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.

Role for androgens in determination of ovarian fate in the common snapping turtle, Chelydra serpentina.

Sex steroids are involved in sex determination in almost all vertebrates, including species with temperature-dependent sex determination (TSD). It is well established that aromatase and estrogens are involved in ovary determination in TSD species. In contrast, the role of non-aromatizable androgens in TSD is less clear. In this study, we used dihydrotestosterone (DHT) and an antagonist of the mammalian androgen receptor (flutamide) to examine the impact of androgens on sex determination in the snapping turtle. We incubated eggs at a male-producing temperature and treated embryos with drug delivery vehicle (5 L ethanol), DHT in vehicle, or flutamide in vehicle during the sex-determining period. We then measured expression of markers for ovarian and testicular development and genes involved in steroidogenesis. A subset of embryos and hatchlings were collected for histological analysis of gonad differentiation and sex determination. DHT and flutamide both induced ovarian development: 100% of vehicle-treated hatchlings had testes, while 60% of DHT-treated and 32% flutamide-treated hatchlings had ovaries. DHT and flutamide treatments also had feminizing effects on gene expression patterns and the structure of embryonic gonads. DHT treatment increased expression of FoxL2, androgen receptor, aromatase and several steroidogenic genes. Flutamide produced a similar, but weaker, pattern of gene expression. Genes involved in testis development (Sox9 and Amh) were influenced by flutamide treatment. Our findings support the hypothesis that androgens and the androgen receptor are involved in ovary determination in the common snapping turtle.

Characterization of the FoxL2 proximal promoter and coding sequence from the common snapping turtle (Chelydra serpentina).

Sex is determined by temperature during embryogenesis in snapping turtles, Chelydra serpentina. Previous studies in this species show that dihydrotestosterone (DHT) induces ovarian development at temperatures that normally produce males or mixed sex ratios. The feminizing effect of DHT is associated with increased expression of FoxL2, suggesting that androgens regulate transcription of FoxL2. To test this hypothesis, we cloned the proximal promoter (1.6kb) and coding sequence for snapping turtle FoxL2 (tFoxL2) in frame with mCherry to produce a fluorescent reporter. The tFoxL2-mCherry fusion plasmid or mCherry control plasmid were stably transfected into mouse KK1 granulosa cells. These cells were then treated with 0, 1, 10, or 100nM DHT to assess androgen effects on tFoxL2-mCherry expression. In contrast to the main hypothesis, DHT did not alter expression of the tFoxL2-mCherry reporter. However, normal serum increased expression of tFoxL2-mCherry when compared to charcoal-stripped serum, indicating that the cloned region of tFoxL2 contains cis regulatory elements. We also used the tFoxL2-mCherry plasmid as an expression vector to test the hypothesis that DHT and tFoxL2 interact to regulate expression of endogenous genes in granulosa cells. While tFoxL2-mCherry and DHT had independent effects on mouse FoxL2, FshR, GnRHR, and StAR expression, tFoxL2-mCherry potentiated low concentration DHT effects on mouse aromatase expression. Further studies will be required to determine whether synergistic regulation of aromatase by DHT and FoxL2 also occurs in turtle gonads during the sex-determining period, which would explain the feminizing effect of DHT in this species.

Transcriptome analysis of the painted lady butterfly, Vanessa cardui during wing color pattern development.

