Evaluating evasion strategies in zebrafish larvae.

An effective evasion strategy allows prey to survive encounters with predators. Prey are generally thought to escape in a direction that is either random or serves to maximize the minimum distance from the predator. Here, we introduce a comprehensive approach to determine the most likely evasion strategy among multiple hypotheses and the role of biomechanical constraints on the escape response of prey fish. Through a consideration of six strategies with sensorimotor noise and previous kinematic measurements, our analysis shows that zebrafish larvae generally escape in a direction orthogonal to the predator's heading. By sensing only the predator's heading, this orthogonal strategy maximizes the distance from fast-moving predators, and, when operating within the biomechanical constraints of the escape response, it provides the best predictions of prey behavior among all alternatives. This work demonstrates a framework for resolving the strategic basis of evasion in predator-prey interactions, which could be applied to a broad diversity of animals.

Soft skeletons transmit force with variable gearing.

A hydrostatic skeleton allows a soft body to transmit muscular force via internal pressure. A human's tongue, an octopus' arm and a nematode's body illustrate the pervasive presence of hydrostatic skeletons among animals, which has inspired the design of soft engineered actuators. However, there is a need for a theoretical basis for understanding how hydrostatic skeletons apply mechanical work. We therefore modeled the shape change and mechanics of natural and engineered hydrostatic skeletons to determine their mechanical advantage (MA) and displacement advantage (DA). These models apply to a variety of biological structures, but we explicitly consider the tube feet of a sea star and the body segments of an earthworm, and contrast them with a hydraulic press and a McKibben actuator. A helical winding of stiff, elastic fibers around these soft actuators plays a critical role in their mechanics by maintaining a cylindrical shape, distributing forces throughout the structure and storing elastic energy. In contrast to a single-joint lever system, soft hydrostats exhibit variable gearing with changes in MA generated by deformation in the skeleton. We found that this gearing is affected by the transmission efficiency of mechanical work (MA×DA) or, equivalently, the ratio of output to input work. The transmission efficiency changes with the capacity to store elastic energy within helically wrapped fibers or associated musculature. This modeling offers a conceptual basis for understanding the relationship between the morphology of hydrostatic skeletons and their mechanical performance.

The science and technology of kinematic measurements in a century of Journal of Experimental Biology.

Kinematic measurements have been essential to the study of comparative biomechanics and offer insight into relationships between technological development and scientific progress. Here, we review the 100 year history of kinematic measurements in Journal of Experimental Biology (JEB) through eras that used film, analog video and digital video, and approaches that have circumvented the use of image capture. This history originated with the career of Sir James Gray and has since evolved over the generations of investigators that have followed. Although some JEB studies have featured technological developments that were ahead of their time, the vast majority of research adopted equipment that was broadly available through the consumer or industrial markets. We found that across eras, an emphasis on high-speed phenomena outpaced the growth of the number of articles published by JEB and the size of datasets increased significantly. Despite these advances, the number of species studied within individual reports has not differed significantly over time. Therefore, we find that advances in technology have helped to enable a growth in the number of JEB studies that have included kinematic measurements, contributed to an emphasis on high-speed phenomena, and yielded biomechanical studies that are more data rich, but are no more comparative now than in previous decades.

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.

The persistent-predation strategy of the red lionfish (Pterois volitans).

The pursuit of prey is vital to the biology of a predator and many aspects of predatory behaviour are well-studied. However, it is unclear how a pursuit can be effective when the prey is faster than a non-cryptic predator. Using kinematic measurements, we considered the strategy of red lionfish (Pterois volitans) as they pursued a faster prey fish (Chromis viridis) under laboratory conditions. Despite swimming about half as fast as C. viridis, lionfish succeeded in capturing prey in 61% of our experiments. This successful pursuit behaviour was defined by three critical characteristics. First, lionfish targeted C. viridis with pure pursuit by adjusting their heading towards the prey's position and not the anticipated point of interception. Second, lionfish pursued prey with uninterrupted motion. By contrast, C. viridis moved intermittently with variation in speed that included slow swimming. Such periods allowed lionfish to close the distance to a prey and initiate a suction-feeding strike at a relatively close distance (less than 9 cm). Finally, lionfish exhibited a high rate of strike success, capturing prey in 74% of all strikes. These characteristics comprise a behaviour that we call the 'persistent-predation strategy', which may be exhibited by a diversity of predators with relatively slow locomotion.

