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It is taken for granted that immature mosquito only breathe atmospheric air through their siphons. However, there is no quantitative study that demonstrated it. We analysed the survival of the last instar larvae ofAedes aegyptifully submerged at different temperatures, and measured oxygen consumption from air and dissolved in water, of larvae and pupae of this species under different conditions. Results revealed that under water, larvae survived much longer than expected, reaching 50% mortality only after 58, 10, and 5 days at 15°, 25° and 35°C, respectively. Interestingly, whereas we registered moults to pupae in larvae with access to air, individuals kept submerged never moulted. When remaining at the water surface, larvae obtained 12.72% of O2from the water, while pupae only 5.32%. When completely submerged, larvae consumed less oxygen than in contact with the surface, but enough for surviving, while pupae did not. At both media, temperature affected larvae respiration rate, with relatively close Q10values. In the related species,Ae. albopictus, a similar pattern of O2consumption were observed. Larvae got 12.14% of their oxygen from the water. Interestingly, no significant differences in total O2consumption were found between water O2consumption, whenAe. albopictuslarvae were submerged, or when they also have access to air (dual O2consumption). Our findings not only challenge the classical idea that mosquito larvae only breathe atmospheric O2, but also force us to reconsider the potential effectiveness of control methods based on asphyxiating larvae by detaching from water surface.Summary statementWe present the first quantitative analysis of mosquito larvae respiration in air and water, unravelling the unknown capacity of larvae of the most cosmopolitan disease vector of breathing underwater.

Lutzomyia longipalpis is known as one of the primary insect vectors of visceral leishmaniasis. For such ectothermic organisms, the ambient temperature is a critical life factor. However, the impact of temperature has been ignored in many induced-stress situations of the vector life. Therefore, this study explored the interaction of Lu. longipalpis with temperature by evaluating its behaviour across a thermal gradient, thermographic recordings during blood-feeding on mice, and the gene expression of heat shock proteins (HSP) when insects were exposed to extreme temperature or infected. The results showed that 72 h after blood ingestion, Lu. longipalpis became less active and preferred relatively low temperatures. However, at later stages of blood digestion, females increased their activity and remained at higher temperatures. Real-time imaging showed that the body temperature of females can adjust rapidly to the host and remain constant until the end of blood-feeding. Insects also increased the expression of HSP90(83) during blood-feeding. Our findings suggest that Lu. longipalpis interacts with temperature by using its behaviour to avoid temperature-induced physiological damage during the gonotrophic cycle. However, the expression of certain HSP might be triggered to mitigate thermal stress in situations where a behavioural response is not the best option.

Pollinating insects can be exposed to temperatures far from ambient air when visiting flowers, reducing their warming tolerance. Typically, such scenario occurs when flowers are exposed to solar radiation. The case of thermogenic flowers is particular because they warm up even when they are not exposed to solar energy. The flowers of Arum attract their pollinators with a deceptive method and trap them for a whole day, thereby imposing elevated temperature to visiting insects. Therefore, we predict a relatively high basal thermal tolerance in those insects. The aim of this study was to assess the thermal tolerance and warming tolerance of females of two fly species (genus Psychoda) pollinating Arum sp. (thermogenic plant). We measured their critical temperature (CTmax) and its response to rate of temperature increase as well as acclimation period to moderate temperature of 25 °C. We found relatively low CTmax (33.7 °C on average) for both species, and a weak response to acclimation period and ramping rate. In general, the thermal tolerance increased with a rapid ramping in temperature. To evaluate the warming tolerance, we compared thermal tolerance limits to flower temperatures measured in the field. We highlighted that the temperature of the thermogenic floral organ could reach values close to the thermal tolerance threshold of pollinators. This discovery raises questions about the sustainability of the interaction between these thermogenic plants and their pollinators.

Blood-sucking arthropods exploit multimodal information for locating and recognising potential hosts. The heat emitted by the body of endothermic vertebrates constitutes a major cue for orientation. To exploit it in a reliable way, insects must be able to deal with two variants of thermal information, that is heat exchange and temperature fluctuations. Evaluating whether or not an object qualifies as a host by its temperature requires solving thermodynamic ambiguities in a context where temperature increase at the receptor level is just one, yet insufficient, piece of information. To be exploitable, this piece of information needs to be integrated with other variables. Here, I discuss the physical constraints associated to thermal orientation, as well as the way different blood-sucking insects acquire and make use of heat to recognise a host.

