Characterizing the thermal preference of fish is important in conservation, environmental and evolutionary physiology and can be determined using a shuttle box system. Initial tank acclimation and trial lengths are important considerations in experimental design, yet systematic studies of these factors are missing. Three different behavioral assay experimental designs were tested to determine the effect of tank acclimation and trial length (12:12, 0:12, 2:2; hours of tank acclimation: behavioral trial) on the temperature preference of juvenile lake whitefish ( Coregonus clupeaformis ), using a shuttle box. Average temperature preferences for the 12:12, 0:12, and 2:2 experimental designs were 16.10±1.07°C, 16.02±1.56°C, 16.12±1.59°C respectively, with no significant differences between experimental designs (p= 0.9337). Ultimately, length of acclimation time and trial length had no significant effect on thermal preference.

ABSTRACT Limbless animals such as snakes, limbless lizards, worms, eels and lampreys move their slender, long bodies in three dimensions to traverse diverse environments. Accurately quantifying their continuous body's 3-D shape and motion is important for understanding body–environment interactions in complex terrain, but this is difficult to achieve (especially for local orientation and rotation). Here, we describe an interpolation method to quantify continuous body 3-D position and orientation. We simplify the body as an elastic rod and apply a backbone optimization method to interpolate continuous body shape between end constraints imposed by tracked markers. Despite over-simplifying the biomechanics, our method achieves a higher interpolation accuracy (∼50% error) in both 3-D position and orientation compared with the widely used cubic B-spline interpolation method. Beyond snakes traversing large obstacles as demonstrated, our method applies to other long, slender, limbless animals and continuum robots. We provide codes and demo files for easy application of our method.

ABSTRACT The capacity to recover after a perturbation is a well-known intrinsic property of physiological systems, including the locomotor system, and can be termed ‘resilience’ . Despite an abundance of metrics proposed to measure the complex dynamics of bipedal locomotion, analytical tools for quantifying resilience are lacking. Here, we introduce a novel method to directly quantify resilience to perturbations during locomotion. We examined the extent to which synchronizing stepping with two different temporal structured auditory stimuli (periodic and 1/ f structure) during walking modulates resilience to a large unexpected perturbation. Recovery time after perturbation was calculated from the horizontal velocity of the body's center of mass. Our results indicate that synchronizing stepping with a 1/ f stimulus elicited greater resilience to mechanical perturbations during walking compared with the periodic stimulus (3.3 s faster). Our proposed method may help to gain a comprehensive understanding of movement recovery behavior of humans and other animals in their ecological contexts.

ABSTRACT The velocity of settling particles is an important determinant of distribution in extinct and extant species with passive dispersal mechanisms, such as plants, corals and phytoplankton. Here, we adapted dynamic scaling, borrowed from engineering, to determine settling velocity. Dynamic scaling leverages physical models with relevant dimensionless numbers matched to achieve similar dynamics to the original object. Previous studies have used flumes, wind tunnels or towed models to examine fluid flow around objects with known velocities. Our novel application uses free-falling models to determine the unknown sinking velocity of planktonic Foraminifera – organisms important to our understanding of the Earth's current and historic climate. Using enlarged 3D printed models of microscopic Foraminifera tests, sunk in viscous mineral oil to match their Reynolds numbers and drag coefficients, we predicted sinking velocity of real tests in seawater. This method can be applied to study other settling particles such as plankton, spores or seeds.

ABSTRACT Negative geotaxis (climbing) performance is a useful metric for quantifying Drosophila health. Manual methods to quantify climbing performance are tedious and often biased, while many available computational methods have challenging hardware or software requirements. We present an alternative: FreeClimber. This open source, Python-based platform subtracts a video's static background to improve detection for flies moving across heterogeneous backgrounds. FreeClimber calculates a cohort's velocity as the slope of the most linear portion of a mean vertical position versus time curve. It can run from a graphical user interface for optimization or a command line interface for high-throughput and automated batch processing, improving accessibility for users with different expertise. FreeClimber outputs calculated slopes, spot locations for follow-up analyses (e.g. tracking), and several visualizations and plots. We demonstrate FreeClimber's utility in a longitudinal study for endurance exercise performance in Drosophila mitonuclear genotypes using six distinct mitochondrial haplotypes paired with a common D. melanogaster nuclear background.

