Global climate is warming rapidly, threatening vertebrates with temperature-dependent sex determination (TSD) by disrupting sex ratios and other traits. Less understood are the effects of increased thermal fluctuations predicted to accompany climate change. Greater fluctuations could accelerate feminization of species that produce females under warmer conditions (further endangering TSD animals), or counter it (reducing extinction risk). Here we use novel experiments exposing eggs of Painted Turtles (Chrysemys picta) to replicated profiles recorded in field nests plus mathematically-modified profiles of similar shape but wider oscillations, and develop a new mathematical model for analysis. We show that broadening fluctuations around naturally male-producing (cooler) profiles feminizes developing embryos, whereas embryos from warmer profiles remain female or die. This occurs presumably because wider oscillations around cooler profiles expose embryos to very low temperatures that inhibit development, and to feminizing temperatures where most embryogenesis accrues. Likewise, embryos incubated under broader fluctuations around warmer profiles experience mostly feminizing temperatures, some dangerously high (which increase mortality), and fewer colder values that are insufficient to induce male development. Therefore, as thermal fluctuations escalate with global warming, the feminization of TSD turtle populations could accelerate, facilitating extinction by demographic collapse. Aggressive global CO2 mitigation scenarios (RCP2.6) could prevent these risks, while intermediate actions (RCP4.5 and RCP6.0 scenarios) yield moderate feminization, highlighting the peril that insufficient reductions of greenhouse gas emissions pose for TSD taxa. If our findings are generalizable, TSD squamates, tuatara, and crocodilians that produce males at warmer temperatures could suffer accelerated masculinization, underscoring the broad taxonomic threats of climate change.

Background. The doublesex and mab-3 related transcription factor 1 (Dmrt1) is a highly conserved gene across numerous vertebrates and invertebrates in sequence and function. Small aminoacid changes in Dmrt1 are associated with turnovers in sex determination in reptiles. Dmrt1 is upregulated in males during gonadal development in many species, including the painted turtle, Chrysemys picta, a reptile with temperaturedependent sex determination (TSD). Dmrt1 is reported to play different roles during sex determination and differentiation, yet whether these functions are controlled by distinct Dmrt1 spliceoforms remains unclear. While Dmrt1 isoforms have been characterized in various vertebrates, no study has investigated their existence in any turtle. Methods. We examine the painted turtle to identify novel Dmrt1 isoforms that may be present during urogenital development using PCR, profile their expression by RNA-seq across five embryonic stages at male-and female-producing temperatures, and validate their expression pattern via qPCR with transcript-specific fluorescent probes. Results. A novel Dmrt1 spliceoform was discovered for the first time in chelonians, lacking exons 2 and 3 (Dmrt1 Ex2Ex3). Dmrt1 canonical and Ex2Ex3 transcripts were differentialy expressed by temperature at stages 19 and 22 in developing gonads of painted turtles, after the onset of sex determination, and displayed a significant malebiased expression pattern. This transcriptional pattern differs from studies in other turtles and vertebrates that reported Dmrt1 differential expression before or at the onset of sex determination. This study provides the first insight into Dmrt1 transcriptional diversity in turtles and opens the door for future functional studies of the alternative Dmrt1 transcript uncovered here. Conclusions. The discovery of an isoform in turtles indicate that alternative splicing may be a common feature of Dmrt1 across vertebrates, as isoforms are also found in crocodilians, birds, mammals and fish, and this variation remains unexplained. The relatively late-onset of Dmrt1 expression observed here contrasts with other turtles, indicating that Dmrt1 is not the topmost male sex -determining factor in C. picta. When placed in a phylogenetic context, this discrepancy underscores the divergent regulation of Dmrt1, and of sexual development more generally, across vertebrates. How to cite this article Mizoguchi B, Valenzuela N. 2020. Alternative splicing and thermosensitive expression of Dmrt1 during urogenital development in the painted turtle, Chrysemys picta. PeerJ 8:e8639 http://doi.org/10.7717/peerj.8639 Mizoguchi and Valenzuela (2020), PeerJ, DOI 10.7717/peerj.8639 2/25 Adolfi MC, Carreira AC, Jesus LW, Bogerd J, Funes RM, Schartl M, Sogayar MC, Borella MI. 2015. Molecular cloning and expression analysis of dmrt1 and sox9 during gonad development and male reproductive cycle in the lambari fish, Astyanax altiparanae. Reproductive Biology and Endocrinology 13:1-15. Alam MA, Kobayashi Y, Horiguchi R, Hirai T, Nakamura M. 2008. Mo...

Sex determination or the commitment of the embryo to its sexual fate is a fundamental developmental process with paramount consequences in ecology and evolution. This process, whether triggered by environmental factors or genotypic constitution, can be derailed by environmental contaminants that alter the endocrine system, which is a key component of the regulatory network underlying vertebrate sex determination. Here, we review the molecular basis of sex determination, the endocrine components of its regulation, the maternal and endogenous sources of hormones to the developing embryo, and the routes through which endocrine disrupting chemicals (EDCs) affect gonadal development in reptiles, especially turtles. Among EDCs, we focus on PCBs, BPA, pesticides like atrazine, nitrates, and heavy metals. We also consider whether adaptation might be possible in the face of persistent chemical insult and conclude that, while plausible, contemporary environmental change may outpace adaptive evolution, particularly for many species that are already endangered and suffer from small population sizes.

BackgroundDNA methylation alters gene expression but not DNA sequence and mediates some cases of phenotypic plasticity. Temperature-dependent sex determination (TSD) epitomizes phenotypic plasticity where environmental temperature drives embryonic sexual fate, as occurs commonly in turtles. Importantly, the temperature-specific transcription of two genes underlying gonadal differentiation is known to be induced by differential methylation in TSD fish, turtle and alligator. Yet, how extensive is the link between DNA methylation and TSD remains unclear. Here we test for broad differences in genome-wide DNA methylation between male and female hatchling gonads of the TSD painted turtle Chrysemys picta using methyl DNA immunoprecipitation sequencing, to identify differentially methylated candidates for future study. We also examine the genome-wide nCpG distribution (which affects DNA methylation) in painted turtles and test for historic methylation in genes regulating vertebrate gonadogenesis.ResultsTurtle global methylation was consistent with other vertebrates (57% of the genome, 78% of all CpG dinucleotides). Numerous genes predicted to regulate turtle gonadogenesis exhibited sex-specific methylation and were proximal to methylated repeats. nCpG distribution predicted actual turtle DNA methylation and was bimodal in gene promoters (as other vertebrates) and introns (unlike other vertebrates). Differentially methylated genes, including regulators of sexual development, had lower nCpG content indicative of higher historic methylation.ConclusionsOurs is the first evidence suggesting that sexually dimorphic DNA methylation is pervasive in turtle gonads (perhaps mediated by repeat methylation) and that it targets numerous regulators of gonadal development, consistent with the hypothesis that it may regulate thermosensitive transcription in TSD vertebrates. However, further research during embryogenesis will help test this hypothesis and the alternative that instead, most differential methylation observed in hatchlings is the by-product of sexual differentiation and not its cause.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0136-2) contains supplementary material, which is available to authorized users.

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.