Physiology& Behavior,Vol. 55, No. 6, pp.

1067-1072, 1994
Copyright© 1994ElsevierScienceLtd
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Incubation Temperature Affects the Behavior

of Adult Leopard Geckos
(Eublepharis macularius)
D E B O R A H FLORES, A L A N T O U S I G N A N T A N D D A V I D C R E W S 1

Institute of Reproductive Biology, Department of Zoology, University of Texas at Austin, Austin, TX 78712

Received 1 September 1993

FLORES, D., A. TOUSIGNANTAND D. CREWS. Incubation temperatureaffects the behaviorof adult leopardgeckos (Euble-
pharis macularius). PHYSIOL BEHAV 55(6) 1067-1072, 1994.--The leopard gecko has temperature-dependentsex determi-
nation (TSD); females are predominantlyproduced when incubatedat 26°C (100%), 30°C (70%), and 34°C (95%), whereas males
are predominantly produced at 32.5°C (75%). Exogenous estradiol can override the effect of temperature on sex determination.
To compare temperature-determinedfemales with hormone-determinedfemales, eggs from the male-biased temperature were
treated with estradiol benzoate during incubation.As adults, animals from a male-biasedincubation temperature were more likely
to exhibit aggression than animals from female-biasedincubation temperatures. Furthermore, females from a male-biased incu-
bation temperature tended to be less attractive than females from female-biasedtemperatures. Hormone-determinedfemales were
both attractive and aggressive. This suggests that incubation temperature is an important developmental determinant of adult
aggressiveness and attractiveness.The 26°C animals ovariectomizedon the day of hatch exhibited more frequent aggression and
were unreceptiveto males, indicating that postnatal ovarian hormones also play a role in adult sociosexualbehaviors.The parallel
between incubation temperature and intrauterine position in laboratory mammals is discussed.

Temperature-dependentsex determination Aggression Attractiveness Ovariectomy Reptile

IN mammals, birds, and many other gonochoristic vertebrates and magnitude of incubation temperature. (ii) Sex determination
(separate sexes in separate individuals), gonadal sex is deter- remains labile to temperature through the early stages of gonadal
mined at fertilization by specific chromosomes. In mammals, differentiation, and initial sex-specific changes in the gonads are
genes on the Y chromosome channel gonadal development to reversible. The temperature-sensitive window corresponds to the
result in testes and the individual to have a male-typical pheno- middle of embryogenesis (sex determination period), beginning
type as an adult; the absence of these genes results in the for- prior to the differentiationof the gonads into recognizable ovaries
marion of ovaries and a female-typical phenotype. This process, or testes but ending well before hatching. (iii) Temperature exerts
referred to as genotypic sex determination (GSD), results in a 1:1 an all-or-none effect on the ovarian or testicular nature of the
sex ratio. gonad and hermaphrodites are rarely produced. (iv) During the
An alternative to GSD is found in a number of reptiles in window of temperature sensitivity, the effect of temperature ap-
which the temperature of the incubating egg determines gonadal pears to be cumulative; the longer an embryo is maintained at a
sex, a condition known as temperature-dependent sex determi- particular temperature, the more extreme the new temperature
nation (TSD). The primary difference is that in GSD, genes, des- must be to override the effect of the original temperature. (v)
ignated SRY, serve to trigger the developmental cascade leading Incubation temperature determines sex by modulating the nature,
to maleness, whereas in TSD, incubation temperature initiates quantity, and activity of steroidogenic enzymes in tissues prox-
the sex-determining cascade. It has been suggested that TSD may imal to the genital ridge, thereby acting on steroid precursors to
have been the precursor to GSD. Species with TSD do not inherit result in localized temperature-specific hormonal milieus as well
from their parents sex chromosomes or a genetic predisposition as regulating the number and type of steroid hormone receptors.
to respond to temperature. Instead, in TSD each individual has Thus, acting together, temperature-regulated enzymes and steroid
an equal ability to become a male or female and the sex ratio of hormone receptors guide the differentiation of the embryonic
a clutch may vary from unity to a single sex depending upon the gonad.
incubation temperature. The leopard gecko (Eublepharis macularius) represents a liz-
The following facts about TSD have been established [see (5) ard animal model for the study of sexual differentiation in a TSD
for review]: (i) Sex determination is sensitive to both the duration species. Individuals are small (40-100 g) and capable of breed-

To whom requests for reprints should be addressed.

