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Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles. Because individual muscles may be used in many different behaviors, MN activity must be flexibly coordinated by dedicated premotor circuitry, the organization of which remains largely unknown. Here, we use comprehensive reconstruction of neuron anatomy and synaptic connectivity from volumetric electron microscopy (i.e., connectomics) to analyze the wiring logic of motor circuits controlling theDrosophilaleg and wing. We find that both leg and wing premotor networks are organized into modules that link MNs innervating muscles with related functions. However, the connectivity patterns within leg and wing motor modules are distinct. Leg premotor neurons exhibit proportional gradients of synaptic input onto MNs within each module, revealing a novel circuit basis for hierarchical MN recruitment. In comparison, wing premotor neurons lack proportional synaptic connectivity, which may allow muscles to be recruited in different combinations or with different relative timing. By comparing the architecture of distinct limb motor control systems within the same animal, we identify common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.

When an animal moves through the world, its brain receives a stream of information about the body’s translational velocity from motor commands and sensory feedback signals. These incoming signals are referenced to the body, but ultimately, they must be transformed into world-centric coordinates for navigation. Here we show that this computation occurs in the fan-shaped body in the brain of Drosophila melanogaster. We identify two cell types, PFNd and PFNv, that conjunctively encode translational velocity and heading as a fly walks. In these cells, velocity signals are acquired from locomotor brain regions and are multiplied with heading signals from the compass system. PFNd neurons prefer forward–ipsilateral movement, whereas PFNv neurons prefer backward–contralateral movement, and perturbing PFNd neurons disrupts idiothetic path integration in walking flies. Downstream, PFNd and PFNv neurons converge onto hΔB neurons, with a connectivity pattern that pools together heading and translation direction combinations corresponding to the same movement in world-centric space. This network motif effectively performs a rotation of the brain’s representation of body-centric translational velocity according to the current heading direction. Consistent with our predictions, we observe that hΔB neurons form a representation of translational velocity in world-centric coordinates. By integrating this representation over time, it should be possible for the brain to form a working memory of the path travelled through the environment.

While insects such as Drosophila are flying, aerodynamic instabilities require that they make millisecond time scale adjustments to their wing motion to stay aloft and on course. These stabilization reflexes can be modeled as a proportional-integral (PI) controller; however, it is unclear how such control might be instantiated in insects at the level of muscles and neurons. Here, we show that the b1 and b2 motor units—prominent components of the fly’s steering muscle system—modulate specific elements of the PI controller: the angular displacement (integral) and angular velocity (proportional), respectively. Moreover, these effects are observed only during the stabilization of pitch. Our results provide evidence for an organizational principle in which each muscle contributes to a specific functional role in flight control, a finding that highlights the power of using top-down behavioral modeling to guide bottom-up cellular manipulation studies.

SUMMARYTo maintain stable flight, animals continuously perform trimming adjustments to compensate for internal and external perturbations. Whereas animals use many different sensory modalities to detect such perturbations, insects rely extensively on optic flow to modify their motor output and remain on course. We studied this behavior in the fruit fly,Drosophila melanogaster, by exploiting the optomotor response, a robust reflex in which an animal steers so as to minimize the magnitude of rotatory optic flow it perceives. Whereas the behavioral and algorithmic structure of the optomotor response has been studied in great detail, its neural implementation is not well-understood. In this paper, we present findings implicating a group of nearly homomorphic descending neurons, the DNg02s, as a core component for the optomotor response in flyingDrosophila. Prior work on these cells suggested that they regulate the mechanical power to the flight system, presumably via connections to asynchronous flight motor neurons in the ventral nerve cord. When we chronically inactivated these cells, we observed that the magnitude of the optomotor response was diminished in proportion to the number of cells silenced, suggesting that the cells also regulate bilaterally asymmetric steering responses via population coding. During an optomotor response, flies coordinate changes in wing motion with movements of their head, abdomen, and hind legs, which are also diminished when the DNg02 cells are silenced. Using two-photon functional imaging, we show that the DNg02 cells respond most strongly to patterns of horizontal motion and that neuronal activity is closely correlated to motor output. However, unilateral optogenetic activation of DNg02 neurons does not elicit the asymmetric changes in wing motion characteristic of the optomotor response to a visual stimulus, but rather generates bilaterally symmetric increases in wingbeat amplitude. We interpret our experiments to suggest that flight maneuvers in flies require a more nuanced coordination of power muscles and steering muscles than previously appreciated, and that the physical flight apparatus of a fly might permit mechanical power to be distributed differentially between the two wings. Thus, whereas our experiments identify the DNg02 cells as a critical component of the optomotor reflex, our results suggest that other classes of descending cells targeting the steering muscle motor neurons are also required for the behavior.

