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Research paper
Methyltestosterone alters sex determination in the American alligator
(Alligator mississippiensis)
Christopher M. Murray
a,
⇑
, Michael Easter
b
, Mark Merchant
c
, Justin L. Rheubert
d
, Kelly A. Wilson
d
,
Amos Cooper
e
, Mary Mendonça
a
, Thane Wibbels
f
, Mahmood Sasa Marin
g
, Craig Guyer
a
a
Department of Biological Sciences, Auburn University, 331 Funchess Hall, Auburn, AL 36849, USA
b
Everglades Holiday Park, Fort Lauderdale, FL 33332, USA
c
Department of Chemistry, McNeese State University, Lake Charles, LA, USA
d
Department of Natural Sciences, The University of Findlay, Findlay, OH 45840, USA
e
J. D. Murphree Wildlife Management Area, Texas Parks and Wildlife Department, Port Arthur, TX 77640, USA
f
Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
g
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
article info
Article history:
Received 11 February 2016
Revised 3 May 2016
Accepted 7 July 2016
Available online 9 July 2016
Keywords:
Methyltestosterone
Temperature-dependent sex determination
Androgen
Crocodilian
abstract
Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic develop-
ment, and/or activational, influencing transitory actions during adulthood. The organizational influence
of endocrine-disrupting contaminants (EDC’s) produces a wide variety of reproductive abnormalities
among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influ-
ences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For
example, 17-
a
methyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex
ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is
reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this
study we test the effects of MT on sexual differentiation in American alligators, which we used as a
surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxi-
cological concentrations. Sexual differentiation was determined by examination of primary and secondary
sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known
to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure
to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of
the clitero-penis (CTP).
Ó2016 Published by Elsevier Inc.
1. Introduction
Recent theory maintains that most vertebrates exist somewhere
along a continuum between strict genetic control of sex determi-
nation and strict environmental control (Sarre et al., 2004) and that
placement along this continuum exists as a function of environ-
mental influence on fitness (Charnov and Bull, 1977). Genotypic
sex determination (GSD) is a preset mechanism of primary and
secondary sexual structure differentiation based on the presence
and subsequent expression of a single gene, or lack of that gene,
resulting in production of a default sex (gene absent) or the alter-
nate sex (gene present). In mammals, for example, the SRY (or
SOX9) gene steers development away from a default female pheno-
type via promotion of a male reproductive tract and simultaneous
suppression of a female tract (Kovacs and Ojeda, 2011). While GSD
allows for a heritable sex, it can be influenced by gonadal sex
disorders (Kovacs and Ojeda, 2011).
Environmental sex determination (ESD) is susceptible to many
more external influences (Wibbels et al., 1994). Temperature
dependent sex determination (TSD), a form of ESD, relies on speci-
fic thermal regimes to dictate the expression of aromatase and/or
reductase, thus governing the sex steroid regime of the developing
embryo and subsequent sex characteristics (Wibbels et al., 1994).
Thermal thresholds or ‘critical’ temperatures, that serve as a thermal
pivot between development of one sex or the other, are
http://dx.doi.org/10.1016/j.ygcen.2016.07.007
0016-6480/Ó2016 Published by Elsevier Inc.
⇑
Corresponding author at: Department of Biological Sciences, Southeastern
Louisiana University, Hammond, LA 70402, USA.
E-mail addresses: christopher.murray-2@selu.edu (C.M. Murray), M.Easter05@
gmail.com (M. Easter), mmerchant@mcneese.edu (M. Merchant), rheubert@findlay.
edu (J.L. Rheubert), amos.cooper@tpwd.state.tx.us (A. Cooper), mendomt@auburn.
edu (M. Mendonça), twibblels@uab.edu (T. Wibbels), msasamarin@gmail.com
(M.S. Marin), guyercr@auburn.edu (C. Guyer).
