Taste Sensilla Research Articles

Measuring Physiological Responses of <em>Drosophila</em> Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors(1,2), ion channels(3-6), and ionotropic receptors(7) that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Insects Can Count: Sensory Basis of Host Discrimination in Parasitoid Wasps Revealed.

The solitary parasitoid Leptopilina heterotoma is one of the best studied organisms concerning the ecology, behaviour and physiology of host discrimination. Behavioural evidence shows that L. heterotoma uses its ovipositor to discriminate not only between parasitized and unparasitized Drosophila melanogaster larvae, but also to discriminate between hosts with different numbers of parasitoid eggs. The existing knowledge about how and when the parasitoid marks the host motivated us to unravel the chemosensory basis of host discrimination by L. heterotoma that allows it to choose the “best” host available. In this paper we report on electrophysiological recordings of multi-neural responses from the single taste sensillum on the tip of the unpaired ovipositor valve. We stimulated this sensillum with haemolymph of unparasitized, one-time-parasitized and two-times-parasitized Drosophila larvae. We demonstrate for the first time that quantitative characteristics of the neural responses to these haemolymph samples differed significantly, implying that host discrimination is encoded by taste receptor neurons in the multi-neuron coeloconic ovipositor sensillum. The activity of three of the six neurons present in the sensillum suffices for host discrimination and support the hypothesis that L. heterotoma females employ an ensemble code of parasitization status of the host.

Taste detection of the non-volatile isothiocyanate moringin results in deterrence to glucosinolate-adapted insect larvae

Isothiocyanates (ITCs), released from Brassicales plants after hydrolysis of glucosinolates, are known for their negative effects on herbivores but mechanisms have been elusive. The ITCs are initially present in dissolved form at the site of herbivore feeding, but volatile ITCs may subsequently enter the gas phase and all ITCs may react with matrix components. Deterrence to herbivores resulting from topically applied volatile ITCs in artificial feeding assays may hence lead to ambiguous conclusions. In the present study, the non-volatile ITC moringin (4-(α-l-rhamnopyranosyloxy)benzyl ITC) and its glucosinolate precursor glucomoringin were examined for effects on behaviour and taste physiology of specialist insect herbivores of Brassicales. In feeding bioassays, glucomoringin was not deterrent to larvae of Pieris napi (Lepidoptera: Pieridae) and Athalia rosae (Hymenoptera: Tenthredinidae), which are adapted to glucosinolates. Glucomoringin stimulated feeding of larvae of the related Pieris brassicae (Lepidoptera: Pieridae) and also elicited electrophysiological activity from a glucosinolate-sensitive gustatory neuron in the lateral maxillary taste sensilla. In contrast, the ITC moringin was deterrent to P. napi and P. brassicae at high levels and to A. rosae at both high and low levels when topically applied to cabbage leaf discs (either 12, 120 or 1200nmol moringin per leaf disc of 1cm diameter). Survival of A. rosae was also significantly reduced when larvae were kept on leaves treated with moringin for several days. Furthermore, moringin elicited electrophysiological activity in a deterrent-sensitive neuron in the medial maxillary taste sensillum of P. brassicae, providing a sensory mechanism for the deterrence and the first known ITC taste response of an insect. In simulated feeding assays, recovery of moringin was high, in accordance with its non-volatile nature. Our results demonstrate taste-mediated deterrence of a non-volatile, natural ITC to glucosinolate-adapted insects.

The Interactions between Bitter and Sweet Taste Processing in Drosophila.

