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Additional factors influence temperature-dependent sex determination in Leopard Geckos

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Additional factors influence temperature-dependent sex determination in Leopard Geckos
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Janes, Daniel E
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Dams ( jstor )
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Estrogens ( jstor )
Female animals ( jstor )
Humidity ( jstor )
Incubation ( jstor )
Reptiles ( jstor )
Sex determination ( jstor )
Sex ratio ( jstor )
Turtles ( jstor )
Dissertations, Academic -- Zoology -- UF
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Zoology thesis, Ph.D
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Thesis (Ph. D.)--University of Florida, 2004.
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Includes bibliographical references.
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Vita.
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by Daniel E. Janes.

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ADDITIONAL FACTORS INFLUENCE TEMPERATURE-DEPENDENT SEX
DETERMINATION IN LEOPARD GECKOS















By

DANIEL E. JANES













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004




















I dedicate this work to my parents. They have always encouraged me to follow my own path and welcomed me back home to recover from my defeats and celebrate my victories.


Also, I dedicate this work to my teachers and students. They have shared their thoughts and ambitions with me. Every one has shown me a slightly different way to see the
world and every one has allowed me to share my own perspective with them. We should all be so lucky.















ACKNOWLEDGMENTS

I extend my deepest gratitude to my co-advisors, Drs. Marta L. Wayne and F. Wayne King, for their support, encouragement, and wisdom. My other committee members, Drs. Karen Bjorndal, Louis Guillette, and Max Nickerson, have maintained an open door policy of which I have taken full advantage. Conversations with my advisors have taught me the value of discussion at all stages of research design and execution and the necessity of collaboration. I wish to thank all of my friends in the Departments of Zoology, Wildlife Ecology and Conservation, and the Florida Museum of Natural History. Their friendship and laughter daily reinforced one of the main reasons I am a scientist and teacher. It is fun. This research has been funded by the Florida Museum of Natural History, Sigma Xi, and William and Marcia Brant of The Gourmet Rodent. Conversations with Ben Bolker helped shape my analyses of data. Data collection assistance was provided by Jason Phillips, Jennifer Comiskey, Sara Reyes, Jennifer Mobberley, Margaret O'Brien, Eric Timauer, Cassandra Pedrosa, Alana Schoenberg, Julia Huang, Craig Ajmo, John Bowden, Erin Taylor, and Kristin Barus. Work was conducted in accordance with University of Florida IACUC protocol Z010.












111















TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS ........................................................................................... iii

ABSTRACT........................................ ........................................................................ vi

CHAPTER

1 GENERAL INTRODUCTION ............................................................................... 1

2 ABIOTIC EFFECTS ON SEX DETERMINATION IN LEOPARD GECKOS.......6

Introduction .................................................. ........................................................ 6
M ethods .................................................................................................................... 10
Animals ....................................................... ................................................10
Sexing Procedure ........................................................... ................................ 11
Analysis............................................................................................................. 12
Results ..................................................................................................... 12
Discussion ..........................................................................................................13

3 QUANTITATIVE GENETIC VARIATION IN SEX-DETERMINING
RESPONSE TO INCUBATION TEMPERATURE IN LEOPARD GECKOS......25

Introduction ........................................................................................................25
M ethods ..............................................................................................................28
Animals ................................................... ............................................... 28
Sexing Procedure ...........................................................................................29
Analysis.............................................................................................................30
Results ................................................................................................................... 30
Discussion ..........................................................................................................31

4 ESTROGEN AND ESTROGEN MIMIC INCREASE PRODUCTION OF MALES
IN A TEMPERATURE-DEPENDENT SEX-DETERMINING SPECIES..........38

Introduction ........................................................................................................38
M ethods ..............................................................................................................40
Animals ................................................... ............................................... 40
Sexing Procedure ...........................................................................................41
Analysis.............................................................................................................42



iv









R esu lts ................................................................................... .............................. 42
D iscussion .......................................... ..................... .... ..................................44

5 GENERAL DISCUSSION............................. ............ ........................ 55

LIST OF REFERENCES ..............................................................................................61

BIOGRAPHICAL SKETCH ........................................................... .....................70















































v















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy


ADDITIONAL FACTORS INFLUENCE TEMPERATURE-DEPENDENT SEX DETERMINATION IN LEOPARD GECKOS


By

Daniel E. Janes

May 2004

Chair: F. Wayne King
Cochair: Marta L. Wayne
Major Department: Zoology


Hatchling sex ratios of reptiles with environmental sex determination (ESD) are influenced by incubation temperature. Leopard Geckos, Eublepharis macularius, nest in a region where they are exposed to a wide range of temperatures. If temperature is the sole determinant of sex in this species, then hatchling sex ratios should be highly correlated with the thermal gradient in different parts of the species' range. In this scenario, a broad thermal gradient would promote regional variation in hatchling sex ratios of E. macularius. However, reports of regional variation in hatchling sex ratios are rare among ESD reptiles. If a secondary factor (or factors) plays a role in sex determination of this species, their hatchling sex ratios in nature would be expected to vary seasonally and regionally with less correlation with thermal gradient. In this study, E. macularius eggs were incubated at three temperatures in two consecutive years. The vi









range of temperatures studied in these experiments includes the species-specific temperatures at which the lowest proportion of males (0.0) and the highest proportion of males (-0.7) are produced per treatment group. Each treatment group consisted of 18 E. macularius eggs. At each temperature, the effects of humidity, quantitative genetic variation, and environmental endocrine disrupters on hatchling sex ratios of treatment groups were measured. Humidity did not significantly affect hatchling sex ratios. Also, results of the quantitative genetics experiment suggest that ESD in E. macularius is a genotype x environment interaction. Both natural and synthetic estrogens affected hatchling sex ratios at both male- and female-producing temperatures. I conclude that ESD is a multi-dimensional trait in E. macularius. The influences of abiotic factors like local profile of environmental hormone and hormone mimic concentrations and biotic factors like quantitative genetic variation may ameliorate the effects of climate change on ESD. Estimates of extinction rates of ESD species as a result of changing climates should be reconsidered in light of these data. As other researchers have concluded for other ESD reptiles, I conclude that the partial genetic control of sex determination in E. macularius is a polygenic trait and responds to temperature fluctuation as a genotype x environment interaction. In light of these conclusions, sex-determining mechanisms of reptiles should be reconsidered as a broad spectrum of multi-dimensional mechanisms instead of a simple dichotomy of ESD and genetic sex determination.












vii














CHAPTER 1
GENERAL INTRODUCTION

The optimal sex ratio of a population may change depending on fluctuating environmental stresses and sex-differential vulnerability to environmental stresses. According to the Trivers-Willard hypothesis, parents may adjust their offspring sex ratio to address sex-differential fitness and garner the greatest reproductive advantage (Trivers and Willard 1973). The Trivers-Willard hypothesis is based on Fisher's theory of equal parental investment in the rearing of sons and daughters (Fisher 1930). If sons and daughters are unequally affected by environmental stress, then offspring sex ratios should be skewed toward the sex that is less negatively affected. According to Fisher, the sex that is most negatively affected by environmental stress will be produced less frequently and will garner more parental input. The cost paid by the parents for the rarer, more energetically expensive offspring sex should balance with the cost they pay for the more common, less energetically expensive offspring sex. For example, sex-differential growth rates would cause an immediate increase in parental investment for one sex over the other. If the immediate investment required by the faster growing offspring can not be made because of resource scarcity or poor maternal condition, then the slower growing sex will be produced in excess of the faster growing sex. The slower growing sex can survive an immediate but short-lived dearth of resources much better than the faster growing sex. Environmental stresses that differentially affect males and females should cause sex ratios to deviate from 0.5. The Trivers-Willard hypothesis is most




1






2


appropriately tested using a species with environmental sex determination. This type of organism will produce offspring sex ratios that can be directly linked to experimentallyinduced environmental conditions without the confounding factor of predominant genetic control of sex determination.

The gonadal sex of all organisms has been attributed to either genetic sex

determination (GSD) or environmental sex determination (ESD). In GSD, the sex of the organism depends on the sex chromosome contributions from the organism's parent(s). In ESD, the sex of the organism depends on the post-fertilization environment experienced by the organism as a developing embryo. Typically, ESD refers to the effects of incubation temperature, although Heiligenberg (1965) reported a sex ratio skewing effect of pH in cichlids. Among vertebrates, the initiation of sex differentiation is predominantly controlled by incubation temperature in tuatara, some fish, turtles, and lizards and all crocodilians (Bull 1980; Conover and Kynard 1981; Ewert and Nelson 1991; Janzen and Paukstis 1991). All other vertebrates appear to be genetically sexdetermined. Incubation temperature can also affect body size and growth (Crews et al. 1998; Janes and King, unpublished data), adult sexuality (Gutzke and Crews 1988), aggressive behavior (Rhen and Crews 1999), and the organization of neural structures (Coomber et al. 1997). Incubation temperature is known to cause the development of an embryo's testes or ovaries; the proximate mechanism is still unknown.

The recognition of ESD as a derived condition is supported by reports of GSD in amphibians, a basal taxon to reptiles. No surveyed amphibians have demonstrated a sexdetermining effect of incubation temperature within the range of temperatures to which their eggs are naturally exposed (Hayes 1998). The adaptive significance of ESD has






3


eluded researchers (Janzen and Paukstis 1991). Concensus favors ESD as either an adaptive mechanism for species in which a fitness advantage oscillates between males and females (Shine 1999) or as a neutral mechanism that has not been counter-selected (Bull and Charnov 1989; Girondot and Pieau 1999).

This study will test the effects of environmental stresses on clutch sex ratios of Leopard Geckos, Eublepharis macularius, a reptile species for which much has been written on their sex-determining response to incubation temperature. Leopard Geckos live in a range of climatic heterogeneity. Their range extends from the grasslands of southeastern Turkey to the forests of southwestern India (Smith 1935). Clutch sex ratios are male-biased when incubated at 32.50C. Cooler and warmer incubation temperatures within the full range of viable incubation temperatures (250 to 35C) cause female-biased clutch sex ratios (Viets et al. 1993). If the ESD mechanism behaves similarly throughout the climatically heterogeneous zones of their range and all other variables are irrelevant, then differences in the primary sex ratios of E. macularius can be expected as a result of different incubation temperatures in different parts of their range (Viets et al. 1993; Crews et al. 1996; Coomber et al. 1997; Rhen et al. 2000). Also, climate change due to global warming or other causes might cause dramatic shifts in population sex ratios of E. macularius and other ESD species.

Based on the observations that (a) among higher vertebrates, the sex of several species of reptiles and fishes appears to be controlled by incubation temperature, (b) populations of environmentally sex-determined species can cover ranges that include more than one climate zone, and (c) sex ratios do not appear to be unbalanced at different extremes of their range, I hypothesize that the sex-determining mechanism in E.






4


macularius is a multi-dimensional mechanism, controlled in part by incubation temperature and in part by at least one of the following parameters. Differences in the timing of incubation (Lance 1989; Guillette et al. 1997), placement and construction of nests (Magnusson et al. 1985; Shine and Harlow 1996), genetic influences (Parker and Orzack 1985; Wachtel 1989), presence of industrial contaminants (Guillette et al. 1997; Crain et al. 1997), or other environmental variables (Webb and Smith 1987) may express partial control over clutch sex ratios in E. macularius. Other variables may counteract the sex ratio skewing effects of ambient temperature and maintain population sex ratios near 0.5 at all points within the range of the species. For this study, chapters 2 and 3 will provide data on the intrinsic responses of the organism to naturally occurring incubation conditions. Chapter 4 will introduce an anthropogenic stress (DDT) to a series of E. macularius eggs in order to test the plasticity of their sex-determining mechanism when faced with an extrinsic stress that is presently found in their home range.

Laboratory incubations at constant temperatures can provide controlled

experimental data but do not accurately represent natural nest environments. The experiments in this study have been designed to offer a more realistic, multi-dimensional representation of the environment experienced by E. macularius eggs during the incubation period when gonadal sex is determined by varying several factors. This study will serve to expand the currently accepted definition of ESD. If gonadal sex is controlled by a multi-dimensional influence of the nest microenvironment, then researchers can investigate potential advantages of maleness and femaleness within a more specifically defined milieu. The Trivers-Willard hypothesis may be supported by






5


the sex-determining responses of E. macularius to environmental factors other than temperature or to interactions of sex-determining factors.

Research Objectives: I tested extrinsic and intrinsic factors in order to determine which, if any, work in concert with incubation temperature to adjust clutch sex ratios in E. macularius. I hypothesized a multi-dimensional sex determination model in which skewing effects from the extremes of sex-determining factor are ameliorated by opposing extremes of another factor or factors.














CHAPTER 2
ABIOTIC EFFECTS ON SEX DETERMINATION IN LEOPARD GECKOS Introduction

Animals and plants have evolved numerous sex-determining mechanisms that are described as either genetic sex determination (GSD) or environmental sex determination (ESD). In GSD, sex is determined by the genetic constitution of an embryo upon conception. In ESD, sex is determined by extrinsic, abiotic factors that define the incubation environment. Regardless of mechanism, Fisher (1930) predicted that primary sex ratios should represent an equal parental investment in male and female offspring. However, Fisher's balanced parental investment may not be represented in ESD species because primary sex ratios may be removed from parental control.

Among squamate reptiles, ESD and GSD are found among closely related

species, suggesting that ESD and GSD have evolved separately on many occasions (Viets et al. 1994). Within the squamate subfamily Eublepharinae, the Leopard Gecko, Eublepharis macularius, and the African Fat-Tail Gecko, Hemitheconyx caudicinctus, are ESD species and the Banded Gecko, Coleonyx mitratus, is a GSD species (Bragg et al. 2000). Environmental sex determination in squamates refers to temperature-dependent sex determination (TSD) specifically, the influence of incubation temperature on sex determination. Other forms of ESD based on photoperiod or nutritional resource availability have been reported in invertebrates but only TSD has thus far been reported in squamates (Bull 1980). Genetic variation in sex-determining responses to incubation




6






7


temperature is seen among and within ESD species like those in Eublepharinae (Elphick and Shine 1999). Thus, the same constant incubation temperature may produce different sex ratios in different populations of the same species.

Most studies report female-biased populations of reptiles exhibiting TSD,

regardless of local nest site temperatures (Bull and Charnov 1989; Ewert and Nelson 1991). In light of this, it appears that the relationship between hatchling sex ratios and the incubation environment has not been adequately defined. Other extrinsic or intrinsic variables can play major or minor roles in the maintenance of female-biased hatchling sex ratios. Aside from temperature, several factors have been implicated in the relationship between the sex determination of an embryo and its immediate environment. Exogenous sex steroid hormones or endocrine active contaminants can alter sex determination. Further, the behavioral and physiological condition of the mother as well as the environments encountered through the life of the mother, and age and sociosexual experience of either parent could influence sex differentiation of ESD species (Crews 2003). Temperature has been identified as a determinant of sex determination in all squamates studied to date that exhibit ESD. Carbon dioxide concentrations and pH (Etchberger et al. 2002) and humidity (Gutzke and Paukstis 1983) also have been identified as having a secondary role in some reptilian species. Here, I consider humidity as a major factor in sex determination of E. macularius.

Eublepharis macularius has a natural range that includes desert and grasslands of the Middle East (Daniel 2002). In this range, nest site temperatures and humidities fluctuate dramatically among seasons, years, and regions. Eublepharis macularius






8


follow a pattern of sex determination in which an increased proportion of males is produced at an intermediate incubation temperature (31 -3 3C) and an increased proportion of females is produced at cooler (26-280C) and warmer (34-35C) incubation temperatures (Viets et al. 1993; Crews 2003).

If E. macularius maintains an evolutionarily stable sex ratio across thermally variant environments, the effects of differing ambient temperature must be mediated either by local adaptation (Mrosovsky 1988; Blackmore and Charnov 1989; Viets et al. 1993), carbon dioxide, pH (Etchberger et al. 2002), maternal affects (Bowden et al. 2002), humidity, or a complex interaction of some or all of these factors. Environmental sex determination could be caused by a wide array of weak influences during incubation (Bull et al. 1982a; Bull et al. 1982b). In addition, sex-determining responses to these influences could be phenotypically plastic, as is widespread in reptilian phenotypes (Shine and Elphick 2001). Population sex ratios of E. macularius can be further altered by short-term weather fluctuations because of the extreme sensitivity of reptiles to environmental variables during embryogenesis (Shine and Elphick 2001).

The influence of humidity on sex determination has been debated in the past. Gutzke and Paukstis (1983) reported that in the Painted Turtle, Chrysemys picta, more males were produced in wet substrates than in dry substrates at typically male-producing temperatures. In a second study, C. picta eggs were again incubated on wet or dry substrates (Paukstis et al. 1984). In the latter study, the reverse pattern was reported. More male turtle hatchlings were produced in dry substrates than in wet substrates. Also, a replication of the experiment by Gutzke and Paukstis (1983) yielded no effect of substrate moisture on C. picta sex determination at any incubation temperature (Packard






9


et al. 1989). Packard's advice to consider the effects of substrate moisture cautiously has been heeded by subsequent researchers (Packard et al. 1989; Janzen and Morjan 2001). Typically, substrate moisture is not considered a potential sex determinant in ESD studies. In previous experiments, humidity of incubation substrate did not affect sex determination in E. macularius (B. Viets, personal communication). Lack of an effect of humidity on ESD has also been reported in the Flatback Turtle, Natator depressus (Hewavisenthi and Parmenter 2000).

Different squamate species have different patterns of water exchange between the egg and the environment and, therefore, different sensitivities to environmental moisture (Ji and Du 2001). If humidity has a partial effect on sex determination in E. macularius, its effect in nature could be significant because of the paucity of moisture in the species' natural range. This species inhabits and builds nests in arid country (Daniel 2002). This habitat would pose a significant challenge to a sex-determining mechanism that is influenced by humidity because the maintenance of a humid nest in an arid habitat is a greater challenge than the maintenance of a dry nest in a humid habitat. Also, humidity may have a more significant effect in the development of more pliable-shelled eggs like those of E. macularius (Ji and Du 2001). The influence of humidity on sex determination could be direct by changing the response of the male- determining factor (Deeming and Ferguson 1989) or indirect by influencing nest temperatures, affecting body size as in C. picta and N. depressus (Cagle et al. 1993; Hewavisenthi and Parmenter 2001), or by influencing the conversion of yolk into fat as in the Cuban Rock Iguana, Cyclura nubila (Christian and Lawrence 1991).






10


In this experiment, E. macularius eggs were treated with substrates of varied

water content and incubated at three temperatures within the species' range of embryonic thermal tolerance. If humidity has an effect on this species' sex determination, then different sex ratios in response to different humidities should be detected most easily at the intermediate incubation temperature. The influence of a weaker determinant of sex is more likely to be detected at an intermediate incubation temperature, because the skewing influence of temperature on the sex ratio is minimized (Bull et al. 1982a). However, extreme temperatures may also interact uniquely with humidity. For these reasons, extreme and intermediate incubation temperatures are studied in this project.

Methods

Animals

On 22 March 2002, Leopard Gecko, Eublepharis macularius, eggs were obtained from The Gourmet Rodent, a reptile breeder in Archer, Florida. Eggs were collected within 24 hrs after oviposition from a breeding colony of -5000 dams. All eggs were candled to test viability. Two hundred and sixteen viable E. macularius eggs were placed in a plastic box filled with vermiculite and transported by car to the University of Florida. They were placed in containers that consisted of six 188 ml plastic cups banded together in rings. Each cup contained 70 ml perlite and tap water and was sealed with a tight fitting lid punctured with one gas-exchange hole. The eggs were divided among 36 egg containers. Each container held six eggs. Each egg was placed individually within one of the six cups in a container. The egg containers were divided into three groups of 12. Each group was placed in an environmental chamber and maintained at 260, 300, or 32.50 C for the duration of the experiment. Chamber temperatures were recorded every minute






11


throughout the experiment with Hobo temperature loggers. Within each chamber, the 12 egg containers were divided into four treatment groups of three containers. Each treatment group, consisting of 18 eggs, was exposed to 20%, 25%, 30%, or 35% humidity. At the beginning of the experiment, humidity was varied by adding 14 ml, 17.5 ml, 21 ml, or 24.5 ml of water to the perlite in the egg cups in the 20%, 25%, 30%, or 35% humidity treatment groups, respectively. Every day, the incubation chambers were opened and the egg containers were removed. Each cup was opened momentarily in order to release metabolic gas waste and check for hatchlings. Eggs that grew mold were discarded upon discovery. Position of egg containers within an incubator was randomized daily.

Sexing Procedure

Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2bromo-2-chloro-1,1,1-trifluoroethane). Geckos were fixed in Bouin's fixative and preserved in 75% ethanol. The reproductive organs were removed from each gecko and prepared for analysis by light microscopy. The reproductive organs are opaque white, cylindrical structures on either side of the posterior end of the dorsal artery. After removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55; Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The resulting paraffin blocks were sectioned at 8 Lm and stained with a modified trichrome of Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from all geckos. If seminiferous tubules were identified within the tissue sections, the gecko was scored as male (Figure 2a). If seminiferous tubules were absent, the gecko was






12


scored as female (Figure 2b). Seminiferous tubules were identified as clusters of simple, circular structures with a narrow lumen (Berman 2003). By relying on histological examination of our specimens, I avoided potential macroscopic misidentification of male and female reproductive organs.

The design was replicated the following year with a new sample of 216 E.

macularius eggs from a separately maintained breeding colony at the Gourmet Rodent. Thus, the sires and dams used in the first year's experiment were not the sires and dams used in the second year's replication. Work was conducted in accordance with University of Florida IACUC protocol Z010. Analysis

Sex ratio data were analyzed by ANOVA with fixed main effects of humidity, incubation temperature, and year, as well as their interactions. Egg containers were nested within year X temperature X humidity, and treated as a random effect. Data were analyzed using SAS version 6.10 for the Macintosh. ANOVAs were performed using PROC GLM.

Results

Temperature and humidity were manipulated simultaneously to assess effects on ESD over the two years. Variances between years were not significantly different (Table 1). Years were analyzed together or separately. Overall, temperature significantly affected hatchling sex ratios as expected for a species exhibiting TSD (MS = 0.527; P<0.05). Humidity did not affect sex ratios when both years were examined together or individually (MS = 0.015; Table 1). In the first year of experimentation, neither temperature nor humidity, examined independently, altered hatchling sex ratios (Figures






13


3 and 4). In the second year, with the exception of the treatment group exposed to 20% humidity, the treatment groups incubated at the coolest temperature (260C) had a lower proportion of males than treatment groups incubated at both warmer temperatures. Temperature significantly affected hatchling sex ratios in year 2 (MS = 0.713; P<0.05; Table 2). The highest proportion of males per treatment group was produced at the warmest incubation temperature (32.5oC; Figures 5 and 6). The order of male proportion per treatment group at 300C in the second year does not follow the order of male proportion per treatment group at 300C in the first year.

Discussion

I have found, using my design, that 1) temperature affected sex ratios, 2) humidity did not affect sex ratios, and 3) humidity and temperature did not interactively affect sex ratios. Previous studies examining the effect of humidity on sex determination in TSD species have produced inconsistent results. Gutzke and Paukstis (1983) found an effect of substrate moisture on sex differentiation in the freshwater turtle, Chrysemys picta, whereas Packard et al. (1989) did not. Clearly, the mechanism and the adaptiveness of the relationship between incubation environments and hatchling sex ratios have not been sufficiently characterized. Deeming and Ferguson (1989) refer to a male-determining factor that is temperature-sensitive. Candidate mechanisms for TSD include temperaturedependent synthesis or activity of enzymes, heat shock proteins, and temperaturesensitive gene expression (Mrosovsky 1994). Some intrinsic mechanism(s) responds to the microclimate of the nest site and determines hatchling sex. Eublepharids select nestsite microclimates that result in higher hatchling survivorship and not necessarily evolutionarily stable sex ratios (Bull et al. 1988a; Bragg et al. 2000). However, if






14


humidity affected both survivorship and to a lesser extent sex determination, then nest site selection could influence population sex ratios. Cultural inheritance of nest site microclimates has also been implicated in the inheritance of hatchling sex ratios (Freedberg and Wade 2001). Nest sites tend to retain their abiotic profiles from year to year. If daughters inherit nest sites from their mothers, then a cultural component exists for the inheritance of hatchling sex ratios in ESD species. However, if the nest sites are found in areas that are thermally different from one breeding season to the next, then cultural inheritance will not fix sex ratios over long periods of time. The microevolution of sex ratio depends in part on maternal choice of thermal qualities of nest sites (Janzen and Morjan 2001).

My results from chapters 3 and 4 suggest that hatchling sex ratios can vary among populations even if incubation temperature remains constant. However, the results of this experiment and previously published conclusions (Janzen and Morjan 2001) suggest that hatchling sex ratios of E. macularius do not vary in relation to nest site humidity. The effect of humidity on hatchling sex ratios in this study was not significantly different between two years of experimentation. In short, sex determination in E. macularius did not respond to humidity, at least as examined by the experimental design I performed. Researchers should note that humidity was not measured throughout this experiment. After initial volumes of water were added at the beginning of trials, humidities may have varied incidentally among the treatment groups. Incidental variation may explain the different effects of temperature on hatchling sex ratios between the first and second years of experimentation. However, incidental variation in temperature is less likely because of constant monitoring of temperature throughout the trials. The lack of effect of






15


temperature on hatchling sex ratios in the first year may also be explained by genetic differences between the colonies of E. macularius used in the two years of experimentation. The breeding facility from which eggs were collected maintains separate breeding colonies that do not interbreed. Drift or local adaptation of the sexdetermining response to incubation temperature may explain the difference between the results from the first and second years of experimentation. This result would suggest intraspecific variation in the effect of abiotic factors on sex determination in E. macularius. Such intraspecific variation of sex-determining mechanisms has only been reported anecdotally for Tokay Geckos, Gekko gecko (F. Janzen, pers. comm.). However, intraspecific variation in the sensitivity of temperature-dependent sex determining mechanisms has been reported for A. mississippiensis (L. Guillette, pers. comm.). In short, the factors that influence ESD within species are not expected to vary but the degree to which fixed factors affect ESD is expected to vary.

A multifactorial sex-determining mechanism would have an impact on

conservation considerations for ESD species. Vogt and Bull (1984) present empirical evidence that vegetation changes influence hatchling sex ratio in Map turtles. Invasive plants are changing vegetation cover over Nile crocodile nesting sites (Leslie and Spotila 2001). The consequent effects on Nile crocodile sex ratios have not yet been reported. Reptile populations with ESD may need to be manipulated if nest site microclimates change too dramatically (Mrosovsky 1994). In the worst case scenario, extinction may result from climate change (Janzen 1994a). If the influence of incubation temperature on hatchling sex ratios is exacerbated or ameliorated by another weaker sex determinant, risk assessments in the face of habitat alteration must be reconsidered.






16


Environmental sex determination could enhance maternal fitness by permitting the production of more offspring of the sex that is best-suited to the incubation environment (Charnov and Bull 1977). Chrysemys picta eggs incubated in wet substrate maintained a higher temperature than eggs in dry substrate but only for the first third of incubation. After the first third, the eggs in drier substrates maintained a higher temperature than those in wet substrates (Gutzke et al. 1987). The temperature-sensitive period for E. macularius in which sex determination is affected by local abiotic factors does not begin until the beginning of the middle third of incubation (Bull 1987). Therefore, we would expect drier treatment groups to be warmer during the temperaturesensitive period and produce more males. This pattern, however, was not observed in this study. The enhanced sensitivity of offspring sex ratio to secondary abiotic factors should be investigated in a wider array of environmentally sex-determined species because of the current lack of detail about the evolution of sex-determining mechanisms. If some species respond similarly to a series of environmental variables, then a new dimension of the mechanism could be considered to characterize the divergence of ESD mechanisms among squamates.








17









0.8 0.7

2 0.6 0 0.5

0

0.4
0.3
c 0.2


0


-0.1

24 26 28 30 32 Temperature (C)






Figure 1. Effect of incubation temperature on hatchling sex ratios of Leopard Geckos.
Control sex ratio data were collected from treatment groups exposed to either no treatment or a 95% EtOH vehicle from the second year of experimentation
described in Chapter 4.






18

















Figure 2. Light microscopy images of reproductive tissue from Leopard Geckos,
Eublepharis macularius. These images are magnified 200X. (a) Male reproductive organs have seminiferous tubules that have a thick, simple
cortex and a narrow lumen. (b) Female reproductive organs lack
seminiferous tubules.







