# Changes in foraging mode caused by a decline in prey size have major bioenergetic consequences for a small pelagic fish

Elisa Thoral, Quentin Queiros, Damien Roussel, Gilbert Dutto, Eric Gasset, David J Mckenzie, Caroline Romestaing, Jean-marc Fromentin, Claire Saraux, Loïc Teulier

► To cite this version:

Elisa Thoral, Quentin Queiros, Damien Roussel, Gilbert Dutto, Eric Gasset, et al.. Changes in foraging mode caused by a decline in prey size have major bioenergetic consequences for a small pelagic fish. Journal of Animal Ecology, 2021, 90, pp.2289-2301. 10.1111/1365-2656.13535. hal-03234605

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Changes in foraging mode caused by a decline in prey size have major bioenergetic consequences for a small pelagic fish

Elisa Thoral¹, Quentin Queiros², Damien Roussel¹, Gilbert Dutto³, Eric Gasset⁴, David J. McKenzie⁵,
Caroline Romestaing¹, Jean-Marc Fromentin², Claire Saraux²,⁶ and Loïc Teulier¹.

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¹Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622, Villeurbanne, France

²MARBEC, Univ Montpellier, CNRS, IFREMER, IRD, Sète, France

³Ifremer (Institut Français de Recherche pour l'Exploitation de la MER), Laboratoire SEA, Chemin de Maguelonne, 34250 Palavas-Les-Flots, France

⁴MARBEC, Univ Montpellier, CNRS, IFREMER, IRD, Palavas-Les-Flots, France

⁵MARBEC, Univ Montpellier, CNRS, IFREMER, IRD, Montpellier, France

⁶IPHC, UMR7178, Université de Strasbourg, CNRS, 67000 Strasbourg, France

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**Corresponding author:** Loïc TEULIER, Université de Lyon, UMR 5023 Ecologie des Hydrosystèmes Naturels et Anthropisés, Université Lyon 1, ENTPE, CNRS, F-69622 Villeurbanne, France, loic.teulier@univ-lyon1.fr

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**Authors' contributions:** QQ, J-MF, GD, EG, DMK, DR, CS and LT conceived and designed the study. ET, QQ, GD, EG, DR, CR, DMK, CS and LT carried out the study. GD & EG took care of the fish and participated to the experimental protocol. ET, QQ, DR, DMK, CS and LT analyzed the data and drafted the manuscript. All authors reviewed and revised the article critically and approved the final version.

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**Acknowledgements:** This study was funded through the MONALISA project, financed by the European Union and the French Ministry of Agriculture through the European Maritime and Fisheries Fund (EMFF/FEAMP) as well as France Filière Pêche. Experimental procedures were approved by the ethics committee for animal experimentation n° 036 of the French Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation, with reference number APAFIS n° 7097-2016093008412692. We warmly thank the reviewers for their constructive remarks and comments on the manuscript.

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**Data availability statement:** Data available from the Dryad Digital Repository:

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https://doi.org/10.5061/dryad.xpnvx0kfp (Thoral *et al.* 2021a)

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**Abstract**

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1. Global warming is causing profound modifications of aquatic ecosystems and one major outcome appears to be a decline in adult size of many fish species. Over the last decade, sardine populations in the Gulf of Lions (NW Mediterranean Sea) have shown severe declines in body size and condition as well as disappearance of the oldest individuals, which could not be related to overfishing, predation pressure or epizootic diseases.

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2. In this study, we investigated whether this situation reflects a bottom-up phenomenon caused by reduced size and availability of prey that could lead to energetic constraints. We fed captive sardines with food items of two different sizes eliciting a change in feeding mode (filter-feeding on small items and directly capturing larger ones) at two different rations for several months, and then assessed their muscle bioenergetics to test for changes in cellular function.

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3. Feeding on smaller items was associated with a decline in body condition, even at high ration, and almost completely inhibited growth by comparison to sardines fed large items at high ration.

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4. Sardines fed on small items presented specific mitochondrial adjustments for energy sparing, indicating a major bioenergetic challenge. Moreover, mitochondria from sardines in poor condition had low basal oxidative activity but high efficiency of ATP production. Notably, when body condition was below a threshold value of 1.07, close to the mean observed in the wild, it was directly correlated with basal mitochondrial activity in muscle.

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5. The results show a link between whole-animal condition and cellular bioenergetics in
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the sardine, and reveal physiological consequences of a shift in feeding mode. They
demonstrate that filter-feeding on small prey leads to poor growth, even under abundant
food and an increase in the efficiency of ATP production. These findings may partially
explain the declines in sardine size and condition observed in the wild.

**Keywords:** Bioenergetics, red muscle, mitochondria, fish shrinking, sardine, foraging
behaviour, food restriction
65 Résumé

66 Le changement global entraîne de profondes modifications des écosystèmes aquatiques, l'une
67 des principales étant le déclin de la taille des adultes chez de nombreuses espèces de poissons.

68 Au cours de la dernière décennie, les populations de sardines du Golfe du Lion (Nord-Ouest de
69 la Méditerranée) ont montré une importante diminution de leur taille et de leur condition
70 corporelle ainsi qu'une disparition des individus les plus âgés, qui n'ont pas pu être liées à la
71 surpêche, à la pression de prédation ou aux épizooties.

72 Dans cette étude, nous avons cherché à savoir si cette situation reflète un phénomène ascendant
73 causé par la réduction de la taille et de la disponibilité des proies qui pourrait entraîner des
74 contraintes énergétiques chez la sardine. Nous avons ainsi nourri des sardines captives avec des
75 granulés de deux tailles différentes provoquant un changement de mode d'alimentation
76 (filtration des petits granulés et capture directe des plus gros) et à deux rations différentes
77 pendant plusieurs mois, puis nous avons évalué leur bioénergétique musculaire pour tester les
78 changements au niveau de leur fonction cellulaire.

79 L'alimentation à base de petits granulés a été associée à un déclin de la condition corporelle,
80 même à une ration élevée, et à une croissance quasiment inhibée par rapport aux sardines
81 nourries avec des plus gros granulés à une ration élevée.