BACKGROUND: Butterfly wing color patterns are an important model system for understanding the evolution and development of morphological diversity and animal pigmentation. Wing color patterns develop from a complex network composed of highly conserved patterning genes and pigmentation pathways. Patterning genes are involved in regulating pigment synthesis however the temporal expression dynamics of these interacting networks is poorly understood. Here, we employ next generation sequencing to examine expression patterns of the gene network underlying wing development in the nymphalid butterfly, Vanessa cardui. RESULTS: We identified 9, 376 differentially expressed transcripts during wing color pattern development, including genes involved in patterning, pigmentation and gene regulation. Differential expression of these genes was highest at the pre-ommochrome stage compared to early pupal and late melanin stages. Overall, an increasing number of genes were down-regulated during the progression of wing development. We observed dynamic expression patterns of a large number of pigment genes from the ommochrome, melanin and also pteridine pathways, including contrasting patterns of expression for paralogs of the yellow gene family. Surprisingly, many patterning genes previously associated with butterfly pattern elements were not significantly up-regulated at any time during pupation, although many other transcription factors were differentially expressed. Several genes involved in Notch signaling were significantly up-regulated during the pre-ommochrome stage including slow border cells, bunched and pebbles; the function of these genes in the development of butterfly wings is currently unknown. Many genes involved in ecdysone signaling were also significantly up-regulated during early pupal and late melanin stages and exhibited opposing patterns of expression relative to the ecdysone receptor. Finally, a comparison across four butterfly transcriptomes revealed 28 transcripts common to all four species that have no known homologs in other metazoans. CONCLUSIONS: This study provides a comprehensive list of differentially expressed transcripts during wing development, revealing potential candidate genes that may be involved in regulating butterfly wing patterns. Some differentially expressed genes have no known homologs possibly representing genes unique to butterflies. Results from this study also indicate that development of nymphalid wing patterns may arise not only from melanin and ommochrome pigments but also the pteridine pigment pathway.

Phenotypic plasticity in the common snapping turtle (Chelydra serpentina): long-term physiological effects of chronic hypoxia during embryonic development.

Studies of embryonic and hatchling reptiles have revealed marked plasticity in morphology, metabolism, and cardiovascular function following chronic hypoxic incubation. However, the long-term effects of chronic hypoxia have not yet been investigated in these animals. The aim of this study was to determine growth and postprandial O2 consumption (V̇o2), heart rate (fH), and mean arterial pressure (Pm, in kPa) of common snapping turtles (Chelydra serpentina) that were incubated as embryos in chronic hypoxia (10% O2, H10) or normoxia (21% O2, N21). We hypothesized that hypoxic development would modify posthatching body mass, metabolic rate, and cardiovascular physiology in juvenile snapping turtles. Yearling H10 turtles were significantly smaller than yearling N21 turtles, both of which were raised posthatching in normoxic, common garden conditions. Measurement of postprandial cardiovascular parameters and O2 consumption were conducted in size-matched three-year-old H10 and N21 turtles. Both before and 12 h after feeding, H10 turtles had a significantly lower fH compared with N21 turtles. In addition, V̇o2 was significantly elevated in H10 animals compared with N21 animals 12 h after feeding, and peak postprandial V̇o2 occurred earlier in H10 animals. Pm of three-year-old turtles was not affected by feeding or hypoxic embryonic incubation. Our findings demonstrate that physiological impacts of developmental hypoxia on embryonic reptiles continue into juvenile life.

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered ß-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

Molecular and morphological differentiation of testes and ovaries in relation to the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina.

Ambient temperatures during embryonic development determine gonadal sex in many reptiles. The temperature sensitive period for sex determination has been defined by shifting eggs between female- and male-producing temperatures in a few species. This phase spans 20-35% of embryogenesis in most species, which makes it difficult to define the mechanisms that transduce temperature into a signal for ovarian versus testicular development. We present an extensive set of studies that define a brief period when high temperature specifies, and then determines, ovarian fate in a northern population of snapping turtles, Chelydra serpentina. We shifted embryos from male to female temperatures, or vice versa, at various stages of development. Gonads in embryos incubated at female temperatures commit to ovarian fate earlier (by stage 18) than gonads in embryos incubated at male temperatures commit to testicular fate (by stages 19-21). In double shift studies, embryos were incubated at a female temperature, exposed to a male temperature for set times, and shifted back to the original temperature, or vice versa. The time required to induce ovarian development (≤6 days at female temperatures) was much shorter than the time required to induce testicular formation (>20 days at male temperatures). Differentiation of the gonads at the histological level occurred after the sex-determining period. Nevertheless, we found that a change in temperature rapidly (within 24h) influenced expression and splicing of WT1 mRNA: the absolute abundance of WT1 mRNA, the relative abundance of +KTS versus -KTS isoforms, as well as the ratio of +KTS:-KTS isoforms was higher in gonads at a male versus a female temperature. In conclusion, ovarian fate is more readily determined than testicular fate in snapping turtle embryos. The short sex-determining period in this species (6-8% of embryogenesis) will facilitate studies of molecular mechanisms for specification and determination of gonad fate by temperature.