19



0.8 0.7
2 0.6


0.5 0.4

C 0.3

S0.2

0.1 --------------0


-0.1
24 26 28 30 32
Temperature (C) Figure 3. Sex ratios of treatment groups of Leopard Geckos in the first of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the solid, dashed, gray stippled, and black
stippled lines respectively. Bars represent standard errors.






20


0.8


O- 0.7
0
oL
(D


M 0.5


Ca 0.4

0.3
0
0
S 0.2
0

a. 0.1



26 30 32.5 Temperature (C)

Figure 4. Sex ratios of treatment groups of Leopard Geckos in the first of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the lightest to darkest bars respectively.
Standard errors are indicated by vertical lines.








21



0.8

0.7

0.6


Z 0.5

0.
0.3


S 0.2 ......
0
0.1
0


-0.1
24 26 28 30 32
Temperature (C) Figure 5: Sex ratios of treatment groups of Leopard Geckos in the second of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the solid, dashed, gray stippled, and black
stippled lines respectively. Bars represent standard error.






22

0.8


0.7
0
C
0.6


0.5




0.3


t 0.2
0
0
L..
a_ 0.1



26 30 32.5

Temperature (C)

Figure 6. Sex ratios of treatment groups of Leopard Geckos in the second of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the lightest to darkest bars respectively.
Standard errors are indicated by vertical lines.






23

Table 1: ANOVA between years for sex ratios of treatment groups of Leopard Geckos.


Source df MS Year 1 0.430 Temperature 2 0.527* Humidity 3 0.015 Year Temperature 2 0.445 Year Humidity 3 0.067 Temperature Humidity 6 0.266 Year Temperature Humidity 6 0.264

*: P < 0.05






24

Table 2. ANOVA within years for sex ratios of treatment groups of Leopard Geckos.

MS
Source df Year 1 Year 2 Temperature 2 0.205 0.713* Humidity 3 0.020 0.058 Temperature Humidity 6 0.214 0.352

*: P < 0.05














CHAPTER 3
QUANTITATIVE GENETIC VARIATION IN SEX-DETERMINING RESPONSE TO INCUBATION TEMPERATURE IN LEOPARD GECKOS


Introduction

The diversity of sex-determining mechanisms in vertebrates can be classified as either genotypic sex determination (GSD) or environmental sex determination (ESD). In this chapter, I will describe quantitative genetic effects on ESD in Leopard Geckos, Eublepharis macularius. Species that follow a GSD pattern are further subdivided into either :XX/d:XY systems in which males are heterogametic (as seen in mammals) or 9:ZW/6:ZZ systems in which females are heterogametic (as seen in birds). Reptiles show a far greater diversity of sex-determining mechanisms than either mammals or birds (Janzen and Paukstis 1991). Reptiles express either XX/XY or ZZ/ZW patterns of GSD or one of several patterns of ESD in which the sex of offspring is predominantly, if not completely, determined by the environment in which offspring develop as embryos.

Great controversy exists about the adaptive significance of different sexdetermining mechanisms. Researchers' ability to predict which GSD or ESD pattern will be followed by a newly discovered species is poor, regardless of what information can be gathered about the species' natural history, phylogeny, or ecology. Although much has been published about advantages of either ESD or GSD, little consensus can be found concerning the differences that would make one mechanism adaptive for one species and another mechanism adaptive for a different species. The adaptive significance of GSD




25






26


has been summarily attributed to its maintenance of balanced sex ratios in natural populations (Fisher 1930). Because the rarer sex gains a fitness advantage over the more common sex, population sex ratios tend to hover near unity (Fisher 1930). The advantages of ESD are more opaque. Hypotheses regarding the advantages of ESD have been organized in five categories: (a) phylogenetic inertia, (b) group-adaptation, (c) inbreeding avoidance, (d) sex-differential fitness, and (e) quasi-neutrality (Shine 1999; Girondot and Pieau 1999). The enigma is further compounded by the existence of closely-related taxa that express very different sex-determining mechanisms. Although the predominant determinants of sex can be readily identified through experimental matings and incubations, the finer points of the relationship between genes and environment in the process of sex determination have not yet been acceptably clarified in many species. The identified dichotomy of ESD and GSD may cause weaker determinants of sex to be overlooked which, if discovered, would more appropriately describe the process by which a bipotential gonad becomes an ovary or a testis.

Additionally, an adaptation that is completely environmentally determined could be invisible to natural selection. In order to describe the evolutionary history of sex determination in vertebrates, researchers must first test hypotheses about the effects of selection on sex-determining mechanisms. Selection may work on the genetic architecture that allows an effect of temperature or other environmental factor on sex determination. Temperature affects the activation of genes that encode steroidogenic enzymes (Crews 2003). Genetic variation most likely affects the sensitivity of genes to thermal stimuli. Models derived for the adaptive evolution of sex ratio assume that genetic variation exists for primary sex ratio and that the variation is caused by genes of






27


modest effect that segregate according to Mendelian rules (Janzen 1992). These models are supported by little empirical evidence because of the difficulty of measuring primary sex ratios and the interference of the predominant effects of sex chromosomes in GSD species. The heritability of offspring sex ratio incubated under constant temperature has been reported in the temperature-dependent sex-determining (TSD) Ouachita Map turtle, Graptemys ouachitensis (Bull et al. 1982a). Bull et al. (1982a) incubated G. ouachitensis eggs from different families at a constant temperature, 29.20C. Their threshold model for ESD yielded a heritability estimate of 0.82 for the hypothetical sex-determining character. They concluded that natural variation in nest temperatures acted against the high heritability of the sex-determining character to maintain natural populations closer to a balanced sex ratio. Also, Janzen found strong genetic variation in the TSD response of the Common Snapping turtle, Chelydra serpetina, to incubation temperature. However, no significant interaction of temperature and family has been reported for C serpentina. Therefore, Janzen (1992) concluded that variation in the sex-determining response to incubation temperature in the TSD species C. serpentina is not due to a gene x environment interaction.

To test for a gene x environment interaction in the sex-determining mechanism of E. macularius, within a broad range of incubation temperatures, I incubated E. macularius eggs at three different temperatures throughout the range of thermal tolerance for the embryos of this species. I followed a half-sib design to record both paternal and maternal effects on TSD response of progeny. Eublepharis macularius are excellent organisms for this study because captive-bred adult virgin females and viable males are available near the University of Florida where this research was conducted. Also, the






28


physiology and ecology of their TSD mechanism have been broadly researched in the recent past (Crews et al. 1996; Rhen et al. 1999; Rhen et al. 2000; Bragg et al. 2000).

Methods

Animals

On 5 January 2002, a group of five male and twenty five female adult Leopard Geckos, Eublepharis macularius, were selected from a colony of > 50,000 at The Gourmet Rodent, a reptile breeding facility in Archer, Florida. The five males were selected based on their previously demonstrated viability and the variation of color patterns on their dorsa. Dorsal coloration is a decent indicator of geographic origin in E. macularius (K. Auffenberg, pers. comm.). The five males were selected because they represented a diversity of regional origins from which the facility's breeding colony is drawn. The 25 females were selected because they were virgin and were considered likely to be viable because of their body sizes. Each male was mated to five females. Each female was housed alone in a cage containing a food bowl, a water bowl, and a nestbox filled with moist vermiculite. Males were mated to females by moving them to a different female's container every day. Males were rotated among their five mates every 24 hours and isolated for 48 hours between rotations. Between 5 January and 15 May, 2002, nestboxes were checked daily. If new eggs were found during daily nestbox inspections, they were placed in a plastic box filled with vermiculite and transported by car to the University of Florida. In the laboratory, the eggs were placed in containers that consisted of six 188 ml plastic cups banded together in rings. Each cup contained 70 ml perlite and 17.5 ml tap water and was sealed with a tight-fitting lid punctured with one gas-exchange hole. The eggs were placed randomly in containers. Each container held






29


six eggs. Each egg was placed individually within one of the six cups in a container. The egg containers were placed randomly in one of three environmental chambers and maintained at either 260, 300, or 32.50 C for the duration of the experiment. The sire, dam, egg container, and egg cup were recorded for each egg. Chamber temperatures were recorded every minute throughout the experiment with Hobo@ temperature loggers. Every day, the environmental chambers were opened and the egg containers were removed. Each cup was opened momentarily in order to release metabolic gas waste and check for hatchlings. Eggs that grew mold were discarded upon discovery. Position of egg containers within an environmental chamber was randomized daily. Sexing procedure

Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2bromo-2-chloro-l,1,1-trifluoroethane). Geckos were fixed in Bouin's fixative and preserved in 75% ethanol. The reproductive organs were removed from each gecko and prepared for analysis by light microscopy. The reproductive organs are opaque white, cylindrical structures on either side of the posterior end of the dorsal artery. After removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55; Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The resulting paraffin blocks were sectioned at 8 gm and stained with a modified trichrome of Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from each gecko. If seminiferous tubules were identified within the tissue sections, the gecko was scored as male (Figure 2a). If seminiferous tubules were absent, the gecko was scored as female (Figure 2b). Seminiferous tubules were identified as clusters of simple,






30


circular structures with a narrow lumen (Berman 2003). By relying on histological examination of our specimens, I avoided potential macroscopic misidentification of male and female reproductive organs.

The experiment was replicated the following year (16 February-5 June, 2003)

with a new group of 5 male and twenty five female adult E. macularius from a separately maintained breeding colony at the Gourmet Rodent. Thus, the sires and dams used in the first year's experiment were not the sires and dams used in the second year's replication. Analysis

Sex ratio data were analyzed by ANOVA with fixed main effects of sire, dam, incubation temperature, and year, as well as their interactions. Egg containers were nested randomly within year X temperature. Data were analyzed using SAS version 6.10 for the Macintosh. ANOVAs were performed using PROC GLM.

Results

As a consequence of allowing this experiment's eggs to develop without variation in any abiotic factors but temperature, the results from this study clearly represent the typical effect of incubation temperature on hatchling sex ratios. Proportion of males per treatment group increase with the temperature at which the group was incubated (MS =

2.361; P = 0.001; Table 3). The effect of temperature on sex ratio did not differ between the two years of experimentation. The sex ratios per sire did not differ significantly between years or among temperatures. The same is true for the sex ratios per dam. However, the offspring sex ratios of couplings of sire and dam differed significantly across incubation temperatures (MS = 0.258; P<0.05; Table 3), indicating a gene x environment interaction. The variation in the hatchling sex ratios per coupling of sire and






31


dam is most apparent at higher incubation temperatures. At each temperature, hatchling sex ratios vary between 100% female and 100% male offspring sex ratios resulting from multiple clutches produced by the same sire and dam. Less variation is observed at the female-producing incubation temperature (260C) than at the two warmer temperatures (300 and 32.50C; Figures 7 and 8).

Discussion

Offspring sex ratios of temperature-dependent sex-determining (TSD) species are highly correlated with mean air temperature during the period in which most clutches are in the middle third of the incubation period; the thermosensitive period (Janzen 1994). As a consequence of this correlation, TSD species are considered vulnerable to climate change (Janzen 1994; Girondot et al. 1998; Leslie and Spotila 2001). Thomas et al. (2004) estimated that climate change will commit 15%-37% of species in their study's sampled regions of the Earth to extinction by 2050. A common hypothesis for the link between climate change and extinction is the incompatibility of TSD to warming climates. Indeed, if dinosaurs expressed TSD, catastrophic climate change could have altered adult sex ratios and made successful mating less common, thus leading to eventual extinction (Paladino et al. 1989). This hypothesis for dinosaur extinction and hypotheses for the effects of current climate change on TSD species stand on the assumed inability of sex ratios of TSD species to adapt with sufficient speed to a rapid and/or drastic temperature shift. For example, if mean July temperature in the central United States rises 40C in the next 100 years as predicted (Manabe and Stouffer 1993) and the sex-determining response of TSD species to ambient temperature is inflexible, the






32


Painted Turtle, Chrysemys picta will become extinct because of an inability to produce males (Janzen 1994).

If Leopard Geckos, E. macularius, or any other TSD species have a chance to

persist in spite of rapid climate change, their continued survival will be attributable to the adaptability of their sex-determining mechanism or their behavioral placement of nests. In order to adapt, the mechanism must have an underlying genetic component. An interaction of temperature and genotype would allow natural populations of TSD species to change the threshold temperatures at which embryos become either male or female. Variation in sex-determining response must vary among individuals and/or among populations. This variation must be caused by variation in genotype. An interaction of temperature and genotype has been reported in an atherinid fish, the Atlantic Silverside, Menidia menidia (Conover and Kynard 1981). In M. menidia, temperature influences offspring sex ratios but the sex-determining mechanisms of progeny from different females respond differently to incubation temperature. Variation in the sex-determining response of offspring from different dams is not attributable to sire because Conover and Kynard used one sire to fertilize all dams in their study. Difference in sex ratio was not attributable to maternal size, clutch size, or level of natural mortality. Conover and Kynard (1981) interpret these results as polygenic sex determination.

In reptiles, evidence of gene x environment interaction for TSD is lacking. If

heritable variation in sex-determining response to incubation temperature exists for TSD species, then local adaptation should be evident. Bull et al. (1982a) tested for local adaptation of threshold incubation temperatures that initiate male or female sex differentiation in six species of turtles of the subfamily Emydinae, genera Graptemys,






33


Pseudemys, and Chrysemys from northern U.S. populations (Wisconsin) and southern U.S. populations (Alabama, Mississippi, and Tennessee). No significant differences between species-specific threshold temperatures were reported for any of their six study species between northern and southern populations. Bull et al. (1982a) concluded that TSD species maintain balanced sex ratios across thermally heterogeneous environments by varying nest construction or timing of oviposition. However, Bull et al. (1982b) calculated a heritability of 0.82 for map turtles. Like Conover and Kynard (1981), Bull et al. (1982b) concluded that TSD is a form of polygenic sex determination. Bull et al. (1982a and 1982b) demonstrated a heritable effect of sex determination among families of map turtles but did not demonstrate local adaptation of sex-determining threshold temperatures between populations of map turtles from different latitudes and climates.

Great variation among the offspring sex ratios of different dams of E. macularius was seen at 300 and 32.50C. Less variation in offspring sex ratios was seen at the femaleproducing temperature, 260C. This fits well with Deeming and Ferguson's (1989) hypothesized male-determining factor that responds directly to thermal cues. My results suggest a gene x environment interaction (Falconer and Mackay 1996). If sex determination in E. macularius is controlled by one gene or a small group of genes, then those genes behave differently in different environments, suggesting that one genotype is favorable in some environments but not in others. Such variation in male-producing response to incubation temperature would allow TSD populations to withstand climate change temporally if not geographically. A broad range of incubation temperatures could conceivably result in mixed sex ratios because of variation among dams in sexdetermining response to temperature. Transplant experiments should follow in which egg






34


clutches of a TSD species from one latitude or climate would be transplanted to an alternate latitude or climate. If a transplanted TSD clutch produces an offspring sex ratio like the population of its origin, then researchers could conclude an effect of genotype on TSD that can be affected by natural selection. If a transplanted TSD clutch produces an offspring sex ratio like the population it has been transplanted to, then researchers could conclude that the heritable component of TSD is too easily overridden to be shaped by natural selection. Although inspired by the conclusions of this study, subsequent experiments would not be most effectively performed with E. macularius. Clutch sizes are small (< 2 eggs), and natural populations are remote and small. Transplant experiments to test the repeatability of TSD response would be performed most appropriately with large clutches from crocodilians or certain turtles with larger clutch sizes.







35









0.9


S0.7


0.5


E 0.3
0

0.1


-0.1
25 26 27 28 29 30 31 32 33 Temperature (C) Figure 7. Offspring sex ratios of Leopard Geckos incubated at one of three
temperatures during one of two years of experimentation. Each line
represents the offspring sex ratio of one of 35 dams used in this study. Many
lines are not visible because they are super-imposed on each other in this
figure. Incubated at the same temperature, different dams produced different
offspring sex ratios. The ranks of the genotypes of different dams changed depending on the environment, indicating a gene x environment interaction.






36




Sire A B C D E F G H I J #Dams 5 5 5 5 5 5 5 5 5


26 12 3 3 4 11 5 7 6 4 9 64
,L
CU 8 8 8 6 4 8 8 6 10 12 78 S30
a

32.5 7 6 6 9 5 11 7 7 6 11 75


27 17 17 19 20 24 22 19 20 32 217 Figure 8. Sample sizes of offspring groups of leopard geckos from
individual dams. Each letter represents a different sire. Each sire was mated to five dams. The offspring of each sire/dam
pairing were divided randomly among three temperature
treatments. Samples sizes differed because of differential
reproduction among the sire/dam pairs.






37

Table 3. ANOVA across years for offspring sex ratios of Leopard Geckos.



Source df MS Temperature 2 2.361*** Dam (Sire) 34 0.190 Temperature Sire Dam 46 0.258*

*: P < 0.05; ***: P = 0.001













CHAPTER 4

ESTROGEN INCREASES PRODUCTION OF MALES IN A TEMPERATUREDEPENDENT SEX-DETERMINING REPTILE



Introduction

Temperature-dependent sex-determining (TSD) species produce hatchling sex ratios that are shaped by the temperature at which the embryonic clutch was exposed during a thermosensitive period of incubation. For example, Leopard Geckos, Eublepharis macularius, follow a pattern of sex determination in which an increased proportion of males is produced at an intermediate incubation temperature (31-33oC) whereas an increased proportion of females is produced at cooler (26-28*C) and warmer (34-35oC) incubation temperatures (Viets et al. 1993; Crews 2003). The masculinizing influence of male-determining incubation temperature can be experimentally disrupted by application of estrogens and estrogen-mimicking compounds (Bull et al. 1988b; Tousignant and Crews 1994). The application of estrogenic compounds to eggs of other temperature-dependent sex-determining species, such as freshwater turtles and crocodilians, also affects sex determination, sex differentiation, organizational and activational development, and sex-specific behavior in resulting hatchlings (Jeyasuria et al. 1994; Sheehan et al. 1999; Willingham et al. 2000). Typically, this disruption takes the form of sex-reversal of males (an override of TSD) or feminization of male reproductive tissues (Fry and Toone 1981; Belaid et al. 2001). However, these purportedly paradigmatic conclusions may only describe one half of an inverted U38






39


shaped distribution of the effects of exogenous estrogenic compounds. The influence of exogenous estrogenic compounds on female reproductive tissues (or reproductive tissues developing at female-producing incubation temperatures) has not been as substantially reported as the influence of those compounds on male reproductive tissues (or reproductive tissues developing at male-producing incubation temperatures). Some endocrine disrupting contaminants (EDCs) have opposing effects on exposed reproductive tissues at high and low concentrations (Parmigiani et al. 2000). For example, prenatal exposure to small amounts of exogenous estradiol or diethystilbesterol (DES) will increase prostate size in neonatal mice. Prenatal exposure to larger amounts of the two compounds will decrease prostate size in neonatal mice (vom Saal et al. 1997).

A growing literature suggests that a wide range of species, including reptiles, are exposed to various environmental contaminants having endocrine disruptive activities (for reviews, see Crain & Guillette, 1998; Guillette & Iguchi, 2003; Tyler et al., 1998). As with the research on mammals, the majority of these studies in reptiles have focused on estrogenic or anti-estrogenic actions. Several studies have documented effects on sex determination in turtles and alligators (Matter et al., 1998; Willingham & Crews, 1999), whereas others have shown no effect on primary sex determination from the chemical(s) tested (Podreka et al., 1998; Portelli et al., 1999). Several studies have reported that various chemicals can bind to a reptilian estrogen receptor (Guillette et al., 2002; Sumida et al., 2001; Vonier et al., 1996) including that from a lizard (Matthews & Zacharewski, 2000). No study to date has examined whether environmental estrogens, such as the pesticide DDT, affect sex determination in a squamate, such as the commonly studied lizard, the Leopard Gecko, Eublepharis macularius.






40


Methods

Animals

Experiment 1

On 26 April 2002, Leopard gecko (E. macularius) eggs were obtained from The Gourmet Rodent, a reptile breeder in Archer, Florida. Eggs were collected < 24 hrs after oviposition from a breeding colony of -5000 dams. All eggs were candled to test viability. Two hundred and thirty four viable E. macularius eggs were placed in a box filled with vermiculite and transported by car to the University of Florida. They were placed in containers that consisted of six 188 ml cups banded together in rings. Each cup contained 70 ml perlite and 17.5 ml tap water and was sealed with a tight-fitting lid punctured with one gas-release hole. The eggs were divided among 39 egg containers. Each container held six eggs. Each egg was placed individually within one of the six cups in a container. The egg containers were divided into three groups of 13. Each group was placed in an environmental chamber and maintained at 260, 300, or 32.50 C for the duration of the experiment. In control treatments, these temperatures are predicted to produce 100% females at 260C, a nearly 1:1 sex ratio at 300C and 70% males at 32.50C. Chamber temperatures were recorded every minute throughout the experiment with Hobo temperature loggers. Within each chamber, the 13 egg containers were divided into five treatment groups. Each treatment group was exposed to (a) 5gl 95% EtOH, the vehicle (12 eggs in containers), (b) 0.014 ppm o,p'-DDT (18 eggs in containers), (c) 0.14 ppm o,p'-DDT (18 eggs in containers), (d) 1.4 ppm o,p'-DDT (18 eggs in containers), or

(e) 10 gg/5 gl estradiol benzoate (2 egg containers). DDT was purchased from Chem Service (lot PS-698). The chemicals of interest in treatments b-e were dissolved in 95%






41


EtOH. Solutions were applied to the shells of eggs in each treatment group < 24 hours after oviposition. After experimental treatments were applied, all treatment groups were placed in their respective environmental chamber at one of the three temperatures defined above. Every day, the chambers were opened and the egg containers were removed. Each cup was opened momentarily in order to release metabolic gas waste and check for hatchlings. Eggs that grew mold were discarded upon discovery. Position of egg containers within the three chambers was randomized daily. Experiment 2

The following year, a new sample of 216 E. macularius eggs were tested. The second year's eggs were oviposited on 26 February 2003. Four treatment groups were exposed to (f) no treatment, (g) 5 pl 95% EtOH, (h) 10 [tg/5 l estradiol benzoate, or (i) 5 jil estradiol -17P. Treatments h and i were dissolved in 95% EtOH. To compare the effect of the timing of treatment of these positive and negative controls on sex determination relative to the data obtained in Experiment 1, we applied these treatments at the beginning of the middle third of the incubation period at each of the three temperatures in contrast to within 24 hr of oviposition as in Experiment 1. Each treatment group consisted of three egg containers housing 6 egg chambers each (N = 18 total / treatment). As in the first year, each egg container held six eggs. Sexing Procedure

Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2bromo-2-chloro-1,1,1-trifluoroethane). Hatchling geckos were fixed in Bouin's fixative and preserved in 75% ethanol. The reproductive organs were removed from each gecko and prepared for analysis by light microscopy. The reproductive organs are opaque






42

white, cylindrical structures on either side of the posterior end of the dorsal artery. After removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55; Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The resulting paraffin blocks were sectioned at 8 gtm and stained with a modified trichrome of Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from all geckos independently. If seminiferous tubules were identified within the tissue sections, the gecko was scored as male (Figure 2a). If seminiferous tubules were absent, the gecko was scored as female (Figure 2b). Seminiferous tubules were identified as clusters of simple, circular structures with narrow lumen (Berman 2003). By relying on histological examination of our specimens, we avoided potential macroscopic misidentification of male and female reproductive organs. Analysis

Sex ratio data were analyzed by ANOVA or unpaired Student's t-test. In both years, treatments were compared against that year's EtOH and/or no treatment negative controls. In Exp. 1, three concentrations of DDT and estradiol benzoate were compared to the EtOH treatment group. Estradiol benzoate was tested as a potential positive control. In Exp. 2, estradiol -17 and estradiol benzoate were tested as potential positive controls and no treatment was administered to a fourth group in order to test EtOH as an effective negative control. Data were analyzed using SAS version 6.10 for the MacIntosh.

Results

Experiment 1 All treatments significantly altered the proportion of males at each temperature as compared to the vehicle control treatment (Figure 9) but did not






43


significantly differ from each other (Tables 1 and 2). That is, all doses of DDT and the positive control E2 benzoate altered the determination of sex in a similar fashion. A sex ratio-reversing effect of DDT was not seen in the treatment groups but a general perturbation appears to have affected all treatment groups that were dosed with either EtOH, DDT, or E2 benzoate within the first 24 hours after oviposition. Experiment 2 The treatment groups in Exp. 2 were exposed to chemical treatments at the beginning of the middle third of incubation, or just prior to the period of sex determination. The timing of exposure of the eggs to potential disrupters of the sexdetermining pathway appears to influence the embryos' sex determination. In Exp. 2, both estradiol treatment groups (E2 benzoate and E2) had a significantly lower proportion of males than the negative control groups (no treatment and 95% EtOH) at the maleproducing incubation temperature (P < 0.001; Tables 3 and 4). Also in Exp. 2, both estradiol treatment groups had a higher proportion of males than the negative control groups at the female-producing incubation temperature (P<0.01; Figure 10). In Exp. 2, the sex ratio of the EtOH treatment group did not differ from the negative control group that received no treatment. Thus, EtOH is a suitable vehicle as it had no effect on the sex determination of the embryos demonstrating that the sex ratio-altering effects seen in the other treatments groups represent specific actions of either estradiol benzoate or estradiol 170, and not a result of general chemical perturbation. No significant differences were found among the hatchling sex ratios of any treatment groups from the 300C environmental chambers.






44


Discussion

Several novel observations were obtained during this study, including: 1)

exogenous estrogens skew the sex ratio toward males at the female-producing incubation temperature and 2) chemical treatment during an early stage of embryonic development produces a generalized perturbing effect on hatchling sex ratios. Wibbels et al. (1991) suggested that lower doses of estrogen would be required to sex-reverse males incubated at temperatures nearer to female-producing incubation temperatures compared to maleproducing incubation temperatures. However, very low doses of estradiol-173 have been shown to sex-reverse turtle embryos as effectively as high doses. Threshold values may not have significance for the effect of exogenous estrogens on sex determination in TSD species (Sheehan et al. 1999).

Extensive experimentation has demonstrated that exogenous estrogens can

decrease the proportion of males in sex ratios of hatchlings incubated at typically maleproducing incubation temperatures in TSD species. Studies of the effects of exogenous estrogens on sex ratios of hatchlings from male-producing incubation temperatures in TSD species have been conducted with American alligators, Alligator mississippiensis, softshell turtles, Apalone spinifera, red-eared slider turtles, Trachemys scripta, European pond turtles, Emys orbicularis, green lacerta, Lacerta viridis, leopard geckos, E. macularius, and others (Bull et al. 1988b; Crews et al. 1991; Tousignant and Crews 1994). The feminizing effect of estrogen mimics has also been reported broadly.

Unlike previous studies, a masculinizing effect of exogenous estrogens at the female-producing incubation temperature was observed in these studies. This effect could represent the second half of an inverted U-shaped response of the sex-determining






45


mechanism to estrogen. Estrogens are responsible for the regression of the medullary region of the developing testis and the proliferation of the cortical region of the bipotential gonad (Crews et al. 1991). The enzyme aromatase is responsible for the conversion of testosterone to estradiol-171. Previous studies with reptiles, specifically turtles and alligators with TSD, have not reported the production of males with any dose of estrogen, androgen, anti-estrogen, anti-androgen or aromatase blocker (for review see Guillette & Crain, 1996). Embryos exposed to aromatase blockers prior to sex determination in the Red-Eared Slider Turtle, Trachemys scripta, develop according to the temperature at which they are incubated (Wibbels & Crews., 1992) whereas in alligators most are female or exhibit an ambiguous gonad (Lance & Bogart, 1992). In contrast, genotypically female larvae of the newt, Pleurodeles waltl, differentiate into functional males following treatment with an aromatase inhibitor (Chardard and Dournon 1999). An estrogen deficit results in male sex differentiation, regardless of genotype in P. waltl. Hayes (1998) reported on sex determination in an amphibian, Rana pipiens, with genetic sex determination finding that low doses of estradiol (< 0.07 IiM) did not affect sex differentiation, whereas high doses (0.07-0.18 [aM) produced 100% females and even higher doses (> 3.69 pM) produced 100% males. A higher dose of estrogen, as is predicted with the administration of exogenous estrogens at a female-producing temperature in E. macularius, suggests that a similar mechanism may occur in this lizard contrary to the observations in other species with TSD. Higher doses of estrogen increased the proportion of male E. macularius produced (at the female-producing temperature), as seen in Hayes' study. The results of this study suggest the hypothesis that an estrogen surplus could alter sex determination in embryos. The mechanism is






46


unknown, but could involve feedback mechanisms either at the level of the ovary or hypothalamo-hypophysial axis. That is, high estrogen concentrations in the gonad or brain could alter the release of stimulatory factors from the brain or the gonad by altering gene expression patterns. Hence, an inverted U-shaped response associated with the sexdetermining mechanism in response to estrogen is supported by these data in E. macularius. That is, at lower or natural levels of estrogen an ovary forms, but at higher levels the genetic mechanism normally induced by estrogen exposure is altered or downregulated leading to the development of a testis. A number of genes are known to be differentially regulated during gonadal differentiation in reptiles (Crews, 2003; Western & Sinclair, 2001). Recent gene knockout studies in mice, suggest that alterations in several of these genes will alter the differentiation of the gonad.

