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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 ofAedes aegyptimosquitoes, vectors of dengue, yellow fever and Zika viruses, to host and plant related odors under different environmental temperatures.

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.

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.

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 STATEMENTWhen 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.

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 (Carolliaspp.) and plants (Piperspp.) 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 inPiper. Through behavioural assays, we foundCarolliaprefer certain compounds, particularly 2-heptanol, which evolved as a unique feature of twoPiperspecies highly consumed by these bats. Thus, we demonstrate that volatile compounds emitted by neotropicalPiperfruits evolved in tandem with seed dispersal by scent-orientedCarolliabats. Specifically, fruit scent chemistry in somePiperspecies 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 inPiper, our results provide some of the first evidence of the effect of bat frugivory on plant chemical diversity.

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 used a real-time 3D tracking system and wind tunnel that allowed careful control of the olfactory and visual environment to quantify the behavior of more than 1.3 million mosquito trajectories. We found that CO2 induces a strong attraction to specific hues, 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. Attraction was 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.

Aedes aegypti have a wide variety of sensory pathways that have supported success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as relevant response behaviors, there has been a lack of studies able to monitor both concurrently. To begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced confocal microscopy, we were able to simultaneously measure live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution. Moreover, by coupling in vivo calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies.Significance StatementUnderstanding mosquito sensory systems and resulting behavior has been a major factor in the advancement of mosquito control innovations. Aedes aegypti larvae offer an effective life stage for further elucidating information on mosquito sensory systems. Due to their relatively simplified nervous system, mosquito larvae are ideal for studying neural signal transduction, coding, and behavior. Moreover, a better understanding of the larval sensory system may enable the development of novel control methodologies able to target mosquitoes before they reach a vector-competent stage. Here we engineer Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s and use this tool to observe links between sensorimotor responses and behavior by exploiting live calcium imaging as well as live tracking based behavioral assays.

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.

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.

Blood-feeding insects, such as the mosquito, Aedes (Ae.) aegypti, use multiple senses to seek out and bite humans [1, 2]. Upon exposure to CO2, the attention of female mosquitoes to potential human targets is greatly increased. Female mosquitoes use vision to assist them in honing in on hosts that may be up to 10 meters away [3–9]. Only after coming into close range do convective heat from skin and odors from volatile organic compounds come into play, allowing female mosquitoes to evaluate 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 compound eyes of Ae. aegypti. Using a cage assay and a wind tunnel assay, we surprisingly 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 to abolish vision-guided target recognition. In contrast to this defect, the double mutants still exhibited normal light attraction. By measuring the optomotor response, we found that the double mutants still recognized moving cues in their environment. In further support of the conclusion that the double mutant is not blind, we found that the animals retained an electrophysiological response to light, although it was diminished. This represents the first perturbation of vision in mosquitoes and indicates that hostseeking by Ae. aegypti depends on redundant rhodopsins.

Significance Nectar feeding by mosquitoes is important for survival and reproduction, and hence disease transmission. However, we know little about the sensory mechanisms that mediate mosquito attraction to sources of nectar, like those of flowers, or how this information is processed in the mosquito brain. Using a unique mutualism between Aedes mosquitoes and Platanthera obtusata orchids, we reveal that the orchid’s scent mediates this mutualism. Furthermore, lateral inhibition in the mosquito’s antennal (olfactory) lobe—via the neurotransmitter GABA—is critical for the representation of the scent. These results have implications for understanding the olfactory basis of mosquito nectar-seeking behaviors., 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.

In Brief Melo et al. show that geosmin mediates egg laying in the yellow fever mosquito Aedes aegypti, which associates geosmin with microbes present in the larval aquatic habitat. The authors further show that geosmin can be used as bait in oviposition traps and that geosmin can be substituted by beetroot peel for mosquito trapping in developing countries., Graphical Abstract

How does the common insect repellent DEET modify a mosquito's ability to detect humans? New research using GCaMP-expressing mosquitoes suggests that DEET works differently for different mosquito species. For An. coluzzii, DEET and other non-volatile repellents mask the mosquitoes' ability to detect odors.

