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Detection of a synthetic sex steroid in the American crocodile ( Crocodylus acutus ): Evidence for a novel environmental androgen

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  • Scales and Tail of Ohio

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Endocrine-disrupting contaminants (EDC's) are well known to alter sexual differentiation among vertebrates via estrogenic effects during development, particularly in organisms characterized by temperature-dependent sex determination. However, substances producing androgenic effects typically lack potency when tested in laboratory settings and are virtually unstudied in field settings. Here, we assay levels of a synthetic androgen, 17α-methyltestosterone (MT), in a heavily male-biased population of American crocodiles in the Tempisque River Basin of Costa Rica based on the recent hypothesis that this chemical is an EDC in developing crocodilian embryos. The presence of MT was documented in all field-collected samples of egg yolk and in plasma of all age classes in among population of crocodiles. Hatchlings exhibited higher plasma MT concentrations (102.1 ± 82.8 ng/mL) than juveniles (33.8 ± 51.5) and adults (25.9 ± 20.8 ng/mL). Among populations, crocodiles captured in the Tempisque River (62.9 ± 73.7 ng/mL) were higher in MT concentration than those from Tarcoles (13.3 ± 11.4 ng/mL) and negative controls (0.001 ± 0.0002 ng/mL). A mechanism for the bio-transport of MT and its subsequent effects is proposed.
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UNCORRECTED PROOF
Chemosphere xxx (2017) xxx-xxx
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Chemosphere
journal homepage: www.elsevier.com
Detection of a synthetic sex steroid in the American crocodile (Crocodylus acutus):
Evidence for a novel environmental androgen
Christopher M. Murraya, , Mark Merchantb, Michael Easterc, Sergio Padillad, Davinia B. Garrigóse,
Mahmood Sasa Marind, f, Craig Guyerg
aDepartment of Biology, Tennessee Technological University, PO Box 5063, Cookeville, TN 38505, USA
bDepartment of Chemistry, McNeese State University, Lake Charles, LA, USA
cScales and Tails of Ohio, Lakewood, OH 44107, USA
dPalo Verde Biological Station, Organization for Tropical Studies, Guanacaste, Costa Rica
eBiodiversity and Evolutionary Biology “Cavanilles” Institute, University of Valencia, Valencia, Spain
fInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
gDepartment of Biological Sciences, Auburn University, 331 Funchess Hall, Auburn, AL 36849, USA
ARTICLE INFO
Article history:
Received 5 December 2016
Received in revised form 3 April 2017
Accepted 4 April 2017
Available online xxx
Handling Editor: Jim Lazorchak
Keywords:
Methyltestosterone
Endocrine disrupting contaminants
Environmental androgen
Crocodylus
ABSTRACT
Endocrine-disrupting contaminants (EDC's) are well known to alter sexual differentiation among vertebrates via estro-
genic effects during development, particularly in organisms characterized by temperature-dependent sex determination.
However, substances producing androgenic effects typically lack potency when tested in laboratory settings and are vir-
tually unstudied in field settings. Here, we assay levels of a synthetic androgen, 17α-methyltestosterone (MT), in a heav-
ily male-biased population of American crocodiles in the Tempisque River Basin of Costa Rica based on the recent hy-
pothesis that this chemical is an EDC in developing crocodilian embryos. The presence of MT was documented in all
field-collected samples of egg yolk and in plasma of all age classes in among population of crocodiles. Hatchlings ex-
hibited higher plasma MT concentrations (102.1 ± 82.8 ng/mL) than juveniles (33.8 ± 51.5) and adults (25.9 ± 20.8 ng/
mL). Among populations, crocodiles captured in the Tempisque River (62.9 ± 73.7 ng/mL) were higher in MT concen-
tration than those from Tarcoles (13.3 ± 11.4 ng/mL) and negative controls (0.001 ± 0.0002 ng/mL). A mechanism for
the bio-transport of MT and its subsequent effects is proposed.
© 2016 Published by Elsevier Ltd.
1. Introduction
Pollutants, referred to as endocrine-disrupting contaminants (ED-
C's), have been shown to alter endocrine function in all vertebrate
classes (Hayes et al., 2002), affecting many organ systems and asso-
ciated processes within each system. The magnitude of this anthro-
pogenic epidemic has spurred pollutant-related operational terminol-
ogy, such as bio-indicators (Hyne et al., 2009) and sentinel species
(Milnes and Guillette, 2008). Commonly, EDC's mimic estrogens or
aromatizable androgens, or act as androgen antagonists, thus biasing
secondary sexual characteristics towards a female morphology and as-
sociated physiology (Guillette et al., 1995). Crocodilian exposure to
environmental estrogens has resulted in hermaphroditic males (fol-
licular growth within seminiferous tubules) or ‘super’ females [rapid
and poorly regulated follicular development, (Guillette et al., 1994)].
To date, the phenomenon of biasing sexual differentiation using an-
drogens is known from lab settings only around pivotal temperatures,
or the temperatures at which sex differentiations changes between
Corresponding author.
Email addresses: cmmurray@tntech.edu (C.M. Murray); mmerchant@mcneese.
edu (M. Merchant); m.easter05@gmail.com (M. Easter); spa_sergio@hotmail.
com (S. Padilla); davinia.beneyto@uv.es (D.B. Garrigós); msasamarin@gmail.
com (M. Sasa Marin); guyercr@auburn.edu (C. Guyer)
male and female (Crews et al., 1994); however, environmental andro-
gens are virtually unknown from field settings, probably as a result
of the aromatizable nature of testosterone and it's structural analogs
that contribute to rapid conversion (Wibbels and Crews, 1995). In lim-
ited fashion, paper-mill effluent was identified as having androgenic
substances associated with the masculinization of female mosquitofish
(Parks et al., 2001), providing support for the only known environ-
mental androgen.
