungi, plants [ 33 ] and released by animals [ 34 ]. Indole may attract female mosquitoes in the contexts of animal host- [ 35 ] and oviposition-seeking [ 36 ]. In Culex mosquitoes, indole has been associated with fermented Bermuda grass infusions, which attract gravid female mosquitoes [ 37 – 39 ]. However, indole alone does not appear to act as an oviposition attractant in Ae. aegypti [ 18 ] or in An. gambiae , while it is slightly repellent to ovipositing Ae. albopictus [ 18 ]. Indole may play a role in human host-seeking as well [ 36 ]. Microbiota on the skin [ 40 ] and in sweat [ 34 ] 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 and provide observational evidence that indole repels female mosquitoes (Fig. 4 ). Whether these doses are ecologically relevant is not known but could provide one explanation as to why different individuals exhibits 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 [ 4 , 5 , 26 – 30 ], and by reducing the volatility of odorants [ 8 ]. In our arm-in-a-cage assay, indole was not applied to the skin but on a physically separate chemical holder, suggesting that the inhibitory effect of indole is not chemically decreasing odorant volatility (‘masking’) as it has been suggested in Anopheles gambiae [ 7 – 9 ]. The number of mosquito visits was the most significant measure of repellency. By contrast, the durations of the visits were highly variable and did not show any significant differences between treatments. Taken together, these results suggest that mosquitoes are repelled before making contact with the net, which is located immediately around the chemical source. This would be consistent with an olfactory-mediated effect, whereas once landed, 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 the fragrance industry, which uses indoles in dilutions of 0.1% or less to create a floral effect in perfumes. In addition, indole may be mixed with other volatiles to enhance this floral effect. Conclusions Our study provides strong support that indole, a mosquito kairomone of unclear ecological significance, is inhibiting 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. Declarations Acknowledgements The authors wish to thank Drs Michael J. Gutnick (The Hebrew University of Jerusalem), Andreas Neef (The University of Göttingen) and Gregory Pask (Bucknell University) for their recommendations and insights on the pharmacological study of ligand-gated ion channels. This research was supported by the ISRAEL SCIENCE FOUNDATION (grant No. 719/21). References Schreck CE, Gilbert IH, Weidhaas DE, Posey KH. Spatial action of mosquito repellents. Journal of economic entomology. 1970,63:1576–8. Khan AA, Maibach HI. A Study of Insect Repellents. 1. Effect on the Flight and Approach by Aedes aegypti . J Econ Entomol. 1972,65:1318–21. Leal WS. The enigmatic reception of DEET - the gold standard of insect repellents. Current Opinion in Insect Science. 2014,6:93–8. Bohbot JD, Dickens JC. Insect repellents: modulators of mosquito odorant receptor activity. Frye MA, editor. PLoS ONE [Internet]. 2010,5:e12138. Available from: http://dx.plos.org/10.1371/journal.pone.0012138 Bohbot JD, Fu L, LE TC, Chauhan KR, Cantrell CL, Dickens JC. Multiple activities of insect repellents on odorant receptors in mosquitoes. Medical and veterinary entomology. 2011,25:436–44. Bohbot JD, Dickens JC. Odorant receptor modulation: Ternary paradigm for mode of action of insect repellents. Neuropharmacology. 2012,62:2086–95. Syed Z, Leal W. Mosquitoes smell and avoid the insect repellent DEET. Proceedings of the National Academy of Sciences [Internet]. 2008,36:13598–603. Available from: http://www.pnas.org/cgi/content/abstract/105/36/13598 Afify A, Betz JF, Riabinina O, Lahondère C, Potter CJ. Commonly used insect repellents hide human odors from Anopheles mosquitoes. Current biology. 2019, Afify A, Potter CJ. Insect repellents mediate species-specific olfactory behaviours in mosquitoes. Malaria J. 2020,19:127. Bohbot J, Pitts RJ, Kwon HW, Rutzler M, Robertson HM, Zwiebel LJ. Molecular characterization of the Aedes aegypti odorant receptor gene family. Insect molecular biology. 2007,16:525–37. Lu T, Qiu YT, Wang G, Kwon JY, Rutzler M, Kwon H-W, et al. Odor coding in the maxillary palp of the malaria vector mosquito Anopheles gambiae . Current biology. 2007,17:1533–44. Bohbot JD, Dickens JC. Characterization of an enantioselective odorant receptor in the yellow fever mosquito Aedes aegypti . Leal WS, editor. PLoS ONE [Internet]. 2009,4:e7032. Dekel A, Pitts RJ, Yakir E, Bohbot JD. Evolutionarily conserved odorant receptor function questions ecological context of octenol role in mosquitoes. Scientific reports. 2016,6:37330. Gibson G, Torr S. Visual and olfactory responses of haematophagous Diptera to host stimuli. Medical and veterinary entomology. 1999,13:2–23. Takken W. The role of olfaction in host-seeking of mosquitoes: A review. International Journal of Tropical Insect Science. 1991,12:287–95. Takken W, Knols BG. Odor-mediated behavior of Afrotropical malaria mosquitoes. Annual review of entomology. 1999,44:131–57. Lindh JM, Borg-Karlson A-K, Faye I. Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteria-containing water. Acta tropica. 2008,107:242–50. Allan SA, Kline DL. Evaluation of organic infusions and synthetic compounds mediating oviposition in Aedes albopictus and Aedes aegypti (Diptera: Culicidae). J Chem Ecol. 1995,21:1847–60. Du Y, Millar J. Electroantennogram and oviposition bioassay responses of Culex quinquefasciatus and Culex tarsalis (Diptera: Culicidae) to chemicals in odors from Bermuda grass infusions. Journal of medical entomology. 1999,36:158–66. Collins L, Blackwell A. Olfactory cues for oviposition behavior in Toxorhynchites moctezuma and Toxorhynchites amboinensis (Diptera: Culicidae). Journal of medical entomology. 2002,39:121–6. Millar JG, Chaney JD, Beehler JW, Mulla MS. Interaction of the Culex quinquefasciatus egg raft pheromone with a natural chemical associated with oviposition sites. J Am Mosq Control Assoc. 1994,10:374–9. Takken W, Loon Jja, Adam W. Effects of gonotrophic development of Anopheles gambiae (Diptera: Culicidae) on physiological and behavioural responses to human odour. Journal of insect physiology. 2001,47:303–10. Team RC. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing, 2021. Xu P, Zeng F, Bedoukian RH, Leal WS. DEET and other repellents are inhibitors of mosquito odorant receptors for oviposition attractants. Insect Biochemistry and Molecular Biology. 2019,113:103224. Xu P, Choo Y-M, Chen Z, Zeng F, Tan K, Chen T-Y, et al. Odorant Inhibition in Mosquito Olfaction. iScience. 2019,19:25–38. Ditzen M, Pellegrino M, Vosshall LB. Insect odorant receptors are molecular targets of the insect repellent DEET. Science (New York, NY). 2008,319:1838–42. Pellegrino M, Steinbach N, Stensmyr MC, Hansson BS, Vosshall LB. A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor. Nature. 2011,478:511–4. Sanford JL, Shields VDC, Dickens JC. Gustatory receptor neuron responds to DEET and other insect repellents in the yellow-fever mosquito, Aedes aegypti . Naturwissenschaften. 2013,100:269–73. Dennis EJ, Goldman OV, Vosshall LB. Aedes aegypti Mosquitoes Use Their Legs to Sense DEET on Contact. Current biology. 2019, Grant GG, Estrera RR, Pathak N, Hall CD, Tsikolia M, Linthicum KJ, et al. Interactions of DEET and novel repellents with mosquito odorant receptors. J Med Entomol. 2020,57:1032–40. Elgaali H, Hamilton-Kemp TR, Newman MC, Collins RW, Yu K, Archbold DD. Comparison of long-chain alcohols and other volatile compounds emitted from food-borne and related Gram positive and Gram negative bacteria. Journal of basic microbiology. 2002,42:373–80. Chen G, Zhang R-R, Liu Y, Sun W-B. Spore dispersal of fetid by feces of mycophagous insects. J Chem Ecol. 2014,40:893–9. Turlings TC, Tumlinson JH, Heath RR, Proveaux AT, Doolittle RE. Isolation and identification of allelochemicals that attract the larval parasitoid, Cotesia marginiventris (Cresson), to the microhabitat of one of its hosts. J Chem Ecol. 1991,17:2235–51. Meijerink J, Braks MAH, Brack AA, Adam W, Dekker T, Posthumus MA, et al. Identification of olfactory stimulants for Anopheles gambiae from human sweat samples. J Chem Ecol. 2000,26:1367–82. Blackwell A, Johnson S. Electrophysiological investigation of larval water and potential oviposition chemo-attractants for Anopheles gambiae s.s. Ann Trop Med Parasitol. 2000,94:389–98. Cork A. Olfactory basis of host location by mosquitoes and other haematophagous Diptera. In: Bock GR, Cardew G, editors. Olfaction in Mosquito‐Host Interactions. Ciba Foundation Symposium 200. Wiley & Sons, Chichester, 1996. p. 71–88. Millar JG, Chaney JD, Mulla MS. Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusions. J Am Mosq Control Assoc. 1992,8:11–7. Blackwell A, Mordue A, Hansson B. A behavioural and electrophysiological study of oviposition cues for Culex quinquefasciatus . Physiological Entomology. 