Asynchronous haltere input drives specific wing and head movements in Drosophila.

Halteres are multifunctional mechanosensory organs unique to the true flies (Diptera). A set of reduced hindwings, the halteres beat at the same frequency as the lift-generating forewings and sense inertial forces via mechanosensory campaniform sensilla. Though haltere ablation makes stable flight impossible, the specific role of wing-synchronous input has not been established. Using small iron filings attached to the halteres of tethered flies and an alternating electromagnetic field, we experimentally decoupled the wings and halteres of flying Drosophila and observed the resulting changes in wingbeat amplitude and head orientation. We find that asynchronous haltere input results in fast amplitude changes in the wing (hitches), but does not appreciably move the head. In multi-modal experiments, we find that wing and gaze optomotor responses are disrupted differently by asynchronous input. These effects of wing-asynchronous haltere input suggest that specific sensory information is necessary for maintaining wing amplitude stability and adaptive gaze control.

Cell lineage analysis reveals signal tracing and compartment characterisation in Drosophila haltere.

Insect halteres, as specialised hind wings, play an important role during aerial manoeuvres. In Drosophila, halteres and wings are homologous appendages with different morphology. Previous studies have focused on the metamorphosis of halteres, while current knowledge about its cell lineage and regional compartmentalization is still limited. In this study, we performed cell-lineage tracing of canonical landmark signals in halteres and present a simple model for haltere development. Cell lineage tracing in wings was used as a reference. The nub showed wing-like expressions in halteres, whereas hth and pnr exhibited different expressions in adult wings and halteres. The lineage tracing revealed that the pouch region gives rise to end-bulb, and hinge cells contribute to proximal haltere formation. Moreover, we demonstrated that twi-expressing cells participate in the cell population of the distal end-bulb. Haematoxylin and eosin staining indicated that muscle cells were present at the distal end-bulb. These results indicated that adult halteres displayed unique cell lineage patterns and the muscle cells are important components of end-bulbs.

MicroRNA-dependent regulation of Hox gene expression sculpts fine-grain morphological patterns in a Drosophila appendage.

Disruptions of normal Hox gene expression can lead to severe morphological defects, revealing a link between the regulation of Hox expression and pattern formation. Here, we explore these links, focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through a combination of bioinformatic and transcriptomic analyses, we identify the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3'UTR sequences is reduced when co-expressed with miR-310C Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, an artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. We propose that miR-310C-mediated repression delimits Ubx fine-grain expression, contributing to the sculpting of complex morphologies in the Drosophila haltere with implications for flight control. Our work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology.

Haltere and visual inputs sum linearly to predict wing (but not gaze) motor output in tethered flying Drosophila.

In the true flies (Diptera), the hind wings have evolved into specialized mechanosensory organs known as halteres, which are sensitive to gyroscopic and other inertial forces. Together with the fly's visual system, the halteres direct head and wing movements through a suite of equilibrium reflexes that are crucial to the fly's ability to maintain stable flight. As in other animals (including humans), this presents challenges to the nervous system as equilibrium reflexes driven by the inertial sensory system must be integrated with those driven by the visual system in order to control an overlapping pool of motor outputs shared between the two of them. Here, we introduce an experimental paradigm for reproducibly altering haltere stroke kinematics and use it to quantify multisensory integration of wing and gaze equilibrium reflexes. We show that multisensory wing-steering responses reflect a linear superposition of haltere-driven and visually driven responses, but that multisensory gaze responses are not well predicted by this framework. These models, based on populations, extend also to the responses of individual flies.

Takeoff diversity in Diptera.

The order Diptera (true flies) are named for their two wings because their hindwings have evolved into specialized mechanosensory organs called halteres. Flies use halteres to detect body rotations and maintain stability during flight and other behaviours. The most recently diverged dipteran monophyletic subsection, the Calyptratae, is highly successful, accounting for approximately 12% of dipteran diversity, and includes common families like house flies. These flies move their halteres independently from their wings and oscillate their halteres during walking. Here, we demonstrate that this subsection of flies uses their halteres to stabilize their bodies during takeoff, whereas non-Calyptratae flies do not. We find that flies of the Calyptratae are able to take off more rapidly than non-Calyptratae flies without sacrificing stability. Haltere removal decreased both velocity and stability in the takeoffs of Calyptratae, but not other flies. The loss of takeoff velocity following haltere removal in Calyptratae (but not other flies) is a direct result of a decrease in leg extension speed. A closely related non-Calyptratae species (D. melanogaster) also has a rapid takeoff, but takeoff duration and stability are unaffected by haltere removal. Haltere use thus allows for greater speed and stability during fast escapes, but only in the Calyptratae clade.