The nervous system of triatomines follows the general plan for insects in terms of organization. Yet, its anatomy is in line with the form of the different regions of the bug’s body. The brain is located in the posterior region of the elongated head, the cavity of which is mostly occupied by the cibarial pump. The ganglia composing the ventral nerve cord exhibit a high degree of fusion, conforming a mass of suboesophageal or gnathal ganglia, a prothoracic ganglion and a unique posterior nervous mass, formed by the fusion of meso- and meta-thoracic and segmental abdominal ganglia. Most of the integration centres (i.e. neuropils) of the brain and ganglia are less structured than in other insects, but they can be easily recognized by histological inspection. In the brain, all major centres are well defined, and in the ganglia it is possible to establish the correspondence of each neuropil with its respective thoracic or abdominal segment. We have tried to keep the nomenclature employed for naming different structures as clear as possible. In some cases, synonymy is indicated, in order to allow the reader to find equivalents in the literature concerning other insects, which could have adopted different criteria. This chapter presents a synthesis of our present knowledge of the neuroanatomy of kissing bugs, underlining its particularities. A general description is presented here, but much work is still needed to elaborate an atlas as detailed as those existing for domestic and fruit flies or honey bees.

Many arthropod species have adopted the blood of vertebrates as their main food. Blood is rich in nutrients and, except for the presence of parasites, otherwise sterile. This food, however, is not freely available nor is its obtention devoid of risk; it circulates inside vessels hidden underneath the skin of mobile hosts, which are able to defend themselves and even predate the insects attempting to feed on them. Thus, the haematophagous lifestyle is associated with major morphological, physiological and behavioural adaptations that have accumulated throughout the evolutionary history of the various lineages of blood-sucking arthropods, including triatomines. These adaptations have, on the other hand, significant consequences for the evolution of parasites, as well as for the epidemiology of vector-transmitted diseases. This review article analyses various aspects of the behaviour of triatomine bugs to illustrate how behavioural traits represent particular adaptations to their close association with hosts, which may easily turn into predators. The aim is to offer to the reader an up-to-date integrative view of the behaviour of Chagas disease vectors from a personal perspective, with the hope of encouraging young and experienced colleagues to explore this fascinating aspect of triatomine biology.

The phlebotomine sand flyLutzomyia longipalpisis the primary insect vector of visceral leishmaniasis in the Americas. For ectothermic organisms such as sand flies, the ambient temperature is a critical factor influencing all aspects of their life. However, the impact of temperature has been ignored in previous investigations of stress-induced responses by the vector, such as taking a blood meal or duringLeishmaniainfection. Therefore, this study explored the interaction ofLu. longipalpiswith temperature by evaluating sand fly behaviour across a thermal gradient after sugar or blood-feeding, and infection withLeishmania mexicana.Thermographic recordings of sand fly females fed on mice were analysed, and the gene expression of heat shock proteins HSP70 and HSP90(83) was evaluated when insects were exposed to extreme temperatures or infected. The results showed that 72h after blood ingestion females ofLu. longipalpisbecame less active and preferred relatively low temperatures. However, at later stages of blood digestion females increased their activity and remained at higher temperatures prior to taking a second blood meal; this behaviour seems to be correlated with the evolution of their oocysts and voracity for a second blood meal. No changes in the temperature preferences of female sand flies were recorded in the presence of a gut infection byLe. mexicana,indicating that this parasite has not triggered behavioural immunity inLu. longipalpis. Real-time imaging showed that the body temperature of female flies feeding on mice increased to the same temperature as the host within a few seconds after landing. The body temperature of females remained around 35 ± 0.5 °C until the end of blood-feeding, revealing a lack of thermoregulatory behaviour. Analysis of expression of heat shock proteins revealed insects increased expression of HSP90(83) when exposed to higher temperatures, such as during blood feeding. Our findings suggest thatLu. longipalpisinteracts with the environmental temperature by using its behaviour to avoid temperature-related physiological damage during the gonotrophic cycle. However, the expression of certain heat shock proteins might be triggered to mitigate against thermal stress in situations where a behavioural response is not the best option.

Feeding on the blood of warm-blooded vertebrates is associated to thermal stress in haematophagous arthropods. It has been demonstrated that blood-sucking insects protect their physiological integrity either by synthesising heat-shock proteins or by means of thermoregulatory mechanisms. In this work, we describe the first thermoregulatory mechanism in a tick species,Ornithodoros rostratus. By performing real-time infrared thermography during feeding on mice we found that this acarian eliminates big amounts of fluid (urine) through their coxal glands; this fluid quickly spreads over the cuticular surface and its evaporation cools-down the body of the tick. The spread of the fluid is possible thanks to capillary diffusion through the sculptured exoskeleton ofOrnithodoros. We discuss our findings in the frame of the adaptive strategies to cope with the thermal stress experienced by blood-sucking arthropods at each feeding event warm-blooded hosts.

