Chrysemys Research Articles

Sub-zero microRNA expression in the liver of the frozen hatchling painted turtle, Chrysemys picta marginata.

The Midland painted turtle (Chrysemys picta marginata) are the highest known vertebrate species to experience and survive freezing and sub-zero temperatures. Painted turtles typically hatch from their eggs in the fall and remain underground in their nests until the following spring. While in these nests over the winter, hatchling turtles withstand over 50 % of their total extracellular body water freezing. Herein, the expression of microRNAs (miRNAs) was investigated in response to freezing stress in the hatchling painted turtle liver. A total of 204 known miRNAs were identified to be expressed in turtles, with 17 being upregulated and 13 being downregulated during freezing. KEGG and GO analyses suggested that upregulated miRNAs inhibit genes of cell cycle and Focal adhesion and Adherens junction, suggesting their role in downregulation of central metabolic processes necessary for metabolic rate depression (MRD) and maintaining the tissue homeostasis. Only 9 of the 36 enriched KEGG pathways were less targeted by miRNAs during freezing, including linoleic acid metabolism and multiple signaling pathways. These predicted upregulated pathways likely promote homeoviscous adaptation and expression of pro-survival/protective proteins for metabolic adaptations necessary for defence of liver during MRD. Overall, miRNA-seq analysis of liver revealed a strong role of miRNA in the adaptive strategy that not only enables hatchlings to substantially suppress their nonessential energy needs but also makes them flexible enough to restore and protect their basal organ functions by activating pro-survival processes.

Demography of a painted turtle intergrade (Chrysemys picta picta X C. p. marginata) population from an altered wetland

The demography of a painted turtle Chrysemys picta picta X C. p. marginata population from a eutrophic habitat was examined at a wetland site in south-central Pennsylvania (USA) during 2011–2019. Males reached sexual maturity at 90 mm carapace length (CL) in half the time taken, but at the same size, as painted turtles studied elsewhere in the north-eastern portion of the United States. Females matured at 130 mm CL at our site, which was larger and began at an earlier age than conspecifics. Our data corroborate findings of faster growth in C. picta juveniles resulting in earlier maturity at body sizes equal to or larger than slower growing juveniles. Our results also conform to previous findings linking wetlands altered by added nutrient input to increased growth patterns of their resident painted turtle population. Rapid growth rates for aquatic turtles are likely to become more common globally as urbanisation continues to expand and alter wetland habitats.

A Cautionary Tale of Sexing by Methylation: Hybrid Bisulfite-Conversion Sequencing of Immunoprecipitated Methylated DNA in Chrysemys picta Turtles with Temperature-Dependent Sex Determination Reveals Contrasting Patterns of Somatic and Gonadal Methylation, but No Unobtrusive Sex Diagnostic.

Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue.

Developing a classification system to assign activity states to two species of freshwater turtles.

Research in ecology often requires robust assessment of animal behaviour, but classifying behavioural patterns in free-ranging animals and in natural environments can be especially challenging. New miniaturised bio-logging devices such as accelerometers are increasingly available to record animal behaviour remotely, and thereby address the gap in knowledge related to behaviour of free-ranging animals. However, validation of these data is rarely conducted and classification model transferability across closely-related species is often not tested. Here, we validated accelerometer and water sensor data to classify activity states in two free-ranging freshwater turtle species (Blanding's turtle, Emydoidea blandingii, and Painted turtle, Chrysemys picta). First, using only accelerometer data, we developed a decision tree to separate motion from motionless states, and second, we included water sensor data to classify the animal as being motionless or in-motion on land or in water. We found that accelerometers separated in-motion from motionless behaviour with > 83% accuracy, whereas models also including water sensor data predicted states in terrestrial and aquatic locations with > 77% accuracy. Despite differences in values separating activity states between the two species, we found high model transferability allowing cross-species application of classification models. Note that reducing sampling frequency did not affect predictive accuracy of our models up to a sampling frequency of 0.0625 Hz. We conclude that the use of accelerometers in animal research is promising, but requires prior data validation and development of robust classification models, and whenever possible cross-species assessment should be conducted to establish model generalisability.