Population genomics provide insight into ancestral relationships and diversity of the feral horses of Theodore Roosevelt National Park.

Theodore Roosevelt National Park (TRNP) manages a herd of feral horses (Equus caballus) which was present on the landscape prior to the establishment of the park. The population presents a unique scenario in that it has experienced fairly intensive and well-documented management since the park's establishment, including herd size reductions, intentional introduction of diversity, and subsequent attempts to remove introduced lineages. This provides an interesting case study on the genetic effects of diverse evolutionary forces on an isolated feral population. To explore the effects of these forces and clarify the relationship of this feral herd with other horses, we used genome-wide markers to examine the population structure of a combined dataset containing common established breeds. Using the Illumina Equine 70k BeadChip, we sampled SNPs across the genome for 118 TRNP horses and evaluated the inbreeding coefficient f and runs of homozygosity (RoH). To identify breed relationships, we compared 23 representative TRNP samples with 792 horses from 35 different breeds using genomic population structure analyses. Mean f of TRNP horses was 0.180, while the mean f for all other breeds in the dataset was 0.116 (SD 0.079). RoH analysis indicates that the TRNP population has experienced recent inbreeding in a timeframe consistent with their management. With Bayesian clustering, PCA, and maximum likelihood phylogeny, TRNP horses show genetic differentiation from other breeds, likely due to isolation, historical population bottlenecks, and genetic drift. However, maximum likelihood phylogeny places them with moderate confidence (76.8%) among draft breeds, which is consistent with the known history of breeds used on early North Dakota ranches and stallions subsequently introduced to the park herd. These findings will help resolve speculation about the origins of the herd and inform management decisions for the TRNP herd.

Plasticity of cardiovascular function in snapping turtle embryos (Chelydra serpentina): chronic hypoxia alters autonomic regulation and gene expression.

Reptile embryos tolerate large decreases in the concentration of ambient oxygen. However, we do not fully understand the mechanisms that underlie embryonic cardiovascular short- or long-term responses to hypoxia in most species. We therefore measured cardiac growth and function in snapping turtle embryos incubated under normoxic (N21; 21% O2) or chronic hypoxic conditions (H10; 10% O2). We determined heart rate (fH) and mean arterial pressure (Pm) in acute normoxic (21% O2) and acute hypoxic (10% O2) conditions, as well as embryonic responses to cholinergic, adrenergic, and ganglionic pharmacological blockade. Compared with N21 embryos, chronic H10 embryos had smaller bodies and relatively larger hearts and were hypotensive, tachycardic, and following autonomic neural blockade showed reduced intrinsic fH at 90% of incubation. Unlike other reptile embryos, cholinergic and ganglionic receptor blockade both increased fH. ß-Adrenergic receptor blockade with propranolol decreased fH, and α-adrenergic blockade with phentolamine decreased Pm. We also measured cardiac mRNA expression. Cholinergic tone was reduced in H10 embryos, but cholinergic receptor (Chrm2) mRNA levels were unchanged. However, expression of adrenergic receptor mRNA (Adrb1, Adra1a, Adra2c) and growth factor mRNA (Igf1, Igf2, Igf2r, Pdgfb) was lowered in H10 embryos. Hypoxia altered the balance between cholinergic receptors, α-adrenoreceptor and ß-adrenoreceptor function, which was reflected in altered intrinsic fH and adrenergic receptor mRNA levels. This is the first study to link gene expression with morphological and cardioregulatory plasticity in a developing reptile embryo.

Temperature-dependent sex determination modulates cardiovascular maturation in embryonic snapping turtles Chelydra serpentina.

