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Sex reversal assessments reveal different vulnerability to endocrine disruption between deeply diverged anuran lineages

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Multiple anthropogenic stressors cause worldwide amphibian declines. Among several poorly investigated causes is global pollution of aquatic ecosystems with endocrine disrupting compounds (EDCs). These substances interfere with the endocrine system and can affect the sexual development of vertebrates including amphibians. We test the susceptibility to an environmentally relevant contraceptive, the artificial estrogen 17α-ethinylestradiol (EE2), simultaneously in three deeply divergent systematic anuran families, a model-species, Xenopus laevis (Pipidae), and two non-models, Hyla arborea (Hylidae) and Bufo viridis (Bufonidae). Our new approach combines synchronized tadpole exposure to three EE2-concentrations (50, 500, 5,000 ng/L) in a flow-through-system and pioneers genetic and histological sexing of metamorphs in non-model anurans for EDC-studies. This novel methodology reveals striking quantitative differences in genetic-male-to-phenotypic-female sex reversal in non-model vs. model species. Our findings qualify molecular sexing in EDC-analyses as requirement to identify sex reversals and state-of-the-art approaches as mandatory to detect species-specific vulnerabilities to EDCs in amphibians.
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Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
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Sex reversal assessments reveal
dierent vulnerability to endocrine
disruption between deeply
diverged anuran lineages
Stephanie Tamschick1, Beata Rozenblut-Kościsty2, Maria Ogielska2, Andreas Lehmann3,
Petros Lymberakis4, Frauke Homann1, Ilka Lutz1, Werner Kloas1 & Matthias Stöck1
Multiple anthropogenic stressors cause worldwide amphibian declines. Among several poorly
investigated causes is global pollution of aquatic ecosystems with endocrine disrupting compounds
(EDCs). These substances interfere with the endocrine system and can aect the sexual development
of vertebrates including amphibians. We test the susceptibility to an environmentally relevant
contraceptive, the articial estrogen 17α-ethinylestradiol (EE2), simultaneously in three deeply
divergent systematic anuran families, a model-species, Xenopus laevis (Pipidae), and two non-models,
Hyla arborea (Hylidae) and Bufo viridis (Bufonidae). Our new approach combines synchronized tadpole
exposure to three EE2-concentrations (50, 500, 5,000 ng/L) in a ow-through-system and pioneers
genetic and histological sexing of metamorphs in non-model anurans for EDC-studies. This novel
methodology reveals striking quantitative dierences in genetic-male-to-phenotypic-female sex
reversal in non-model vs. model species. Our ndings qualify molecular sexing in EDC-analyses as
requirement to identify sex reversals and state-of-the-art approaches as mandatory to detect species-
specic vulnerabilities to EDCs in amphibians.
Amphibians face a global ongoing decline1,2. Anthropogenic causes such as industrial agriculture3, habitat
destruction4,5, invasive species6, climate change7, land use8 and infectious diseases9, including several forms of
chytridiomycosis10,11, are among the major threats. However, the sum of multiple stressors1,7, some of which
poorly known, is considered to be the true reason for the massive population declines. One potential cause rep-
resents endocrine disrupting compounds (EDCs)12. Besides pesticides, EDCs comprise either natural products
or synthetic chemicals that mimic, enhance (an agonist), or inhibit (an antagonist) the action of hormones and
in this way interfere with the synthesis, secretion, transport, binding, action, or elimination of natural hor-
mones, which are responsible for the maintenance of homeostasis, reproduction, development, and/or behav-
ior13. Considerable amounts of EDCs are globally found in waste and surface waters14,15 and can easily enter the
body of aquatic organisms and impair their natural hormonal pathways. EDCs are well known for their negative
impacts on the sexual development of aquatic organisms such as sh16,17 and are suspected to cause fertility
problems in humans18,19. However, their impact to non-model amphibians with aquatic larvae is not well studied,
despite recent evidence for high EDC-relevance to suburban frog populations20. One globally relevant EDC is
17α -ethinylestradiol (EE2), a synthetically stabilized estrogen and main ingredient of many female contraceptive
pills. e inert EE2 is then excreted and insuciently eliminated by sewage plants and hence reaches aquatic
ecosystems14. It is a main hormonal pollutant, resistant to degradation, that accumulates in sediments and biota14.
Concentrations from 24 to 831 ng/L have been detected in European and American surface waters21–23. Such con-
centrations have been shown to alter behavior and somatic and sexual development in sh and amphibians12,14,15.
Due to their semi-aquatic life cycle, oen aquatic reproduction and a highly permeable skin, amphibians are
1Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 301 & 310, D-12587 Berlin,
Germany. 2Department of Evolutionary Biology and Conservation of Vertebrates, Wroclaw University, Sienkiewicza
21, 50-335 Wroclaw, Poland. 3Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Str. 11,
D-12489 Berlin, Germany. 4Natural History Museum of Crete, University of Crete, Knossou Ave., 71409 Heraklion,
Crete, Greece. Correspondence and requests for materials should be addressed to M.S. (email: matthias.stoeck@
igb-berlin.de)
Received: 09 December 2015
Accepted: 15 March 2016
Published: 31 March 2016
OPEN
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Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
especially sensitive to EDCs. Effects on development and reproduction are best examined in clawed frogs,
Xenopus laevis and X. tropicalis. In these amphibian models, EE2-concentrations as low as 0.3 ng/L have been
shown to aect calling behavior and mating success24. Higher but still environmentally relevant amounts of EE2
(29 to 840 ng/L) have been shown to aect body morphology, metamorphosis and hemoglobin catabolism25,26.