Summary The ability to keep track of one’s location in space is a critical behavior for animals navigating to and from a salient location, and its computational basis is now beginning to be unraveled. Here, we tracked flies in a ring-shaped channel as they executed bouts of search triggered by optogenetic activation of sugar receptors. Unlike experiments in open field arenas, which produce highly tortuous search trajectories, our geometrically constrained paradigm enabled us to monitor flies’ decisions to move toward or away from the fictive food. Our results suggest that flies use path integration to remember the location of a food site even after it has disappeared, and flies can remember the location of a former food site even after walking around the arena one or more times. To determine the behavioral algorithms underlying Drosophila search, we developed multiple state transition models and found that flies likely accomplish path integration by combining odometry and compass navigation to keep track of their position relative to the fictive food. Our results indicate that whereas flies re-zero their path integrator at food when only one feeding site is present, they adjust their path integrator to a central location between sites when experiencing food at two or more locations. Together, this work provides a simple experimental paradigm and theoretical framework to advance investigations of the neural basis of path integration.

SummaryLike many insect species, Drosophila melanogaster are capable of maintaining a stable flight trajectory for periods lasting up to several hours(1, 2). Because aerodynamic torque is roughly proportional to the fifth power of wing length(3), even small asymmetries in wing size require the maintenance of subtle bilateral differences in flapping motion to maintain a stable path. Flies can even fly straight after losing half of a wing, a feat they accomplish via very large, sustained kinematic changes to the both damaged and intact wings(4). Thus, the neural network responsible for stable flight must be capable of sustaining fine-scaled control over wing motion across a large dynamic range. In this paper, we describe an unusual type of descending neurons (DNg02) that project directly from visual output regions of the brain to the dorsal flight neuropil of the ventral nerve cord. Unlike most descending neurons, which exist as single bilateral pairs with unique morphology, there is a population of at least 15 DNg02 cell pairs with nearly identical shape. By optogenetically activating different numbers of DNg02 cells, we demonstrate that these neurons regulate wingbeat amplitude over a wide dynamic range via a population code. Using 2-photon functional imaging, we show that DNg02 cells are responsive to visual motion during flight in a manner that would make them well suited to continuously regulate bilateral changes in wing kinematics. Collectively, we have identified a critical set of DNs that provide the sensitivity and dynamic range required for flight control.

Despite the ecological importance of long-distance dispersal in insects, its mechanistic basis is poorly understood in genetic model species, in which advanced molecular tools are readily available. One critical question is how insects interact with the wind to detect attractive odor plumes and increase their travel distance as they disperse. To gain insight into dispersal, we conducted release-and-recapture experiments in the Mojave Desert using the fruit fly, Drosophila melanogaster. We deployed chemically baited traps in a 1 km radius ring around the release site, equipped with cameras that captured the arrival times of flies as they landed. In each experiment, we released between 30,000 and 200,000 flies. By repeating the experiments under a variety of conditions, we were able to quantify the influence of wind on flies’ dispersal behavior. Our results confirm that even tiny fruit flies could disperse ∼12 km in a single flight in still air and might travel many times that distance in a moderate wind. The dispersal behavior of the flies is well explained by an agent-based model in which animals maintain a fixed body orientation relative to celestial cues, actively regulate groundspeed along their body axis, and allow the wind to advect them sideways. The model accounts for the observation that flies actively fan out in all directions in still air but are increasingly advected downwind as winds intensify. Our results suggest that dispersing insects may strike a balance between the need to cover large distances while still maintaining the chance of intercepting odor plumes from upwind sources.

