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.

The north-south divide? Macroalgal functional trait diversity and redundancy varies with intertidal aspect.

BACKGROUND AND AIMS: Marine macroalgae ('seaweeds') are critical to coastal ecosystem structure and function, but also vulnerable to the many environmental changes associated with anthropogenic climate change (ACC). The local habitat conditions underpinning observed and predicted ACC-driven changes in intertidal macroalgal communities are complex and probably site-specific and operate in addition to more commonly reported regional factors such as sea surface temperatures. METHODS: We examined how the composition and functional trait expression of macroalgal communities in SW England varied with aspect (i.e. north-south orientation) at four sites with opposing Equator- (EF) and Pole-facing (PF) surfaces. Previous work at these sites had established that average annual (low tide) temperatures vary by 1.6 °C and that EF-surfaces experience six-fold more frequent extremes (i.e. >30 °C). KEY RESULTS: PF macroalgal communities were consistently more taxon rich; 11 taxa were unique to PF habitats, with only one restricted to EF. Likewise, functional richness and dispersion were greater on PF-surfaces (dominated by algae with traits linked to rapid resource capture and utilization, but low desiccation tolerance), although differences in both taxon and functional richness were probably driven by the fact that less diverse EF-surfaces were dominated by desiccation-tolerant fucoids. CONCLUSIONS: Although we cannot disentangle the influence of temperature variation on algal ecophysiology from the indirect effects of aspect on species interactions (niche pre-emption, competition, grazing, etc.), our study system provides an excellent model for understanding how environmental variation at local scales affects community composition and functioning. By virtue of enhanced taxonomic diversity, PF-aspects supported higher functional diversity and, consequently, greater effective functional redundancy. These differences may imbue PF-aspects with resilience against environmental perturbation, but if predicted increases in global temperatures are realized, some PF-sites may shift to a depauperate, desiccation-tolerant seaweed community with a concomitant loss of functional diversity and redundancy.

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.

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.

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.

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.

Do aquatic ectotherms perform better under hypoxia after warm acclimation?

Aquatic animals increasingly encounter environmental hypoxia due to climate-related warming and/or eutrophication. Although acute warming typically reduces performance under hypoxia, the ability of organisms to modulate hypoxic performance via thermal acclimation is less understood. Here, we review the literature and ask whether hypoxic performance of aquatic ectotherms improves following warm acclimation. Interpretation of thermal acclimation effects is limited by reliance on data from experiments that are not designed to directly test for beneficial or detrimental effects on hypoxic performance. Most studies have tested hypoxic responses exclusively at test temperatures matching organisms' acclimation temperatures, precluding the possibility of distinguishing between acclimation and acute thermal effects. Only a few studies have applied appropriate methodology to identify beneficial thermal acclimation effects on hypoxic performance, i.e. acclimation to different temperatures prior to determining hypoxic responses at standardised test temperatures. These studies reveal that acute warming predominantly impairs hypoxic performance, whereas warm acclimation tends to be either beneficial or have no effect. If this generalises, we predict that warm-acclimated individuals in some species should outperform non-acclimated individuals under hypoxia. However, acclimation seems to only partially offset acute warming effects; therefore, aquatic ectotherms will probably display overall reduced hypoxic performance in the long term. Drawing on the appropriate methodology, future studies can quantify the ability of organisms to modulate hypoxic performance via (reversible) thermal acclimation and unravel the underlying mechanisms. Testing whether developmental acclimation and multigenerational effects allow for a more complete compensation is essential to allow us to predict species' resilience to chronically warmer, hypoxic environments.

Universal metabolic constraints shape the evolutionary ecology of diving in animals.

Diving as a lifestyle has evolved on multiple occasions when air-breathing terrestrial animals invaded the aquatic realm, and diving performance shapes the ecology and behaviour of all air-breathing aquatic taxa, from small insects to great whales. Using the largest dataset yet assembled, we show that maximum dive duration increases predictably with body mass in both ectotherms and endotherms. Compared to endotherms, ectotherms can remain submerged for longer, but the mass scaling relationship for dive duration is much steeper in endotherms than in ectotherms. These differences in diving allometry can be fully explained by inherent differences between the two groups in their metabolic rate and how metabolism scales with body mass and temperature. Therefore, we suggest that similar constraints on oxygen storage and usage have shaped the evolutionary ecology of diving in all air-breathing animals, irrespective of their evolutionary history and metabolic mode. The steeper scaling relationship between body mass and dive duration in endotherms not only helps explain why the largest extant vertebrate divers are endothermic rather than ectothermic, but also fits well with the emerging consensus that large extinct tetrapod divers (e.g. plesiosaurs, ichthyosaurs and mosasaurs) were endothermic.