Representation of Haltere Oscillations and Integration with Visual Inputs in the Fly Central Complex.

The reduced hindwings of flies, known as halteres, are specialized mechanosensory organs that detect body rotations during flight. Primary afferents of the haltere encode its oscillation frequency linearly over a wide bandwidth and with precise phase-dependent spiking. However, it is not currently known whether information from haltere primary afferent neurons is sent to higher brain centers where sensory information about body position could be used in decision making, or whether precise spike timing is useful beyond the peripheral circuits that drive wing movements. We show that in cells in the central brain, the timing and rates of neural spiking can be modulated by sensory input from experimental haltere movements (driven by a servomotor). Using multichannel extracellular recording in restrained flesh flies (Sarcophaga bullata of both sexes), we examined responses of central complex cells to a range of haltere oscillation frequencies alone, and in combination with visual motion speeds and directions. Haltere-responsive units fell into multiple response classes, including those responding to any haltere motion and others with firing rates linearly related to the haltere frequency. Cells with multisensory responses showed higher firing rates than the sum of the unisensory responses at higher haltere frequencies. They also maintained visual properties, such as directional selectivity, while increasing response gain nonlinearly with haltere frequency. Although haltere inputs have been described extensively in the context of rapid locomotion control, we find haltere sensory information in a brain region known to be involved in slower, higher-order behaviors, such as navigation.SIGNIFICANCE STATEMENT Many animals use vision for navigation; however, these cues must be interpreted in the context of the body's position. In mammalian brains, hippocampal cells combine visual and vestibular information to encode head direction. A region of the arthropod brain, known as the central complex (CX), similarly encodes heading information, but it is unknown whether proprioceptive information is integrated here as well. We show that CX neurons respond to input from halteres, specialized proprioceptors in flies that detect body rotations. These neurons also respond to visual input, providing one of the few examples of multiple sensory modalities represented in individual CX cells. Haltere stimulation modifies neural responses to visual signals, providing a mechanism for integrating vision with proprioception.

Timing precision in fly flight control: integrating mechanosensory input with muscle physiology.

Animals rapidly collect and act on incoming information to navigate complex environments, making the precise timing of sensory feedback critical in the context of neural circuit function. Moreover, the timing of sensory input determines the biomechanical properties of muscles that undergo cyclic length changes, as during locomotion. Both of these issues come to a head in the case of flying insects, as these animals execute steering manoeuvres at timescales approaching the upper limits of performance for neuromechanical systems. Among insects, flies stand out as especially adept given their ability to execute manoeuvres that require sub-millisecond control of steering muscles. Although vision is critical, here I review the role of rapid, wingbeat-synchronous mechanosensory feedback from the wings and structures unique to flies, the halteres. The visual system and descending interneurons of the brain employ a spike rate coding scheme to relay commands to the wing steering system. By contrast, mechanosensory feedback operates at faster timescales and in the language of motor neurons, i.e. spike timing, allowing wing and haltere input to dynamically structure the output of the wing steering system. Although the halteres have been long known to provide essential input to the wing steering system as gyroscopic sensors, recent evidence suggests that the feedback from these vestigial hindwings is under active control. Thus, flies may accomplish manoeuvres through a conserved hindwing circuit, regulating the firing phase-and thus, the mechanical power output-of the wing steering muscles.

Single mechanosensory neurons encode lateral displacements using precise spike timing and thresholds.

During locomotion, animals rely on multiple sensory modalities to maintain stability. External cues may guide behaviour, but they must be interpreted in the context of the animal's own body movements. Mechanosensory cues that can resolve dynamic internal and environmental conditions, like those from vertebrate vestibular systems or other proprioceptors, are essential for guided movement. How do afferent proprioceptor neurons transform movement into a neural code? In flies, modified hindwings known as halteres detect forces produced by body rotations and are essential for flight. However, the mechanisms by which haltere neurons transform forces resulting from three-dimensional body rotations into patterns of neural spikes are unknown. We use intracellular electrodes to record from haltere primary afferent neurons during a range of haltere motions. We find that spike timing activity of individual neurons changes with displacement and propose a mechanism by which single neurons can encode three-dimensional haltere movements during flight.

