Higher temperatures directly increase germ cell number, promoting feminization of red-eared slider turtles.

In many reptile species, gonadal sex is affected by environmental temperature during a critical period of embryonic development-a process known as temperature-dependent sex determination (TSD).1 The oviparous red-eared slider turtle, Trachemys scripta, has a warm-female/cool-male TSD system and is among the best-studied members of this group.2 When incubated at low temperatures, the somatic cells of the bipotential gonad differentiate into Sertoli cells, the support cells of the testis, whereas at high temperatures, they differentiate into granulosa cells, the support cells of the ovary.3 Here, we report the unexpected finding that temperature independently affects the number of primordial germ cells (GCs) in the embryonic gonad at a time before somatic cell differentiation has initiated. Specifically, embryos incubated at higher, female-inducing temperatures have more GCs than those incubated at the male-inducing temperature. Furthermore, elimination of GCs in embryos incubating at intermediate temperatures results in a strong shift toward male-biased sex ratios. This is the first evidence that temperature affects GC number and the first evidence that GC number influences sex determination in amniotes. This observation has two important implications. First, it supports a new model in which temperature can impact sex determination in incremental ways through multiple cell types. Second, the findings have important implications for a major unresolved question in the fields of ecology and evolutionary biology-the adaptive significance of TSD. We suggest that linking high GC number with female development improves female reproductive potential and provides an adaptive advantage for TSD.

Long-Term Resilience of Primary Sex Ratios in a Species with Temperature-Dependent Sex Determination after Decades of Climate Warming.

AbstractSpecies with environmental sex determination (ESD) have persisted through deep time, despite massive environmental perturbation in the geological record. Understanding how species with temperature-dependent sex determination (TSD), a type of ESD, persist through climate change is particularly timely given the current climate crisis, as highly biased sex ratios and extinction are predicted. Since 1982, we have studied primary sex ratios of a reptile with TSD (Chelydra serpentina). Primary sex ratios remained unchanged over time, despite warming in the environment. Resilience of the primary sex ratio occurred via a portfolio effect, realized through remarkable intra-annual variation in nest-level sex ratios, leading to a relatively consistent mean annual sex ratio. Intra-annual variation in nest-level sex ratios was related to variation in egg burial depth coupled with large clutch sizes, creating thermal gradients in the nest and promoting mixed-sex clutches. Furthermore, both locally and globally, sustained increases in nighttime air temperature contribute more to warming than increases in daily maximum temperature, but development rate was affected more strongly by maximum daily air temperature, conferring additional resilience to overall warming. Our study suggests that some TSD species may be resilient to warming and provides an example of how ESD may persist under environmental change.

A Cautionary Tale of Sexing by Methylation: Hybrid Bisulfite-Conversion Sequencing of Immunoprecipitated Methylated DNA in Chrysemys picta Turtles with Temperature-Dependent Sex Determination Reveals Contrasting Patterns of Somatic and Gonadal Methylation, but No Unobtrusive Sex Diagnostic.

Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue.

Metabolic Rates and Thermal Thresholds of Embryonic Flatback Turtles (Natator depressus) from the North West Shelf of Australia.

AbstractNest microclimates influence embryonic development and survival in many lineages, including reptiles with temperature-dependent sex determination. These microclimates are dependent on physical drivers and biological processes, such as embryonic metabolism, that generate heat. The flatback turtle (Natator depressus) has among the largest hatchlings of the seven extant sea turtle species, making it an excellent candidate for quantifying the contribution of embryonic metabolism to the nest microclimate. Consequently, we measured embryonic metabolic rates, development rates, and the relationship between temperature and sex determination for a N. depressus population nesting at Cemetery Beach in Western Australia, a mainland beach characterized by high sand temperatures. Total oxygen consumed at 29.5°C during an average 52-d incubation period was 2,622 mL, total carbon dioxide produced was 1,886 mL, and estimated embryonic heat production reached 38 mW at 90% of development. Adjustment of metabolic rates to 32°C and 34°C increased peak heat production by 18% and 27%, respectively. The pivotal temperature (TPIV) producing an equal sex ratio was 30.3°C, mixed sexes were produced between 29.3°C and 31.2°C, and only females were produced above 31.2°C. The TPIV was similar (within 0.2°C) to that of an island rookery within the same genetic stock (North West Shelf), but the peak development rate (2.5% d-1) was estimated to be achieved at a temperature ~2.5°C higher (34.7°C) than the island rookery. Our results add to a growing consensus that thermal thresholds vary among sea turtle populations, even within the same genetic stock. Furthermore, we show that metabolic heat will have an appreciable impact on the nest microclimate, which has implications for embryonic survival and fitness under a future climate with warmer sand temperatures.

Predicting the effects of climate change on incubation in reptiles: methodological advances and new directions.

The unprecedented advancement of global climate change is affecting thermal conditions across spatial and temporal scales. Reptiles with temperature-dependent sex determination (TSD) are uniquely vulnerable to even fine-scale variation in incubation conditions and are a model system for investigating the impacts of shifting temperatures on key physiological and life-history traits. The ways in which current and predicted future climatic conditions translate from macro- to ultra-fine scale temperature traces in subterranean nests is insufficiently understood. Reliably predicting the ways in which fine-scale, daily and seasonally fluctuating nest temperatures influence embryonic development and offspring phenotypes is a goal that remains constrained by many of the same logistical challenges that have persisted throughout more than four decades of research on TSD. However, recent advances in microclimate and developmental modeling should allow us to move farther away from relatively coarse metrics with limited predictive capacity and towards a fully mechanistic model of TSD that can predict incubation conditions and phenotypic outcomes for a variety of reptile species across space and time and for any climate scenario.