Active searching for vertebrate blood is a necessary activity for haematophagous insects, and it can be assumed that this search should also be costly in terms of energetic expenditure. Whether by swimming, walking, running or flying, active movement requires energy, increasing metabolic rate relative to resting situations. We analysed the respiratory pattern and energetic cost of pedestrian locomotion in the blood-sucking bug

Active searching for vertebrate blood is a necessary activity for haematophagous insects, and it can be assumed that this search should also be costly in terms of energetic expenditure. Either if it is by swimming, walking, running or flying, active movement requires energy, increasing metabolic rates relative to resting situations. We analysed the respiratory pattern and the energetic cost of pedestrian locomotion in the blood-sucking bug Rhodnius prolixus using flow-through respirometry, by measuring carbon dioxide emission and water loss before, during and after walking. We observed an increase of up to 1.7-fold in the metabolic rate during walking as compared to resting in male R. prolixus and 1.5-fold in females, as well as a change in their respiratory pattern. The last switched from cyclic during resting to continuous, when the insects started to walk, remaining this condition unchanged during locomotion and for several minutes after stopping. Walking induced a significant loss of weight in both, males and females. This can be explained by the increase in both, the metabolic rate and the water loss during walking. These data constitute the first metabolic measures of active hematophagous insects and provide the first insights on the energetic expenditure associated to the active search for blood in this group.

Lice from pinnipeds, i.e. sea lions, seals, and walruses, are the only insects capable of surviving marine dives. Along their evolutionary history, they adapted to tolerate hypoxia, high salinity, low temperature and, in particular, to tolerate conditions of high hydrostatic pressure. To understand the limits of the capacity of lice to survive during host deep dives, we conducted a series of controlled experiments in the laboratory. We collected lice from elephant seals and submitted the different life stages to high pressure conditions. Lice were first exposed to one of four hydrostatic pressures: (a) 30 Kg/cm2; (b) 80 Kg/cm2; (c) 150 Kg/cm2, or (d) 200 Kg/cm2. They were then exposed a second time to higher or lower hydrostatic pressure conditions to test for the impact of the first experience, which could either be deleterious or trigger physiological adaption, allowing them a better tolerance to high pressure. We found that lice from elephant seals can tolerate hydrostatic pressures higher than 200 Kg/cm2 (close to 200 atm), equivalent to 2000 m depth. Adults exhibited lower recovery times than nymphs after immersion at high hydrostatic pressure. Our findings show that lice developed unique adaptations to endure extreme marine conditions. We discuss these extreme performances as related to the morphological characteristics and physiological responses to diving in these insects.

The increase in body temperature over that of the environment has been frequently reported in insects, in particular in relation with flight activity. Scarab beetles of the genus Cyclocephala living in tropical areas are known to exploit the heat produced by thermogenic plants, also producing heat by endothermy. Here, we report the first case of endothermy in a species of this genus living in a temperate region, Cyclocephala signaticollis. We characterised the phenomenon in this beetle using infrared thermography and exposing them to different thermal conditions. We evaluated the frequency of endothermic bouts, the nature of their periodic occurrence and their association with the activity cycles of the beetles. We found that endothermy occurs in both males and females in a cyclic fashion, at the beginning of the night, around 21:00 local time. The mean temperature increase was of 9 °C, and the mean duration of the bouts was 7 min. During endothermic bouts, the temperature of the thorax was on average 3.6 °C higher than that of the head and 4.8 °C above that of the abdomen. We found no differences between females and males in the maximum temperature attained and in the duration of the endothermy bouts. The activity period of the beetles extends throughout the whole night, with maximum activity between 22:00 and 23:00. By subjecting the beetles to different light regimes we were able to determine that the rhythm of endothermy is not controlled by the circadian system. Finally, we experimentally tested if by performing endothermy the scarabs try to reach a particular body temperature or if they invest a given amount of energy in heating up, instead. Our results indicate that at lower ambient temperature beetles show higher increase in body temperature, and that endothermy bouts last longer than at relatively higher ambient temperatures. We discuss our findings in relation to the ecology and behaviour of this beetle pest. Fil: Zermoglio, Paula Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina Fil: Castelo, Marcela Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina Fil: Lazzari, Claudio Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universite de Tours; Francia

The heat emitted by the host body constitutes a short distance orientation cue for most blood-sucking insects, as is the case of the kissing-bug Rhodnius prolixus. We evaluated here how kissing bugs assess the distance to a warm target, in order to reach it by displaying the Proboscis Extension Reflex (PER). We confronted blind-folded insects to a thermal source either at 35° or at 40 °C under both, open- and closed-loop conditions. The results showed that nymphs were able to estimate the distance to a thermal source just using thermal information. Free walking insects displayed PER with a maximum frequency at 5 mm from the object, even without touching it. Yet, our experiments showed that the insects need to walk freely to estimate the distance to the source accurately, i.e. performing the PER at a distance allowing them to reach the target with the tip of the proboscis. The distance at which PER was triggered was independent of the temperature of the thermal source (35° or 40 °C). Moreover, our results also unravelled that mechanical stimuli can be integrated with thermal cues, being capable of affecting the triggering of PER in kissing bugs. This is the first study providing evidence that blood-sucking vector insects use mechanoreception for eliciting their bites. We discuss our findings in the light of present models explaining the ability of kissing bugs to estimate the distance and the temperature of a potential food sources.

