Multisensory integration in Anopheles mosquito swarms: The role of visual and acoustic information in mate tracking and collision avoidance.

Malaria mosquitoes mate in swarms. Here, they must rely on multiple sensory cues in shaping their individual responses, such as during mate recognition, swarm maintenance, and collision avoidance. While male mosquitoes are known to use faint female flight tones for recognizing their mates, the role of other sensory modalities remains less explored. By combining free-flight and tethered flight simulator experiments with Anopheles coluzzii, we demonstrate that swarming mosquitoes integrate visual and acoustic information to track conspecifics and avoid collisions. In tethered experiments, acoustic stimuli gated male steering responses to visual objects simulating nearby female mosquitoes, whereas visual cues alone triggered changes in wingbeat amplitude and frequency. Free-flight experiments show that mosquitoes modulate their flight responses to nearby conspecifics similarly to tethered animals, allowing for collision avoidance within swarms. These findings suggest that combined visual and acoustic information contributes to conspecific recognition within swarms, and, for males, permits female tracking while avoiding collisions.

Ingested histamine and serotonin interact to alter Anopheles stephensi feeding and flight behavior and infection with Plasmodium parasites.

Blood levels of histamine and serotonin (5-HT) are altered in human malaria, and, at these levels, we have shown they have broad, independent effects on Anopheles stephensi following ingestion by this invasive mosquito. Given that histamine and 5-HT are ingested together under natural conditions and that histaminergic and serotonergic signaling are networked in other organisms, we examined effects of combinations of these biogenic amines provisioned to A. stephensi at healthy human levels (high 5-HT, low histamine) or levels associated with severe malaria (low 5-HT, high histamine). Treatments were delivered in water (priming) before feeding A. stephensi on Plasmodium yoelii-infected mice or via artificial blood meal. Relative to effects of histamine and 5-HT alone, effects of biogenic amine combinations were complex. Biogenic amine treatments had the greatest impact on the first oviposition cycle, with high histamine moderating low 5-HT effects in combination. In contrast, clutch sizes were similar across combination and individual treatments. While high histamine alone increased uninfected A. stephensi weekly lifetime blood feeding, neither combination altered this tendency relative to controls. The tendency to re-feed 2 weeks after the first blood meal was altered by combination treatments, but this depended on mode of delivery. For blood delivery, malaria-associated treatments yielded higher percentages of fed females relative to healthy-associated treatments, but the converse was true for priming. Female mosquitoes treated with the malaria-associated combination exhibited enhanced flight behavior and object inspection relative to controls and healthy combination treatment. Mosquitoes primed with the malaria-associated combination exhibited higher mean oocysts and sporozoite infection prevalence relative to the healthy combination, with high histamine having a dominant effect on these patterns. Compared with uninfected A. stephensi, the tendency of infected mosquitoes to take a second blood meal revealed an interaction of biogenic amines with infection. We used a mathematical model to project the impacts of different levels of biogenic amines and associated changes on outbreaks in human populations. While not all outbreak parameters were impacted the same, the sum of effects suggests that histamine and 5-HT alter the likelihood of transmission by mosquitoes that feed on hosts with symptomatic malaria versus a healthy host.

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.

Mosquito biology: Scents and selectability.

Malaria-transmitting mosquitoes are skilled human hunters, selectively choosing their prey based on a complex array of sensory cues. A new study unveils a distinct pattern of preference for human-associated olfactory cues that underlies the selective behavior of these mosquitoes.

Experiments and Analysis of Mosquito Flight Behaviors in a Wind Tunnel: An Introduction.

Mosquitoes detect and navigate to important resources, like a host, using combinations of different sensory stimuli. The relative importance of the sensory cues can change as the mosquito gets closer to their target. Other factors, both internal and external, can also influence the mosquito behavior. A mechanistic understanding of these sensory stimuli, and how they impact mosquito navigation, can now be readily studied using wind tunnels and associated computer vision systems. In this introduction, we present a behavioral paradigm using a wind tunnel for flight behavior analysis. The wind tunnel's large size with its associated cameras and software system for analysis of the mosquito flight tracks can be sophisticated and sometimes cost-prohibitive. Nevertheless, the wind tunnel's flexibility in allowing the testing of multimodal stimuli and scaling of environmental stimuli makes it possible to reproduce conditions from the field and test them in the laboratory, while also allowing the observation of natural flight kinematics.

Conducting an Analysis of Mosquito Flight Behaviors in a Wind Tunnel.

