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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.

Simple Summary Sucking lice are permanent and obligate ectoparasites throughout their whole life cycle. Echinophthiriids escorted their mammal hosts during their passage from fully terrestrial to amphibian life. Seal lice synchronize their reproduction cycle with that of their mammalian hosts. Echinophthiriids tolerate long immersion periods and extreme hydrostatic pressures. Diving lice can reach kilometers under the surface and survive, during the months their hosts remain in the open ocean. In the present work, we describe and discuss how some of these adaptations allow seal lice to cope with the amphibious habits of their hosts and how they can help us to understand why insects are so rare in the ocean. Abstract Insects are the most evolutionarily and ecologically successful group of living animals, being present in almost all possible mainland habitats; however, they are virtually absent in the ocean, which constitutes more than 99% of the Earth’s biosphere. Only a few insect species can be found in the sea but they remain at the surface, in salt marshes, estuaries, or shallow waters. Remarkably, a group of 13 species manages to endure long immersion periods in the open sea, as well as deep dives, i.e., seal lice. Sucking lice (Phthiraptera: Anoplura) are ectoparasites of mammals, living while attached to the hosts’ skin, into their fur, or among their hairs. Among them, the family Echinophthiriidae is peculiar because it infests amphibious hosts, such as pinnipeds and otters, who make deep dives and spend from weeks to months in the open sea. During the evolutionary transition of pinnipeds from land to the ocean, echinophthiriid lice had to manage the gradual change to an amphibian lifestyle along with their hosts, some of which may spend more than 80% of the time submerged and performing extreme dives, some beyond 2000 m under the surface. These obligate and permanent ectoparasites have adapted to cope with hypoxia, high salinity, low temperature, and, in particular, conditions of huge hydrostatic pressures. We will discuss some of these adaptations allowing seal lice to cope with their hosts’ amphibious habits and how they can help us understand why insects are so rare in the ocean.

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.

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.

The temperature of the environment is one of the most important abiotic factors affecting the life of insects. As poikilotherms, their body temperature is not constant, and they rely on various strategies to minimize the risk of thermal stress. They have been thus able to colonize a large spectrum of habitats. Mosquitoes, such as Ae. aegypti and Ae. albopictus, vector many pathogens, including dengue, chikungunya, and Zika viruses. The spread of these diseases has become a major global health concern, and it is predicted that climate change will affect the mosquitoes’ distribution, which will allow these insects to bring new pathogens to naïve populations. We synthesize here the current knowledge on the impact of temperature on the mosquito flight activity and host-seeking behavior (1); ecology and dispersion (2); as well as its potential effect on the pathogens themselves and how climate can affect the transmission of some of these pathogens (3).

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.

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; 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.

National audience; Despite the drastic consequences it may have on the transmission of parasites, the ability of disease vectors to learn and retain information has just begun to be characterised. The kissing bug Rhodnius prolixus, a vector of Chagas disease, is an excellent model, particularly because conditioning the proboscis extension response (PER) constitutes a valuable paradigm to study their cognitive abilities under carefully controlled conditions. Another characteristic of these bugs is the temporal organisation of their different activities in a bimodal endogenous daily rhythm. This offers the opportunity to address the implication of the circadian system in learning and memory. Using aversive conditioning of the PER, we tested whether the ability of kissing bugs to learn and remember information varies during the day. We found that bugs perform well during the night, but not during the day: their ability to acquire information – but not their ability to retrieve it – is modulated by time. When the bugs were kept under constant conditions in order to analyse the origin of this rhythm, the rhythm continued to free run, showing its endogenous and truly circadian nature. These results are the first to evince the effect of the circadian system on the learning abilities of disease vectors and one of the few in insects in general.

