Comparisons of stable isotope composition among tissues of green turtles.

Ecologists often need to make choices about what body parts (tissues or organs) of an animal to sample. The decision is typically guided by the need to treat animals as humanely as possible, as well as the information that different body parts can provide. When using stable isotopes, decisions are also influenced by whether specimens would require preservation, and whether they have properties (such as high lipid concentrations) that would influence measurements. Sometimes we cannot use a preferred tissue (for example, because of ethical or logistical constraints), and in such cases an ability to reliably predict stable isotope composition for one tissue from data yielded by another would be useful. METHODS: In this study we analysed multiple tissues (skin, whole blood, red blood cells, plasma and nail) from green turtles (Chelonia mydas) to evaluate variation in C:N ratios, and test hypotheses about the intercept and slope of regressions of stable carbon and nitrogen isotope compositions among tissues. RESULTS: Regression models revealed that linear relationships were present for most comparisons, except those involving the δ13 C of skin, and the slopes (ß1 ) of most regressions were different from unity. The C:N ratios of skin were significantly higher and more variable than those of other tissues. The δ13 C and δ15 N of nail were highly correlated with those of the whole blood, red blood cells and plasma. Nail and red blood cells showed low variation in C:N. CONCLUSIONS: The patterns in slopes of regressions indicate that comparisons of measurements yielded by different tissues of wild animals are complicated by the fact that the tissues are unlikely to be in isotopic equilibrium with their diet. Of the tissues used in this study, nail is simple to collect, requires minimal disturbance to the animal and no special preservation; these traits should make it attractive to turtle ecologists, but more information is needed on aspects such as growth rates.

Changes of loggerhead turtle (Caretta caretta) dive behavior associated with tropical storm passage during the inter-nesting period.

To improve conservation strategies for threatened sea turtles, more knowledge on their ecology, behavior, and how they cope with severe and changing weather conditions is needed. Satellite and animal motion datalogging tags were used to study the inter-nesting behavior of two female loggerhead turtles in the Gulf of Mexico, which regularly has hurricanes and tropical storms during nesting season. We contrast the behavioral patterns and swimming energetics of these two turtles, the first tracked in calm weather and the second tracked before, during and after a tropical storm. Turtle 1 was highly active and swam at the surface or submerged 95% of the time during the entire inter-nesting period, with a high estimated specific oxygen consumption rate (0.95 ml min-1 kg-0.83). Turtle 2 was inactive for most of the first 9 days of the inter-nesting period, during which she rested at the bottom (80% of the time) with low estimated oxygen consumption (0.62 ml min-1 kg-0.83). Midway through the inter-nesting period, turtle 2 encountered a tropical storm and became highly active (swimming 88% of the time during and 95% after the storm). Her oxygen consumption increased significantly to 0.97 ml min-1 kg-0.83 during and 0.98 ml min-1 kg-0.83 after the storm. However, despite the tropical storm, turtle 2 returned to the nesting beach, where she successfully re-nested 75 m from her previous nest. Thus, the tropical storm had a minor effect on this female's individual nesting success, even though the storm caused 90% loss nests at Casey Key.

Biomarkers reveal sea turtles remained in oiled areas following the Deepwater Horizon oil spill.

Assessments of large-scale disasters, such as the Deepwater Horizon oil spill, are problematic because while measurements of post-disturbance conditions are common, measurements of pre-disturbance baselines are only rarely available. Without adequate observations of pre-disaster organismal and environmental conditions, it is impossible to assess the impact of such catastrophes on animal populations and ecological communities. Here, we use long-term biological tissue records to provide pre-disaster data for a vulnerable marine organism. Keratin samples from the carapace of loggerhead sea turtles record the foraging history for up to 18 years, allowing us to evaluate the effect of the oil spill on sea turtle foraging patterns. Samples were collected from 76 satellite-tracked adult loggerheads in 2011 and 2012, approximately one to two years after the spill. Of the 10 individuals that foraged in areas exposed to surface oil, none demonstrated significant changes in foraging patterns post spill. The observed long-term fidelity to foraging sites indicates that loggerheads in the northern Gulf of Mexico likely remained in established foraging sites, regardless of the introduction of oil and chemical dispersants. More research is needed to address potential long-term health consequences to turtles in this region. Mobile marine organisms present challenges for researchers to monitor effects of environmental disasters, both spatially and temporally. We demonstrate that biological tissues can reveal long-term histories of animal behavior and provide critical pre-disaster baselines following an anthropogenic disturbance or natural disaster.

