Despite evidence from anatomy, behavior and genomics indicating that the sense of smell in turtles is important, our understanding of chemical communication in this group is still rudimentary. Our aim was to describe the microanatomy of mental glands (MGs) in a freshwater turtle,
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, 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 most common.
Chemical signals have been extensively studied in certain groups of invertebrates such as insects (
Several lines of evidence indicate that chelonians have a well-developed olfactory system. First, the draft genomes of soft-shell (
In this article we report the chemical composition and microanatomy of the MGs of the Spanish terrapin,
The individuals of
The CRARC holds Catalan permit B2100126 for Zoo Facilities to maintain reptiles, including
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 (
Small fragments of MGs used for TEM were fixed in Karnovsky’s fixative (
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.
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 taking secretion), as well as samples of water from the turtle enclosures to check for potential contaminants.
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 μL of acetonitrile and 50 μL 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 μL 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 μm 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 μL was performed at injection port heated to 280 °C. The MS transfer line temperature was set to 280 °C and ion source 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).
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 (
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;
Analysis and some plots were performed in R version 3.4.4 (
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 (
(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 same.
(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
(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.
Fine scale examination of MG tissue in TEM showed that the glandular epithelium comprises an engrossed basal layer of epithelium (
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
(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.
After filtration steps (see “Material and Methods”) a total of 61 chemical compounds were identified, at least to class, in the MG secretions of
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 (
Name | Abbr. | Ratios (mean) | SD | M (mean) | F (mean) | P Adj. | F (num.) | M (num.) | Percentage (mean) |
---|---|---|---|---|---|---|---|---|---|
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 derivative | Aol-3 | 0.109 | 0.200 | 0.114 | 0.102 | 0.907 | 7 | 10 | 0.350 |
1-O-Octadecylglycerol, - bis(trimethylsilyl) ether derivative | 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 ester | 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 |
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 | 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 | 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 | 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 | 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 |
5α-Cholestan-3β-ol, trimethylsilyl derivative | Std-3 | 0.989 | 1.727 | 0.835 | 1.192 | 16 | 11 | 4.437 | |
5α-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 | 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 |
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 identified.
The number of compounds was similar between sexes (Wilcoxon rank sum test with continuity correction;
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:
(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
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 trimethylsilyl ether (
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. Significant values after adjustment for multiple comparisons are marked with an asterisk. Compound abbreviations are as in
This study provides a comprehensive assessment of MG anatomy and chemistry in a freshwater turtle species. The MGs of adult
Steroids and carboxylic acids were the most common chemical compounds in MGs of the Spanish terrapin, a pattern also found in other reptiles (
Previous studies on gopher tortoises (
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
MGs consist of heavily keratinized invaginations in females. Histological evidence 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 (
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 (
Two steroids (5α-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, 5α-cholestan-3β-ol can be metabolically converted from cholesterol (
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
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
This study showed that MGs in male
Non-metric multidimensional scaling (NMDS) plot based on Bray Curtis dissimilarity. A) NMDS plot considering all compounds (stress = 0.098). B) NMDS plot excluding cholesterol trimethylsilyl ether (stress = 0.17). In this analysis percentages were used instead of the relative areas in respect to the internal standard (see “METHODS”). 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.
Click here for additional data file.
Mean ± 95% CI (estimated by bootstrapping) for the relative areas of the distinct classes are shown. Steroids include cholesterol. Others include an inorganic acid, a nucleoside, two amines and two sugar alcohols.
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Non- metric multidimensional scaling (NMDS) plot calculated from Bray Curtis dissimilarity among samples and compounds (excluding cholesterol). Blue points represent year 2018, and red points represent 2019. Closer points represent more similar compositions in individual turtles. Only males were considered for this analysis. Ellipses were calculated with the function ordiellipse (package Vegan) and represent 95% confidence interval (based on standard error) for the years. Stress = 0.13
Click here for additional data file.
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. Rafał Piprek for his 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.
The authors declare that they have no competing interests.
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
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,
- RAW chromatographic data (GC-MS) for the article: “The chemistry and histology of sexually dimorphic mental glands in the freshwater turtle,
- Research datasets of the article: “The chemistry and histology of sexually dimorphic mental glands in the freshwater turtle,