1067
1068 FLORES. T()USIGNANT AND CREWS

ing at 45 weeks of age. Incubation of leopard gecko eggs at 26°C

produces only female hatchlings, whereas 30°C produces a fe-
male-biased sex ratio (30:70) and 32.5°C produces a male-biased
(75:25) sex ratio; an incubation temperature of 34°C and above
produces predominantly females (5:95) (15) (Fig. 1). An all- ,\o
male-producing temperature has yet to be identified, although !,
presumably it lies somewhere between 30°C and 32.5°C.

/
Incubation temperature modifies the endocrine physiology of /
¢~ 60
the adult leopard gecko. As would be expected, there is a differ-
ence in the nature and pattern of concentrations of sex steroids
\
between the sexes. Further, among females, androgen levels are
significantly higher, and estradiol levels significantly lower, in
females incubated at male-biased incubation temperatures com- 40

pared to females from a female-biased temperature (9). Similarly,

males from a female-biased incubation temperature have signif-
icantly higher levels of estradiol than males from a male-biased
/°
incubation temperature (13).
Two important questions become apparent and were the ob-
jectives of this study. (i) Is the sexual behavior of individuals
from temperatures that produce a mixed-sex ratio equivalent to
the sexual behavior of individuals from incubation temperatures o

i
- -

i '
/
i i 1 i i i
°~o
i i~
that produce predominantly or exclusively one sex? That is, do 26 28 29 30 31,5 325 34 35
all females (or males) behave similarly? (ii) Is the temperature
that produces primarily one sex more potent in its masculinizing Incubation Temperature (°C)
or feminizing abilities than the temperature that produces the FIG. 1. Percentage of male hatchlings as a function of constant incuba-
opposite sex? In other words, could certain temperatures serve tion temperature in the leopard gecko (Eublepharis macularius) [modi-
as an organizer in TSD species in a fashion similar to the role of fied from (15)].
steroid hormones in GSD species?

METHOD
During the first 10 weeks of age, animals were exposed to a
controlled environment (14:10 LD photic cycle, 30°C constant
Animals temperature) that maximizes hatchling survival. From 10 weeks
of age and older, animals were exposed to a 14:10 LD photic
All animals were hatched from eggs obtained from controlled cycle and a corresponding 30:18°C daily thermal cycle. This vari-
matings in the laboratory. Eggs were incubated individually in able thermal cycle more accurately reflects natural conditions.
covered plastic cups containing moist vermiculite (1.5:1, wa-
ter:vermiculite) at a constant temperature (--_0.2°C) of 26°C (n =
28), 30°C (n = t8), and 32.5°C (n = 10). Animals incubated at Ovariectomy
34°C (n = 12) were shipped to us 2 weeks after hatching from A subset of animals from the 26°C incubation temperature
Brian Viets and Craig Nelson at Indiana University. The incu- were either gonadectomized (OVEX) (n = 8) or received a sham
bation technique used at Indiana University was different in that operation (SHAM) (n = 8) on the day of hatching. Surgery was
the eggs were placed in airtight plastic Rubbermaid~ boxes (38 performed using hypothermia anesthesia. Because it is difficult
× 26 × 13 cm) and flushed with air daily. They were also shifted to determine the sex of a hatchling, tissue from the ovariectom-
from 32°(2 to 34°C after the first day of incubation and then ized females was preserved in Bouin's fixative and later pro-
shifted back to 32°C for 1-3 days before hatching. These addi- cessed for histological analysis to confirm that the gonads were
tional steps improved survivorship because 34°C is at or near the ovarian in nature.
lethal temperature for this species.
Hormone Manipulation
Housing and Maintenance
Eggs (n = 8) from the 32.5°C incubation temperature were
Newborn geckos were housed individually in a 12 × 30 × 6 spotted with 10 mg of estradiol benzoate in 5 ml of ethanol on
cm plastic shoebox. The box contained a water dish and a plastic day 5 of incubation (12). Exogenous estrogen will override the
shelter. After 65 weeks, each animal was transferred into a larger effects of a male-producing temperature in reptiles with temper-
polycarbonate cage measuring 43 x 22 x 20 cm containing a ature-dependent sex determination (7).
water dish, a large plastic shelter, and a brick. A 60-W incan-
descent light was positioned directly above the individual cages Behavior Testing
and cycled on and off 1 h after and 1 h before the fluorescent
lights, respectively. This allowed for individual thermoregula- All animals were tested on six occasions over a 5-week period
tion. for both homotypical and heterotypical sexual behavior. Aggres-
Hatchling leopard geckos were maintained on live crickets sive and submissive behavior was also quantified. Each animal
and mealworms; the diet of juvenile (>10 weeks) and adult was tested in three trials each with two different types of stimulus
geckos was supplemented with neonatal mice once a week. An- animals. The stimulus animals used in female tests were intact
imals were fed three times a week and water was available ad adult males from a 32.5°C incubation temperature and females
lib. The following supplements were added to the food: vitamin- from a 26°C incubation temperature. The stimulus animals used
mineral powder (Petco Animal Supplies) and calcium diphos- to test males were adult reproductively active females from a
phate (Texas Gulf Supplies). 26°C incubation temperature and intact adult males from a 32.5°(2
F U N C T I O N A L O U T C O M E S OF TSD 1069

