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This paper highlighted the annual distribution, seasonality and reproduction status of two species of sea turtles in Negeri Sembilan, Peninsular Malaysia between January 2016 and July 2020 (55 months). These data were officially provided by the Department of Fisheries Malaysia (Negeri Sembilan's state), as a part of a conservation effort made by them to protect the endangered species, as the turtle's population residing in Negeri Sembilan is one of the smallest in Malaysia. Current status shows that the hawksbill turtles, Eretmochelys Imbricata population in Negeri Sembilan is in stable decline with an average of <30 nests per year, and an average of <10 nests per year for the green turtle, Chelonia mydas. The overall sum was 122 nests collected for both species from 2016-2020. This paper updated the status of the hawksbill and green turtle after 30 years from the first study recorded by Mortimer et al. in 1993.

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It has been widely demonstrated that air and sand temperatures influence the anatomy of sea turtle hatchlings. We examined the impact of precipitation during the nesting season on the hatchling body size of loggerhead and green turtles from 37 beaches worldwide. Longitudinal data collected between 2012 and 2018 from Florida (US) and from a sample on Bõa Vista Island (Cabo Verde) carried out in 2019 showed that loggerhead body size at hatching was negatively correlated with precipitation, while precipitation was not correlated with hatchling body size in green turtles. A meta-analysis revealed that precipitation is positively correlated with hatchling mass in loggerhead turtles, while it is positively correlated with straight carapace length and width in green turtle hatchlings. The strongest influence of precipitation was found in the middle of the incubation period of loggerhead turtles in Cabo Verde, and we posit that this is due to an increase in the uptake of water for embryonic growth. These findings highlight the great importance of understanding the correlated effects of regional environmental variables, such as precipitation, on the development of sea turtle hatchlings and will have an impact on the evaluation of ongoing conservation and climate change discussions.

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Successful embryonic development depends on the interaction between genetic factors and environmental variables. Congenital malformations in sea turtles can result from extreme conditions during the incubation period, reducing hatching success and potentially impeding population recovery. We aimed to characterize the congenital malformations found in green turtle nests, determine their prevalence and severity, and understand their drivers during the 2022 nesting season on Samandag beach on northern Mediterranean nesting beaches. A total of 2986 examples of congenital malformations were observed in 362 out of 907 green turtle nests. The prevalence of congenital malformations per nest was 39%, and the severity (the number of malformed individuals per nest) was 3.8%. Nests with congenital malformations exhibited a lower mean distance from the sea, a shorter incubation duration (a proxy for incubation temperature), lower hatching success, a larger clutch size, and higher mortality at late embryonic and hatchling stages than nests without congenital malformations. There was no significant difference in total mortality between these two nest types. A total of 52 different congenital malformations were recorded, 2 of which were observed for the first time in sea turtles and 28 for the first time in green turtles. The results suggest that congenital malformations may be related to nest temperature and clutch size, while overall mortality may be independent of malformations. Pigmentation disorders and craniofacial malformations typically coexist in cases of multiple malformations. Long-term monitoring of congenital malformations is crucial, as it can provide clues about the health status of the nesting beach and nesting colony.

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Important foraging and nesting habitats for Caribbean green sea turtles (Chelonia mydas) exist within the Mesoamerican Reef System in the Mexican Caribbean. During the last 25 years, urban development and touristic activities have drastically increased in Quintana Roo, Mexico. Moreover, in the last decade, massive pelagic sargasso blooms have also afflicted this region; however, information about the biochemical responses of Caribbean green turtles to these inputs is absent. This study aimed to assess if the oxidative stress indicators in the red blood cells of green turtles are valuable biomarkers of the extent of the anthropic impact in this region. Persistent organic pollutants (POPs) were also measured in the plasma of free-living green turtles during 2015-2018 to characterize these habitats further. As biochemical biomarkers, the production rate of superoxide radical (O2•-), carbonylated protein content, and lipid peroxidation (TBARS) levels, and the activities of superoxide dismutase, glutathione S-transferase (GST), catalase, glutathione peroxidase were measured in erythrocytes. A 15 % occurrence of fibropapillomatosis (FP) was revealed, with tumor size being positively correlated with CAT activity in the affected individuals. A multivariate analysis embracing all oxidative stress markers discriminated green turtles between years of capture (p < 0.001), with those sampled during 2015 presenting the highest production of O2•- (p = 0.001), activities of GST (p < 0.001), levels of TBARS (p < 0.001) and carbonylated proteins (p = 0.02). These local and temporal biochemical responses coincided with the first massive Sargassum spp. bloom reported in the region. The results of this study corroborate the utility of the oxidative stress indicators as biomarkers of environmental conditions (sargasso blooms and POPs) in the green turtle as sentinel species.

