Abstract Feeding behavior in blood-sucking insects relies on gustatory evaluation to decide on sustained ingestion, yet quantifying this process from electromyogram (EMG) recordings is labor-intensive. Here we developed MyoRec, an automated computational framework employing machine learning to analyse EMG signals from the triatomine bug Rhodnius prolixus . Using recordings under appetitive and aversive conditions, a convolutional neural network detected ingestion events with 97.7% accuracy. Automated analysis revealed distinct feeding dynamics, with prolonged ingestion and higher pumping frequency under appetitive stimuli, compared to rapid feeding cessation under aversive stimuli. MyoRec substantially reduces analysis time while maintaining accuracy, providing a scalable tool to investigate how gustatory cues modulate feeding decisions in hematophagous insects.

Kissing bugs are known for being skilled blood-feeders on warm-blooded hosts, using cues like carbon dioxide, infrared radiation, and skin odors to locate them. They depend on heat to direct their bites and use gustatory signals for feeding decisions. Recent evidence suggests that they also feed on cold-blooded animals and insects. This implies that they may respond to other sensory cues, such as slight temperature changes, vibrations, and/or odors, to trigger biting attempts. Besides, new studies reveal that triatomines can ingest plant or fruit fluids, showing a broader dietary flexibility than previously recognized. Future research should focus on how triatomines detect and respond to sensory cues from poikilothermic hosts or plant fluids, and how these sensory inputs influence their feeding behavior. Understanding their sensory abilities and dietary flexibility could have important implications for vector surveillance and control, offering insights into the evolutionary shift from predatory to blood-feeding behaviors.

Hematophagous arthropods rely on taste mechanisms to navigate host selection, feeding, mating, and oviposition. These behaviors are driven by environmental taste cues, which shape acceptance or aversion depending on their valence. Positive stimuli, like low concentrations of salts, sugars, amino acids, and nucleotides, promote feeding and oviposition, while negative stimuli, including high salt, bitter compounds, and nociceptive chemicals, trigger avoidance to prevent hazards. Species-specific adaptations enable blood feeders to overcome ecological challenges. Understanding their behavioral, neuronal, and molecular taste mechanisms aids in developing targeted vector control strategies, such as repellents, toxic baits, and oviposition deterrents, to disrupt disease transmission.

Chemical repellents play a crucial role in personal protection, serving as essential elements in reducing the transmission of vector-borne diseases. A biorational perspective that extends beyond the olfactory system as the classical target may be a promising direction to move. The taste system provides reliable information regarding food quality, helping animals to discriminate between nutritious and potentially harmful food sources, often associated with a bitter taste. Understanding how bitter compounds affect feeding in blood-sucking insects could unveil novel molecules with the potential to reduce biting and feeding. Here, we investigated the impact of two naturally occurring bitter compounds, caffeine and quinine, on the feeding decisions in female Aedes aegypti mosquitoes at two distinctive phases: (1) when the mosquito explores the biting substrate using external taste sensors and (2) when the mosquito takes a sip of food and tastes it using internal taste receptors. We assessed the aversiveness of bitter compounds through both an artificial feeding condition (artificial feeder test) and a real host (arm-in-cage test). Our findings revealed different sensitivities in the external and internal sensory pathways responsible for detecting bitter taste in Ae. aegypti. Internal detectors exhibited responsiveness to lower doses compared to the external sensors. Quinine exerted a more pronounced negative impact on biting and feeding activity than caffeine. The implications of our findings are discussed in the context of mosquito food recognition and the potential practical implications for personal protection.

Background Obligate blood-feeding insects obtain the nutrients and water necessary to ensure survival from the vertebrate blood. The internal taste sensilla, situated in the pharynx, evaluate the suitability of the ingested food. Here, through multiple approaches, we characterized the pharyngeal organ (PO) of the hematophagous kissing bug Rhodnius prolixus to determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises eight taste sensilla that become bathed with the incoming blood. Results We showed that these taste sensilla house gustatory receptor neurons projecting their axons through the labral nerves to reach the subesophageal zone in the brain. We found that these neurons are electrically activated by relevant appetitive and aversive gustatory stimuli such as NaCl, ATP, and caffeine. Using RNA-Seq, we examined the expression of sensory-related gene families in the PO. We identified gustatory receptors, ionotropic receptors, transient receptor potential channels, pickpocket channels, opsins, takeouts, neuropeptide precursors, neuropeptide receptors, and biogenic amine receptors. RNA interference assays demonstrated that the salt-related pickpocket channel Rproppk014276 is required during feeding of an appetitive solution of NaCl and ATP. Conclusions We provide evidence of the role of the pharyngeal organ in food evaluation. This work shows a comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

