The western painted turtle, Chrysemys picta bellii, has the greatest tolerance to anoxia of any tetrapod studied to date. These turtles reside in the northern United States and southern Canada, and survive months of anoxia while submerged in ice-locked ponds and bogs. Reference genomes provide an important resource for elucidating the molecular bases for such unique physiological traits. The initial reference genome for this species, published in 2013, is highly fragmented, thereby limiting downstream analyses and biological interpretation. We created a new and improved assembly by combining PacBio HiFi, 10 × Genomics Chromium, Hi-C sequence data and Bionano optical mapping derived from a single individual to generate a new haplotype-resolved chromosome-level reference assembly for C. picta bellii: “SLU_Cpb5.0”. The genome size of the primary assembly is 2.372 Gb with a scaffold N50 of 133.6 Mb, a 6.5-fold improvement over the previous assembly. Annotation of SLU_Cpb5.0 revealed 12,242 novel genes compared to previous assemblies. PacBio Iso-Seq RNA sequencing using twelve tissues identified more than 100,000 novel transcript isoforms and 3,910 novel genes not previously annotated. This new genome assembly and annotation will support future comparative genomics studies, and the distinct patterns of tissue-specific isoform expression create a robust foundation for future characterization of the functions of these genes. Furthermore, to better understand the genetic basis of C. picta bellii’s extreme physiological adaptations and other aspects of its biology, we utilize existing RNA-seq data to identify dozens of novel, differentially expressed genes in the heart and brain of anoxic painted turtles.

Cultured neuronal models for non-mammalian vertebrates are uncommon but could prove useful for investigating mechanisms of exceptional physiological performance, such as anoxia tolerance. We describe a procedure to isolate, culture and characterize cerebrocortical neurons of extremely anoxia-tolerant painted turtles; we then imposed anoxia while recording reactive oxygen species (ROS) using fluorescence photometry. Cerebrocortical sheets from hatchlings were dissociated enzymatically and mechanically, and cultured for ≤7 days. Within 24 h, most cells possessed classic neuronal morphology with identifiable soma and fine neurites. Immunocytochemistry showed MAP2-positive cells accounted for 90.9% of DAPI-positive cells. Ca2+ recordings (Fura-2) demonstrated neurons were immediately excitable with KCl and glutamate, but not acetylcholine. ROS recordings (CM-H2DCFDA) showed they avoided excessive ROS production post-anoxia, like other turtle brain preparations, but with higher signal and temporal resolution. These approaches should extend previous work in other brain preparations to isolated cells that possess the morphological and functional features of neurons.

ABSTRACT North American pond turtles (Emydidae) are renowned for their ability to survive extreme hypoxia and anoxia, which enables several species to overwinter in ice-locked, anoxic freshwater ponds and bogs for months. Centrally important for surviving these conditions is a profound metabolic suppression, which enables ATP demands to be met entirely with glycolysis. To better understand whether anoxia limits special sensory functions, we recorded evoked potentials in a reduced brain preparation, in vitro, that was perfused with severely hypoxic artificial cerebral spinal fluid (aCSF). For recordings of visual responses, an LED was flashed onto retinal eyecups while evoked potentials were recorded from the retina or the optic tectum. For recordings of auditory responses, a piezomotor-controlled glass actuator displaced the tympanic membrane while evoked potentials were recorded from the cochlear nuclei. We found that visual responses decreased when perfused with hypoxic perfusate (aCSF PO2<4.0 kPa). In contrast, the evoked response within the cochlear nuclei was unattenuated. These data provide further support that pond turtles have a limited ability to sense visual information in their environment even while moderately hypoxic, but that auditory input may become a principal avenue of sensory perception during extreme diving in this species such as occurs during anoxic submergence.

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.