Flies tune the activity of their multifunctional gyroscope.

Members of the order Diptera, the true flies, are among the most maneuverable flying animals. These aerial capabilities are partially attributed to flies' possession of halteres, tiny club-shaped structures that evolved from the hindwings and play a crucial role in flight control. Halteres are renowned for acting as biological gyroscopes that rapidly detect rotational perturbations and help flies maintain a stable flight posture. Additionally, halteres provide rhythmic input to the wing steering system that can be indirectly modulated by the visual system. The multifunctional capacity of the haltere is thought to depend on arrays of embedded mechanosensors called campaniform sensilla that are arranged in distinct groups on the haltere's dorsal and ventral surfaces. Although longstanding hypotheses suggest that each array provides different information relevant to the flight control circuitry, we know little about how the haltere campaniforms are functionally organized. Here, we use in vivo calcium imaging during tethered flight to obtain population-level recordings of the haltere sensory afferents in specific fields of sensilla. We find that haltere feedback from both dorsal fields is continuously active, modulated under closed-loop flight conditions, and recruited during saccades to help flies actively maneuver. We also find that the haltere's multifaceted role may arise from the steering muscles of the haltere itself, regulating haltere stroke amplitude to modulate campaniform activity. Taken together, our results underscore the crucial role of efferent control in regulating sensor activity and provide insight into how the sensory and motor systems of flies coevolved.

Development and evaluation of haltere-mimicking gyroscope for three-axis angular velocity sensing using a haltere-mimicking structure pair.

This paper presents a three-axis biomimetic gyroscope, mimicking the haltere of Diptera. Diptera use a club-shaped mechanosensory organ called the haltere to get the three-axis angular velocity information, namely roll, pitch and yaw axes, for flight control. One pair of halteres is physically connected to the wings of Diptera that vibrate in antiphase to the flapping wings in ambient air. They sense the Coriolis force and relay angular velocity information to the Diptera. As an alternative to the conventional micro-electro-mechanical system gyroscopes which are widely used in robotics, many research groups have attempted to mimic the haltere. However, no previous study succeeded in measuring all three-axis components of angular velocity, due to various shortcomings. In this paper, we developed the first three-axis haltere-mimicking gyroscope. Two perpendicularly positioned haltere-mimicking structures that can vibrate at a 180° amplitude were mechanically integrated into a robot actuator. Two accelerometers, placed at the tip of each structure, were employed to measure the Coriolis force. The performance of the novel biomimetic gyroscope was measured in all rotational directions, using a motion capture system as the ground truth. One-axis input experiments were performed 240 times at different input magnitudes and directions, and the measured orientation error was less than ±2.0% in all experiments. In 80 three-axis input experiments, the orientation error was less than ±3.5%.

Cell-type-specific Hox regulatory strategies orchestrate tissue identity.

Hox proteins are homeodomain transcription factors that diversify serially homologous segments along the animal body axis, as revealed by the classic bithorax phenotype of Drosophila melanogaster, in which mutations in Ultrabithorax (Ubx) transform the third thoracic segment into the likeness of the second thoracic segment. To specify segment identity, we show that Ubx both increases and decreases chromatin accessibility, coinciding with its dual role as both an activator and repressor of transcription. However, the choice of transcriptional activity executed by Ubx is spatially regulated and depends on the availability of cofactors, with Ubx acting as a repressor in some populations and as an activator in others. Ubx-mediated changes to chromatin accessibility positively and negatively affect the binding of Scalloped (Sd), a transcription factor that is required for appendage development in both segments. These findings illustrate how a single Hox protein can modify complex gene regulatory networks to transform the identity of an entire tissue.

Wings and halteres act as coupled dual oscillators in flies.

The mechanics of Dipteran thorax is dictated by a network of exoskeletal linkages that, when deformed by the flight muscles, generate coordinated wing movements. In Diptera, the forewings power flight, whereas the hindwings have evolved into specialized structures called halteres, which provide rapid mechanosensory feedback for flight stabilization. Although actuated by independent muscles, wing and haltere motion is precisely phase-coordinated at high frequencies. Because wingbeat frequency is a product of wing-thorax resonance, any wear-and-tear of wings or thorax should impair flight ability. How robust is the Dipteran flight system against such perturbations? Here, we show that wings and halteres are independently driven, coupled oscillators. We systematically reduced the wing length in flies and observed how wing-haltere synchronization was affected. The wing-wing system is a strongly coupled oscillator, whereas the wing-haltere system is weakly coupled through mechanical linkages that synchronize phase and frequency. Wing-haltere link acts in a unidirectional manner; altering wingbeat frequency affects haltere frequency, but not vice versa. Exoskeletal linkages are thus key morphological features of the Dipteran thorax that ensure wing-haltere synchrony, despite severe wing damage.

