Mosquitoes do not Like Bitter.

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

Response to thermal and infection stresses in an American vector of visceral leishmaniasis.

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.

Thermal tolerance of two Diptera that pollinate thermogenic plants.

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 as fuel: the metabolic cost of pedestrian locomotion in Rhodnius prolixus.

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 Rhodnius prolixus using flow-through respirometry, by measuring carbon dioxide emission and water loss before, during and after walking. We observed an increase in the metabolic rate during walking as compared with resting of up to 1.7-fold in male R. prolixus and 1.5-fold in females, as well as a change in their respiratory pattern, which switched from cyclic during rest to continuous when the insects started to walk, remaining in this condition during locomotion and for several minutes after stopping. Walking induced a significant loss of mass in both males and females. This can be explained by an increase in both metabolic rate and water loss during walking. These data constitute the first metabolic measures of active haematophagous insects and provide the first insights into the energetic expenditure associated with the active search for blood in this group.

Under pressure: the extraordinary survival of seal lice in the deep sea.

Lice from pinnipeds - sea lions, seals and walruses - are the only insects capable of surviving marine dives. Throughout their evolutionary history, they have 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: 30, 80, 150 or 200 kg cm-2 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 cm-2 (close to 200 atm), which is equivalent to 2000 m depth. Adults exhibited lower recovery times than nymphs after immersion at high hydrostatic pressure. Our findings show that lice have developed unique adaptations to endure extreme marine conditions. We discuss these extreme performances in relation to the morphological characteristics and physiological responses to diving in these insects.

Glyphosate impairs learning in Aedes aegypti mosquito larvae at field-realistic doses.

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.

Haematophagy is costly: respiratory patterns and metabolism during feeding in Rhodnius prolixus.

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.

Circadian modulation of learning ability in a disease vector insect, Rhodnius prolixus.

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.

Thermal effect of blood feeding in the telmophagous fly Glossina morsitans morsitans.

During feeding on warm-blooded hosts, haematophagous insects are exposed to thermal stress due to the ingestion of a meal which temperature may highly exceed their own body temperature. In order to avoid overheating and its subsequent deleterious effects, these insects respond by setting up molecular protective mechanisms such as heat shock proteins synthesis or by using thermoregulative strategies. Moreover, the duration of contact with the host depends on the way of feeding displayed by the different species (either telmophagous or solenophagous) and thus also impacts their exposure to heat. Solenophagous insects feed directly on blood vessels and are relatively slow feeders while telmophagous insects by lacerating capillaries, facilitate their access to blood and thus feed more quickly. The aim of this work was to investigate to what extent strictly telmophagous insects such as tsetse flies are exposed to thermal stress during feeding and consequently to evaluate the impact of the feeding strategy on the exposition to overheating in haematophagous insects in general. Real time thermographic analysis during feeding revealed that the flies' body significantly heat up quite homogeneously. At the end of feeding, however, a marked regional heterothermy occurs as a consequence of the alary muscles warm up that precedes take-off. Feeding strategies, either solenophagy or telmophagy, thus appear to have a great impact on both exposition to predation risks and to thermal stress.

Infrared detection without specialized infrared receptors in the bloodsucking bug Rhodnius prolixus.

Bloodsucking bugs use infrared radiation (IR) for locating warm-blooded hosts and are able to differentiate between infrared and temperature (T) stimuli. This paper is concerned with the neuronal coding of IR in the bug Rhodnius prolixus. Data obtained are from the warm cells in the peg-in-pit sensilla (PSw cells) and in the tapered hairs (THw cells). Both warm cells responded to oscillating changes in air T and IR with oscillations in their discharge rates. The PSw cells produced stronger responses to T oscillations than the THw cells. Oscillations in IR did the reverse: they stimulated the latter more strongly than the former. The reversal in the relative excitability of the two warm cell types provides a criterion to distinguish between changes in T and IR. The existence of strongly responsive warm cells for one or the other stimulus in a paired comparison is the distinguishing feature of a "combinatory coding" mechanism. This mechanism enables the information provided by the difference or the ratio between the response magnitudes of both cell types to be utilized by the nervous system in the neural code for T and IR. These two coding parameters remained constant, although response strength changed when the oscillation period was altered. To discriminate between changes in T and IR, two things are important: which sensory cell responded to either stimulus and how strong was the response. The label warm or infrared cell may indicate its classification, but the functions are only given in the context of activity produced in parallel sensory cells.