The studies presented here indicate that when administered outside of the

thermosensitive period, exogenous estrogens do not significantly alter sex determination of E. macularius at male- or female-producing temperatures. We do, however, show evidence of a general perturbation of the sex ratio in response to administration of estrogen, diphenyltrichloroethane (DDT), and ethanol during earlier stages of embryonic development. In E. macularius, sex determination occurs over an interval within the middle third of incubation (stages 32-37; Bull 1987). During stage 32 (the onset of sex determination), overall embryonic morphology corresponds to stage 15 in turtles. Stage 15 in turtles also marks the onset of sex determination (Yntema 1979). In snapping turtles, Chelydra serpentina (a TSD species), administration of exogenous estrogens only sex-reversed embryos when applied between stages 10-22. Outside of this middle third of incubation, no hormonal effects on gonadal development were evident (Gutzke and






47

Chymiy 1988). The chemicals we administered to E. macularius eggs within 24 hours after oviposition, could have been sequestered in the yolk as they are lipophilic and disrupted gonadal differentiation in a more variable manner depending on size, relative rate of development or concentration of maternally-derived sex steroids in individual eggs. Female painted turtles, Chrysemys picta, donate varying concentrations of sex steroid hormones depending on follicle size. In C. picta, estradiol levels in eggs decrease with increased follicle size (Bowden et al. 2002).

Industrial pollutants, herbicides, fungicides, and pesticides, including DDT, cause anomalous plasma steroid concentrations and gonadal aberrations in neonates and offspring following embryonic exposure (Crain et al. 1997; Rooney 1998; Willingham et al. 2000; Matter et al. 1998). Degradation products of DDT act antagonistically or agonistically with estrogen at estrogen receptors (Rooney & Guillette 2000). Recognizing the ability or lack of ability of DDT and other contaminants to sex-reverse embryos, as opposed to only feminizing male-specific tissues allows more effective evaluation of the threat posed by those contaminants on natural populations of TSD species. In effect, populations of a TSD species with unusually high proportions of females despite incubation at a male-producing temperature still consist of reproductively viable individuals. If DDT, or other common contaminants, feminizes male tissues or masculinizes female tissues without sex-reversing individuals to a full female or male phenotype, then the threat posed to the population by the presence of the contaminant in the natal sites of TSD species is far greater. Our results do not show a significant effect of three ecologically relevant (Kannan et al. 1995) concentrations of DDT on hatchling sex ratios of E. macularius at any of the three temperatures studied. Therefore, we






48


conclude that the impact of this contaminant does not lead to abnormal hatchling sex ratios that would be a far lesser concern than increased reproductive abnormalities.






49




0.8


0.7
0

E 0.6


2 0.5 !

C" 0.4

o
c 0.3 0o
0
t 0.2


0.1


0

26 30 32.5 Temperature (C)

Figure 9. Sex ratios of Leopard Geckos in the first year of experimentation. In the first
year, treatments were administered to eggs within 24 hrs after oviposition.
The treatment groups of 5ul 95% Ethanol, 0.014 ppm DDT, 0.14 ppm DDT,
1.4 ppm DDT, and 10ug / 5ul Estradiol Benzoate are indicated by the
checkered, light, dark, and darkest gray bars respectively. The 10ug/5ul
Estradiol Benzoate treatment groups contained no males so no bar is shown for
that group.






50


0.8


0

0.6
E



m 0.4 a)

O
C
0
0.2
0


00


26
30 32.5

Temperature (C)

Figure 10. Sex ratios of Leopard Geckos in the second year of experimentation. In the
second year, treatments were administered to eggs at the beginning of the
middle third of incubation. The treatment groups exposed to no treatment, 5 gl
95% EtOH, 10 g/ 5 pl estradiol benzoate, or 5 pl estradiol 170 are indicated
by the light, dark, and darkest gray and black respectively. The 10 Oug/5ul
Estradiol Benzoate treatment groups contained no males so no bar is shown for
that group.






51



Table 1. ANOVA for sex ratios of Leopard Geckos from Experiment 1. All treatment groups are considered separately in this analysis.


Source df MS Treatment 4 0.246 Temperature 2 0.418 Temperature Treatment 14 0.254






52


Table 2. ANOVA for sex ratios of Leopard Geckos from Experiment 1. The three concentrations of DDT treatment are considered together as one variable in this analysis.
Source df MS Treatment 2 0.393 Temperature 2 0.238 Temperature Treatment 4 0.065






53


Table 3. ANOVA for sex ratios of Leopard Geckos from Experiment 2. All treatment groups are considered separately in this analysis.
Source df MS Treatment 3 0.174 Temperature 2 0.190 Temperature Treatment 11 0.515**

**: P = 0.002






54


Table 4. ANOVA for sex ratios of Leopard Geckos from Experiment 2. Both estradiol treatments are considered together as one variable. Both control treatments are considered together as one control group.
Source df MS
Treatment 1 0.133 Temperature 2 0.214 Temperature Treatment 50.915***

***: P < 0.001














CHAPTER 5
GENERAL DISCUSSION

Although dioecy, the trait of having two sexes, is almost universal within the

animal kingdom, great variation exists for the derivation of two sexes from a bipotential embryo. Understanding the evolution of sex depends on a clear historical record of the evolution of sex-determining mechanisms. Species under different selective regimes have evolved different genomic mechanisms leading to dioecy. Elucidating the basic dichotomy of ESD and GSD informs the controversy of adaptive significance for either mechanism or mechanisms that draw on both genetic and environmental cues.

Sex determination in E. macularius responds to environmental cues. As shown in chapter 2, temperature influences sex determination and humidity does not. Differential effects of temperature and humidity suggest the structure of the molecular action that initiates sex determination in this species. Temperature may be an indicator of habitat quality. If dams use ESD to control their offspring sex ratios in heterogenous environments in order to produce more offspring of the sex that is most likely to be fit in the immediate habitat, temperature and humidity would be clear and immediate indicators of microhabitat quality. Temperature does influence clutch sex ratios. Humidity does not.

In chapter 3, a gene x environment interaction was demonstrated in the sexdetermining mechanism of E. macularius. This result addresses concerns about the stability of ESD in the face of rapid climate change. Having a genetic component may




55






56


allow ESD species to adapt to changing nest site temperatures and avoid extinction by way of unbalanced sex ratios, as has been hypothesized in the past (Leslie and Spotila 2001). Remarkably, the variation in sex-determining response to incubation temperatures is derived from dam but not sire. Compared to the sex determination of mammals in which the sex of offspring depends on the genetic contribution of the sire to progeny, sex determination of E. macularius seems anomalous. If ESD is partially controlled by the genetic contribution of dams, dams have more control over offspring sex ratios than sires. Male-biased sex ratios are expected from dams in polygynous or promiscuous mating systems (McGinley 1984). Stressed females are also expected to produce male-biased sex ratios when litter size is small. Female-biased sex ratios are considered adaptive in social species in which female offspring and siblings will compete less for resources than male offspring or siblings would (Schwarz 1994). A synthesis of ESD mechanisms with behavioral ecology and evolutionary biology of sex ratios should begin with the dam's improved fitness. For example, E. macularius dams could benefit more from malebiased offspring sex ratios because of the environmental stress indicated by temperatures of 32.50C. The adaptive significance of female production at temperatures higher than 32.50C has not yet been considered. The maternal benefit of male-biased sex ratios diffuses at temperatures higher than 32.50C. Future studies should identify females that are more likely to produce a higher proportion of females at male-producing temperatures. The fitness of several dams of known sex-determining disposition at a constant incubation temperature over several reproductive seasons should be followed empirically. Benefits of offspring sex ratios should be considered according to the fitness






57


advantage of the dam because data from this study suggest that the mother's genetic contribution is what interacts with environmental cues to shape offspring sex ratios.

In chapter 4, I reported the influences of three concentrations of DDT, estradiol benzoate, estradiol 17,, and EtOH. In the first experiment, eggs were treated within 24 hours of oviposition with either DDT, EtOH, or estradiol benzoate. A general perturbing effect was found in which sex-determining responses to incubation temperature differed from the classic collinear increase of males with temperature up to and including 32.50C. However, a pattern of perturbation could not be attributed to any of the treatments from Exp. 1. Instead, I concluded that any chemical treatment could affect developmental processes if administered at an early stage of embryogenesis. In Exp. 2, E. macularius eggs were treated at the beginning of the thermosensitive period of embryonic development with estradiol 17f3, estradiol benzoate, EtOH, and a negative control treatment in which no chemicals were administered to eggs. The two estradiol treatments increased male production at the female-producing incubation temperature, 260C. The estradiol treatments also decreased male production at the male-producing temperature, 32.50C. The decreased male production at 32.50C was expected, judging from past results of the effects of exogenous estrogens on sex-determination in ESD turtles at maleproducing incubation temperatures. The increased male production at 260C suggests negative feedback inhibition on aromatase. Aromatase converts testosterone to estrogen. A surplus of estrogen early in development may prematurely inhibit the action of aromatase, thereby causing the decreased production of estrogen later in the thermosensitive period. An induced decrease in estrogen within the clutch would cause the clutch to develop with a male-biased sex ratio.






58


I conclude that ESD is a multi-dimensional trait in E. macularius. An influence of temperature, quantitative genetic variation, and estrogen concentration on sex ratios was demonstrated in this species. The influences of other abiotic and biotic factors offer ameliorative effects on sex determination in the face of climate change. Estimates of extinction rates of ESD species should be reconsidered in light of these data. Research into the adaptive significance of ESD should be focused on the benefits to the dam as the dam invests the partial controlling element of genetic variation. As others have concluded for other reptiles, I conclude that the partial genetic control of sex determination in E. macularius is a polygenic mechanism and responds to temperature fluctuation as a classic gene x environment interaction. Lastly, the effect of superfluous estrogen on sex determination at the female-producing temperature should encourage researchers to test the effects of aromatase inhibitors at female-producing temperatures. The hypothetical male-determining factor is most likely an aromatase inhibitor that can be stimulated by superfluous estrogen at the beginning of the thermosensitive period of embryonic development.

In summary, I have clarified a more specific effect of the nest environment on hatchling sex ratios in E. macularius. Elucidation of this kind is necessary for the continuing effort to identify patterns in the evolution of sex-determining mechanisms. Environmental sex determination may have arisen in response to changes in the environment that have not yet been considered. Fisherian sex ratios may be shaped by a broad spectrum of environmental stresses. Natural selection favors parents that invest equally in sons and daughters (Fisher 1930). However, depending on local resource availability, sons and daughters have different reproductive potentials. Males in good






59


condition are expected to outreproduce their sisters but females are expected to outreproduce their brothers if both are in poor condition (Trivers and Willard 1973). Poor condition could be defined as poor individual health or poor resource availability that will result in poor individual health. Several factors in the nest could serve as indicators of local resource availability and therefore sex-differential fitness. Sexdifferential fitness could result from fluctuations in temperature and/or any other factor in the habitat as described in chapter 1. Temperature may be only one of many factors to which ESD species adapted. At present, no categorization of species can separate ESD species from GSD species. In light of these data, ESD species should be categorized by their differential sex-determining response to humidity, quantitative genetic variation, and exogenous estrogens. Different species can yield different patterns of sexdetermining responses to these variables. Different patterns would inform the evolution of sex-determining mechanisms.

Researchers' ability to compare and contrast sex-determining mechanisms among eublepharids, within Squamata and across Chordata has been hindered by insufficient characterizations of the mechanisms. Comparisons of ESD species have not effectively described the evolutionary history of ESD By comparing the effects of temperature on sex determination, researchers have differentiated Type I, Type II, and Type III patterns of temperature-dependent sex determination (TSD). Type I TSD species like A. mississippiensis produce larger proportions of males at higher incubation temperatures (Bull 1983). Type II TSD species like Green Sea Turtles, Chelonia mydas produce larger proportions of males at low temperatures (Mrosovsky 1988). Type 1I TSD species like E. macularius produce larger proportions of males at intermediate temperatures and






60


lower proportions of males at lower or higher temperatures (Viets et al. 1993). Further resolution of the differences among types of TSD should be obtained to track its evolutionary history. At present, detail is lacking from the descriptions of sexdetermining mechanisms and how species-specific mechanisms differ from each other. Detail is also lacking in the general classification of sex-determining mechanisms. At present, sex-determining mechanisms are characterized by a simple dichotomy of ESD and GSD. Data from my experiments suggest a broad spectrum of influences and degrees to which different influences affect sex determination in a species-specific manner. My results should encourage a reclassification of sex-determining mechanisms that acknowledges their responses to much more than temperature or genetics.















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70







BIOGRAPHICAL SKETCH

I was born on 27 October 1974 and raised in Everett, Massachusetts. I took an avid interest in biology and performance art at an early age. My thrill for the arts was tempered by a fascination with the patterns and mechanisms of the natural world. At an early age, I envisioned a life where I could orate, continue to improve my understanding of biology, and contribute to the existing body of knowledge about biology. My subsequent experiences solidified my ambitions. As an undergraduate student at Boston University, I spent a semester in Ecuador studying tropical ecology. The experience gave me a broad fundamental grasp of modern ecological paradigms and environmental crises. During my field semester in Ecuador, I decided that the most effective use of my professional time as a concerned citizen and appreciator of the natural sciences is to teach and research the guiding principles of evolution, physiology, and ecology. Since then, I have taught biology courses from the level of 2nd grade through graduate courses in ecophysiology and molecular evolution. I returned to Ecuador upon receiving my B.A. from Boston University. I taught biology and environmental science for two years at a private high school in Quito that is affiliated with Boston University's School of Education. Concurrently, I became certified to teach secondary-level biology in Massachusetts and completed an Ed.M. in teaching and curriculum design from Boston University.

After my graduate program in education, I moved from Quito to Memphis,

Tennessee, to complete an M.S. in biology. From Memphis, I moved to Gainesville and






71


the University of Florida to complete a Ph.D. Throughout my experiences, I have been fortunate enough to consistently pursue my two loves of education and basic biological research. I look forward to an academic career that will allow me to continue this pattern.









I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



F. Wae g Chair t
Professor of Zoology



I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Marta L. Wayne, CoChair
Assistant Professor of Zoology



I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Louis"J G e
Distinguished Professor of Zoology



I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Karen A. Bjornd //
Professor of Zoology









I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Max A. Nickerson
Professor of Wildlife Ecology and Conservation



This dissertation was submitted to the Graduate Faculty of the Department of
Zoology in the College of Liberal Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.


May 2004
Dean, Graduate School









I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Max A. Nickerson
Professor of Wildlife Ecology and Conservation



This dissertation was submitted to the Graduate Faculty of the Department of
Zoology in the College of Liberal Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.


May 2004
Dean, Graduate School




Full Text
Proportion of Males / Treatment Group
21
24 26 28 30 32
Temperature (C)
Figure 5: Sex ratios of treatment groups of Leopard Geckos in the second of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the solid, dashed, gray stippled, and black
stippled lines respectively. Bars represent standard error.


57
advantage of the dam because data from this study suggest that the mothers genetic
contribution is what interacts with environmental cues to shape offspring sex ratios.
In chapter 4,1 reported the influences of three concentrations of DDT, estradiol
benzoate, estradiol 17(8, and EtOH. In the first experiment, eggs were treated within 24
hours of oviposition with either DDT, EtOH, or estradiol benzoate. A general perturbing
effect was found in which sex-determining responses to incubation temperature differed
from the classic collinear increase of males with temperature up to and including 32.5C.
However, a pattern of perturbation could not be attributed to any of the treatments from
Exp. 1. Instead, I concluded that any chemical treatment could affect developmental
processes if administered at an early stage of embryogenesis. In Exp. 2, E. macularius
eggs were treated at the beginning of the thermosensitive period of embryonic
development with estradiol 178, estradiol benzoate, EtOH, and a negative control
treatment in which no chemicals were administered to eggs. The two estradiol treatments
increased male production at the female-producing incubation temperature, 26C. The
estradiol treatments also decreased male production at the male-producing temperature,
32.5C. The decreased male production at 32.5C was expected, judging from past
results of the effects of exogenous estrogens on sex-determination in ESD turtles at male-
producing incubation temperatures. The increased male production at 26C suggests
negative feedback inhibition on aromatase. Aromatase converts testosterone to estrogen.
A surplus of estrogen early in development may prematurely inhibit the action of
aromatase, thereby causing the decreased production of estrogen later in the
thermosensitive period. An induced decrease in estrogen within the clutch would cause
the clutch to develop with a male-biased sex ratio.


31
dam is most apparent at higher incubation temperatures. At each temperature, hatchling
sex ratios vary between 100% female and 100% male offspring sex ratios resulting from
multiple clutches produced by the same sire and dam. Less variation is observed at the
female-producing incubation temperature (26C) than at the two warmer temperatures
(30 and 32.5C; Figures 7 and 8).
Discussion
Offspring sex ratios of temperature-dependent sex-determining (TSD) species are
highly correlated with mean air temperature during the period in which most clutches are
in the middle third of the incubation period; the thermosensitive period (Janzen 1994).
As a consequence of this correlation, TSD species are considered vulnerable to climate
change (Janzen 1994; Girondot et al. 1998; Leslie and Spotila 2001). Thomas et al.
(2004) estimated that climate change will commit 15%-37% of species in their studys
sampled regions of the Earth to extinction by 2050. A common hypothesis for the link
between climate change and extinction is the incompatibility of TSD to warming
climates. Indeed, if dinosaurs expressed TSD, catastrophic climate change could have
altered adult sex ratios and made successful mating less common, thus leading to
eventual extinction (Paladino et al. 1989). This hypothesis for dinosaur extinction and
hypotheses for the effects of current climate change on TSD species stand on the assumed
inability of sex ratios of TSD species to adapt with sufficient speed to a rapid and/or
drastic temperature shift. For example, if mean July temperature in the central United
States rises 4C in the next 100 years as predicted (Manabe and Stouffer 1993) and the
sex-determining response of TSD species to ambient temperature is inflexible, the


CHAPTER 2
ABIOTIC EFFECTS ON SEX DETERMINATION IN LEOPARD GECKOS
Introduction
Animals and plants have evolved numerous sex-determining mechanisms that are
described as either genetic sex determination (GSD) or environmental sex determination
(ESD). In GSD, sex is determined by the genetic constitution of an embryo upon
conception. In ESD, sex is determined by extrinsic, abiotic factors that define the
incubation environment. Regardless of mechanism, Fisher (1930) predicted that primary
sex ratios should represent an equal parental investment in male and female offspring.
However, Fishers balanced parental investment may not be represented in ESD species
because primary sex ratios may be removed from parental control.
Among squamate reptiles, ESD and GSD are found among closely related
species, suggesting that ESD and GSD have evolved separately on many occasions (Viets
et al. 1994). Within the squamate subfamily Eublepharinae, the Leopard Gecko,
Eublepharis macularius, and the African Fat-Tail Gecko, Hemitheconyx caudicinctus, are
ESD species and the Banded Gecko, Coleonyx mitratus, is a GSD species (Bragg et al.
2000). Environmental sex determination in squamates refers to temperature-dependent
sex determination (TSD) specifically, the influence of incubation temperature on sex
determination. Other forms of ESD based on photoperiod or nutritional resource
availability have been reported in invertebrates but only TSD has thus far been reported
in squamates (Bull 1980). Genetic variation in sex-determining responses to incubation
6


24
Table 2. ANOVA within years for sex ratios of treatment groups of Leopard Geckos.
MS
Source
df
Year 1
Year 2
Temperature
2
0.205
0.713*
Humidity
3
0.020
0.058
Temperature *
Humidity 6
0.214
0.352
*: P < 0.05


Proportion of Males / Treatment Group
22
0.8 -i
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
0.2 -
26
Temperature (C)
32.5
Figure 6. Sex ratios of treatment groups of Leopard Geckos in the second of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the lightest to darkest bars respectively.
Standard errors are indicated by vertical lines.


Proportion of Males / Treatment Group
49
0.8 -i
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
0.2 -
0.1 -
0 T
26 30 32.5
Temperature (C)
Figure 9. Sex ratios of Leopard Geckos in the first year of experimentation. In the first
year, treatments were administered to eggs within 24 hrs after oviposition.
The treatment groups of 5ul 95% Ethanol, 0.014 ppm DDT, 0.14 ppm DDT,
1.4 ppm DDT, and lOug / 5ul Estradiol Benzoate are indicated by the
checkered, light, dark, and darkest gray bars respectively. The 10ug/5ul
Estradiol Benzoate treatment groups contained no males so no bar is shown for
that group.


44
Discussion
Several novel observations were obtained during this study, including: 1)
exogenous estrogens skew the sex ratio toward males at the female-producing incubation
temperature and 2) chemical treatment during an early stage of embryonic development
produces a generalized perturbing effect on hatchling sex ratios. Wibbels et al. (1991)
suggested that lower doses of estrogen would be required to sex-reverse males incubated
at temperatures nearer to female-producing incubation temperatures compared to male-
producing incubation temperatures. However, very low doses of estradiol-17(3 have been
shown to sex-reverse turtle embryos as effectively as high doses. Threshold values may
not have significance for the effect of exogenous estrogens on sex determination in TSD
species (Sheehan et al. 1999).
Extensive experimentation has demonstrated that exogenous estrogens can
decrease the proportion of males in sex ratios of hatchlings incubated at typically male-
producing incubation temperatures in TSD species. Studies of the effects of exogenous
estrogens on sex ratios of hatchlings from male-producing incubation temperatures in
TSD species have been conducted with American alligators, Alligator mississippiensis,
softshell turtles, Apalone spinifera, red-eared slider turtles, Trachemys scripta, European
pond turtles, Emys orbicularis, green lacerta, Lacerta viridis, leopard geckos, E.
macularius, and others (Bull et al. 1988b; Crews et al. 1991; Tousignant and Crews
1994). The feminizing effect of estrogen mimics has also been reported broadly.
Unlike previous studies, a masculinizing effect of exogenous estrogens at the
female-producing incubation temperature was observed in these studies. This effect
could represent the second half of an inverted U-shaped response of the sex-determining


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ADDITIONAL FACTORS INFLUENCE TEMPERATURE-DEPENDENT SEX
DETERMINATION IN LEOPARD GECKOS
By
Daniel E. Janes
May 2004
Chair: F. Wayne King
Cochair: Marta L. Wayne
Major Department: Zoology
Hatchling sex ratios of reptiles with environmental sex determination (ESD) are
influenced by incubation temperature. Leopard Geckos, Eublepharis macularius, nest in
a region where they are exposed to a wide range of temperatures. If temperature is the
sole determinant of sex in this species, then hatchling sex ratios should be highly
correlated with the thermal gradient in different parts of the species range. In this
scenario, a broad thermal gradient would promote regional variation in hatchling sex
ratios of E. macularius. However, reports of regional variation in hatchling sex ratios are
rare among ESD reptiles. If a secondary factor (or factors) plays a role in sex
determination of this species, their hatchling sex ratios in nature would be expected to
vary seasonally and regionally with less correlation with thermal gradient. In this study,
E. macularius eggs were incubated at three temperatures in two consecutive years. The
vi


ACKNOWLEDGMENTS
I extend my deepest gratitude to my co-advisors, Drs. Marta L. Wayne and F.
Wayne King, for their support, encouragement, and wisdom. My other committee
members, Drs. Karen Bjomdal, Louis Guillette, and Max Nickerson, have maintained an
open door policy of which I have taken full advantage. Conversations with my advisors
have taught me the value of discussion at all stages of research design and execution and
the necessity of collaboration. I wish to thank all of my friends in the Departments of
Zoology, Wildlife Ecology and Conservation, and the Florida Museum of Natural
History. Their friendship and laughter daily reinforced one of the main reasons I am a
scientist and teacher. It is fun. This research has been funded by the Florida Museum of
Natural History, Sigma Xi, and William and Marcia Brant of The Gourmet Rodent.
Conversations with Ben Bolker helped shape my analyses of data. Data collection
assistance was provided by Jason Phillips, Jennifer Comiskey, Sara Reyes, Jennifer
Mobberley, Margaret OBrien, Eric Timauer, Cassandra Pedrosa, Alana Schoenberg,
Julia Huang, Craig Ajmo, John Bowden, Erin Taylor, and Kristin Barns. Work was
conducted in accordance with University of Florida LACUC protocol Z010.


48
conclude that the impact of this contaminant does not lead to abnormal hatchling sex
ratios that would be a far lesser concern than increased reproductive abnormalities.


34
clutches of a TSD species from one latitude or climate would be transplanted to an
alternate latitude or climate. If a transplanted TSD clutch produces an offspring sex ratio
like the population of its origin, then researchers could conclude an effect of genotype on
TSD that can be affected by natural selection. If a transplanted TSD clutch produces an
offspring sex ratio like the population it has been transplanted to, then researchers could
conclude that the heritable component of TSD is too easily overridden to be shaped by
natural selection. Although inspired by the conclusions of this study, subsequent
experiments would not be most effectively performed with E. macularius. Clutch sizes
are small (< 2 eggs), and natural populations are remote and small. Transplant
experiments to test the repeatability of TSD response would be performed most
appropriately with large clutches from crocodilians or certain turtles with larger clutch
sizes.


40
Methods
Animals
Experiment 1
On 26 April 2002, Leopard gecko (E. macularius) eggs were obtained from The
Gourmet Rodent, a reptile breeder in Archer, Florida. Eggs were collected < 24 hrs after
oviposition from a breeding colony of-5000 dams. All eggs were candled to test
viability. Two hundred and thirty four viable E. macularius eggs were placed in a box
filled with vermiculite and transported by car to the University of Florida. They were
placed in containers that consisted of six 188 ml cups banded together in rings. Each cup
contained 70 ml perlite and 17.5 ml tap water and was sealed with a tight-fitting lid
punctured with one gas-release hole. The eggs were divided among 39 egg containers.
Each container held six eggs. Each egg was placed individually within one of the six
cups in a container. The egg containers were divided into three groups of 13. Each
group was placed in an environmental chamber and maintained at 26, 30, or 32.5 C for
the duration of the experiment. In control treatments, these temperatures are predicted to
produce 100% females at 26C, a nearly 1:1 sex ratio at 30C and 70% males at 32.5C.
Chamber temperatures were recorded every minute throughout the experiment with
Hobo temperature loggers. Within each chamber, the 13 egg containers were divided
into five treatment groups. Each treatment group was exposed to (a) 5pl 95% EtOH, the
vehicle (12 eggs in containers), (b) 0.014 ppm o,p-DDT (18 eggs in containers), (c) 0.14
ppm o,p-DDT (18 eggs in containers), (d) 1.4 ppm o,p-DDT (18 eggs in containers), or
(e) 10 pg/5 pi estradiol benzoate (2 egg containers). DDT was purchased from Chem
Service (lot PS-698). The chemicals of interest in treatments b-e were dissolved in 95%


13
3 and 4). In the second year, with the exception of the treatment group exposed to 20%
humidity, the treatment groups incubated at the coolest temperature (26C) had a lower
proportion of males than treatment groups incubated at both warmer temperatures.
Temperature significantly affected hatchling sex ratios in year 2 (MS = 0.713; P<0.05;
Table 2). The highest proportion of males per treatment group was produced at the
warmest incubation temperature (32.5C; Figures 5 and 6). The order of male proportion
per treatment group at 30C in the second year does not follow the order of male
proportion per treatment group at 30C in the first year.
Discussion
I have found, using my design, that 1) temperature affected sex ratios, 2) humidity
did not affect sex ratios, and 3) humidity and temperature did not interactively affect sex
ratios. Previous studies examining the effect of humidity on sex determination in TSD
species have produced inconsistent results. Gutzke and Paukstis (1983) found an effect
of substrate moisture on sex differentiation in the freshwater turtle, Chrysemys picta,
whereas Packard et al. (1989) did not. Clearly, the mechanism and the adaptiveness of
the relationship between incubation environments and hatchling sex ratios have not been
sufficiently characterized. Deeming and Ferguson (1989) refer to a male-determining
factor that is temperature-sensitive. Candidate mechanisms for TSD include temperature-
dependent synthesis or activity of enzymes, heat shock proteins, and temperature-
sensitive gene expression (Mrosovsky 1994). Some intrinsic mechanism(s) responds to
the microclimate of the nest site and determines hatchling sex. Eublepharids select nest-
site microclimates that result in higher hatchling survivorship and not necessarily
evolutionarily stable sex ratios (Bull et al. 1988a; Bragg et al. 2000). However, if


Proportion of Males / Treatment Group
20
0.8 -i
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
30 32.5
Temperature (C)
Figure 4. Sex ratios of treatment groups of Leopard Geckos in the first of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the lightest to darkest bars respectively.
Standard errors are indicated by vertical lines.