Compounds from the metasternal and Brindley's glands of the blood-sucking bug, Triatoma infestans, were identified by solid phase microextraction (SPME) and gas chromatography-mass spectrometry. Volatile compounds released by adult bugs during copulation or after mechanical disturbance were also characterized. Six compounds were identified and found consistently in all samples from metasternal glands. The most abundant were 3-pentanone, 2-methylbutanol, 3-pentanol, and an unidentified compound. The metasternal gland blends did not differ qualitatively between sexes. Compounds found in Brindley's glands were short chain acids, alcohols, esters, and a ketone with no qualitative differences between sexes. Isobutyric acid was the main component of this blend, and two new confirmed compounds were described as products of these glands: 2-butanone and 2-methylbutyric acid. 3-Pentanone was collected from the headspace over 33% of the copulating pairs of T. infestans. Volatiles found in the headspace of disturbed T. infestans adults included short-chain fatty acids, alcohols, esters, and ketones, with no qualitative differences between sexes. Both types of glands apparently discharge their contents after disturbance. However, most of the volatiles released by bugs after disturbance came from Brindley's glands. The locomotor activity of fourth instars increased significantly after stimulation with the odors emitted by disturbed adults, as compared with larvae stimulated by the odor of undisturbed adults or by clean air. We also studied the directional behavioral response of fifth instars to the disturbance scent in a locomotion compensator. Larvae exposed to volatiles released by disturbed adults walked away from the direction of the odor. The results suggest that this blend or part of it functions as an alarm pheromone for T. infestans. We suggest that the metasternal glands of this species are involved both in the sexual and the alarm contexts, and that the Brindley's glands probably have both alarm and defensive roles.

We studied the spectral sensitivity of the visual system of the blood-sucking bug Triatoma infestans, one of the main vectors of Chagas Disease in South America. We quantified the photonegative reaction of this insect in a rectangular arena, half of which was kept dark and the other half illuminated with various intensities of different monochromatic lights (or broadband stimuli for lambda665 nm). As a behavioral parameter of the photonegative response, we measured the time each insect spent in the dark half of the arena. We found that low intensity levels (under 0.06 microW/cm(2)) of monochromatic lights of 397, 458, 499, and 555 nm evoked a statistically significant (i.e., different from that of control groups) photonegative reaction. Insects were less sensitive to monochromatic lights of 357 nm (UV) and 621 nm (dark orange), and to broadband stimuli in the red part of the spectrum (665-695 nm). These findings indicate that the visual system of T. infestans is sensitive to broader regions of the spectrum than those previously reported.

The effect of the relative humidity (RH) of the environment on water loss is studied in Triatoma brasiliensis. Starved or recently fed early stages of T. brasiliensis are exposed to 15, 37, 46 and 73% RH. Water loss is estimated by weighing individual insects daily. The results obtained with starved insects show that the group maintained at 15% RH loses more water and shows a higher mortality compared with the others. An experiment in which recently fed nymphs are exposed to different RHs shows that all groups of insects lose the greatest amount of water during the first day. The group exposed to 15% RH show a higher water loss than the others before and after ecdysis. The survival of recently fed nymphs is not affected by the level of RH to which they are exposed. The results are discussed in relation to the water balance in this species.

The role of short-chain fatty acids in the host-seeking behaviour of Triatoma infestans larvae was investigated using a locomotion compensator. Several short-chain fatty acids were tested alone over a wide range of doses, or in combination with L-lactic acid (L-LA; 100 microg). Bugs showed no attractive response to single carboxylic acids, but when L-LA was added to airstreams carrying specific intensities of either propionic (C3; 100 microg), butyric (C4; 1 microg) or valeric acid (C5; 1 microg), these mixtures elicited an attractive response, evincing a synergistic effect. No orientation response was observed when caproic acid (C6) was offered with L-LA at the doses tested. Two blends were created: (1) C3, C4 and C5 combined at the effective doses when added with L-LA [C3C4C5 (1)], and (2) C3, C4 and C5 combined at a third of those intensities [C3C4C5 (2)]. Both blends were tested alone, with L-LA (100 microg), with a sub-threshold concentration of CO(2) (300 p.p.m. above the ambient level), and combined with both compounds together. Oriented responses of bugs were only observed with the blend (2) added with L-LA and with the combination of this lure with CO(2). This last combination evoked a behavioural response similar in intensity to that induced by a live mouse.