Importantly, EE2 can lead to impaired sexual development as mirrored by gonad histomorphology, demonstrat-
ing that male clawed frogs (X. laevis) develop mixed sex (= ‘intersex’, see below) gonads or even show complete
phenotypic sex reversal26–29. e undierentiated anuran gonad is bipotential and can develop into either ovary
or testis30. erefore, exogenous hormones can override the primary genetic sex determination signal and lead to
developmental disturbances, mixed sexes or complete sex reversal. One major obstacle of studying EDC-eects
in amphibians has been the mostly inaccessible information about genetic sex. In most previous EDC-studies,
sex reversal had to be inferred by comparing sex ratios of control and exposed frogs, assuming a normal 1:1 pro-
portion, which may have easily led to wrong conclusions about EDC-impacts on sex ratios. While all amphibian
species investigated show genetic sex determination31, exhibiting either male (XX/XY) or female (ZZ/ZW) heter-
ogamety, an extrapolated 96% of all species have microscopically indistinguishable sex chromosomes32, requiring
molecular sexing methods. Although EDC-studies with molecular sexing were applied to the model Xenopus26,33,
sex markers have become only recently available for some non-model anurans32,34–38, and have not been used in
EDC-experiments.
Using a high-standard ow-through-system and the rst direct experimental approach of its kind, we simul-
taneously exposed European tree frogs (H. arborea), green toads (B. viridis) and the well investigated but deeply
diverged model-species X. laevis to EE2, applied molecular sexing followed by histological analysis and compared
impacts on their sexual development. We found striking dierences in the susceptibility to sex reversal between
model and non-model species, showing that state-of-the-art approaches are an important prerequisite to detect
species-specic vulnerabilities to EDCs in amphibians.
Results
Phenotypic sex reversal of genetic males. Among all three anuran species, simultaneous exposure to
three EE2-concentrations under ow-through-conditions resulted in dierent proportions of male-to-female sex
reversal, ranging from 15 to 100% (Table1 and Fig.1), which was solely revealed when comparing genetic and
phenotypic sex of experimental animals. Importantly, no sex reversal occurred in control groups. While sex rever-
sal (Figs1 and 2) was generally correlated to EE2-concentration, interspecies dierences (p 0.010) between
clawed frogs (X. laevis) and tree frogs (H. arborea) were found at all concentrations, and between clawed frogs
and green toads (B. viridis) at the highest concentration (5,000 ng/L; p 0.001). While EE2-treatment produced
similar percentages of sex-reversed tree frogs and green toads (15 to 36%), clawed frogs appeared most suscepti-
ble (up to 100%). At the lowest concentration (50 ng/L) 31.3% of genetically male clawed frogs developed female
phenotypes, i.e. ovaries, while no sex reversal occurred in the non-model species (H. arborea, B. viridis). As
expected for a feminizing EDC, sex reversal occurred always from genetic male to phenotypic female. According
to gross morphological observation and histological evidence, sex-reversed genetic male frogs and toads devel-
oped ovaries that showed no dierence to those of genetic control and untreated39 females.
Mixed sex gonads. In addition to sex reversals, EE2-treatment provoked the development of various per-
centages of mixed sex40 gonads (equivalent to ‘intersexes’ of some authors41–43) that were histologically recorded
in all three species (Fig.3 and Table1). Such altered gonads are characterized by the presence of ovarian within
testicular tissue in genetic males and were never found in control groups. In contrast to the sex reversal analy-
ses, X. laevis formed fewer mixed sex gonads than B. viridis (p 0.026). No signicant susceptibility dierences
between H. arborea and the model species were found. Both non-model species also diered in their susceptibil-
ity at the lowest concentration (50 ng/L; p 0.015).
Discussion
Using a new combination of experimental features, we provide evidence for different quantities of
genetic-male-to-phenotypic-female sex reversal in three amphibian species, diverged between 78 million years44
(Hyla, Bufo) and 206 My (Xenopus), under exposure to the estrogen EE2. is synthetic substance is globally of
high relevance for EDC-pollution of aquatic ecosystems14,15. Our new approach combined simultaneous exposure
of tadpoles to three EE2-concentrations in a ow-through-system and genetic sexing of metamorphs of model
and non-model experimental anurans. We applied environmentally (pollution) and physiologically (expected
eects in X. laevis) relevant concentrations of EE2. Genetic sexing of metamorphosed tree frogs and green toads
revealed these two non-model species to have similar susceptibilities to sex reversal among each other, while
both signicantly diered from X. laevis. is model-species, in which genetic sex is governed by a female heter-
ogametic (ZZ/ZW) chromosome system45, proved to be more sensitive to EE2 with a lower dose provoking sex
reversals and more aected animals (Table1). On the other hand, B. viridis and H. arborea, both diverged 206 My
from X. laevis and possessing male heterogametic (XX/XY) sex chromosomes32,35, showed higher percentages of
mixed sex individuals than X. laevis.
All of this suggests that species-specic developmental stages, sex determination systems or endocrine path-
ways, shaped by long separate evolutionary histories, were dierently aected by EE2, and such a wide spectrum
of eects can be generally expected also for other EDCs among diverged anuran lineages.
e occurrence of more than 50% of genetic females among the randomly chosen hatchlings in several of
our test tanks underlines the importance of genetic sexing. Unavailability of genetic sexing, as in many previous
studies, could easily lead to wrong conclusions about the strength of feminizing (or masculinizing) eects of
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EDCs when determining “no observed eect concentration” (NOEC) and “lowest observed eect concentration
(LOEC) for endocrine active substances.
Different estrogenic compounds with concentrations reaching from the low nanogram- to the high
microgram-per-liter range have been shown to provoke phenotypic male-to-female sex reversals in X. laevis46–48
models. To our knowledge, only one previous study26 has examined sex reversals aer EE2-exposure using molec-
ular sexing in X. laevis, examining a similar range of concentrations (90, 840, 8,810 ng/L). In contrast to our study,
male-to-female sex reversals were not detected under the 90 ng/L treatment, and at the higher concentrations
with only 7 and 17%, respectively. However, these authors used a static and not a ow-through-system, which
may explain the deviating results to our study, as EE2-concentrations may stronger uctuate due to eects of met-
abolic activity of microorganisms in tanks49,50, due to greater biomass sorption of EE251, or due to simple adsorp-
tion to surfaces of exposure tanks. Beyond the synthetic EE2, on which we focused due to its high environmental
relevance, previous sex reversal estimates in X. laevis, evaluating only sex ratios, involved the natural, ephemeral
X. laevis
Genet.