General and Comparative Endocrinology 236 (2016) 63–69
Contents lists available at ScienceDirect
General and Comparative Endocrinology
journal homepage: www.elsevier.com/locate/ygcen
species-specific and vary widely among vertebrates with TSD
(Janzen and Paukstis, 1991). TSD mechanisms also vary among
taxa and three main mechanisms exist; male production at low
temperatures and female at high, female production at low tem-
peratures and male at high, and female at low, male at intermedi-
ate, and female at high temperatures (Valenzuela, 2004). A
remarkable series of sex steroid and temperature manipulations
have elucidated some constants among TSD mechanisms; 1) A crit-
ical period exists at which an embryo’s sex determination is sensi-
tive to both temperature and sex steroids; 2) feminization and
masculinization are mediated by steroid-specific receptors; 3) aro-
matase and reductase inhibitors can manipulate sex regardless of
temperature, however with unequal potency; 4) response to estro-
gens can produce female embryos at male-producing tempera-
tures, but androgens can only masculinize embryos at threshold
temperatures; and 5) mixed sex ratio clutches are produced at
threshold temperatures rather than hermaphrodites (Wibbels
et al., 1991, 1994; Wibbels and Crews, 1994; Crews et al., 1994;
Wibbels and Crews, 1995; Crews; 1996).
Our understanding of how hormone sources, storage, and inter-
nal feedback mechanisms affect sexual differentiation mechanisms
has greatly improved as a result of research in temperature-
dependent steroid expression and utilization (Kamel and
Kubajak, 1987; Janzen et al., 1998; Paitz and Bowden, 2011;
Pfannkuche et al., 2011; Paitz et al., 2012). Unfortunately, so has
our understanding of anthropogenic influences on such processes
(Guillette et al., 1995). Numerous industrial and agricultural com-
pounds, when introduced to natural systems, have endocrine-
disrupting affects. Endocrine-disrupting compounds (EDCs) have
negative activational effects on endocrine systems, but more
daunting is the organizational role they play as hormone mimics
during sexual differentiation and embryonic organization
(Guillette et al., 1995). Because sexual differentiation mechanisms
vary among reptiles, these taxa have become model indicators of
EDC exposure. Among reptiles, contaminants that mimic estrogen
are common (e.g. PCBs, Dioxin, Furans, DDE), while environmental
androgens are far more rare, presumably as a result of the aroma-
tizable nature of testosterone and the dominance of the estrogen
pathway associated with TSD (Wibbels and Crews, 1995).
Among reptiles the most notable sentinel taxa for EDCs are
crocodilians (Milnes and Guillette, 2008). Their popularity as
‘model’ organisms emerged because of the Lake Apopka superfund
site, where dicofol, DDT and subsequent metabolites were discov-
ered as contaminants in 1980 (Guillette et al., 1994). Such contam-
inants were deemed potent environmental estrogens after female
alligators displayed unnaturally high 17b-estradiol plasma con-
centrations, polyovular follicles, polynuclear oocytes (Guillette
et al., 1994), and reduced gonadal-adrenal mesonephros (GAM)
aromatase activity (Crain et al., 1997). Males exhibited decreased
plasma testosterone, reduced phallus size (Guillette et al., 1996)
and poorly organized testes (Guillette et al., 1994). This case study
exemplified the utility of crocodilians in understanding the activa-
tional and organizational effects of EDCs.
In Guanacaste, Costa Rica, three large tilapia farms utilize 17-
a
methyltestosterone (MT) to produce all-male offspring that grow
faster and reach larger maximum size than females. Preliminary
data on MT persistence in water and soil was noted during initial
testing of this fish farming practice, however, lipid persistence of
the compound and its effects on vertebrates other than fish are
unknown (Phelps and Popma, 2000; Gupta and Acosta, 2004).
The nearby Tempisque Basin harbors a rapidly expanding popula-
tion of American crocodiles (Crocodylus acutus) that exhibits a
male-biased sex ratio (Bolaños-Montero, 2012; Murray et al.,
2015). Hatchling sex ratios from this population do not match
the ratios predicted by clutch thermal regimes and this sex
ratio bias differs among clutches, with some clutches being
male-biased and others not (Murray et al., 2016). Here, we test
the potential for MT to produce male crocodilian embryos at
female-producing temperatures and histologically analyze
organizational effects of urogenital development from MT exposure
during the experimental assay.
2. Materials and methods
2.1. Experimental assay
For this experiment, 108 American alligator (Alligator mississip-
piensis) eggs were collected from five clutches in June 2013 and 76
eggs from three clutches in June 2014. All eggs were collected at J.