In Drosophila , the detection of sweet and bitter compounds is mediated, for the most part, by distinct gustatory receptors (Grs) expressed in sweet and bitter gustatory receptor neurons (GRNs), respectively. The GRNs are intermixed within hair-like structures called taste sensilla that are

Antennal gustatory perception and behavioural responses in Trissolcus brochymenae females

Trissolcus brochymenae (Hymenoptera: Platygastridae) is an egg parasitoid that could be used to control stink bugs like Murgantia histrionica (Heteroptera: Pentatomidae), a pest of brassicaceous crops. Before laying their eggs, parasitoid females spend considerable time examining the substrate with their antennae, which are also used during feeding and mating behaviours. This suggests that contact chemoreception plays a prominent role in many aspects of parasitoid ecology. Therefore, we examined the sensitivity of antennal taste sensilla chaetica to several classical tastants including those that are appetitive or aversive. In addition we studied the taste preferences of wasps when presented with these chemicals alone or mixed. The sensilla chaetica of T. brochymenae responded to serial concentrations of sucrose, salts, and quinine, but no concentration-dependent effect was observed when testing sinigrin, a secondary metabolite found in many brassicaceae. However, both sinigrin and quinine inhibited responses to 0.1M sucrose when mixed with this sugar. Behavioural taste preference assays confirmed that wasps showed a dose dependent preference for sucrose over agarose. In addition, a behavioural avoidance of sucrose solutions containing quinine was observed. This effect was not observed when sinigrin was used as a feeding deterrent. In the two-choice tests the wasp did not discriminate between sucrose solutions mixed with salts and sucrose alone. Further no preference for salts or sinigrin compared to agarose alone was observed. This work represents the first step towards the identification of gustatory receptor neurons implicated in the detection of different types of chemical cues in egg parasitoids.

Enhancing Perception of Contaminated Food through Acid-Mediated Modulation of Taste Neuron Responses

Natural foods contain not only nutrients, but also nonnutritious and potentially harmful chemicals. Thus, animals need to evaluate food content in order to make adequate feeding decisions. Here, we investigate the effects of acids on the taste neuron responses and on taste behavior of desirable, nutritious sugars and sugar/bitter compound mixtures in Drosophila melanogaster. Using Ca2+ imaging, we show that acids activate neither sweet nor bitter taste neurons in tarsal taste sensilla. However, they suppress responses to bitter compounds in bitter-sensing neurons. Moreover, acids reverse suppression of bitter compounds exerted on sweet-sensing neurons. Consistent with these observations, behavioral analyses show that bitter-compound-mediated inhibition on feeding behavior is alleviated by acids. To investigate the cellular mechanism by which acids modulate these effects, we silenced bitter-sensing gustatory neurons. Surprisingly, this intervention had little effect on acid-mediated derepression of sweet neuron or feeding responses to either sugar/bitter compound mixtures or sugar/bitter compound/acid mixtures, suggesting that there are two independent pathways by which bitter compounds are sensed. Our investigations reveal that acids, when presented in dietary relevant concentrations, enhance the perception of sugar/bitter compound mixtures. Drosophila's natural food sources-fruits and cohabitating yeast-are rich in sugars and acids but are rapidly colonized by microorganisms, such as fungi, protozoan parasites, and bacteria, many of which produce bitter compounds. We propose that the acids present in most fruits counteract the inhibitory effects of these bitter compounds during feeding.

Hygienic grooming is induced by contact chemicals in Drosophila melanogaster.

In social insects, grooming is considered as a behavioral defense against pathogen and parasite infections since it contributes to remove microbes from their cuticle. However, stimuli which trigger this behavior are not well characterized yet. We examined if activating contact chemoreceptive sensilla could trigger grooming activities in Drosophila melanogaster. We monitored the grooming responses of decapitated flies to compounds known to activate the immune system, e.g., dead Escherichia coli (Ec) and lipopolysaccharides (LPS), and to tastants such as quinine, sucrose, and salt. LPS, quinine, and Ec were quite effective in triggering grooming movements when touching the distal border of the wings and the legs, while sucrose had no effect. Contact chemoreceptors are necessary and sufficient to elicit such responses, as grooming could not be elicited by LPS in poxn mutants deprived of external taste sensilla, and as grooming was elicited by light when a channel rhodopsin receptor was expressed in bitter-sensitive cells expressing Gr33a. Contact chemoreceptors distributed along the distal border of the wings respond to these tastants by an increased spiking activity, in response to quinine, Ec, LPS, sucrose, and KCl. These results demonstrate for the first time that bacterial compounds trigger grooming activities in D. melanogaster, and indicate that contact chemoreceptors located on the wings participate in the detection of such chemicals.

