A novel application of motion analysis for detecting stress responses in embryos at different stages of development

Abstract

BackgroundMotion analysis is one of the tools available to biologists to extract biologically relevant information from image datasets and has been applied to a diverse range of organisms. The application of motion analysis during early development presents a challenge, as embryos often exhibit complex, subtle and diverse movement patterns. A method of motion analysis able to holistically quantify complex embryonic movements could be a powerful tool for fields such as toxicology and developmental biology to investigate whole organism stress responses. Here we assessed whether motion analysis could be used to distinguish the effects of stressors on three early developmental stages of each of three species: (i) the zebrafish Danio rerio (stages 19 h, 21.5 h and 33 h exposed to 1.5% ethanol and a salinity of 5); (ii) the African clawed toad Xenopus laevis (stages 24, 32 and 34 exposed to a salinity of 20); and iii) the pond snail Radix balthica (stages E3, E4, E6, E9 and E11 exposed to salinities of 5, 10 and 15). Image sequences were analysed using Sparse Optic Flow and the resultant frame-to-frame motion parameters were analysed using Discrete Fourier Transform to quantify the distribution of energy at different frequencies. This spectral frequency dataset was then used to construct a Bray-Curtis similarity matrix and differences in movement patterns between embryos in this matrix were tested for using ANOSIM.ResultsSpectral frequency analysis of these motion parameters was able to distinguish stage-specific effects of environmental stressors in most cases, including Xenopus laevis at stages 24, 32 and 34 exposed to a salinity of 20, Danio rerio at 33 hpf exposed to 1.5% ethanol, and Radix balthica at stages E4, E9 and E11 exposed to salinities of 5, 10 and 15. This technique was better able to distinguish embryos exposed to stressors than analysis of manual quantification of movement and within species distinguished most of the developmental stages studied in the control treatments.ConclusionThis innovative use of motion analysis incorporates data quantifying embryonic movements at a range of frequencies and so provides an holistic analysis of an embryo’s movement patterns. This technique has potential applications for quantifying embryonic responses to environmental stressors such as exposure to pharmaceuticals or pollutants, and also as an automated tool for developmental staging of embryos.

Challenges and opportunities in developmental integrative physiology

“…, 96 well plates) development for individual embryos of model ( D. rerio , X. laevis ) and emerging-model organisms ( R. balthica ) provides an excellent avenue to replicate and examine changes in the timing of developmental events in response to physiological stress ( e.g. , temperature or oxygen level changes) (Tills et al, 2013a). These types of data allow for more explicit links to be generated between heterochrony and heterokairy that to date are, unfortunately, largely treated as separate phenomena in the literature (Spicer and Rundle, 2007).…”

“…Testing models for the importance of heterokairy is difficult for such species, given the limitations of working with preserved specimens or making observations from live embryos within opaque eggs. Recent advances in bio-imaging technology, however, allow the simultaneous in vivo observation and videoing of multiple, individual embryos, including analytical techniques that can be used to quantify physiological responses to stress in species such as R. balthica , Danio rerio and Xenopus laevis (Tills et al, 2013a). Video footage at high temporal and spatial resolution enables an integrative approach whereby physiological, behavioral and morphological traits and their time of onset and offset can be measured.…”

Multi‐generational responses of a marine polychaete to a rapid change in seawater pCO2

“…Throughout the experiment, all wells were observed daily using a custom‐built bio‐imaging system for aquatic animals (Tills et al. ). Polychaetes were fed daily ad libitum on minced spinach (Massamba‐N'Siala et al.…”

Automated high-throughput measurement of body movements and cardiac activity of Xenopus tropicalis tadpoles

“…This approach allowed us to extract important features such as buccal pumping rates and locomotor activity time. The incorporation of holistic spectral analysis of the motion analysis signals [45] could enable the automatic detection of many more movement parameters such as tail beat frequencies or temporal patterns of movements. By using tracking and motion analysis, we analyzed basal activity of tadpoles and found that tadpoles in 24-well plates are mainly immobile while performing buccal pumping at variable frequency.…”