Despite the ecological importance of long-distance dispersal in insects, its mechanistic basis is poorly understood in genetic model species, in which advanced molecular tools are readily available. One critical question is how insects interact with the wind to detect attractive odor plumes and increase their travel distance as they disperse. To gain insight into dispersal, we conducted release-and-recapture experiments in the Mojave Desert using the fruit fly, Drosophila melanogaster. We deployed chemically baited traps in a 1 km radius ring around the release site, equipped with cameras that captured the arrival times of flies as they landed. In each experiment, we released between 30,000 and 200,000 flies. By repeating the experiments under a variety of conditions, we were able to quantify the influence of wind on flies’ dispersal behavior. Our results confirm that even tiny fruit flies could disperse ∼12 km in a single flight in still air and might travel many times that distance in a moderate wind. The dispersal behavior of the flies is well explained by an agent-based model in which animals maintain a fixed body orientation relative to celestial cues, actively regulate groundspeed along their body axis, and allow the wind to advect them sideways. The model accounts for the observation that flies actively fan out in all directions in still air but are increasingly advected downwind as winds intensify. Our results suggest that dispersing insects may strike a balance between the need to cover large distances while still maintaining the chance of intercepting odor plumes from upwind sources.

Carbon dioxide is produced by many organic processes and is a convenient volatile cue for insects1 that are searching for blood hosts2, flowers3, communal nests4, fruit5 and wildfires6. Although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments7-12 suggest that walking flies avoid CO2. Here we resolve this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when they are in an active state associated with foraging. Their aversion to CO2 at low-activity levels may be an adaptation to avoid parasites that seek CO2, or to avoid succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature13,14. In contrast to CO2, flies are attracted to ethanol in all behavioural states, and invest twice the time searching near ethanol compared to CO2. These behavioural differences reflect the fact that ethanol is a unique signature of yeast fermentation, whereas CO2 is generated by many natural processes. Using genetic tools, we determined that the evolutionarily conserved ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved in this attraction. Our study lays the foundation for future research to determine the neural circuits that underlie both state- and odorant-dependent decision-making in Drosophila.

SUMMARYThe ability to keep track of one’s location in space is a critical behavior for animals navigating to and from a salient location, and its computational basis is now beginning to be unraveled. Here, we tracked flies in a ring-shaped channel as they executed bouts of search triggered by optogenetic activation of sugar receptors. Unlike experiments in open field arenas, which produce highly tortuous search trajectories, our geometrically constrained paradigm enabled us to monitor flies’ decisions to move toward or away from the fictive food. Our results suggest that flies use path integration to remember the location of a food site even after it has disappeared, and that flies can remember the location of a former food site even after walking around the arena one or more times. To determine the behavioral algorithms underlying Drosophila search, we developed multiple state transition models and found that flies likely accomplish path integration by combining odometry and compass navigation to keep track of their position relative to the fictive food. Our results indicate that whereas flies re-zero their path integrator at food when only one feeding site is present, they adjust their path integrator to a central location between sites when experiencing food at two or more locations. Together, this work provides a simple experimental paradigm and theoretical framework to advance investigations of the neural basis of path integration.

Despite the ecological importance of long-distance dispersal in insects, its underlying mechanistic basis is poorly understood. One critical question is how insects interact with the wind to increase their travel distance as they disperse. To gain insight into dispersal using a species amenable to further investigation using genetic tools, we conducted release-and-recapture experiments in the Mojave Desert using the fruit fly,Drosophila melanogaster. We deployed chemically-baited traps in a 1 km-radius ring around the release site, equipped with machine vision systems that captured the arrival times of flies as they landed. In each experiment, we released between 30,000 and 200,000 flies. By repeating the experiments under a variety of conditions, we were able to quantify the influence of wind on flies’ dispersal behavior. Our results confirm that even tiny fruit flies could disperse ∼15 km in a single flight in still air, and might travel many times that distance in a moderate wind. The dispersal behavior of the flies is well explained by a model in which animals maintain a fixed body orientation relative to celestial cues, actively regulate groundspeed along their body axis, and allow the wind to advect them sideways. The model accounts for the observation that flies actively fan out in all directions in still air, but are increasingly advected downwind as winds intensify. In contrast, our field data do not support a Lévy flight model of dispersal, despite the fact that our experimental conditions almost perfectly match the core assumptions of that theory.Significance StatementFlying insects play a vital role in terrestrial ecosystems, and their decline over the past few decades has been implicated in a collapse of many species that depend upon them for food. By dispersing over large distances, insects transport biomass from one region to another and thus their flight behavior influences ecology on a global scale. Our experiments provide key insight into the dispersal behavior of insects, and suggest that these animals employ a single algorithm that is functionally robust in both still air and under windy conditions. Our results will make it easier to study the ecologically important phenomenon of long-distance dispersal in a genetic model organism, facilitating the identification of cellular and genetic mechanisms.

