Aedes albopictus colonies from different geographic origins differ in their sleep and activity levels but not in the time of peak activity.

Mosquitoes occupy a wide range of habitats where they experience various environmental conditions. The ability of some species, such as the tiger mosquito, Aedes albopictus, to adapt to local conditions certainly contributes to their invasive success. Among traits that remain to be examined, mosquitoes' ability to time their activity with that of the local host population has been suggested to be of significant epidemiological importance. However, whether different populations display heritable differences in their chronotype has not been examined. Here, we compared laboratory strains originating from 8 populations from 3 continents, monitored their spontaneous locomotor activity patterns, and analyzed their sleep-like states. Overall, all strains showed conserved diurnal activity concentrated in the hours preceding the crepuscule. Similarly, they all showed increased sleep levels during the morning and night hours. However, we observed strain-specific differences in the activity levels at each phase of the day. We also observed differences in the fraction of time that each strain spends in a sleep-like state, explained by variations in the sleep architecture across strains. Human population density and the latitude of the site of geographic origin of the tested strain showed significant effects on sleep and activity patterns. Altogether, these results suggest that Ae. albopictus mosquitoes adapt to local environmental conditions via heritable adaptations of their chronotype.

Population-specific responses to developmental temperature in the arboviral vector Aedes albopictus: Implications for climate change.

The increase of environmental temperature due to current global warming is not only favouring the expansion of the distribution range of many insect species, but it is also changing their phenology. Insect phenology is tightly linked to developmental timing, which is regulated by environmental temperatures. However, the degree to which the effects of developmental temperatures extend across developmental stages and their inter-stage relationships have not been thoroughly quantified in mosquitoes. Here, we used the mosquito Aedes albopictus, which is an aggressive invasive species and an arboviral vector, to study how developmental temperature influences fitness across developmental stages, thermal traits, energy reserves, transcriptome and Wolbachia prevalence in laboratory-reared populations originally collected from either temperate or tropical regions. We show that hatchability, larval and pupal viability and developmental speed are strongly influenced by temperature, and these effects extend to wing length, body mass, longevity and content of water, protein and lipids in adults in a population-specific manner. On the contrary, neither adult thermal preference nor heat resistance significantly change with temperature. Wolbachia density was generally lower in adult mosquitoes reared at 18°C than at other tested temperatures, and transcriptome analysis showed enrichment for functions linked to stress responses (i.e. cuticle proteins and chitin, cytochrome p450 and heat shock proteins) in mosquitoes reared at both 18 and 32°C. Our data showed an overall reduced vector fitness performance when mosquitoes were reared at 32°C, and the absence of isomorphy in the relationship between developmental stages and temperature in the laboratory population deriving from larvae collected in northern Italy. Altogether, these results have important implications for reliable model projections of the invasion potentials of Ae. albopictus and its epidemiological impact.

Sugar feeding by invasive mosquito species on ornamental and wild plants.

Feeding on plant-derived sugars is an essential component of mosquito biology, affecting key aspects of their lives such as survival, metabolism, and reproduction. Among mosquitoes, Aedes aegypti and Aedes albopictus are two invasive mosquito species in the US, and are vectors of diseases such as dengue fever, chikungunya, and Zika. These species live in heavily populated, urban areas, where they have high accessibility to human hosts as well as to plants in backyards and public landscapes. However, the range of plants that are suitable sugar hosts for these species remains to be described, despite the importance of understanding what plants may attract or repel mosquitoes to inform citizens and municipal authorities accordingly. Here, we tested whether Ae. aegypti and Ae. albopictus would sugar-feed on eleven commonly planted ornamental plant species. We confirmed feeding activity using the anthrone method and identified the volatile composition of plant headspace using gas-chromatography mass-spectroscopy. These chemical analyses revealed that a broad range of olfactory cues are associated with plants that mosquitoes feed on. This prompted us to use plant DNA barcoding to identify plants that field-caught mosquitoes feed on. Altogether, results show that native and invasive mosquito species can exploit a broader range of plants than originally suspected, including wild and ornamental plants from different phyla throughout the Spring, Summer and Fall seasons.

The role of Culex territans mosquitoes in the transmission of Batrachochytrium dendrobatidis to amphibian hosts.