We investigated sex differences in cardiovascular maturation in embryos of the snapping turtle Chelydra serpentina, a species with temperature-dependent sex determination. One group of eggs was incubated at 26.5°C to produce males. Another group of eggs was incubated at 26.5°C until embryos reached stage 17; eggs were then shifted to 31°C for 6 days to produce females, and returned to 26.5°C for the rest of embryogenesis. Thus, males and females were at the same temperature when autonomic tone was determined and for most of development. Cholinergic blockade increased resting blood pressure (P(m)) and heart rate (f(H)) in both sexes at 75% and 90% of incubation. However, the magnitude of the f(H) response was enhanced in males compared with females at 90% of incubation. ß-adrenergic blockade increased P(m) at 75% of incubation in both sexes but had no effect at 90% of incubation. ß-adrenergic blockade reduced f(H) at both time points but produced a stronger response at 90% versus 75% of incubation. We found that α-adrenergic blockade decreased P(m) in both sexes at 75% and 90% of incubation and decreased f(H) at 75% of incubation in both sexes. At 90% of incubation, f(H) decreased in females but not males. Although these data clearly demonstrate sexual dimorphism in the autonomic regulation of cardiovascular physiology in embryos, further studies are needed to test whether differences are caused by endocrine signals from gonads or by a hormone-independent temperature effect.

Major locus on ECA18 influences effectiveness of GonaCon vaccine in feral horses.

Contraceptive vaccines are used to reduce birth rates in wild and feral animal populations. While the immunocontraceptive GonaCon-Equine has proven effective in reducing fertility among female feral horses, there is individual variation in the duration of infertility following treatment. To identify genetic factors influencing the effectiveness of GonaCon-Equine, we conducted a genome-wide association study of 88 mares from a feral population genotyped using the Illumina GGP Equine 70k SNP array. Contraceptive treatment schedules and long-term foaling rates have been recorded for each individual. We used mixed linear models to control for relatedness among mares. We found a significant association (p < 5 ×10-8) with a locus on equine chromosome 18. The most likely candidate genes in this region are STAT1 and STAT4, which are both involved in immune system function. Variation in STAT function could affect the immune response to the vaccine, leading to variation in contraceptive efficacy. Additional SNPs reaching a less stringent threshold of significance (p < 5 ×10-6) were located on other chromosomes near known immune system genes, supporting the hypothesis that variation in immunocontraceptive efficacy can be attributed to genetic variation in immune response rather than fertility genes.

Sensitivity of a Model Reptile, the Common Snapping Turtle (Chelydra serpentina), to In Ovo Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin and Other Dioxin-Like Chemicals.

Reptiles represent the least-studied group of vertebrates with regards to ecotoxicology and no empirical toxicity data existed for dioxin-like chemicals (DLCs). This lack of toxicity data represents a significant uncertainty in ecological risk assessments of this taxon. Therefore, the present study assessed early-life sensitivity to select DLCs and developed relative potencies in the common snapping turtle (Chelydra serpentina) as a model reptile. Specifically, survival to hatch and incidence of pathologies were assessed in common snapping turtle exposed in ovo to serial concentrations of the prototypical reference congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and three other DLCs of environmental relevance, namely, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). In ovo exposure to TCDD, PeCDF, TCDF, and PCB 126 caused a dose-dependent increase in early-life mortality, with median lethal doses (LD50s) of 14.9, 11.8, 29.6, and 185.9 pg/g-egg, respectively. Except for abnormal vasculature development, few pathologies were observed. Based on the measured LD50, common snapping turtle is more sensitive to TCDD in ovo than other species of oviparous vertebrates investigated to date. The potencies of PeCDF, TCDF, and PCB 126 relative to TCDD were 1.3, 0.5, and 0.08, respectively. These relative potencies are within an order of magnitude of World Health Organization (WHO) TCDD-equivalency factors (TEFs) for both mammals and birds supporting these TEFs as relevant for assessing ecological risk to reptiles. The great sensitivity to toxicities of the common snapping turtle, and potentially other species of reptiles, suggests a clear need for further investigation into the ecotoxicology of this taxon. Environ Toxicol Chem 2022;41:175-183. © 2021 SETAC.

The oxysterol 27-hydroxycholesterol regulates α-synuclein and tyrosine hydroxylase expression levels in human neuroblastoma cells through modulation of liver X receptors and estrogen receptors--relevance to Parkinson's disease.

