ISTS42 Program/Agenda

The Loggerhead Sea turtle, Caretta caretta, is exposed to numerous natural and anthropogenic threats during its long and complex life cycle. The potential toxicity of contaminants, in particular marine litter, has received the interest of the scientific community due to their possible impacts on the survival of this sea turtle species worldwide. Therefore, C. caretta has been identified as the official bioindicator for the Descriptor 10 "Marine Litter" in the European Union’s Marine Strategy Framework Directive and UNEP/MAP Barcelona Convention.

The presence of microplastics (MPs) could act in synergy with other contaminants and biotic factors impairing the health condition of sea turtles starting from the embryonic phase, one of the most sensitive phases of its entire life cycle.

Consequently, this study aimed to investigate the presence of MPs in unhatched embryos. From 180 unhatched eggs collected from nests laid along the Northwestern Mediterranean coast, only 17 unhatched embryos at stage 30 of development were considered suitable for MPs extraction and following analysis. Alkaline digestion and Raman microspectroscopy were performed on yolk and liver samples to investigate the maternal transfer and the absorption of MPs. All microplastics were categorized in terms of abundance, size, shape, color and polymer. All microplastics identified were smaller than 5µm, the more abundant shapes were spheres and fragments and the main polymers were Acrylonitrile butadiene styrene, polyvinyl chloride and polyethylene.

Furthermore, to determine the effects of MPs on the embryo's health status, selected biomarkers, defined as any measurable parameter indicating stress status (such as melanomacrophages and cortisol levels in the liver), inflammatory processes (such as IL-1β levels in the liver) and exposure to contaminants (such as CYP4501A1 levels in the liver), were evaluated. These biomarkers were assessed through histology, immunohistochemistry and western blotting performed on liver samples.

The effects of MPs on the embryo’s health status was confirmed by the statistical correlation analysis between the expression of these biomarkers and the number of microplastics. In particular, the abundance of melanomacrophages could represent an optimal biomarker for MPs presence due to the very strong correlation.

Further studies should be performed to increase the knowledge about the possible effects of MPs and associated pollutants on embryonic development and provide standardized protocols for future assessments.

Microplastics (MPs) are a pervasive marine environmental pollutant, posing serious threats to marine ecosystems and organisms at all trophic levels. Loggerhead sea turtles (Caretta caretta) have been identified as a promising plastic indicator species to monitor MP pollution globally; these long lived, highly mobile, wide-ranging species show high site fidelity and are particularly susceptible to marine MP pollution due to their visual feeding strategies and backward facing oesophageal papillae. The impacts of plastic pollution on marine turtles are well documented for ingestion, entanglement, and habitat degradation (e.g., coral reef and nesting beaches). Direct (i.e., mistaken for/ mixed with natural prey items) and indirect (i.e., trophic transfer) ingestion of plastics can have many possible lethal and sublethal impacts, with sublethal effects being far more difficult to detect and likely more frequent.

In marine turtles, plastics can remain in the gut between 41 days and up to four months. During these long gut residency times it is possible that MPs and their associated toxicants can translocate past the gastrointestinal canal, contributing to a suite of sub-lethal effects. The ability of small MP particles (<10µm) to translocate across the gastro-intestinal membrane and accumulate in distal body tissues has been demonstrated in fish, mice, and humans. Microplastics contain additives and organic pollutants from production (e.g., PAHs, PCBs, PBDEs and BPAs) and readily associate with chemical contaminants from the environment (e.g., heavy metals and persistent organic pollutants). These can be released during digestion, transmitted in the circulatory system, gradually bioaccumulating in body tissues. Many of these contaminants possess endocrine disrupting activity and can lead to disruption of fundamental processes such as: growth and development, impacting fecundity, offspring viability and reproductive output, possibly leading to late sexual maturation and transmitting epigenetic transgenerational inheritance. All potential outcomes could have long-term demographic ramifications for the stability of marine turtle populations.

A recent call for research highlighted that a better understanding of the translocation and bioaccumulation potential of MPs in marine turtles is required, to fully assess the impact that this anthropogenic pollutant poses to protected and vulnerable populations globally. Loggerhead turtles in the Mediterranean are an excellent candidate for this analysis, with record levels of MP concentrations reported in the Mediterranean Sea at 1.25 million fragments per km². Here we present the first comprehensive analysis of MP accumulation in several organs of 10 loggerhead turtles, examining the kidney, liver, spleen, heart, skeletal muscle, subcutaneous fat, oesophagus, stomach, small intestine, large intestine, and reproductive organs from deceased individuals received at the Oceanogràfic Foundation through the sea turtle stranding network in Valencia, Spain, between April 2022 – April 2023. Microplastic extraction was conducted using chemical tissue digestion (KOH 10%). Suspected MPs have, so far, been identified in the tissues of many individuals: intestine (n=2), kidney (n=5), lung (n=2), reproductive organs (n=6), spleen (n=8), faeces (n=2) and heart (n=2); MP types include fibres, fragments, and spheres, with polymer types to be confirmed using FTIR- ATR spectroscopy. We will compare relative MP concentrations to identify which organs have higher levels of bioaccumulation.

