ISTS42 Program/Agenda

In sea turtles, the hormonal profile is an effective and minimally invasive method that provides basic information about reproductive state and sex identification in some species; however, hormone concentrations and their utility as demographic predictors are unknown for the green turtle (Chelonia mydas) population of the Mexican Caribbean coast.

The objectives of this study were to determine hormonal concentrations of estradiol (E₂), testosterone (T), and thyroxine (T₄) and to evaluate their usefulness for sex and maturity stage classification.

Length (standard curve carapace length CCL) was measured and a blood sample was collected from 150 free-ranging green turtles. Individuals were classified by stage of maturity (immature and adults) and by sex, according to their size, morphological characters (adults) and inguinal ultrasounds (immature). The hormone concentrations matched the expected steroid levels for the species and proposed age/size classes for other foraging populations.

E₂ and T levels discriminated reproductive stages, with 100% correct classification of immatures, when using the minimum nesting size (86 cm CCL) (Wilk´s λ= 0.37, p= 0.01). CCL and E₂ discriminated adult green turtles of known sex from immatures with suggested sex (ultrasound) (Wilk´s λ= 0.43, p= 0.0001). However, sex identified morphologically was not discriminated by hormones or size in adult turtles. Hormones and size of adults correctly predicted 83% of females and 78% of males by a logistic regression analysis, meanwhile, only the 69% females and 56% males were accurate predicted by the variables when immatures of suggested sex (ultrasound) was analyzed in tandem.

Results indicate that the hormonal profile and size are useful to discriminate the reproductive stage of green turtles when used simultaneously during modelling. More evaluations are required to understand the relationship between hormone profiles and sex of the green turtle population inhabiting in the coast of Quintana Roo, Mexico.

Imperiled sea turtle species with temperature-dependent sex determination (warm female-cool male) are especially vulnerable to climate change. A small increase in nest temperature can produce highly female-biased sex ratios, potentially threatening population stability due to a lack of males. Currently, there is no simple or non-invasive approach to identify sex of a sea turtle hatchling. A previous study used Western blots to identify a sex-specific protein, Anti-Müllerian Hormone (AMH), in male blood plasma of loggerhead (Caretta caretta) and red-ear slider (Trachemys scripta) hatchlings from controlled temperatures. This method relied on the use of a human AMH polyclonal antibody. However, when tested in loggerhead hatchlings from in situ nests, several inconsistencies emerged: (1) the “AMH” band in Western blots occurred in both sexes, and (2) the antibody interacted with other proteins resulting in multiple bands (non-specific binding). Consequently, we aimed to refine previous methods to identify hatchling sex from in situ nests. To increase specificity, a monoclonal antibody was produced based on a partial AMH sequence obtained using male green turtle (Chelonia mydas) gonad biopsies. Sequence comparison demonstrated that the nucleotide and the translated AMH amino acid sequence is highly conserved among sea turtle species and other freshwater turtles. Based on this information, an antigen was designed and hybridoma technology was employed (ProMab Biotechnologies) to generate stable cell lines (clones) each producing a distinct monoclonal antibody. Testing of these different antibody clones in loggerhead hatchlings incubated under controlled temperatures showed two prominent protein bands, a ~60kDa band previously identified as AMH, and a lower band of around 30kD. We hypothesize that, as in humans, there is a cleavage site within the sea turtle AMH protein, resulting in two bands corresponding to the pro-peptide and the active form of AMH. Both appear to be detected in males and females.

The eastern coastline of the USA is highly urbanized, which has contributed to a significant anthropogenic output of pollutants (such as heavy metals) entering the environment and washing out to the ocean. This is of particular concern for long-lived marine species like sea turtles. Sea turtles, therefore, make useful indicator species because they incorporate environmental and dietary heavy metals as they migrate through marine habitats. Scute samples (from the carapace) can be collected in a relatively non-invasive manner and can reflect the environment and diet of sea turtles within 4-6 years of their life. To better understand trophic accumulation of heavy metals in loggerhead sea turtles (Caretta caretta), we collected scute samples during necropsies of cold-stunned loggerhead turtles from Cape Cod Bay, Massachusetts (CCB; n=17), as well as from live loggerhead turtles in the Mid-Atlantic Bight (MAB; n=37) and off the coast of North Carolina (NC; n=9). The three loggerhead turtle groups are of different life stages and exposure duration, with CCB having the smallest loggerhead turtles and MAB having the largest loggerhead turtles. Therefore, the heavy metal concentrations in their scutes act as indicators of what these sea turtles were exposed to in the environments they experienced and their diet at different stages of their lives. We also collected several commonly known prey items of loggerhead turtles including whelk (Buccinum undatum; n=12), Atlantic scallop (Placopecten magellanicus; n=10) and Jonah crab (Cancer borealis; n=5) from the Mid-Atlantic Bight region. The concentrations of silver (Ag), aluminum (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn) were analyzed using an Inductively Coupled Plasma Mass Spectrometry (ICP-MS). NC loggerhead turtles had higher heavy metal concentrations than other locations except for cadmium (mean ± SD μg g^-1 wet weight; CCB 0.256 ± 0.150; NC=0.103 ± 0.042; MAB=0.095 ± 0.040) and zinc (CCB=201.79 ± 0.50.97; NC=184.66 ± 70.85; MAB=172.92 ± 52.16), where CCB loggerhead turtles were higher. As NC and CCB loggerhead turtles’ scute samples are probably still reflecting heavy metal concentrations from their juvenile omnivorous diets, the higher NC heavy metal concentrations are likely indicative of the heavy metals bioaccumulating in the larger NC turtles. On the other hand, NC turtles having higher heavy metal concentrations than MAB turtles indicates that MAB turtles’ scute samples are probably reflecting heavy metal concentrations from their carnivorous adult loggerhead diet. We found that all heavy metals except silver, cadmium, and lead appear to be biomagnified (TTF>1) in loggerhead turtles. This study provided baseline information on heavy metal concentrations in loggerhead scute samples and their prey in east coast US.