100=
TABLE 1
BEHAVIORAL SCORING SYSTEM FOR EVALUATINGMALE-TYPICAL 0 Agg~ssiveness/O~*
SEXUAL AND AGGRESSIVEBEHAVIOR IN THE LEOPARD GECKO 80,
• Attractiveness/ O
(Eublepharis macularius)
Aggressive/Submissive
Sexual Behavior

No response
Score

0 Fleeing
Behavior Score

-2 0 0 ~ 0
/
Tail and/or back lick +1 Low posture/tail flicker - 1
Tail vibration +2 No response 0 20,
Tail grip/head shake +3 High posture/tail wave +1
Body, neck grip/head shake +4 Attack +2
Position for copulation +5 • u • i • i • | • i • i • u • i • i • l •

26 30 32.5 34
Each individual was tested with females from a 26"C incubation tem- Incubation Temperature (°C)
perature and males from a 32.5*(2 incubation temperature as stimulus FIG. 2. Aggressiveness and attractiveness among female leopard geckos
animals. Females do not display obvious sexual behavior. Attractiveness (Eublepharis macularius) from different incubation temperatures. Dis-
is based on the degree of sexual behavior displayed by the male stimulus played is the percentage of females responding aggressively to male
animals toward an experimental female animal. stimulus animals (aggressiveness), and percentage of stimulus males
courting these females (attractiveness). The asterisk indicates significant
differences in pairwise comparisons with 260C females.
incubation temperature. Therefore, each experimental animal
was tested with six stimulus animals and no stimulus animal was
used more than once for each experimental animal. animal (p < .24). There was no statistical difference in the num-
Each test consisted of observing a subject/stimulus pair for 5 ber of males that courted adult female stimulus animals (Fig. 3).
min following the introduction of the stimulus animal into the
subject's home cage. Table 1 denotes the behaviors monitored Attractiveness
during the test and the scores associated with them. Individuals
were considered to exhibit high-intensity aggression if they re- Although attractiveness to a male stimulus did not differ sig-
sponded with a score of + 1 or more on two or more of the three nificantly among unmanipulated females from the four incuba-
tests or if they attacked (+2) the stimulus animal on any trial. tion temperatures (p < 0.309), its pattern was inverse to that of
Females were considered to be attractive if a male stimulus ani- aggression (Fig. 2). That is, fewer females from the 32.5°C in-
mal displayed a courtship score of + 2 (tail vibration) or more on cubation temperature were attractive (30%) to male stimulus an-
at least two of the three trials (Table 1). The tests were terminated imals compared to females from 26°C (67%), 30°C (39%), or
at the end of 5 min or if an attack or attempted copulation oc- 34°C (50%) incubation temperatures.
curred. Tests were performed during the late afternoon coinciding
with the onset of daily activity cycles as observed in communal Ovariectomy
breeding cages. The sequence of tests was randomized. Females from a 26*(2 incubation temperature that were ovar-
iectomized on the day of hatch (OVEX) were significantly more
Statistics aggressive toward males (p ~ 0.02; Fisher's Exact test), but not
toward females (p < 0.28) compared with sham-operated fe-
The behavioral tests produced nonparametric, ordinal-scored males from 260C (SHAM) (Fig. 4). The difference in attractive-
data. Statistical analysis consisted of the likelihood ratio chi-
square test and the two-tailed Fisher's Exact test.

