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.

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.

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.

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.

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.

Regional adaptation defines sensitivity to future ocean acidification.

Physiological responses to temperature are known to be a major determinant of species distributions and can dictate the sensitivity of populations to global warming. In contrast, little is known about how other major global change drivers, such as ocean acidification (OA), will shape species distributions in the future. Here, by integrating population genetics with experimental data for growth and mineralization, physiology and metabolomics, we demonstrate that the sensitivity of populations of the gastropod Littorina littorea to future OA is shaped by regional adaptation. Individuals from populations towards the edges of the natural latitudinal range in the Northeast Atlantic exhibit greater shell dissolution and the inability to upregulate their metabolism when exposed to low pH, thus appearing most sensitive to low seawater pH. Our results suggest that future levels of OA could mediate temperature-driven shifts in species distributions, thereby influencing future biogeography and the functioning of marine ecosystems.

An in situ assessment of local adaptation in a calcifying polychaete from a shallow CO2 vent system.

Ocean acidification (OA) is likely to exert selective pressure on natural populations. Our ability to predict which marine species will adapt to OA and what underlies this adaptive potential is of high conservation and resource management priority. Using a naturally low-pH vent site in the Mediterranean Sea (Castello Aragonese, Ischia) mirroring projected future OA conditions, we carried out a reciprocal transplant experiment to investigate the relative importance of phenotypic plasticity and local adaptation in two populations of the sessile, calcifying polychaete Simplaria sp. (Annelida, Serpulidae, Spirorbinae): one residing in low pH and the other from a nearby ambient (i.e. high) pH site. We measured a suite of fitness-related traits (i.e. survival, reproductive output, maturation, population growth) and tube growth rates in laboratory-bred F2 generation individuals from both populations reciprocally transplanted back into both ambient and low-pH in situ habitats. Both populations showed lower expression in all traits, but increased tube growth rates, when exposed to low-pH compared with high-pH conditions, regardless of their site of origin suggesting that local adaptation to low-pH conditions has not occurred. We also found comparable levels of plasticity in the two populations investigated, suggesting no influence of long-term exposure to low pH on the ability of populations to adjust their phenotype. Despite high variation in trait values among sites and the relatively extreme conditions at the low pH site (pH < 7.36), response trends were consistent across traits. Hence, our data suggest that, for Simplaria and possibly other calcifiers, neither local adaptations nor sufficient phenotypic plasticity levels appear to suffice in order to compensate for the negative impacts of OA on long-term survival. Our work also emphasizes the utility of field experiments in natural environments subjected to high level of pCO 2 for elucidating the potential for adaptation to future scenarios of OA.

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.

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.

Home advantage? Decomposition across the freshwater-estuarine transition zone varies with litter origin and local salinity.

Expected increases in the frequency and intensity of storm surges and river flooding may greatly affect the relative salinity of estuarine environments over the coming decades. In this experiment we used detritus from three contrasting environments (marine Fucus vesiculosus; estuarine Spartina anglica; terrestrial Quercus robur) to test the prediction that the decomposition of the different types of litter would be highest in the environment with which they are associated. Patterns of decomposition broadly fitted our prediction: Quercus detritus decomposed more rapidly in freshwater compared with saline conditions while Fucus showed the opposite trend; Spartina showed an intermediate response. Variation in macro-invertebrate assemblages was detected along the salinity gradient but with different patterns between estuaries, suggesting that breakdown rates may be linked in part to local invertebrate assemblages. Nonetheless, our results suggest that perturbation of salinity gradients through climate change could affect the process of litter decomposition and thus alter nutrient cycling in estuarine transition zones. Understanding the vulnerability of estuaries to changes in local abiotic conditions is important given the need to better integrate coastal proceses into a wider management framework at a time when coastlines are increasingly threatened by human activities.

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.

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.

Development of cardiovascular function in the marine gastropod Littorina obtusata (Linnaeus).

The molluscan cardiovascular system typically incorporates a transient extracardiac structure, the larval heart, early in development, but the functional importance of this structure is unclear. We documented the ontogeny and regulatory ability of the larval heart in relation to two other circulatory structures, the true heart and the velum, in the intertidal gastropod Littorina obtusata. There was a mismatch between the appearance of the larval heart and the velum. Velar lobes appeared early in development (day 4), but the larval heart did not begin beating until day 13. The beating of the larval heart reached a maximum on day 17 and then decreased until the structure itself disappeared (day 24). The true heart began to beat on day 17. Its rate of beating increased as that of the larval heart decreased, possibly suggesting a gradual shift from a larval heart-driven to a true heart-driven circulation. The true heart was not sensitive to acutely declining P(O(2)) shortly after it began to beat, but increased in activity in response to acutely declining P(O(2)) by day 21. Larval heart responses were similar to those of the true heart, with early insensitivity to declining P(O(2)) (day 13) followed by a response by day 15. Increased velum-driven rotational activity under acutely declining P(O(2)) was greatest in early developmental stages. Together, these findings point to cardiovascular function in L. obtusata larvae being the result of a complex interaction between velum, larval and true heart activities, with the functions of the three structures coinciding but their relative importance changing throughout larval development.

Exposure to elevated temperature and Pco(2) reduces respiration rate and energy status in the periwinkle Littorina littorea.

In the future, marine organisms will face the challenge of coping with multiple environmental changes associated with increased levels of atmospheric Pco(2), such as ocean warming and acidification. To predict how organisms may or may not meet these challenges, an in-depth understanding of the physiological and biochemical mechanisms underpinning organismal responses to climate change is needed. Here, we investigate the effects of elevated Pco(2) and temperature on the whole-organism and cellular physiology of the periwinkle Littorina littorea. Metabolic rates (measured as respiration rates), adenylate energy nucleotide concentrations and indexes, and end-product metabolite concentrations were measured. Compared with values for control conditions, snails decreased their respiration rate by 31% in response to elevated Pco(2) and by 15% in response to a combination of increased Pco(2) and temperature. Decreased respiration rates were associated with metabolic reduction and an increase in end-product metabolites in acidified treatments, indicating an increased reliance on anaerobic metabolism. There was also an interactive effect of elevated Pco(2) and temperature on total adenylate nucleotides, which was apparently compensated for by the maintenance of adenylate energy charge via AMP deaminase activity. Our findings suggest that marine intertidal organisms are likely to exhibit complex physiological responses to future environmental drivers, with likely negative effects on growth, population dynamics, and, ultimately, ecosystem processes.