17α-methyltestosterone (MT) is a synthetic androgen used widely
in the commercial farming of fish (Phelps and Popma, 2000). It is
commonly applied to fish fry to divert sexual differentiation away
from female development and towards male, the more profitable sex
in most cases. The suggested aquaculture concentrations for the treat-
ment of 300,000 fry is 60 mg/kg feed (Popma and Green, 1990), al-
though the strict application of this concentration among facilities
and countries is unknown. The half-life of MT in water and soil is
short, suggesting that MT is unlikely to be an EDC. However, the
persistence of this compound in a hydrophobic environment, such as
lipids, is poorly known (Phelps and Popma, 2000; Gupta and Acosta,
2004; Murray et al., 2016a) while potential for negative ecological ef-
fects have been suggested (Mlalila et al., 2015). Use of this synthetic
androgen is a regular practice in tilapia farming, perhaps the most
prominent New World fish industry, where it is applied liberally via
feed (Gupta and Acosta, 2004). Containment regulations of the com-
pound, either by water or animal exchange with the surrounding envi
http://dx.doi.org/10.1016/j.chemosphere.2017.04.020
0045-6535/© 2016 Published by Elsevier Ltd.
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2 Chemosphere xxx (2017) xxx-xxx
ronment, vary widely by region and detailed studies of MT fate in the
environment are not available (Phelps and Popma, 2000).
A uniquely male-biased sex ratio of American crocodiles (Croco-
dylus acutus) in the Tempisque drainage of Costa Rica has been pre-
viously described (Bolaños-Montero, 2012; Murray et al., 2015). This
is one of multiple reports that describe recent male-biased crocodil-
ian populations throughout Central America (Charruau et al., 2005;
Escobedo-Galván, 2008). Specific to the Tempisque drainage, ther-
mal data from nests demonstrate that the male-bias is not a function
of temperature effects on hatchling sex determination (Murray et al.,
2016b). Additionally, exposure of crocodilian eggs to MT generates
male hatchlings when incubated at a temperature that should produce
only female offspring (Murray et al., 2016a). The goal of this study is
to determine if MT is present in eggs as well as free-ranging hatchling,
juvenile and adult crocodiles on the North Pacific versant of Costa
Rica. In addition, a mechanism for its physiological action in the Tem-
pisque system is proposed.
2. Methods
2.1. Sample collection
Between January 2013 and January 2016, we hand captured Amer-
ican crocodiles at Palo Verde National Park and surrounding areas.
Juveniles and adults were captured in four locations within the Park
and three outside of it. El Humedal (10.341128°, −85.343311°), La
Bocana (10.335950°, −85.278837°), Nicaragua Lagoon (10.328573°,
−85.273901°) and Varillal Lagoon (10.403402°, −85.358544°) are all
seasonally flooded wetlands within the Park. Varillal Lagoon is in
very close proximity to the Tempisque River while the other wetlands
are all relatively equidistant and farther from the permanent Temp-
isque and Bebedero waterways (Fig. 1). We also surveyed waterway
banks of a drainage canal along the northwestern border of Palo Verde
National Park (10.393632°, −85.396221°), as well as the Bebedero
River near its junction with the Tempisque River (10.327898°,
−85.206055°), in order to find fresh nests. These banks were then
surveyed frequently enough to assure that hatchlings were captured
within days of hatching. Other locations sampled included the Tar-
coles River south of Guanacaste (9.800419°, −84.606156°). Tarcoles
was hypothesized to be a negative control population, lacking MT ex-
posure, based on its distance from aquaculture facilities. For each indi-
vidual sampled we extracted 0.5–2.5 mL of whole blood (18–27 gauge
needles, depending on the size of the individual) from the spinal vein.
Needles and syringes were flushed with 0.5% heparin solution to pre-
vent coagulation. Whole blood was kept on ice for no more than 2 h
before being centrifuged so that plasma supernatant could be removed
and frozen (−20 °C).
During the 2015 nesting season, six eggs were collected from five
nests late in the first trimester or early in the second trimester of in-
cubation. Eggs were obtained from the waterway banks of a drainage
canal along the northwestern border of Palo Verde National Park.
Canal banks were monitored multiple times per week as part of an-
other study to ensure accuracy of deposition and hatching date to
within two days. Eggs were frozen at −20 °C. Upon capture, sex was
determined for individuals via inspection of the secondary sex organ.
Individuals were diagnosed as male is the clitero-penis exhibited all
of the following characteristics: bi-lobed structure, extensive vascu-
larization, and length extending the length of the vent (Murray et al.,
2015).
Fig. 1. A. Map of general sampling localities in Costa Rica including the Palo Verde
National Park region (Area 1) and Tarcoles River [Area 2, (9.800419°, −84.606156°).
B. Map of Area 1 including egg sampling locality [A (10.393632°, −85.396221°)],
hatchling sampling localities [A, B (10.327898°, −85.206055°)], Varillal Lagoon [C,
(10.403402°, −85.358544°) Humedal Palo Verde [D, (10.341128°, −85.343311°)], La
Bocana [E, (10.335950°, −85.278837°)] and Nicaragua Lagoon [F, (10.328573°,
−85.273901°)]. Specific tilapia farm localities are not included to protect privacy of the
organizations.
2.2. Sample preparation and analysis
Steroid hormones were extracted from egg yolk and plasma us-
ing a 3:2 solution (volume: volume) of ethyl acetate and hexane, re-
spectively. Samples were dried under vacuum at 25 °C and the re-
sulting residue was dissolved in 100 μL assay buffer supplemented
with 10 μL of dimethyl sulfoxide (DMSO) to encourage dissolution.