A new species of Chrysemys (Emydidae: Deirochelyinae) from the latest Miocene-Early Pliocene of Tennessee, USA and its implications for the evolution of painted turtles

Abstract Chrysemys, commonly known as painted turtles, have the largest native biogeographic range of all North American turtles. The presence of a new species, Chrysemys corniculata sp. nov., in the Late Hemphillian-Early Blancan North American Land Mammal Age (latest Miocene-Early Pliocene) of Tennessee provides further data on the evolution of Chrysemys, deirochelyines and emydids. The new fossil species lies basally in Deirochelyinae and suggests that either Chrysemys represents a basal deirochelyine morphology and is one of the oldest genera in the family, or that similar basal morphologies have evolved multiple times throughout deirochelyine evolution. Its occurrence at the same time as Chrysemys picta, during the Hemphillian-Early Blancan, a time of high biodiversity in emydid turtles, suggests either multiple species of Chrysemys during the Late Hemphillian-Early Blancan (at least one in the mid-west and one farther east), or multiple lineages with basal morphologies during this time. Early fossil deirochelyines occur after the greenhouse conditions of the Eocene and the Mid-Miocene Climatic Optimum. Vicariance led to deirochelyines becoming more speciose, including the occurrence of C. corniculata, after the Mid-Miocene Climatic Optimum, potentially suggesting cooler temperatures aided in the evolution of the subfamily and their speciation during the Hemphillian and into the Early Blancan.

Protective down-regulated states in the human brain: A possible lesson from COVID-19

The COVID-19 pandemic has created a large population of patients who are slow to recover consciousness following mechanical ventilation and sedation in the intensive care unit. Few clinical scenarios are comparable. Possible exceptions are the rare patients in post-cardiac arrest coma with minimal to no structural brain injuries who recovered cognitive and motor functions after prolonged delays. A common electroencephalogram (EEG) signature seen in these patients is burst suppression [8]. Biophysical modeling has shown that burst suppression is likely a signature of a neurometabolic state that preserves basic cellular function "during states of lowered energy availability." These states likely act as a brain protective mechanism [9]. Similar EEG patterns are observed in the anoxia resistant painted turtle [24]. We present a conceptual analysis to interpret the brain state of COVID-19 patients suffering prolonged recovery of consciousness. We begin with the Ching model and integrate findings from other clinical scenarios and studies of the anoxia-tolerant physiology of the painted turtle. We postulate that prolonged recovery of consciousness in COVID-19 patients could reflect the effects of modest hypoxic injury to neurons and the unmasking of latent neuroprotective mechanisms in the human brain. This putative protective down-regulated state appears similar to that observed in the painted turtle and suggests new approaches to enhancing coma recovery [12].

Evidence of successful hatching by introduced Red-eared Slider (<i>Trachemys scripta elegans</i>) in British Columbia, Canada

Globally, competition and disease from introduced Red-eared Slider (Trachemys scripta elegans) is a threat to co-existing native turtles. Red-eared Slider has been introduced throughout south coastal British Columbia (BC), mainly as pet turtle releases. Urban centres receive the most individuals, particularly in the Lower Mainland area outlying Vancouver, on southern Vancouver Island, and on the Sunshine Coast. The range of Red-eared Sliders in BC overlaps that of the Threatened Pacific Coast population of Western Painted Turtle (Chrysemys picta bellii). Herein we report on a survey for both species, noting presence, assessed population sizes, and nesting activity. Across 19 sites in the south coast occupied by both turtle species, we found the median abundance of Red-eared Sliders to be 2.5 times larger than that of Western Painted Turtles (Mann–Whitney U = 104, n1 = n2 = 19, Z-Score = −2.2188, P = 0.02642, two-tailed). There had been no evidence of Red-eared Sliders successfully hatching in the wild in BC until our study. We observed complete development, with 19 neonates from three different nesting sites between 2015 and 2017. Thus, Red-eared Slider is indeed established and able to breed in BC and thus competition and disease introduction from the species likely contributes to the decline of the Pacific Coast population of Western Painted Turtle, particularly at sites with low painted turtle numbers. The scale and mechanisms of impact requires further investigation.