D. Murphree Wildlife Management Area, Port Arthur, TX, within
five days of deposition, as assessed by daily nest monitoring and
the width and length of banding (Masser, 1993). Eggs were trans-
ported to an Auburn University live animal facility and incubated
at 28 °C, a female-producing temperature (Lang and Andrews,
1994). In 2013, eggs were misted with water daily in an incubator
(Fisher Scientific, Isotemp model 655D) to maintain humidity.
However, 50 eggs failed to complete development, likely because
of dehydration. Therefore, eggs in 2014 were maintained at
approximately 100% humidity using a vermiculite substrate and
steam heating. Each year, four eggs were opened periodically to
stage the embryos as described by Ferguson (1985), resulting
in 126 experimental eggs in total. Prior to the temperature-
sensitive period each year (stage 20, when sex determination
occurs; Lang and Andrews, 1994), eggs were randomly assigned
to one of five treatments using a random number generator. Eggs
were randomly dispersed among plastic bins in the incubator with
10–14 eggs per treatment per year. Two treatment groups served
as controls. One control received no treatment while the other
received 5
l
l of 95% ethanol (ETOH) to control for effects of the
vehicle used to deliver MT to all treatment groups. Treatment
groups received 4
l
g/ml, 40
l
g/ml, or 400
l
g/ml of 17
a
-MTin
95% ETOH. These treatments exposed eggs to between 10 and
1000 times the natural amount of testosterone in alligator egg yolk
(Conley et al., 1997), a range of doses standard for ecotoxicological
dose-response assays with sex steroid hormones or related
endocrine-disrupting compounds (Wibbels and Crews, 1995;
Crain et al., 1997). Treatments were applied topically as 5
l
lof
solution deposited on the surface of an egg at stage 21, a technique
that is used to transport compounds inside reptilian eggshells
(Crews et al., 1991,Paitz et al., 2012). Using this method, Crews
et al. (1991) found that at least 90% of applied compound was
incorporated into the embryo. Additional eggs were incubated
separately at male-producing temperatures (32 °C) to serve as
control males for primary and secondary sex organ comparison.
2.2. Methyltestosterone quantification
Upon hatching, yolk samples were collected and frozen to quan-
tify the concentration of 17
a
- MT that reached the embryo. Steroid
hormones were extracted from egg yolk using a 3:2 volume solu-
tion of ethyl acetate and hexane, respectively. Samples were dried
under vacuum at 25 °C and dissolved in 100
l
L assay buffer
supplemented with 10
l
L of DMSO to encourage dissolution.
17
a
-methyltestosterone concentrations were quantified using a
sandwich ELISA kit (MaxSignal
Ò
methyltestosterone kit, Bioo
Scientific, Austin, TX; Rabbit; polyclonal). Cross-reactivity with
testosterone was 0.3% and samples were not analyzed in duplicate
as all other wells were occupied for another study. Optical density
was determined using a Benchmark Plus microtiter plate spec-
trophotometer (Bio-Rad, Hercules, CA) at 450 nm. Hatchlings were
individually marked via caudal scute removal, and snout-to-vent
64 C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69
length and total length were recorded. The sex of each hatchling
was determined by cloacal examination with an MDS 105 2.7 mm
endoscope. A hatchling was determined to be male if the clitero-
penis (CTP) possessed all of the following character states:
bi-lobed structure, extensive vascularization, and length equal to
or greater than length of the vent (Fig. 2;Allsteadt and Lang, 1995).
2.3. Sexual differentiation analysis
A subset of twenty hatchings was euthanized and preserved for
detailed CTP examination using a Leica M165C stereoscope with
Leica DFC425 camera attachment. These sacrificed individuals
were examined histologically for internal verification of sex and
description of testicular and ovular development among treat-
ments. The remaining surviving hatchlings (n = 16) were housed
at Auburn University and McNeese State University so that sex of
each individual could be verified at five months of growth.
Whole animals were fixed in 10% neutral buffered formalin and
preserved in 70% EtOH. The developing urogenital tissues were
excised under a stereomicroscope and placed in 70% EtOH for fur-
ther manipulation. Tissues were washed with deionized water,
dehydrated in a series of increasing concentrations of EtOH, and
placed in paraffin wax overnight to allow the wax to fully infiltrate
the tissues. Tissues were then placed in embedding molds with
paraffin wax and allowed to cure for 24 h. The urogenital tracts
were then serial sectioned sagittally on an American Optical rotary
microtome, and sections were then placed on albuminized slides
and stained with Ehrlich’s Hematoxylin and Eosin.