Drosophila melanogaster is an established model for neuroscience research with relevance in biology and medicine. Until recently, research on the Drosophila brain was hindered by the lack of a complete and uniform nomenclature. Recognizing this, Ito et al. (2014) produced an authoritative nomenclature for the adult insect brain, using Drosophila as the reference. Here, we extend this nomenclature to the adult thoracic and abdominal neuromeres, the ventral nerve cord (VNC), to provide an anatomical description of this major component of the Drosophila nervous system. The VNC is the locus for the reception and integration of sensory information and involved in generating most of the locomotor actions that underlie fly behaviors. The aim is to create a nomenclature, definitions, and spatial boundaries for the Drosophila VNC that are consistent with other insects. The work establishes an anatomical framework that provides a powerful tool for analyzing the functional organization of the VNC.

For almost 100 years, biologists have made fundamental discoveries using a handful of model organisms that are not representative of the rich diversity found in nature. The advent of CRISPR genome editing now opens up a wide range of new organisms to mechanistic investigation. This increases not only the taxonomic breadth of current research but also the scope of biological problems that are now amenable to study, such as population control of invasive species, management of disease vectors such as mosquitoes, the creation of chimeric animal hosts to grow human organs and even the possibility of resurrecting extinct species such as passenger pigeons and mammoths. Beyond these practical applications, work on non-model organisms enriches our basic understanding of the natural world. This special issue addresses a broad spectrum of biological problems in non-model organisms and highlights the utility of genome editing across levels of complexity from development and physiology to behaviour and evolution.

The fruit fly, Drosophila melanogaster, is an established and powerful model system for neuroscience research with wide relevance in biology and medicine. Until recently, research on the Drosophila brain was hindered by the lack of a complete and uniform nomenclature. Recognising this problem, the Insect Brain Name Working Group produced an authoritative hierarchical nomenclature system for the adult insect brain, using Drosophila melanogaster as the reference framework, with other taxa considered to ensure greater consistency and expandability (Ito et al., 2014). Here, we extend this nomenclature system to the sub-gnathal regions of the adult Drosophila nervous system, thus providing a systematic anatomical description of the ventral nervous system (VNS). This portion of the nervous system includes the thoracic and abdominal neuromeres that were not included in the original work and contains the motor circuits that play essential roles in most fly behaviours.

To navigate through the world, animals must stabilize their path against disturbances and change direction to avoid obstacles and to search for resources [1 ; 2]. Locomotion is thus guided by sensory cues but also depends on intrinsic processes, such as motivation and physiological state. Flies, for example, turn with the direction of large-field rotatory motion, an optomotor reflex that is thought to help them fly straight [3; 4 ; 5]. Occasionally, however, they execute fast turns, called body saccades, either spontaneously or in response to patterns of visual motion such as expansion [6; 7 ; 8]. These turns can be measured in tethered flying Drosophila [ 3; 4 ; 9], which facilitates the study of underlying neural mechanisms. Whereas there is evidence for an efference copy input to visual interneurons during saccades [10], the circuits that control spontaneous and visually elicited saccades are not well known. Using two-photon calcium imaging and electrophysiological recordings in tethered flying Drosophila, we have identified a descending neuron whose activity is correlated with both spontaneous and visually elicited turns during tethered flight. The cell’s activity in open- and closed-loop experiments suggests that it does not underlie slower compensatory responses to horizontal motion but rather controls rapid changes in flight path. The activity of this neuron can explain some of the behavioral variability observed in response to visual motion and appears sufficient for eliciting turns when artificially activated. This work provides an entry point into studying the circuits underlying the control of rapid steering maneuvers in the fly brain.

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.

