# The heart's energy secret: impact of DHA deficiency on cardiac mitochondrial efficiency and thermal performance in sardine (Sardina pilchardus)

Léopold Ghinter, Mathilde Bertrand, Karine Salin, Christophe Lebigre, Philippe Soudant, Victor Simon, Fabienne Le Grand, Laure Pecquerie, Marie Vagner

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## To cite this version:

Léopold Ghinter, Mathilde Bertrand, Karine Salin, Christophe Lebigre, Philippe Soudant, et al.. The heart's energy secret: impact of DHA deficiency on cardiac mitochondrial efficiency and thermal performance in sardine (Sardina pilchardus). Journal of Experimental Biology, 2025, 228 (23), 10.1242/jeb.251331. hal-05387487

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Submitted on 28 Nov 2025

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**The heart's energy secret: Impact of a DHA deficiency on cardiac mitochondrial efficiency and thermal performance in sardine (Sardina pilchardus)**

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Léopold Ghinter¹,²,*, Mathilde Bertrand¹, Karine Salin¹, Christophe Lebigre², Philippe Soudant¹, Victor Simon¹, Fabienne Le Grand¹, Laure Pecquerie¹, Marie Vagner¹

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¹ Univ Brest, CNRS, IRD, Ifremer, LEMAR, IUEM, F-29280 Plouzané, France

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² UMR DECOD (Ecosystem Dynamics and Sustainability), Ifremer, INRAE, L'Institut Agro, Brest, France

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* Corresponding author: leopold.ghinter@hotmail.fr

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**Abstract**

Climate-induced changes in marine ecosystems are predicted to reduce the production of omega-3 long-chain polyunsaturated fatty acids (n-3 LC PUFA), such as docosahexaenoic acid (DHA), in primary producers. This decline can spread through food webs, reducing DHA availability in small pelagic fish like sardines. DHA is essential for mitochondrial membrane integrity and ATP production. We tested whether dietary DHA deficiency impairs cardiac mitochondrial function in sardines, limiting their ability to cope with rapid temperature changes. Wild adult sardines reared under laboratory conditions were fed either a control or a DHA-deficient diet at natural fluctuating temperature. After 6 months, we measured *in vitro* cardiac mitochondria performance from nine individuals per group at 12°C, 15°C, 18°C, and 21°C. DHA-deficient sardines had lower DHA in cardiac cell membranes, leading to reduced mitochondrial efficiency, with increased proton leakage, reduced oxidative phosphorylation and ATP production. High inter-individual variability suggests DHA alone does not fully explain mitochondrial efficiency changes, with additional metabolic mechanisms involved. While mitochondria maintained respiratory capacity up to 21°C, ATP synthesis efficiency declined with temperature, suggesting DHA-deficient sardines may face greater energetic constraints *at moderately elevated temperatures*. These findings highlight how dietary changes alter the metabolic resilience in marine ectotherms facing rapid environmental fluctuations, *although responses under more extreme temperatures remain to be explored*.

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**Keywords:** Docosahexaenoic acid (DHA), cardiac mitochondrial efficiency, thermal performance, dietary deficiency, climate change, small pelagic fish

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**Introduction**
Marine ectotherms are particularly vulnerable to climate change, as rapid shifts in environmental conditions can disrupt their metabolism and life history traits¹,². For example, rising ocean temperatures, compounded by the increasing frequency and intensity of marine heatwaves, not only push ectotherms beyond their optimal thermal windows¹,³,⁴ but also threaten their ability to meet increasing metabolic demands. During acute extreme thermal events, animals face a dual challenge: increased energy demands to sustain cellular function and a reduction in oxygen solubility in warmer water, potentially limiting oxygen availability and leaving little time for physiological adjustment. Additionally, warming oceans are widely expected to lead to a decrease in omega-3 long-chain polyunsaturated fatty acids (n-3 LC PUFA), i.e. fatty acids (FA) with ≥20 carbon atoms and multiple double bonds, especially docosahexaenoic acid (DHA, 22:6n-3), at the base of the food web⁵,⁶. Vertebrates, including marine fish, have a limited capacity to biosynthesize n-3 LC PUFA and therefore rely primarily on their dietary intake to get these molecules. N-3 LC PUFA are essential components of cell membranes and influence their fluidity, permeability⁷ and the activity of embedded proteins⁸,⁹. In marine fish, dietary deficiencies in n-3 LC PUFA have been associated with impaired growth¹⁰, reproduction¹¹, swimming performance¹², thermal tolerance¹³, and survival¹⁴. Understanding how a dietary DHA deficiency influences energy-producing cellular processes and impacts the metabolic resilience of ectotherms to acute thermal extremes is essential for assessing their ability to cope with a rapid changing environment.