82 Les sardines nourries avec des petits granulés ont également présenté des ajustements
83 mitochondriaux spécifiques pour économiser de l'énergie, indiquant un défi bioénergétique
84 majeur. De plus, les mitochondries des sardines en mauvaise condition présentaient une faible
85 activité oxydative basale, mais une efficacité élevée de production d'ATP. Notamment, lorsque
86 la condition corporelle était inférieure à une valeur seuil de 1,07, proche de la moyenne observée
87 dans la nature, elle était directement corrélée à l'activité mitochondriale basale dans le muscle.

88 Ces résultats montrent un lien entre la condition de l'animal entier et la bioénergétique cellulaire
89 chez la sardine, et révèlent les conséquences physiologiques d'un changement de mode
90 d'alimentation. Ils démontrent que le nourrissage via la filtration de petites proies entraîne une
91 faible croissance, même en cas de nourriture abondante, et une augmentation de l'efficacité de
92 la production d'ATP. Ces résultats peuvent expliquer en partie le déclin de la taille et de la
93 condition des sardines observé dans la nature.
## 1. Introduction

Marine ecosystems face strong pressures from a combination of climate forcing, anthropogenic disturbance (pollution, navigation, recreational activities, by-catch, climate change) and anthropogenic competition for resources (e.g. fisheries) (Halpern et al., 2008), all of which can impact organismal phenotypes. While water warming and a relaxation of density-dependence phenomena through fishing should promote an increase in growth rates of ectothermic organisms (Morrongiello, Sweetman, & Thresher, 2019), there is evidence that the final adult size of many aquatic ectotherms is declining globally (Daufresne, Lengfellner, & Sommer, 2009; Forster, Hirst, & Atkinson, 2012). Whereas recently debated (Audzijonyte et al., 2020), this size reduction occurs within and amongst species, throughout the food web, from top predators (Sibert et al., 2006) to primary producers (Sommer, Paul, & Moustaka-Gouni, 2015). The decrease in size could result from fishery-induced evolution (as fisheries targeting mostly larger organisms should favour the selection of slow growth and smaller phenotypes, Conover & Munch, 2002; Jørgensen et al., 2007) and/or from within-generation plastic physiological responses. It could also result from direct effects of warming on physiology (e.g. the temperature-size rule; Atkinson, 1994) or global-change-derived modifications of primary production (Bopp et al., 2013).

There has been a profound decline in size and body condition of sardines *Sardina pilchardus* and anchovies *Engraulis encrasicolus* in the Gulf of Lions (northwestern Mediterranean Sea), due to lower growth and disappearance of larger and older individuals (Brosset et al., 2017; Van Beveren et al., 2014). Extensive research has established that this is not due to top-down effects, such as predation or fishing pressure, nor to pathogens (reviewed in Saraux et al., 2019). Rather, a bottom-up process appears to be at play, linked to a shift in plankton communities that the fishes feed upon, towards smaller zooplankton species (Brosset et al., 2016a; Queiros et al., 2019). When maintained in captivity on artificial feeds, wild
119 sardines exhibited high growth rates and condition, indicating that the decline in size and
120 condition was a plastic rather than an evolutionary response (Saraux et al., 2019). While it is
121 obvious that food quantity can have a significant impact on growth and body condition in fishes
122 (Lee et al., 2018; Sun & Chen, 2009), the potential implications of prey size are less well
123 known. They deserve to be investigated because of climate-change related regime shifts in food
124 webs (Garzke, Ismar, & Sommer, 2015). A reduction in prey size is often accompanied by
125 lower energy content (Zarubin et al., 2014), which may present a major energetic challenge to
126 predators, even for sardines that are opportunistic planktivores (Palomera et al., 2007; Rumolo
127 et al., 2016).

128 It has been demonstrated that, for rations of similar energy content, sardines fed small
129 prey have lower growth and condition than those fed larger prey (Queiros et al., 2019). The
130 sardine modifies its feeding behaviour with prey size, switching from filter feeding on small
131 prey to direct capture of larger prey, regardless of prey availability (Costalago, Garrido, &
132 Palomera, 2015; Garrido et al., 2007). Foraging duration was much higher for sardines fed on
133 small food items than those fed on larger ones (Queiros et al., 2019). Thus, a possible
134 mechanism for the poor growth and condition may relate to high energy requirements for
135 extended swimming activity during filter-feeding, compared to shorter bouts of swimming to
136 capture large items (Costalago & Palomera, 2014; Crowder, 1985).

137 Foraging behaviour in fishes is sustained by skeletal muscle activity, with the muscles
138 being structurally separated into discrete slow-twitch oxidative 'red' muscle, used for steady
139 aerobic swimming, and fast-twitch glycolytic 'white' muscle, used for short bursts of high-
140 speed swimming (Bone, 1978; Webb, 1998). Muscular contraction is powered by ATP but, its
141 production depends on muscle type: while red muscle generates this aerobically by
142 mitochondrial oxidative phosphorylation using substrates and oxygen delivered in the
143 bloodstream, white muscle generates ATP mostly through anaerobic glycolysis of intracellular
substrates (Fig. 1, Bone, 1978; Webb, 1998). In the sardine, swimming muscle represents up to 144 70% of body mass and, although the red muscle mass only represents ~10% of the body mass, 145 its mitochondrial activity accounts for 70% of muscle metabolic activity (Teulier et al., 2019). 146

Mitochondrial oxidative phosphorylation is a series of reactions that couple substrate oxidation 147 (catabolism of carbohydrates, lipids or proteins) and oxygen consumption with the generation 148 of a proton motive force, which is then used by ATP synthase to synthesize ATP (Fig. 1). A 149 significant proportion of mitochondrial oxygen consumption is not, however, coupled to ATP 150 synthesis but is used to compensate for proton leakage across the inner mitochondrial 151 membrane. Mitochondrial proton leak is a ubiquitous waste of potential energy, which reduces 152 the efficiency of ATP production (i.e. the ATP/O ratio) (Brand, 2005; Fig. 1). The ATP/O ratio 153 defines how much oxygen and nutrients are needed to meet cell ATP demands and, ultimately, 154 sustain animal performance (Brand, 2005). For instance, mitochondrial ATP/O has been 155 positively related to muscle performance (Conley et al., 2013; Distefano et al., 2018) and 156 growth efficiency (Salin et al., 2019; Toyomizu et al., 2011). Mitochondrial efficiency is also 157 flexible, varying among individuals over time, notably in response to environmental and 158 physiological constraints such as temperature, oxygen or food availability (Roussel & Voituron, 159 2020; Salin et al., 2015; Salin et al., 2012; Thoral et al., 2021b). For example, mitochondrial 160 efficiency can increase to enhance energy conservation during food deprivation (Bourguignon 161 et al., 2017; Monternier et al., 2017) or decrease to promote heat generation, as in brown 162 adipose tissue of cold-adapted mammals (Nedergaard & Cannon, 2018). The maintenance of a 163 high ATP/O ratio and related efficiency may carry significant costs, especially increased 164 production of reactive oxygen species (Brand, 2000), and/or a decreased carbon flux from 165 mitochondria that can compromise aspects of cellular biosynthesis (Rolfe & Brand, 1997). 166

