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

Motion analysis is one of the tools available to biologists to extract biologically relevant information from image datasets and has been applied to a diverse range of organisms. The application of motion analysis during early development presents a challenge, as embryos often exhibit complex, subtle and diverse movement patterns. A method of motion analysis able to holistically quantify complex embryonic movements could be a powerful tool for fields such as toxicology and developmental biology to investigate whole organism stress responses. Here we assessed whether motion analysis could be used to distinguish the effects of stressors on three early developmental stages of each of three species: (i) the zebrafish Danio rerio (stages 19 h, 21.5 h and 33 h exposed to 1.5% ethanol and a salinity of 5); (ii) the African clawed toad Xenopus laevis (stages 24, 32 and 34 exposed to a salinity of 20); and iii) the pond snail Radix balthica (stages E3, E4, E6, E9 and E11 exposed to salinities of 5, 10 and 15). Image sequences were analysed using Sparse Optic Flow and the resultant frame-to-frame motion parameters were analysed using Discrete Fourier Transform to quantify the distribution of energy at different frequencies. This spectral frequency dataset was then used to construct a Bray-Curtis similarity matrix and differences in movement patterns between embryos in this matrix were tested for using ANOSIM

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

Development of cardiac function and control of larval and adult heart in embryos of the marine gastropod Littorina obtusata (Linnaeus)

In many gastropod larvae, an extracardiac structure termed the larval heart appears during the veliger stage. The adult heart develops later and there is a transitional phase during which both hearts beat at the same time before the larval heart degenerates. Detailed information about the function and ontogeny of the two structures is lacking. Here, I used descriptive and experimental approaches to investigate the ontogeny of cardiovascular function and control in Littorina obtusata, an intertidal gastropod with direct development. Based on an earlier hypothesis that the function of the larval heart is directly linked to that of the velum, I examined the relative timing of appearance and disappearance of the two structures. Additionally, physiological (larval and adult heart rate) and behavioural (spinning) responses to declining levels of dissolved oxygen were examined in different developmental stages. There was a considerable mismatch between the appearance of the velum and the appearance of the larval heart: The velum started developing on day 4, but the larval heart did not begin to beat until day 12. This mismatch is in contrast to that observed in planktonic larvae where the appearance of the two structures is much more attuned. Physiological and behavioural responses to hypoxia differed remarkably between developmental stages. Larval and adult hearts were insensitive to hypoxia during the first 3-4 days of their appearance but after this time showed a bradycardic response. Once the adult heart began to beat, the larval heart again became insensitive to hypoxia. Embryos of all examined developmental stages increased time spent spinning under hypoxia, although the increase in time spent spinning generally decreased with progressing development. Together, these findings indicate that circulation through the larval heart alone sufficed in early larval stages, where embryos were small in size and had a high ability to spin. With progressive development, consequent increasing metabolic demands and a decreasing ability to spin, oxygen supply became critical under hypoxic conditions, expressed as a bradycardia of the larval heart at that stage. The additional support provided by the adult heart assisted in meeting the increased energetic demands, even under hypoxic conditions.Faculty of Science & Technolog

Six data files containing Crinia georgiana embryonic, tadpole and metamorph traits from sire-by-dam 90 families

Data files contain Crinia georgiana traits (survival, development rate, tadpole and metamorph morphology and metamorph jumping performance) for 90 sire-by-dam families. Fixed effects were water depth treatment, ovum size and corticosterone concentrations in the yolk. Random effects were sire, dam, block (and all interactions). Please see Materials and Methods for detailed description

Data from: Environmental stress increases the magnitude of non-additive genetic variation in offspring fitness in the frog Crinia georgiana

