The pharyngeal taste organ of a blood-feeding insect functions in food recognition.

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.

Mosquitoes do not Like Bitter.

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.

Sensory discrimination between aversive salty and bitter tastes in an haematophagous insect.

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.

Plasticity in Insect Olfaction: To Smell or Not to Smell?

In insects, olfaction plays a crucial role in many behavioral contexts, such as locating food, sexual partners, and oviposition sites. To successfully perform such behaviors, insects must respond to chemical stimuli at the right moment. Insects modulate their olfactory system according to their physiological state upon interaction with their environment. Here, we review the plasticity of behavioral responses to different odor types according to age, feeding state, circadian rhythm, and mating status. We also summarize what is known about the underlying neural and endocrinological mechanisms, from peripheral detection to central nervous integration, and cover neuromodulation from the molecular to the behavioral level. We describe forms of olfactory plasticity that have contributed to the evolutionary success of insects and have provided them with remarkable tools to adapt to their ever-changing environment.

Pheromone modulates plant odor responses in the antennal lobe of a moth.

In nature, male moths are exposed to a complex plant odorant environment when they fly upwind to a sex pheromone source in their search for mates. Plant odors have been shown to affect responses to pheromone at various levels but how does pheromone affects plant odor perception? We recorded responses from neurons within the non-pheromonal "ordinary glome ruli" of the primary olfactory center, the antennal lobe (AL), to single and pulsed stimulations with the plant odorant heptanal, the pheromone, and their mixture in the male moth Agrotis ipsilon. We identified 3 physiological types of neurons according to their activity patterns combining excitatory and inhibitory phases. Both local and projection neurons were identified in each physiological type. Neurons with excitatory responses to heptanal responded also frequently to the pheromone and showed additive responses to the mixture. Moreover, the neuron's ability of resolving successive pulses generally improved with the mixture. Only some neurons with combined excitatory/inhibitory, or purely inhibitory responses to heptanal, also responded to the pheromone. Although individual mixture responses were not significantly different from heptanal responses in these neurons, pulse resolution was improved with the mixture as compared with heptanal alone. These results demonstrate that the pheromone and the general odorant subsystems interact more intensely in the moth AL than previously thought.

Bitter stimuli modulate the feeding decision of a blood-sucking insect via two sensory inputs.

The gustatory system of animals is involved in food quality assessment and controls the feeding decision of an individual confronted with a potential alimentary source. Triatomines are haematophagous insects that feed on vertebrate blood. Once they reach a potential host, they walk over the host skin searching for an adequate site to pierce. Then, they insert their stylets and take a first sampling gorge to decide whether food is acceptable. Our work reveals that the presence of bitter compounds inhibits the feeding behavior of these bugs. Firstly, triatomines decreased their feeding behavior if substrates spread with quinine or caffeine were detected by external receptors localized exclusively in the antennae. Morphological inspections along with electrophysiological recordings revealed the existence of four gustatory sensilla located in the tip of the antenna that respond to both bitter tastants. The absence of these bitter detectors by antennal ablation reversed the observed feeding inhibition evoked by bitter compounds. Secondly, once triatomines pumped the first volume of food with bitter compounds (quinine, caffeine, berberine, salicin), a decrease in their feeding behavior was observed. Morphological inspections revealed the existence of eight gustatory sensilla located in the pharynx that might be responsible for the internal bitter detection. Finally, we found that a brief pre-exposure to bitter compounds negatively modulates the motivation of bugs to feed on an appetitive solution. Results presented here highlight the relevance of bitter taste perception in the modulation of the feeding behavior of a blood-sucking insect.

Brief predator sound exposure elicits behavioral and neuronal long-term sensitization in the olfactory system of an insect.

Modulation of sensitivity to sensory cues by experience is essential for animals to adapt to a changing environment. Sensitization and adaptation to signals of the same modality as a function of experience have been shown in many cases, and some of the neurobiological mechanisms underlying these processes have been described. However, the influence of sensory signals on the sensitivity of a different modality is largely unknown. In males of the noctuid moth, Spodoptera littoralis, the sensitivity to the female-produced sex pheromone increases 24 h after a brief preexposure with pheromone at the behavioral and central nervous level. Here we show that this effect is not confined to the same sensory modality: the sensitivity of olfactory neurons can also be modulated by exposure to a different sensory stimulus, i.e., a pulsed stimulus mimicking echolocating sounds from attacking insectivorous bats. We tested responses of preexposed male moths in a walking bioassay and recorded from neurons in the primary olfactory center, the antennal lobe. We show that brief exposure to a bat call, but not to a behaviorally irrelevant tone, increases the behavioral sensitivity of male moths to sex pheromone 24 h later in the same way as exposure to the sex pheromone itself. The observed behavioral modification is accompanied by an increase in the sensitivity of olfactory neurons in the antennal lobe. Our data provide thus evidence for cross-modal experience-dependent plasticity not only on the behavioral level, but also on the central nervous level, in an insect.