Overall, there is strong evidence that mitochondrial efficiency (ATP/O ratio) can be adjusted 167
plastically, in response to metabolic constraints and energy demands, leading to a dynamic interplay with the biological condition of an organism (Koch et al., 2021).

In this study we investigated whether aerobic muscle energy metabolism and, in particular, mitochondrial function, might represent a mechanistic nexus for how sardine foraging mode impacts upon their physical condition (Fig. 1). We thus investigated the oxidative activity of red muscle fibres and the coupling efficiency of mitochondrial oxidative phosphorylation in sardines fed for 7 months with either small or large food items (0.1mm and 1.2mm diameter commercial pellets), at either low or high ration (0.3% and 0.6% of fish mass per day). We predicted that fish fed on small items would show specific mitochondrial adjustments for energy sparing, with decreased oxygen consumption but enhanced efficiency (ATP/O ratio), which would indicate that increased metabolic costs of foraging mode represented an energetic challenge.

# 2. Material and methods

## 2.1 Experimental conditions

All details of the experiment can be found in Queiros *et al.*, 2019. Briefly, wild sardines were captured off Sète (South of France) in October 2016 and brought back to the Ifremer Palavas-les-Flots research station. The mean age of sardines was 1.24 years. After acclimation and weaning onto commercial pellets, sardines were weighed, measured and marked individually using RFID tags (Biolog-id, Bernay, France). Thereafter, they were distributed among 8 tanks (vol. 300L, 56-57 sardines per tank) with similar mean (± SD) of mass and length in each (mass: 14.1 ± 3.2g; length: 11.9 ± 0.7 cm). A 7-month feeding experiment was performed, from 14/11/2016 to 15/6/2017, with natural photoperiod and water temperature (water was pumped directly from the sea). Animals were fed four times a day with commercial pellets (except on weekends where one daily meal was provided). The experiment included the
reproductive season, which occurs once a year between December and March. As detailed in Queiros *et al.*, 2019, the daily weight gain of sardines *in natura* was 0.2% with good condition and 0.1% with bad condition. A preliminary study found that these growth rates were achieved with daily feeding rates of 0.6% and 0.3% of fish mass. Thus, the combination of two pellet sizes (0.1mm and 1.2mm, within the natural range of sardine prey size) (Le Bourg *et al.*, 2015) and two rations (0.3% and 0.6% of fish biomass per day) resulted in four treatments: (1) small items in small quantity (SI-SQ), (2) small items in large quantity (SI-LQ), (3) large items in small quantity (LI-SQ), (4) large items in large quantity (LI-LQ). The two sizes elicited two distinct foraging modes, filtration vs. particulate capture (Queiros *et al.*, 2019). Each treatment was randomly assigned to two tanks for replicates. Biometries were performed each month to correct food quantity against increasing biomass, adjusted each week based on linear extrapolation of biomass across successive biometries.

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## 2.2 Sardine sacrifices and morphometric measurements

At the end of the experiment, 1 or 2 sardines were randomly removed from each tank every day over a period of 1 week, i.e. 11 to 12 fish per group, and killed by an overdose of benzocaine (1000 ppm). Sardines were fasted for 24h before euthanasia. Mass and size of each sardine were recorded. Intramuscular fat was estimated by fatmeter (Distell Fish Fatmeter MFM-992, as described in Brosset *et al.*, 2015a) and sex was determined visually. Experiments were performed in June when sardines were out of the breeding season (Brosset *et al.*, 2016b). Cumulative growth was estimated based on the increase in size divided by the experiment duration (mm/day), and the relative growth (in %) was calculated as follows:

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$$ \text{Relative growth} = \frac{\text{Final size} - \text{Initial size}}{\text{Initial size}} $$

216 Where initial and final sizes are expressed in mm.
Body condition was calculated following the Le Cren index K_n as estimated by Brosset et al., 2015b (based on more than 24,000 wild sardines sampled over the last 50 years):

$$K_n = \frac{BM}{0.00607 \times TL^{3.057}}$$

where TL is the total length (cm) and BM is the wet body mass (g). This index compares the actual wet mass of an individual to the mass predicted by the relationship of mass to length for the entire wild population. As such, K_n < 1 (or K_n > 1) means that body condition is lower (or higher) than the long-term average observed in the wild population

## 2.3 Mitochondrial respiration

Oxygen consumption measurements were performed in red muscle at two levels: in muscle fibres and in isolated mitochondria. At the mitochondrial level, oxygen consumption was expressed in nmol O.min⁻¹.mg⁻¹ of protein, and it was expressed in nmol O.min⁻¹.mg⁻¹ of red muscle at the fibre level. The oxygen consumption was then estimated at the muscle level by multiplying the oxygen consumption obtained at the fibre level by the red muscle mass (µmol O.min⁻¹) or by the relative red muscle mass (nmol O.min⁻¹.g⁻¹ of fish) (see Table 2).