Scaling and development of elastic mechanisms: the tiny strikes of larval mantis shrimp.

Latch-mediated spring actuation (LaMSA) is used by small organisms to produce high acceleration movements. Mathematical models predict that acceleration increases as LaMSA systems decrease in size. Adult mantis shrimp use a LaMSA mechanism in their raptorial appendages to produce extremely fast strikes. Until now, however, it was unclear whether mantis shrimp at earlier life-history stages also strike using elastic recoil and latch mediation. We tested whether larval mantis shrimp (Gonodactylaceus falcatus) use LaMSA and, because of their smaller size, achieve higher strike accelerations than adults of other mantis shrimp species. Based on microscopy and kinematic analyses, we discovered that larval G. falcatus possess the components of, and actively use, LaMSA during their fourth larval stage, which is the stage of development when larvae begin feeding. Larvae performed strikes at high acceleration and speed (mean: 4.133×105 rad s-2, 292.7 rad s-1; 12 individuals, 25 strikes), which are of the same order of magnitude as for adults - even though adult appendages are up to two orders of magnitude longer. Larval strike speed (mean: 0.385 m s-1) exceeded the maximum swimming speed of similarly sized organisms from other species by several orders of magnitude. These findings establish the developmental timing and scaling of the mantis shrimp LaMSA mechanism and provide insights into the kinematic consequences of scaling limits in tiny elastic mechanisms.

The sensory basis of schooling by intermittent swimming in the rummy-nose tetra (Hemigrammus rhodostomus).

Schooling is a collective behaviour that enhances the ability of a fish to sense and respond to its environment. Although schooling is essential to the biology of a diversity of fishes, it is generally unclear how this behaviour is coordinated by different sensory modalities. We used experimental manipulation and kinematic measurements to test the role of vision and flow sensing in the rummy-nose tetra (Hemigrammus rhodostomus), which swims with intermittent phases of bursts and coasts. Groups of five fish required a minimum level of illuminance (greater than 1.5 lx) to achieve the necessary close nearest-neighbour distance and high polarization for schooling. Compromising the lateral line system with an antibiotic treatment caused tetras to swim with greater nearest-neighbour distance and lower polarization. Therefore, vision is both necessary and sufficient for schooling in H. rhodostomus, and both sensory modalities aid in attraction. These results can serve as a basis for understanding the individual roles of sensory modalities in schooling for some fish species.

Pursuit predation with intermittent locomotion in zebrafish.

The control of a predator's locomotion is critical to its ability to capture prey. Flying animals adjust their heading continuously with control similar to guided missiles. However, many animals do not move with rapid continuous motion, but rather interrupt their progress with frequent pauses. To understand how such intermittent locomotion may be controlled during predation, we examined the kinematics of zebrafish (Danio rerio) as they pursued larval prey of the same species. Like many fishes, zebrafish move with discrete burst-and-coast swimming. We found that the change in heading and tail excursion during the burst phase was linearly related to the prey's bearing. These results suggest a strategy, which we call intermittent pure pursuit, that offers advantages in sensing and control. This control strategy is similar to perception and path-planning algorithms required in the design of some autonomous robots and may be common to a diversity of animals.

The pursuit strategy of predatory bluefish ( Pomatomus saltatrix).

A predator's ability to capture prey depends critically on how it coordinates its approach in response to a prey's motion. Flying insects, bats and raptors are capable of capturing prey with a strategy known as parallel navigation, which allows a predator to move directly towards the anticipated point of interception. It is unclear if predators using other modes of locomotion are employing this strategy when pursuing evasive prey. Using kinematic measurements and mathematical modelling, we tested whether bluefish ( Pomatomus saltatrix) pursue prey fish ( Fundulus heteroclitus) with parallel navigation. We found that the directional changes of bluefish were not consistent with this strategy, but rather were predicted by a strategy known as deviated pursuit. Although deviated pursuit requires few sensory cues and relatively modest motor coordination, a comparison of mathematical models suggested negligible differences in path length from parallel navigation, largely owing to the acceleration exhibited by bluefish near the end of a pursuit. Therefore, the strategy of bluefish is unlike flying predators, but offers comparable performance with potentially more robust control that may be well suited to the visual system and habitat of fishes. These findings offer a foundation for understanding the sensing and locomotor control of predatory fishes.

