Jeffrey W. Lang

PHENOTYPIC PLASTICITY FOR GROWTH IN THE COMMON SNAPPING TURTLE: EFFECTS OF INCUBATION TEMPERATURE, CLUTCH, AND THEIR INTERACTION

We examined a critical component of the Charnov-Bull hypothesis of temperature-dependent sex determination (TSD) by determining the reaction norms of hatchling growth to embryonic incubation temperature in the common snapping turtle, Chelydra serpentina Hormone manipulations of eggs produced females at male temperatures and vice versa, which thereby permitted same-sex comparisons of hatchling growth across a range of incubation temperatures. In this way, the normally confounded effects of incubation temperature and sex were dissociated experimentally. The resultant hatchlings, including controls and experimentals, exhibited normal gonadal structure and sex steroid profiles. The subsequent growth of hatchlings monitored for 6 mo was strongly affected by embryonic incubation temperature but not by sex As predicted, growth was enhanced at incubation temperatures that produced males. Clutch effects and interaction effects (clutch by incubation temperature) on growth were significant. In addition, there was a positive genetic covariance among incubation temperatures, but incubation temperature effects varied among clutches. The variation in growth plasticity among clutches was consistent with Charnov-Bull predictions. In this TSD species, incubation temperature is likely to have differential fitness effects on the sexes mediated via differences in growth.

AMONG-FAMILY VARIATION FOR ENVIRONMENTAL SEX DETERMINATION IN REPTILES

Unlike birds and mammals, in many reptiles the temperature experienced by a developing embryo determines its gonadal sex. To understand how temperature‐dependent sex determination (TSD) evolves, we must first determine the nature of genetic variation for sex ratio. Here, we analyze among‐family variation for sex ratio in three TSD species: the American alligator (Alligator mississipiensis), the common snapping turtle (Chelydra serpentina) and the painted turtle (Chrysemys picta). Significant family effects and significant temperature effects were detected in all three species. In addition, family‐by‐temperature interactions were evident in the alligator and the snapping turtle, but not in the painted turtle. Overall, the among‐family variation detected in this study indicates potential for sex‐ratio evolution in at least three reptiles with TSD. Consequently, climate change scenarios that are posited on the presumption that sex‐ratio evolution in TSD reptiles is genetically constrained may require reevaluation.

Geographic variation in the pattern of temperature-dependent sex determination in the American snapping turtle (Chelydra serpentina)

Sex ratios of the American snapping turtleChelydraserpentina are female-biased at cool temperatures, male-biased at moderate temperatures, and only females are produced at warm temperatures. The laboratory incubation of eggs at several constant temperatures yields patterns of skewed sex ratios. In this study, incubated eggs represented regional samples of C. serpentina from six different latitudes. The sample from the highest latitude yielded malebiased sex ratios across the broadest range of cool temperatures. This male-biased zone and those of the other five samples collectively contributed to a nested arrangement that is approximately symmetrical around a temperature of 24.5 ◦ C. Two indices of temperature-dependent sex determination (TSD) patterns (MF pivotal temperature, width of the male-biased zone) were significantly associated with latitude and with published data on several climatic variables (e.g. sunshine and rainfall). At high latitudes, females nest in exposed locations, locally the warmest places. At low latitudes females place their nests in shaded locations. Such nest site selection renders high latitude nests warmer than low latitude nests, even if only intermittently. Given that constant conditions show faster embryogenesis in high vs low latitude eggs, exposed nest sites seem to be an additional adaptation to hasten embryogenesis in high latitude nests. At low latitudes, shading prevents nests from overheating. We propose that a trade-off between evolution, (1) to balance sex ratio and (2) to maintain male-biased sex ratios in a temperature range that favours male fitness, leads to the observed arrangement of concentric TSD patterns centred around male production at 24.5 ◦ C.

Yolk hormone levels in the eggs of snapping turtles and painted turtles.

Although yolk steroids appear to play important roles in the development, growth, and behavior of some birds, their effects in oviparous reptiles are largely unknown. These investigations were initiated to determine initial levels of steroid hormones in the yolks of eggs from two turtle species. Clutches of snapping turtle (Chelydra serpentina) and of painted turtle (Chrysemys picta) eggs were collected and individual egg yolks were analyzed for estradiol-17beta (E(2)) and testosterone (T) using specific RIAs. E(2) and T levels differed significantly between species, the mean E(2) value in snapping turtles was 2.78+/-0.095 (ng/g) compared to 0.89+/-0.064 (ng/g) for painted turtles, and the mean value for T in snapping turtle yolks was 2.56+/-0.098 (ng/g) compared to 0.68+/-0.045 (ng/g) for painted turtles. In addition, E(2) levels were greater than T levels in both species. Within each species, there were significant differences among clutches from different females. E(2) levels in the snapping turtle yolks varied from a clutch mean of 1.38 to 4.55 ng/g and in painted turtles, the clutch means for E(2) varied from 0.34 to 1.34 ng/g. T levels demonstrated similar phenomena within species, with levels in snapping turtles varying from a clutch mean of 0.68 to 4.71 ng/g. Painted turtle levels of T varied from a clutch mean of 0.22 to 0.72 ng/g. There were also significant differences in the E(2)/T ratio, however, E(2)/T ratios did not differ between species. Painted turtle follicles of different sizes showed significant differences in levels of both E(2) and T, and these differences may reflect differing deposition patterns of these steroids in the egg yolk of this turtle during vitellogenesis. The differences in E(2) and T concentration reported here could have important implications for development, growth, and behavior in oviparous reptilian species.

