Anidación de la tortuga marina Lepidochelys olivacea (Testudinata: Cheloniidae) en Campamento Tortuguero Cedeño, Honduras (2011-2021) / Nesting of olive ridley sea turtle Lepidochelys olivacea (Testudinata: Cheloniidae) in Campamento Tortuguero Cedeño, Honduras (2011-2021)

Introducción: El Campamento Tortuguero de Cedeño ha sido el sitio menos investigado del Golfo de Fonseca, donde se protege a la tortuga golfina en Honduras desde 1975. Objetivo: Evaluar la anidación de la tortuga Golfina (Lepidochelys olivacea) durante la temporada de veda entre el 2011 y 2021 en Campamento Tortuguero Cedeño, Choluteca, Honduras. Métodos: Entre 2011 a 2021, se llevó a cabo el monitoreo diario de las actividades de anidación durante la veda del 1 al 25 de septiembre. Los patrullajes se realizaron entre las 6:00-18:00 h, y las 18:00-5:00 h. Se registró el número total de tortugas que anidaban y se recogieron sus huevos, que se transportaron al criadero, donde se tabularon los resultados de las puestas y las crías. Resultados: Se registró un total 1 065 tortugas de L. olivacea, 95 051 huevos recolectados, 1 065 nidos marcados en tres playas que fueron reubicados en viveros artificiales y una eclosión exitosa de 62 747 neonatos. La playa Las Doradas fue el sitio con el mayor número de tortugas anidadoras, seguido de Los Delgaditos y por último Cedeño. El promedio de la frecuencia de anidación fue de 96 nidos. Del 2011 al 2021 el esfuerzo de recolección de los nidos aumentó en un 91.6 %, pasando de 84 a 161 nidos. El número de personas patrullando se asoció con la cantidad de nidos detectados en las playas. Conclusión: Los esfuerzos de monitoreo y conservación para la especie han indicado que ha habido un incremento en la anidación de L. olivacea en las tres playas, con un mayor incremento en Playa Las Doradas. Este escenario comprueba la funcionalidad de la veda en esta zona.

Introduction: The Cedeño Turtle Camp has been the least researched site in the Fonseca Gulf, where Olive Ridley Turtles in Honduras have been protected since 1975. Objective: To evaluate the nesting of Olive Ridley turtles (Lepidochelys olivacea) during the closed season from 2011 to 2021 in Campamento Tortuguero Cedeño, Choluteca, Honduras. Methods: From 2011 to 2021, daily monitoring of nesting activities was conducted during the closed season from the 1st to 25th of September. Patrols were conducted between 6:00-18:00 h, and 18:00-5:00 h. The total number of nesting turtles was recorded, and their eggs were collected and transported to the hatchery, where clutch and hatchling performance were tabulated. Results: A total of 1 065 L. olivacea turtles were recorded, 95 051 eggs collected, 1 065 nests marked on three beaches that were relocated in artificial hatcheries and a successful hatching of 62 747 hatchlings. Las Doradas beach was the site with the highest number of nesting turtles, followed by Los Delgaditos and lastly Cedeño. The average nesting frequency was 96 nests. From 2011 to 2021 the nest collection effort increased by 91.6 %, from 84 to 161 nests. The number of people patrolling was associated with the number of nests detected on the beaches. Conclusion: Monitoring and conservation efforts for L. olivacea in the Campamento Tortuguero Cedeño show a positive trend in nesting with a greater increase in Playa Las Doradas. This scenario proves the functionality of the closed season in this area.

Incubating green turtle (Chelonia mydas) eggs at constant temperatures: Hatching success, hatchling morphology and post-hatch growth.

