Temperature-dependent Sex Determination Pattern Research Articles

Temperature regulates sex determination and growth in the paralichthid flatfishCalifornia halibut.

California halibut (Paralichthys californicus) is a candidate species for aquaculture and stock enhancement. These applications rely on sex control, either to maximize the production of faster growing females or to match sex ratios in the wild. Other paralichthids exhibit temperature-dependent sex determination (TSD), but the presence and pattern of TSD is not well defined in California halibut. Juvenile California halibut were cultured at three distinct temperatures (15°C, 19°C, and 23°C) through the developmental period presumed to be thermosensitive based on findings from congeners. Sex ratios were quantified in each treatment using phenotypic sex identification techniques applied early (molecular biomarkers; 51-100 mm total length [TL]) and late (visual examination of the gonads; ≥100 mm TL) in the juvenile phase. Both techniques indicated similar sex determination trends at each temperature, with overall sex ratios assessed as 49.9% maleat 15°C, 74.5% maleat 19°C, and 98.2% maleat 23°C. Growth rates were highest at 23°C and lowest at 15°C, with intrinsically fast- and slow-growing individuals at all temperatures. At 15°C and 19°C, females comprised a higher proportion among the fast growers than they did among the slow growers. These data show that California halibut exhibit TSD, with temperatures of 19°C and 23°C masculinizing fish while 15°C appears to produce a 1:1 sex ratio. This study will help optimize sex ratios and growth in hatcheries through thermal manipulation. Furthermore, the developed biomolecular tools and identified temperature thresholds will be important in future work to understand the influence of global warming on wild population demographics.

Assessment of Acetylcholinesterase Enzyme (AChE) Activity in the Developing Brain of TSD Lizard, Calotes Versicolor (Daud.)

Acetylcholinesterase (AChE) is a critical enzyme in the neuronal cholinergic system of vertebrates. It is one of the efficient cholinesterases that is involved in the termination of acetylcholine-mediated neurotransmission, rapidly hydrolyzing acetylcholine into acetate and choline. Barring a few reports on the distribution/presence of AChE enzyme activity in the adult reptilian species there are nil reports on the presence of this enzyme activity in the developing brain. Hence, the present investigation aims to evaluate AChE enzyme activity in the developing brain of an oviparous lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The eggs were collected during breeding season, padded with moist cotton, and incubated in an incubator at 30o C. The whole brain from the embryos was collected during different developmental stages from oviposition (Stage 27) to hatching (stage 42). The AChE enzyme activity was quantified in the brain, according to Ellman’s protocol using a spectrophotometer. The findings reveal that the onset of AChE enzyme activity is observed as early as at oviposition, which reflects the early action of AChE activity in the developing brain. The observed results albeit indirectly suggest the involvement of AChE enzyme activity in the morphogenetic process. Further, an exponential increase in AChE activity during the post-gonadal differentiation phase indicates age-related elevation in AChE enzyme activity which in turn unveils its involvement in neuronal transmission of the embryonic brain. Besides, this specific AChE enzyme activity in the developing brain of Calotes versicolor represents a conserved pattern for the cholinergic system in vertebrates.

Sexually dimorphic expression of AMH facilitates testis differentiation pathway in the tropical lizard, Calotes versicolor (Daud.)

The Anti-mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a glycoprotein that belongs to transforming growth factor β superfamily. The significance of AMH during gonadal differentiation is not clearly deciphered in reptiles. Hence, current study aims to know the onset of AMH secretion and its functional role in Mullerian duct regression gonadal differentiation in tropical lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The Immunohistochemistry and qRT-PCR techniques were employed to analyze the gonadal expression profile of AMH during different stages of embryonic development. The eggs of the lizard were incubated at both male-producing temperature (MPT: 25.5 ± 0.5 °C) and female-producing temperatures (FPT: 31.5 ± 0.5 °C). The results reveal that the onset of AMH gene expression was observed as early as oviposition prior to the immunolocalization of AMH protein at early-TSP (Temperature-sensitive period). The substantial rise in the intensity of the immunoreaction of AMH protein in the cytoplasm confining to Sertoli cells of seminiferous cords at MPT with low level of expression at FPT during gonadal sex differentiation, specify sexually dimorphic expression of AMH protein. Further, with the onset of sexual differentiation, the developing testis immensely expresses AMH gene which is 7-fold greater than that of transcripts levels in female embryos; signifies its conserved role in Mullerian duct regression thereby promoting testis differentiation. The robust immunnoexpression of AMH protein during post-gonadal differentiation coincides with the onset of the regression of Mullerian duct point out a positive correlation between testis differentiation and Mullerian duct regression, thus facilitating testis differentiation pathway. Based on the immunoexpression pattern of AMH protein and transcript levels of AMH gene, it is inferred that AMH plays a significant role in Mullerian duct regression, favoring testis differentiation.