### 2.3.a Red muscle fibre preparation and oxidative activity

After dissection, the total mass of red muscle was measured and a ~5 mg sample of fibres was withdrawn (Fig. 1). Fibre respiration was monitored at 20°C using high resolution respirometers (Oxygraph-2K, Oroboros® Instruments – WGT Austria) in air-saturated respiration buffer (Mir05: 110 mM sucrose, 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 1 g/L fatty acid-free bovine serum albumin and 20 mM Hepes, pH 7.1) using different combination of respiratory substrates (5 mM glutamate/2.5 mM malate; 40 µM palmitoyl-carnitine/2.5 mM malate; 5 mM pyruvate/2.5 mM malate/5 mM succinate) with a protocol adapted from Teulier et al., 2019. These different
combinations of substrates enabled us to assay mitochondrial respiration using substrates derived from amino acid (glutamate), carbohydrate (pyruvate) and lipid (palmitoyl-carnitine) metabolic pathways. In addition, the mixture of NADH- and FADH₂-linked substrates (pyruvate/malate and succinate) allowed a full activation of mitochondrial oxidative activity by providing electrons to complex I and complex II of the electron transport system (ETS). Phosphorylating respiration was then initiated by adding 1mM ADP, and the integrity of mitochondria within muscle fibres was subsequently tested by adding 10µM cytochrome c. Finally, a sequential addition of 1 µM p-trifluoromethoxy-carbonyl-cyanide-phenyl hydrazine (FCCP) was performed to measure maximal respiratory activity of the fibres.

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252 **2.3.b Mitochondrial isolation, coupling efficiency, respiratory capacity and content**

Mitochondria were isolated from red skeletal muscle in an ice-cold isolation buffer containing (100 mM sucrose, 50 mM KCl, 5 mM EDTA, 50 mM Tris-base, pH 7.4 at 4°C), according to previously described protocol for small tissue samples (Boël *et al.*, 2019), involving potter homogenizer, partial protease digestion, and differential centrifugation with mitochondria pelleted at 9000 × g, all steps at 4°C. Protein content of the preparation was assayed in duplicate at 540nm using the biuret method, with bovine serum albumin as standard (Gornall *et al.*, 1949). Absorbance of the same volume of mitochondria was also assayed at 540nm in a solution containing 0.6% Na-K-tartrate and 3% NaOH and subtracted, to correct for any contamination with pigments absorbing at 540nm.

Mitochondrial oxidative phosphorylation efficiency was assessed at 20°C by measuring the rates of ATP synthesis and oxygen consumption in respiratory buffer (120 mM KCl, 5 mM KH₂PO₄, 1 mM EGTA, 2 mM MgCl₂, 0.3% fatty acid-free bovine serum albumin (w/v), and 3mM HEPES, pH 7.4) supplemented with 20 mM glucose, 1.5 U/mL hexokinase (Colinet, Renault, & Roussel, 2017; Teulier *et al.*, 2010). Mitochondria were energized with a mixture
267 of 5 mM pyruvate, 2.5 mM malate, 5 mM succinate to fully activate the ETS. ADP (500 µM)
268 was added to initiate ATP synthesis. After recording the phosphorylating respiration rate using
269 a Clark electrode (Rank Brother Ltd, Cambridge, UK), four 100 µL samples of mitochondrial
270 suspension were withdrawn from the respiratory chamber every 2 min and immediately
271 quenched in 100µL of ice-cold perchloric acid solution (10% HCLO4 and 25mM EDTA).
272 Production of ATP was determined from the slope of the linear accumulation of glucose-6-
273 phosphate content over the sampling time interval (6 min) as described previously (Colinet et
274 al., 2017; Teulier et al., 2010). To make sure that ATP synthesis rate was specific to
275 mitochondrial ATP synthase activity, we determined oxygen consumption and ATP synthesis
276 rates in the presence of oligomycin (1µg/mL) that inhibits mitochondrial ATP synthase. These
277 values were taken into account (i.e. subtracted from the raw ATP synthesis) to calculate the rate
278 of mitochondrial ATP synthesis (Teulier et al., 2010). Of note, the basal non-phosphorylating
279 oxygen consumption recorded in the presence of oligomycin is a measure of the maximal rate
280 of proton leak across the inner membrane of mitochondria. Due to the linear relationship
281 between ATP synthesis and oxygen consumption in isolated mitochondria from different tissues
282 and organisms (Beavis & Lehninger, 1986; Fontaine et al., 1996; Nogueira et al., 2002; Roussel
283 et al., 2015; Teulier et al., 2010), we presented these data on a single graph by drawing a linear
284 relationship between the basal non-phosphorylating oxygen consumption activity and the
285 maximal oxidative phosphorylation activity, which is related to maximal rates of oxygen
286 consumption and corresponding ATP synthesis (Fig. 1B).

287 Maximal activity of the electron transport system (i.e. the respiratory capacity) was
288 measured at 20°C by adding 2 µM FCCP in the respiratory buffer supplemented with 5mM
289 pyruvate, 2.5 mM malate, 5 mM succinate, and 1 µg/mL oligomycin. Mitochondrial content of
290 skeletal muscle was estimated from the ratio between the maximal oxygen consumption rates
291 of muscle fibres (expressed per gram of muscle) and mitochondria (expressed per milligram of protein).
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294 **2.5. Statistical analyses**

295 Linear mixed-models (LMM) were used to evaluate the effects of food size and quantity
296 on phenotypic traits, red muscle mitochondrial respiration and efficiency. These models all
297 followed a similar structure, with food size and quantity as well as their interaction as fixed
298 effects, while sex was included as a random effect to remove any potential bias associated to
299 differences between males and females. When an interaction between size and quantity of items
300 was significant (*P* < 0.05) or tended to be significant (*P* < 0.07), post-hoc tests comparing all
301 four treatments (lsmeans) were performed. When the interaction was not significant, it was
302 removed from the model and the p- values obtained in the simplified model were reported in
303 the results section.

304 The relationship between individual body condition and basal mitochondrial respiration
305 was analysed by piecewise linear regression (Fig. 3). Once the breakpoint was estimated, linear
306 regressions were evaluated over each segment of condition (i.e. before and after the breakpoint).

307 Data are presented as means ± SE. Sample sizes varied among models because some
308 data were missing for some fish, or at the fibre level, data were removed from analyses if
309 addition of cytochrome c caused oxygen consumption to increase by 30% or more (see 2.3.a).
310 Statistical analyses were performed in *R* v. 3.6.1. with the packages *lme4* and *lsmeans*.