Meiotic chromosome dynamics and double strand break formation in reptiles.

During meiotic prophase I, tightly regulated processes take place, from pairing and synapsis of homologous chromosomes to recombination, which are essential for the generation of genetically variable haploid gametes. These processes have canonical meiotic features conserved across different phylogenetic groups. However, the dynamics of meiotic prophase I in non-mammalian vertebrates are poorly known. Here, we compare four species from Sauropsida to understand the regulation of meiotic prophase I in reptiles: the Australian central bearded dragon (Pogona vitticeps), two geckos (Paroedura picta and Coleonyx variegatus) and the painted turtle (Chrysemys picta). We first performed a histological characterization of the spermatogenesis process in both the bearded dragon and the painted turtle. We then analyzed prophase I dynamics, including chromosome pairing, synapsis and the formation of double strand breaks (DSBs). We show that meiosis progression is highly conserved in reptiles with telomeres clustering forming the bouquet, which we propose promotes homologous pairing and synapsis, along with facilitating the early pairing of micro-chromosomes during prophase I (i.e., early zygotene). Moreover, we detected low levels of meiotic DSB formation in all taxa. Our results provide new insights into reptile meiosis.

Complete mitochondrial genome of the river cooter (Pseudemys concinna, Testudines: Emydidae) in Korea

The complete mitochondrial genome of Pseudemys concinna in Korea was sequenced and characterized. The mitochondrial genome is constituted of 37 genes (13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes) and a noncoding region. Phylogenetic analysis based on the complete mitochondrial genome showed that P. concinna has closer relationship with Chrysemys picta than Trachemys scripta elegans. This is the first case for complete mitochondrial genome from P. concinna in Korea, which will provide information for biogeographical studies and management plan for invasive species.

Silky dogwood (Swida amomum) seed dispersal by freshwater turtles.

Effective seed dispersal is essential to the success of plant species. Swida amomum (silky dogwood) has a seed-dispersal syndrome characteristic of autumn-ripening shrubs with fleshy fruits; attached fruits are ingested and defecated by birds, while fallen fruits are consumed by ground-foraging birds and mammals. We documented that fallen fruits of this shrub were consumed by two aquatic turtle species (eastern painted turtle [Chrysemys picta] and red-eared slider [Trachemys scripta]) and that their seeds were defecated. We compared germination success (percentage of seeds germinated) of defecated seeds, seeds collected from a pond surface, and seeds removed from shrubs. While four seed species were identified in fecal samples, seeds of S. amomum were the most frequent (93%) among samples and the most numerous (106 seeds) in any sample. Average proportion of fecal seeds germinated (85.99%) exceeded that of seeds from the pond surface (82.76%) and from shrubs (60.24%), albeit the difference in germination success was insignificant. When analyzed using fecal samples from painted turtles only, the difference in germination success between fecal seeds and those collected from pond or shrub became significant. Our findings represent the first report of S. amomum seeds being dispersed by turtle gut passage and suggest aquatic turtles could be an important part of a secondary seed dispersal process influencing woody plant community composition in temperate wetland ecosystems.

Developmental asynchrony might not reduce fitness in early life in painted turtles.