Freshwater turtles found in higher latitudes can experience extreme challenges to acid-base homeostasis while overwintering, due to a combination of cold temperatures along with the potential for environmental hypoxia. Histidine containing dipeptides (HCDs; carnosine, anserine and balenine) may facilitate pH regulation in response to these challenges, through their role as pH buffers. We measured the HCDs content of three tissues (liver, cardiac muscle and skeletal muscle) from the anoxia-tolerant painted turtle (Chrysemys picta bellii) acclimated to either 3 or 20°C. HCDs were detected in all tissues, with the highest content shown in the skeletal muscle. Turtles acclimated to 3°C had more HCD in their skeletal muscle than those acclimated to 20°C (carnosine = 20.8±4.5 vs 12.5±5.9 mmol·kg DM-1; ES = 1.59 (95%CI: 0.16 – 3.00), P = 0.013). The higher HCD content observed in the skeletal muscle of the cold-acclimated turtles suggests a role in acid-base regulation in response to physiological challenges associated with living in the cold, with the increase possibly related to the temperature sensitivity of carnosine’s dissociation constant and buffering power of the skeletal muscle during anoxic submergence. Highlights pH regulation is a major challenge for overwintering freshwater turtles. Histidine containing dipeptides are important intracellular buffers. Turtles acclimated to 3°C had higher HCD content than those at 20°C. HCDs may be important pH regulators in cold-acclimated turtles.

Oxygen deprivation swiftly damages tissues in most animals, yet some species show remarkable abilities to tolerate little or even no oxygen. Painted turtles exhibit a development-dependent tolerance that allows adults to survive anoxia ∼4x longer than hatchlings: adults survive ∼170 days and hatchlings survive ∼40 days at 3°C. We hypothesized this difference is related to development-dependent differences in ventricular gene expression. Using a comparative ontogenetic approach, we examined whole transcriptomic changes before, during, and five days after a 20-day bout of anoxic submergence at 3°C. Ontogeny accounted for more gene expression differences than treatment (anoxia or recovery): 1,175 vs. 237 genes, respectively. Of the 237 differences, 93 could confer protection against anoxia and reperfusion injury, 68 could be injurious, and 20 may be constitutively protective. Especially striking during anoxia was the expression pattern of all 76 annotated ribosomal protein (R-protein) mRNAs, which decreased in anoxia-tolerant adults, but increased in anoxia-sensitive hatchlings, suggesting adult-specific regulation of translational suppression. These genes, along with 60 others that decreased their levels in adults and either increased or remained unchanged in hatchlings, implicate antagonistic pleiotropy as a mechanism to resolve the long-standing question about why hatchling painted turtles overwinter in terrestrial nests, rather than emerge and overwinter in water during their first year. In sum, developmental differences in the transcriptome of the turtle ventricle revealed potentially protective mechanisms that contribute to extraordinary adult-specific anoxia tolerance, and provide a unique perspective on differences between the anoxia-induced molecular responses of anoxia-tolerant or anoxia-sensitive phenotypes within a species.

The involvement of mineralized tissues in acid-base homeostasis was likely important in the evolution of terrestrial vertebrates. Extant reptiles encounter hypercapnia when submerged in water, but early tetrapods may have experienced hypercapnia on land due to their inefficient mode of lung ventilation (likely buccal pumping, as in extant amphibians). Extant amphibians rely on cutaneous carbon dioxide elimination on land, but early tetrapods were considerably larger forms, with an unfavourable surface area to volume ratio for such activity, and evidence of a thick integument. Consequently, they would have been at risk of acidosis on land, while many of them retained internal gills and would not have had a problem eliminating carbon dioxide in water. In extant tetrapods, dermal bone can function to buffer the blood during acidosis by releasing calcium and magnesium carbonates. This review explores the possible mechanisms of acid-base regulation in tetrapod evolution, focusing on heavily armoured, basal tetrapods of the Permo-Carboniferous, especially the physiological challenges associated with the transition to air-breathing, body size and the adoption of active lifestyles. We also consider the possible functions of dermal armour in later tetrapods, such as Triassic archosaurs, inferring palaeophysiology from both fossil record evidence and phylogenetic patterns, and propose a new hypothesis relating the archosaurian origins of the four-chambered heart and high systemic blood pressures to the perfusion of the osteoderms. This article is part of the theme issue 'Vertebrate palaeophysiology'.