Chemical repellents play a crucial role in personal protection, serving as essential elements in reducing the transmission of vector-borne diseases. A biorational perspective that extends beyond the olfactory system as the classical target may be a promising direction to move. The taste system provides reliable information regarding food quality, helping animals discriminate between nutritious and potentially harmful food sources, often associated with a bitter taste. Understanding how bitter compounds affect feeding in blood-sucking insects could unveil novel molecules with the potential to reduce biting and feeding. Here, we investigated the impact of two naturally occurring bitter compounds, caffeine and quinine, on the feeding decisions in female Aedes aegypti mosquitoes at two distinctive phases: (1) when the mosquito explores the biting substrate using external taste sensors and (2) when the mosquito takes a sip of food and tastes it using internal taste receptors. We assessed the aversiveness of bitter compounds through both an artificial feeding condition (artificial feeder test) and a real host (arm-in-cage test). Our findings revealed different sensitivities in the external and internal sensory pathways responsible for detecting bitter taste in Ae. aegypti . Internal detectors exhibited responsiveness to lower doses compared to the external sensors. Quinine exerted a more pronounced negative impact on biting and feeding activity than caffeine. The implications of our findings are discussed in the context of mosquito food recognition and the potential practical implications for personal protection.

Triatomines are vectors of Chagas disease. Due to failures in their control, there is an urgent need for more efficient and environmentally friendly monitoring and control tools. These hematophagous insects rely heavily on chemical information from the environment to detect hosts and cues/signals from conspecifics. Chemical ecology includes the elucidation of the functional role of chemicals mediating interactions between organisms. Studies on the chemical ecology of triatomines are leading to novel methods for their monitor and control. Thus, laboratory tests to develop chemical attractants and repellents are promissory and have led to the design of, for example, efficient baited traps. However, the monitoring and control tools proposed until now have not been as effective in the field.

Many hematophagous insects acquire medical and veterinary relevance because they transmit disease causing pathogens to humans. Hematophagy is only fulfilled once a blood feeder successfully locates a vertebrate host by means of fine sensory systems. In nature, blood-sucking insects can exploit environments with differential association with their hosts. Given the relevance of the sensory systems during host searching, we review the current state of knowledge of the sensory machinery of four blood-sucking insects: human lice, bed bugs, kissing bugs and mosquitoes. Each one is representative of highly anthropophilic behaviours and a different degree of association with human hosts. We compare the number, arrangement and functional type of cuticular sensory structures dispersed on the main sensory organs. We also compare the genetic machinery potentially involved in the detection of host stimuli. Finally, we discuss the sensory diversity of the insects studied here.

Background Obligate blood-feeding insects obtain the nutrients and water necessary to ensure survival from the vertebrate blood. The internal taste sensilla, situated in the pharynx, evaluate the suitability of the ingested food. Here, through multiple approaches, we characterized the pharyngeal organ (PO) of the hematophagous kissing bug Rhodnius prolixus to determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises 8 taste sensilla that become bathed with the incoming blood. Results We showed that these taste sensilla house gustatory receptor neurons projecting their axons through the labral nerves to reach the subesophageal zone in the brain. We found that these neurons are electrically activated by relevant appetitive and aversive gustatory stimuli such as NaCl, ATP and caffeine. Using RNA-Seq, we examined the expression of sensory-related gene families in the PO. We identified gustatory receptors, ionotropic receptors, transient receptor potential channels, pickpocket channels, opsins, takeouts, neuropeptide precursors, neuropeptide receptors and biogenic amine receptors. RNA interference assays demonstrated that the pickpocket channel Rproppk014276 is necessary for salt detection during feeding. Conclusion We provide evidence of the role of the pharyngeal organ in food evaluation. This work shows the first comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

Salts are essential nutrients required for many physiological processes, and accordingly, their composition and concentration are tightly regulated. Taste is the ultimate sensory modality involved in resource quality assessment, resulting in acceptance or rejection. Here we found that high salt concentrations elicit feeding avoidance in the blood-sucking bug Rhodnius prolixus and elucidate the molecular and neurophysiological mechanisms involved. We found that high-salt avoidance is mediated by a salt-sensitive antennal gustatory receptor neuron (GRN). Using RNAi, we demonstrate that this process requires two amiloride-sensitive pickpocket channels (PPKs; RproPPK014276 and RproPPK28) expressed within these cells. We found that antennal GRNs project to the insect primary olfactory center, the antennal lobes, revealing these centers as potential sites for the integration of taste and olfactory host-derived cues. Moreover, the identification of the gustatory basis of high-salt detection in a hematophagous insect suggests novel targets for the prevention of biting and feeding.