BACKGROUND: Mosquitoes are the deadliest organisms in the world, killing an estimated 750,000 people per year due to the pathogens they can transmit. Mosquitoes also pose a major threat to other vertebrate animals. Culex territans is a mosquito species found in temperate zones worldwide that feeds almost exclusively on amphibians and can transmit parasites; however, little is known about its ability to transmit other pathogens, including fungi. Batrachochytrium dendrobatidis (Bd) is a topical pathogenic fungus that spreads through contact. With amphibian populations around the world experiencing mass die-offs and extinctions due to this pathogen, it is critical to study all potential modes of transmission. Because Cx. territans mosquitoes are in contact with their hosts for long periods of time while blood-feeding, we hypothesize that they can transmit and pick up Bd. METHODS: In this study, we first assessed Cx. territans ability to transfer the fungus from an infected surface to a clean one under laboratory conditions. We also conducted a surveillance study of Bd infections in frogs and mosquitoes in the field (Mountain Lake Biological station, VA, USA). In parallel, we determined Cx. territans host preference via blood meal analysis of field caught mosquitoes. RESULTS: We found that this mosquito species can carry the fungus to an uninfected surface, implying that they may have the ability to transmit Bd to their amphibian hosts. We also found that Cx. territans feed primarily on green frogs (Rana clamitans) and bullfrogs (Rana catesbeiana) and that the prevalence of Bd within the frog population at our field site varied between years. CONCLUSIONS: This study provides critical insights into understanding the role of amphibian-biting mosquitoes in transmitting pathogens, which can be applied to disease ecology of susceptible amphibian populations worldwide.

Recent advances in insect thermoregulation.

Ambient temperature (Ta) is a critical abiotic factor for insects that cannot maintain a constant body temperature (Tb). Interestingly, Ta varies during the day, between seasons and habitats; insects must constantly cope with these variations to avoid reaching the deleterious effects of thermal stress. To minimize these risks, insects have evolved a set of physiological and behavioral thermoregulatory processes as well as molecular responses that allow them to survive and perform under various thermal conditions. These strategies range from actively seeking an adequate environment, to cooling down through the evaporation of body fluids and synthesizing heat shock proteins to prevent damage at the cellular level after heat exposure. In contrast, endothermy may allow an insect to fight parasitic infections, fly within a large range of Ta and facilitate nest defense. Since May (1979), Casey (1988) and Heinrich (1993) reviewed the literature on insect thermoregulation, hundreds of scientific articles have been published on the subject and new insights in several insect groups have emerged. In particular, technical advancements have provided a better understanding of the mechanisms underlying thermoregulatory processes. This present Review aims to provide an overview of these findings with a focus on various insect groups, including blood-feeding arthropods, as well as to explore the impact of thermoregulation and heat exposure on insect immunity and pathogen development. Finally, it provides insights into current knowledge gaps in the field and discusses insect thermoregulation in the context of climate change.

Effect of Temperature on Mosquito Olfaction.

Mosquitoes use a wide range of cues to find a host to feed on, eventually leading to the transmission of pathogens. Among them, olfactory cues (e.g., host-emitted odors, including CO2, and skin volatiles) play a central role in mediating host-seeking behaviors. While mosquito olfaction can be impacted by many factors, such as the physiological state of the insect (e.g., age, reproductive state), the impact of environmental temperature on the olfactory system remains unknown. In this study, we quantified the behavioral responses of Aedes aegypti mosquitoes, vectors of dengue, yellow fever, and Zika viruses, among other pathogens, to host and plant-related odors under different environmental temperatures.

Soap application alters mosquito-host interactions.

To find nutrients, mosquitoes use volatile organic compounds (VOCs) emitted by plants and animal hosts. These resources overlap in their chemical composition, and an important layer of information resides in VOCs' relative abundance in the headspace of each resource. In addition, a large majority of the human species regularly uses personal care products such as soaps and perfumes, which add plant-related VOCs to their olfactory signature. Using headspace sampling and gas chromatography-mass spectrometry, we quantified how human odor is modified by soap application. We showed that soaps alter mosquito host selection, with some soaps increasing the attractiveness of the host and some soaps reducing it. Analytical methods revealed the main chemicals associated with these changes. These results provide proof-of-concept that data on host-soap valences can be reverse-engineered to produce chemical blends for artificial baits or mosquito repellents, and evince the impact of personal care products on host selection processes.