Highlights, challenges, and perspectives in basic and applied chemical ecology of triatomines.

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.

Plant odour stimuli reshape pheromonal representation in neurons of the antennal lobe macroglomerular complex of a male moth.

Male moths are confronted with complex odour mixtures in a natural environment when flying towards a female-emitted sex pheromone source. Whereas synergistic effects of sex pheromones and plant odours have been observed at the behavioural level, most investigations at the peripheral level have shown an inhibition of pheromone responses by plant volatiles, suggesting a potential role of the central nervous system in reshaping the peripheral information. We thus investigated the interactions between sex pheromone and a behaviourally active plant volatile, heptanal, and their effects on responses of neurons in the pheromone-processing centre of the antennal lobe, the macroglomerular complex, in the moth Agrotis ipsilon. Our results show that most of these pheromone-sensitive neurons responded to the plant odour. Most neurons responded to the pheromone with a multiphasic pattern and were anatomically identified as projection neurons. They responded either with excitation or pure inhibition to heptanal, and the response to the mixture pheromone + heptanal was generally weaker than to the pheromone alone, showing a suppressive effect of heptanal. However, these neurons responded with a better resolution to pulsed stimuli. The other neurons with either purely excitatory or inhibitory responses to all three stimuli did not exhibit significant differences in responses between stimuli. Although the suppression of the pheromone responses in AL neurons by the plant odour is counter-intuitive at first glance, the observed better resolution of pulsed stimuli is probably more important than high sensitivity to the localization of a calling female.

The closer the better: Sensory tools and host-association in blood-sucking insects.

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.

Molecular and functional basis of high-salt avoidance in a blood-sucking 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; Rpro PPK014276 and Rpro PPK28) 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.

Mating-induced differential coding of plant odour and sex pheromone in a male moth.

Innate behaviours in animals can be influenced by several factors, such as the environment, experience, or physiological status. This behavioural plasticity originates from changes in the underlying neuronal substrate. A well-described form of plasticity is induced by mating. In both vertebrates and invertebrates, males experience a post-ejaculatory refractory period, during which they avoid new females. In the male moth Agrotis ipsilon, mating induces a transient inhibition of responses to the female-produced sex pheromone. To understand the neural bases of this inhibition and its possible odour specificity, we carried out a detailed analysis of the response characteristics of the different neuron types from the periphery to the central level. We examined the response patterns of pheromone-sensitive and plant volatile-sensitive neurons in virgin and mated male moths. By using intracellular recordings, we showed that mating changes the response characteristics of pheromone-sensitive antennal lobe (AL) neurons, and thus decreases their sensitivity to sex pheromone. Individual olfactory receptor neuron (ORN) recordings and calcium imaging experiments indicated that pheromone sensory input remains constant. On the other hand, calcium responses to non-pheromonal odours (plant volatiles) increased after mating, as reflected by increased firing frequencies of plant-sensitive AL neurons, although ORN responses to heptanal remained unchanged. We suggest that differential processing of pheromone and plant odours allows mated males to transiently block their central pheromone detection system, and increase non-pheromonal odour detection in order to efficiently locate food sources.

Evolution of the Insect PPK Gene Family.

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.

Switching attraction to inhibition: mating-induced reversed role of sex pheromone in an insect.

In the moth, Agrotis ipsilon, newly mated males cease to be attracted to the female-produced sex pheromone, preventing them from re-mating until the next night, by which time they would have refilled their reproductive glands for a potential new ejaculate. The behavioural plasticity is accompanied by a decrease in neuron sensitivity within the primary olfactory centre, the antennal lobe (AL). However, it was not clear whether the lack of the sexually guided behaviour results from the absence of sex pheromone detection in the ALs, or if they ignore it in spite of detection, or if the sex pheromone itself inhibits attraction behaviour after mating. To test these hypotheses, we performed behavioural tests and intracellular recordings of AL neurons to non-pheromonal odours (flower volatiles), different doses of sex pheromone and their mixtures in virgin and newly mated males. Our results show that, although the behavioural and AL neuron responses to flower volatiles alone were similar between virgin and mated males, the behavioural response of mated males to flower odours was inhibited by adding pheromone doses above the detection threshold of central neurons. Moreover, we show that the sex pheromone becomes inhibitory by differential central processing: below a specific threshold, it is not detected within the AL; above this threshold, it becomes inhibitory, preventing newly mated males from responding even to plant odours. Mated male moths have thus evolved a strategy based on transient odour-selective central processing, which allows them to avoid the risk-taking, energy-consuming search for females and delay re-mating until the next night for a potential new ejaculate.

