The C. lectularius colony utilized in this study was originated from Ft. Dix, New Jersey, USA. This strain is susceptible to pyrethroid insecticides (Romero et al., 2007). All the common bed bugs were reared at 25 ± 2°C under a photoperiod of 12:12 (L: D). DEET and compounds were purchased from Sigma-Aldrich at high purity and diluted (v/v for liquid and w/v for solid) in dimethyl sulfoxide (DMSO) as indicated. Chemical Abstracts Service (CAS) numbers and purity are as follows: DMSO (67-68-5, 100%); pentanal (110-62-3, 97%); 2-butanone (78-93-3, 99.7%); 2-heptanone (107-87-9, 99%); 2-hexanone (591-78-6, 96%); hexanal (66-25-1, 98%); heptanal (111-71-7, 92%); octenal (124-13-0, 99%); nonanal (124-19-6, 95%); decanal (112-31-2, 98%); toluene (108-88-3, 99.8%); xylene (106-42-3, 99.5%); styrene (100-42-5, 99%); propylbenzene (103-65-1, 98%); ethylbenzene (100-41-4, 99%); 2,4-dimethylhexane (589-43-5, 99%); propylamine (107-10-8, 99%); butylamine (109-73-9, 99.5%); DEET (134-62-3, 97%); eugenol (97-53-0, 99%); carvacrol (499-75-2, 98%); linalyl acetate (115-95-7, 97%); menthyl acetate (89-48-5,97%); (−)-linalool (126-91-0, 95%); (+)-menthone (3391-87-5, 98.5%); citral (5392-40-5, 96%); geranyl acetate (105-87-3, 98%); 1S-(+)-3-carene (498-15-7, 99%); (+)-β-pinene (19902-08-0, 95%); α-terpineol (10482-56-1, 96%).

Single sensillum recordings were performed as described previously (Liu et al., 2014; Liu and Liu, 2015). Female adult bed bug were selected for experiment at least 5 days after blood feeding.

To test the responses to DEET, 10 μl desired concentration of DEET was applied onto a filter paper strip (3 × 40 mm) and inserted into a Pasteur pipette to create the stimulus cartridge. While in investigating the potential influence of DEET on bed bug's response to odorants, we followed the method of Pellegrino et al. (2011) by pipetting undiluted DEET (10 μl) onto a filter paper strip and the desired concentration of odorants (10 μl) onto a second filter paper strip (Pellegrino et al., 2011). Both filter paper strips were then carefully inserted into a glass Pasteur pipette. A constant airflow across the antennae was maintained at 20 ml/s throughout the experiment. Humidified air was delivered to the antenna through a glass tube with a small hole, about 10 cm from the end of the tube. Stimulation was achieved by inserting the tip of the stimulus cartridge into the hole on the glass tube. A stimulus controller (Syntech, Germany) diverted a portion of the air stream (0.5 l/min) to flow through the stimulus cartridge for 0.5 s, thus delivering the stimulus to the sensilla. At least six replicates for each recording experiment with different stimuli were conducted on different individuals. As a high number of ORNs are co-located in each sensillum type, we did not attempt to calculate the firing rate for each ORN within the same sensillum. Instead, the total numbers of action potentials were counted off-line in a 500 ms period before and after stimulation for the whole sensillum. The number of action potentials after stimulation was subtracted from the number of action potentials before stimulation and multiplied by two in order to quantify the firing rate change in one sensillum in spikes per second.

Bed bug ORs and ORCO (odorant receptor co-receptor) were amplified as previous descried (Liu et al., 2017). Basciallly, the ORs and ORCO were cloned into pT7Ts vector (a gift from Dr. Wang, Institute of Plant Protection in Chinese Academy of Agricultural Science, Beijing, China). The constructed vectors were linearized with specific restriction enzyme and cRNAs were synthesized from linearized vectors with mMESSAGE mMACHINE T7 (Ambion, Carlsbad, CA).