SummaryMosquitoes 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 behaviours in mediating mosquito-host interactions [1], the role of visual cues remains 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 LED arena, which allowed for quantitative control over the stimuli, to show that CO2exposure affects target-tracking responses, but not responses to large-field visual stimuli. In addition, we show that CO2modulates behavioural responses to visual objects in a time-dependent manner. 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 the majority of ROIs in the lobula region of the optic lobe exhibited strong responses to small-field stimuli, but showed little response to a large-field stimulus. Approximately 20% of the neurons we imaged were modulated when an attractive odour preceded the visual stimulus; these same neurons also elicited a small response when the odour was presented alone. By contrast, imaging in the antennal lobe revealed no modulation when visual stimuli were presented before or after the olfactory stimulus. Together, our results are the first to reveal the dynamics of olfactory modulation in visually evoked behaviours of mosquitoes, and suggest that coupling between these sensory systems is asymmetrical and time-dependent.

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 flyDrosophila melanogaster, geosmin is decoded in a remarkably precise fashion and induces aversion, presumably signaling the presence of harmful microbes. We have here investigated the effect of geosmin on the behavior of the yellow fever mosquitoAedes aegypti. In contrast to flies, geosmin is not aversive in mosquitoes but stimulates egg-laying site selection. Female mosquitoes could associate geosmin with microbes, including cyanobacteria consumed by larvae, who also find geosmin – as well as geosmin producing cyanobacteria – attractive. Usingin vivomultiphoton imaging from mosquitoes with pan-neural expression of the calcium reporter GCaMP6s, we show thatAe. aegypticode geosmin in a similar fashion to flies, i.e. with extreme sensitivity and with a high degree of selectivity. 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 mean of mosquito control and surveillance in developing countries.

Mosquitoes are prolific disease vectors that affect public health around the world. Although many studies have investigated search strategies used by host-seeking adult mosquitoes, little is known about larval search behavior. Larval behavior affects adult body size and fecundity, and thus the capacity of individual mosquitoes to find hosts and transmit disease. Understanding vector survival at all life stages is crucial for improving disease control. In this study we use experimental and computational methods to investigate the chemical ecology and search behavior ofAedes aegyptimosquito larvae. We show that larvae do not respond to several olfactory cues used by adultAe. aegyptito assess larval habitat quality, but perceive microbial RNA as a potent foraging attractant. Second, we demonstrate thatAe. aegyptilarvae use a strategy consistent with chemokinesis, rather than chemotaxis, to navigate chemical gradients. Using computational modeling, we further show that chemokinesis is more efficient than chemotaxis for avoiding repellents in ecologically relevant larval habitat sizes. Finally, we use experimental observations and computational analyses to demonstrate that larvae respond to starvation pressure by optimizing exploration behavior. Our results identify key characteristics of foraging behavior in a disease vector mosquito, including the identification of a surprising foraging attractant and an unusual behavioral mechanism for chemosensory preference. In addition to implications for better understanding and control of disease vectors, this work establishes mosquito larvae as a tractable model for chemosensory behavior and navigation.

