Variations in isotope incorporation rates and trophic discrimination factors of carbon and nitrogen stable isotopes in scales from three European sea bass (Dicentrarchus labrax) populations

Highlights

• We determined δ¹³C and δ¹⁵N values in scales of Dicentrarchus labrax.

• Each fish was sampled 17 times without deleterious effects on growth or survival.

• Incorporation rates were higher in males and strongly variable among individuals.

• Trophic discrimination factors were strongly variable among individuals.

• Individual variability should be taken into account in field-based studies.

Abstract

Carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope analyses are used in marine ecology to study trophic relationships and migrations of species since they reflect dietary sources consumed which may vary geographically. However, better estimations of isotope incorporation rates and trophic discrimination factors (TDF) under controlled conditions are required. Moreover, variability of isotope incorporation rates and TDF among and within populations has been poorly described, especially in fish scales, whereas the use of non-lethal method is becoming a standard. This study aimed to experimentally assess whether carbon and nitrogen isotope incorporation rates (λC and λN, respectively) and TDF of scales vary in the European sea bass (Dicentrarchus labrax) among (1) Atlantic, West Mediterranean and East Mediterranean populations, (2) sexes and (3) individuals. Fish were reared under controlled conditions and switched from a diet 1 to a diet 2 with different δ¹³C and δ¹⁵N values. Scales were sampled repeatedly on 16 fish within the three populations, from the day of diet change (day 0) to the end of the experiment (day 217). Isotope incorporation rates of scales and TDF were determined using a time-dependent model. Isotopic carbon and nitrogen half-lives (t₅₀C and t₅₀N) were similar among the three populations but males had significantly lower t₅₀C and t₅₀N than females (29 ± 2 and 35 ± 2 days vs. 53 ± 7 and 80 ± 11 days, respectively). Females had higher growth rates but lower catabolic rates than males. Variability of λC and λN was large within sexes: t₅₀C ranged from 17 to 159 days and t₅₀N ranged from 18 to 342 days among individuals. Thus, variability between sexes and among individuals must be considered to avoid misinterpretation in field-based studies. For the 48 fish, TDF were 4.91 ± 0.03 and 2.46 ± 0.06‰ for carbon and nitrogen, respectively, and similar between sexes and among populations. Besides, TDF varied among individuals from 2.95 to 5.59‰ and from 0.93 to 3.55‰ for carbon and nitrogen, respectively. Empirical mixing models were run to estimate how different TDF influenced estimation of the contributions of food sources to diet of their consumer. The output differed considerably when using TDF from fish literature or those estimated herein, which confirms that a tissue-specific TDF must be used to avoid misinterpretation in field-based studies. Individual variation in TDF did not, however, influence estimation of the contributions of food sources, confirming that scales are a valid tissue for non-lethal sampling.

Introduction

Analysis of carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotopes has proven to be a powerful tool in marine ecology, to study trophic relationships and migrations of various species through time and space (Hansson et al., 1997; Perga and Gerdeaux, 2003; Dempson et al., 2010; Sweeting, 2010). The δ¹³C and δ¹⁵N values of organisms reflect those of assimilated dietary sources as it is generally accepted that consumers are enriched by 1.0 and 3.5‰ in ¹³C and ¹⁵N, respectively, relative to their diets (Fry and Arnold, 1982; Minagawa and Wada, 1984). However, these assumptions are not universal and reconstruction of diet history as well as quantification of trophic relationships of organisms in their environment require better estimations of both carbon and nitrogen isotope incorporation rates and trophic discrimination factors (TDF) under controlled conditions (Wolf et al., 2009; Martínez del Rio and Carleton, 2012).

Isotope incorporation rate is defined as the time required by an organism to acquire the isotopic composition of its new diet (Martínez del Rio and Carleton, 2012). This variable is essential to determine the temporal window in which stable isotope data can be used to elucidate the diet of an animal (Perga and Gerdeaux, 2005; Wolf et al., 2009). Stable isotope values of an organism in a situation of disequilibrium, after a change in diet, do not represent either the past or the present diet (Sweeting, 2010). It is well established that isotope incorporation rates are higher in metabolically active species, organisms or tissues, depending on both growth rate (i.e., anabolic rate or adjunction of new tissues) and catabolic rate (i.e., replacement of tissues; Hesslein et al., 1993; MacNeil et al., 2006). Generally, isotope incorporation rates are higher in liver and plasma than in muscle and red blood cells of fishes (Carleton and Martínez del Rio, 2010). Moreover, isotope incorporation rates vary with environmental conditions such as temperature, food quantity and quality as well as the physiological state of the animal, such as ontogenetic stage or level of stress (Witting et al., 2004; Carleton and Martínez del Rio, 2010; Bloomfield et al., 2011; Carter et al., 2019).