Slides were viewed at various magnifications using an Olympus
microscope to identify structures of the developing urogenital
system and to determine if gonads were of the male or female cat-
egory for each of the above-mentioned treatments. Representative
slides from each treatment along with any abnormalities were
photographed using an attached digital camera and images were
compiled into composite micrographs using Adobe Photoshop CS5.
2.4. Statistics
G tests for independence were performed on the proportion
of male hatchlings among treatments, the proportion of male
hatchlings among clutches, the proportion of survivors among
treatments, and the proportion of survivors among clutches. A
two-sample T test was used to test for difference in size between
control or treatment individuals. Welch’s two- sample T-tests were
used to determine difference in CTP length and width (Fig. 1)
between treated individuals and control individuals because
sample sizes were low among individual experimental groups.
3. Results
3.1. Sexual differentiation of secondary sex organ
Of 126 experimental or control eggs, only 36 survived long
enough to be categorized as to sex. Survival did not differ among
experimental groups but did differ among clutches (G = 28.3,
df = 7, p = 0.0002). Alligator eggs incubated at a female-producing
temperature and treated with 17
a
- MT produced a significantly
higher proportion of male hatchlings than control groups
(G = 20.2, df = 4, p = 0.0005; Fig. 2). There was no difference in pro-
portion of males among clutches. CTP lengths, but not widths, were
significantly larger in treatment (pooled across treatment groups)
versus the pooled control groups (t = 2.65, df = 10.72, p = 0.02,
Fig. 3). Sixteen surviving treatment eggs were housed for sex con-
firmation five months later. All but one of these was confirmed to
have the same sex as morphologically determined at hatching. The
only hatchling whose sex diagnosis differed was deemed a female
at hatching and a male five months later.
Fig. 1. Schematic illustrating cliteropenis (CTP) width and length measurements
during stereoscopic examination in both A) control females and B) masculinized
CTP at 40
l
g/ml.
Fig. 2. Sex ratios among control, ETOH, 4
l
g/ml, 40
l
g/ml and 400
l
g/ml exper-
imental groups. Percent male based on morphological sexing at hatching was
significantly different among groups (G = 20.2, df = 4, p = 0.0005).
C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69 65
Stereoscope examination of twenty individuals sacrificed at
hatching revealed novel CTP characters for differentiating males
from females as well as treatment effects on CTP development.
Control females exhibited flatter, non-vascularized structures that
extended from the dorsal cloacal surface in a low-lying ‘‘T” shape
(Fig. 4 A, B). Control male CTPs exhibited a basal bi-lobed structure
with a large projection characterized by a mid-sagittal groove,
presumably for sperm delivery. Four to four hundred
l
g/ml MT
treatment concentrations present a gradient from minimal to
extreme vascularization, bi-lobed shape and projection length
(Fig. 4). Such characters are more pronounced within the 4
l
g/ml
treatment than in control females and nearly mirror control males
for individuals in the 400
l
g/ml treatment group.
3.2. Sexual differentiation of primary sex organ
3.2.1. Control specimens
Alligators sacrificed for histological examination had gonads
that had completely separated from the mesonephros and had
begun differentiation. In controls incubated at female-producing
temperatures, the ovaries extended along the medial border of
the developing mesonephros but were distinctly separated from
the mesonephros by a band of loose connective tissue. Along the
posterior border of the mesonephros separation between the ovary
from the mesonephros was more distinct (Fig. 5A).
Histological analysis of the gonads showed that embryos
incubated at female-producing temperatures had a gonad that is
characterized by a thick band of primordial germ cells (Fig 5A,
Pgc) along the cortical region of the gonad. The medullary region
of the gonad was filled with large open lacunae (Fig 5A, La) and
sparsely arranged cells. Embryos incubated at male producing
temperatures had a gonad that was characterized by a lack of
accumulation of primordial germ cells in the cortical region and
large germ cells organized into seminiferous tubules (Fig 5B, St)
where the germ cells are arranged on the periphery of the tube
to form the lumen (Fig 5C, St).