Animals rely on multiple sensory modalities to perform even the simplest ecological tasks (Burkhardt 1989; Siemers and Schnitzler 2000; Holland et al. 2005; Knaden and Graham 2016). One of the main goals of behavioral and sensory ecology lies in understanding the ability of different species to employ and modulate their sensory modes in the context of different environmental cues, and how the resulting behavioral decisions ultimately affect their ecology and evolution. Frugivores use various cues, including components of fruit color (Burkhardt 1989; Hiramatsu et al. 2008; Melin et al. 2008; Osorio and Vorobyev 2008; Valenta et al. 2013), shape (Von Helversen and Von Helversen 1999; Kalko and Condon 1998), and scent (Sanchez et al. 2006; Valenta et al. 2013) to find and select ripe fruit, and exhibit corresponding sensory specializations in their visual, auditory, and/or olfactory systems to target those cues (Catania 1999; Muller et al. 2007; Vanderelst et al. 2010). Little is known, however, about what scenarios facilitate or constrain sensory use and modulation during fruit location, selection, and acquisition in vertebrate frugivores. Insight into these processes would provide mechanistic understanding of the behaviors underlying their foraging ecology. Frugivorous and omnivorous Neotropical leaf-nosed bats (Phyllostomidae) use three major senses, echolocation, olfaction, and vision, for navigation and foraging (e.g., Reiger and Jakob 1988; Gutierrez et al. 2018; Thiele and Winter 2005; Kalko and Condon 1998), which makes these organisms an exceptional system for investigating the relative role of different sensory modes during ecologically important tasks. Although there has been some work on phyllostomid vision, including comparisons of their short- and long-wave opsins (Muller et al. 2007, 2009; Kries et al. 2018; Sadier et al. 2018; Gutierrez et al. 2018), the importance of vision in ripe fruit location and selection has not been experimentally tested in phyllostomids. By contrast, experimental evidence strongly suggests that omnivorous phyllostomids rely primarily on echolocation to locate fruits (Kalko and Condon 1998), and specialized frugivores employ either olfaction or a combination of olfaction and echolocation to locate ripe fruit (Kalko and Schnitzler 1993; Korine and Kalko 2005; Hodgkison et al. 2007, 2013). The relative importance of different sensory modes for fruit detection may depend on which plant cues can be readily perceived within a specific environmental context. To date, little is known about whether and how frugivorous phyllostomids integrate their primary sensory modes (echolocation and olfaction) conditional on which plant cues are present. While plant scents can travel over short or long distances (Riffell et al. 2014), they are rarely directional and may be difficult to detect in saturated scent environments such as rainforests. Conversely, echolocation allows for highly precise prey detection (Schnitzler and Kalko 2001; Brinklov et al. 2009; Jakobsen et al. 2013), but phyllostomids emit short, high frequency, and low intensity calls (Thies et al. 1998; Korine and Kalko 2005). The echoes from these calls provide information about size, shape, texture, range, and position of an object in space relative to the bat (Simmons et al. 1975, 1983; Simmons and Stein 1980; Neuweiler 1989; Schmidt et al. 2000; Schnitzler and Kalko 2001), but these types of information are only effective at very short distances because low intensity, high frequency calls attenuate rapidly in warm, humid environments (e.g., 45–90 kHz attenuate at 1.4–4 dB/m at 25°C and 80% humidity; Jakobsen et al. 2013). Additionally, surrounding foliage can produce acoustic masking effects that may complicate fruit detection (Arlettaz et al. 2001; Korine and Kalko 2005). Therefore, flexibility in olfaction versus echolocation use could be highly beneficial for frugivorous bats given the limitations of each sensory mode within complex forest environments. Here, we study the primary plant cues from vegetation and fruits and how they relate to the roles of echolocation and olfaction for fruit detection and localization in the chestnut short-tailed fruit bat, Carollia castanea, a highly abundant frugivore and ecologically important seed disperser that inhabits forests in Central and South America (Bonaccorso 1979; Fleming 1991; Fig. 1). This research builds upon the seminal work of Thies et al. (1998), which demonstrated the importance of olfaction for fruit detection in Carollia; here we investigate the relative contributions of vegetative and fruit scent cues that drive C. castanea’s foraging decisions, and how varying foraging scenarios (i.e., changing of plant scent cues) may affect the relative reliance of these bats on olfaction versus echolocation for fruit detection. Evaluating the effect of vegetative scents on frugivore foraging behavior is particularly important because background scents from neighboring plants can impact the ability of animals to discriminate fruit or flower odors (Riffell et al. 2014) or, if vegetative scents are from the same plant, they can amplify the scent of the food source and make it more potent (e.g., Raguso and Willis 2005; Rusch et al. 2016; Stutz et al. 2016; McArthur et al. 2019). Open in a separate window Fig. 1 Study organisms, Carollia castanea (left) and Piper sancti-felicis (right). Photo credit: S.E. Santana.

Plants experience often opposing energetic demands and selective pressures-for instance, where plants need to attract an insect that is both the pollinator and herbivore, or alternately, where plants attract prey (due to limited resources) and pollinators. Together, these selective pressures can modify the volatile signals available to the plant's mutualistic and antagonistic partners. Nevertheless, it remains an open question how changes in the information content of volatile signals modify behavioral responses in mutualists and antagonists, and what the underlying neural bases of these behaviors are. This review focuses on two systems to explore the impact of herbivory and resource availability on plant-pollinator interactions: hawkmoth-pollinated hostplants (where herbivory is common), and carnivorous bee-pollinated pitcher plants (where the plants differentially attract bee pollinators and other insect prey). We focus on (1) the volatile signals emitted from these plants because these volatiles operate as long-distance signals to attract, or deter, insect partners, (2) how this information is processed in the hawkmoth olfactory system, and (3) how volatile information changes spatiotemporally. In both the plants and their respective insect partner(s), volatile signaling, reception and behavior are dynamic and plastic, providing flexibility an ever-changing environment.