Multichannel stroboscopic videography (MSV): a technique for visualizing multiple channels for behavioral measurements.

Biologists commonly visualize different features of an organism using distinct sources of illumination. Such multichannel imaging has largely not been applied to behavioral studies because of the challenges posed by a moving subject. We address this challenge with the technique of multichannel stroboscopic videography (MSV), which synchronizes multiple strobe lights with video exposures of a single camera. We illustrate the utility of this approach with kinematic measurements of a walking cockroach (Gromphadorhina portentosa) and calculations of the pressure field around a swimming fish (Danio rerio). In both, transmitted illumination generated high-contrast images of the animal's body in one channel. Other sources of illumination were used to visualize the points of contact for the feet of the cockroach and the water flow around the fish in separate channels. MSV provides an enhanced potential for high-throughput experimentation and the capacity to integrate changes in physiological or environmental conditions in freely-behaving animals.

A faster escape does not enhance survival in zebrafish larvae.

An escape response is a rapid manoeuvre used by prey to evade predators. Performing this manoeuvre at greater speed, in a favourable direction, or from a longer distance have been hypothesized to enhance the survival of prey, but these ideas are difficult to test experimentally. We examined how prey survival depends on escape kinematics through a novel combination of experimentation and mathematical modelling. This approach focused on zebrafish (Danio rerio) larvae under predation by adults and juveniles of the same species. High-speed three-dimensional kinematics were used to track the body position of prey and predator and to determine the probability of behavioural actions by both fish. These measurements provided the basis for an agent-based probabilistic model that simulated the trajectories of the animals. Predictions of survivorship by this model were found by Monte Carlo simulations to agree with our observations and we examined how these predictions varied by changing individual model parameters. Contrary to expectation, we found that survival may not be improved by increasing the speed or altering the direction of the escape. Rather, zebrafish larvae operate with sufficiently high locomotor performance due to the relatively slow approach and limited range of suction feeding by fish predators. We did find that survival was enhanced when prey responded from a greater distance. This is an ability that depends on the capacity of the visual and lateral line systems to detect a looming threat. Therefore, performance in sensing, and not locomotion, is decisive for improving the survival of larval fish prey. These results offer a framework for understanding the evolution of predator-prey strategy that may inform prey survival in a broad diversity of animals.

Fish prey change strategy with the direction of a threat.

Predation is a fundamental interaction between species, yet it is unclear what escape strategies are effective for prey survival. Classical theory proposes that prey should either escape in a direction that conforms to a performance optimum or that is random and therefore unpredictable. Here, we show that larval zebrafish (Danio rerio) instead use a mixed strategy that may be either random or directed. This was determined by testing classic theory with measurements of the escape direction in response to a predator robot. We found that prey consistently escaped in a direction contralateral to the robot when approached from the side of the prey's body. At such an orientation, the predator appeared in the prey's central visual field and the contralateral response was consistent with a model of strategy that maximizes the distance from the predator. By contrast, when the robot approached the rostral or caudal ends of the body, and appeared in the prey's peripheral vision, the escape showed an equal probability of a contralateral or ipsilateral direction. At this orientation, a contralateral response offered little strategic advantage. Therefore, zebrafish larvae adopt an escape strategy that maximizes distance from the threat when strategically beneficial and that is otherwise random. This sensory-mediated mixed strategy may be employed by a diversity of animals and offers a new paradigm for understanding the factors that govern prey survival.

Zebrafish learn to forage in the dark.