1993,18:343–8. Beehler J, Millar J, Mulla M. Synergism between chemical attractants and visual cues influencing oviposition of the mosquito, Culex quinquefasciatus (Diptera: Culicidae). J Chem Ecol. 1993,19:635-644. Bernier UR, Kline DL, Barnard DR, Schreck CE, Yost RA. Analysis of human skin emanations by gas chromatography/mass spectrometry. 2. Identification of volatile compounds that are candidate attractants for the yellow fever mosquito ( Aedes aegypti ). Anal Chem. 2000,72:747–56. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.pdf Additional file 1: Fig. S1. Diagrams of the odor delivery system. A) The odor delivery system is composed of interlocking top and bottom rings attached to a removable chemical holder (overview, top, bottom and side views are provided). Dimensions are provided in mm. B) The rings are locked in place across the experimenter glove. The glove within the inner area of the ring is removed exposing the skin surface. A plastic net is intercalated between the rings and serves to physically protect the skin from mosquito bites. Additionalfile2.stl Additional file 2: Hand rings.stl Additionalfile3.stl Additional file 3: Chemical holder.stl Additionalfile4.docx Additional file 4: Table S1. Scripts and functions used to calculate flight parameters. Additionalfile5.xlsx Additional file 5: Table S2. EC 50 values (Log [M]) of AaegOR8-Orco concentration-response relationships Additionalfile6.m4v Additional file 6: Video S1. Representative videos of arm-in-cage assays. Side-by-side recorded sessions of the first 5 minutes of a control experiment (vehicle) and an indole (0.1 M) treatment. Additionalfile7.pdf Additional file 7: Fig. S2. High indole concentrations exhibit significant repellency over time. Cumulative number of mosquito landings per elapsed minute (see Fig. 4 B). Points represented are mean ± SEM (n = 3). Statistical significance was determined using a non-parametric test followed by a Dunn’s multiple comparisons test ( P values shown on the histograms). Additionalfile8.pdf Additional file 8: Fig. S3. Representative single flight trajectories of female mosquitoes exposed to CO 2 , 1-octen-3-ol and indole. A) Schematic of the flight tunnel and the overall region of interest (ROI) located between X-500-1300 mm (grey). The three odor treatments are color-coded. B) Example trajectories of mosquitoes exposed to CO 2 or to a combination of CO 2 + 1-octen-3-ol, or CO 2 + 1-octen-3-ol + indole. These trajectories were recorded in the ROI within the flight tunnel and projected into each of the three 2-dimensional planes (from left to right, Y-X, Z-X, and Z-Y). Speed is color-coded according to the speed index. The last coordinate of the trajectory is black and marked with an arrow. Trajectory segments outside the ROI are symbolized by a dashed line. Cite Share Download PDF Status: Under Review Version 2 posted Editorial decision: Major revision 13 Sep, 2022 Reviews received at journal 13 Sep, 2022 Reviewers agreed at journal 15 Aug, 2022 Reviewers agreed at journal 29 Jul, 2022 Reviewers invited by journal 27 Jul, 2022 Editor assigned by journal 21 Jul, 2022 Submission checks completed at journal 21 Jul, 2022 First submitted to journal 17 Jul, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1135352","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-01-18 17:26:46","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":126828125,"identity":"b9af249e-1a19-4b42-9aed-2f42b42bf6dd","order_by":0,"name":"Amir Dekel","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amir","middleName":"","lastName":"Dekel","suffix":""},{"id":126828126,"identity":"1cd44e4f-2f4c-47ef-9dc9-114b2f189582","order_by":1,"name":"Evyatar Sar-Shalom","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Evyatar","middleName":"","lastName":"Sar-Shalom","suffix":""},{"id":126828127,"identity":"12b040fd-1d17-44b7-b407-99550170f64d","order_by":2,"name":"Yuri Vainer","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuri","middleName":"","lastName":"Vainer","suffix":""},{"id":126828128,"identity":"5bfb4baa-5a69-4ed2-bd76-5f9424cfed95","order_by":3,"name":"Esther Yakir","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Esther","middleName":"","lastName":"Yakir","suffix":""},{"id":126828129,"identity":"76cb3bef-2a6a-4fcb-97f3-7d66cb5e4beb","order_by":4,"name":"Jonathan D. Bohbot","email":"data:image/png;base64,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","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"D.","lastName":"Bohbot","suffix":""}],"badges":[],"createdAt":"2021-12-02 15:46:20","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1135352/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1135352/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24920602,"identity":"f76168e6-0253-49dd-bc84-89948049067a","added_by":"auto","created_at":"2022-08-08 13:44:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":341211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIndole inhibits OR8-Orco activation by (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol is an alkenyl alcohol\u0026nbsp;and a potent ligand of OR8. Indole is an\u0026nbsp;aromatic\u0026nbsp;bicyclic organic compound. \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-Diethyl-\u003cem\u003emeta\u003c/em\u003e-toluamide (DEET) is a benzyl-ester sharing a benzene ring and a nitrogen atom with indole (red features). \u003cstrong\u003eB\u003c/strong\u003e) Representative current traces of AaegOR8-Orco activation by 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (black arrowheads) or by a mixture of 10\u003csup\u003e-3\u003c/sup\u003e M indole (red arrowhead) and 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (black arrowhead). Normalized responses of AaegOR8-Orco to 10\u003csup\u003e−7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol alone (control) or in combination with 10\u003csup\u003e−3\u003c/sup\u003e M indole (indole). Statistical significance was determined using the Wilcoxon–Mann–Whitney test (n = 5-6). Orco-injected oocytes respond to 10\u003csup\u003e-3\u003c/sup\u003e M of the Orco agonist VUAA1 but not to 10\u003csup\u003e-3\u003c/sup\u003e M indole (n = 4).\u003cstrong\u003e C\u003c/strong\u003e)\u0026nbsp;Representative current traces of AaegOR8-Orco activation by (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol alone or in the presence of 10\u003csup\u003e-4\u003c/sup\u003e M, 10\u003csup\u003e-3\u003c/sup\u003e M or 10\u003csup\u003e-2\u003c/sup\u003e M indole. Arrowheads above the traces indicate the onset of the odorant stimulus, black arrowheads represent (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol and red arrowheads represent indole. \u003cstrong\u003eD\u003c/strong\u003e) Concentration-response relationships of AaegOR8-Orco in response to increasing indole concentrations. Current amplitudes were normalized to the maximum response. Statistical significance was determined using a One-Way ANOVA (\u003cem\u003eP \u003c/em\u003e= 0.0142) followed by a Dunn’s multiple comparisons test (n = 4-7). \u003cstrong\u003eE\u003c/strong\u003e) Non-normalized concentration-current response relationships (n = 4-7). \u003cstrong\u003eF\u003c/strong\u003e) Histogram of the depolarization current response amplitudes of AaegOR8-Orco in response to (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol alone (black) or in combination with various indole concentrations (shades of red). Statistical significance was determined using multiple t-test and statistical significance determined by the Holm-Sidak method (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, n = 3-7). \u003cstrong\u003eG\u003c/strong\u003e) Concentration-response relationships of indole-induced hyperpolarization currents exhibited by AaegOR8-Orco-injected oocytes.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/7cbb9ed67c7ec5de450f42e6.png"},{"id":24920601,"identity":"0a0c9225-6e90-4ee1-9236-fef36bd88a09","added_by":"auto","created_at":"2022-08-08 13:44:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh indole concentrations elicit both depolarization and hyperpolarization currents.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) Representative current traces of AaegOR8-Orco activation by 8 sec stimulations of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol alone or increasing indole concentrations (10\u003csup\u003e-6\u003c/sup\u003e-10\u003csup\u003e-2\u003c/sup\u003e M). Arrowheads above the traces indicate the onset of the odorant stimulus, black arrowheads represent (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol and red arrowheads represent indole. \u003cstrong\u003eB\u003c/strong\u003e) Representative current trace of AaegOR8-Orco activation by 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (black arrowhead) and by 10\u003csup\u003e-2\u003c/sup\u003e M indole (red arrowhead). Statistical significance was determined using the Wilcoxon–Mann–Whitney test (n = 7). \u003cstrong\u003eC\u003c/strong\u003e) (\u003cem\u003eR\u003c/em\u003e)-1-Octen-3-ol is required to elicit significant indole-induced hyperpolarization currents. Nanomolar concentrations of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol induce larger indole-induced depolarization currents than indole alone. Representative traces are shown above. Statistical significance was determined using a t-test (n = 7-12).