The chemistry and histology of sexually
dimorphic mental glands in the freshwater
turtle, Mauremys leprosa
, Albert Martínez-Silvestre
, Dagmara Podkowa
and Maciej Pabijan
1Department of Comparative Anatomy, Institute of Zoology and Biomedical Research
Jagiellonian University, Krakow, Poland
2Catalonian Reptile and Amphibian Rescue Centre-CRARC, Masquefa, Spain
3Department of Analytical Chemistry, Laboratory for Forensic Chemistry, Faculty of Chemistry,
Jagiellonian University, Krakow, Poland
Despite evidence from anatomy, behavior and genomics indicating that the sense of
smell in turtles is important, our understanding of chemical communication in this
groupisstillrudimentary.Ouraimwastodescribe the microanatomy of mental glands
(MGs) in a freshwater turtle, Mauremys leprosa (Geoemydidae), and to assess the
chemical composition of their secretions with respect to variation among individuals
and between sexes. MGs are paired sac-like organs on the gular region of the neck and
are dimorphic in this species with males having fully functional holocrine glands
while those of females appear non-secretory and vestigial. In adult males, the glandular
epithelium of the inner portion of the gland provides exocytotic products as well as
cellular debris into the lumen of the gland. The contents of the lumen can be secreted
through the narrow duct portion of the gland ending in an oriﬁce on the surface of the
skin. Females have invaginated structures similar in general outline to male glands, but
lack a glandular epithelium. Using gas chromatography coupled to mass spectrometry,
we identiﬁed a total of 61 compounds in mental gland secretions, the most numerous
being carboxylic acids, carbohydrates, alkanes, steroids and alcohols. The number of
compounds per individual varied widely (mean (median) ± SD = 14.54 (13) ± 8.44;
relative abundances of only six chemicals were different between the sexes, although
males tended to have larger amounts of particular compounds. Although the lipid
fraction of mental gland secretions is rich in chemical compounds, most occur in both
sexes suggesting that they are metabolic byproducts with no role in chemical signaling.
However, the relative amounts of some compoundstendedtobehigherinmales,with
signiﬁcantly larger amounts of two carboxylic acids and one steroid, suggesting their
putative involvement in chemical communication.
Subjects Animal Behavior, Zoology, Histology
Keywords Geoemydidae, Mental glands, GC-MS, Lipids, Secretions, Semiochemicals, TEM
Animals communicate with other members of their species in a wide variety of contexts
over their lifetimes. Communication is essential for many crucial activities, as for example,
How to cite this article Ibáñez A, Martínez-Silvestre A, Podk owa D, Woźniakiewicz A, Woźniakiewicz M, Pabijan M. 2020. The chemistry
and histology of sexually dimorphic mental glands in the freshwater turtle, Mauremys leprosa.PeerJ 8:e9047 DOI 10.7717/peerj.9047
Submitted 14 January 2020
Accepted 2 April 2020
Published 15 May 2020
Additional Information and
Declarations can be found on
2020 Ibáñez et al.
Creative Commons CC-BY 4.0
avoidance of aggressive encounters among conspeciﬁcs in territorial species and/or
choosing mating partners for reproduction. Given the relevance of the process, animals
have developed the ability to exploit several signaling channels or pathways to transmit
pertinent information, with acoustic, chemical, tactile and visual signals being among the
Chemical signals have been extensively studied in certain groups of invertebrates
such as insects (Symonds & Elgar, 2008). Chemical communication in vertebrates is less
understood; however, a relatively large array of molecules has been described in a limited
set of taxa, indicating an underlying complexity of chemical signaling in this group
(Weldon, Flachsbarth & Schulz, 2008;Wyatt, 2014;Apps, Weldon & Kramer, 2015).
In some cases—especially in mammals—the chemical compounds have been isolated and
their functions are known. For instance, major urinary proteins are involved in individual
identity in wild house mice Mus domesticus (Hurst et al., 2001). However, for most
vertebrates chemical compounds with a (potential) signaling role have not been identiﬁed.
Reptilian chemosignals have been studied in a taxonomically restricted set of species
(Martín & López, 2011). Femoral glands in lacertid and iguanid lizards and the skin of
colubrid snakes are known to secrete sex-speciﬁc chemosignals (Weldon, Flachsbarth &
Schulz, 2008;Houck, 2009). For example, skin-borne methyl ketones released by female
red-sided garter snakes (Thamnophis sirtalis parietalis) are used as male attractants and
might convey information on female size and indicate fecundity (Lemaster & Mason,
2002). Furthermore, the variation in methyl ketone composition between different
species within the genus Thamnophis is species-speciﬁc and thus could be a driver of
speciation (Uhrig, LeMaster & Mason, 2014). Other compounds such as vitamin E might
be reliable signals of male quality in lizards (Martín & López, 2010;García-Roa et al., 2017;
Kopena, López & Martín, 2017). These results indicate that assessing the level of
inter-individual and sex-speciﬁc variation in chemical signals is an essential step in
understanding the functionality and evolution of semiochemical compounds in
Several lines of evidence indicate that chelonians have a well-developed olfactory
system. First, the draft genomes of soft-shell (Pelodiscus sinensis) and green sea
(Chelonia mydas) turtles have revealed extensive and independent expansion of functional
olfactory receptor genes (Wang et al., 2013). Second, a number of behavioral studies have
provided indirect evidence for the importance of olfactory cues in intra and intersexual
recognition (Rose, 1970;Weaver, 1970;Muñoz, 2004;Poschadel, Meyer-Lucht & Plath,
2006;Lewis et al., 2007;Mason & Parker, 2010;Ibáñez, López & Martín, 2012).
For instance, freshwater turtles might detect chemicals from other conspeciﬁcs that can be
relevant to mate ﬁnding or for establishing dominance between members of their own
species (Poschadel, Meyer-Lucht & Plath, 2006;Ibáñez, López & Martín, 2012;Ibáñez et al.,
2013). Third, some turtle species have specialized secretory organs such as cloacal glands
and/or Rathke’s glands in the inguinal or axillary regions (Waagen, 1972;Ehrenfeld &
Ehrenfeld, 1973;Mason & Parker, 2010). In addition, numerous chelonian
species within the superfamily Testudinoidea possess mental glands (MGs), also called
subdentary or chin glands, located in the integument of the gular part of the neck
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 2/24
(Winokur & Legler, 1975). A fourth line of evidence comes from chemical analysis of the
contents of MGs in desert-dwelling gopher tortoises (Gopherus) in which these glands are
particularly pronounced (Rose, Drotman & Weaver, 1969;Rose, 1970;Weaver, 1970;
Alberts, Rostal & Lance, 1994). Functionally, it has been demonstrated that secretions
produced by MGs might mediate sex recognition and male interactions in G. berlandieri
and G. agassizii (Rose, 1970;Weaver, 1970;Alberts, Rostal & Lance, 1994). Chemical
analysis revealed the presence of proteins of varying molecular size as well as lipid
compounds such as cholesterol, phospholipids, triglycerides and free fatty acids (Rose,
Drotman & Weaver, 1969;Rose, 1970;Alberts, Rostal & Lance, 1994). Overall, the available
data indicates that chemical communication in chelonians is widespread and occurs
through the production of chemical compounds in different types of secreting organs.
However, mental gland microanatomy has only been assessed superﬁcially using standard
light microscopy, while the ultrastructure of these organs remains unknown (Rose,
Drotman & Weaver, 1969;Weaver, 1970;Winokur & Legler, 1975). Furthermore, with the
exception of electrophoretic and chromatographic studies on secretions in Gopherus
(Rose, Drotman & Weaver, 1969;Rose, 1970;Alberts, Rostal & Lance, 1994), nearly nothing
is known of the chemical compounds produced by MG secretions.
In this article we report the chemical composition and microanatomy of the MGs of the
Spanish terrapin, M. leprosa (Geoemydidae, former Bataguridae), a sexually dimorphic
and predominantly freshwater species distributed in northwestern Africa and the Iberian
peninsula into southern France (Díaz-Paniagua, Andreu & Keller, 2015;Bertolero &
Busack, 2017). Our aims are to (i) characterize the chemical composition of MG secretions
in this species, and (ii) test if there are sex-speciﬁc differences in MG gland components
and gland structure in M. leprosa, known for using chemical cues to discriminate
among potential partners and avoid competitors (Ibáñez, López & Martín, 2012;Ibáñez
et al., 2013). We confront the chemical proﬁles of the freshwater M. leprosa MGs with
those of Gopherus spp. inhabiting xeric environments. In addition, under the assumption
that MG secretions play a role in the reproduction of this species, we expect that
male MGs are structurally more complex than female glands, and that they produce
sex-speciﬁc compounds absent in the latter. We use gas chromatography coupled to mass
spectrometry (GC–MS) to characterize MG chemical compositions in both sexes of this
species and apply a combination of light microscopy (LM) and transmission electron
microscopy (TEM) to provide a detailed assessment of the structure of these glands.