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
ABSTRACT vi
CHAPTER
1 GENERAL INTRODUCTION 1
2 ABIOTIC EFFECTS ON SEX DETERMINATION IN LEOPARD GECKOS 6
Introduction 6
Methods 10
Animals 10
Sexing Procedure 11
Analysis 12
Results 12
Discussion 13
3 QUANTITATIVE GENETIC VARIATION IN SEX-DETERMINING
RESPONSE TO INCUBATION TEMPERATURE IN LEOPARD GECKOS 25
Introduction 25
Methods 28
Animals 28
Sexing Procedure 29
Analysis 30
Results 30
Discussion 31
4 ESTROGEN AND ESTROGEN MIMIC INCREASE PRODUCTION OF MALES
IN A TEMPERATURE-DEPENDENT SEX-DETERMINING SPECIES 38
Introduction 38
Methods 40
Animals 40
Sexing Procedure 41
Analysis 42
IV


52
Table 2. ANOVA for sex ratios of Leopard Geckos from Experiment 1. The three
concentrations of DDT treatment are considered together as one variable in this analysis.
Source
df
MS
Treatment
2
0.393
Temperature
2
0.238
Temperature Treatment
4
0.065


27
modest effect that segregate according to Mendelian rules (Janzen 1992). These models
are supported by little empirical evidence because of the difficulty of measuring primary
sex ratios and the interference of the predominant effects of sex chromosomes in GSD
species. The heritability of offspring sex ratio incubated under constant temperature has
been reported in the temperature-dependent sex-determining (TSD) Ouachita Map turtle,
Graptemys ouachitensis (Bull et al. 1982a). Bull et al. (1982a) incubated G. ouachitensis
eggs from different families at a constant temperature, 29.2C. Their threshold model for
ESD yielded a heritability estimate of 0.82 for the hypothetical sex-determining
character. They concluded that natural variation in nest temperatures acted against the
high heritability of the sex-determining character to maintain natural populations closer
to a balanced sex ratio. Also, Janzen found strong genetic variation in the TSD response
of the Common Snapping turtle, Chelydra serpetina, to incubation temperature.
However, no significant interaction of temperature and family has been reported for C.
serpentina. Therefore, Janzen (1992) concluded that variation in the sex-determining
response to incubation temperature in the TSD species C. serpentina is not due to a gene
x environment interaction.
To test for a gene x environment interaction in the sex-determining mechanism of
E. macularius, within a broad range of incubation temperatures, I incubated E.
macularius eggs at three different temperatures throughout the range of thermal tolerance
for the embryos of this species. I followed a half-sib design to record both paternal and
maternal effects on TSD response of progeny. Eublepharis macularius are excellent
organisms for this study because captive-bred adult virgin females and viable males are
available near the University of Florida where this research was conducted. Also, the


Temperature (C)
36
Sye
A
B
c
D
E
F
G
H
I
J
Dams
5
5
5
5
5
5
5
5
5
26
12
3
3
4
11
5
7
6
4
9
30
8
8
8
6
4
8
8
6
10
12
32.5
7
6
6
9
5
11
7
7
6
11
27
17
17
19
20
24
22
19
20
32
Figure 8. Sample sizes of offspring groups of leopard geckos from
individual dams. Each letter represents a different sire. Each
sire was mated to five dams. The offspring of each sire/dam
pairing were divided randomly among three temperature
treatments. Samples sizes differed because of differential
reproduction among the sire/dam pairs.


60
lower proportions of males at lower or higher temperatures (Viets et al. 1993). Further
resolution of the differences among types of TSD should be obtained to track its
evolutionary history. At present, detail is lacking from the descriptions of sex
determining mechanisms and how species-specific mechanisms differ from each other.
Detail is also lacking in the general classification of sex-determining mechanisms. At
present, sex-determining mechanisms are characterized by a simple dichotomy of ESD
and GSD. Data from my experiments suggest a broad spectrum of influences and
degrees to which different influences affect sex determination in a species-specific
manner. My results should encourage a reclassification of sex-determining mechanisms
that acknowledges their responses to much more than temperature or genetics.


71
the University of Florida to complete a Ph.D. Throughout my experiences, I have been
fortunate enough to consistently pursue my two loves of education and basic biological
research. I look forward to an academic career that will allow me to continue this pattern.


9
et al. 1989). Packards advice to consider the effects of substrate moisture cautiously has
been heeded by subsequent researchers (Packard et al. 1989; Janzen and Moijan 2001).
Typically, substrate moisture is not considered a potential sex determinant in ESD
studies. In previous experiments, humidity of incubation substrate did not affect sex
determination in E. macularius (B. Viets, personal communication). Lack of an effect of
humidity on ESD has also been reported in the Flatback Turtle, Natator depressus
(Hewavisenthi and Parmenter 2000).
Different squamate species have different patterns of water exchange between the
egg and the environment and, therefore, different sensitivities to environmental moisture
(Ji and Du 2001). If humidity has a partial effect on sex determination in E. macularius,
its effect in nature could be significant because of the paucity of moisture in the species
natural range. This species inhabits and builds nests in arid country (Daniel 2002). This
habitat would pose a significant challenge to a sex-determining mechanism that is
influenced by humidity because the maintenance of a humid nest in an arid habitat is a
greater challenge than the maintenance of a dry nest in a humid habitat. Also, humidity
may have a more significant effect in the development of more pliable-shelled eggs like
those of E. macularius (Ji and Du 2001). The influence of humidity on sex determination
could be direct by changing the response of the male- determining factor (Deeming and
Ferguson 1989) or indirect by influencing nest temperatures, affecting body size as in C.
picta and N. depressus (Cagle et al. 1993; Hewavisenthi and Parmenter 2001), or by
influencing the conversion of yolk into fat as in the Cuban Rock Iguana, Cyclura nubila
(Christian and Lawrence 1991).


14
humidity affected both survivorship and to a lesser extent sex determination, then nest
site selection could influence population sex ratios. Cultural inheritance of nest site
microclimates has also been implicated in the inheritance of hatchling sex ratios
(Freedberg and Wade 2001). Nest sites tend to retain their abiotic profiles from year to
year. If daughters inherit nest sites from their mothers, then a cultural component exists
for the inheritance of hatchling sex ratios in ESD species. However, if the nest sites are
found in areas that are thermally different from one breeding season to the next, then
cultural inheritance will not fix sex ratios over long periods of time. The microevolution
of sex ratio depends in part on maternal choice of thermal qualities of nest sites (Janzen
and Moijan 2001).
My results from chapters 3 and 4 suggest that hatchling sex ratios can vary among
populations even if incubation temperature remains constant. However, the results of this
experiment and previously published conclusions (Janzen and Moijan 2001) suggest that
hatchling sex ratios of E. macularius do not vary in relation to nest site humidity. The
effect of humidity on hatchling sex ratios in this study was not significantly different
between two years of experimentation. In short, sex determination in E. macularius did
not respond to humidity, at least as examined by the experimental design I performed.
Researchers should note that humidity was not measured throughout this experiment.
After initial volumes of water were added at the beginning of trials, humidities may have
varied incidentally among the treatment groups. Incidental variation may explain the
different effects of temperature on hatchling sex ratios between the first and second years
of experimentation. However, incidental variation in temperature is less likely because of
constant monitoring of temperature throughout the trials. The lack of effect of


Proportion of Males / Treatment Group
19
24 26 28 30 32
Temperature (C)
Figure 3. Sex ratios of treatment groups of Leopard Geckos in the first of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the solid, dashed, gray stippled, and black
stippled lines respectively. Bars represent standard errors.


12
scored as female (Figure 2b). Seminiferous tubules were identified as clusters of simple,
circular structures with a narrow lumen (Berman 2003). By relying on histological
examination of our specimens, I avoided potential macroscopic misidentification of male
and female reproductive organs.
The design was replicated the following year with a new sample of 216 i?.
macularius eggs from a separately maintained breeding colony at the Gourmet Rodent.
Thus, the sires and dams used in the first years experiment were not the sires and dams
used in the second years replication. Work was conducted in accordance with
University of Florida IACUC protocol Z010.
Analysis
Sex ratio data were analyzed by ANOVA with fixed main effects of humidity,
incubation temperature, and year, as well as their interactions. Egg containers were
nested within year X temperature X humidity, and treated as a random effect. Data were
analyzed using SAS version 6.10 for the Macintosh. ANOVAs were performed using
PROC GLM.
Results
Temperature and humidity were manipulated simultaneously to assess effects on
ESD over the two years. Variances between years were not significantly different (Table
1). Years were analyzed together or separately. Overall, temperature significantly
affected hatchling sex ratios as expected for a species exhibiting TSD (MS = 0.527;
P<0.05). Humidity did not affect sex ratios when both years were examined together or
individually (MS = 0.015; Table 1). In the first year of experimentation, neither
temperature nor humidity, examined independently, altered hatchling sex ratios (Figures


16
Environmental sex determination could enhance maternal fitness by permitting
the production of more offspring of the sex that is best-suited to the incubation
environment (Chamov and Bull 1977). Chrysemys picta eggs incubated in wet substrate
maintained a higher temperature than eggs in dry substrate but only for the first third of
incubation. After the first third, the eggs in drier substrates maintained a higher
temperature than those in wet substrates (Gutzke et al. 1987). The temperature-sensitive
period for E. macularius in which sex determination is affected by local abiotic factors
does not begin until the beginning of the middle third of incubation (Bull 1987).
Therefore, we would expect drier treatment groups to be warmer during the temperature-
sensitive period and produce more males. This pattern, however, was not observed in
this study. The enhanced sensitivity of offspring sex ratio to secondary abiotic factors
should be investigated in a wider array of environmentally sex-determined species
because of the current lack of detail about the evolution of sex-determining mechanisms.
If some species respond similarly to a series of environmental variables, then a new
dimension of the mechanism could be considered to characterize the divergence of ESD
mechanisms among squamates.


33
Pseudemys, and Chrysemys from northern U.S. populations (Wisconsin) and southern
U.S. populations (Alabama, Mississippi, and Tennessee). No significant differences
between species-specific threshold temperatures were reported for any of their six study
species between northern and southern populations. Bull et al. (1982a) concluded that
TSD species maintain balanced sex ratios across thermally heterogeneous environments
by varying nest construction or timing of oviposition. However, Bull et al. (1982b)
calculated a heritability of 0.82 for map turtles. Like Conover and Kynard (1981), Bull et
al. (1982b) concluded that TSD is a form of polygenic sex determination. Bull et al.
(1982a and 1982b) demonstrated a heritable effect of sex determination among families
of map turtles but did not demonstrate local adaptation of sex-determining threshold
temperatures between populations of map turtles from different latitudes and climates.
Great variation among the offspring sex ratios of different dams of E. macularius
was seen at 30 and 32.5C. Less variation in offspring sex ratios was seen at the female-
producing temperature, 26C. This fits well with Deeming and Fergusons (1989)
hypothesized male-determining factor that responds directly to thermal cues. My results
suggest a gene x environment interaction (Falconer and Mackay 1996). If sex
determination in E. macularius is controlled by one gene or a small group of genes, then
those genes behave differently in different environments, suggesting that one genotype is
favorable in some environments but not in others. Such variation in male-producing
response to incubation temperature would allow TSD populations to withstand climate
change temporally if not geographically. A broad range of incubation temperatures could
conceivably result in mixed sex ratios because of variation among dams in sex
determining response to temperature. Transplant experiments should follow in which egg


56
allow ESD species to adapt to changing nest site temperatures and avoid extinction by
way of unbalanced sex ratios, as has been hypothesized in the past (Leslie and Spotila
2001). Remarkably, the variation in sex-determining response to incubation temperatures
is derived from dam but not sire. Compared to the sex determination of mammals in
which the sex of offspring depends on the genetic contribution of the sire to progeny, sex
determination of E. macularius seems anomalous. If ESD is partially controlled by the
genetic contribution of dams, dams have more control over offspring sex ratios than sires.
Male-biased sex ratios are expected from dams in polygynous or promiscuous mating
systems (McGinley 1984). Stressed females are also expected to produce male-biased
sex ratios when litter size is small. Female-biased sex ratios are considered adaptive in
social species in which female offspring and siblings will compete less for resources than
male offspring or siblings would (Schwarz 1994). A synthesis of ESD mechanisms with
behavioral ecology and evolutionary biology of sex ratios should begin with the dams
improved fitness. For example, E. macularius dams could benefit more from male-
biased offspring sex ratios because of the environmental stress indicated by temperatures
of 32.5C. The adaptive significance of female production at temperatures higher than
32.5C has not yet been considered. The maternal benefit of male-biased sex ratios
diffuses at temperatures higher than 32.5C. Future studies should identify females that
are more likely to produce a higher proportion of females at male-producing
temperatures. The fitness of several dams of known sex-determining disposition at a
constant incubation temperature over several reproductive seasons should be followed
empirically. Benefits of offspring sex ratios should be considered according to the fitness


I dedicate this work to my parents. They have always encouraged me to follow my own
path and welcomed me back home to recover from my defeats and celebrate my victories.
Also, I dedicate this work to my teachers and students. They have shared their thoughts
and ambitions with me. Every one has shown me a slightly different way to see the
world and every one has allowed me to share my own perspective with them. We should
all be so lucky.


70
BIOGRAPHICAL SKETCH
I was bom on 27 October 1974 and raised in Everett, Massachusetts. I took an
avid interest in biology and performance art at an early age. My thrill for the arts was
tempered by a fascination with the patterns and mechanisms of the natural world. At an
early age, I envisioned a life where I could orate, continue to improve my understanding
of biology, and contribute to the existing body of knowledge about biology. My
subsequent experiences solidified my ambitions. As an undergraduate student at Boston
University, I spent a semester in Ecuador studying tropical ecology. The experience gave
me a broad fundamental grasp of modem ecological paradigms and environmental crises.
During my field semester in Ecuador, I decided that the most effective use of my
professional time as a concerned citizen and appreciator of the natural sciences is to teach
and research the guiding principles of evolution, physiology, and ecology. Since then, I
have taught biology courses from the level of 2nd grade through graduate courses in
ecophysiology and molecular evolution. I returned to Ecuador upon receiving my B.A.
from Boston University. I taught biology and environmental science for two years at a
private high school in Quito that is affiliated with Boston Universitys School of
Education. Concurrently, I became certified to teach secondary-level biology in
Massachusetts and completed an Ed.M. in teaching and curriculum design from Boston
University.
After my graduate program in education, I moved from Quito to Memphis,
Tennessee, to complete an M.S. in biology. From Memphis, I moved to Gainesville and


67
Rhen, T., and D. Crews. 1999. Embryonic temperature and gonadal sex organize male-
typical sexual and aggressive behavior in a lizard with temperature-dependent sex
determination. Endocrinology 140:4501-4508.
Rhen, T., J.T. Sakata, M. Zeller, and D. Crews. 2000. Sex steroid levels across the
reproductive cycle of female leopard geckos, Eublepharis macularius, from
different incubation temperatures. General and Comparative Endocrinology
118:322-331.
Rooney, A.A. 1998. Variation in the endocrine and immune system of juvenile alligators:
environmental influence on physiology. Ph.D. Dissertation. University of Florida.
Rooney, A.A. and L.J. Guillette. 2000. Contaminant interactions with steroid receptors:
Evidence for receptor binding. In L.J. Guillette and D.A. Crain (eds.) Endocrine
Disrupting Contaminants: An Evolutionary Perspective, p. 82-125. Francis and
Taylor, Inc. Philadelphia.
Schwarz, M.P. 1994. Female-biased sex ratios in a facultatively social bee and their
implications for social evolution. Evolution 48:1684-1697.
Sheehan, D.M., E. Willingham, D. Gaylor, J.M. Bergeron, and D. Crews. 1999. No
threshold dose for estradiol-induced sex reversal of turtle embryos: How little is
too much? Environmental Health Perspectives 107:155-159.
Shine, R. 1999. Why is sex determined by nest temperature in many reptiles. Trends in
Ecology and Evolution 14:186-189.
Shine, R., and M.J. Elphick. 2001. The effect of short-term weather fluctuations on
temperatures inside lizard nests, and on the phenotypic traits of hatchling lizards.
Biological Journal of the Linnean Society 72:555-565.
Shine, R., and P.S. Harlow. 1996. Maternal manipulation of offspring phenotypes
via nest-site selection in an oviparous lizard. Ecology 77:1808-1817.
Smith, M.A. 1935. The fauna of British India. Taylor and Francis. London.
Sumida, K., N. Ooe, K. Saito, and H. Kaneko. 2001. Molecular cloning and
characterization of reptilian estrogen receptor cDNAs. Molecular and Cellular
Endocrinology 183: 33-39.
Thomas, C.D., A. Cameron, R.E. Green, M. Bakkenes, L.J. Beaumont, Y.C. Collingham,
B.F.N. Erasmus, M.F. de Siqueira, A. Grainger, L. Hannah, L. Hughes, B.
Huntley, A.S. van Jaarsveld, G.F. Midgley, L. Miles, M.A. Ortega-Huerta, A.T.
Peterson, O.L. Phillips, and S.E. Williams. 2004. Extinction risk from climate
change. Nature 427:145-148.


10
In this experiment, E. macularius eggs were treated with substrates of varied
water content and incubated at three temperatures within the species range of embryonic
thermal tolerance. If humidity has an effect on this species sex determination, then
different sex ratios in response to different humidities should be detected most easily at
the intermediate incubation temperature. The influence of a weaker determinant of sex is
more likely to be detected at an intermediate incubation temperature, because the
skewing influence of temperature on the sex ratio is minimized (Bull et al. 1982a).
However, extreme temperatures may also interact uniquely with humidity. For these
reasons, extreme and intermediate incubation temperatures are studied in this project.
Methods
Animals
On 22 March 2002, Leopard Gecko, Eublepharis macularius, eggs were obtained
from The Gourmet Rodent, a reptile breeder in Archer, Florida. Eggs were collected
within 24 hrs after oviposition from a breeding colony of -5000 dams. All eggs were
candled to test viability. Two hundred and sixteen viable E. macularius eggs were placed
in a plastic box filled with vermiculite and transported by car to the University of Florida.
They were placed in containers that consisted of six 188 ml plastic cups banded together
in rings. Each cup contained 70 ml perlite and tap water and was sealed with a tight
fitting lid punctured with one gas-exchange hole. The eggs were divided among 36 egg
containers. Each container held six eggs. Each egg was placed individually within one
of the six cups in a container. The egg containers were divided into three groups of 12.
Each group was placed in an environmental chamber and maintained at 26, 30, or 32.5
C for the duration of the experiment. Chamber temperatures were recorded every minute


5
the sex-determining responses of E. macularius to environmental factors other than
temperature or to interactions of sex-determining factors.
Research Objectives: I tested extrinsic and intrinsic factors in order to determine
which, if any, work in concert with incubation temperature to adjust clutch sex ratios in
E. macularius. I hypothesized a multi-dimensional sex determination model in which
skewing effects from the extremes of sex-determining factor are ameliorated by opposing
extremes of another factor or factors.


LIST OF REFERENCES
Belaid, B., and N. Richard-Mercier, C. Pieau, and M. Dorizzi. 2001. Sex reversal and
aromatase in the European pond turtle: treatment with letrozole after the
thermosensitive period for sex determination. Journal of Experimental Zoology
290:490-497.
Berman, I. 2003. Color atlas of basic histology. McGraw-Hill. New York.
Blackmore, M.S., and E.L. Chamov. 1989. Adaptive variation in environmental
sex determination in a nematode. American Naturalist 134:817-823.
Bowden, R.M., M.A. Ewert, S. Freedberg, and C.E. Nelson. 2002.
Maternally derived yolk hormones vary in follicles of the Painted Turtle,
Chrysemys picta. Journal of Experimental Zoology 293:67-72.
Bragg, W.K., J.D. Fawcett, and T.B. Bragg. 2000. Nest-site selection in two eublepharid
geckos species with temperature-dependent sex determination and one with
genotypic sex determination. Biological Journal of the Linnean Society 69:319-
332.
Bull, J.J. 1980. Sex determination in reptiles. Quarterly Review of Biology 55:3-21.
Bull, J.J. 1987. Temperature-sensitive periods of sex determination in a lizard:
similarities with turtles and crocodilians. Journal of Experimental Zoology
241:143-148.
Bull, J.J., and E.L. Chamov. 1989. Enigmatic reptilian sex ratios. Evolution 43:
1561-1566.
Bull, J.J., W.H.N. Gutzke, and M.G. Bulmer. 1988a. Nest choice in a captive
lizard with temperature-dependent sex determination. Journal of Evolutionary
Biology 2:177-184.
Bull, J.J., W.H.N. Gutzke, and D. Crews. 1988b. Sex reversal by estradiol in three
reptilian orders. General and Comparative Endocrinology 70:425-428.
Bull, J.J., R.C. Vogt, and M.G. Bulmer. 1982a. Heritability of sex ratio in turtles with
environmental sex determination. Evolution 36:333-341.
61


26
has been summarily attributed to its maintenance of balanced sex ratios in natural
populations (Fisher 1930). Because the rarer sex gains a fitness advantage over the more
common sex, population sex ratios tend to hover near unity (Fisher 1930). The
advantages of ESD are more opaque. Hypotheses regarding the advantages of ESD have
been organized in five categories: (a) phylogenetic inertia, (b) group-adaptation, (c)
inbreeding avoidance, (d) sex-differential fitness, and (e) quasi-neutrality (Shine 1999;
Girondot and Pieau 1999). The enigma is further compounded by the existence of
closely-related taxa that express very different sex-determining mechanisms. Although
the predominant determinants of sex can be readily identified through experimental
matings and incubations, the finer points of the relationship between genes and
environment in the process of sex determination have not yet been acceptably clarified in
many species. The identified dichotomy of ESD and GSD may cause weaker
determinants of sex to be overlooked which, if discovered, would more appropriately
describe the process by which a bipotential gonad becomes an ovary or a testis.
Additionally, an adaptation that is completely environmentally determined could
be invisible to natural selection. In order to describe the evolutionary history of sex
determination in vertebrates, researchers must first test hypotheses about the effects of
selection on sex-determining mechanisms. Selection may work on the genetic
architecture that allows an effect of temperature or other environmental factor on sex
determination. Temperature affects the activation of genes that encode steroidogenic
enzymes (Crews 2003). Genetic variation most likely affects the sensitivity of genes to
thermal stimuli. Models derived for the adaptive evolution of sex ratio assume that
genetic variation exists for primary sex ratio and that the variation is caused by genes of


45
mechanism to estrogen. Estrogens are responsible for the regression of the medullary
region of the developing testis and the proliferation of the cortical region of the
bipotential gonad (Crews et al. 1991). The enzyme aromatase is responsible for the
conversion of testosterone to estradiol-17(5. Previous studies with reptiles, specifically
turtles and alligators with TSD, have not reported the production of males with any dose
of estrogen, androgen, anti-estrogen, anti-androgen or aromatase blocker (for review see
Guillette & Crain, 1996). Embryos exposed to aromatase blockers prior to sex
determination in the Red-Eared Slider Turtle, Trachemys scripta, develop according to
the temperature at which they are incubated (Wibbels & Crews., 1992) whereas in
alligators most are female or exhibit an ambiguous gonad (Lance & Bogart, 1992). In
contrast, genotypically female larvae of the newt, Pleurodeles waltl, differentiate into
functional males following treatment with an aromatase inhibitor (Chardard and Doumon
1999). An estrogen deficit results in male sex differentiation, regardless of genotype in
P. waltl. Hayes (1998) reported on sex determination in an amphibian, Rana pipiens,
with genetic sex determination finding that low doses of estradiol (< 0.07 pM) did not
affect sex differentiation, whereas high doses (0.07-0.18 pM) produced 100% females
and even higher doses (> 3.69 pM) produced 100% males. A higher dose of estrogen, as
is predicted with the administration of exogenous estrogens at a female-producing
temperature in E. macularius, suggests that a similar mechanism may occur in this lizard
contrary to the observations in other species with TSD. Higher doses of estrogen
increased the proportion of male E. macularius produced (at the female-producing
temperature), as seen in Hayes study. The results of this study suggest the hypothesis
that an estrogen surplus could alter sex determination in embryos. The mechanism is


CHAPTER 4
ESTROGEN INCREASES PRODUCTION OF MALES IN A TEMPERATURE-
DEPENDENT SEX-DETERMINING REPTILE
Introduction
Temperature-dependent sex-determining (TSD) species produce hatchling sex
ratios that are shaped by the temperature at which the embryonic clutch was exposed
during a thermosensitive period of incubation. For example, Leopard Geckos,
Eublepharis macularius, follow a pattern of sex determination in which an increased
proportion of males is produced at an intermediate incubation temperature (31-33C)
whereas an increased proportion of females is produced at cooler (26-28C) and warmer
(34-35C) incubation temperatures (Viets et al. 1993; Crews 2003). The masculinizing
influence of male-determining incubation temperature can be experimentally disrupted by
application of estrogens and estrogen-mimicking compounds (Bull et al. 1988b;
Tousignant and Crews 1994). The application of estrogenic compounds to eggs of other
temperature-dependent sex-determining species, such as freshwater turtles and
crocodilians, also affects sex determination, sex differentiation, organizational and
activational development, and sex-specific behavior in resulting hatchlings (Jeyasuria et
al. 1994; Sheehan et al. 1999; Willingham et al. 2000). Typically, this disruption takes
the form of sex-reversal of males (an override of TSD) or feminization of male
reproductive tissues (Fry and Toone 1981; Belaid et al. 2001). However, these
purportedly paradigmatic conclusions may only describe one half of an inverted U-
38


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
MhcA.
Max A. Nickerson
Professor of Wildlife Ecology and
Conservation
This dissertation was submitted to the Graduate Faculty of the Department of
Zoology in the College of Liberal Arts and Sciences and to the Graduate School and was
accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.
May 2004
Dean, Graduate School


18
Figure 2. Light microscopy images of reproductive tissue from Leopard Geckos,
Eublepharis macularius. These images are magnified 200X. (a) Male
reproductive organs have seminiferous tubules that have a thick, simple
cortex and a narrow lumen, (b) Female reproductive organs lack
seminiferous tubules.


Proportion of Males / Treatment Group
17
24 26 28 30 32
Temperature (C)
Figure 1. Effect of incubation temperature on hatchling sex ratios of Leopard Geckos.
Control sex ratio data were collected from treatment groups exposed to either
no treatment or a 95% EtOH vehicle from the second year of experimentation
described in Chapter 4.