Floral scents act as long-distance signals to attract pollinators, but volatiles emitted from the vegetation and neighboring plant community may modify this mutualistic communication system. What impact does the olfactory background have on pollination systems and their evolution? We consider recent behavioral studies that address the context of when and where volatile backgrounds influence a pollinator's perception of floral blends. In parallel, we review neurophysiological studies that show the importance of blend composition and background in modifying the representation of floral blends in the pollinator brain, as well as experience-dependent plasticity in increasing the representation of a rewarding odor. Here, we suggest that the efficacy of the floral blend in different environments may be an important selective force shaping differences in pollinator olfactory receptor expression and underlying neural mechanisms that mediate flower visitation and plant reproductive isolation.

Background and aims Growing experimental evidence that floral scent is a key contributor to pollinator attraction supports its investigation as a component of the suite of floral traits that result from pollinator-mediated selection. Yet, the fate of floral scent during the transition out of biotic into abiotic pollination has rarely been tested. In the case of wind pollination, this is due not only to its rarer incidence among flowering plants compared with insect pollination, but also to the scarcity of systems amenable to genus-level comparisons. Thalictrum (Ranunculaceae), with its multiple transitions from insect to wind pollination, offers a unique opportunity to test interspecific changes in floral fragrance and their potential impact on pollinator attraction. Methods First, the Thalictrum phylogeny was revised and the ancestral character state of pollination mode was reconstructed. Then, volatile organic compounds (VOCs) that comprise the scent bouquets of flowers from 11 phylogenetically representative wind- and insect-pollinated species were characterized and compared. Finally, to test the hypothesis that scent from insect-pollinated flowers elicits a significantly greater response from potential pollinators than that from wind-pollinated flowers, electroantennograms (EAGs) were performed on Bombus impatiens using whole flower extracts. Key results Phylogenetic reconstruction of the pollination mode recovered 8-10 transitions to wind pollination from an ancestral insect pollination state and two reversals to insect pollination. Biochemical and multivariate analysis showed that compounds are distinct by species and only partially segregate with pollination mode, with no significant phylogenetic signal on scent composition. Floral VOCs from insect-pollinated Thalictrum elicited a larger antennal response from potential insect pollinators than those from wind-pollinated congeners. Conclusions An evolutionary scenario is proposed where an ancestral ability of floral fragrance to elicit an insect response through the presence of specific compounds was independently lost during the multiple evolutionary transitions to wind pollination in Thalictrum.

Mosquitoes are best known for their proclivity towards biting humans and transmitting bloodborne pathogens, but there are over 3500 species, including both blood-feeding and non-blood-feeding taxa. The diversity of host preference in mosquitoes is exemplified by the feeding habits of mosquitoes in the genus Malaya that feed on ant regurgitation or those from the genus Uranotaenia that favor amphibian hosts. Host preference is also by no means static, but is characterized by behavioral plasticity that allows mosquitoes to switch hosts when their preferred host is unavailable and by learning host cues associated with positive or negative experiences. Here we review the diverse range of host-preference behaviors across the family Culicidae, which includes all mosquitoes, and how adaptations in neural circuitry might affect changes in preference both within the life history of a mosquito and across evolutionary time-scales.

Natural decision-making often involves extended decision sequences in response to variable stimuli with complex structure. As an example, many animals follow odor plumes to locate food sources or mates, but turbulence breaks up the advected odor signal into intermittent filaments and puffs. This scenario provides an opportunity to ask how animals use sparse, instantaneous, and stochastic signal encounters to generate goal-oriented behavioral sequences. Here we examined the trajectories of flying fruit flies (Drosophila melanogaster) and mosquitoes (Aedes aegypti) navigating in controlled plumes of attractive odorants. While it is known that mean odor-triggered flight responses are dominated by upwind turns, individual responses are highly variable. We asked whether deviations from mean responses depended on specific features of odor encounters, and found that odor-triggered turns were slightly but significantly modulated by two features of odor encounters. First, encounters with higher concentrations triggered stronger upwind turns. Second, encounters occurring later in a sequence triggered weaker upwind turns. To contextualize the latter history dependence theoretically, we examined trajectories simulated from three normative tracking strategies. We found that neither a purely reactive strategy nor a strategy in which the tracker learned the plume centerline over time captured the observed history dependence. In contrast, “infotaxis”, in which flight decisions maximized expected information gain about source location, exhibited a history dependence aligned in sign with the data, though much larger in magnitude. These findings suggest that while true plume tracking is dominated by a reactive odor response it might also involve a history-dependent modulation of responses consistent with the accumulation of information about a source over multi-encounter timescales. This suggests that short-term memory processes modulating decision sequences may play a role in natural plume tracking., Author summary Many important behaviors require animals to make extended decision sequences in response to complex stimuli. In this study we investigated the sequences of navigational decisions made by fruit flies and mosquitoes while tracking odor plumes, which are generally subject to turbulence. By examining video-taped 3D trajectories of these insects flying in a wind tunnel containing an attractive odor plume, we asked what features of encounters with the plume influenced odor-triggered flight decisions. Most notably, we found that although the average response was dominated by a reflexive upwind turn, its strength was modulated by the history of prior plume encounters throughout the trajectory. While no theoretical strategy we simulated captured all aspects of the data, the algebraic sign of the history dependence was only recapitulated in a model where a simulated tracking agent maximizes information about the position of the plume source. This suggests that real odor tracking may involve short-term memory processes over multi-encounter timescales that are consistent with the accumulation of information about source location.