Striking muscle adaptations induced by volume-dependent repeated bouts of low-intensity eccentric exercise of the elbow flexors.

We investigated the effects of repeating 30 low-intensity eccentric contractions with a dumbbell corresponding to 10% maximal isometric strength (10%EC) on muscle strength and hypertrophy, and muscle damage after 30 maximal eccentric contractions (MaxEC) of the elbow flexors. Young men were placed into 1 of 3 experimental groups that performed 10%EC either once, twice a week for 4 (8 bouts) or 8 weeks (16 bouts) before MaxEC, or a control group that performed 2 bouts of MaxEC separated by 2 weeks (n = 13/group). Repeating 16 bouts of 10%EC increased (P < 0.05) maximal voluntary contraction strength (30 ± 21%) and muscle thickness (4.2 ± 2.3%) greater than 8 bouts (16 ± 4%, 1.9 ± 1.3%). Changes in the muscle damage markers after MaxEC were smaller (P < 0.05) for the experimental groups than the control group, and the magnitude of muscle damage protection was greater (P < 0.05) after 16 bouts (65 ± 30%) than 8 bouts (55 ± 33%), followed by 1 bout (34 ± 27%). The protection by 16 bouts was similar (P = 0.81) to that shown by the second MaxEC of the control group. These results showed that 10%EC produced potent muscle adaptation effects accumulatively and conferred muscle damage protection, but 1 bout of 10%EC was still effective for conferring approximately 20% of the protection of that by 16 bouts. Novelty: Repeating low-intensity eccentric exercise induces large increases in muscle strength and hypertrophy. Low-intensity eccentric exercise protects muscle damage induced by maximal eccentric contractions, and the protection is reinforced by repeating it. These are especially beneficial for individuals who are frail and cannot tolerate high-intensity resistance training.

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 microRNA-306/abrupt regulatory axis controls wing and haltere growth in Drosophila.

Growth control relies on extrinsic and intrinsic mechanisms that regulate and coordinate the size and pattern of organisms. This control is crucial for a homeostatic development and healthy physiology. The gene networks acting in this process are large and complex: factors involved in growth control are also important in diverse biological processes and these networks include multiple regulators that interact and respond to intra- and extra-cellular inputs that may ultimately converge in the control of the cell cycle. In this work we have studied the function of the Drosophila abrupt gene, coding for a BTB-ZF protein and previously reported to be required for wing vein pattern, in the control of haltere and wing growth. We have found that inactivation of abrupt reduces the size of the wing and haltere. We also found that the microRNA miR-306 controls abrupt expression and that miR-306 and abrupt genetically interact to control wing size. Moreover, the reduced appendage size due to abrupt inactivation is rescued by overexpression of Cyclin-E and by inactivation of dacapo. These findings define a miR-306-abrupt regulatory axis that controls wing and haltere size, whereby miR-306 maintains appropriate levels of abrupt expression which, in turn, regulates the cell cycle. Thus, our results uncover a novel function of abrupt in the regulation of the size of Drosophila appendages during development and contribute to the understanding of the coordination between growth and pattern as well as to the understanding of abrupt oncogenic function in flies.

Coriolis and centrifugal forces drive haltere deformations and influence spike timing.