Gustatory sensitivity and food acceptance in two phylogenetically closely related papilionid species: Papilio hospiton and Papilio machaon.

In herbivorous insects, food selection depends on sensitivity to specific chemical stimuli from host-plants as well as to secondary metabolites (bitter) and to sugars (phagostimulatory). Bitter compounds are noxious, unpalatable or both and evoke an aversive feeding response. Instead, sugars and sugar alcohols play a critical role in determining and enhancing the palatability of foods. We assumed that peripheral taste sensitivity may be related to the width of the host selection. Our model consists of two closely phylogenetically related Papilionid species exhibiting a difference in host plant choice: Papilio hospiton and Papilio machaon. The spike activity of the lateral and medial maxillary styloconic taste sensilla was recorded following stimulation with several carbohydrates, nicotine and NaCl, with the aim of characterizing their gustatory receptor neurons and of comparing their response patterns in the light of their different acceptability in feeding behaviour. The results show that: a) each sensillum houses phagostimulant and phagodeterrent cells; b) the spike activity of the gustatory neurons in response to different taste stimuli is higher in P. hospiton than in P. machaon; c) sugar solutions inhibit the spike activity of the deterrent and salt cells, and the suppression is higher in P. machaon than in P. hospiton. In conclusion, we propose that the different balance between the phagostimulant and phagodeterrent inputs from GRNs of maxillary sensilla may contribute in determining the difference in food choice and host range.

The molecular and cellular basis of taste coding in the legs of Drosophila.

To understand the principles of taste coding, it is necessary to understand the functional organization of the taste organs. Although the labellum of the Drosophila melanogaster head has been described in detail, the tarsal segments of the legs, which collectively contain more taste sensilla than the labellum, have received much less attention. We performed a systematic anatomical, physiological, and molecular analysis of the tarsal sensilla of Drosophila. We construct an anatomical map of all five tarsal segments of each female leg. The taste sensilla of the female foreleg are systematically tested with a panel of 40 diverse compounds, yielding a response matrix of ∼500 sensillum-tastant combinations. Six types of sensilla are characterized. One type was tuned remarkably broadly: it responded to 19 of 27 bitter compounds tested, as well as sugars; another type responded to neither. The midleg is similar but distinct from the foreleg. The response specificities of the tarsal sensilla differ from those of the labellum, as do n-dimensional taste spaces constructed for each organ, enhancing the capacity of the fly to encode and respond to gustatory information. We examined the expression patterns of all 68 gustatory receptors (Grs). A total of 28 Gr-GAL4 drivers are expressed in the legs. We constructed a receptor-to-sensillum map of the legs and a receptor-to-neuron map. Fourteen Gr-GAL4 drivers are expressed uniquely in the bitter-sensing neuron of the sensillum that is tuned exceptionally broadly. Integration of the molecular and physiological maps provides insight into the underlying basis of taste coding.

Function and central projections of gustatory receptor neurons on the antenna of the noctuid moth Spodoptera littoralis

Chemosensory information is crucial for most insects to feed and reproduce. Olfactory signals are mainly used at a distance, whereas gustatory stimuli play an important role when insects directly contact chemical substrates. In noctuid moths, although the antennae are the main olfactory organ, they also bear taste sensilla. These taste sensilla detect sugars and hence are involved in appetitive learning but could also play an important role in food evaluation by detecting salts and bitter substances. To investigate this, we measured the responses of individual taste sensilla on the antennae of Spodoptera littoralis to sugars and salts using tip recordings. We also traced the projections of their neuronal axons into the brain. In each sensillum, we found one or two neurons responding to sugars: one NaCl-responsive and one water-sensitive neuron. Responses of these neurons were dose-dependent and similar across different locations on the antenna. Responses were dependent on the sex for sucrose and on both sex and location for glucose and fructose. We did not observe a spatial map for the projections from specific regions of the antennae to the deutocerebrum or the tritocerebrum/suboesophageal ganglion complex. In accordance with physiological recordings, back-fills from individual sensilla revealed up to four axons, in most cases targeting different projection zones.

