Incubation environment and parental identity affect sea turtle development and hatchling phenotype.

For reptiles, the incubation environment experienced by embryos during development plays a major role in many biological processes. The unprecedented rate of climate change makes it critical to understand the effects that the incubation environment has on developing embryos, particularly in imperiled species such as chelonians. Consequently, a number of studies have focused on the effects of different environmental conditions on several developmental processes and hatchling phenotypic traits. In addition to the incubation environment, it is also essential to understand how parental contributions can influence hatchling quality. This is the first study that investigates the effects of parental origin and incubation conditions on sea turtle embryonic development and hatchling phenotype in nests incubating in the field (rather than under controlled laboratory conditions). Here, we used the loggerhead sea turtle (Caretta caretta) to investigate the effects of parental origin (clutch), incubation temperature, and the nest hydric environment on embryonic growth, incubation durations, hatching success, and hatchling phenotype. Our results show that nest moisture and temperature affect embryo mass towards the last third of development, with hatchling size positively correlated with nest moisture content, and maternal origin had a strong impact on hatching success and hatchling size regardless of the incubation conditions. The results from this experiment identify multiple factors that affect turtle embryonic development under field incubation conditions, a fundamental consideration when interpreting the potential impacts of climate change on reptilian development.

A new approach for measuring temperature inside turtle eggs.

For turtles, the thermal environment experienced during development plays critical roles in many biological processes. While the temperature inside an egg is assumed to match the substrate temperature, many factors such as evaporative cooling, metabolic heating and the insulating properties of extra-embryonic components can lead to thermal differences. However, no method developed to date has allowed for measurement of the embryonic temperature in live chelonian eggs. We designed a thermocouple-based technique to measure embryonic temperature, achieving 94% survival in Trachemys scripta This methodology may be applicable to other reptile species. We found that, while the temperature in the substrate adjacent to the eggshell accurately reflects the internal egg temperature, it differs from air temperature (∼2°C) in a moisture-dependent manner. Our results demonstrate that external egg temperature, but not air temperature, is suitable for assessing the effects of temperature on biological processes, which could be critical when considering that processes such as temperature-dependent sex determination in turtles occurs within a 4°C window.

Identifying Sex of Neonate Turtles with Temperature-dependent Sex Determination via Small Blood Samples.

Temperature-dependent sex determination, present in most turtle species, is a mechanism that uses temperature to direct the sex of the embryo. The rapid increase of global temperatures highlights the need for a clear assessment of how sex ratios of organisms with TSD are affected. In turtles with TSD, quantifying primary sex ratios is challenging because they lack external dimorphism and heteromorphic sex chromosomes. Here we describe a new technique used to identify sex in neonate turtles of two TSD species, a freshwater turtle (Trachemys scripta) and a marine turtle (Caretta caretta) via analysis of small blood samples. We used an immunoassay approach to test samples for the presence of several proteins known to play an important role in sex differentiation. Our results show that Anti-Mullerian Hormone (AMH) can be reliably detected in blood samples from neonate male turtles but not females and can be used as a sex-specific marker. Verification of sex via histology or laparoscopy revealed that this method was 100% reliable for identifying sex in both T. scripta and C. caretta 1-2 day-old hatchlings and 90% reliable for identifying sex in 83-177 day-old (120-160 g) loggerhead juveniles. The method described here is minimally invasive, and for the first time, greatly enhances our ability to measure neonate turtle sex ratios at population levels across nesting sites worldwide, a crucial step in assessing the impact of climate change on imperiled turtle species.

Hydric environmental effects on turtle development and sex ratio.

Experimental and field studies of different turtle species suggest that moisture influences embryonic development and sex ratios, wetter substrates tend to produce more males, and drier substrates produce more females. In this study, we used Trachemys scripta elegans to test the effect of moisture on embryonic development and sex ratios. T. s. elegans eggs were incubated under different temperature and moisture regimes. We monitored embryonic development until stage 22 (after sex determination) and, for the first time, we estimated sex ratios using a male-specific transcriptional molecular marker, Sox9. Among treatments, we found differences in developmental rates, egg mass, and sex ratio. Embryos developed slowly in cooler and wetter sand substrate while water uptake by the eggs was significantly greater on wetter substrates. Developmental differences were due to moisture interacting with temperature where increased water content of the sand resulted in temperatures that were 2-3°C lower than air temperatures. The coolest and the wettest substrates produced 100% males compared to 42% males from the warmest and driest treatment. Further, we found that embryonic growth appears to be more sensitive to temperature at earlier stages of development and to moisture at later stages. This study shows how moisture may change the incubation conditions inside nests by changing the temperature experienced by eggs, which affects development, growth and sex ratios. The results of this study highlight the importance of including moisture conditions when predicting embryo growth and sex ratios and in developing proxies of embryonic development.

An Immunohistochemical Approach to Identify the Sex of Young Marine Turtles.

Marine turtles exhibit temperature-dependent sex determination (TSD). During critical periods of embryonic development, the nest's thermal environment directs whether an embryo will develop as a male or female. At warmer sand temperatures, nests tend to produce female-biased sex ratios. The rapid increase of global temperature highlights the need for a clear assessment of its effects on sea turtle sex ratios. However, estimating hatchling sex ratios at rookeries remains imprecise due to the lack of sexual dimorphism in young marine turtles. We rely mainly upon laparoscopic procedures to verify hatchling sex; however, in some species, morphological sex can be ambiguous even at the histological level. Recent studies using immunohistochemical (IHC) techniques identified that embryonic snapping turtle (Chelydra serpentina) ovaries overexpressed a particular cold-induced RNA-binding protein in comparison to testes. This feature allows the identification of females vs. males. We modified this technique to successfully identify the sexes of loggerhead sea turtle (Caretta caretta) hatchlings, and independently confirmed the results by standard histological and laparoscopic methods that reliably identify sex in this species. We next tested the CIRBP IHC method on gonad samples from leatherback turtles (Dermochelys coriacea). Leatherbacks display delayed gonad differentiation, when compared to other sea turtles, making hatchling gonads difficult to sex using standard H&E stain histology. The IHC approach was successful in both C. caretta and D. coriacea samples, offering a much-needed tool to establish baseline hatchling sex ratios, particularly for assessing impacts of climate change effects on leatherback turtle hatchlings and sea turtle demographics. Anat Rec, 300:1512-1518, 2017. © 2017 Wiley Periodicals, Inc.