Heavy metals bioaccumulate and biomagnify in sea turtles and their food webs. High concentrations of these contaminants in turtle tissues may pose risks to the health of these protected animals. We studied the rate of increase for six heavy metals in captive-reared marine turtles at a head-starting facility off the coast of Rayong province in eastern Thailand. We collected blood samples from 193 hawksbill turtles and four green turtles fed frozen fish and commercial pellet food. Blood samples were pooled for individuals under the age of 12 months, as the required amount of blood for analysis was greater than 1% total blood volume. We recorded body size (minimum curved carapace length and curved carapace width), mass, and approximate age for each animal. We measured cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), and zinc (Zn) concentrations in whole blood via microwave plasma atomic emission spectrometry (MP-AES). A multiple analysis of covariance (MANCOVA) model treating log-transformed body size (geometric mean of carapace length and width) as a covariate showed that heavy metals increased more with body size than age. We therefore removed age from the final model. Overall, heavy metal concentrations differed significantly between the species (P = 0.032), and increased with body size (P < 0.001). Post-hoc univariate ANCOVAs indicated that only one metal differed between the species, with cadmium concentrations lower in green turtles than hawksbills (P = 0.034). Three of the six metals (Cu, Fe, Zn) increased significantly with body size while one (Pb) increased slightly with body size (all P < 0.001). Curve-fitting regression specified a linear relationship with body size for Zn (r² = 0.49) and a quadratic relationship for Cu, Fe, and Pb (power equations: r² = 0.63, 0.81, and 0.17, respectively). For animals ≥ 12 months old (non-pooled samples), a multiple regression model revealed that no relationship existed between body size and heavy metal concentrations (P = 0.89, adjusted R² = 0.00). Heavy metal concentrations were substantially higher in the food sources than the water (N = 4 for each) for all metals (Mann-Whitney tests: P = 0.029) except for Pb (P = 1.00). While we know that metals increased with body size, we do not yet understand how metals may impact growth rates and general health. The identical foods fed to all turtles at the facility may also have implications for metal accumulation in green turtles that naturally intake a largely plant-based diet. Further study is needed to assess whether heavy metal accumulation has implications for turtle health and growth both within the facility and after release.

Temperature insulation in sea turtle nests plays a crucial role in monitoring the survival of embryonic development inside the eggs. The climate change phenomenon has been shown to influence the morphological and physiological traits of reptiles, including the sea turtle population, where it has significantly contributed to the increasing turtle nest temperature affecting the incubated eggs and hatchlings. In addition to the external temperatures, metabolic heat generated by the incubated hatchlings within the nest could potentially alter their phenotype and morphological traits, resulting in the production of abnormalities. The impact of this metabolic heat is substantial and is influencing the decline of their habitat range worldwide. Hence, this study aims to investigate the effect of incubation temperature and the presence of metabolic heat produced by the hatchlings on their digging performance and general fitness. Temperature data loggers were deployed in three different clutch sizes of green turtle nests, varying from 50, 30, and 15 sizes. The time taken for the hatchlings to emerge from their nest was recorded to observe their digging duration. The findings revealed that larger clutch sizes were associated with slightly shorter digging duration, and hatchlings with smaller body size, lower body weight, larger umbilical scar size, lower glucose level, and quicker self-righting time compared to hatchlings with smaller clutch sizes emerged. This study provides evidence that the presence of metabolic heat within sea turtle nests could potentially alter the temperature along the nest column, adversely affecting emergence hatchlings. Warmer nests are highly exposed to risks such as increased predation due to smaller body sizes, decreased physical responses, and reduced overall fitness. This condition may introduce the hatchlings to dehydration and possible inflammation or stress, increasing the risks to their survival. As temperatures continue to rise, sea turtle hatchlings are at increasing risk of developing suboptimal physiological features that would negatively impact their overall fitness and survival. This study was crucially important to understand the circumstances of rising environmental temperatures, coupled with metabolic heat in incubated sea turtle nests on the hatchlings. Such insights are crucial for the development of effective sea turtle management plans in the face of changing environmental conditions.