Comparison of Adult Testis and Ovary MicroRNA Expression Profiles in Reeves' Pond Turtles (Mauremys reevesii) With Temperature-Dependent Sex Determination.

Some differentially expressed genes (DEGs) that encode key enzymes involved in steroidogenic biosynthesis (CYP19A1) and key molecules related to gonadal functions (DMRT1, SOX9, AMH, FOXL2, WNT4, RSPO2, and GDF9) have been identified in adult gonadal RNA-seq studies of Reeves' pond turtle (Mauremys reevesii) with temperature-dependent sex determination (TSD). Gonadal functional maintenance and gametogenesis comprises a highly regulated and coordinated biological process, and increasing evidence indicates that microRNAs (miRNAs) may be involved in this dynamic program. However, it is not clear how the regulatory network comprising miRNAs changes the expression levels of these genes. In this study, miRNA sequencing of adult testis and ovary tissues from M. reevesii detected 25 known and 379 novel miRNAs, where 60 miRNAs were differentially expressed in the testis and ovary. A total of 1,477 target genes based on the differentially expressed miRNAs were predicted, where 221 target genes also exhibited differential expression. To verify the accuracy of the sequencing data, 10 differentially expressed miRNAs were validated by quantitative reverse transcription real-time PCR, and were found to be consistent with the transcriptome sequencing results. Moreover, several miRNA/target gene pairs, i.e., mre-let-7a-5p/mre-let-7e-5p and CYP19A1, mre-miR-200a-3p and DMRT1, mre-miR-101-3p and SOX9, and mre-miR-138-5p and AMH were identified. To explore the regulatory role of miRNAs, we conducted target gene enrichment analysis of the miRNAs and 221 target genes in the regulatory network. The signaling pathways related to gonadal functional maintenance and gametogenesis based on the DEGs and target genes were then compared. Our findings provide crucial information to facilitate further research into the regulatory mechanisms involving miRNAs in turtle species with TSD.

The Duplicated Y-specific amhy Gene Is Conserved and Linked to Maleness in Silversides of the Genus Odontesthes.

Sex-determining genes have been successively isolated in several teleosts. In Odontesthes hatcheri and O. bonariensis, the amhy gene has been identified as a master sex-determining gene. However, whether this gene is conserved along related species is still unknown. In this study, the presence of amhy and its association with phenotypic sex was analyzed in 10 species of Odontesthes genus. The primer sets from O. hatcheri that amplify both amhs successfully generated fragments that correspond to amha and amhy in all species. The full sequences of amhy and amha isolated for four key species revealed higher identity values among presumptive amhy, including the 0.5 Kbp insertion in the third intron and amhy-specific insertions/deletions. Amha was present in all specimens, regardless of species and sex, whereas amhy was amplified in most but not all phenotypic males. Complete association between amhy-homologue with maleness was found in O. argentinensis, O. incisa, O. mauleanum, O. perugiae, O. piquava, O. regia, and O. smitti, whereas O. humensis, O. mirinensis, and O. nigricans showed varied degrees of phenotypic/genotypic sex mismatch. The conservation of amhy gene in Odontesthes provide an interesting framework to study the evolution and the ecological interactions of genotypic and environmental sex determination in this group.

The Influence of Temperature on Embryonic Respiration, Growth, and Sex Determination in a Western Australian Population of Green Turtles (Chelonia mydas).

The thermal environment of sea turtle embryos has marked effects on many aspects of their development and energetics and has consequences for posthatching stages. Here we incubated Chelonia mydas embryos from Ningaloo Reef in Western Australia at a range of temperatures (27°, 29°, 30°, 31°, 32°, and 30° ± 5°C) to determine development rates and the pivotal temperature for sex determination. We also measured embryonic growth, oxygen consumption, and carbon dioxide production throughout development at 27° and 31°C. Metabolic rates were higher at 31°C than at 27°C, but total energy expenditure was greater at 27°C, with 2,281 mL of oxygen consumed compared with 1,992 mL at 31°C. Respiration at both temperatures showed a pattern typical of sea turtle embryos, with peak rates occurring at approximately 85% of development and then declining toward hatching. Hatchlings produced at higher incubation temperatures developed faster, were smaller, and had larger residual yolk masses than hatchlings produced at lower temperatures. The pivotal temperature that produced an equal sex ratio was 29.2°C, with mixed sexes produced between 27.9° and 30.4°C. Our results showed that the Ningaloo population of C. mydas has somewhat different thermal sensitivities than other C. mydas populations and justified why is it necessary to collect population-specific data to accurately project the impacts of global warming on focal populations.

Temperature Effects on Development and Phenotype in a Free-Living Population of Western Pond Turtles (Emys marmorata).

Changes in temperature regimes are occurring globally due to climate change as well as habitat alterations. Temperatures are expected to continue to rise in the future, along with a greater degree of climatic instability. Such changes could have potentially serious consequences for oviparous ectotherms, especially those with temperature-dependent sex determination. To investigate the effects of temperature on a range of developmental phenomena in a population of western pond turtles (Emys marmorata), we placed temperature sensors on top of each layer of eggs within nests and recorded temperatures hourly through the first 2-3 mo of incubation. These methods allowed us to look at in situ nest temperatures with high resolution. We found that mean incubation temperatures were similar between different nests and at different levels within nests but that incubation temperature fluctuations and maximum incubation temperatures differed greatly in both cases. The hatchling turtles were more likely to be female if they spent 30% or more of their sex-determining period of incubation above 29°C. Hatching success was best predicted by the maximum incubation temperature. We also found that incubation duration tended to be shorter as the mean temperature increased. However, exposure to either extremely high or low temperatures extended incubation times.