Past studies applying constant-temperature incubation of eggs have involved all species of sea turtles, but rarely can we find a single one incubating eggs at three or more temperatures. Here, we incubated green turtle (Chelonia mydas) eggs from Ganquan Island, South China Sea, at five constant temperatures (26, 28, 30, 32 and 34 °C) to determine hatching success, incubation length and hatchling phenotype at each test temperature and temperatures optimal for embryonic development. Temperature affected hatching success, incubation length and all seven examined hatchlings traits, and clutch origin affected three (head length, fore-flipper length and hind-flipper length) of the seven. Hatching success was lowest at 34 °C and none of hatchlings hatched at this temperature was normal and survived over one week. The rate of embryonic development and the rate of post-hatch growth both were lowest at 26 °C. Given that low survival and growth rates can translate into reduced individual fitness, we conclude that both 26 °C and 34 °C are unsuitable for incubation of C. mydas eggs. Post-hatch growth was fastest in hatchlings incubated at 30 °C, and eggs of C. mydas incubated at temperatures around 30 °C are more likely to produce mixed sexes. Accordingly, we conclude that temperatures within the range from 28 °C to 32 °C are generally optimal for embryonic development of C. mydas.

Prenatal hypoxia affects scaling of blood pressure and arterial wall mechanics in the common snapping turtle, Chelydra serpentina.

In reptiles, exposure to hypoxia during embryonic development affects several cardiovascular parameters. These modifications may impose different mechanical stress to the arterial system, and we speculated that the arterial wall of major outflow vessels would be modified accordingly. Since non-crocodilian reptiles possess a partially divided ventricle, ensuing similar systemic and pulmonary systolic pressures, we investigated how morphological and mechanical properties of segments from the left aortic arch (LAo) and the proximal and distal segments of the left pulmonary artery (LPAp and LPAd, respectively) change as body mass (Mb) increases. Eggs from common snapping turtles, Chelydra serpentina, were incubated under normoxia (21% O2; N21) or hypoxia (10% O2; H10), hatched and maintained in normoxia thereafter. Turtles (0.11-6.85 kg) were cannulated to measure arterial pressures, and an injection of adrenaline was used to increase pressures. Portions of the LAo, LPAp and LPAd were fixed under physiological hydrostatic pressures for histology and mechanical assessment. Arterial pressures increased with Mb for N21 but not for H10. Although mechanical and functional characteristics from the LPAp and LPAd were similar between N21 and H10, wall thickness from LAo did not change with Mb in the H10 group, thus wall stress increased in larger turtles. This indicates that larger H10 turtles probably experience an elevated probability of arterial wall rupture without concomitant changes in the cardiovascular system to prevent it. Finally, collagen content of the LPAp and LAo was smaller than in LPAd, suggesting a more distensible arterial wall could attenuate higher pressures from larger turtles.

Thermosensitive sex chromosome dosage compensation in ZZ/ZW softshell turtles, Apalone spinifera.

Sex chromosome dosage compensation (SCDC) overcomes gene-dose imbalances that disturb transcriptional networks, as when ZW females or XY males are hemizygous for Z/X genes. Mounting data from non-model organisms reveal diverse SCDC mechanisms, yet their evolution remains obscure, because most informative lineages with variable sex chromosomes are unstudied. Here, we discovered SCDC in turtles and an unprecedented thermosensitive SCDC in eukaryotes. We contrasted RNA-seq expression of Z-genes, their autosomal orthologues, and control autosomal genes in Apalone spinifera (ZZ/ZW) and Chrysemys picta turtles with temperature-dependent sex determination (TSD) (proxy for ancestral expression). This approach disentangled chromosomal context effects on Z-linked and autosomal expression, from lineage effects owing to selection or drift. Embryonic Apalone SCDC is tissue- and age-dependent, regulated gene-by-gene, complete in females via Z-upregulation in both sexes (Type IV) but partial and environmentally plastic via Z-downregulation in males (accentuated at colder temperature), present in female hatchlings and a weakly suggestive in adult liver (Type I). Results indicate that embryonic SCDC evolved with/after sex chromosomes in Apalone's family Tryonichidae, while co-opting Z-gene upregulation present in the TSD ancestor. Notably, Apalone's SCDC resembles pygmy snake's, and differs from the full-SCDC of Anolis lizards who share homologous sex chromosomes (XY), advancing our understanding of how XX/XY and ZZ/ZW systems compensate gene-dose imbalance. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

Influence of incubation temperature on embryo development, hatchling morphology and early growth rate in red-footed tortoise (Chelonoidis carbonaria).