Many animals use visual cues like object shape, color, and motion to detect and pursue conspecific mates. Contrast is another possibly informative visual cue, but has not been studied in great detail. In this study, we presented male Drosophila melanogaster with small, fly-sized, moving objects painted either black, white, or grey to test if they use contrast cues to identify mates. We found that males frequently chased grey objects and rarely chased white or black objects. Although males started chasing black objects as often as grey objects, the resulting chases were much shorter. To test whether the attraction to grey objects was mediated via contrast, we fabricated black and grey behavioral chambers. However, wildtype males almost never chased any objects in these darkly colored chambers. To circumvent this limitation, we increased baseline levels of chasing by thermogenetically activating P1 neurons to promote courtship. Males with thermogenetically activated P1 neurons maintained a similar preference for grey objects despite elevated levels of courtship behavior. When placed in a black chamber, males with activated P1 neurons switched their preference and chased black objects more than grey objects. We also tested whether males use contrast cues to orient to particular parts of the female's body during courtship. When presented with moving objects painted two colors, males positioned themselves next to the grey half regardless of whether the other half was painted black or white. These results suggest that males can use contrast to recognize potential mates and to position themselves during courtship.

Foraging animals may benefit from remembering the location of a newly discovered food patch while continuing to explore nearby [1, 2]. For example, after encountering a drop of yeast or sugar, hungry flies often perform a local search [3, 4]. That is, rather than remaining on the food or simply walking away, flies execute a series of exploratory excursions during which they repeatedly depart and return to the resource. Fruit flies, Drosophila melanogaster, can perform this food-centered search behavior in the absence of external landmarks, instead relying on internal (idiothetic) cues [5]. This path-integration behavior may represent a deeply conserved navigational capacity in insects [6, 7], but its underlying neural basis remains unknown. Here, we used optogenetic activation to screen candidate cell classes and found that local searches can be initiated by diverse sensory neurons. Optogenetically induced searches resemble those triggered by actual food, are modulated by starvation state, and exhibit key features of path integration. Flies perform tightly centered searches around the fictive food site, even within a constrained maze, and they can return to the fictive food site after long excursions. Together, these results suggest that flies enact local searches in response to a wide variety of food-associated cues and that these sensory pathways may converge upon a common neural system for navigation. Using a virtual reality system, we demonstrate that local searches can be optogenetically induced in tethered flies walking on a spherical treadmill, laying the groundwork for future studies to image the brain during path integration. VIDEO ABSTRACT.

Many animals use visual cues such as object shape, color and motion to detect and pursue conspecific mates. Contrast is another possibly informative visual cue, but has not been studied in great detail. In this study, we presented male

Many casual observers typecast Drosophila melanogaster as a stationary pest that lurks around fruit and wine. However, the omnipresent fruit fly, which thrives even in desert habitats, likely established and maintained its cosmopolitan status via migration over large spatial scales. To perform long-distance dispersal, flies must actively maintain a straight compass heading through the use of external orientation cues, such as those derived from the sky. In this Review, we address how D. melanogaster accomplishes long-distance navigation using celestial cues. We focus on behavioral and physiological studies indicating that fruit flies can navigate both to a pattern of linearly polarized light and to the position of the sun – the same cues utilized by more heralded insect navigators such as monarch butterflies and desert ants. In both cases, fruit flies perform menotaxis, selecting seemingly arbitrary headings that they then maintain over time. We discuss how the fly's nervous system detects and processes this sensory information to direct the steering maneuvers that underlie navigation. In particular, we highlight recent findings that compass neurons in the central complex, a set of midline neuropils, are essential for navigation. Taken together, these results suggest that fruit flies share an ancient, latent capacity for celestial navigation with other insects. Furthermore, they illustrate the potential of D. melanogaster to help us to elucidate both the cellular basis of navigation and mechanisms of directed dispersal on a landscape scale.

Localizing the sources of stimuli is essential. Most organisms cannot eat, mate, or escape without knowing where the relevant stimuli originate. For many, if not most, animals, olfaction plays an essential role in search. While microorganismal chemotaxis is relatively well understood, in larger animals the algorithms and mechanisms of olfactory search remain mysterious. In this symposium, we will present recent advances in our understanding of olfactory search in flies and rodents. Despite their different sizes and behaviors, both species must solve similar problems, including meeting the challenges of turbulent airflow, sampling the environment to optimize olfactory information, and incorporating odor information into broader navigational systems.