RESULTS

Aggression °
["7 30 "C (n=5)
e~
[7'7~ 32.5 "C (n=9)
The likelihood of aggression exhibited by unmanipulated fe- e~ ///
males toward a male (p < 0.01) or a female (p < 0.02) stimulus
animal differed significantly as a function of incubation temper- ///
ature (likelihood ratio chi-square test). Fewer females from the
26°C incubation temperature responded aggressively with male
stimulus animals (33%) than did females from the 30°C (56%),
N
the 32.5°C (90%), and the 34°C (83%) incubation temperatures //I
(Fig. 2).
The likelihood of aggression exhibited by males from 30°(2
and 32.5°C incubation temperatures toward an intact female stim- Aggressive C ourt s hi p Aggressive
to26"C to 26"C to32.5'C
ulus animal also differed significantly (p < 0.003; Fisher's Exact females females males
test) (Fig. 3). None of the males from 30°(2 incubation temper-
ature responded aggressively toward females, whereas 88% of Type of Response
the males from 32.5°(2 incubation temperature were aggressive FIG. 3. Comparison of aggressive and courting behaviors of male leopard
to female stimulus animals. There was no statistical difference geckos (Eublepharis macu/ar/us) from a female-biased incubation tem-
between these groups in their response toward a male stimulus perature (30"C) and a male-biased incubation temperature (32.5°C).
1070 FLORES. T()USIGNANf ANI) (!REWS

I00
In mammals, the embryonic environment influences behav-
ioral expression in adults. Sociosexual behavior of adults varies
according to intrauterine positioning of male and female fetuses
>. [] S h a m (n=8)
7. For example, in lab mice, females situated next to males in utero
[] ()vex (n=8)
//~/, (2M) have a masculinized adult phenotype compared to females
oJ~ 60 /////
situated next to females (0M) (16). Further, 2M females exhibit
.< "///~//
/////.
a higher intensity of aggression toward female stimulus animals,
e"
~Y/,2 and a decrease in attractiveness to male stimulus animals, com-
4o