Mating-induced transient inhibition of responses to sex pheromone in a male moth is not mediated by octopamine or serotonin.

In the male moth, Agrotis ipsilon, mating induces a transient inhibition of behavioural and central nervous responses to sex pheromone. Newly mated males are not attracted to sex pheromone, and the sensitivity of their antennal lobe (AL) neurons is lower than in virgin males. This rapid transient olfactory inhibition prevents them from re-mating unsuccessfully until they have refilled their sex glands. We hypothesized that this olfactory 'switch off' might be controlled by neuromodulators such as biogenic amines. To test our hypothesis, we studied the effects of octopamine (OA) and serotonin (5-hydroxytryptamine, 5-HT) on the coding properties of pheromone-sensitive AL neurons in virgin and newly mated males. We show that AL neuron sensitivity increased in newly mated males after injection of OA or 5-HT, but only OA treatment affected certain response characteristics of AL neurons in virgin males. Whereas all measured AL neuron response characteristics were different between virgin and newly mated males, amine treatment in newly mated males restored only the latency and spike frequency, but not the duration of excitatory and inhibitory phases, which were initially found in virgin males. Additionally, we investigated the behavioural effects of OA and 5-HT treatments in virgin and mated males. Although OA and 5-HT enhanced the general flight activity of newly mated males, amine treatments did not restore the behavioural pheromone response of mated moths. Altogether, these results show that, although biogenic amines modulate the olfactory system in moths, OA and 5-HT are probably not involved in the post-mating inhibition of responses to sex pheromone in A. ipsilon males.

Impact of alkaloids in food consumption, metabolism and survival in a blood-sucking insect.

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.

Mouthpart sensory structures of the human head louse Pediculus humanus capitis.

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.

Author Correction: The use of Leaf Surface Contact Cues During Oviposition Explains Field Preferences in the Willow Sawfly Nematus Oligospilus.

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

Age-dependent plasticity of sex pheromone response in the moth, Agrotis ipsilon: combined effects of octopamine and juvenile hormone.

Male moths use sex pheromones to find their mating partners. In the moth, Agrotis ipsilon, the behavioral response and the neuron sensitivity within the primary olfactory centre, the antennal lobe (AL), to sex pheromone increase with age and juvenile hormone (JH) biosynthesis. By manipulating the JH level, we previously showed that JH controls this age-dependent neuronal plasticity, and that its effects are slow (within 2 days). We hypothesized that the hormonal effect might be indirect, and one neuromodulator candidate, which might serve as a mediator, is octopamine (OA). Here, we studied the effects of OA and an OA receptor antagonist, mianserin, on behavioral and AL neuron responses of mature and immature males during stimulation with sex pheromone. Our results indicate that, although OA injections enhanced the behavioral pheromone response in mature males, OA had no significant effect on behavior in immature males. However, mianserin injections decreased the behavioral response in mature males. AL neuron sensitivity increased after OA treatment in immature males, and decreased after mianserin treatment in mature males. Determination of OA levels in ALs of immature and mature males did not reveal any difference. To study the possible interactive effects of JH and OA, the behavioral pheromone response was analyzed in JH-deprived mature males injected with OA, and in immature males injected with fenoxycarb, a JH agonist, and mianserin. Results show that both JH and OA are necessary to elicit a behavioral response of A. ipsilon males to sex pheromone.

Food recognition in hematophagous insects.

Hematophagous insects use heat, odors, visual cues and humidity emitted by vertebrate hosts to find them in space and time. Once they reach a host, they integrate multimodal information from its skin, and decide whether to bite or not. If skin conditions fulfil the insect's expectations, it bites and pumps a small quantity of blood. Again, only if the sampled blood fulfils the insect's feeding requirements, it continues with a full ingestion. Taste is involved in both timely linked evaluation processes via contact chemoreceptors located in different parts of their bodies, driving jointly food acceptance or rejection. However, the whole picture of how blood-sucking insects evaluate the quality of a potential host is poorly understood. Here, I summarize the actual knowledge about the feeding decision-making in blood-sucking insects. Being typically involved in the transmission of diseases to humans or livestock, a deeper understanding about factors affecting an essential process as feeding in these insects could help us to find new strategies to reduce interactions.