Antenna-Biased Odorant Receptor PstrOR17 Mediates Attraction of Phyllotreta striolata to (S)-Cis-Verbenol and (-)-Verbenone.

Phyllotreta striolata, the striped flea beetle, is one of the most destructive pests in Brassicaceae plants worldwide. Given the drawbacks associated with long-term use of chemical insecticides, green strategies based on chemical ecology are an effective alternative for beetle control. However, the lack of information on beetle ecology has hindered the development of effective biocontrol strategies. In this report, we identified two odorants, (S)-cis-verbenol and (-)-verbenone, which displayed significant attraction for P. striolata (p < 0.05), indicating their great potential for P. striolata management. Using the Drosophila "empty neuron" system, an antenna-biased odorant receptor, PstrOR17, was identified as responsible for the detection of (-)-verbenone and (S)-cis-verbenol. Furthermore, the interactions between PstrOR17 and (-)-verbenone or (S)-cis-verbenol were predicted via modeling and molecular docking. Finally, we used RNAi to confirm that PstrOR17 is essential for the detection of (-)-verbenone and (S)-cis-verbenol to elicit an attraction effect. Our results not only lay a foundation for the development of new and effective nonchemical insecticide strategies based on (S)-cis-verbenol and (-)-verbenone, but also provide new insight into the molecular basis of odorant recognition in P. striolata.

Dynamic antennal positioning allows honeybee followers to decode the dance.

The honeybee waggle dance has been widely studied as a communication system, yet we know little about how nestmates assimilate the information needed to navigate toward the signaled resource. They are required to detect the dancer's orientation relative to gravity and duration of the waggle phase and translate this into a flight vector with a direction relative to the sun1 and distance from the hive.2,3 Moreover, they appear capable of doing so from varied, dynamically changing positions around the dancer. Using high-speed, high-resolution video, we have uncovered a previously unremarked correlation between antennal position and the relative body axes of dancer and follower bees. Combined with new information about antennal inputs4,5 and spatial encoding in the insect central complex,6,7 we show how a neural circuit first proposed to underlie path integration could be adapted to decoding the dance and acquiring the signaled information as a flight vector that can be followed to the resource. This provides the first plausible account of how the bee brain could support the interpretation of its dance language.

Neuromorphic antennal sensory system.

Insect antennae facilitate the nuanced detection of vibrations and deflections, and the non-contact perception of magnetic or chemical stimuli, capabilities not found in mammalian skin. Here, we report a neuromorphic antennal sensory system that emulates the structural, functional, and neuronal characteristics of ant antennae. Our system comprises electronic antennae sensor with three-dimensional flexible structures that detects tactile and magnetic stimuli. The integration of artificial synaptic devices adsorbed with solution-processable MoS2 nanoflakes enables synaptic processing of sensory information. By emulating the architecture of receptor-neuron pathway, our system realizes hardware-level, spatiotemporal perception of tactile contact, surface pattern, and magnetic field (detection limits: 1.3 mN, 50 µm, 9.4 mT). Vibrotactile-perception tasks involving profile and texture classifications were accomplished with high accuracy (> 90%), surpassing human performance in "blind" tactile explorations. Magneto-perception tasks including magnetic navigation and touchless interaction were successfully completed. Our work represents a milestone for neuromorphic sensory systems and biomimetic perceptual intelligence.

Sensory pathway in aquatic basal polyneoptera: Antennal sensilla and brain morphology in stoneflies.

Aquatic insects represent a great portion of Arthropod diversity and the major fauna in inland waters. The sensory biology and neuroanatomy of these insects are, however, poorly investigated. This research aims to describe the antennal sensilla of nymphs of the stonefly Dinocras cephalotes using scanning electron microscopy and comparing them with the adult sensilla. Besides, central antennal pathways in nymphs and adults are investigated by neuron mass-tracing with tetramethylrhodamine, and their brain structures are visualized with an anti-synapsin antibody. No dramatic changes occur in the antennal sensilla during nymphal development, while antennal sensilla profoundly change from nymphs to adults when switching from an aquatic to an aerial lifestyle. However, similar brain structures are used in nymphs and adults to process diverging sensory information, perceived through different sensilla in water and air. These data provide valuable insights into the evolution of aquatic heterometabolous insects, maintaining a functional sensory system throughout development, including a distinct adaptation of the peripheral olfactory systems during the transition from detection of water-soluble chemicals to volatile compounds in the air. From a conservation biology perspective, the present data contribute to a better knowledge of the biology of stoneflies, which are very important bioindicators in rivers.