When organisms encounter heterogeneous environments, selection may favor the ability of individuals to tailor their phenotypes to suit the prevailing conditions. Understanding the genetic basis of plastic responses is therefore vital for predicting whether susceptible populations can adapt and persist under new selection pressures. Here, we investigated whether there is potential for adaptive plasticity in development time in the quacking frog Crinia georgiana, a species experiencing a drying climate. Using a North Carolina II breeding design, we exposed 90 family groups to two water depth treatments (baseline and low-water) late in larval development. We estimated the contribution of additive and non-additive sources of genetic variation to early offspring fitness under both environments. Our results revealed a marked decline in larval fitness under the stressful (low-water) rearing environment, but also that additive genetic variation was negligible for all traits. However, in most cases we found significant sire-by-dam interactions, indicating the importance of non-additive genetic variation for offspring fitness. Moreover, sire-by-dam interactions were modified by the treatment, indicating that patterns of non-additive genetic variance depend on environmental context. For all traits, we found higher levels of non-additive genetic variation (relative to total phenotypic variation) when larvae were reared under stressful conditions, suggesting that the fitness costs associated with incompatible parental crosses (e.g. homozygous deleterious recessive alleles) will only be expressed when water availability is low. Taken together, our results highlight the need to consider patterns of non-additive genetic variation under contrasting selective regimes when considering the resilience of species to environmental change

Data from: A genome‐wide search for local adaptation in a terrestrial‐breeding frog reveals vulnerability to climate change

Terrestrial‐breeding amphibians are likely to be vulnerable to warming and drying climates, as their embryos require consistent moisture for successful development. Adaptation to environmental change will depend on sufficient genetic variation existing within or between connected populations. Here, we use Single Nucleotide Polymorphism (SNP) data to investigate genome‐wide patterns in genetic diversity, gene flow and local adaptation in a terrestrial‐breeding frog (Pseudophryne guentheri) subject to a rapidly drying climate and recent habitat fragmentation. The species was sampled across 12 central and range‐edge populations (192 samples), and strong genetic structure was apparent, as were high inbreeding coefficients. Populations showed differences in genetic diversity, and one population lost significant genetic diversity in a decade. More than 500 SNP loci were putatively under directional selection, and 413 of these loci were correlated with environmental variables such as temperature, rainfall, evaporation and soil moisture. One locus showed homology to a gene involved in the activation of maturation in Xenopus oocytes, which may facilitate rapid development of embryos in drier climates. The low genetic diversity, strong population structuring and presence of local adaptation revealed in this study shows why management strategies such as targeted gene flow may be necessary to assist isolated populations to adapt to future climates

Sympatric and novel species SNP data

The file contains Single Nucleotide Polymorphism (SNP) data for each individual from the sympatric species (Pseudophryne occidentalis) and the novel species studied

Pseudophryne guentheri SNP and environmental data

The file contains Single Nucleotide Polymorphism (SNP) data for each individual from each population studied. The average rainfall, soil moisture, temperature and evaporation for each study site, calculated over a 37 year period (1980-2017) are also provided

Multidimensional scaling plot in two dimensions using Bray and Curtis similarities, performed on logarithmically transformed data consisting of 18 frequency bins for each of (i) negative angle, (ii) positive angle, (iii) centre of mass – rho, and (iv) centre of mass – theta.

<p>The mean values of each of the five groups (pre-exposure, ethanol 1 and 2, recovery 1 and 2) were used. Dissimilarities in the motion of embryos within the five experimental periods is signified by the distance between them. The bubble size reflects the average frequency of tail flicks of the 15 embryos for each group.</p

Schematic representation of the experimental design.

<p>One cycle represents the time period taken to record all 15 embryos once (7.5 min). The experiment is divided into five time periods: ”pre-exposure period” (recording cycle 1–4: aerated, deionized water), “ethanol period” (divided into two groups “ethanol 1” (recording cycle 5–8) and “ethanol 2” (recording cycle 9–12); 1.5% ethanol solution) and “recovery period” (divided into two groups “recovery 1” (recording cycle 13–16) and “recovery 2” (recording cycle 17–20); aerated, deionized water).</p