A laboratory investigation into features of morphology and physiology for their potential to predict reproductive success in male frogs

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 μg/L)/linuron (9 or 45 μg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.


Supplementary methods S2 -Rearing conditions
Tadpoles were exposed to nominal concentrations of linuron ('low' -32 nM/9 µg/L or 'high' -181 nM/45 µg/L) or flutamide (181 nM/50 µg/L: purity > 98 %, Sigma Aldrich, USA) in acetone (0.0008%) or to acetone only (0.0008%: hereaf er referred to as 'controls') using a semi-static system (50% water change, x3 per week) to maintain good water quality 1 . Aquatic half life for flutamide has been reported to be 49 days 2 , though equivalent information for flutamide was not available. Flutamide and linuron levels in the control and exposure water were measured using gas chromatography/mass spectroscopy (see table S2). Triplicate tanks were used for each experimental group (total = 12 tanks) and the placing of tanks in the experimental room was randomised. Tanks were placed in water channels to maintain constant temperature, and this was checked daily (26 ± 1 °C), together with conductivity of water used for water changes (505 ± 20 µS/cm). The photoperiod cycle was 12:12 light:dark.
Tank water temperature, dissolved oxygen, pH and nitrite/ammonia levels were measured weekly. Flutamide and linuron concentrations were analysed using GCMS before water changes and immediately af er water changes. For flutamide, analysis of the tank water concentrations were done bi-weekly in flutamide and control tanks (5 before + 5 af er, see: 3 ) and for linuron, analysis of tank water concentrations were done every three weeks in linuron and control tanks (3 before + 1 af er, see: 4 ). The chemical analyses were undertaken at Umea University (flutamide) and at the Swedish University of agricultural sciences (linuron). At completion of metamorphosis (NF stage 66), exposure ceased and individuals were placed in new tanks containing test substance-free water (32 per tank, water flowthrough). The effects of treatment on sex ratio, sex organ weights, gonadal histomorphology, breeding behaviour, fertility and on secondary sexual characteristics (nuptial pad size/colour/histomorphology and forelimb width in males) were investigated in sexually mature frogs (6 months post-metamorphosis). In adult females, ovarian histomorphology was analysed in a sub-sample that were sacrificed prior to breeding. In adult males gonadal histomorphology was conducted on a sub-sample of individuals immediately following breeding.
Supplementary methods S1 -Animal husbandry Adult male and female X.tropicalis were obtained from Xenopus 1 (Dexter, USA) and housed in glass tanks in a flowthrough system (12:12 light:dark cycle, 26 ± 1 °C, conductivity 505 ± 20 µS/cm) for approximately two years prior to this study. Four pairs of frogs were induced to mate using human chorionic gonadotropin (hCG) as previously described 1 . Thirty-eight tadpoles (18 hours post-fertilisation at 22 0 C, stage 40) were selected from the 2 pairs of frogs with highest fertilisation rates (estimated, > 50%) and placed in experimental tanks (15 L, n = 76 tadpoles per tank).
Tadpoles were initially fed Sera micron and fish flakes (Sera vipan baby, Sera, Heinsberg, Germany) until forelimbs could be observed, when their food was supplemented with Frog & Tadpole bites (HBH Pet Products, Springville USA).
Pettersson, I., Arukwe, A., Lundstedt-Enkel, K., Mortensen, A. S. & Berg, C. Persistent sex-reversal and oviducal agenesis in adult Xenopus (Silurana) tropicalis frogs following larval exposure to the environmental pollutant ethynylestradiol. Aquatic Toxicology 79, 356-365 (2006 Figure S1. Measurement of forelimb width and length. Before the second boosting injection, each male frog was photographed. A piece of graph paper was held beneath the forearm. The camera (Nicon D70, objective AF micro Nikkor 60 mm 1:2:8D) was held by a camera support, approximately 30 cm over a bench, facing it, and a torch was used to illuminate the arm. The forelimb length and width of the male frogs were measured in the photos using an image analysis program, ImageJ. The graph paper beneath the forelimb on the picture was used as a scale. The length was measured on the inside of the forearm, from the bend at the elbow, down to the wrist. The arm width was measured by rotating this drawn line 90°, then moving it to where the "length line" (first measured line described above) ended at the elbow end, and the width was measured at this point. Figure S2. Measurement of nuptial pad size and colour.
The same photograph was used for forelimb width and nuptial pad measurements. The size and colour intensity of the nuptial pad were analysed using Adobe Photoshop CS6. The nuptial pad was selected using the Quick Selection Tool, and the area (in number of pixels) and colour intensities were recorded. Colour intensity ranged from 0 (black) to 255 (white). Reflections on the arm from water drops have an intense white colour which interferes with the colour intensity measurements. To eliminate this artefact the reflections were removed using the Spot Healing Brush Tool. This tool allows the removal of the reflections by replacing these areas with a composite colour sampled from the skin surrounding the reflection. For each photo the area was calculated by comparing the number of pixels in the selected part with the number of pixels in a 1x2 mm selected area in the graph paper. Each photograph was analysed twice without knowledge of treatment and the mean of these measurements was used for data analysis.
Supplementary methods S3 -Fertility determination Two factors were taken into account during optimisation: 1. Practicality: Development over 20 hours after amplexus had ceased (~26 hours after initiation of amplexus) allowed time to remove the fertilized eggs and get the tanks ready for the next batch of eggs for the following breeding trials (depending on amplexus behaviour, there could be as little as 30 minutes between the two). 2. Optimal timing: As embryo development starts directly at fertilization, 20 hours after amplexus ceased fertilized oocytesd should have reached at least Nieuwkoop and Faber 25 to 33 (depending on temperature) and fertilized eggs should be obvious/easy to identify.
Fertilisation success was measured at 18, 19, 20 and 21 hours after amplexus ceased. Variability in fertility rate between aquarias was observed, with higher levels in aquaria 1 than aquaria 2. However, in both In aquarium 1 ( Figure 1) and aquarium 2 ( Figure 2) 20 hours was shown to be sufficient to capture the vast majority of the fertilisation that had occurred.   Table S1.   Table S2. Covariates included in models and justification for inclusion for modelling Table S3. Variable groups for modelling Table S4 Models for achieving amplexus in breeding 1. For each variable, "1" refers to frog 1 of the pair and "2" refers to frog 2 of the pair, highlighted indicates significance whereby effects were seen for both frogs in the pair. Table S5 Models for achieving amplexus in breeding 2 for morphological endpoints. For each variable, "1" refers to frog 1 of the pair and "2" refers to frog 2 of the pair, highlighted indicates significance whereby effects were seen for both frogs in the pair. Table S6 Models for achieving amplexus in breeding 2 for nuptial pad histomorphology endpoints. For each variable, "1" refers to frog 1 of the pair and "2" refers to frog 2 of the pair, highlighted indicates significance whereby effects were seen for both frogs in the pair. No. = number, SM = Special Mucous Glands Table S7 Models for achieving amplexus in breeding 2 for testicular morphology endpoints. For each variable, "1" refers to frog 1 of the pair and "2" refers to frog 2 of the pair. Red = positive association for both individuals in the pair and thus excluded from results reporting. Table S8 Models for achieving amplexus in breeding 2 for testicular histomorphology endpoints. For each variable, "1" refers to frog 1 of the pair and "2" refers to frog 2 of the pair.
Table S9 Models for achieving amplexus in breeding 2 for androgens/androgen receptor endpoints. For each variable, "1" refers to frog 1 of the pair and "2" refers to frog 2 of the pair.
Table S10 Models for achieving amplexus in breeding 2 for corticosterone/glucocorticoid receptor endpoints. For each variable, "1" refers to frog 1 of the pair and "2" refers to frog 2 of the pair.   S13 Models for fertility in breeding 2 for nuptial pad/testicular histomorphology and testicular morphology. No. = number Table S14 Models for fertility in breeding 2 for androgens/corticosterone and androgen/glucocorticoid receptor. Table S15. Correlations between nuptial pad colour (darker = lower number) and features of morphology and physiology. Model with highest AIC value shown.