Resources are often sparsely clustered in nature. Thus, foraging animals may benefit from remembering the location of a newly discovered food patch while continuing to explore nearby [1, 2]. For example, after encountering a drop of yeast or sugar, hungry flies often perform a local search consisting of frequent departures and returns to the food site [3, 4]. Fruit flies, Drosophila melanogaster, can perform this food-centered search behavior in the absence of external stimuli or landmarks, instead relying solely on internal (idiothetic) cues to keep track of their location [5]. This path integration behavior may represent a deeply conserved navigational capacity in insects [6, 7], but the neural pathways underlying food-triggered searches remain unknown. Here, we used optogenetic activation to screen candidate cell classes and found that local searches can be initiated by diverse sensory neurons including sugar-sensors, water-sensors, olfactory-receptor neurons, as well as hunger-signaling neurons of the central nervous system. Optogenetically-induced searches resemble those triggered by actual food and are modulated by starvation state. Furthermore, search trajectories exhibit key features of path integration: searches remain tightly centered around the fictive-food site, even during long periods without reinforcement, and flies re-center their searches when they encounter a new fictive-food site. Flies can even perform elaborate local searches within a constrained maze. Together, these results suggest that flies enact local searches in response to a wide variety of food-associated cues, and that these sensory pathways may converge upon a common neural system for path integration. Optogenetically induced local searches in Drosophila can now serve as a tractable system for the study of spatial memory and navigation in insects.

Many insects exploit skylight polarization as a compass cue for orientation and navigation. In the fruit fly,Drosophila melanogaster, photoreceptors R7 and R8 in the dorsal rim area (DRA) of the compound eye are specialized to detect the electric vector (e-vector) of linearly polarized light. These photoreceptors are arranged in stacked pairs with identical fields of view and spectral sensitivities, but mutually orthogonal microvillar orientations. As in larger flies, we found that the microvillar orientation of the distal photoreceptor R7 changes in a fan-like fashion along the DRA. This anatomical arrangement suggests that the DRA constitutes a detector for skylight polarization, in which different e-vectors maximally excite different positions in the array. To test our hypothesis, we measured responses to polarized light of varying e-vector angles in the terminals of R7/8 cells using genetically encoded calcium indicators. Our data confirm a progression of preferred e-vector angles from anterior to posterior in the DRA, and a strict orthogonality between the e-vector preferences of paired R7/8 cells. We observed decreased activity in photoreceptors in response to flashes of light polarized orthogonally to their preferred e-vector angle, suggesting reciprocal inhibition between photoreceptors in the same medullar column, which may serve to increase polarization contrast. Together, our results indicate that the polarization-vision system relies on a spatial map of preferred e-vector angles at the earliest stage of sensory processing.SIGNIFICANCE STATEMENTThe fly's visual system is an influential model system for studying neural computation, and much is known about its anatomy, physiology, and development. The circuits underlying motion processing have received the most attention, but researchers are increasingly investigating other functions, such as color perception and object recognition. In this work, we investigate the early neural processing of a somewhat exotic sense, called polarization vision. Because skylight is polarized in an orientation that is rigidly determined by the position of the sun, this cue provides compass information. Behavioral experiments have shown that many species use the polarization pattern in the sky to direct locomotion. Here we describe the input stage of the fly's polarization-vision system.

Multifunctional motor systems produce distinct output patterns that are dependent on behavioral context, posing a challenge to underlying neuronal control. Flies use their wings for flight and the production of a patterned acoustic signal, the male courtship song, employing in both cases a small set of wing muscles and corresponding motor neurons. We took first steps toward elucidating the neuronal control mechanisms of this multifunctional motor system by live imaging of muscle ensemble activity patterns during song and flight, and we established the functional role of a comprehensive set of wing muscle motor neurons by silencing experiments. Song and flight rely on distinct configurations of neuromuscular activity, with most, but not all, flight muscles and their corresponding motor neurons contributing to song and shaping its acoustic parameters. The two behaviors are exclusive, and the neuronal command for flight overrides the command for song. The neuromodulator octopamine is a candidate for selectively stabilizing flight, but not song motor patterns.