3.2.2. Treatment specimens
Upon exposure to MT the gonads of embryos incubated at
female producing temperatures developed masculinizing charac-
teristics that seem to follow a continuum with increasing concen-
trations of MT exposure. Individuals treated with 4 mcg/mL of MT
and incubated at female producing temperatures had a gonad that
showed a small band (compared to the control female) of primor-
dial germ cells. The medullary region still contained lacunae
(Fig 5D, La) with sparsely spaced cells but a few accumulations of
cells arranged in tubules (Fig 5D, St) are observed. Individuals trea-
ted with 40 mcg/mL of MT and incubated at female producing tem-
peratures lacked an accumulation of primordial germ cells in the
cortical region (Fig 5E) and a medullary region with large open
lacunae (Fig 5E, La) and instances of germ cell organization similar
to that of seminiferous tubule development (Fig 5E, St). A high dose
of MT of 400 mcg/mL of MT to embryos incubated at female pro-
ducing temperatures exhibited a gonad that was more similar to
the control individuals incubated at male producing temperatures
than the controls individuals incubated at female producing tem-
peratures. The gonad lacked an accumulation of primordial germ
cells in the cortical region, had sparsely located lacunae (Fig 5F,
La) and an accumulation of germ cells organized into seminiferous
tubules.
3.3. Yolk methyltestosterone concentration assay
Sample for yolk analysis were limited due to hatching and cost
logistics. Small samples sizes and prohibited construction of a dose
response curve. However, it is notable that all salvaged yolk sam-
ples (n = 7) contained 17
a
- MT including samples from all three
treatments. The average methyltestosterone (MT) concentration
in yolk samples was 0.05 ± 0.014 ng/g yolk. Yolk MT concentrations
ranged from 0.00004 to 0.09 ng/g yolk. Concentrations did not differ
among treatment groups (F = 1.61, DF = 3, p = 0.35). Yolk was not
obtained from control eggs for comparison. All concentrations
Fig. 3. Clitero-penis (CTP) lengths significantly larger in treatment groups than
control individuals (t = 2.65, df = 10.72, p = 0.02).
Fig. 4. Stereoscope images of experimental alligator clitero-penises at hatching
including control females (A, B), 4 g/ml treatment group (C), 40
l
g/ml treatment
group (D), 400
l
g/ml treatment group (E) and natural male hatchling incubated at
male producing temperatures (F).
66 C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69
among treatment groups were on the order of ng/mL compared to
the initial
l
g/mL dosage.
4. Discussion
Results presented here indicate that MT has a masculinizing
effect on crocodilian embryos during sexual differentiation and
produces male hatchling alligators at female-producing tempera-
tures. This response is observable in both the gonad and sec-
ondary sex structure (CTP). Additionally, the effect of MT on
developing embryos is dose-dependent. MT does not affect sur-
vivorship of exposed individuals and, regardless of exposure con-
centrations, is barely detectable in yolk late in development.
Further, methodology used here to determine the sex of hatch-
lings was conservative in the sense that individuals deemed male
were unlikely to be female, but some deemed female could have
been male. This method proved to be accurate when assessing
male versus non-males at hatching (15 out of 16) including the
potential diagnosis of hermaphroditic individuals if some, but
not all, male character criteria are met. However, hatchlings used
to determine the accuracy (male versus non-male) of our
methodology after five months were not histologically analyzed
for hermaphroditic gonads.
Stereoscopic and histological examination reveals a gradient of
characters among treatment individuals from feminine to mascu-
line. As exposure concentrations increase, so does vascularization,
lobature and length of the CTP. Control female CTP morphology
appears to be a non-vascularized laterally folded tissue that is
slightly thickened medially. Control male CTP morphology appears
to be twice as long as control female CTP and is highly vascularized
with a low-lying second lobe posterior to the lengthy primary
structure. The primary structure is characterized by a medial, ante-
rior groove that may serve to assist sperm transport. The gradient
of secondary sex characters noted among MT treatment groups
appears to mirror the relationship between MT exposure and uro-
genital differentiation. Gonads of control hatchlings exhibit differ-
entiated gonads with associated tissues and precursors to gamete
Fig. 5. Histological micrographs of the gonads from control and treatment Alligator mississippiensis embryos. A.) Gonad of a control embryo incubated at female producing
temperature showing an enlarged cortical region of the gonad with a thick band of primordial germ cells (Pgc) and a medullary region with enlarged lacunae. B.) Gonad of
control embryo incubated at male producing temperature showing seminiferous tubule (St) development and a lack of an enlarged cortical region with an accumulation of
primordial germ cells. C.) Enlarged micrograph of the gonad of a male showing the arrangement of cells into seminiferous tubules. D.) Gonad of embryo treated with 4
l
g/ml
of MT showing a medullary region with lacuna (La) that appear less developed than in the control and sparsely located accumulations of cells arranged in tubules (St). E.)