2
appropriately tested using a species with environmental sex determination. This type of
organism will produce offspring sex ratios that can be directly linked to experimentally-
induced environmental conditions without the confounding factor of predominant genetic
control of sex determination.
The gonadal sex of all organisms has been attributed to either genetic sex
determination (GSD) or environmental sex determination (ESD). In GSD, the sex of the
organism depends on the sex chromosome contributions from the organisms parent(s).
In ESD, the sex of the organism depends on the post-fertilization environment
experienced by the organism as a developing embryo. Typically, ESD refers to the
effects of incubation temperature, although Heiligenberg (1965) reported a sex ratio
skewing effect of pH in cichlids. Among vertebrates, the initiation of sex differentiation
is predominantly controlled by incubation temperature in tuatara, some fish, turtles, and
lizards and all crocodilians (Bull 1980; Conover and Kynard 1981; Ewert and Nelson
1991; Janzen and Paukstis 1991). All other vertebrates appear to be genetically sex-
determined. Incubation temperature can also affect body size and growth (Crews et al.
1998; Janes and King, unpublished data), adult sexuality (Gutzke and Crews 1988),
aggressive behavior (Rhen and Crews 1999), and the organization of neural structures
(Coomber et al. 1997). Incubation temperature is known to cause the development of an
embryos testes or ovaries; the proximate mechanism is still unknown.
The recognition of ESD as a derived condition is supported by reports of GSD in
amphibians, a basal taxon to reptiles. No surveyed amphibians have demonstrated a sex
determining effect of incubation temperature within the range of temperatures to which
their eggs are naturally exposed (Hayes 1998). The adaptive significance of ESD has


32
Painted Turtle, Chrysemys picta will become extinct because of an inability to produce
males (Janzen 1994).
If Leopard Geckos, E. macularius, or any other TSD species have a chance to
persist in spite of rapid climate change, their continued survival will be attributable to the
adaptability of their sex-determining mechanism or their behavioral placement of nests.
In order to adapt, the mechanism must have an underlying genetic component. An
interaction of temperature and genotype would allow natural populations of TSD species
to change the threshold temperatures at which embryos become either male or female.
Variation in sex-determining response must vary among individuals and/or among
populations. This variation must be caused by variation in genotype. An interaction of
temperature and genotype has been reported in an atherinid fish, the Atlantic Silverside,
Menidia menidia (Conover and Kynard 1981). In M. menidia, temperature influences
offspring sex ratios but the sex-determining mechanisms of progeny from different
females respond differently to incubation temperature. Variation in the sex-determining
response of offspring from different dams is not attributable to sire because Conover and
Kynard used one sire to fertilize all dams in their study. Difference in sex ratio was not
attributable to maternal size, clutch size, or level of natural mortality. Conover and
Kynard (1981) interpret these results as polygenic sex determination.
In reptiles, evidence of gene x environment interaction for TSD is lacking. If
heritable variation in sex-determining response to incubation temperature exists for TSD
species, then local adaptation should be evident. Bull et al. (1982a) tested for local
adaptation of threshold incubation temperatures that initiate male or female sex
differentiation in six species of turtles of the subfamily Emydinae, genera Graptemys,


range of temperatures studied in these experiments includes the species-specific
temperatures at which the lowest proportion of males (0.0) and the highest proportion of
males (~0.7) are produced per treatment group. Each treatment group consisted of 18 E.
macularius eggs. At each temperature, the effects of humidity, quantitative genetic
variation, and environmental endocrine disrupters on hatchling sex ratios of treatment
groups were measured. Humidity did not significantly affect hatchling sex ratios. Also,
results of the quantitative genetics experiment suggest that ESD in E. macularius is a
genotype x environment interaction. Both natural and synthetic estrogens affected
hatchling sex ratios at both male- and female-producing temperatures. I conclude that
ESD is a multi-dimensional trait in E. macularius. The influences of abiotic factors like
local profile of environmental hormone and hormone mimic concentrations and biotic
factors like quantitative genetic variation may ameliorate the effects of climate change on
ESD. Estimates of extinction rates of ESD species as a result of changing climates
should be reconsidered in light of these data. As other researchers have concluded for
other ESD reptiles, I conclude that the partial genetic control of sex determination in E.
macularius is a polygenic trait and responds to temperature fluctuation as a genotype x
environment interaction. In light of these conclusions, sex-determining mechanisms of
reptiles should be reconsidered as a broad spectrum of multi-dimensional mechanisms
instead of a simple dichotomy of ESD and genetic sex determination.
vii


58
I conclude that ESD is a multi-dimensional trait in E. macularius. An influence
of temperature, quantitative genetic variation, and estrogen concentration on sex ratios
was demonstrated in this species. The influences of other abiotic and biotic factors offer
ameliorative effects on sex determination in the face of climate change. Estimates of
extinction rates of ESD species should be reconsidered in light of these data. Research
into the adaptive significance of ESD should be focused on the benefits to the dam as the
dam invests the partial controlling element of genetic variation. As others have
concluded for other reptiles, I conclude that the partial genetic control of sex
determination in E. macularius is a polygenic mechanism and responds to temperature
fluctuation as a classic gene x environment interaction. Lastly, the effect of superfluous
estrogen on sex determination at the female-producing temperature should encourage
researchers to test the effects of aromatase inhibitors at female-producing temperatures.
The hypothetical male-determining factor is most likely an aromatase inhibitor that can
be stimulated by superfluous estrogen at the beginning of the thermosensitive period of
embryonic development.
In summary, I have clarified a more specific effect of the nest environment on
hatchling sex ratios in E. macularius. Elucidation of this kind is necessary for the
continuing effort to identify patterns in the evolution of sex-determining mechanisms.
Environmental sex determination may have arisen in response to changes in the
environment that have not yet been considered. Fisherian sex ratios may be shaped by a
broad spectrum of environmental stresses. Natural selection favors parents that invest
equally in sons and daughters (Fisher 1930). However, depending on local resource
availability, sons and daughters have different reproductive potentials. Males in good


62
Bull, J.J., R.C. Vogt, and C.J. McCoy. 1982b. Sex determining temperatures in
turtles: a geographic comparison. Evolution 36:326-332.
Cagle, K.D., G.C. Packard, K. Miller, and M.J. Packard. 1993. Effects of
the microclimate in natural nests on development of embryonic Painted Turtles,
Chrysemys picta. Functional Ecology 7:653-660.
Chardard, D., and C. Doumon. 1999. Sex reversal by aromatase inhibitor treatment in the
newt Pleurodeles waltl. Journal of Experimental Zoology 283:43-50.
Chamov, E.L., and J.J. Bull. 1977. When is sex environmentally determined?
Nature 266:828-830.
Christian, K.A., and W.T. Lawrence. 1991. Microclimatic conditions in nests of
the Cuban Iguana (Cyclura nubila). Biotropica 23:287-293.
Conover, D.O., and B.E. Kynard. 1981. Environmental sex determination: interaction of
temperature and genotype in a fish. Science 213:577-579.
Coomber, P., D. Crews, and F. Gonzalez-Lima. 1997. Independent effects of incubation
temperature and gonadal sex on the volume and metabolic capacity of brain nuclei
in the leopard gecko (Eublepharis macularius), a lizard with temperature-
dependent sex determination. Journal of Comparative Neurology 380:409-421.
Crain, D.A. and L.J. Guillette. 1998. Reptiles as models of contaminant-induced
endocrine disruption. Animal Reproduction Science 53: 77-86.
Crain, D.A., L.J. Guillette, A.A. Rooney, and D.B. Pickford. 1997. Alterations in
steroidogenesis in Alligators {Alligator mississippiensis) exposed naturally and
experimentally to environmental contaminants. Environmental Health
Perspectives 105:528-533.
Crews, D. 2003. Sex determination: where environment and genetics meet. Evolution and
Development 5:50-55.
Crews, D., J.J. Bull, and T. Wibbels. 1991. Estrogen and sex reversal in turtles: a dose-
dependent phenomenon. General and Comparative Endocrinology 81:357-364.
Crews, D., A.R. Cantu, T. Rhen, and R. Vohra. 1996. The relative effectiveness of
estrone, estradiol-17/3, and estriol in sex reversal in the red-eared slider
{Trachemys scripta), a turtle with temperature-dependent sex determination.
General and Comparative Endocrinology 102:317-326.


4
macularius is a multi-dimensional mechanism, controlled in part by incubation
temperature and in part by at least one of the following parameters. Differences in the
timing of incubation (Lance 1989; Guillette et al. 1997), placement and construction of
nests (Magnusson et al. 1985; Shine and Harlow 1996), genetic influences (Parker and
Orzack 1985; Wachtel 1989), presence of industrial contaminants (Guillette et al. 1997;
Crain et al. 1997), or other environmental variables (Webb and Smith 1987) may express
partial control over clutch sex ratios in E. macularius. Other variables may counteract
the sex ratio skewing effects of ambient temperature and maintain population sex ratios
near 0.5 at all points within the range of the species. For this study, chapters 2 and 3 will
provide data on the intrinsic responses of the organism to naturally occurring incubation
conditions. Chapter 4 will introduce an anthropogenic stress (DDT) to a series of E.
macularius eggs in order to test the plasticity of their sex-determining mechanism when
faced with an extrinsic stress that is presently found in their home range.
Laboratory incubations at constant temperatures can provide controlled
experimental data but do not accurately represent natural nest environments. The
experiments in this study have been designed to offer a more realistic, multi-dimensional
representation of the environment experienced by E. macularius eggs during the
incubation period when gonadal sex is determined by varying several factors. This study
will serve to expand the currently accepted definition of ESD. If gonadal sex is
controlled by a multi-dimensional influence of the nest microenvironment, then
researchers can investigate potential advantages of maleness and femaleness within a
more specifically defined milieu. The Trivers-Willard hypothesis may be supported by


11
throughout the experiment with Hobo temperature loggers. Within each chamber, the
12 egg containers were divided into four treatment groups of three containers. Each
treatment group, consisting of 18 eggs, was exposed to 20%, 25%, 30%, or 35%
humidity. At the beginning of the experiment, humidity was varied by adding 14 ml,
17.5 ml, 21 ml, or 24.5 ml of water to the perlite in the egg cups in the 20%, 25%, 30%,
or 35% humidity treatment groups, respectively. Every day, the incubation chambers
were opened and the egg containers were removed. Each cup was opened momentarily
in order to release metabolic gas waste and check for hatchlings. Eggs that grew mold
were discarded upon discovery. Position of egg containers within an incubator was
randomized daily.
Sexing Procedure
Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2-
bromo-2-chloro-l,l,l-trifluoroethane). Geckos were fixed in Bouins fixative and
preserved in 75% ethanol. The reproductive organs were removed from each gecko and
prepared for analysis by light microscopy. The reproductive organs are opaque white,
cylindrical structures on either side of the posterior end of the dorsal artery. After
removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of
ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55;
Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The
resulting paraffin blocks were sectioned at 8 pm and stained with a modified trichrome of
Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from
all geckos. If seminiferous tubules were identified within the tissue sections, the gecko
was scored as male (Figure 2a). If seminiferous tubules were absent, the gecko was


51
Table 1. ANOVA for sex ratios of Leopard Geckos from Experiment 1. All treatment
groups are considered separately in this analysis.
Source
df
MS
Treatment
4
0.246
Temperature
2
0.418
Temperature Treatment
14
0.254


30
circular structures with a narrow lumen (Berman 2003). By relying on histological
examination of our specimens, I avoided potential macroscopic misidentification of male
and female reproductive organs.
The experiment was replicated the following year (16 February-5 June, 2003)
with a new group of 5 male and twenty five female adult E. macularius from a separately
maintained breeding colony at the Gourmet Rodent. Thus, the sires and dams used in the
first years experiment were not the sires and dams used in the second years replication.
Analysis
Sex ratio data were analyzed by ANOVA with fixed main effects of sire, dam,
incubation temperature, and year, as well as their interactions. Egg containers were
nested randomly within year X temperature. Data were analyzed using SAS version 6.10
for the Macintosh. ANOVAs were performed using PROC GLM.
Results
As a consequence of allowing this experiments eggs to develop without variation
in any abiotic factors but temperature, the results from this study clearly represent the
typical effect of incubation temperature on hatchling sex ratios. Proportion of males per
treatment group increase with the temperature at which the group was incubated (MS =
2.361; P = 0.001; Table 3). The effect of temperature on sex ratio did not differ between
the two years of experimentation. The sex ratios per sire did not differ significantly
between years or among temperatures. The same is true for the sex ratios per dam.
However, the offspring sex ratios of couplings of sire and dam differed significantly
across incubation temperatures (MS = 0.258; P<0.05; Table 3), indicating a gene x
environment interaction. The variation in the hatchling sex ratios per coupling of sire and


CHAPTER 5
GENERAL DISCUSSION
Although dioecy, the trait of having two sexes, is almost universal within the
animal kingdom, great variation exists for the derivation of two sexes from a bipotential
embryo. Understanding the evolution of sex depends on a clear historical record of the
evolution of sex-determining mechanisms. Species under different selective regimes
have evolved different genomic mechanisms leading to dioecy. Elucidating the basic
dichotomy of ESD and GSD informs the controversy of adaptive significance for either
mechanism or mechanisms that draw on both genetic and environmental cues.
Sex determination in E. macularius responds to environmental cues. As shown in
chapter 2, temperature influences sex determination and humidity does not. Differential
effects of temperature and humidity suggest the structure of the molecular action that
initiates sex determination in this species. Temperature may be an indicator of habitat
quality. If dams use ESD to control their offspring sex ratios in heterogenous
environments in order to produce more offspring of the sex that is most likely to be fit in
the immediate habitat, temperature and humidity would be clear and immediate indicators
of microhabitat quality. Temperature does influence clutch sex ratios. Humidity does
not.
In chapter 3, a gene x environment interaction was demonstrated in the sex
determining mechanism of E. macularius. This result addresses concerns about the
stability of ESD in the face of rapid climate change. Having a genetic component may
55


41
EtOH. Solutions were applied to the shells of eggs in each treatment group < 24 hours
after oviposition. After experimental treatments were applied, all treatment groups were
placed in their respective environmental chamber at one of the three temperatures defined
above. Every day, the chambers were opened and the egg containers were removed.
Each cup was opened momentarily in order to release metabolic gas waste and check for
hatchlings. Eggs that grew mold were discarded upon discovery. Position of egg
containers within the three chambers was randomized daily.
Experiment 2
The following year, a new sample of 216 E. macularius eggs were tested. The
second years eggs were oviposited on 26 February 2003. Four treatment groups were
exposed to (/) no treatment, (g) 5 pi 95% EtOH, (h) 10 pg/5 pi estradiol benzoate, or (i) 5
pi estradiol -170. Treatments h and i were dissolved in 95% EtOH. To compare the
effect of the timing of treatment of these positive and negative controls on sex
determination relative to the data obtained in Experiment 1, we applied these treatments
at the beginning of the middle third of the incubation period at each of the three
temperatures in contrast to within 24 hr of oviposition as in Experiment 1. Each
treatment group consisted of three egg containers housing 6 egg chambers each (N = 18
total / treatment). As in the first year, each egg container held six eggs.
Sexing Procedure
Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2-
bromo-2-chloro-l,l,l-trifluoroethane). Hatchling geckos were fixed in Bouins fixative
and preserved in 75% ethanol. The reproductive organs were removed from each gecko
and prepared for analysis by light microscopy. The reproductive organs are opaque


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Zoology t. X
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Marta L. Wayne, CoChair
Assistant Professor of Zoology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Louis'TTuuTTlelte
Distinguished Professor of Zoology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Karen A. BjomdaJ
Professor of Zoology


28
physiology and ecology of their TSD mechanism have been broadly researched in the
recent past (Crews et al. 1996; Rhen et al. 1999; Rhen et al. 2000; Bragg et al. 2000).
Methods
Animals
On 5 January 2002, a group of five male and twenty five female adult Leopard
Geckos, Eublepharis macularius, were selected from a colony of > 50,000 at The
Gourmet Rodent, a reptile breeding facility in Archer, Florida. The five males were
selected based on their previously demonstrated viability and the variation of color
patterns on their dorsa. Dorsal coloration is a decent indicator of geographic origin in E.
macularius (K. Auffenberg, pers. comm.). The five males were selected because they
represented a diversity of regional origins from which the facilitys breeding colony is
drawn. The 25 females were selected because they were virgin and were considered
likely to be viable because of their body sizes. Each male was mated to five females.
Each female was housed alone in a cage containing a food bowl, a water bowl, and a
nestbox filled with moist vermiculite. Males were mated to females by moving them to a
different females container every day. Males were rotated among their five mates every
24 hours and isolated for 48 hours between rotations. Between 5 January and 15 May,
2002, nestboxes were checked daily. If new eggs were found during daily nestbox
inspections, they were placed in a plastic box filled with vermiculite and transported by
car to the University of Florida. In the laboratory, the eggs were placed in containers that
consisted of six 188 ml plastic cups banded together in rings. Each cup contained 70 ml
perlite and 17.5 ml tap water and was sealed with a tight-fitting lid punctured with one
gas-exchange hole. The eggs were placed randomly in containers. Each container held


15
temperature on hatchling sex ratios in the first year may also be explained by genetic
differences between the colonies of E. macularius used in the two years of
experimentation. The breeding facility from which eggs were collected maintains
separate breeding colonies that do not interbreed. Drift or local adaptation of the sex
determining response to incubation temperature may explain the difference between the
results from the first and second years of experimentation. This result would suggest
intraspecific variation in the effect of abiotic factors on sex determination in E.
macularius. Such intraspecific variation of sex-determining mechanisms has only been
reported anecdotally for Tokay Geckos, Gekko gecko (F. Janzen, pers. comm.).
However, intraspecific variation in the sensitivity of temperature-dependent sex
determining mechanisms has been reported for A. mississippiensis (L. Guillette, pers.
comm.). In short, the factors that influence ESD within species are not expected to vary
but the degree to which fixed factors affect ESD is expected to vary.
A multifactorial sex-determining mechanism would have an impact on
conservation considerations for ESD species. Vogt and Bull (1984) present empirical
evidence that vegetation changes influence hatchling sex ratio in Map turtles. Invasive
plants are changing vegetation cover over Nile crocodile nesting sites (Leslie and Spotila
2001). The consequent effects on Nile crocodile sex ratios have not yet been reported.
Reptile populations with ESD may need to be manipulated if nest site microclimates
change too dramatically (Mrosovsky 1994). In the worst case scenario, extinction may
result from climate change (Janzen 1994a). If the influence of incubation temperature on
hatchling sex ratios is exacerbated or ameliorated by another weaker sex determinant,
risk assessments in the face of habitat alteration must be reconsidered.


65
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68
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43
significantly differ from each other (Tables 1 and 2). That is, all doses of DDT and the
positive control E2 benzoate altered the determination of sex in a similar fashion. A sex
ratio-reversing effect of DDT was not seen in the treatment groups but a general
perturbation appears to have affected all treatment groups that were dosed with either
EtOH, DDT, or E2 benzoate within the first 24 hours after oviposition.
Experiment 2 The treatment groups in Exp. 2 were exposed to chemical treatments at
the beginning of the middle third of incubation, or just prior to the period of sex
determination. The timing of exposure of the eggs to potential disrupters of the sex
determining pathway appears to influence the embryos sex determination. In Exp. 2,
both estradiol treatment groups (E2 benzoate and E2) had a significantly lower proportion
of males than the negative control groups (no treatment and 95% EtOH) at the male-
producing incubation temperature (P < 0.001; Tables 3 and 4). Also in Exp. 2, both
estradiol treatment groups had a higher proportion of males than the negative control
groups at the female-producing incubation temperature (P<0.01; Figure 10). In Exp. 2,
the sex ratio of the EtOH treatment group did not differ from the negative control group
that received no treatment. Thus, EtOH is a suitable vehicle as it had no effect on the sex
determination of the embryos demonstrating that the sex ratio-altering effects seen in the
other treatments groups represent specific actions of either estradiol benzoate or estradiol
17p, and not a result of general chemical perturbation. No significant differences were
found among the hatchling sex ratios of any treatment groups from the 30C
environmental chambers.


59
condition are expected to outreproduce their sisters but females are expected to
outreproduce their brothers if both are in poor condition (Trivers and Willard 1973).
Poor condition could be defined as poor individual health or poor resource availability
that will result in poor individual health. Several factors in the nest could serve as
indicators of local resource availability and therefore sex-differential fitness. Sex-
differential fitness could result from fluctuations in temperature and/or any other factor in
the habitat as described in chapter 1. Temperature may be only one of many factors to
which ESD species adapted. At present, no categorization of species can separate ESD
species from GSD species. In light of these data, ESD species should be categorized by
their differential sex-determining response to humidity, quantitative genetic variation,
and exogenous estrogens. Different species can yield different patterns of sex
determining responses to these variables. Different patterns would inform the evolution
of sex-determining mechanisms.
Researchers ability to compare and contrast sex-determining mechanisms among
eublepharids, within Squamata and across Chordata has been hindered by insufficient
characterizations of the mechanisms. Comparisons of ESD species have not effectively
described the evolutionary history of ESD By comparing the effects of temperature on
sex determination, researchers have differentiated Type I, Type and Type HI patterns
of temperature-dependent sex determination (TSD). Type I TSD species like A.
mississippiensis produce larger proportions of males at higher incubation temperatures
(Bull 1983). Type II TSD species like Green Sea Turtles, Chelonia mydas produce larger
proportions of males at low temperatures (Mrosovsky 1988). Type III TSD species like
E. macularius produce larger proportions of males at intermediate temperatures and


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
MhcA.
Max A. Nickerson
Professor of Wildlife Ecology and
Conservation
This dissertation was submitted to the Graduate Faculty of the Department of
Zoology in the College of Liberal Arts and Sciences and to the Graduate School and was
accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.
May 2004
Dean, Graduate School


Proportion of Males / Treatment Group
50
30 32.5
Temperature (C)
Figure 10. Sex ratios of Leopard Geckos in the second year of experimentation. In the
second year, treatments were administered to eggs at the beginning of the
middle third of incubation. The treatment groups exposed to no treatment, 5 pi
95% EtOH, 10 pg/ 5 pi estradiol benzoate, or 5 pi estradiol 17/3 are indicated
by the light, dark, and darkest gray and black respectively. The 10ug/5ul
Estradiol Benzoate treatment groups contained no males so no bar is shown for
that group.


3
eluded researchers (Janzen and Paukstis 1991). Concensus favors ESD as either an
adaptive mechanism for species in which a fitness advantage oscillates between males
and females (Shine 1999) or as a neutral mechanism that has not been counter-selected
(Bull and Chamov 1989; Girondot and Pieau 1999).
This study will test the effects of environmental stresses on clutch sex ratios of
Leopard Geckos, Eublepharis macularius, a reptile species for which much has been
written on their sex-determining response to incubation temperature. Leopard Geckos
live in a range of climatic heterogeneity. Their range extends from the grasslands of
southeastern Turkey to the forests of southwestern India (Smith 1935). Clutch sex ratios
are male-biased when incubated at 32.5C. Cooler and warmer incubation temperatures
within the full range of viable incubation temperatures (25 to 35C) cause female-biased
clutch sex ratios (Viets et al. 1993). If the ESD mechanism behaves similarly throughout
the climatically heterogeneous zones of their range and all other variables are irrelevant,
then differences in the primary sex ratios of E. macularius can be expected as a result of
different incubation temperatures in different parts of their range (Viets et al. 1993;
Crews et al. 1996; Coomber et al. 1997; Rhen et al. 2000). Also, climate change due to
global warming or other causes might cause dramatic shifts in population sex ratios of E.
macularius and other ESD species.
Based on the observations that (a) among higher vertebrates, the sex of several
species of reptiles and fishes appears to be controlled by incubation temperature, (b)
populations of environmentally sex-determined species can cover ranges that include
more than one climate zone, and (c) sex ratios do not appear to be unbalanced at different
extremes of their range, I hypothesize that the sex-determining mechanism in E.


64
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reptiles. Proceedings of the National Academy of Sciences USA 91:7487-7490.


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54
Table 4. ANOVA for sex ratios of Leopard Geckos from Experiment 2. Both estradiol
treatments are considered together as one variable. Both control treatments are
considered together as one control group.
Source
df
MS
Treatment
1
0.133
Temperature
2
0.214
Temperature Treatment
50.915***
***: P< 0.001


46
unknown, but could involve feedback mechanisms either at the level of the ovary or
hypothalamo-hypophysial axis. That is, high estrogen concentrations in the gonad or
brain could alter the release of stimulatory factors from the brain or the gonad by altering
gene expression patterns. Hence, an inverted U-shaped response associated with the sex
determining mechanism in response to estrogen is supported by these data in E.
macularius. That is, at lower or natural levels of estrogen an ovary forms, but at higher
levels the genetic mechanism normally induced by estrogen exposure is altered or down-
regulated leading to the development of a testis. A number of genes are known to be
differentially regulated during gonadal differentiation in reptiles (Crews, 2003; Western
& Sinclair, 2001). Recent gene knockout studies in mice, suggest that alterations in
several of these genes will alter the differentiation of the gonad.
The studies presented here indicate that when administered outside of the
thermosensitive period, exogenous estrogens do not significantly alter sex determination
of£. macularius at male- or female-producing temperatures. We do, however, show
evidence of a general perturbation of the sex ratio in response to administration of
estrogen, diphenyltrichloroethane (DDT), and ethanol during earlier stages of embryonic
development. In E. macularius, sex determination occurs over an interval within the
middle third of incubation (stages 32-37; Bull 1987). During stage 32 (the onset of sex
determination), overall embryonic morphology corresponds to stage 15 in turtles. Stage
15 in turtles also marks the onset of sex determination (Yntema 1979). In snapping
turtles, Chelydra serpentina (a TSD species), administration of exogenous estrogens only
sex-reversed embryos when applied between stages 10-22. Outside of this middle third
of incubation, no hormonal effects on gonadal development were evident (Gutzke and


53
Table 3. ANOVA for sex ratios of Leopard Geckos from Experiment 2. All treatment
Source
df
MS
Treatment
3
0.174
Temperature
2
0.190
Temperature Treatment
11 0.515**
**: P = 0.002


66
Matter, J.M., D.A. Crain, C. Sills-McMurry, D.B. Pickford, T.R. Rainwater, K.D.
Reynolds, A.A. Rooney, R.L. Dickerson, and LJ. Guillette. 1998. Effects of
endocrine-disrupting contaminants in reptiles: alligators, pp. 267-289. In: Kendall,
R.J., R.L. Dickerson, J.P. Giesy, and W.A. Suk (eds.). 1998. Principles and
processes for evaluating endocrine disruption in wildlife. SETAC Press,
Pensacola, Florida.
McGinley, M.A. 1984. The adaptive value of male-biased sex ratios among stressed
animals. American Naturalist 124:597-599.
Mrosovsky, N. 1988. Pivotal temperatures for loggerhead turtles (Caretta caretta)
from northern and southern nesting beaches. Canadian Journal of Zoology
66:661-669.
Mrosovsky, N. 1994. Sex ratios of sea turtles. Journal of Experimental Zoology 270:16-
27.
Packard, G.C., M.J. Packard, and G.F. Birchard. 1989. Sexual differentiation and
hatching success by Painted Turtles incubating in different thermal and hydric
environments. Herpetologica 45:385-392.
Paladino, F.V., P. Dodson, J.K. Hammond, J.R. Spotila. 1989. Temperature-dependent
sex determination in dinosaurs? Implications for population dynamics and
extinction. In: Farlow, J.O. (ed.). Paleobiology of the dinosaurs: Boulder,
Colorado, Geological Society of America Special Paper 238:63-70.
Parker, E.D., and S.H. Orzack. 1985. Genetic variation for the sex ratio in Nasonia
vitripennis. Genetics 110:93-105.
Parmigiani, S., P. Palanza, and F.S. vom Saal. 2000. Ethotoxicology: An evolutionary
approach to behavioral toxicology, pp. 217-233. In: Guilllette, L.J. and D.A.
Crain (eds.). 2000. Environmental endocrine disruptors. Taylor and Francis. New
York.
Paukstis, G.L., W.H.N. Gutzke, and G.C. Packard. 1984. Effects of substrate
water potential and fluctuating temperatures on sex ratios of hatchling Painted
Turtles (Chrysemyspicta). Canadian Journal ofZoology 62:1491-1494.
Podreka, S., A. Georges, B. Maher, and C.J. Limpus. 1998. The environmental
contaminant DDE fails to influence the outcome of sexual differentiation in the
marine turtle Chelonia mydas. Environmental Health Perpectives 106: 185-188.
Portelli, M.J., S.R. de Sola, R.J. Brooks, and C.A. Bishop. 1999. Effect of
dichlorodiphenyltrichloroethane on sex determination of the common snapping
turtle (Chelydra serpentina serpentina). Ecotoxicology and Environmental Safety
43: 284-291.