Triple electroantennography captures the range and spatial arrangement of olfactory sensory neuron response on an insect antenna.

BACKGROUND: Electroantennography (EAG) is a basic neuroscientific tool that is widely used to measure olfactory responses in insects. It is particularly adapted to probing the olfactory systems of non-model insect species in chemical ecology and evolutionary biology. As currently practiced, EAG measures have varying degrees of correlation with olfactory responses, especially for insects whose olfactory sensory neurons (OSNs) are arranged in zones on the antennae. This limitation was shown to be partly due to the fact that only a single antennal position was recorded. NEW METHODS: We describe a setup using triple electroantennography (EAG3), whereby three antennal positions are recorded simultaneously. The spatial arrangement of the electrodes ensures the mechanical stability of the assembly. The EAG3 detector was coupled to a gas chromatograph (GC-EAD3), customized using a Dean's switch to improve the EAG signals by chopper modulation. EAG3 signals were analysed through a current point model to estimate olfactory responses across the antenna. RESULTS: Recordings were performed on Tephritidae and Drosophila species, which have antennae of different shapes and sizes. We confirmed that the spatio-temporal pattern of antennal activation was stimulus dependent and allowed us to quantify the antennal olfactory response. COMPARISONS WITH EXISTING METHOD: Compared to typical single-probe EAG, we show that EAG3 improves response quantification and increases the range of compounds for which a sensory response is detected. CONCLUSIONS: Our EAG3 setup is an original low-cost and easy-to-use method. It offers a useful bridge between comprehensive neurophysiological investigations and the broader themes explored in chemical ecology.

Active antennal movements in Drosophila can tune wind encoding.

Insects use their antennae to smell odors,1,2 detect auditory cues,3,4 and sense mechanosensory stimuli such as wind5 and objects,6,7,8 frequently by combining sensory processing with active movements. Genetic access to antennal motor systems would therefore provide a powerful tool for dissecting the circuit mechanisms underlying active sensing, but little is known about how the most genetically tractable insect, Drosophila melanogaster, moves its antennae. Here, we use deep learning to measure how tethered Drosophila move their antennae in the presence of sensory stimuli and identify genetic reagents for controlling antennal movement. We find that flies perform both slow adaptive movements and fast flicking movements in response to wind-induced deflections, but not the attractive odor apple cider vinegar. Next, we describe four muscles in the first antennal segment that control antennal movements and identify genetic driver lines that provide access to two groups of antennal motor neurons and an antennal muscle. Through optogenetic inactivation, we provide evidence that antennal motor neurons contribute to active movements with different time courses. Finally, we show that activation of antennal motor neurons and muscles can adjust the gain and acuity of wind direction encoding by antennal displacement. Together, our experiments provide insight into the neural control of antennal movement and suggest that active antennal positioning in Drosophila may tune the precision of wind encoding.

Behavioural function and development of body-to-limb proportions and active movement ranges in three stick insect species.

Overall body proportions and relative limb length are highly characteristic for most insect taxa. In case of the legs, limb length has mostly been discussed with regard to parameters of locomotor performance and, in particular cases, as an adaptation to environmental factors or to the mating system. Here, we compare three species of stick and leaf insects (Phasmatodea) that differ strongly in the length ratio between antennae and walking legs, with the antennae of Medauroidea extradentata being much shorter than its legs, nearly equal length of antennae and legs in Carausius morosus, and considerably longer antennae than front legs in Aretaon asperrimus. We show that that relative limb length is directly related to the near-range exploration effort, with complementary function of the antennae and front legs irrespective of their length ratio. Assuming that these inter-species differences hold for both sexes and all developmental stages, we further explore how relative limb length differs between sexes and how it changes throughout postembryonic development. We show that the pattern of limb-to-body proportions is species-characteristic despite sexual dimorphism, and find that the change in sexual dimorphism is strongest during the last two moults. Finally, we show that antennal growth rate is consistently higher than that of front legs, but differs categorically between the species investigated. Whereas antennal growth rate is constant in Carausius, the antennae grow exponentially in Medauroidea and with a sudden boost during the last moult in Aretaon.

Odour motion sensing enhances navigation of complex plumes.