Body and head lice are known to be exclusive ectoparasites of human hosts. Current genomic and transcriptomic data suggest that both louse types represent ecotypes of the same species. They exhibit morphological and physiological differences that probably arose from living in different biotopes. Sensory traits represent suitable candidates to be affected by environmental heterogeneity. Therefore, through scanning electron microscope studies, this study analysed and compared the sensory structures of both ecotypes' antennae. Seven types of sensory structures were identified in both ecotypes: bristles, tuft organs, pore organs, single pore and three morphotypes of sensilla basiconica. Notably, the single pore and the morphotypes of sensilla basiconica were described for the first time in the body louse antenna. This study's comparative analysis mainly revealed size differences across the sensory structures of the ecotypes. Bristles of the flagellomere 2 of the body louse antenna were longer than the head louse bristles. In addition, the pore organs of the head louse antenna presented a higher diameter than those of the body louse. The possible relevance of size differences regarding the biotopes exploited by the body louse and the head louse is discussed. Yet, physiological studies may help to fully understand the phenotypical differences of both ecotypes.

Salts are essential nutrients required for many physiological processes, and deficient or excessive salt results in adverse health problems. Taste is the ultimate sensory modality involved in resource quality assessment, resulting in acceptance or rejection. Here, we show that detection of high-salt substrates by a salt-sensitive antennal gustatory receptor neuron, S1-GRN, results in feeding avoidance in the hematophagous bug Rhodnius prolixus . Knock-down of two antennal-expressed amiloride-sensitive pickpocket channel receptors (PPKs; RproPPK014276 and RproPPK28 ) using RNA interference, prevents avoidance of bugs to high-salt substrates. Tracing antennal GRNs to the central nervous system reveals the antennal lobes as a gustatory processing center. The identification of the gustatory basis of high-salt detection in a blood feeder provides novel targets to prevent biting and feeding, as well as to promote substrate avoidance in a relevant disease vector. Significance Statement Detection of aversive gustatory stimuli induces avoidance responses in animals. Avoidance acquires particular interest if it reduces the biting rates of blood-feeding insects of medical relevance. Here we describe the molecular and physiological basis of high-salt detection in the blood-sucking disease vector Rhodnius prolixus . We show that detection of high-salt substrates through two PPK receptors expressed in an antennal gustatory receptor neuron produces feeding avoidance. Understanding these gustatory-driven aversive responses allows the hitherto overlooked use of gustatory molecules as a complement to known olfactory repellents.

Insect pickpocket (PPK) receptors mediate diverse functions, among them the detection of mechano- and chemo-sensory stimuli. Notwithstanding their relevance, studies on their evolution only focused on Drosophila. We have analyzed the genomes of 26 species of eight orders including holometabolous and hemimetabolous insects (Blattodea, Orthoptera, Hemiptera, Phthiraptera, Hymenoptera, Lepidoptera, Coleoptera, and Diptera), to characterize the evolution of this gene family. PPKs were detected in all genomes analyzed, with 578 genes distributed in seven subfamilies. According to our phylogeny, ppk17 is the most divergent member, composing the new subfamily VII. PPKs evolved under a gene birth-and-death model that generated lineage-specific expansions usually located in clusters, while purifying selection affected several orthogroups. Subfamily V was the largest, including a mosquito-specific expansion that can be considered a new target for pest control. PPKs present a high gene turnover generating considerable variation. On one hand, Musca domestica (59), Aedes albopictus (51), Culex quinquefasciatus (48), and Blattella germanica (41) presented the largest PPK repertoires. On the other hand, Pediculus humanus (only ppk17), bees, and ants (6-9) had the smallest PPK sets. A subset of prevalent PPKs was identified, indicating very conserved functions for these receptors. Finally, at least 20% of the sequences presented calmodulin-binding motifs, suggesting that these PPKs may amplify sensory responses similarly as proposed for Drosophila melanogaster ppk25. Overall, this work characterized the evolutionary history of these receptors revealing relevant unknown gene sequence features and clade-specific expansions.

Sensory systems mediate behaviors such as food, mate and shelter search, habitat selection, avoidance of environmental risks, escape from predators, and communication with conspecifics. Yet, immersed in an environment full of flowing information, triatomines, as other living organisms, must recognize and make use of adequate and sparse sensory stimuli. Specialized sensory structures allow the detection of a great variety of stimuli, such as visual cues, water vapor, heat, infrared radiation, vibratory signals, and a plethora of volatile and nonvolatile chemicals. Thus, different and specialized sensory capacities allow these animals an efficient exploitation of their environments according to their evolutionary history in specific ecotopes and particular selective pressures.

