Comparative phenomics: a new approach to study heterochrony

Understanding the links between development and evolution is one of the major challenges of biology. ‘Heterochronies’, evolutionary alterations in the timings of development are posited as a key mechanism of evolutionary change, but their quantification requires gross simplification of organismal development. Consequently, how changes in event timings influence development more broadly is poorly understood. Here, we measure organismal development as spectra of energy in pixel values of video, creating high-dimensional landscapes integrating development of all visible form and function. This approach we termed ‘Energy proxy traits’ (EPTs) is applied alongside previously identified heterochronies in three freshwater pulmonate molluscs (Lymnaea stagnalis, Radix balthica and Physella acuta). EPTs were calculated from time-lapse video of embryonic development to construct a continuous functional time series. High-dimensional transitions in phenotype aligned with major sequence heterochronies between species. Furthermore, differences in event timings between conspecifics were associated with changes in high-dimensional phenotypic space. We reveal EPTs as a powerful approach to considering the evolutionary importance of alterations to developmental event timings. Reimagining the phenotype as energy spectra enabled continuous quantification of developmental changes in high-dimensional phenotypic space, rather than measurement of timings of discrete events. This approach has the possibility to transform how we study heterochrony and development more generally

De novo transcriptome assembly of the amphipod Gammarus chevreuxi exposed to chronic hypoxia

publisher: Elsevier articletitle: De novo transcriptome assembly of the amphipod Gammarus chevreuxi exposed to chronic hypoxia journaltitle: Marine Genomics articlelink: http://dx.doi.org/10.1016/j.margen.2017.01.006 content_type: article copyright: © 2017 Elsevier B.V. All rights reserved

HeartCV: a tool for transferrable, automated measurement of heart rate and heart rate variability in transparent animals.

Heart function is a key component of whole-organismal physiology. Bioimaging is commonly, but not exclusively, used for quantifying heart function in transparent individuals, including early developmental stages of aquatic animals, many of which are transparent. However, a central limitation of many imaging-related methods is the lack of transferability between species, life-history stages and experimental approaches. Furthermore, locating the heart in mobile individuals remains challenging. Here, we present HeartCV: an open-source Python package for automated measurement of heart rate and heart rate variability that integrates automated localization and is transferrable across a wide range of species. We demonstrate the efficacy of HeartCV by comparing its outputs with measurements made manually for a number of very different species with contrasting heart morphologies. Lastly, we demonstrate the applicability of the software to different experimental approaches and to different dataset types, such as those corresponding to longitudinal studies

Both maternal and embryonic exposure to mild hypoxia influence embryonic development of the intertidal gastropod Littorina littorea (Linnaeus, 1758)

There is growing evidence that maternal exposure to environmental stressors can alter offspring phenotype and increase fitness. Here, we investigate the relative and combined effects of maternal and developmental exposure to mild hypoxia (65% and 74% air saturation respectively) on the growth and development of embryos of the marine gastropod Littorina littorea. Differences in embryo morphological traits were driven by the developmental environment, whereas the maternal environment and interactive effects of maternal and developmental environment were the main driver of differences in the timing of developmental events. While developmental exposure to mild hypoxia significantly increased the area of an important respiratory organ, the velum, it significantly delayed hatching of veliger larvae and reduced their size at hatching and overall survival. Maternal exposure had a significant effect on these traits, and interacted with developmental exposure to influence the time of appearance of morphological characters, suggesting that both are important in affecting developmental trajectories. A comparison between embryos that successfully hatched and those that died in mild hypoxia revealed that survivors exhibited hypertrophy in the velum and associated pre-oral cilia suggesting these traits are linked with survival in low oxygen environments. We conclude that both maternal and developmental environments shape offspring phenotype in a species with a complex, developmental life history, and that plasticity in embryo morphology arising from exposure to even small reductions in oxygen tensions impacts the hatching success of these embryos.</jats:p

A high-throughput and open-source platform for embryo phenomics

<div><p>Phenomics has the potential to facilitate significant advances in biology but requires the development of high-throughput technologies capable of generating and analysing high-dimensional data. There are significant challenges associated with building such technologies, not least those required for investigating dynamic processes such as embryonic development, during which high rates of temporal, spatial, and functional change are inherently difficult to capture. Here, we present EmbryoPhenomics, an accessible high-throughput platform for phenomics in aquatic embryos comprising an Open-source Video Microscope (OpenVIM) that produces high-resolution videos of multiple embryos under tightly controlled environmental conditions. These videos are then analysed by the Python package Embryo Computer Vision (EmbryoCV), which extracts phenomic data for morphological, physiological, behavioural, and proxy traits during the process of embryonic development. We demonstrate the broad-scale applicability of EmbryoPhenomics in a series of experiments assessing chronic, acute, and multistressor responses to environmental change (temperature and salinity) in >30 million images of >600 embryos of two species with markedly different patterns of development—the pond snail <i>Radix balthica</i> and the marine amphipod <i>Orchestia gammarellus</i>. The challenge of phenomics is significant but so too are the rewards, and it is particularly relevant to the urgent task of assessing complex organismal responses to current rates of environmental change. EmbryoPhenomics can acquire and process data capturing functional, temporal, and spatial responses in the earliest, most dynamic life stages and is potentially game changing for those interested in studying development and phenomics more widely.</p></div