sexed Females Males Females Males Sex-reversed Mixed sex
N N N % % N % N %
Control 35 24 11 68.6 31.4 0 0 0 0
50 ng/L 38 22 16 57.9 42.1 5*31.3*0x0.0x
500 ng/L 37 20 17 54.1 45.9 13*,# 76.5*,# 1 12.5
5,000 ng/L 38 21 17 55.3 44.7 17*,x, # 100*,x, # 0x0.0x
H. arborea
Control 36 13 23 36.1 63.9 0 0 0 0
50 ng/L 36 15 21 41.7 58.3 0*0.0*0° 0.0°
500 ng/L 41 22 19 53.7 46.3 6*,# 31.6*,# 3 30
5,000 ng/L 37 17 20 45.9 54.1 3*15.0*3 27.3
B. viridis
Control 25 13 12 52 48 0 0 0 0
50 ng/L 24 11 13 45.8 54.2 0 0 4x,° 57.1x,°
500 ng/L 27 15 12 55.6 44.4 4 36.4 4#80.0#
5,000 ng/L 27 12 15 44.4 55.6 5x33.3x9x,# 69.2x,#
Table 1. Eects of three 17α-ethinylestradiol (EE2) concentrations (50, 500 and 5,000 ng/L) on the sexual
development of model and non-model amphibian species. Species comprised African clawed frogs (Xenopus
laevis), European tree frogs (Hyla arborea), and European green toads (Bufo viridis); numbers and percentages
of genetically sexed individuals, sex-reversed males and mixed sex individuals. Signicant inter-species
susceptibility dierences occurred at all concentrations, resulting in genetic-male-to-phenotypic-female sex
reversal and development of mixed sex individuals. *Signicant dierence between clawed frogs and tree frogs;
xbetween clawed frogs and green toads; °between tree frogs and green toads; #signicant dierence between
treatment and control groups within the same species.
Figure 1. Quantities of sex reversal (contradiction between genetic and phenotypic sex) under the
inuence of 17α-ethinylestradiol (EE2) in three deeply diverged anuran amphibians. Percentages of
genetic-male-to-phenotypic-female sex reversal in African clawed frogs (Xenopus laevis, red), European tree
frogs (Hyla arborea, green), and European green toads (Bufo viridis, blue) exposed to three concentrations
of EE2 and in control animals; pooled data from two replicate experiments for each treatment or control.
Susceptibility dierences in genetic-male-to-phenotypic-female sex reversal occurred at all concentrations:
(*) signicant dierences between clawed frogs and tree frogs (p 0.010); (x) signicant dierences between
clawed frogs and green toads (p 0.001). Statistical analyses were conducted using cross-tabulation, Chi square
and Fisher´s exact tests (α = 0.05).
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17β -estradiol (E2). Such E2-treatments provoked skewed sex ratios40,48,52–54 or complete feminization46,53,55,56. In
H. arborea and B. viridis eects have only been studied56 at the very high 100,000 ng/L E2-concentration. In both
species, no female-biased sex ratios but a high percentage (59.3%) of undierentiated gonads in B. viridis were
found. Since gonad dierentiation in bufonid toads is slower compared to the other species at this developmental
stage39, we assume that the time of dissection at metamorphosis may have inuenced these results.
Several inconsistent outcomes in the literature may be explicable by the potentially wrong assumption of
initial 1:1 sex ratios of experimental amphibians. Based on our data, we strongly recommend genetic sexing,
whenever available, as a hallmark of appropriate evaluation of EDC-eects in amphibians. is demand can be
extended to other vertebrates and generalized to EDC-research in organisms with homomorphic sex chromo-
somes, including invertebrates57. Otherwise, as shown here, complete sex reversal as a very profound EDC-eect,
occurring at low concentrations, may be completely overlooked. Furthermore, deep phylogenetic dierences may
result in strong susceptibility dierences towards EDCs. ough we do not advocate the extensive use of endan-
gered amphibians, we conclude that results gained from earlier studies in X. laevis in general and without genetic
sex information specically should not be uncritically extrapolated to other anuran species.
Figure 2. Histological sections of three anuran species under the inuence of 17α-ethinylestradiol (EE2).
(a–c) Normal male, normal female and phenotypically sex-reversed gonad of African clawed frog (Xenopus laevis).
(d–f) Normal male, normal female and phenotypically sex-reversed gonad of European green toad (Bufo viridis).
(g–i) Normal male, normal female and phenotypically sex-reversed gonad of European tree frog (Hyla arborea). Bo
– Bidder’s organ, characteristic of bufonid gonads (for details: Methods);  – fat body; o – ovary; t – testis; arrows
indicate seminiferous tubules; *ovarian cavity; arrowheads – diplotene oocytes. Scale bars are 100 micrometers.
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Methods
Animals. is experiment was approved by the German State Oce of Health and Social Aairs (LaGeSo,
Berlin, Germany; G0359/12); all methods were carried out in accordance with approved guidelines. Xenopus
laevis tadpoles were obtained from the stock at the Leibniz-Institute for Freshwater Ecology and Inland Fisheries.
Induction of spawning and tadpole husbandry followed standard methods58. Parental animals of B. viridis and H.
arborea were caught at several localities in Greece (Supplementary Table 1), and non-invasively DNA-sampled59.
Parts of their clutches were transferred to IGB (permit 115790/229) and acclimated at 22 ± 1 °C in 10 L Milli-Q
grade water, supplemented with 2.5 g marine salt (Tagis, Germany).
