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.

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.

Sleep: An Essential and Understudied Process in the Biology of Blood-Feeding Arthropods.

Understanding the biology of blood-feeding arthropods is critical to managing them as vectors of etiological agents. Circadian rhythms act in the regulation of behavioral and physiological aspects such as blood feeding, immunity, and reproduction. However, the impact of sleep on these processes has been largely ignored in blood-feeding arthropods, but recent studies in mosquitoes show that sleep-like states directly impact host landing and blood feeding. Our focus in this review is on discussing the relationship between sleep and circadian rhythms in blood-feeding arthropods along with how unique aspects such as blood gluttony and dormancy can impact sleep-like states. We highlight that sleep-like states are likely to have profound impacts on vector-host interactions but will vary between lineages even though few direct studies have been conducted. A myriad of factors, such as artificial light, could directly impact the time and levels of sleep in blood-feeding arthropods and their roles as vectors. Lastly, we discuss underlying factors that make sleep studies in blood-feeding arthropods difficult and how these can be bypassed. As sleep is a critical factor in the fitness of animal systems, a lack of focus on sleep in blood-feeding arthropods represents a significant oversight in understanding their behavior and its role in pathogen transmission.

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.

Tethered Preparation for the Analysis of Mosquito Visual-Motor Responses Using Modular Visual Displays.

In the present protocol, we describe methods to assess mosquito visual-motor responses using the Reiser-Dickinson light-emitting diode (LED) panels arranged in a cylindrical arena and fixed-tethered preparations where the insect cannot adjust its orientation relative to the visual display. Variations around this approach might be better adapted for the specific requirements of each research project and must be considered by the investigators. Other types of displays may provide other stimulation possibilities (e.g., color range, refresh rate, field of view). Also, other types of preparations, such as rotating (magneto-tethered) preparations where the insect can rotate around a vertical axis and reorient relative to the visual display, may reveal other aspects of mosquito optomotor responses. Finally, the methods described here are applicable to multiple species and were used to produce data published previously using 6-d-old Aedes aegypti females.

Retrospective Review of and Introduction to the Analysis of Mosquito Optomotor Responses.

Adult hematophagous female mosquitoes require nutrients and proteins from vertebrate blood to produce progeny. To find these hosts, mosquitoes rely on olfactory, thermal, and visual cues. Among these sensory modalities, vision has received far less attention than olfaction, in part because of a lack of experimental tools providing sufficient control on the delivery of visual stimuli and the recording of mosquito responses. Although free-flight experiments (e.g., wind tunnel and cage) ensure higher ecological relevance and allow the observation of more natural flight dynamics, tethered flight assays offer a greater level of control on the suite of sensory stimuli experienced by mosquitoes. In addition, these tethered assays provide a stepping stone toward understanding the neural underpinnings of mosquito optomotor behavior. Advances in computer vision tracking systems and programmable light-emitting diode displays have permitted significant discoveries in models such as the fly Drosophila melanogaster Here, we introduce the use of these methods with mosquitoes.

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.

Visual threats reduce blood-feeding and trigger escape responses in Aedes aegypti mosquitoes.

The diurnal mosquitoes Aedes aegypti are vectors of several arboviruses, including dengue, yellow fever, and Zika viruses. To find a host to feed on, they rely on the sophisticated integration of olfactory, visual, thermal, and gustatory cues emitted by the hosts. If detected by their target, this latter may display defensive behaviors that mosquitoes need to be able to detect and escape in order to survive. In humans, a typical response is a swat of the hand, which generates both mechanical and visual perturbations aimed at a mosquito. Here, we used programmable visual displays to generate expanding objects sharing characteristics with the visual component of an approaching hand and quantified the behavioral response of female mosquitoes. Results show that Ae. aegypti is capable of using visual information to decide whether to feed on an artificial host mimic. Stimulations delivered in a LED flight arena further reveal that landed Ae. aegypti females display a stereotypical escape strategy by taking off at an angle that is a function of the direction of stimulus introduction. Altogether, this study demonstrates that mosquitoes landed on a host mimic can use isolated visual cues to detect and avoid a potential threat.

Behavioral and postural analyses establish sleep-like states for mosquitoes that can impact host landing and blood feeding.