Determining origin in a migratory marine vertebrate: a novel method to integrate stable isotopes and satellite tracking.

Stable isotope analysis is a useful tool to track animal movements in both terrestrial and marine environments. These intrinsic markers are assimilated through the diet and may exhibit spatial gradients as a result of biogeochemical processes at the base of the food web. In the marine environment, maps to predict the spatial distribution of stable isotopes are limited, and thus determining geographic origin has been reliant upon integrating satellite telemetry and stable isotope data. Migratory sea turtles regularly move between foraging and reproductive areas. Whereas most nesting populations can be easily accessed and regularly monitored, little is known about the demographic trends in foraging populations. The purpose of the present study was to examine migration patterns of loggerhead nesting aggregations in the Gulf of Mexico (GoM), where sea turtles have been historically understudied. Two methods of geographic assignment using stable isotope values in known-origin samples from satellite telemetry were compared: (1) a nominal approach through discriminant analysis and (2) a novel continuous-surface approach using bivariate carbon and nitrogen isoscapes (isotopic landscapes) developed for this study. Tissue samples for stable isotope analysis were obtained from 60 satellite-tracked individuals at five nesting beaches within the GoM. Both methodological approaches for assignment resulted in high accuracy of foraging area determination, though each has advantages and disadvantages. The nominal approach is more appropriate when defined boundaries are necessary, but up to 42% of the individuals could not be considered in this approach. All individuals can be included in the continuous-surface approach, and individual results can be aggregated to identify geographic hotspots of foraging area use, though the accuracy rate was lower than nominal assignment. The methodological validation provides a foundation for future sea turtle studies in the region to inexpensively determine geographic origin for large numbers of untracked individuals. Regular monitoring of sea turtle nesting aggregations with stable isotope sampling can be used to fill critical data gaps regarding habitat use and migration patterns. Probabilistic assignment to origin with isoscapes has not been previously used in the marine environment, but the methods presented here could also be applied to other migratory marine species.

Stable isotopic comparison between loggerhead sea turtle tissues.

RATIONALE: Stable isotope analysis has been used extensively to provide ecological information about diet and foraging location of many species. The difference in isotopic composition between animal tissue and its diet, or the diet-tissue discrimination factor, varies with tissue type. Therefore, direct comparisons between isotopic values of tissues are inaccurate without an appropriate conversion factor. We focus on the loggerhead sea turtle (Caretta caretta), for which a variety of tissues have been used to examine diet, habitat use, and migratory origin through stable isotope analysis. We calculated tissue-to-tissue conversions between two commonly sampled tissues. METHODS: Epidermis and scute (the keratin covering on the carapace) were sampled from 33 adult loggerheads nesting at two beaches in Florida (Casey Key and Canaveral National Seashore). Carbon and nitrogen stable isotope ratios were measured in the epidermis and the youngest portion of the scute tissue, which reflect the isotopic composition of the diet and habitat over similar time periods of the order of several months. RESULTS: Significant linear relationships were observed between the δ(13)C and δ(15)N values of these two tissues, indicating they can be converted reliably. CONCLUSIONS: Whereas both epidermis and scute samples are commonly sampled from nesting sea turtles to study trophic ecology and habitat use, the data from these studies have not been comparable without reliable tissue-to-tissue conversions. The equations provided here allow isotopic datasets using the two tissues to be combined in previously published and subsequent studies of sea turtle foraging ecology and migratory movement. In addition, we recommend that future isotopic comparisons between tissues of any organism utilize linear regressions to calculate tissue-to-tissue conversions.