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/41bac1a9eb597960f4508ab6.png"},{"id":24921203,"identity":"da514a41-b771-4b01-9c41-235f66034b41","added_by":"auto","created_at":"2022-08-08 13:49:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIndole and insect repellents increase the baseline current state of AaegOR8-Orco-injected oocytes.\u003c/strong\u003e Representative current traces of AaegOR8-Orco successively exposed to 100 nM (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol under ND96 (black trace), continuous perfusion under indole (0.005 M), DEET (0.01 M), and IR3535 (0.01 M) perfusions (red traces) with increasing concentrations of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol, ND96 perfusion, and 100 nM (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol. The same protocol was applied to water-injected oocytes as well. In AaegOR8-Orco-injected oocytes, perfusion buffer transitions from ND96 to indole, DEET or IR3535 are shown as solid red arrows, while the reverse perfusion transitions 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 exhibit significant increases and decreases compared to water-injected oocytes. Statistical significance was determined using the Wilcoxon–Mann–Whitney test (n = 5-9). \u003cstrong\u003eB\u003c/strong\u003e) Under saline perfusion (ND96), AaegOR8-Orco elicits a stable current baseline. A stimulus pulse of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol activates AaegOR8-Orco, which translates into a transient whole cell depolarization current. A pulse of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (low concentration) and indole (high concentration) mixture transiently induces a hyperpolarization current. Increasing (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol concentrations reverses indole blockade. High indole concentrations elicit small depolarization and hyperpolarization currents. Under indole perfusion, the current baseline is increased.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/17bd7d90bc02b8ae90ee013d.png"},{"id":24920607,"identity":"c75e7f19-7ba1-41e7-be37-1352ec818f50","added_by":"auto","created_at":"2022-08-08 13:44:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIndole repels human-seeking female \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) Diagram of the arm-in-cage assay (inset). The gloved experimenter’s hand is introduced inside a cage containing 15 \u003cem\u003eAe. aegypti\u003c/em\u003e female mosquitoes. The exposed skin area was monitored by a recording camera (see Additional file 6: Video S1 for a representative video).\u0026nbsp;The number of mosquito visits were counted and normalized as a function of treatments, including diethyl ether (vehicle), DEET 1 M and seven indole concentrations.\u0026nbsp;All treatment\u0026nbsp;groups\u0026nbsp;were analyzed using a Wilcoxon rank-sum test (n = 3, see methods section for more details). Percentage of inhibition are indicated below the graph in the grey shaded area. \u003cstrong\u003eB\u003c/strong\u003e) Hyperbola line fitting of the cumulative number of mosquito landings over time. Points represented are mean ± SEM (n = 3). See Additional file 7: Fig. S2. \u003cstrong\u003eC\u003c/strong\u003e) 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. Shapiro-Wilk normality test (W = 0.95056, \u003cem\u003eP \u003c/em\u003e= 0.2212) was performed\u0026nbsp;on the square\u0026nbsp;root of the dependent variable (n = 3).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/631944c8d13656697095ebe4.png"},{"id":24921344,"identity":"32032cd2-7d45-4956-934b-78238fe8f1a7","added_by":"auto","created_at":"2022-08-08 13:54:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":461137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIndole reduces anemotactic behavior induced by CO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and 1-octen-3-ol.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) Schematic of the flight tunnel. The region of interest (ROI) was equally divided into three sections represented by differentially shaded volumes (1-3). \u003cstrong\u003eB\u003c/strong\u003e) Kernel density estimations of mosquito coordinates within the first 3 min of odorant-induced flight within the ROI. Statistical significance is in parentheses. \u003cstrong\u003eC\u003c/strong\u003e) Bird eye view of the collective flight trajectories elicited by the three odor treatments. Each visible dot represents a trajectory coordinate. Speed levels along the trajectory are color-coded according to the speed index (mm/sec). Graded areas below the X-axes represent the three sections of the region of interest. \u003cstrong\u003eD\u003c/strong\u003e) Violin plot of mosquito speed in ROIs 1, 2 and 3. Median speeds are provided in the table below the plot as well as the statistical significance. \u003cstrong\u003eE\u003c/strong\u003e) Speed in ROI (1+2) as a function of the odor treatments. \u003cstrong\u003eF\u003c/strong\u003e) Upwind velocity in the ROI (1+2) as a function of the odor treatments. \u003cstrong\u003eG\u003c/strong\u003e) Tortuosity in ROI (1+2) as a function of the odor treatments. The CO\u003csub\u003e2\u003c/sub\u003e treatment is green colored, the CO\u003csub\u003e2\u003c/sub\u003e + 1-octen-3-ol treatment is colored in blue, and the CO\u003csub\u003e2\u003c/sub\u003e + 1-octen-3-ol + indole treatment is colored in maroon. CO\u003csub\u003e2\u003c/sub\u003e, 600 ppm, 1-octen-3-ol, 168 mg, indole, 29 mg. ROI, region of interest, Stat. sig., statistical significance.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/9eadaa807231fe048a39303e.png"},{"id":24921346,"identity":"d4fe5e54-cd3e-4147-8685-923c19e98506","added_by":"auto","created_at":"2022-08-08 13:54:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":572312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/3e6f4cf1-6450-4870-acce-8a5ffd6ca6f1.pdf"},{"id":24921205,"identity":"b96b3b65-9e6c-41bb-a2c0-64ab97e2a2e5","added_by":"auto","created_at":"2022-08-08 13:49:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3110413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Fig. S1. Diagrams of the odor delivery system.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) The odor delivery system is composed of interlocking top and bottom rings attached to a removable chemical holder (overview, top, bottom and side views are provided). Dimensions are provided in mm. \u003cstrong\u003eB\u003c/strong\u003e) The rings are locked in place across the experimenter glove. The glove within the inner area of the ring is removed exposing the skin surface. A plastic net is intercalated between the rings and serves to physically protect the skin from mosquito bites.\u003c/p\u003e","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/ea31a554fdc3861bef166477.pdf"},{"id":24920610,"identity":"e019d584-c320-47c3-8350-20d18e2444a1","added_by":"auto","created_at":"2022-08-08 13:44:52","extension":"stl","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":399284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Hand rings.stl\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile2.stl","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/720492af9bc4396ad5fb075c.stl"},{"id":24921204,"identity":"4d02077e-bca2-439c-8cab-1a5ff00b61b5","added_by":"auto","created_at":"2022-08-08 13:49:52","extension":"stl","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":39084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: Chemical holder.stl\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile3.stl","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/1ce84dd3c64028d76a4849f8.stl"},{"id":24920603,"identity":"340f81c8-b05c-4b47-b7d8-ba1c31f36ef7","added_by":"auto","created_at":"2022-08-08 13:44:52","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: Table S1. Scripts and functions used to calculate flight parameters.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile4.docx","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/bafbf1d10b97dcf9386182d1.docx"},{"id":24920609,"identity":"54af55c6-f98e-4781-a32c-04d4406e87b0","added_by":"auto","created_at":"2022-08-08 13:44:52","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5: Table S2. EC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e values (Log [M]) of AaegOR8-Orco concentration-response relationships\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/9b09c1bcec8e7a2300ffb838.xlsx"},{"id":24920613,"identity":"6c2bddc9-ab31-4b2f-867c-8d5550fb361b","added_by":"auto","created_at":"2022-08-08 13:44:52","extension":"m4v","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":6145992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 6: Video S1. Representative videos of arm-in-cage assays.\u003c/strong\u003e Side-by-side recorded sessions of the first 5 minutes of a control experiment (vehicle) and an indole (0.1 M) treatment.