Identification of a Drosophila Glucose Receptor Using Ca2+ Imaging of Single Chemosensory Neurons

Evaluation of food compounds by chemosensory cells is essential for animals to make appropriate feeding decisions. In the fruit fly Drosophila melanogaster, structurally diverse chemicals are detected by multimeric receptors composed of members of a large family of Gustatory receptor (Gr) proteins. Putative sugar and bitter receptors are expressed in distinct subsets of Gustatory Receptor Neurons (GRN) of taste sensilla, thereby assigning distinct taste qualities to sugars and bitter tasting compounds, respectively. Here we report a Ca2+ imaging method that allows association of ligand-mediated responses to a single GRN. We find that different sweet neurons exhibit distinct response profiles when stimulated with various sugars, and likewise, different bitter neurons exhibit distinct response profiles when stimulated with a set of bitter chemicals. These observations suggest that individual neurons within a taste modality are represented by distinct repertoires of sweet and bitter taste receptors, respectively. Furthermore, we employed this novel method to identify glucose as the primary ligand for the sugar receptor Gr61a, which is not only expressed in sweet sensing neurons of classical chemosensory sensilla, but also in two supersensitive neurons of atypical taste sensilla. Thus, single cell Ca2+ imaging can be employed as a powerful tool to identify ligands for orphan Gr proteins.

Functional structure of antennal sensilla in the myrmecophilous beetle Paussus favieri (Coleoptera, Carabidae, Paussini)

The evolution of a myrmecophilous lifestyle in beetles is often associated with morphological alterations. In particular, the antennae of all members of the myrmecophilous ground beetle tribe Paussini are greatly modified, with flagellomeres flattened or crassate, frequently reduced in number from 9 to 5 or even 1 single “antennal club”. The enhanced glandular function of the antennal club has been recently described by scanning (SEM) and transmission (TEM) electron microscopy in Paussus favieri Fairmaire, 1851, where the antenna has become a complex glandular organ, supplying rewarding substances to the ants. In the present work, the antennal sensilla of P. favieri are investigated by SEM, TEM and focused ion beam (FIB/SEM) technology. Most sensilla of scape and antennal club are highly modified mechanoreceptors (i.e. multipointed, fringed, branched, brush-like, sickle-shaped), singly or grouped in tufts (“antennal symphilous organs”). These “trichomes”, here assigned to 8 different morphotypes of sensilla chaetica (Ch.1–Ch.8), show a variable number of basal pores (present also at the base of the taste sensilla Ch.9), which spread dense substances of unknown chemical composition on the seta. Although hygro-, thermo- and chemoreceptors are reduced in number as compared with non-myrmecophilous relatives, and mainly relegated to the apex of the antennal club, their diversity is comparable to that of other carabid beetles: two types of sensilla trichodea (Tr.1–Tr.2); three types of basiconica (Ba.1–Ba.3); one type of campaniformia (Ca); one type of coeloconica (Co) and one type of Böhm sensilla (Bo). Contrary to the hypothesis that Paussus species lack a Johston's organ, a non-connective chordotonal organ composed of 9 groups of scolopidia has been found inside the pedicel. A comparison between sensilla of P. favieri and those of other non-myrmecophilous and myrmecophilous ground beetle species is provided.