A large diversity of fishes struggle early in life to forage on zooplankton while under the threat of predation. Some species, such as zebrafish (Danio rerio), acquire an ability to forage in the dark during growth as larvae, but it is unclear how this is achieved. We investigated the functional basis of this foraging by video-recording larval and juvenile zebrafish as they preyed on zooplankton (Artemia sp.) under infrared illumination. We found that foraging improved with age, to the extent that 1-month-old juveniles exhibited a capture rate that was an order of magnitude greater than that of hatchlings. At all ages, the ability to forage in the dark was diminished when we used a chemical treatment to compromise the cranial superficial neuromasts, which facilitate flow sensing. However, a morphological analysis showed no developmental changes in these receptors that could enhance sensitivity. We tested whether the improvement in foraging with age could instead be a consequence of learning by raising fish that were naïve to the flow of prey. After 1 month of growth, both groups foraged with a capture rate that was significantly less than that of fish that had the opportunity to learn and indistinguishable from that of fish with no ability to sense flow. This suggests that larval fish learn to use water flow to forage in the dark. This ability could enhance resource acquisition under reduced competition and predation. Furthermore, our findings offer an example of learning in a model system that offers promise for understanding its neurophysiological basis.

The kinematics of directional control in the fast start of zebrafish larvae.

Larval fish use the 'fast start' escape response to rapidly evade the strike of a predator with a three-dimensional (3D) maneuver. Although this behavior is essential for the survival of fishes, it is not clear how its motion is controlled by the motor system of a larval fish. As a basis for understanding this control, we measured the high-speed kinematics of the body of zebrafish (Danio rerio) larvae when executing the fast start in a variety of directions. We found that the angular excursion in the lateral direction is correlated with the yaw angle in the initial stage of bending (stage 1). In this way, larvae moved in a manner similar to that reported for adult fish. However, larvae also have the ability to control the elevation of a fast start. We found that escapes directed downwards or upwards were achieved by pitching the body throughout the stages of the fast start. Changes in the pitching angle in each stage were significantly correlated with the elevation angle of the trajectory. Therefore, as a larva performs rapid oscillations in yaw that contribute to undulatory motion, the elevation of an escape is generated by more gradual and sustained changes in pitch. These observations are consistent with a model of motor control where elevation is directed through the differential activation of the epaxial and hypaxial musculature. This 3D motion could serve to enhance evasiveness by varying elevation without slowing the escape from a predator.

The sensitivity of lateral line receptors and their role in the behavior of Mexican blind cavefish (Astyanax mexicanus).

The characid fish species Astyanax mexicanus offers a classic comparative model for the evolution of sensory systems. Populations of this species evolved in caves and became blind while others remained in streams (i.e. surface fish) and retained a functional visual system. The flow-sensitive lateral line receptors, called superficial neuromasts, are more numerous in cavefish than in surface fish, but it is unclear whether individual neuromasts differ in sensitivity between these populations. The aims of this study were to determine whether the neuromasts in cavefish impart enhanced sensitivity relative to surface fish and to test whether this aids their ability to sense flow in the absence of visual input. Sensitivity was assessed by modeling the mechanics and hydrodynamics of a flow stimulus. This model required that we measure the dimensions of the transparent cupula of a neuromast, which was visualized with fluorescent microspheres. We found that neuromasts within the eye orbit and in the suborbital region were larger and consequently about twice as sensitive in small adult cavefish as in surface fish. Behavioral experiments found that these cavefish, but not surface fish, were attracted to a 35 Hz flow stimulus. These results support the hypothesis that the large superficial neuromasts of small cavefish aid in flow sensing. We conclude that the morphology of the lateral line could have evolved in cavefish to permit foraging in a cave environment.

Prey fish escape by sensing the bow wave of a predator.

Prey fish possess a remarkable ability to sense and evade an attack from a larger fish. Despite the importance of these events to the biology of fishes, it remains unclear how sensory cues stimulate an effective evasive maneuver. Here, we show that larval zebrafish (Danio rerio) evade predators using an escape response that is stimulated by the water flow generated by an approaching predator. Measurements of the high-speed responses of larvae in the dark to a robotic predator suggest that larvae respond to the subtle flows in front of the predator using the lateral line system. This flow, known as the bow wave, was visualized and modeled with computational fluid dynamics. According to the predictions of the model, larvae direct their escape away from the side of their body exposed to more rapid flow. This suggests that prey fish use a flow reflex that enables predator evasion by generating a directed maneuver at high speed. These findings demonstrate a sensory-motor mechanism that underlies a behavior that is crucial to the ecology and evolution of fishes.

Cooperative transport in sea star locomotion.