Reptile embryos respond to temperature changes with metabolic and physiological adjustments that influence hatchling success, phenotype, behaviour, and growth rate. Climate change and global warming can affect the reptile population by altering the frequencies of hatchling survival and phenotypes. Therefore, previous studies proposed artificial incubation as a potential strategy for mitigating these effects. Red-footed tortoise (Chelonoidis carbonaria) eggs were collected and incubated at constant temperatures of 27.5 °C and 29.5 °C to investigate the physiological effects of temperature on embryo development, hatchling morphology, and early post-hatch growth rate. The direct effect of temperature on the incubation period, egg mass loss, hatching success, hatchling size, and mass was evaluated at hatching and three months of age. Hatchlings from 29.5 °C presented a shorter incubation period (141 days) than those from 27.5 °C (201 days; p < 0.05). Egg mass loss, hatchling mass, and size at hatching were not different between the incubation temperatures (p > 0.05). However, the hatching success (survival rate) was lower (64.5% versus 100%) in eggs incubated at 29.5 °C, but the hatchling mass and straight plastron width were higher at three months of age than those from eggs incubated at 27.5 °C (p < 0.05). These results indicate that incubation temperature influences hatching success and hatchling size and mass in the first months by influencing the early growth rate.

Vasa expression is associated with sex differentiation in the Asian yellow pond turtle, Mauremys mutica.

Vasa, one of the best-studied germ cell markers plays a critical role in germ cell development and differentiation in animals. Vasa deficiency would lead to male-specific sterility in most vertebrates, but female sterility in the fly. However, the role of the vasa gene involved in germ cell differentiation is largely elusive. Here, we first characterized the expression profile of vasa products in the Asian yellow pond turtle by quantitative reverse-transcription polymerase chain reaction and fluorescence immunostaining. The results showed that vasa messenger RNA (mRNA) is initially detected in embryos at stage 16, and then dramatically increased in embryos at stage 19. In particular, like the sex-related genes, vasa mRNA exhibited differential expression in embryos between the male-producing temperature (MPT, 25°C) and the female-producing temperature (FPT, 33°C), whereas there was no difference in methylation levels of vasa promoter detected between FPT and MPT. In contrast, in the adult Asian yellow pond, the level of vasa mRNA was much higher in the testis than ovary. Moreover, the immunostaining on testicular sections and cells showed that Vasa protein was exclusively expressed in germ cells: Weak but detectable in spermatogonia, highest in spermatocytes, moderate and concentrated in chromatid bodies in spermatids and spermatozoa, and bare in somatic cells. The expression profile of Vasa protein is similar in turtle species studied so far but distinct from those in fish species in this study. The findings of this study would provide new insights into our understanding of the conservation and divergence of the vasa gene, even other germ cell genes across phyla.

Cardiovascular responses to putative chemoreceptor stimulation of embryonic common snapping turtles (Chelydra serpentina) chronically incubated in hypoxia (10% O2).