We quantified 17α-methyltestosterone using commercially available
sandwich ELISA kits (MaxSignal®methyltestosterone kit, Bioo Sci-
entific, Austin, TX). This competitive enzyme immunoassay reliably
quantifies methyltestosterone concentrations in feed, fish, shrimp and
meat from skeletal muscle, urine and serum samples with a high re-
covery rate (>80%), high sensitivity (0.1 ng/g) and low detection limit
(0.3 ng/mg). Cross-reactivity with testosterone was listed as <0.1%
and samples for 1 kit were not analyzed in duplicate. However, fif-
teen samples from kit 1 were also run in kit 2 to test the variabil-
ity within samples among kits. The average difference of sample
UNCORRECTED PROOF
Chemosphere xxx (2017) xxx-xxx 3
MT concentrations between kits was 27% (3.52 ng/mL). All samples
in kit 2 were run in duplicate. Optical density was determined using
a Benchmark Plus microtiter plate spectrophotometer (Bio-Rad, Her-
cules, CA) at 450 nm. Six negative control samples were added to the
analysis to detect false positive readings. These included six captive
American alligators from McNeese State University, Lake Charles,
LA.
2.3. Statistical analysis
MT concentration data did not meet the assumptions for the
Shapiro-Wilk test of normality. A Wilcoxon signed rank test was per-
formed to assess difference in means between yolk MT concentrations
of alligator eggs given doses of MT known to cause sex reversal from
female to male (Murray et al., 2016a) and wild yolk concentrations
collected for crocodiles sampled during this study. Concentrations of
MT in blood plasma and eggs were compared among crocodile co-
horts using Kruskal-Wallis H Test. Dunn's post-hoc test was used to
elucidate differences among cohorts. Among localities, all Palo Verde
field sites were pooled and plasma MT concentrations were compared
to Tarcoles samples and negative controls via a Kruskal-Wallis H
Test. Dunn's post-hoc test was used to elucidate differences among
locations. This test determined whether exposure is spatially acute to
the Tempisque or widespread. Within cohorts, Wilcoxon signed rank
tests were used to assess statistical differences in MT concentrations
between morphological sexes. Standard deviation was used as a vari-
ability estimator and is reported in association with treatment means.
All statistical analyses were performed in program R (R Development
Core Team, 2011).
2.4. Spike-recovery
Efficiency for the extraction method above was determined by
spiking 300 μL egg yolk samples to 1, 15, and 50 ng/mL with MT.
Each sample was spiked and extracted in triplicate, and the results
compared to un-spiked samples. The samples were vortexed vigor-
ously for 1 min to ensure the absorption of the MT spike into the
yolk prior to extraction. Recovery concentrations were measured to be
0.82 ± 0.04, 14.6 ± 0.9, 56.2 ± 8.1, respectively, for 1, 15, and 50 ng/
mL of MT. Likewise, fresh plasma samples from American alligators
were spiked 1, 15, and 50 ng/mL MT. Recovery of spiked samples
were found to be 0.9 ± 0.1, 16.0 ± 2.0, 52.4 ± 4.2, respectively, in the
plasma samples. For all yolk and plasma samples that were not spiked,
the concentrations of MT were determined to be below the detection
limit of 0.1 ng/mL.
3. Results
MT concentrations in yolk sampled from Crocodylus acutus eggs
in natural nests (n = 6) did not differ from concentrations known from
yolk of alligator eggs dosed with MT concentrations known to cause
masculinization in the lab (n = 7) (Fig. 2, [Murray et al., 2016a]). MT
was detected in every egg yolk sample analyzed.
Hatchlings, juveniles and adults differed significantly in plasma
MT concentrations (χ2= 29.52, DF = 3, p < 0.0001; Fig. 3). Hatch-
lings exhibited higher plasma MT concentrations (102.1 ± 82.8 ng/
mL, n = 35) than juveniles and adults, who did not differ from each
other 33.8 ± 51.5 (n = 32) and 25.9 ± 20.8 ng/mL (n = 10) respec-
tively). All cohorts were significantly higher than negative control
concentrations (0.001 ± 0.0002 ng/mL, n = 6). Individuals were iden-
tified to sex via inspection of the secondary sex organ and MT con-
centrations did not differ between males and females within any co
Fig. 2. Bar graph comparing yolk MT concentrations between experimental eggs from
Murray et al. (2016a) and wild eggs collected for this study. MT concentrations did not
differ indicating the potential for biasing sexual differentiation towards males in wild
eggs in Palo Verde. Standard deviation is used as the variability estimator.
Fig. 3. Bar graph showing variation in plasma MT concentrations among crocodile
age cohorts with associated standard deviation (χ = 29.52, df = 3, p < 0.0001). Coding
above bars indicate post-hoc Dunn's statistical difference and sample size, respectively.
hort. All plasma samples contained a measureable level of MT higher
than the highest negative control concentration and MT concentrations
of hatchlings were much greater than those in yolk of eggs producing
those hatchlings (mean = 0.04 ± 0.19 ng/g).
Concentrations of plasma MT differed significantly among collec-
tion locations (χ2= 14.3, df = 3, p = 0.002) (Fig. 4). Tempisque sam-
ples (62.9 ± 73.7 ng/mL, n = 73) were higher in MT concentration
than negative controls (0.001 ± 0.0002 ng/mL, n = 6) however not sta-
tistically different than Tarcoles (13.3 ± 11.4 ng/mL, n = 3).