On the origin of patterns of temperature-dependent sex determination.

Species with temperature-dependent sex determination (TSD) exhibit significant variation in the relationship between incubation temperatures and the sex ratios they produce, making this an ideal system for comparing processes producing variation above and below the species level. Furthermore, a deeper mechanistic understanding of TSD macro- and microevolution may help reveal the currently unknown adaptive significance of this variation or of TSD as a whole. Here, we probe these topics by examining the evolutionary dynamics of this sex-determining mechanism in turtles. Our ancestral state reconstructions of discrete patterns of TSD suggest that producing females at cool incubation temperatures is derived and potentially adaptive. However, the ecological irrelevance of these cool temperatures and a strong genetic correlation across the sex-ratio reaction norm in Chelydra serpentina both contradict this interpretation. We further find the phenotypic consequence of this genetic correlation in C. serpentina reflected across all turtle species, suggesting that a single genetic architecture underlies both intra- and interspecific variation in TSD in this clade. This correlated architecture can explain the macroevolutionary origin of discrete TSD patterns without assigning cool-temperature female production an adaptive value. However, this architecture may also constrain adaptive microevolutionary responses to ongoing climate change.

Latitudinal Variation in the Pattern of Temperature-Dependent Sex Determination in the Japanese Gecko, Gekko japonicus.

Simple SummaryIn egg-laying lizards, sex is determined by genetic factors in species with sex chromosomes or egg incubation temperatures in species without sex chromosomes, i.e., temperature-dependent sex determination (TSD). Surprisingly, recent studies find sex chromosomes and TSD co-occur in the same species in some lizards. The Japanese gecko from Japan may be this case. However, Japanese gecko with TSD from a Chinese population does not have sex chromosomes, suggesting that the pattern of TSD in this gecko may vary among populations. We incubated gecko eggs from three populations in China at constant temperatures of 24, 26, 28, 30, and 32 °C to quantify the sex determination pattern. We found that the temperature yielding an equal number of sons and daughters of the low-latitude population was lower than that of the two high-latitude populations. Moreover, the low-latitude population had a narrower temperature range producing mixed sex offspring at lower temperatures, but a wider range at higher temperatures. Sex ratio was almost 1:1 for the low-latitude population when incubated from 26 to 30 °C. Conversely, more male offspring were produced at 28 or 30 °C in the two high-latitude populations. Our study may provide an interesting system to explore the evolution of sex determination mechanisms in animals.Identifying latitudinal variation in the pattern of temperature-dependent sex determination (TSD) may provide insight into the evolution of sex determining system in vertebrates, but such studies remain limited. Here, we quantified TSD patterns of three geographically separated populations of the Japanese gecko (Gekko japonicus) along the latitudinal cline of China. We incubated gecko eggs from the three populations at constant temperatures of 24, 26, 28, 30, and 32 °C to quantify the TSD pattern. Our study demonstrated that G. japonicus exhibited a FMF pattern of TSD, with the low and high incubation temperatures yielding significantly female-biased hatchlings, and the medium temperatures producing male-biased hatchlings. More interestingly, we found latitudinal variations in the TSD pattern in terms of pivotal temperatures (Tpivs), transitional range of temperatures (TRT), and the sex ratios at the medium temperatures. The Tpivs for the low-latitude population were lower than those for the two high-latitude populations. The low-latitude population has a narrower FM TRT, but a wider MF TRT. The sex ratio is almost 50:50 for the low-latitude population when eggs were incubated from 26 to 30 °C. Conversely, the sex ratio is male-biased for the two high-latitude populations at 28 or 30 °C. Therefore, G. japonicus may provide an interesting system to explore the evolution of TSD in reptiles given the diversity of TSD patterns among populations.