Rhodnius prolixusis able to cool down the ingested blood during feeding on a warm-blooded host. This is possible because of a counter-current heat exchanger located in its head, which transfers heat from the warm blood to the insect haemolymph and can dissipate through the head cuticle. Given the key role haemolymph circulation in thermoregulation, we investigated the modulation of the activity of the heart during the warmed meal intake. We evaluated the impact of meal temperature on the heart rate and found that feeding led to an increase in the frequency of heart contractions, which increases with increasing food temperature. We also found that females have a higher heart rate during feeding compare to males.HIGHLIGHTSFeeding increases the heart rate ofRhodnius prolixusThe higher the meal temperature, the higher the heart rate becomesFemales have a higher heart rate than males

Blood feeding in arthropods has evolved in multiple lineages. This feeding preference provides a source of ample proteins and lipids for egg production and survival, but ingestion of a large warm blood-meal can boost the arthropod’s body temperature 15°–20°C within seconds to minutes. This represents one of, if not the most, rapid thermal change documented under a natural setting. Here, we describe mechanisms of thermoregulation and thermotolerance in arthropods during blood feeding. The ability to prevent blood-induced thermal damage is a fundamental physiological adaptation linked to the use of warm-blooded vertebrates as food sources. Specific functional and comparative studies have identified unique and divergent mechanisms that suppress or repair thermal stress during blood feeding. These mechanisms include countercurrent heat exchange, evaporative cooling, and upregulation of stress associated proteins.

Glyphosate is the most widely used herbicide in the world. Over the past few years, the number of studies revealing deleterious effects of glyphosate on non-target species has been increasing. Here, we studied the impact of glyphosate at field-realistic doses on learning in mosquito larvae (

Glyphosate is the most widely used herbicide in the world. Over the past few years, the number of studies revealing deleterious effects of glyphosate on non-target species has been increasing. Here, we studied the impact of glyphosate at field-realistic doses on learning in mosquito larvae (Aedes aegypti). Larvae of A. aegypti live in small bodies of water and perform a stereotyped escape response when a moving object projects its shadow on the water surface. Repeated presentations of an innocuous visual stimulus induce a decrease in response due to habituation, a non-associative form of learning. In this study, different groups of larvae were reared in water containing different concentrations of glyphosate that are commonly found in the field (50 µg l−1, 100 µg l−1, 210 µg l−1 and 2 mg l−1). Larvae reared in a glyphosate solution of 2 mg l−1 (application dose) could complete their development. However, glyphosate at a concentration of 100 µg l−1 impaired habituation. A dose-dependent deleterious effect on learning ability was observed. This protocol opens new avenues to further studies aimed at understanding how glyphosate affects non-target organisms, such as insects. Habituation in mosquito larvae could serve as a parameter for testing the impact of pollutants in the water.

Kissing bugs possess a highly developed thermal sense and when starved, they attempt to bite any object which temperature is close to that of a warm-blooded host. At each feeding event, these insects take massive meals in just a few minutes. One could then expect fed-bugs being heated-up by the ingested warm blood and so becoming attractive to starved conspecifics. This is not however the case, arising the question about why cannibalism is very rare among these insects. Recently, the ability of thermoregulating during feeding has been demonstrated in Rhodnius prolixus. These bugs possess a countercurrent heat-exchanger that cools down the ingested blood, before it reaches the abdomen. We hypothesise that avoiding thermal stress is not the only adaptive advantages of this mechanism, but could also help avoiding cannibalism. We tested this hypothesis by quantifying cannibalism by never-fed first-instar larvae on: (1) just-fed 5th instar bugs, (2) artificially heated just-fed bugs, (3) heated or (4) non-heated objects of the same size. In line with our hypothesis, non-heated just-fed bugs were not attacked by the 1st instar larvae, whereas heated bugs and object triggered biting behaviour in starved bugs, which performed either cleptohaematophagy or haemolymphagy on heated bugs. We conclude that cannibalism triggered by thermal stimuli has been one of the selection pressures that gave origin to thermoregulation during feeding on kissing bugs.

Triatominae are blood-sucking insects that localise their hosts using a range of sensory signals to find food, and among them, the heat emitted by the hosts. Heat is one of the main short-range cues in vertebrate hosts, able to trigger alone the Proboscis Extension Response (PER) that precedes the bite. Previous studies demonstrated that heat responsiveness of fifth-instar nymphs is maximum to moderate temperatures (30–35°C) compatible with those of their vertebrate host’s body surface. This study investigated whether this thermal preference for biting is maintained along the life cycle of R. prolixus, from the first larval instar to male and female adults. The results showed that PER rates were at maximum around 30–35°C and decreased for a warmer temperature. The same thermal preference was maintained all along the life cycle, despite the increase in the size of the antennae linked to the growth. Interestingly, a decreased thermal responsiveness was stated in males as compared with larval instars and females. This decrease might reveal a lower motivation for host-seeking and might have an impact on males’s vectorial competence.