Odour plumes in the wild are spatially complex and rapidly fluctuating structures carried by turbulent airflows1-4. To successfully navigate plumes in search of food and mates, insects must extract and integrate multiple features of the odour signal, including odour identity5, intensity6 and timing6-12. Effective navigation requires balancing these multiple streams of olfactory information and integrating them with other sensory inputs, including mechanosensory and visual cues9,12,13. Studies dating back a century have indicated that, of these many sensory inputs, the wind provides the main directional cue in turbulent plumes, leading to the longstanding model of insect odour navigation as odour-elicited upwind motion6,8-12,14,15. Here we show that Drosophila melanogaster shape their navigational decisions using an additional directional cue-the direction of motion of odours-which they detect using temporal correlations in the odour signal between their two antennae. Using a high-resolution virtual-reality paradigm to deliver spatiotemporally complex fictive odours to freely walking flies, we demonstrate that such odour-direction sensing involves algorithms analogous to those in visual-direction sensing16. Combining simulations, theory and experiments, we show that odour motion contains valuable directional information that is absent from the airflow alone, and that both Drosophila and virtual agents are aided by that information in navigating naturalistic plumes. The generality of our findings suggests that odour-direction sensing may exist throughout the animal kingdom and could improve olfactory robot navigation in uncertain environments.

Insect antennae: Coupling blood pressure with cuticle deformation to control movement.

Insect antennae are hollow, blood-filled fibers with complex shape. Muscles in the two basal segments control antennal movement, but the rest (flagellum) is muscle-free. The insect can controllably flex, twist, and maneuver its antennae laterally. To explain this behavior, we performed a comparative study of structural and tensile properties of the antennae of Periplaneta americana (American cockroach), Manduca sexta (Carolina hawkmoth), and Vanessa cardui (painted lady butterfly). These antennae demonstrate a range of distinguishable tensile properties, responding either as brittle or strain-adaptive fibers that stiffen when stretched. Scanning electron microscopy and high-speed imaging of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation in antennae. A generalized Lamé theory of solid mechanics was developed to include the force-driven deformation of blood-filled antennal tubes. We validated the theory against experiments with artificial antennae with no adjustable parameters. Blood pressure increased when the insect inflated its antennae or decreased below ambient pressure when an external tensile load was applied to the antenna. The pressure-cuticle coupling can be controlled through changes of the blood volume in the antennal lumen. In insects that do not fill the antennal lumen with blood, this blood pressure control is lacking, and the antennae react only by muscular activation. We suggest that the principles we have discovered for insect antennae apply to other appendages that share a leg-derived ancestry. Our work offers promising new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the same fluid on demand. STATEMENT OF SIGNIFICANCE: Insect antennae are blood-filled, segmented fibers with muscles in the two basal segments. The long terminal segment is muscle-free but can be flexed. To explain this behavior, we examined structure-function relationships of antennae of cockroaches, hawkmoths, and butterflies. Hawkmoth antennae behaved as brittle fibers, but butterfly and cockroach antennae showed strain-adaptive behavior like fibers that stiffen when stretched. Videomicroscopy of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation. Our solid mechanics model explains this behavior. Because antennae are leg-derived appendages, we suggest that the principles we found apply to other appendages of leg-derived ancestry. Our work offers new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the fluid on demand.

Spatial perception mediated by insect antennal mechanosensory system.

Animals perceive their surroundings using various modalities of sensory inputs to guide their locomotion. Nocturnal insects such as crickets use mechanosensory inputs mediated by their antennae to orient in darkness. Spatial information is acquired via voluntary antennal contacts with surrounding objects, but it remains unclear whether the insects modulate behaviors mediated by other sensory organs based on that information. Crickets exhibit escape behavior in response to a short air puff, which is detected by the abdominal mechanosensory organs called cerci and is perceived as a 'predator approach' signal. We placed objects of different shapes at different locations with which the cricket actively made contact using its antennae. We then examined the effects on wind-elicited escape behavior. The crickets changed their movement trajectory in response to nearby objects such as walls so that they could avoid collision with these obstacles even during the cercal-mediated behavior. For instance, when a wall was placed in front of the crickets so that it was detected by one antenna, the escape trajectory in response to a stimulus from behind was significantly biased toward the side opposite the wall. Even when the antenna on the free side without the wall was ablated, this collision avoidance was also observed, suggesting that the mechanosensory inputs from one antennae detecting an object edge would be sufficient to perceive the location of obstacle in front. This study demonstrated that crickets were able to use the spatial information acquired with their antennal system to modify their behavior mediated by other sensory organs.