Developmental hypoxia has been shown to result in significant changes in cardiovascular development of American alligators and common snapping turtles. These include similar effects on cardiac mass and aspects of cardiovascular function. However, given the distant phylogenetic relationship between crocodilians and chelonians, we hypothesized that snapping turtles would also exhibit differences in the effects of developmental hypoxia on cardiovascular regulation. This hypothesis was based in part on prior studies that documented differences in plasticity of vagal tone on the heart between alligators and snapping turtles incubated in hypoxic conditions. To test this hypothesis, we investigated how 10% O2 exposure over final 80% of incubation altered the heart rate and blood pressure response to two chemical manipulations of the "chemoreflex" in common snapping turtles at 70% and 90% of incubation. NaCN injections produced a dose dependent bradycardia that was mediated by cholinergic receptor stimulation. This reflex was relatively unaffected by hypoxic incubation conditions in snapping turtle embryos. Injections of the 5-HT3 agonist phenylbiguanide (PBG) caused a pronounced bradycardia that decreased in intensity at 90% of incubation in embryos from the normoxic group while the heart rate response was unchanged in the hypoxic group. This differs from the previously reported diminished heart rate response of embryonic alligators incubated in 10% O2, suggesting plasticity in this chemoreflex response differs between the species. Our data also indicate the cardiovascular response is mediated by a secondary cholinergic receptor stimulation however the inability of ganglionic blockade to inhibit the PBG response leaves the location of the receptors antagonized by PBG in question in embryonic snapping turtles. Primarily, our findings refute the hypothesis that hypoxic incubation decreases the "chemoreflex' response of snapping turtle embryos.

Comparative study of collagen distribution in the dermis of the embryonic carapace of soft- and hard-shelled cryptodiran turtles.

Turtles are characterized by their typical carapace, which is primarily composed of corneous beta proteins in the horny part and collagen in the dermal part. The formation of the extracellular matrix in the dermis of the carapace in a hard-shelled and a soft-shelled turtle has been compared. The study examines carapace development, with an emphasis on collagen accumulation, in the soft-shelled turtle Pelodiscus sinensis and hard-shelled turtle Trachemys scripta elegans, using comparative morphological and embryological analyses. The histological results showed that collagen deposition in the turtle carapace increased as the embryos developed. However, significant differences were observed between the two turtle species at the developmental stages examined. The microstructure of the dermis of the carapace of P. sinensis showed light and dark banding of collagen bundles, with a higher overall collagen content, whereas the carapacial matrix of T. scripta was characterized by loosely packed and thinner collagenous fiber bundles with a lower percentage of type I collagen. Overall, the formation and distribution of collagen fibrils at specific developmental stages are different between the soft-and hard-shelled turtles. These results indicate that the pliable epidermis of the soft-shelled turtle is supported by a strong dermis that is regularly distributed with collagen and that it allows improved maneuvering, whereas a strong but inflexible epidermis as observed in case of hard-shelled turtles limits movement.

Development of scleral ossicles in Podocnemis expansa (Testudines: Podocnemididae) embryos exposed to atrazine.

Understanding the effects of atrazine exposure on embryo development in oviparous animals may provide important data regarding the impacts of agrochemical use on wildlife and the ecosystem. This study set out to determine the effects of embryonic atrazine exposure on the development of osseous and cartilaginous components of scleral ossicles in Podocnemis expansa. Eggs were collected at the Environmental Protection Area Meandros do Rio Araguaia, Brazil, and artificially incubated in sand treated with solutions containing 2, 20 or 200 µg/L of atrazine. Sixty embryos were collected per treatment throughout the incubation period. Embryos were diaphanized with potassium hydroxide (KOH) and stained with Alizarin Red S and Alcian blue (bone and cartilage tissue respectively). Scleral ossicles were then counted and examined for skeletal abnormalities at different stages of embryonic development. Scleral ossicle counts were significantly reduced in P. expansa embryos treated with 200 µg/L atrazine solution. Rudimentary ossicles and gaps were also noted in embryos exposed to atrazine concentrations of 2 µg/L or 200 µg/L. Findings of this study emphasize the relevance of ecotoxicological investigations in determining the impacts of agrochemicals on native fauna.

A developmental staging system and musculoskeletal development sequence of the common musk turtle (Sternotherus odoratus).