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.

The effects of elevated temperature and PCO2 on the energetics and haemolymph pH homeostasis of juveniles of the European lobster, Homarus gammarus.

Regulation of extracellular acid-base balance, while maintaining energy metabolism, is recognised as an important aspect when defining an organism's sensitivity to environmental changes. This study investigated the haemolymph buffering capacity and energy metabolism (oxygen consumption, haemolymph [l-lactate] and [protein]) in early benthic juveniles (carapace length <40 mm) of the European lobster, Homarus gammarus, exposed to elevated temperature and PCO2 At 13°C, H. gammarus juveniles were able to fully compensate for acid-base disturbances caused by the exposure to elevated seawater PCO2  at levels associated with ocean acidification and carbon dioxide capture and storage (CCS) leakage scenarios, via haemolymph [HCO3-] regulation. However, metabolic rate remained constant and food consumption decreased under elevated PCO2 , indicating reduced energy availability. Juveniles at 17°C showed no ability to actively compensate haemolymph pH, resulting in decreased haemolymph pH particularly under CCS conditions. Early benthic juvenile lobsters at 17°C were not able to increase energy intake to offset increased energy demand and therefore appear to be unable to respond to acid-base disturbances due to increased PCO2 at elevated temperature. Analysis of haemolymph metabolites suggests that, even under control conditions, juveniles were energetically limited. They exhibited high haemolymph [l-lactate], indicating recourse to anaerobic metabolism. Low haemolymph [protein] was linked to minimal non-bicarbonate buffering and reduced oxygen transport capacity. We discuss these results in the context of potential impacts of ongoing ocean change and CCS leakage scenarios on the development of juvenile H. gammarus and future lobster populations and stocks.

Does sex really matter? Explaining intraspecies variation in ocean acidification responses.

Ocean acidification (OA) poses a major threat to marine ecosystems globally, having significant ecological and economic importance. The number and complexity of experiments examining the effects of OA has substantially increased over the past decade, in an attempt to address multi-stressor interactions and long-term responses in an increasing range of aquatic organisms. However, differences in the response of males and females to elevated pCO2 have been investigated in fewer than 4% of studies to date, often being precluded by the difficulty of determining sex non-destructively, particularly in early life stages. Here we highlight that sex can significantly impact organism responses to OA, differentially affecting physiology, reproduction, biochemistry and ultimately survival. What is more, these impacts do not always conform to ecological theory based on differential resource allocation towards reproduction, which would predict females to be more sensitive to OA owing to the higher production cost of eggs compared with sperm. Therefore, non-sex-specific studies may overlook subtle but ecologically significant differences in the responses of males and females to OA, with consequences for forecasting the fate of natural populations in a near-future ocean.

Differences in the timing of cardio-respiratory development determine whether marine gastropod embryos survive or die in hypoxia.

Physiological plasticity of early developmental stages is a key way by which organisms can survive and adapt to environmental change. We investigated developmental plasticity of aspects of the cardio-respiratory physiology of encapsulated embryos of a marine gastropod, Littorina obtusata, surviving exposure to moderate hypoxia (PO2 =8 kPa) and compared the development of these survivors with that of individuals that died before hatching. Individuals surviving hypoxia exhibited a slower rate of development and altered ontogeny of cardio-respiratory structure and function compared with normoxic controls (PO2 >20 kPa). The onset and development of the larval and adult hearts were delayed in chronological time in hypoxia, but both organs appeared earlier in developmental time and cardiac activity rates were greater. The velum, a transient, 'larval' organ thought to play a role in gas exchange, was larger in hypoxia but developed more slowly (in chronological time), and velar cilia-driven, rotational activity was lower. Despite these effects of hypoxia, 38% of individuals survived to hatching. Compared with those embryos that died during development, these surviving embryos had advanced expression of adult structures, i.e. a significantly earlier occurrence and greater activity of their adult heart and larger shells. In contrast, embryos that died retained larval cardio-respiratory features (the velum and larval heart) for longer in chronological time. Surviving embryos came from eggs with significantly higher albumen provisioning than those that died, suggesting an energetic component for advanced development of adult traits.