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312 **3. Results**

313 **3.1. Effect of food size and quantity on body condition and growth**

314 For the effect of food size and quantity on body condition, fat content, growth and final
315 mass, the interaction between size and quantity was always significant or close to significance
(LMMs: *P* = 0.025-0.061). LI-LQ sardines showed the highest body condition, fat content, body mass, cumulative and relative growth compared to the other three groups (post-hoc *lsmeans*: *P* < 0.010), while LI-SQ, SI-LQ and SI-SQ did not differ significantly (Table 1). By contrast, there was no interaction between food size and quantity for effects on red muscle mass, whether in absolute terms (F = 2.359; *P* = 0.133, *n* = 9) or relative to body mass (F = 0.002; *P* = 0.962, *n* = 9). Still, LI sardines had greater absolute red muscle mass than SI sardines (F = 15.152; *P* < 0.001), as did LQ compared to SQ sardines (F = 4.551; *P* = 0.039). Finally, the size of items also had an effect on relative red muscle mass (F = 16.515; *P* < 0.001) with LI sardines being higher than SI (Table 1).

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## 3.2. Oxidative metabolism at different levels of organisation

The phosphorylating respiration state of skeletal muscle fibres obtained with different substrates is presented in Supplementary Material (Figure S1). Apart from glutamate/malate, there was no effect of food size, quantity or their interaction on the phosphorylating state of fibres (all *P* > 0.05). Regarding glutamate/malate, the interaction between food size and quantity tended to be significant (F = 3.614, *P* = 0.066), and muscle phosphorylating respiration was affected by food size (F = 4.220, *P* = 0.048, post-hoc tests: SI-LQ < LI-LQ, *P* = 0.040).

Table 2 reports the maximal respiration state following addition of FCCP, as measured in isolated mitochondria and muscle fibres, and further estimated in whole red muscle and fish. Maximal respiration of isolated mitochondria was only affected by food size (F = 9.334; *P* = 0.004), and was significantly higher in LI than in SI sardines (Table 2). Similarly, mitochondrial content was only affected by food size (F = 4.604; *P* = 0.040) but in an opposite direction, being significantly lower in LI groups. The interaction between food size and quantity was close to significance (F = 3.228; *P* = 0.082), suggesting that the negative effect of food size on mitochondrial content was mostly observed in sardines fed in small quantity. At the fibre level,
while the interaction between food size and quantity was significant for the maximal respiration state measured at this level (F = 6.697; *P* = 0.014), no differences in fibre respiration were highlighted among groups. Finally, the food treatments had no effect on maximal respiration rates at the red muscle or whole fish level (*P* > 0.05 for size, quantity, and its interaction).

### 3.3. Mitochondrial oxidative phosphorylation efficiency

At the mitochondrial level, there was no interaction between food size and quantity on oxidative phosphorylation activity (i.e. the rates of oxygen consumption and associated ATP synthesis). However, activity was higher in sardines fed large food items (oxygen consumption: F = 9.807; *P* = 0.003; ATP production: F = 10.638; *P* = 0.002), regardless of quantity (*P* > 0.05, Fig. 2A). Basal non-phosphorylating respiration state measured in the presence of oligomycin (i.e. the intercept with the x-axis) was also significantly higher in LI sardines (F = 11.746; *P* = 0.002, see box in Fig. 2A). There was no significant interaction of size and quantity, and no effect of size or quantity on the slopes of the relationships between rates of ATP synthesis and oxygen consumption (Size*Quantity: F = 0.014; *P* = 0.906; Size: F = 0.784; *P* = 0.382; Quantity: F = 2.017; *P* = 0.164). Thus, these slopes were similar between SI and LI sardines (Slope SI: 3.19 ± 0.15; Slope LI: 3.03 ± 0.10). Altogether, these results indicate that the regression line relating ATP synthesis to oxygen consumption was shifted to the right in LI compared to SI groups, meaning that mitochondria from LI sardines had to consume more oxygen to produce the same amount of ATP, due to a higher basal non-phosphorylating respiration or “leak” respiration (Fig. 2A).

When mitochondrial function was quantified for the entire red muscle, oxidative phosphorylation activity did not differ among treatments except for LI-LQ sardines, which exhibited higher rates of ATP synthesis and corresponding phosphorylating respiration, and higher basal non-phosphorylating respiration than the other groups (*P* < 0.05, Fig. 2B).
However, as the slopes were similar between LI-LQ and the other groups (Slope LI-LQ: 3.17 ± 0.19; Slope other groups: 2.96 ± 0.18; P > 0.05), red muscles of LI-LQ sardines needed to consume more oxygen to produce the same amount of ATP compared to the sardines from the other three groups (Fig. 2B).

## 3.4. Basal respiration in relation to body condition

The rate of basal non-phosphorylating respiration at the mitochondrial level was significantly related to Le Cren body condition (Fig. 3). The segmented regression analysis showed a significant increase in basal non-phosphorylating respiration rate with body condition, until a condition threshold of 1.07 (R² = 0.45; P < 0.001) beyond which no significant relation was found (P > 0.05, Fig. 3).

## 4. Discussion

The results demonstrate clear links between sardine red muscle energetics and foraging modes, indicating effects of both food size and ration. Reducing the size and/or quantity of food led to a series of mitochondrial energy sparing mechanisms (decrease in maximum oxygen consumption and ATP production, enhanced ATP production efficiency and reduced waste of oxygen linked to proton leak), presumably to maintain energy homeostasis. Such energy saving processes suggest that sardines feeding on small food items or small food quantities faced an energetic challenge. These metabolic processes were linked across different levels, from macroscopic integrative traits such as body mass and body condition to intracellular mechanisms such as mitochondrial efficiency. The effects of food ration were stronger at the whole animal level than at the tissue or mitochondrial level, whereas food size also induced marked mitochondrial adjustments beyond the effects at tissue or whole animal scale (Fig. 4). This shows that energy saving processes were not adequate to counteract detrimental effects of
391 foraging on small particles. Indeed, despite an enhanced mitochondrial efficiency to produce 392 ATP, SI sardines still exhibited lower body condition, body mass and growth performance, 393 regardless of the ration provided (Queiros *et al.*, 2019). Our results therefore support the 394 hypothesis that filter-feeding behaviour is more costly than particulate-feeding in 395 Mediterranean sardines (Queiros *et al.*, 2019), unlike in other small pelagic species (Gibson & 396 Ezzi, 1992; Sanderson & Cech, 1992; Van Der Lingen, 1995), and that this induces plastic 397 responses in cellular energetics. 398