MATERIALS AND METHODS
The individuals of M. leprosa examined in this study came from wild Iberian populations
and were housed outdoors in semi-natural conditions in the facilities of the Catalonian
Reptile and Amphibian Rescue Center (CRARC). Turtles were fed regularly and were part
of a reproductive program aimed at releasing individuals into wetlands that are recovering
ecologically. The turtles’diet included ﬁsh, crabs, algae and aquatic plants, at times
supplemented by chicken, ﬁsh and lettuce from the market. All individuals used in this
study were adults sexed according to external morphological features.
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 3/24
The CRARC holds Catalan permit B2100126 for Zoo Facilities to maintain reptiles,
including M. leprosa, in captivity. The sampling of MGs was accepted by the institutional
board of CRARC. Following European Union directive 2010/63/UE, the extraction of
mental gland exudates does not qualify as an experimental procedure because it does not
puncture the tissue or harm the turtle. The sampling protocol was performed following
standard rules of animal welfare and certiﬁcation CPISR-1 C29052019 granted by the
Departament de Territori i Sostenibilitat, Generalitat de Catalunya (Spain) to Albert
Dissection and histology of mental glands
Mental glands from four adult turtle specimens (three males and one female) from CRARC
were used for histological examination. MGs were dissected from freshly dead turtles
and stored in chemical buffers (see below). Turtles died from trauma-related injuries
and the necropsy procedure was performed at CRARC by a specialized veterinarian
(A Martínez-Silvestre). MG structure was examined using LM and TEM.
For examination in LM, entire MGs were ﬁxed in Bouin’s solution immediately after
excision. Dehydration was done in an alcohol gradient followed by clearing in xylene
and embedding in parafﬁn as previously described (Piprek et al., 2012). Sectioning of
parafﬁn blocks was conducted on a ZEISS HYRAX microtome. Parafﬁn sections (6 mm
thick) were stained with Harris’s hematoxylin and eosin to visualize the general structure
of the glands. Alcian blue staining was used to detect acidic polysaccharides. Periodic
acid-Schiff (PAS) stain was used to detect polysaccharides. Mallory’s trichrome stain was
used for the visualization of collagen (Kiernan, 1990).
Small fragments of MGs used for TEM were ﬁxed in Karnovsky’sﬁxative (Karnowsky,
1965). The material was washed in 0.1 M cacodylate buffer and post-ﬁxed in 1% osmium
tetroxide. Dehydration was carried out in a series of graded ethanol solutions. Then
the material was embedded in epoxy resin (EPON-812) as previously described (Piprek
et al., 2017). Semithin sections (0.5 mm) of the resin were stained with methylene blue and
Azure II (1:1) for LM examination. Ultrathin sections (60–70 nm) were contrasted
with uranyl acetate and lead citrate for TEM examination. Images were collected with a
JEOL-2100HT transmission electron microscope in the Department of Cell Biology and
Imaging, Jagiellonian University (Kraków, Poland).
LM and TEM images were processed in CorelDRAW and Corel Photo-Paint to create
the layout of the ﬁgures. Basic adjustments on image brightness, contrast, intensity and
tone were performed if needed.
Collection of mental gland secretions for chemical analysis
We sampled live turtles of both sexes in two different years or seasons (August–September
2018 and March 2019) at CRARC. Secretions from a total of 38 individuals were sampled
and analyzed using GC–MS but only 37 (21 males and 16 females) were included in
the statistical analysis (see below). Samples were refrigerated after sample collection and
stored in cold conditions (−20 C) until chemical analysis. We additionally collected blank
control samples (opened and handled the same way as other samples but without
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 4/24
taking secretion), as well as samples of water from the turtle enclosures to check for
The procedure for MG sampling of live turtles (applicable to all small- and
medium-sized chelonians possessing MGs) is outlined below. First, the head should be
pulled out from inside the shell and immobilized. Second, the mouth should be carefully
opened by using an oral avian speculum to pry apart the jaws. Third, mechanical
pressure needs to be applied to the MGs from within the oral cavity by using forceps with
curved tips. Usually secretions are then released from the oriﬁces of the glands located in
the gular region, but mechanical pressure can also be applied at the margins of the
gland with forceps to squeeze the glands. The duration of the procedure was less than
10 min per turtle. Secretions can be gathered directly into collection vials, but usually they
are collected using forceps if in solid state, or pipetted by glass microcapillary tubes if
in liquid state and then deposited in glass vials. Forceps and other tools used for the
collection of secretions should be cleaned with dichloromethane before the sampling
process to minimize contamination. Glass microcapillary tubes should be used only once,
then disposed of. MG exudates were collected in glass vials previously ﬁlled with
dichloromethane and closed with silicone/PTFE screw caps.
Before the GC–MS analysis, all samples were subjected to a derivatization protocol to
introduce a trimethylsilyl functional group to the compound of interest. First, samples
were warmed up to ambient temperature and dichloromethane was evaporated to dryness
under a stream of nitrogen at 40 C. Then, 10 mL of acetonitrile and 50 mL of 99% N,O-bis
(trimethylsilyl)triﬂuoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)
mixture were added into the dry residue. The amber glass vials were tightly closed and the
derivatization process was carried out at 60 C for 1 h. Afterwards, the vials were opened
and the derivatization solution was evaporated under a stream of nitrogen at 60 C
and the dry residue was dissolved in 25 mL of dichloromethane. Water from the pond was
extracted using dichloromethane and ethyl acetate. Brieﬂy, extracting solvent and water
samples were mixed in a ratio of 2:1, respectively shaken for 10 min, then phases
were separated by centrifugation and the organic layer was collected. Three consecutive
extractions were performed for every sample, and the separated organic solvents were put
into one vial (separate for each solvent type). The collected solvent was evaporated to
dryness under a stream of nitrogen at 40 C and the dry residue was subjected to the
derivatization procedure described above.
All samples were analyzed by a GC–MS system consisting of a 6850 Series II gas
chromatograph and a 5975C MSD mass spectrometer (Agilent Technologies, Santa Clara,
CA, USA) equipped with a HP-5ms capillary column (30 m long, 0.25 mm i.d., 0.25 mm
ﬁlm thickness, Agilent Technologies, Santa Clara, CA, USA). The oven temperature
program was set up to hold 50 C for 10 min, then ramp the temperature up to 280 C with
a rate of 5 C/min, and then hold for 30 min. Helium (5.0) was used as a carrier gas with a
ﬂow rate of 1.0 mL/min. Splitless injection of two mL was performed at injection port
heated to 280 C. The MS transfer line temperature was set to 280 C and ion source
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 5/24
temperature to 230 C. EI source operated at 70 eV, and the mass range for the MS detector
in scan mode was from 39 to 400 m/z (from 5 min) and from 39 to 600 m/z (from 20 min).
The identiﬁcation of all detected compounds was based on a semi-automatic library
search (all results were inspected by the operator) with the NIST 11 database (NIST,
Gaithersburg, MA, USA).
Data filtering and statistical analysis
Compounds were tentatively identiﬁed on the basis of their mass spectra match and the
retention times of detected peaks were additionally used to compare samples. First,
we excluded unmatched compounds as well as compounds with a match lower than
850, considering them as unidentiﬁed. Afterwards, we constructed a database with all the
identiﬁed compounds and ﬁltered out compounds that appeared only in one sample
(including potential contaminants). Thus, only compounds appearing in at least two
samples were retained. Likewise, substances occurring in two or more control tubes
were considered as contaminants and excluded from the downstream analysis. In several
instances, compounds considered here as contaminants were non-natural products
(e.g., phthalates). In a few cases compounds that could naturally appear in MG secretions
were excluded as they were found in at least two control samples. After all ﬁltration steps,
one of the samples contained only cholesterol trimethylsilyl ether and was therefore
excluded from the statistical analysis. In several cases some non-derivatized parent
compounds were found present next to their derivatives. In such cases, only trimethylsilyl
derivatives were taken into account for statistical analysis, and thus, non-derived
compounds were excluded. Substances resistant to derivatization (sylilation process), for
example, alkanes, were considered in their parent form.