4. Discussion
Analysis of crocodile egg yolk and blood plasma from samples col-
lected in the Tempisque Basin and Tarcoles River indicated that ani-
mals experience heavy MT exposure and/or retain MT as they grow.
The presence of MT in blood plasma of juveniles and adults, how-
ever, suggests that exposure to this synthetic hormone is chronic. If
exposure to MT were acute within individuals, a steroid of this nature
would be filtered by the liver and stored (Guillette et al., 1995), mak-
ing acute past exposure of MT undetectable in plasma. Further, degra-
dation of MT in the environment is rapid (Gupta and Acosta, 2004)
potentially lending support for a bio-transporter.
UNCORRECTED PROOF
4 Chemosphere xxx (2017) xxx-xxx
Fig. 4. Bar graph showing variation in plasma MT concentrations among crocodile sam-
pling localities with associated standard deviation (χ = 14.3, df = 3, p = 0.002). Coding
above bars indicate post-hoc Dunn's statistical difference and sample size, respectively.
Our observation that yolk MT concentrations do not differ between
wild eggs and those experimentally dosed with MT (Murray et al.,
2016a) suggests that Palo Verde crocodile eggs contain MT at levels
known to produce male offspring despite incubation at female-produc-
ing temperatures (Murray et al., 2016b). In fact, of 357 eggs monitored
at Palo Verde, 225 were estimated to produce females and 132 were
estimated to produce males, based on temperature alone (Murray et al.,
2016b). Data collected by Murray et al., 2016a suggests that 60% of
the females would convert to males in the presence of MT. Thus, 75%
of eggs incubated at field temperatures known from Palo Verde would
produce males in the presence of known levels of MT in the yolk. This
value approaches the 80% male bias for hatchling crocodiles recorded
at Palo Verde (Murray et al., 2015).
Concentrations of plasma MT for adults at Palo Verde do not dif-
fer from levels measured at Tarcoles samples. Initially, we thought
that crocodiles at Palo Verde were affected by tilapia farms located
just outside the Park boundary. Therefore, the Tarcoles population was
sampled to serve as an uncontaminated control site. However, our re-
sults instead demonstrate either that MT is more widespread in Costa
Rica or that Palo Verde crocodiles are dispersing south. The Tarcoles
River drains San Jose, the major industrial and residential center of
Costa Rica. It is known to be a highly polluted river, accumulating and
concentrating waste from many sources (Rainwater et al., 2011) and
may very well be accumulating MT input from other pollution sources
or smaller unknown fish farming operations. Further, restaurants and
tourists surround the site, and fish waste deposited in the river may be
frequent despite not being directly downstream from a tilapia farm.
Among cohorts, eggs collected after one-third of incubation ex-
hibit concentrations of MT that are barely detectable, while hatch-
lings exhibit extremely high concentrations of MT in their blood
plasma without sufficient time for exposure after hatching. Juveniles
and adults exhibit lower MT concentrations than hatchlings. This may
be indicative of less frequent exposure and/or more rapid utiliza-
tion or storage of this exogenous androgen in growing or reproduc-
tively mature individuals. Based on these results we propose a likely
mechanism for chronic crocodile exposure to MT in the Tempisque
Basin. Initially, tilapia bio-accumulate MT in adipose tissue, a com-
mon storehouse for cholesterol-based exogenous steroids (Guillette
et al., 1995), from the supplied feed. Retention of MT in exposed
tilapia has been documented at 67 ng/g fish for ≥21 days (Goudie
et al., 1986), a potent concentration for endocrine-disrupting effects
(Murray et al., 2016a). Since the escape of tilapia from farms to sur-
rounding natural waterways is observable and tilapia are now com-
mon the Tempisque Basin, and crocodiles readily move in and out of
tilapia farm ponds, tilapia has become an available food source for
crocodiles in the system. Crocodiles, like the tilapia, sequester the MT
acquired via the fish consumption, and must also use adipose tissue
for storage after plasma circulation. Some processes during folliculo-
genesis likely mobilize MT stored in adipose tissue during the prepa-
ration and deposition of yolk, allowing deposit of MT in eggs (Paitz
and Bowden, 2010). Such processes have been suggested for andro-
gens (Guillette et al., 1995) and the detectability of MT in hatchlings
and documented masculinizing effects (Murray et al., 2016a) imply
that MT is not aromatizable in Alligator mississippiensis or Crocody-
lus acutus, or is aromatized at a low rate. Supplied doses are known
to bias sexual differentiation in crocodilians (Murray et al., 2016a)
and act as an environmental androgen within the eggs in the Temp-
isque basin, resulting in the male-biased sex ratio previously recov-
ered (Murray et al., 2015).
Of interest is the low concentration of MT detected in egg yolk and
high concentration detected in hatchling plasma before the hatchlings
were large enough to consume tilapia. The persistence of MT in water
and soil is on the order of hours or days (Phelps and Popma, 2000),
so environmental exposure without a bio-transporter is unlikely. Be-
cause the concentrations of MT recovered in wild eggs in this study is
comparable to the concentrations of experimentally-dosed crocodilian
eggs from (Murray et al., 2016a), it is likely that much MT persists
within developing eggs but is unrecognizable in our analysis. Paitz
and Bowden (2011) and Paitz et al. (2012) posit that maternally-de-
rived steroid hormones (in their case, estradiol) are conjugated to a
water-soluble sulphates in painted turtles, a reptilian species that also
exhibits TSD. Such conjugates are transported from yolk to embryo
and utilized later in development, while the intended embryonic re-
sponse to the modified steroid is the same as if it lacked conjugation.
When experimentally treated, conjugation of sex steroid hormones can
occur within hours (Paitz and Bowden, 2015). Such a process would
make MT undetectable to our analyses but result in high hatchling
concentrations when (and if) the steroid is biologically reactivated.
