Real-Time Tracking of Multiple Moving Mosquitoes.

Tracking mosquitoes in real time, as opposed to recording video files and performing the tracking step later, is useful for two reasons. The first is efficiency. Real-time tracking requires less storage because video images do not need to be saved and followed by a tracking step. The second is that tracking data can be used to interact with the animal in some way, such as triggering the approach of a looming object. In this protocol, we discuss the use of Braid, free software for performing real-time, multicamera, multianimal tracking. We describe a setup with four cameras capable of tracking the three-dimensional (3D) position of mosquitoes at 100 frames per second in a volume of 30 cm × 30 cm × 60 cm with millimeter accuracy. The specific hardware configuration is flexible and can be substituted using different or additional components to adjust the tracking parameters as needed.

Tracking the Body, Wing, and Leg Kinematics of Moving Mosquitoes.

In this protocol, we discuss general techniques for tracking the three-dimensional (3D) locations of the mosquito body, wings, legs, or other features of interest using videos. Tracking data must be acquired to produce detailed kinematics of moving mosquitoes. The software of focus for this protocol, DLTdv, was chosen for its widespread use and excellent support and because it is open-source. In addition, DLTdv allows both manual and automatic tracking. The automatic tracking can be done using a classic machine vision or machine-learning algorithm. The software supports both single-camera analysis and multicamera systems and can take advantage of sophisticated calibration algorithms, both for intrinsic lens distortion correction and for 3D DLT-based reconstruction. For this protocol, we assume all kinematic data is acquired post hoc through video analysis.

Designing a Generic Videography Experiment for Studying Mosquito Behavior.

In this protocol, we describe the basic design considerations and general method to set up a videography system to study mosquito behavior. A basic videography system to study mosquito behavior requires one or more cameras with an optical lens, camera lighting, a calibration setup, and a system to record the video data or otherwise control the camera. Here, we define two types of systems: (1) a real-time videography-based tracking system for determining the position of multiple moving (flying) mosquitoes, and (2) a high-fidelity videography system that can track the detailed movements of body, wings, and legs of a single mosquito at high spatial and temporal resolutions. These high-fidelity trackers are divided into single-camera systems for studying two-dimensional (2D) movements, and multicamera systems that can reconstruct three-dimensional (3D) movements of the mosquito.

Using Videography to Study the Biomechanics and Behavior of Freely Moving Mosquitoes.

Female mosquitoes of most species require a blood meal for egg development. When biting a human host to collect this blood meal, they can spread dangerous diseases such as malaria, yellow fever, or dengue. Researchers use videography to study many aspects of mosquito behavior, including in-flight host-seeking, takeoff, and landing behaviors, as well as probing and blood feeding, and more. Here, we introduce protocols on how to use videography to capture and analyze mosquito movements at high spatial and temporal resolution, in two and three dimensions.

Diurnal and nocturnal mosquitoes escape looming threats using distinct flight strategies.

Flying insects have evolved the ability to evade looming objects, such as predators and swatting hands. This is particularly relevant for blood-feeding insects, such as mosquitoes that routinely need to evade the defensive actions of their blood hosts. To minimize the chance of being swatted, a mosquito can use two distinct strategies-continuously exhibiting an unpredictable flight path or maximizing its escape maneuverability. We studied how baseline flight unpredictability and escape maneuverability affect the escape performance of day-active and night-active mosquitoes (Aedes aegypti and Anopheles coluzzii, respectively). We used a multi-camera high-speed videography system to track how freely flying mosquitoes respond to an event-triggered rapidly approaching mechanical swatter, in four different light conditions ranging from pitch darkness to overcast daylight. Results show that both species exhibit enhanced escape performance in their natural blood-feeding light condition (daylight for Aedes and dark for Anopheles). To achieve this, they show strikingly different behaviors. The enhanced escape performance of Anopheles at night is explained by their increased baseline unpredictable erratic flight behavior, whereas the increased escape performance of Aedes in overcast daylight is due to their enhanced escape maneuvers. This shows that both day and night-active mosquitoes modify their flight behavior in response to light intensity such that their escape performance is maximum in their natural blood-feeding light conditions, when these defensive actions by their blood hosts occur most. Because Aedes and Anopheles mosquitoes are major vectors of several deadly human diseases, this knowledge can be used to optimize vector control methods for these specific species.

