Machine learning reveals the control mechanics of an insect wing hinge.

Insects constitute the most species-rich radiation of metazoa, a success that is due to the evolution of active flight. Unlike pterosaurs, birds and bats, the wings of insects did not evolve from legs1, but are novel structures that are attached to the body via a biomechanically complex hinge that transforms tiny, high-frequency oscillations of specialized power muscles into the sweeping back-and-forth motion of the wings2. The hinge consists of a system of tiny, hardened structures called sclerites that are interconnected to one another via flexible joints and regulated by the activity of specialized control muscles. Here we imaged the activity of these muscles in a fly using a genetically encoded calcium indicator, while simultaneously tracking the three-dimensional motion of the wings with high-speed cameras. Using machine learning, we created a convolutional neural network3 that accurately predicts wing motion from the activity of the steering muscles, and an encoder-decoder4 that predicts the role of the individual sclerites on wing motion. By replaying patterns of wing motion on a dynamically scaled robotic fly, we quantified the effects of steering muscle activity on aerodynamic forces. A physics-based simulation incorporating our hinge model generates flight manoeuvres that are remarkably similar to those of free-flying flies. This integrative, multi-disciplinary approach reveals the mechanical control logic of the insect wing hinge, arguably among the most sophisticated and evolutionarily important skeletal structures in the natural world.

Descending control and regulation of spontaneous flight turns in Drosophila.

The clumped distribution of resources in the world has influenced the pattern of foraging behavior since the origins of locomotion, selecting for a common search motif in which straight movements through resource-poor regions alternate with zig-zag exploration in resource-rich domains. For example, during local search, flying flies spontaneously execute rapid flight turns, called body saccades, but suppress these maneuvers during long-distance dispersal or when surging upstream toward an attractive odor. Here, we describe the key cellular components of a neural network in flies that generate spontaneous turns as well as a specialized pair of neurons that inhibits the network and suppresses turning. Using 2-photon imaging, optogenetic activation, and genetic ablation, we show that only four descending neurons appear sufficient to generate the descending commands to execute flight saccades. The network is organized into two functional units-one for right turns and one for left-with each unit consisting of an excitatory (DNae014) and an inhibitory (DNb01) neuron that project to the flight motor neuropil within the ventral nerve cord. Using resources from recently published connectomes of the fly, we identified a pair of large, distinct interneurons (VES041) that form inhibitory connections to all four saccade command neurons and created specific genetic driver lines for this cell. As predicted by its connectivity, activation of VES041 strongly suppresses saccades, suggesting that it promotes straight flight to regulate the transition between local search and long-distance dispersal. These results thus identify the key elements of a network that may play a crucial role in foraging ecology.

Machine learning reveals the control mechanics of an insect wing hinge.

Insects constitute the most species-rich radiation of metazoa, a success due to the evolution of active flight. Unlike pterosaurs, birds, and bats, the wings of insects did not evolve from legs 1 , but are novel structures attached to the body via a biomechanically complex hinge that transforms tiny, high-frequency oscillations of specialized power muscles into the sweeping back-and-forth motion of the wings 2 . The hinge consists of a system of tiny, hardened structures called sclerites that are interconnected to one another via flexible joints and regulated by the activity of specialized control muscles. Here, we imaged the activity of these muscles in a fly using a genetically encoded calcium indicator, while simultaneously tracking the 3D motion of the wings with high-speed cameras. Using machine learning approaches, we created a convolutional neural network 3 that accurately predicts wing motion from the activity of the steering muscles, and an encoder-decoder 4 that predicts the role of the individual sclerites on wing motion. By replaying patterns of wing motion on a dynamically scaled robotic fly, we quantified the effects of steering muscle activity on aerodynamic forces. A physics-based simulation that incorporates our model of the hinge generates flight maneuvers that are remarkably similar to those of free flying flies. This integrative, multi-disciplinary approach reveals the mechanical control logic of the insect wing hinge, arguably among the most sophisticated and evolutionarily important skeletal structures in the natural world.

Descending control and regulation of spontaneous flight turns in Drosophila.