Mitochondria, the powerhouse of animal cells, generate over 90% of the Adenosine Tri-Phosphate (ATP) through oxidative phosphorylation (OXPHOS)¹⁵. This process occurs at the inner mitochondrial membrane, where energy substrates are oxidized in the electron transport system (ETS), generating a proton gradient for ATP synthesis to phosphorylate Adenosine Di-Phosphate (ADP) into ATP. The efficiency of this process can be assessed *in vitro* by measuring OXPHOS respiration and ATP production rates¹⁶,¹⁷. However, part of the oxygen consumed by the mitochondria is dissipated across the inner mitochondrial membrane, due to proton leakage (LEAK)¹⁶,¹⁸. This phenomenon, inherent to the composition in FA and proteins of the inner membrane, leads to a decrease of mitochondrial coupling efficiency¹⁹. This efficiency can be calculated as either the OxCE (Oxidative Coupling Efficiency, i.e. the proportion of mitochondrial respiration supporting ATP synthesis to that required to offset the proton leak²⁰) or as ATP/O ratio (i.e. the amount of ATP generated per oxygen (O) consumed in OXPHOS conditions²¹). A low OxCE indicates that the mitochondria waste a lot of energy to compensate for proton leakage, while a low ATP/O ratio reflects inefficiency in ATP production from OXPHOS respiration. Either ratio may be depressed with temperature increases because of the loss in function of enzymatic complexes²² or an excessive LEAK without a parallel increase in OXPHOS respiration and ATP production. As a result,
mitochondrial functions are highly temperature-sensitive<sup>23,24</sup>, and thermal stress can lead to impaired tissue function<sup>23,25</sup> and reduced overall physiological performance<sup>20,26-28</sup>.

The susceptibility of mitochondrial coupling efficiency to thermal stress is not only driven by enzymatic dynamics, but also by the biophysical properties of the inner mitochondrial membrane, particularly its lipid composition. Mitochondrial membranes are particularly sensitive to temperature changes because their structure and function rely on the physical state and composition of their lipid bilayer<sup>29,30</sup>, in particular the degree of unsaturation of fatty acyl chains and the balance of phospholipid headgroups<sup>31</sup>. Among PUFA that determine these properties, DHA is especially important due to its high number of double bonds, which enhances membrane flexibility and stabilizes protein-lipid interactions during thermal fluctuations<sup>32</sup>. Therefore, any reduction in DHA availability may affect mitochondrial membrane structure and compromise its bioenergetic functions, particularly under varying thermal conditions.

Dietary FA intake strongly determines mitochondrial membrane composition in fish<sup>33-35</sup> and alterations in n-3 LC PUFA levels can affect mitochondrial bioenergetics in diverse and sometimes contradictory ways. Some studies suggest that reduced membrane unsaturation level enhances mitochondrial efficiency and ATP production<sup>35,36</sup>, while others report an impaired mitochondrial performance<sup>33,37</sup> or no significant effect<sup>37</sup>. Even when the mitochondrial function is affected, the specific traits impacted by the n-3 LC PUFA deficiency vary across studies. Some findings suggest that changes in mitochondrial efficiency stem from alterations in the structural properties of the inner mitochondrial membrane, leading to either reduced<sup>35</sup> or increased proton leakage<sup>37</sup>. In other cases, functional changes related to enzymatic activity within the inner membrane appear to be the primary driver<sup>36</sup>, while some studies report a combined effect on both structural integrity and functional capacity<sup>33</sup>. These conflicting results suggest that mitochondrial function is shaped by a complex interplay of factors related to mitochondrial membrane properties and FA composition. Literature shows that mitochondrial efficiency under PUFA-limited conditions may be driven by (1) the ability of tissues to mitigate reductions in membrane n-3 LC PUFA levels<sup>37</sup>, (2) the regulation of dietary FA assimilation to maintain essential mitochondrial functions<sup>33,35</sup>, (3) additional mechanisms involving protein abundance and activity within the mitochondrial inner membrane, and (4) shifts in metabolic substrate use<sup>33,36</sup>.