37
Table 3. ANOVA across years for offspring sex ratios of Leopard Geckos.
Source df MS
Temperature 2 2.361***
Dam (Sire) 34 0.190
Temperature Sire Dam 46 0.258*
*: P < 0.05; ***: P = 0.001


7
temperature is seen among and within ESD species like those in Eublepharinae (Elphick
and Shine 1999). Thus, the same constant incubation temperature may produce different
sex ratios in different populations of the same species.
Most studies report female-biased populations of reptiles exhibiting TSD,
regardless of local nest site temperatures (Bull and Chamov 1989; Ewert and Nelson
1991). In light of this, it appears that the relationship between hatchling sex ratios and
the incubation environment has not been adequately defined. Other extrinsic or intrinsic
variables can play major or minor roles in the maintenance of female-biased hatchling
sex ratios. Aside from temperature, several factors have been implicated in the
relationship between the sex determination of an embryo and its immediate environment.
Exogenous sex steroid hormones or endocrine active contaminants can alter sex
determination. Further, the behavioral and physiological condition of the mother as well
as the environments encountered through the life of the mother, and age and sociosexual
experience of either parent could influence sex differentiation of ESD species (Crews
2003). Temperature has been identified as a determinant of sex determination in all
squamates studied to date that exhibit ESD. Carbon dioxide concentrations and pH
(Etchberger et al. 2002) and humidity (Gutzke and Paukstis 1983) also have been
identified as having a secondary role in some reptilian species. Here, I consider humidity
as a major factor in sex determination of E. macularius.
Eublepharis macularius has a natural range that includes desert and grasslands of
the Middle East (Daniel 2002). In this range, nest site temperatures and humidities
fluctuate dramatically among seasons, years, and regions. Eublepharis macularius


Proportion of Males / Dam
35
Temperature (C)
Figure 7. Offspring sex ratios of Leopard Geckos incubated at one of three
temperatures during one of two years of experimentation. Each line
represents the offspring sex ratio of one of 35 dams used in this study. Many
lines are not visible because they are super-imposed on each other in this
figure. Incubated at the same temperature, different dams produced different
offspring sex ratios. The ranks of the genotypes of different dams changed
depending on the environment, indicating a gene x environment interaction.


29
six eggs. Each egg was placed individually within one of the six cups in a container. The
egg containers were placed randomly in one of three environmental chambers and
maintained at either 26, 30, or 32.5 C for the duration of the experiment. The sire,
dam, egg container, and egg cup were recorded for each egg. Chamber temperatures
were recorded every minute throughout the experiment with Hobo temperature loggers.
Every day, the environmental chambers were opened and the egg containers were
removed. Each cup was opened momentarily in order to release metabolic gas waste and
check for hatchlings. Eggs that grew mold were discarded upon discovery. Position of
egg containers within an environmental chamber was randomized daily.
Sexing procedure
Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2-
bromo-2-chloro-l,l,l-trifluoroethane). Geckos were fixed in Bouins fixative and
preserved in 75% ethanol. The reproductive organs were removed from each gecko and
prepared for analysis by light microscopy. The reproductive organs are opaque white,
cylindrical structures on either side of the posterior end of the dorsal artery. After
removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of
ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55;
Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The
resulting paraffin blocks were sectioned at 8 pm and stained with a modified trichrome of
Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from
each gecko. If seminiferous tubules were identified within the tissue sections, the gecko
was scored as male (Figure 2a). If seminiferous tubules were absent, the gecko was
scored as female (Figure 2b). Seminiferous tubules were identified as clusters of simple,


8
follow a pattern of sex determination in which an increased proportion of males is
produced at an intermediate incubation temperature (31-33C) and an increased
proportion of females is produced at cooler (26-28C) and warmer (34-35C) incubation
temperatures (Viets et al. 1993; Crews 2003).
If E. macularius maintains an evolutionarily stable sex ratio across thermally
variant environments, the effects of differing ambient temperature must be mediated
either by local adaptation (Mrosovsky 1988; Blackmore and Chamov 1989; Viets et al.
1993), carbon dioxide, pH (Etchberger et al. 2002), maternal affects (Bowden et al.
2002), humidity, or a complex interaction of some or all of these factors. Environmental
sex determination could be caused by a wide array of weak influences during incubation
(Bull et al. 1982a; Bull et al. 1982b). In addition, sex-determining responses to these
influences could be phenotypically plastic, as is widespread in reptilian phenotypes
(Shine and Elphick 2001). Population sex ratios of E. macularius can be further altered
by short-term weather fluctuations because of the extreme sensitivity of reptiles to
environmental variables during embryogenesis (Shine and Elphick 2001).
The influence of humidity on sex determination has been debated in the past.
Gutzke and Paukstis (1983) reported that in the Painted Turtle, Chrysemyspicta, more
males were produced in wet substrates than in dry substrates at typically male-producing
temperatures. In a second study, C. picta eggs were again incubated on wet or dry
substrates (Paukstis et al. 1984). In the latter study, the reverse pattern was reported.
More male turtle hatchlings were produced in dry substrates than in wet substrates. Also,
a replication of the experiment by Gutzke and Paukstis (1983) yielded no effect of
substrate moisture on C. picta sex determination at any incubation temperature (Packard


23
Table 1: ANOVA between years for sex ratios of treatment groups of Leopard Geckos.
Source
df MS
Year
1
0.430
Temperature
2
0.527
Humidity
3
0.015
Year* Temperature
2
0.445
Year Humidity
3
0.067
Temperature Humidity
6
0.266
Year Temperature Humidity 6
0.264
*: P < 0.05


ADDITIONAL FACTORS INFLUENCE TEMPERATURE-DEPENDENT SEX
DETERMINATION IN LEOPARD GECKOS
By
DANIEL E. JANES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2004

I dedicate this work to my parents. They have always encouraged me to follow my own
path and welcomed me back home to recover from my defeats and celebrate my victories.
Also, I dedicate this work to my teachers and students. They have shared their thoughts
and ambitions with me. Every one has shown me a slightly different way to see the
world and every one has allowed me to share my own perspective with them. We should
all be so lucky.

ACKNOWLEDGMENTS
I extend my deepest gratitude to my co-advisors, Drs. Marta L. Wayne and F.
Wayne King, for their support, encouragement, and wisdom. My other committee
members, Drs. Karen Bjomdal, Louis Guillette, and Max Nickerson, have maintained an
open door policy of which I have taken full advantage. Conversations with my advisors
have taught me the value of discussion at all stages of research design and execution and
the necessity of collaboration. I wish to thank all of my friends in the Departments of
Zoology, Wildlife Ecology and Conservation, and the Florida Museum of Natural
History. Their friendship and laughter daily reinforced one of the main reasons I am a
scientist and teacher. It is fun. This research has been funded by the Florida Museum of
Natural History, Sigma Xi, and William and Marcia Brant of The Gourmet Rodent.
Conversations with Ben Bolker helped shape my analyses of data. Data collection
assistance was provided by Jason Phillips, Jennifer Comiskey, Sara Reyes, Jennifer
Mobberley, Margaret OBrien, Eric Timauer, Cassandra Pedrosa, Alana Schoenberg,
Julia Huang, Craig Ajmo, John Bowden, Erin Taylor, and Kristin Barns. Work was
conducted in accordance with University of Florida LACUC protocol Z010.

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
ABSTRACT vi
CHAPTER
1 GENERAL INTRODUCTION 1
2 ABIOTIC EFFECTS ON SEX DETERMINATION IN LEOPARD GECKOS 6
Introduction 6
Methods 10
Animals 10
Sexing Procedure 11
Analysis 12
Results 12
Discussion 13
3 QUANTITATIVE GENETIC VARIATION IN SEX-DETERMINING
RESPONSE TO INCUBATION TEMPERATURE IN LEOPARD GECKOS 25
Introduction 25
Methods 28
Animals 28
Sexing Procedure 29
Analysis 30
Results 30
Discussion 31
4 ESTROGEN AND ESTROGEN MIMIC INCREASE PRODUCTION OF MALES
IN A TEMPERATURE-DEPENDENT SEX-DETERMINING SPECIES 38
Introduction 38
Methods 40
Animals 40
Sexing Procedure 41
Analysis 42
IV

Results 42
Discussion 44
5 GENERAL DISCUSSION 55
LIST OF REFERENCES 61
BIOGRAPHICAL SKETCH 70
v

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ADDITIONAL FACTORS INFLUENCE TEMPERATURE-DEPENDENT SEX
DETERMINATION IN LEOPARD GECKOS
By
Daniel E. Janes
May 2004
Chair: F. Wayne King
Cochair: Marta L. Wayne
Major Department: Zoology
Hatchling sex ratios of reptiles with environmental sex determination (ESD) are
influenced by incubation temperature. Leopard Geckos, Eublepharis macularius, nest in
a region where they are exposed to a wide range of temperatures. If temperature is the
sole determinant of sex in this species, then hatchling sex ratios should be highly
correlated with the thermal gradient in different parts of the species range. In this
scenario, a broad thermal gradient would promote regional variation in hatchling sex
ratios of E. macularius. However, reports of regional variation in hatchling sex ratios are
rare among ESD reptiles. If a secondary factor (or factors) plays a role in sex
determination of this species, their hatchling sex ratios in nature would be expected to
vary seasonally and regionally with less correlation with thermal gradient. In this study,
E. macularius eggs were incubated at three temperatures in two consecutive years. The
vi

range of temperatures studied in these experiments includes the species-specific
temperatures at which the lowest proportion of males (0.0) and the highest proportion of
males (~0.7) are produced per treatment group. Each treatment group consisted of 18 E.
macularius eggs. At each temperature, the effects of humidity, quantitative genetic
variation, and environmental endocrine disrupters on hatchling sex ratios of treatment
groups were measured. Humidity did not significantly affect hatchling sex ratios. Also,
results of the quantitative genetics experiment suggest that ESD in E. macularius is a
genotype x environment interaction. Both natural and synthetic estrogens affected
hatchling sex ratios at both male- and female-producing temperatures. I conclude that
ESD is a multi-dimensional trait in E. macularius. The influences of abiotic factors like
local profile of environmental hormone and hormone mimic concentrations and biotic
factors like quantitative genetic variation may ameliorate the effects of climate change on
ESD. Estimates of extinction rates of ESD species as a result of changing climates
should be reconsidered in light of these data. As other researchers have concluded for
other ESD reptiles, I conclude that the partial genetic control of sex determination in E.
macularius is a polygenic trait and responds to temperature fluctuation as a genotype x
environment interaction. In light of these conclusions, sex-determining mechanisms of
reptiles should be reconsidered as a broad spectrum of multi-dimensional mechanisms
instead of a simple dichotomy of ESD and genetic sex determination.
vii

CHAPTER 1
GENERAL INTRODUCTION
The optimal sex ratio of a population may change depending on fluctuating
environmental stresses and sex-differential vulnerability to environmental stresses.
According to the Trivers-Willard hypothesis, parents may adjust their offspring sex ratio
to address sex-differential fitness and gamer the greatest reproductive advantage (Trivers
and Willard 1973). The Trivers-Willard hypothesis is based on Fishers theory of equal
parental investment in the rearing of sons and daughters (Fisher 1930). If sons and
daughters are unequally affected by environmental stress, then offspring sex ratios should
be skewed toward the sex that is less negatively affected. According to Fisher, the sex
that is most negatively affected by environmental stress will be produced less frequently
and will gamer more parental input. The cost paid by the parents for the rarer, more
energetically expensive offspring sex should balance with the cost they pay for the more
common, less energetically expensive offspring sex. For example, sex-differential
growth rates would cause an immediate increase in parental investment for one sex over
the other. If the immediate investment required by the faster growing offspring can not
be made because of resource scarcity or poor maternal condition, then the slower growing
sex will be produced in excess of the faster growing sex. The slower growing sex can
survive an immediate but short-lived dearth of resources much better than the faster
growing sex. Environmental stresses that differentially affect males and females should
cause sex ratios to deviate from 0.5. The Trivers-Willard hypothesis is most
1

2
appropriately tested using a species with environmental sex determination. This type of
organism will produce offspring sex ratios that can be directly linked to experimentally-
induced environmental conditions without the confounding factor of predominant genetic
control of sex determination.
The gonadal sex of all organisms has been attributed to either genetic sex
determination (GSD) or environmental sex determination (ESD). In GSD, the sex of the
organism depends on the sex chromosome contributions from the organisms parent(s).
In ESD, the sex of the organism depends on the post-fertilization environment
experienced by the organism as a developing embryo. Typically, ESD refers to the
effects of incubation temperature, although Heiligenberg (1965) reported a sex ratio
skewing effect of pH in cichlids. Among vertebrates, the initiation of sex differentiation
is predominantly controlled by incubation temperature in tuatara, some fish, turtles, and
lizards and all crocodilians (Bull 1980; Conover and Kynard 1981; Ewert and Nelson
1991; Janzen and Paukstis 1991). All other vertebrates appear to be genetically sex-
determined. Incubation temperature can also affect body size and growth (Crews et al.
1998; Janes and King, unpublished data), adult sexuality (Gutzke and Crews 1988),
aggressive behavior (Rhen and Crews 1999), and the organization of neural structures
(Coomber et al. 1997). Incubation temperature is known to cause the development of an
embryos testes or ovaries; the proximate mechanism is still unknown.
The recognition of ESD as a derived condition is supported by reports of GSD in
amphibians, a basal taxon to reptiles. No surveyed amphibians have demonstrated a sex
determining effect of incubation temperature within the range of temperatures to which
their eggs are naturally exposed (Hayes 1998). The adaptive significance of ESD has

3
eluded researchers (Janzen and Paukstis 1991). Concensus favors ESD as either an
adaptive mechanism for species in which a fitness advantage oscillates between males
and females (Shine 1999) or as a neutral mechanism that has not been counter-selected
(Bull and Chamov 1989; Girondot and Pieau 1999).
This study will test the effects of environmental stresses on clutch sex ratios of
Leopard Geckos, Eublepharis macularius, a reptile species for which much has been
written on their sex-determining response to incubation temperature. Leopard Geckos
live in a range of climatic heterogeneity. Their range extends from the grasslands of
southeastern Turkey to the forests of southwestern India (Smith 1935). Clutch sex ratios
are male-biased when incubated at 32.5C. Cooler and warmer incubation temperatures
within the full range of viable incubation temperatures (25 to 35C) cause female-biased
clutch sex ratios (Viets et al. 1993). If the ESD mechanism behaves similarly throughout
the climatically heterogeneous zones of their range and all other variables are irrelevant,
then differences in the primary sex ratios of E. macularius can be expected as a result of
different incubation temperatures in different parts of their range (Viets et al. 1993;
Crews et al. 1996; Coomber et al. 1997; Rhen et al. 2000). Also, climate change due to
global warming or other causes might cause dramatic shifts in population sex ratios of E.
macularius and other ESD species.
Based on the observations that (a) among higher vertebrates, the sex of several
species of reptiles and fishes appears to be controlled by incubation temperature, (b)
populations of environmentally sex-determined species can cover ranges that include
more than one climate zone, and (c) sex ratios do not appear to be unbalanced at different
extremes of their range, I hypothesize that the sex-determining mechanism in E.

4
macularius is a multi-dimensional mechanism, controlled in part by incubation
temperature and in part by at least one of the following parameters. Differences in the
timing of incubation (Lance 1989; Guillette et al. 1997), placement and construction of
nests (Magnusson et al. 1985; Shine and Harlow 1996), genetic influences (Parker and
Orzack 1985; Wachtel 1989), presence of industrial contaminants (Guillette et al. 1997;
Crain et al. 1997), or other environmental variables (Webb and Smith 1987) may express
partial control over clutch sex ratios in E. macularius. Other variables may counteract
the sex ratio skewing effects of ambient temperature and maintain population sex ratios
near 0.5 at all points within the range of the species. For this study, chapters 2 and 3 will
provide data on the intrinsic responses of the organism to naturally occurring incubation
conditions. Chapter 4 will introduce an anthropogenic stress (DDT) to a series of E.
macularius eggs in order to test the plasticity of their sex-determining mechanism when
faced with an extrinsic stress that is presently found in their home range.
Laboratory incubations at constant temperatures can provide controlled
experimental data but do not accurately represent natural nest environments. The
experiments in this study have been designed to offer a more realistic, multi-dimensional
representation of the environment experienced by E. macularius eggs during the
incubation period when gonadal sex is determined by varying several factors. This study
will serve to expand the currently accepted definition of ESD. If gonadal sex is
controlled by a multi-dimensional influence of the nest microenvironment, then
researchers can investigate potential advantages of maleness and femaleness within a
more specifically defined milieu. The Trivers-Willard hypothesis may be supported by

5
the sex-determining responses of E. macularius to environmental factors other than
temperature or to interactions of sex-determining factors.
Research Objectives: I tested extrinsic and intrinsic factors in order to determine
which, if any, work in concert with incubation temperature to adjust clutch sex ratios in
E. macularius. I hypothesized a multi-dimensional sex determination model in which
skewing effects from the extremes of sex-determining factor are ameliorated by opposing
extremes of another factor or factors.

CHAPTER 2
ABIOTIC EFFECTS ON SEX DETERMINATION IN LEOPARD GECKOS
Introduction
Animals and plants have evolved numerous sex-determining mechanisms that are
described as either genetic sex determination (GSD) or environmental sex determination
(ESD). In GSD, sex is determined by the genetic constitution of an embryo upon
conception. In ESD, sex is determined by extrinsic, abiotic factors that define the
incubation environment. Regardless of mechanism, Fisher (1930) predicted that primary
sex ratios should represent an equal parental investment in male and female offspring.
However, Fishers balanced parental investment may not be represented in ESD species
because primary sex ratios may be removed from parental control.
Among squamate reptiles, ESD and GSD are found among closely related
species, suggesting that ESD and GSD have evolved separately on many occasions (Viets
et al. 1994). Within the squamate subfamily Eublepharinae, the Leopard Gecko,
Eublepharis macularius, and the African Fat-Tail Gecko, Hemitheconyx caudicinctus, are
ESD species and the Banded Gecko, Coleonyx mitratus, is a GSD species (Bragg et al.
2000). Environmental sex determination in squamates refers to temperature-dependent
sex determination (TSD) specifically, the influence of incubation temperature on sex
determination. Other forms of ESD based on photoperiod or nutritional resource
availability have been reported in invertebrates but only TSD has thus far been reported
in squamates (Bull 1980). Genetic variation in sex-determining responses to incubation
6

7
temperature is seen among and within ESD species like those in Eublepharinae (Elphick
and Shine 1999). Thus, the same constant incubation temperature may produce different
sex ratios in different populations of the same species.
Most studies report female-biased populations of reptiles exhibiting TSD,
regardless of local nest site temperatures (Bull and Chamov 1989; Ewert and Nelson
1991). In light of this, it appears that the relationship between hatchling sex ratios and
the incubation environment has not been adequately defined. Other extrinsic or intrinsic
variables can play major or minor roles in the maintenance of female-biased hatchling
sex ratios. Aside from temperature, several factors have been implicated in the
relationship between the sex determination of an embryo and its immediate environment.
Exogenous sex steroid hormones or endocrine active contaminants can alter sex
determination. Further, the behavioral and physiological condition of the mother as well
as the environments encountered through the life of the mother, and age and sociosexual
experience of either parent could influence sex differentiation of ESD species (Crews
2003). Temperature has been identified as a determinant of sex determination in all
squamates studied to date that exhibit ESD. Carbon dioxide concentrations and pH
(Etchberger et al. 2002) and humidity (Gutzke and Paukstis 1983) also have been
identified as having a secondary role in some reptilian species. Here, I consider humidity
as a major factor in sex determination of E. macularius.
Eublepharis macularius has a natural range that includes desert and grasslands of
the Middle East (Daniel 2002). In this range, nest site temperatures and humidities
fluctuate dramatically among seasons, years, and regions. Eublepharis macularius

8
follow a pattern of sex determination in which an increased proportion of males is
produced at an intermediate incubation temperature (31-33C) and an increased
proportion of females is produced at cooler (26-28C) and warmer (34-35C) incubation
temperatures (Viets et al. 1993; Crews 2003).
If E. macularius maintains an evolutionarily stable sex ratio across thermally
variant environments, the effects of differing ambient temperature must be mediated
either by local adaptation (Mrosovsky 1988; Blackmore and Chamov 1989; Viets et al.
1993), carbon dioxide, pH (Etchberger et al. 2002), maternal affects (Bowden et al.
2002), humidity, or a complex interaction of some or all of these factors. Environmental
sex determination could be caused by a wide array of weak influences during incubation
(Bull et al. 1982a; Bull et al. 1982b). In addition, sex-determining responses to these
influences could be phenotypically plastic, as is widespread in reptilian phenotypes
(Shine and Elphick 2001). Population sex ratios of E. macularius can be further altered
by short-term weather fluctuations because of the extreme sensitivity of reptiles to
environmental variables during embryogenesis (Shine and Elphick 2001).
The influence of humidity on sex determination has been debated in the past.
Gutzke and Paukstis (1983) reported that in the Painted Turtle, Chrysemyspicta, more
males were produced in wet substrates than in dry substrates at typically male-producing
temperatures. In a second study, C. picta eggs were again incubated on wet or dry
substrates (Paukstis et al. 1984). In the latter study, the reverse pattern was reported.
More male turtle hatchlings were produced in dry substrates than in wet substrates. Also,
a replication of the experiment by Gutzke and Paukstis (1983) yielded no effect of
substrate moisture on C. picta sex determination at any incubation temperature (Packard

9
et al. 1989). Packards advice to consider the effects of substrate moisture cautiously has
been heeded by subsequent researchers (Packard et al. 1989; Janzen and Moijan 2001).
Typically, substrate moisture is not considered a potential sex determinant in ESD
studies. In previous experiments, humidity of incubation substrate did not affect sex
determination in E. macularius (B. Viets, personal communication). Lack of an effect of
humidity on ESD has also been reported in the Flatback Turtle, Natator depressus
(Hewavisenthi and Parmenter 2000).
Different squamate species have different patterns of water exchange between the
egg and the environment and, therefore, different sensitivities to environmental moisture
(Ji and Du 2001). If humidity has a partial effect on sex determination in E. macularius,
its effect in nature could be significant because of the paucity of moisture in the species
natural range. This species inhabits and builds nests in arid country (Daniel 2002). This
habitat would pose a significant challenge to a sex-determining mechanism that is
influenced by humidity because the maintenance of a humid nest in an arid habitat is a
greater challenge than the maintenance of a dry nest in a humid habitat. Also, humidity
may have a more significant effect in the development of more pliable-shelled eggs like
those of E. macularius (Ji and Du 2001). The influence of humidity on sex determination
could be direct by changing the response of the male- determining factor (Deeming and
Ferguson 1989) or indirect by influencing nest temperatures, affecting body size as in C.
picta and N. depressus (Cagle et al. 1993; Hewavisenthi and Parmenter 2001), or by
influencing the conversion of yolk into fat as in the Cuban Rock Iguana, Cyclura nubila
(Christian and Lawrence 1991).

10
In this experiment, E. macularius eggs were treated with substrates of varied
water content and incubated at three temperatures within the species range of embryonic
thermal tolerance. If humidity has an effect on this species sex determination, then
different sex ratios in response to different humidities should be detected most easily at
the intermediate incubation temperature. The influence of a weaker determinant of sex is
more likely to be detected at an intermediate incubation temperature, because the
skewing influence of temperature on the sex ratio is minimized (Bull et al. 1982a).
However, extreme temperatures may also interact uniquely with humidity. For these
reasons, extreme and intermediate incubation temperatures are studied in this project.
Methods
Animals
On 22 March 2002, Leopard Gecko, Eublepharis macularius, eggs were obtained
from The Gourmet Rodent, a reptile breeder in Archer, Florida. Eggs were collected
within 24 hrs after oviposition from a breeding colony of -5000 dams. All eggs were
candled to test viability. Two hundred and sixteen viable E. macularius eggs were placed
in a plastic box filled with vermiculite and transported by car to the University of Florida.
They were placed in containers that consisted of six 188 ml plastic cups banded together
in rings. Each cup contained 70 ml perlite and tap water and was sealed with a tight
fitting lid punctured with one gas-exchange hole. The eggs were divided among 36 egg
containers. Each container held six eggs. Each egg was placed individually within one
of the six cups in a container. The egg containers were divided into three groups of 12.
Each group was placed in an environmental chamber and maintained at 26, 30, or 32.5
C for the duration of the experiment. Chamber temperatures were recorded every minute

11
throughout the experiment with Hobo temperature loggers. Within each chamber, the
12 egg containers were divided into four treatment groups of three containers. Each
treatment group, consisting of 18 eggs, was exposed to 20%, 25%, 30%, or 35%
humidity. At the beginning of the experiment, humidity was varied by adding 14 ml,
17.5 ml, 21 ml, or 24.5 ml of water to the perlite in the egg cups in the 20%, 25%, 30%,
or 35% humidity treatment groups, respectively. Every day, the incubation chambers
were opened and the egg containers were removed. Each cup was opened momentarily
in order to release metabolic gas waste and check for hatchlings. Eggs that grew mold
were discarded upon discovery. Position of egg containers within an incubator was
randomized daily.
Sexing Procedure
Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2-
bromo-2-chloro-l,l,l-trifluoroethane). Geckos were fixed in Bouins fixative and
preserved in 75% ethanol. The reproductive organs were removed from each gecko and
prepared for analysis by light microscopy. The reproductive organs are opaque white,
cylindrical structures on either side of the posterior end of the dorsal artery. After
removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of
ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55;
Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The
resulting paraffin blocks were sectioned at 8 pm and stained with a modified trichrome of
Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from
all geckos. If seminiferous tubules were identified within the tissue sections, the gecko
was scored as male (Figure 2a). If seminiferous tubules were absent, the gecko was

12
scored as female (Figure 2b). Seminiferous tubules were identified as clusters of simple,
circular structures with a narrow lumen (Berman 2003). By relying on histological
examination of our specimens, I avoided potential macroscopic misidentification of male
and female reproductive organs.
The design was replicated the following year with a new sample of 216 i?.
macularius eggs from a separately maintained breeding colony at the Gourmet Rodent.
Thus, the sires and dams used in the first years experiment were not the sires and dams
used in the second years replication. Work was conducted in accordance with
University of Florida IACUC protocol Z010.
Analysis
Sex ratio data were analyzed by ANOVA with fixed main effects of humidity,
incubation temperature, and year, as well as their interactions. Egg containers were
nested within year X temperature X humidity, and treated as a random effect. Data were
analyzed using SAS version 6.10 for the Macintosh. ANOVAs were performed using
PROC GLM.
Results
Temperature and humidity were manipulated simultaneously to assess effects on
ESD over the two years. Variances between years were not significantly different (Table
1). Years were analyzed together or separately. Overall, temperature significantly
affected hatchling sex ratios as expected for a species exhibiting TSD (MS = 0.527;
P<0.05). Humidity did not affect sex ratios when both years were examined together or
individually (MS = 0.015; Table 1). In the first year of experimentation, neither
temperature nor humidity, examined independently, altered hatchling sex ratios (Figures

13
3 and 4). In the second year, with the exception of the treatment group exposed to 20%
humidity, the treatment groups incubated at the coolest temperature (26C) had a lower
proportion of males than treatment groups incubated at both warmer temperatures.
Temperature significantly affected hatchling sex ratios in year 2 (MS = 0.713; P<0.05;
Table 2). The highest proportion of males per treatment group was produced at the
warmest incubation temperature (32.5C; Figures 5 and 6). The order of male proportion
per treatment group at 30C in the second year does not follow the order of male
proportion per treatment group at 30C in the first year.
Discussion
I have found, using my design, that 1) temperature affected sex ratios, 2) humidity
did not affect sex ratios, and 3) humidity and temperature did not interactively affect sex
ratios. Previous studies examining the effect of humidity on sex determination in TSD
species have produced inconsistent results. Gutzke and Paukstis (1983) found an effect
of substrate moisture on sex differentiation in the freshwater turtle, Chrysemys picta,
whereas Packard et al. (1989) did not. Clearly, the mechanism and the adaptiveness of
the relationship between incubation environments and hatchling sex ratios have not been
sufficiently characterized. Deeming and Ferguson (1989) refer to a male-determining
factor that is temperature-sensitive. Candidate mechanisms for TSD include temperature-
dependent synthesis or activity of enzymes, heat shock proteins, and temperature-
sensitive gene expression (Mrosovsky 1994). Some intrinsic mechanism(s) responds to
the microclimate of the nest site and determines hatchling sex. Eublepharids select nest-
site microclimates that result in higher hatchling survivorship and not necessarily
evolutionarily stable sex ratios (Bull et al. 1988a; Bragg et al. 2000). However, if

14
humidity affected both survivorship and to a lesser extent sex determination, then nest
site selection could influence population sex ratios. Cultural inheritance of nest site
microclimates has also been implicated in the inheritance of hatchling sex ratios
(Freedberg and Wade 2001). Nest sites tend to retain their abiotic profiles from year to
year. If daughters inherit nest sites from their mothers, then a cultural component exists
for the inheritance of hatchling sex ratios in ESD species. However, if the nest sites are
found in areas that are thermally different from one breeding season to the next, then
cultural inheritance will not fix sex ratios over long periods of time. The microevolution
of sex ratio depends in part on maternal choice of thermal qualities of nest sites (Janzen
and Moijan 2001).
My results from chapters 3 and 4 suggest that hatchling sex ratios can vary among
populations even if incubation temperature remains constant. However, the results of this
experiment and previously published conclusions (Janzen and Moijan 2001) suggest that
hatchling sex ratios of E. macularius do not vary in relation to nest site humidity. The
effect of humidity on hatchling sex ratios in this study was not significantly different
between two years of experimentation. In short, sex determination in E. macularius did
not respond to humidity, at least as examined by the experimental design I performed.
Researchers should note that humidity was not measured throughout this experiment.
After initial volumes of water were added at the beginning of trials, humidities may have
varied incidentally among the treatment groups. Incidental variation may explain the
different effects of temperature on hatchling sex ratios between the first and second years
of experimentation. However, incidental variation in temperature is less likely because of
constant monitoring of temperature throughout the trials. The lack of effect of

15
temperature on hatchling sex ratios in the first year may also be explained by genetic
differences between the colonies of E. macularius used in the two years of
experimentation. The breeding facility from which eggs were collected maintains
separate breeding colonies that do not interbreed. Drift or local adaptation of the sex
determining response to incubation temperature may explain the difference between the
results from the first and second years of experimentation. This result would suggest
intraspecific variation in the effect of abiotic factors on sex determination in E.
macularius. Such intraspecific variation of sex-determining mechanisms has only been
reported anecdotally for Tokay Geckos, Gekko gecko (F. Janzen, pers. comm.).
However, intraspecific variation in the sensitivity of temperature-dependent sex
determining mechanisms has been reported for A. mississippiensis (L. Guillette, pers.
comm.). In short, the factors that influence ESD within species are not expected to vary
but the degree to which fixed factors affect ESD is expected to vary.
A multifactorial sex-determining mechanism would have an impact on
conservation considerations for ESD species. Vogt and Bull (1984) present empirical
evidence that vegetation changes influence hatchling sex ratio in Map turtles. Invasive
plants are changing vegetation cover over Nile crocodile nesting sites (Leslie and Spotila
2001). The consequent effects on Nile crocodile sex ratios have not yet been reported.
Reptile populations with ESD may need to be manipulated if nest site microclimates
change too dramatically (Mrosovsky 1994). In the worst case scenario, extinction may
result from climate change (Janzen 1994a). If the influence of incubation temperature on
hatchling sex ratios is exacerbated or ameliorated by another weaker sex determinant,
risk assessments in the face of habitat alteration must be reconsidered.