Trophic discrimination factor, the difference between stable isotope values of a consumer and its diet when at isotopic equilibrium, also fluctuates considerably. For fishes, carbon and nitrogen TDF vary from 0.2 to 4.0‰ and from −0.4 to 5.5‰, respectively (Sweeting et al., 2007a, Sweeting et al., 2007b). Accurate values are required to interpret relationships across trophic levels. A robust estimation of TDF is also a fundamental requirement for mixing models that predict the proportional composition of consumers' diets from stable isotope data (Phillips et al., 2014). Physiological mechanisms underlying TDF are not thoroughly understood but result from the balance between processes of assimilation and excretion of light versus heavy elements acquired in the food (Minagawa and Wada, 1984; Ponsard and Averbuch, 1999; Olive et al., 2003). Thus, TDF is influenced by both dietary and non-dietary factors (Trueman et al., 2005; Barnes et al., 2007; Matley et al., 2016; Nuche-Pascual et al., 2018).

In isotope-based studies, variation among individuals has been evaluated as the variance of δ¹³C and δ¹⁵N values. For fishes, δ¹³C and δ¹⁵N values of tissues have been shown to vary within and among populations (Barnes et al., 2008), and especially with sex (Kim et al., 2012; Marcus et al., 2019). To date, however, studies of variation in isotope incorporation rates and TDF within and among populations are scarce. When distinct isotope values appear among individuals, the assumption commonly made is that they must have been feeding on distinct food sources (e.g. Grey, 2001). However, several studies revealed an inherent variability between individuals fed with a same diet (Matthews and Mazumder, 2004; Araújo et al., 2007; Barnes et al., 2008). These studies underscored the need to take this variability into account in field-studies before concluding that individuals have distinct feeding habits. Differences in individual physiology are suggested to be the cause of this inherent individual variability (Bearhop et al., 2004). Isotope incorporation rates are estimated by changing from one isotopically distinct experimental diet to another, and then sampling tissues over time. In fishes, most isotope incorporation rate studies have used dorsal white muscle, so individuals must be sacrificed at each sampling point to monitor stable isotope values (Hesslein et al., 1993; German and Miles, 2010; Madigan et al., 2012). This precludes the study of individual variation in the kinetics of isotopic incorporation. Using non-lethal methods, such as sampling of red blood cells, plasma, fins and scales, would permit multiple sequential samplings on the same individuals, to study variation in isotope incorporation rates. Moreover, the development of non-lethal methods is desirable from a perspective of animal welfare (European Union, 2010; Australian Government, 2013; US Government, 2015). To our knowledge, the few studies that have sampled the same individuals over time after diet change have shown marked variation in carbon and nitrogen isotope incorporation rates (Hilderbrand et al., 1996; Voigt et al., 2003; Evans Ogden et al., 2004; Kim et al., 2012). Similarly, individual variation in TDF was revealed within different species (Lecomte et al., 2011; Kurle et al., 2014; Galván et al., 2016).

In the present study, we estimated isotope incorporation rates and TDF of carbon and nitrogen stable isotopes in the scales of 48 European sea bass (Dicentrarchus labrax) from three distinct populations (Atlantic AT, West Mediterranean WM and East Mediterranean EM; Guinand et al., 2017) reared under controlled conditions. European sea bass is highly prized by both commercial and sports fishermen, but a severe decline in stocks has recently raised concern about the conservation status of the species (de Pontual et al., 2019). The use of carbon and nitrogen stable isotope analyses is a good tool to improve the management of this species because it can reveal its feeding habitats and migrations (Cambiè et al., 2016). We assessed whether there were differences in isotope incorporation rates and TDF among (1) the three populations, (2) sexes and (3) individuals. Finally, empirical stable isotope mixing models were run with different carbon and nitrogen TDF to assess their influence on dietary predictions in field-based studies. Scales are a superposition of an organic layer mainly composed of proteins (mostly collagen) and an inorganic layer which is a carbonate salt (Hutchinson and Trueman, 2006). However, scales are not an inalterable record: several studies support the existence of a catabolic activity in scales with the destruction and the renewal of collagen by cells within the scale structure, respectively named osteoclasts and osteoblasts (Sire et al., 1990; Suzuki et al., 2000). Thus, it can be hypothesized that isotope incorporation rate in scale might not be only driven by growth.