Differential effects of ambient temperature on warm cell responses to infrared radiation in the bloodsucking bug Rhodnius prolixus.

Thermoreceptors provide animals with background information about the thermal environment, which is at least indirectly a prerequisite for thermoregulation and assists bloodsucking insects in the search for their host. Recordings from peg-in-pit sensilla and tapered hairs on the antennae of the bug Rhodnius prolixus revealed two physiologically different types of warm cells. Both types responded more strongly to temperature pulses produced by switching between two air streams at different constant temperatures than to infrared radiation pulses employed in still air. In addition, both warm cells were better able to discriminate small changes in air temperature than in infrared radiation. As convective and radiant heat determines the discharge, it is impossible for a single warm cell to signal the nature of the stimulus unequivocally. Individual responses are ambiguous, not with regard to temperature change, but with regard to its source. We argue that the bugs use mechanical flow information to differentiate between pulses of convective and radiant heat. However, if pulses of radiant heat occur together with a constant temperature air stream, the mechanical cues would not allow avoiding ambiguity that convective heat introduces into radiant heat stimulation. In this situation, the warm cell in the tapered hairs produced stronger responses than those in the peg-in-pit sensilla. The reversal in the excitability of the two types of warm cells provides a criterion by which to distinguish the combination of convective and radiant heat from the stimuli presented alone.

Habituation leads to short but not long term memory formation in mosquito larvae.

In animals, memory allows to remember important locations and conserve energy by not responding to irrelevant stimuli. However, memory formation and maintenance are metabolically costly, making it worthwhile to understand the mechanisms underlying different types of memory and their adaptive value. In this study, we investigated the memory persistence of Aedes aegypti mosquito larvae, after habituation to a visual stimulus. We used an automated tracking system for quantifying the response of mosquito larvae to the passage of a shadow, simulating an approaching predator. First, we compared different retention times, from 4 min to 24 h, and found that mosquito larvae only exhibited memory capabilities less than 3 h after training. Secondly, we investigated the role of inter-trial intervals in memory formation. In contrast to other aquatic invertebrates, mosquito larvae showed no long-term memory even at long inter-trial intervals (i.e., 5 min and 10 min). Our results are discussed in relation to the ecological constraints.

Challenging Popular Belief, Mosquito Larvae Breathe Underwater.

Immature mosquitoes are thought to breathe only atmospheric air through their siphons despite reports of prolonged submerged survival. We studied the survival of last-instar larvae of Aedes aegypti fully submerged at different temperatures and measured the oxygen consumption from air and water-dissolved larvae and pupae of this species under different conditions. Larvae survived much longer than expected, reaching 50% mortality only after 58, 10, and 5 days at 15°, 25°, and 35 °C, respectively. Larval to pupa molt was only observed in larvae with access to air, whereas individuals kept submerged never molted. Although most of the oxygen was obtained from the air, larvae obtained 12.72% of their oxygen from the water, while pupae took only 5.32%. In both media, temperature affected the respiration rate of the larvae, with relatively close Q10 values (1.56 and 1.83 for water and air, respectively). A similar pattern of O2 consumption was observed in Ae. albopictus, whose larvae obtained 12.14% of their oxygen from the water. The detailed quantification of oxygen consumption by mosquito larvae showed that water-dissolved oxygen is not negligible and physiologically relevant, challenging the idea that mosquito larvae only breathe atmospheric oxygen.

Learning and memory in Rhodnius prolixus: habituation and aversive operant conditioning of the proboscis extension response.

It has been largely accepted that the cognitive abilities of disease vector insects may have drastic consequences on parasite transmission. However, despite the research effort that has been invested in the study of learning and memory in haematophagous insects, hitherto few conclusive results have been obtained. Adapting procedures largely validated in Drosophila, honeybees and butterflies, we demonstrate here that the proboscis extension response (PER) of the haematophagous insect Rhodnius prolixus can be modulated by non-associative (habituation) and associative (aversive conditioning) learning forms. Thermal stimuli were used as both unconditional stimulus (appetitive temperatures) and negative reinforcement (thermal shock). In the first part of this work, the PER was habituated and dishabituated to thermal stimuli, demonstrating the true central processing of information and discarding motor fatigue or sensory adaptation. Habituation was revealed to be modulated by the spatial context. In the second part, bugs that were submitted to aversive operant conditioning stopped responding with PER to thermal stimulation more quickly than by habituation. They were able to use their training experience when tested up to 72 h later. Our work constitutes the first demonstration of PER habituation and conditioning in a blood-sucking insect and provides reproducible experimental tools for the study of the mechanisms underlying learning and memory in disease vectors.