\u003c/p\u003e","description":"","filename":"Additionalfile6.m4v","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/1579dda792de3798a881aa98.m4v"},{"id":24921207,"identity":"798c2d9a-3b57-4664-82f7-22e8416af982","added_by":"auto","created_at":"2022-08-08 13:49:52","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":454146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 7: Fig. S2. High indole concentrations exhibit significant repellency over time.\u003c/strong\u003e Cumulative number of mosquito landings per elapsed minute (see Fig. 4 B). Points represented are mean ± SEM (n = 3). Statistical significance was determined using a non-parametric test followed by a Dunn’s multiple comparisons test (\u003cem\u003eP\u003c/em\u003e values shown on the histograms).\u003c/p\u003e","description":"","filename":"Additionalfile7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/da488eb085ad656eb1a4c728.pdf"},{"id":24921345,"identity":"7ea7bccb-b26e-4084-92df-75f067f1bf07","added_by":"auto","created_at":"2022-08-08 13:54:52","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":850189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 8: Fig. S3. Representative single flight trajectories of female mosquitoes exposed to CO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, 1-octen-3-ol and indole.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) Schematic of the flight tunnel and the overall region of interest (ROI) located between X-500-1300 mm (grey). The three odor treatments are color-coded. \u003cstrong\u003eB\u003c/strong\u003e) Example trajectories of mosquitoes exposed to CO\u003csub\u003e2\u003c/sub\u003e or to a combination of CO\u003csub\u003e2\u003c/sub\u003e + 1-octen-3-ol, or CO\u003csub\u003e2\u003c/sub\u003e + 1-octen-3-ol + indole. These trajectories were recorded in the ROI within the flight tunnel and projected into each of the three 2-dimensional planes (from left to right, Y-X, Z-X, and Z-Y). Speed is color-coded according to the speed index. The last coordinate of the trajectory is black and marked with an arrow. Trajectory segments outside the ROI are symbolized by a dashed line.\u003c/p\u003e","description":"","filename":"Additionalfile8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1135352/v2/cd3803b28fecdc63c08be074.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The oviposition cue indole inhibits animal-host attraction in Aedes aegypti (Diptera: Culicidae) mosquitoes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMosquitoes are a major source of infectious diseases worldwide due to their ability to transmit pathogens and are also a major source of annoyance. While N,N-Diethyl-meta-toluamide (DEET) and other topical repellents provide sufficient personal protection to ward off these insects, these compounds act at a short range [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite the protection 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 extended protection range such as volatile odorant repellents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Unlike topical repellents, odorants such as geraniol, citral, citronellal, eugenol, and anisaldehyde have been shown to exhibit various degrees of spatial repellency against mosquitoes (Hao et al. 2008). In the future, these plant volatiles may play a major role in the development of spatial repellent technology.\u003c/p\u003e \u003cp\u003eMosquito repellents such as DEET or Insect Repellent 3535 (IR3535) exhibit low volatility and act at a short range. These compounds exhibit complex interactions with odorant receptors (ORs), activating some ORs while inhibiting others [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notwithstanding its odor masking properties [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], DEET alone activates the \u003cem\u003eAe. aegypti\u003c/em\u003e indole receptor AaegOR2 and also inhibits (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol-activated AaegOR8 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The \u003cem\u003eOR8\u003c/em\u003e gene is expressed in basiconic sensilla on the maxillary palps of mosquitoes belonging to species of the Anopheline and Culicine families [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This phylogenetically-conserved receptor specifically responds to the (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol enantiomer in the nanomolar range [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and is believed to synergize the effect of CO\u003csub\u003e2\u003c/sub\u003e in the attraction to human-hosts [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIndole alone does not mediate oviposition in \u003cem\u003eAnopheles gambiae\u003c/em\u003e (Diptera: Culicidae) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and \u003cem\u003eAe. aegypti\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] unless combined with 3-methyl-1-butanol [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] in \u003cem\u003eAn. gambiae\u003c/em\u003e, and nonanal, p-cresol, phenol and dimethyltrisulfide in \u003cem\u003eCulex spp.\u003c/em\u003e (Diptera: Culicidae) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. High indole dosages have been demonstrated to repel the oviposition of mosquitoes such as \u003cem\u003eToxorhynchites spp.\u003c/em\u003e (Diptera: Culicidae) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], \u003cem\u003eAedes spp.\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNoting that DEET and indole exhibit overlapping chemical features, including the presence of an aromatic ring and a neighboring nitrogen atom (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we surmised that indole might interfere with OR8 function. Using the two-electrode voltage clamp of \u003cem\u003eXenopus laevis\u003c/em\u003e oocytes expressing \u003cem\u003eAe. aegypti OR8\u003c/em\u003e, we show that indole inhibits OR8-mediated current depolarizations elicited by (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol. We also show that sustained exposures to indole and repellents generate an increase of the current baseline. We observed that co-administration of indole and (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol evokes significant hyperpolarizations that are concentration-dependent. In light of these pharmacological results, we tested the potential behavioral effect of indole on animal-host-seeking female \u003cem\u003eAedes aegypti\u003c/em\u003e using an arm-in-a-cage assay, and provide evidence that indole acts as a repellent at various doses. Using a wind tunnel, we show that indole decreases anemotactic flight elicited by a synthetic human host blend composed of CO\u003csub\u003e2\u003c/sub\u003e and 1-octen-3-ol. Our findings suggest that volatile compounds such as indole may be discovered based on the pharmacological knowledge of ORs. The volatility of indole and the phylogenetic conservation of the CO\u003csub\u003e2\u003c/sub\u003e 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eAedes aegypti\u003c/span\u003e \u003cb\u003emosquitoes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe mosquito laboratory-reared colony developed by Prof. Joel Margalit is maintained in an air-controlled insect chamber with a temperature of 26\u003csup\u003eo\u003c/sup\u003eC, 80% relative humidity, and 12:12h light/dark photoperiod. Mosquito larvae were kept in plastic pans containing one liter of water. Larvae were fed with a ground mixture of Novocrabs feed (JBL GmbH \u0026amp; Co, Germany), mice feed and dry yeast until adult emergence. Pupae were transferred into plastic cages and eclosed adults were allowed to mate and feed \u003cem\u003ead libitum\u003c/em\u003e with a 10% sucrose solution. Females were fed with cow blood using a membrane feeding system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAaegOR8-Orco mRNA expression in\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eXenopus laevis\u003c/span\u003e \u003cb\u003eoocytes\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e transcription and two-microelectrode voltage-clamp electrophysiological recordings were carried out as previously described (Dekel et al., 2016). Both AaegOR8 and AaegOrco were synthesized using the mMESSAGE mMACHINE\u0026reg; SP6 kit (ThermoFisher Scientific) from the linearized pSP64tRFA expression vector. \u003cem\u003eX. laevis\u003c/em\u003e oocytes were manually collected, separated, and treated for 30 min with a 8mg/mL collagenase solution at 18\u0026deg;C to remove the follicular layer. Stage V-VI oocytes were rinsed in Ringer solution (96 mM NaCl, 2mM KCl, 5mM MgCl\u003csub\u003e2\u003c/sub\u003e and 5mM HEPES, pH 7.6) and microinjected with a mixture of 1 \u0026micro;L AaegOR2 (3 \u0026micro;g/\u0026micro;L), 1 \u0026micro;L AaegOrco (3 \u0026micro;g/\u0026micro;L) and 1 \u0026micro;L of double-distilled water. Injected oocytes were maintained at 18\u0026deg;C for 3 days in Ringer\u0026rsquo;s solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.8 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 5 mM HEPES, pH 7.6) supplemented with 5% dialyzed horse serum, 50 \u0026micro;g/mL tetracycline, 100 \u0026micro;g/mL streptomycin and 550 \u0026micro;g/mL sodium pyruvate.