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Background and Aim: The International Union for the Conservation of Nature and Natural Resources lists the green turtle as endangered. Green turtle nesting behavior in the Gulf of Thailand has decreased to <50% of the 1995 level. The population structure of green turtles in the Gulf of Thailand has not yet been studied. This study aimed to characterize the genetic diversity of green turtles in the Gulf of Thailand based on comparisons of mitochondrial DNA (mtDNA) control region with sequences of Indo-Pacific management units (MUs) and rookeries, to investigate population structures, and to explore phylogeographic relationships. Materials and Methods: Blood samples (1 mL each) from 91 stranded green turtles were collected from four parts of the Gulf of Thailand (eastern, upper, central, and lower). The control mtDNA region was amplified by polymerase chain reaction using LCM15382 and H950 primer. The obtained 384-bp or 770-bp sequences were analyzed for haplotype, clade, and haplotype and nucleotide diversities and were used to construct a phylogenetic tree and haplotype network diagram, respectively. In addition, we analyzed genetic differentiation within and among populations of green turtles in the Gulf of Thailand and between green turtles in the Gulf of Thailand and other Indo-Pacific MUs and rookeries. Results: In total, 12 (based on 384 bp) or 13 (based on 770 bp) haplotypes and two clades (clades VII and VIII) were identified, with nine or 10 haplotypes belonging to clade VIII and three haplotypes belonging to clade VII. Of the new haplotypes, four or five were identified and classified as clade VII (two haplotypes, for both fragment lengths) and clade VIII (two or three haplotypes, for 384 bp or 770 bp fragments, respectively). The overall haplotype and nucleotide diversity of green turtles in the Gulf of Thailand were high (0.755 ± 0.039 and 0.01146 ± 0.00248, respectively). Based on the analysis of molecular variance, green turtles in the Gulf of Thailand could be divided into two subpopulations (UC-Eastern Gulf of Thailand [UC-EGT] and lower Gulf of Thailand [LGT]). Comparisons with other MUs and rookeries in the Indo-Pacific showed that UC-EGT was not genetically different from the Peninsular Malaysia and Eastern Taiwan (Lanyu) MUs and the Terrangganu and Mersing rookeries, and LGT were not genetically different from Peninsular Malaysia, Sipadan, Brunei Bay, Eastern Taiwan (Lanyu), Scott Reef and Browse Island, and Gulf of Carpentaria MUs and the Perak, Perhentain Island, Redang, Pahang, and Vietnam rookeries. Conclusion: To the best of our knowledge, this is the first report to identify the haplotypes and clades of green turtles in the Gulf of Thailand and to show that the populations in the Gulf of Thailand not only present high genetic diversity but also have haplotypic endemism. Longer mtDNA fragments (770 bp) increased the resolution of the stock structure. Clade VII is a unique clade not only for Japan but also for Thailand and Malaysia, and CmP82 is a unique haplotype for both the Gulf of Thailand and Malaysia. Conservation and management of these populations are important to preserve the genetic diversity, biological diversity, and evolutionary potential of green turtles in the Gulf of Thailand.

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Mercury (Hg) is a highly toxic pollutant that endangers several marine animals, including green sea turtles (Chelonia mydas), particularly in their foraging grounds along Brazilian coastal waters. Environmental Hg levels differ along this extensive littoral, rendering mandatory Hg long-term assessments of the different Hg fate in different sectors of the Brazilian coast. This study quantifies total Hg concentrations in the liver and muscle of green sea turtle populations from three foraging coastal regions in northeastern Brazil and analyzes Hg concentration differences given the locality and size of 61 juvenile individuals sampled. The results showed wide variations in Hg concentrations in the liver (81-3135 ng g-1) and muscle (10.1-8569 ng g-1). There was no significant correlation between animal size and Hg concentrations. Also, no difference was found among areas, reflecting the opportunistic feeding habit of juveniles of this species. This suggests that, in the case of green turtles, the ontogenetic change of diet plays an important role in influencing Hg concentrations found in this species.