Spatiotemporal Expression of Aromatase (CYP19) During Gonadal Sex Differentiation in the Oviparous Lizard Calotes versicolor

Steroidal sex hormones play critical rolest during gonadal sex differentiation and development. Among the sex steroids, estradiol acts at the central level in the sexual differentiation of many reptilian species exhibiting temperature-dependent sex determination (TSD). In recent years, the growing amount of information has highlighted the importance of aromatase (CYP19) in irreversibly catalyzing conversion of androgens into estrogens by aromatization during ovarian morphogenesis. To elucidate the involvement of aromatase in this process we investigated the ontogenic and cellular expression of aromatase in the embryos of the lizard Calotes versicolor that exhibits a unique pattern of TSD. The eggs of this lizard were incubated at 31.5±0.5°C which is 100% Female-Producing Temperature (FPT). The torso of embryos containing Adrenal- Kidney-Gonadal (AKG) complex was collected during different stages of development and subjected to Western blotting and immunohistochemical analysis. Western blot analysis revealed a single immunoreactive protein at 58 kDa for aromatase in the total protein extracts of AKG complex during early Thermo-Sensitive Period (TSP) suggesting early action of aromatase. A very weak but specific immunoexpression of aromatase noticed in the cytoplasm of adrenocortical cells, mesonephric kidney tubules (stage 28) and medullary region of the gonad (stage 30) during early TSP suggests early onset of aromatase activity which in turn indicates that these are the sites of estrogen biosynthesis. Further, a remarkable increase in the intensity of immunoreaction for aromatase during late TSP and gonadal differentiation stage shows it’s up-regulation and estrogen biosynthesis resulting in ovary differentiation. It is inferred that high expression level of aromatase at FPT is associated with proliferation of cortex facilitating differentiation of ovary in C. versicolor .

Modeling Temperature-Dependent Sex Determination in Oviparous Species Using a Dynamical Systems Approach.

In many oviparous species, the incubation temperature of the egg determines the sex of the offspring. This is known as temperature-dependent sex determination (TSD). The probability of the hatched offspring being male or female varies across the incubation temperature range. This leads to the appearance of different TSD patterns in species such as FM pattern where females are predominately born at lower temperature and males at higher temperature, FMF pattern where the probability of female being born is higher at extreme temperatures and of the male being born is high at intermediate temperatures. We analyze an enzymatic reaction system proposed in the literature involving sex hormones with positive feedback effect to understand the emergence of different TSD patterns. The nonlinearity in the model is accounted through temperature sensitivity of the reaction rates affecting the catalytic mechanism in the reaction system. We employ a dynamical systems approach of singularity theory and bifurcation analysis to divide the parameter plane of temperature sensitivities into different regions where different TSD patterns are observed. Bifurcation analysis in association with the delineation of the parameter space for different TSD pattern has led to the identification of a subspace where all the TSD patterns observed in nature can be realized. We also show how modulation of the sex hormone in the species can be used to change the probability of occurrence of a specific sex, thereby preventing the extinction of endangered species.

Revisiting the first report of temperature‐dependent sex determination in a vertebrate, the African redhead agama

AbstractVertebrates display a wide variety of sex‐determining mechanisms (SDMs). Identifying SDMs for different taxa is important for understanding the origins and phylogenetic history of sex‐determining mechanisms. Temperature‐dependent sex determination (TSD) is found in several vertebrate and invertebrate species, and has been studied most extensively in reptiles. The initial discovery of TSD was in a lizard, the African redhead agama (Agama agama), over 50 years ago. The original study of A. agama used only two incubation temperature treatments and had a small sample size, thus it could not fully characterize the pattern of TSD in this species. The goal of this study is to revisit this research to better characterize the pattern of TSD. We also assessed the effect of fluctuating incubation temperatures on offspring sex ratio to provide insight into how variation in offspring sex ratio is produced under natural conditions. After incubating eggs across six constant temperature treatments, we demonstrated that females are produced at relatively cool temperatures and males at warmer temperatures (30°C); fluctuating thermal regimes showed this same pattern. However, some females are again produced at even warmer temperatures, resulting in relatively balanced sex ratios toward the end of the sex‐determining reaction norm. Though the shape of the sex‐determining reaction norm shown here is unusual, it shows some similarities to that of other agamid lizards. Future studies that explore the diversity of TSD reaction norms and underlying physiological mechanisms will provide new insights into the evolution of TSD.

