Johan L. van Leeuwen

Swimming of larval zebrafish: ontogeny of body waves and implications for locomotory development

SUMMARY Fish larvae, like most adult fish, undulate their bodies to propel themselves. A detailed kinematic study of the larval body wave is a prerequisite to formulate a set of functional requirements that the locomotor system must fulfil to generate the observed swimming kinematics. Lateral displacement and curvature profiles were obtained for zebrafish (Danio rerio) larvae at 2–21 days post-fertilisation for three swimming behaviours (cyclic swimming, slow starts and fast startle responses) using high-speed video. During cyclic swimming, fish larvae maintain tail beat frequencies of up to 100 Hz. The corresponding longitudinal strains, estimated from the peak curvatures of the midline, reach up to 0.19 in superficial tissue. The strain rate can reach 120 s–1. The wave of curvature travels along the body at a near-constant rate. Posterior to the stiff head, body-lengthspecific curvature is high and rises gently along the entire trunk to a maximum value of 6. Burst-and-coast swimming generates similar peak curvatures to cyclic swimming, but curvature rises more steeply from head to tail. Fish larvae exhibit phase shifts of 57–63° between the wave of lateral displacement and the wave of curvature, resulting in a 1:1.2 ratio of body wave length to curvature wave length. During C-starts, muscle strain can reach 0.19 and superficial longitudinal strain rates approach 30 s–1. Fish larvae do not initiate their escape response with a standing wave of curvature, although their C-starts approach a standing wave as the larvae grow older. The performance demands derived from swimming kinematics suggest that larval axial muscles have very short contraction cycles (10 ms), experience considerable strains (up to 0.2) and strain rates (up to 30 s–1 in white muscle fibres) yet are able to power swimming for several seconds.

Automated visual tracking for studying the ontogeny of zebrafish swimming

SUMMARY The zebrafish Danio rerio is a widely used model organism in studies of genetics, developmental biology, and recently, biomechanics. In order to quantify changes in swimming during all stages of development, we have developed a visual tracking system that estimates the posture of fish. Our current approach assumes planar motion of the fish, given image sequences taken from a top view. An accurate geometric fish model is automatically designed and fit to the images at each time frame. Our approach works across a range of fish shapes and sizes and is therefore well suited for studying the ontogeny of fish swimming, while also being robust to common environmental occlusions. Our current analysis focuses on measuring the influence of vertebra development on the swimming capabilities of zebrafish. We examine wild-type zebrafish and mutants with stiff vertebrae (stocksteif) and quantify their body kinematics as a function of their development from larvae to adult (mutants made available by the Hubrecht laboratory, The Netherlands). By tracking the fish, we are able to measure the curvature and net acceleration along the body that result from the fishs body wave. Here, we demonstrate the capabilities of the tracking system for the escape response of wild-type zebrafish and stocksteif mutant zebrafish. The response was filmed with a digital high-speed camera at 1500 frames s–1. Our approach enables biomechanists and ethologists to process much larger datasets than possible at present. Our automated tracking scheme can therefore accelerate insight in the swimming behavior of many species of (developing) fish.

Adaptive bone formation in acellular vertebrae of sea bass(Dicentrarchus labrax L.)

