Open Access
Issue
Knowl. Manag. Aquat. Ecosyst.
Number 420, 2019
Article Number 2
Number of page(s) 8
DOI https://doi.org/10.1051/kmae/2018040
Published online 10 January 2019

© N. Bélouard et al., Published by EDP Sciences 2019

Licence Creative Commons
This is an Open Access article distributed under the terms of the Creative Commons Attribution License CC-BY-ND (http://creativecommons.org/licenses/by-nd/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. If you remix, transform, or build upon the material, you may not distribute the modified material.

1 Introduction

Stable isotope analyses (SIA) are now widely used by ecologists to study the trophic-related issues (Fry, 2006; Nielsen et al., 2018). These natural biomarkers provide time-integrated information on the trophic status of organisms and insights into species interactions notably because the isotopic signature of a consumer's tissues reflects its resources (Fry, 2006; Michener and Lajtha, 2007; Layman et al., 2012). More particularly, the stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) are the most commonly used tracers in food web studies (Fry, 2006). They can be obtained from whole bodies or internal tissues (e.g. muscle, liver or bones) when their size is sufficient for SIA, although non-destructive tissue collection is preferable for both ethical and epistemological reasons, especially when studying rare or vulnerable species (Hette-Tronquart et al., 2012). Non-lethal sampling methods also allow for repeated measurements on the same individuals, hence the possibility to address additional trophic questions. Exploring the path of alternative non-lethally sampled tissues is therefore highly needed. However, stable isotope ratios vary among tissues within a same individual depending on several factors, including isotope routing, amino acid composition and turnover rates (Fry, 2006; Michener and Lajtha, 2007; Martinez del Rio et al., 2009). It is thus essential to test whether alternative tissues provide stable isotope measurements similar to those of reference tissues in order to ensure that they can actually be substituted. In practice, food web studies require sampling tissues with low variation in stable isotope values, so that muscle became routinely used for SIA in vertebrates (Pinnegar and Polunin, 1999). It is now virtually replaced by non-destructively sampled tissues in vertebrates, such as fish fins and scales (e.g. Jardine et al., 2011; Fincel et al., 2012; Hette-Tronquart et al., 2012), bird claws (Bearhop et al., 2003), reptile claws (Marques et al., 2011) and cetacean fins (Todd et al., 1997).

Surprisingly enough, research on the trophic ecology of amphibians is lagging behind that of other vertebrate taxa (Altig et al., 2007). Yet, amphibians likely play a pivotal role in the structure and functioning of aquatic food webs because they are both predators and preys (Schiesari et al., 2009; Solé and Rödder, 2010). Furthermore, the remarkable biphasic life history of many amphibians contributes to energy flows between terrestrial and aquatic habitats (Solé and Rödder, 2010; Trakimas et al., 2011). Amphibians also suffer severe global population declines as a result of global changes (habitat destruction, pollution, or introduction of non-native species, etc.; Beebee and Griffiths, 2005) and are one of the most threatened vertebrate taxa (Baillie et al., 2004). Therefore, any information on their trophic ecology and its variation in fluctuating environments is relevant and of potential use for their conservation. The diet of tadpoles has been more investigated since Altig et al. (2007) called for more research (see e.g. Trakimas et al., 2011; Schriever and Williams, 2013; Arribas et al., 2015; Carreira et al., 2016), but as for now relatively few SIA studies are related to the trophic ecology of amphibians. This is probably partly because non-lethal sampling methods are rarely used (but see e.g. Sepulveda et al., 2012; Gillespie, 2013; Remon et al., 2016, Lejeune et al., 2018). Only one study thoroughly explored the relevance of tail clips (i.e. the whole distal part of the tail, including bones, muscle and skin) for SIA, and showed that the carbon and nitrogen isotope ratios of the distal part of the tail were correlated to the carcass ratios in three salamander species, but with variable success (Milanovich and Maerz, 2012). As compared to tail clips, the tail fin of amphibians appears as an ideal candidate for SIA because it is a homogeneous tissue, and its regeneration is frequently recorded in wild populations following injuries by predators (Nunes et al., 2010). Validating the relevance of fin biopsies for performing SIA in amphibians is therefore highly needed.

The major objectives of this study were twofold: (1) to test whether the tail fin of amphibian species is relevant as a surrogate for muscle tissues and provide fin-derived corrective equations to estimate stable isotope ratios of carbon and nitrogen, and to address additional practical issues related to (2) the use of a topical anaesthetic, recommended for minor surgeries (Gentz, 2007; Mitchell, 2009) likely to modify stable isotope ratios, and (3) the minimum quantities of fin needed to successfully perform SIA while limiting the severity of tail fin sampling. In doing so, we want to provide a non-destructive sampling method for stable isotope research in amphibians.

2 Materials and Methods

2.1 Study area and sample collection

An extensive amphibian study took place over more than 150 ponds in the Regional Natural Park of Brière, northwestern France (47°23′N, 02°12′W), in 2015 and 2016 (see Tréguier et al., 2018 for a detailed description of the study area). Two types of funnel traps were set over the night period: wire mesh traps (Tréguier et al., 2018) and Ortmann's traps (Drechsler et al., 2010; Fig. 1). Despite appropriate fieldwork precautions, 107 amphibians were found dead in the traps in 24 ponds, partly predated on by non-targeted trapped predators or for unknown causes, from 14 April to 22 May 2015 and from 21 April to 10 June 2016. We used all these animals in the present study. They were agile frog tadpoles (Rana dalmatina, N = 29, one to 16 per pond from four ponds), European tree frog tadpoles (Hyla arborea, N = 14 from one pond), adult marbled newts (Triturus marmoratus, N = 30, one to nine per pond from nine ponds) and palmate newts (Lissotriton helveticus, N = 34, one to six per pond from 12 ponds), altogether representing <1% of the amphibians caught over two years. The developmental stages of tadpoles ranged from 26 to 42 (i.e. development of hind limbs; Gosner, 1960). All the collected life stages were commonly present in the ponds for a time period long enough to play a critical role in aquatic food webs. As specimens were collected from different populations in ponds displaying a variety of environmental conditions, the consistency of correlation of stable isotope ratios of carbon and nitrogen between fin and muscle across amphibian species would be a first step before transferring such relationships to other study sites. Specimens were preserved by freezing at –20°C immediately after collection for further analysis.

thumbnail Fig. 1

Habitat and fin sampling of amphibians: (a) Ortmann's traps set in a typical pond of the study area; (b) and (c): sampling of the fin of an agile frog tadpole and a marbled newt adult, respectively; (d): regeneration of the fin of a marbled newt, 10 days after sampling.

2.2 Sample preparation and stable isotope analyses

In the laboratory, tail fins were rinsed with deionized water and then sampled using 2.5- and 3-mm diameter biopsy punches (Fig. 1 and Tab. 1 for sample sizes). In tadpoles and palmate newts, biopsy samples were taken on the well-developed dorsal part of the tail fin. In marbled newts, the sampling of the ventral part of the fin was preferred so as not to damage the dorsal part which is of great importance for males during courtship. One to six samples were taken per individual when possible depending on the species (agile frog: 1–4, European tree frog: 3–5, palmate newt: 3–4, marbled newt: 3–6) to ensure that enough biological material was collected. Then, amphibians were dissected to sample their tail muscle. All samples were rinsed with deionized water before proceeding to the subsequent steps of sample preparation.

Local anaesthetics can be used for minor surgical procedures such as fin biopsy for amphibians, to limit the handling time of animals and risks as compared to water anaesthetics (Gentz, 2007; Mitchell, 2009). A prerequisite is to check beforehand whether the anaesthetic affects stable isotope values. The effect of lidocaine, a common topical anaesthetic recommended for amphibians (Wright, 2001; Gentz, 2007) was assessed by comparing the carbon and nitrogen isotope ratios of samples treated with the anaesthetic to those of control samples. Because we used most of the fin for exploring the relationships between fin and muscle, notably in palmate newts, the anaesthetic experiment was done on an approximately 1-cm section of an intact part of the tail of individuals of the two newt species (N = 21, 8 marbled newts and 13 palmate newts). For each individual, the tail sample was divided into two parts. On one part, lidocaine gel (2% solution) was applied on the integument for 5 min (the recommended latent period), and then it was rinsed with deionized water to remove any lidocaine residues. The other part was only rinsed with deionized water and used as a control.

All samples were freeze-dried for 48 h. Then they were ground to a homogeneous fine powder, except fin samples because of the small amount of tissue available. For a subset of individuals (17 agile frogs, 10 European tree frogs, 29 marbled newts and 7 palmate newts), fin samples were individually weighted after freeze-drying to calculate mean density values (μg/mm2) and ultimately to estimate the minimal fin surface required to successfully perform SIA. Fin samples were then gathered to constitute a unique sample per individual, except in nine marbled newts for which, due to large material supply, we analysed three different samples of fin to assess intra-individual variations in the stable isotope values (i.e. to explore whether the fin is a homogeneous tissue). Each sample was packed into a tin capsule (mean ± SD = 415.6 ± 17.3 μg). SIA were performed by continuous-flow isotope-ratio mass spectrometry at the Stable Isotopes in Nature Laboratory (Fredericton, New Brunswick, Canada). Isotope ratios were reported as delta (δ) notations relatively to international standards (Vienna Peedee Belemnite for carbon, and atmospheric air for nitrogen). Maximum standard deviations of replicates from the International Atomic Energy Agency and working standards (i.e. the analytical errors) were ±0.10‰ and 0.25‰ for δ13C and δ15N, respectively. C:N was calculated as the ratio between elemental composition (%C:%N) of each sample.

Table 1

Mean differences in δ13C and δ15N between fin and muscle values, and linear models (equations, 95% confidence intervals (95% CI) of the estimate and the intercept) fitting muscle δ13C and δ15N as a function of the isotope ratios in fin tissue for each species or group of species (anuran tadpoles and urodele adults). δ13C values were corrected for the lipid content according to Caut et al. (2013). The significance of the mean differences and linear equations is indicated: NS: non-significant, *p ≤ 0.05, **p < 0.01, ***p < 0.001.

2.3 Data analysis

Variations in lipid content among tissues are known to bias δ13C values because lipid-rich tissues are depleted in 13C. The C:N ratio is routinely used as a proxy of the lipid content of a tissue, so that when working on aquatic animal tissues with C:N > 3.50, it is common to correct δ13C values using normalization equations (e.g. Post et al., 2007; Logan et al., 2008). The mean C:N values of fin and muscle were 3.47–3.88 and 3.37–3.53, respectively, across the four species. Two normalization equations were tested in parallel: one developed for aquatic animals (Post et al., 2007) and a second one specifically designed for amphibians (Caut et al., 2013). Both of them led to very similar results (differences <1% in the resulting δ13C values), so we used the amphibian-specific equation of Caut et al. (2013) for subsequent calculations. Results from the other method are provided in Supplementary Table S1.

Statistical analyses were performed with R software, version 3.3.5 (R core Team, 2018). The significance threshold was set to p = 0.05. We first tested whether δ13C and δ15N values differed between fin and muscle for each species using paired t-tests (or Wilcoxon signed rank tests when data did not meet assumptions of normality) and also calculated the mean difference (±SD) between tissues. Linear models were used to test the link between muscle and fin isotope ratios for each species (specific models) and for grouped anuran or grouped urodele species (hereafter called generic models). The same statistical procedure was used to test the differences and then the link between treated and control samples in the anaesthetic experiment. Finally, we assessed the importance of intra-individual variation in isotope ratios in marbled newts using one-way ANOVA with the individuals as factors.

3 Results

After correcting δ13C for lipid contents (the range of the corrections was 0.11–0.40‰ for fin and 0.10–0.23‰ for muscle), fin and muscle differed in their isotope ratios by −1.44 to 0.44‰ for δ13C and by −0.84 to 0.43‰ for δ15N, depending on the species (Tab. 1). These differences were significant in all species, except for δ13C in the marbled newt and for δ15N in the agile frog (Tab. 1). Whether fins were enriched or depleted in stable isotopes as compared to muscle varied among the species and between the elements (Fig. 2): δ13C in the fin of anuran tadpoles and specifically of agile frogs was generally higher than in muscle, whereas it was the opposite in palmate newts; δ15N was generally higher in the fin of newts than in muscle, as opposed to European tree frogs.

The isotope ratios of fin and muscle were strongly correlated independently of the species (Fig. 2, Tab. 1). A substantial proportion of the total variance was explained by the specific models, ranging from 0.67 to 0.96 (>0.80 in five out of eight models, Tab. 1). Despite no significant difference in δ13C between tissues in the marbled newt, the total variance of the model was the lowest across species. The generic models also fitted well (explained variance: 0.87–0.95, see Tab. 1).

The application of lidocaine caused differences (mean ± SD) in isotope ratios between the two tissues of –0.07 ± 0.16 (p = 0.06) for δ13C and –0.09 ± 0.11‰ (p = 0.002) for δ15N. The equations linking control and treated samples (Fig. 3) were δ13Ccontrol = 1.01 × δ13Clidocaine + 0.37 (r 2 = 0.98, p < 0.001) and δ15Ncontrol = 1.02 × δ15Nlidocaine − 0.03 (r 2 = 1, p < 0.001).

Fin matter densities varied greatly among species, and were on average 2.75 to 7 times higher in adult newts than in tadpoles (Tab. 2). Differences in matter density were also recorded within species (see values in Tab. 2). In practice, average numbers of two 2.5-mm diameter fin biopsies were required to produce 100 μg of freeze-dried matter (see Sect. 4 regarding the minimum required mass to achieve accurate stable isotope measurements) for agile frog tadpoles, two 3-mm diameter fin biopsies for European tree frog tadpoles and only one 2-mm diameter fin biopsy for adult marbled and palmate newts. Finally, the intra-individual variations in isotope values recorded in marbled newts were very low (1.6% for δ13C and 1.7% for δ15N, 3 fin replicates × 9 newts; one-way ANOVA, p < 0.001).

thumbnail Fig. 2

Linear regressions of δ13C (left) and δ15N (right) between muscle and fin in (a) anuran tadpoles and (b) newt adults. Empty circles on a dotted line represent the European tree frog, full circles on a dashed line the agile frog, empty squares on a dotted line and full squares on a dashed line the palmate newt and marbled newts, respectively. Plain lines represent y = x.

thumbnail Fig. 3

Relationships between control and lidocaine-treated samples for δ13C and δ15N. Filled squares, marbled newt samples; empty squares, palmate newt samples. Dashed lines represent the linear model and plain lines indicate y = x.

Table 2

Density of amphibian tail fin, and conversion into the fin surface required to produce 100 μg of freeze-dried matter (i.e. the minimum mass required for nitrogen-rich biological material for stable isotope measurements).

4 Discussion

The tail fin of amphibians was found to be a highly relevant substitute for muscle tissue for conducting SIA. The strong relationships between the tail fin and muscle isotope ratios suggest that the two tissues had similar turnover rates and integrated comparable diet information. Mean differences in δ15N between the two tissues were low compared to common trophic discrimination factors (TDF) found in several amphibian species: 3.80 ± 0.46‰ in Caut et al. (2013), 3.66 ± 0.48‰ and 2.23 ± 0.49‰ in San Sebastián et al. (2015). Moreover, these differences were marginal compared to other sources of error, such as the variability in the TDF that can be used to characterize trophic networks (see the values above, and also Martinez del Rio et al., 2009), even if the error was higher in the European tree frog than in other species. The same reasoning can apply to δ13C: the mean differences δ13C between the two tissues were lower than the TDF values reported for amphibians in the literature and comparable to the variability among them (1.19 ± 0.31 in Caut et al. (2013), 1.56 ± 0.64 and 0.91 ± 0.49 in San Sebastián et al. (2015)), except in the palmate newt. As a result, this potential error due to fin–muscle differences were probably too low to cause important misinterpretations in food web studies (see also Jardine et al., 2011); but this was not true for all species, especially for δ13C in the palmate newt and to a lesser extent for δ15N in the European tree frog. These specific results remain unexplained.

Both the specific and the generic equations were adequate to estimate muscle values from fin samples. By comparison, existing corrective equations built for tail clips in three salamander species were less powerful (Milanovich and Maerz, 2012). One potential explanation could be that the tissue composition was more heterogeneous in their case. The equations we used for the two anuran species were very similar, and so were the equations for the two newt species. This is not surprising given that both anuran tadpoles are essentially phytophagous, and both newt species are predators. The fact that the amphibians came from different ponds with various environmental conditions (except the European tree frog) did not seem to affect the results. Therefore, it can be hypothesized that the relationship between fin and muscle was robust for other study areas, although further exploration of this question would be welcome. We recommend building specific equations for any new target species; nevertheless, the generic equations we implemented represent a valuable alternative depending on the diet of the species.

The present study also answers important practical questions. Topical anaesthetics are used for potentially painful surgical procedures on amphibians, but their application could influence the isotope ratios of the tissues. The deviations in δ13C and δ15N due to the application of lidocaine (2% solution) were negligible as they did not exceed the commonly recorded analytical errors. Nonetheless, the adverse effects of anaesthetics on the skin of amphibians are poorly known, and when a biopsy sample is very small, it is recommended to proceed without applying any chemical anaesthetics (Wright, 2001; Canadian Council on Animal Care, 2004). This is the case for fin biopsies dedicated to SIA, because the minimal quantity of tail fin required to accurately measure δ13C and δ15N can be as low as 100 μg for nitrogen-rich samples (depending on the analytic optimization of SIA platforms), corresponding to fin surfaces as small as 10–15 mm2 for tadpoles and 2 mm2 for newts. We recommend sampling these minimal fin areas, or slightly greater areas in accordance with the instructions of SIA laboratories, because these areas are far below the 30% tail clipping that did not affect larval survival in salamanders (Segev et al., 2015). Moreover, the tail of amphibians is a lure for predators and is routinely injured in the wild (Nunes et al., 2010). The low intra-individual variations in δ13C or δ15N in the marbled newt indicate that the fin is a relatively homo genous tissue, and we therefore suggest that sampling can be done on the part of the fin that is considered to be the least detrimental for the animals. Finally, in additional field work, we observed the complete regeneration of the tail fin in marbled newts, and sampled individuals were unrecognizable within 3 weeks after sampling (unpublished data, Fig. 1).

Using SIA to address trophic issues used to be highly constrained in amphibians because it often implied destructive sampling techniques. The equations implemented in this study allow us to accurately estimate δ13C or δ15N in muscle, a reference tissue in SIA, from the non-lethal fin sampling of larvae and adults in several amphibian species. We hope that our findings will encourage new investigations on the trophic ecology of amphibians. Furthermore, exploring SIA using fin biopsy opens onto promising prospects, such as tracking trophic status changes at the individual level over time. More generally, a better understanding of the trophic ecology of amphibian populations is critical for predicting the consequences of global changes, and is undoubtedly of great interest for their conservation.

Acknowledgements

This study was funded by the Agence Française pour la Biodiversité (research program supervised by J.M.P.) and the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (Ph.D. grant to N.B.). This work was carried out under the licenses 08/2015 and 07/2016 delivered by the Préfecture de la Loire-Atlantique. We thank the Parc Natural Régional de Brière, notably J.P. Damien, for supporting our research activities on amphibian ecology. The authors declare that they have no conflict of interest. We are also grateful to two anonymous reviewers for their constructive comments on an earlier version of the manuscript. A. Buchwalter edited the final version of the article.

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Cite this article as: Bélouard N, Petit EJ, Huteau D, Oger A, Paillisson J-M. 2019. Fins are relevant non-lethal surrogates for muscle to measure stable isotopes in amphibians. Knowl. Manag. Aquat. Ecosyst., 420, 2.

Supplementary Material

Supplementary Table S1. Mean difference in δ13C and δ15N between fin and muscle values, and linear models (equations, 95% confidence intervals (95% CI) of the estimate and the intercept) fitting muscle δ13C and δ15N as a function of the isotope ratios in fin tissue for each species or group of species (anuran tadpoles and urodele adults). δ13C values were corrected for lipid content according to Post et al. (2007). The significance of the mean differences and linear equations is indicated. NS: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. (Access here)

All Tables

Table 1

Mean differences in δ13C and δ15N between fin and muscle values, and linear models (equations, 95% confidence intervals (95% CI) of the estimate and the intercept) fitting muscle δ13C and δ15N as a function of the isotope ratios in fin tissue for each species or group of species (anuran tadpoles and urodele adults). δ13C values were corrected for the lipid content according to Caut et al. (2013). The significance of the mean differences and linear equations is indicated: NS: non-significant, *p ≤ 0.05, **p < 0.01, ***p < 0.001.

Table 2

Density of amphibian tail fin, and conversion into the fin surface required to produce 100 μg of freeze-dried matter (i.e. the minimum mass required for nitrogen-rich biological material for stable isotope measurements).

All Figures

thumbnail Fig. 1

Habitat and fin sampling of amphibians: (a) Ortmann's traps set in a typical pond of the study area; (b) and (c): sampling of the fin of an agile frog tadpole and a marbled newt adult, respectively; (d): regeneration of the fin of a marbled newt, 10 days after sampling.

In the text
thumbnail Fig. 2

Linear regressions of δ13C (left) and δ15N (right) between muscle and fin in (a) anuran tadpoles and (b) newt adults. Empty circles on a dotted line represent the European tree frog, full circles on a dashed line the agile frog, empty squares on a dotted line and full squares on a dashed line the palmate newt and marbled newts, respectively. Plain lines represent y = x.

In the text
thumbnail Fig. 3

Relationships between control and lidocaine-treated samples for δ13C and δ15N. Filled squares, marbled newt samples; empty squares, palmate newt samples. Dashed lines represent the linear model and plain lines indicate y = x.

In the text