The ecology and evolution of temperature-dependent reaction norms for sex determination in reptiles: a mechanistic conceptual model.

Sex-determining mechanisms are broadly categorised as being based on either genetic or environmental factors. Vertebrate sex determination exhibits remarkable diversity but displays distinct phylogenetic patterns. While all eutherian mammals possess XY male heterogamety and female heterogamety (ZW) is ubiquitous in birds, poikilothermic vertebrates (fish, amphibians and reptiles) exhibit multiple genetic sex-determination (GSD) systems as well as environmental sex determination (ESD). Temperature is the factor controlling ESD in reptiles and temperature-dependent sex determination (TSD) in reptiles has become a focal point in the study of this phenomenon. Current patterns of climate change may cause detrimental skews in the population sex ratios of reptiles exhibiting TSD. Understanding the patterns of variation, both within and among populations and linking such patterns with the selection processes they are associated with, is the central challenge of research aimed at predicting the capacity of populations to adapt to novel conditions. Here we present a conceptual model that innovates by defining an individual reaction norm for sex determination as a range of incubation temperatures. By deconstructing individual reaction norms for TSD and revealing their underlying interacting elements, we offer a conceptual solution that explains how variation among individual reaction norms can be inferred from the pattern of population reaction norms. The model also links environmental variation with the different patterns of TSD and describes the processes from which they may arise. Specific climate scenarios are singled out as eco-evolutionary traps that may lead to demographic extinction or a transition to either male or female heterogametic GSD. We describe how the conceptual principles can be applied to interpret TSD data and to explain the adaptive capacity of TSD to climate change as well as its limits and the potential applications for conservation and management programs.

Geographic Variation in Sex Determination Patterns in the River TurtlePodocnemis lewyana: Implications for Global Warming

Abstract Species with temperature-dependent sex determination (TSD) are more vulnerable to climate change than species with genetically based sex determination because an increase in average environmental temperature could lead to biased offspring sex ratios. In TSD species, the relationship between sex ratio and constant incubation temperature is characterized by two parameters: the pivotal temperature (Tpiv) and the transitional range of temperature (TRT) that produces both sexes. Species with variation in either of these parameters should have more potential to adapt to new climatic conditions, therefore putting them at lower risk to global warming. In this study, we incubated eggs of the turtle Podocnemis lewyana (Testudines: Podocnemididae) from two geographically isolated populations (Magdalena and Sinu rivers) under controlled microclimatic conditions to corroborate the TSD pattern previously reported for this species and to estimate the degree of inter- and intrapopulation variation in Tpiv and TR...

Differential expression of estrogen receptor alpha in the embryonic adrenal–kidney–gonadal complex of the oviparous lizard, Calotes versicolor (Daud.)

Estrogen signalling is critical for ovarian differentiation in reptiles with temperature-dependent sex determination (TSD). To elucidate the involvement of estrogen in this process, adrenal–kidney–gonadal (AKG) expression of estrogen receptor (ERα) was studied at female-producing temperature (FPT) in the developing embryos of the lizard, Calotes versicolor which exhibits a distinct pattern of TSD. The eggs of this lizard were incubated at 31.5±0.5°C (100% FPT). The torso of embryos containing adrenal–kidney–gonadal complex (AKG) was collected during different stages of development and subjected to Western blotting and immunohistochemistry analysis.The ERα antibody recognized two protein bands with apparent molecular weight ∼55 and ∼45kDa in the total protein extracts of embryonic AKG complex of C. versicolor. The observed results suggest the occurrence of isoforms of ERα. The differential expression of two different protein isoforms may reveal their distinct role in cell proliferation during gonadal differentiation. This is the first report to reveal two isoforms of the ERα in a reptile during development.Immunohistochemical studies reveal a weak, but specific, cytoplasmic ERα immunostaining exclusively in the AKG during late thermo-sensitive period suggesting the responsiveness of AKG to estrogens before gonadal differentiation at FPT.Further, cytoplasmic as well as nuclear expression of ERα in the medulla and in oogonia of the cortex (faint activity) at gonadal differentiation stage suggests that the onset of gonadal estrogen activity coincides with sexual differentiation of gonad. Intensity and pattern of the immunoreactions of ERα in the medullary region at FPT suggest endogenous production of estrogen which may act in a paracrine fashion to induce neighboring cells into ovarian differentiation pathway.