Although the effects of dietary n-3 LC PUFA deficiency on mitochondrial efficiency remain variable across studies, they may become particularly critical under thermal stress, when energetic demands rise sharply. Given DHA's role in maintaining membrane structure and supporting mitochondrial function<sup>33,36,37</sup>, its dietary deficiency could impair ATP production capacity during acute
temperature challenges. Moreover, mitochondrial function varies markedly among individuals and has been associated with traits related to temperature tolerance³⁸, locomotion³⁹, and growth³⁷,⁴⁰, suggesting its potential relevance for organismal performance. For instance, Salin et al. (2021)³⁶ reported up to a 2-fold variation in ATP/O ratios among golden grey mullet (*Chelon aurata*) individuals fed the same n-3 LC PUFA-deficient diet, which was linked to differences in membrane FA composition. Such inter-individual variation in mitochondrial efficiency may influence thermal sensitivity but remains poorly understood.

Here, we investigated whether a dietary deficiency in DHA alters cardiac mitochondrial efficiency in fish, and how this effect may interact with acute thermal stress. This addresses a gap in understanding how nutritional stress modulates temperature-dependent mitochondrial performance in marine ectotherms. We focused on the European sardine *Sardina pilchardus*, which has exhibited a significant and unexplained decline in size-at-age and body condition over the past 15 years in both the Atlantic and Mediterranean Seas⁴¹,⁴². We targeted cardiac mitochondria because heart function is a key determinant of thermal tolerance and performance⁴³⁻⁴⁵ and reduced ATP production in this tissue has been associated with compromised cardiovascular function²³,⁴³,⁴⁶ and even failure under thermal stress²³,²⁴,⁴³. Because membrane structure and function are sensitive to thermal fluctuations, especially under conditions of altered lipid composition, a deficiency in DHA may compromise mitochondrial performance when fish experience acute temperature changes. Given that mitochondrial membranes are highly sensitive to temperature and rely on PUFA-rich lipids for structural and functional integrity, we hypothesized that a dietary DHA deficiency compromises cardiac mitochondrial efficiency (lower OxCE and ATP/O) by altering membrane lipid composition. Such a reduction in mitochondrial efficiency could limit the energy available for sustaining cardiac function during acute temperature fluctuations. To test this hypothesis, we measured *in vitro* cardiac mitochondrial performance from fish fed either a DHA-deficient diet or a control diet for six months. We assessed cardiac mitochondrial function (OXPHOS and LEAK respiration, ATP production, and ATP/O ratio) at four assay temperatures (12, 15, 18, and 21°C), representative of the thermal range experienced by sardines in the Bay of Biscay⁴⁷. We examined inter-individual variability in FA composition and mitochondrial performance within each dietary treatment to assess whether sardines could mitigate the effects of reduced DHA by adjusting membrane FA composition, thereby maintaining mitochondrial efficiency.

## 2. Materials and methods

### 2.1. Fish capture and husbandry
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Sardines were kept at Ifremer's facility in Plouzané (France). All fish handling was performed under anaesthesia using benzocaine (0.1 mL·L⁻¹, ethyl ester of p-aminobenzoic acid) to minimize animal suffering during procedures.

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### 2.1.1 Fish capture and acclimation

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Approximately 150 adult sardines (total length ± standard deviation SD: 187 ± 9 mm, mass: 46 ± 8 g, aged 2 years-old according to otolith reading carried out after the end of the experiment) were caught with a commercial seine in Douarnenez Bay in December 2022 (Brittany, France). Sardines were transported in seawater tanks where the oxygen concentration was regularly monitored (between 11 and 14 mg·L⁻¹, < 200%) and then transferred to the facilities one by one using beakers (no exposure to air, no friction). Fish underwent a quarantine period in a 1 500 L open-circuit seawater tank. Seawater was pumped and filtered from Brest harbour, providing natural temperature with an average winter temperature (December–March) of 11.1 ± 0.7°C (± SD), an average summer temperature (June–July) of 18.1 ± 0.8°C, and a transitional phase between the two periods. The photoperiod was set every week to match environmental conditions. Fish were fasted for two days to allow them to recover from fishing and then fed once a day with 10-15 g of a 50/50 mixture of Al1 (0.4 and 0.7 mm) and Al2 (0.7 to 1.0 mm) commercial pellets (Le Gouessant, Lamballe, France).