The escalating threat of pollution has breached critical ecological thresholds, heralding severe consequences for marine ecosystems and essential processes. While our understanding of contaminants’ impacts remains an ongoing challenge, the effect of pollutants on the health of marine organisms, particularly sea turtles, is gaining more attention. This study aimed to assess the health implications due to contaminant uptake in three foraging populations of green turtles (Chelonia mydas) in the southern Great Barrier Reef of Australia. A comprehensive field study conducted health assessments, including visual, biochemical, and haematological evaluations, on 45 juvenile and sub-adult green sea turtles across offshore and inshore foraging sites. An in vitro cytotoxicity assay using green turtle fibroblasts was used to test the toxicity of blood extracts. Additionally, trace element analysis via ICP-MS identified concentrations of 14 essential and non-essential elements in whole blood. These analyses revealed associations between increased cobalt, molybdenum and manganese concentrations and diminished kidney function and overall body condition. Surprisingly, our findings indicated higher cytotoxicity in the two offshore foraging sites. While these sites were anticipated to have low contaminant exposure, individuals displayed an increased level of overall toxicity despite showing no visible signs of decreased health and no significant changes in health parameters. This discrepancy underscores the intricate and complex nature of ecotoxicology and health assessments in marine turtles and highlights the need to learn from inevitable research oversights. Moving forward, this study emphasises the need for future investigations delving deeper into immune responses and endocrine disruption, essential for a more wholistic comprehension on the impact of contaminants on sea turtle health. This research illustrates the intricate relationship between contaminants, health, and the environment, signifying both the challenges and the crucial importance of further exploration for the sustainability of marine ecosystems.

Breeding strategies are important to understand when utilizing conservation approaches to protect endangered species, such as the Hawksbill sea turtle (Eretmochelys imbricata). Two opposing strategies often compared include capital and income breeding. Income breeders continuously forage during their reproductive season, whereas capital breeders fuel reproduction from energy stored prior to reproductive events. The goal of this research is to explore which strategy best fits the habits of the Hawksbill sea turtle. To determine how foraging is linked to reproduction, we are analyzing the concentration of β-hydroxybutyrate (BHB), triglycerides (TRG), total protein (TP), and testosterone (T) in nesting Hawksbill sea turtles. Blood samples from nesting adult female Hawksbill sea turtles were collected May-August 2023 in Boca del Drago, Panama. Blood was drawn from the dorsal cervical sinus with 21-gauge 1.5-inch needles and heparinized syringes. Using Inconel and PIT tags, the flippers were tagged for identification. After the blood samples were collected, the blood was centrifuged at 10,000 RPM for 10 minutes. The plasma was then stored frozen until assaying. In total, 49 samples were collected from 26 different turtles over 75 days. There were 12 ‘returning’ Hawksbill turtles that were sampled at least two times. To analyze trends in the metabolites, the season was arbitrarily parsed into three 25-day periods: early season, mid-season, and late season. Averages from the samples were calculated from each of the relative times.

The average T concentration (pg/mL) for early, mid, and late season were 2,329.4, 1,920.9, and 1,525.4, respectively. The average TRG concentration (mg/dl) for early, mid, and late season were 875.9, 606.6, and 533.8, respectively. The average BHB concentration (uM) for early, mid, and late season are 484.8, 737.4, and 683.8, respectively. The average TP concentration (mg/mL) for early, mid, and late season are 25.4, 22.1, and 22.8, respectively. Results suggest Hawksbill turtles are capital breeders, which is consistent with decreasing TRG concentration and increasing BHB concentration. Decreasing T concentration across the nesting season are consistent with literature. The results of this study serve to narrow the location of the most important foraging grounds for this population of Hawksbill turtles. The capital breeding strategy suggests these turtles are forgaing prior to their reproductive migration. Due to their conservation status, protecting their feeding grounds is critical. Learning when they are acquiring most of their energy fueling reproduction will help to locate the most important feeding grounds for these animals.