Loss of dopaminergic neurons and α-synuclein accumulation are the two major pathological hallmarks of Parkinson's disease. Currently, the mechanisms governing depletion of dopamine content and α-synuclein accumulation are not well understood. We showed that the oxysterol 27-hydroxycholesterol (27-OHC) reduces the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and increases α-synuclein levels in SH-SY5Y cells. However, the cellular mechanisms involved in 27-OHC effects were not elucidated. In this study, we demonstrate that 27-OHC regulates TH and α-synuclein expression levels through the estrogen receptors (ER) and liver X receptors (LXR). We specifically show that inhibition of ERß mediates 27-OHC-induced decrease in TH expression, an effect reversed by the ER agonist estradiol. We also show that 27-OHC and the LXR agonist GW3965 increase α-synuclein while the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate significantly attenuated the 27-OHC-induced increase in α-synuclein expression. We further demonstrate that LXRß positively regulates α-synuclein expression and 27-OHC increases LXRß-mediated α-synuclein transcription. Our results demonstrate the involvement of two distinct pathways that are involved in the 27-OHC regulation of TH and α-synuclein levels. Concomitant activation of ERß and inhibition of LXRß prevent 27-OHC effects and may therefore reduce the progression of Parkinson's disease by precluding TH reduction and α-synuclein accumulation.

Evolutionary Turnover in Wnt Gene Expression but Conservation of Wnt Signaling during Ovary Determination in a TSD Reptile.

Temperature-dependent sex determination (TSD) is a well-known characteristic of many reptilian species. However, the molecular processes linking ambient temperature to determination of gonad fate remain hazy. Here, we test the hypothesis that Wnt expression and signaling differ between female- and male-producing temperatures in the snapping turtle Chelydra serpentina. Canonical Wnt signaling involves secretion of glycoproteins called WNTs, which bind to and activate membrane bound receptors that trigger ß-catenin stabilization and translocation to the nucleus where ß-catenin interacts with TCF/LEF transcription factors to regulate expression of Wnt targets. Non-canonical Wnt signaling occurs via 2 pathways that are independent of ß-catenin: one involves intracellular calcium release (the Wnt/Ca2+ pathway), while the other involves activation of RAC1, JNK, and RHOA (the Wnt/planar cell polarity pathway). We screened 20 Wnt genes for differential expression between female- and male-producing temperatures during sex determination in the snapping turtle. Exposure of embryos to the female-producing temperature decreased expression of 7 Wnt genes but increased expression of 2 Wnt genes and Rspo1 relative to embryos at the male-producing temperature. Temperature also regulated expression of putative Wnt target genes in vivo and a canonical Wnt reporter (6x TCF/LEF sites drive H2B-GFP expression) in embryonic gonadal cells in vitro. Results indicate that Wnt signaling was higher at the female- than at the male-producing temperature. Evolutionary analyses of all 20 Wnt genes revealed that thermosensitive Wnts, as opposed to insensitive Wnts, were less likely to show evidence of positive selection and experienced stronger purifying selection within TSD species.

Developmental programming of DNA methylation and gene expression patterns is associated with extreme cardiovascular tolerance to anoxia in the common snapping turtle.

BACKGROUND: Environmental fluctuation during embryonic and fetal development can permanently alter an organism's morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species. RESULTS: Genome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such as SCN5A may account for differences in heart rate, while genes such as TNNT2 and TPM3 may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated. CONCLUSIONS: Our data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

Embryonic Temperature Programs Phenotype in Reptiles.

Reptiles are critically affected by temperature throughout their lifespan, but especially so during early development. Temperature-induced changes in phenotype are a specific example of a broader phenomenon called phenotypic plasticity in which a single individual is able to develop different phenotypes when exposed to different environments. With climate change occurring at an unprecedented rate, it is important to study temperature effects on reptiles. For example, the potential impact of global warming is especially pronounced in species with temperature-dependent sex determination (TSD) because temperature has a direct effect on a key phenotypic (sex) and demographic (population sex ratios) trait. Reptiles with TSD also serve as models for studying temperature effects on the development of other traits that display continuous variation. Temperature directly influences metabolic and developmental rate of embryos and can have permanent effects on phenotype that last beyond the embryonic period. For instance, incubation temperature programs post-hatching hormone production and growth physiology, which can profoundly influence fitness. Here, we review current knowledge of temperature effects on phenotypic and developmental plasticity in reptiles. First, we examine the direct effect of temperature on biophysical processes, the concept of thermal performance curves, and the process of thermal acclimation. After discussing these reversible temperature effects, we focus the bulk of the review on developmental programming of phenotype by temperature during embryogenesis (i.e., permanent developmental effects). We focus on oviparous species because eggs are especially susceptible to changes in ambient temperature. We then discuss recent work probing the role of epigenetic mechanisms in mediating temperature effects on phenotype. Based on phenotypic effects of temperature, we return to the potential impact of global warming on reptiles. Finally, we highlight key areas for future research, including the identification of temperature sensors and assessment of genetic variation for thermosensitivity.