BACKGROUND: The extremely derived body plan of turtles has sparked a great interest in studying their developmental biology. Here, we describe the embryonic development of the Stinkpot, or common musk turtle (Sternotherus odoratus), a small aquatic turtle from the family Kinosternidae. RESULTS: We identify 20 distinct developmental stages, some comparable to stages described by previous studies on other turtles and some in between these, improving the resolution of the generalities of turtle development. We provide a detailed account of both the external morphology and skeletal development, as well as a general look at the early stages of muscular development until the attainment of the adult muscular anatomical pattern. CONCLUSIONS: Several potential skeletal and muscular apomorphies of turtles are identified or elaborated. The musk turtle, with its small size and hard-shelled egg, could become an important species for the study of turtle evolution and development, suitable for in ovo experimentation and late stage imaging of well-advanced anatomical features.

Thermosensitive period for sex determination of the tropical freshwater turtle Malayemys macrocephala.

Many egg-laying reptiles possess temperature-dependent sex determination (TSD) in which outcome of gonadogenesis is determined by incubation temperature during a temperature-sensitive period of development. Prior studies on Malayemys macrocephala showed that incubation temperatures influence gonadal development and suggested that M. macrocephala exhibits TSD. However, information on the temperature-sensitivity period in this species was unknown until the current study. Turtle eggs were collected from rice fields in central Thailand from December 2016 to February 2017. In the laboratory, eggs were incubated at male-biased temperature (26 °C) and shifted to female-biased temperature (32 °C), or vice versa. Single shift experiments were performed systematically during embryonic stages 13-21. After hatching, sex of individual turtles was determined by histological analysis. We found that the sex determination of M. macrocephala is affected by temperature up to stage 16 of embryonic development.

The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles.

Sea turtle embryos at high-density nesting beaches experience relative high rates of early stage embryo death. One hypothesis to explain this high mortality rate is that there is an increased probability that newly constructed nests are located close to maturing clutches whose metabolising embryos cause low oxygen levels, high carbon dioxide levels, and high temperatures. Although these altered environmental conditions are well tolerated by mature embryos, early stage embryos, i.e. embryos in eggs that have only been incubating for less than a week, may not be as tolerant leading to an increase in their mortality. To test this hypothesis, we incubated newly laid sea turtle eggs over a range of temperatures in different combinations of oxygen and carbon dioxide concentrations and assessed embryo development and death rates. We found that gas mixtures of decreased oxygen and increased carbon dioxide, similar to those found in natural sea turtle nests containing mature embryos, slowed embryonic development but did not influence the mortality rate of early stage embryos. We found incubation temperature had no effect on early embryo mortality but growth rate at 27°C and 34°C was slower than at 30°C and 33°C. Our findings indicate that low oxygen and high carbon dioxide partial pressures are not the cause of the high early stage embryo mortality observed at high-density sea turtle nesting beaches, but there is evidence suggesting high incubation temperatures, particularly above 34°C are harmful. Any management strategies that can increase the spacing between nests or other strategies such as shading or irrigation that reduce sand temperature are likely to increase hatching success at high-density nesting beaches.

Regulation of the limb shape during the development of the Chinese softshell turtles.

Interdigital cell death is an important mechanism employed by amniotes to shape their limbs; inhibiting this process leads to the formation of webbed fingers, as seen in bats and ducks. The Chinese softshell turtle Pelodiscus sinensis (Reptilia: Testudines: Trionychidae) has a distinctive limb morphology: the anterior side of the limbs has partially webbed fingers with claw-like protrusions, while the posterior fingers are completely enclosed in webbings. Here, P. sinensis embryos were investigated to gain insights on the evolution of limb-shaping mechanisms in amniotes. We found cell death and cell senescence in their interdigital webbings. Spatial or temporal modulation of these processes were correlated with the appearance of indentations in the webbings, but not a complete regression of this tissue. No differences in interdigital cell proliferation were found. In subsequent stages, differential growth of the finger cartilages led to a major difference in limb shape. While no asymmetry in bone morphogenetic protein signaling was evident during interdigital cell death stages, some components of this pathway were expressed exclusively in the clawed digit tips, which also had earlier ossification. In addition, a delay and/or truncation in the chondrogenesis of the posterior digits was found in comparison with the anterior digits of P. sinensis, and also when compared with the previously published pattern of digit skeletogenesis of turtles without posterior webbings. In conclusion, modulation of cell death, as well as a heterochrony in digit chondrogenesis, may contribute to the formation of the unique limbs of the Chinese softshell turtles.