\u003c/p\u003e \u003cp\u003eWhole-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, Hamden, CT, USA) and exposed for 8 seconds to different concentrations of indole (CAS 120-72-9, Sigma-Aldrich), (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (CAS 3687-48-7, 98.2%, Bedoukian Research Inc.), N,N-Diethyl-m-toluamide (DEET; CAS number 134-62-3, Sigma-Aldrich), and IR3535 (CAS 52304-36-6, Merck). All compounds were solubilized in 200 \u0026micro;L of dimethyl sulfoxide (DMSO) prior to dilutions in Ringer\u0026rsquo;s buffer. \u003cem\u003eOrco\u003c/em\u003e alone was expressed in oocytes and exposed to \u003cem\u003eN\u003c/em\u003e-(4-Ethylphenyl)-2-{[4-ethyl-5-(pyridin-3-yl)-4\u003cem\u003eH\u003c/em\u003e-1,2,4-triazol-3-yl]sulfanyl}acetamide (VUAA1, from Innovapharm Ltd) as a positive control. Currents were allowed to return to baseline between odorant applications. Data acquisition were carried out with a Digidata 1550A and pCLAMP10 (Molecular Devices, Sunnyvale, CA, USA). Plotting and statistical analyses of the data were performed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman-host seeking arm-in-cage bioassay\u003c/h2\u003e \u003cp\u003eTo investigate the potential role of indole in human host-seeking behavior, we developed an arm-in-cage bioassay in which the experimenter\u0026rsquo;s hand is presented to fifteen 5\u0026ndash;10-day-old post emergence adult females. The bioassay is comprised of a custom-designed 3D-printed 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: Fig. S1.; Additional files 2\u0026amp;3). Mosquitoes are introduced in a 20.3 cm\u003csup\u003e3\u003c/sup\u003e metal cage located inside an incubator for at least 10 minutes before each experiment (26\u0026deg;C, 80% RH) for acclimatation. The cage is then placed at room temperature of (23\u0026ndash;25 \u0026deg;C) under a video camera (Canon, EOS 70D, lance: MACRO 0.25/0.8ft) to monitor and quantify the numbers of mosquito visits and their duration. On a blank filter discs, 25 \u0026micro;L of the solvent diethyl ether (vehicle), indole or DEET were deposited on the filter paper and allowed to evaporate for 2 minutes prior to mosquito exposure. We used 10-fold dilutions of indole ranging from 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e-10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e M, and 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e M DEET. The experimenter rubbed the ring-mounted glove against the shirt and skin. All experiments were conducted between 10:00 and 13:00 and lasted 10 minutes. 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 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({x}^{\\text{\u0026#039;}}= \\frac{x-\\text{m}\\text{i}\\text{n}\\left(x\\right)}{\\text{max}\\left(x\\right)-\\text{m}\\text{i}\\text{n}\\left(x\\right)}*100)\\)\u003c/span\u003e\u003c/span\u003e. 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 using Wilcoxon rank sum exact test (\u003cem\u003eP\u003c/em\u003e value adjustment method: Benjamini-Hochberg (BH)). A one-way ANOVA analysis was used to analyze mosquito duration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWind tunnel bioassay\u003c/h2\u003e \u003cp\u003eA wind tunnel system was used to measure the response of female \u003cem\u003eAe. aegypti\u003c/em\u003e to CO\u003csub\u003e2\u003c/sub\u003e alone, 1-octen-3-ol and indole stimuli. The wind tunnel is a rectangularly-shaped chamber (2200 x 500 x 500 mm), which is composed of three compartments, including release, flight, and odor delivery chambers. The release chamber holds the mosquitoes prior to the experiment, while the odorant chamber releases odor from a clean air delivery system (Sigma Scientific LLC., USA). Ten females (same age as previously described) were transferred to a release box (200 x 160 x 200 mm) inside the release chamber (500 mm x 200 mm x 500 mm) and allowed to acclimate for 1 hour in darkness with 27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003eo\u003c/sup\u003eC. Experiments were conducted between 10:00 and 13:00 local time. Three experiments were conducted for each treatment. The flight tunnel (1500 x 500 x 500 mm) was illuminated with 5 infra-red lights (RT VAR2-i2-1, Raytec Ltd., United Kingdom) 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 the experiments) with compressed air (4 L/m), which delivered the stimulus. Immediately afterwards, flight coordinates were recorded using TrackIt (SciTrackS GmbH, 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTracking software and data analysis\u003c/h2\u003e \u003cp\u003eFlight 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 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 90614 raw X,Y,Z coordinates. Coordinates located outside the flight tunnel (reflection) were removed. The visual field was cropped to define a 800 x 500 x 500 region of interest (ROI, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Coordinates within the first three minutes 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 using Wilcoxon rank sum exact test (\u003cem\u003eP\u003c/em\u003e value adjustment method: Bonferroni).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eOdorant stimuli\u003c/h2\u003e \u003cp\u003eThe injected stimuli included (i) CO\u003csub\u003e2\u003c/sub\u003e (600\u0026thinsp;\u0026plusmn;\u0026thinsp;20 ppm)\u0026thinsp;+\u0026thinsp;25 \u0026micro;L of DEE, (ii) CO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 \u0026micro;L of DEE\u0026thinsp;+\u0026thinsp;2 \u0026micro;L of 1-octen-3-ol (168 mg), and (iii) CO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 \u0026micro;L of DEE\u0026thinsp;+\u0026thinsp;2 \u0026micro;L of 1-octen-3-ol (168 mg) with indole 5 M (29 mg). Each stimulus was deposited on a double Whatman filter paper (WHA10016508, Merck) as described above in the arm-in-cage bioassay. To allow DEE to evaporate, the holder was placed for 2 minutes in a separate room. Afterwards, the chemical holder was placed inside an OSI-4550 inline volatile collection chamber (Sigma Scientific LLC., USA) connected between the clean air delivery system and the injector. Carbon dioxide was delivered by a steel compressed gas cylinder. The CO\u003csub\u003e2\u003c/sub\u003e content was monitored at the injector site using a TES-1370 CO\u003csub\u003e2\u003c/sub\u003e analyser (TES electrical electronic Corp., Taiwan). During all these procedures, the experimenter wore laboratory gloves and a mask.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIndole inhibits AaegOR8 activation by (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the chemical similarities between indole and DEET (Fig. 1A), we surmised that indole exerts an inhibitory effect on AaegOR8. A 10\u003csup\u003e-3\u003c/sup\u003e M indole concentration reduced by approximately 30% the AaegOR8 current amplitude elicited by 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (Fig. 1B). This effect was not observed in \u003cem\u003eOrco\u003c/em\u003e-injected oocytes.\u003c/p\u003e\n\u003cp\u003eTo 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 (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol alone or combined with 10\u003csup\u003e-4\u003c/sup\u003e M, 10\u003csup\u003e-3\u003c/sup\u003e M or 10\u003csup\u003e-2\u003c/sup\u003e M indole (Fig. 1C). Two types of currents were observed, including the expected agonist-induced depolarization currents and unusual indole-dependent hyperpolarization or reduction in baseline currents.\u003c/p\u003e\n\u003cp\u003eThe interpolated EC\u003csub\u003e50\u003c/sub\u003e values for octenol alone versus 10\u003csup\u003e-4\u003c/sup\u003e M and 10\u003csup\u003e-3\u003c/sup\u003e M indole were not statistically significant (Fig. 1D, Additional file 5: Table S2). The EC\u003csub\u003e50\u003c/sub\u003e value elicited by 10\u003csup\u003e-2\u003c/sup\u003e M indole was significantly different from (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol alone but was moderate. These findings suggest that indole does not have an important effect on the sensitivity of this receptor for (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol. Contrary to our initial results (Fig. 1B), we did not observe any significant inhibition of the amplitude response as the indole concentration increased (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 the current responses to the initial 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol exposure (Fig. 1C). 10\u003csup\u003e-2\u003c/sup\u003e M indole consistently reduced the current amplitude across the concentration of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol, excluding 10\u003csup\u003e-10\u003c/sup\u003e M. We confirmed that 10\u003csup\u003e-3\u003c/sup\u003e M indole significantly reduced the response amplitude of AaegOR8 to 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (Fig. 1F).