Non-synaptic interactions between olfactory receptor neurons, a possible key feature of odor processing in flies.

When flies explore their environment, they encounter odors in complex, highly intermittent plumes. To navigate a plume and, for example, find food, they must solve several challenges, including reliably identifying mixtures of odorants and their intensities, and discriminating odorant mixtures emanating from a single source from odorants emitted from separate sources and just mixing in the air. Lateral inhibition in the antennal lobe is commonly understood to help solving these challenges. With a computational model of the Drosophila olfactory system, we analyze the utility of an alternative mechanism for solving them: Non-synaptic ("ephaptic") interactions (NSIs) between olfactory receptor neurons that are stereotypically co-housed in the same sensilla. We find that NSIs improve mixture ratio detection and plume structure sensing and do so more efficiently than the traditionally considered mechanism of lateral inhibition in the antennal lobe. The best performance is achieved when both mechanisms work in synergy. However, we also found that NSIs decrease the dynamic range of co-housed ORNs, especially when they have similar sensitivity to an odorant. These results shed light, from a functional perspective, on the role of NSIs, which are normally avoided between neurons, for instance by myelination.

Experience-dependent plasticity modulates ongoing activity in the antennal lobe and enhances odor representations.

Ongoing neural activity has been observed across several brain regions and is thought to reflect the internal state of the brain. Yet, it is important to understand how ongoing neural activity interacts with sensory experience and shapes sensory representations. Here, we show that the projection neurons of the fruit fly antennal lobe exhibit spatiotemporally organized ongoing activity. After repeated exposure to odors, we observe a gradual and cumulative decrease in the amplitude and number of calcium events occurring in the absence of odor stimulation, as well as a reorganization of correlations between olfactory glomeruli. Accompanying these plastic changes, we find that repeated odor experience decreases trial-to-trial variability and enhances the specificity of odor representations. Our results reveal an odor-experience-dependent modulation of ongoing and sensory-evoked activity at peripheral levels of the fruit fly olfactory system.

Multisensory-motor integration in olfactory navigation of silkmoth, Bombyx mori, using virtual reality system.

Most animals survive and thrive due to navigational behavior to reach their destinations. In order to navigate, it is important for animals to integrate information obtained from multisensory inputs and use that information to modulate their behavior. In this study, by using a virtual reality (VR) system for an insect, we investigated how the adult silkmoth integrates visual and wind direction information during female search behavior (olfactory behavior). According to the behavioral experiments using a VR system, the silkmoth had the highest navigational success rate when odor, vision, and wind information were correctly provided. However, the success rate of the search was reduced if the wind direction information provided was different from the direction actually detected. This indicates that it is important to acquire not only odor information but also wind direction information correctly. When the wind is received from the same direction as the odor, the silkmoth takes positive behavior; if the odor is detected but the wind direction is not in the same direction as the odor, the silkmoth behaves more carefully. This corresponds to a modulation of behavior according to the degree of complexity (turbulence) of the environment. We mathematically modeled the modulation of behavior using multisensory information and evaluated it using simulations. The mathematical model not only succeeded in reproducing the actual silkmoth search behavior but also improved the search success relative to the conventional odor-source search algorithm.

Interspecific variation of antennal lobe composition among four hornet species.

Olfaction is a crucial sensory modality underlying foraging, social and mating behaviors in many insects. Since the olfactory system is at the interface between the animal and its environment, it receives strong evolutionary pressures that promote neuronal adaptations and phenotypic variations across species. Hornets are large eusocial predatory wasps with a highly developed olfactory system, critical for foraging and intra-specific communication. In their natural range, hornet species display contrasting ecologies and olfactory-based behaviors, which might match to adaptive shifts in their olfactory system. The first olfactory processing center of the insect brain, the antennal lobe, is made of morphological and functional units called glomeruli. Using fluorescent staining, confocal microscopy and 3D reconstructions, we compared antennal lobe structure, glomerular numbers and volumes in four hornet species (Vespa crabro, Vespa velutina, Vespa mandarinia and Vespa orientalis) with marked differences in nesting site preferences and predatory behaviors. Despite a conserved organization of their antennal lobe compartments, glomeruli numbers varied strongly between species, including in a subsystem thought to process intraspecific cuticular signals. Moreover, specific adaptations involving enlarged glomeruli appeared in two species, V. crabro and V. mandarinia, but not in the others. We discuss the possible function of these adaptations based on species-specific behavioral differences.

