Through the looking glass: attempting to predict future opportunities and challenges in experimental biology.

To celebrate its centenary year, Journal of Experimental Biology (JEB) commissioned a collection of articles examining the past, present and future of experimental biology. This Commentary closes the collection by considering the important research opportunities and challenges that await us in the future. We expect that researchers will harness the power of technological advances, such as '-omics' and gene editing, to probe resistance and resilience to environmental change as well as other organismal responses. The capacity to handle large data sets will allow high-resolution data to be collected for individual animals and to understand population, species and community responses. The availability of large data sets will also place greater emphasis on approaches such as modeling and simulations. Finally, the increasing sophistication of biologgers will allow more comprehensive data to be collected for individual animals in the wild. Collectively, these approaches will provide an unprecedented understanding of 'how animals work' as well as keys to safeguarding animals at a time when anthropogenic activities are degrading the natural environment.

Small-amplitude head oscillations result from a multimodal head stabilization reflex in hawkmoths.

In flying insects, head stabilization is an essential reflex that helps to reduce motion blur during fast aerial manoeuvres. This reflex is multimodal and requires the integration of visual and antennal mechanosensory feedback in hawkmoths, each operating as a negative-feedback-control loop. As in any negative-feedback system, the head stabilization system possesses inherent oscillatory dynamics that depend on the rate at which the sensorimotor components of the reflex operate. Consistent with this expectation, we observed small-amplitude oscillations in the head motion (or head wobble) of the oleander hawkmoth, Daphnis nerii, which are accentuated when sensory feedback is aberrant. Here, we show that these oscillations emerge from the inherent dynamics of the multimodal reflex underlying gaze stabilization, and that the amplitude of head wobble is a function of both the visual feedback and antennal mechanosensory feedback from the Johnston's organs. Our data support the hypothesis that head wobble results from a multimodal, dynamically stabilized reflex loop that mediates head positioning.

Evolutionary constraints on flicker fusion frequency in Lepidoptera.

Flying insects occupy both diurnal and nocturnal niches, and their visual systems encounter distinct challenges in both conditions. Visual adaptations, such as superposition eyes of moths, enhance sensitivity to low light levels but trade off with spatial and temporal resolution. Conversely, apposition eyes of butterflies enable high spatial resolution but are poorly sensitive in dim light. Although diel activity patterns of insects influence visual processing, their role in evolution of visual systems is relatively unexplored. Lepidopteran insects present an excellent system to study how diel activity patterns and phylogenetic position influence the visual transduction system. We addressed this question by comparing electroretinography measurements of temporal response profiles of diverse Lepidoptera to light stimuli that were flickering at different frequencies. Our data show that the eyes of diurnal butterflies are sensitive to visual stimuli of higher temporal frequencies than nocturnal moths. Hesperiid skippers, which are typically diurnal or crepuscular, exhibit intermediate phenotypes with peak sensitivity across broader frequency range. Across all groups, species within families exhibited similar phenotypes irrespective of diel activity. Thus, Lepidopteran photoreceptors may have diversified under phylogenetic constraints, and shifts in their sensitivity to higher temporal frequencies occurred concomitantly with the evolution of diurnal lifestyles.

Gene Regulation and Species-Specific Evolution of Free Flight Odor Tracking in Drosophila.

The flying ability of insects has coevolved with the development of organs necessary to take-off from the ground, generate, and modulate lift during flight in complex environments. Flight orientation to the appropriate food source and mating partner depends on the perception and integration of multiple chemical signals. We used a wind tunnel-based assay to investigate the natural and molecular evolution of free flight odor tracking in Drosophila. First, the comparison of female and male flies of several populations and species revealed substantial sex-, inter-, and intra-specific variations for distinct flight features. In these flies, we compared the molecular structure of desat1, a fast-evolving gene involved in multiple aspects of Drosophila pheromonal communication. We manipulated desat1 regulation and found that both neural and nonneural tissues affect distinct flight features. Together, our data suggest that desat1 is one of the genes involved in the evolution of free-flight odor tracking behaviors in Drosophila.