To calculate the relative amounts of the compounds, we used the ratio of the area
of an identiﬁed compound divided by the area of a compound present in all samples.
The contaminant: phthalic acid, hept-4-yl isobutyl ester was chosen as a compound likely
originating from the dichloromethane bottle sealing used during sampling and it was
present in all samples. The ratios of the compounds were used for further analysis.
Potential sexual differences were tested using ANOSIM on the ratio matrix. A distance
matrix (vegdist function, Bray–Curtis option) was calculated using the ratio matrix as
input. The distance matrix was used for non-metric multidimensional scaling (NMDS)
plots to visualize inter-individual and sexual variation in chemical composition.
NMDS plots were done using the function metaMDS in the vegan package (Oksanen et al.,
2019). Cholesterol trimethylsilyl ether was found in all samples, but its relative amount
varied greatly among samples. This compound was excluded from some analyses to avoid
its potentially confounding effect (see “Results”). Lipid proﬁles were examined in more
detail to explore sexual differences in speciﬁc compounds. Only alcohols, alkanes,
carboxylic acids and steroids (excluding cholesterol) were selected for this analysis
(carbohydrates and other classes were not included). Differences between the sexes in
the amounts of selected compounds were assessed with a Wilcoxon rank sum test
with continuity correction using the R-function wilcox.test. The pvalues were adjusted
for multiple comparisons using the R function p.adjust (method = “fdr”)
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 6/24
(Benjamini & Hochberg, 1995). Temporal variation in MG chemistry was explored only for
males due to the higher number of available samples.
The method used here, that is, the use of an internal standard to calculate the relative
amounts of the compounds, has the advantage of not being affected by the number of
peaks present in the sample. However, as we did not measure or weigh the secretions, it is
likely that different amounts of secretions were collected for each individual. Therefore we
also calculated the relative amounts of the compounds by another commonly used
method. The percentage of each compound was calculated as the area of the compound
divided by the total area (summation) of the rest of the identiﬁed compounds (i.e., area of
the compound divided by the total area of the identiﬁed compounds in the sample).
We used percentages to visualize sexual differences. A similar pattern was observed
with percentages (see NMDS plot; Fig. S1) compared to the relative amounts calculated
with the internal standard and therefore no further analyses using percentages were carried
Analysis and some plots were performed in R version 3.4.4 (R Core Team, 2018) using
the interface Rstudio. Chemical proﬁles were visualized using the software Instant Clue
(Nolte et al., 2018).
General structure of mental glands in males
In external morphology, MGs in males consist of two bulges located on both sides of the
gular region (ventral surface of the neck) and are relatively prominent (Fig. 1A). A pair of
oriﬁces (openings), leading to the MGs are present on each side of the neck. Oriﬁces
are hardly visible to the naked eye in males (see Figs. 1B and 1C for a comparison with
females, in which openings are more visible). LM showed that each of these four external
oriﬁces leads to sac-shaped elongated epidermal invaginations (Fig. 2A). There is no
evidence from our histological examination that these invaginations are interconnected,
therefore they are likely separate units, that is, each turtle possesses four separate exocrine
MGs with separate openings (two on each side of the neck). The histological structure
of each gland is similar, although one of each pair is slightly larger than the other. MGs are
sac-shaped, simple acinar glands consisting of two parts: (1) an excretory duct opening
with an outlet (oriﬁce) at the skin surface, and (2) an inner-most secretory portion
(Fig. 2A). The outlet and the excretory duct are lined by a keratin layer (stratum corneum)
which is continuous with the outer keratinized layer of the epidermis at the surface of the
neck (Figs. 2A and 2B). The excretory duct is narrow and possesses a lumen inside
(Figs. 2A–2D). The gland outlet is plugged with secretion and exfoliated keratinized
epithelium (Fig. 2B). The excretory duct, as a narrow tube, opens into the lumen of the
wide, sac-shaped basal part of the gland (Figs. 2A and 2D). At the interface between the
excretory duct and secretory portion (Fig. 2C), the keratinized layer becomes thin,
ultimately disappearing, and is replaced by glandular epithelium in the secretory portion.
The secretory portion is lined exclusively with thick, multilayer glandular epithelium
(Fig. 2E). The lumen of the secretory portion is ﬁlled with a mix of the contents of
glandular cells and their derived products, demonstrating that MGs are holocrine (Fig. 2E).
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 7/24
When MG tissue was tested with PAS (Figs. 2F and 2G), the holocrine secretion showed a
weak positive reaction suggesting that carbohydrates or polysaccharides are likely present
(Fig. 2G). MG tissue did not stain with alcian blue, indicating a lack of acidic
Fine scale examination of MG tissue in TEM showed that the glandular epithelium
comprises an engrossed basal layer of epithelium (stratum basale) consisting of dividing
cells that successively differentiate toward the next intermediate cell layer (similar to
the prickle cell layer, that is, stratum spinosum in normal skin epidermis) until maturing
and disintegrating into the glandular lumen (Fig. 3). TEM observations revealed that
the inner-most layer of the glandular epithelium (i.e., basal layer) contains basal cells that
have thick bundles of keratin as well as a nucleus rich in chromatin (Figs. 3B and 3C).
Basal cells are anchored to the dermis by numerous cytoplasmic protrusions that fold into
the basement membrane (Fig. 3B). The stratum spinosum possesses polyhedral cells
that are more elongated than basal cells (Figs. 3A and 3D). Prickle cells are loosely
arranged, and there are intercellular spaces between their cytoplasmic projections. Toward
the lumen, cells from this intermediate layer are more tightly packed, and their cytoplasms
Figure 1 Images showing the macroscopic aspect of mental glands in Mauremys leprosa.(A) Lateral
view of the head in female (♀) and male (♂); more prominent mental glands (arrows) are noticeable in
the male. (B and C) Ventral view of the gular region of a female (♀) (B) and male (♂) (C), the oriﬁces
(openings) of the glands are clearly visible in females and are ﬁlled by brownish plugs (arrowheads),
unlike males in which oriﬁces are not easily visible. Specimens pictured in (A), (B) and (C) are not the
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 8/24
form protrusions connected by desmosomes (Fig. 3D). These cells are rich in
mitochondria. Close to the lumen of the gland, cells differentiate into mature holocrine
cells that are swollen and irregular in shape (Figs. 3E and 3F). Cells disintegrate,
nuclei become fragmented and the cytoplasm together with other cell products is
discharged into the lumen (Figs. 3E–3H). The holocrine secretions are therefore composed
of the excretion products of the cell as well as organellular debris and cellular membrane
fragments (Fig. 3G). Electron-light bodies and small electron-dense bodies were also
detected in the lumen of the gland (Figs. 3G and 3H). Mature cells have abundant Golgi
apparatus that probably play a key role in exocytosis occurring in mature cells (Fig. 3H).
General structure of mental glands in females
Mental glands of females are much more reduced and less prominent compared to
male glands (roughly one third of the length of male glands based on a single specimen,
see Figs. 1,2A and 4A for a rough comparison). Moreover, histological examination of
Figure 2 Histological structure of a mental gland in a male of M. leprosa.(A) Section of the full
mental gland, hematoxylin-eosin (HE) staining (scale is approximate). (B) Detail of the outlet (opening),
the outlet is plugged with secretion and exfoliated keratinized epithelium, HE staining. (C) Detail of the
transition between the excretory duct and the secretory portion, HE staining. (D) Detail of the excretory
duct and secretory portion, Mallory’s Trichrome; note the presence of a keratinized layer in the excretory
duct stained red (arrowheads); connective tissue in the dermis is stained blue. (E) Detail of the glandular
epithelium and the secretory portion (see Fig. 3 for detail on the ﬁne structure of the different cell layers
in the glandular epithelium), HE staining. (F) PAS staining in the excretory duct of the mental gland.
(G) Positive PAS staining (intense purple color) of the holocrine secretion in the secretory portion of
the mental gland. d, excretory duct; de, dermis; ep, epidermis; ge, glandular epithelium; ho, holocrine
secretion; kl, keratinized layer; lu, lumen; out, plugged outlet of the gland; sp, secretory portion.
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 9/24
female MGs also showed a lower degree of complexity (Fig. 4A). The female MG is a
simple, short invagination of the epidermis without a wide secretory portion (Figs.