Forty years of monitoring increasing sea turtle relative abundance in the Gulf of Mexico.

Longitudinal data sets for population abundance are essential for studies of imperiled organisms with long life spans or migratory movements, such as marine turtles. Population status trends are crucial for conservation managers to assess recovery effectiveness. A direct assessment of population growth is the enumeration of nesting numbers and quantifying nesting attempts (successful nests/unsuccessful attempts) and emergence success (number of hatchlings leaving the nest) because of the substantial annual variations due to nest placement, predation, and storm activity. We documented over 133,000 sea turtle crawls for 50.9 km of Florida Gulf of Mexico coastline from 1982 to 2021 for a large loggerhead turtle nesting aggregation and a recovering remnant population of green sea turtles. Over time both species have emerged to nest significantly earlier in the year and green sea turtle nesting seasons have extended. Nest counts and hatchling production for both species have significantly increased, but the rate of emergence success of hatchlings leaving nests has not changed for loggerheads and has declined for green sea turtles. Sea level rise and coastal developments undoubtedly influence coastal habitats in the long-term, impacting nest site selection and potential recruitment from the loss of emerged hatchlings. However, the present indications for steady Gulf of Mexico recovery of loggerhead and green sea turtles counter findings of the Florida Atlantic coasts. This study indicates that effective conservation practices can be detected within time scales of 1-2 turtle generations.

Underwater hearing in the loggerhead turtle (Caretta caretta): a comparison of behavioral and auditory evoked potential audiograms.

The purpose of this study was to compare underwater behavioral and auditory evoked potential (AEP) audiograms in a single captive adult loggerhead sea turtle (Caretta caretta). The behavioral audiogram was collected using a go/no-go response procedure and a modified staircase method of threshold determination. AEP thresholds were measured using subdermal electrodes placed beneath the frontoparietal scale, dorsal to the midbrain. Both methods showed the loggerhead sea turtle to have low frequency hearing with best sensitivity between 100 and 400 Hz. AEP testing yielded thresholds from 100 to 1131 Hz with best sensitivity at 200 and 400 Hz (110 dB re. 1 µPa). Behavioral testing using 2 s tonal stimuli yielded underwater thresholds from 50 to 800 Hz with best sensitivity at 100 Hz (98 dB re. 1 µPa). Behavioral thresholds averaged 8 dB lower than AEP thresholds from 100 to 400 Hz and 5 dB higher at 800 Hz. The results suggest that AEP testing can be a good alternative to measuring a behavioral audiogram with wild or untrained marine turtles and when time is a crucial factor.

Age-specific growth and maturity estimates for the flatback sea turtle (Natator depressus) by skeletochronology.