Bumblebees land rapidly and robustly using a sophisticated modular flight control strategy.

When approaching a landing surface, many flying animals use visual feedback to control their landing. Here, we studied how foraging bumblebees (Bombus terrestris) use radial optic expansion cues to control in-flight decelerations during landing. By analyzing the flight dynamics of 4,672 landing maneuvers, we showed that landing bumblebees exhibit a series of deceleration bouts, unlike landing honeybees that continuously decelerate. During each bout, the bumblebee keeps its relative rate of optical expansion constant, and from one bout to the next, the bumblebee tends to shift to a higher, constant relative rate of expansion. This modular landing strategy is relatively fast compared to the strategy described for honeybees and results in approach dynamics that is strikingly similar to that of pigeons and hummingbirds. The here discovered modular landing strategy of bumblebees helps explaining why these important pollinators in nature and horticulture can forage effectively in challenging conditions; moreover, it has potential for bio-inspired landing strategies in flying robots.

Effects of drop height, conveyor belt speed, and acceleration on the welfare of broiler chickens in early and later life.

During automated processing in commercial hatcheries, day-old chicks are subjected to a range of possible mental and physical stressors. Three determinants of the processing line seem to have the potential to affect the birds in particular: drop height from one conveyor belt to another, conveyor belt speed, and acceleration. The aim of this study was to evaluate the effects of these 3 factors on chicken health and welfare in early and later life. In a first trial, chickens were tested on an experimental processing line that was adjusted to different levels of drop heights, belt speeds, and accelerations separately (n = 14 animals per factor and increment). Besides the assessment of several indicators for disorientation during the treatment, postmortem radiographic images were created and analyzed with focus on traumatic injuries. The number of chickens changing their orientation after the drop was affected by drop height (P < 0.01), whereas body posture changes were affected both by drop height (P < 0.01) and belt speed (P < 0.01). Traumatic injuries were found only sporadically and were not related to a certain treatment. In a second trial, chickens that were exposed to a combination of the 3 processing factors were compared with an untreated control group (n = 63 per group) until 15 d of age. There were no differences between the 2 groups regarding BW, welfare scores, and fear-related responses in a novel object and in a tonic immobility test. The present results suggest that the treatments on the experimental conveyor belts affected the birds' health, welfare, and behavior to a limited extend. However, starting at a drop height of 280 mm and a conveyor belt speed of 27 m/min, significantly more chickens were not able to maintain their initial body position on the belt. This indicates that there may be scope for discomfort and welfare impairment if commercial systems are operated with considerably larger drop heights and at higher speeds.

Visualizing trypanosomes in a vertebrate host reveals novel swimming behaviours, adaptations and attachment mechanisms.

Trypanosomes are important disease agents of humans, livestock and cold-blooded species, including fish. The cellular morphology of trypanosomes is central to their motility, adaptation to the host's environments and pathogenesis. However, visualizing the behaviour of trypanosomes resident in a live vertebrate host has remained unexplored. In this study, we describe an infection model of zebrafish (Danio rerio) with Trypanosoma carassii. By combining high spatio-temporal resolution microscopy with the transparency of live zebrafish, we describe in detail the swimming behaviour of trypanosomes in blood and tissues of a vertebrate host. Besides the conventional tumbling and directional swimming, T. carassii can change direction through a 'whip-like' motion or by swimming backward. Further, the posterior end can act as an anchoring site in vivo. To our knowledge, this is the first report of a vertebrate infection model that allows detailed imaging of trypanosome swimming behaviour in vivo in a natural host environment.