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This study analyzed the concentrations of 15 (Al, As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Se, V, Zn) toxicologically important trace elements in the livers of 110 green turtles stranded in two areas of the Brazilian coast. These areas are essential for the refuge, feeding, and reproduction of the species, and the information obtained is intended to support the development of conservation strategies. Higher concentrations were observed in the Região dos Lagos, RJ in almost all elements, except for Al, Mo, Pb, and V. This location showed statistically higher differences in the concentrations of Cd (4.66 ± 2.33 µg.g-1), Fe (846.62 ± 583.06 µg.g-1), and Zn (27.17 ± 10.90 µg.g-1). The differences in trace element concentration patterns between the two study areas are likely influenced by multiple factors, including the bioavailability of trace elements, oceanic upwelling events, anthropogenic activities, habitat characteristics, and organism-specific metabolic processes.

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Contamination in marine ecosystems is of the most critical threats to marine turtles. The identification of useful biomarkers to detect and monitor the physiological and clinical effects of pollutants on these populations will allow early detection of alterations (e.g., mutagenic damages) that could risk their viability or favor the development of diseases, thus threatening the biodiversity of these ecosystems and human population. This study is aimed at describing and quantifying nuclear anomalies in peripheral blood erythrocytes of green turtles (Chelonia mydas) from three distinct foraging areas in Mexico (Akumal, Xcalak, and Punta Herrero). We developed a novel morphological index that could be used as a biomarker to identify abnormal nuclei in peripheral blood erythrocytes. Here we describe for the first time in C. mydas, with a species-specific staining protocol, distinct nuclear abnormalities such as blebbed, lobed, notched, eight shape nuclei, and binucleated cells. These nuclear abnormalities were present in > 90 % of the subjects (n = 30). Moreover, 50 % of the organisms presented erythrocytes with micronuclei. The number of nuclear abnormalities did not correlate with size of the green turtles or differ between sites, or health status. We found a higher frequency of green turtles with nuclear abnormalities in the southern region (Punta Herrero and Xcalak) with the highest frequency of micronucleus and buds. The former could be associated to the constant exposure to chemical pollutants of oceanographic origin in the southern coast of Quintana Roo. Furthermore, the increasing anthropogenic pollution in Akumal could also explain the highest variability in the number of nuclear abnormalities presented in resident individuals. We propose that a long-term monitoring programs of green turtle populations in the Mexican Caribbean that include a micronucleus test could be a useful to determine possible mutagenic damage in these animals.

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BACKGROUND: The green sea turtle, Chelonia mydas, is a migratory species with a strong natal homing behavior leading to a complex population structure worldwide. The species has suffered severe declines in local populations; it is therefore crucial to understand its population dynamics and genetic structure to adopt appropriate management policies. Here, we describe the development of 25 new microsatellite markers specific to C. mydas and suitable for these analyses. METHODS AND RESULTS: They were tested on 107 specimens from French Polynesia. An average allelic diversity of 8 alleles per locus was reported and observed heterozygosity ranged from 0.187 to 0.860. Ten loci were significantly deviant from the Hardy-Weinberg equilibrium, and 16 loci showed a moderate to high level of linkage disequilibrium (4-22%). The overall Fis was positive (0.034, p-value < 0.001), and sibship analysis revealed 12 half- or full-sibling dyads, suggesting possible inbreeding in this population. Cross-amplification tests were performed on two other marine turtle species, Caretta caretta and Eretmochelys imbricata. All loci successfully amplified on these two species, though 1 to 5 loci were monomorphic. CONCLUSION: These new markers will not only be relevant for further analyses on the population structure of the green turtle and the two other species, but they will also be invaluable for parentage studies, for which a high number of polymorphic loci are necessary. This can provide important insight into male reproductive behavior and migration, an aspect of sea turtle biology that is of critical importance for the conservation of the species.

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A female juvenile green turtle (Chelonia mydas), found alive in Guanabara Bay, Rio de Janeiro, Brazil, was weak, dehydrated and cachectic, with a healed fracture in the caudal portion of the carapace. Despite supportive treatment, the animal died after 9 days. At necropsy the main lesions were pallor of visceral organs, arthritis and deposits of whitish granular material in the wall of large arteries and the trachea. Histopathological analysis revealed mild to severe deposition of crystals, consistent with calcium oxalate, in both kidneys and the spleen, heart, small intestine, pancreas, thymus and salt gland, as well as bacterial meningitis, septic arthritis, spirorchidiasis and a fibropapilloma on the nictitating membrane. The main pathological findings were suggestive of septic shock, mainly due to the bacterial meningitis and septic arthritis, with systemic oxalosis and spirorchidiasis as contributing lesions. Although renal oxalosis has been described in green turtles as an incidental finding, presumably due to ingestion of oxalate-containing plants, this turtle had an unusual systemic deposition of oxalate crystals.