Incubation temperature and sex affect mass and energy reserves of hatchling snapping turtles, Chelydra serpentina

Temperature during embryonic development irreversibly determines gonadal sex in many oviparous reptiles. Although embryonic temperature also influences a number of other traits in these species, it is unclear whether such effects are primarily due to incubation temperature or to gonadal sex. Here we dissociated these normally confounding effects via hormonal manipulations of snapping turtle embryos (Chelydra serpentina), a species with temperature-dependent sex determination. We then ascertained temperature and sex effects on residual yolk mass, abdominal fat body mass, and total mass in neonates shortly after hatching or after one month without feeding. Yolk mass was initially affected by incubation temperature, clutch identity. and their interaction, but not gonadal sex. Yolk mass was not influenced by any of these variables 30 d after hateching. Fat mass was initially affected by temperature and clutch, but not sex. Gonadal sex did, however, affect fat mass at 30 d of age as did temperature and clutch. Incubation temperature affected total mass in a complex manner. There were significant clutch by temperature interactions initially, but no main effect of temperature. These effects changed so that the temperature effect was significant but interaction effects were not detected at 30 d of age. Repeated measures analysis of total mass after hatching and at 30 d of age indicated that turtles from lower temperatures lost more mass than turtles from higher incubation temperatures. and that females lost more mass than males. These data indicate that some hatchling traits were influenced directly by incubation temperature, whereas others were independently affected by gonadal sex. These results are consistent with a leading hypothesis for the evolution of temperature-dependent sex determination.

Dynamics of yolk steroid hormones during development in a reptile with temperature-dependent sex determination

Many oviparous reptiles exhibit temperature-dependent sex determination (TSD); i.e., the temperature at which the egg is incubated determines the sex of the offspring. In TSD reptiles, yolk steroids not only may influence sex determination, but also may mediate hormonal effects on subsequent growth and behavior, as in some avian species. We investigated changes in the levels of estradiol (E(2)) and testosterone (T) during development in yolks of snapping turtle eggs, examined how incubation temperature affects hormone levels, and determined how hormones in turtle eggs are influenced by individual females (=clutch effects). Results indicate significant decreases in both hormones (>50% decline) by the end of the sex-determining period, when two-thirds of the development is complete. The declines in both E(2) and T were significantly affected by incubation temperature, but in different ways. Eggs incubated at female-producing temperatures maintained high levels, those incubated at male-producing temperatures had low E(2) values, and eggs incubated at pivotal temperatures had intermediate levels of E(2). At all three temperatures, T values underwent significant but approximately equal declines, except during the developmental stages just after the sex-determining period, when T levels decreased more at the male-producing temperature than at either of the other two temperatures. Initially, there were significant clutch effects in both hormones, but such differences, attributable to individual females, were maintained only for E(2) later in development. Here we report for the first time that incubation temperature significantly affects the hormonal environment of the developing embryo of a turtle with temperature-dependent sex determination. Based on this and related findings, we propose that yolk sex steroids influence sexual differentiation in these TSD species and play a role in sex determination at pivotal temperatures.

Sex‐reversed and normal turtles display similar sex steroid profiles

In many oviparous reptiles, incubation temperature determines hatchling sex. Manipulation of the embryonic hormonal environment can result in sex reversal. We measured circulating estradiol 17-β (E2) and testosterone (T) levels in temperature-determined and sex-reversed snapping turtles (Chelydra serpentina). Eight-month-old turtles have sex steroid profiles that are sex specific and are consistent with gonadal sex. Males had higher levels of T than females, both before and after treatment with follicle stimulating hormone (FSH). In males, T levels increased approximately 11-fold after FSH challenge, whereas in females, T levels remained low. In contrast, levels of E2 after FSH challenge were higher in females than in males, but showed no difference before treatment. Sex-reversed individuals appear to have normal sex steroid profiles of T and E2, both at basal levels and in response to gonadotropin challenge. Our study indicates that the steroidogenic capability of these juvenile turtles was consistent with gonadal morphology and suggests that these sex-reversed individuals have normal gonadal function.

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.

Sex Ratios of Wild American Alligator Hatchlings in Southwest Louisiana

Abstract The sex of American Alligator (Alligator mississippiensis) hatchlings is determined by the egg temperature during the middle third of the 9–12 week incubation period. As a consequence, predictable sex ratios are possible for clutches incubated in constant temperatures in the laboratory, but naturally occurring sex ratios of American Alligator hatchlings from wild nests exposed to fluctuating temperatures are not well documented. Over a 5-year period (1995–1999), we determined the sex of American Alligator hatchlings from wild nests left in the field until after sex was irreversibly determined. A total of 6226 hatchlings from 232 naturally incubated wild nests showed a strong female bias (71.9% females, yearly range = 62.3–89.4% females). Most nests (64.2%) produced hatchlings of both sexes. Of the remaining clutches that produced exclusively one sex (83 nests), 78 nests produced all females, and 5 nests produced only male hatchlings. For the 2 years in which nest-cavity temperatures were known, higher temperatures led to production of significantly more male hatchlings (P < 0.001 for both 1997 and 1999). Knowledge of natural sex ratios of hatchlings can aid in the management and harvest of this commercially valuable species, and in understanding sex-ratio bias in American Alligator populations.