Effect of temperature on mosquito olfaction.

Mosquitoes use a wide range of cues to find a host to feed on, eventually leading to the transmission of pathogens. Among them, olfactory cues ( e.g. , host emitted odors, including CO 2 , and skin volatiles) play a central role in mediating host seeking behaviors. While mosquito olfaction can be impacted by many factors, such as the physiological state of the insect ( e.g. , age, reproductive state), the impact of environmental temperature on the olfactory system remains unknown. In this study, we quantified the behavioral responses of Aedes aegypti mosquitoes, vectors of dengue, yellow fever and Zika viruses, to host and plant related odors under different environmental temperatures.

Discovering Aethina tumida responses to attractant and repellent molecules: A potential basis for future management strategies.

Small hive beetle (Aethina tumida) management has been highly dependent upon chemical and mechanical control over the past two decades; however, many of these methods have not been consistently effective or safe for European honey bee (Apis mellifera) colonies. Here we explore the behavioral and physiological effects of the attractants isopentyl acetate and pollen patty upon A. tumida adults, and also investigate the mixture of attractants with repellent compounds, which were previously untested against A. tumida. Electroantennograms established sensitivity of A. tumida antennae to both attractants and all repellents with the exception of DEET, with antennae displaying greatest sensitivity to the repellent pyrrolidine. A walking-response olfactometer, designed specifically for A. tumida, was used for all behavioral experiments. It was found that both pollen patty and isopentyl acetate were attractive to A. tumida adults; conversely, mixes of attractants and repellent volatiles led to less attraction or avoidance of what was previously a significantly attractive source. Of all repellents tested, pyrrolidine was found to be the most repelling molecule, with significant avoidance of the attractive source at a 10 mg treatment of pyrrolidine. The results of this study indicate that, at the behavioral level, the repellent compounds pyrrolidine and 1,4-dimethylpiperazine resulted in a negative preference index indicating a repellent behavioral response. By strategically implementing a repellent source in an apiary environment, A. tumida adults could be deterred from entering and invading hives.

Neuromodulation and differential learning across mosquito species.

Mosquitoes can change their feeding behaviours based on past experiences, such as shifting from biting animals to biting humans or avoiding defensive hosts (Wolff & Riffell 2018 J. Exp. Biol. 221, jeb157131. (doi:10.1242/jeb.157131)). Dopamine is a critical neuromodulator for insects, allowing flexibility in their feeding preferences, but its role in the primary olfactory centre, the antennal lobe (AL), remains unclear (Vinauger et al. 2018 Curr. Biol. 28, 333-344.e8. (doi:10.1016/j.cub.2017.12.015)). It is also unknown whether mosquitoes can learn some odours and not others, or whether different species learn the same odour cues. We assayed aversive olfactory learning in four mosquito species with different host preferences, and found that they differentially learn odours salient to their preferred host. Mosquitoes that prefer humans learned odours found in mammalian skin, but not a flower odour, and a nectar-feeding species only learned a floral odour. Comparing the brains of these four species revealed significantly different innervation patterns in the AL by dopaminergic neurons. Calcium imaging in the Aedes aegypti AL and three-dimensional image analyses of dopaminergic innervation show that glomeruli tuned to learnable odours have significantly higher dopaminergic innervation. Changes in dopamine expression in the insect AL may be an evolutionary mechanism to adapt olfactory learning circuitry without changing brain structure and confer to mosquitoes an ability to adapt to new hosts.

Warm Blood Meal Increases Digestion Rate and Milk Protein Production to Maximize Reproductive Output for the Tsetse Fly, Glossina morsitans.