Alternatively, it may be a result of low extraction efficiency of MT
from the hydrophobic environment of the yolk. However, this seems
very unlikely given that the extraction of androgens from avian eggs
has been shown to be efficient with the use of hexane as a solvent
(Von Engelhardt and Groothuis, 2005).
5. Conclusions
Results presented here, in combination with previous experimental
studies, suggest that 17α-methyltestosterone (MT) acts as a non-arom-
atizable environmental androgen on crocodiles in the Tempisque and,
likely, surrounding basins in Costa Rica and is responsible for a
male-biased sex ratio. Interestingly, because the short environmen-
tal half-life of MT limits exposure to bio-transport and accumulation,
and MT concentrations are highly detectable in juveniles and adults
as well as newborn hatchlings, MT likely follows a rare pathway for
maternally supplied exogenous sex steroids or their mimics in devel-
oping embryos. Further hypothesis testing regarding the physiologi-
cal mechanism of maternal supply, and bio-reactivation during late
development, is needed. Specifically, analysis of MT concentration
among yolking follicles within female crocodiles is critical to our pro-
posed mechanism. Additionally, study of the travel and conjugation
of MT within eggs among yolk, albumin, and embryo is needed. En-
vironmentally, it is necessary to quantify MT in tilapia and other po-
tential crocodile food sources to isolate the mechanism of bioaccu
UNCORRECTED PROOF
Chemosphere xxx (2017) xxx-xxx 5
mulation. Lastly, the sex ratios and endocrine profiles of other verte-
brates in the area require quantification to fully understand the breadth
and mechanism of effects of this environmental androgen. If the pro-
posed mechanism is isolated to diet, then predatory fish and piscivorus
birds may likely be affected, either in sex ratio and/or endocrine pro-
files. However, if MT is widely present in the food chain then one may
expect a male-bias in the TSD mud turtle population or other aquatic
reptiles and amphibians. Testing of such hypotheses is critical in falsi-
fying the proposed model.
Acknowledgements
We thank the Palo Verde Biological Station staff and MINAET
for logistic support and permitting as well as J. Bolaños for guidance
and logistic support. We also acknowledge J. Goessling for proof-
reading and A. Cooper for logistic support and thank Vicerrectoría
de Investigación Universidad de Costa Rica VI 741-B5-270 and the
Organization for Tropical Studies for permitting assistance and fund-
ing (OTS Fund 507). The authors declare they have no actual or poten-
tial competing financial interests. Institutional guidelines for the care
and use of animals were followed under approved IACUC proposal
2013–2247.
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... American crocodile populations have been reported to have male-skewed sex ratios in some Costa Rican drainages, despite living under female-producing temperature regimes [138][139][140]. The levels of accumulated MT in all the field collected eggs and in the plasma of wild crocodile hatchlings were similar to those that found in experimentally masculinized hatchlings, even when eggs were exposed to a female-producing temperature [139]. ...
... The levels of accumulated MT in all the field collected eggs and in the plasma of wild crocodile hatchlings were similar to those that found in experimentally masculinized hatchlings, even when eggs were exposed to a female-producing temperature [139]. These results strongly suggest that the observed natural bias could result from the masculinizing effects of this synthetic androgen [139][140][141]. ...
... A possible mechanism to explain chronic exposure to MT and subsequent effects on crocodile sex ratios in the Tempisque and surrounding basins in Costa Rica relies on tilapia as a possible biotransporter of the environmental androgen [140]. Since 2004, Costa Rica has become an important Nile tilapia producer, with 15,500 tons in 2014 and 25,000 tons in 2013 [12]. ...
Chapter
Tilapias are the second largest group of fish produced worldwide, due to their great plasticity and ideal aquaculture traits, particularly the Nile tilapia. On most tilapia farms, sex control is necessary to increase profitability, due to early and continuous reproduction and female mouth‐brooding, and to benefit from the males’ faster growth rate. This review presents the different ways to produce all‐male populations by genetic approaches, such as the use of YY males (or ZZ females in the blue tilapia), and hormonal or high temperature sex reversal treatments, presenting the advantages and drawbacks of each method, as well as protocols for their use. Androgen treatment is still the predominant means to generate monosex male offspring, due to its simplicity, efficiency and price. The hormone amounts currently used worldwide, and its consequences on biodiversity, are discussed, regarding the sustainability of tilapia farming and taking into account growing consumer awareness. Results regarding 17α‐methyltestosterone (MT) accumulation and the possible use of MT‐degrading bacteria are discussed. More sustainable alternative methodologies can be potentiated. Current research status on the sex‐determining loci in Nile, blue, Mozambique and black chin tilapias is presented. Finally, we show the available genetic and phenotypic sex markers that can accelerate progeny testing, rapidly identifying particular genotypes and phenotypes of interest such as YY males or ZZ females, as well as being important for selection programs.
... American crocodile populations have been reported to have male-skewed sex ratios in some Costa Rican drainages, despite living under female-producing temperature regimes [138][139][140]. The levels of accumulated MT in all the field collected eggs and in the plasma of wild crocodile hatchlings were similar to those that found in experimentally masculinized hatchlings, even when eggs were exposed to a female-producing temperature [139]. ...
... The levels of accumulated MT in all the field collected eggs and in the plasma of wild crocodile hatchlings were similar to those that found in experimentally masculinized hatchlings, even when eggs were exposed to a female-producing temperature [139]. These results strongly suggest that the observed natural bias could result from the masculinizing effects of this synthetic androgen [139][140][141]. ...