\u003c/p\u003e\n\u003cp\u003eIndole concentration of 10\u003csup\u003e-3\u003c/sup\u003e M and 10\u003csup\u003e-2\u003c/sup\u003e M elicited hyperpolarization currents (reductions in current baseline) in the presence of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol concentrations ranging from 10\u003csup\u003e-10\u003c/sup\u003e M to 10\u003csup\u003e-7\u003c/sup\u003e M (Fig. 1C). These hyperpolarization currents were concentration-dependent and were surmounted at higher (\u003cem\u003eR\u003c/em\u003e)-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 (Fig. 2A). 10\u003csup\u003e-6\u003c/sup\u003e to 10\u003csup\u003e-4\u003c/sup\u003e M indole evoked small depolarization currents. The two highest indole concentrations elicited either depolarization or hyperpolarization currents that were a fraction of the initial 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol stimulation. To better characterize these small yet inconsistent effects, we focused on the currents elicited by 10\u003csup\u003e-2\u003c/sup\u003e M indole and consistently observed these small hyperpolarization and hyperpolarization currents (Fig. 2B). By comparison, this same concentration of indole, in the presence of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol at concentrations as low as 10\u003csup\u003e-10\u003c/sup\u003e M, elicited currents reaching the initial response to 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol, suggesting that the significant indole-induced hyperpolarization currents require the presence of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol (Fig. 2C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndole modifies the OR8-mediated current baseline of the oocyte membrane.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cause for the observed hyperpolarization currents caused by indole in the presence of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol was intriguing. It mirrored a phenomenon previously documented with AaegOR8, AaegOR2 and AaegOR10 [4,6] and more recently with additional ORs from \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e, \u003cem\u003eAedes aegypti\u003c/em\u003e, and \u003cem\u003eAnopheles gambiae\u0026nbsp;\u003c/em\u003e[24]. 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 solution to a 5.10\u003csup\u003e-3\u003c/sup\u003e M indole perfusion. We also administered increasing ten-fold dilutions of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol before reversing the perfusion solution back to ND96 (Fig. 3A). Prior and after the two perfusion buffer exchanges, the oocyte was exposed with a transient stimulation of 10\u003csup\u003e-7\u003c/sup\u003e M (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol for control purposes. The switch from ND96 to indole elicited a stable decrease in the baseline current not observed in water-injected oocytes (Fig. 3A). (\u003cem\u003eR\u003c/em\u003e)-1-Octen-3-ol produced very little depolarization currents at all tested concentrations. By \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ecomparison, DEET and IR3535 evoked larger currents. The opposite buffer switch exhibited a stable decrease in baseline current most pronounced in the case of DEET and IR3535 as well (Fig. 3A). In these experiments, we treated the oocytes with a lower indole concentration, as compared to DEET and IR3535, because 10\u003csup\u003e-2\u003c/sup\u003e M indole consistently killed the perfused oocytes. All observed currents elicited by (\u003cem\u003eR\u003c/em\u003e)-1-Octen-3-ol, indole, a mixture of these two ligands, and buffer switch are summarized in Fig. 3B.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndole inhibits close-range human-host attraction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the behavioral role of indole, we exposed a human hand to female mosquitoes using an arm-in-a-cage assay (Fig. 4A insert). The hand was covered with a protective glove allowing 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: Fig. S1., Additional files 2\u0026amp;3). Increasing doses of indole ranging from 10\u003csup\u003e-6\u003c/sup\u003e to 10\u003csup\u003e-1\u003c/sup\u003e 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 different from vehicle and indole 10\u003csup\u003e-6\u003c/sup\u003e M\u003cins cite=\"mailto:Evyatar%20Sar-Shalom\" datetime=\"2022-07-16T23:01\"\u003e\u0026nbsp;\u003c/ins\u003e(Fig. 4A) (Kruskal-Wallis H test, \u003cem\u003eH\u003c/em\u003e = 106.11, \u003cem\u003edf\u003c/em\u003e = 8, p-value \u0026lt; 0.0001). All indole treatments, except 10\u003csup\u003e-6\u003c/sup\u003e M, were significantly different from 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\u003csup\u003e-6\u003c/sup\u003e M indole concentration but this effect was not statistically significant. Looking at the accumulated landing numbers, vehicle and 10\u003csup\u003e-6\u003c/sup\u003e M indole elicited overlapping temporal dynamic (Fig. 4B). Indole at 10\u003csup\u003e-1\u003c/sup\u003e 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 \u0026nbsp;observed between the vehicle and 1M indole (Additional file 7: Fig. S2). In terms of visit durations, mosquitoes spent on average the same amount of time on the open area when landing occurred (Fig. 4C) (ANOVA, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(8,18) =\u0026nbsp;\u003c/sub\u003e1.219, \u003cem\u003eP\u003c/em\u003e = 0.343).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndole reduces 1-octen-3-ol-mediated attraction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile we have circumstantial pharmacological evidence that indole may in part affect OR8-mediated detection, we do not have any direct indication that indole affects 1-octen-3-ol-mediated attraction in the context of human host-seeking. 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\u003csub\u003e2\u003c/sub\u003e and 1-octen-3-ol. We used three odor treatments, including CO\u003csub\u003e2\u003c/sub\u003e alone or in combination with 1-octen-3-ol and indole. We divided the ROI 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 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 ROIs, speed was higher and more consistent in response to CO\u003csub\u003e2\u003c/sub\u003e in ROI-1-2 than in ROI-3 (Fig. 5D) (Kruskal-Wallis H test, \u003cem\u003eH\u003c/em\u003e = 86.8, \u003cem\u003edf\u003c/em\u003e\u0026nbsp; = 2, P \u0026lt; 0.0001). As a result, we focused on ROI-1\u0026amp;2 for further analyses. As reflected in Fig. 5C, the addition of 1-octen-3-ol elicited higher speeds than with CO\u003csub\u003e2\u003c/sub\u003e alone (Fig. 5E). The addition of indole elicited significant decreases in speed compared to those observed with CO\u003csub\u003e2\u003c/sub\u003e alone or in combination with 1-octen-3-ol (Kruskal-Wallis H test, \u003cem\u003eH\u003c/em\u003e = 301.2, \u003cem\u003edf\u003c/em\u003e\u0026nbsp; = 2, P \u0026lt; 0.0001). While upwind velocity did not show any statistical differences between CO\u003csub\u003e2\u003c/sub\u003e and 1-octen-3-ol, indole elicited lower upwind velocities (Fig. 5F) (Kruskal-Wallis H test, \u003cem\u003eH\u003c/em\u003e = 35.5 , \u003cem\u003edf\u003c/em\u003e\u0026nbsp; = 2, P \u0026lt; 0.0001). Finally, indole-induced tortuosity was significantly higher than those elicited by CO\u003csub\u003e2\u003c/sub\u003e alone or in combination with 1-octen-3-ol (Fig. 5G) (Kruskal-Wallis H test, \u003cem\u003eH\u003c/em\u003e = 20.9 , \u003cem\u003edf\u003c/em\u003e\u0026nbsp; = 2, P \u0026lt; 0.0001).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe 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, and indole generated (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol-dependent hyperpolarization currents (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). 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. This finding indicates that the affinity of indole for OR8 is higher than for 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.\u003c/p\u003e \u003cp\u003eWe had previously reported on these currents \u0026ndash; elicited by the highest concentrations of DEET, IR3535 and picaridin \u0026ndash; without providing a molecular mechanism underlying this phenomenon [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recently, similar observations were observed with \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e and \u003cem\u003eAn. gambiae\u003c/em\u003e ORs suggesting that these hyperpolarization currents may be mediated by chloride influx [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The reversible decrease of the baseline current in OR8-injected oocyte suggests that these hyperpolarization currents may not be generated by the activation and subsequent opening of the OR8 ion channel but may reflect a reduction of its constitutive activity. This interpretation is consistent with the moderate yet significant increase in baseline current.