Sleep is an evolutionarily conserved process that has been described in different animal systems. For insects, sleep characterization has been primarily achieved using behavioral and electrophysiological correlates in a few systems. Sleep in mosquitoes, which are important vectors of disease-causing pathogens, has not been directly examined. This is surprising as circadian rhythms, which have been well studied in mosquitoes, influence sleep in other systems. In this study, we characterized sleep in mosquitoes using body posture analysis and behavioral correlates, and quantified the effect of sleep deprivation on sleep rebound, host landing and blood-feeding propensity. Body and appendage position metrics revealed a clear distinction between the posture of mosquitoes in their putative sleep and awake states for multiple species, which correlated with a reduction in responsiveness to host cues. Sleep assessment informed by these posture analyses indicated significantly more sleep during periods of low activity. Night-time and daytime sleep deprivation resulting from the delivery of vibration stimuli induced sleep rebound in the subsequent phase in day and night active mosquitoes, respectively. Lastly, sleep deprivation suppressed host landing in both laboratory and field settings, and impaired blood feeding of a human host when mosquitoes would normally be active. These results suggest that quantifiable sleep states occur in mosquitoes and highlight the potential epidemiological importance of mosquito sleep.

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.

Modeling the effects of Aedes aegypti's larval environment on adult body mass at emergence.

Mosquitoes vector harmful pathogens that infect millions of people every year, and developing approaches to effectively control mosquitoes is a topic of great interest. However, the success of many control measures is highly dependent upon ecological, physiological, and life history traits of mosquito species. The behavior of mosquitoes and their potential to vector pathogens can also be impacted by these traits. One trait of interest is mosquito body mass, which depends upon many factors associated with the environment in which juvenile mosquitoes develop. Our experiments examined the impact of larval density on the body mass of Aedes aegypti mosquitoes, which are important vectors of dengue, Zika, yellow fever, and other pathogens. To investigate the interactions between the larval environment and mosquito body mass, we built a discrete time mathematical model that incorporates body mass, larval density, and food availability and fit the model to our experimental data. We considered three categories of model complexity informed by data, and selected the best model within each category using Akaike's Information Criterion. We found that the larval environment is an important determinant of the body mass of mosquitoes upon emergence. Furthermore, we found that larval density has greater impact on body mass of adults at emergence than on development time, and that inclusion of density dependence in the survival of female aquatic stages in models is important. We discuss the implications of our results for the control of Aedes mosquitoes and on their potential to spread disease.

Do Mosquitoes Sleep?

Sleep is a phenomenon conserved across the animal kingdom, where studies on Drosophila melanogaster have revealed that sleep phenotypes and molecular underpinnings are similar to those in mammals. However, little is known about sleep in blood-feeding arthropods, which have a critical role in public health as disease vectors. Specifically, sleep studies in mosquitoes are lacking despite considerable focus on how circadian processes, which have a central role in regulating sleep/wake cycles, impact activity, feeding, and immunity. Here, we review observations which suggest that sleep-like states likely occur in mosquitoes and discuss the potential role of sleep in relation to mosquito biology and their ability to function as disease vectors.

Linking Mosquito Ecology, Traits, Behavior, and Disease Transmission.

Mosquitoes are considered to be the deadliest animals on Earth because the diseases they transmit claim at least a million human lives every year globally. Here, we discuss the scales at which the effects of ecological factors cascade to influence epidemiologically relevant behaviors of adult mosquitoes. In particular, we focused our review on the environmental conditions (coarse-scale variables) that shape the life-history traits of larvae and adult mosquitoes (fine-scale traits), and how these factors and their association, in turn, modulate adult behaviors to influence mosquito-borne disease transmission. Finally, we explore the integration of physical, physiological, and behavioral information into predictive models with epidemiological applications.

Mechanism and plasticity of vectors' host-seeking behavior.

The host-seeking behavior of disease vector insects is central to the transmission of pathogens. In this context, an improved understanding of the mechanisms that allow vectors to detect, identify and locate a potential host will be crucial to refine existing control strategies and invent new ones. Host-seeking is mediated by the integration of cues that are processed by multiple sensory modalities, and provide robust information about host location and quality. Responses to these cues are plastic and vary as a function of the vector's internal state, age, and previous experience. Vectors also integrate other factors such as time of day, or even the level of defensiveness of the host. Here, we review the most recent advances on the molecular basis of host-seeking behavior, with a particular emphasis on disease vector mosquitoes.

The olfactory basis of orchid pollination by mosquitoes.