Investigating the effects of nest shading on the green turtle (Chelonia mydas) hatchling phenotype in the Ogasawara islands using a field-based split clutch experiment.

The Ogasawara Islands are an important rookery for the green turtle (Chelonia mydas) in the North Pacific. Green turtles possess temperature-dependent sex determination, and warmer incubation temperatures produce more females than males. Therefore, conservation practices such as nest shading may be required for this population to mitigate the effect of global warming on their sex ratio. To consider the application of such conservation practices in the Ogasawara population, it is fundamental to understand how artificially modified nest environments will affect green turtle hatchling phenotypes that influence their fitness. Here, we investigated the effects of nest shading on green turtle hatchling phenotypes in the Ogasawara population by using a split clutch experiment equally separating the clutch, relocating each half-clutch into an outdoor hatchery area either with or without shading, and observing the subsequent hatchling phenotype. Our results showed that the shading treatment produced hatchlings with a better self-righting response and a larger carapace size. Additionally, the shading treatment mostly reduced the production of hatchlings with a nonmodal scute pattern and produced hatchlings with a smaller unabsorbed yolk sac, which may be associated with their residual yolk mass. These results suggest that conservation practices such as shading could alter not only the sex ratio but also the hatchling phenotype that influences their fitness. Hence, our results suggest that applications of such conservation strategies must be carefully considered.

Transcriptomic similarities and differences between the limb bud AER and unique carapacial ridge of turtle embryos.

Evolutionary innovation may arise via major departures from an ancestral condition. Turtle shell morphogenesis depends on a unique structure known as the carapacial ridge (CR). This lateral tissue protrusion in turtle embryos exhibits similar properties as the apical ectodermal ridge (AER)-a well-known molecular signaling center involved in limb development. Still, how the CR influences shell morphogenesis is not entirely clear. The present study aimed to describe the CR transcriptome shortly before ribs were halted within its mesenchyme, as required for shell development. Analyses exposed that the mesenchymal marker VIM was one of the most highly co-expressed genes and numerous appendage formation genes were situated within the core of CR and AER co-expression networks. However, there were tissue-specific differences in the activity of these genes. For instance, WNT5A was most frequently assigned to appendage-related annotations of the CR network core, but not in the AER. Several homeobox transcription factors known to regulate limb bud patterning exhibited their highest expression levels in the AER, but were underexpressed in the CR. The results of this study corroborate that novel body plans often originate via alterations of pre-existing genetic networks. Altogether, this exploratory study enhances the groundwork for future experiments on the molecular underpinnings of turtle shell development and evolution.

Introduction of Developmental Biology at Utkal University, (Odisha, India).

The paper deals with the background and the establishment of a Developmental Biology Laboratory in Utkal University in Odisha state. It describes the process from a humble beginning with limited facilities into a leading research centre, initially for amphibians and later for the endangered olive ridley (Lepidochelys olivacea) turtle. Starting from the biology, reproduction and development in many anurans, the laboratory took up research on regeneration, especially on super-regeneration in tadpoles under the influence of morphogens such as vitamin A (retinoids). Treatment with vitamin A after amputation of the tail inhibited tail regeneration but unexpectedly induced homeotic transformation of tails into limbs in many anurans, starting with the marbled balloon frog Uperodon systoma. This was the first observation of homeotic transformation in any vertebrate. The laboratory continues research on histological and molecular aspects of this phenomenon. In addition, taking advantage of the largest rookery of olive ridley sea turtles in Gahirmatha, in the same state the laboratory has contributed significantly to the biology, breeding patterns, development and especially the temperature-dependent sex determination phenomenon (TSD). This research was extended to biochemical and ultrastructural aspects during development for the first time for any sea turtle. The laboratory has contributed significantly to the conservation of olive ridleys as well as the saltwater crocodile (Crocodylus porosus). Recognition and awards for the laboratory have been received from both national and international bodies.