Reorientation and propulsion in fast-starting zebrafish larvae: an inverse dynamics analysis.

Most fish species use fast starts to escape from predators. Zebrafish larvae perform effective fast starts immediately after hatching. They use a C-start, where the body curls into a C-shape, and then unfolds to accelerate. These escape responses need to fulfil a number of functional demands, under the constraints of the fluid environment and the larva's body shape. Primarily, the larvae need to generate sufficient escape speed in a wide range of possible directions, in a short-enough time. In this study, we examined how the larvae meet these demands. We filmed fast starts of zebrafish larvae with a unique five-camera setup with high spatiotemporal resolution. From these videos, we reconstructed the 3D swimming motion with an automated method and from these data calculated resultant hydrodynamic forces and, for the first time, 3D torques. We show that zebrafish larvae reorient mostly in the first stage of the start by producing a strong yaw torque, often without using the pectoral fins. This reorientation is expressed as the body angle, a measure that represents the rotation of the complete body, rather than the commonly used head angle. The fish accelerates its centre of mass mostly in stage 2 by generating a considerable force peak while the fish 'unfolds'. The escape direction of the fish correlates strongly with the amount of body curvature in stage 1, while the escape speed correlates strongly with the duration of the start. This may allow the fish to independently control the direction and speed of the escape.

Estimating the maximum attachment performance of tree frogs on rough substrates.

Tree frogs can attach to smooth and rough substrates using their adhesive toe pads. We present the results of an experimental investigation of tree frog attachment to rough substrates, and of the role of mechanical interlocking between superficial toe pad structures and substrate asperities in the tree frog species Litoria caerulea and Hyla cinerea. Using a rotation platform setup, we quantified the adhesive and frictional attachment performance of whole frogs clinging to smooth, micro-, and macrorough substrates. The transparent substrates enabled quantification of the instantaneous contact area during detachment by using frustrated total internal reflection. A linear mixed-effects model shows that the adhesive performance of the pads does not differ significantly with roughness (for nominal roughness levels of 0-15 µm) in both species. This indicates that mechanical interlocking does not contribute to the attachment of whole animals. Our results show that the adhesion performance of tree frogs is higher than reported previously, emphasising the biomimetic potential of tree frog attachment. Overall, our findings contribute to a better understanding of the complex interplay of attachment mechanisms in the toe pads of tree frogs, which may promote future designs of tree-frog-inspired adhesives.

Light-dark rhythms during incubation of broiler chicken embryos and their effects on embryonic and post hatch leg bone development.

There are indications that lighting schedules applied during incubation can affect leg health at hatching and during rearing. The current experiment studied effects of lighting schedule: continuous light (24L), 12 hours of light, followed by 12 hours of darkness (12L:12D), or continuous darkness (24D) throughout incubation of broiler chicken eggs on the development and strength of leg bones, and the role of selected hormones in bone development. In the tibiatarsus and femur, growth and ossification during incubation and size and microstructure at day (D)0, D21, and D35 post hatching were measured. Plasma melatonin, growth hormone, and IGF-I were determined perinatally. Incidence of tibial dyschondroplasia, a leg pathology resulting from poor ossification at the bone's epiphyseal plates, was determined at slaughter on D35. 24L resulted in lower embryonic ossification at embryonic day (E)13 and E14, and lower femur length, and lower tibiatarsus weight, length, cortical area, second moment of area around the minor axis, and mean cortical thickness at hatching on D0 compared to 12L:12D especially. Results were long term, with lower femur weight and tibiatarsus length, cortical and medullary area of the tibiatarsus, and second moment of area around the minor axis, and a higher incidence of tibial dyschondroplasia for 24L. Growth hormone at D0 was higher for 24D than for 12L:12D, with 24L intermediate, but plasma melatonin and IGF-I did not differ between treatments, and the role of plasma melatonin, IGF-I, and growth hormone in this process was therefore not clear. To conclude, in the current experiment, 24L during incubation of chicken eggs had a detrimental effect on embryonic leg bone development and later life leg bone strength compared to 24D and 12L:12D, while the light-dark rhythm of 12L:12D may have a stimulating effect on leg health.