Incubation temperature and satiety influence general locomotor and exploratory behaviors in the common snapping turtle (Chelydra serpentina).

Temperature during embryogenesis determines sex and has been shown to influence other physiological traits in reptiles. The common snapping turtle (Chelydra serpentina) is an ideal model for testing how temperature impacts behavior in species that display temperature-dependent sex determination. Behavioral assays are crucial to understanding how a changing climate may affect whole organism function in the snapping turtle. Currently, there are few behavioral assays for semi-aquatic vertebrates like turtles. In this study, we used digital cameras to record behavior of fed and fasted hatchling turtles from different incubation temperatures in an open field setting for 20 min in 2018 and repeated the experiment in 2019. Open fields were circular tanks filled with water to a depth of 3.5 cm. Each field was split into four quadrants and two zones (inner and outer). The amount of time turtles spent actively moving, total distance travelled, and several other measures were collected and summarized automatically from videos with open source image analysis software (ImageJ). Satiety and incubation temperature had significant effects on total distance moved, time spent moving, and time moving in the outer zone. These findings indicate that temperature during embryogenesis has a long-lasting effect on neural mechanisms underlying exploratory or general locomotor behavior in turtles.

Draft Genome of the Common Snapping Turtle, Chelydra serpentina, a Model for Phenotypic Plasticity in Reptiles.

Turtles are iconic reptiles that inhabit a range of ecosystems from oceans to deserts and climates from the tropics to northern temperate regions. Yet, we have little understanding of the genetic adaptations that allow turtles to survive and reproduce in such diverse environments. Common snapping turtles, Chelydra serpentina, are an ideal model species for studying adaptation to climate because they are widely distributed from tropical to northern temperate zones in North America. They are also easy to maintain and breed in captivity and produce large clutch sizes, which makes them amenable to quantitative genetic and molecular genetic studies of traits like temperature-dependent sex determination. We therefore established a captive breeding colony and sequenced DNA from one female using both short and long reads. After trimming and filtering, we had 209.51Gb of Illumina reads, 25.72Gb of PacBio reads, and 21.72 Gb of Nanopore reads. The assembled genome was 2.258 Gb in size and had 13,224 scaffolds with an N50 of 5.59Mb. The longest scaffold was 27.24Mb. BUSCO analysis revealed 97.4% of core vertebrate genes in the genome. We identified 3.27 million SNPs in the reference turtle, which indicates a relatively high level of individual heterozygosity. We assembled the transcriptome using RNA-Seq data and used gene prediction software to produce 22,812 models of protein coding genes. The quality and contiguity of the snapping turtle genome is similar to or better than most published reptile genomes. The genome and genetic variants identified here provide a foundation for future studies of adaptation to climate.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development.

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-alpha and PdgfR-beta in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-alpha mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-beta mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-alpha and PdgfR-beta in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle.

Developmental plasticity in reptiles: Critical evaluation of the evidence for genetic and maternal effects on temperature-dependent sex determination.

We critically review literature that examines variation in temperature-dependent sex determination (TSD) within species. Although variation in sex ratio among clutches of eggs from different females is common in reptiles with TSD, the underlying mechanism that causes this variation is not clear. Authors have made claims about genetic variation in TSD and maternal effects on TSD. The latter type of study usually focuses on maternally derived steroids in egg yolk. Here, we outline the types of experiments and data required to unequivocally demonstrate that variation in sex ratio among clutches (1) has a genetic basis, (2) is caused by maternally derived steroids, or (3) is influenced by both factors. To date, few studies have met these requirements.