ABSTRACT Studies of tardigrade biology have been severely limited by the sparsity of appropriate quantitative techniques, informative on a single-organism level. Therefore, many studies rely on motility-based survival scoring and quantifying reproductive success. Measurements of O 2 respiration rates, as an integrating expression of the metabolic activity of single tardigrades, would provide a more comprehensive insight into how an individual tardigrade is responding to specific environmental factors or changes in life stages. Here, we present and validate a new method for determining the O 2 respiration rate (nmol O 2 mg −1 h −1 ) of single tardigrades under steady state, using O 2 microsensors. As an example, we show that the O 2 respiration rate determined in MilliQ water for individuals of Richtersius coronifer and of Macrobiotus macrocalix at 22°C was 10.8±1.84 and 13.1±2.19 nmol O 2 mg −1 h −1 , respectively.

ABSTRACT Three-dimensional (3D) magnetic resonance microscopy (MRM) is a modality of magnetic resonance imaging (MRI) optimized for the best resolution. Metamorphosis of the Carniolan worker honey bee ( Apis mellifera carnica ) was studied in vivo under controlled temperature and humidity conditions from sealed larvae until the emergence of an adult. The 3D images were analyzed by volume rendering and segmentation, enabling the analysis of the body, tracheal system and gastrointestinal tract through the time course of volume changes. Fat content sensitivity enabled the analysis of flight muscles transformation during the metamorphosis by the signal histogram and gray level co-occurrence matrix (GLCM). Although the transformation during metamorphosis is well known, MRM enables an alternative insight to this process, i.e. 3D in vivo , which has relatively high spatial and temporal resolutions. The developed methodology can easily be adapted for studying the metamorphosis of other insects or any other incremental biological process on a similar spatial and temporal scale.

ABSTRACT Three-dimensional studies of range of motion currently plot joint poses in a ‘Euler space’ whose axes are angles measured in the joint's three rotational degrees of freedom. Researchers then compute the volume of a pose cloud to measure rotational mobility. However, pairs of poses that are equally different from one another in orientation are not always plotted equally far apart in Euler space. This distortion causes a single joint's mobility to change when measured based on different joint coordinate systems and precludes fair comparison among joints. Here, we present two alternative spaces inspired by a 16th century map projection – cosine-corrected and sine-corrected Euler spaces – that allow coordinate-system-independent comparison of joint rotational mobility. When tested with data from a bird hip joint, cosine-corrected Euler space demonstrated a 10-fold reduction in variation among mobilities measured from three joint coordinate systems. This new quantitative framework enables previously intractable, comparative studies of articular function.

ABSTRACT Marker tracking is a major bottleneck in studies involving X-ray reconstruction of moving morphology (XROMM). Here, we tested whether DeepLabCut, a new deep learning package built for markerless tracking, could be applied to videoradiographic data to improve data processing throughput. Our novel workflow integrates XMALab, the existing XROMM marker tracking software, and DeepLabCut while retaining each program's utility. XMALab is used for generating training datasets, error correction and 3D reconstruction, whereas the majority of marker tracking is transferred to DeepLabCut for automatic batch processing. In the two case studies that involved an in vivo behavior, our workflow achieved a 6 to 13-fold increase in data throughput. In the third case study, which involved an acyclic, post-mortem manipulation, DeepLabCut struggled to generalize to the range of novel poses and did not surpass the throughput of XMALab alone. Deployed in the proper context, this new workflow facilitates large scale XROMM studies that were previously precluded by software constraints.

ABSTRACT We present a simple device to mechanically immobilize motile cells such as ciliates. It can be used in particular for intracellular electrophysiology and microinjection. A transparent filter with holes smaller than the specimen is stretched over an outlet. A flow is induced by either a peristaltic pump or a depressurized tank, mechanically entraining cells to the bottom, where they are immobilized against the filter. The cells start swimming again as soon as the flow is stopped. We demonstrate the device by recording action potentials in Paramecium and injecting a fluorescent dye into the cytosol.