Sex Ratio Bias and Extinction Risk in an Isolated Population of Tuatara (Sphenodon punctatus)

Understanding the mechanisms underlying population declines is critical for preventing the extinction of endangered populations. Positive feedbacks can hasten the process of collapse and create an ‘extinction vortex,’ particularly in small, isolated populations. We provide a case study of a male-biased sex ratio creating the conditions for extinction in a natural population of tuatara (Sphenodon punctatus) on North Brother Island in the Cook Strait of New Zealand. We combine data from long term mark-recapture surveys, updated model estimates of hatchling sex ratio, and population viability modeling to measure the impacts of sex ratio skew. Results from the mark-recapture surveys show an increasing decline in the percentage of females in the adult tuatara population. Our monitoring reveals compounding impacts on female fitness through reductions in female body condition, fecundity, and survival as the male-bias in the population has increased. Additionally, we find that current nest temperatures are likely to result in more male than female hatchlings, owing to the pattern of temperature-dependent sex determination in tuatara where males hatch at warmer temperatures. Anthropogenic climate change worsens the situation for this isolated population, as projected temperature increases for New Zealand are expected to further skew the hatchling sex ratio towards males. Population viability models predict that without management intervention or an evolutionary response, the population will ultimately become entirely comprised of males and functionally extinct. Our study demonstrates that sex ratio bias can be an underappreciated threat to population viability, particularly in populations of long-lived organisms that appear numerically stable.

Sex ratios in naturally incubating Macrochelys temminckii nests, a species with type II temperature-dependent sex determination

The alligator snapping turtle, Macrochelys temminckii, exhibits type II temperature-dependent sex determination (TSD), wherein females are produced at high and low incubation temperatures. This TSD pattern is well studied at constant temperatures, but little work has focused on sex ratios in natural nests that experience daily and seasonal temperature fluctuations. We monitored nesting activity of reintroduced Macrochelys temminckii at Tishomingo National Wildlife Refuge in 2010–2011. Nests located prior to predation were excavated to determine clutch size and the eggs were reburied with a temperature data logger to collect nest temperatures. Overall, 24% of nests were protected with wire mesh prior to predation, and the average clutch size in intact nests was 22.4 eggs. Nest predation rates in the study population will likely approach 100% if nest protection efforts do not continue. Temperature profiles were used to compare estimated sex ratios using two methods—mean nest temperature during middle third of incubation and the degree-day model—to actual sex ratios in naturally incubated Macrochelys temminckii nests. The sex ratio in all 2010 recruits was female-biased (91.8% female); 2011 nests did not produce any hatchlings, likely the result of severe drought. The predicted sex ratios based on mean nest temperature and the degree-day model matched actual sex ratios in the warmer nests (0% male), but the degree-day model estimate proved more accurate in the cooler nest. A strongly skewed population sex ratio could become a threat to this reintroduced population if the strongly female-biased sex ratio in 2010 reflects a long-term trend.

Microclimate of American crocodile nests in Banco Chinchorro biosphere reserve, Mexico: Effect on incubation length, embryos survival and hatchlings sex

Crocodilians have temperature-dependent sex determination (TSD) in which incubation temperature determines sex of embryo. Global warming is expected to alter hatchling sex ratio, leading to the extinction of small populations. Regional climate influence on crocodile nest microclimate and hatchlings' characteristics is poorly known. Here, microclimate in natural nests of American crocodile ( Crocodylus acutus) and its relation with incubation length, hatchling sex and nesting success was studied in Banco Chinchorro Biosphere Reserve (Mexico) from 2007 to 2010. Temperature and relative humidity in different locations within and outside the nests were registered by data loggers. Incident solar radiation above nest was calculated from hemispheric photographs. Incubation length, proportion of hatchling reaching complete development and hatchling sex were determined at hatching. Nest temperatures exhibited a cyclic daily fluctuation due to solar radiation, which is the major heat source for nests. Clutch temperature was relatively stable and its daily amplitude was negatively correlated with clutch depth and size. Rainfall was the major source of clutch temperature decrease. Clutch and metabolic temperatures increased significantly during incubation. A small sample size failed to demonstrate a statistical relationship between length of incubation and mean clutch temperature. Proportion of embryos reaching complete development depended on maximum and minimum clutch temperature, maximum daily amplitude of clutch temperature and maximum decrease in clutch temperature on a period ≤4 day. Results confirmed a Female–Male–Female TSD pattern for C. acutus, with 31 and 32.5 °C as possible pivotal temperatures. Population and hatchling sex ratios were male-biased and fate of crocodiles of Banco Chinchorro could depend on the magnitude of temperature increase in the future.