Keywords: Endangered species, Physiology, Nest escaping, Conservation management, Hatchery

The fibropapillomatosis (FP) is a deadly neoplastic disease recorded in all of the seven sea turtle species but it is more frequently observed in green turtles. At the first stages, this disease is characterized by external tumors on soft tissues but could evolve in internal tumors, blood parameter and behavior disorder which could potentially lead to the death of the animal. The Chelonid Herpesvirus 5 (ChHV5) has been associated to FP tumors and could be the main aetiologic agent of the disease. FP could spread through mechanical vectors such as marine leeches or cleaner fishes for instance. Transmission of the disease could also occur during physical interactions between turtles.

The recent increasing in FP prevalence despite a million years coexistence between turtles and ChHV5 suggests a significant role played by environmental cofactors. Sea surface temperature and salinity seems to affect FP prevalence. Moreover, FP is frequently associated with pollution of coastal waters close to human activity. Metallic contaminants, persistent organic pollutants and eutrophication associated with a high rate of arginine in sea turtle foraging resources are suspected to facilitate the turtle sensibility to FP.

In Martinique, FP has been observed since 2011 in immature green turtles. These turtles recruit in Martinique at around 25cm carapace length and find here ideal condition to perform their development to adult stage. These juveniles contribute widely to Atlantic population and their conservation is therefore crucial. Thus, occurrence of FP in Martinique green turtles highlights the need to understand the characteristic of the disease in these foraging grounds. A preliminary study (Roost et al., 2022) demonstrated that prevalence vary between sites and could reach 50% of the population.

It is therefore critical to provide knowledges on 1) the dynamics of FP in Martinique juvenile green turtle population and on the triggering factors of the disease, on 2) the genetic variations of ChHV5 between geographic locality and their implications on the etiology and symptomatology of the disease, and on 3) the demographic, physiological and behavioral consequences of the disease on green turtles. In order to meet these objectives, the study rely mostly on a long-term capture-mark-recapture protocol started in 2013, during which individual measurements, health status and biopsies are collected, and on environmental quality assessment (benthic biocenosis cartography, coastal environment contamination by pollutants). First results and attached conservation implications will be detailed in this presentation.

The Mexican Caribbean region located at the Mesoamerican Reef System is home to some of the most important foraging and nesting habitats for green sea turtles (Chelonia mydas) and is characterized for vast expanses of seagrass beds. During the last 25 years, urban development and tourism have increased in the Quintana Roo state. Additionally, this region has been ecologically affected in the last decade by pelagic Sargassum blooms. Information about the biochemical responses of green turtles from the Caribbean is limited, impeding the use of the oxidative stress biomarkers for the evaluation of the health status of this population. The goal of this study was to quantify the biochemical oxidative stress indicators in intracellular material of red blood cells of green turtles inhabiting the coast of Mexican Caribbean and characterize the variations in relation to biotic (sex, size, age class, fibropapilloma) and abiotic (years, areas and sampling duration) factors. Free-living green turtles (n=195) were captured in four foraging bays during 2015-2018. Activities of glutathione peroxidase (GPx r=-0.61, p=0.03) and superoxide dismutase (SOD r=0.54, p=0.04) were correlated with handling time prior to venipuncture in Akumal and Xcalak, respectively. Activity of SOD was correlated with body size of the individuals (r=0.21, p=0.04), with green turtle adults captured in the northern region (Punta Arenas) presenting with the highest activity of this enzyme (p=0.02). Levels of carbonyl proteins presented a decreasing pattern from north (Punta Arenas) to south (Xcalak). Annual variability in oxidative stress responses of green turtles from Akumal and Punta Herrero was found; in the former sites, individuals captured during 2015 were characterized for presenting highest activities of glutathione-S-transferase (GST), GPx, and highest levels of thiobarbituric acid reactive substances (TBARS). A multivariate analysis discriminated the annual biochemical responses of green turtles captured in Akumal bay (Wilks’ Lambda=0.06, p<0.001), where their production rate of superoxide radical (O2^•-), activities of SOD, GPx, GST, and the levels of TBARS were highest in 2015 than the following years. Biochemical responses of green turtles captured during 2015 in Akumal and Punta Herrero Bays coincided with one of the first peaks reported of massive atypical influx of pelagic Sargassum, which in turn decreased in 2016 and 2017. Results of this study corroborates the utility of the oxidative stress indicators as biomarkers of environmental conditions in this sentinel’s species.