... A possible mechanism to explain chronic exposure to MT and subsequent effects on crocodile sex ratios in the Tempisque and surrounding basins in Costa Rica relies on tilapia as a possible biotransporter of the environmental androgen [140]. Since 2004, Costa Rica has become an important Nile tilapia producer, with 15,500 tons in 2014 and 25,000 tons in 2013 [12]. ...
... Other sources include livestock waste runoff (Yost et al., 2013), effluent from human wastewater (Belfroid et al., 1999;Sun et al., 2013), pulp and paper mills (Jenkins et al., 2001;Jenkins et al., 2003), and crop production in intensive agricultural areas (crops are sources of naturallyoccurring phytoestrogens) (Kolpin et al., 2010). Disruption of the endocrine system by environmental EDCs is an established phenomenon in wildlife and plants (Adeel et al., 2017;Murray et al., 2017;UNEP/ WHO, 2012). Evidence of adverse effects on human reproductive and thyroid systems is also growing (Annamalai and Namasivayam, 2015;UNEP/WHO, 2012), and EDCs are hypothesized risk factors for some hormone-related malignancies (Adeel et al., 2017;Street et al., 2018). ...
... They have a poor organoleptic quality, because they are killed after maturation. Environmental risk requires more attention [233], knowing that MT has already been detected in the aquatic environment, where it can act as an endocrine disruptor [234], even at low doses [235]. ...
Book
The Salmonidae family comprises three sub‐families (Coregoninae, Thymallinae and Salmoninae), with 11 genera and about 66 species. Salmonid farming has grown considerably in the last fifty years, with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) being the main farmed species, and with an increasing production of large size fish that has required the development of all‐female or sterile populations. Salmonids are gonochoristic fishes with a genotypic sex determination (GSD) system, classically described as male heterogametic (XX/XY). Some temperature effects (GSD + TE) have been reported in a limited number of cases. The master sex determining gene, sdY (sexually dimorphic on the Y chromosome), has been characterized in rainbow trout, and is conserved in many salmonid species. sdY is expressed very early (around hatching in rainbow trout) during gonadal differentiation, long before the initiation of histological sex differentiation. In addition, other genes exhibit a sex‐dimorphic expression, particularly those involved in steroid synthesis, leading to a hypothesis in favor of a key role of estrogens in inducing and maintaining ovarian differentiation in salmonids. Sex control in salmonids is mostly carried out by producing XX neomales that give rise to all‐female populations after crossing with wild‐type females. These XX neomales are usually produced by masculinization of females, using an androgen treatment (the most commonly used being 17α‐methyltestosterone) administered in the food from the first feeding of larvae. Limits and concerns of such methods are discussed, and other potential approaches are foreseen.
... Moreover, environmental androgens are detrimental to the reproductive development of aquatic organisms, affecting the structure and function of microbial communities [5][6][7][8]. Environmental androgens have a wide range of sources [9][10][11][12][13][14] and can migrate to subterranean and surrounding environments through infiltration and runoff [15,16], thereby increasing the risk and scope of contamination. ...
Article
Full-text available
: In the present study, the accumulation and degradation of testosterone by Chlorella vulgaris were studied. The results showed that C. vulgaris has a significant ability to eliminate testosterone by bioaccumulation and biodegradation, and during the 96 h experimental period, the data demonstrated that the accumulation of testosterone followed a sigmoidal accumulation pattern. At the end of the experiment, the bioconcentration percentages of testosterone by C. vulgaris in the high-concentration group and the low-concentration group were 11.49 ± 2.78% and 40.10 ± 1.98%, respectively, and the biodegradation percentages of testosterone were 69.64 ± 4.33% and 42.48 ± 1.92%, respectively. The rate of biodegradation of testosterone by C. vulgaris mainly depended on the relative initial concentration of testosterone. When the relative initial concentration of testosterone increases, the degradation may gradually change from zero-order kinetics to second-order kinetics.
... The dominant fish being caught and consumed by gharials participating in these feeding aggregations is tilapia, and typically many fish are consumed by individual animals, on any given day when conditions are favorable Kumar 2013, 2016, andunpublished observations). Elsewhere, the production of a strong male-bias in populations of the American crocodile (Crocodylus acutus) has been attributed to a novel environmental androgen added to fish feed (methyl testosterone) for rapid growth in Tilapia aquaculture operations adjacent to the natural riverine habitats of the affected crocodiles (Murray et al. 2017). The possible effects of tilapia consumption, a relatively new food source for Chambal gharial, have not yet been studied, but may be noteworthy, with reference to the recent mass die-off, as well as to environmental influences on sex determination (Lang and Andrews 1994;Andrews and Whitaker 2004), growth and overall health. ...
