Shelter selection in the kissing bugs Triatoma infestans and Rhodnius prolixus: Intra- and inter-specific interactions and quality evaluation.

Triatomines display most of their activities during the night. Before sunrise, they search, select and occupy adequate shelters to stay during the photophase, avoiding exposure to diurnal predators. In this work, we first explored the interactions between individuals of the same or different species in the shelter selection process of Triatoma infestans (Klug 1834) and Rhodnius prolixus (Stål 1859). When two groups of insects (either of different nutritional status, nymphal instar or species) were released together over an experimental arena containing two identical shelters, all nymphs were distributed randomly, suggesting the absence of intra- and inters-pecific interactions. Secondly, we analysed their preferences for particular features of shelters by releasing one group of insects (either T. infestans or R. prolixus) over an arena containing two different refuges. Nymphs exhibited preferences for darker shelters with a vertical orientation of its substrate and elevated from the ground, highlighting the importance of such features in a shelter selection context. We conclude that these species disregard the presence of other individuals but evaluate certain characteristics of the shelters to choose them. This information may contribute to understanding the colonization/recolonization dynamic processes of these Chagas disease vectors.

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.

The Sensory Machinery of the Head Louse Pediculus humanus capitis: From the Antennae to the Brain.

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.

Learning Spatial Aversion Is Sensory-Specific in the Hematophagous Insect Rhodnius prolixus.

Even though innate behaviors are essential for assuring quick responses to expected stimuli, experience-dependent behavioral plasticity confers an advantage when unexpected conditions arise. As being rigidly responsive to too many stimuli can be biologically expensive, adapting preferences to time-dependent relevant environmental conditions provide a cheaper and wider behavioral reactivity. According to their specific life habits, animals prioritize different sensory modalities to maximize environment exploitation. Besides, when mediating learning processes, the salience of a stimulus usually plays a relevant role in determining the intensity of an association. Then, sensory prioritization might reflect an heterogeneity in the cognitive abilities of an individual. Here, we analyze in the kissing bug Rhodnius prolixus if stimuli from different sensory modalities generate different cognitive capacities under an operant aversive paradigm. In a 2-choice walking arena, by registering the spatial distribution of insects over an experimental arena, we evaluated firstly the innate responses of bugs confronted to mechanical (rough substrate), visual (green light), thermal (32°C heated plate), hygric (humidified substrate), gustatory (sodium chloride), and olfactory (isobutyric acid) stimuli. In further experimental series bugs were submitted to an aversive operant conditioning by pairing each stimulus with a negative reinforcement. Subsequent tests allowed us to analyze if the innate behaviors were modulated by such previous aversive experience. In our experimental setup mechanical and visual stimuli were neutral, the thermal cue was attractive, and the hygric, gustatory and olfactory ones were innately aversive. After the aversive conditioning, responses to the mechanical, the visual, the hygric and the gustatory stimuli were modulated while responses to the thermal and the olfactory stimuli remained rigid. We present evidences that the spatial learning capacities of R. prolixus are dependent on the sensory modality of the conditioned stimulus, regardless their innate valence (i.e., neutral, attractive, or aversive). These differences might be given by the biological relevance of the stimuli and/or by evolutionary aspects of the life traits of this hematophagous insect.

Kissing bugs can generalize and discriminate between different bitter compounds.

