Dev-ResNet: automated developmental event detection using deep learning.

Delineating developmental events is central to experimental research using early life stages, permitting widespread identification of changes in event timing between species and environments. Yet, identifying developmental events is incredibly challenging, limiting the scale, reproducibility and throughput of using early life stages in experimental biology. We introduce Dev-ResNet, a small and efficient 3D convolutional neural network capable of detecting developmental events characterised by both spatial and temporal features, such as the onset of cardiac function and radula activity. We demonstrate the efficacy of Dev-ResNet using 10 diverse functional events throughout the embryonic development of the great pond snail, Lymnaea stagnalis. Dev-ResNet was highly effective in detecting the onset of all events, including the identification of thermally induced decoupling of event timings. Dev-ResNet has broad applicability given the ubiquity of bioimaging in developmental biology, and the transferability of deep learning, and so we provide comprehensive scripts and documentation for applying Dev-ResNet to different biological systems.

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.

Phenomics as an approach to Comparative Developmental Physiology.

The dynamic nature of developing organisms and how they function presents both opportunity and challenge to researchers, with significant advances in understanding possible by adopting innovative approaches to their empirical study. The information content of the phenotype during organismal development is arguably greater than at any other life stage, incorporating change at a broad range of temporal, spatial and functional scales and is of broad relevance to a plethora of research questions. Yet, effectively measuring organismal development, and the ontogeny of physiological regulations and functions, and their responses to the environment, remains a significant challenge. "Phenomics", a global approach to the acquisition of phenotypic data at the scale of the whole organism, is uniquely suited as an approach. In this perspective, we explore the synergies between phenomics and Comparative Developmental Physiology (CDP), a discipline of increasing relevance to understanding sensitivity to drivers of global change. We then identify how organismal development itself provides an excellent model for pushing the boundaries of phenomics, given its inherent complexity, comparably smaller size, relative to adult stages, and the applicability of embryonic development to a broad suite of research questions using a diversity of species. Collection, analysis and interpretation of whole organismal phenotypic data are the largest obstacle to capitalising on phenomics for advancing our understanding of biological systems. We suggest that phenomics within the context of developing organismal form and function could provide an effective scaffold for addressing grand challenges in CDP and phenomics.

A phenomics approach reveals interspecific differences in integrated developmental responses to chronic elevated temperatures.

Phenomics, high-dimensional organismal phenotyping, is advanced as a solution to quantifying complex developmental responses to elevated temperatures. 'Energy proxy traits' (EPTs) measure the phenotype as a spectrum of energy values across different temporal frequencies from pixel value fluctuations of video. Although they have proven effective in measuring the biology of complex and dynamic developing organisms, their utility in assessing environmental sensitivity of different species is untested. Using EPTs, we assess the relative thermal sensitivities of embryos of three species of freshwater snail with marked differences in their developmental event timings. Embryos of Lymnaea stagnalis, Radix balthica and Physella acuta were videoed hourly for the duration of their embryonic development at two temperatures: 20°C and 25°C. The video was used to calculate EPTs for the duration of their embryonic development, and during discrete physiological windows in development. Changes in energy spectra during development identified marked differences in thermal sensitivities between species, and suggest a relatively heightened sensitivity of gross rates of embryonic physiology and behaviour in embryos of R. balthica, developmental-window-specific thermal responses that reflect ontogenetic differences in observable physiologies, and temperature-induced changes in physiological event timing. EPTs enabled comparison of high-dimensional spectral phenotypes, providing a unique capability for assessing sensitivity continuously in developing individuals. Such integrative and scalable phenotyping is a prerequisite for improved understanding of the sensitivity of early life stages of different species.

Phenomics enables measurement of complex responses of developing animals to global environmental drivers.

Phenomics offers technological advances for high-dimensional phenotyping, facilitating rapid, high-throughput assessment of physiological performance and has proven invaluable in global research challenges including drug discovery and food security. However, this rapidly growing discipline has remained largely inaccessible to the increasingly urgent challenge of assessing organismal functional sensitivity to global change drivers. Here, we investigate the response of an ecologically important marine invertebrate to multiple environmental drivers using Energy Proxy Traits (EPTs), a new approach for measuring complex phenotypes captured on video as a spectrum of energy levels across different temporal frequencies in fluctuating pixel values. We imaged three developmental stages of the common prawn Palaemon serratus at different salinities and temperatures, and measured EPTs and heart rate, a major proxy of physiological performance in ectotherms present across stages. Significant interactions were detected between temperature, developmental stage and salinity in frequency-specific energy levels. Despite cardiac activity being a significant contributor to the EPT spectra, treatment interactions were different from those observed on EPTs, highlighting additional phenotypic drivers of EPTs. Elevated temperature resulted in a shift of the EPT spectra towards higher frequency signals, indicating a reallocation of resources within the phenome. Using a non-linear dimensionality reduction, we interrogated the responses of EPT spectra in high-dimensional space. We discovered complex developmental-stage specific sensitivities, highlighting both the complexity of phenotypic responses, and the limits of using univariate approaches with pre-selected traits to assess responses to multiple global environmental drivers. EPTs are a high-dimensional, transferrable method of phenotyping, and are therefore highly relevant to addressing the current limitations of traditional methods of phenotyping applied to assessing biological sensitivity to drivers of global change. We predict that EPTs will become an important tool for indiscriminate phenotyping, transferrable between species, developmental stages and experimental designs.

