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In reptiles, such as the red-eared slider turtle ( Trachemys scripta elegans), gonadal sex determination is highly dependent on the environmental temperature during embryonic stages. This complex process, which leads to differentiation into either testes or ovaries, is governed by the finely tuned expression of upstream genes, notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2. Recent studies have identified epigenetic regulation as a crucial factor in testis development, with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T. s. elegans. However, whether KDM6B alone can induce testicular differentiation remains unclear. In this study, we found that overexpression of Kdm6b in T. s. elegans embryos induced the male development pathway, accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C, a temperature typically resulting in female development. Notably, this sex reversal could be entirely rescued by Dmrt1 knockdown. These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway, underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

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The problem of the origin of the bony shell in turtles has a two-century history and still has not lost its relevance. First, this concerns the issues of the homology, the sources of formation and the ratio of bones of different nature, that is, thecal and epithecal, in particular. This article analyzes various views on the nature of the shell elements, and proposes their typification, based on modern data on developmental biology. It is proposed that the defining characteristic of the types of shell ossifications is not the level of their anlage in the dermis (thecality or epithecality), but, first of all, the primary sources of their formation: (1) neural crest (nuchal and plastral plates); (2) vertebral and rib periosteum (neural and costal plates); and (3) dermal mesenchyme (peripheral, suprapygal and pygal plates, as well as epithecal elements). In addition, there is complete correspondence between these types of ossifications and the sequence of their appearance in the turtle ontogenesis. The data show fundamental coincidence of the modifications of the ontogenetic development and evolutionary formation of the shell ossifications and are in agreement with a stepwise model for the origin of the turtle body plan. Particular attention is paid to the origin of the epithecal elements of the turtle shell, which correspond to the additional or supernumerary ossifications and seem to have wider distribution among turtles, than previously thought.

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Ovarian development was traditionally recognized as a "default" sexual outcome and therefore received much less scientific attention than testis development. In turtles with temperature-dependent sex determination (TSD), how the female pathway is initiated to induce ovary development remains unknown. In this study, we have found that phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3) and Foxl2 exhibit temperature-dependent sexually dimorphic patterns and tempo-spatial coexpression in early embryos of the red-eared slider turtle (Trachemys scripta elegans). Inhibition of pSTAT3 at a female-producing temperature of 31 °C induces 64.7% female-to-male sex reversal, whereas activation of pSTAT3 at a male-producing temperature of 26 °C triggers 75.6% male-to-female sex reversal. In addition, pSTAT3 directly binds to the locus of the female sex-determining gene Foxl2 and promotes Foxl2 transcription. Overexpression or knockdown of Foxl2 can rescue the sex reversal induced by inhibition or activation of pSTAT3. This study has established a direct genetic link between warm temperature-induced STAT3 phosphorylation and female pathway initiation in a TSD system, highlighting the critical role of pSTAT3 in the cross talk between female and male pathways.

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Successful embryonic development depends on the interaction between genetic factors and environmental variables. Congenital malformations in sea turtles can result from extreme conditions during the incubation period, reducing hatching success and potentially impeding population recovery. We aimed to characterize the congenital malformations found in green turtle nests, determine their prevalence and severity, and understand their drivers during the 2022 nesting season on Samandag beach on northern Mediterranean nesting beaches. A total of 2986 examples of congenital malformations were observed in 362 out of 907 green turtle nests. The prevalence of congenital malformations per nest was 39%, and the severity (the number of malformed individuals per nest) was 3.8%. Nests with congenital malformations exhibited a lower mean distance from the sea, a shorter incubation duration (a proxy for incubation temperature), lower hatching success, a larger clutch size, and higher mortality at late embryonic and hatchling stages than nests without congenital malformations. There was no significant difference in total mortality between these two nest types. A total of 52 different congenital malformations were recorded, 2 of which were observed for the first time in sea turtles and 28 for the first time in green turtles. The results suggest that congenital malformations may be related to nest temperature and clutch size, while overall mortality may be independent of malformations. Pigmentation disorders and craniofacial malformations typically coexist in cases of multiple malformations. Long-term monitoring of congenital malformations is crucial, as it can provide clues about the health status of the nesting beach and nesting colony.