![Figure][1] ‘Gray hair is a crown of glory, which is won by a virtuous life’ (Proverbs 16:31). Donald C. Jackson (1937–2020) analyzing lactate concentration in the shell of anoxic (and edible) Burgundy snail ( Helix pomatia ) during a research visit to Aarhus University in the summer

The involvement of mineralized tissues in acid–base homeostasis was likely important in the evolution of terrestrial vertebrates. Extant reptiles encounter hypercapnia when submerged in water, but early tetrapods may have experienced hypercapnia on land due to their inefficient mode of lung ventilation (likely buccal pumping, as in extant amphibians). Extant amphibians rely on cutaneous carbon dioxide elimination on land, but early tetrapods were considerably larger forms, with an unfavourable surface area to volume ratio for such activity, and evidence of a thick integument. Consequently, they would have been at risk of acidosis on land, while many of them retained internal gills and would not have had a problem eliminating carbon dioxide in water. In extant tetrapods, dermal bone can function to buffer the blood during acidosis by releasing calcium and magnesium carbonates. This review explores the possible mechanisms of acid–base regulation in tetrapod evolution, focusing on heavily armoured, basal tetrapods of the Permo-Carboniferous, especially the physiological challenges associated with the transition to air-breathing, body size and the adoption of active lifestyles. We also consider the possible functions of dermal armour in later tetrapods, such as Triassic archosaurs, inferring palaeophysiology from both fossil record evidence and phylogenetic patterns, and propose a new hypothesis relating the archosaurian origins of the four-chambered heart and high systemic blood pressures to the perfusion of the osteoderms. This article is part of the theme issue ‘Vertebrate palaeophysiology’.

The painted turtle, Chrysemys picta, is the most anoxia‐tolerant tetrapod known, surviving over 170 days without oxygen at 3°C. A key component of C. picta's anoxia tolerance is metabolic depression: ATP use is dramatically reduced in the brain, in large part by suppression of nervous activity and channel arrest. This differs from mammals, which lack the ability to enter metabolic depression and withstand anoxia. Developing new tools to understand the neurobiology of the painted turtle has the potential to provide novel insight into the biology of anoxia‐tolerant vertebrates. While nearly all studies of anoxic turtle neuron function have been conducted in cortical sheets, the isolation and subsequent culture of primary neurons, to our knowledge, has not been conducted in this species. The goal of this study was to culture primary neurons from hatchling painted turtles and to develop a functional model for studying the neurobiology of anoxia tolerance. Primary neurons were dissociated from the cerebrocortex using enzymatic treatment and mechanical trituration and cultured in neurobasal medium supplemented with FBS, Penicillin/Streptomycin, B27, and L‐glutamine. Culturing the neurons on glass coverslips coated with laminin and poly‐D‐lysine at 37°C with 5% CO2 results in high neuronal survival and growth of neurites. Isolated neurons exhibit morphologies stereotypical of normal vertebrate neurons, with neuritic processes clearly identifiable. Importantly, the isolated cells showed functional responses characteristic of neurons, including calcium transients when excitatory factors, such as high concentration of KCl, were applied. This work represents the first in vitro primary culture of neurons from anoxia‐tolerant painted turtles that show normal vertebrate neuronal morphology and function.Support or Funding InformationThis work is supported by the NSF CAREER award 1253939 to Dr. Warren and the Saint Louis University Start‐Up Fund to Dr. Xu.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adsorbed and structurally incorporated carbonate in bioapatite, the primary mineral phase of bone, is observed across vertebrates, typically at 2-8 wt%, and supports critical physiological and biochemical functions. Several turtle species contain elevated bone-associated carbonate, a property linked to pH buffering and overwintering survival. Prior studies of turtle bone utilized bulk analyses, which do not provide spatial resolution of carbonate. Using Raman spectroscopy, the goals of this study were to: (1) quantify and spatially resolve carbonate heterogeneity within the turtle shell; (2) determine if cortical and trabecular bone contain distinct carbonate concentrations; and (3) assess if simulated overwintering conditions result in decreased bioapatite carbonation. Here, we demonstrate the potential for Raman spectroscopic analysis to spatially resolve bioapatite carbonation, using the western painted turtle as a model species. Carbonate concentration was highly variable within cortical and trabecular bone, based on calibrated Raman spot analyses and mapping, suggesting heterogeneous carbonate distribution among crystallites. Mean carbonate concentration did not significantly differ between cortical and trabecular bone, which indicates random distribution of crystallites with elevated and depleted carbonate. Carbonate concentrations (range: 5-22 wt%) were not significantly different in overwintering and control animals, deviating from previous bulk analyses. In reconciling bulk and Raman analyses, two hypotheses explain how overwintering turtles potentially access carbonate: (1) mobilization of mineral-associated, surface components of bone crystallites; and (2) selective, dispersed crystallite dissolution. Elevated bioapatite carbonate in the western painted turtle, averaging 11.8 wt%, represents the highest carbonation observed in vertebrates, and is one physiological trait that facilitates overwintering survival.