399 **4.1. Food size induces subcellular bioenergetic adjustments.**

400 Why would the size of food items affect traits like body condition, body mass or growth 401 performance? Part of the answer may reside in the most striking result of our study, which is 402 that small food items triggered bioenergetic adjustments in sardine muscle mitochondria. The 403 effects of food size overwhelmed any potential effect of quantity. Small food items may have 404 induced an increased utilisation of lipids, as intramuscular lipids were ~50% lower in SI-SQ 405 compared to LI-LQ sardines. Moreover, a reduction in glutamate oxidation is a known protein- 406 sparing mechanism, which suggests that protein catabolism may have been occurring. This, 407 together, may explain why SI sardine exhibited lower red muscle mass both in absolute and 408 relative terms. 409

410 Feeding on small food items was also associated with decreased oxidative capacity and 411 reduced energy (ATP) outflow, from mitochondria up to whole muscle. This involved a severe 412 loss of mitochondrial power, *i.e.* a ~50% decrease in both maximal rates of ATP synthesis and 413 oxygen consumption. These reduced maximal rates were balanced by a higher coupling 414 efficiency; mitochondria of SI sardines consumed less oxygen than LI sardines to produce a 415 given amount of ATP. The lower maximal respiratory capacity of skeletal muscle and economic 416 management of cellular resources are energy saving mechanisms, decreasing the quantity of
substrates required to produce ATP. Similar adjustments have been reported in skeletal muscle of birds at a critical stage of fasting, when endogenous lipid stores are depleted (Bourguignon *et al.*, 2017; Monternier *et al.*, 2017), and also in skeletal muscle of mammals fed on protein-deficient diets (Zangarelli *et al.*, 2006). Overall, these results indicate that mitochondrial efficiency improves when energy balance is severely threatened in the sardine. Consequently, our results indicate that sardines were unable to cope with the high energy expenditure imposed by filter-feeding on small items, even when these were provided at high ration.

423

424 **4.2. Food quantity mainly influenced the whole organism.**

It is well established that food shortage triggers adaptive behavioural, physiological and cellular responses to lower costs of living, reducing metabolic rates, spontaneous activity and somatic growth (Metcalfe, Van Leeuwen, & Killen, 2016). For example, the standard metabolic rate of juvenile brown trout *Salmo trutta* changes as a function of food availability, decreasing or increasing when fish are fed on a low or high ration, respectively (Auer *et al.*, 2016), which was closely related to their growth rate (Auer *et al.*, 2015). In our study, a 50% reduction in ration negatively impacted body condition (-26%), body mass (-33%) and dramatically slowed growth rate of LI sardines (-64%, similarly to what Queiros *et al.*, 2019 found). While a similar trend was observed for growth rate in SI sardines when comparing between rations (-68%) the difference was not significant, presumably because the energetic challenge of foraging on small items transcended any effect of ration (Table 1).

Energy conservation during food shortage is also enacted at a cellular level, by reductions in energy demanding processes and oxidative activity of metabolically active tissues, such as liver and skeletal muscles (McCue, 2010). For instance, the oxidative activity of brown trout muscle is positively correlated with food intake and maximum metabolic rate (Salin *et al.*, 2016a; 2016b). In the sardines, bioenergetics of whole red muscle in the LI group was positively
441 correlated with food availability, which is likely due to muscle mass. Because of lower body
442 mass and red muscle mass in food-restricted fish, maximal oxygen consumption and ATP
443 synthesis rates of the muscle are depressed. Down regulation of aerobic metabolic pathways
444 has been reported in the red muscle of food-restricted gilthead sea bream Sparus aurata
445 (Bermejo-Nogales et al., 2011).

446 The lowered respiratory capacity of whole muscle in LI-SQ sardines appears mainly to
447 be due to decreased mitochondrial content rather than to changes in mitochondrial activity or
448 skeletal muscle protein content. Changes in mitochondrial content could be caused by
449 dysregulation of autophagy and biogenesis. The former could be induced by caloric restriction
450 (reviewed in Mehrabani et al., 2020) while the latter, which is extremely costly, is probably
451 inhibited to conserve energy. It is also possible that the decrease in mitochondrial content and
452 associated loss of metabolic power in the sardines is linked to decreased use of slow-twitch
453 aerobic red muscle for a particulate capture feeding mode (Hood, 2009; Novak et al., 2005), as
454 prey capture tends to use fast-twitch anaerobic white muscle (Bone, 1978).
455

455

456 **4.3. Body condition index and skeletal muscle mitochondria.**

457 The energetic constraints associated with the filtering mode led to very low body
458 condition in the sardines. It is fascinating that, below a threshold value of 1.07, declines in body
459 condition index were strongly correlated with declines in basal non-phosphorylating respiration
460 rate of muscle mitochondria. This threshold is surprisingly close to the mean body condition of
461 sardines observed in the wild, around 1 (Brosset et al., 2015b), which may imply that wild
462 populations are also at a threshold of bioenergetic disequilibrium. Basal non-phosphorylating
463 respiration represents maximal proton leakage across the inner mitochondrial membrane.
464 Limiting proton leak lowers costs of basal maintenance (Boutilier & St-Pierre, 2002) but may
465 carry an oxidative risk, such as an increased reactive oxygen species production (Salin et al.,
466 2018). A reduction of maintenance costs could also be achieved by decreasing mitochondrial
467 content (Guderley, 2004), as observed in LI-SQ sardines (Fig 2b). That is, a decrease in
468 mitochondrial content seems less harmful than directly reducing basal respiration rate itself, as
469 LI-SQ body condition stayed closer to 1 than the SI groups.

470 Hence, the decline in mitochondrial energy wastage in sardines with poor condition
471 suggests that modifying mitochondrial function provides an economical phenotype when
472 metabolic costs of foraging are high compared to energy acquired.