Hormone exposure and experimental conditions. 17α -Ethinylestradiol (Sigma-Aldrich, Germany),
dissolved in dimethyl sulfoxide (DMSO; Roth, Germany), was applied in nominal concentrations of 50, 500 and
5,000 ng/L (Supplementary Fig. 1 and Supplementary Table 2, for measurements during the experiment); control
animals received 0.00001% DMSO. EE2-concentrations in test tanks were checked weekly by high performance
liquid chromatography/mass spectrometry (HPLC-MS/MS), and adjusted if required. In order to minimize
adsorption or release of EDCs, we used glass tanks and all connections of the ow through system consisted of
inert materials involving mainly PTFE (Polytetrauoroethylene, “Teon”)-coating or Platinum-cured Silicon tub-
ing (Cole-Parmer). Exposure of tadpoles started at Gosner60-stage 22–23 in B. viridis and H. arborea, equivalent
to Nieuwkoop-Faber61 stage 42–44 in X. laevis, distinctly prior to the sensitive phase of sex determination in all
species30,62. Twenty randomly chosen individuals per species and treatment were transferred into each test tank
in a high-standard ow-through-system (details52). Two replicates per exposure group (including control) com-
prised in total 160 tadpoles per species. Stock solutions and water were piped via a peristaltic pump into a mixing
chamber, mixed to nal EE2-concentrations, and supplied to a cluster of three test tanks each. Concentrations
were thus identical for all three species in each treatment group. Tadpoles were reared in a 12/12 h light/dark cycle
at constantly 22 ± 1 °C in suciently aerated and regularly cleaned tanks. Weekly monitored water parameters
comprised: dissolved oxygen, nitrate, ammonium, pH, conductivity, and hardness; values were adequate as in
previous studies involving the same equipment40. Tadpoles were fed SeraMicron (Sera, Germany), H. arborea and
B. viridis were additionally supplied with TetraMin (Tetra, Germany). To imitate natural conditions under which
H. arborea and B. viridis leave water at metamorphosis, animals were transferred to glass terraria at Gosner stage
46. Xenopus laevis were dissected at equivalent Nieuwkoop-Faber stage 66; hylids and bufonids aer sucient
post-metamorphic dierentiation39.
Phenotypic sexing based on gonad gross morphology and histology. Animals were anesthetized
by immersion in tricaine methanesulfonate (MS 222; Sigma-Aldrich), decapitated and dissected under a binoc-
ular microscope (Olympus SZX7). Gonadal anatomy served for preliminary phenotypic sexing and detection of
underdeveloped gonads. To improve visualization, a drop of Bouin’s solution (Sigma-Aldrich) was added; and
in situ anatomical photographs were taken (Olympus DT5 camera). For histology, gonads were carefully dissected,
separated from adjacent tissue, xed in Bouin (24 h) and subsequently rinsed several rounds in 70% ethanol.
Histological sections were prepared for 50% of study animals (from each tank 10 randomly chosen individuals,
i.e. 20 per treatment group). Analyses were performed according to established protocols30,39,63. Using Stemi SV11
(Zeiss) microscope and camera, separated gonads were photographed, embedded in paraplast, sectioned into
7 m longitudinal slices, stained with Mallory’s trichrome, and examined using Zeiss Axioskop 20 microscope.
Images were acquired by a cooled Carl Zeiss AxioCam HRc CCD camera. Histological sections were screened
slide by slide to establish phenotypic sex. Ovaries were recognized by the presence of ovarian cavities, early mei-
otic oocytes and/or diplotenes, and testes by spermatogonia, spermatocytes and/or seminiferous cords or tubules.
Figure 3. Histological sections of mixed sex gonads of three anuran species under the inuence of
17α-ethinylestradiol (EE2). (a) African clawed frog (Xenopus laevis), (b) European green toad (Bufo viridis),
(c) European tree frog (Hyla arborea); Fig.2 for control and sex-reversed individuals. Bo – Bidder’s organ,
specic of bufonid toads’ gonads;  – fat body; m – meiocytes; o – ovary; st – seminiferous tubules; t – testis;
*a cavity separating testicular and ovarian parts of the mixed sex gonad; white arrow indicates ovarian cavity in
the ovarian portion of the mixed gonad; white arrowheads show diplotene oocytes; yellow dotted lines separate
testicular and ovarian parts of the mixed sex gonads. Scale bars represent 100 micrometers.
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In the case of B. viridis, the most anterior part of both male and female gonads is Bidder’s organ, an ovary-like
structure, characteristic of bufonids64. In B. viridis mixed sex was dened when ovarian meiocytes were found
inside male testicular tissue behind the physiological transition region between Bidder’s organ and the actual
gonad. All phenotypic sexing was performed without prior knowledge about genetic sex of animals.
Genotypic sex determination. DNA extraction involved the BioSprint robotic workstation with
its 96 DNA Plant Kit (Qiagen, Germany) according to the manufacturer’s protocol. To establish genetic sex,
species-specic polymerase chain reactions (PCRs) were conducted on Eppendorf Mastercyclers (Ep Gradient S).
For X. laevis, two genes were amplied45,65: DMRT1 and the female-specic DM-W (Supplementary Table 3).
Genetic sexing of non-model species involved microsatellites WHa5–201 and Ha-H10834,36 (H. arborea) and
C20132,66, HNRNPD and CHD167 (B. viridis; Supplementary Table 3). Genotypes were analyzed on a sequencer
(3500 × L Genetic Analyzer, Applied Biosystems) and G v. 4.0 was used for visualization of peaks.
DNA quality issues (four H. arborea) and homomorphy of microsatellites (33 B. viridis) prevented genetic sexing
in these individuals that were excluded from sex reversal analyses.
Detection of complete sex reversal and mixed sex. Phenotypic sexing of all animals was based on
gross morphology and histology of gonads39,52. Complete sex reversal was stated if genetic males showed a phe-
notypically female gonad, irrespective of the degree of its dierentiation and not diering from those of control
females. Mixed sex gonads were detected by the presence of ovarian and testicular tissue in the same gonad.
Statistics. All data were analyzed with SPSS Statistics 22 (IBM, Armonk, NY). Intra- and inter-specic dif-
ferences in EE2-susceptibility were examined. For evaluations of sex reversal and mixed sex, we rst compared
both replicates per species and parameter using Fisher’s exact test. If no dierences (exact p 0.05) were found,
both replicates per treatment were pooled in order to compare control and exposure groups within and between
species using cross-tabulations with 2-sided Chi square tests (α = 0.05). Post-hoc Fishers exact tests (2-sided)
were applied for pairwise comparisons including False Discovery Rate corrections68.
