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.

Body size of ectotherms constrains thermal requirements for reproductive activity in seasonal environments

Body size may influence ectotherm behaviour by influencing heating and cooling rates, thereby constraining the time of day that some individuals can be active. The time of day at which turtles nest, for instance, is hypothesized to vary with body size at both inter- and intra-specific levels because large individuals have greater thermal inertia, retaining preferred body temperatures for a longer period of time. We use decades of data on thousands of individual nests from Algonquin Park, Ontario, Canada, to explore how body size is associated with nesting behaviour in Painted Turtles (Chrysemys picta (Schneider, 1783); small bodied) and Snapping Turtles (Chelydra serpentina (Linnaeus, 1758); large bodied). We found that (i) between species, Painted Turtles nest earlier in the evening and at higher mean temperatures than Snapping Turtles, and (ii) within species, relatively large individuals of both species nest at cooler temperatures and that relatively larger Painted Turtles nest later in the evening compared with smaller Painted Turtles. Our data support the thermal inertia hypothesis and may help explain why turtles in general exhibit geographic clines in body size: northern environments experience more daily variation in temperature, and larger size may evolve, in part, for retention of preferred body temperature during terrestrial forays.

Wait and snap: eastern snapping turtles ( Chelydra serpentina ) prey on migratory fish at road-stream crossing culverts

There is growing evidence that culverts at road-stream crossings can increase fish density by reducing stream width and fish movement rates, making these passageways ideal predator ambush locations. In this study, we used a combination of videography and δ 13 C stable isotope analyses to investigate predator–prey interactions at a road-stream crossing culvert. Eastern snapping turtles ( Chelydra serpentina ) were found to regularly reside within the culvert to ambush migratory river herring ( Alosa spp.). Resident fish species displayed avoidance of the snapping turtles, resulting in zero attempted attacks on these fish. In contrast, river herring did not display avoidance and were attacked by a snapping turtle on 79% of approaches with a 15% capture rate. Stable isotope analyses identified an apparent shift in turtle diet to consumption of river herring in turtles from culvert sites that was not observed in individuals from non-culvert sites. These findings suggest that anthropogenic barriers like culverts that are designed to allow passage may create predation opportunities by serving as a bottleneck to resident and migrant fish movement.

A Trapping Survey Targeting Head-Started Alligator Snapping Turtles in Southwest Louisiana

The alligator snapping turtle Macrochelys temminckii is the largest freshwater turtle in North America and humans seek it as a food source, primarily in Louisiana. Scientists point to decades of intensive commercial harvest of alligator snapping turtles as a cause of population declines. The Louisiana Department of Wildlife and Fisheries initiated a head-start program for alligator snapping turtles and released 53 head-started juveniles at seven sites along an approximately 5.7-km stretch of Bundick Creek in southwest Louisiana between November 2015 and October 2016. Before release, department personnel measured, weighed, and marked all alligator snapping turtles with both an internal passive integrated transponder tag and a numbered external tag. In 2018, the U.S. Geological Survey initiated a turtle trapping survey at those seven release sites targeting the head-started alligator snapping turtles. In 1 wk of trapping effort at each site, we recorded 69 turtle captures comprising seven species, including 15 alligator snapping turtles (representing 12 individuals). Of those 12 individuals, 8 were head-started juveniles and 4 were native to the creek. A landowner captured an additional head-started juvenile alligator snapping turtle during our trapping and we took measurements before its release. A minimum of 17% of head-started alligator snapping turtles survived since release, and we trapped most captured head-started individuals near their release site; the captured individuals exhibited growth consistent with other studies, indicating acclimatization to their new environment. Three head-started alligator snapping turtles had their external tags entangled in the net mesh, and two of these turtles drowned. An additional two head-started individuals lost their external tags in the natural environment prior to their capture in this study. The Louisiana Department of Wildlife and Fisheries discontinued the use of external tags based on our findings, as these tags were detrimental to the health of head-started turtles.