To address a major knowledge gap for flatback sea turtles (Natator depressus), a species endemic to Australia and considered 'Data Deficient' for IUCN Red List assessment, we present the first-ever skeletochronology-derived age and growth rate estimates for this species. Using a rare collection of bone samples gathered from across northern Australia, we applied skeletochronology and characterized the length-at-age relationship, established baseline growth rates from the hatchling to adult life stages, and produced empirical estimates of age-at- and size-at-sexual-maturation (ASM, SSM). We analyzed humeri from 74 flatback sea turtles ranging in body size from 6.0-96.0 cm curved carapace length (CCL), and recovered from Western Australia (n = 48), Eastern Australia (n = 13), central Australia (n = 8; Northern Territory n = 3, the Gulf of Carpentaria n = 5), and unknown locations (n = 5). We identified the onset of sexual maturity for 29 turtles, based on rapprochement growth patterns in the bones. Estimates for ASM ranged from 12.0 to 23.0 years (mean: 16.3 ± 0.53 SE), SSM ranged from 76.1 to 94.0 cm CCL (mean: 84.9 ± 0.90 SE), and maximum observed reproductive longevity was 31 years for a 45-year old male flatback. Growth was modeled as a smoothing spline fit to the size-at-age relationship and at the mean SSM (84.9 cm CCL) corresponded with a spline-predicted maturity age of 18 years (95% CI: 16 to 24), while mean nesting sizes reported in the literature (86.4 to 94 cm CCL) corresponded to estimated ages of 24+ years. A bootstrapped von Bertalanffy growth model was also applied and showed consistencies with the spline curve, yielding an estimated upper size limit, Linf, at 89.2 ± 0.04 cm (95% CI: 85.5 to 95.9 cm) with the intrinsic growth rate parameter, k, at 0.185 ± 0.0004 (0.16 to 0.22); at the same mean SSM (84.9 cm CCL) the estimated ASM was 16.3 ± 0.05 years (95% CI: 12.8 to 27.7 years). Lastly, four of the samples analyzed were collected from deceased adult females that had previous sizes known from on-going mark/recapture studies at nesting sites in Western Australia. The paired CCL data (measured at nesting and back-calculated) did not significantly differ (p = 0.875). This first skeletochronology study for flatback sea turtles generates valuable empirical estimates for ongoing conservation and management efforts.

Age prediction of green turtles with an epigenetic clock.

Age is a fundamental life history attribute that is used to understand the dynamics of wild animal populations. Unfortunately, most animals do not have a practical or nonlethal method to determine age. This makes it difficult for wildlife managers to carry out population assessments, particularly for elusive and long-lived fauna such as marine turtles. In this study, we present an epigenetic clock that predicts the age of marine turtles from skin biopsies. The model was developed and validated using DNA from known-age green turtles (Chelonia mydas) from two captive populations, and mark-recapture wild turtles with known time intervals between captures. Our method, based on DNA methylation levels at 18 CpG sites, was highly accurate with a median absolute error of 2.1 years (4.7% of maximum age in data set). This is the first epigenetic clock developed for a reptile and illustrates their broad applicability across a broad variety of vertebrate species. It has the potential to transform marine turtle management through a nonlethal and inexpensive method to provide key life history information.

Multivariate analysis of biologging data reveals the environmental determinants of diving behaviour in a marine reptile.

Diving behaviour of 'surfacers' such as sea snakes, cetaceans and turtles is complex and multi-dimensional, thus may be better captured by multi-sensor biologging data. However, analysing these large multi-faceted datasets remains challenging, though a high priority. We used high-resolution multi-sensor biologging data to provide the first detailed description of the environmental influences on flatback turtle (Natator depressus) diving behaviour, during its foraging life-history stage. We developed an analytical method to investigate seasonal, diel and tidal effects on diving behaviour for 24 adult flatback turtles tagged with biologgers. We extracted 16 dive variables associated with three-dimensional and kinematic characteristics for 4128 dives. K-means and hierarchical cluster analyses failed to identify distinct dive types. Instead, principal component analysis objectively condensed the dive variables, removing collinearity and highlighting the main features of diving behaviour. Generalized additive mixed models of the main principal components identified significant seasonal, diel and tidal effects on flatback turtle diving behaviour. Flatback turtles altered their diving behaviour in response to extreme tidal and water temperature ranges, displaying thermoregulation and predator avoidance strategies while likely optimizing foraging in this challenging environment. This study demonstrates an alternative statistical technique for objectively interpreting diving behaviour from multivariate collinear data derived from biologgers.

Lifespan estimation in marine turtles using genomic promoter CpG density.