SUMMARY Mammalian bone is an active tissue in which osteoblasts and osteoclasts balance bone mass. This process of adaptive modelling and remodelling is probably regulated by strain-sensing osteocytes. Bone of advanced teleosts is acellular yet, despite the lack of osteocytes, it is capable of an adaptive response to physical stimuli. Strenuous exercise is known to induce lordosis. Lordosis is a ventrad curvature of the vertebral column, and the affected vertebrae show an increase in bone formation. The effects of lordosis on the strain distribution in sea bass (Dicentrarchus labrax L.) vertebrae are assessed using finite element modelling. The response of the local tissue is analyzed spatially and ontogenetically in terms of bone volume. Lordotic vertebrae show a significantly increased strain energy due to the increased load compared with normal vertebrae when loaded in compression. High strain regions are found in the vertebral centrum and parasagittal ridges. The increase in strain energy is attenuated by a change in architecture due to the increased bone formation. The increased bone formation is seen mainly at the articular surfaces of the vertebrae, although some extra bone is formed in the vertebral centrum. Regions in which the highest strains are found do not spatially correlate with regions in which the most extensive bone apposition occurs in lordotic vertebrae of sea bass. Mammalian-like strain-regulated bone modelling is probably not the guiding mechanism in adaptive bone modelling of acellular sea bass vertebrae. Chondroidal ossification is found at the articular surfaces where it mediates a rapid adaptive response, potentially attenuating high stresses on the dorsal zygapophyses.

Extremely high-power tongue projection in plethodontid salamanders.

SUMMARY Many plethodontid salamanders project their tongues ballistically at high speed and for relatively great distances. Capturing evasive prey relies on the tongue reaching the target in minimum time, therefore it is expected that power production, or the rate of energy release, is maximized during tongue launch. We examined the dynamics of tongue projection in three genera of plethodontids (Bolitoglossa, Hydromantes and Eurycea), representing three independent evolutionary transitions to ballistic tongue projection, by using a combination of high speed imaging, kinematic and inverse dynamics analyses and electromyographic recordings from the tongue projector muscle. All three taxa require high-power output of the paired tongue projector muscles to produce the observed kinematics. Required power output peaks in Bolitoglossa at values that exceed the greatest maximum instantaneous power output of vertebrate muscle that has been reported by more than an order of magnitude. The high-power requirements are likely produced through the elastic storage and recovery of muscular kinetic energy. Tongue projector muscle activity precedes the departure of the tongue from the mouth by an average of 117 ms in Bolitoglossa, sufficient time to load the collagenous aponeuroses within the projector muscle with potential energy that is subsequently released at a faster rate during tongue launch.

The Recording and Interpretation of Pressures in Prey-Sucking Fish

Possible pitfalls of techniques used to record pressures in prey-sucking fish have been analysed by applying control systems and hydrodynamic theory. Fourier analysis revealed a bandwidth of 1 kHz to be generally sufficient for an accurate recording of the overall pressure waveform. The exact bandwidth needed depends on the species, specimen size and intensity of the feeding act. A bandwidth greater than 1 kHz may be needed when secondary fluctuations (e.g. due to vortices) are also to be recorded accurately. Large errors (i.e. of the same order of magnitude as the real pressure) due to the dimensions of the transducer itself cannot be excluded on theoretical grounds. Most reliable pressure records were made with catheter tip pressure transducers, possessing small dimensions and a broad bandwidth (about 3 kHz). Pressure records were made for Amia calva, Salmo gairdneri, Esox lucius, Gadus morhua and Perca fluviatilis. The largest negative pressure peak was measured in Gadus (-42 kPa). Accurate simulations of the pressure records were made using the hydrodynamic model of suction feeding of MULLER, et al. (1982) to which new boundary conditions were added. This model takes into account the unsteady nature of the flow. The good correlation between measured and simulated pressures suggests, but does not prove, that: 1) the model is a good description of the process of suction feeding; 2) the errors in the pressure records obtained with catheter tip pressure transducers are small. The pressure inside the mouth of a prey-sucking fish has velocity and acceleration components due to head expansion and forward motion of the fish. Forward motion strongly influences the pressure profile in feeding events of the two fast-swimming fish studied, Salmo gairdneri and Esox lucius. The literature on pressure measurements in prey-sucking fish is reviewed.

A 3D analysis of flight behavior of Anopheles gambiae sensu stricto malaria mosquitoes in response to human odor and heat.