Three-dimensional analysis of the fast-start escape response of the least killifish, Heterandria formosa.

Fish make C-starts to evade predator strikes. Double-bend (DB) C-starts consist of three stages: Stage 1, in which the fish rapidly bends into a C-shape; Stage 2, in which the fish bends in the opposite direction; and a variable Stage 3. In single-bend (SB) C-starts, the fish immediately straightens after Stage 1. Despite fish moving in three-dimensional (3D) space, fast-start responses of adult fish have mainly been studied in a horizontal plane. Using automated 3D tracking of multi-camera high-speed video sequences, we show that both SB and DB fast-starts by adult female least killifish (Heterandria formosa) often contain a significant vertical velocity component, and large changes in pitch (DB up to 43 deg) and roll (DB up to 77 deg) angles. Upwards and downwards elevation changes are correlated with changes in pitch angle of the head; movement in the horizontal plane is correlated with changes in yaw angle of the head. With respect to the stimulus, escape heading correlates with the elevation of the fish at the onset of motion. Irrespective of the initial orientation, fish can escape in any horizontal direction. In many cases, the centre of mass barely accelerates during Stage 1. However, it does accelerate in the final direction of the escape in other instances, indicating that Stage 1 can serve a propulsive role in addition to its preparatory role for Stage 2. Our findings highlight the importance of large-scale 3D analyses of fast-start manoeuvres of adult fish in uncovering the versatility of fish escape repertoire.

Automated Reconstruction of Three-Dimensional Fish Motion, Forces, and Torques.

Fish can move freely through the water column and make complex three-dimensional motions to explore their environment, escape or feed. Nevertheless, the majority of swimming studies is currently limited to two-dimensional analyses. Accurate experimental quantification of changes in body shape, position and orientation (swimming kinematics) in three dimensions is therefore essential to advance biomechanical research of fish swimming. Here, we present a validated method that automatically tracks a swimming fish in three dimensions from multi-camera high-speed video. We use an optimisation procedure to fit a parameterised, morphology-based fish model to each set of video images. This results in a time sequence of position, orientation and body curvature. We post-process this data to derive additional kinematic parameters (e.g. velocities, accelerations) and propose an inverse-dynamics method to compute the resultant forces and torques during swimming. The presented method for quantifying 3D fish motion paves the way for future analyses of swimming biomechanics.

Refraction corrected calibration for aquatic locomotion research: application of Snell's law improves spatial accuracy.

Images of underwater objects are distorted by refraction at the water-glass-air interfaces and these distortions can lead to substantial errors when reconstructing the objects' position and shape. So far, aquatic locomotion studies have minimized refraction in their experimental setups and used the direct linear transform algorithm (DLT) to reconstruct position information, which does not model refraction explicitly. Here we present a refraction corrected ray-tracing algorithm (RCRT) that reconstructs position information using Snell's law. We validated this reconstruction by calculating 3D reconstruction error-the difference between actual and reconstructed position of a marker. We found that reconstruction error is small (typically less than 1%). Compared with the DLT algorithm, the RCRT has overall lower reconstruction errors, especially outside the calibration volume, and errors are essentially insensitive to camera position and orientation and the number and position of the calibration points. To demonstrate the effectiveness of the RCRT, we tracked an anatomical marker on a seahorse recorded with four cameras to reconstruct the swimming trajectory for six different camera configurations. The RCRT algorithm is accurate and robust and it allows cameras to be oriented at large angles of incidence and facilitates the development of accurate tracking algorithms to quantify aquatic manoeuvers.