Conserved <i>cis</i>-regulatory elements of two odorant-binding protein genes, <i>Obp57d</i> and <i>Obp57e</i>, in <i>Drosophila</i>

Insect odorant-binding proteins function in the sensing of odors, tastes, and pheromones. Genes encoding two odorant-binding proteins, Obp57d and Obp57e, were identified to be involved in the behavioral adaptation of Drosophila sechellia to its host plant. The two genes are expressed in cells associated with taste sensilla on the legs, and the expression pattern in the legs is conserved among closely related species. To identify the cis-regulatory elements necessary for the expression in the leg sensilla, the promoter sequences of Obp57d and Obp57e were compared among species. Two types of conserved sequence-motifs were found as candidate cis-regulatory elements. Functions of these conserved elements in the promoters of D. melanogaster Obp57d and Obp57e were examined by using a newly constructed vector that combines the advantages of φC31 integrase-based transformation and gypsy transposable-element-derived insulators. By GFP-reporter assay using the new vector, it was confirmed that these conserved elements are necessary for the expression in the legs, working synergistically with each other to affect the expression level. Single-nucleotide substitutions in these elements dramatically changed the promoter activity. These results provide insight into the molecular mechanism for evolution of adaptive behavior via modulation of OBP expression levels.

Electrophysiological responses of the chemoreceptor neurones in the antennal taste sensilla to plant alkaloids and glucosides in a granivorous ground beetle

The electrophysiological response of chemoreceptor neurones from the antennal chaetoid taste sensilla of the omnivorous ground beetle Pterostichus oblongopunctatus to several plant alkaloids and glucosides is investigated. A quinine‐sensitive neurone responding to quinine and quinine hydrochloride is found, most probably related to the granivorous feeding habit of P. oblongopunctatus. The response to quinine hydrochloride is concentration‐dependent at 0.001–50 mm, with the response threshold at 0.01 mm and a maximum rate of firing of 67 spikes/s at 50 mm. The stimulatory effect of caffeine is very weak, where the firing rate increases by only 1.4 spikes/s at a concentration of 10 mm compared with that evoked by a control stimulus. In addition, both quinine and quinine hydrochloride strongly inhibit spike production by the salt‐ and pH‐sensitive neurones when presented in mixtures with 10 mm NaCl. Several tested plant secondary compounds (i.e. salicin, sinigrin, caffeine and nicotine), which have only little or no effect on the firing rate of the quinine‐sensitive neurone, greatly reduce the responses of the salt‐ and pH‐sensitive neurones. The results of the present study suggest that the antennal taste sensilla of P. oblongopunctatus may detect plant defensive compounds both through the activation of a quinine‐sensitive neurone and via peripheral inhibition of other chemoreceptor neurones of the taste sensillum.

A gustatory receptor involved in host plant recognition for oviposition of a swallowtail butterfly

Swallowtail butterflies belonging to the family of Papilionidae selectively utilize a limited number of plants from a single or a few families. Female butterflies lay eggs on their host only when they detect specific chemicals through their foreleg chemosensilla while drumming on the leaf surface. Here we show that the butterfly, Papilio xuthus, uses a gustatory receptor specific for synephrine to select its host in oviposition behaviour. We identify a gustatory receptor gene involved in the recognition of an oviposition stimulant, synephrine, from the P. xuthus by a combination of in silico, in vitro and in vivo approaches. The receptor, PxutGr1, responds specifically to synephrine in Sf9 cells. The sensitivity of tarsal taste sensilla to synephrine and the oviposition behaviour in response to synephrine are strongly reduced after injecting double-stranded RNA of PxutGr1 into pupae. These observations indicate that the receptor PxutGr1 represents a key factor in host specialization in P. xuthus.

Electrophysiological Recording from Drosophila Taste Sensilla

INTRODUCTIONThe chemical senses smell and taste, detect and discriminate an enormous diversity of environmental stimuli and provide fascinating but challenging models to investigate how sensory cues are represented in the brain. Important stimulus-coding events occur in peripheral sensory neurons, which express specific combinations of chemosensory receptors with defined ligand-response profiles. These receptors convert ligand recognition into spatial and temporal patterns of neural activity that are transmitted to and interpreted in central brain regions. Drosophila provides an attractive model to study chemosensory coding, because it possesses relatively simple peripheral olfactory and gustatory systems that display many organizational parallels to those of vertebrates. Moreover, virtually all of the peripheral chemosensory neurons are easily accessible for physiological analysis, as they are exposed on the surface of sensory organs in specialized sensory hairs called sensilla. In recent years, improvements in microscopy and instrumentation for electrode manipulation have opened up the much smaller Drosophila system to electrophysiological techniques, powerfully complementing many years of molecular genetic studies. As with most electrophysiological methods, there is probably no substitute for learning this technique directly from a laboratory in which it is already established. This protocol describes the basics of setting up the electrophysiology rig and stimulus delivery device, sample preparation, and performing and analyzing recordings of stimulus-evoked activity from Drosophila taste sensilla.