Sensory aversion is essential for avoiding prospective dangers. We studied the chemical perception of aversive compounds of different gustatory modalities (salty, bitter) in the haematophagous bug, Rhodnius prolixus . Over a walking arena, insects avoided a substrate embedded with 1M NaCl or KCl if provided with water as an alternative. However, no preferences were expressed when both salts were opposed to each other. A pre‐exposure to amiloride interfered with the repellency of NaCl and KCl equally, suggesting that amiloride‐sensitive receptors are involved in the detection of both salts. Discriminative experiments were then performed to determine whether R. prolixus can distinguish between these salts. An aversive operant conditioning involving either NaCl or KCl modulated the repellency of the conditioned salt, but also of the novel salt. Repellency levels of both salts were rigid to a chemical pre‐exposure to any of both salts. When gustatory modalities were crossed by presenting as a choice NaCl and a bitter molecule as caffeine (Caf), no innate preferences were expressed. Aversive operant conditionings with either NaCl or Caf rendered unspecific changes in the repellency of both compounds. A chemical pre‐exposure to Caf modulated the response to Caf but not to NaCl, suggesting the existence of two independent neural pathways for the detection of salts and bitter compounds. Overall results suggest that R. prolixus cannot discriminate molecules of the same gustatory modality (i.e. salty), but can distinguish between salty and bitter tastes. The potential use of aversive gustatory stimuli as a complement of commercially available olfactory repellents is discussed.

Head lice are exclusive human parasitic blood-sucking insects. Distributed worldwide among school-age children, this parasitosis generates scalp irritation and sometimes social prejudice. Understanding how head lice detect and perceive their human hosts is crucial to control transmission. Here, we describe the sensory structures present on the mouthparts of Pediculus humanus capitis and their possible contribution to the feeding decision-making process. On the anterior zone of the clypeus around the haustellum two morphological types of sensilla, invariable in location and number, were identified: fourteen short clypeus bristles (SCB) and six long clypeus bristles (LCB). During feeding these structures contact the host skin but not its blood. Located antero-dorsally on the everted haustellum and between the epipharyngeal teeth, a third sensillar type was identified: about four short peg epipharyngeal (SPE) sensilla. These structures are bathed with the incoming blood, when head lice feed, so may have a gustatory role. In behavioural experiments antennectomy of lice did not interfere with feeding behaviour, suggesting that the sensory structures on the mouthparts could be involved in host assessment.

The sense of taste provides information about the "good" or "bad" quality of a food source, which may be potentially nutritious or toxic. Most alkaloids taste bitter to humans, and because bitter taste is synonymous of noxious food, they are generally rejected. This response may be due to an innate low palatability or due to a malaise that occurs after food ingestion, which could even lead to death. We investigated in the kissing bug Rhodnius prolixus, whether alkaloids such as quinine, caffeine and theophylline, are merely distasteful, or if anti-appetitive responses are caused by a post-ingestion physiological effect, or both of these options. Although anti-appetitive responses were observed for the three alkaloids, only caffeine and theophylline affect metabolic and respiratory parameters that reflected an underlying physiological stress following their ingestion. Furthermore, caffeine caused the highest mortality. In contrast, quinine appears to be a merely unpalatable compound. The sense of taste helps insects to avoid making wrong feeding decisions, such as the intake of bitter/toxic foods, and thus avoid potentially harmful effects on health, a mechanism preserved in obligate hematophagous insects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insect antennae are sophisticated sensory organs, usually covered with sensory structures responsible for the detection of relevant signals of different modalities coming from the environment. Despite the relevance of the head louse Pediculus humanus capitis as a human parasite, the role of its antennal sensory system in the highly dependent relation established with their hosts has been barely studied. In this work, we present a functional description of the antennae of these hematophagous insects by applying different approaches, including scanning electron microscopy (SEM), anterograde antennal fluorescent backfills, and behavioral experiments with intact or differentially antennectomized lice. Results constitute a first approach to identify and describe the head louse antennal sensilla and to determine the role of the antenna in host recognition. SEM images allowed us to identify a total of 35–40 sensilla belonging to seven different morphological types that according to their external architecture are candidates to bear mechano-, thermo-, hygro-, or chemo-receptor functions. The anterograde backfills revealed a direct neural pathway to the ipsilateral antennal lobe, which includes 8–10 glomerular-like diffuse structures. In the two-choice behavioral experiments, intact lice chose scalp chemicals and warm surfaces (i.e., 32°C) and avoided wet substrates. Behavioral preferences disappeared after ablation of the different flagellomeres of their antenna, allowing us to discuss about the location and function of the different identified sensilla. This is the first study that integrates morphological and behavioral aspects of the sensory machinery of head lice involved in host perception.