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Tropical oceans host a high diversity of species, including large marine consumers. In these oligotrophic ecosystems, oceanic islands often favour the aggregation of species and biomass as they provide feeding opportunities related to the mechanisms of island mass effect. As such, the waters surrounding La Reunion (Southwest Indian Ocean) host seabirds, large pelagic teleosts, elasmobranchs, delphinids and sea turtles. Isotopic niche partitioning and comparison of trophic levels among these species (n = 21) were investigated using stable carbon (δ13C) and nitrogen (δ15N) isotope analysis. Overall, δ13C values were highly variable among taxa, indicating that the species exploit multiple foraging habitats along a coast-open ocean gradient. Overlap in δ15N values was limited, except for teleost species, the two species of sea turtles and two species of delphinids, the Indo-pacific bottlenose dolphin (Tursiops aduncus) and the Spinner dolphin (Stellena longirostris). Stable isotope analyses of samples collected over a 9-years period on different tissues with different integration times provide a consistent picture of the structure of the community of large marine vertebrates species around La Reunion and highlight the underlying mechanisms to limit the competition between species. The wide range of isotopic values confirms that large marine vertebrates have different trophic roles in coastal marine food webs around this oceanic island, which limits their potential of competitive interactions for resources.

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Long-term monitoring programs of species at risk are efficacious tools to assess population changes, evaluate conservation strategies, and improve management practices to ensure populations reach levels at which they can fulfill their ecological roles. For sea turtles, annual nesting beach surveys are the most accessible method to estimating the population abundance and reproductive output, especially when these are done in primary nesting sites. However, little data exist on the long-term assessment of these parameters. Here, we present the trends of the nest abundance, female size, hatching, and emergence success of hawksbill (Eretmochelys imbricata) and green (Chelonia mydas) turtles at key nesting beaches in the southern Gulf of Mexico over 31 years (from 1990 to 2021). The nest abundance showed an increasing trend in both species as a result of the sustained protection and conservation effort, but there was no significant temporal trend in the annual female size, clutch size, hatching, and emergence success. However, these indicators showed decreasing mean values over the last decade and should be closely monitored. We suggest these decreases link to the combined effects of ocean warming and anthropogenic pressures affecting the sea turtle foraging grounds. Aside from protecting key nesting sites, protecting and restoring crucial foraging habitats should be an immediate priority requiring international cooperation.

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Tortuguero, Costa Rica is considered the second largest green turtle (Chelonia mydas) rookery in the world. By 1950, Tortuguero was one of the sites with the greatest take of green turtles in the Caribbean. Currently, Tortuguero is a worldwide example for ecotourism-based on sea turtle conservation. However, illegal take of nesting turtles still occurs. We aimed to describe the illegal take at Tortuguero, estimating the minimum number of sea turtles taken using data collected during daily and weekly track surveys from 2005 to 2021. Additionally, we conducted 12 semi-structured interviews with key informants to obtain a better understanding of this activity. We documented 735 nesting turtles illegally taken at Tortuguero, being the green turtle the most affected species; these findings were also supported by our interviewees. Respondents stated that in Tortuguero the take of sea turtles has always occurred and traditions regarding sea turtle meat consumption are still present, even though it is considered shameful in the village. However, our interviewees affirmed that most of the sea turtles taken are traded to other locations away from Tortuguero. Our findings represent the minimum of illegal take (documented only at the beach), as not all the sea turtles taken were observed. Finally, despite long-standing conservation efforts carried out in Tortuguero, further changes in the National Park's management plans are needed, including more personnel and increased law enforcement. This may be necessary to reduce the impact on the Tortuguero green turtle nesting population in the near future.