In spite of the mosquito Aedes aegypti being a vector of several infectious diseases, a limited number of studies has been undertaken on learning in this species. Moreover, larval stages have been neglected as model organisms, although they are active, aquatic and perform stereotyped behavioural responses, e.g. the escape response when disturbed. To study the learning abilities of mosquito larvae, we focused on habituation, a form of non-associative learning widely studied in vertebrates and invertebrates. Habituation was defined as the progressive and reversible decrease in response to a reiterative stimulus. We first aimed at confirming habituation of the escape response in mosquito larvae (4th instar). Then, we determined whether a mnesic trace was established. Larvae were individually stimulated with a visual danger stimulus inducing the escape response. We set up a protocol for testing larvae individually, allowing the control of different parameters that are crucial for the study of cognitive abilities. After 15 trials, the escape response of mosquitoes was significantly lower. A disturbance stimulus presented after the 15th trial, induced the escape response and reversed habituation. Retention was confirmed up to 1h after the last habituation trial. This original bioassay can be adapted for studying the physiology of learning and memory in mosquito larvae, for analysing the effects of chemicals in the water, the characterisation of the cognitive abilities related to the life history of different mosquito species across preimaginal stages.

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The blood of endothermic vertebrates constitutes the main, or even the only food for many arthropod species. Even though blood is a food rich in nutrients and in most cases sterile, its consumption is associated to many stressing factors. Energetic, thermal, osmotic and oxidative stresses are among the consequences for arthropods of the rapid ingestion of large amounts of warm blood. To cope with these stressors, these animals have developed different physiological and molecular mechanisms allowing the reduction of the stress or the reparation of the infringed damage. Among the first, specific mechanisms of thermoregulation and rapid excretion have been identified. The rapid synthesis of HSP following each feeding event make parts of the mechanisms of molecular reparation. The increase in the HSP70 levels varies across species from about 3 to around 17 times the basal level. This variability in the molecular response is plausibly associated to the occurrence or not of complementary mechanisms for reducing the effect of the stressor, as for instance, thermoregulation. The reduction of HSP70 or HSP70/HSC70 expression does not affect the blood meal size, but impairs blood digestion by the insect.

Insects sense thermal cues mainly through thermoreceptors located in the antenna. To analyse the impact of antennectomy on the thermal behaviour of the haematophagous bug Triatoma infestans, we studied the distribution of intact and antennectomised bugs in an experimental arena where a temperature gradient was established, as well as the biting response of insects with and without antennae to objects at the temperature of a potential host. Antennectomy did not abolish thermopreference, but modified the temperature at which the insects preferred to stay. In the arena, antennectomised insects chose to remain at a higher temperature (ca. 3 °C higher in unfed bugs), and exhibited a larger dispersion around that preferred temperature, than intact bugs. In addition, ablated insects temporarily lost their ability to bite an object at the temperature of a potential host, but that ability was gradually recovered after the fifth day post-antennectomy. Results presented here show that thermoreceptors other than those located on the antennae can also guide thermal behaviours. We conclude that the function of antennal thermoreceptors can be taken over by other receptors located in different regions of the body. Those receptors have a different sensitivity and confer the insects with a different responsiveness.

International audience; Major emergency efforts are being mounted for each vector-borne disease epidemiological crisis anew, while knowledge about the biology of arthropods vectors is dwindling slowly but continuously, as is the number of field entomologists. The discrepancy between the rates of production of knowledge and its use and need for solving crises is widening, in particular due to the highly differing time spans of the two concurrent processes. A worldwide web based search using multiple key words and search engines of onsite and online courses in English, Spanish, Portuguese, French, Italian and German concerned with the biology of vectors identified over 140 courses. They are geographically and thematically scattered, the vast majority of them are on-site, with very few courses using the latest massive open online course (MOOC) powerfulness. Over two third of them is given in English and Western Africa is particularity poorly represented. The taxonomic groups covered are highly unbalanced towards mosquitoes. A worldwide unique portal to guide students of all grades and levels of expertise, in particular those in remote locations, is badly needed. This is the objective a new activity supported by the Special Programme for Research and Training in Tropical Diseases (TDR).

Learning and memory plays an important role in host preference and parasite transmission by disease vector insects. Historically there has been a dearth of standardized protocols that permit testing their learning abilities, thus limiting discussion on the potential epidemiological consequences of learning and memory to a largely speculative extent. However, with increasing evidence that individual experience and associative learning can affect processes such as oviposition site selection and host preference, it is timely to review the recently acquired knowledge, identify research gaps and discuss the implication of learning in disease vector insects in perspective with control strategies.