Mature healthy oocytes (stage V–VII) (Nasco, Salida, CA) were harvested from the African Clawed Frog (Xenopus laevis) and treated with collagenase I (GIBCO, Carlsbad, CA) in washing buffer [96 mM NaCl, 2 mM KCl, 5 mM MgCl₂, and 5 mM HEPES (pH = 7.6)] for about 1 h at room temperature. After being cultured overnight at 18°C, oocytes were microinjected with 10 ng cRNAs of both OR and ORCO. After injection, oocytes were incubated for 4–7 days at 18°C in 1 × Ringer's solution [96 mM NaCl, 2 mM KCl, 5 mM MgCl₂, 0.8 mM CaCl₂, and 5 mM HEPES (pH = 7.6)] supplemented with 5% dialyzed horse serum, 50 mg/ml tetracycline, 100 mg/ml streptomycin, and 550 mg/ml sodium pyruvate.

Whole-cell currents were recorded from the injected Xenopus oocytes with two-electrode voltage clamp. Odorant-induced currents were recorded with an OC-725C oocyte clamp (Warner Instruments, Hamden, CT) at a holding potential of −80 mV. Odorants and DEET were dissolved in DMSO at a 1:10 ratio to make stock solutions that were diluted in 1 × Ringer's solution to the indicated concentrations (Wang et al., 2010). Between stimulations, oocytes were allowed to return to their resting potential by washing out the odorants or DEET using Ringer's solution. Recovery time was determined according to the time required for agonist-induced responses to abate and to reach pre-stimulation levels. Data acquisition and analysis were carried out with Digidata 1440A and pCLAMP 10.2 software (Axon Instruments Inc., CA).

The olfactometer bioassay was conducted by following the procedure described by Gries et al. (2015) with only minor modifications. Bioassays were conducted in dual-choice olfactometers consisting of two lateral Petri dishes (with lid) and a central dish (without lid) (3 × 9 cm inner diameter). The central dish was connected to the two lateral dishes via a plastic tube (2.5 cm long × 0.5 cm inner diameter). The dishes in this olfactometer mimic the natural still-air shelters in which bed bugs spend the day. Prior to the start of each bioassay, a disc of filter paper (9 cm diameter) was placed into each dish and a strip of filter paper (24 × 0.6 cm) inserted into the connecting tubing to provide traction for walking bed bugs. In addition, a piece of filter paper was placed into each lateral dish and covered with a piece of cardboard (2.2 × 2.2 cm) as a refuge for bioassay insects. An inverted lid of a 4-ml vial was placed on top of the corrugated cardboard shelter in the randomly assigned treatment dish of the olfactometer. All these olfactometers were placed in a small room with excellent air circulation. Before adding the stimulus or DMSO, the connected tube was sealed using a small piece of Parafilm membrane (Sigma) and a single male or female adult bed bug released into the central chamber of each of 20–60 olfactometers for each experiment at the end of the 12-h photophase. The chemical stimulus formulated in equal amounts in DMSO was then pipetted into the lid of experimental treatment, while in the control treatment only DMSO was added into the inverted lid. The bed bug in each olfactometer was then allowed to explore the central dish for 1 h of darkness, after which the Parafilm membrane was removed in the connected tubes, enabling the bed bugs to detect the odorants in either side of the dish. After the 12-h darkness period, the bed bug's position within each olfactometer was recorded. Any insect not found in a lateral chamber was recorded as a non-responder. Olfactometers were washed with unscented detergent (Beaumont Products, GA, USA), rinsed with distilled water, and dried at room temperature between each bioassay.

This study was carried out in accordance with the recommendations of the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. The protocol was approved by the Auburn University Animal Care and Use committee (Policy number is 2016-2987).

To test if bed bugs are able to sense DEET, we screened all types of olfactory sensillum on the bed bug antennae, including Dα, Dβ, Dγ, C, E1, and E2 sensilla, via single sensillum recording. The results showed that DEET elicited excitatory response from Dα and Dβ sensilla, particularly at high concentrations. For example, pure DEET was able to stimulate the Dα and Dβ sensilla,with the firing rate of 50 ± 4.8 and 53 ± 3.7 spikes/s, respectively (Figures 1A,B).