Prior to conducting wind tunnel experiments, mosquitoes must be prepared for testing. Important factors and state-dependent processes of the mosquito-like the sex, age, infection status, reproductive status, or nutritional status-should be evaluated and motivated by questions and hypotheses one seeks to address. Other critical external factors that can impact the mosquitoes' behavior and should be controlled for both in the colony and in the room where the wind tunnel experiments take place include the circadian rhythm, room temperature, light intensity, and relative humidity. Together, the internal and external factors, and wind tunnel design, ultimately control the behavior of the mosquito and, hence, the success of the experiments. In the present protocol, we describe methods using a standard wind tunnel design in which the fan pulls the air through the working section of the wind tunnel and the mosquito behavior is recorded by a multicamera system. Variations around the camera tracking system can be adapted according to the research questions being asked and include real-time tracking for both closed-loop and open-loop control of the stimulus environment or recording of the videos for off-line digitization and analysis. Within the working section, the sensory environment (odor, visual, wind) can be controlled to test the mosquito responses to different stimuli, and below we include different equipment and tools for modifying the stimuli the mosquito experiences during flight. Finally, the methods described here are applicable to multiple mosquito species, although the experiment parameters may need to be changed (e.g., ambient luminosity).

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.

Anopheles stephensi Feeding, Flight Behavior, and Infection With Malaria Parasites are Altered by Ingestion of Serotonin.

Approximately 3.4 billion people are at risk of malaria, a disease caused by infection with Plasmodium spp. parasites, which are transmitted by Anopheles mosquitoes. Individuals with severe falciparum malaria often exhibit changes in circulating blood levels of biogenic amines, including reduced serotonin or 5-hydroxytryptamine (5-HT), and these changes are associated with disease pathology. In insects, 5-HT functions as an important neurotransmitter for many behaviors and biological functions. In Anopheles stephensi, we show that 5-HT is localized to innervation in the head, thorax, and midgut, suggesting a gut-to-brain signaling axis that could support the effects of ingested 5-HT on mosquito biology and behavioral responses. Given the changes in blood levels of 5-HT associated with severe malaria and the key roles that 5-HT plays in insect neurophysiology, we investigated the impact of ingesting blood with healthy levels of 5-HT (1.5 µM) or malaria-associated levels of 5-HT (0.15 µM) on various aspects of A. stephensi biology. In these studies, we provisioned 5-HT and monitored fecundity, lifespan, flight behavior, and blood feeding of A. stephensi. We also assessed the impact of 5-HT ingestion on infection of A. stephensi with the mouse malaria parasite Plasmodium yoelii yoelii 17XNL and the human malaria parasite Plasmodium falciparum. Our data show that ingestion of 5-HT associated with severe malaria increased mosquito flight velocity and investigation of visual objects in response to host odor (CO2). 5-HT ingestion in blood at levels associated with severe malaria also increased the tendency to take a second blood meal 4 days later in uninfected A. stephensi. In mosquitoes infected with P. y. yoelii 17XNL, feeding tendency was decreased when midgut oocysts were present but increased when sporozoites were present. In addition to these effects, treatment of A. stephensi with 5-HT associated with severe malaria increased infection success with P. y. yoelii 17XNL compared to control, while treatment with healthy levels of 5-HT decreased infection success with P. falciparum. These changes in mosquito behavior and infection success could be used as a basis to manipulate 5-HT signaling in vector mosquitoes for improved control of malaria parasite transmission.

The olfactory gating of visual preferences to human skin and visible spectra in mosquitoes.

Mosquitoes track odors, locate hosts, and find mates visually. The color of a food resource, such as a flower or warm-blooded host, can be dominated by long wavelengths of the visible light spectrum (green to red for humans) and is likely important for object recognition and localization. However, little is known about the hues that attract mosquitoes or how odor affects mosquito visual search behaviors. We use a real-time 3D tracking system and wind tunnel that allows careful control of the olfactory and visual environment to quantify the behavior of more than 1.3 million mosquito trajectories. We find that CO2 induces a strong attraction to specific spectral bands, including those that humans perceive as cyan, orange, and red. Sensitivity to orange and red correlates with mosquitoes' strong attraction to the color spectrum of human skin, which is dominated by these wavelengths. The attraction is eliminated by filtering the orange and red bands from the skin color spectrum and by introducing mutations targeting specific long-wavelength opsins or CO2 detection. Collectively, our results show that odor is critical for mosquitoes' wavelength preferences and that the mosquito visual system is a promising target for inhibiting their attraction to human hosts.

Human attractive cues and mosquito host-seeking behavior.