ABSTRACT Animal flight requires aerodynamic power, which is challenging to determine accurately in vivo . Existing methods rely on approximate calculations based on wake flow field measurements, inverse dynamics approaches, or invasive muscle physiological recordings. In contrast, the external mechanical work required for terrestrial locomotion can be determined more directly by using a force platform as an ergometer. Based on an extension of the recent invention of the aerodynamic force platform, we now present a more direct method to determine the in vivo aerodynamic power by taking the dot product of the aerodynamic force vector on the wing with the representative wing velocity vector based on kinematics and morphology. We demonstrate this new method by studying a slowly flying dove, but it can be applied more generally across flying and swimming animals as well as animals that locomote over water surfaces. Finally, our mathematical framework also works for power analyses based on flow field measurements.

ABSTRACT Disassembling parasitic spores and acquiring the main subunits for analysis is a prerequisite for a deep understanding of the basic biology of parasites. Herein, we present a fast and efficient method to dissect myxospores in a few steps, which mainly involves sonication, and sucrose and Percoll density gradient ultracentrifugation. We tested our method on three myxozoan species and demonstrate that this method allows the dismembering of myxospores, and the isolation of intact and clean nematocysts and shell valves within 2 h at low cost. This new tool will facilitate subsequent analyses and enable a better understanding of the ecological and evolutionary significance of parasitic spores.

ABSTRACT Muscle force–length dynamics are governed by intrinsic contractile properties, motor stimulation and mechanical load. Although intrinsic properties are well characterised, physiologists lack in vitro instrumentation to account for combined effects of limb inertia, musculoskeletal architecture and contractile dynamics. We introduce in vitro virtual reality ( in vitro -VR) which enables in vitro muscle tissue to drive a musculoskeletal jumping simulation. In hardware, muscle force from a frog plantaris was transmitted to a software model where joint torques, inertia and ground reaction forces were computed to advance the simulation at 1 kHz. To close the loop, simulated muscle strain was returned to update in vitro length. We manipulated (1) stimulation timing and (2) the virtual muscle's anatomical origin. This influenced interactions among muscular, inertial, gravitational and contact forces dictating limb kinematics and jump performance. We propose that in vitro -VR can be used to illustrate how neuromuscular control and musculoskeletal anatomy influence muscle dynamics and biomechanical performance.

ABSTRACT Predicting the sensitivity of reef-building corals to disturbance, including bleaching, requires an understanding of physiological responses to stressors, which may be limited by destructive sampling and the capacity of common methodologies to characterize early life history stages. We developed a new methodology using laser scanning confocal microscopy (LSCM) to measure and track the physiological condition of corals. In a thermal stress experiment, we used LSCM to track coral condition during bleaching in adults and juveniles of two species, Montipora capitata and Pocillopora acuta . Depth of fluorescence in coral tissues provides a proxy measure of tissue thickness, whereas Symbiodiniaceae population fluorescence relates to both population density and chlorophyll a content. In response to thermal stress, there were significant shifts in tissue thickness and Symbiodiniaceae fluorescence with differences between life stages. This method is particularly well suited for detecting shifts in physiological condition of living corals in laboratory studies, especially in small juvenile colonies.

ABSTRACT Physiological investigations of fish gills have traditionally centred on the two principal functions of the gills: gas exchange and ion regulation. Mitochondrion-rich cells (MRCs) are primarily found within the gill filaments of fish, and are thought to proliferate in order to increase the ionoregulatory capacity of the gill in response to environmentally induced osmotic challenges. However, surprisingly little attention has been paid to the metabolic function of mitochondria within fish gills. Here, we describe and validate a simple protocol for the permeabilization of fish gills and subsequent measurement of mitochondrial respiration rates in vitro . Our protocol requires only small tissue samples (8 mg), exploits the natural structure of fish gills, does not require mechanical separation of the gill tissue (so is relatively quick to perform), and yields accurate and highly reproducible measurements of respiration rates. It offers great potential for the study of mitochondrial function in gills over a wide range of fish sizes and species.