20
Q2 6 C female
//////
//////
,/////
/////~
7///',,;
32.5 (' male
pared to 0M females. The effect of incubation temperature on
adult sociosexual behavior in a TSD species is similar in principle
to the intrauterine positioning phenomenon. Female leopard
geckos from a male-biased incubation temperature are signifi-
cantly larger and masculinized in their physiology and behavior
compared to females from female-biased incubation tempera-
tures (9,13). The sociosexual behavior of females also differed
Stimulus Animal according to incubation temperature. For example, the likelihood
FIG. 4. Influence of neonatal ovariectomy on the aggressive behavior of of aggression exhibited in standardized tests increased as a func-
adult females obtained from a 26°C incubation temperature. tion of an individual's incubation temperature. The fact that fe-
males from a 34°C incubation temperature did not exhibit statis-
tically higher frequencies of aggression compared to females
ness between OVEX- and SHAM-treated animals was not statis- from a 32.5°C incubation temperature could indicate a ceiling
tically significant (p -< 0.50). effect. It is also possible that the different incubation techniques
used by Indiana University resulted in a different adult pheno-
type. Finally, the fact that the animals from Indiana University
Hormone Manipulation were from a different genetic pool than the animals at the Uni-
versity of Texas may have been a factor.
Hormone-determined females (from eggs incubated at 32.5°C
In mice, body weight is positively correlated with dominance
and prenatally treated with estradiol benzoate) were significantly
(1). Larger females tend to be more aggressive and offensively
more attractive to males than were unmanipulated females from
attack juvenile males and other females, but not larger males (19).
a 32.5°C incubation temperature (p ~ 0.02; Fisher's Exact test),
A similar relationship between body size and aggression may
but not more attractive than females from a 26°C incubation tem-
occur in the leopard gecko. Females from a 32.5°C (male-biased)
perature (p -< 0.30). There was no difference in the likelihood of
incubation temperature are larger than females from cooler (fe-
aggressive encounters with male stimulus animals between un-
male-biased) incubation temperatures (13). These females show
manipulated temperature-determined females and hormone-de-
differences in the likelihood of aggression toward other females
termined females from the 32.5°C incubation temperature (p -<
and they are unreceptive and exhibit defensive aggressive behav-
0.70); both were significantly more aggressive to males com-
ior toward males attempting to court them. It is perhaps signifi-
pared to females from the 26°C incubation temperature (p - 0.01
cant that, regardless of incubation temperature, females do not
and p < 0.002, respectively). Finally, there were more aggressive
exhibit offensive aggression or initiate attacks, which is a male-
encounters with female stimulus animals by the hormone-deter-
mined females from the 32.5°(3 incubation temperature compared
to females from the 26°C incubation temperature (p ~ 0.05), but
not when compared to unmanipulated temperature-determined
32 5 C. tmmanipulated
females from the 32.5°C incubation temperature (p -< 0.20) a 2 5 1 bB treated
(Fig. 5).
1~)

DISCUSSION 90

8O
The present study indicates that incubation temperature has
.~ 70
an organizing effect on the sociosexual behavior of the leopard
gecko, a species with TSD. For example, female and male leop- "~ 60
O
ard geckos from a male-biased incubation temperature exhibit 50
higher frequencies of aggressive encounters than do animals from e~
female-biased incubation temperatures. Hormone-determined fe- 40
males from a male-biased incubation temperature were found to 317
be more attractive than temperature-determined females from the
20
same incubation temperature, and equally as attractive as females
from the female-biased temperature (26°C). However, there were
no differences between the hormone-determined and tempera-
ture-determined females from the male-biased temperature in the
Attractivity Aggression towards Aggression towards
likelihood of aggression toward male stimulus animals. Lastly,
males females
gonadal hormones after hatching may also play a role in the de-
velopment of behaviors in adult animals. Females from an all-
Type of Response
female incubation temperature (26°C) and gonadectomized on
the day of hatch were more aggressive than were sham-operated FIG. 5. Comparison of attractiveness and aggressiveness in temperature-
females. determined and hormone-determined female leopard geckos.
FUNCTIONAL OUTCOMES OF TSD 1071