Female mosquitoes use chemical and physical cues, including vision, smell, heat, and humidity, to orient toward hosts. Body odors are produced by skin resident bacteria that convert metabolites secreted in sweat into odorants that confer the characteristic body scent. Mosquitoes detect these compounds using olfactory receptors in their antennal olfactory receptor neurons. Such information is further integrated with the senses of temperature and humidity, as well as vision, processed in the brain into a behavioral output, leading to host finding. Knowledge of human scent components unveils a variety of odorants that are attractive to mosquitoes, but also odor-triggering repellency. Finding ways to divert human-seeking behavior by female mosquitoes using odorants can possibly mitigate mosquito-borne pathogen transmission.

Fruit odorants mediate co-specialization in a multispecies plant-animal mutualism.

Despite the widespread notion that animal-mediated seed dispersal led to the evolution of fruit traits that attract mutualistic frugivores, the dispersal syndrome hypothesis remains controversial, particularly for complex traits such as fruit scent. Here, we test this hypothesis in a community of mutualistic, ecologically important neotropical bats (Carollia spp.) and plants (Piper spp.) that communicate primarily via chemical signals. We found greater bat consumption is significantly associated with scent chemical diversity and presence of specific compounds, which fit multi-peak selective regime models in Piper. Through behavioural assays, we found Carollia prefer certain compounds, particularly 2-heptanol, which evolved as a unique feature of two Piper species highly consumed by these bats. Thus, we demonstrate that volatile compounds emitted by neotropical Piper fruits evolved in tandem with seed dispersal by scent-oriented Carollia bats. Specifically, fruit scent chemistry in some Piper species fits adaptive evolutionary scenarios consistent with a dispersal syndrome hypothesis. While other abiotic and biotic processes likely shaped the chemical composition of ripe fruit scent in Piper, our results provide some of the first evidence of the effect of bat frugivory on plant chemical diversity.

Elimination of vision-guided target attraction in Aedes aegypti using CRISPR.

Blood-feeding insects, such as the mosquito, Aedes (Ae.) aegypti, use multiple senses to seek out and bite humans.1,2 Upon exposure to the odor of CO2, the attention of female mosquitoes to potential targets is greatly increased. Female mosquitoes are attracted to high-contrast visual cues and use skin olfactory cues to assist them in homing in on targets several meters away.3-9 Within close range, convective heat from skin and additional skin odors further assist the mosquitoes' evaluation as to whether the object of interest might be a host.10,11 Here, using CRISPR-Cas9, we mutated the gene encoding Op1, which is the most abundant of the five rhodopsins expressed in the eyes of Ae. aegypti. Using cage and wind-tunnel assays, we found that elimination of op1 did not impair CO2-induced target seeking. We then mutated op2, which encodes the rhodopsin most similar to Op1, and also found that there was no impact on this behavior. Rather, mutation of both op1 and op2 was required for abolishing vision-guided target attraction. In contrast, the double mutants exhibited normal phototaxis and odor-tracking responses. By measuring the walking optomotor response, we found that the double mutants still perceived optic flow. In further support of the conclusion that the double mutant is not blind, the animals retained an electrophysiological response to light, although it was diminished. This represents the first genetic perturbation of vision in mosquitoes and indicates that vision-guided target attraction by Ae. aegypti depends on two highly related rhodopsins.

Histamine Ingestion by Anopheles stephensi Alters Important Vector Transmission Behaviors and Infection Success with Diverse Plasmodium Species.

An estimated 229 million people worldwide were impacted by malaria in 2019. The vectors of malaria parasites (Plasmodium spp.) are Anopheles mosquitoes, making their behavior, infection success, and ultimately transmission of great importance. Individuals with severe malaria can exhibit significantly increased blood concentrations of histamine, an allergic mediator in humans and an important insect neuromodulator, potentially delivered to mosquitoes during blood-feeding. To determine whether ingested histamine could alter Anopheles stephensi biology, we provisioned histamine at normal blood levels and at levels consistent with severe malaria and monitored blood-feeding behavior, flight activity, antennal and retinal responses to host stimuli and lifespan of adult female Anopheles stephensi. To determine the effects of ingested histamine on parasite infection success, we quantified midgut oocysts and salivary gland sporozoites in mosquitoes infected with Plasmodium yoelii and Plasmodium falciparum. Our data show that provisioning An. stephensi with histamine at levels consistent with severe malaria can enhance mosquito behaviors and parasite infection success in a manner that would be expected to amplify parasite transmission to and from human hosts. Such knowledge could be used to connect clinical interventions by reducing elevated histamine to mitigate human disease pathology with the delivery of novel lures for improved malaria control.

Visuo-Motor Feedback Modulates Neural Activities in the Medulla of the Honeybee, Apis mellifera.