Blood-sucking vectors must overcome thermal stress caused by intake of proportionally large amounts of warm blood from their hosts. In response to this, Heat Shock Proteins (HSPs) such as the widely studied HSP70 family (the inducible HSP70 and the cognate form HSC70, known for their role in preserving essential cellular functions) are rapidly up-regulated in their tissues. The triatomine Rhodnius prolixus is an important vector of Trypanosoma cruzi , the causative pathogen of Chagas’ disease, and is also a model organism for studying insect biology and physiology. In this work, we observed that the expression of Rhodnius prolixus HSP70 was rapidly up-regulated in response to thermal shocks (0 °C and 40 °C) and also during the first hours after feeding on blood. HSP70/HSC70 RNAi knockdown elicited important alterations in R. prolixus physiological responses triggered by blood meal and starvation. HSP70/HSC70 knockdown insects showed lower resistance to prolonged starvation in comparison to appropriate controls, dying between 32 and 40 days after dsRNA injection. After blood feeding, the physiological effects of HSP70/HSC70 knockdown were more prominent and the insects died even earlier, within 14–20 days after feeding (21–27 days after dsRNA injection). These bugs showed impaired blood processing and digestion, reduced energetic metabolism and the midgut immune responses were compromised. Our findings suggest that HSP70/HSC70 depletion affected R. prolixus in starvation or fed conditions. After feeding, the arrival of blood in the digestive tract of knockdown insects fails to activate essential signaling pathways involved in blood processing, producing several alterations in their physiological processes enough to generate a premature death.

International audience; Feeding on the blood of vertebrates is a risky task for haematophagous insects and it can be reasonably assumed that it should also be costly in terms of energetic expenditure. Blood circulates inside vessels and it must be pumped through narrow tubular stylets to be ingested. We analysed the respiratory pattern and the energetic cost of taking a blood meal in Rhodnius prolixus using flow-through and stop-flow respirometry to measure carbon dioxide emission, oxygen consumption and water loss before and during feeding. We observed an increase of up to 17-fold in the metabolic rate during feeding and a change in the respiratory pattern, which switched from a discontinuous cyclic pattern during resting to a continuous pattern when the insects started to feed, remaining in this condition unchanged for several hours. The energetic cost of taking a meal was significantly higher when bugs fed on a living host, compared with feeding on an artificial feeder. No differences were observed between feeding on blood or on saline solution in vitro, revealing that the substrate for feeding (vessels versus membrane) and not the nature of the fluid was responsible for such a difference in the energetic cost. Water loss significantly increased during feeding, but did not vary with feeding method or type of food. The mean respiratory quotient in resting bugs was 0.83, decreasing during feeding to 0.52. These data constitute the first metabolic measures of an insect during blood feeding and provide the first insights into the energetic expenditure associated with haematophagy.

International audience; It has been largely assumed that the individual experience of disease vector insects may affect host choice and, as a consequence, have an important influence on parasite transmission. In particular, it is speculated that vector insects should be able to learn and remember the most and/or less defensive hosts, shifting their preference accordingly. Nevertheless, despite the invested efforts in testing the capacity to learn and remember information of blood-sucking insects, only little conclusive information has been obtained hitherto. Recently, the ability of Rhodnius prolixus to associate a behaviourally neutral odour to the perspective of either obtaining a blood-meal or being punished has been demonstrated, the same odour becoming attractant or repellent for the bugs, respectively, according to the individual previous experience. The present work represents a step forward in the study of the cognitive abilities of Chagas disease vectors and their influence on host choice. We tested whether or not bugs bias their choice for a host based on the association of its odour with a negative experience. Our results show that whereas naïve bugs presented no preference when confronted to the odour of two different hosts, bugs previously exposed to the contingency of the odour of one host and a mechanical perturbation mimicking defensive behaviour, biased their preference towards the other host. This constitutes the first evidence of olfactory conditioning to host odours in triatomine bugs, vectors of Chagas disease and one of the few available up to date on haematophagous insects. The epidemiological significance of this finding is discussed.

International audience; The adaptation to light of compound eyes in insects has been extensively documented and their adaptive role is well understood. Much less attention has been paid, however, to the control of ocelli sensitivity, a study which could help us to understand the functional role of these simple eyes. We analyzed the dynamic changes in the distribution of screening pigments which occur in the ocelli of the haematophagous bug, Triatoma infestans, when the insects are subjected either to light/dark cycles (LD), to constant darkness (DD) or constant light (LL). We then compared these changes with those occurring in the compound eyes of the same individuals and found that, while compound eyes are subject to the control of an endogenous circadian clock, the adaptation of the ocelli is entirely dependent on environmental illumination. In addition, we have observed that environmental temperature is not involved in the control of screening pigments in either ocelli or compound eyes as a direct stimulus, nor as a Zeitgeber. The existence of a differential control in the components of the dual visual system represents an adaptive advantage in the adjustment of visual sensitivity in insects exposed to quick changes in lighting conditions in their natural habitat. We discuss the implications of our findings with regards to the biology of triatomines and with respect to the general understanding the functional role of insect ocelli.