Animals make use of contact chemoreception structures to examine the quality of potential food sources. During this evaluation they can detect nutritious compounds that promote feeding and recognize toxins that trigger evasive behaviors. Although animals can easily distinguish between stimuli of different gustatory qualities (bitter, salty, sweet, etc.), their ability to discriminate between compounds of the same quality may be limited. Numerous plants produce alkaloids, compounds that elicit aversive behaviors in phytophagous insects and almost uniformly evoke a bitter taste for man. In hematophagous insects, however, the effect of feeding deterrent molecules has been barely studied. Recent studies showed that feeding in Rhodnius prolixus can be negatively modulated by the presence of alkaloids such as quinine (QUI) and caffeine (CAF), compounds that elicit similar aversive responses. Here, we applied associative and non-associative learning paradigms to examine under two behavioral contexts the ability of R. prolixus to distinguish, discriminate and/or generalize between these two bitter compounds, QUI and CAF. Our results show that bugs innately repelled by bitter compounds can change their behavior from avoidance to indifference or even to preference according to their previous experiences. After an aversive operant conditioning with QUI or CAF, R. prolixus modified its behavior in a direct but also in a cross-compound manner, suggesting the occurrence of a generalization process between these two alkaloids. Conversely, after a long pre-exposure to each alkaloid, bugs decreased their avoidance to the compound used during pre-exposure but still expressed an avoidance of the novel compound, proving that QUI and CAF are detected separately. Our results suggest that R. prolixus is able to discriminate between QUI and CAF, although after an associative conditioning they express a symmetrical cross-generalization. This kind of studies adds insight into the gustatory sense of a blood-sucking model but also into the learning abilities of hematophagous insects.

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.

The main component of an alarm pheromone of kissing bugs plays multiple roles in the cognitive modulation of the escape response.

Innate responses in animals can be modulated by experience. Disturbed adults of the triatomine bug Triatoma infestans release an alarm pheromone (AP) that elicits an escape response in conspecific larvae. The main component of this AP, the isobutyric acid (IsoAc), alone has already shown to generate an escape response in this species. However, not much is known about the modulation of this behavior by non-associative and associative cognitive processes. We present here evidences of the cognitive capacities of T. infestans larvae in an escape context under different conditioning paradigms, including IsoAc in different roles. We show that: (1) the duration of a pre-exposure to IsoAc plays a main role in determining the type of non-associative learning expressed: short time pre-exposures elicit a sensitization while a longer pre-exposure time triggers a switch from repellence to attractiveness; (2) a simple pre-exposure event is enough to modulate the escape response of larvae to the AP and to its main component: IsoAc; (3) IsoAc and the AP are perceived as different chemical entities; (4) an association between IsoAc and an aversive stimulus can be created under a classical conditioning paradigm; (5) an association between IsoAc and a self-action can be generated under an operant conditioning. These results evince that IsoAc can attain multiple and different cognitive roles in the modulation of the escape response of triatomines and show how cognitive processes can modulate a key behavior for surviving, as it is the escaping response in presence of a potential danger in insects.

Dislodgement effect of natural semiochemicals released by disturbed triatomines: a possible alternative monitoring tool.

The quick detection of domestic and peridomestic triatomines in their environments becomes difficult without the use of dislodgement substances that flush them out from their shelters. At present, tetramethrin 0.2% is being widely used in control programs. Although it is an efficient dislodging agent, its toxicity might affect the health of captured triatomines, of other insects and, to a lesser extent, of other animals, including humans. Here, we tested if semiochemicals released by disturbed adults of Triatoma infestans and/or Rhodnius prolixus can make larvae of the same species exit from their refuges. In a walking olfactometer we found that: 1) larvae of T. infestans were repelled by the odors released by disturbed adults of their own species and of R. prolixus, 2) larvae of R. prolixus did not change their behavior in the presence of odors released by adults of both species, and 3) activity levels were not modulated by these odors in any of both species. Besides, in pseudo-natural conditions we found an increased flushing-out activity of larvae of T. infestans when their shelters were sprayed with isobutyric acid or 3-pentanol, and of larvae of R. prolixus when sprayed with 3-methyl-1-butanol. We succeeded in this work to dislodge larvae of triatomines from artificial shelters using natural volatile compounds, allowing the capture of live bugs for further investigations (e.g., xenodiagnosis or genetic studies) and favoring ecological aspects (e.g., minimizing environmental insecticide-contamination and non-targeted mortality).

Flight dispersal of the Chagas disease vectors Triatoma brasiliensis and Triatoma pseudomaculata in northeastern Brazil.