It is unclear how animals with radial symmetry control locomotion without a brain. Using a combination of experiments, mathematical modeling, and robotics, we tested the extent to which this control emerges in sea stars (Protoreaster nodosus) from the local control of their hundreds of feet and their mechanical interactions with the body. We discovered that these animals compensate for an experimental increase in their submerged weight by recruiting more feet that synchronize in the power stroke of the locomotor cycle during their bouncing gait. Mathematical modeling of the mechanics of a sea star replicated this response to loading without a central controller. A robotic sea star was found to similarly recruit more actuators under higher loads through purely decentralized control. These results suggest that an array of biological or engineered actuators are capable of cooperative transport where the actuators are dynamically recruited by the mechanics of the body. In particular, the body's vertical oscillations serve to recruit feet in greater numbers to overcome the weight to propel the body forward. This form of distributed control contrasts the conventional view of animal locomotion as governed by the central nervous system and offers inspiration for the design of engineered devices with arrays of actuators.

Zebrafish larvae evade predators by sensing water flow.

The ability of fish to evade predators is central to the ecology and evolution of a diversity of species. However, it is largely unclear how prey fish detect predators in order to initiate their escape. We tested whether larval zebrafish (Danio rerio) sense the flow created by adult predators of the same species. When placed together in a cylindrical arena, we found that larvae were able to escape 70% of predator strikes (mean escape probability P(escape)=0.7, N=13). However, when we pharmacologically ablated the flow-sensitive lateral line system, larvae were rarely capable of escape (mean P(escape)=0.05, N=11). In order to explore the rapid events that facilitate a successful escape, we recorded freely swimming predators and prey using a custom-built camera dolly. This device permitted two-dimensional camera motion to manually track prey and record their escape response with high temporal and spatial resolution. These recordings demonstrated that prey were more than 3 times more likely to evade a suction-feeding predator if they responded before (P(escape)=0.53, N=43), rather than after (P(escape)=0.15, N=13), a predator's mouth opened, which is a highly significant difference. Therefore, flow sensing plays an essential role in predator evasion by facilitating a response prior to a predator's strike.

Coordinated ventilation and spiracle activity produce unidirectional airflow in the hissing cockroach, Gromphadorhina portentosa.

Insects exchange respiratory gases via an extensive network of tracheal vessels that open to the surface of the body through spiracular valves. Although gas exchange is known to increase with the opening of these spiracles, it is not clear how this event relates to gas flow through the tracheal system. We examined the relationship between respiratory airflow and spiracle activity in a ventilating insect, the hissing cockroach, Gromphadorhina portentosa, to better understand the complexity of insect respiratory function. Using simultaneous video recordings of multiple spiracular valves, we found that abdominal spiracles open and close in unison during periods of ventilation. Additionally, independent recordings of CO2 release from the abdominal and thoracic regions and observations of hyperoxic tracer gas movement indicate that air is drawn into the thoracic spiracles and expelled from the abdominal spiracles. Our video recordings suggest that this unidirectional flow is driven by abdominal contractions that occur when the abdominal spiracles open. The spiracles then close as the abdomen relaxes and fills with air from the thorax. Therefore, the respiratory system of the hissing cockroach functions as a unidirectional pump through the coordinated action of the spiracles and abdominal musculature. This mechanism may be employed by a broad diversity of large insects that respire by active ventilation.

Evaluating Evasion Strategies in Zebrafish Larvae.

An effective evasion strategy allows prey to survive encounters with predators. Prey are generally thought to escape in a direction that is either random or serves to maximize the minimum distance from the predator. Here we introduce a comprehensive approach to determine the most likely evasion strategy among multiple hypotheses and the role of biomechanical constraints on the escape response of prey fish. Through a consideration of six strategies with sensorimotor noise and previous kinematic measurements, our analysis shows that zebrafish larvae generally escape in a direction orthogonal to the predator's heading. By sensing only the predator's heading, this orthogonal strategy maximizes the distance from fast-moving predators, and, when operating within the biomechanical constraints of the escape response, it provides the best predictions of prey behavior among all alternatives. This work demonstrates a framework for resolving the strategic basis of evastion in predator-prey interactions, which could be applied to a broad diversity of animals.