International audience; Heat is the principal host-associated cue for the blood-sucking bug Rhodnius prolixus. It is both necessary and sufficient to trigger the ''proboscis extension response'' (PER), an essential element of the feeding behaviour of this insect. The aim of this study was to determine whether the temperature of an object itself or the thermal contrast between the object and the environmental background is responsible for triggering the PER. Thermal stimuli at 25-50 C were presented in thermal environments of 20-40 C. The results showed that stimuli at 30 and 35 C trigger the highest rates of response, provided that they were presented in thermal backgrounds at temperatures of below 35 C. Thus, bugs display a preference for objects at temperatures corresponding to those at the surface of their vertebrate hosts (birds and mammals). However, this preference disappears if no heat exchange between the bug and its potential host is possible (i.e. if they are at the same temperature) and may even become negative if the insect and the surrounding environment are at a temperature above 35 C). In these situations, and when the object was too warm to be a potential host, PER rates were much lower. These findings have potential implications for the feeding strategies adopted by triatomine bugs in the natural tropical areas they inhabit.

SUMMARY After having demonstrated that blood-sucking bugs are able to associate a behaviourally neutral odour (l-lactic acid) with positive reinforcement (i.e. appetitive conditioning) in the first part of this study, we tested whether these insects were also able to associate the same odour with a negative reinforcement (i.e. aversive conditioning). Learned aversion to host odours has been repeatedly suggested as a determinant for the distribution of disease vectors among host populations. Nevertheless, no experimental evidence has been obtained so far. Adapting a classical conditioning approach to our haematophagous model, we trained larvae of Rhodnius prolixus to associate l-lactic acid, an odour perceived by bugs but behaviourally neutral when presented alone, with a mechanical perturbation (i.e. negative reinforcement). Naive bugs and bugs exposed to CS, punishment, or CS and punishment without contingency remained indifferent to the presence of an air stream loaded with l-lactic acid (random orientation on a locomotion compensator), whereas the groups previously exposed to the contingency CS–punishment were significantly repelled by l-lactic acid. In a companion paper, the opposite, i.e. attraction, was induced in bugs exposed to the contingency of the same odour with a positive reinforcement. These constitute the first pieces of evidence of olfactory conditioning in triatomine bugs and the first demonstration that the same host odour can be used by insects that are disease vectors to learn to recognize either a host to feed on or a potentially defensive one. The orientation mechanism during repulsion is also discussed in light of our results.

Artificial baits constitute important tools for the detection and sampling of blood-sucking arthropods, in particular those that are vectors of parasites affecting human health. At present, many different devices have been proposed to attract blood-sucking arthropods, mostly based on the attractiveness of particular chemicals or blends. However, most of them revealed themselves as unpractical (e.g. they require an electrical supply), expensive (e.g. gas bottles) or not efficient enough. On the other hand, the use of living baits is as effective but it has practical constraints and/or raises ethical questions. We present here a multimodal lure to attract blood-sucking arthropods designed taking into account both practical constraints and costs. The main characteristics of our bait are: (1) artificiality (no living-host); (2) multimodality (it associates heat, carbon dioxide and chemical attractants); (3) independency from any energy source; (4) no need for gas bottles; (5) easy to prepare and use in the field; (6) low cost. We tested the ability of the bait to attract blood-sucking arthropods in the laboratory and in the field, using capture sticky-traps. Our bait evinced to be almost as efficient as live hosts (mice) for the capture of Chagas disease and Borrelia vectors in Bolivia. The multimodal lure here presented is a generalist bait, i.e. effective for attracting different haematophagous species.

International audience; Optophysiological imaging methods can be used to record the activity in vivo of groups of neurons from particular areas of the nervous system (e.g. the brain) or of cell cultures. Such methods are used, for example, in the spatio-temporal coding and processing of sensory information. However, the data generated by optophysiological methods must be processed carefully if relevant results are to be obtained. The raw fluorescence data must be digitally filtered and analyzed appropriately to obtain activity maps and fluorescence time course for single spots.We used a Matlab® environment to implement the necessary procedures in a user-friendly manner.We developed OpenFluo, a program for people inexperienced in optophysiological methods and for advanced users wishing to perform simple, rapid data analyses without the need for complex, time-consuming programming procedures. This program will be made available as stand-alone software and as an open-source Matlab® tool. It will therefore be possible for experienced users to integrate their own routines. We validated this software by assessing its ability to process both artificial recordings and real biological data corresponding to recordings of the honeybee brain.

SUMMARYVertebrate blood is essential for the growth and the reproduction of haematophagous insects. Provided that hosts play the double role of food sources and predators, feeding on their blood exposes these insects to a high predation risk. Therefore, it is expected that host seeking occurs only when insects need to feed. In the present study, we analyse how the feeding status affects the response to host-associated cues in the blood-sucking insect Rhodnius prolixus. We show that the responsiveness to host-associated cues, such as CO2 and heat, and that the motivation to feed depend on the time elapsed since a blood meal. Depending on the time elapsed after feeding, the same concentration of CO2 may attract or repel the insects. As far as we know, this is the first time that a host signal has been shown to be repellent for a haematophagous insect. The response to heat is also modulated but no repellence was observed. When blood was replaced by saline solution as food, a significant reduction of the response to both signals was evinced but repellence was not observed. The injection of haemolymph from fed insects into starved insects inhibited the response of the receiver insects to both signals but this was not observed after the injection of saline solution or of haemolymph from non-fed insects. This is the first time that the modulation of feeding behaviour by the feeding status has been analysed in a hemimetabolous blood-sucking insect, fully excluding any effect of other processes, such as reproduction.