Impacts of toxic substances from oil production in the Alberta oil sands (AOS), such as polycyclic aromatic hydrocarbons (PAHs), have been widely debated. Studies have been largely restricted to exposures from surface mining in aquatic species. We measured PAHs in Woodland caribou (Rangifer tarandus caribou), moose (Alces americanus), and Grey wolf (Canis lupus) across three areas that varied in magnitude of in situ oil production. Our results suggest a distinction of PAH level and source profile (petro/pyrogenic) between study areas and species. Caribou samples indicated pyrogenic sourced PAHs in the study area previously devastated by forest fire. Moose and wolf samples from the high oil production area demonstrated PAH ratios indicative of a petrogenic source and increased PAHs, respectively. These findings emphasize the importance of broadening monitoring and research programs in the AOS.

What is Neuroecology? Animal behavior mediates many critical ecological processes that, in turn, have implications for the evolution of organismal interactions. Because the peripheral and central nervous systems ultimately control behavior, research in neuroecology seeks to link the neural basis of behavior with behavioral control of ecological interactions, and to determine how specific processes (e.g., environmental and genetic constraints, ecological and evolutionary forces) operating to alter nervous system function might constrain or facilitate adaptive behavior. Our goal for this symposium was to promote a general framework for neuroecology by exploring fundamental questions germane to this new area of research, and to develop a "toolbox" of techniques and approaches for addressing those questions. In the following series of papers, we provide a starting point for future work on neuroecology, including evolutionary context, the role of plasticity in shaping nervous system function and behavior, and an exploration of various sensorimotor systems that control ecological interactions. By promoting an integration of observational and experimental approaches at different levels of organization, we can reveal much about how the neural bases of behaviors influence interactions that occur under ecologically relevant contexts that would otherwise be impossible from isolated physiological, behavioral, or ecological components.

Honeybees are well-known models for the study of visual learning and memory. Whereas most of our knowledge of learned responses comes from experiments using free-flying bees, a tethered preparation would allow fine-scale control of the visual stimuli as well as accurate characterization of the learned responses. Unfortunately, conditioning procedures using visual stimuli in tethered bees have been limited in their efficacy. Here in this study, using a novel virtual reality environment and a differential training protocol in tethered walking bees, we show that the majority of honeybees learn visual stimuli, and need only six paired training trials to learn the stimulus. We found that bees readily learn visual stimuli that differ in both shape and colour. However, bees learn certain components over others (colour versus shape), and visual stimuli are learned in a non-additive manner with the interaction of specific colour and shape combinations being critical for learned responses. To better understand which components of the visual stimuli the bees learned, the shape-colour association of the stimuli were reversed either during or after training. Results showed that maintaining the visual stimuli in training and testing phases was necessary to elicit visual learning, suggesting that bees learn multiple components of the visual stimuli. Together, our results demonstrate a protocol for visual learning in restrained bees that provides a powerful tool for understanding how components of a visual stimulus elicits learned responses as well as elucidating how visual information is processed in the honeybee brain.

Most plant-pollinator interactions occur during specific periods during the day. To facilitate these interactions, many flowers are known to display their attractive qualities, such as scent emission and petal opening, in a daily rhythmic fashion. However, less is known about how the internal timing mechanisms (the circadian clocks) of plants and animals influence their daily interactions. We examine the role of the circadian clock in modulating the interaction between Petunia and one of its pollinators, the hawkmoth Manduca sexta. We find that desynchronization of the Petunia circadian clock affects moth visitation preference for Petunia flowers. Similarly, moths with circadian time aligned to plants show stronger flower-foraging activities than moths that lack this alignment. Moth locomotor activity is circadian clock-regulated, although it is also strongly repressed by light. Moths show a time-dependent burst increase in flight activity during subjective night. In addition, moth antennal responsiveness to the floral scent compounds exhibits a 24-hour rhythm in both continuous light and dark conditions. This study highlights the importance of the circadian clocks in both plants and animals as a crucial factor in initiating specialized plant-pollinator relationships., Scientific Reports, 8 (1), ISSN:2045-2322