The present paper reports for the first time the capture of wild Triatoma brasiliensis and Triatoma pseudomaculata by means of light traps in Brazil. We tested commercially available lighting devices powered by batteries to attract the bugs to a white piece of cloth in the field. Two main findings showed to be significant: first, the results presented here show that light traps can be used for sampling these species in wild environments; second, they reveal that house colonization by triatomines may also happen as a consequence of the arrival of flying sylvatic bugs guided by artificial light sources. In addition, we discuss the effect of some environmental and biological factors on triatomine flight activity modulation.

Take-off activity and orientation of triatomines (Heteroptera: Reduviidae) in relation to the presence of artificial lights.

We analysed the flying activity of Triatoma infestans and Rhodnius prolixus when confronted to artificial lights of different spectral quality. We found that the presence of light sources (white or ultraviolet) did not affect their spontaneous take-off rate. The comparison between species showed that R. prolixus was more prone to fly than T. infestans. Females of T. infestans initiated flight more frequently than males of the same species. Although the same tendency was observed in R. prolixus, no significant differences were assessed between sexes. Concerning the orienting behaviour of triatomines at take-off in relation to the position of the light source, T. infestans showed a significant tendency to fly towards white light, but a non-oriented response when confronted to UV light or in the absence of a light source. R. prolixus also preferred to fly towards a source of white light and exhibited a non-oriented response with no light. However, when the UV light was presented, these bugs exhibited a bimodal attraction/repellence-behaviour. Our results support true attraction by white light rather than menotaxis or arrival by chance. These findings are discussed in relation to the colonization of human dwellings by Chagas disease vectors.

Circadian rhythm of behavioural responsiveness to carbon dioxide in the blood-sucking bug Triatoma infestans (Heteroptera: Reduviidae).

The temporal modulation of the behavioural response to carbon dioxide and its chronobiological basis were investigated in larvae of Triatoma infestans. We analysed the orientation towards CO(2) of insects kept under three different illumination regimes: (1) 12 h light/12 h darkness cycles (L/D), (2) constant darkness (D/D) and (3) constant light (L/L). When maintained under L/D conditions, insects exhibited an oriented response towards airstreams added with 1500 ppm of CO(2) during the first hours of the scotophase only. Bugs maintained under D/D also showed a positive orientation response towards CO(2) during the first hours of the subjective night, while bugs kept under L/L did not show a rhythmic oriented behaviour. Thus, T. infestans displayed a daily rhythm of orientation towards CO(2) (i.e. a potential food source) only at the beginning of the scotophase. The persistence of the rhythm under constant darkness reveals the existence of an endogenous circadian control of this behaviour.

Chronobiological basis of thermopreference in the haematophagous bug Triatoma infestans.

The chronobiological basis of the daily dynamics of thermopreference was tested in adults of Triatoma infestans, continuously registering the preferred temperature of insects released over a temperature gradient for 13 days. We found that the thermopreference in T. infestans is a dynamic process that depends on the time of the day and the post-feeding time. When submitted to a 12:12 h light/darkness cycle (L/D), the preferred temperature reached the highest and the lowest values at the end of the light and dark phases, respectively. This daily rhythm persisted under constant conditions of illumination (D/D and L/L), suggesting the existence of an internal oscillator controlling this behaviour. Statistical analysis revealed that the thermopreference of insects kept under L/D exhibited a ca. 24 h periodicity, while insects kept in D/D and L/L showed free-running periods of tau((D/D))=23.35 and tau((L/L))=27.35 h, respectively. The persistence of a cyclic pattern of thermopreference under constant conditions, and free-running periods, close to, but different from 24 h, demonstrate the existence of an endogenous control of the thermopreference in this species. The biological relevance of these results is discussed in the light of the hypothesis that both the length of time elapsed since feeding and the time of the day modify thermopreference in these bugs. The gradual decline in preferred temperature following feeding may be associated with energy conservation during starvation. The rhythmic modulation of thermopreference may be associated with the daily rhythm of locomotion activity shown by these bugs.