Behavioural responses of Triatoma infestans larvae to carbon dioxide and other odours of vertebrate origin were investigated in a locomotion compensator. T. infestans oriented towards airstreams enriched with carbon dioxide exhibiting a threshold response between 300 and 400 p.p.m. above the ambient CO(2) background. The accuracy of the oriented response to carbon dioxide improved with stimulus intensity. Remarkably, insects did not show any change in their sensitivity threshold to carbon dioxide with the starvation time. The attractiveness to carbon dioxide depended on the time of the day, i.e. these nocturnal bugs only oriented towards carbon dioxide-loaded airstreams during the first hours of the scotophase. L-lactic acid did not evoke oriented responses when it was presented as a single stimulus in a wide range of intensities. However, a marked synergism was evident when L-lactic acid was combined with a sub-threshold concentration of carbon dioxide. Under this condition, the threshold response to carbon dioxide decreased to 75-150 p.p.m. above ambient CO(2) background. The isomer D-lactic acid evoked no response, either alone or in combination with carbon dioxide. When insects were stimulated with 1-octen-3-ol a significant positive orientation was found. This response was not modified by the addition of carbon dioxide.

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.

The host-vector-parasite interaction offers a clear illustration of the adaptive value of biological rhythms. In this review, we summarise some of the information currently available on daily rhythms of insect vectors, particularly those responsible for the transmission of parasites to humans. Included amongst the cases described are circadian rhythms of locomotor and flight activity, and of eclosion and oviposition in tsetse flies, blood-sucking hemipterans, mosquitoes and other haematophagous insects. Both published and new data are presented, and they indicate that a study of the rhythms in disease-vector insects can provide an understanding of the value of this application of chronobiology to ecology and applied sciences. Related to this, the possibility of strategies for the control of insects based on their temporal characteristics is proposed; for example, the use of insecticides could be restricted to those phases of the day when the susceptibility of the insects to them is increased.

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 τ(D/D)=23.35 and τ(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.

The effects of temperature on the development of early stages and the thermopreference of nymphs and adults were analysed in the haematophagous bug Triatoma brasiliensis Neiva, 1911 (Hemiptera, Reduviidae). Egg hatching, mortality of nymphs, feeding and moulting success of the early stages of T. brasiliensis were all affected by temperature. While high rates of egg hatching were observed between 25 and 27 °C, no hatching occurred at 12, 19 and 38 °C. The mortality of first-instar nymphs was highest at 38 °C, at which no insects survived after 10 days of exposure. Feeding success was only affected at the lowest temperature (12 °C). No ecdysis was observed in the groups exposed to 12, 19 and 21 °C. Recently fed fourth-instar nymphs preferred to stay at a temperature of approximately 30 °C. The preferred temperature began to decline gradually to approximately 27 °C during ecdysis, reaching 26 °C at 30 days after ecdysis. After a second blood meal, the insects' preferred temperature was again approximately 30 °C. The thermopreference pattern of females was similar to that of nymphs. Nymphs and females showed a daily fluctuation in their preferred temperature, moving towards higher values at the beginning of the dark phase, and choosing lower ones after this time interval, at which they remained until the end of the light phase. The females laid their eggs in all sectors of the arena, although the largest numbers of eggs were found between 28 and 32 °C.

The behavioural response to water vapour of the haematophagous bug Triatoma infestans was analysed. Dry or humid discrete sources at different temperatures were used as stimuli for insects walking on a locomotion compensator. Humidity significantly increased the tendency of these bugs to orientate towards thermal sources. Furthermore, humid sources at room temperature were attractive to T. infestans, but this effect was limited to short-range distances. On the other hand, dynamic sources, i.e. airstreams carrying different water vapour contents did not affect the spontaneous anemotactic behaviour of this species, neither in sign (positive) nor in intensity. The anemotactic behaviour was also not influenced by the physiological water balance state of the bugs. Results are discussed in relation to the cues released by living hosts of triatomine bugs and in relation to their responses to air-currents.