Article
Full-text available
Gharial were historically distributed throughout the major channels of the Indus, Ganges, Mahanadi, Brahmaputra-Meghna and possibly Irrawaddy drainages, to elevations of <500 m, an estimated combined linear river distance of >20,000 km, or an historic occupancy area of 80,000 km² (using Red List standard 4 km² resolution). The species is currently extirpated from the Indus, Irrawaddy and most rivers and tributaries of the Ganges and Brahmaputra-Meghna systems, but persists in fourteen sites within the Ganges drainage (6 major and 8 minor), with confirmed breeding at only five locations (Table 1 in the Supplementary Information). Surveys and counts in 2010-2017 indicate an adult global metapopulation conservatively estimated at 650 (median) with a range of 300-900. The largest and most populous location, the protected National Chambal Sanctuary in north India, spans 625 river km, with approximately 500 mature adults, comprising 77% of the global total, producing >410 nests annually, or >86% of the global total. Five other locations reporting breeding are small and highly disturbed. The remaining 8 minor locations together contain less than 10% of the world population (<50 mature adults), with no recent breeding. During the next decade, gharial will likely be extirpated from some of the minor/non-breeding sites, including three sanctuaries in India designed for their protection (Son, Ken, Satkosia Gorge), as well as the Padma-Jamuna, Brahmaputra-Meghna, and Bhagirathi-Hooghly drainages, based on the infrequent sightings in these regions. Generation time is estimated at 25 years giving a period of decline of 75 years or since 1943. Cause of declines have been principally dams and barrages disrupting river hydrology, mortality in fishing nets, and historically, unregulated hide-hunting. Current serious threats include major water control and extraction activities, mortality in fishing gear, and increased anthropogenic river-bank disruptions, especially sand mining and boulder removal. These threats are known, continuing and not reversible, therefore criterion A2 applies. The expansion of threats and major declines have intensified since the 1950s, and continue presently with increasing demand for river resources. Past population levels are inferred to be >20,000 adult gharialglobally. Calculating across the range of current and past population estimates, exponential declines are 94% or greater. Even a very conservative calculation of 5,000 in 1943 and 1,000 currently gives a decline of 80%. Decline in 3 generations is confidently inferred to exceed the criterion A2bc 80% for Critically Endangered. Toxins/pollutants were strongly suspected to play a role in the death of >110 gharial in the size range of 2-4m during the winter of 2008-2009 on the lower Chambal (Whitaker et al. 2008), and possibly the loss of additional gharial in 2012 (<10-15 individuals; Nair et al. 2013). This initial major loss suggested a species-specific sensitivity to whatever caused the deaths, which were localized geographically, most occurring rapidly within 2-15 weeks. These deaths were related to articular and visceral gout, related to kidney failure, associated with low ambient temperatures. Despite the absence of a re-occurrence, the susceptibility of gharial to whatever resulted in gout in the first instance warrants the application of criterion A2e. Extent of occurrence (EOO) exceeded 80,000km² historically. Area of occupancy (AOO) is generously estimated for the six major locations with sizeable resident populations at 4,400 km², a reduction of 94%. A population estimate of 300-900 mature individuals meets criterion C1 for Endangered if we infer that the decline is continuing at a rate of at least 20% in two generations/50 years i.e. since 1967. Gharial meets criterion D1 Vulnerable (population size <1,000 mature individuals). The species does not meet criteria E.
... They have a poor organoleptic quality, because they are killed after maturation. Environmental risk requires more attention [233], knowing that MT has already been detected in the aquatic environment, where it can act as an endocrine disruptor [234], even at low doses [235]. ...
Chapter
The Salmonidae family comprises three sub‐families (Coregoninae, Thymallinae and Salmoninae), with 11 genera and about 66 species. Salmonid farming has grown considerably in the last fifty years, with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) being the main farmed species, and with an increasing production of large size fish that has required the development of all‐female or sterile populations. Salmonids are gonochoristic fishes with a genotypic sex determination (GSD) system, classically described as male heterogametic (XX/XY). Some temperature effects (GSD + TE) have been reported in a limited number of cases. The master sex determining gene, sdY (sexually dimorphic on the Y chromosome), has been characterized in rainbow trout, and is conserved in many salmonid species. sdY is expressed very early (around hatching in rainbow trout) during gonadal differentiation, long before the initiation of histological sex differentiation. In addition, other genes exhibit a sex‐dimorphic expression, particularly those involved in steroid synthesis, leading to a hypothesis in favor of a key role of estrogens in inducing and maintaining ovarian differentiation in salmonids. Sex control in salmonids is mostly carried out by producing XX neomales that give rise to all‐female populations after crossing with wild‐type females. These XX neomales are usually produced by masculinization of females, using an androgen treatment (the most commonly used being 17α‐methyltestosterone) administered in the food from the first feeding of larvae. Limits and concerns of such methods are discussed, and other potential approaches are foreseen.
... The dominant fish being caught and consumed by gharials participating in these feeding aggregations is tilapia, and typically many fish are consumed by individual animals, on any given day when conditions are favorable Kumar 2013, 2016, andunpublished observations). Elsewhere, the production of a strong male-bias in populations of the American crocodile (Crocodylus acutus) has been attributed to a novel environmental androgen added to fish feed (methyltestosterone) for rapid growth in Tilapia aquaculture operations adjacent to the natural riverine habitats of the affected crocodiles (Murray et al. 2017). The possible effects of tilapia consumption, a relatively new food source for Chambal gharial, have not yet been studied, but may be noteworthy, with reference to the recent mass die-off, as well as to other environmental influences, e.g., on sex determination (Lang & Andrews 1994;Andrews & Whitaker 2004), growth, and overall health. ...
Article
Full-text available
Reason(s) for Change in Red List Category from the Previous Assessment: Status is unchanged, but the criteria have been modified based on recent information by removing criterion C1 and adding criterion A2e. This species is the sole living representative of the family Gavialidae, which represents an ancient third lineage in the order Crocodylia (in addition to Alligatoridae and Crocodylidae). Therefore, gharial extinction would eliminate the only remaining representative of an Archosaur group related to birds and dinosaurs.
Article
Full-text available
American Crocodile Crocodylus acutus has most recently been assessed for The IUCN Red List of Threatened Species in 2020. Crocodylus acutus is listed as Vulnerable under criteria A2cd.