\u003c/p\u003e \u003cp\u003eWhat is the ecological role of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol? \u003cem\u003eOR8\u003c/em\u003e is expressed in the maxillary palps of adult mosquitoes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It is selectively activated by nanomolar concentrations of (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol when expressed in \u003cem\u003eXenopus\u003c/em\u003e oocytes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The evidence presented in this study suggest 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, indole-induced repellency may have no direct relevance to 1-octen-3-ol/OR8-mediated behavior. The high concentrations used here (up to 1 M) are unlikely to be found in nature. However, at the lower used concentrations, we also overserved 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 observed with DEET [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIndole is synthesized by bacteria [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], fungi, plants [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and released by animals [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Indole may attract female mosquitoes in the contexts of animal host- [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and oviposition-seeking [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In \u003cem\u003eCulex\u003c/em\u003e mosquitoes, indole has been associated with fermented Bermuda grass infusions, which attract gravid female mosquitoes [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, indole alone does not appear to act as an oviposition attractant in \u003cem\u003eAe. aegypti\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] or in \u003cem\u003eAn. gambiae\u003c/em\u003e, while it is slightly repellent to ovipositing \u003cem\u003eAe. albopictus\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIndole may play a role in human host-seeking as well [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Microbiota on the skin [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and in sweat [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] 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 and provide observational evidence that indole repels female mosquitoes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Whether these doses are ecologically relevant is not known but could provide one explanation as to why different individuals exhibits different levels of attractiveness towards mosquitoes.\u003c/p\u003e \u003cp\u003eTo test whether indole-mediated OR8 inhibition would elicit repellency, we tested its effect against blood-seeking female \u003cem\u003eAe. aegypti\u003c/em\u003e. Our results suggest that indole acts as an olfactory repellent in a broad range of doses (1 M-10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M) and loses its repellent activity at low doses (10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e 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 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and by reducing the volatility of odorants [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In our arm-in-a-cage assay, indole was not applied to the skin but on a physically separate chemical holder, suggesting that the inhibitory effect of indole is not chemically decreasing odorant volatility (\u0026lsquo;masking\u0026rsquo;) as it has been suggested in \u003cem\u003eAnopheles gambiae\u003c/em\u003e [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The number of mosquito visits was the most significant measure of repellency. By contrast, the durations of the visits were highly variable and did not show any significant differences between treatments. Taken together, these results suggest that mosquitoes are repelled before making contact with the net, which is located immediately around the chemical source. This would be consistent with an olfactory-mediated effect, whereas once landed, olfactory information may be downplayed by the brain while other senses, such as taste or close range chemosensation take precedence.\u003c/p\u003e \u003cp\u003eThe 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\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M elicit significant repellency at short range. These concentrations are consistent with the fragrance industry, which uses indoles in dilutions of 0.1% or less to create a floral effect in perfumes. In addition, indole may be mixed with other volatiles to enhance this floral effect.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study provides strong support that indole, a mosquito kairomone of unclear ecological significance, is inhibiting human-host seeking \u003cem\u003eAe. aegypti\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors wish to thank Drs Michael J. Gutnick (The Hebrew University of Jerusalem), Andreas Neef (The University of G\u0026ouml;ttingen) and Gregory Pask (Bucknell University) for their recommendations and insights on the pharmacological study of ligand-gated ion channels. This research was supported by the ISRAEL SCIENCE FOUNDATION (grant No. 719/21).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchreck CE, Gilbert IH, Weidhaas DE, Posey KH. Spatial action of mosquito repellents. Journal of economic entomology. 1970,63:1576\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eKhan AA, Maibach HI. A Study of Insect Repellents. 1. Effect on the Flight and Approach by \u003cem\u003eAedes aegypti\u003c/em\u003e. J Econ Entomol. 1972,65:1318\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eLeal WS. The enigmatic reception of DEET - the gold standard of insect repellents. Current Opinion in Insect Science. 2014,6:93\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eBohbot JD, Dickens JC. Insect repellents: modulators of mosquito odorant receptor activity. Frye MA, editor. PLoS ONE [Internet]. 2010,5:e12138. Available from: http://dx.plos.org/10.1371/journal.pone.0012138\u003c/li\u003e\n\u003cli\u003eBohbot JD, Fu L, LE TC, Chauhan KR, Cantrell CL, Dickens JC. Multiple activities of insect repellents on odorant receptors in mosquitoes. Medical and veterinary entomology. 2011,25:436\u0026ndash;44. \u003c/li\u003e\n\u003cli\u003eBohbot JD, Dickens JC. Odorant receptor modulation: Ternary paradigm for mode of action of insect repellents. Neuropharmacology. 2012,62:2086\u0026ndash;95. \u003c/li\u003e\n\u003cli\u003eSyed Z, Leal W. Mosquitoes smell and avoid the insect repellent DEET. Proceedings of the National Academy of Sciences [Internet]. 2008,36:13598\u0026ndash;603. Available from: http://www.pnas.org/cgi/content/abstract/105/36/13598\u003c/li\u003e\n\u003cli\u003eAfify A, Betz JF, Riabinina O, Lahond\u0026egrave;re C, Potter CJ. Commonly used insect repellents hide human odors from \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes. Current biology. 2019, \u003c/li\u003e\n\u003cli\u003eAfify A, Potter CJ. Insect repellents mediate species-specific olfactory behaviours in mosquitoes. Malaria J. 2020,19:127. \u003c/li\u003e\n\u003cli\u003eBohbot J, Pitts RJ, Kwon HW, Rutzler M, Robertson HM, Zwiebel LJ. Molecular characterization of the \u003cem\u003eAedes aegypti\u003c/em\u003e odorant receptor gene family. Insect molecular biology. 2007,16:525\u0026ndash;37. \u003c/li\u003e\n\u003cli\u003eLu T, Qiu YT, Wang G, Kwon JY, Rutzler M, Kwon H-W, et al. Odor coding in the maxillary palp of the malaria vector mosquito \u003cem\u003eAnopheles gambiae\u003c/em\u003e. Current biology. 2007,17:1533\u0026ndash;44. \u003c/li\u003e\n\u003cli\u003eBohbot JD, Dickens JC. Characterization of an enantioselective odorant receptor in the yellow fever mosquito \u003cem\u003eAedes aegypti\u003c/em\u003e. Leal WS, editor. PLoS ONE [Internet]. 2009,4:e7032.\u003c/li\u003e\n\u003cli\u003eDekel A, Pitts RJ, Yakir E, Bohbot JD. Evolutionarily conserved odorant receptor function questions ecological context of octenol role in mosquitoes. Scientific reports. 2016,6:37330. \u003c/li\u003e\n\u003cli\u003eGibson G, Torr S. Visual and olfactory responses of haematophagous Diptera to host stimuli. Medical and veterinary entomology. 1999,13:2\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eTakken W. The role of olfaction in host-seeking of mosquitoes: A review. International Journal of Tropical Insect Science. 1991,12:287\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eTakken W, Knols BG. Odor-mediated behavior of Afrotropical malaria mosquitoes. Annual review of entomology. 1999,44:131\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eLindh JM, Borg-Karlson A-K, Faye I. Transstadial and horizontal transfer of bacteria within a colony of \u003cem\u003eAnopheles gambiae\u003c/em\u003e (Diptera: Culicidae) and oviposition response to bacteria-containing water. Acta tropica. 2008,107:242\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eAllan SA, Kline DL. Evaluation of organic infusions and synthetic compounds mediating oviposition in \u003cem\u003eAedes albopictus\u003c/em\u003e and \u003cem\u003eAedes aegypti\u003c/em\u003e (Diptera: Culicidae). J Chem Ecol. 1995,21:1847\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eDu Y, Millar J. Electroantennogram and oviposition bioassay responses of \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e and \u003cem\u003eCulex tarsalis\u003c/em\u003e (Diptera: Culicidae) to chemicals in odors from Bermuda grass infusions. Journal of medical entomology. 