Temperature-dependent sex determination is mediated by pSTAT3 repression of Kdm6b.

In many reptiles, including the red-eared slider turtle Trachemys scripta elegans (T. scripta), sex is determined by ambient temperature during embryogenesis. We previously showed that the epigenetic regulator Kdm6b is elevated at the male-producing temperature and essential to activate the male pathway. In this work, we established a causal link between temperature and transcriptional regulation of Kdm6b We show that signal transducer and activator of transcription 3 (STAT3) is phosphorylated at the warmer, female-producing temperature, binds the Kdm6b locus, and represses Kdm6b transcription, blocking the male pathway. Influx of Ca2+, a mediator of STAT3 phosphorylation, is elevated at the female temperature and acts as a temperature-sensitive regulator of STAT3 activation.

The impact of sand moisture on the temperature-sex ratio responses of developing loggerhead (Caretta caretta) sea turtles.

All species of sea turtles exhibit a cooler male/warmer female temperature-sex ratio response. Field and experimental studies on loggerhead sea turtle sex ratios suggest that increased sand moisture impacts sea turtle sex ratios with, and perhaps beyond, a cooling effect. This study examines how varying sand moisture impacts the embryo's response to temperature. Across three years, loggerhead (Caretta caretta) sea turtle eggs were incubated at temperatures ranging from 28.0°C to 33.0°C. Groups of eggs were incubated in one of three volumetric moisture contents: low, medium, or high. Temperatures inside the group of eggs were recorded throughout incubation. Hatchlings were raised for 2-3 months and sex was identified laparoscopically. We calculated temperature response curves for groups of eggs incubated at each moisture level. Pivotal temperatures did not different among eggs incubated in different sand moistures. The transitional ranges of temperatures (TRT) for eggs incubated in high moisture and low moisture were narrower than the TRT for eggs incubated in medium moisture. The results of this study are crucial for understanding how sea turtle embryos respond to temperature directly or indirectly under different moisture conditions. Current sex ratio predictions rely on the embryos response to temperature only and may inaccurately estimate sex ratios especially during periods of heavy rainfall or drought.

Sex and Incubation Temperature Independently Affect Embryonic Development and Offspring Size in a Turtle with Temperature-Dependent Sex Determination.

Developmental environments can have lasting effects on an individual's phenotype. In many reptiles, for example, egg incubation temperature permanently determines offspring sex (temperature-dependent sex determination, TSD) and also influences a suite of morphological, physiological, and behavioral traits. Thus, the contributions of sex and incubation temperature to phenotypic variation are difficult to identify because these factors are confounded under TSD. We used chemical manipulations to experimentally decouple gonadal sex and incubation temperature in a turtle with TSD (Chrysemys picta) to examine their relative and interactive effects on variation in incubation duration and offspring size. We show that warm incubation temperature accelerates development as expected and that exogenous estradiol treatment to eggs further shortens incubation duration across all incubation temperatures. Moreover, estradiol unexpectedly induced male development, resulting in male offspring hatching sooner than female offspring. Variation in offspring size was also influenced by incubation temperature and gonadal sex, but interactions between these two variables were relatively small or nonsignificant. The fitness consequences of these effects are unknown, but we provide preliminary results from our attempts at examining the long-term and sex-specific effects of incubation temperature. Manipulative experimental approaches, combined with longer-term experiments that track individuals through reproduction, will provide novel insights into the adaptive significance of developmental plasticity in long-lived organisms.