Effectiveness and efficiency of two distinct mechanisms for take-off in a derbid planthopper insect.

Analysis of the kinematics of take-off in the planthopper Proutista moesta (Hemiptera, Fulgoroidea, family Derbidae) from high-speed videos showed that these insects used two distinct mechanisms involving different appendages. The first was a fast take-off (55.7% of 106 take-offs by 11 insects) propelled by a synchronised movement of the two hind legs and without participation of the wings. The body was accelerated in 1 ms or less to a mean take-off velocity of 1.7 m s-1 while experiencing average forces of more than 150 times gravity. The power required from the leg muscles implicated a power-amplification mechanism. Such take-offs propelled the insect along its trajectory a mean distance of 7.9 mm in the first 5 ms after take-off. The second and slower take-off mechanism (44.3% of take-offs) was powered by beating movements of the wings alone, with no discernible contribution from the hind legs. The resulting mean acceleration time was 16 times slower at 17.3 ms, the mean final velocity was six times lower at 0.27 m s-1, the g forces experienced were 80 times lower and the distance moved in 5 ms after take-off was 7 times shorter. The power requirements could be readily met by direct muscle contraction. The results suggest a testable hypothesis that the two mechanisms serve distinct behavioural actions: the fast take-offs could enable escape from predators and the slow take-offs that exert much lower ground reaction forces could enable take-off from more flexible substrates while also displacing the insect in a slower and more controllable trajectory.

Odor source localization in complex visual environments by fruit flies.

Flying insects routinely forage in complex and cluttered sensory environments. Their search for a food or a pheromone source typically begins with a whiff of odor, which triggers a flight response, eventually bringing the insect near the odor source. However, pinpointing the precise location of an odor source requires use of both visual and olfactory modalities, aided by odor plumes. Here, we investigated odor-tracking behavior in fruit flies (Drosophila melanogaster) presented with low- or high-contrast visual landmarks, either paired with or separate from an attractive odor cue. These experiments were conducted either in a gentle air stream which generated laminar odor plumes or in still air in which odor dissipates uniformly in all directions. Trajectories of flies revealed several novel features of their odor-tracking behavior in addition to those previously documented. First, in both moving and still air, odor-seeking flies rely on the co-occurrence of visual landmarks with olfactory cues to guide them to odorant objects. Second, flies abruptly decelerate upon encountering an odor plume, thereafter steering towards the nearest visual objects that had no inherent salience in the absence of odor. Thus, interception of an attractive odor increases their salience to nearby high-contrast visual landmarks. Third, flies adopt distinct odor-tracking strategies during flight in moving versus still air. Whereas they weave in and out of plumes towards an odor source in airflow, their approach is more incremental in still air. Both strategies are robust and flexible, and enable flies to reliably find odor sources under diverse visual and airflow environments.

Biomechanical basis of wing and haltere coordination in flies.

The spectacular success and diversification of insects rests critically on two major evolutionary adaptations. First, the evolution of flight, which enhanced the ability of insects to colonize novel ecological habitats, evade predators, or hunt prey; and second, the miniaturization of their body size, which profoundly influenced all aspects of their biology from development to behavior. However, miniaturization imposes steep demands on the flight system because smaller insects must flap their wings at higher frequencies to generate sufficient aerodynamic forces to stay aloft; it also poses challenges to the sensorimotor system because precise control of wing kinematics and body trajectories requires fast sensory feedback. These tradeoffs are best studied in Dipteran flies in which rapid mechanosensory feedback to wing motor system is provided by halteres, reduced hind wings that evolved into gyroscopic sensors. Halteres oscillate at the same frequency as and precisely antiphase to the wings; they detect body rotations during flight, thus providing feedback that is essential for controlling wing motion during aerial maneuvers. Although tight phase synchrony between halteres and wings is essential for providing proper timing cues, the mechanisms underlying this coordination are not well understood. Here, we identify specific mechanical linkages within the thorax that passively mediate both wing-wing and wing-haltere phase synchronization. We demonstrate that the wing hinge must possess a clutch system that enables flies to independently engage or disengage each wing from the mechanically linked thorax. In concert with a previously described gearbox located within the wing hinge, the clutch system enables independent control of each wing. These biomechanical features are essential for flight control in flies.