The morphology and fine structure of the ocelli of Triatoma infestans have been analyzed by means of light and electron microscopy. The two dorsal ocelli of this species are located behind the compound eyes, looking dorsally and frontally. Externally, the ocelli are marked by the corneal lenses virtually spherical in form and limited internally by a cuticular apodeme. The lens focuses the incoming rays beyond the retina. A single layer of corneagen cells lies below the cuticular lens. The corneagen cells and photoreceptors are arranged in a cup-like fashion beneath the cuticular lens. A distal retinal zone comprises the rhabdoms, which are laterally connected in an hexagonal meshwork. A middle retinal zone comprises the receptor cell segment free of rhabdom, and a proximal zone their axons. In the middle zone, the oviform nuclei and spheroids are located. Screening pigment granules are present within the retinal cell. Spherical mitochondria are homogeneously distributed in the cytoplasm of the cell body. In the axonal zone, mitochondria are found in the peripheral region. Axons from receptor cells extend into the ocellar neuropile at the base of the ocelli, to synapse with second order neurons. The large axons of second order neurons are bundled by glial cells. The ocellar plexus exhibits a high diversity of synaptic unions (i.e. axo-dendritic, axo-axonic, dendro-axonic, and dendro-dendritic).

The thermopreference of resting P. megistus (Burmeister, 1834) adults was studied in a temperature gradient. We also determined the thermopreference for oviposition and ecdysis. Thermopreference of resting individuals depended on the feeding state of the insects. Results demonstrated an initial resting preference of ≈29°C, which gradually changed to 26–27°C with increasing starvation. A daily variation in thermopreference was also observed with this species. The insects showed a daily maximum preference at the beginning of the dark phase, and a minimum preference during the first half of the light phase. Ecdysis took place all along the gradient, but exhibited a maximum frequency at 30°C. Most eggs were found between 25 and 29°C. Results are discussed in relation to the ecology of P. megistus as compared with other related species, and the existence of mechanisms of behavioral thermoregulation in the group.

SUMMARY We analysed dynamic changes in the ommatidial structure of the compound eyes of Triatoma infestans. This nocturnal insect possesses open-rhabdom eyes, in which a ring of six rhabdomeres from retinula cells 1–6 (R1–6) surrounds a central pair of rhabdomeres from retinula cells 7 and 8 (R7–8). Screening pigments are located in all the photoreceptors and in the primary (PPC) and secondary (SPC) pigment cells. During the day, pigments within R1–6 and the PPCs form a small ‘pupil’ above the rhabdom and pigments within R7–8 are clustered around the central rhabdomere, allowing light to reach only the central rhabdomere. At night, the ‘pupil’ widens, and pigments inside R7–8 concentrate in the proximal region of the cells, allowing light to reach the peripheral rhabdomeres. In addition, the distance between the cornea and the rhabdom decreases. These rhythmic changes adapt the sensitivity of the eye by controlling the amount of light reaching and travelling within the rhabdom. Furthermore, the rhythm persists under conditions of constant darkness (DD), i.e. it is controlled by an endogenous oscillator. Remarkably, there are differences in pigment movements between the retinula cells of a single ommatidium. The migration of pigments in R1–6 is regulated by a circadian input, while that in R7–8 is regulated by both direct light and circadian inputs. The rhythm vanishes under constant-light conditions (LL). In this species, the circadian rhythm of photonegative behaviour persists in both DD and LL conditions, suggesting that these two rhythms, in retinal morphology and visual behaviour, may be generated by different circadian oscillators.

International audience; Triatominae are blood-sucking insects that localise their hosts with their multimodal host associatedperceptive signals. Among that sensory information, one of the main short-range cue is heat which, evenin isolation, is able to trigger the Proboscis Extension Response (PER) preceding the bite. Previous studieshave demonstrated a rhythmic variation of the response to host signals compatible with the nocturnalhabits of triatomine insects. This study was aimed at determining whether the PER to heat was alsomodulated by a daily variation and, if so, if it was an endogenous or exogenous rhythm. We also investigatedwhat was used as a synchroniser between light and temperature. The results showed that alternationof light and darkness was required to ensure a high level of responsiveness to heat in earlyscotophase while the temperature cycle appeared with a weaker synchronisation power. We concludedthat the responsiveness to heat was an exogenous rhythm synchronised by a complementary control oflight and temperature cycles. The involvement of two synchronisers hierarchically organised might haveincreased the plasticity of the host-seeking behaviour to cope with environmental conditions