Oxidative stress, photodamage and the role of screening pigments in insect eyes.

Using red-eyed mutant triatomine bugs (Hemiptera: Reduvidae), we tested the hypothesis of an alternative function of insect screening pigments against oxidative stress. To test our hypothesis, we studied the morphological and physiological changes associated with the mutation. We found that wild-type eyes possess a great amount of brown and red screening pigment inside the primary and secondary pigment cells as well as in the retinular cells. Red-eyed mutants, however, have only scarce red granules inside the pigmentary cells. We then compared the visual sensitivity of red-eyed mutants and wild types by measuring the photonegative responses of insects reared in light:dark cycles [12 h:12 h light:dark (LD)] or constant darkness (DD). Finally, we analyzed both the impact of oxidative stress associated with blood ingestion and photodamage of UV light on the eye retina. We found that red-eyed mutants reared in DD conditions were the most sensitive to the light intensities tested. Retinae of LD-reared mutants were gradually damaged over the life cycle, while for DD-reared insects retinae were conserved intact. No retinal damage was observed in non-fed mutants exposed to UV light for 2 weeks, whereas insects fed on blood prior to UV exposure showed clear signs of retinal damage. Wild-type insects exposed to UV light showed a marked increase in the amount and density of screening pigments.

Assessing learning in mosquito larvae using video-tracking.

Mosquito larvae display a stereotyped escape response when they rest attached to the water surface. It consists in detaching from the surface and diving, to return to the surface after a brief time. It has been shown that this response can be evoked several times, by repeatedly presenting a moving shadow. Diving triggered by a potential danger revealed as a simple bioassay for investigating behavioural responses in mosquito larvae, in particular their ability to learn. In the present work, we describe an automated system, based on video-tracking individuals, and extracting quantitative data of their movements. We validated our system, by reinvestigating the habituation response of larvae of Aedes aegypti reared in the laboratory, and providing original data on field-collected larvae of genera Culex and Anopheles. Habituation could be demonstrated to occur in all the species, even though it was not possible to induce dishabituation in Culex and Anopheles mosquitoes. In addition to non-associative learning, we characterised motor activity in the studied species, thanks to the possibility offered by the tracking system to extract multiple variables. The here-described system and algorithms can be easily adapted to multiple experimental situations and variables of interest.

Learning the way to blood: first evidence of dual olfactory conditioning in a blood-sucking insect, Rhodnius prolixus. I. Appetitive learning.

It has been largely assumed that the individual experience of insects that are disease vectors might not only contribute to animal fitness, but also have an important influence on parasite transmission. Nevertheless, despite the invested efforts in testing the capacity to learn and remember information in blood-sucking insects, only little conclusive information has been obtained to date. Adapting a classical conditioning approach to our haematophagous model, we trained larvae of Rhodnius prolixus to associate L-lactic-acid, an odour perceived by these bugs but behaviourally neutral when presented alone, with food (i.e. positive reinforcement). Naive bugs--those exposed either to a conditioned stimulus (CS, L-lactic acid), unconditioned stimulus (US, heat) and reward (blood) alone or CS, US and reward in the absence of contingency--remained indifferent to the presence of an air stream loaded with L-lactic acid when tested in an olfactometer (random orientation), whereas the groups previously exposed to the contingency CS-US-reward (blood) were significantly attracted by L-lactic-acid. In a companion paper, the opposite, i.e. repellence, was induced in bugs exposed to the contingency of the same odour with a negative reinforcement. This constitutes the first evidence of olfactory conditioning in triatomine bugs, vectors of Chagas disease, and one of the few substantiations available to date of olfactory conditioning in haematophagous insects.

Learning the way to blood: first evidence of dual olfactory conditioning in a blood-sucking insect, Rhodnius prolixus. II. Aversive learning.

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.

How Did Seal Lice Turn into the Only Truly Marine Insects?

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.

Triggering the proboscis extension reflex (PER) in Rhodnius prolixus.

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.