4B–4D). The entire female MG is lined with a keratinized epidermis similar to the
epithelium of the excretory duct in the male MG. Thus, female MGs more closely resemble
the regular skin epidermis and are notably different than those of males (Figs. 4C and 4D).
As there was no evidence of secretion in females, PAS reaction was not carried out.
Characterization of chemical compounds in mental glands
After ﬁltration steps (see “Material and Methods”) a total of 61 chemical compounds
were identiﬁed, at least to class, in the MG secretions of M. leprosa (Table 1). The number
of compounds per individual was highly variable (mean (median) ± SD = 14.54 (13) ± 8.44;
min = 3; max = 40). The most common compound was cholesterol trimethylsilyl ether
(averaged relative area: 17.79). Although cholesterol trimethylsilyl ether was the only
compound found in all samples, the amount per sample was extremely variable, ranging
from a maximum of 105.6 to a minimum of 1.63. Besides cholesterol, the most abundant
compounds were 5a-cholestan-3β-ol trimethylsilyl derivative (averaged relative
Figure 3 Fine structure of the glandular epithelium of mental glands of M. leprosa males.
(A) Semithin section showing the different layers of the glandular epidermis, methylene blue–Azure
II. (B) Section (TEM) of the basal layer showing basal cells with protrusions invaginating through the
connective tissue. (C) Detail (TEM) of basal cells. (D) Section (TEM) of polyhedral cells constituting the
prickled cell layer. (E) Semithin section of the mature cells and holocrine secretion, methylene
blue-azure II. (F) Mature cells (TEM) that disintegrate in the lumen with abundant vacuoles. (G) Detail
of cytoplasm fragments and electron-light bodies (white arrowheads). (H) Mature cell (TEM) in the
lumen of the gland. Note the presence of Golgi apparatus, exocytotic vesicles (black arrowheads), free
electron-dense bodies and fragments of cell membranes in the lumen of the gland. bl, basal cell layer; bc,
basal cell; bm, basal membrane; cd, cytoplasmic discharge; des, desmosome; lu, lumen; er, endoplasmic
reticulum; ga, Golgi apparatus; kf, keratin ﬁbers; ho, holocrine secretion; pcl, prickled cell layer; nu,
nucleus; m, mitochondria; mc, mature cell; pr, protusion; va, vacuoles.
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 10/24
area = 0.99), 3-((trimethylsilyl)oxy)lanosta-9(11),24-diene (0.66), carbohydrate
unidentiﬁed 8 trimethylsilyl derivative (0.51) and campesterol trimethylsilyl ether (0.43)
(see Table 1).
By far the most abundant class of compounds were steroids (mean of the summed
relative amounts of steroids per sample = 20.49), followed by carboxylic acids (2.14),
carbohydrates (1.51) alkanes (0.40) and alcohols (0.20). In addition, two amines (0.09),
two sugar-alcohols (0.002), one inorganic acid (0.28) and one nucleoside (0.07) were also
Sexual variation in chemical composition
The number of compounds was similar between sexes (Wilcoxon rank sum test with
continuity correction; P= 0.33; males: mean ± SD = 15.67 ± 8.36; females: mean ±
SD = 13.06 ± 8.59). Males and females differed in the amounts of the main compound
classes, with a tendency for males to have larger amounts of particular chemicals. However,
this difference was only signiﬁcant in the case of carbohydrates, for which males had
statistically larger amounts (Wilcoxon rank sum test with continuity correction; P= 0.04;
Taking into account the relative abundances of compounds, the chemical composition
of MG secretions did not differ clearly between males and females when including all
61 constituents (ANOSIM: R= 0.053, P= 0.095; Fig. 5A). The relative amount of
cholesterol trimethylsilyl ether was highly variable among individuals, but amounts
between sexes were similar (Fig. 5B). Indeed, a second NMDS plot excluding cholesterol
showed a clear pattern discriminating between the sexes as revealed by different centroids
and non-overlapping conﬁdence intervals (ANOSIM: R= 0.31, P= 0.001; Fig. 5C).
Figure 4 Histological and ﬁne structure of mental glands in a female of M. leprosa.(A) Lateral
section stained with HE. (B) Superﬁcial region of the gland (semithin section) (C) Detail of the basal part
of the gland (semithin section). (D) Basal part of the gland (TEM). de, dermis; ep, epidermis; ker,
keratinocyte; kl, keratin layer; lu, lumen; out, outlet of the gland.
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 11/24
Table 1 Identiﬁed chemical compounds in Mauremys leprosa mental glands from the GC-MS. Relative amounts (Ratios; Mean and SD) of the
compounds, calculated by using an internal standard (phthalic acid, hept-4-yl isobutyl ester), a non-natural compound appearing in all samples
(see details in “Materials and Methods”). Relative amounts for males (mean) and females (mean), together with adjusted signiﬁcance value for
multiple comparisons (PAdj.) are shown—only alcohols, alkanes, carboxylic acids and steroids (except cholesterol trimethylsilyl ether) were
considered in this analysis. Signiﬁcant differences after adjustment are highlighted in bold. Abbreviations (Abrr.) as in Fig. 6. The number of males
and females in which the compound was detected is provided in columns “M (num.)”and “F (num.)”. Percentage (mean) for each compound was
calculated based on the area of the focal compound in respect to the total area of identiﬁed compounds.
Name Abbr. Ratios
1-Hexadecanol, trimethylsilyl ether Aol-1 0.046 0.207 0.075 0.007 0.817 1 2 0.112
Octadec-9Z-enol, trimethylsilyl ether derivative Aol-2 0.022 0.082 0.039 0.000 0.250 0 4 0.061
1-O-Hexadecylglycerol, - bis(trimethylsilyl) ether
Aol-3 0.109 0.200 0.114 0.102 0.907 7 10 0.350
1-O-Octadecylglycerol, - bis(trimethylsilyl) ether
Aol-4 0.021 0.072 0.022 0.019 0.907 2 2 0.049
Tricosane Alk-1 0.019 0.085 0.026 0.010 0.907 2 2 0.053
Tetracosane Alk-2 0.045 0.135 0.055 0.031 0.891 4 4 0.144
Pentacosane Alk-3 0.055 0.183 0.057 0.052 0.350 5 2 0.187
Hexacosane Alk-4 0.060 0.244 0.069 0.049 0.285 4 1 0.170
Heptacosane Alk-5 0.066 0.238 0.077 0.052 0.483 4 2 0.183
Octacosane Alk-6 0.060 0.220 0.071 0.047 0.624 3 2 0.169
Nonacosane Alk-7 0.057 0.222 0.070 0.040 0.624 3 2 0.155
Triacontane Alk-8 0.041 0.185 0.050 0.029 0.907 1 1 0.092
1-Dodecanamine, N,N-dimethyl- –0.073 0.226 –––5 2 0.379
1-Tetradecanamine, N,N-dimethyl- –0.020 0.081 –––3 1 0.095
Carbohydrate Unidentiﬁed 1, trimetylsilyl derivative –0.004 0.019 –––1 1 0.012
Carbohydrate Unidentiﬁed 2, trimetylsilyl derivative –0.009 0.035 –––3 1 0.031
Carbohydrate Unidentiﬁed 3, trimetylsilyl derivative –0.002 0.008 –––1 1 0.003
Carbohydrate Unidentiﬁed 5, trimetylsilyl derivative –0.101 0.286 –––1 7 0.789
Carbohydrate Unidentiﬁed 8, trimethylsilyl derivative –0.509 0.656 –––11 18 3.596
Carbohydrate Unidentiﬁed 9, trimethylsilyl derivative –0.213 0.321 –––4 14 1.827
Carbohydrate Unidentiﬁed 10, trimethylsilyl derivative –0.006 0.024 –––0 3 0.083
Carbohydrate Unidentiﬁed 11, trimethylsilyl derivative –0.096 0.318 –––0 4 0.450
Carbohydrate Unidentiﬁed 12, trimethylsilyl derivative –0.037 0.107 –––0 6 0.219
Carbohydrate Unidentiﬁed 13, trimethylsilyl derivative –0.104 0.263 –––0 8 0.697
Carbohydrate Unidentiﬁed 14, trimethylsilyl derivative –0.127 0.386 –––0 4 0.469
Carbohydrate Unidentiﬁed 6, trimetylsilyl derivative –0.286 0.394 –––11 16 1.883
Carbohydrate Unidentiﬁed 7, trimetylsilyl derivative –0.012 0.037 –––5 4 0.061
Propanoic acid, 2-[(trimethylsilyl)oxy]-, trimethylsilyl
Cac-1 0.055 0.331 0.098 0.000 0.420 0 2 0.191
Benzoic acid, trimethylsilyl ester Cac-2 0.010 0.024 0.014 0.004 0.591 3 6 0.040
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 12/24
Lipid proﬁles were examined through statistical tests for a further 41 compounds to
detect potential differences between the sexes. Sexual differences occurred in three
carboxylic acids and three steroids. Males had larger amounts of tetradecanoic acid
trimethylsilyl ester, hexadecenoic acid trimethylsilyl ester (isomer 1) and campesterol
Table 1 (continued ).