ABSTRACT Open-source technology has been increasingly used for developing low-cost animal-borne bio-loggers; however, a gap remains for a bio-logger that records both inertial movement and GPS positions. We address this need with the Tapered Wings Logger (TWLogger), an archival bio-logger that records high-resolution (e.g. 50 Hz) tri-axial accelerometry and magnetometry, temperature and GPS. The TWLogger can be built for 90 USD, accepts user-defined sampling parameters, and with a 500 mA h battery weighs 25 g. We provide publicly available build instructions and custom analysis scripts. Bench tests recorded 50 Hz inertial movement and 2 min GPS for 31.8±2.2 h (mean±s.d., n =6) with GPS accuracy within 10.9±13.6 m. Field deployments on a medium-sized bird of prey in the wild achieved similar results ( n =13). The customizable TWLogger has wide-ranging application across systems and thus offers a practical solution for eco-physiology applications.

ABSTRACT Subterranean digging behaviors provide opportunities for protection, access to prey, and predator avoidance for a diverse array of vertebrates, yet studies of the biomechanics of burrowing have been limited by the technical challenges of measuring kinetics and kinematics of animals moving within a medium. We describe a new system for measuring 3D reaction forces during burrowing, called a ‘tunnel-tube’, which is composed of two, separately instrumented plastic tubes: an ‘entry tube’ with no medium, in series with a ‘digging tube’ filled with medium. Mean reaction forces are measured for a digging bout and Fourier analysis is used to quantify the amplitude of oscillatory digging force as a function of frequency. In sample data from pocket gophers digging in artificial and natural media, the mean ground reaction force is constant, whereas Fourier analysis resolves a reduced amplitude of oscillatory force in the artificial medium with lower compaction strength.

ABSTRACT Animals experience complex odorant stimuli that vary widely in composition, intensity and temporal properties. However, stimuli used to study olfaction in the laboratory are much simpler. This mismatch arises from the challenges in measuring and controlling them precisely and accurately. Even simple pulses can have diverse kinetics that depend on their molecular identity. Here, we introduce a model that describes how stimulus kinetics depend on the molecular identity of the odorant and the geometry of the delivery system. We describe methods to deliver dynamic odorant stimuli of several types, including broadly distributed stimuli that reproduce some of the statistics of naturalistic plumes, in a reproducible and precise manner. Finally, we introduce a method to calibrate a photo-ionization detector to any odorant it can detect, using no additional components. Our approaches are affordable and flexible and can be used to advance our understanding of how olfactory neurons encode real-world odor signals.

ABSTRACT The critical oxygen tension ( P crit ) for fishes is the oxygen level below which the rate of oxygen consumption ( Ṁ O 2 ) becomes dependent upon ambient oxygen partial pressure ( P O 2 ). We compare multiple curve-fitting approaches to estimate P crit of the Gulf killifish, Fundulus grandis , during closed and intermittent-flow respirometry. Fitting two line segments of Ṁ O 2 versus P O 2 produced high and variable estimates of P crit , as did nonlinear regression using a hyperbolic (Michaelis–Menten) function. Using nonlinear regression fit to an exponential (modified Weibull) function, or linear regression of Ṁ O 2 versus P O 2 at low P O 2 , and determining P crit as the P O 2 when Ṁ O 2 equals standard metabolic rate (SMR) yielded values that were consistent across fish and among experimental trials. The magnitude of the difference in P crit determined by alternative calculation methods exceeded the differences determined in closed and intermittent-flow respirometry, highlighting the need to standardize analytical as well as experimental approaches in determining P crit .

ABSTRACT Zooplankton play critical roles in marine ecosystems, yet their fine-scale behavior remains poorly understood because of the difficulty in studying individuals in situ . Here, we combine biologging with supervised machine learning (ML) to propose a pipeline for studying in situ behavior of larger zooplankton such as jellyfish. We deployed the ITAG, a biologging package with high-resolution motion sensors designed for soft-bodied invertebrates, on eight Chrysaora fuscescens in Monterey Bay, using the tether method for retrieval. By analyzing simultaneous video footage of the tagged jellyfish, we developed ML methods to: (1) identify periods of tag data corrupted by the tether method, which may have compromised prior research findings, and (2) classify jellyfish behaviors. Our tools yield characterizations of fine-scale jellyfish activity and orientation over long durations, and we conclude that it is essential to develop behavioral classifiers on in situ rather than laboratory data.