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Acknowledgements
This work was supported by the German Science Foundation (DFG; grant Sto 493/3-1) and a Heisenberg-
Fellowship (Sto 493/2-1, Sto 493/2-2) to MS. We thank the Ministry of the Environment of Greece that provided
the permit (115790/229) to collect the specimens. We thank M. Papadimitrakis for help during the eld work, W.
Kleiner, J. Garmshausen, M. Brehm, and A. Weißhuhn for animal care and/or laboratory assistance, J.F. Gerchen
for unpublished primers, M. Kazmierczak for help with microphotography, E. Serwa for excellent histology, I.
Haufe for detailed statistical advice, J. Plötner for access to the DNA extraction work station and M. Monaghan
and K. Preuss for access to the IGB genotyping facility. e publication of this article was funded by the Open
Access Fund of the Leibniz Association.
Author Contributions
M.S. and W.K. conceived and designed the research. M.S. and P.L. did field work; S.T., M.S., F.H. and I.L.
conducted the experiments. S.T. performed genotyping and, supported by F.H. statistical analyses. S.T. and M.S.
wrote the paper; B.R.K. and M.O. performed histology and evaluated, supported by I.L. gonadal dierentiation.
A.L. analyzed water samples. All authors contributed to the nal manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Tamschick, S. et al. Sex reversal assessments reveal dierent vulnerability to endocrine
disruption between deeply diverged anuran lineages. Sci. Rep. 6, 23825; doi: 10.1038/srep23825 (2016).
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
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unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
... Sex reversal occurs in fish, amphibians and reptiles in nature (Alho et al., 2010;Baroiller & D'Cotta, 2016;Lambert et al., 2019;Nemesházi et al., 2020;Whiteley et al., 2021;Xu et al., 2021), and theoretical studies caution that it may have far-reaching consequences including skewed sex ratios, sex-chromosome evolution and even population extinction (Bókony et al., 2017;Grossen et al., 2011;Perrin, 2009;Schwanz et al., 2020;Wedekind, 2017). Laboratory experiments show that sex reversal can be induced by anthropogenic stressors such as chemical pollution and elevated temperature (Flament, 2016;Lambert et al., 2018;Mikó et al., 2021;Tamschick et al., 2016), and thus we may expect that the contemporary and future increase in the levels of anthropogenic stressors will influence the rates of sex reversal in free-living populations of ectothermic vertebrates. Whether this influence would be an increased or decreased sex-reversal frequency in anthropogenic environments is not a trivial question, for the following reasons. ...
... Assessing sex-reversal frequencies in wild populations has been hindered by the difficulty of diagnosing sex reversal in nonmodel organisms. Due to the high evolutionary lability and homomorphy of sex chromosomes in ectothermic vertebrates, genetic sexing methods are available only for a small fraction of species (e.g., Alho et al., 2010;Baroiller & D'Cotta, 2016;Lambert et al., 2019;Nemesházi et al., 2020;Tamschick et al., 2016;Whiteley et al., 2021;Xu et al., 2021). In two such species, recently developed genetic sex markers have been used to investigate whether sex reversal is more prevalent in anthropogenic habitats, and they reported contradictory answers: yes in one frog species (Nemesházi et al., 2020) but no in another (Lambert et al., 2019). ...
... Both these EDCs may cause male-to-female sex reversal based on their effects on oestrogenic enzymatic activities, female-skewed sex ratios and intersex gonads (Bhandari et al., 2015;Howe et al., 2004;Lanctôt et al., 2014;Tamschick et al., 2016). As both chemicals have been in use for about half a century, we can expect resistance to have potentially evolved in populations chronically exposed to these pollutants. ...
Article
Full-text available
Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable, since environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex‐ratio distortion and population decline. Despite these implications, we have scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex‐reversing stimuli more frequently, which may lead to higher sex‐reversal rate, or alternatively, these populations may adapt to resist sex reversal. We developed PCR‐based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous estrogenic pollutants in a common‐garden experiment. We found negligible sex‐reversal frequency in free‐living adults despite the presence of various endocrine‐disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg/L 17α‐ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng/L EE2 and a glyphosate‐based herbicide with 3 µg/L or 3 mg/L glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex‐reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species‐specific resistance to sex reversal.
... Sex reversal occurs in fish, amphibians, and reptiles in nature (Alho, Matsuba, & Merilä, 2010;Baroiller & D'Cotta, 2016;Lambert, Tran, Kilian, Ezaz, & Skelly, 2019;Nemesházi et al., 2020;Whiteley et al., 2021;Xu et al., 2021), and theoretical studies caution that it may have far-reaching consequences including skewed sex ratios, sex-chromosome evolution, and even population extinction (Bókony, Kövér, Nemesházi, Liker, & Székely, 2017;Grossen, Neuenschwander, & Perrin, 2011;Nemesházi, Kövér, & Bókony, 2021;Perrin, 2009;Schwanz, Georges, Holleley, & Sarre, 2020;Wedekind, 2017). Laboratory experiments show that sex reversal can be induced by anthropogenic stressors like chemical pollution and elevated temperature (Flament, 2016;Lambert, Smylie, Roman, Freidenburg, & Skelly, 2018;Mikó et al., 2021;Tamschick et al., 2016), thus, we may expect that the contemporary and future increase in the levels of anthropogenic stressors will influence the rates of sex reversal in freeliving populations of ectothermic vertebrates. This influence is conceivable in at least two ways. ...
... Due to the high evolutionary lability and homomorphy of sex chromosomes in ectothermic vertebrates, genetic sexing methods are available only for a handful of species (e.g. Alho et al., 2010;Baroiller & D'Cotta, 2016;Tamschick et al., 2016;Lambert et al., 2019;Nemesházi et al., 2020;Whiteley et al., 2021;Xu et al., 2021). In two of those species, recently developed genetic sex markers have been used to investigate whether sex reversal is more prevalent in anthropogenic habitats, and they reported contradictory answers: yes in one frog species (Nemesházi et al., 2020) but no in another (Lambert et al., 2019). ...