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Fifty years ago, researchers discovered a link between ambient temperature and the sex of turtle embryos. More recently, significant progress has been made in understanding the influence of temperature on freshwater turtles. However, our understanding of the key genetic factors in other turtle groups, such as sea turtles, remains limited. To address this gap, we conducted RNA-seq analyses on embryonic tissues from the sea olive ridley turtle during the thermosensitive period (stages 21-26) at temperatures known to produce males (26 °C) and females (33 °C). Our findings revealed that incubation temperatures primarily influence genes with broad expression across tissues due to differential cell division rates and later have an effect regulating gonad-specific transcripts. This effect is mostly related to gene activation rather than transcription repression. We performed transcriptome analyses following shifts in incubation temperatures of bi-potential gonads. This approach allowed us to identify genes that respond rapidly and may be closer to the beginning of the temperature-sensing pathway. Notably, we observed swift adaptations in the expression levels of chromatin modifiers JARID2 and KDM6B, as well as the splicing factor SRSF5, and transcription regulators THOC2, DDX3X and CBX3, but little impact in the overall gonad-specific pathways, indicating that temperature-sensing genes may change rapidly but the rewiring of the gonad's developmental fate is complex and resilient. AUTHOR SUMMARY: Sea turtles, one of the most iconic creatures of our oceans, confront a troubling reality of endangerment, a peril magnified by the looming specter of climate change. This climatic shift is gradually increasing the temperature of the nesting beaches thus causing dramatic male/female population biases. Conservation efforts will need genetic and molecular information to reverse the negative effects of climate change on the populations. In this study, we conducted the first transcriptomic analysis of embryonic tissues, including gonads, brain, liver, and mesonephros, in the olive ridley sea turtle during the critical thermosensitive period spanning stages 21-26. We examined both male-producing (26 °C) and female-producing (33 °C) temperatures and found that incubation temperatures influence temperature-sensitive genes that are either expressed globally or specifically associated with the gonads. These findings indicate that incubation temperatures predominantly sway genes with broad expression patterns due to differential cell division rates. This natural process was opted in the gonads to drive sex determination. We also identified genes that are rapidly capable of sensing temperature changes and that could play a role in the activation of the sex determination pathway. Overall, our study sheds light on the intricate interplay between temperature and gene expression during sea turtle development, revealing dynamic changes in the transcriptome and highlighting the involvement of key genetic players in sex determination.

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Background: The developmental biology of Kinosternon scorpioides is described, based on the phenotype. This species is important for the flora because they are excellent seed disseminators. In addition, basic embryological information is not yet fully clarified, and this research provides unprecedented information on the chelonian embryology of the Amazonian fauna. Aim: The present study aims to identify the embryology of K. scorpioides in captivity during different periods. Methods: Females were monitored throughout the reproductive cycle, by video monitoring, to identify nests and the presence of newly laid eggs. At regular weekly intervals, embryo samples were collected fixed in a 4% paraformol solution and preserved in 70% alcohol. For the embryonic characterization, we used a stereomicroscope and the scanning electron microscopy method. Results: We describe 15 embryonic stages for a 15-week (105-day) incubation process. Only at 42 days (6th week) was the morphological characterization of a chelonian observed and at the 12th week (Stage XII), the phenotypic characterization of the species K. scorpioides. Conclusion: In view of the evidence, we found that these phases are similar to the other turtles, with structural variations in the appearance and disappearance of structures due to the specific characteristics of the species.

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Sea turtles, with their global distribution and complex life cycle, often accumulate pollutants such as metals and metalloids due to their extended lifespan and feeding habits. However, there are limited studies exploring the impact of metal pollution on the reproductive health of female sea turtles, specifically focusing on the quality of their eggs, which has significant implications for the future generations of these charismatic animals. São Tomé Island, a crucial nesting and feeding habitat for green sea turtles, underscores the urgent need for comprehensive research in this ecologically significant area. This study aimed to investigate whether metals and metalloids in the blood of nesting female green sea turtles induce genotoxic effects in their erythrocytes and affect their egg morphometric characteristics and the composition of related compartments. Additionally, this study aimed to evaluate whether the quality of energetic reserves for embryo development (fatty acids in yolk's polar and neutral lipids) is influenced by the contamination status of their predecessors. Results revealed correlations between Cu and Hg levels and increased "lobed" erythrocytes, while As and Cu negatively influenced shell thickness. In terms of energy reserves, both polar and neutral lipid fractions contained primarily saturated and monounsaturated fatty acids, with prevalent 18:1n-9, 18:0, 16:0, 14:0, and 12:0 fatty acids in yolk samples. The yolk polar fraction was more susceptible to contaminant levels in female sea turtles, showing consistent negative correlations between pollution load index and essential n3 fatty acids, including linolenic, eicosatrienoic, eicosapentaenoic, and docosapentaenoic acids, crucial for embryonic development. These metals accumulation, coupled with the reduced availability of these key fatty acids, may disrupt the eicosanoid and other important pathways, affecting reproductive development. This study reveals a negative correlation between metal contamination in female sea turtles' blood and egg lipid reserves, raising concerns about embryonic development and the species' future generations.

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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.

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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.

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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)'.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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