Chapter
Wildlife is exposed to a diverse range of natural and man-made chemicals. Some environmental chemicals possess specific endocrine disrupting properties, which have the potential to disrupt reproductive and developmental process in certain animals. There is growing evidence that exposure to endocrine disrupting chemicals plays a key role in reproductive disorders in fish, amphibians, mammals, reptiles and invertebrates. This evidence comes from field-based observations and laboratory based exposure studies, which provide substantial evidence that environmental chemicals can cause adverse effects at environmentally relevant doses. There is particular concern about wildlife exposures to cocktails of biologically active chemicals, which combined with other stressors, may play an even greater role in reproductive disorders than can be reproduced in laboratory experiments. Regulation of chemicals affords some protection to animals of the adverse effects of exposure to legacy chemicals but there continues to be considerable debate on the regulation of emerging pollutants.
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Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic development, 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 influences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For example, 17-αmethyltestosterone (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 ecotoxicological 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).
Article
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A male-biased sex ratio of 3:1 has been reported for a population of American crocodiles (Crocodylus acutus) in the Tempisque River Basin, Guanacaste, Costa Rica. If confirmed, this would constitute one of the largest male-biased sex ratios reported for any population of a member of the genus Crocodylus. Here, we examine the aforementioned population of C. acutus and report on sex ratios of hatchling, juvenile, and adult age classes within a sample of 474 crocodiles captured in the Tempisque Basin between May 2012 and June 2014. Hatchling sex ratio is exceptionally male biased (3.5:1), an imbalance that is maintained in juveniles but is reduced in adults (1.5:1). Mark–recapture data document that juvenile males disperse from the study site, potentially to avoid competition, a process that reduces male bias in the adult age class. An increased role of males in human–crocodile conflict may be a result of juvenile males dispersing to human-inhabited areas.
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Vertebrate embryos develop in the presence of maternally derived steroids. While these steroids can influence development, embryonic enzymes are thought to buffer some steroid sensitive processes, such as gonadal differentiation, from the effects of maternal steroids. Many of these same enzymes may also buffer the embryo from chemicals present in the environment, but this may alter their capacity to metabolize maternal steroids. Here, we characterized the ability of red-eared slider (Trachemys scripta) embryos to metabolize oestrone immediately following oviposition and tested whether a prevalent environmental chemical, Bisphenol A (BPA), would affect the in ovo conversion of oestrone to oestrone sulfate. We found that tritiated oestrone applied at the time of oviposition is mostly converted to oestrone sulfate within 6 h. However, when BPA is present, that conversion is inhibited, resulting in elevated oestrone levels. Our finding of rapid in ovo metabolism of steroids suggests that maternally derived enzymes are present in the egg and can alter embryonic exposure to exogenous chemicals. The disruption of this metabolism by BPA demonstrates how environmental chemicals might change embryonic exposure to endogenous substances within the egg. Taken together, these findings highlight the dynamic nature of the early endocrine environment in developing vertebrates. © 2015 The Author(s) Published by the Royal Society. All rights reserved.
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In recent years, all-male cultures of Nile tilapia (Oreochromis niloticus) have been the most preferred mode of production in aquaculture industry. All-male individuals achieve higher somatic growth rate and shut high energy losses associated with gonadal development and reproduction. The economic advantages of culturing all-male tilapia have led to the development of procedures for producing unisex cultures, using 17α-methyltestosterone (MT). Despite widespread use of the MT in tilapia farming, the implications of hormone treatment in relation to human health and the environment have raised a number of concerns in the scientific community. In this review, the hormonal application processes, economic and ecological significance of MT, food safety and residual MT, comparative uses of steroids in aquaculture, animal husbandry, and medicine have been briefly reviewed for regulatory guidelines, and finally, future research perspectives have been addressed. The review can be used as policy-making guidelines in aquaculture framework development as can be emphasized in African continent, among others. The most important conclusion to draw is that the quantity of MT used in conventional practice is large compared to the actual dose required for sex reversal, fish produced are safe for human consumptions, and the environmental hazards should be further emphasized.
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Evaluation of population status of Caiman crocodilus (Linnaeus, 1758) in Caño Negro Wildlife Refuge started in 1986. However, only information related with abundance and size of the population is known. The objective of this study was to evaluate population structure and sex ratio of C. crocodilus. We captured 145 caimans in 22 nocturnal spotlight surveys from May/2004 to May/2005. Most of caimans were less than 60 cm in length and between 150 to 180 cm. The average sex ratio was 1:6.06 female/male, being one of the highest differences reported for this species. If this sex ratio persists, there is going to be a decrease in the population viability in a short and long term period.
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Tilapias are an important component of subsistence fisheries for thousand of years but have gained prominence in recent years in areas where they are not endemic. Introductions of better performing tilapia species/strains and development of techniques to manage unwanted reproduction have spurred significant developments that led to success in tilapia farming. Of the 70 species of tilapias, 9 are used in farming and of these, Nile tilapia (Oreochromis niloticus) is the main cultured species and responsible for the significant increase in global tilapia aquaculture production. Tilapia contributed 1.27 million metric tons in 2000 or 3.57% of global aquaculture production. The major producing countries are China, Egypt, Thailand, Philippines and Indonesia.
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Wildlife species have been recognized as sentinels of environmental health for decades. In fact, ecological data on various wildlife populations provided the impetus for banning some organochlorine pesticides over the last few decades. Alligators are important sentinels of ecosystem health in the wetlands of the southeastern United States. Over the last 15 years, a series of studies have demonstrated that environmental exposure to a complex mixture of contaminants from agricultural and municipal activities alters the development and functioning of alligators' reproductive and endocrine systems. Further studies of basic developmental and reproductive endocrinology in alligators and exposure studies performed under controlled laboratory conditions support the role of contaminants as causal agents of abnormalities in gonadal steroidogenesis and in reproductive tract development. These studies offer potential insight into environmentally induced defects reported in other wildlife and human populations exposed to a wide array of endocrine-disruptive contaminants.