The clumped distribution of resources in the world has influenced the pattern of foraging behavior since the origins of life, selecting for a common locomotor search motif in which straight movements through resource-poor regions alternate with zig -zag exploration in resource-rich domains. For example, flies execute rapid changes in flight heading called body saccades during local search, but suppress these turns during long-distance dispersal or when surging upwind after encountering an attractive odor plume. Here, we describe the key cellular components of a neural network in flies that generates spontaneous turns as well as a specialized neuron that inhibits the network to promote straight flight. Using 2-photon imaging, optogenetic activation, and genetic ablation, we show that only four descending neurons appear sufficient to generate the descending commands to execute flight saccades. The network is organized into two functional couplets-one for right turns and one for left-with each couplet consisting of an excitatory (DNae014) and inhibitory (DNb01) neuron that project to the flight motor neuropil within the ventral nerve cord. Using resources from recently published connectomes of the fly brain, we identified a large, unique interneuron (VES041) that forms inhibitory connections to all four saccade command neurons and created specific genetic driver lines for this cell. As suggested by its connectivity, activation of VES041 strongly suppresses saccades, suggesting that it regulates the transition between local search and long-distance dispersal. These results thus identify the critical elements of a network that not only structures the locomotor behavior of flies, but may also play a crucial role in their natural foraging ecology.

Single-cell type analysis of wing premotor circuits in the ventral nerve cord of Drosophila melanogaster.

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, we used a combinatorial genetic technique (split-GAL4) to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, we systematically characterized the cell types targeted in our collection. Taken together, the resources and results presented here form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.

Neuromuscular embodiment of feedback control elements in Drosophila flight.

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.

A population of descending neurons that regulates the flight motor of Drosophila.

Similar to many insect species, Drosophila melanogaster is 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 both the 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 study, we describe an unusual type of descending neuron (DNg02) that projects directly from visual output regions of the brain to the dorsal flight neuropil of the ventral nerve cord. Unlike many 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 two-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 descending neurons that provides the sensitivity and dynamic range required for flight control.

Transforming representations of movement from body- to world-centric space.

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 navigation1,2. 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 PFNv3-5, that conjunctively encode translational velocity and heading as a fly walks. In these cells, velocity signals are acquired from locomotor brain regions6 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 flies7. 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 environment8-10.

Drosophila re-zero their path integrator at the center of a fictive food patch.

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.

Steinernema carpocapsae jumps with greater velocity and acceleration than previously reported.

Infective juveniles of the insect-parastic nematode Steinernema carpocapsae canjump greater than 6 times their height, a striking evolved novelty in some species of this genus. Using high-speed videography, we observed the kinematics of Steinernema carpocapsae spontaneousjumping behavior. Our analysis places a lower bound on the velocity and acceleration of these worms.

The long-distance flight behavior of Drosophila supports an agent-based model for wind-assisted dispersal in insects.

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.

Flies Regulate Wing Motion via Active Control of a Dual-Function Gyroscope.

Flies execute their remarkable aerial maneuvers using a set of wing steering muscles, which are activated at specific phases of the stroke cycle [1-3]. The activation phase of these muscles-which determines their biomechanical output [4-6]-arises via feedback from mechanoreceptors at the base of the wings and structures unique to flies called halteres [7-9]. 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 [10-15]. 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.

The effects of target contrast on Drosophila courtship.

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 Drosophila melanogaster with small, fly-sized, moving objects painted either black, white or gray to test whether they use contrast cues to identify mates. We found that males frequently chased gray objects and rarely chased white or black objects. Although males started chasing black objects as often as gray objects, the resulting chases were much shorter. To test whether the attraction to gray objects was mediated via contrast, we fabricated black and gray behavioral chambers. However, wild-type 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 gray 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 gray 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 gray 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.

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

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

Diverse Food-Sensing Neurons Trigger Idiothetic Local Search in Drosophila.

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.

Celestial navigation in Drosophila.

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.

Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila.

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.

Imaging neural activity in the ventral nerve cord of behaving adult Drosophila.

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because limb premotor and motor circuits have been largely inaccessible for large-scale recordings 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 (VNC) of behaving adult Drosophila 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 previously uncharacterized class of locomotion-associated A1 descending neurons. Finally, we develop a genetic reagent to destroy the indirect flight muscles and to facilitate experimental access to the VNC. Taken together, these approaches enable the direct investigation of circuits associated with complex limb movements.

Sun Navigation Requires Compass Neurons in Drosophila.

Despite their small brains, insects can navigate over long distances by orienting using visual landmarks [1], skylight polarization [2-9], 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 [17-19], 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.