Mechanics of the thorax in flies.

Insects represent more than 60% of all multicellular life forms, and are easily among the most diverse and abundant organisms on earth. They evolved functional wings and the ability to fly, which enabled them to occupy diverse niches. Insects of the hyper-diverse orders show extreme miniaturization of their body size. The reduced body size, however, imposes steep constraints on flight ability, as their wings must flap faster to generate sufficient forces to stay aloft. Here, we discuss the various physiological and biomechanical adaptations of the thorax in flies which enabled them to overcome the myriad constraints of small body size, while ensuring very precise control of their wing motion. One such adaptation is the evolution of specialized myogenic or asynchronous muscles that power the high-frequency wing motion, in combination with neurogenic or synchronous steering muscles that control higher-order wing kinematic patterns. Additionally, passive cuticular linkages within the thorax coordinate fast and yet precise bilateral wing movement, in combination with an actively controlled clutch and gear system that enables flexible flight patterns. Thus, the study of thoracic biomechanics, along with the underlying sensory-motor processing, is central in understanding how the insect body form is adapted for flight.

Visual feedback influences antennal positioning in flying hawk moths.

Insect antennae serve a variety of sensory functions including tactile sensing, olfaction and flight control. For all of these functions, the precise positioning of the antenna is essential to ensure the proper acquisition of sensory feedback. Although antennal movements in diverse insects may be elicited or influenced by multimodal sensory stimuli, the relative effects of these cues and their integration in the context of antennal positioning responses are not well understood. In previous studies, we have shown that fields of Böhm's bristles located at the base of the antennae provide crucial mechanosensory input for antennal positioning in flying hawk moths. Here, we present electrophysiological and behavioral evidence to show that, in addition to the Böhm's bristles, antennal muscles of hawk moths also respond to bilateral visual input. Moreover, in contrast to the mechanosensory-motor circuit, which is entirely contained within the ipsilateral side, visual feedback influences antennal positioning on both contralateral and ipsilateral sides. Electromyograms recorded from antennal muscles show that the latency of muscle responses to visual stimulation ranged from 35 to 60 ms, considerably slower than their responses to mechanosensory stimuli (<10 ms). Additionally, the visual inputs received by antennal muscles are both motion-sensitive and direction-selective. We characterized the influence of visual feedback on antennal positioning by presenting open-loop translational and rotational visual stimuli to tethered flying moths. During rotational stimuli, we observed that the antenna contralateral to the direction of the turn moved forward through larger angles than the ipsilateral antenna. These observations suggest that whereas input from the Böhm's bristles mediates rapid corrections of antennal position, visual feedback may be involved in slower, bilaterally coordinated movements of the antenna during visually guided flight maneuvers. Thus, visual feedback can modulate the set point at which the antenna is held during flight in hawk moths.

Encoding properties of the mechanosensory neurons in the Johnston's organ of the hawk moth, Manduca sexta.

Antennal mechanosensors play a key role in control and stability of insect flight. In addition to the well-established role of antennae as airflow detectors, recent studies have indicated that the sensing of antennal vibrations by Johnston's organs also provides a mechanosensory feedback relevant for flight stabilization. However, few studies have addressed how the individual units, or scolopidia, of the Johnston's organs encode these antennal vibrations and communicate it to the brain. Here, we characterize the encoding properties of individual scolopidia from the Johnston's organs in the hawk moth, Manduca sexta, through intracellular neurophysiological recordings from axons of the scolopidial neurons. We stimulated the flagellum-pedicel joint using a custom setup that delivered mechanical stimuli of various (step, sinusoidal, frequency and amplitude sweeps) waveforms. Single units of the Johnston's organs typically displayed phaso-tonic responses to step stimuli with short (3-5 ms) latencies. Their phase-locked response to sinusoidal stimuli in the 0.1-100 Hz frequency range showed high fidelity (vector strengths>0.9). The neurons were able to encode different phases of the stimulus motion and were also extremely sensitive to small amplitude (<0.05 deg) deflections with some indication of directional tuning. In many cases, the firing frequency of the neurons varied linearly as a function of the stimulus frequency at wingbeat and double wingbeat frequencies, which may be relevant to their role in flight stabilization. Iontophoretic fills of these neurons with fluorescent dyes showed that they all projected in the antennal mechanosensory and motor center (AMMC) area of the brain. Taken together, these results showcase the speed and high sensitivity of scolopidia of the Johnston's organs, and hence their ability to encode fine antennal vibrations.

