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.

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.

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.

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.

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.

Soft ticks perform evaporative cooling during blood-feeding.

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 of Ornithodoros. 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 on warm-blooded hosts.

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.

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.

The thermal sense of blood-sucking insects: why physics matters.

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.

Thermoprotective adaptations are critical for arthropods feeding on warm-blooded hosts.

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.

Effects of the Environmental Temperature on Aedes aegypti and Aedes albopictus Mosquitoes: A Review.

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 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.

Endothermy in the temperate scarab Cyclocephala signaticollis.

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.

Keeping cool: Kissing bugs avoid cannibalism by thermoregulating.

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.

Countercurrent heat exchange and thermoregulation during blood-feeding in kissing bugs.

Blood-sucking insects experience thermal stress at each feeding event on endothermic vertebrates. We used thermography to examine how kissing-bugs Rhodnius prolixus actively protect themselves from overheating. During feeding, these bugs sequester and dissipate the excess heat in their heads while maintaining an abdominal temperature close to ambient. We employed a functional-morphological approach, combining histology, µCT and X-ray-synchrotron imaging to shed light on the way these insects manage the flow of heat across their bodies. The close alignment of the circulatory and ingestion systems, as well as other morphological characteristics, support the existence of a countercurrent heat exchanger in the head of R. prolixus, which decreases the temperature of the ingested blood before it reaches the abdomen. This kind of system has never been described before in the head of an insect. For the first time, we show that countercurrent heat exchange is associated to thermoregulation during blood-feeding.

Associative visual learning by tethered bees in a controlled visual environment.

Free-flying honeybees exhibit remarkable cognitive capacities but the neural underpinnings of these capacities cannot be studied in flying insects. Conversely, immobilized bees are accessible to neurobiological investigation but display poor visual learning. To overcome this limitation, we aimed at establishing a controlled visual environment in which tethered bees walking on a spherical treadmill learn to discriminate visual stimuli video projected in front of them. Freely flying bees trained to walk into a miniature Y-maze displaying these stimuli in a dark environment learned the visual discrimination efficiently when one of them (CS+) was paired with sucrose and the other with quinine solution (CS-). Adapting this discrimination to the treadmill paradigm with a tethered, walking bee was successful as bees exhibited robust discrimination and preferred the CS+ to the CS- after training. As learning was better in the maze, movement freedom, active vision and behavioral context might be important for visual learning. The nature of the punishment associated with the CS- also affects learning as quinine and distilled water enhanced the proportion of learners. Thus, visual learning is amenable to a controlled environment in which tethered bees learn visual stimuli, a result that is important for future neurobiological studies in virtual reality.