The ingestion of blood represents a significant burden that immediately increases water, oxidative, and thermal stress, but provides a significant nutrient source to generate resources necessary for the development of progeny. Thermal stress has been assumed to solely be a negative byproduct that has to be alleviated to prevent stress. Here, we examined if the short thermal bouts incurred during a warm blood meal are beneficial to reproduction. To do so, we examined the duration of pregnancy and milk gland protein expression in the tsetse fly, Glossina morsitans, that consumed a warm or cool blood meal. We noted that an optimal temperature for blood ingestion yielded a reduction in the duration of pregnancy. This decline in the duration of pregnancy is due to increased rate of blood digestion when consuming warm blood. This increased digestion likely provided more energy that leads to increased expression of transcript for milk-associated proteins. The shorter duration of pregnancy is predicted to yield an increase in population growth compared to those that consume cool or above host temperatures. These studies provide evidence that consumption of a warm blood meal is likely beneficial for specific aspects of vector biology.

North American House Sparrows Are Competent for Usutu Virus Transmission.

Usutu virus (USUV, Flaviviridae) is an emerging mosquito-borne virus that has been implicated in neuroinvasive disease in humans and epizootic deaths in wild birds. USUV is maintained in an enzootic cycle between ornithophilic mosquitoes, primarily Culex spp., and wild birds, predominantly passerine species. However, limited experimental data exist on the species competent for USUV transmission. Here, we demonstrate that house sparrows are susceptible to multiple USUV strains. Our study also revealed that Culex quinquefasciatus mosquitoes are susceptible to USUV, with a significantly higher infection rate for the Netherlands 2016 USUV strain compared to the Uganda 2012 USUV strain at 50% and 19%, respectively. To assess transmission between avian host and mosquito vector, we allowed mosquitoes to feed on either juvenile chickens or house sparrows inoculated with USUV. Both bird models transmitted USUV to C. quinquefasciatus mosquitoes. Linear regression analyses indicated that C. quinquefasciatus infection rates were positively correlated with avian viremia levels, with 3 to 4 log10 PFU/mL representing the minimum avian viremia threshold for transmission to mosquitoes. Based on the viremia required for transmission, house sparrows were estimated to more readily transmit the Netherlands 2016 strain compared to the Uganda 2012 strain. These studies provide insights on a competent reservoir host of USUV. IMPORTANCE Usutu virus (USUV) is a zoonotic mosquito-borne virus that can cause neuroinvasive disease, including meningitis and encephalitis, in humans and has resulted in hundreds of thousands of deaths in wild birds. The perpetuation of USUV in nature is dependent on transmission between Culex spp. mosquitoes and various avian species. To date, few experimental data exist for determining which bird species are important for the maintenance of USUV. Our studies showed that house sparrows can transmit infectious Usutu virus, indicating their role as a competent host species. By identifying reservoir species of USUV, we can predict areas of USUV emergence and mitigate its impacts on global human and wildlife health.

Mosquito Electroantennogram Recordings.

Electroantennography (EAG) is a simple, yet powerful technique that can be used to indicate that chemical compounds are detected by mosquito antennae. When coupled with gas chromatography (GC), it allows for the identification of detectable compounds within complex mixtures, including those in plant and animal headspaces that might influence mosquito behavior. EAGs and GC-EAGs reveal compounds that can be tested in behavioral assays with the goal of developing new repellents or attractants for mosquito control.

Mosquito Electroantennography.

Mosquitoes use multiple cues to locate food sources (animal and plants), mates, and oviposition sites. The sense of smell plays an important role in these behaviors, and olfactory cues are detected primarily by the appendages on the head-in particular, the antennae. Thus, mosquito olfaction can be studied by conducting electroantennogram (EAG) recordings. EAGs have emerged as a reliable technique to screen for bioactive compounds and are useful in the development of attractants and repellents for mosquito population control. Here, we focus on comparing the two main experimental approaches used in the literature (whole-body and whole-head mounting) and highlight key considerations for conducting EAGs in multiple species and for obtaining reliable and reproducible data.

Thermal biology of invasive Aedes mosquitoes in the context of climate change.

The increasing incidence of arboviral diseases in tropical endemic areas and their emergence in new temperate countries is one of the most important challenges that Public Health agencies are currently facing. Because mosquitoes are poikilotherms, shifts in temperature influence physiological functions besides egg viability. These traits impact not only vector density, but also their interaction with their hosts and arboviruses. As such the relationship among mosquitoes, arboviral diseases and temperature is complex. Here, we summarize current knowledge on the thermal biology of Aedes invasive mosquitoes, highlighting differences among species. We also emphasize the need to expand knowledge on the variability in thermal sensitivity across populations within a species, especially in light of climate change that encompasses increase not only in mean environmental temperature but also in the frequency of hot and cold snaps. Finally, we suggest a novel experimental approach to investigate the molecular architecture of thermal adaptation in mosquitoes.