Behavioral and Antennal Responses of Tribolium confusum to Varronia globosa Essential Oil and Its Main Constituents: Perspective for Their Use as Repellent.

Essential oils of aromatic plants represent an alternative to classical pest control with synthetic chemicals. They are especially promising for the alternative control of stored product pest insects. Here, we tested behavioral and electrophysiological responses of the stored product pest Tribolium confusum, to the essential oil of a Brazilian indigenous plant, Varronia globosa, collected in the Caatinga ecosystem. We analyzed the essential oil by GC-MS, tested the effects of the entire oil and its major components on the behavior of individual beetles in a four-way olfactometer, and investigated responses to these stimuli in electroantennogram recordings (EAG). We could identify 25 constituents in the essential oil of V. globosa, with anethole, caryophyllene and spathulenole as main components. The oil and its main component anethole had repellent effects already at low doses, whereas caryophyllene had only a repellent effect at a high dose. In addition, the essential oil abolished the attractive effect of the T. confusum aggregation pheromone. EAG recordings revealed dose-dependent responses to the individual components and increasing responses to the blend and even more to the entire oil. Our study reveals the potential of anethole and the essential oil of V. globosa in the management of stored product pests.

Agonistic experience during development establishes inter-individual differences in approach-avoidance behaviour of crickets.

Members of numerous animal species show consistent inter-individual differences in behaviours, but the forces generating animal "personality" or individuality remain unclear. We show that experiences gathered solely from social conflict can establish consistent differences in the decision of male crickets to approach or avoid a stimulus directed at one antenna. Adults isolated for 48 h from a colony already exhibit behavioural differences. Prior to staging a single dyadic contest, prospective winners approached the stimulus whereas prospective losers turned away, as they did also after fighting. In contrast, adults raised as nymphs with adult males present but isolated from them as last instar nymphs, all showed avoidance. Furthermore, adults raised without prior adult contact, showed no preferred directional response. However, following a single fight, winners from both these groups showed approach and losers avoidance, but this difference lasted only one day. In contrast, after 6 successive wins or defeats, the different directional responses of multiple winners and losers remained consistent for at least 6 days. Correlation analysis revealed examples of consistent inter-individual differences in the direction and magnitude of turning responses, which also correlated with individual aggressiveness and motility. Together our data reveal that social subjugation, or lack thereof, during post-embryonic and early adult development forges individuality and supports the notion of a proactive-reactive syndrome in crickets.

Effects of Mono-2-ethylhexyl Phthalate on the Neural Transmission of PNs in Drosophila Antennal Lobe.

Long-term exposure to different types of chemicals is hazardous to human health. Di(2-ethylhexyl) phthalate (DEHP) could exert pleiotropic deleterious effects on nervous systems. Mono(2-ethylhexyl) phthalate (MEHP), as one of the most toxic metabolites of DEHP, may have similar effects on nervous systems. However, no effects of MEHP on neural circuits have been reported. To uncover the regulation of MEHP on neural transmission, the functional changes of neural excitability and synaptic plasticity of projection neurons (PNs) have been assessed. In the current study, we recorded the action potentials (APs), stimulate action potentials (sti-APs), mini excitement postsynaptic current (mEPSC), calcium currents, and sodium currents from PNs of isolated whole brain of Drosophila model utilizing patch clamp recordings. We found that MEHP-300 (at the concentration of 300 µM), but not MHEP-100 (at the concentration of 100 µM), significantly decreased the frequency and amplitude of APs. Besides, the amplitude and anti-amplitude of sti-APs were reduced with the application of MEHP-300. Meanwhile, MEHP-300 reduced the frequency of mEPSC, but not the amplitude. Furthermore, MEHP-300 reduced the peak current densities of sodium and calcium channels. Therefore, our results indicated that MEHP could alter the neural excitability and synaptic plasticity of PNs by inhibiting the ion channels activities, revealing the potential modulation of MEHP on neural transmission of PNs.

Input of hair field afferents to a descending interneuron.