Synchronous hatching and emergence of turtles from nests may be adaptive in predator avoidance during dispersal. However, little is known about the phenotypic consequences of such synchrony or the generality of predator avoidance in driving the evolution of this trait. Colbert et al. (2010) found that less advanced embryos hatched early in the presence of more advanced sibs, sustaining a persistent reduction in neuromuscular function. In this study, we experimentally assessed the influence of such accelerated embryonic development on hatching success, winter survival, and survival during terrestrial dispersal from the nest. Although we predicted that shortened incubation periods would reduce survival, early-hatching individuals suffered no detectable fitness costs at any stage considered in this study. Incubation temperature did not affect hatching success, and offspring sex did not affect survival across treatment groups. Incubation regime influenced offspring body size and was negatively correlated with dispersal time, however, there was no effect on survival during winter or terrestrial dispersal. Lack of a detectable fitness cost in these key early-life stages associated with hatching synchrony is consistent with a single, predator avoidance origin for this trait and retention in C. picta and other derived turtles via phylogenetic inertia.

Spatial Capture–Recapture Derived Turtle Capture Probabilities and Densities in the Chesapeake and Ohio Canal

More than half of turtle species worldwide are threatened because of habitat loss, invasive species, environmental pollution, disease, unsustainable use, and global climate change. However, some turtles are capable of existing in highly modified habitats, including structures designed to benefit human populations such as reservoirs and canal systems. Examining turtle distributions in large canal systems can inform conservation plans protecting turtle populations within a potential reservoir network and expand our understanding of underlying mechanisms regulating populations. We conducted spatial capture–recapture on turtles inhabiting sections of the Chesapeake and Ohio Canal. We developed a Bayesian spatial capture–recapture model to estimate densities, sex ratios, and associated capture probability parameters for Chrysemys picta (Painted Turtle), Chelydra serpentina (Common Snapping Turtle), Sternotherus odoratus (Eastern Musk Turtle), and Pseudemys rubriventris (Red-Bellied Turtle) captured at 12 sites along 28 km of the canal. We examined the impact of canal depth and forest cover on population densities and the variation in capture probability between sites and sampling days. We found population densities to vary between sites and the associated sex ratios to vary between species, as did the effect of depth and forest cover. Overall capture rates decreased each day, but there was trap-happy behavior from all species except S. odoratus. Our information can set a baseline for understanding turtle populations and inform management in the Chesapeake and Ohio Canal National Historical Park. It is also one of the first studies to establish methods for using new spatial capture–recapture to quantify densities and aquatic space use of turtles.

Complete mitochondrial genome of the peninsula cooter (Pseudemys peninsularis, Testudines: Emydidae) in Korea

The complete mitochondrial genome of Pseudemys peninsularis in Korea was sequenced and characterized. The mitochondrial genome is constituted of 37 genes (13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes) and a noncoding region. Phylogenetic analysis based on the 13 protein-coding gene sequences showed that P. peninsularis has closer relationship with Chrysemys picta than Trachemys scripta elegans. This is the first case for a complete mitochondrial genome from P. peninsularis, which will provide information for biogeographical studies and management plans for invasive species.

Cadmium Ecotoxic Effects on Embryonic Dmrt1 and Aromatase Expression in Chrysemys picta Turtles May Implicate Changes in DNA Methylation

Temperature-dependent sex determination (TSD) decides the sex fate of an individual based on incubation temperature. However, other environmental factors, such as pollutants, could derail TSD sexual development. Cadmium is one such contaminant of soils and water bodies known to affect DNA methylation, an epigenetic DNA modification with a key role in sexual development of TSD vertebrate embryos. Yet, whether cadmium alters DNA methylation of genes underlying gonadal formation in turtles remains unknown. Here, we investigated the effects of cadmium on the expression of two gene regulators of TSD in the painted turtle, Chrysemys picta, incubated at male-producing and female-producing temperatures using qPCR. Results revealed that cadmium alters transcription of Dmrt1 and aromatase, overriding the normal thermal effects during embryogenesis, which could potentially disrupt the sexual development of TSD turtles. Results from a preliminary DNA methylation-sensitive PCR assay implicate changes in DNA methylation of Dmrt1 as a potential cause that requires further testing (aromatase methylation assays were precluded).

Review: A history and perspective of mitochondria in the context of anoxia tolerance.

Symbiosis is found throughout nature, but perhaps nowhere is it more fundamental than mitochondria in all eukaryotes. Since mitochondria were discovered and mechanisms of oxygen reduction characterized, an understanding gradually emerged that these organelles were involved not just in the combustion of oxygen, but also in the sensing of oxygen. While multiple hypotheses exist to explain the mitochondrial involvement in oxygen sensing, key elements are developing that include potassium channels and reactive oxygen species. To understand how mitochondria contribute to oxygen sensing, it is informative to study a model system which is naturally adapted to survive extended periods without oxygen. Amongst air-breathing vertebrates, the most highly adapted are western painted turtles (Chrysemys picta bellii), which overwinter in ice-covered and anoxic water bodies. Through research of this animal, it was postulated that metabolic rate depression is key to anoxic survival and that mitochondrial regulation is a key aspect. When faced with anoxia, excitatory neurotransmitter receptors in turtle brain are inhibited through mitochondrial calcium release, termed "channel arrest". Simultaneously, inhibitory GABAergic signalling contributes to the "synaptic arrest" of excitatory action potential firing through a pathway dependent on mitochondrial depression of ROS generation. While many pathways are implicated in mitochondrial oxygen sensing in turtles, such as those of adenosine, ATP turnover, and gaseous transmitters, an apparent point of intersection is the mitochondria. In this review we will explore how an organelle that was critical for organismal complexity in an oxygenated world has also become a potentially important oxygen sensor.

Apparent winterkill of Painted Turtle (<i>Chrysemys picta</i>)

Around the margin of an artificial pond in Ottawa, Ontario, we found 25 Painted Turtles (Chrysemys picta) that appeared to have died over the course of two winters (17 during the first winter and eight during the second). We examined meteorological data to try to determine the cause of the mortality. Summer and fall rains were only slightly below normal in both years, suggesting water levels should have been close to normal. The winter air temperature was warmer than normal and winter snowfall was slightly above normal in both years. Unseasonable weather does not appear to be responsible for the winter mortality and the pond’s maximum depth of 1.7 m should prevent freezing to the bottom. It is possible that the artificial nature of the pond creates suboptimal overwintering habitat, rendering the site an ecological trap; however, there is no direct evidence to support this theory. It is also possible that winter mortality of turtles is widespread at temperate wetlands, but that dead turtles were more detectable at this site because of the bare shoreline around the pond. Winter mass mortality events, if common, may represent an additional threat to turtle populations, which are declining from various anthropogenic threats.

Aural Abscesses in Wild Midland Painted Turtles (Chrysemys picta marginata) Admitted to a Wildlife Hospital in Ontario, Canada, 2011–20

Between 2011 and 2020, the Ontario Turtle Conservation Centre's veterinary hospital admitted 6,335 turtles of eight different species, with 3,246 being midland painted turtles (Chrysemys picta marginata). At the time of admission from the wild, aural abscesses were present in 26 of the painted turtles (0.8%). Aural abscesses were never observed in juvenile turtles and were not found in any other species of turtle admitted to the hospital. An equal number of males and females were affected. All cases showed signs of chronicity. Abscesses alone did not appear to affect the overall health of turtles regardless of clinical signs. All turtles with abscesses as the sole cause for admission were released after successful treatment.

EVALUATION OF PROGNOSTIC INDICATORS FOR INJURED TURTLES PRESENTING TO A WILDLIFE CLINIC.

The Turtle Rescue Team (TRT) at North Carolina State University College of Veterinary Medicine currently sees over 500 chelonian cases a year. Each turtle receives a thorough physical examination as the main evaluation of its health because further diagnostics are cost prohibitive in a donation-based facility. Thus, there is a need to further understand how a few economical diagnostics can evaluate the prognosis of chelonians. The objectives were to evaluate lactate, PCV, total solids, glucose, temperature, and HR as prognostic indicators in multiple chelonian species and to determine how the change in lactate concentrations over a 24 h period affects prognosis. Eastern box turtles (Terrapene carolina carolina), yellow-bellied sliders (Trachemys scripta scripta), river cooters (Pseudemys concinna), painted turtles (Chrysemys picta), and common snapping turtles (Chelydra serpentina) were evaluated because of prevalence of presentation to the TRT, differences in habitat, and differences in physiology. Hyperlactatemia, an increased lactate over 24 h when the initial lactate concentration was greater than 5 mM/L, and anemia were determined to be significant prognostic indicators of mortality for all turtles, and hypoglycemia was a significant prognostic indicator of mortality for male turtles (P < 0.05).

Cardiovascular contributions and energetic costs of thermoregulation in ectothermic vertebrates.

Ectothermic vertebrates use a suite of physiological and behavioral mechanisms to thermoregulate, which result in various thermoregulatory strategies from thermoconformity to thermoregulation. Here, we present a novel synthesis of theoretical and empirical methods to determine cardiovascular contributions to heat transfer in free-living ectothermic vertebrates. We start by identifying the fundamental components of heat transfer and the cardiovascular mechanisms for physiological modulation of heat exchange, and then integrate these components into a single, integrative framework: the cardiovascular heat exchange framework (CHEF). We demonstrate that this framework can identify details of the thermoregulatory strategy in two turtle species, most notably the preponderance of instances where turtles use physiological mechanisms to avoid overheating, suggesting vulnerability to climate change. As modulated physiological contributions to heat flow incur a greater energy demand than relying on unmodulated passive heat transfer, we then asked whether we could characterize the energetic costs of thermoregulation. We measured field metabolic rate (FMR) in free-living turtles and used the CHEF to determine FMR while actively or passively thermoregulating. Comparing an individual's actual FMR to the rate calculated assuming absence of thermoregulation revealed that painted turtles, a partial thermoregulator, elevate their daily energy expenditure (DEE) by about 25%, while box turtles, a thermoconformer, have a DEE that is nearly unchanged as a result of thermoregulation. This integrative framework builds a new paradigm that provides a mechanism to explain correlations between energy demand and thermoregulatory strategy, quantifies the energetic costs of thermoregulation, and identifies the role of cardiovascular contributions to thermoregulation in free-living animals.

A Genomic Perspective on the Evolutionary Diversification of Turtles.

To examine phylogenetic heterogeneity in turtle evolution, we collected thousands of high-confidence single-copy orthologs from 19 genome assemblies representative of extant turtle diversity and estimated a phylogeny with multispecies coalescent and concatenated partitioned methods. We also collected next-generation sequences from 26 turtle species and assembled millions of biallelic markers to reconstruct phylogenies based on annotated regions from the western painted turtle (Chrysemys picta bellii) genome (coding regions, introns, untranslated regions, intergenic, and others). We then measured gene tree-species tree discordance, as well as gene and site heterogeneity at each node in the inferred trees, and tested for temporal patterns in phylogenomic conflict across turtle evolution. We found strong and consistent support for all bifurcations in the inferred turtle species phylogenies. However, a number of genes, sites, and genomic features supported alternate relationships between turtle taxa. Our results suggest that gene tree-species tree discordance in these data sets is likely driven by population-level processes such as incomplete lineage sorting. We found very little effect of substitutional saturation on species tree topologies, and no clear phylogenetic patterns in codon usage bias and compositional heterogeneity. There was no correlation between gene and site concordance, node age, and DNA substitution rate across most annotated genomic regions. Our study demonstrates that heterogeneity is to be expected even in well-resolved clades such as turtles, and that future phylogenomic studies should aim to sample as much of the genome as possible in order to obtain accurate phylogenies for assessing conservation priorities in turtles. [Discordance; genomes; phylogeny; turtles.].