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.
Highlights
Motion 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
Time series of the output parameters produced by optic flow (Figure 2) revealed patterns that accurately reflected the organism’s movement seen during manual observation of the same image sequence, e.g. tail flicks in zebrafish embryos resulted in peaks in the motion parameters recorded by the optic flow motion analysis
Subsequent analysis of the frame-to-frame motion analysis parameters was successful in distinguishing individuals exposed to environmental stress from those under control conditions within many of the developmental stages studied (Table 1), and to distinguish, within species, individuals under control conditions at different stages of development (Table 2)
Summary
Motion 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. Among the many methods of automated image analysis available to biologists is motion analysis, a technique that has been used for quantifying the behavior of mammals [7], fish [8], sperm [9], larvae [10], algal spores [11,12] and bacteria [13] These applications measure parameters including distance travelled, speed and angular velocity and they rely on an observable forward motion in the sample studied. The application of these motion analysis systems to quantify embryonic movement effectively is limited, as the movement patterns embryos exhibit are often complex, subtle and diverse (e.g. muscle flexing, spinning, heart beat, blood flow and tail flicking), which creates challenges in quantifying embryonic movements in a holistic way. We demonstrate a technique for quantifying motion patterns in embryos that can distinguish the effects of environmental stressors on embryos at different developmental stages
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