Insect odorant receptors (ORs) in the dendrite membrane of ORNs are responsible for sensitizing odorants and producing the neuronal firings (action potential) (Leal, 2013). To further identify potential bed bug odorant receptor(s) that are activated by DEET, previously reported 15 bed bug ORs expressed in the Xenopus oocytes were challenged by DEET with two-electrode voltage clamp. The results showed that at least three of these ORs, OR20, OR36, and OR37, showed remarkable current responses (≥100 nA) to DEET (Figure 2A). The responses of these ORs to DEET also followed a dose-dependent pattern, with EC₅₀ values of 7.5 × 10⁻⁶, 7.1 × 10⁻⁶, and 6.5 × 10⁻⁶, respectively (Figures 2B–D).

Of particular interest is the observation that all these ORs (OR20, OR36, and OR37) that were activated by DEET were even more sensitive to certain terpenes/terpenoids than odorants from other chemical classes. Particularly, OR37 was mainly activated by terpenes/terpenoids (Liu et al., 2017). Comparatively, OR20, OR36, and OR37 showed much stronger responses to (−)-linlool, (−)-menthone and citral than to DEET (Figure 3). When we mixed DEET with (−)-linalool (Figure 4A) or α-terpineol (Figure 4B) in stimulating OR20/ORCO, the response to either (−)-linalool or α-terpineol was reduced when high dose of DEET was applied, which suggested that a competitive effect may exist between DEET and (−)-linalool/α-terpineol. As all these terpenes/terpenoids are major components of essential oils or other botanical repellents that are repulsive for blood-feeding mosquitoes, we then continued to test the behavioral response of bed bugs to DEET and terpenes/terpenoids.

To test the behavioral responses of bed bugs to DEET and terpenes/terpenoids, we applied a dual-choice olfactometer bioassay as described by Gries et al. (2015). With this method, we found that both male and female bed bugs were significantly repelled by pure DEET but not 10% DEET solution, which suggests that only high doses of DEET elicit an aversive response in both male (Figure 5A) and female bed bugs (Figure 5B). To test if DEET's repellency to bed bugs was due to excessive concentrations applied, we chose other two terpenoids, eugenol, and carvacrol at the concentration of ~100%, which did not activate any DEET-sensitive ORs. Unsurprisingly, both of eugenol and carvacrol showed absolutely no activity in repelling the bed bugs (Figures 6A,B), which suggested that bed bugs did sense DEET and repelled by it but not due to other physical interference. For the terpenes and terpenoids which displayed strong activation on the DEET-sensitive ORs, much more potency was observed in repelling bed bugs than that of DEET. For instance, 1% of linalyl acetate, menthyl acetate, (−)-linalool (Figures 6C–E), (+)-menthone (Figure S1A) and 5% of citral, geranyl acetate and 1S-(+)-3-carene (Figures S1B–D) already displayed very strong repellency to bed bugs. Another terpene odorant, (+)-β-pinene, which demonstrated a closing activating effect as DEET on the receptors, displayed a similar repellency to bed bugs only at high concentrations (Figure 6F).