Flies execute their remarkable aerial maneuvers using a set of wing steering muscles, which are activated at specific phases of the stroke cycle [1, 2, 3]. The activation phase of these muscles—which determines their biomechanical output [4, 5, 6]—arises via feedback from mechanoreceptors at the base of the wings and structures unique to flies called halteres [7, 8, 9]. Evolved from the hindwings, the tiny halteres oscillate at the same frequency as the wings, although they serve no aerodynamic function [10] and are thought to act as gyroscopes [10, 11, 12, 13, 14, 15]. Like the wings, halteres possess minute control muscles whose activity is modified by descending visual input [16], raising the possibility that flies control wing motion by adjusting the motor output of their halteres, although this hypothesis has never been directly tested. Here, using genetic techniques possible in Drosophila melanogaster, we tested the hypothesis that visual input during flight modulates haltere muscle activity and that this, in turn, alters the mechanosensory feedback that regulates the wing steering muscles. Our results suggest that rather than acting solely as a gyroscope to detect body rotation, halteres also function as an adjustable clock to set the spike timing of wing motor neurons, a specialized capability that evolved from the generic flight circuitry of their four-winged ancestors. In addition to demonstrating how the efferent control loop of a sensory structure regulates wing motion, our results provide insight into the selective scenario that gave rise to the evolution of halteres.

SUMMARYIn most animals, the brain controls the body via a set of descending neurons (DNs) that traverse the neck and terminate in post-cranial regions of the nervous system. This critical neural population is thought to activate, maintain and modulate locomotion and other behaviors. Although individual members of this cell class have been well-studied across species ranging from insects to primates, little is known about the overall connectivity pattern of DNs as a population. We undertook a systematic anatomical investigation of descending neurons in the fruit fly,Drosophila melanogaster, and created a collection of over 100 transgenic lines targeting individual cell types. Our methods allowed us to describe the morphology of roughly half of an estimated 400 DNs and create a comprehensive map of connectivity between the sensory neuropils in the brain and the motor neuropils in the ventral nerve cord. Like the vertebrate spinal cord, our results show that the fly nerve cord is a highly organized, layered system of neuropils, an organization that reflects the fact that insects are capable of two largely independent means of locomotion – walking and fight – using distinct sets of appendages. Our results reveal the basic functional map of descending pathways in flies and provide tools for systematic interrogation of sensory-motor circuits.

In contrast to larger species, little is known about the flight of the smallest flying insects, such as thrips and fairyflies. These tiny animals range from 300 to 1000 microns in length and fly at Reynolds numbers ranging from about 4 to 60. Previous work with numerical and physical models have shown that the aerodynamics of these diminutive insects is significantly different from that of larger animals, but most of these studies have relied on two-dimensional approximations. There can, however, be significant differences between two- and three-dimensional flows, as has been found for larger insects. To better understand the flight of the smallest insects, we have performed a systematic study of the forces and flow structures around a three-dimensional revolving elliptical wing. We used both a dynamically scaled physical model and a three-dimensional computational model at Reynolds numbers ranging from 1 to 130 and angles of attacks ranging from 0&deg, to 90&deg, The results of the physical and computational models were in good agreement and showed that dimensionless drag, aerodynamic efficiency, and spanwise flow all decrease with decreasing Reynolds number. In addition, both the leading and trailing edge vortices remain attached to the wing over the scales relevant to the smallest flying insects. Overall, these observations suggest that there are drastic differences in the aerodynamics of flight at the scale of the smallest flying animals.

Despite their small brains, insects can navigate over long distances by orienting using visual landmarks [1], skylight polarization [2, 3, 4, 5, 6, 7, 8, 9], and sun position [3, 4, 6, 10]. Although Drosophila are not generally renowned for their navigational abilities, mark-and-recapture experiments in Death Valley revealed that they can fly nearly 15 km in a single evening [11]. To accomplish such feats on available energy reserves [12], flies would have to maintain relatively straight headings, relying on celestial cues [13]. Cues such as sun position and polarized light are likely integrated throughout the sensory-motor pathway [14], including the highly conserved central complex [4, 15, 16]. Recently, a group of Drosophila central complex cells (E-PG neurons) have been shown to function as an internal compass [17, 18, 19], similar to mammalian head-direction cells [20]. Using an array of genetic tools, we set out to test whether flies can navigate using the sun and to identify the role of E-PG cells in this behavior. Using a flight simulator, we found that Drosophila adopt arbitrary headings with respect to a simulated sun, thus performing menotaxis, and individuals remember their heading preference between successive flights—even over several hours. Imaging experiments performed on flying animals revealed that the E-PG cells track sun stimulus motion. When these neurons are silenced, flies no longer adopt and maintain arbitrary headings relative to the sun stimulus but instead exhibit frontal phototaxis. Thus, without the compass system, flies lose the ability to execute menotaxis and revert to a simpler, reflexive behavior.

