Anthropogenic contaminants can have a variety of adverse effects on exposed organisms, including genotoxicity in the form of DNA damage. One of the most commonly used methods to evaluate genotoxicity in exposed organisms is the micronucleus (MN) assay. It provides an efficient assessment of chromosomal impairment due to either chromosomal rupture or mis-segregation during mitosis. However, evaluating chromosomal damage in the MN assay through manual microscopy is a highly time-consuming and somewhat subjective process. High-throughput evaluation with automated image analysis could reduce subjectivity and increase accuracy and throughput. In this study, we optimised and streamlined the HiTMiN assay, adapting the MN assay to a miniaturised, 96-well plate format with reduced steps, and applied it to primary cells from green turtle fibroblasts (GT12s-p). Image analysis using both commercial (Columbus) and freely available (CellProfiler) software automated the scoring of MN, with improved precision and drastically reduced time compared to manual scoring and other available protocols. The assay was validated through exposure to two inorganic (chromium and cobalt) and one organic (the herbicide metolachlor) compounds, which are genotoxicants of concern in the marine environment. All test compounds induced MN formation below cytotoxic concentrations. Once validated, genotoxicity of turtle blood extracts were then assessed from captured turtles of three major foraging grounds within Port Curtis, Gladstone, Queensland. All sampling sites induced MN formation above the detection limit and below cytotoxic concentrations with spatial variability observed. The HiTMiN assay presented here greatly increases the suitability of the MN assay as a quick, affordable, sensitive and accurate assay to measure genotoxicity of environmental samples in cultured cell lines.

Sea turtles are constantly exposed to changes in oxygen tension derived from extended breath-hold diving, but the cellular-level response to such changes is not completely understood. Most studies in hypoxia-tolerant reptiles have focused on the freshwater turtle Trachemys scripta, which responds to anoxia by inducing metabolic rewiring and preserving mitochondrial integrity, facilitating efficient mitochondrial respiration during reoxygenation. Here, we derived primary dermal fibroblasts from loggerhead sea turtles and western fence lizards to study the bioenergetic cellular response to hypoxia exposure in diving and non-diving reptiles. Cells from both species stained positive for the fibroblast markers vimentin and PDGFRb and responded to hypoxia exposure (0.1% O₂) by accumulating HIF-1α. We measured oxygen consumption rates (OCR) in intact cells using Seahorse technology. We found that basal, maximal, and spare respiratory capacity was lower in lizard than in loggerhead cells at 26^oC under normoxic (room air) conditions (p < 0.0001). Exposure to short- (1h) and long-term hypoxia (24h) altered mitochondrial function in both species. Maximal and spare respiratory capacity were lower in loggerhead than in lizard cells (p < 0.01) after 1h hypoxia followed by 1h reoxygenation. In contrast, basal and maximal respiratory capacity was higher in loggerhead compared to lizard cells after 24h hypoxia (p < 0.04). Nonetheless, the overall OCR after 24h hypoxia in both species was lower than that observed after 1h hypoxia. Overall, there was a significant reduction in cellular respiration in both species from short-term to long-term hypoxia exposure. In lizard cells, basal OCR decreased by a factor of three while maximal OCR decreased by a factor of 4.5. In loggerhead cells, both the basal and maximal OCR decreased by a factor of two. Our results demonstrate species-specific regulation of cellular respiration after hypoxia exposure. Remarkably, loggerhead cells show a steady downregulation of cellular respiration in response to hypoxia exposure and faster recovery after extended hypoxia (24h) than lizard cells, aligning with the remarkable hypoxic tolerance exhibited by sea turtles, which can endure up to 7 hours of breath-holding underwater. Understanding the cellular mechanisms that drive hypoxic tolerance in diving animals can provide insights for treating human conditions characterized by hypoxia signaling.

The health status of critically endangered hawksbill sea turtles from the Indian Ocean area is largely unknown as few studies have been performed on this population. We evaluated clinical indices of health in juvenile hawksbill sea turtles (n=36) from Maldivian waters in order to 1) establish reference intervals for plasma biochemical and hematological analytes, 2) perform physical and neurological examinations, 3) assess anatomically available structures by ultrasonic study, and 4) collect colonic and cloacal samples for gut microbial evaluation. All work was subjected to independent animal welfare review and all work was performed under permit from the Maldivian government. Blood analytes included complete blood counts with manual differentiation, PCV, TS, Glucose, SDMA, Creatinine, BUN, Uric Acid, Phosphorus, Calcium, Total Protein, Albumin, Globulin, Albumin:Globulin, ALT, ALP, GGT, Total Bilirubin, Cholesterol, Amylase, Lipase, and BHB. Full physical examinations were performed on each animal, including neurological examination and heart rate measurement by doppler. Ultrasonic examinations were performed using pre-femoral fossa access and renal and lower intestinal features were evaluated. Samples were collected from the cloaca and distal colon using a specialized technique validated for this project. Gut samples were preserved and stored for subsequent microbial analysis. All animals were returned to the wild without incident. This is the first comprehensive health assessment of a population of critically endangered hawksbill sea turtles in the Maldives.