The motor apparatus of head movements in the Oleander hawkmoth (Daphnis nerii, Lepidoptera).

Head movements of insects play a vital role in diverse locomotory behaviors including flying and walking. Because insect eyes move minimally within their sockets, their head movements are essential to reduce visual blur and maintain a stable gaze. As in most vertebrates, gaze stabilization behavior in insects requires the integration of both visual and mechanosensory feedback by the neck motor neurons. Although visual feedback is derived from the optic flow over the retina of their compound eyes, mechanosensory feedback is derived from their organs of balance, similar to the vestibular system in vertebrates. In Diptera, vestibular feedback is derived from the halteres-modified hindwings that evolved into mechanosensory organs-and is integrated with visual feedback to actuate compensatory head movements. However, non-Dipteran insects, including Lepidoptera, lack halteres. In these insects, vestibular feedback is obtained from the antennal Johnston's organs but it is not well-understood how it integrates with visual feedback during head movements. Indeed, although head movements are well-studied in flies, the underlying motor apparatus in non-Dipteran taxa has received relatively less attention. As a first step toward understanding compensatory head movements in the Oleander hawkmoth Daphnis nerii, we image the anatomy and architecture of their neck joint sclerites and muscles using X-ray microtomography, and the associated motor neurons using fluorescent dye fills and confocal microscopy. Based on these morphological data, we propose testable hypotheses about the putative function of specific neck muscles during head movements, which can shed light on their role in neck movements and gaze stabilization.

Generating controlled gust perturbations using vortex rings.

To understand the locomotory mechanisms of flying and swimming animals, it is often necessary to develop assays that enable us to measure their responses to external gust perturbations. Typically, such measurements have been carried out using a variety of gusts which are difficult to control or characterize owing to their inherently turbulent nature. Here, we present a method of generating discrete gusts under controlled laboratory conditions in the form of a vortex rings which are well-characterized and highly controllable. We also provide the theoretical guidelines underlying the design of gust generators for specific applications. As a case study, we tested the efficacy of this method to study the flight response of freely-flying soldier flies Hermetia illucens. The vortex ring based method can be used to generate controlled gusts to study diverse phenomena ranging from a natural flight in insects to the artificial flight of insect-sized drones and micro-aerial vehicles.

Insect Flight: State of the Field and Future Directions.

The evolution of flight in an early winged insect ancestral lineage is recognized as a key adaptation explaining the unparalleled success and diversification of insects. Subsequent transitions and modifications to flight machinery, including secondary reductions and losses, also play a central role in shaping the impacts of insects on broadscale geographic and ecological processes and patterns in the present and future. Given the importance of insect flight, there has been a centuries-long history of research and debate on the evolutionary origins and biological mechanisms of flight. Here, we revisit this history from an interdisciplinary perspective, discussing recent discoveries regarding the developmental origins, physiology, biomechanics, and neurobiology and sensory control of flight in a diverse set of insect models. We also identify major outstanding questions yet to be addressed and provide recommendations for overcoming current methodological challenges faced when studying insect flight, which will allow the field to continue to move forward in new and exciting directions. By integrating mechanistic work into ecological and evolutionary contexts, we hope that this synthesis promotes and stimulates new interdisciplinary research efforts necessary to close the many existing gaps about the causes and consequences of insect flight evolution.

Vestibular feedback for flight control in hawkmoths.

Flying insects require mechanosensory feedback to rapidly generate compensatory responses to unexpected perturbations. Such feedback is critical in insects such as moths, which fly under low light levels, compromising their ability to visually compensate for aerial perturbations. Here, we describe how diverse mechanosensory organs have adapted to provide vestibular feedback in various insects, with particular focus on hawkmoths.