Maximum lifespan for most animal species is difficult to define. This is challenging for wildlife management as it is critical for estimating important aspects of population biology such as mortality rate, population viability, and period of reproductive potential. Recently, it has been shown cytosine-phosphate-guanine (CpG) density is predictive of maximum lifespan in vertebrates. This has made it possible to predict lifespan in long-lived species, which are generally the most intractable. In this study, we use gene promoter CpG density to predict the lifespan of five marine turtle species. Marine turtles are a particularly difficult group for lifespan estimation because of their migratory behaviour, longevity and high juvenile mortality rates, which all restrict individual tracking over their lifespan. Sanger sequencing was used to determine the CpG density in selected promoters. We predicted the lifespans for marine turtle species ranged from 50.4 years (flatback turtle, Natator depressus) to 90.4 years (leatherback turtle, Dermochelys coriacea). These lifespan predictions have broad applications in marine turtle research such as better understanding life cycles and determining population viability.

Patterns of metal accumulation in osteoderms of the Australian freshwater crocodile, Crocodylus johnstoni.

The concentrations of 15 metals were measured in the osteoderms (dermal bones) of 30 freshwater crocodiles (Crocodylus johnstoni) from a single population in the Lynd River, northeastern Australia (17 degrees 50'S, 144 degrees 20'E), that were well characterised with respect to site fidelity, reproductive status and age. Multiple linear regression analyses were used to determine the effects of crocodile size (snout-vent length, 25-128 cm), age (0.7-62.7 years), gender (male or female) and reproductive status (sexually mature or immature) on osteoderm metal concentrations. Gender and reproductive status were not significant (P > 0.05) co-predictors of the osteoderm concentration of any metal. In contrast, size, age and osteoderm calcium concentration were highly significant (P < 0.001) systematic predictors of the osteoderm concentrations of all metals, except Na and K. Osteoderm metal concentrations were inversely related (P < 0.001) to both size (r(2) = 0.52-0.92) and age (r(2) = 0.52-0.84), but positively related (P < 0.001) to osteoderm calcium concentration (r(2) = 0.67-0.92). Relative to calcium concentration, the rates of metal accumulation in the osteoderms of C. johnstoni were inversely related to the solubility constant (log K(sp)) of the metal as a phosphate; however this relationship was not linear. This finding is consistent with that previously established for the flesh of freshwater bivalves, which like the crocodilian osteoderm, have a calcium phosphate repository in the form of extracellular granules. The constancy of this relationship between rate of metal accumulation and relative solubility for calcium phosphate deposits, despite contrasting Ca accumulation regimes and taxonomic dissimilarity, points to a potential underlying principle that warrants investigation in a greater range of biota. The implications for using the osteoderms of C. johnstoni as an indicator of metal levels in freshwater ecosystems are also discussed.

Interactions between ecology, demography, capture stress, and profiles of corticosterone and glucose in a free-living population of Australian freshwater crocodiles.

In this study we examined three aspects pertaining to adrenocortical responsiveness in free-ranging Australian freshwater crocodiles (Crocodylus johnstoni). First, we examined the ability of freshwater crocodiles to produce corticosterone in response to a typical capture-stress protocol. A second objective addressed the relationship between capture stress, plasma glucose and corticosterone. Next we examined if variation in basal and capture-stress-induced levels of plasma corticosterone was linked to ecological or demographic factors for individuals in this free-ranging population. Blood samples obtained on three field trips were taken from a cross-sectional sample of the population. Crocodiles were bled once during four time categories at 0, 0.5, 6, and 10h post-capture. Plasma corticosterone increased significantly with time post-capture. Plasma glucose also significantly increased with duration of capture-stress and exhibited a positive and significant relationship with plasma corticosterone. Significant variation in basal or stress induced levels of corticosterone in crocodiles was not associated with any ecological or demographic factors including sex, age class or the year of capture that the crocodiles were sampled from. However, three immature males had basal levels of plasma corticosterone greater than 2 standard deviations above the mean. While crocodiles exhibited a pronounced adrenocortical and hyperglycaemic response to capture stress, limited variation in adrenocortical responsiveness due to ecological and demographic factors was not evident. This feature could arise in part because this population was sampled during a period of environmental benigness.