There are seven extant species of sea turtles, all of which are imperiled. As such, large efforts are aimed at understanding the effects of climate change on these animals to aid conservation efforts. Currently, a large knowledge gap exists across hatchling and post hatchling baseline health values and the potential impact of elevated incubation temperatures. Filling these knowledge gaps is critical for these imperiled species, especially leatherbacks. All leatherback populations are considered endangered, but isolated populations of leatherbacks are at higher risk of extinction. The Eastern Pacific population is anticipated to become extinct within the next 100 years. As such, maintaining this population of leatherbacks in temporary human care may be necessary to rescue the population. But to do this successfully, we need to have baseline information, including baseline blood and microbiota parameters. Here we present baseline blood data, preliminary skin microbiota data, and their relation to incubation temperatures in leatherback neonates. We analyzed the effects of incubation temperature on key blood values and found that hatchlings emerging from warmer nests had higher A/G ratios, higher alpha-1 globulin values and lower beta globulin values. All values were assessed again in 3-4 weeks and these alterations persisted. We also assessed skin microbiota at both time points and found significant differences in microbial diversity on turtles emerging from hotter nests. These alterations suggest that increasing nest temperatures are physiologically straining hatchlings at emergence and continue to cause physiologic differences up to a month of age. Suboptimal physiologic states, such as dehydration, inflammation, or skin dysbiosis negatively impact overall fitness and survival of a hatchling. These data provide initial information to begin assessing hatchling health and possibly elucidate a cause for decreased survival in hatchlings incubated at higher temperatures. Ultimately, these findings can be useful for developing management strategies to mitigate the effects of elevated incubation temperatures in nature.

Green turtles are exposed to, and accumulate, chemical contaminants such as trace elements and organic chemicals (e.g., industrial chemicals, pesticides, pharmaceuticals) from the marine environment. Their high foraging site fidelity and long-lived nature make them excellent sentinel species for chemical pollution. Trace element exposure is best assessed using well-developed, sensitive, and inexpensive analytical techniques that are relatively comprehensive. Analytical techniques are less comprehensive for organic contaminants. There can be thousands of organic chemicals present in a sample and each group of chemicals (e.g., polycyclic aromatic hydrocarbons, polychlorinated biphenyls, organochlorine pesticides) requires a different analysis, which can quickly become expensive (>$2000/sample). These limitations can be overcome by testing the overall toxicity of complex chemical mixtures of organic contaminants using in vitro bioassays, which are relatively inexpensive. For sea turtles, this is accomplished by extracting organic chemical mixtures from turtle blood and testing the toxicity of this extract on sea turtle cell cultures. In this study, trace element chemical analysis and in vitro bioassays for organic chemicals were used to examine differences in chemical exposure and effect of green sea turtles foraging in three locations in Queensland, Australia: the Port of Gladstone, the northern Capricorn Bunker, and the southern Capricorn Bunker. The trace element analysis revealed that turtles foraging in Gladstone typically had significantly higher trace element concentrations, with concentrations indicating potential risk to this population. A small number of trace elements were significantly higher in turtle blood from the Capricorn Bunker locations (cadmium, arsenic, selenium) with no significant difference between north and south, potentially indicating inshore/offshore differences. Only vanadium was significantly higher in the southern Capricorn Bunker turtles, requiring further investigation. The in vitro bioassays indicated that organic contaminant concentrations in the turtles foraging in Gladstone and the northern Capricorn Bunker were similar and at levels high enough to cause risk to these turtles. The bioassay results for the southern Capricorn Bunker indicated low toxicity of blood extracts from these turtles. The low toxicity and low variation in results at this location indicate its potential as a reference location for organic contaminants in the southern Great Barrier Reef. Together, these results from the trace element analysis and bioassays indicate that offshore locations may experience different, but not necessarily lower, chemical risk and highlight the importance of characterising this risk to sea turtle populations.