How mosquitoes determine which individuals to bite has important epidemiological consequences. This choice is not random; most mosquitoes specialize in one or a few vertebrate host species, and some individuals in a host population are preferred over others. Mosquitoes will also blood feed from other hosts when their preferred is no longer abundant, but the mechanisms mediating these shifts between hosts, and preferences for certain individuals within a host species, remain unclear. Here, we show that olfactory learning may contribute to Aedes aegypti mosquito biting preferences and host shifts. Training and testing to scents of humans and other host species showed that mosquitoes can aversively learn the scent of specific humans and single odorants and learn to avoid the scent of rats (but not chickens). Using pharmacological interventions, RNAi, and CRISPR gene editing, we found that modification of the dopamine-1 receptor suppressed their learning abilities. We further show through combined electrophysiological and behavioral recordings from tethered flying mosquitoes that these odors evoke changes in both behavior and antennal lobe (AL) neuronal responses and that dopamine strongly modulates odor-evoked responses in AL neurons. Not only do these results provide direct experimental evidence that olfactory learning in mosquitoes canplay an epidemiological role, but collectively, they also provide neuroanatomical and functional demonstration of the role of dopamine in mediating this learning-induced plasticity, for the first time in a disease vector insect.

How do plants use scent to attract pollinators while preventing herbivory? New research reveals the genetic basis by which tobacco plants control the temporal emission of volatile attractants from flowers and leaves, enabling attraction of the predators of pests during the day and pollinators at night.

Mosquitoes transmit many debilitating diseases including malaria, dengue and Zika. Odors mediate behaviors that directly impact disease transmission (blood-feeding) as well as life history events that contribute to mosquito survival and fitness (mating and oviposition, nectar foraging, larval foraging and predator avoidance). In addition to innate olfaction-mediated behaviors, mosquitoes rely on olfactory experience throughout their life to inform advantageous choices in many of these important behaviors. Previous reviews have addressed either the chemical ecology of mosquitoes, or olfactory-driven behaviors including host-feeding or oviposition. Adding to this literature, we use a holistic life history perspective to integrate and compare innate and learned olfactory behavior at various stages of mosquito development.

How hawkmoths sniff out a flower Pollinators such as butterflies and bees are the true targets of the flower odors we love so much. Though we might imagine insects “following their noses,” the wealth of odors in the real world can drown out the smell of a flower, making it hard to find. Riffel et al. found that hawkmoths find angel's trumpets by creating a neuronal picture within their antennal lobe, the part of the moth brain that receives olfactory signals from the antennae (see the Perspective by Szyszka). The picture represents both the flower and the background odors. Finding a flower involves a complex reading of both background and target odors, and changes in the background odors—including human pollutants—can hinder the process. Science , this issue p. 1515 ; see also p. 1454

Mosquitoes are prolific disease vectors that affect public health around the world. Although many studies have investigated search strategies used by host-seeking adult mosquitoes, little is known about larval search behaviour. Larval behaviour affects adult body size and fecundity, and thus the capacity of individual mosquitoes to find hosts and transmit disease. Understanding vector survival at all life stages is crucial for improving disease control. In this study, we use experimental and computational methods to investigate the chemical ecology and search behaviour of Aedes aegypti mosquito larvae. We first show that larvae do not respond to several olfactory cues used by adult Ae. aegypti to assess larval habitat quality, but perceive microbial RNA as a potent foraging attractant. Second, we demonstrate that Ae. aegypti larvae use chemokinesis, an unusual search strategy, to navigate chemical gradients. Finally, we use computational modelling to demonstrate that larvae respond to starvation pressure by optimizing exploration behaviour—possibly critical for exploiting limited larval habitat types. Our results identify key characteristics of foraging behaviour in an important disease vector mosquito. In addition to implications for better understanding and control of disease vectors, this work establishes mosquito larvae as a tractable model for chemosensory behaviour and navigation.