Thermodynamic effects drive countergradient responses in the thermal performance of Littorina saxatilis across latitude.

Thermal performance curves (TPCs) provide a powerful framework to assess the evolution of thermal sensitivity in populations exposed to divergent selection regimes across latitude. However, there is a lack of consensus regarding the extent to which physiological adjustments that compensate for latitudinal temperature variation (metabolic cold adaptation; MCA) may alter the shape of TPCs, including potential repercussion on upper thermal limits. To address this, we compared TPCs for cardiac activity in latitudinally-separated populations of the intertidal periwinkle Littorina saxatilis. We applied a non-linear TPC modelling approach to explore how different metrics governing the shape of TPCs varied systematically in response to local adaptation and thermal acclimation. Both critical upper limits, and the temperatures at which cardiac performance was maximised, were higher in the northernmost (cold-adapted) population and displayed a countergradient latitudinal trend which was most pronounced following acclimation to low temperatures. We interpret this response as a knock-on consequence of increased standard metabolic rate in high latitude populations, indicating that physiological compensation associated with MCA may indirectly influence variation in upper thermal limits across latitude. Our study highlights the danger of assuming that variation in any one aspect of the TPC is adaptive without appropriate mechanistic and ecological context.

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.

Divergence in Thermal Physiology Could Contribute to Vertical Segregation in Intertidal Ecotypes of Littorina saxatilis.

AbstractThermal stress is a potentially important selective agent in intertidal marine habitats, but the role that thermal tolerance might play in local adaptation across shore height has been underexplored. Northwest Spain is home to two morphologically distinct ecotypes of the periwinkle Littorina saxatilis, separated by shore height and subject to substantial differences in thermal stress exposure. However, despite other biotic and abiotic drivers of ecotype segregation being well studied, their thermal tolerance has not been previously characterized. We investigated thermal tolerance across multiple life history stages by employing the thermal death time (TDT) approach to determine (i) whether the two ecotypes differ in thermal tolerance and (ii) how any differences vary with life history stage. Adults of the two ecotypes differed in their thermal tolerance in line with their shore position: the upper-shore ecotype, which experiences more extreme temperatures, exhibited greater endurance of thermal stress compared with the lower-shore ecotype. This difference was most pronounced at the highest temperatures tested. The proximate physiological basis for these differences is unknown but likely due to a multifarious interaction of traits affecting different parts of the TDT curve. Differences in tolerance between ecotypes were less pronounced in early life history stages but increased with ontogeny, suggesting partial divergence of this trait during development. Thermal tolerance could potentially play an important role in maintaining population divergence and genetic segregation between the two ecotypes, since the increased thermal sensitivity of the lower-shore ecotype may limit its dispersal onto the upper shore and so restrict gene flow.

Spectral phenotyping of embryonic development reveals integrative thermodynamic responses.

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 & 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.

Both maternal and embryonic exposure to mild hypoxia influence embryonic development of the intertidal gastropod Littorina littorea.

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 that 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 affects the hatching success of these embryos.

Moderate reductions in dissolved oxygen may compromise performance in an ecologically-important estuarine invertebrate.

Coastal ecosystems, including estuaries, are increasingly pressured by expanding hypoxic regions as a result of human activities such as increased release of nutrients and global warming. Hypoxia is often defined as oxygen concentrations below 2 mL O2 L-1. However, taxa vary markedly in their sensitivity to hypoxia and can be affected by a broad spectrum of low oxygen levels. To better understand how reduced oxygen availability impacts physiological and molecular processes in invertebrates, we investigated responses of an estuarine amphipod to an ecologically-relevant level of moderate hypoxia (~2.6 mL O2 L-1) or severe hypoxia (~1.3 mL O2 L-1). Moderate hypoxia elicited a reduction in aerobic scope, and widespread changes to gene expression, including upregulation of metabolic genes and stress proteins. Under severe hypoxia, a marked hyperventilatory response associated with maintenance of aerobic performance was accompanied by a muted transcriptional response. This included a return of metabolic genes to baseline levels of expression and downregulation of transcripts involved in protein synthesis, most of which indicate recourse to hypometabolism and/or physiological impairment. We conclude that adverse ecological effects may occur under moderate hypoxia through compromised individual performance and, therefore, even modest declines in future oxygen levels may pose a significant challenge to coastal ecosystems.