Behavioral and internal-state modulation of sensory processing has been described in several organisms. In insects, visual neurons in the optic lobe are modulated by locomotion, but the degree to which visual-motor feedback modulates these neurons remains unclear. Moreover, it also remains unknown whether self-generated and externally generated visual motion are processed differently. Here, we implemented a virtual reality system that allowed fine-scale control over visual stimulation in relation to animal motion, in combination with multichannel recording of neural activity in the medulla of a female honeybee (Apis mellifera). We found that this activity was modulated by locomotion, although, in most cases, only when the bee had behavioral control over the visual stimulus (i.e., in a closed-loop system). Moreover, closed-loop control modulated a third of the recorded neurons, and the application of octopamine (OA) evoked similar changes in neural responses that were observed in a closed loop. Additionally, in a subset of modulated neurons, fixation on a visual stimulus was preceded by an increase in firing rate. To further explore the relationship between neuromodulation and adaptive control of the visual environment of the bee, we modified motor gain sensitivity while locally injecting an OA receptor antagonist into the medulla. Whereas female honeybees were tuned to a motor gain of -2 to 2 (between the heading of the bee and its visual feedback), local disruption of the OA pathway in the medulla abolished this tuning, resulting in similar low levels of response across levels of motor gain. Our results show that behavioral control modulates neural activity in the medulla and ultimately impacts behavior.SIGNIFICANCE STATEMENT When moving, an animal generates the motion of the visual scene over its retina. We asked whether self-generated and externally generated optic flow are processed differently in the insect medulla. Our results show that closed-loop control of the visual stimulus modulates neural activity as early as the medulla and ultimately impacts behavior. Moreover, blocking octopaminergic modulation further disrupted object-tracking responses. Our results suggest that the medulla is an important site for context-dependent processing of visual information and that placing the animal in a closed-loop environment may be essential to understanding its visual cognition and processing.

The neuroecology of insect-plant interactions: the importance of physiological state and sensory integration.

Natural behaviorally important stimuli are combinations of cues that are integrated by the nervous system to elicit behavior. Nonetheless, these cues dynamically change in time and space. In turn, the animal's internal state can cause changes in the encoding and representation of these stimuli. Despite abundant behavioral examples, links between the neural bases of sensory integration and the internal state-dependency of these responses remains an active study area. Recent studies in different insect models have provided new insights into how plasticity and the insect's internal state may influence odor representation. These studies show that complex stimuli are represented in unique percepts that are different from their sensory channels and that the representations may be modulated by physiological state.

Distinct navigation behaviors in Aedes, Anopheles and Culex mosquito larvae.

Mosquitoes spread deadly diseases that impact millions of people every year. Understanding mosquito physiology and behavior is vital for public health and disease prevention. However, many important questions remain unanswered in the field of mosquito neuroethology, particularly in our understanding of the larval stage. In this study, we investigate the innate exploration behavior of six different species of disease vector mosquito larvae. We show that these species exhibit strikingly different movement paths, corresponding to a wide range of exploration behaviors. We also investigated the response of each species to an appetitive food cue, aversive cue or neutral control. In contrast to the large differences in exploration behavior, all species appeared to gather near preferred cues through random aggregation rather than directed navigation, and exhibited slower speeds once encountering food patches. Our results identify key behavioral differences among important disease vector species, and suggest that navigation and exploration among even closely related mosquito species may be much more distinct than previously thought.

Geosmin Attracts Aedes aegypti Mosquitoes to Oviposition Sites.

Geosmin is one of the most recognizable and common microbial smells on the planet. Some insects, like mosquitoes, require microbial-rich environments for their progeny, whereas for other insects such microbes may prove dangerous. In the vinegar fly Drosophila melanogaster, geosmin is decoded in a remarkably precise fashion and induces aversion, presumably signaling the presence of harmful microbes [1]. We have here investigated the effect of geosmin on the behavior of the yellow fever mosquito Aedes aegypti. In contrast to flies, geosmin is not aversive but mediates egg-laying site selection. Female mosquitoes likely associate geosmin with microbes, including cyanobacteria consumed by larvae [2], who also find geosmin-as well as geosmin-producing cyanobacteria-attractive. Using in vivo multiphoton calcium imaging from transgenic PUb-GCaMP6s mosquitoes, we show that Ae. aegypti code geosmin in a qualitatively similar fashion to flies, i.e., through a single olfactory channel with a high degree of sensitivity for this volatile. We further demonstrate that geosmin can be used as bait under field conditions, and finally, we show that geosmin, which is both expensive and difficult to obtain, can be substituted by beetroot peel extract, providing a cheap and viable potential mean for mosquito control and surveillance in developing countries.