Name Abbr. Ratios
Nonanoic acid, trimethylsilyl ester Cac-3 0.006 0.021 0.004 0.007 0.591 2 1 0.057
Dodecanoic acid, trimethylsilyl ester Cac-4 0.020 0.054 0.034 0.001 0.197 1 7 0.052
Azelaic acid, bis(trimethylsilyl) ester Cac-5 0.015 0.059 0.021 0.006 0.817 1 2 0.046
Tetradecanoic acid, trimethylsilyl ester Cac-6 0.312 0.624 0.519 0.040 0.028 2 14 0.972
Pentadecanoic acid, trimethylsilyl ester. Isomer 1 Cac-7 0.021 0.068 0.036 0.002 0.420 1 4 0.048
Pentadecanoic acid, trimethylsilyl ester. Isomer 2 Cac-8 0.039 0.188 0.067 0.003 0.591 1 3 0.063
Pentadecanoic acid, trimethylsilyl ester. Isomer 3 Cac-9 0.015 0.064 0.027 0.000 0.420 0 2 0.023
Hexadecenoic acid, trimethylsilyl ester. Isomer 1 Cac-10 0.051 0.112 0.089 0.000 0.037 0 9 0.190
Hexadecenoic acid, trimethylsilyl ester. Isomer 2 Cac-11 0.329 0.734 0.515 0.085 0.285 7 12 0.953
Heptadecanoic acid, trimethylsilyl ester. Isomer 1 Cac-12 0.007 0.029 0.012 0.000 0.420 0 2 0.010
Heptadecenoic acid, trimethylsilyl ester Cac-13 0.010 0.047 0.018 0.000 0.420 0 2 0.029
Heptadecanoic acid, trimethylsilyl ester. Isomer 2 Cac-14 0.042 0.101 0.068 0.007 0.285 2 7 0.155
Octadecadienoic acid, trimethylsilyl ester Cac-15 0.329 0.641 0.488 0.120 0.247 5 12 1.033
Octadecenoic acid, trimethylsilyl ester. Isomer 2 Cac-16 0.179 0.252 0.210 0.139 0.624 7 11 0.676
Octadecenoic acid, trimethylsilyl ester. Isomer 3 Cac-17 0.366 2.133 0.641 0.006 0.350 1 5 0.657
Octadecenoic acid, trimethylsilyl ester. Isomer 4 Cac-18 0.095 0.436 0.168 0.000 0.420 0 2 0.153
Arachidonic acid, trimethylsilyl ester Cac-19 0.061 0.193 0.058 0.065 0.624 3 2 0.173
Eicosenoic acid, trimethylsilyl ester. Isomer 1 Cac-20 0.019 0.101 0.029 0.007 0.907 1 1 0.039
Eicosanoic acid, trimethylsilyl ester Cac-21 0.109 0.244 0.055 0.180 0.236 8 4 0.456
Docosanoic acid, trimethylsilyl ester Cac-22 0.051 0.130 0.014 0.101 0.047 7 1 0.236
Phosphoric acid, trimethylsilyl ester –0.275 1.474 –––4 6 1.015
Uridine, 2′,3′,5′-tris-O-TMS –0.066 0.154 –––3 7 0.362
Steroid Unidentiﬁed 1, trimethylsilyl derivative Std-1 0.322 0.594 0.122 0.584 0.047 11 4 1.425
Steroid Unidentiﬁed 2, trimethylsilyl derivative Std-2 0.010 0.030 0.000 0.023 0.107 4 0 0.088
Cholesterol trimethylsilyl ether –17.789 19.815 –––16 21 69.749
5a-Cholestan-3β-ol, trimethylsilyl derivative Std-3 0.989 1.727 0.835 1.192 0.028 16 11 4.437
5a-Cholest-7-en-3β-ol, trimethylsilyl derivative Std-4 0.230 0.490 0.225 0.236 0.591 6 12 0.788
Campesterol, trimethylsilyl ether Std-5 0.431 0.708 0.652 0.141 0.037 6 19 1.545
β-Sitosterol, trimethylsilyl ether Std-6 0.062 0.209 0.108 0.000 0.197 0 5 0.219
3-[(Trimethylsilyl)oxy]lanosta-9(11),24-diene Std-7 0.656 1.710 0.803 0.464 0.591 8 7 1.784
D-Sorbitol, hexakis (trimethylsilyl) ether –0.001 0.005 –––1 1 0.013
Sugar alcohol 1, trimethylsilyl derivative –0.001 0.005 –––1 1 0.002
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 13/24
trimethylsilyl ether (Table 1;Fig. 6). On the other hand, docosanoic acid trimethylsilyl
ester, unidentiﬁed steroid 1 trimethylsilyl derivative and 5a-cholestan-3β-ol, trimethylsilyl
derivative were signiﬁcantly more abundant in females (Table 1;Fig. 6). Alcohol and
alkane proﬁles were similar between males and females (Fig. 6). The chemical composition
of male MG secretions showed a signiﬁcant but weak difference between the seasons (i.e.,
sampling occasions; year 2018 vs year 2019) when considering all compounds, including
cholesterol trimethylsilyl ether (ANOSIM: R= 0.176; P= 0.044). However, when
cholesterol trimethylsilyl ether was excluded there was no clear seasonal pattern in
chemical composition (ANOSIM: R= 0.141, P= 0.063; Fig. S3).
-1.0 -0.5 0.0 0.5 1.0
Cholesterol TMS (Rel. area)
-0.4 -0.2 0.0 0.2 0.4
Figure 5 Plots showing sexual variation in chemical composition of mental glands of Mauremys
leprosa.(A) Non-metric multidimensional scaling plots (NMDS) based on Bray Curtis dissimilarity
considering all compounds (stress = 0.07). (B) Boxplot showing the amount (relative area) of cholesterol
trimethylsilyl ether (TMS) in males and females. Median, interquartile range and outliers/extreme values
are shown. (C) Non-metric multidimensional scaling plots (NMDS) based on Bray Curtis dissimilarity
excluding cholesterol trimethylsilyl ether (stress = 0.21). In (A) and (C), gray points represent females
and black points males. Closer points represent more similar compositions in individual turtles. Ellipses
were calculated with the function ordiellipse (package Vegan) and represent 95% conﬁdence interval
(based on standard error) for the sexes. Full-size
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 14/24
This study provides a comprehensive assessment of MG anatomy and chemistry in a
freshwater turtle species. The MGs of adult M. leprosa consist of four independent
sac-shaped invaginations opening in the gular region of the neck. Glands might differ
Amount (Rel. area) Amount (Rel. area)
Amount (Rel. area)
Figure 6 Chemical proﬁles of M. leprosa.Amount (mean and 0.95 conﬁdence interval estimated by
bootstrapping) of: (A) Alcohols (Aol). (B) Alkanes (Alk). (C) Carboxylic acids (Cac). (D) Steroids (Std;
except cholesterol trimethylsilyl ether). Males are represented by black and females by gray color. Sig-
niﬁcant values after adjustment for multiple comparisons are marked with an asterisk. Compound
abbreviations are as in Table 1.Full-size
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 15/24
slightly in size in a given individual but they show similar structure. In terms of general
morphological complexity, the MGs of M. leprosa lie halfway between the simple
invaginations present in emydid turtles and large, compound and elaborate glands of
the desert tortoise Gopherus agassizi and the aquatic turtle Siebenrockiella crassicollis
(Winokur & Legler, 1975). Histological examination showed that the glands of males are
more complex than those present in females. In addition, chemical analysis revealed a
relatively high diversity of compounds in MGs. Sexual differences in gland chemistry were
driven by six of the compounds present in MGs, as well as a tendency for higher relative
amounts in many other compounds in males. Three compounds were found in
signiﬁcantly larger amounts in males than in females and three others showed the
Chemical compounds in mental gland secretions
Steroids and carboxylic acids were the most common chemical compounds in MGs of
the Spanish terrapin, a pattern also found in other reptiles (Weldon, Flachsbarth &
Schulz, 2008;Martín & López, 2011). The most prevalent compound was cholesterol
(trimethylsilyl ether), a steroid occurring in cell membranes of vertebrate tissues as well as
in epidermal and glandular secretions of reptiles (Weldon, Flachsbarth & Schulz, 2008).