International audience; It has been demonstrated in several insect species that a circadian clock makes the whole of antennal chemoreceptors more sensitive during a particular temporal window every day. This assessment raises the question about how insects exhibiting bimodal activity handle their sensitivity to odours which are relevant at different moments of the day. To shed some light on this problem, we studied in Rhodnius prolixus the daily dynamics of their responsiveness to CO2 (host-associated cue) and aggregation cues (refuge-associated), which are relevant at dusk and dawn, respectively. We analysed: (1) whether a temporal modulation of the responsiveness to odours does exist in R. prolixus, (2) if this modulation is a general one or it is specific for each type of volatile, and (3) if it is controlled by exogenous or endogenous mechanisms. We found that the responsiveness to CO2 only occurs at dusk and that to assembling odours is restricted to dawn. Experiments under free-running conditions revealed that only the responsiveness to CO2 is controlled by a circadian clock, but not that to assembling signals. Thus, by combining endogenous and exogenous mechanisms, sensitivities to different odours are adjusted according to their associated behavioural context and moment of the day.

The thermal sense of triatomine bugs, vectors of Chagas disease, is unique among insects. Not only do these bugs exhibit the highest sensitivity to heat known in any animal up to date, but they can also perceive the infrared radiation emitted by the body of their warm-blooded hosts. The sensory basis of this capacity has just started to be unravelled. To shed additional light on our understanding of thermosensation, we initiated an analysis of the genetic basis of the thermal sense in Rhodnius prolixus. We tested the hypothesis that a TRPV (transient receptor potential vanilloid) channel receptor is involved in the evaluation of heat in this species. Two different approaches were adopted. Initially, we analysed the expression of a TRPV candidate for this function, i.e., RproIav, in different tissues. Subsequently, we tested the effects of capsaicin and capsazepine, two molecules known to interact with mammal TRPV1, using three different behavioural protocols for evaluating thermal responses: (1) proboscis extension response (PER), (2) thermopreference in a temperature gradient and (3) spatial learning in an operant conditioning context. Bioinformatic analyses confirmed that the characteristic features typical of the TRPV channel subfamily are found in the RproIav protein sequence. Molecular analysis showed that RproIav is expressed in R. prolixus, not only in the antennae, but also in other body structures bearing sensory organs. Behavioural experiments consistently revealed that capsaicin treated insects are less responsive to heat stimuli and prefer lower temperatures than non-treated insects, and that they fail to orient in space. Conversely, capsazepine induces the opposite behaviours. The latter data suggest that triatomine thermoreception is based on the activation of a TRP channel, with a similar mechanism to that described for mammal TRPV1. The expression of RproIav in diverse sensory structures suggests that this receptor channel is potentially involved in bug thermoreception. This constitutes solid evidence that thermosensation could be based on the activation of TRP receptors that are expressed in different tissues in R. prolixus. Whether RproIav channel is a potential target for the compounds tested and whether it mediates the observed effects on behaviour still deserves to be confirmed by further research. Fil: Zermoglio, Paula Florencia. Université François Rabelais de Tours; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina Fil: Latorre Estivalis, Jose Manuel. Fundación Oswaldo Cruz; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Crespo, José Emilio. Université François Rabelais de Tours; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina Fil: Lorenzo, Marcelo Gustavo. Fundación Oswaldo Cruz; Brasil Fil: Lazzari, Claudio Ricardo. Université François Rabelais de Tours; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Insect repellents are known since many decades ago and constitute a major tool for personal protection against the biting of mosquitoes. Despite their wide use, the understanding of why and how repellents repel is relatively recent. In particular, the question about to what extent insects other than mosquitoes are repulsed by repellents remains open. We developed a series of bioassays aimed to test the performance of well established as well as potential repellent molecules on the Chagas disease vector Rhodnius prolixus. Besides testing their ability to prevent biting, we tested the way in which they act, i.e., by obstructing the detection of attractive odours or by themselves. By using three different experimental protocols (host-biting, open-loop orientation to odours and heat-triggered proboscis extension response) we show that DEET repels bugs both in the presence and in the absence of host-associated odours but only at the highest quantities tested. Piperidine was effective with or without a host and icaridine only repelled in the absence of a living host. Three other molecules recently proposed as potential repellents due to their affinity to the Ir40a+ receptor (which is also activated by DEET) did not evoke significant repellency. Our work provides novel experimental tools and sheds light on the mechanism behind repellency in haematophagous bugs. Fil: Zermoglio, Paula Florencia. Centre National de la Recherche Scientifique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina Fil: Martin Herrou, Hadrien. Centre National de la Recherche Scientifique; Francia Fil: Bignon, Yohan. Centre National de la Recherche Scientifique; Francia Fil: Lazzari, Claudio Ricardo. Centre National de la Recherche Scientifique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina