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Abstract BackgroundEnvironmental 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. ResultsGenome-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 asSCN5Amay account for differences in heart rate, while genes such asTNNT2andTPM3may 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. ConclusionsOur 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. 

Temperature during embryogenesis determines sex and has been shown to influence other physiological traits in reptiles. The common snapping turtle (Chelydra serpentina) is an ideal model for testing how temperature impacts behavior in species that display temperature-dependent sex determination. Behavioral assays are crucial to understanding how a changing climate may affect whole organism function in the snapping turtle. Currently, there are few behavioral assays for semi-aquatic vertebrates like turtles. In this study, we used digital cameras to record behavior of fed and fasted hatchling turtles from different incubation temperatures in an open field setting for 20 minutes in 2018 and repeated the experiment in 2019. Open fields were circular tanks filled with water to a depth of 3.5 cm. Each field was split into four quadrants and two zones (inner and outer). The amount of time turtles spent actively moving, total distance travelled, and several other measures were collected and summarized automatically from videos with open source image analysis software (ImageJ). Satiety and incubation temperature had significant effects on total distance moved, time spent moving, and time moving in the outer zone. These findings indicate that temperature during embryogenesis has a long-lasting effect on neural mechanisms underlying exploratory or general locomotor behavior in turtles.