Gonad of embryo treated with 40
l
g/ml of MT showing a reduced cortical region and a diminished accumulation of primordial germ cells (Pgc). The lacunae are enlarged and
are disorganized in appearance with an accumulation of cells arranged in tubules resembling those of seminiferous tubules (St). F.) Gonad of an embryo treated with 400
l
g/
ml of MT showing the lack of a cortical region and primordial germ cells, sparse lacunae (La) and multiple developments of seminiferous tubules (St).
C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69 67
production. Low MT exposure at female-producing temperatures
results in slight masculinization of the ovary or underdevelopment
of both kidney and gonad. This underdevelopment may be a result
of a both being exposed to low amounts of steroid binding which
could then have an effect on organ development trajectories and
a result in subsequent delay in development. Exposure to higher
concentrations promotes a qualitatively higher ratio of seminifer-
ous tubules to follicles with more gonadal masculinization at higher
MT concentrations. Although the sexing of hatchlings by CTP
morphology was conservative for this study, the gradient of CTP
masculinization appears to be correlated with gonadal masculin-
ization at a fine scale. In future studies, determination of sex based
upon CTP may prove to be more accurate in determining male
versus non-male hatchling than previously thought (See Ziegler
and Olbort, 2007 and Otaño et al., 2010 for review).
The ability of MT to bias structural differentiation towards male
morphology indicates that it is not aromatizable and is, therefore, a
potent androgen. Its effective use in fish farming indicates affinity
as an androgen at the cellular level via steroid ‘swamping,’ or forcing
masculinization by changing relative concentrations of sex steroids
from estrogen rich to androgen rich while the concentrations of
estrogens stays the same (Phelps and Popma, 2000). Results
presented here indicate a potent organizational role for MT as a
sex steroid in vertebrates with temperature-dependent sex
determination.
The minimal detection of MT among experimental yolks
suggests one of two possibilities. First, a limited amount of MT
may have reached the yolk of each treatment egg. Given that
dose-dependence was not observed within yolks, but was observed
in other aspects of this study, limited crossing of MT into the egg
seems unlikely. More likely is a scenario in which we detected only
a portion of MT in yolk samples. Paitz et al. (2012) discovered the
conjugation of maternally-supplied steroid hormones within the
egg of a TSD reptile and further noted accumulation of such
hormones in the albumin. In our analysis, conjugation of experi-
mentally supplied MT may make it invisible to our assay and higher
detected concentrations may be evident in albumin as opposed to
yolk. Because we were unable to sample albumin, this possibility
awaits further testing.
5. Conclusions
Results presented here contradict two observations previously
regarded as constants in the mechanics of TSD. The efficiency of
androgens at producing males has previously been restricted to
threshold temperatures. In addition, our data contradict the
expected result of mixed-sex clutches. Instead, the production of
hermaphrodites was observed in this study. Instead of these pat-
terns, we demonstrate the experimental production of male alliga-
tors at a strong female-producing temperature, as well as
production of dose-dependent hermaphroditic individuals. This
finding is likely a result of the non-aromatizable nature of MT as
opposed to natural androgens used in prior studies. Crews
(1996), in a review of TSD mechanisms, notes the production of
male-biased clutches in turtles as a result of exogenous application
of the non-aromatizable dihydrotestosterone. The male-biased sex
ratio documented in the Tempisque Basin, Costa Rica appears
unrelated to nest thermal regimes (Murray et al., 2016). However,
MT utilized by local tilapia farms is capable of acting as a potent
androgen yielding male-biased sex ratios at hatching. If this repre-
sents the mechanism by which the population of crocodiles of the
Tempisque Basin became male biased, then we predict wild croco-
diles from this site will demonstrate exposure to MT and evidence
of hermaphroditism among females.
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