typical characteristic. Hormone-determined females from a male- display high-intensity courtship behavior toward females from a
biased temperature were shown to be equally as aggressive as 32.5°C incubation temperature. However, hormone-determined
temperature-determined females. This suggests that the females from a 32.5°C incubation temperature were shown to be
aggressive behavior in a female leopard gecko is less affected by significantly more attractive than the temperature-determined fe-
ovarian hormones than by incubation temperature. males. DOmer et al. (8) found that female rats treated with estra-
Comparison of the aggression exhibited by males from two diol benzoate on the first day of life had an increased attractive-
incubation temperatures indicated that males from a female-bi- ness to male stimulus animals. Such females exhibit persistent
ased incubation temperature (30°C) were less likely to be ag- vaginal estrus, perhaps accounting for the increase in attractive-
gressive toward female stimulus animals compared to males from ness. Thus, female leopard geckos from a male-biased incubation
a male-biased incubation temperature (32.5°C). As is the case temperature may have masculinized skin lipid ratios, and prenatal
with females, males from 30°C incubation temperature are treatment of eggs with estrogen at this temperature may skew the
smaller than males from a 32.5°C (13). chemical composition of these lipids toward female-like skin
In the gerbil, intrauterine position affects the age of maturity lipid ratios. This would explain the change in the behavior of the
of females; 0M females mature early whereas 2M females mature male stimulus animal toward hormone-determined females.
late (4). Early maturing (0M) females have lower androgen levels The modern perspective of sexual differentiation was devel-
and higher estradiol levels than do late-maturing (2M) females oped from work on species with GSD. Whalen (18) suggested
(3). Studies have indicated that the onset of sexual maturity and an orthogonal model of sexual differentiation in which male and
plasma steroid levels in leopard geckos also vary as a function female represent separate dimensions or continuua of masculinity
of the incubation temperature of the egg. Females from a 32.5°C (masculinization~demasculinization)and femininity (feminiza-
incubation temperature take longer to reach sexual maturity (14) tionodefeminization).Although these often covary, they are sep-
and have significantly higher circulating concentrations of tes- arable processes. Reinisch (11) proposed the oblique model,
tosterone and significantly lower estradiol levels (9). Males from which suggests that there is not complete independence between
a female-biased 30°C incubation temperature have significantly masculinity and feminity (as in the orthogonal model), but rather
higher levels of estrogen as adults than do males from a male- some degree of correlation between masculinization/defemini-
biased 32.5°C incubation temperature (13). zation and feminization/demasculinization.A common analogy
Although testicular androgens play a major organizational is that the brain mechanisms underlying sexually dimorphic be-
role in the development of adult aggression in mammals, ovarian haviors are like the accessory sex structures; both are initially
estrogens may be an important postnatal regulator in aggressive bipotential and only later acquire sexually dimorphic expres-
behavior in the leopard gecko. Females from a female-biased sions. Although individuals predominantly exhibit reproductive
incubation temperature that were ovariectomized on the day of behaviors characteristic of their own gonadal sex (homotypical
hatch were never receptive and were highly aggressive toward sexual behaviors), in many species these same individuals can,
males in a manner similar to intact females from a higher, male- and often do, perform behavior patterns typical of the opposite
biased incubation temperature. These neonatally ovariectomized sex (heterotypical sexual behaviors).
females grew significantly larger than did sham-operated females It is important to keep in mind that, in GSD species, male and
(13), suggesting that ovarian hormones play an important role in female individuals differ genetically. In TSD, each individual has
female growth after hatching. an equal probability of developing as a male or as a female.
In lab mice, adult 0M females are more attractive than 2M Because gonad determination depends fundamentally on the in-
females (17). Male leopard geckos extrude their tongues more cubation temperature experienced during the mid-trimester of
frequently and vibrate their tails when placed in a cage previously embryonic development and is an all-or-none process (intersexes
inhabited by a female, but become agitated if the previous in- are not produced), is the pattern of sexual differentiation similarly
habitant had been a male (2). As in snakes, tongue extrusions are affected by incubation temperature (6)? The available evidence
used to perceive chemical signals. Mason and Gutzke (10) found suggests that the answer is yes. As the present experiments in-
differences in the chemical composition of the skin-derived lipids dicate, the extent to which an individual is masculine (or femi-
between male and female leopard geckos, suggesting olfaction- nine) is synonymous with the extent to which it is feminine (or
based sex recognition. Although not statistically significant, it is masculine), suggesting that sexual differentiation in TSD species
interesting to note that females from a predominantly male in- may consist of only a single male,-,female continuum.
cubation temperature were less attractive than females from fe-
ACKNOWLEDGEMENTS
male-producing incubation temperatures and that the trend in at-
tractiveness was pronounced and opposite to that of aggression. We thank John Branch and Kira Wennstromfor technical assistance,
The lack of statistical significance could be due to the number of and Tony Alexander and Jami Milton for histology and maintenanceof
individuals in each group and/or the measure of attractiveness. the gecko colony. D. L. Flores was supported by ADAMHASupplement
Grant for Underrepresented Minorities in Biomedical and Behavioral
All stimulus males were sexually experienced and strong court- Sciences for MH R37 41770 (to D.C.). A. Tousignant was supported by
ers, and were thus likely to recognize and court females regard- NIMH NRSA 5F31-09901. D. Crews was supported by NIMH Research
less of variation in female quality. Males were not as likely to Scientist Award 00135.

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