Female mosquitoes use odor and heat as cues to navigate to a suitable landing site on their blood host. The way these cues affect flight behavior and modulate anemotactic responses, however, is poorly understood. We studied in-flight behavioral responses of females of the nocturnal malaria mosquito Anopheles gambiae sensu stricto to human odor and heat. Flight-path characteristics in a wind tunnel (flow 20 cm/s) were quantified in three dimensions. With wind as the only stimulus (control), short and close to straight upwind flights were recorded. With heat alone, flights were similarly short and direct. The presence of human odor, in contrast, caused prolonged and highly convoluted flight patterns. The combination of odor+heat resulted in longer flights with more landings on the source than to either cue alone. Flight speed was greatest (mean groundspeed 27.2 cm/s) for odor+heat. Odor alone resulted in decreased flight speed when mosquitoes arrived within 30 cm of the source whereas mosquitoes exposed to odor+heat maintained a high flight speed while flying in the odor plume, until they arrived within 15 cm of the source. Human odor evoked an increase in crosswind flights with an additive effect of heat at close range (<15 cm) to the source. This was found for both horizontal and vertical flight components. However, mosquitoes nevertheless made upwind progress when flying in the odor+heat generated plume, suggesting that mosquitoes scan their environment intensively while they progress upwind towards their host. These observations may help to improve the efficacy of trapping systems for malaria mosquitoes by (1) optimizing the site of odor release relative to trap entry and (2) adding a heat source which enhances a landing response.

Postnatal development of collagen structure in ovine articular cartilage

BackgroundArticular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries) as our model animal.ResultsPredominant collagen orientation is parallel to the articular surface throughout the tissue depth for perinatal cartilage. This remodels to the Benninghoff structure before the sheep reach sexual maturity. Remodelling of predominant collagen orientation starts at a depth just below the future transitional zone. Tissue retardance shows a minimum near the articular surface at all ages, which indicates the presence of zonal differentiation at all ages. The absolute position of this minimum does change between birth and maturity. Between different anatomical sites, we find differences in the dynamics of collagen remodelling, but no differences in adult collagen structure.ConclusionsThe collagen network in articular cartilage remodels between birth and sexual maturity from a network with predominant orientation parallel to the articular surface to a Benninghoff network. The retardance minimum near, but not at, the articular surface at all ages shows that a zonal differentiation is already present in the perinatal animals. In these animals, the zonal differentiation can not be correlated to the collagen network orientation. We find no difference in adult collagen structure in the nearly congruent metacarpophalangeal joint, but we do find differences in the dynamics of collagen network remodelling.

Bird song: Superfast muscles control dove's trill

Bird songs frequently contain trilling sounds that demand extremely fast vocalization control. Here we show that doves control their syrinx, a vocal organ that is unique to birds, by using superfast muscles. These muscles, which are similar to those that operate highly specialist acoustic organs such as the rattle of the rattlesnake, are among the fastest vertebrate muscles known and could be much more widespread than previously thought if they are the principal muscle type used to control bird songs.

Vertical forces on the horse's back in sitting and rising trot

In equestrian sports, it is generally assumed that rising and sitting trot load the horses back differently. The objective of this study was to quantify the load on the horses back in these riding techniques. Kinematic data of 13 riders were collected in rising and sitting trot. The time-history of the position of the riders centre of mass (CoM) was calculated, and differentiated twice to obtain the acceleration of the CoM. The reaction force between the rider and the horses back was calculated from the acceleration. Forces were divided by the body weight of the rider to obtain dimensionless forces. As expected, the computed average vertical force did not differ between riding techniques and was not significantly different from the body weight of the riders. At trot, two force peaks were present during one stride cycle. Both peaks in rising trot were significantly lower compared to sitting trot (peak 1: 2.54+/-0.30 versus 2.92+/-0.29; p<0.001; peak 2: 1.95+/-0.34 versus 3.03+/-0.32; p<0.001). This supports the general assumption that rising trot is less demanding for the horse than sitting trot.

Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio)

Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.