Species-Specificity in Thermopreference and CO2-Gated Heat-Seeking in Culex Mosquitoes.

Combining thermopreference (Tp) and CO2-gated heat-seeking assays, we studied the thermal preferendum and response to thermal cues in three Culex mosquito species exhibiting differences in native habitat and host preference (e.g., biting cold and/or warm-blooded animals). Results show that these species differ in both Tp and heat-seeking behavior. In particular, we found that Culex territans, which feed primarily on cold-blood hosts, did not respond to heat during heat-seeking assays, regardless of the CO2 concentration, but exhibited an intermediate Tp during resting. In contrast, Cx. quinquefasciatus, which feeds on warm blooded hosts, sought the coolest locations on a thermal gradient and responded only moderately to thermal stimuli when paired with CO2 at higher concentrations. The third species, Cx. tarsalis, which has been shown to feed on a wide range of hosts, responded to heat when paired with high CO2 levels and exhibited a high Tp. This study provides the first insights into the role of heat and CO2 in the host seeking behavior of three disease vectors in the Culex genus and highlights differences in preferred resting temperatures.

Development of an Attractive Toxic Sugar Bait for the Control of Aedes j. japonicus (Diptera: Culicidae).

Both female and male mosquitoes consume sugar meals to obtain carbohydrates used for energy. This behavior has recently been identified as a possible mosquito control target, as the World Health Organization has urged for the development of integrated vector management. This is critical as many medically important mosquito species are developing insecticide resistance, resulting in current control strategies becoming less effective. Additionally, the traditional use of insecticides is detrimental to many beneficial insects such as pollinators. The main goal of this study was to develop an attractive toxic sugar bait (ATSB) to limit the populations of a local invasive mosquito, Aedes j. japonicus (Theobald) (Diptera: Culicidae). An ATSB is a lure bait composed of an attractant odorant, a toxic component, and sugar that the mosquitoes can feed on. ATSBs are cost-effective, sustainable, environmentally friendly, and can be species-specific. Mosquitoes were isolated into cages or cups and each group had access to either a toxic sugar solution (containing boric acid), a control solution or a choice between the two. We tested multiple fruits, including mango, peach, blueberries, and blackberries, as well as a soda and grape juice and monitored their survival for 96 h. We found that this species fed on all tested fruit solutions and that the groups that imbibed toxic solutions died within 48 h, indicating that boric acid is an effective oral toxin against Ae. j. japonicus. Further experiments will be conducted in the field to determine the ATSBs efficacy and to monitor potential effects on off-target species.

A Step-by-Step Guide to Mosquito Electroantennography.

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

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.

Beat the heat: Culex quinquefasciatus regulates its body temperature during blood feeding.

Mosquitoes are regarded as one of the most dangerous animals on earth. Because they are responsible for the spread of a wide range of both human and animal pathogens, research of the underlying mechanisms of their feeding behavior and physiology is critical. Among disease vector mosquitoes, Culex quinquefasciatus, a known carrier of West Nile virus and Western Equine Encephalitis, remains relatively understudied. As blood-sucking insects, adaptations (either at the molecular or physiological level) while feeding on warm blood are crucial to their survival, as overheating can result in death due to heat stress. Our research aims to determine how Cx. quinquefasciatus copes with the heat associated with warm blood meal ingestion and possibly uncover the adaptations this species uses to avoid thermal stress. Through the use of thermographic imaging, we analyzed the body temperature of Cx. quinquefasciatus while blood feeding. Infrared thermography has allowed us to identify a cooling strategy, evaporative cooling via the production of fluid droplets, and an overall low body temperature in comparison to the blood temperature during feeding. Understanding Cx. quinquefasciatus' adaptations and the strategies they employ to reduce their body temperature while blood feeding constitutes the first step towards discovering potential targets that could be used for their control.