The role of cholesterol in intraspeciﬁc signaling is unclear, with previous studies suggesting
that it might be species-speciﬁc. One hypothesis is that cholesterol could act as an
unreactive matrix protecting semiochemical functional compounds, found in smaller
amounts, from accelerated degradation due to the effect of high temperatures in hot
environments (Escobar et al., 2003). In the Spanish terrapin, we observed extremely high
inter-individual variation in cholesterol trimethylsilyl ether that indeed masked differences
between sexes in chemical compounds. Therefore, it is unlikely that cholesterol is
directly involved in intraspeciﬁc communication in M. leprosa.
Previous studies on gopher tortoises (Rose, Drotman & Weaver, 1969;Rose, 1970)
reported a different chemical composition of MG secretions compared to those of Spanish
terrapins. Although distinctive chemical proﬁles of MG secretions could be expected
due to the strongly divergent habitats occupied by both species, the different methods
used in both studies (e.g., chemical analysis procedure and/or derivatization agents)
render the comparison preliminary. Nonetheless, some interesting patterns emerge. First,
cholesterol and saturated and unsaturated fatty acids in the range of C
chain lengths have been identiﬁed in secretions of Gopherus berlandieri, whereas MGs of
M. leprosa contained saturated and unsaturated C
carboxylic acids, as well as
shorter compounds such as benzoic and propanoic acids (the latter occurring only in two
males). Therefore, Spanish terrapin secretions encompass a larger chain length range
of carboxylic acids than those of G. berlandieri. The xeric terrestrial environment inhabited
by G. berlandieri might explain the lack of highly volatile compounds such as relatively
short chain carboxylic acids that would fade out quickly due to the degradative effect
of elevated temperatures (Alberts, 1992;Van Oudenhove et al., 2011;Martín & López,
2013). In contrast, Spanish terrapins are predominantly aquatic—but may also be
active on land (see below)—and therefore occupy habitats associated with more mesic
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 16/24
conditions than Gopherus, where more volatile compounds such as relatively short
carboxylic acids (e.g., benzoic and propanoic acids) could persist for a longer time. Second,
some compounds present in Spanish terrapin MGs such as alcohols, alkanes and
carbohydrates have not been reported in G. berlandieri (Rose, 1970). However, it is
important to point out that methodological differences between the studies could
affect the results and therefore a comparision between our ﬁndings and the previous
studies needs to be considered carefully. Compounds with relatively high polarity, such as
carbohydrates, can solve better in water and thus facilitate the reception of other
compounds that might be chemosignals from glandular secretions by turtles that are
actively mating. These compounds are potentially important since copulations in
M. leprosa typically, but not exclusively, occur underwater (Bertolero & Busack, 2017)
and experiments indicate that turtles might use water-borne chemical cues to
communicate with other conspeciﬁcs (Ibáñez, López & Martín, 2012;Ibáñez et al., 2013).
Sexual dimorphism in mental gland anatomy and chemistry
MGs differ clearly in terms of anatomical and structural complexity between males
and females. In males, the oriﬁce of the gland is followed by a simple duct connected to
the lumen in which secretion accumulates. Secretions had a positive (weak) reaction to
PAS indicating the presence of carbohydrates and/or neutral mucosubstances, in line
with Gopherus males, in which highly vacuolated cells present in MGs showed a
PAS-positive reaction (Winokur & Legler, 1975). The presence of well-developed lipid
droplets was not observed in M. leprosa glands. However, electron-light bodies are present
in the lumen of the gland but the chemical nature of these remains unknown. We detected
a relatively high degree of exocytotic vacuolization in mature cells that disintegrate in
the lumen, indicating that MGs in male M. leprosa are active and produce holocrine
secretions composed of cellular and organellar fragments as well as exocytotic products.
MGs consist of heavily keratinized invaginations in females. Histological evidence
in Gopherus berlandieri showed that MGs are active in both sexes (Weaver, 1970).
However, inactive glands have been described in females and juveniles of other chelonians,
including species of the family Geoemydidae (Winokur & Legler, 1975). Similarly, the
histology and ﬁne structure of MGs in female M. leprosa suggest that they might be
inactive or at least reduced. No evidence of holocrine secretion in female glands was
observed in this study. Female glands are likely vestigial or primordium organs in
An interesting feature of MG chemistry in Spanish terrapins is the presence of
carbohydrates, found in larger amounts in males than in females. One explanation is that
sugars found in MGs are involved in glycosylation of secreted proteins, a process affecting
the three dimensional conformation of the molecules and therefore their function
(Moremen, Tiemeyer & Nairn, 2012). Although the protein fraction of chelonian MGs
is nearly unknown (Alberts, Rostal & Lance, 1994), proteins could function as potential
signals (Wyatt, 2014). It is tempting to speculate that the sugars found in MG secretions
of M. leprosa are involved in the glycosylation and regulation of sexually-mediating
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 17/24
glycoproteins expressed in male glands. Proteomic analysis of MGs should shed light on
Some compounds appeared in larger amounts in one of the sexes and deserve special
attention as they could potentially be involved in chemical signaling. Males had relatively
larger amounts of campesterol trimethylsilyl ether, a steroid, as well as hexadecenoic
acid trimethylsilyl ester (isomer 1) and tetradecanoic acid trimethylsilyl ester, both of
which are carboxylic acids. A possible explanation is that male terrapins allocate these
compounds from their fat stores to the MGs, where they accumulate in the secretions
until being released. However, most of the research on chemical communication in aquatic
or semiaquatic turtles focuses on the freshwater environment (Muñoz, 2004;Poschadel,
Meyer-Lucht & Plath, 2006;Lewis et al., 2007;Ibáñez, López & Martín, 2012) and it is
unclear how carboxylic acids could be transmitted in this media. Spanish terrapins might
also be able to communicate in the terrestrial environment as they spend long periods
basking (Díaz-Paniagua, Andreu & Keller, 2015;Bertolero & Busack, 2017). Carboxylic
acids might be used as olfactory signals on land for mating and/or intrasexual interactions
as postulated for G. berlandieri (Rose, 1970). This could be important in male–male
interactions during basking site competition, as optimal sites for thermoregulation are
a limiting factor in natural habitats (Cadi & Joly, 2003;Polo-Cavia, López & Martín, 2010).
Campesterol is a phytosterol present in the epidermis and femoral secretions of squamates
(Weldon, Flachsbarth & Schulz, 2008). Phytosterols are typically of plant origin and
given that M. leprosa is omnivorous, campesterol could be obtained from the intake of
plant material. Vertebrates might excrete steroids that serve as olfactory cues involved in
social communication (Doyle & Meeks, 2018). In goldﬁsh, steroid-derived pheromones
are released into the water to modulate reproductive behavior by synchronizing
female–male cycles (Dulka et al., 1987). However, the potential mechanism of action of
these compounds in turtles is unknown. In M. leprosa, chemosignal detection is dependent
on concentration (Ibáñez et al., 2014). However, without a behavioral bioassay or a
physiological experiment, we cannot establish whether these compounds are involved in
sexual signaling. Nonethless, our study provides the groundwork for future empirical
assessments of the roles of these compounds in chemical communication in turtles.
Two steroids (5a-cholestan-3β-ol trimethylsilyl derivative and an unidentiﬁed steroid)
and one carboxylic acid (docosanoic acid trimethylsilyl ester) were found in larger
amounts in female glands. We hypothesize that these come from metabolism of other
compounds and/or are present in large amounts in turtle skin. In fact, 5a-cholestan-3β-ol
can be metabolically converted from cholesterol (Shefer, Milch & Mosbach, 1964;Werbin,
Chaikoff & Phillips, 1964). A plausible explanation is that chemical compounds found
in female glands originate from the excess of accumulated keratin in corneocytes, as
carboxylic acids—especially very long chain (C
) ones—and cholesterol among other
compounds are found in abundance in these cells (Downing, 1992). However, we cannot
rule out that these compounds are used by females to signal their quality to potential
partners (Ibáñez, López & Martín, 2012), although the simple structure of MGs in females
and a lack of glandular epithelium argues against it. It is also possible that chemical signals
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 18/24
in turtles are produced by other kinds of secretory organs such as cloacae and/or Rathke’s
We note that other compounds might also be important in sexual communication but
could be overlooked or masked by the high level of inter-individual variability in chemical
composition shown by Spanish terrapins. In general, and in line with the rudimentary
character of female MGs, males tended to have a higher compound diversity and relatively
elevated amounts of most compounds. Several compounds were present in males but
were entirely absent in females. However, only one compound was absent in males but was
present in some females (unidentiﬁed steroid 2, trimethylsilyl derivative; see Table 1).