Ectothermic vertebrates living at extreme latitudes are often forced to endure extended periods with their body temperatures at or just above the freezing point of water. Cold temperatures impose a number of physiological and biochemical challenges directly related to the thermodynamic effects on biological matter, including metabolic enzyme kinetics and even the physical properties of blood. The painted turtle, Chrysemys picta, is a pond turtle that can experience temperatures just above freezing for 3–5 months as adults and can tolerate freezing or even subzero temperatures as hatchlings. In the present study, we characterized how cold acclimation to 3°C affected the ventricular transcriptome in both adult and hatchling turtles. Because hatchlings must tolerate subzero temperatures when they overwinter in their nest, we sought to determine if they might also exhibit a unique gene expression pattern related to possible cryoprotective mechanisms. Despite differences in life history, adults and hatchlings responded similarly to cold acclimation at the transcriptomic level; out of the 1304 genes that were differentially expressed due to temperature, 1272 showed similar expression patterns in adults and hatchlings, the majority of which were upregulated. In adults and hatchlings combined, 776 genes were upregulated at 3°C, 550 genes were downregulated, of which 22 fell into both categories due to development‐specific expression patterns. Of these upregulated genes, 51 encoded for transcription factors, 27 for histone modification proteins, and 26 for mRNA processing and translational regulation enzymes. These results suggest that the ventricle actively remodels chromatin structure and modulates both gene expression and mRNA processing in response to cold acclimation.Support or Funding InformationThis work was supported by NSF CAREER grant 1253939 awarded to DEWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myoglobins (Mb) are ubiquitous proteins found in striated muscle of nearly all vertebrate taxa. Although their function is most commonly associated with facilitating oxygen storage and diffusion, Mb has also been implicated in cellular antioxidant defense. The oxidized (Fe3+) form of Mb (metMB) can react with hydrogen peroxide (H2O2) to produce ferrylMb. FerrylMb can be reduced back to metMb for another round of reaction with H2O2. In the present study, we have shown that horse skeletal muscle Mb displays peroxidase activity using 2,2′-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, as well as the biologically-relevant substrates NADH/NADPH, ascorbate, caffeic acid, and resveratrol. We have also shown that ferrylMb can be reduced by both ethanol and acetaldehyde, which are known to accumulate in some vertebrate tissues under anaerobic conditions, such as anoxic goldfish and crucian carp, implying a potential mechanism for ethanol detoxification in striated muscle. We found that metMb peroxidase activity is pH-dependent, increasing as pH decreases from 7.4 to 6.1, which is biologically relevant to anaerobic vertebrate muscle when incurring intracellular lactic acidosis. Finally, we found that metMb reacts with hypochlorite in a heme-dependent fashion, indicating that Mb could play a role in hypochlorite detoxification. Taken together, these data suggest that Mb peroxidase activity might be an important antioxidant mechanism in vertebrate cardiac and skeletal muscle under a variety of physiological conditions, such as those that might occur in contracting skeletal muscle or during hypoxia.

Western painted turtles (Chrysemys picta bellii) tolerate anoxic submergence longer than any other tetrapod, surviving more than 170 days at 3°C. This ability is due, in part, to the shell and skeleton simultaneously releasing calcium and magnesium carbonates, and sequestering lactate and H+ to prevent lethal decreases in body fluid pH. We evaluated the effects of anoxic submergence at 3°C on various material properties of painted turtle bone after 60, 130, and 167-170 days, and compared them to normoxic turtles held at the same temperature for the same time periods. To assess changes in the mechanical properties, beams (4×25 mm) were milled from the plastron and broken in a three-point flexural test. Bone mineral density, CO2 concentration (a measure of total bone HCO3−/CO32-), and elemental composition were measured using microCT, HCO3−/CO32- titration, and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Tissue mineral density of the sampled bone beams were not significantly altered by 167-170 days of aquatic overwintering in anoxic or normoxic water, but bone CO2 and Mg were depleted in anoxic compared normoxic turtles. At this time point, the plastron beams from anoxic turtles yielded at stresses that were significantly smaller and strains significantly greater than the plastron beams of normoxic turtles. When data from anoxic and normoxic turtles were pooled, plastron beams had a diminished elastic modulus after 167-170 days compared to control turtles sampled on Day 1, indicating an effect of prolonged housing of the turtles in 3°C water without access to basking sites. There were no changes in the mechanical properties of the plastron beams at any of the earlier time points in either group. We conclude that anoxic hibernation can weaken the painted turtle's plastron, but likely only after durations that exceed what it might naturally experience. The duration of aquatic overwintering, regardless of oxygenation state, is likely to be an important factor determining the mechanical properties of the turtle shell during spring emergence.