Drosophila Evaluates and Learns the Nutritional Value of Sugars

Living organisms need to search for and ingest nutritional chemicals, and gustation plays a major role in detecting and discriminating between chemicals present in the environment. Using Drosophila as a model organism, we asked whether animals have the ability to evaluate the nutritional value of sugars. In flies, chemosensilla on the tarsi and labellum are the gustatory organs used to discriminate between edible and nonedible compounds [1, 2]. We noticed that Drosophila do not assign nutritional values to all sweet chemicals. D-arabinose is sweet to flies, but it provides them with no nutrition. By contrast, the sugar alcohol D-sorbitol is not sensed as sweet, but flies can live on it. We performed behavioral and electrophysiological measurements to confirm these gustatory and feeding responses. We found that Drosophila can learn the nutritional value of nonsweet D-sorbitol when it is associated with an odor cue. The learning process involved the synapsin molecule, suggesting that a neuronal mechanism is involved. We propose that Drosophila uses neural machinery to detect, evaluate, and learn the nutritional value of foods after ingestion.

Taste receptor activity and feeding behaviour reveal mechanisms of white spruce natural resistance to Eastern spruce budworm Choristoneura fumiferana

The pattern of feeding of Eastern spruce budworm Choristoneura fumiferana (Clem.) (Lepidoptera, Tortricidae) is compared on foliage from white spruce Picea glauca (Moench) Voss. (Pinaceae) trees previously determined to be susceptible and resistant to defoliation by budworm. No differences are observed in electrophysiological responses from taste sensilla to aqueous extracts of the two foliage types, nor is there a preference for either extract type in a choice test. Acetone extracts from the two foliage types are both preferred to a control sucrose solution, although neither elicits a preference relative to the other. These results suggest that there is no difference in phagostimulatory power of internal leaf contents of the two foliage types. Longer‐term observation of feeding behaviour in a no‐choice situation shows no difference in meal duration, confirming the lack of difference in phagostimulatory power. However, on average, intermeal intervals are twice as long on the resistant foliage, leading to an overall lower food consumption during the assay. This result suggests an anti‐digestive or toxic effect of the resistant foliage that slows behaviour and limits food intake. Previous research has shown that waxes of the resistant foliage deter initiation of feeding by the spruce budworm and that this foliage contains higher levels of tannins and monoterpenes. The data suggest that the resistant foliage contains a post‐ingestive second line of defence against the spruce budworm.

Avoiding DEET through Insect Gustatory Receptors

DEET is the most widely used insect repellent worldwide. In Drosophila olfactory receptor neurons (ORNs), DEET is detected through a mechanism employing the olfactory receptor, OR83b. However, it is controversial as to whether ORNs respond directly to DEET or whether DEET blocks the response to attractive odors. Here, we showed that DEET suppressed feeding behavior in Drosophila, and this effect was mediated by gustatory receptor neurons (GRNs). DEET was potent in suppressing feeding as <0.1% DEET elicited aversive behavior. Inhibition of feeding required multiple gustatory receptors (GRs) expressed in inhibitory GRNs. DEET stimulated action potentials in GRNs that respond to aversive compounds, and this response was lost in the Gr32a, Gr33a, and Gr66a mutants. Since 0.02% DEET elicited action potentials, we conclude that DEET directly activates of GRNs. We suggest that the effectiveness of DEET in pest control owes to its dual action in inducing avoidance simultaneously via GRNs and ORNs.