In many instances, these “exclusive”compounds were found in only a small number of
males. Therefore, it could be that other factors such as age, body size or health status of
male turtles inﬂuence the chemical composition of MGs.
Slight seasonal differences in chemical composition were observed in male turtles
but this was mostly driven by variable levels of the main compound—cholesterol, and
therefore we conclude that there is no clear pattern of temporal variation in chemical
composition of MG secretions. MG size (volume) in male G. agassizii changes through the
year being larger during the mating season (Alberts, Rostal & Lance, 1994). Although
our results in male M. leprosa do not show a clear difference between seasons, our
sampling occasions, August–September 2018 and March 2019, might have encompassed
reproductive periods in this species (Díaz-Paniagua, Andreu & Keller, 2015), and thus
were not designed to discern seasonal variation in semiochemical production.
This study showed that MGs in male M. leprosa are complex structures producing
holocrine secretions. In contrast, female MGs resemble inactive or rudimentary organs.
The bulk of the MG secretion is composed of steroids, especially cholesterol, as in other
reptilian epidermal glands. Males and females showed qualitatively similar types of
compounds in MGs, suggesting that many chemicals could be metabolic byproducts
excreted from skin cells with no obvious role as sexual signals. However, the relative
amounts of some compounds were higher in males, with signiﬁcantly larger amounts
of three compounds. Moreover, signiﬁcant amounts of carbohydrates were found in
glandular secretions, especially in males; their role is unknown but they could be important
in protein glycosylation. Through biossays, behavioral or physiological experiments,
future research should test whether any of the compounds identiﬁed in this study play a
role in intraspeciﬁc communication. In parallel, studies on the protein fraction should be
done to identify proteins used in chemosignalling. Furthermore, a uniform chemical
identiﬁcation method applied to MG secretions from a wider set of turtle species could
address the effect of environment in shaping interspeciﬁc variation in chelonian chemical
We would like to thank the personnel who helped during the sampling at CRARC,
especially Adrian Melero and Joaquim Soler. We are grateful to Dr. RafałPiprek for his
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 19/24
contribution in preparing histological ﬁgures and text as well as numerous suggestions that
helped to improve this manuscript. We thank Emilia Rydzy for helping to prepare part of
the histological material. We thank the editor and three anonymous referees for the
comments and suggestions improved the manuscript.
ADDITIONAL INFORMATION AND DECLARATIONS
Financial support was obtained from the Polish National Science Center (Narodowe
Centrum Nauki, NCN, https://www.ncn.gov.pl/), OPUS grant no. UMO-2017/25/B/NZ8/
01498 to Alejandro Ibáñez. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
The following grant information was disclosed by the authors:
Polish National Science Center.
OPUS Grant: UMO-2017/25/B/NZ8/01498.
The authors declare that they have no competing interests.
Alejandro Ibáñez conceived and designed the experiments, performed the experiments,
analyzed the data, prepared ﬁgures and/or tables, authored or reviewed drafts of the
paper, and approved the ﬁnal draft.
Albert Martínez-Silvestre performed the experiments, authored or reviewed drafts of the
paper, and approved the ﬁnal draft.
Dagmara Podkowa analyzed the data, prepared ﬁgures and/or tables, authored or
reviewed drafts of the paper, and approved the ﬁnal draft.
Aneta Woźniakiewicz analyzed the data, authored or reviewed drafts of the paper, and
approved the ﬁnal draft.
MichałWoźniakiewicz analyzed the data, authored or reviewed drafts of the paper, and
approved the ﬁnal draft.
Maciej Pabijan conceived and designed the experiments, authored or reviewed drafts of
the paper, and approved the ﬁnal draft.
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
The Catalonian Reptile and Amphibian Rescue Center (CRARC) holds Catalan permit
B2100126 for Zoo Facilities to maintain reptiles, including Mauremys leprosa, in captivity.
The sampling of MGs was accepted by the institutional board of CRARC. Following
European Union directive 2010/63/UE, the extraction of mental gland exudates does not
qualify as an experimental procedure because it does not puncture the tissue or harm the
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 20/24
turtle. The sampling protocol was performed following standard rules of animal welfare
and certiﬁcation CPISR-1 C29052019 granted by the Departament de Territori i
Sostenibilitat, Generalitat de Catalunya (Spain) to Albert Martínez-Silvestre.
The following information was supplied regarding data availability:
The raw data is available at the Jagiellonian University Repository (RUJ):
- Raw images for histology pictures of the article: “The chemistry and histology
of sexually dimorphic mental glands in the freshwater turtle, Mauremys leprosa”:
- RAW chromatographic data (GC-MS) for the article: “The chemistry and histology
of sexually dimorphic mental glands in the freshwater turtle, Mauremys leprosa”:
- Research datasets of the article: “The chemistry and histology of sexually dimorphic
mental glands in the freshwater turtle, Mauremys leprosa”:
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
Alberts AC. 1992. Constraints on the design of chemical communication systems in terrestrial
vertebrates. American Naturalist 139:S62–S89 DOI 10.1086/285305.
Alberts AC, Rostal DC, Lance VA. 1994. Studies on the chemistry and social signiﬁcance of chin
gland secretions in the desert tortoise, Gopherus agassizii.Herpetological Monographs 8:116–124
Apps PJ, Weldon PJ, Kramer M. 2015. Chemical signals in terrestrial vertebrates: search for design
features. Natural Product Reports 32(7):1131–1153 DOI 10.1039/C5NP00029G.
Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a practical and powerful
approach to multiple testing. Journal of the Royal Statistical Society: Series B (Methodological)
57(1):289–300 DOI 10.1111/j.2517-6161.1995.tb02031.x.
Bertolero A, Busack S. 2017. Mauremys leprosa (Schoepff in Schweigger 1812)-Mediterranean
pond turtle, Spanish terrapin, Mediterranean stripe-necked terrapin. In: Rhodin AGJ,
Iverson JB, Dijk PP, Buhlmann KA, Pritchard PCH, Mittermeier RA, eds. Conservation Biology
of Freshwater Turtles And Tortoises: A Compilation Project of the Iucn/ssc Tortoise and
Freshwater Turtle Specialist Group. Lunenburg: Chelonian Research Monographs, 102.1–102.19.
Cadi A, Joly P. 2003. Competition for basking places between the endangered European pond
turtle (Emys orbicularis galloitalica) and the introduced red-eared slider (Trachemys scripta
elegans). Canadian Journal of Zoology 81(8):1392–1398 DOI 10.1139/z03-108.
Downing DT. 1992. Lipid and protein structures in the permeability barrier of mammalian
epidermis. Journal of Lipid Research 33:301–313.
Doyle WI, Meeks JP. 2018. Excreted steroids in vertebrate social communication.
Journal of Neuroscience 38(14):3377–3387 DOI 10.1523/JNEUROSCI.2488-17.2018.
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 21/24
Dulka J, Stacey N, Sorensen PW, Van Der Kraak G. 1987. A steroid sex pheromone synchronizes
male–female spawning readiness in goldﬁsh. Nature 325(6101):251–253
Díaz-Paniagua C, Andreu AC, Keller C. 2015. Galápago leproso—Mauremys leprosa.
In: Salvador A, Marco A, eds. Enciclopedia Virtual de los Vertebrados Españoles. Madrid: Museo
Nacional de Ciencias Naturales. Available at http://www.vertebradosibericos.org/.
Ehrenfeld JG, Ehrenfeld DW. 1973. Externally secreting glands of freshwater and sea turtles.
Copeia 305-314(2):305–314 DOI 10.2307/1442969.
Escobar CM, Escobar CA, Labra A, Niemeyer HM. 2003. Chemical composition of precloacal
secretions of two Liolaemus fabiani populations: are they different? Journal of Chemical Ecology
29(3):629–638 DOI 10.1023/A:1022858919037.
