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Floating aquatic vegetation and algal blooms are increasing with global warming, potentially reducing UVB exposure and, consequently, vitamin D (vit-D) synthesis in freshwater turtles. Vit-D mediates calcium metabolism and overall health, yet the effects of floating aquatic vegetation on vit-D levels remain unclear, as is whether turtles actively avoid habitats with abundant floating vegetation. Here, we address these questions by quantifying vit-D3 levels in the blood of adult female painted turtles (Chrysemys picta) exposed to high-vegetation (darker/colder) or clear-water (lighter/warmer) treatments for one month outdoors and one month indoors at a single temperature during late summer and early fall. The observed circulating vit-D3 levels resembled those reported for other freshwater turtles, declined over time in both treatments, and were marginally lower under high vegetation after 60 days compared to clear water. However, this difference disappeared after correcting for lymph contamination and multiple comparisons, suggesting that perhaps adult females are robust to the effect of floating vegetation, but whether they were buffered by vit-D3 stores in lipids is unclear. Additionally, in subsequent years, females were exposed to habitat choice experiments and exhibited a strong preference for high floating vegetation over clear water, both as a group (outdoors) and individually (outdoors, and indoors at 21 °C and 26 °C), consistent with known benefits conferred by floating vegetation (food, predator avoidance). While no ill effects of high vegetation nor behavioral avoidance were detected here, longer experiments at different seasons on both sexes and varying ages are warranted before concluding whether painted turtles are truly resilient in their vit-D levels or if, instead, a tradeoff exists between the known benefits of floating vegetation and potential [yet unidentified] detrimental effects (lower dissolved oxygen or vit-D) when vegetation is overgrown for extended periods. 

Abstract: Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research. 

Maternal hormones such as estrogens deposited into the yolk of turtle eggs follow circulating levels in adult females, and they may alter the sexual fate of developing embryos in species with temperature-dependent sex determination (TSD). In temperate regions, this deposition occurs during the spring when estrogens increase in adult females as ambient temperatures rise, drop after the first clutch, and peak again (albeit less) in the fall. Global warming alters turtle nesting phenology (inducing earlier nesting), but whether it affects circulating hormones remains unknown, hindering our understanding of all potential challenges posed by climate change and the adaptive potential (or lack thereof) of turtle populations. Here, we addressed this question in painted turtles (Chrysemys picta) by quantifying estradiol, estrone, and testosterone via mass spectrometry in the blood of wild adult females exposed to 26 °C and 21 °C in captivity between mid-August and mid-October (15 females per treatment). Results from ANOVA and pairwise comparisons revealed no differences between treatments in circulating hormones measured at days 0, 2, 7, 14, 28, and 56 of the experiment. Further research is warranted (during the spring, using additional temperatures) before concluding that females are truly buffered against the indirect risk of climate change via maternal hormone allocation. 

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).