Seed ingestion by frugivorous vertebrates commonly benefits plants by moving seeds to locations with fewer predators and pathogens than under the parent. For plants with high local population densities, however, movement from the parent plant is unlikely to result in ‘escape’ from predators and pathogens. Changes to seed condition caused by gut passage may also provide benefits, yet are rarely evaluated as an alternative. Here, we use a common bird-dispersed chilli pepper (Capsicum chacoense) to conduct the first experimental comparison of escape-related benefits to condition-related benefits of animal-mediated seed dispersal. Within chilli populations, seeds dispersed far from parent plants gained no advantage from escape alone, but seed consumption by birds increased seed survival by 370% – regardless of dispersal distance – due to removal during gut passage of fungal pathogens and chemical attractants to granivores. These results call into question the pre-eminence of escape as the primary advantage of dispersal within populations and document two overlooked mechanisms by which frugivores can benefit fruiting plants.

A Varied Bouquet Pollinators display innate attractions to odor, but can also learn to associate odor with a nectar reward. Riffell et al. (p. 200 , published online 6 December; see the Perspective by Knaden and Hansson ) characterized the odor profile for flowers to which hawkmoths are innately attracted and found that the majority contain a distinct chemical profile, which is uniquely represented on their olfactory lobe. The moths could also be trained to associate nonattractive odors with a reward and thus learn novel odor attractions. Though learning altered neurons within the antennal lobe, the innate preferences were not changed.

Sperm selection by females is an important process influencing fertilization and, particularly in broadcast-spawning organisms, often occurs before sperm reach the egg. Waterborne sperm chemoattractants are one mechanism by which eggs selectively influence conspecific sperm behavior, but it remains an open question whether the eggs from different females produce different amounts of sperm chemoattractant, and how that might influence sperm behavior. Here, we quantify the differences in attractant production between females of the sea urchin species Lytechinus pictus and use computational models and microfluidic sperm chemotaxis assays to determine how differences in chemoattractant production between females affects their ability to attract sperm. Our study demonstrates that there is significant individual female variation in egg chemoattractant production, and that this variation changes the scope and strength of sperm attraction. These results provide evidence for the importance of individual female variability in differential sperm attraction and fertilization success.

Plant volatiles play vital roles in signaling with their insect associates. Empirical studies show that both pollinators and herbivores exert strong selective pressures on plant phenotypes. While studies often evoke the assumption that volatiles from floral and vegetative tissues are distinct due to strong pollinator-mediated selection operating on the flowers or selection from herbivores acting on the leaves, explicit tests of these assumptions are often lacking. In this study, we examined the evolution of floral and vegetative volatiles in the North American (NA) pitcher plants (Sarraceniaceae). In these taxa, insects are attracted for both pollination and prey capture, providing an ideal opportunity to understand the evolution of scent compounds across different plant organs. We collected a comprehensive dataset of floral and vegetative volatiles from across the NA Sarraceniaceae. We used multivariate analysis methods to examine whether volatile profiles are distinct between plant tissues, and investigated rates of scent evolution in these unique taxa. Our major findings revealed that (i) flowers and pitchers produced highly distinct scent profiles, consistent with the hypothesis that volatiles alleviate trade-offs due to incidental pollinator consumption; (ii) across species, floral scent separated into distinct regions of scent space, while pitchers showed little evidence of clustering – this may be due to convergence on a generalist strategy for insect capture; and (iii) rates of scent evolution depended on tissue type, suggesting that pollinators and herbivores differentially influence the evolution of chemical traits. We emphasize the need for additional functional studies to further distinguish between volatile functions.

Learning and memory plays an important role in host preference and parasite transmission by disease vector insects. Historically there has been a dearth of standardized protocols that permit testing their learning abilities, thus limiting discussion on the potential epidemiological consequences of learning and memory to a largely speculative extent. However, with increasing evidence that individual experience and associative learning can affect processes such as oviposition site selection and host preference, it is timely to review the recently acquired knowledge, identify research gaps and discuss the implication of learning in disease vector insects in perspective with control strategies.