1999,36:158\u0026ndash;66.\u003c/li\u003e\n\u003cli\u003eCollins L, Blackwell A. Olfactory cues for oviposition behavior in \u003cem\u003eToxorhynchites moctezuma\u003c/em\u003e and \u003cem\u003eToxorhynchites amboinensis\u003c/em\u003e (Diptera: Culicidae). Journal of medical entomology. 2002,39:121\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eMillar JG, Chaney JD, Beehler JW, Mulla MS. Interaction of the \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e egg raft pheromone with a natural chemical associated with oviposition sites. J Am Mosq Control Assoc. 1994,10:374\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eTakken W, Loon Jja, Adam W. Effects of gonotrophic development of \u003cem\u003eAnopheles gambiae\u003c/em\u003e (Diptera: Culicidae) on physiological and behavioural responses to human odour. Journal of insect physiology. 2001,47:303\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eTeam RC. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing, 2021.\u003c/li\u003e\n\u003cli\u003eXu P, Zeng F, Bedoukian RH, Leal WS. DEET and other repellents are inhibitors of mosquito odorant receptors for oviposition attractants. Insect Biochemistry and Molecular Biology. 2019,113:103224. \u003c/li\u003e\n\u003cli\u003eXu P, Choo Y-M, Chen Z, Zeng F, Tan K, Chen T-Y, et al. Odorant Inhibition in Mosquito Olfaction. iScience. 2019,19:25\u0026ndash;38. \u003c/li\u003e\n\u003cli\u003eDitzen M, Pellegrino M, Vosshall LB. Insect odorant receptors are molecular targets of the insect repellent DEET. Science (New York, NY). 2008,319:1838\u0026ndash;42. \u003c/li\u003e\n\u003cli\u003ePellegrino M, Steinbach N, Stensmyr MC, Hansson BS, Vosshall LB. A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor. Nature. 2011,478:511\u0026ndash;4. \u003c/li\u003e\n\u003cli\u003eSanford JL, Shields VDC, Dickens JC. Gustatory receptor neuron responds to DEET and other insect repellents in the yellow-fever mosquito, \u003cem\u003eAedes aegypti\u003c/em\u003e. Naturwissenschaften. 2013,100:269\u0026ndash;73. \u003c/li\u003e\n\u003cli\u003eDennis EJ, Goldman OV, Vosshall LB. \u003cem\u003eAedes aegypti\u003c/em\u003e Mosquitoes Use Their Legs to Sense DEET on Contact. Current biology. 2019, \u003c/li\u003e\n\u003cli\u003eGrant GG, Estrera RR, Pathak N, Hall CD, Tsikolia M, Linthicum KJ, et al. Interactions of DEET and novel repellents with mosquito odorant receptors. J Med Entomol. 2020,57:1032\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eElgaali H, Hamilton-Kemp TR, Newman MC, Collins RW, Yu K, Archbold DD. Comparison of long-chain alcohols and other volatile compounds emitted from food-borne and related Gram positive and Gram negative bacteria. Journal of basic microbiology. 2002,42:373\u0026ndash;80.\u003c/li\u003e\n\u003cli\u003eChen G, Zhang R-R, Liu Y, Sun W-B. Spore dispersal of fetid by feces of mycophagous insects. J Chem Ecol. 2014,40:893\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eTurlings TC, Tumlinson JH, Heath RR, Proveaux AT, Doolittle RE. Isolation and identification of allelochemicals that attract the larval parasitoid, \u003cem\u003eCotesia marginiventris\u003c/em\u003e (Cresson), to the microhabitat of one of its hosts. J Chem Ecol. 1991,17:2235\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eMeijerink J, Braks MAH, Brack AA, Adam W, Dekker T, Posthumus MA, et al. Identification of olfactory stimulants for \u003cem\u003eAnopheles gambiae\u003c/em\u003e from human sweat samples. J Chem Ecol. 2000,26:1367\u0026ndash;82. \u003c/li\u003e\n\u003cli\u003eBlackwell A, Johnson S. Electrophysiological investigation of larval water and potential oviposition chemo-attractants for \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.s. Ann Trop Med Parasitol. 2000,94:389\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003eCork A. Olfactory basis of host location by mosquitoes and other haematophagous Diptera. In: Bock GR, Cardew G, editors. Olfaction in Mosquito‐Host Interactions. Ciba Foundation Symposium 200. Wiley \u0026amp; Sons, Chichester, 1996. p. 71\u0026ndash;88.\u003c/li\u003e\n\u003cli\u003eMillar JG, Chaney JD, Mulla MS. Identification of oviposition attractants for \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e from fermented Bermuda grass infusions. J Am Mosq Control Assoc. 1992,8:11\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eBlackwell A, Mordue A, Hansson B. A behavioural and electrophysiological study of oviposition cues for \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e. Physiological Entomology. 1993,18:343\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eBeehler J, Millar J, Mulla M. Synergism between chemical attractants and visual cues influencing oviposition of the mosquito,\u003cem\u003eCulex quinquefasciatus\u003c/em\u003e (Diptera: Culicidae). J Chem Ecol. 1993,19:635-644.\u003c/li\u003e\n\u003cli\u003eBernier UR, Kline DL, Barnard DR, Schreck CE, Yost RA. Analysis of human skin emanations by gas chromatography/mass spectrometry. 2. Identification of volatile compounds that are candidate attractants for the yellow fever mosquito (\u003cem\u003eAedes aegypti\u003c/em\u003e). Anal Chem. 2000,72:747\u0026ndash;56. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aedes aegypti, indole, DEET, IR3535, (R)-1-octen-3-ol, OR8, repellent ","lastPublishedDoi":"10.21203/rs.3.rs-1135352/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1135352/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMosquitoes represent a major source of disease transmission worldwide. They possess the uncanny ability to discriminate between different ecological resources, including nectar sources, animal-hosts, and oviposition sites, a feature mediated by their exquisite 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 available for personal protection against mosquitoes are topically applied on the skin and operate at a short range. In our search for potential long-range inhibitors of human-host attractants, we have hypothesized that the shared chemical similarities between indole and DEET may confer the former the ability to block odorant receptor function and inhibit human-host attraction.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe used the two-electrode voltage clamp of \u003cem\u003eXenopus laevis\u003c/em\u003e oocytes as a pharmacological platform, to compare the pharmacological effect of commercially-available insect repellents and indole on the \u003cem\u003eAedes aegypti\u003c/em\u003e (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol receptor OR8, a receptor involved in the decision of female mosquitoes to identify human hosts. We conducted an arm-in-a-cage and a wind-tunnel bioassays to explore the effect of indole on human-host seeking female \u003cem\u003eAedes aegypti\u003c/em\u003e mosquitoes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe provide evidence that indole inhibits the \u003cem\u003eAedes aegypti\u003c/em\u003e (\u003cem\u003eR\u003c/em\u003e)-1-octen-3-ol receptor OR8, a receptor involved in the decision of female mosquitoes to identify human hosts. In our arm-in-a-cage assay, one molar 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 one micromolar. In our long-range bioassay, indole elicited on average 27.5% lower speed, 42.3% lower upwind velocity and 30.4% higher tortuosity compared to our synthetic blend.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIndole significantly inhibits OR8 activation by (\u003cem\u003eR\u003c/em\u003e)-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.\u003c/p\u003e","manuscriptTitle":"The oviposition cue indole inhibits animal-host attraction in Aedes aegypti (Diptera: Culicidae) mosquitoes","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-08-08 13:44:50","doi":"10.21203/rs.3.rs-1135352/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-09-13T15:52:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-09-13T10:09:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8b294512-d2e6-4dc3-8d1e-ac4fd620274c","date":"2022-08-15T11:07:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"da15cf94-6173-4252-93d8-df6a75154f86","date":"2022-07-29T14:20:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-27T05:20:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-21T18:37:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-21T18:31:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2022-07-17T13:58:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8559805a-7700-4e16-ad88-da38a6bbeb75","owner":[],"postedDate":"August 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-07T13:14:26+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-08 13:44:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1135352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1135352","identity":"rs-1135352","version":["v2"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}