Although we confirmed that DEET can activate ORNs or ORs of bed bugs directly, many previous studies on fruit fly and mosquito have also indicated that DEET might exert an interfering effect on neuronal responses to the odorants (Ditzen et al., 2008; Pellegrino et al., 2011). To investigate whether DEET displayed the same function on bed bugs' olfactory systems, we characterized the neuronal response of Dγ and C sensilla to the combination of DEET and human odorants. Human odorants from different classes were chosen based on their strong stimulation on the Dγ or C sensilla, demonstrated in our previous study (Liu and Liu, 2015). Interestingly, we found that the responses of bed bugs to human odorants were totally “scrambled” when DEET was added into the stimulation (Figure 7 and Figures S2, S3). For example, Dγ sensilla showed no significant difference in dose-dependent response to combinations of DEET and hexanal or DEET and toluene compared to hexanal or toluene alone (Figure 7B and Figure S2A). However, for some other aldehyde chemicals, including heptanal, octanal, nonanal, and decanal, a very significant blocking effect was observed in the neuronal responses to the mixtures when DEET was added, with the dose-dependent curves being much lower than those of the aldehydes alone (Figures 7C–F). It is worth noting that while the majority of the strongest blocking effects appeared at the highest concentration of human odorants, a few were presented at some medium concentration, such as heptanal at the concentration of 10⁻⁴, and octanal at 10⁻³(Figures 7C,D), suggesting that high concentration of heptanal and octanal can overcome the blocking effect of DEET on the ORNs, which was consistent with previous finding in Drosophila (Pellegrino et al., 2011). Although DEET showed an extensive blocking effect on the neuronal responses of Dγ sensilla to certain chemicals, no interfering effect was observed on the response of C sensilla to two amines tested, propylamine and butylamine (Figure S3).

The temporal dynamic of neuronal responses is also considered an important feature for odorant encoding or recognition. To test whether DEET showed any influence on the temporal characteristics of neuronal response to human odorants, we compared the temporal dynamics of the bed bugs' response to combinations of DEET and odorants with those for solely odorants. We found that DEET did change the temporal structure of the neuronal response to certain odorants and this change was both odorant-specific and dose-specific (Figure 8). For instance, DEET showed a huge modification on the temporal structure of responses to nonanal by inhibiting the peak firing from the ORNs housed in the Dγ sensillum (Figure 8C), but had no effect on the temporal structure of responses to hexanal (Figure 8A). For another odorants, DEET had a large impact on the response to heptanal, with the temporal structure shifting from more tonic to more phasic (Figure 8B). However, when the concentration of heptanal increased, DEET's effect was diminished, providing further evidence for high concentration of odorants could overcome DEET's interference on the temporal structure of neuronal responses.

Previous studies on mosquitoes have proved that DEET affects the ion channel formed by the complex of odorant receptors and co-receptors, disturbing the recognition of odorant receptors to their ligands (Bohbot et al., 2011; Bohbot and Dickens, 2012). To investigate whether a similar mechanism is also involved in the repulsive effect of DEET for bed bugs, we tested the current responses of a bed bug odorant receptor (OR19) to several human-odor stimuli (pentanal, butanal, 2-butanone, 2-pentanone, 2-hexanone) both with and without DEET added into the perfusion. The results showed that all the stimuli with no DEET elicited typical strong responses in OR19/ORCO. For example, 2-butanone and 2-pentanone triggered remarkable current responses in OR19/ORCO, even though the responses decreased slightly when challenged repeatedly with high concentrations of odorants (Figure 9A). However, when DEET was added to either 2-butanone or 2-pentanone solutions, the current responses of OR19 to both stimuli decreased dramatically (Figure 9B) and this pattern was repeated in all the other human-odor stimuli tested (Figure 9C).

The effect of DEET on the dose-dependent response of OR19/ORCO to 2-butanone and 2-pentanone was also investigated in this study. We found that DEET showed a very clear antagonistic effect to the dose-dependent responses of OR19/ORCO to both 2-butanone and 2-pentanone (Figures 10A,B). The concentrations of DEET also had a major impact on the antagonistic effect of DEET: increasing the concentration of DEET significantly enhanced the antagonistic effect. For example, the antagonistic effect of DEET on the dose-dependent response of OR19/ORCO to 2-pentanone and 2-butanone was significantly stronger at the concentration of 10⁻³ than 10⁻⁴ (Figure 10C).

These results clearly indicate that DEET interacts with bed bug odorant receptors to inhibit the current response of specific receptors to human odors. This antagonistic effect of DEET on one or more odorant receptors probably creates the blocking effect on the ORNs in response to human odors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.