Validation of Y-tube olfactometer function
Prior to testing the effectiveness of catnip oil as a mosquito repellent, we first evaluated our Y-tube olfactometer (designed according to WHO guidance13; Supplementary Fig. S1) for use in repellence assay experiments (Fig. 1; Supplementary Table S1). We first established whether the Aedes aegypti mosquitoes displayed a preference for a particular branch of the olfactometer, irrespective of the use of attractants or repellents. Twenty mosquitoes were released from the holding port into the apparatus and after 30 seconds, the number of mosquitoes in each branch port was recorded. A total of six replicates were performed. The distribution of mosquitoes in the olfactometer showed that an average of 13% travelled to the end of each collecting branch, while the remaining mosquitoes were distributed in the base leg or holding port (Fig. 1A; Supplementary Table S1). These findings indicate a random, uniform and repeatable distribution pattern of mosquitoes in both olfactometer branches. Thus, the Y-tube olfactometer is suitable for conducting unbiased repellence assays.
We next tested our apparatus to confirm that the presence of a human hand in one of the branches acted as an attractant to mosquitoes. In line with this, we found that an average of 57% of mosquitoes were attracted to the branch containing the human hand while 15% were found in the empty control branch and 28% remained in the holding port or base leg of the olfactometer (Fig.1B; Supplementary Table S1).
We then performed a series of experiments to evaluate the performance of the mosquito repellent N,N-diethyl-3-methylbenzamide (DEET) using our olfactometer. We first placed an open vial containing 100µl of 20% DEET in one branch and left the other branch empty. An average of 85% of mosquitoes remained in the holding tube/ base leg, while 14% were found in the empty branch and <1% were found in the branch containing DEET (Fig. 1C; Supplementary Table S1). In the next experiment we placed a human hand in one branch as an attractant and a vial containing 100µl of 20% DEET in the other branch. We found that 49% of mosquitoes were attracted to the human hand, while 51% remained in the holding tube/ base leg. No mosquitoes were found in the branch containing DEET (Fig. 1D; Supplementary Table S1). Finally, we placed a human hand in one branch and a human hand with a vial containing 100µl of 20% DEET in front of it in the other branch. An average of 62% of mosquitoes were found in the branch containing the human hand, while 33% remained in the holding tube/ base leg and 5% were found in the branch containing the human hand + 20% DEET (Fig. 1E; Supplementary Table S1). These data confirm that human hands act as an attractant to Aedes aegypti mosquitoes and that DEET functions as an effective repellent using our Y-tube olfactometer and experimental methodology.
Testing efficacy of commercially available repellents with the Y-tube olfactometer
We tested our experimental setup and testing procedure with commercially available insect repellents Peaceful Sleep (15% DEET) and Odomos, which contains 12% N,N-diethylbenzamide, a compound closely related to DEET, that has been shown to be effective in repelling Anopheles stephensi and Aedes aegypti mosquitoes14. We found that Peaceful Sleep-treated human hands attracted an average of 14% of responding Aedes aegypti mosquitoes (ie. only those mosquitoes that moved to either branch and did not remain in the base leg/ holding port) while the untreated control hand attracted an average of 86% of mosquitoes (Fig.2; Supplementary Table S2; Supplementary Fig. S2). Likewise, Odomos-treated human hands attracted an average of 14% of mosquitoes while the untreated control hand attracted an average of 86% mosquitoes (Fig.2; Supplementary Table S2; Supplementary Fig. S2). These data confirm that our experimental design and apparatus can replicate the known mosquito repellence quality of DEET and N,N-diethylbenzamide, establishing that it is suitable for analysing the repellence qualities of other compounds.
Testing efficacy of catnip oil in repelling Aedes aegypti
We next investigated the effectiveness of different concentrations of catnip essential oil in repelling Aedes aegypti mosquitoes. We used essential oil obtained by hydro-distillation of leaves, stems and flowers of a variety of Nepeta cataria designated as Chemotype A, which has previously been shown to contain the 4aS,7S,7aR isomer of nepetalactone7. In line with this, our GC-MS analysis showed that the oil contained ~97.5% nepetalactone, ~1.5% caryophyllene and <1% caryophyllene oxide (Table 1).
We assessed the efficacy of a series of catnip oil dilutions ranging from 100% catnip oil with no diluent (olive oil), to 0% catnip oil with 100% diluent. A human hand was placed at the control branch of the Y-tube olfactometer, while in the other branch a 100 µl aliquot of catnip oil or diluent was placed in a vial cap in front of the human hand. We found that catnip oil at concentrations ranging from 100% to just 2% provided significant repellence of mosquitoes, with the percentage of responding mosquitoes in the control branch ranging from 74% to 90%, and the percentage of mosquitoes in the branch containing repellent ranging from 26% to 10% (Fig.3A; Supplementary Tables S3 and S4; Supplementary Fig. S3). Catnip oil diluted to 1%, or diluent alone, did not show any significant repellence properties. These data demonstrate the catnip oil is effective in repelling Aedes aegypti mosquitoes at a broad range of concentrations, with 2% representing the lower limit of efficacy.
Assessing the duration of mosquito repellence using 2% catnip oil
Having established that 2% catnip oil provided substantial Aedes aegypti mosquito repellence, we next investigated the duration of repellence after the initial application to human skin. We tested a 2% dilution of catnip oil, again using olive oil as the diluent, and the effect of 2% catnip oil in a standard hand lotion formulation (see Materials and Methods for ingredients). We performed the repellence assay over a 5-hour period and found that 2% catnip oil diluted in olive oil gave significant repellence for up to one hour after application to skin, with 85% of responding mosquitoes found in the branch containing the human hand and 15% of mosquitoes found in the branch containing the human hand and 2% catnip oil one hour after application (Fig.3B; Supplementary Table S5 and S6; Supplementary Fig. S3). Some repellence was observed at later timepoints, with ~60% of responding mosquitoes found in the branch containing the human hand and ~40% of mosquitoes found in the branch containing the human hand and 2% catnip oil, but these results were not statistically significant. For 2% catnip oil added to a hand lotion, we observed significant repellence for up to 4 hours after application to the skin, with 82% of responding mosquitoes found in the branch containing the human hand and 18% in the branch containing the human hand and 2% catnip lotion 1 hour after application, and 61% and 39% respectively after 4 hours (Fig.3C; Supplementary Table S7 and S8; Supplementary Fig. S3). Hence, 2% catnip oil has significant repellence properties for variable amounts of time after the initial application, depending on the diluent used.