Climate warming and environmental sex determination in tuatara: the Last of the Sphenodontians?

Climate warming and environmental sex determination in tuatara: the Last of the Sphenodontians?

Effects of incubation temperature on embryonic development rate, sex ratio and post-hatching growth in the Chinese three-keeled pond turtle, Chinemys reevesii

Understanding the effects of incubation temperature on embryos and hatchlings may have important implications for husbandry and conservation in turtles. Unfortunately, such knowledge is deficient for most Asian turtles. We incubated eggs of the Chinese three-keeled pond turtle (Chinemys reevesii) at 6 constant temperatures (24, 26, 28, 30, 32 and 34 °C) to test for the effects of embryonic thermal environment on incubation duration, hatching success, hatchling size and mass, sex ratio and post-hatching growth. Incubation duration (ID) decreased nonlinearly as the temperature (T) increased, and could be estimated by the equation: ID=42.74⁎exp (4.30/(T−17.28). Eggs incubated at 32 °C and 34 °C had lower hatching success than those at 24 °C, 26 °C and 28 °C. The turtle showed a Male–Female pattern of temperature-dependent sex determination (MF or TSDIa), with male bias at low temperatures (24 °C and 26 °C), and female bias at high temperatures (30 °C, 32 °C, and 34 °C). The relationship between sex ratio (SR) and temperature (T) could be estimated by a nonlinear equation, SR=0.025+0.923/(1+exp (−(T−30.03)/0.009))^0.006. Hatchlings from eggs incubated at 24, 26 and 28 °C were larger and heavier than their counterparts at 30 °C, 32 °C and 34 °C. However, the temperature influence on hatchling size disappeared when the turtles were 3 months old, while most hatchlings from eggs incubated at 34 °C did not survive. After 3 months, female turtles from 30 °C and 32 °C grew faster than did male turtles from 24 °C and 26 °C; females from 28 °C grew significantly faster than males from the same temperature. In contrast, there was no difference in growth rate either within females or within males from different temperatures. The dichotomy of growth rate between turtles from high vs low temperatures are thus largely attributed to between-sex difference rather than temperature effects. Taken together, our results indicate that the temperatures ranging from 28 to 30 °C are most suitable for egg incubation in C. reevesii, because of the high hatchability and post-hatching survival, and the fastest growth of hatchlings in these thermal regimes. Small hatchling turtles may catch up in the subsequent post-hatching growth if provided with a suitable husbandry environment, given that turtle size at hatching is not a determinant of growth in this species.

Temperature-Dependent Sex Determination in Hd-rR Medaka Oryzias latipes: Gender Sensitivity, Thermal Threshold, Critical Period, and DMRT1 Expression Profile

The developmental time and thermal threshold for temperature-dependent sex determination (TSD), gender differences in temperature sensitivity, the fertility of thermally sex reversed fish, and the effect of temperature on the expression of two major sex determination/differentiation genes (DMY/DMRT1bY and DMRT1) were examined in the Hd-rR strain of medaka, Oryzias latipes. Fertilized eggs were exposed from either shortly after fertilization (8–16 cells; embryonic stages 5–6) or from middle embryogenesis (heart development stage; stage 36) until hatching to temperatures ranging from 17°C to 34°C. Secondary sexual characteristics, gonadal histology, progeny testing, sex-linked body coloration and gene expression were used to determine phenotypic and genotypic sex. Sex determination was unaffected by low or high temperatures in genotypic (XY) males. In contrast, genotypic (XX) females treated from stages 5–6 showed increasing rates of sex reversal into phenotypic males at temperatures above 27°C up to 100% at 34°C. Thermal manipulation of sex was ineffective after stage 36, indicating that gonadal fate in medaka is determined considerably earlier than histological differentiation (stage 39). High temperature induced DMRT1 expression in genotypic females, which was observed already from stage 36. Sex-reversed males had histologically normal testes, were capable of sexual courtship and, with the exception of fish from 34°C, sired viable progeny when mating with fertile females. These results clarify the pattern of TSD in medaka and provide important clues to understand the mechanism of sex determination in this species. They also suggest that a brief exposure to high temperature early in life could impair the fertility of medaka as adults.