Mosquitoes are important vectors of disease and require sources of carbohydrates for reproduction and survival. Unlike host-related behaviors of mosquitoes, comparatively less is understood about the mechanisms involved in nectar-feeding decisions, or how this sensory information is processed in the mosquito brain. Here we show that Aedes spp. mosquitoes, including Aedes aegypti, are effective pollinators of the Platanthera obtusata orchid, and demonstrate this mutualism is mediated by the orchid's scent and the balance of excitation and inhibition in the mosquito's antennal lobe (AL). The P. obtusata orchid emits an attractive, nonanal-rich scent, whereas related Platanthera species-not visited by mosquitoes-emit scents dominated by lilac aldehyde. Calcium imaging experiments in the mosquito AL revealed that nonanal and lilac aldehyde each respectively activate the LC2 and AM2 glomerulus, and remarkably, the AM2 glomerulus is also sensitive to N,N-diethyl-meta-toluamide (DEET), a mosquito repellent. Lateral inhibition between these 2 glomeruli reflects the level of attraction to the orchid scents. Whereas the enriched nonanal scent of P. obtusata activates the LC2 and suppresses AM2, the high level of lilac aldehyde in the other orchid scents inverts this pattern of glomerular activity, and behavioral attraction is lost. These results demonstrate the ecological importance of mosquitoes beyond operating as disease vectors and open the door toward understanding the neural basis of mosquito nectar-seeking behaviors.

Visual-Olfactory Integration in the Human Disease Vector Mosquito Aedes aegypti.

Mosquitoes rely on the integration of multiple sensory cues, including olfactory, visual, and thermal stimuli, to detect, identify, and locate their hosts [1-4]. Although we increasingly know more about the role of chemosensory behaviors in mediating mosquito-host interactions [1], the role of visual cues is comparatively less studied [3], and how the combination of olfactory and visual information is integrated in the mosquito brain remains unknown. In the present study, we used a tethered-flight light-emitting diode (LED) arena, which allowed for quantitative control over the stimuli, and a control theoretic model to show that CO2 modulates mosquito steering responses toward vertical bars. To gain insight into the neural basis of this olfactory and visual coupling, we conducted two-photon microscopy experiments in a new GCaMP6s-expressing mosquito line. Imaging revealed that neuropil regions within the lobula exhibited strong responses to objects, such as a bar, but showed little response to a large-field motion. Approximately 20% of the lobula neuropil we imaged were modulated when CO2 preceded the presentation of a moving bar. By contrast, responses in the antennal (olfactory) lobe were not modulated by visual stimuli presented before or after an olfactory stimulus. Together, our results suggest that asymmetric coupling between these sensory systems provides enhanced steering responses to discrete objects.

Vector cognition and neurobiology.

The processing and integration of sensory information are central to the ability of disease vector insects to find their hosts, and eventually transmit diseases. Deciphering the underlying mechanisms and the modulation of their behavioral responses to host cues is likely to reveal molecular pathways and neural processes, which could then be targeted for reducing the transmission rates of pathogens. In addition, the double role of prey and predator played by hosts imposes unique challenges on vectors, making them an underexploited model to study the evolution of sensory neurobiology and of cognitive processes in miniature brains. Here, I review the most recent advances on the cognitive abilities of triatomine bugs and mosquitoes, with a particular emphasis on their ability to learn and remember information.

Odor-Specific Daily Rhythms in the Olfactory Sensitivity and Behavior of Aedes aegypti Mosquitoes.

Many biological processes and behaviors in mosquitoes display rhythmic patterns, allowing for fine tuning to cyclic environmental conditions. In mosquitoes, vector-host interactions are primarily mediated by olfactory signals. Previous studies have established that, in the malaria vector Anopheles gambiae, rhythmic expression of odorant binding proteins and takeout proteins in the antenna resulted in a corresponding rhythm in olfactory sensitivity to relevant host odors. However, it remained unclear how rhythms observed in olfactory sensitivity affect or explain rhythms in behavioral output, which ultimately impacts disease transmission. In order to address this knowledge gap, we quantified and compared patterns in locomotor activity, olfactory sensitivity, and olfactory behaviors in adult female Aedes aegypti mosquitoes. Here, we demonstrate an odorant-specific modulation of olfactory sensitivity in Ae. aegypti, decoupled from rhythms in olfactory behavior. Additionally, behavioral assays performed herein represent the first evidence of a time-dependence of the olfactory activation of behavior in Ae. aegypti mosquitoes. Results suggest that olfactory behavior of Aedes mosquitoes is modulated at both the peripheral (antenna) and central levels. As such, this work serves as a foundation for future studies aimed at further understanding the neural and molecular mechanisms underlying behavioral plasticity.