García-Roa R, Sáiz J, Gómara B, López P, Martín J. 2017. Dietary constraints can preclude the
expression of an honest chemical sexual signal. Scientiﬁc Reports 7(1):6073
Houck LD. 2009. Pheromone communication in amphibians and reptiles. Annual Review of
Physiology 71(1):161–176 DOI 10.1146/annurev.physiol.010908.163134.
Hurst JL, Payne CE, Nevison CM, Marie AD, Humphries RE, Robertson DH, Cavaggioni A,
Beynon RJ. 2001. Individual recognition in mice mediated by major urinary proteins. Nature
414(6864):631–634 DOI 10.1038/414631a.
Ibáñez A, López P, Martín J. 2012. Discrimination of conspeciﬁcs’chemicals may allow Spanish
terrapins to ﬁnd better partners and avoid competitors. Animal Behaviour 83(4):1107–1113
Ibáñez A, Marzal A, López P, Martín J. 2013. Boldness and body size of male Spanish terrapins
affect their responses to chemical cues of familiar and unfamiliar males. Behavioral Ecology and
Sociobiology 67(4):541–548 DOI 10.1007/s00265-012-1473-6.
Ibáñez A, Marzal A, López P, Martín J. 2014. Chemosensory assessment of rival body size is based
on chemosignal concentration in male Spanish terrapins. Behavioral Ecology and Sociobiology
68(12):2005–2012 DOI 10.1007/s00265-014-1806-8.
Karnowsky MJ. 1965. A formaldehyde-glutaraldehyde ﬁxative of high osmolarity for use in
electron microscopy. Journal of Cell Biology 27:137A–138A.
Kiernan JA. 1990. Histological and histochemical methods: theory and practice. Second Edition.
Oxford: Pergamon Press.
Kopena R, López P, Martín J. 2017. Immune challenged male Iberian green lizards may increase
the expression of some sexual signals if they have supplementary vitamin E. Behavioral Ecology
and Sociobiology 71(12):173 DOI 10.1007/s00265-017-2401-6.
Lemaster MP, Mason RT. 2002. Variation in a female sexual attractiveness pheromone controls
male mate choice in garter snakes. Journal of Chemical Ecology 28(6):1269–1285
Lewis CH, Molloy SF, Chambers RM, Davenport J. 2007. Response of common musk turtles
(Sternotherus odoratus) to intraspeciﬁc chemical cues. Journal of Herpetology 41(3):349–353
Martín J, López P. 2010. Multimodal sexual signals in male ocellated lizards Lacerta lepida:
vitamin E in scent and green coloration may signal male quality in different sensory channels.
Naturwissenschaften 97(6):545–553 DOI 10.1007/s00114-010-0669-8.
Martín J, López P. 2011. Pheromones and reproduction in Reptiles. In: Lopez KH, Norris DO, eds.
Hormones and Reproduction in Vertebrates. CA: Academic Press, 141–167.
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 22/24
Martín J, López P. 2013. Effects of global warming on sensory ecology of rock lizards: increased
temperatures alter the efﬁcacy of sexual chemical signals. Functional Ecology 27(6):1332–1340
Mason RT, Parker MR. 2010. Social behavior and pheromonal communication in reptiles.
Journal of Comparative Physiology A 196(10):729–749 DOI 10.1007/s00359-010-0551-3.
Moremen KW, Tiemeyer M, Nairn AV. 2012. Vertebrate protein glycosylation: diversity,
synthesis and function. Nature Reviews Molecular Cell Biology 13(7):448–462
Muñoz A. 2004. Chemo-orientation using conspeciﬁc chemical cues in the stripe-necked terrapin
(Mauremys leprosa). Journal of Chemical Ecology 30(3):519–530
Nolte H, MacVicar TD, Tellkamp F, Krüger M. 2018. Instant clue: a software suite for interactive
data visualization and analysis. Scientiﬁc Reports 8(1):12648 DOI 10.1038/s41598-018-31154-6.
Oksanen J, Blanchet G, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O’Hara RB,
Simpson GL, Solymos P, Henry M, Stevens H, Szoecs E, Wagner H. 2019. Vegan: Community
ecology package. R package version 2.5-4. Available at https://CRAN.R-project.org/
Piprek RP, Kloc M, Tassan J-P, Kubiak JZ. 2017. Development of Xenopus laevis bipotential
gonads into testis or ovary is driven by sex-speciﬁc cell–cell interactions, proliferation rate, cell
migration and deposition of extracellular matrix. Developmental Biology 432(2):298–310
Piprek RP, Pecio A, Kubiak JZ, Szymura JM. 2012. Differential effects of busulfan on gonadal
development in ﬁve divergent anuran species. Reproductive Toxicology 34(3):393–401
Polo-Cavia N, López P, Martín J. 2010. Competitive interactions during basking between native
and invasive freshwater turtle species. Biological Invasions 12(7):2141–2152
Poschadel JR, Meyer-Lucht Y, Plath M. 2006. Response to chemical cues from conspeciﬁcs reﬂects
male mating preference for large females and avoidance of large competitors in the European
pond turtle, Emys orbicularis.Behaviour 143(5):569–587 DOI 10.1163/156853906776759510.
R Core Team. 2018. R: a language and environment for statistical computing. Vienna:
R Foundation for Statistical Computing. Available at http://www.R-project.org/.
Rose FL. 1970. Tortoise chin gland fatty acid composition: behavioral signiﬁcance.
Comparative Biochemistry and Physiology 32(3):577–580 DOI 10.1016/0010-406X(70)90475-5.
Rose FL, Drotman R, Weaver WG. 1969. Electrophoresis of chin gland extracts of Gopherus
(tortoises). Comparative Biochemistry and Physiology 29(2):847–851
Shefer S, Milch S, Mosbach EH. 1964. Biosynthesis of 5a-cholestan-3β-ol in the rabbit and guinea
pig. Journal of Biological Chemistry 239:1731–1736.
Symonds MR, Elgar MA. 2008. The evolution of pheromone diversity. Trends in Ecology &
Evolution 23(4):220–228 DOI 10.1016/j.tree.2007.11.009.
Uhrig EJ, LeMaster MP, Mason RT. 2014. Species speciﬁcity of methyl ketone proﬁles in the skin
lipids of female garter snakes, genus Thamnophis.Biochemical Systematics and Ecology 53:51–58
Van Oudenhove L, Billoir E, Boulay R, Bernstein C, Cerdá X. 2011. Temperature limits trail
following behaviour through pheromone decay in ants. Naturwissenschaften 98(12):1009–1017
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 23/24
Waagen GN. 1972. Musk glands in recent turtles. M.S. Thesis. University of Utah, Utah, USA.
Wang Z, Pascual-Anaya J, Zadissa A, Li W, Niimura Y, Huang Z, Li C, White S, Xiong Z,
Fang D, Wang B, Ming Y, Chen Y, Zheng Y, Kuraku S, Pignatelli M, Herrero J, Beal K,
Nozawa M, Li Q, Wang J, Zhang H, Yu L, Shigenobu S, Wang J, Liu J, Flicek P, Searle S,
Wang J, Kuratani S, Yin Y, Aken B, Zhang G, Irie N. 2013. The draft genomes of soft-shell
turtle and green sea turtle yield insights into the development and evolution of the turtle-speciﬁc
body plan. Nature Genetics 45(6):701–706 DOI 10.1038/ng.2615.
Weaver WG. 1970. Courtship and combat behavior in Gopherus berlandieri.Bulletin of the Florida
State Museum: Biological Sciences 15:1–43.
Weldon PJ, Flachsbarth B, Schulz S. 2008. Natural products from the integument of nonavian
reptiles. Natural Product Reports 25(4):738–756 DOI 10.1039/b509854h.
Werbin H, Chaikoff I, Phillips BP. 1964. Conversion of cholesterol to 5a-cholestan-3β-ol in
germfree guinea pigs. Biochemistry 3(10):1558–1563 DOI 10.1021/bi00898a029.
Winokur RM, Legler JM. 1975. Chelonian mental glands. Journal of Morphology 147(3):275–291
Wyatt TD. 2014. Proteins and peptides as pheromone signals and chemical signatures.
Animal Behaviour 97:273–280 DOI 10.1016/j.anbehav.2014.07.025.
Ibáñez et al. (2020), PeerJ, DOI 10.7717/peerj.9047 24/24