In Southwestern USA, the jimsonweed Datura wrightii and the nocturnal sphinx moth Manduca sexta form a pollinator-plant and herbivore-plant association. While certain plant volatile organic compounds (VOCs) attract moths for oviposition, it is likely that other host-derived olfactory cues, such as herbivore-induced VOCs, repel moths for oviposition. Here, we studied the oviposition preference of female M. sexta towards intact and damaged host plants of three species: D. wrightii, D. discolor (a less preferred feeding resource but also used by females for oviposition), and Solanum lycopersicum-tomato-(used by moths as an oviposition resource only). Damage was inflicted to the plants either by larval feeding or artificial damage. Mated females were exposed to an intact plant and a damaged plant and allowed to lay eggs for 10 min. Oviposition preferences of females were highly heterogeneous in all cases, but a larger proportion of moths laid significantly fewer eggs on feeding-damaged and artificially damaged plants of S. lycopersicum. Many females also avoided feeding-damaged D. discolor and D. wrightii plants induced by treatment with methyl jasmonate. Chemical analyses showed a significant increase in the total amount of VOCs released by vegetative tissues of feeding-damaged plants, as well as species-specific increases in emission of certain VOCs. In particular, feeding-damaged S. lycopersicum plants emitted (-)-linalool, an odorant that repels moths for oviposition. Finally, the emission of D. wrightii floral VOCs, which are important in mediating feeding by adult moths (and hence pollination), did not change in plants damaged by larval feeding. We propose that the observed differential effects of herbivory on oviposition choice are due to different characteristics (i.e., mutually beneficial or parasitic) of the insect-plant interaction.

The need to detect and process sensory cues varies in different behavioral contexts. Plasticity in sensory coding can be achieved by the context-specific release of neuromodulators in restricted brain areas. The context of aversion triggers the release of dopamine in the insect brain, yet the effects of dopamine on sensory coding are unknown. In this study, we characterize the morphology of dopaminergic neurons that innervate each of the antennal lobes (ALs; the first synaptic neuropils of the olfactory system) of the moth Manduca sexta and demonstrate with electrophysiology that dopamine enhances odor-evoked responses of the majority of AL neurons while reducing the responses of a small minority. Because dopamine release in higher brain areas mediates aversive learning we developed a naturalistic, ecologically inspired aversive learning paradigm in which an innately appetitive host plant floral odor is paired with a mimic of the aversive nectar of herbivorized host plants. This pairing resulted in a decrease in feeding behavior that was blocked when dopamine receptor antagonists were injected directly into the ALs. These results suggest that a transient dopaminergic enhancement of sensory output from the AL contributes to the formation of aversive memories. We propose a model of olfactory modulation in which specific contexts trigger the release of different neuromodulators in the AL to increase olfactory output to downstream areas of processing.

Studies of plant–pollinator interactions have often documented species differences in preferences for floral advertisements and rewards. However, the contribution of intraspecific variation in behaviours, especially between sexes, remains less understood. We explored resource preference and resource use by male and female Manduca sexta hawkmoths, relative to two important nectar resources in southern Arizona, U.S.A. Manduca sexta is the major pollinator of one of these species (Datura wrightii, Solanaceae). Because females must also seek out D. wrightii as an oviposition resource, females were predicted to feed upon it more than would males, which should be free to choose the best nectar resource. Using naive laboratory-reared moths in flight arena experiments, we found that both sexes preferred Datura wrightii over Agave palmeri (Agavaceae). Exposure to only one species and an odourless paper control, however, revealed sex-specific differences in foraging behaviour, with females feeding longer from A. palmeri and males feeding longer from D. wrightii, leading us to reject our hypothesis. Differences in feeding preferences directly translated into differences in energy intake. Females gained significantly more energy than did males by feeding from A. palmeri. We also examined whether behavioural preferences of moths in the laboratory translated into foraging behaviour in the field. Pollen load analysis of moths caught in 2004 showed that females carried significantly more A. palmeri pollen than did males, whereas males carried more D. wrightii pollen than did females. Whereas most studies examine pollination associations at the species level, our results highlight the potential importance of between-sex variation in floral visits.

In southwestern USA, the jimsonweed Datura wrightii and the nocturnal moth Manduca sexta form a pollinator–plant and herbivore–plant association. Because the floral scent is probably important in mediating this interaction, we investigated the floral volatiles that might attract M. sexta for feeding and oviposition. We found that flower volatiles increase oviposition and include small amounts of both enantiomers of linalool, a common component of the scent of hawkmoth-pollinated flowers. Because (+)-linalool is processed in a female-specific glomerulus in the primary olfactory centre of M. sexta , we hypothesized that the enantiomers of linalool differentially modulate feeding and oviposition. Using a synthetic mixture that mimics the D. wrightii floral scent, we found that the presence of linalool was not necessary to evoke feeding and that mixtures containing (+)- and/or (−)-linalool were equally effective in mediating this behaviour. By contrast, females oviposited more on plants emitting (+)-linalool (alone or in mixtures) over control plants, while plants emitting (−)-linalool (alone or in mixtures) were less preferred than control plants. Together with our previous investigations, these results show that linalool has differential effects in feeding and oviposition through two neural pathways: one that is sexually isomorphic and non-enantioselective, and another that is female-specific and enantioselective.