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus, the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark (Hemiscyllium ocellatum), crucian carp (Carassius carassius), western painted turtle (Chrysemys picta bellii), and leopard frog (Rana pipiens) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia. Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia. Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.

Adult Western painted turtles, Chrysemys picta bellii, are the most anoxia‐tolerant tetrapod known and is capable of surviving over 170 days of anoxia at 3°C. Hatchling turtles, however can survive only 41 days at 4°C. To investigate the transcriptomic basis for this difference, we compared the anoxia‐induced transcriptional responses in the ventricle of adult and hatchling painted turtles exposed to four different conditions: 20 °C acclimation, five weeks at 3 °C, 20 days of anoxia at 3 °C, and re‐exposure to oxygen at 3 °C. At each time point, adult and hatchling turtles were sampled and RNA extractions were performed on the frozen, homogenized ventricular tissue. Illumina HiSeq 4000 was used to sequence mRNA, and transcript expression levels were compared between treatments to determine the changes attributed to temperature, anoxia exposure and development.Support or Funding InformationThis work was supported by a CAREER award from the National Science Foundation.

We aimed to determine how increases in intracellular H+ and inorganic phosphate (P-i) to levels observed during anoxic submergence affect contractility in ventricular muscle of the anoxia-tolerant Western painted turtle, Chrysemys picta bellii. Skinned multicellular preparations were exposed to six treatments with physiologically relevant levels of pH (7.4, 7.0, 6.6) and P-i (3 and 8 mmol l(-1)). Each preparation was tested in a range of calcium concentrations (pCa 9.0-4.5) to determine the pCa-tension relationship for each treatment. Acidosis significantly decreased contractility by decreasing Ca2+ sensitivity (pCa(50)) and tension development (P<0.001). Increasing [P-i] also decreased contractility by decreasing tension development at every pH level (P<0.001) but, alone, did not affect Ca2+ sensitivity (P=0.689). Simultaneous increases in [H+] and [P-i] interacted to attenuate the decreased tension development and Ca2+ sensitivity (P<0.001), possibly reflecting a decreased sensitivity to P-i when it is present as the dihydrogen phosphate form, which increases as pH decreases. Compared with that of mammals, the ventricle of turtles exhibits higher Ca2+ sensitivity, which is consistent with previous studies of ectothermic vertebrates.

The painted turtle is known for its extreme tolerance to anoxia, but it is unknown whether previous experience with anoxic stress might alter physiological performance during or following a test bout of anoxia. Repeatedly subjecting 25°C-acclimated painted turtles to 2h of anoxic stress every other day for 19days (10 submergence bouts total) caused resting levels of liver glycogen to decrease by 17% and liver citrate synthase (CS) and cytochrome oxidase (COX) activities to increase by 33% and 112%, respectively. When the repeatedly submerged turtles were studied during a subsequent anoxic stress test, liver COX and CS activities decreased during anoxia to the same levels of naïve turtles, which were unchanged, and remained there throughout metabolic recovery. There were no effects of the repeated anoxia treatment on any of the other measured variables, which included lactate dehydrogenase and phosphofructokinase activities in liver, skeletal muscle, and ventricle, blood acid-base status, hemoglobin, hematocrit and plasma ion (Na, K, Ca, Mg, Cl) and metabolite concentrations (lactate, glucose, free-fatty acids), before, during, or after the anoxic stress test. We conclude that although painted turtles can show a physiological reaction to repeated anoxic stress, the changes appear to have no measurable effect on anaerobic physiological performance or ability to recover from anoxia.