16
Environmental sex determination could enhance maternal fitness by permitting
the production of more offspring of the sex that is best-suited to the incubation
environment (Chamov and Bull 1977). Chrysemys picta eggs incubated in wet substrate
maintained a higher temperature than eggs in dry substrate but only for the first third of
incubation. After the first third, the eggs in drier substrates maintained a higher
temperature than those in wet substrates (Gutzke et al. 1987). The temperature-sensitive
period for E. macularius in which sex determination is affected by local abiotic factors
does not begin until the beginning of the middle third of incubation (Bull 1987).
Therefore, we would expect drier treatment groups to be warmer during the temperature-
sensitive period and produce more males. This pattern, however, was not observed in
this study. The enhanced sensitivity of offspring sex ratio to secondary abiotic factors
should be investigated in a wider array of environmentally sex-determined species
because of the current lack of detail about the evolution of sex-determining mechanisms.
If some species respond similarly to a series of environmental variables, then a new
dimension of the mechanism could be considered to characterize the divergence of ESD
mechanisms among squamates.

Proportion of Males / Treatment Group
17
24 26 28 30 32
Temperature (C)
Figure 1. Effect of incubation temperature on hatchling sex ratios of Leopard Geckos.
Control sex ratio data were collected from treatment groups exposed to either
no treatment or a 95% EtOH vehicle from the second year of experimentation
described in Chapter 4.

18
Figure 2. Light microscopy images of reproductive tissue from Leopard Geckos,
Eublepharis macularius. These images are magnified 200X. (a) Male
reproductive organs have seminiferous tubules that have a thick, simple
cortex and a narrow lumen, (b) Female reproductive organs lack
seminiferous tubules.

Proportion of Males / Treatment Group
19
24 26 28 30 32
Temperature (C)
Figure 3. Sex ratios of treatment groups of Leopard Geckos in the first of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the solid, dashed, gray stippled, and black
stippled lines respectively. Bars represent standard errors.

Proportion of Males / Treatment Group
20
0.8 -i
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
30 32.5
Temperature (C)
Figure 4. Sex ratios of treatment groups of Leopard Geckos in the first of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the lightest to darkest bars respectively.
Standard errors are indicated by vertical lines.

Proportion of Males / Treatment Group
21
24 26 28 30 32
Temperature (C)
Figure 5: Sex ratios of treatment groups of Leopard Geckos in the second of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the solid, dashed, gray stippled, and black
stippled lines respectively. Bars represent standard error.

Proportion of Males / Treatment Group
22
0.8 -i
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
0.2 -
26
Temperature (C)
32.5
Figure 6. Sex ratios of treatment groups of Leopard Geckos in the second of two years
of experimental incubations. The 20%, 25%, 30%, and 35% humidity
treatment groups are indicated by the lightest to darkest bars respectively.
Standard errors are indicated by vertical lines.

23
Table 1: ANOVA between years for sex ratios of treatment groups of Leopard Geckos.
Source
df MS
Year
1
0.430
Temperature
2
0.527
Humidity
3
0.015
Year* Temperature
2
0.445
Year Humidity
3
0.067
Temperature Humidity
6
0.266
Year Temperature Humidity 6
0.264
*: P < 0.05

24
Table 2. ANOVA within years for sex ratios of treatment groups of Leopard Geckos.
MS
Source
df
Year 1
Year 2
Temperature
2
0.205
0.713*
Humidity
3
0.020
0.058
Temperature *
Humidity 6
0.214
0.352
*: P < 0.05

CHAPTER 3
QUANTITATIVE GENETIC VARIATION IN SEX-DETERMINING RESPONSE TO
INCUBATION TEMPERATURE IN LEOPARD GECKOS
Introduction
The diversity of sex-determining mechanisms in vertebrates can be classified as
either genotypic sex determination (GSD) or environmental sex determination (ESD). In
this chapter, I will describe quantitative genetic effects on ESD in Leopard Geckos,
Eublepharis macularius. Species that follow a GSD pattern are further subdivided into
either $:XX/':XY systems in which males are heterogametic (as seen in mammals) or
$:ZW/(J:ZZ systems in which females are heterogametic (as seen in birds). Reptiles
show a far greater diversity of sex-determining mechanisms than either mammals or birds
(Janzen and Paukstis 1991). Reptiles express either XX/XY or ZZ/ZW patterns of GSD
or one of several patterns of ESD in which the sex of offspring is predominantly, if not
completely, determined by the environment in which offspring develop as embryos.
Great controversy exists about the adaptive significance of different sex
determining mechanisms. Researchers ability to predict which GSD or ESD pattern will
be followed by a newly discovered species is poor, regardless of what information can be
gathered about the species natural history, phylogeny, or ecology. Although much has
been published about advantages of either ESD or GSD, little consensus can be found
concerning the differences that would make one mechanism adaptive for one species and
another mechanism adaptive for a different species. The adaptive significance of GSD
25

26
has been summarily attributed to its maintenance of balanced sex ratios in natural
populations (Fisher 1930). Because the rarer sex gains a fitness advantage over the more
common sex, population sex ratios tend to hover near unity (Fisher 1930). The
advantages of ESD are more opaque. Hypotheses regarding the advantages of ESD have
been organized in five categories: (a) phylogenetic inertia, (b) group-adaptation, (c)
inbreeding avoidance, (d) sex-differential fitness, and (e) quasi-neutrality (Shine 1999;
Girondot and Pieau 1999). The enigma is further compounded by the existence of
closely-related taxa that express very different sex-determining mechanisms. Although
the predominant determinants of sex can be readily identified through experimental
matings and incubations, the finer points of the relationship between genes and
environment in the process of sex determination have not yet been acceptably clarified in
many species. The identified dichotomy of ESD and GSD may cause weaker
determinants of sex to be overlooked which, if discovered, would more appropriately
describe the process by which a bipotential gonad becomes an ovary or a testis.
Additionally, an adaptation that is completely environmentally determined could
be invisible to natural selection. In order to describe the evolutionary history of sex
determination in vertebrates, researchers must first test hypotheses about the effects of
selection on sex-determining mechanisms. Selection may work on the genetic
architecture that allows an effect of temperature or other environmental factor on sex
determination. Temperature affects the activation of genes that encode steroidogenic
enzymes (Crews 2003). Genetic variation most likely affects the sensitivity of genes to
thermal stimuli. Models derived for the adaptive evolution of sex ratio assume that
genetic variation exists for primary sex ratio and that the variation is caused by genes of

27
modest effect that segregate according to Mendelian rules (Janzen 1992). These models
are supported by little empirical evidence because of the difficulty of measuring primary
sex ratios and the interference of the predominant effects of sex chromosomes in GSD
species. The heritability of offspring sex ratio incubated under constant temperature has
been reported in the temperature-dependent sex-determining (TSD) Ouachita Map turtle,
Graptemys ouachitensis (Bull et al. 1982a). Bull et al. (1982a) incubated G. ouachitensis
eggs from different families at a constant temperature, 29.2C. Their threshold model for
ESD yielded a heritability estimate of 0.82 for the hypothetical sex-determining
character. They concluded that natural variation in nest temperatures acted against the
high heritability of the sex-determining character to maintain natural populations closer
to a balanced sex ratio. Also, Janzen found strong genetic variation in the TSD response
of the Common Snapping turtle, Chelydra serpetina, to incubation temperature.
However, no significant interaction of temperature and family has been reported for C.
serpentina. Therefore, Janzen (1992) concluded that variation in the sex-determining
response to incubation temperature in the TSD species C. serpentina is not due to a gene
x environment interaction.
To test for a gene x environment interaction in the sex-determining mechanism of
E. macularius, within a broad range of incubation temperatures, I incubated E.
macularius eggs at three different temperatures throughout the range of thermal tolerance
for the embryos of this species. I followed a half-sib design to record both paternal and
maternal effects on TSD response of progeny. Eublepharis macularius are excellent
organisms for this study because captive-bred adult virgin females and viable males are
available near the University of Florida where this research was conducted. Also, the

28
physiology and ecology of their TSD mechanism have been broadly researched in the
recent past (Crews et al. 1996; Rhen et al. 1999; Rhen et al. 2000; Bragg et al. 2000).
Methods
Animals
On 5 January 2002, a group of five male and twenty five female adult Leopard
Geckos, Eublepharis macularius, were selected from a colony of > 50,000 at The
Gourmet Rodent, a reptile breeding facility in Archer, Florida. The five males were
selected based on their previously demonstrated viability and the variation of color
patterns on their dorsa. Dorsal coloration is a decent indicator of geographic origin in E.
macularius (K. Auffenberg, pers. comm.). The five males were selected because they
represented a diversity of regional origins from which the facilitys breeding colony is
drawn. The 25 females were selected because they were virgin and were considered
likely to be viable because of their body sizes. Each male was mated to five females.
Each female was housed alone in a cage containing a food bowl, a water bowl, and a
nestbox filled with moist vermiculite. Males were mated to females by moving them to a
different females container every day. Males were rotated among their five mates every
24 hours and isolated for 48 hours between rotations. Between 5 January and 15 May,
2002, nestboxes were checked daily. If new eggs were found during daily nestbox
inspections, they were placed in a plastic box filled with vermiculite and transported by
car to the University of Florida. In the laboratory, the eggs were placed in containers that
consisted of six 188 ml plastic cups banded together in rings. Each cup contained 70 ml
perlite and 17.5 ml tap water and was sealed with a tight-fitting lid punctured with one
gas-exchange hole. The eggs were placed randomly in containers. Each container held

29
six eggs. Each egg was placed individually within one of the six cups in a container. The
egg containers were placed randomly in one of three environmental chambers and
maintained at either 26, 30, or 32.5 C for the duration of the experiment. The sire,
dam, egg container, and egg cup were recorded for each egg. Chamber temperatures
were recorded every minute throughout the experiment with Hobo temperature loggers.
Every day, the environmental chambers were opened and the egg containers were
removed. Each cup was opened momentarily in order to release metabolic gas waste and
check for hatchlings. Eggs that grew mold were discarded upon discovery. Position of
egg containers within an environmental chamber was randomized daily.
Sexing procedure
Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2-
bromo-2-chloro-l,l,l-trifluoroethane). Geckos were fixed in Bouins fixative and
preserved in 75% ethanol. The reproductive organs were removed from each gecko and
prepared for analysis by light microscopy. The reproductive organs are opaque white,
cylindrical structures on either side of the posterior end of the dorsal artery. After
removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of
ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55;
Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The
resulting paraffin blocks were sectioned at 8 pm and stained with a modified trichrome of
Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from
each gecko. If seminiferous tubules were identified within the tissue sections, the gecko
was scored as male (Figure 2a). If seminiferous tubules were absent, the gecko was
scored as female (Figure 2b). Seminiferous tubules were identified as clusters of simple,

30
circular structures with a narrow lumen (Berman 2003). By relying on histological
examination of our specimens, I avoided potential macroscopic misidentification of male
and female reproductive organs.
The experiment was replicated the following year (16 February-5 June, 2003)
with a new group of 5 male and twenty five female adult E. macularius from a separately
maintained breeding colony at the Gourmet Rodent. Thus, the sires and dams used in the
first years experiment were not the sires and dams used in the second years replication.
Analysis
Sex ratio data were analyzed by ANOVA with fixed main effects of sire, dam,
incubation temperature, and year, as well as their interactions. Egg containers were
nested randomly within year X temperature. Data were analyzed using SAS version 6.10
for the Macintosh. ANOVAs were performed using PROC GLM.
Results
As a consequence of allowing this experiments eggs to develop without variation
in any abiotic factors but temperature, the results from this study clearly represent the
typical effect of incubation temperature on hatchling sex ratios. Proportion of males per
treatment group increase with the temperature at which the group was incubated (MS =
2.361; P = 0.001; Table 3). The effect of temperature on sex ratio did not differ between
the two years of experimentation. The sex ratios per sire did not differ significantly
between years or among temperatures. The same is true for the sex ratios per dam.
However, the offspring sex ratios of couplings of sire and dam differed significantly
across incubation temperatures (MS = 0.258; P<0.05; Table 3), indicating a gene x
environment interaction. The variation in the hatchling sex ratios per coupling of sire and

31
dam is most apparent at higher incubation temperatures. At each temperature, hatchling
sex ratios vary between 100% female and 100% male offspring sex ratios resulting from
multiple clutches produced by the same sire and dam. Less variation is observed at the
female-producing incubation temperature (26C) than at the two warmer temperatures
(30 and 32.5C; Figures 7 and 8).
Discussion
Offspring sex ratios of temperature-dependent sex-determining (TSD) species are
highly correlated with mean air temperature during the period in which most clutches are
in the middle third of the incubation period; the thermosensitive period (Janzen 1994).
As a consequence of this correlation, TSD species are considered vulnerable to climate
change (Janzen 1994; Girondot et al. 1998; Leslie and Spotila 2001). Thomas et al.
(2004) estimated that climate change will commit 15%-37% of species in their studys
sampled regions of the Earth to extinction by 2050. A common hypothesis for the link
between climate change and extinction is the incompatibility of TSD to warming
climates. Indeed, if dinosaurs expressed TSD, catastrophic climate change could have
altered adult sex ratios and made successful mating less common, thus leading to
eventual extinction (Paladino et al. 1989). This hypothesis for dinosaur extinction and
hypotheses for the effects of current climate change on TSD species stand on the assumed
inability of sex ratios of TSD species to adapt with sufficient speed to a rapid and/or
drastic temperature shift. For example, if mean July temperature in the central United
States rises 4C in the next 100 years as predicted (Manabe and Stouffer 1993) and the
sex-determining response of TSD species to ambient temperature is inflexible, the

32
Painted Turtle, Chrysemys picta will become extinct because of an inability to produce
males (Janzen 1994).
If Leopard Geckos, E. macularius, or any other TSD species have a chance to
persist in spite of rapid climate change, their continued survival will be attributable to the
adaptability of their sex-determining mechanism or their behavioral placement of nests.
In order to adapt, the mechanism must have an underlying genetic component. An
interaction of temperature and genotype would allow natural populations of TSD species
to change the threshold temperatures at which embryos become either male or female.
Variation in sex-determining response must vary among individuals and/or among
populations. This variation must be caused by variation in genotype. An interaction of
temperature and genotype has been reported in an atherinid fish, the Atlantic Silverside,
Menidia menidia (Conover and Kynard 1981). In M. menidia, temperature influences
offspring sex ratios but the sex-determining mechanisms of progeny from different
females respond differently to incubation temperature. Variation in the sex-determining
response of offspring from different dams is not attributable to sire because Conover and
Kynard used one sire to fertilize all dams in their study. Difference in sex ratio was not
attributable to maternal size, clutch size, or level of natural mortality. Conover and
Kynard (1981) interpret these results as polygenic sex determination.
In reptiles, evidence of gene x environment interaction for TSD is lacking. If
heritable variation in sex-determining response to incubation temperature exists for TSD
species, then local adaptation should be evident. Bull et al. (1982a) tested for local
adaptation of threshold incubation temperatures that initiate male or female sex
differentiation in six species of turtles of the subfamily Emydinae, genera Graptemys,

33
Pseudemys, and Chrysemys from northern U.S. populations (Wisconsin) and southern
U.S. populations (Alabama, Mississippi, and Tennessee). No significant differences
between species-specific threshold temperatures were reported for any of their six study
species between northern and southern populations. Bull et al. (1982a) concluded that
TSD species maintain balanced sex ratios across thermally heterogeneous environments
by varying nest construction or timing of oviposition. However, Bull et al. (1982b)
calculated a heritability of 0.82 for map turtles. Like Conover and Kynard (1981), Bull et
al. (1982b) concluded that TSD is a form of polygenic sex determination. Bull et al.
(1982a and 1982b) demonstrated a heritable effect of sex determination among families
of map turtles but did not demonstrate local adaptation of sex-determining threshold
temperatures between populations of map turtles from different latitudes and climates.
Great variation among the offspring sex ratios of different dams of E. macularius
was seen at 30 and 32.5C. Less variation in offspring sex ratios was seen at the female-
producing temperature, 26C. This fits well with Deeming and Fergusons (1989)
hypothesized male-determining factor that responds directly to thermal cues. My results
suggest a gene x environment interaction (Falconer and Mackay 1996). If sex
determination in E. macularius is controlled by one gene or a small group of genes, then
those genes behave differently in different environments, suggesting that one genotype is
favorable in some environments but not in others. Such variation in male-producing
response to incubation temperature would allow TSD populations to withstand climate
change temporally if not geographically. A broad range of incubation temperatures could
conceivably result in mixed sex ratios because of variation among dams in sex
determining response to temperature. Transplant experiments should follow in which egg

34
clutches of a TSD species from one latitude or climate would be transplanted to an
alternate latitude or climate. If a transplanted TSD clutch produces an offspring sex ratio
like the population of its origin, then researchers could conclude an effect of genotype on
TSD that can be affected by natural selection. If a transplanted TSD clutch produces an
offspring sex ratio like the population it has been transplanted to, then researchers could
conclude that the heritable component of TSD is too easily overridden to be shaped by
natural selection. Although inspired by the conclusions of this study, subsequent
experiments would not be most effectively performed with E. macularius. Clutch sizes
are small (< 2 eggs), and natural populations are remote and small. Transplant
experiments to test the repeatability of TSD response would be performed most
appropriately with large clutches from crocodilians or certain turtles with larger clutch
sizes.

Proportion of Males / Dam
35
Temperature (C)
Figure 7. Offspring sex ratios of Leopard Geckos incubated at one of three
temperatures during one of two years of experimentation. Each line
represents the offspring sex ratio of one of 35 dams used in this study. Many
lines are not visible because they are super-imposed on each other in this
figure. Incubated at the same temperature, different dams produced different
offspring sex ratios. The ranks of the genotypes of different dams changed
depending on the environment, indicating a gene x environment interaction.

Temperature (C)
36
Sye
A
B
c
D
E
F
G
H
I
J
Dams
5
5
5
5
5
5
5
5
5
26
12
3
3
4
11
5
7
6
4
9
30
8
8
8
6
4
8
8
6
10
12
32.5
7
6
6
9
5
11
7
7
6
11
27
17
17
19
20
24
22
19
20
32
Figure 8. Sample sizes of offspring groups of leopard geckos from
individual dams. Each letter represents a different sire. Each
sire was mated to five dams. The offspring of each sire/dam
pairing were divided randomly among three temperature
treatments. Samples sizes differed because of differential
reproduction among the sire/dam pairs.

37
Table 3. ANOVA across years for offspring sex ratios of Leopard Geckos.
Source df MS
Temperature 2 2.361***
Dam (Sire) 34 0.190
Temperature Sire Dam 46 0.258*
*: P < 0.05; ***: P = 0.001

CHAPTER 4
ESTROGEN INCREASES PRODUCTION OF MALES IN A TEMPERATURE-
DEPENDENT SEX-DETERMINING REPTILE
Introduction
Temperature-dependent sex-determining (TSD) species produce hatchling sex
ratios that are shaped by the temperature at which the embryonic clutch was exposed
during a thermosensitive period of incubation. For example, Leopard Geckos,
Eublepharis macularius, follow a pattern of sex determination in which an increased
proportion of males is produced at an intermediate incubation temperature (31-33C)
whereas an increased proportion of females is produced at cooler (26-28C) and warmer
(34-35C) incubation temperatures (Viets et al. 1993; Crews 2003). The masculinizing
influence of male-determining incubation temperature can be experimentally disrupted by
application of estrogens and estrogen-mimicking compounds (Bull et al. 1988b;
Tousignant and Crews 1994). The application of estrogenic compounds to eggs of other
temperature-dependent sex-determining species, such as freshwater turtles and
crocodilians, also affects sex determination, sex differentiation, organizational and
activational development, and sex-specific behavior in resulting hatchlings (Jeyasuria et
al. 1994; Sheehan et al. 1999; Willingham et al. 2000). Typically, this disruption takes
the form of sex-reversal of males (an override of TSD) or feminization of male
reproductive tissues (Fry and Toone 1981; Belaid et al. 2001). However, these
purportedly paradigmatic conclusions may only describe one half of an inverted U-
38

39
shaped distribution of the effects of exogenous estrogenic compounds. The influence of
exogenous estrogenic compounds on female reproductive tissues (or reproductive tissues
developing at female-producing incubation temperatures) has not been as substantially
reported as the influence of those compounds on male reproductive tissues (or
reproductive tissues developing at male-producing incubation temperatures). Some
endocrine disrupting contaminants (EDCs) have opposing effects on exposed
reproductive tissues at high and low concentrations (Parmigiani et al. 2000). For
example, prenatal exposure to small amounts of exogenous estradiol or diethystilbesterol
(DES) will increase prostate size in neonatal mice. Prenatal exposure to larger amounts
of the two compounds will decrease prostate size in neonatal mice (vom Saal et al. 1997).
A growing literature suggests that a wide range of species, including reptiles, are
exposed to various environmental contaminants having endocrine disruptive activities
(for reviews, see Crain & Guillette, 1998; Guillette & Iguchi, 2003; Tyler et al., 1998).
As with the research on mammals, the majority of these studies in reptiles have focused
on estrogenic or anti-estrogenic actions. Several studies have documented effects on sex
determination in turtles and alligators (Matter et al., 1998; Willingham & Crews, 1999),
whereas others have shown no effect on primary sex determination from the chemical(s)
tested (Podreka et al., 1998; Portelli et al., 1999). Several studies have reported that
various chemicals can bind to a reptilian estrogen receptor (Guillette et al., 2002; Sumida
et al., 2001; Vonier et al., 1996) including that from a lizard (Matthews & Zacharewski,
2000). No study to date has examined whether environmental estrogens, such as the
pesticide DDT, affect sex determination in a squamate, such as the commonly studied
lizard, the Leopard Gecko, Eublepharis macularius.

40
Methods
Animals
Experiment 1
On 26 April 2002, Leopard gecko (E. macularius) eggs were obtained from The
Gourmet Rodent, a reptile breeder in Archer, Florida. Eggs were collected < 24 hrs after
oviposition from a breeding colony of-5000 dams. All eggs were candled to test
viability. Two hundred and thirty four viable E. macularius eggs were placed in a box
filled with vermiculite and transported by car to the University of Florida. They were
placed in containers that consisted of six 188 ml cups banded together in rings. Each cup
contained 70 ml perlite and 17.5 ml tap water and was sealed with a tight-fitting lid
punctured with one gas-release hole. The eggs were divided among 39 egg containers.
Each container held six eggs. Each egg was placed individually within one of the six
cups in a container. The egg containers were divided into three groups of 13. Each
group was placed in an environmental chamber and maintained at 26, 30, or 32.5 C for
the duration of the experiment. In control treatments, these temperatures are predicted to
produce 100% females at 26C, a nearly 1:1 sex ratio at 30C and 70% males at 32.5C.
Chamber temperatures were recorded every minute throughout the experiment with
Hobo temperature loggers. Within each chamber, the 13 egg containers were divided
into five treatment groups. Each treatment group was exposed to (a) 5pl 95% EtOH, the
vehicle (12 eggs in containers), (b) 0.014 ppm o,p-DDT (18 eggs in containers), (c) 0.14
ppm o,p-DDT (18 eggs in containers), (d) 1.4 ppm o,p-DDT (18 eggs in containers), or
(e) 10 pg/5 pi estradiol benzoate (2 egg containers). DDT was purchased from Chem
Service (lot PS-698). The chemicals of interest in treatments b-e were dissolved in 95%

41
EtOH. Solutions were applied to the shells of eggs in each treatment group < 24 hours
after oviposition. After experimental treatments were applied, all treatment groups were
placed in their respective environmental chamber at one of the three temperatures defined
above. Every day, the chambers were opened and the egg containers were removed.
Each cup was opened momentarily in order to release metabolic gas waste and check for
hatchlings. Eggs that grew mold were discarded upon discovery. Position of egg
containers within the three chambers was randomized daily.
Experiment 2
The following year, a new sample of 216 E. macularius eggs were tested. The
second years eggs were oviposited on 26 February 2003. Four treatment groups were
exposed to (/) no treatment, (g) 5 pi 95% EtOH, (h) 10 pg/5 pi estradiol benzoate, or (i) 5
pi estradiol -170. Treatments h and i were dissolved in 95% EtOH. To compare the
effect of the timing of treatment of these positive and negative controls on sex
determination relative to the data obtained in Experiment 1, we applied these treatments
at the beginning of the middle third of the incubation period at each of the three
temperatures in contrast to within 24 hr of oviposition as in Experiment 1. Each
treatment group consisted of three egg containers housing 6 egg chambers each (N = 18
total / treatment). As in the first year, each egg container held six eggs.
Sexing Procedure
Upon hatching, geckos were euthanized by exposure to halothane (Fluothane: 2-
bromo-2-chloro-l,l,l-trifluoroethane). Hatchling geckos were fixed in Bouins fixative
and preserved in 75% ethanol. The reproductive organs were removed from each gecko
and prepared for analysis by light microscopy. The reproductive organs are opaque

42
white, cylindrical structures on either side of the posterior end of the dorsal artery. After
removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of
ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55;
Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The
resulting paraffin blocks were sectioned at 8 pm and stained with a modified trichrome of
Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from
all geckos independently. If seminiferous tubules were identified within the tissue
sections, the gecko was scored as male (Figure 2a). If seminiferous tubules were absent,
the gecko was scored as female (Figure 2b). Seminiferous tubules were identified as
clusters of simple, circular structures with narrow lumen (Berman 2003). By relying on
histological examination of our specimens, we avoided potential macroscopic
misidentification of male and female reproductive organs.
Analysis
Sex ratio data were analyzed by ANOVA or unpaired Students Mest. In both
years, treatments were compared against that years EtOH and/or no treatment negative
controls. In Exp. 1, three concentrations of DDT and estradiol benzoate were compared
to the EtOH treatment group. Estradiol benzoate was tested as a potential positive
control. In Exp. 2, estradiol -17(3 and estradiol benzoate were tested as potential positive
controls and no treatment was administered to a fourth group in order to test EtOH as an
effective negative control. Data were analyzed using SAS version 6.10 for the Macintosh.
Results
Experiment 1 All treatments significantly altered the proportion of males at each
temperature as compared to the vehicle control treatment (Figure 9) but did not

43
significantly differ from each other (Tables 1 and 2). That is, all doses of DDT and the
positive control E2 benzoate altered the determination of sex in a similar fashion. A sex
ratio-reversing effect of DDT was not seen in the treatment groups but a general
perturbation appears to have affected all treatment groups that were dosed with either
EtOH, DDT, or E2 benzoate within the first 24 hours after oviposition.
Experiment 2 The treatment groups in Exp. 2 were exposed to chemical treatments at
the beginning of the middle third of incubation, or just prior to the period of sex
determination. The timing of exposure of the eggs to potential disrupters of the sex
determining pathway appears to influence the embryos sex determination. In Exp. 2,
both estradiol treatment groups (E2 benzoate and E2) had a significantly lower proportion
of males than the negative control groups (no treatment and 95% EtOH) at the male-
producing incubation temperature (P < 0.001; Tables 3 and 4). Also in Exp. 2, both
estradiol treatment groups had a higher proportion of males than the negative control
groups at the female-producing incubation temperature (P<0.01; Figure 10). In Exp. 2,
the sex ratio of the EtOH treatment group did not differ from the negative control group
that received no treatment. Thus, EtOH is a suitable vehicle as it had no effect on the sex
determination of the embryos demonstrating that the sex ratio-altering effects seen in the
other treatments groups represent specific actions of either estradiol benzoate or estradiol
17p, and not a result of general chemical perturbation. No significant differences were
found among the hatchling sex ratios of any treatment groups from the 30C
environmental chambers.