The Hawksbill (Eretmochelys imbricata) is a critically endangered sea turtle that is pan-tropically distributed. Of the seven sea turtle species, Hawksbills have one of the longest histories of exploitation by humans that have resulted in an estimated 80% decrease in their global populations in the last century. Understanding the intricate interplay between reproductive biomarkers such as testosterone, estradiol, and vitellogenin (VTG) and reproductive output is crucial for informing conservation strategies and management practices. This study investigates the temporal relationship between these reproductive biomarkers and reproductive output throughout the nesting season which will provide pertinent information about sea turtle reproductive physiology. VTG is a pivotal lipoprotein integral to oviparous vertebrate reproduction and plays a crucial role in the formation of egg yolk within ovarian follicles. The role of VTG in free-ranging sea turtles remains understudied, making this research particularly pertinent. Blood samples were collected from nesting Hawksbills between May and August 2023 along the Caribbean coast of Panama in Bocas del Drago. We utilized commercial steroid ELISAs (i.e., estradiol and testosterone) and an in-house VTG ELISA to quantify the concentrations of each biomarker. Our initial hypothesis was a gradual decrease in both sex steroid and VTG concentrations as the nesting season progressed. Preliminary findings indicate a consistent decline in testosterone concentrations, aligning with expectations. Given correlative observations in other organisms, including sea turtles, we anticipate that VTG concentrations will follow a similar trend. While further analysis is necessary, early data suggests a parallel trend in reproductive output. I expect that the reproductive biomarkers will provide an improved understanding of the physiological mechanisms governing the number of eggs per clutch laid by female Hawksbill turtles throughout the nesting season. The results of this study will enhance our fundamental understanding of sea turtle reproductive physiology and will also carry implications for conservation and management efforts in the future.

Yolk to nurture growing embryos starts being deposited into vitellogenic ovarian follicles 8 to 12 months prior to sea turtle reproduction. Yolk deposition is believed to be completed prior to reproductive migration in leatherback (Dermochelys coriacea) and Kemp’s ridley turtles (Lepidochelys kempii). However, follicle size and circulating concentrations of yolk precursor indicate that yolk deposition is completed during the nesting season of green turtles (Chelonia mydas) in the Indian and northwest Atlantic oceans, respectively.

From 2021 to 2023, I collected the ovaries of 69 green turtles depredated by jaguars (Panthera onca) while nesting at Tortuguero National Park, Costa Rica, and I counted and measured the diameter of all vitellogenic follicles present in each ovary. When I compared the number of vitellogenic follicles in both ovaries with the average clutch size and frequency for green turtles nesting at Tortuguero, it was clear that a vitellogenic follicle size hierarchy existed. The diameter of small follicles increases up to 13 mm prior to ovulation due to yolk deposition during the nesting season at Tortuguero. Is the increase in size due only to water deposition or were lipids and/or protein being deposited as well? To answer this question, I dehydrated small (~ 23mm) and large (~29mm) vitellogenic follicles at 70^o C and weighed sequentially until stable mass was achieved.

Prior to dehydration, small follicles (n = 376) weighed an average of 8.8 g (± 3.5) and large follicles (n = 397) weighed an average of 14.4 g (± 3.5). After dehydration, large follicles lost an average of 47.1% (± 4.1) of the initial weight, whereas small follicles lost an average of 49.6% (± 4.1) of the initial weight. The yolk of small follicles had significantly higher proportion of water than the yolk of large follicles (Kruskal-Wallis test, H₁ = 172.8, p < 0.001). Because the yolk in small follicles was more diluted than the yolk in large follicles, the increase in diameter of green turtle vitellogenic follicles during the nesting season at Tortuguero is due to the deposition of not only water, but mainly lipids and/or protein. I am currently quantifying lipid and protein content in the samples, which we expect will not differ between small and large follicles. These results will be ready to present at the ISTS42.

Though there is evidence that females will eat opportunistically during the nesting season, sea turtles are considered capital breeders that amass all the energy required for reproduction at foraging grounds. Here, we show that provisioning of vitellogenic follicles, a major energy expenditure associated with reproduction, was still underway when female green turtles arrived at the nesting beach in Tortuguero. It remains to be seen whether food intake at the nesting beach provides energy for Tortuguero green turtles to produce the egg yolk that complements small vitellogenic follicles. This would enhance our understanding about the energetic requirements for reproduction of this important population of endangered green turtles, which is even more crucial in light of the rapid worldwide decline of seagrass meadows, its main food source.