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.

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because the portion of the nervous system that contains limb premotor and motor circuits is largely inaccessible to large-scale recording techniques in intact, moving animals – a constraint that is true for both vertebrate and invertebrate models. Here, we introduce a method for 2-photon functional imaging from the ventral nerve cord of behaving adultDrosophila melanogaster. We use this method to reveal patterns of activity across nerve cord populations during grooming and walking and to uncover the functional encoding of moonwalker ascending neurons (MANs), moonwalker descending neurons (MDNs), and a novel class of locomotion-associated descending neurons. This new approach enables the direct investigation of circuits associated with complex limb movements.

Animals must use external cues to maintain a straight course over long distances. In this study, we investigated how the fruit fly

Animals must use external cues to maintain a straight course over long distances. In this study, we investigated how the fruit fly, Drosophila melanogaster, selects and maintains a flight heading relative to the axis of linearly polarized light, a visual cue produced by the atmospheric scattering of sunlight. To track flies’ headings over extended periods, we used a flight simulator that coupled the angular velocity of dorsally presented polarized light to the stroke amplitude difference of the animal's wings. In the simulator, most flies actively maintained a stable heading relative to the axis of polarized light for the duration of 15 minute flights. We found that individuals selected arbitrary, unpredictable headings relative to the polarization axis, which demonstrates that Drosophila can perform proportional navigation using a polarized light pattern. When flies flew in two consecutive bouts separated by a 5 minute gap, the two flight headings were correlated, suggesting individuals retain a memory of their chosen heading. We found that adding a polarized light pattern to a light intensity gradient enhanced flies’ orientation ability, suggesting Drosophila use a combination of cues to navigate. For both polarized light and intensity cues, flies’ capacity to maintain a stable heading gradually increased over several minutes from the onset of flight. Our findings are consistent with a model in which each individual initially orients haphazardly but then settles on a heading which is maintained via a self-reinforcing process. This may be a general dispersal strategy for animals with no target destination.

Carbon dioxide is produced by many organic processes, and is a convenient volatile cue for insects1 searching for blood hosts2, flowers3, communal nests4, fruit5, and wildfires6. Curiously, although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments suggest that walking flies avoid CO27–12. Here, we resolve this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when in an active state associated with foraging. Aversion at low activity levels may be an adaptation to avoid CO2-seeking-parasites, or succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature13,14. In contrast to CO2, flies are attracted to ethanol in all behavioral states, and invest twice the time searching near ethanol compared to CO2. These behavioral differences reflect the fact that whereas CO2 is generated by many natural processes, ethanol is a unique signature of yeast fermentation. Using genetic tools, we determined that the evolutionarily ancient ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved. Our study lays the foundation for future research to determine the neural circuits underlying both state- and odorant- dependent decision making in Drosophila.

The remarkable alkali fly, Ephydra hians , deliberately crawls into the alkaline waters of Mono Lake to feed and lay eggs. These diving flies are protected by an air bubble that forms around their superhydrophobic cuticle upon entering the lake. To study the physical mechanisms underlying this process we measured the work required for flies to enter and leave various aqueous solutions. Our measurements show that it is more difficult for the flies to escape from Mono Lake water than from fresh water, due to the high concentration of Na 2 CO 3 which causes water to penetrate and thus wet their setose cuticle. Other less kosmotropic salts do not have this effect, suggesting that the phenomenon is governed by Hofmeister effects as well as specific interactions between ion pairs. These effects likely create a small negative charge at the air–water interface, generating an electric double layer that facilitates wetting. Compared with six other species of flies, alkali flies are better able to resist wetting in a 0.5 M Na 2 CO 3 solution. This trait arises from a combination of factors including a denser layer of setae on their cuticle and the prevalence of smaller cuticular hydrocarbons compared with other species. Although superbly adapted to resisting wetting, alkali flies are vulnerable to getting stuck in natural and artificial oils, including dimethicone, a common ingredient in sunscreen and other cosmetics. Mono Lake’s alkali flies are a compelling example of how the evolution of picoscale physical and chemical changes can allow an animal to occupy an entirely new ecological niche.