The neural mechanisms of antennal positioning in flying moths.

In diverse insects, the forward positioning of the antenna is often among the first behavioral indicators of the onset of flight. This behavior may be important for the proper acquisition of the mechanosensory and olfactory inputs by the antennae during flight. Here, we describe the neural mechanisms of antennal positioning in hawk moths from behavioral, neuroanatomical and neurophysiological perspectives. The behavioral experiments indicated that a set of sensory bristles called Böhm's bristles (or hair plates) mediate antennal positioning during flight. When these sensory structures were ablated from the basal segments of their antennae, moths were unable to bring their antennae into flight position, causing frequent collisions with the flapping wing. Fluorescent dye-fills of the underlying sensory and motor neurons revealed that the axonal arbors of the mechanosensory bristle neurons spatially overlapped with the dendritic arbors of the antennal motor neurons. Moreover, the latency between the activation of antennal muscles following stimulation of sensory bristles was also very short (<10 ms), indicating that the sensorimotor connections may be direct. Together, these data show that Böhm's bristles control antennal positioning in moths via a reflex mechanism. Because the sensory structures and motor organization are conserved across most Neoptera, the mechanisms underlying antennal positioning, as described here, are likely to be conserved in these diverse insects.

Integration of visual and antennal mechanosensory feedback during head stabilization in hawkmoths.

During flight maneuvers, insects exhibit compensatory head movements which are essential for stabilizing the visual field on their retina, reducing motion blur, and supporting visual self-motion estimation. In Diptera, such head movements are mediated via visual feedback from their compound eyes that detect retinal slip, as well as rapid mechanosensory feedback from their halteres - the modified hindwings that sense the angular rates of body rotations. Because non-Dipteran insects lack halteres, it is not known if mechanosensory feedback about body rotations plays any role in their head stabilization response. Diverse non-Dipteran insects are known to rely on visual and antennal mechanosensory feedback for flight control. In hawkmoths, for instance, reduction of antennal mechanosensory feedback severely compromises their ability to control flight. Similarly, when the head movements of freely flying moths are restricted, their flight ability is also severely impaired. The role of compensatory head movements as well as multimodal feedback in insect flight raises an interesting question: in insects that lack halteres, what sensory cues are required for head stabilization? Here, we show that in the nocturnal hawkmoth Daphnis nerii, compensatory head movements are mediated by combined visual and antennal mechanosensory feedback. We subjected tethered moths to open-loop body roll rotations under different lighting conditions, and measured their ability to maintain head angle in the presence or absence of antennal mechanosensory feedback. Our study suggests that head stabilization in moths is mediated primarily by visual feedback during roll movements at lower frequencies, whereas antennal mechanosensory feedback is required when roll occurs at higher frequency. These findings are consistent with the hypothesis that control of head angle results from a multimodal feedback loop that integrates both visual and antennal mechanosensory feedback, albeit at different latencies. At adequate light levels, visual feedback is sufficient for head stabilization primarily at low frequencies of body roll. However, under dark conditions, antennal mechanosensory feedback is essential for the control of head movements at high frequencies of body roll.

The biomechanics of fast prey capture in aquatic bladderworts.

Carnivorous plants match their animal prey for speed of movements and hence offer fascinating insights into the evolution of fast movements in plants. Here, we describe the mechanics of prey capture in aquatic bladderworts Utricularia stellaris, which prey on swimming insect larvae or nematodes to supplement their nitrogen intake. The closed Utricularia bladder develops lower-than-ambient internal pressures by pumping out water from the bladder and thus setting up an elastic instability in bladder walls. When the external sensory trigger hairs on their trapdoor are mechanically stimulated by moving prey, the trapdoor opens within 300-700 µs, causing strong inward flows that trap their prey. The opening time of the bladder trapdoor is faster than any recorded motion in carnivorous plants. Thus, Utricularia have evolved a unique biomechanical system to gain an advantage over their animal prey.