Support for a rare pattern of temperature-dependent sex determination in archaic reptiles: evidence from two species of tuatara (Sphenodon).

BackgroundThe sex of many reptiles is determined by the temperature an embryo experiences during its development. Three patterns of temperature-dependent sex determination (TSD) have been defined, but one pattern where only males are produced above an upper temperature threshold (Type IB) is controversial. Here we report new data on the relationship between constant temperature incubation and sexual phenotype in two species of tuatara (Sphenodon), archaic reptiles of enormous zoological significance as the sole representatives of a once widespread reptilian order.ResultsIn both species, the pattern observed with constant incubation temperatures from 18 to 23°C (or 24°C) supported a female→male (FM or Type IB) pattern of TSD: in Sphenodon guntheri males were produced above a pivotal temperature of 21.6°C, and in S. punctatus (unnamed subspecies on Stephens Island, Cook Strait), males were produced above a pivotal temperature of 22.0°C. The pivotal temperatures and scaling parameters differed between species (p < 0.001). The thermosensitive period (TSP), where temperature influences gonad morphogenesis, occurs between 0.25 and 0.55 of embryonic development. While it is possible that the more common female→male→female (FMF or Type II) pattern exists, with a second pivotal temperature above 23–24°C, we review several lines of evidence to the contrary. Most notably, we show that in S. punctatus, the warmest natural nests during the TSP produce predominantly males.ConclusionAn FM pattern of TSD could be currently adaptive in promoting sexual size dimorphism in tuatara. However, an FM pattern has particularly serious consequences for S. guntheri because current patterns of global warming could exacerbate the male bias already present in the relic population.

Assessment of patterns of temperature-dependent sex determination using maximum likelihood model selection

Sex determination in some reptiles is independent of egg incubation temperature and is called genotypic sex determination (GSD). In many other reptiles, sexual phenotype is dependent on incubation temperature. This phenomenon is called temperature-dependent sex determination (TSD). TSD is categorized by three patterns, based on the majority sex produced at lower and higher incubation temperatures, named MF for Male-Female, FM for Female-Male, or FMF for Female-Male-Female. When large numbers of eggs are incubated at many different incubation temperatures, the assessment of TSD pattern is unambiguous, but when few eggs or few incubation temperatures are used, the categorization of TSD pattern is less straightforward. We propose a new methodology based on maximum likelihood model selection that evaluates and ranks the performance of four descriptive models of sex determination for discrete datasets. This method has the added benefit of giving standardized definitions of two commonly reported parameters of TSD: the pivotal temperature and the transitional range of temperature. Standardization of analyses will help facilitate cross-species meta-analyses of TSD in reptiles.

Significant difference of temperature-dependent sex determination between French Guiana (Atlantic) and Playa Grande (Costa-Rica, Pacific) leatherbacks ( Dermochelys coriacea)

Temperature-dependent sex determination has been recently characterized for leatherbacks ( Dermochelys coriacea) from Playa Grande, on the Pacific coast of Costa Rica. The authors concluded that the pattern of TSD in leatherbacks from Pacific Costal Rica is the same as for leatherbacks from French Guiana, in the Atlantic. However, no statistical tests were performed to validate their conclusion. Here, we use a maximum-likelihood test to look for a possible difference between the populations. We found that the pattern of temperature-dependent sex determination in Atlantic and Pacific leatherbacks was significantly different. The temperature producing 50 % of each sex was not significantly different in both groups, but the range of temperatures producing both sexes was significantly narrower for the French Guiana population. We hypothesize that this difference could reflect a lower genetic polymorphism for temperature-dependent sex determination in this population. A low genetic diversity in the Guiana population compared to the Playa Grande population has been already observed for mitochondrial haplotypes. Our results emphasize the importance of statistical analyses in studies of temperaturedependent sex determination.