Living on the Edge and Beyond of Anoxia: Evolutionary Ecological Insights From Inside Crayfish Burrows

Burrowing is a common trait among crayfish thought to help species deal with adverse environmental challenges. Here we used in-vivo experimental data and in-silico modelling of oxygen saturation in a virtual burrow inhabited by crayfish. Except for the entrance 200 mm region, the burrow microenvironment becomes anoxic, on average, within 8 hours, and 12-hour day-night multiple cycles were not sufficient for refreshing the burrow microenvironment even with temporary lack of crayfish. We asked whether the ecological category of crayfish burrowing activity is reflected in the physiological ability to cope with hypoxia and anoxia. As dissolved oxygen declined, respiration patterns of primary burrowers differed from those of secondary and tertiary burrowers, showing also the highest variability in anoxia tolerance. Secondary burrowers showed consistent tolerance with all species exhibiting a mean survival of > 3h anoxic conditions. Tertiary burrowers were variable, exhibiting moderate to zero tolerance of anoxia. The adaptive mechanisms to cope with hypoxia might be a basal legacy from the crayfish monophyletic ancestors – lobsters, traveller crustaceans often reaching deep depths in the ocean. These results challenge the current understanding of crayfish ecology, opening an evolutionary ecological perspective which might be relevant for the next generation of phylogenetical approaches.

Developmental programming of DNA methylation and gene expression patterns is associated with extreme cardiovascular tolerance to anoxia in the common snapping turtle

Background Environmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species. Results Genome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such as SCN5A may account for differences in heart rate, while genes such as TNNT2 and TPM3 may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated. Conclusions Our data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

Cytoskeletal Arrest: An Anoxia Tolerance Mechanism

Polymerization of actin filaments and microtubules constitutes a ubiquitous demand for cellular adenosine-5′-triphosphate (ATP) and guanosine-5′-triphosphate (GTP). In anoxia-tolerant animals, ATP consumption is minimized during overwintering conditions, but little is known about the role of cell structure in anoxia tolerance. Studies of overwintering mammals have revealed that microtubule stability in neurites is reduced at low temperature, resulting in withdrawal of neurites and reduced abundance of excitatory synapses. Literature for turtles is consistent with a similar downregulation of peripheral cytoskeletal activity in brain and liver during anoxic overwintering. Downregulation of actin dynamics, as well as modification to microtubule organization, may play vital roles in facilitating anoxia tolerance. Mitochondrial calcium release occurs during anoxia in turtle neurons, and subsequent activation of calcium-binding proteins likely regulates cytoskeletal stability. Production of reactive oxygen species (ROS) formation can lead to catastrophic cytoskeletal damage during overwintering and ROS production can be regulated by the dynamics of mitochondrial interconnectivity. Therefore, suppression of ROS formation is likely an important aspect of cytoskeletal arrest. Furthermore, gasotransmitters can regulate ROS levels, as well as cytoskeletal contractility and rearrangement. In this review we will explore the energetic costs of cytoskeletal activity, the cellular mechanisms regulating it, and the potential for cytoskeletal arrest being an important mechanism permitting long-term anoxia survival in anoxia-tolerant species, such as the western painted turtle and goldfish.

Cold-acclimation induces life stage-specific responses in the cardiac proteome of Western painted turtles (Chrysemys picta bellii): implications for anoxia tolerance

Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3oC survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. Since protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold-acclimation would evoke preparatory changes in protein expression to support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20oC (warm) or 3oC (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis. The relative abundances of 1316 identified proteins were compared between temperatures and developmental stages. The effect of cold-acclimation on the cardiac proteome was only evident in the context of an interaction with life stage, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope with age that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of the life stage- and temperature-induced changes to the cardiac proteome, including reduced complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stage-specific anoxia tolerance in turtles.

Cold-acclimation induces life stage-specific responses in the cardiac proteome of Western painted turtles (Chrysemys picta bellii): implications for anoxia tolerance

Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3°C survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. Since protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold-acclimation would evoke preparatory changes in protein expression that would support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20°C (warm) or 3°C (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis using labeled isobaric tags and mass spectrometry. The relative abundances of 1316 identified proteins were compared between temperatures and developmental stages. The effect of cold-acclimation on the cardiac proteome was most evident when life stage was included as a covariable, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart from its hatchling to adult form and, only after this maturation occurs, will cold-acclimation induce protein expression changes appropriate for supporting heart function during prolonged anoxia. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of life stage- and temperature-induced changes to the cardiac proteome, including reduced complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stagespecific anoxia tolerance in turtles.

GABA metabolism is crucial for long-term survival of anoxia in annual killifish embryos

ABSTRACTIn most vertebrates, a lack of oxygen quickly leads to irreparable damages to vital organs, such as the brain and heart. However, there are some vertebrates that have evolved mechanisms to survive periods of no oxygen (anoxia). The annual killifish (Austrofundulus limnaeus) survives in ephemeral ponds in the coastal deserts of Venezuela and their embryos have the remarkable ability to tolerate anoxia for months. When exposed to anoxia, embryos of A. limnaeus respond by producing significant amounts of γ-aminobutyric acid (GABA). This study aims to understand the role of GABA in supporting the metabolic response to anoxia. To explore this, we investigated four developmentally distinct stages of A. limnaeus embryos that vary in their anoxia tolerance. We measured GABA and lactate concentrations across development in response to anoxia and aerobic recovery. We then inhibited enzymes responsible for the production and degradation of GABA and observed GABA and lactate concentrations, as well as embryo mortality. Here, we show for the first time that GABA metabolism affects anoxia tolerance in A. limnaeus embryos. Inhibition of enzymes responsible for GABA production (glutamate decarboxylase) and degradation (GABA-transaminase and succinic acid semialdehyde dehydrogenase) led to increased mortality, supporting a role for GABA as an intermediate product and not a metabolic end-product. We propose multiple roles for GABA during anoxia and aerobic recovery in A. limnaeus embryos, serving as a neurotransmitter, an energy source, and an anti-oxidant.