Spectral phenotyping of embryonic development reveals integrative thermodynamic responses

Abstract Background Energy proxy traits (EPTs) are a novel approach to high dimensional organismal phenotyping that quantify the spectrum of energy levels within different temporal frequencies associated with mean pixel value fluctuations from video. They offer significant potential in addressing the phenotyping bottleneck in biology and are effective at identifying lethal endpoints and measuring specific functional traits, but the extent to which they might contribute additional understanding of the phenotype remains unknown. Consequently, here we test the biological significance of EPTs and their responses relative to fundamental thermodynamic principles. We achieve this using the entire embryonic development of Radix balthica, a freshwater pond snail, at different temperatures (20, 25 &amp; 30 °C) and comparing responses against predictions from Arrhenius’ equation (Q10 = 2). Results We find that EPTs are thermally sensitive and their spectra of frequency response enable effective high-dimensional treatment clustering throughout organismal development. Temperature-specific deviation in EPTs from thermodynamic predictions were evident and indicative of physiological mitigation, although they differed markedly in their responses from manual measures. The EPT spectrum was effective in capturing aspects of the phenotype predictive of biological outcomes, and suggest that EPTs themselves may reflect levels of energy turnover. Conclusions Whole-organismal biology is incredibly complex, and this contributes to the challenge of developing universal phenotyping approaches. Here, we demonstrate the biological relevance of a new holistic approach to phenotyping that is not constrained by preconceived notions of biological importance. Furthermore, we find that EPTs are an effective approach to measuring even the most dynamic life history stages. </jats:sec

Thermal strategies vary with life history stage.

With both global surface temperatures and the incidence and intensity of extreme temperature events projected to increase, the assessment of species' sensitivity to chronic and acute changes in temperature has become crucial. Sensitivity predictions are based predominantly on adult responses, despite the fact that early life stages may be more vulnerable to thermal challenge. Here, we compared the sensitivity of different life history stages of the intertidal gastropod Littorina obtusata using thermal death time curves, which incorporate the intensity and duration of heat stress, and used these to calculate upper critical thermal limits (CTmax) and sensitivity to temperature change (z). Early (larval) life stages had both a lower CTmax and a lower z than adults, suggesting they are less good at withstanding short-term extreme thermal challenges but better able to survive moderate temperatures in the long term. This result supports the predicted trade-off between acute and chronic tolerance to thermal stress, and is consistent with the different thermal challenges that these stages encounter in the intertidal zone. We conclude that different life history stages employ different thermal strategies that may be adaptive. Our findings caution against the use of predictions of the impact of global warming that are based on only adult responses and, hence, which may underestimate vulnerability

Transcriptomic responses to predator kairomones in embryos of the aquatic snail Radix balthica

The ability of organisms to respond to predation threat by exhibiting induced defenses is well documented, but studies on the potential mechanistic basis for such responses are scarce. Here, we examine the transcriptomic response to predator kairomones of two functionally distinct developmental stages in embryos of the aquatic snail Radix balthica: E8—the stage at which a range‐finding trial indicated that kairomone‐induced accelerated growth and development first occurred; and E9—the stage at which embryos switched from ciliary‐ to crawling‐driven locomotion. We tested whether expression profiles were influenced by kairomones and whether this influence varied between stages. We also identified potential candidate genes for investigating mechanisms underpinning induced responses. There were 6,741 differentially expressed transcripts between developmental stages, compared to just five in response to predator kairomones. However, on examination of functional enrichment in the transcripts responding to predator kairomones and adopting a less stringent significance threshold, 206 transcripts were identified relating to muscle function, growth, and development, with this response being greater at the later E9 stage. Furthermore, these transcripts included putative annotations for genes identified as responding to predator kairomones in other taxa, including C1q, lectin, and actin domains. Globally, transcript expression appeared reduced in response to predator kairomones and we hypothesize that this might be a result of metabolic suppression, as has been reported in other taxa in response to predation threat

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

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. 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