... We focused on the sex-reversing effects of two endocrine disrupting chemical (EDC) compounds with high prevalence in surface water in agricultural and urban areas, respectively: glyphosate, the most used herbicide worldwide (Brovini et al., 2021), and 17α-ethinylestradiol (EE2), a common ingredient of contraceptives that pollutes natural water bodies via wastewater (Bhandari et al., 2015). Both EDCs may cause male-to-female sex reversal based on their effects on estrogenic enzymatic activities, female-skewed sex ratios, and intersex gonads (Bhandari et al., 2015;Howe et al., 2004;Lanctôt et al., 2014;Tamschick et al., 2016). As both chemicals have been in use for about half a century, we can expect resistance to have potentially evolved in populations chronically exposed to these pollutants. ...
Preprint
Full-text available
Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable, since their sexual development can be disrupted by environmental changes, which can cause sex reversal, a mismatch between genetic and phenotypic sex, potentially leading to sex-ratio distortion and population decline. Despite these implications, we have scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may experience sex reversal more frequently, or alternatively, they may adapt to resist sex reversal. To test these alternative hypotheses, we developed PCR-based genetic sex markers for the common toad (Bufo bufo) . We assessed the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous estrogenic pollutants in a common-garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg/L 17α- ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng/L EE2 and a glyphosate-based herbicide with 3 µg/L or 3 mg/L glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the surprisingly low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.
... [12] Detailed searching methods are described in Supplement 1, and the data extracted in Supplementary Table 1. We found only four experiments in which anuran species with both male and female heterogamety were studied for sex-reversal propensity, [22,[49][50][51] although heterogamety was not in their focus. Other studies were usually restricted to a single species. ...
... Therefore, mortalities and sample sizes should always be clearly reported. Preferably, sex-reversed individuals should be identified by genetic sexing, [49,57,64] and for this, development of genetic sex markers for those many thousands of species where such markers are not yet available is an inevitable challenge. ...
Article
Full-text available
Sex reversal, a mismatch between phenotypic and genetic sex, can be induced by chemical and thermal insults in ectotherms. Therefore, climate change and environmental pollution may increase sex‐reversal frequency in wild populations, with wide‐ranging implications for sex ratios, population dynamics, and the evolution of sex determination. We propose that reconsidering the half‐century old theory “Witschi's rule” should facilitate understanding the differences between species in sex‐reversal propensity and thereby predicting their vulnerability to anthropogenic environmental change. The idea is that sex reversal should be asymmetrical: more likely to occur in the homogametic sex, assuming that sex‐reversed heterogametic individuals would produce new genotypes with reduced fitness. A review of the existing evidence shows that while sex reversal can be induced in both homogametic and heterogametic individuals, the latter seem to require stronger stimuli in several cases. We provide guidelines for future studies on sex reversal to facilitate data comparability and reliability. Environment‐induced sex reversal might catalyze demographic changes and sex‐chromosome evolution in ectotherms. We propose a modified view of the half‐century old observation called Witschi's rule and propose methodological standards, following which would facilitate our understanding on how sex‐chromosome systems may differ in their vulnerability to anthropogenic environmental change.
... Detailed searching methods are described in Supplement 1, and the data extracted in Supplementary Table 1. We found only four experiments in which anuran species with both male and female heterogamety were studied for sex-reversal propensity [30,[42][43][44] , although heterogamety was not in their focus. Other studies were usually restricted to a single species. ...
... Therefore, mortalities and sample sizes should always be clearly reported. Preferably, sex-reversed individuals should be identified by genetic sexing [42,50,54] , and for this, development of genetic sex markers for those many thousands of species where such markers are not yet available is an inevitable challenge. ...
... These disrupting compounds cause, for example, a decline of certain species (i.e. amphibians and alligators), and sex changes in fish and shellfish, among other problems [16,17]. This group of ECs includes a wide variety of natural compounds and anthropogenic chemicals such as natural or synthetic estrogens, phthalates and, particularly, parabens [16,18]. ...
Article
The presence of emerging contaminants (ECs) in aquatic systems and, particularly, in wastewater (WW) has become a major concern over the past years. Among these contaminants, parabens, belonging to the group of endocrine disruptors, are used on a daily basis as preservatives and constantly enter the environment, being called pseudo-persistent contaminants. Parabens have the potential to bioaccumulate and can be toxic to aquatic species. Unfortunately, the traditional methods used in wastewater treatment plants (WWTPs), namely the adsorption process, activated sludge and advanced oxidation processes (AOPs) are not effective in removing this type of contaminants. These treatment methods generate wastes with high concentrations of parabens adsorbed in activated carbon, large amounts of activated sludge containing parabens and/or chemically unstable by-products. To overcome these limitations, microalgae-based bioremediation has aroused great interest as an effective and sustainable process where parabens can be used in the microalgae metabolism as a carbon source (diauxic growth). However, several factors that affect microalgae growth and, consequently, their bioremediation capacity, must be considered for effective implementation of this biological treatment. This study reviews the impact of parabens on aquatic environments (ecotoxicity, bioaccumulation and persistence) as well as the limitations of the current methods applied in WWTPs considering the removal mechanisms and by-product formation. Moreover, it also addresses the metabolic pathways and the environmental factors (i.e. carbon and nutrients concentration, irradiation, photoperiod, pH and temperature) that can affect the parabens removal. As such, this review provides a set of conditions that can influence microalgae-based bioremediation, highlighting their ability for parabens removal and the requirement for supplementary research.
... Des preuves de féminisations dues aux PE existent bien, in natura et en laboratoire. L'exposition à l'atrazine (composant de pesticides) ou à l'EE2 chez différents amphibiens provoque ainsi une féminisation partielle (intersexués) à totale Tamschick et al., 2016). Au contraire, l'exposition à des PE peut conduire à une masculinisation des êtres affectés, comme en témoigne l'apparition de pénis chez des femelles ours polaires vivant dans des régions polluées par du PCB ainsi que l'apparition de pénis (imposex) et d'intersexués chez différents mollusques femelles touchés par le TBT (tributylétain des peintures antifouling) (Bryan et al., 1986;Smith and McVeagh, 1991;Matthiessen and Gibbs, 1998). ...