473

474 **4.4. Conclusions**

475 Within our experimental conditions, we provide clear evidence that, in sardines, feeding
476 on small prey can present a greater energetic challenge than reduced availability of large prey.
477 Feeding sardines a restricted ration of large food items reduced their body condition, growth
478 rate and body mass, revealing an unbalanced energy budget, but did not stimulate plastic
479 metabolic responses within mitochondria. By contrast, feeding small items, whatever the
480 quantity, induced energy-saving adjustments in mitochondria that are typical of a severe
481 energetic challenge (Bourguignon et al., 2017; Costalago & Palomera, 2014). The food size
482 effect seems to be mediated by feeding mode in the sardines, with the switch from prey capture
483 to filtering. Therefore, impacts of prey size on population condition could be profoundly non-
484 linear. Prey size might only have minor influence on condition as long as sardines use a capture
485 feeding mode, but when prey species are small enough to trigger a change to filtering mode,
486 there are dramatic consequences for body condition.

487 While this study focuses on sardines in the north-western Mediterranean Sea, it may be
488 indicative of a widespread phenomenon linked to climate change. That is, if global warming
489 causes systematic reductions in prey size (Daufresne et al., 2009), the extra time and energy
490 required to forage could cause energy challenges at multiple levels of the food web. Coupled
491 with increased metabolic demands of ectotherms in a warmer environment, this could lead to
492 reduced growth rates and be a major contributor to declining final adult sizes in fishes (Gardner
493 et al., 2011).

494

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FIGURES

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**Figure 1.** Illustration of the integrative levels of the red muscle bioenergetics in sardine, from isolated mitochondria, to the whole tissue.

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Substrates are oxidized by the complexes of the electron transport system (ETS) and ATP is produced through a phosphorylation process by the ATP synthase. The ETS and ATP synthase are enclosed in the inner membrane of the mitochondria (panel A). Mitochondrial respiration rates associated to either basal non-phosphorylating state (BASAL) and/or phosphorylating state (PHOSPHO) were assessed at different levels of integration. 1) Mitochondrial oxygen consumption was coupled to the amount of ATP produced, to obtain the mitochondrial efficiency which is the slope of the linear regression between oxygen consumption and ATP production, called P/O ratio (panel B). 2) The muscle fibre respiration rates were induced by addition of different substrates to discriminate the metabolic pathways fuelling the red muscle (panel C). 3) The whole red muscle oxidation phosphorylation activity was calculated to estimate the relative importance of the whole muscle bioenergetics. The red muscle is positioned along the lateral line and is clearly separated from the white muscle (panel D). ETS: Electron transport system; ATP: Adenosine triphosphate; ADP: adenosine diphosphate; Pi: Phosphate inorganic; IMS: intermembrane space of the mitochondrion; M: matrix of the mitochondrion. See the "Material and Methods" section for further details.

<figure><img src="image_2.png" /></figure>
**Figure 2.** Mitochondrial efficiency of sardine that received different quantities and sizes of food items, at the mitochondrial level (Panel A; LI-LQ: large food items in large quantities, n = 11; LI-SQ: large food items in small quantities, n = 12; SI-LQ: small food items in large quantities, n = 11; SI-SQ: small food items in small quantities, n = 9) **and red muscle level** (Panel B, LI-LQ: n = 11, LI-SQ: n = 11; SI-LQ: n = 10; SI-SQ: n = 9). In panel A, the two trend curves drawn are those of the “large food items” (LI: y = 3.03x – 44.40) and “small food items (SI: y = 3.19x – 37.96) groups, and in panel B, the trend curves are those of LI-LQ group (y = 3.17x – 4.52) and of the other groups together (y = 2.97x – 1.77). These data were analysed using a linear-mixed effects model with size and quantity of items as fixed-effects and sex as random effect. The values shown are means ± s.e.m. (the error bars of the basal oxygen consumption are small enough that they are hidden by the points, see zoom boxes). An asterisk represents a significantly difference in maximal oxygen consumption, ATP production and basal oxygen consumption (see zoom boxes) between diets composed of different size food items (Panel A, difference between LI and SI) and between LI-LQ sardines and other groups (Panel B; \*P < 0.05; \*\*P < 0.01).
<figure><img src="image_3.png" /></figure>

Oxygen consumption rate (nmol O. min⁻¹. mg protein⁻¹)

<figure><img src="image_4.png" /></figure>

Red muscle oxygen consumption rate (µmol O. min⁻¹)
**Figure 3.** Basal oxygen consumption at the mitochondrial level as a function of sardine body condition index. Fish of each treatment are represented by a coloured circle. Sardines fed with large food items are represented in blue for large quantity (LI-LQ, n = 11) and in green for small quantity (LI-SQ, n = 12). Yellow and red circles represent sardines fed with small food items, in large (SI-LQ, n = 10) and small (SI-SQ, n = 11) quantities, respectively. Basal mitochondrial respiration in nmol O.min⁻¹.mg protein⁻¹ is positively correlated to the body condition index when the BCI is less than 1.07 (P < 0.001). Above this threshold, there is no longer a regression between these two parameters.

<figure><img src="image_5.png" /></figure>
**Figure 4.** Whole-organism, muscle, and subcellular consequences of the quantity and/or size of food. In order to investigate the effect of size and/or quantity of food, a fully factorial protocol has been set up. Size and quantity impacted the whole-organism and muscle parameters, whereas subcellular bioenergetics have been modified only by size of food items. Significant effects are illustrated by an asterisk. As foraging mode depends on food size (Costalago *et al.*, 2015; Garrido *et al.*, 2007), the effect described by size is called “foraging mode” (FM). Lowering the quantity of food by 50% induces a severe “caloric restriction” (CR). [1] Queiros *et al.*, 2019

<figure><img src="image_6.png" /></figure>
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**Table 1. Characteristics of sardines used to measure the respiration of red muscle fibres.** These data correspond to the measurements made

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on the day sardines were euthanised. Values are mean ± s.e.m. N corresponds to the number of individuals randomly split into 4 treatments, sorted

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in three sex categories (male, female and indeterminate). Cumulative growth represents the total length gain in mm, over the entire experiment.