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Fibropapillomatosis (FP) is a tumor disease that affects all sea turtle species but is mainly seen in green turtles Chelonia mydas. The pathology of FP has been described extensively, but its dynamics in populations over time have been less studied. We analyzed the dynamics of FP in a population of green turtles in Akumal Bay on the central coast of the Mexican Caribbean. A total of 475 green turtles were captured over 15 yr (2004-2018). The highest prevalence of FP was found in the largest turtles, and there was a positive relationship between FP prevalence and size of turtles. FP was first detected in 2008 at a prevalence of 1.6%, and annual prevalence increased markedly from 17.9% in 2015 to 54% by 2018. Likewise, severity of FP increased over time, with most turtles falling into moderately to severely diseased categories (tumor score 2). The average size of turtles with FP was significantly larger than the size of individuals without FP. Regression of tumors was seen in 21% of turtles, tumor score was higher in smaller individuals, and only tumor score 2 was present in the largest sea turtles. An increase in the prevalence and tumor score of FP coincided with the massive arrival of Sargassum in 2015, suggesting that altered environmental conditions may have played a role. The increased prevalence of FP in Akumal Bay prompts the need to explain what might be driving this phenomenon and how widespread it is in the Caribbean.

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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 both primary cells from green turtle fibroblasts (GT12s-p) and a freshwater fish hepatoma cell line (PLHC-1). 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 compounds tested induced MN formation below cytotoxic concentrations. 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 different cell lines.

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Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min-1mg·protein-1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.

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Fibropapillomatosis (FP), a debilitating, infectious neoplastic disease, is rarely reported in endangered Kemp's ridley sea turtles (Lepidochelys kempii). With this study, we describe FP and the associated chelonid alphaherpesvirus 5 (ChHV5) in Kemp's ridley turtles encountered in the United States during 2006-2020. Analysis of 22 case reports of Kemp's ridley turtles with FP revealed that while the disease was mild in most cases, 54.5% were adult turtles, a reproductively valuable age class whose survival is a priority for population recovery. Of 51 blood samples from tumor-free turtles and 12 tumor samples from turtles with FP, 7.8% and 91.7%, respectively, tested positive for ChHV5 DNA via quantitative polymerase chain reaction (qPCR). Viral genome shotgun sequencing and phylogenetic analysis of six tumor samples show that ChHV5 sequences in Kemp's ridley turtles encountered in the Gulf of Mexico and northwestern Atlantic cluster with ChHV5 sequences identified in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles from Hawaii, the southwestern Atlantic Ocean, and the Caribbean. Results suggest an interspecific, spatiotemporal spread of FP among Kemp's ridley turtles in regions where the disease is enzootic. Although FP is currently uncommon in this species, it remains a health concern due to its uncertain pathogenesis and potential relationship with habitat degradation.

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Among the various pollutants released into the environment, there are persistent organic pollutants (POPs). Chelonia mydas are one of the species that can be exposed to these pollutants and it is classified in the IUCN Red List as "endangered". The present study evaluated the occurrence of POPs in 49liver tissue samples of C. mydas juveniles collected on the southeastern Brazilian coast. Furthermore, the concentrations were correlated with carbon and nitrogen isotopic ratio, biometrics, and ecological factors. The main POPs found were Æ´-HCH and PCBs. Overall, the concentrations found were low and there were no significant correlations among POPs, isotopic ratios, size and weight, which may be related to the fact that the studied individuals are juveniles and occupy similar trophic positions despite the individual variations found. Despite the low concentrations, the presence of POPs, mainly PCBs, in the sea turtles' liver indicates their exposure to these compounds.

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There is a strong correlation between degraded marine habitats and the prevalence of diseases such as green turtle fibropapillomatosis (GTFP) in coastal populations. In GTFP, small to large tumors grow on the turtle's soft tissues and shell, while internal nodules may also occur. The disease primarily affects juvenile green sea turtles (Chelonia mydas) that reside in nearshore waters. As a link has been shown between environmental pollution and immune suppression in a variety of animals, the objective of our research was to compare innate and adaptive immune responsiveness in green sea turtles from a severely degraded and a more pristine habitat, which differ greatly in rates of GTFP. We quantified phagocytosis by flow cytometry and performed in vitro stimulation analysis to measure activity of both the innate and adaptive immune systems in wild-caught Florida green turtles. Sea turtles from the degraded environment, both with and without visible cutaneous tumors, exhibited significantly reduced phagocytosis and stimulation indices than did those from the less polluted environment. Our results suggest that environmental factors may contribute to the development of GTFP and thus can impact the health of sea turtle populations.

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