The halteres of flies are mechanosensory organs that serve a crucial role in the control of agile flight, providing sensory input for rapid course corrections to perturbations. Derived from hind wings, halteres are actively flapped and are thus subject to a variety of inertial forces as the fly undergoes complex flight trajectories. Previous analyses of halteres modelled them as a point mass, showing that Coriolis forces lead to subtle deflections orthogonal to the plane of flapping. By design, these models could not consider the effects of force gradients associated with a mass distribution, nor could they reveal three-dimensional spatio-temporal patterns of strain that result from those forces. In addition, diversity in the geometry of halteres, such as shape and asymmetries, could not be simply modelled with a point mass on a massless rod. To study the effects of mass distributions and asymmetries, we examine the haltere subject to both flapping and body rotations using three-dimensional finite-element simulations. We focus on a set of simplified geometries, in which we vary the stalk and bulb shape. We find that haltere mass distribution gives rise to two unreported deformation modes: (i) halteres twist with a magnitude that strongly depends on stalk and bulb geometry and (ii) halteres with an asymmetric mass distribution experience out-of-plane bending due to centrifugal forces, independent of body rotation. Since local strains at the base of the haltere drive deformations of mechanosensory neurons, we combined measured neural encoding mechanisms with our structural analyses to predict the spatial and temporal patterns of neural activity. This activity depends on both the flapping and rotation dynamics, and we show how the timing of neural activity is a viable mechanism for rotation-rate encoding. Our results provide new insights in haltere dynamics and show the viability for timing-based encoding of fly body rotations by halteres.

Specific expression and function of the Six3 optix in Drosophila serially homologous organs.

Organ size and pattern results from the integration of two positional information systems. One global information system, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. However, the mechanisms of the Hox-signal integration in this well-studied system are incomplete. Here, we have investigated this issue by studying the expression and function of the Six3 transcription factor optix during Drosophila wing and haltere development. We find that in both organs, Dpp defines the expression domain of optix through repression, and that the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. We show that optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. In Drosophila, optix is necessary for the growth of wing and haltere. In the wing, optix is required for the growth of the most anterior/proximal region (the 'marginal cell') and for the correct formation of sensory structures along the proximal anterior wing margin; the halteres of optix mutants are also significantly reduced. In addition, in the haltere, optix is necessary for the suppression of sensory bristles.

Origins and Specification of the Drosophila Wing.

The insect wing is a key evolutionary innovation that was essential for insect diversification. Yet despite its importance, there is still debate about its evolutionary origins. Two main hypotheses have been proposed: the paranotal hypothesis, which suggests that wings evolved as an extension of the dorsal thorax, and the gill-exite hypothesis, which proposes that wings were derived from a modification of a pre-existing branch at the dorsal base (subcoxa) of the leg. Here, we address this question by studying how wing fates are initially specified during Drosophila embryogenesis, by characterizing a cis-regulatory module (CRM) from the snail (sna) gene, sna-DP (for dorsal primordia). sna-DP specifically marks the early primordia for both the wing and haltere, collectively referred to as the DP. We found that the inputs that activate sna-DP are distinct from those that activate Distalless, a marker for leg fates. Further, in genetic backgrounds in which the leg primordia are absent, the DP are still partially specified. However, lineage-tracing experiments demonstrate that cells from the early leg primordia contribute to both ventral and dorsal appendage fates. Together, these results suggest that the wings of Drosophila have a dual developmental origin: two groups of cells, one ventral and one more dorsal, give rise to the mature wing. We suggest that the dual developmental origins of the wing may be a molecular remnant of the evolutionary history of this appendage, in which cells of the subcoxa of the leg coalesced with dorsal outgrowths to evolve a dorsal appendage with motor control.

Modelling of soldier fly halteres for gyroscopic oscillations.

Nature has evolved a beautiful design for small-scale vibratory rate-gyro in the form of dipteran halteres that detect body rotations via Coriolis acceleration. In most Diptera, including soldier fly, Hermetia illucens, halteres are a pair of special organs, located in the space between the thorax and the abdomen. The halteres along with their connecting joint with the fly's body constitute a mechanism that is used for muscle-actuated oscillations of the halteres along the actuation direction. These oscillations lead to bending vibrations in the sensing direction (out of the haltere's actuation plane) upon any impressed rotation due to the resulting Coriolis force. This induced vibration is sensed by the sensory organs at the base of the haltere in order to determine the rate of rotation. In this study, we evaluate the boundary conditions and the stiffness of the anesthetized halteres along the actuation and the sensing direction. We take several cross-sectional SEM (scanning electron microscope) images of the soldier fly haltere and construct its three dimensional model to get the mass properties. Based on these measurements, we estimate the natural frequency along both actuation and sensing directions, propose a finite element model of the haltere's joint mechanism, and discuss the significance of the haltere's asymmetric cross-section. The estimated natural frequency along the actuation direction is within the range of the haltere's flapping frequency. However, the natural frequency along the sensing direction is roughly double the haltere's flapping frequency that provides a large bandwidth for sensing the rate of rotation to the soldier flies.

Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila.

In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). Here we report our studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, we observed dramatic increase in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, our results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ.