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. 2026 Mar 16;109(1):189–200. doi: 10.1111/jfb.70376

Fatty acid profiles of muscle and liver reveal ontogenetic shifts in diet and reproductive investment in an undulate ray from the Bay of Biscay (northeast Atlantic)

Pablo Brosset 1,✉, Tino Jamme 1, Bastien Sadoul 1, Manuel Vlach 2, Pascal Lorance 1, Vincent Rioux 2, Salomé Fabri‐Ruiz 1
PMCID: PMC13397247  PMID: 41837321

Abstract

Elasmobranchs, with their slow growth, long lifespans and low reproductive output, are particularly sensitive to fluctuations in nutritional status and energy availability throughout ontogeny. Fatty acids serve as valuable biomarkers for studying trophic ecology, ontogenetic dietary shifts and energy allocation during key life periods such as growth and reproduction. This study examined fatty acid profiles in Raja undulata by analysing muscle and liver tissues to investigate ontogenetic dietary shifts, assess niche breadth and overlap between juveniles and adults, and explore how reproductive development influences fatty acid allocation after sexual maturity. The muscle fatty acid profiles of R. undulata varied across life stages, with adults showing a reduced trophic niche and a diet composed of higher‐trophic‐level prey compared to juveniles. This pattern suggested a dietary shift from a diverse, invertebrate‐based diet in juveniles to more specialized predation on fish and larger crustaceans in adults. The liver fatty acid profiles varied across reproductive stages, with an increasing proportion of omega‐3 (C22:6n‐3, docosahexaenoic acid [DHA]; 22:5n‐3, docosapentaenoic acid) and omega‐6 polyunsaturated (C20:4n‐6, arachidonic acid [ARA]) fatty acids, alongside a marked decline in eicosapentaenoic acid (C20:5n‐3), likely reflecting its direct allocation to the ovaries. Additionally, the elevated monounsaturated fatty acids levels in adults and their decline during gametogenesis highlighted their key role as energy sources for oocyte development, supporting previous findings on reproductive energy mobilization in elasmobranchs. While DHA and ARA proportions increased, their stable absolute values suggested a different allocation pattern compared to viviparous species, potentially due to R. undulata's oviparous strategy. Our findings, in line with previous studies, showed that analysing different tissues is key to understanding how diet and energy use are linked in elasmobranchs.

Keywords: biochemical tracer, elasmobranch, northeast Atlantic, Raja undulata, ray, trophic ecology

1. INTRODUCTION

Elasmobranchs (sharks, rays and skates) are pivotal components of marine ecosystems, playing diverse ecological roles that range from apex predators to keystone mesopredators and planktivores (Pimiento et al., 2023). Through these roles, they help to regulate prey populations, shape food web dynamics and maintain ecosystem structure and function (Heithaus et al., 2008). Given their long lifespans, slow growth rates and low reproductive output, individuals must efficiently exploit a variety of energy sources at different life stages to meet metabolic demands and sustain survival and reproduction over decades (Frisk et al., 2001). Despite its importance for reproductive performance, energetic investment is rarely considered in studies of elasmobranch reproduction (Rangel et al., 2021a). Yet, gonadal maturation requires substantial energy investment, especially in species with nutrient‐rich reproductive modes, such as placentotrophy and histotrophy (Blackburn & Hughes, 2024). The nutritional composition of prey is a well‐known driver of elasmobranch fitness (Munroe et al., 2022). Studying the trophic ecology of elasmobranchs is therefore crucial to understand life‐history trade‐offs and how elasmobranchs meet energetic demands across ontogeny. Moreover, energy acquisition through diet and its allocation to reproduction also have far‐reaching ecological implications, affecting population dynamics and the species' ability to fulfil its ecological roles.

Fatty acids have proven to be valuable tools for understanding the trophic ecology, ontogenetic shifts and energy allocation across marine and terrestrial taxa, habitats and biomes (Couturier et al., 2020). These compounds are vital components of cellular membranes and energy reserves, and are critical for various physiological functions, including growth, reproduction and energy storage (Parrish, 2009). Furthermore, they are acquired directly from diet with minimal modification (even if some elasmobranchs can modify fatty acids), making most fatty acids reliable indicators of dietary intake and physiological adjustments in marine animals (Dalsgaard et al., 2003; Pethybridge et al., 2018). Indeed, the relative abundance of these fatty acids in their tissues reflects their prey's nutritional profiles (as distinct fatty acids are synthesized by different producers), enabling researchers to infer diet, habitat use and changes in feeding patterns over time (e.g. using fatty acid ratio markers of carnivory; Beckmann et al., 2013; Burke et al., 2024; Lipscombe et al., 2024; Meyer et al., 2021, Meyer et al., 2019). Furthermore, elasmobranchs, like other vertebrates, have very limited capacities to synthesize long‐chain polyunsaturated fatty acids (PUFAs) de‐novo, such as arachidonic acid (ARA, C20:4n‐6), eicosapentaenoic acid (EPA, C20:5n‐3) and docosahexaenoic acid (DHA, C22:6n‐3), which are essential for processes like gonad development and reproduction (Parrish, 2009). Fatty acid analysis also provides insights into the influence of ontogenetic energy allocation shifts, as elasmobranchs transition through physiological adaptations to different life or maturation stages with differing energy allocation patterns (Rangel et al., 2021b). For instance, early‐stage individuals may rely on PUFA‐rich prey for growth and neural development, while during gonad maturation, adults alter their fatty acid profiles to support gametogenesis and reproductive success (Rangel et al., 2021b). These life stage‐specific demands underscore the role of fatty acids in linking diet to energy allocation strategies, but remain mostly unknown in elasmobranchs.

By examining tissue‐specific fatty acid profiles, researchers can track short‐ and long‐term dietary patterns and assess changes in diet and fatty acid allocation to reproductive processes (Dalsgaard et al., 2003). The liver, acting as a storage organ, reflects last weeks of feeding and its fatty acid profiles are strongly influenced by individual physiology and can be used to investigate fatty acid allocation across maturation stages (Davidson et al., 2014; Pethybridge, Daley, Virtue, Butler, et al., 2010; Pethybridge, Daley, Virtue, & Nichols, 2010). Conversely, muscle tissue fatty acid profiles, reflecting last months of feeding, have close resemblance to prey fatty acid profiles for elasmobranch as they are less influenced by physiological factors such as reproduction and offers the advantage of reduced intra‐ and inter‐individual variability (Davidson et al., 2014; Pethybridge, Daley, Virtue, Butler, et al., 2010; Pethybridge, Daley, Virtue, & Nichols, 2010). Nonetheless, muscle fatty acid composition can be influenced by both external factors, such as temperature, and internal factors, including migratory activity. As such, fatty acids can reveal diet, trophic niche breadth and overlap, habitat use and energy allocation patterns, with short‐term information (~30 days) gained from metabolically active tissues like liver, while less active tissue like muscle provides longer‐term (months) diet information (Lipscombe et al., 2024). This is why fatty acids are increasingly used to complement stomach content analysis, offering broader ecologically relevant spatial and temporal insights compared to brief snapshots from stomach content analysis (Carlisle et al., 2021; Meyer et al., 2021).

Elasmobranchs, and especially rays, are among the most threatened families of vertebrates (Dulvy et al., 2014, 2021; Pimiento et al., 2023). During the 20th century, the market value of elasmobranchs steadily increased, leading to intensified fishing pressure on these species (Dulvy et al., 2017; Dulvy et al., 2021). The Atlantic waters of the European Union are no exception to this global trend, experiencing a decline in ray landings during this period, although the extent of this decline varied across regions (Alonso‐Fernández et al., 2019). Consequently, the undulate ray (Raja undulata, Lacepède, 1802) was listed as Endangered on the International Union for Conservation of Nature Red List of Threatened Species. In 2009, the European Commission implemented a ban on R. undulata fishing due to insufficient data. In 2014, however, the species was removed from the list of prohibited species in northeast Atlantic waters (ICES subareas 7–9), and a precautionary total allowable catch (TAC) was set at 25 tonnes for the Bay of Biscay in 2015. This TAC was subsequently increased to 30 tonnes in 2017 and 33 tonnes in 2019, with quotas allocated to France, Portugal and Spain while undulate ray landings represented 76–111 tonnes per year in the Bay of Biscay before 2009. Acquiring more biological and ecological data, particularly for data‐deficient and vulnerable species such as R. undulata (Coelho & Erzini, 2006; Daban et al., 2024; Ellis et al., 2012; Moura et al., 2008), is thus essential to improve their management and sustainable exploitation. Similar protection regulations were implemented in the English Channel from 2009 to 2022. In this area, the species abundance increased from the early 2010s and the TAC was set to 3192 tonnes in 2023 and has remained at similar levels since (ICES, 2022). Recent abundance trends in the Bay of Biscay are unknown.

The present study uses fatty acid profiles of muscle and liver in R. undulata to investigate the allocation of fatty acids at different life and reproductive stages. Specifically, we (1) investigated ontogenic shift in diet from juveniles to adults using both muscle and liver fatty acids profiles, (2) assessed trophic niche breadth and overlap between juveniles and adults based on muscle fatty acid profiles, and (3) explored how reproductive stages development influences individuals' liver fatty acid profiles. These findings will enhance our understanding of ontogenetic shifts in resource use and reproductive energy allocation in R. undulata, and more broadly, in elasmobranchs.

2. MATERIALS AND METHODS

2.1. Species of interest, sample collection and processing

R. undulata is a medium‐sized oviparous skate, producing up to 70 egg cases per year (Serra‐Pereira et al., 2015). This species is found in continental shelf waters at depths of up to 150 m, although it is more frequently encountered at depths of less than 50 m, preferring soft and sandy substrates (Ellis et al., 2012; Stehmann & Bürkel, 1984). Its distribution extends from the southern North Sea and south of Ireland to the Gulf of Guinea, with occasional occurrences in the Mediterranean Sea (Coelho & Erzini, 2006; Ellis et al., 2024; Serena et al., 2020; Stehmann & Bürkel, 1984).

A total of 150 specimens were sampled in the Bay of Bourgneuf, situated in the northern part of the Bay of Biscay near the Loire estuary (Figure 1), at 38 distinct sampling stations during a dedicated research survey aboard a small‐scale commercial trawler (24 April to 5 May 2023). The vessel was chartered by the scientific project and required to target undulate ray with a bottom trawl. Total length (ranging from 13 to 103 cm in length with a mode at 89 cm) and weight were recorded immediately after capture. Liver and dorsal muscle tissue samples (~1 g) were collected and rapidly preserved in liquid nitrogen before being stored at −80°C for laboratory analyses. Due to limited sampling time and the initial focus on R. undulata trophic ecology, gonads were not sampled, and sex differences in fatty acid mobilization patterns were not investigated.

FIGURE 1.

FIGURE 1

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Map of Raja undulata sampling stations (n = 38) in the Bay of Biscay, corresponding to trawling events conducted between 24 April and 5 May 2023.

Sex was determined onboard through visual inspection, and both sexual maturity and reproductive stages were assessed visually based on macroscopical gonad development, classifying individuals into one of four stages: immature, maturing, mature or active (ICES, 2013; Serra‐Pereira et al., 2015). Life‐stage determination was based on macroscopic assessment of gonad development, classifying individuals as immatures or adults (i.e. including three reproductive stages: maturing [first time maturing], mature [spawned already once] and active [actively reproducing] individuals). This separation occurred around 78 cm total length, with juveniles below and adults above. This threshold is consistent with length‐at‐maturity estimates reported for nearby areas: 86.2 cm for females and 76.8 cm for males in northern Portuguese waters (Serra‐Pereira et al., 2015), and 76.2 cm for females and 73.6 cm for males in southern Portuguese waters (Coelho & Erzini, 2006). Seventy juveniles and 80 adults were identified (see Table S1 for details on sub‐group sample sizes and mean lengths).

2.2. Ethics statement

The care and use of experimental animals complied with French animal welfare laws, guidelines and policies as approved by Ifremer Ethics Advisory Committee (2010/63/UE).

2.3. Fatty acid analysis

Lipid extractions and derivations into fatty acid methyl esters (FAMEs) were carried out using approximately 100 mg of freeze‐dried tissue (muscle or liver), which was first homogenized into a fine powder. Lipids were extracted from the samples using a dimethoxymethane/methanol mixture (4:1, v/v), after adding margaric acid (C17:0, H3500, purity > 98%; Sigma Aldrich) as an internal standard (12 μg in muscle samples and 36 μg in liver samples) in the solvent mixture. Samples were first homogenized in this mixture with an Ultra‐Turrax, then stirred on a rotary wheel at 40 rpm for 20 min and finally centrifuged at 250 × g for 1 min at room temperature. The supernatants containing the lipids were transferred to clean tubes, and solvents were evaporated under a nitrogen stream at 55°C. The extracted total lipids were then saponified by adding 1 mL of 0.5 M NaOH in methanol and incubated at 70°C for 10 min, leading to fatty acid soaps. Methylation was subsequently carried out by directly adding 1 mL of boron trifluoride (BF₃, 14% w/v in methanol) to the fatty acid soaps, followed by an additional 10 min incubation at 70°C (Guillocheau et al., 2019), leading to the synthesis of FAMEs.

FAMEs were extracted with 4 mL of pentane and washed with 4 mL of 0.9% NaCl solution. The samples were stirred on a rotary wheel at 40 rpm for 20 min and centrifuged at 250 × g for 1 min at room temperature. The upper organic phases containing the FAMEs were transferred to new tubes and evaporated under a nitrogen stream at 55°C. FAMEs were then resuspended in 200 μL of hexane and transferred to 2 mL vials compatible with the gas chromatography–mass spectrometry (GC–MS) autosampler. The FAMEs were analysed using GC–MS in electronic impact mode (GC–MS EI). GC–MS analysis was conducted using an Agilent 7890A system (Agilent Technologies, Inc.) equipped with a bonded silica capillary column (BPX 70, 60 m × 0.25 mm), with helium as the carrier gas at a constant flow of 1.8 mL/min. The column temperature was programmed to start at 130°C and gradually increased at 4°C /min until it reached 250°C, and then remained at 250°C for 10 min. Mass spectra were recorded with an Agilent Technologies 5975C inert MSD fitted with a triple‐axis detector. The mass spectrometer was operated under electron impact ionization conditions (electron energy 70 eV, source temperature 230°C). Data were obtained in full‐scan mode with a mass range of m/z 50–550 amu. FAMEs were identified based on the retention times of methyl ester standards when available and mass spectra data collected (Ciesielski et al., 2025). FAME analyses were performed using MassHunter Workstation Software Qualitative Analysis (version B.07.00 for Windows; Agilent Technologies) with results expressed as percentages of total fatty acids and in mg g−1 of dry weight tissue, calculated using the known amounts of the C17:0 internal standard added at the beginning.

Two fatty acid ratio markers of carnivory (DHA/EPA and 18:1n‐9/18:1n‐7; Couturier et al., 2020) were also used in muscle fatty acids profiles to describe potential ontogenic shifts in R. undulata diet. Indeed, R. undulata and related Rajidae species occurring in the Bay of Biscay are known to be exclusive carnivores, larger individuals feeding on larger preys of higher trophic level with a higher contribution of fish and cephalopods to their diet (Farias et al., 2006; Holden & Tucker, 1974; Kadri et al., 2014). As DHA is conserved throughout the food web (i.e. is accumulated through increasing trophic level), the ratio of DHA/EPA should be higher at higher trophic levels (Dalsgaard et al., 2003). The ratio of DHA/EPA in muscle will also reflect the basal origins of prey items, that is, which primary producers are at the base of the trophic network of R. undulata.

2.4. Statistical analysis

Only fatty acids contributing to more than 0.1% to the total fatty acid profile at least in one of both tissues were retained for statistical analysis. Fatty acid profiles of muscle and liver tissues were analysed using a multi‐step statistical approach relying on a multivariate statistical analysis. Proportions of fatty acids were arcsine‐square‐root‐transformed, avoiding over‐emphasis of extreme values prior to statistical analysis, and all ordination and tests were based on Euclidean distance matrices (Happel et al., 2017; Iverson, 2009).

To test whether fatty acid composition differed between life stages (juveniles or adults), reproductive stages (maturing, mature or active) and sex (male or female), we ran a one‐way permutational multivariate analysis of variance (PERMANOVA) using a Type III sum of squares (9999 permutations). p values were determined after 9999 permutations, after stepwise selection of explanatory variables using R 2 as selection criterion.

When PERMANOVA tests showed significant differences for a factor (e.g. life stages), a similarity percentage (SIMPER) test with a contribution cut‐off point of 70% (Clarke 1993) was used to identify the main fatty acids contributing to the dissimilarity among factors (e.g. between juveniles and adults or between the various reproductive stages). To assess the contribution of individual fatty acids to overall changes in the fatty acid profile related to ontogeny or diet, we calculated the mean percentage contribution of each fatty acid indicator across all pairwise comparisons. The variation in fatty acid composition of R. undulata from different life stages or reproductive stages was visualized by non‐metric multidimensional scaling (nMDS).

The nMDS ordinations were used from the fatty acid dataset of muscle tissue to measure fatty acids niche metrics (breadth and overlap) as proxies of the size of the feeding niche of both juveniles and adults and potential trophic competition. Niche breadths were estimated and compared using Bayesian analyses of muscle fatty acid profiles (two‐dimensional nMDS ordinations) at 10,000 runs with a probability level of α = 0.95. Ellipses of trophic niche breadth were generated using a random 25 draws to create two‐dimensional niche projections. The size of the trophic niche was calculated based on the parameters μ and ∑ in a Bayesian context following several fatty acid trophic niche studies (Burke et al., 2024; Roberts et al., 2024; Sardenne et al., 2019). nMDS ordinations were also used to calculate the overlap of muscle fatty acid niche space based on the percentage of the adults' niche area that overlaps with that of juveniles, providing a probability that the adults' trophic niche overlaps with that of juveniles. A permutational analysis of multidimensional dispersion (PERMDISP) was used to determine significant differences between the niche breadth among life stages.

To investigate whether fatty‐acid‐based carnivory markers in muscle (DHA/EPA and 18:1n‐9/18:1n‐7 ratios) reflect potential ontogenetic shifts in the diet of R. undulata, we fitted linear models using ordinary least squares. Each ratio was modelled as a function of ray length, either using all individuals together or separating them by life‐stage group. This approach allowed us to assess whether changes occur progressively throughout development or are specific to particular life stages. We evaluated model assumptions using standard diagnostic plots. Assumptions of normality, homoscedasticity and linearity were adequately met for all models.

All statistical analyses were performed with R v.4.2.2 using vegan and nicheROVER packages (Dixon, 2003; Swanson et al., 2015). Values are given as mean ± standard error, and statistical tests were considered significant at p < 0.05.

3. RESULTS

Thirty‐six fatty acids were detected in muscle and liver tissues of the R. undulata analysed, 27 of which contributed >0.1% to the total fatty acid profile at least in one of both tissues (Table 1). Total fatty acid concentrations were approximately 10 times lower in the muscle than in the liver, with values of 3.46 and 31.71 mg g−1, respectively.

TABLE 1.

Mean percentages of fatty acids (>0.1%) in muscle and liver tissues from Raja undulata sampled in the Bay of Biscay (n = 150, sampled between 24 April and 5 May 2023).

Fatty acid Raja undulata
Muscle Liver
14:0 0.13 ± 0.18 1.41 ± 0.53
15:0 0.34 ± 0.38 0.55 ± 0.12
16:0 20.16 ± 2.22 18.04 ± 2.58
18:0 7.07 ± 1.03 6.62 ± 1.11
20:0 0.04 ± 0.03 0.17 ± 0.08
∑SFA 27.7 ± 1.12 26.79 ± 0.84
16:1n‐5 0.04 ± 0.02 0.17 ± 0.04
16:1n‐7 1.36 ± 0.17 5.18 ± 1.29
16:1n‐9 0.33 ± 0.11 0.24 ± 0.09
16:1n‐10 0.26 ± 0.13 0.35 ± 0.09
17:1n‐8 0.17 ± 0.15 0.61 ± 0.22
18:1n‐4 0.03 ± 0.12 0.43 ± 0.13
18:1n‐6 0.02 ± 0.01 0.20 ± 0.04
18:1n‐7 4.15 ± 0.97 6.04 ± 0.71
18:1n‐9 9.24 ± 3.01 9.55 ± 1.30
20:1n‐7 0.28 ± 0.23 0.70 ± 0.25
20:1n‐9 0.45 ± 0.20 0.97 ± 0.29
∑MUFA 16.24 ± 0.41 24.44 ± 0.23
18:2n‐6 1.55 ± 0.54 1.49 ± 0.31
18:3n‐3 0.20 ± 0.33 0.44 ± 0.22
20:2n‐6 0.42 ± 0.17 1.00 ± 1.00
20:4n‐3 0.08 ± 0.02 0.19 ± 0.07
20:4n‐6 5.81 ± 1.40 4.39 ± 1.20
20:5n‐3 5.78 ± 1.78 7.57 ± 2.92
21:5n‐3 0.12 ± 0.03 0.34 ± 0.05
22:4n‐6 0.78 ± 0.27 0.79 ± 0.29
22:5n‐6 0.80 ± 0.22 1.01 ± 0.33
22:5n‐3 2.81 ± 0.65 3.13 ± 0.94
22:6n‐3 35.99 ± 4.58 25.56 ± 4.34
∑PUFA 54.26 ± 2.12 45.57 ± 2.48
∑FA 3.46 ± 1.09 31.71 ± 8.42
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Abbreviations: ∑FA, total fatty acids quantity (mg g−1 of dry tissue); ∑MUFA, total monounsaturated fatty acids; ∑PUFA, total polyunsaturated fatty acids; ∑SFA, total saturated fatty acids.

3.1. Muscle fatty acid profiles across life and reproductive stages

Although total muscle fatty acid concentrations were similar between juveniles and adults (3.39 ± 0.88 vs. 3.51 ± 1.45 mg g−1, respectively; Table S2), fatty acid profiles differed significantly between life stages (PERMANOVA, p < 0.001; Tables S3 and S4) but were not affected by sex (p > 0.05). According to SIMPER analysis, fatty acids 18:1n‐9, DHA, 18:2n‐6, EPA, 16:1n‐7 and ARA were the most common drivers of dissimilarity among life stages (explaining 39% of the dissimilarities due to life stage; Table 2). Non‐metric multidimensional scaling confirmed that juveniles had higher proportions of 18:1n‐9, EPA and 16:1n‐7 fatty acids in muscle samples compared to adults, but lower ARA and DHA (Figure 2). Additionally, when focusing on the muscle fatty acid profiles of adults and accounting for length, sex and reproductive stage, only length had a significant effect (PERMANOVA, p < 0.05 only for length). Regarding the DHA:EPA and 18:1n‐9/18:1n‐7 ratios, they increased significantly with R. undulata length (linear model, p = 0.007, F = 32.2, df = 148 and r 2 = 0.18 for the DHA:EPA ratio and p = 8.10−8, F = 7.3, df = 148 and r 2 = 0.05 for the 18:1n‐9/18:1n‐7 ratio; Figure 3). However, when life stages were analysed separately, this relationship remained significant only in juveniles (linear model, p = 0.03, F = 5.2, df = 68 and r 2 = 0.05 for the DHA:EPA ratio and p = 0.02, F = 5.3, df = 68 and r 2 = 0.05 for the 18:1n‐9/18:1n‐7 ratio, respectively).

TABLE 2.

Outputs of analyses on fatty acids in muscle tissue from Raja undulata sampled in the Bay of Biscay.

SIMPER contributing variables Comparison of average percentage between life stages
Juveniles (%) vs. (%) adults
18:1n‐9 (7.6%) 10.6 ± 3.35 vs. 8.1 ± 2.15
22:6n‐3 (6.7%) 34.7 ± 5.28 vs. 37.1 ± 3.57
18:2n‐6 (6.6%) 2.0 ± 2.07 vs. 1.2 ± 0.73
20:5n‐3 (6.2%) 6.2 ± 1.38 vs. 5.4 ± 2.01
16:1n‐7 (6%) 1.7 ± 0.85 vs. 1.1 ± 0.79
20:4n‐6 (5.6%) 5.1 ± 0.98 vs. 6.5 ± 1.38
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Note: The percentages in the contributing column representing each fatty acid's relative contribution to the dissimilarity between life stages (juveniles [n = 70] vs. adults [gathering first time maturing, spawned already once and actively reproducing individuals, n = 80]).

FIGURE 2.

FIGURE 2

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Non‐metric multidimensional scaling (nMDS) representing variation of the fatty acid composition of Raja undulata muscle tissue over life stages. Overlaid vectors represent fatty acid tracers strongly contributing to juvenile (n = 70) versus adult (first time maturing, spawned already once and actively reproducing, n = 80) fatty acid profiles separation to indicate which tracers are contributing to the position of individuals in the two‐dimensional space. Two‐dimensional nMDS stress was 0.14. In the NMDS plot, vector direction shows the association of variables with the ordination axes and vector length represents the strength of the correlation.

FIGURE 3.

FIGURE 3

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Linear model describing the relationship between muscle fatty acid ratios (as indicators of carnivory/prey trophic level) and Raja undulata total length, considering all individuals (n = 150). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid.

3.2. Niche metrics using muscle fatty acid profiles

Standard ellipse areas estimated using the nMDS ordinations of fatty acid data revealed different‐sized niches among life stages (PERMDISP test, p < 0.05) with the largest fatty acid niche space observed for juveniles (0.050 ± 0.006) compared to adults (0.027 ± 0.003; Figure S1). Substantial niche overlap was also predicted between adult and juvenile fatty acid niche space, as adults had a high probability (0.87) of being found in the large juvenile fatty acid niche space while the probability for juveniles of being found in adult fatty acids niche space was weaker (0.55).

3.3. Liver fatty acid profiles across life stages and reproductive stages

Although total liver fatty acid concentrations were similar between juveniles and adults (29 ± 9.27 vs. 33 ± 7.87 mg g−1, respectively), fatty acid profiles significantly varied among life stages (PERMANOVA, p < 0.001) but were not influenced by sex (PERMANOVA, p > 0.05). According to SIMPER analysis, fatty acids EPA, DHA, 16:1n‐7, 22:5n‐3 (docosapentaenoic acid, DPA), ARA and 18:1n‐9 were the most common drivers of differences among life stages when considering liver fatty acid profiles (explaining 41% of the dissimilarities due to life stage; Table 3). Non‐metric multidimensional scaling confirmed that liver samples collected in juveniles had higher proportions of EPA and lower proportions of 22:5n‐3 compared to adults (Figure 4). Other fatty acids varied along the second nMDS axis but did not significantly discriminate juveniles from adults (Figure 4). The liver DHA:EPA and 18:1n‐9/18:1n‐7 ratios did not show significant variation with R. undulata length (linear model, p > 0.05), whether life stages were analysed collectively or separately.

TABLE 3.

Outputs of analyses on fatty acids in liver tissue from Raja undulata sampled in the Bay of Biscay.

SIMPER contributing variables Comparison of average percentage between life stages
Juveniles (%) vs. (%) adults
20:5n‐3 (12.2%) 9.5 ± 2.05 vs. 5.9 ± 2.57
22:6n‐3 (8.1%) 26.5 ± 4.59 vs. 23.8 ± 3.97
16:1n‐7 (5.7%) 4.7 ± 1.37 vs. 5.6 ± 1.03
22:5n‐3 (5.5%) 2.6 ± 0.67 vs. 3.6 ± 0.88
20:4n‐6 (5.2%) 4.8 ± 1.28 vs. 4.0 ± 1.00
18:1n‐9 (4.4%) 9.7 ± 1.42 vs. 9.4 ± 1.20
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Note: The percentages in the contributing column representing each fatty acid's relative contribution to the dissimilarity between life stages (juveniles [n = 70] vs. adults [gathering first time maturing, spawned already once and actively reproducing individuals, n = 80]).

FIGURE 4.

FIGURE 4

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Non‐metric multidimensional scaling (nMDS) representing variation of the fatty acid composition of adult Raja undulata liver tissue over juvenile and adult life stages. Overlaid vectors represent fatty acid tracers strongly contributing to juvenile (n = 70) versus adult (first time maturing, spawned already once and actively reproducing, n = 80) fatty acid profile separation to indicate which tracers are contributing to the position of individuals in the two‐dimensional space. Two‐dimensional nMDS stress was 0.13. In the NMDS plot, vector direction shows the association of variables with the ordination axes and vector length represents the strength of the correlation. AD, adults; Juv, juveniles.

When analysing only adults, liver fatty acid profiles differed significantly across reproductive stages (PERMANOVA, p < 0.001; Table S5) but were not influenced by sex or length (PERMANOVA, p > 0.05). SIMPER analysis identified EPA, DHA, ARA, 16:1n‐7, 22:5n‐3 (n‐3DPA), 22:5n‐6 (n‐6DPA) and 16:0 as the primary contributors to differences in liver fatty acid composition among reproductive stages (explaining in average 41% of the dissimilarities). The most notable shifts occurred as EPA levels declined from maturing to active stage (Table 4), whereas DHA and ARA increased primarily between stages maturing and mature and then remained relatively stable until stage active (Table 4). Non‐metric multidimensional scaling further confirmed that liver of individuals in early reproductive stage (i.e. maturing stage) exhibited higher EPA proportions and lower DHA and ARA proportions compared to later stages (Figure 5).

TABLE 4.

Outputs of analyses on adult fatty acids in liver tissue from Raja undulata sampled in the Bay of Biscay (adults gather maturing, first time maturing (n = 14); mature, spawned already once (n = 34); active, actively reproducing individuals, n = 32).

Reproductive stages SIMPER contributing variables Comparison of average percentage between reproductive stages
Maturing (%) vs. (%) mature
Maturing vs. mature 20:5n‐3 (14%) 9.42 ± 2.68 vs. 5.40 ± 1.78
22:6n‐3 (9.7%) 21.7 ± 3.77 vs. 23.1 ± 3.56
22:5n‐3 (6.7%) 2.83 ± 0.99 vs. 3.81 ± 0.79
22:5n‐6 (5%) 0.75 ± 0.30 vs. 1.23 ± 0.28
16:1n‐7 (4.7%) 6.46 ± 1.02 vs. 5.39 ± 0.89
20:4n‐6 (4.3%) 3.74 ± 0.65 vs. 4.15 ± 0.93
Mature (%) vs. (%) active
Mature vs. active 22:6n‐3 (8.5%) 23.1 ± 3.56 vs. 24.7 ± 3.83
20:5n‐3 (8.3%) 5.4 ± 1.78 vs. 5 ± 1.93
16:0 (6.4%) 18.04 ± 2.65 vs. 19.28 ± 3.92
20:4n‐6 (5.6%) 4.15 ± 0.93 vs. 4.03 ± 1.19
16:1n‐7 (5%) 5.39 ± 0.89 vs. 5.49 ± 1.02
22:5n‐3 (4.5%) 3.81 ± 0.79 vs. 3.73 ± 0.76
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Note: The percentages in the contributing column representing each fatty acid's relative contribution to the dissimilarity between reproductive stages.

FIGURE 5.

FIGURE 5

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Non‐metric multidimensional scaling (nMDS) representing variation of the fatty acid composition of adult Raja undulata liver tissue across reproductive stages (maturing, first time maturing; mature, spawned already once; active, actively reproducing, n = 80). Overlaid vectors represent fatty acid tracers strongly contributing to reproductive stages separation to indicate which tracers are contributing to the position of individuals in the two‐dimensional space. Two‐dimensional nMDS stress was 0.15. In the NMDS plot, vector direction shows the association of variables with the ordination axes and vector length represents the strength of the correlation.

4. DISCUSSION

This study investigated R. undulata ontogenetic dietary shifts and trophic niche overlap between juveniles and adults based on muscle fatty acids profiles and evaluated the influence of reproductive development on adult liver fatty acid composition. Consistent with previous studies on elasmobranch, muscle fatty acid profiles of R. undulata differed across life stages, with adults exhibiting a narrower trophic niche and a diet consisting of higher‐trophic‐level prey compared to juveniles. This study also highlighted the difference in DHA, ARA, EPA and n‐3DPA percentages in adult liver across reproductive stages, probably reflecting how R. undulata fuels gonad maturation and yolk provisioning.

4.1. Raja undulata trophic changes along life

Muscle tissue analysis reveals longer‐term (months) dietary patterns, highlighting the basal origins of prey items and demonstrating ontogenetic shifts in R. undulata diet. Juveniles exhibited lower levels of DHA and ARA compared to adults, while adults showed reduced proportions of EPA relative to juveniles. These differences likely reflect age‐related changes in dietary intake, with juveniles and adults potentially feeding on different prey. As individuals were collected in the same small area with rays of all lengths caught in the same trawl hauls the difference in diets can be ascribed to prey selection rather than to different habitats. This prey selection might be associated with the catching and ingesting ability, where small rays cannot catch and ingest large prey, rather than preference. This supports previous findings based on stomach content analyses, which suggest that both life stages consume different prey despite occupying the same location (Ellis et al., 2012; Moura et al., 2008). DHA and ARA have been associated with gonad formation and maturation at sexual maturity (Tocher, 2003), thereby influencing reproductive success and offspring survival (Henrotte et al., 2010). This suggests that these fatty acids may be selectively retained in adults for later mobilization during reproduction (Parrish, 2009), which could help explain part of the observed pattern.

These dietary differences are likely driven by ontogenetic shifts toward consuming higher‐trophic‐level prey. The fatty acid ratios indicative of carnivory and higher trophic level (DHA/EPA, 18:1n‐9/18:1n‐7) increased with R. undulata length, with the steepest slope observed within the juvenile length range. This pattern aligns with previous studies on R. undulata feeding ecology, which document a dietary shift toward higher trophic levels with ontogeny. Adults progressively specialize in predation on fish (e.g. European hake, gobies and flatfishes) and larger crustaceans (Ellis et al., 2012; Moura et al., 2008). Meanwhile, juveniles consume a more varied diet, including diverse benthic invertebrates such as brachyuran crabs, crangonid and other natantid shrimps. Furthermore, we observed a reduction in adult trophic niche breadth, approximately half the width of that observed in juveniles. This supports the notion that juveniles maintain a more generalized diet before transitioning to a more specialized, higher‐trophic‐level diet as adults (Costa‐Pereira et al., 2019). This trend mirrors broader patterns in elasmobranchs, where developmental changes in morphology, hunting efficiency and energetic demands drive dietary shifts and specialization in adulthood (Wilga et al., 2007). This could also be regarded as an effect of prey–predator size ratio, which may be conserved throughout the length range of R. undulata, implying that organisms such as shrimps, Amphipods, Euphausiids and Mysids are only fed upon by small rays. Larger rays feed upon larger preys, which larvae and juveniles may also be fed upon by small rays.

Our findings reinforce the understanding of R. undulata as an opportunistic predator that adapts its feeding strategy with length and age. This differentiation in diet among life stages underscores the importance of studying marine predator diets across all life stages, as variations in resource availability may influence life stages differently according to their feeding preferences and ecological roles.

4.2. Trophic changes are highlighted by both muscle and liver tissues

It is commonly assumed that different tissues’ fatty acid profiles bring different information. In elasmobranchs, muscle and liver fatty acid profiles differ due to the distinct physiological roles and metabolic functions (Meyer et al., 2017, 2019). The liver serves as the primary site for lipid metabolism, storage and mobilization, leading to a dynamic fatty acid composition that reflects recent dietary intake (over the last weeks; Ballantyne, 1997) and metabolic processes such as lipid oxidation and biosynthesis. In contrast, muscle primarily functions as an energy reservoir for sustained activity, with a more stable fatty acid composition that integrates long‐term dietary assimilation (over months; Ballantyne, 1997). The results we obtained with liver (generally considered less accurate to study elasmobranch trophic ecology) support the diet differences along life stages that we highlighted with muscle tissue. Similar fatty acids in muscle and liver tissues were found to discriminate juveniles and adults (except the 22:5n‐3 in liver tissue that was not highlighted in muscle tissue), with juveniles having more EPA and less DHA and ARA than adults. The convergence of results from both tissues reinforces the dietary differences observed between life stages and advocate that R. undulata diet change between both life stages could be very rapid, likely due to changing physiological requirements. However, not all dietary shifts were detectable in the liver, since the increase in prey trophic level with length, indicated by the fatty acid ratio proxy of carnivory (DHA/EPA, 18:1n‐9/18:1n‐7) in juvenile muscle, was not reflected in liver tissue. Metabolic processes such as lipid oxidation and biosynthesis may have masked this dietary signal of an increase of animal prey (Meyer et al., 2017).

4.3. Fatty acid allocation during gonadal development

The elasmobranch liver is a key storage organ during vitellogenesis (i.e. is the deposition of yolk in developing oocytes), accumulating lipid reserves and essential fatty acids to support gonad maturation and yolk provisioning (Pethybridge, Daley, Virtue, & Nichols, 2010; Rossouw, 1987). In R. undulata, the primary pattern observed in liver fatty acid profiles during gonadal development (particularly between maturing and mature stages) is the higher proportion of DHA, ARA and n‐3DPA (i.e. docosapentaenoic acid, C22:5n‐3), alongside the lower proportions of both EPA and 16:1n‐7, similar to other studies on fatty acid changes through reproductive development (De Sousa Rangel et al., 2020; Rangel et al., 2021a). However, when considering absolute values, the observed changes are primarily driven by a depletion of EPA during gonadal development, while DHA and ARA remained relatively stable and the n‐3 DPA slightly increased. The decline in EPA aligns with the known energy allocation patterns during vitellogenesis, a period when n‐3 PUFAs are preferentially transferred to the ovaries. These fatty acids are crucial for supporting key physiological processes in the developing offspring, particularly brain and eye development, and can influence offspring survival (Henrotte et al., 2010). An alternative explanation for the reduction of EPA in the later stages of gonadal development could be its enzymatic elongation to form n‐3 DPA, a fatty acid typically present in low concentrations in marine plankton. n‐3 DPA is recognized for its benefits to brain and eye development in aquatic organisms and plays a key role in early‐life development (Mejri et al., 2021), suggesting that its selective retention may be linked to its allocation for the synthesis of the amount of yolk necessary for the production of 50 to 70 egg cases per year (Serra‐Pereira et al., 2015; Villagra et al., 2022). However, the only slight increase in n‐3 DPA observed (Table S6) makes it more likely that EPA is directly allocated to vitellogenesis rather than being extensively converted into n‐3 DPA.

Despite their increase in proportion, the quite stable quantities of DHA and ARA contrasted with literature. DHA and ARA levels classically decrease throughout embryonic development and gonadal maturation, being allocated to gamete production (De Sousa Rangel et al., 2020; Rangel et al., 2021b; Tocher, 2003). This is related to the critical role of essential long‐chain PUFAs, particularly DHA (Pethybridge et al., 2011), at the onset of embryonic development (Henrotte et al., 2010). However, previous studies have focused on viviparous elasmobranch species, where DHA and ARA are supplied from the gravid females to the developing embryos during gestation (De Sousa Rangel et al., 2020; Henningsen et al., 2004; Iverson, 2009). These long‐chain PUFAs underpin critical facets of embryogenesis, supporting immune and inflammatory regulation and enhancing growth, survival and stress resistance (Tocher, 2010). Because R. undulata is oviparous, it likely follows a different strategy of fatty acid allocation to vitellogenesis.

Beyond long‐chain PUFAs, monounsaturated fatty acids (MUFAs) play a crucial role as a primary energy source during oogenesis, particularly in mature follicles before ovulation in most elasmobranchs (Tocher, 2010). The dominance of MUFAs, specifically 16:1n‐7, 18:1n‐9 and 20:1n‐9, stems from their higher metabolic availability, as they are more efficiently catabolized for energy production compared to saturated fatty acids and PUFAs (Sargent et al., 1999). Adults R. undulata exhibited higher MUFA proportions and absolute values (Table S6) compared to juveniles, likely reflecting increased energetic demands associated with reproductive development. Furthermore, a decrease in MUFA absolute values was observed throughout gametogenesis in mature adults (Table S6), suggesting mobilization of liver reserves to support follicular maturing and future embryonic development. These findings are consistent with previous studies emphasizing the importance of MUFAs in fuelling reproductive processes, even if these studies were mainly conducted on viviparous elasmobranch species (Pethybridge et al., 2011). These observed changes in liver fatty acid profiles during gonadal development suggest a potential dietary shift in reproductive individuals to support gametogenesis and/or an internal regulatory mechanism controlling essential fatty acid composition, alongside increased energy demands during maturation.

4.4. Study limitations

While this study provides valuable insights, some limitations should be noted. First, samples were collected over a limited temporal window, which prevents the assessment of seasonal or interannual variability in diet. Second, although fatty acids provide integrative information on trophic ecology, the absence of a complementary dataset on prey fatty acids limits our ability to directly link R. undulata dietary composition to prey availability, potentially obscuring finer‐scale dietary preferences or trophic shifts between life stages.

5. CONCLUSION

Consistent with previous studies, our findings highlight the importance of analysing different tissues to gain a comprehensive understanding of the complex relationships between diet, trophic interactions and energy allocation in elasmobranchs (Meyer et al., 2017). Despite the absence of a significant sex effect in this study, further research is needed to better understand sex‐specific fatty acid allocation to gonads and energetic demands during reproductive development. Our findings underscore the need to further investigate the significance of individual fatty acids for estimating the contributions of specific prey items to elasmobranch diets, as well as to continue sampling to better understand temporal dietary shifts (at both seasonal and interannual scales) in order to fully comprehend feeding ecology and energy allocation throughout ontogeny.

AUTHOR CONTRIBUTIONS

P.B. contributed to the study design, data analysis and manuscript preparation. T.J, M.V. and V.R. were responsible for fieldwork, sample processing and data generation. B.S. provided conceptual input, and contributed to the study design and to the manuscript preparation. S.F‐R and P.L were responsible for fieldwork, supervised the project and secured funding. All authors contributed to the final version of the manuscript and approved its submission.

FUNDING INFORMATION

This study was conducted as part of the AGEPPOP project, funded by France Filière Pêche.

Supporting information

DATA S1 Supporting Information.

JFB-109-189-s001.docx (229.6KB, docx)

ACKNOWLEDGEMENTS

This study was conducted as part of the AGEPPOP project (Genetic and physiological approaches to assess the health status of ray populations), funded by France Filière Pêche. The authors gratefully acknowledge the fishermen for their valuable assistance and support in the sampling of R. undulata aboard the fishing vessel Déesses de L'Océan.

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