44
Discussion
Several novel observations were obtained during this study, including: 1)
exogenous estrogens skew the sex ratio toward males at the female-producing incubation
temperature and 2) chemical treatment during an early stage of embryonic development
produces a generalized perturbing effect on hatchling sex ratios. Wibbels et al. (1991)
suggested that lower doses of estrogen would be required to sex-reverse males incubated
at temperatures nearer to female-producing incubation temperatures compared to male-
producing incubation temperatures. However, very low doses of estradiol-17(3 have been
shown to sex-reverse turtle embryos as effectively as high doses. Threshold values may
not have significance for the effect of exogenous estrogens on sex determination in TSD
species (Sheehan et al. 1999).
Extensive experimentation has demonstrated that exogenous estrogens can
decrease the proportion of males in sex ratios of hatchlings incubated at typically male-
producing incubation temperatures in TSD species. Studies of the effects of exogenous
estrogens on sex ratios of hatchlings from male-producing incubation temperatures in
TSD species have been conducted with American alligators, Alligator mississippiensis,
softshell turtles, Apalone spinifera, red-eared slider turtles, Trachemys scripta, European
pond turtles, Emys orbicularis, green lacerta, Lacerta viridis, leopard geckos, E.
macularius, and others (Bull et al. 1988b; Crews et al. 1991; Tousignant and Crews
1994). The feminizing effect of estrogen mimics has also been reported broadly.
Unlike previous studies, a masculinizing effect of exogenous estrogens at the
female-producing incubation temperature was observed in these studies. This effect
could represent the second half of an inverted U-shaped response of the sex-determining

45
mechanism to estrogen. Estrogens are responsible for the regression of the medullary
region of the developing testis and the proliferation of the cortical region of the
bipotential gonad (Crews et al. 1991). The enzyme aromatase is responsible for the
conversion of testosterone to estradiol-17(5. Previous studies with reptiles, specifically
turtles and alligators with TSD, have not reported the production of males with any dose
of estrogen, androgen, anti-estrogen, anti-androgen or aromatase blocker (for review see
Guillette & Crain, 1996). Embryos exposed to aromatase blockers prior to sex
determination in the Red-Eared Slider Turtle, Trachemys scripta, develop according to
the temperature at which they are incubated (Wibbels & Crews., 1992) whereas in
alligators most are female or exhibit an ambiguous gonad (Lance & Bogart, 1992). In
contrast, genotypically female larvae of the newt, Pleurodeles waltl, differentiate into
functional males following treatment with an aromatase inhibitor (Chardard and Doumon
1999). An estrogen deficit results in male sex differentiation, regardless of genotype in
P. waltl. Hayes (1998) reported on sex determination in an amphibian, Rana pipiens,
with genetic sex determination finding that low doses of estradiol (< 0.07 pM) did not
affect sex differentiation, whereas high doses (0.07-0.18 pM) produced 100% females
and even higher doses (> 3.69 pM) produced 100% males. A higher dose of estrogen, as
is predicted with the administration of exogenous estrogens at a female-producing
temperature in E. macularius, suggests that a similar mechanism may occur in this lizard
contrary to the observations in other species with TSD. Higher doses of estrogen
increased the proportion of male E. macularius produced (at the female-producing
temperature), as seen in Hayes study. The results of this study suggest the hypothesis
that an estrogen surplus could alter sex determination in embryos. The mechanism is

46
unknown, but could involve feedback mechanisms either at the level of the ovary or
hypothalamo-hypophysial axis. That is, high estrogen concentrations in the gonad or
brain could alter the release of stimulatory factors from the brain or the gonad by altering
gene expression patterns. Hence, an inverted U-shaped response associated with the sex
determining mechanism in response to estrogen is supported by these data in E.
macularius. That is, at lower or natural levels of estrogen an ovary forms, but at higher
levels the genetic mechanism normally induced by estrogen exposure is altered or down-
regulated leading to the development of a testis. A number of genes are known to be
differentially regulated during gonadal differentiation in reptiles (Crews, 2003; Western
& Sinclair, 2001). Recent gene knockout studies in mice, suggest that alterations in
several of these genes will alter the differentiation of the gonad.
The studies presented here indicate that when administered outside of the
thermosensitive period, exogenous estrogens do not significantly alter sex determination
of£. macularius at male- or female-producing temperatures. We do, however, show
evidence of a general perturbation of the sex ratio in response to administration of
estrogen, diphenyltrichloroethane (DDT), and ethanol during earlier stages of embryonic
development. In E. macularius, sex determination occurs over an interval within the
middle third of incubation (stages 32-37; Bull 1987). During stage 32 (the onset of sex
determination), overall embryonic morphology corresponds to stage 15 in turtles. Stage
15 in turtles also marks the onset of sex determination (Yntema 1979). In snapping
turtles, Chelydra serpentina (a TSD species), administration of exogenous estrogens only
sex-reversed embryos when applied between stages 10-22. Outside of this middle third
of incubation, no hormonal effects on gonadal development were evident (Gutzke and

47
Chymiy 1988). The chemicals we administered to E. macularius eggs within 24 hours
after oviposition, could have been sequestered in the yolk as they are lipophilic and
disrupted gonadal differentiation in a more variable manner depending on size, relative
rate of development or concentration of maternally-derived sex steroids in individual
eggs. Female painted turtles, Chrysemys picta, donate varying concentrations of sex
steroid hormones depending on follicle size. In C. picta, estradiol levels in eggs decrease
with increased follicle size (Bowden et al. 2002).
Industrial pollutants, herbicides, fungicides, and pesticides, including DDT, cause
anomalous plasma steroid concentrations and gonadal aberrations in neonates and
offspring following embryonic exposure (Crain et al. 1997; Rooney 1998; Willingham et
al. 2000; Matter et al. 1998). Degradation products of DDT act antagonistically or
agonistically with estrogen at estrogen receptors (Rooney & Guillette 2000).
Recognizing the ability or lack of ability of DDT and other contaminants to sex-reverse
embryos, as opposed to only feminizing male-specific tissues allows more effective
evaluation of the threat posed by those contaminants on natural populations of TSD
species. In effect, populations of a TSD species with unusually high proportions of
females despite incubation at a male-producing temperature still consist of reproductively
viable individuals. If DDT, or other common contaminants, feminizes male tissues or
masculinizes female tissues without sex-reversing individuals to a full female or male
phenotype, then the threat posed to the population by the presence of the contaminant in
the natal sites of TSD species is far greater. Our results do not show a significant effect
of three ecologically relevant (Kannan et al. 1995) concentrations of DDT on hatchling
sex ratios of E. macularius at any of the three temperatures studied. Therefore, we

48
conclude that the impact of this contaminant does not lead to abnormal hatchling sex
ratios that would be a far lesser concern than increased reproductive abnormalities.

Proportion of Males / Treatment Group
49
0.8 -i
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
0.2 -
0.1 -
0 T
26 30 32.5
Temperature (C)
Figure 9. Sex ratios of Leopard Geckos in the first year of experimentation. In the first
year, treatments were administered to eggs within 24 hrs after oviposition.
The treatment groups of 5ul 95% Ethanol, 0.014 ppm DDT, 0.14 ppm DDT,
1.4 ppm DDT, and lOug / 5ul Estradiol Benzoate are indicated by the
checkered, light, dark, and darkest gray bars respectively. The 10ug/5ul
Estradiol Benzoate treatment groups contained no males so no bar is shown for
that group.

Proportion of Males / Treatment Group
50
30 32.5
Temperature (C)
Figure 10. Sex ratios of Leopard Geckos in the second year of experimentation. In the
second year, treatments were administered to eggs at the beginning of the
middle third of incubation. The treatment groups exposed to no treatment, 5 pi
95% EtOH, 10 pg/ 5 pi estradiol benzoate, or 5 pi estradiol 17/3 are indicated
by the light, dark, and darkest gray and black respectively. The 10ug/5ul
Estradiol Benzoate treatment groups contained no males so no bar is shown for
that group.

51
Table 1. ANOVA for sex ratios of Leopard Geckos from Experiment 1. All treatment
groups are considered separately in this analysis.
Source
df
MS
Treatment
4
0.246
Temperature
2
0.418
Temperature Treatment
14
0.254

52
Table 2. ANOVA for sex ratios of Leopard Geckos from Experiment 1. The three
concentrations of DDT treatment are considered together as one variable in this analysis.
Source
df
MS
Treatment
2
0.393
Temperature
2
0.238
Temperature Treatment
4
0.065

53
Table 3. ANOVA for sex ratios of Leopard Geckos from Experiment 2. All treatment
Source
df
MS
Treatment
3
0.174
Temperature
2
0.190
Temperature Treatment
11 0.515**
**: P = 0.002

54
Table 4. ANOVA for sex ratios of Leopard Geckos from Experiment 2. Both estradiol
treatments are considered together as one variable. Both control treatments are
considered together as one control group.
Source
df
MS
Treatment
1
0.133
Temperature
2
0.214
Temperature Treatment
50.915***
***: P< 0.001

CHAPTER 5
GENERAL DISCUSSION
Although dioecy, the trait of having two sexes, is almost universal within the
animal kingdom, great variation exists for the derivation of two sexes from a bipotential
embryo. Understanding the evolution of sex depends on a clear historical record of the
evolution of sex-determining mechanisms. Species under different selective regimes
have evolved different genomic mechanisms leading to dioecy. Elucidating the basic
dichotomy of ESD and GSD informs the controversy of adaptive significance for either
mechanism or mechanisms that draw on both genetic and environmental cues.
Sex determination in E. macularius responds to environmental cues. As shown in
chapter 2, temperature influences sex determination and humidity does not. Differential
effects of temperature and humidity suggest the structure of the molecular action that
initiates sex determination in this species. Temperature may be an indicator of habitat
quality. If dams use ESD to control their offspring sex ratios in heterogenous
environments in order to produce more offspring of the sex that is most likely to be fit in
the immediate habitat, temperature and humidity would be clear and immediate indicators
of microhabitat quality. Temperature does influence clutch sex ratios. Humidity does
not.
In chapter 3, a gene x environment interaction was demonstrated in the sex
determining mechanism of E. macularius. This result addresses concerns about the
stability of ESD in the face of rapid climate change. Having a genetic component may
55

56
allow ESD species to adapt to changing nest site temperatures and avoid extinction by
way of unbalanced sex ratios, as has been hypothesized in the past (Leslie and Spotila
2001). Remarkably, the variation in sex-determining response to incubation temperatures
is derived from dam but not sire. Compared to the sex determination of mammals in
which the sex of offspring depends on the genetic contribution of the sire to progeny, sex
determination of E. macularius seems anomalous. If ESD is partially controlled by the
genetic contribution of dams, dams have more control over offspring sex ratios than sires.
Male-biased sex ratios are expected from dams in polygynous or promiscuous mating
systems (McGinley 1984). Stressed females are also expected to produce male-biased
sex ratios when litter size is small. Female-biased sex ratios are considered adaptive in
social species in which female offspring and siblings will compete less for resources than
male offspring or siblings would (Schwarz 1994). A synthesis of ESD mechanisms with
behavioral ecology and evolutionary biology of sex ratios should begin with the dams
improved fitness. For example, E. macularius dams could benefit more from male-
biased offspring sex ratios because of the environmental stress indicated by temperatures
of 32.5C. The adaptive significance of female production at temperatures higher than
32.5C has not yet been considered. The maternal benefit of male-biased sex ratios
diffuses at temperatures higher than 32.5C. Future studies should identify females that
are more likely to produce a higher proportion of females at male-producing
temperatures. The fitness of several dams of known sex-determining disposition at a
constant incubation temperature over several reproductive seasons should be followed
empirically. Benefits of offspring sex ratios should be considered according to the fitness

57
advantage of the dam because data from this study suggest that the mothers genetic
contribution is what interacts with environmental cues to shape offspring sex ratios.
In chapter 4,1 reported the influences of three concentrations of DDT, estradiol
benzoate, estradiol 17(8, and EtOH. In the first experiment, eggs were treated within 24
hours of oviposition with either DDT, EtOH, or estradiol benzoate. A general perturbing
effect was found in which sex-determining responses to incubation temperature differed
from the classic collinear increase of males with temperature up to and including 32.5C.
However, a pattern of perturbation could not be attributed to any of the treatments from
Exp. 1. Instead, I concluded that any chemical treatment could affect developmental
processes if administered at an early stage of embryogenesis. In Exp. 2, E. macularius
eggs were treated at the beginning of the thermosensitive period of embryonic
development with estradiol 178, estradiol benzoate, EtOH, and a negative control
treatment in which no chemicals were administered to eggs. The two estradiol treatments
increased male production at the female-producing incubation temperature, 26C. The
estradiol treatments also decreased male production at the male-producing temperature,
32.5C. The decreased male production at 32.5C was expected, judging from past
results of the effects of exogenous estrogens on sex-determination in ESD turtles at male-
producing incubation temperatures. The increased male production at 26C suggests
negative feedback inhibition on aromatase. Aromatase converts testosterone to estrogen.
A surplus of estrogen early in development may prematurely inhibit the action of
aromatase, thereby causing the decreased production of estrogen later in the
thermosensitive period. An induced decrease in estrogen within the clutch would cause
the clutch to develop with a male-biased sex ratio.

58
I conclude that ESD is a multi-dimensional trait in E. macularius. An influence
of temperature, quantitative genetic variation, and estrogen concentration on sex ratios
was demonstrated in this species. The influences of other abiotic and biotic factors offer
ameliorative effects on sex determination in the face of climate change. Estimates of
extinction rates of ESD species should be reconsidered in light of these data. Research
into the adaptive significance of ESD should be focused on the benefits to the dam as the
dam invests the partial controlling element of genetic variation. As others have
concluded for other reptiles, I conclude that the partial genetic control of sex
determination in E. macularius is a polygenic mechanism and responds to temperature
fluctuation as a classic gene x environment interaction. Lastly, the effect of superfluous
estrogen on sex determination at the female-producing temperature should encourage
researchers to test the effects of aromatase inhibitors at female-producing temperatures.
The hypothetical male-determining factor is most likely an aromatase inhibitor that can
be stimulated by superfluous estrogen at the beginning of the thermosensitive period of
embryonic development.
In summary, I have clarified a more specific effect of the nest environment on
hatchling sex ratios in E. macularius. Elucidation of this kind is necessary for the
continuing effort to identify patterns in the evolution of sex-determining mechanisms.
Environmental sex determination may have arisen in response to changes in the
environment that have not yet been considered. Fisherian sex ratios may be shaped by a
broad spectrum of environmental stresses. Natural selection favors parents that invest
equally in sons and daughters (Fisher 1930). However, depending on local resource
availability, sons and daughters have different reproductive potentials. Males in good

59
condition are expected to outreproduce their sisters but females are expected to
outreproduce their brothers if both are in poor condition (Trivers and Willard 1973).
Poor condition could be defined as poor individual health or poor resource availability
that will result in poor individual health. Several factors in the nest could serve as
indicators of local resource availability and therefore sex-differential fitness. Sex-
differential fitness could result from fluctuations in temperature and/or any other factor in
the habitat as described in chapter 1. Temperature may be only one of many factors to
which ESD species adapted. At present, no categorization of species can separate ESD
species from GSD species. In light of these data, ESD species should be categorized by
their differential sex-determining response to humidity, quantitative genetic variation,
and exogenous estrogens. Different species can yield different patterns of sex
determining responses to these variables. Different patterns would inform the evolution
of sex-determining mechanisms.
Researchers ability to compare and contrast sex-determining mechanisms among
eublepharids, within Squamata and across Chordata has been hindered by insufficient
characterizations of the mechanisms. Comparisons of ESD species have not effectively
described the evolutionary history of ESD By comparing the effects of temperature on
sex determination, researchers have differentiated Type I, Type and Type HI patterns
of temperature-dependent sex determination (TSD). Type I TSD species like A.
mississippiensis produce larger proportions of males at higher incubation temperatures
(Bull 1983). Type II TSD species like Green Sea Turtles, Chelonia mydas produce larger
proportions of males at low temperatures (Mrosovsky 1988). Type III TSD species like
E. macularius produce larger proportions of males at intermediate temperatures and

60
lower proportions of males at lower or higher temperatures (Viets et al. 1993). Further
resolution of the differences among types of TSD should be obtained to track its
evolutionary history. At present, detail is lacking from the descriptions of sex
determining mechanisms and how species-specific mechanisms differ from each other.
Detail is also lacking in the general classification of sex-determining mechanisms. At
present, sex-determining mechanisms are characterized by a simple dichotomy of ESD
and GSD. Data from my experiments suggest a broad spectrum of influences and
degrees to which different influences affect sex determination in a species-specific
manner. My results should encourage a reclassification of sex-determining mechanisms
that acknowledges their responses to much more than temperature or genetics.

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70
BIOGRAPHICAL SKETCH
I was bom on 27 October 1974 and raised in Everett, Massachusetts. I took an
avid interest in biology and performance art at an early age. My thrill for the arts was
tempered by a fascination with the patterns and mechanisms of the natural world. At an
early age, I envisioned a life where I could orate, continue to improve my understanding
of biology, and contribute to the existing body of knowledge about biology. My
subsequent experiences solidified my ambitions. As an undergraduate student at Boston
University, I spent a semester in Ecuador studying tropical ecology. The experience gave
me a broad fundamental grasp of modem ecological paradigms and environmental crises.
During my field semester in Ecuador, I decided that the most effective use of my
professional time as a concerned citizen and appreciator of the natural sciences is to teach
and research the guiding principles of evolution, physiology, and ecology. Since then, I
have taught biology courses from the level of 2nd grade through graduate courses in
ecophysiology and molecular evolution. I returned to Ecuador upon receiving my B.A.
from Boston University. I taught biology and environmental science for two years at a
private high school in Quito that is affiliated with Boston Universitys School of
Education. Concurrently, I became certified to teach secondary-level biology in
Massachusetts and completed an Ed.M. in teaching and curriculum design from Boston
University.
After my graduate program in education, I moved from Quito to Memphis,
Tennessee, to complete an M.S. in biology. From Memphis, I moved to Gainesville and

71
the University of Florida to complete a Ph.D. Throughout my experiences, I have been
fortunate enough to consistently pursue my two loves of education and basic biological
research. I look forward to an academic career that will allow me to continue this pattern.

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Zoology t. X
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Marta L. Wayne, CoChair
Assistant Professor of Zoology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Louis'TTuuTTlelte
Distinguished Professor of Zoology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Karen A. BjomdaJ
Professor of Zoology

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
MhcA.
Max A. Nickerson
Professor of Wildlife Ecology and
Conservation
This dissertation was submitted to the Graduate Faculty of the Department of
Zoology in the College of Liberal Arts and Sciences and to the Graduate School and was
accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.
May 2004
Dean, Graduate School

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
MhcA.
Max A. Nickerson
Professor of Wildlife Ecology and
Conservation
This dissertation was submitted to the Graduate Faculty of the Department of
Zoology in the College of Liberal Arts and Sciences and to the Graduate School and was
accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.
May 2004
Dean, Graduate School



CHAPTER 3
QUANTITATIVE GENETIC VARIATION IN SEX-DETERMINING RESPONSE TO
INCUBATION TEMPERATURE IN LEOPARD GECKOS
Introduction
The diversity of sex-determining mechanisms in vertebrates can be classified as
either genotypic sex determination (GSD) or environmental sex determination (ESD). In
this chapter, I will describe quantitative genetic effects on ESD in Leopard Geckos,
Eublepharis macularius. Species that follow a GSD pattern are further subdivided into
either $:XX/':XY systems in which males are heterogametic (as seen in mammals) or
$:ZW/(J:ZZ systems in which females are heterogametic (as seen in birds). Reptiles
show a far greater diversity of sex-determining mechanisms than either mammals or birds
(Janzen and Paukstis 1991). Reptiles express either XX/XY or ZZ/ZW patterns of GSD
or one of several patterns of ESD in which the sex of offspring is predominantly, if not
completely, determined by the environment in which offspring develop as embryos.
Great controversy exists about the adaptive significance of different sex
determining mechanisms. Researchers ability to predict which GSD or ESD pattern will
be followed by a newly discovered species is poor, regardless of what information can be
gathered about the species natural history, phylogeny, or ecology. Although much has
been published about advantages of either ESD or GSD, little consensus can be found
concerning the differences that would make one mechanism adaptive for one species and
another mechanism adaptive for a different species. The adaptive significance of GSD
25


39
shaped distribution of the effects of exogenous estrogenic compounds. The influence of
exogenous estrogenic compounds on female reproductive tissues (or reproductive tissues
developing at female-producing incubation temperatures) has not been as substantially
reported as the influence of those compounds on male reproductive tissues (or
reproductive tissues developing at male-producing incubation temperatures). Some
endocrine disrupting contaminants (EDCs) have opposing effects on exposed
reproductive tissues at high and low concentrations (Parmigiani et al. 2000). For
example, prenatal exposure to small amounts of exogenous estradiol or diethystilbesterol
(DES) will increase prostate size in neonatal mice. Prenatal exposure to larger amounts
of the two compounds will decrease prostate size in neonatal mice (vom Saal et al. 1997).
A growing literature suggests that a wide range of species, including reptiles, are
exposed to various environmental contaminants having endocrine disruptive activities
(for reviews, see Crain & Guillette, 1998; Guillette & Iguchi, 2003; Tyler et al., 1998).
As with the research on mammals, the majority of these studies in reptiles have focused
on estrogenic or anti-estrogenic actions. Several studies have documented effects on sex
determination in turtles and alligators (Matter et al., 1998; Willingham & Crews, 1999),
whereas others have shown no effect on primary sex determination from the chemical(s)
tested (Podreka et al., 1998; Portelli et al., 1999). Several studies have reported that
various chemicals can bind to a reptilian estrogen receptor (Guillette et al., 2002; Sumida
et al., 2001; Vonier et al., 1996) including that from a lizard (Matthews & Zacharewski,
2000). No study to date has examined whether environmental estrogens, such as the
pesticide DDT, affect sex determination in a squamate, such as the commonly studied
lizard, the Leopard Gecko, Eublepharis macularius.


CHAPTER 1
GENERAL INTRODUCTION
The optimal sex ratio of a population may change depending on fluctuating
environmental stresses and sex-differential vulnerability to environmental stresses.
According to the Trivers-Willard hypothesis, parents may adjust their offspring sex ratio
to address sex-differential fitness and gamer the greatest reproductive advantage (Trivers
and Willard 1973). The Trivers-Willard hypothesis is based on Fishers theory of equal
parental investment in the rearing of sons and daughters (Fisher 1930). If sons and
daughters are unequally affected by environmental stress, then offspring sex ratios should
be skewed toward the sex that is less negatively affected. According to Fisher, the sex
that is most negatively affected by environmental stress will be produced less frequently
and will gamer more parental input. The cost paid by the parents for the rarer, more
energetically expensive offspring sex should balance with the cost they pay for the more
common, less energetically expensive offspring sex. For example, sex-differential
growth rates would cause an immediate increase in parental investment for one sex over
the other. If the immediate investment required by the faster growing offspring can not
be made because of resource scarcity or poor maternal condition, then the slower growing
sex will be produced in excess of the faster growing sex. The slower growing sex can
survive an immediate but short-lived dearth of resources much better than the faster
growing sex. Environmental stresses that differentially affect males and females should
cause sex ratios to deviate from 0.5. The Trivers-Willard hypothesis is most
1


42
white, cylindrical structures on either side of the posterior end of the dorsal artery. After
removal, they were stored in 75% ethanol, dehydrated by increasing concentrations of
ethanol, cleared in two changes of Citrosolv, and infiltrated with paraffin (Fisher 55;
Fisher Biotech, Orangeburg, NY) under increasing pressure (12, 15, 21, 23.5 lb/in2). The
resulting paraffin blocks were sectioned at 8 pm and stained with a modified trichrome of
Harris (Humason 1997). Two researchers analyzed sections of reproductive tissue from
all geckos independently. If seminiferous tubules were identified within the tissue
sections, the gecko was scored as male (Figure 2a). If seminiferous tubules were absent,
the gecko was scored as female (Figure 2b). Seminiferous tubules were identified as
clusters of simple, circular structures with narrow lumen (Berman 2003). By relying on
histological examination of our specimens, we avoided potential macroscopic
misidentification of male and female reproductive organs.
Analysis
Sex ratio data were analyzed by ANOVA or unpaired Students Mest. In both
years, treatments were compared against that years EtOH and/or no treatment negative
controls. In Exp. 1, three concentrations of DDT and estradiol benzoate were compared
to the EtOH treatment group. Estradiol benzoate was tested as a potential positive
control. In Exp. 2, estradiol -17(3 and estradiol benzoate were tested as potential positive
controls and no treatment was administered to a fourth group in order to test EtOH as an
effective negative control. Data were analyzed using SAS version 6.10 for the Macintosh.
Results
Experiment 1 All treatments significantly altered the proportion of males at each
temperature as compared to the vehicle control treatment (Figure 9) but did not


ADDITIONAL FACTORS INFLUENCE TEMPERATURE-DEPENDENT SEX
DETERMINATION IN LEOPARD GECKOS
By
DANIEL E. JANES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2004


Results 42
Discussion 44
5 GENERAL DISCUSSION 55
LIST OF REFERENCES 61
BIOGRAPHICAL SKETCH 70
v


47
Chymiy 1988). The chemicals we administered to E. macularius eggs within 24 hours
after oviposition, could have been sequestered in the yolk as they are lipophilic and
disrupted gonadal differentiation in a more variable manner depending on size, relative
rate of development or concentration of maternally-derived sex steroids in individual
eggs. Female painted turtles, Chrysemys picta, donate varying concentrations of sex
steroid hormones depending on follicle size. In C. picta, estradiol levels in eggs decrease
with increased follicle size (Bowden et al. 2002).
Industrial pollutants, herbicides, fungicides, and pesticides, including DDT, cause
anomalous plasma steroid concentrations and gonadal aberrations in neonates and
offspring following embryonic exposure (Crain et al. 1997; Rooney 1998; Willingham et
al. 2000; Matter et al. 1998). Degradation products of DDT act antagonistically or
agonistically with estrogen at estrogen receptors (Rooney & Guillette 2000).
Recognizing the ability or lack of ability of DDT and other contaminants to sex-reverse
embryos, as opposed to only feminizing male-specific tissues allows more effective
evaluation of the threat posed by those contaminants on natural populations of TSD
species. In effect, populations of a TSD species with unusually high proportions of
females despite incubation at a male-producing temperature still consist of reproductively
viable individuals. If DDT, or other common contaminants, feminizes male tissues or
masculinizes female tissues without sex-reversing individuals to a full female or male
phenotype, then the threat posed to the population by the presence of the contaminant in
the natal sites of TSD species is far greater. Our results do not show a significant effect
of three ecologically relevant (Kannan et al. 1995) concentrations of DDT on hatchling
sex ratios of E. macularius at any of the three temperatures studied. Therefore, we


63
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a scincid lizard species. Oecologia 118:431-437.
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dioxide influences sex determination in two species of turtles. Amphibia-Reptilia
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Ewert, M.A., and C.E. Nelson. 1991. Sex determination in turtles: diverse patterns
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