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RM/BM ratio corresponds to the relative red muscle mass, which is the proportion of the red muscle mass over the body mass. Data were analysed

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with a linear mixed-effect model with size and quantity as fixed-effects and sex as random effect. Data with different superscripts are significantly

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different at P < 0.05. The boxes with the asterisks indicated a global effect of item size (**P < 0.001).
<table><thead><tr><th></th><th>LI-LQ</th><th>LI-SQ</th><th>SI-LQ</th><th>SI-SQ</th><th>Statistical analysis</th></tr></thead><tbody><tr><td>N</td><td>8-11</td><td>10-12</td><td>9-11</td><td>10-12</td><td></td></tr><tr><td>(M/F/indeterminate)</td><td>(6/3/2)</td><td>(5/6/1)</td><td>(3/5/2)</td><td>(3/8/1)</td><td></td></tr><tr><td>Final BCI</td><td>1.17 ± 0.04<sup>a</sup></td><td>0.98 ± 0.02<sup>b</sup></td><td>0.93 ± 0.05<sup>b</sup></td><td>0.87 ± 0.02<sup>b</sup></td><td>Size: P < 0.001 824<br>Quantity: P = 0.001 825<br>Size*Quantity: P = 0.061 826</td></tr><tr><td>Fat content (%)</td><td>13.46 ± 1.40<sup>a</sup></td><td>8.86 ± 0.76<sup>b</sup></td><td>8.12 ± 0.81<sup>b</sup></td><td>6.93 ± 0.37<sup>b</sup></td><td>Size: P < 0.001 827<br>Quantity: P = 0.002 828<br>Size*Quantity: P = 0.055 829</td></tr><tr><td>Cumulative growth (mm)</td><td>14.29 ± 1.68<sup>a</sup></td><td>5.05 ± 1.07<sup>b</sup></td><td>5.27 ± 1.42<sup>b</sup></td><td>1.64 ± 0.46<sup>b</sup></td><td>Size: P < 0.001 830<br>Quantity: P < 0.001 831<br>Size*Quantity: P = 0.025 832</td></tr><tr><td>Relative growth (%)</td><td>12.55 ± 1.56<sup>a</sup></td><td>4.35 ± 0.91<sup>b</sup></td><td>4.60 ± 1.29<sup>b</sup></td><td>1.34 ± 0.36<sup>b</sup></td><td>Size: P < 0.001 833<br>Quantity: P < 0.001 834<br>Size*Quantity: P = 0.029 835</td></tr><tr><td>Body mass (g)</td><td>19.23 ± 1.38<sup>a</sup></td><td>13.98 ± 0.62<sup>b</sup></td><td>14.22 ± 1.19<sup>b</sup></td><td>12.81 ± 0.73<sup>b</sup></td><td>Size: P = 0.005 836<br>Quantity: P = 0.001 837<br>Size*Quantity: P = 0.058 838</td></tr><tr><td>Red muscle mass (g)</td><td>1.59 ± 0.17<sup>a</sup></td><td>1.18 ± 0.06<sup>a,b</sup></td><td>0.98 ± 0.13<sup>b</sup></td><td>0.91 ± 0.07<sup>b</sup></td><td>Size: P < 0.001 839<br>Quantity: P = 0.039 840<br>Size: P < 0.001 841</td></tr><tr><td>RM/BM (%)</td><td>8.11 ± 0.39</td><td>8.46 ± 0.20</td><td>6.70 ± 0.45</td><td>7.09 ± 0.30</td><td></td></tr></tbody></table>

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Table 2. Maximal respiration ETS activity and mitochondrial content of sardine red muscle for each feeding treatment. The maximal respiration rate of ETS induced by FCCP is represented at three different levels of organisation: at the subcellular level (nmol O.min⁻¹.mg proteins⁻¹) obtained through respiration measurements of isolated mitochondria; at the red muscle fibre level (pmol O₂.s⁻¹.mg muscle⁻¹) obtained through respiration measurements of a red muscle sample, and at the red muscle level estimated by multiplying maximal respiration rate at the fibre level by red muscle mass or relative red muscle mass (total: pmol O₂.s⁻¹; specific: pmol O₂.s⁻¹.g fish⁻¹). Values are mean ± s.e.m. The mitochondrial content of red muscle is expressed in milligram of mitochondrial proteins per gram of red muscle and was estimated as the ratio between respiration rates at the fibre level and mitochondrial level. Data were analysed with a linear mixed-effects model with size and quantity as fixed-effects and sex as random effect. Different letters indicate a significant difference among groups at P < 0.05. The boxes with the asterisks on the measurements indicates a global effect of item size (*P < 0.05; **P < 0.01).

<table><thead><tr><th></th><th>LI-LQ</th><th>LI-SQ</th><th>SI-LQ</th><th>SI-SQ</th><th>Statistical analysis</th></tr></thead><tbody><tr><td>N</td><td>9-11</td><td>11-12</td><td>9-11</td><td>9-12</td><td></td></tr><tr><td>Maximal respiration ETS activity</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mitochondrial level (nmol O.min⁻¹.mg protein⁻¹)</td><td>90 ± 9</td><td>96 ± 7</td><td>59 ± 12</td><td>65 ± 12</td><td>Size: P = 0.004</td></tr><tr><td>Red muscle fibre level (nmol O.min⁻¹.mg muscle⁻¹)</td><td>5 ± 0.8<sup>a</sup></td><td>3.5 ± 0.4<sup>a</sup></td><td>3.3 ± 0.4<sup>a</sup></td><td>5.3 ± 0.9<sup>a</sup></td><td>Size*Quantity: P = 0.014</td></tr><tr><td>Red muscle level</td><td>6.3 ± 0.9<sup>a</sup></td><td>4.4 ± 0.7<sup>a</sup></td><td>3.6 ± 0.7<sup>a</sup></td><td>4.1 ± 0.8<sup>a</sup></td><td>n.s.</td></tr><tr><td>Specific (nmol O.min⁻¹.g fish⁻¹)</td><td>350 ± 44<sup>a</sup></td><td>306 ± 38<sup>a</sup></td><td>245 ± 34<sup>a</sup></td><td>307 ± 58<sup>a</sup></td><td>n.s.</td></tr><tr><td>Mitochondrial content (mg protein. g muscle⁻¹)<sup>X</sup></td><td>50.9 ± 7.7</td><td>34.8 ± 5.4</td><td>52.6 ± 8.2</td><td>62.2 ± 7.6</td><td>Size: P = 0.040</td></tr></tbody></table>