What can an ecophysiological approach tell us about the physiological responses of marine invertebrates to hypoxia?

Hypoxia (low O2) is a common and natural feature of many marine environments. However, human-induced hypoxia has been on the rise over the past half century and is now recognised as a major problem in the world's seas and oceans. Whilst we have information on how marine invertebrates respond physiologically to hypoxia in the laboratory, we still lack understanding of how they respond to such stress in the wild (now and in the future). Consequently, here the question 'what can an ecophysiological approach tell us about physiological responses of marine invertebrates to hypoxia' is addressed. How marine invertebrates work in the wild when challenged with hypoxia is explored using four case studies centred on different hypoxic environments. The recent integration of the various -omics into ecophysiology is discussed, and a number of advantages of, and challenges to, successful integration are suggested. The case studies and -omic/physiology integration data are used to inform the concluding part of the review, where it is suggested that physiological responses to hypoxia in the wild are not always the same as those predicted from laboratory experiments. This is due to behaviour in the wild modifying responses, and therefore more than one type of 'experimental' approach is essential to reliably determine the actual response. It is also suggested that assuming it is known what a measured response is 'for' can be misleading and that taking parodies of ecophysiology seriously may impede research progress. This review finishes with the suggestion that an -omics approach is, and is becoming, a powerful method of understanding the response of marine invertebrates to environmental hypoxia and may be an ideal way of studying hypoxic responses in the wild. Despite centring on physiological responses to hypoxia, the review hopefully serves as a contribution to the discussion of what (animal) ecophysiology looks like (or should look like) in the 21st century.

(1)H NMR metabolomics reveals contrasting response by male and female mussels exposed to reduced seawater pH, increased temperature, and a pathogen.

Human activities are fundamentally altering the chemistry of the world's oceans. Ocean acidification (OA) is occurring against a background of warming and an increasing occurrence of disease outbreaks, posing a significant threat to marine organisms, communities, and ecosystems. In the current study, (1)H NMR spectroscopy was used to investigate the response of the blue mussel, Mytilus edulis, to a 90-day exposure to reduced seawater pH and increased temperature, followed by a subsequent pathogenic challenge. Analysis of the metabolome revealed significant differences between male and female organisms. Furthermore, males and females are shown to respond differently to environmental stress. While males were significantly affected by reduced seawater pH, increased temperature, and a bacterial challenge, it was only a reduction in seawater pH that impacted females. Despite impacting males and females differently, stressors seem to act via a generalized stress response impacting both energy metabolism and osmotic balance in both sexes. This study therefore has important implications for the interpretation of metabolomic data in mussels, as well as the impact of environmental stress in marine invertebrates in general.

The embryonic thermal environment has positive but weak effects on thermal tolerance later in life in the aquatic invertebrate Gammarus chevreuxi.

Recent evidence suggests that the adult phenotype is influenced by temperatures experienced in early life. However, our understanding of the extent to which the embryonic environment can modulate thermal tolerance later in life is limited, owing to the paucity of studies with appropriate experimental designs to test for this form of developmental plasticity. We investigated whether the thermal environment experienced during embryonic development affects thermal limits in later life. Embryos of the estuarine amphipod Gammarus chevreuxi were incubated until hatching to 15 °C, 20 °C and 25 °C, then reared under a common temperature. Using thermal ramping assays, we determined upper thermal limits in juveniles, four weeks post-hatch. Individuals exposed to higher temperatures during embryonic development displayed greater thermal tolerance as juveniles (acclimation response ratio ≈ 0.10-0.25 for upper lethal temperature). However, we suggest that the degree of developmental plasticity observed is limited, and will provide little benefit under future climate change scenarios.

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

BACKGROUND: 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. RESULTS: Spectral 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. CONCLUSION: This 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.

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.

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.

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 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.