Thesis
Full-text available
La différenciation sexuelle des Isopodes dépend d'une hormone sexuelle protéique, l'hormone androgène (HA), caractéristique des Malacostracés. Cet Insulin-Like Peptide suffit à induire par sa présence la différenciation mâle de ces Crustacés. Nous avons identifié in silico le transporteur circulant de l'HA, l'IGFBP-rP1, chez de nombreuses espèces d'Isopodes ainsi qu'à l'échelle des Crustacés. De la même façon, nous avons identifié deux récepteurs transmembranaires, l'IR1 et l'IR2, issus d'une duplication de gène spécifique des Malacostracés. Les patrons d'expression de ces gènes ont été étudiés sur notre espèce modèle, Armadillidium vulgare. Av-IGFBP-rP1 et Av-IR1 sont exprimés de manière ubiquiste et tout au long du développement. Av-IR2 est aussi exprimé à chaque stade de la différenciation mais ce transcrit est quasi-spécifique des glandes androgènes et ovaires. Une approche par ARNi a confirmé l'implication de ces trois protéines dans la voie de signalisation de l'HA. En effet, l'inhibition de l'HA, Av-IGFBP-rP1 et Av-IR1 provoquent l'hypertrophie des glandes androgènes, suggérant leur implication dans une boucle de rétro-contrôle de l'HA. L'inhibition de Av-IR2 semble seulement provoquer la différenciation d'ouvertures génitales femelles. Ces phénotypes sont comparables à ceux des intersexués mâles induits par la bactérie féminisante endogène Wolbachia. Nous montrons cependant que la bactérie altère seulement l'expression de l'HA et pas celle des récepteurs. Enfin, nous avons testé l'effet du bisphénol A mais nous n'observons pas d'altération de la différenciation sexuelle des larves lors d'expositions à ce perturbateur endocrinien exogène.
... 5,10 Amphibians are useful for assessing the potential effects of contaminant exposure and have been used extensively as physiological and behavioral research models. 13,14 However, amphibians are a large and diverse group of species with a range of morphological, physiological, and life history traits which can lead to varying species-specific responses to EDCs. 15 For example, Tamschick et al. 16 observed species-specific differences in responses among X. laevis, Hyla arborea, and Bufo viridis following larval exposure of 17⊍-ethinylestradiol (EE2). ...
Article
Background: The herbicide atrazine has been proposed as a potential endocrine disrupting compound (EDC) for amphibians. Using atrazine concentrations below or at those typically found in surface waters (0.5, 5.0, 50 μg/L), we exposed Acris blanchardi (Blanchard's cricket frog) larvae throughout development until metamorphosis (i.e. Gosner stages 26-45). An additional 50 μg/L treatment (50s μg/L) was utilized where supplemented algae was added to control for indirect atrazine effects from reduced food sources. In addition to atrazine, experimental groups also included a negative control and two positive controls, 17β-estradiol (E2) at 2.3 and 25 μg/L. At 60 days post-metamorphosis, A. blanchardi metamorphs were euthanized for analysis of gross and histopathological development. Results: Atrazine did not significantly influence mortality (mean recovery of 54% across treatments), sex ratio, body mass (BM), snout-vent length (SVL), gonad size, nor gonad development of A. blanchardi. Females exposed to 50s μg/L atrazine had 29% less mass, were 10% shorter, and had a 29% lower mean ovary area (mm2 ) as compared to negative controls, suggesting algae enrichment had a significant negative effect. Males exposed to estradiol (25 μg/L) showed an increased level of oviduct development. Ovary area was also significantly influenced by estradiol treatment at 2.3 and 25 μg/L. Conclusion: Overall, estradiol had much less effect than predicted based on other model species (e.g. Xenopus laevis). Development of A. blanchardi, overall, was not affected by long-term exposure to environmentally relevant concentrations of atrazine. However, this species also was largely insensitive to exogenous estradiol in this test system. © 2022 Society of Chemical Industry.
... boulengeri) adults had undergone sex reversal, making the frequency of sex reversal much lower than that in the European common frog (R. temporaria) population. No studies have assessed whether endocrine disruption in wild amphibians results in sex reversal, although the response has been exhibited using chemicals with sex-linked markers in laboratory experiments [36,37]. Future areas of study could include the use of amphibian sex-linked genetic markers to investigate sex reversal in wild populations in the context of natural environment or anthropogenically induced factors. ...
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We used genotyping-by-sequencing (GBS) to identify sex-linked markers in 43 wild-collected spiny frog (Quasipaa boulengeri) adults from a single site. We identified a total of 1049 putatively sex-linked GBS-tags, 98% of which indicated an XX/XY system, and finally confirmed 574 XY-type sex-linked loci. The sex specificity of five markers was further validated by PCR amplification using a large number of additional individuals from 26 populations of this species. A total of 27 sex linkage markers matched with the Dmrt1 gene, showing a conserved role in sex determination and differentiation in different organisms from flies and nematodes to mammals. Chromosome 1, which harbors Dmrt1, was considered as the most likely candidate sex chromosome in anurans. Five sex-linked SNP makers indicated sex reversals, which are sparsely present in wild amphibian populations, in three out of the one-hundred and thirty-three explored individuals. The variety of sex-linked markers identified could be used in population genetics analyses requiring information on individual sex or in investigations aimed at drawing inferences about sex determination and sex chromosome evolution.
... Additionally, female-dominant sex ratios were documented in suburban and urban environments in Connecticut ponds contaminated with estrogenic EDCs (Lambert et al., 2015). Evidence of female-biased sex ratios following exposures to xenoestrogens has been intensively studied, with particular emphasis on the potent synthetic estrogen, 17α-ethynylestradiol (Tamschick et al., 2016). Exposure to 17α-ethynylestradiol at concentrations ≤5 nM has been associated a female-biased sex ratio in X. laevis (Villalpando and Merchant-Larios, 1990), R. pipiens (Hogan et al., 2008), and S. tropicalis (Pettersson et al., 2006). ...