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

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 Radix balthica and the marine amphipod Orchestia gammarellus. 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.

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.

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.

Short-term acclimation in adults does not predict offspring acclimation potential to hypoxia.

The prevalence of hypoxic areas in coastal waters is predicted to increase and lead to reduced biodiversity. While the adult stages of many estuarine invertebrates can cope with short periods of hypoxia, it remains unclear whether that ability is present if animals are bred and reared under chronic hypoxia. We firstly investigated the effect of moderate, short-term environmental hypoxia (40% air saturation for one week) on metabolic performance in adults of an estuarine amphipod, and the fitness consequences of prolonged exposure. We then reared the offspring of hypoxia-exposed parents under hypoxia, and assessed their oxyregulatory ability under declining oxygen tensions as juveniles and adults. Adults from the parental generation were able to acclimate their metabolism to hypoxia after one week, employing mechanisms typically associated with prolonged exposure. Their progeny, however, did not develop the adult pattern of respiratory regulation when reared under chronic hypoxia, but instead exhibited a poorer oxyregulatory ability than their parents. We conclude that species apparently hypoxia-tolerant when tested in short-term experiments, could be physiologically compromised as adults if they develop under hypoxia. Consequently, we propose that the increased prevalence of hypoxia in coastal regions will have marked effects in some species currently considered hypoxia tolerant.

Embryonic transcriptome of the brackishwater amphipod Gammarus chevreuxi.

Environmental change can dramatically alter the development of aquatic organisms. While the effect of such change on physiological and morphological ontogenies is becoming clearer, the molecular mechanisms underpinning them are largely unexplored. Characterizing these mechanisms is often limited by the lack of molecular resources. We have applied Illumina HiSeq sequencing to RNA isolated from different developmental stages of the brackishwater amphipod Gammarus chevreuxi. Over 52.6M paired-end reads were assembled de novo into 172,081 contigs, representing 118,812 potential genes. The assembly generated constitutes a reference embryonic transcriptome for an ecologically-important aquatic shredder species. This resource will contribute to our understanding of the mechanisms underpinning the development of physiological function through functional, comparative and quantitative expression studies. It will also allow the identification of candidate biomarkers for assessing the impact of environmental stressors in estuarine systems.

An embryonic transcriptome of the pulmonate snail Radix balthica.

The pond snail, Radix balthica (Linnaeus 1758), is an emerging model species within ecological developmental biology. While its development has been characterised in detail, genomic resources for embryonic stages are lacking. We applied Illumina MiSeq RNA-seq to RNA isolated from pools of embryos at two points during development. Embryos were cultured in either the presence or absence of predator kariomones to increase the diversity of the transcripts assembled. Sequencing produced 47.2M paired-end reads, assembled into 54,360 contigs of which 73% were successfully annotated. This transcriptome provides an invaluable resource to build a mechanistic understanding of developmental plasticity.

Combining motion analysis and microfluidics--a novel approach for detecting whole-animal responses to test substances.

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.

Parent--offspring similarity in the timing of developmental events: an origin of heterochrony?

Understanding the link between ontogeny (development) and phylogeny (evolution) remains a key aim of biology. Heterochrony, the altered timing of developmental events between ancestors and descendants, could be such a link although the processes responsible for producing heterochrony, widely viewed as an interspecific phenomenon, are still unclear. However, intraspecific variation in developmental event timing, if heritable, could provide the raw material from which heterochronies originate. To date, however, heritable developmental event timing has not been demonstrated, although recent work did suggest a genetic basis for intraspecific differences in event timing in the embryonic development of the pond snail, Radix balthica. Consequently, here we used high-resolution (temporal and spatial) imaging of the entire embryonic development of R. balthica to perform a parent-offspring comparison of the timing of twelve, physiological and morphological developmental events. Between-parent differences in the timing of all events were good predictors of such timing differences between their offspring, and heritability was demonstrated for two of these events (foot attachment and crawling). Such heritable intraspecific variation in developmental event timing could be the raw material for speciation events, providing a fundamental link between ontogeny and phylogeny, via heterochrony.