In insects the tactile sense is important for near-range orientation and is involved in various behaviors. Nocturnal insects, such as the stick insect Carausius morosus, continuously explore their surroundings by actively moving their antennae when walking. Upon antennal contact with objects, stick insects show a targeted front-leg movement. As this reaction occurs within 40 ms, descending transfer of information from the brain to the thorax needs to be fast. So far, a number of descending interneurons have been described that may be involved in this reach-to-grasp behavior. One of these is the contralateral ON-type velocity-sensitive neuron (cONv). cONv was found to encode antennal joint-angle velocity during passive movement. Here, we characterize the transient response properties of cONv, including its dependence on joint angle range and direction. As antennal hair field afferent terminals were shown to arborize close to cONv dendrites, we test whether antennal hair fields contribute to the joint-angle velocity encoding of cONv. To do so, we conducted bilateral extracellular recordings of both cONv interneurons per animal before and after hair field ablations. Our results show that cONv responses are highly transient, with velocity-dependent differences in delay and response magnitude. As yet, the steady state activity level was maintained until the stop of antennal movement, irrespective of movement velocity. Hair field ablation caused a moderate but significant reduction of movement-induced cONv firing rate by up to 40%. We conclude that antennal proprioceptive hair fields contribute to the velocity-tuning of cONv, though further antennal mechanoreceptors must be involved, too.NEW & NOTEWORTHY Active tactile exploration and tactually induced behaviors are important for many animals. They require descending information transfer about tactile sensor movement to thoracic networks. Here, we investigate response properties and afferent input to the identified descending interneuron cONv in stick insects. cONv may be involved in tactually induced reach-to-grasp movements. We show that cONv response delay, transient and steady state are velocity-dependent and that antennal proprioceptive hair fields contribute to the velocity encoding of cONv.

Olfactory coding in the antennal lobe of the bumble bee Bombus terrestris.

Sociality is classified as one of the major transitions in evolution, with the largest number of eusocial species found in the insect order Hymenoptera, including the Apini (honey bees) and the Bombini (bumble bees). Bumble bees and honey bees not only differ in their social organization and foraging strategies, but comparative analyses of their genomes demonstrated that bumble bees have a slightly less diverse family of olfactory receptors than honey bees, suggesting that their olfactory abilities have adapted to different social and/or ecological conditions. However, unfortunately, no precise comparison of olfactory coding has been performed so far between honey bees and bumble bees, and little is known about the rules underlying olfactory coding in the bumble bee brain. In this study, we used in vivo calcium imaging to study olfactory coding of a panel of floral odorants in the antennal lobe of the bumble bee Bombus terrestris. Our results show that odorants induce reproducible neuronal activity in the bumble bee antennal lobe. Each odorant evokes a different glomerular activity pattern revealing this molecule's chemical structure, i.e. its carbon chain length and functional group. In addition, pairwise similarity among odor representations are conserved in bumble bees and honey bees. This study thus suggests that bumble bees, like honey bees, are equipped to respond to odorants according to their chemical features.

Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi.

American trypanosomiasis, or Chagas disease, is transmitted by both domestic and sylvatic species of Triatominae which use sensory cues to locate their vertebrate hosts. Among them, odorants have been shown to play a key role. Previous work revealed morphological differences in the sensory apparatus of different species of Triatomines, but to date a comparative functional study of the olfactory system is lacking. After examining the antennal sensilla with scanning electronic microscopy (SEM), we compared olfactory responses of Rhodnius prolixus and the sylvatic Rhodnius brethesi using an electrophysiological approach. In electroantennogram (EAG) recordings, we first showed that the antenna of R. prolixus is highly responsive to carboxylic acids, compounds found in their habitat and the headspace of their vertebrate hosts. We then compared responses from olfactory sensory neurons (OSNs) housed in the grooved peg sensilla of both species, as these are tuned to these compounds using single-sensillum recordings (SSRs). In R. prolixus, the SSR responses revealed a narrower tuning breath than its sylvatic sibling, with the latter showing responses to a broader range of chemical classes. Additionally, we observed significant differences between these two species in their response to particular volatiles, such as amyl acetate and butyryl chloride. In summary, the closely related, but ecologically differentiated R. prolixus and R. brethesi display distinct differences in their olfactory functions. Considering the ongoing rapid destruction of the natural habitat of sylvatic species and the likely shift towards environments shaped by humans, we expect that our results will contribute to the design of efficient vector control strategies in the future.