Article
Endocrine disrupting chemicals (EDCs) are ubiquitous in aquatic and terrestrial environments. The main objective of this review was to summarize the current knowledge of the impacts of EDCs on reproductive success in wildlife and humans. The examples selected often include a retrospective assessment of the knowledge of reproductive impacts over time to discern how the effects of EDCs have changed over the last several decades. Collectively, the evidence summarized here within reinforce the concept that reproduction in wildlife and humans is negatively impacted by anthropogenic chemicals, with several altering endocrine system function. These observations of chemicals interfering with different aspects of the reproductive endocrine axis are particularly pronounced for aquatic species and are often corroborated by laboratory-based experiments (i.e. fish, amphibians, birds). Noteworthy, many of these same indicators are also observed in epidemiological studies in mammalian wildlife and humans. Given the vast array of reproductive strategies used by animals, it is perhaps not surprising that no single disrupted target is predictive of reproductive effects. Nevertheless, there are some general features of the endocrine control of reproduction, and in particular, the critical role that steroid hormones play in these processes that confer a high degree of susceptibility to environmental chemicals. New research is needed on the implications of chemical exposures during development and the potential for long-term reproductive effects. Future emphasis on field-based observations that can form the basis of more deliberate, extensive, and long-term population level studies to monitor contaminant effects, including adverse effects on the endocrine system, are key to addressing these knowledge gaps.
Chapter
Hormone-active substances are those compounds which behave like hormone activity, irrespective of mechanism. Hormone system is controlled by endocrine system in organism's body; therefore, they are also called as endocrine-active substances (EASs). Endocrine system, important communication system, comprises of endocrine glands that secrete “hormones” in blood stream (in response to stimulus) to regulate body function. Endocrine-disrupting chemicals disturb inadvertently the complex communication system and interfere with synthesis and secretion of bodily hormones. Endocrine substance influences the regular activity hormones. If these substances cause negative change in body, then they are also called endocrine disrupters. Endocrine disruptors are exogenous substances or mixture of substances that alter functions of endocrine system that eventually cause adverse impact on health of intact organism, its progeny, and subpopulations. Endocrine disrupters/EASs affect the health of exposed human and animals by entering into ecosystem via different sources. EAS could be naturally occurring (like phytoestrogen in soya) or synthetic. The artificially or synthetic EASs are man-made such as pesticides, dioxins, PCBs, Biphenyl A, and other environmental pollutants. Endocrine disrupters are also considered as environmental micropollutants or toxins that could harm the organism's health. These disrupting toxins (chemicals) could affect the endocrine system and cause diseases and dysfunctions across the whole life span of organisms. They are omnipresent, so they enter into body via different sources and pathways. Some environmental toxins are heavy metals, dioxins, pesticides, and polychlorinated biphenyl and other atmospheric pollutants (ozone, smog). The severity of effects on exposed organisms depends on dose-response relationship. The amount or concentration of dose is proportional to effect. These EASs induce toxic impacts on animals and plant's health by entering into food chain. In this chapter, different toxic impacts of hormone-active substances on animals, plants, and human health are mentioned in detail.
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Significance We focus on a critical issue, the influence of human-derived contaminants on wildlife populations. Endocrine disrupting chemicals that act through hormonal pathways are capable of having large influences even when concentrations are relatively low. While there is evidence that such endocrine disruption can result from the application of agricultural pesticides and through exposure to wastewater effluent, we have identified a diversity of endocrine disrupting chemicals within suburban neighborhoods. Sampling populations of a local frog species, we found a strong association between the degree of landscape development and frog offspring sex ratio. Our study points to rarely studied contamination sources, like vegetation landscaping and impervious surface runoff, that may be associated with endocrine disruption environments around suburban homes.
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Bidder's organ is an ovary-like structure, which develops from the anterior part of the gonadal ridge in anuran amphibians belonging to the Bufonidae family. Bidder's organs form in both males and females. Because Bidder's organ contains female germ cells (oocytes), the bufonid males are de facto hermaphrodites. Due to similarity with the undeveloped ovary, Bidder's organ was, in early literature, described, inaccurately, as a structure present only in males. Due to the fact that Bidder's organ is a unique structure present only in Bufonidae, it is not well studied and its function still remains a mystery. Here we describe the development and structure of Bidder's organs, summarize the knowledge on gene expression and steroidogenic activity in these organs, and present hypotheses regarding Bidder's organ function.
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Human activities, especially conversion and degradation of habitats, are causing global biodiversity declines. How local ecological assemblages are responding is less clear[mdash]a concern given their importance for many ecosystem functions and services. We analysed a terrestrial assemblage database of unprecedented geographic and taxonomic coverage to quantify local biodiversity responses to land use and related changes. Here we show that in the worst-affected habitats, these pressures reduce within-sample species richness by an average of 76.5%, total abundance by 39.5% and rarefaction-based richness by 40.3%. We estimate that, globally, these pressures have already slightly reduced average within-sample richness (by 13.6%), total abundance (10.7%) and rarefaction-based richness (8.1%), with changes showing marked spatial variation. Rapid further losses are predicted under a business-as-usual land-use scenario; within-sample richness is projected to fall by a further 3.4% globally by 2100, with losse
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Endocrine disruption is a named field of research which has been very active for over 10 years, although the effects of endocrine disruptors in wildlife have been studied mainly in vast since the 1940s. A large number of chemicals have been identified as endocrine disruptors and humans can be exposed to them either due to their occupations or through dietary and environmental exposure (water, soil and air). Endocrine disrupting chemicals are compounds that alter the normal functioning of the endocrine system of both humans and wildlife. In order to understand the vulnerability and risk factors of people due to endocrine disruptors as well as the remedies for these, methods need to be developed in order to predict effects on populations and communities from the knowledge of effects on individuals. For several years there have been a growing interest on the mechanism and effect of endocrine disruptors and their relation with environment and human health effect. This paper, based on extensive literature survey, briefly studies the progress mainly in human to provide information concerning causative substances, mechanism of action, ubiquity of effects and important issues related to endocrine disruptors. It also reviews the current knowledge of the potential impacts of endocrine disruptors on human health so that the effects can be known and remedies applied for the problem as soon as possible. Copyright © 2015 Elsevier B.V. All rights reserved.