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### 2.1.2 Fish housing

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After the quarantine, sardines were anaesthetised with benzocaine (0.1 mL·L⁻¹) and Stress Coat (0.26 mL·L⁻¹, API) was added to limit stress and scale loss during manipulation. Fish were weighed and measured in a small basin containing seawater and individually marked with subcutaneous transponder (8 mm x 1.4 mm, Biolog ID) inserted in the white muscle upstream of the dorsal fin. Fish were randomly distributed in six cylindrical tanks of 500 L (height 72 cm x radius 57 cm; flow rate of 450 L·h⁻¹; n = 21 sardines per tank) with the same water conditions, mirroring natural environmental conditions. In each tank, temperature and oxygen saturations were recorded daily (95% < O₂ < 100%) and pH was recorded weekly (8.0 < pH < 8.1). Sardines were reared under these conditions until August 2023. Over the last month of the experiment, the temperature of the tanks was 18.5 ± 0.35°C (SD).

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### 2.1.3 Dietary treatment

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From the 31st of January 2023 onwards, we conditioned the sardines for 6 months under two experimental conditions in triplicate: control pellets or DHA-deficient pellets. The transition from commercial quarantine pellets to experimental pellets took place over four consecutive days (75%/25%; 50%/50%; 25%/75%; 0%/100% of commercial/experimental pellets, respectively). The

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daily amount of food represented 1 to 2% of the total fish biomass in the tank and was provided three times a day by an automatic feeder (11:00 am, 2:00 pm, and 5:00 pm). In addition, from the 19th of June 2023 onwards (i.e. one months before the fish sampling), when the temperature increased as the summer started, we reduced the amount of food to 50% of the estimated *ad libitum*, i.e. approximately 0.8% of the total fish biomass in the tank, so that an excess of food would not compensate the deficiency. *Ad libitum* corresponded to the amount of food given when the sardines stopped feeding (visual estimation: number of swimming bursts, number of direction changes and reduced speed). Experimental diets were manufactured at INRAE NUMEA facilities (Donzacq, France). Pellets were isolipidic and isoproteic, but the control diet was made from DHA-rich fish oil, while the DHA deficient diet was made from a DHA-free nanochloropsis oil (Polaris, France). As the dietary requirements for sardines are not known, the control diet was formulated according to the eicosapentaenoic acid (EPA 20:5n-3) and DHA needs known for juvenile sea bass⁴⁸, despite their nutritional needs between the two species may differ. In addition, the control diet FA composition was in line with the composition of the commercial diet used by Queiros et al⁴⁹ for sardine. The control pellets contained 5.6 mg of DHA.g⁻¹ compared with 1.3 mg of DHA.g⁻¹ for the DHA-deficient pellets (see Table S1 and S2 for the FA and ingredient composition of the two experimental diets, respectively). At the end of the dietary treatment period, fish were fasted for 24 h before being sampled.

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## 2.2. Fish sampling

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### 2.2.1 Sardines biometry

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After ca. 6 months of dietary conditioning, i.e. from July 27 to August 8 2023, 18 sardines (9 from each dietary treatment, i.e. 3 from each tank) were euthanized using an overdose of benzocaine (> 0.1 mL L⁻¹). Each fish was immediately weighed (± 0.1 g), measured (total length ± 0.1 cm) and their heart was removed, lightly squeezed and weighed. Fish were sexed after euthanasia by macroscopic visual inspection of their gonads. As the sardines were adults, males and females could be reliably identified.

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### 2.2.2 Hearts sampling and homogenisation

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Immediately after being collected, hearts were cleaned of the venous sinus, drained of blood, washed in ice-cold respirometry buffer, and transferred to a clean ice-cold respirometry buffer (MiR05: 20 mM Taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM D-sucrose, 60 mM K-lactobionate, pH 7.2 at 20°C) without Bovine Serum Albumin (BSA). Hearts were then weighed (range: 81.9 – 153.4

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197 mg) and subsequently homogenized for ca. 1 min with scissors' pre-grinding, followed by four gentle
198 passages in a 10 mL Potter-Elvehjem homogenizer. Resulting homogenates were diluted in MiR05
199 (with 1 g.L⁻¹ BSA) to obtain final homogenates with a cardiac tissue concentration of 1 mg.mL⁻¹ for
200 high-resolution respirometry analysis. The remainder of the homogenized mixture without BSA was
201 used for further FA analyses. All extraction steps were conducted on ice and the duration between
202 fish culling and the start of mitochondrial assays was 15 ± 5 min (SD).

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204 2.3. Mitochondrial high resolution respirometry

205 Mitochondrial respiration and ATP production in heart homogenates were measured
206 following the methods described in Salin et al. (2016b)¹⁷. Oxygen consumption and magnesium green
207 fluorescence were recorded simultaneously using high-resolution respirometry chambers equipped
208 with fluorescent sensors (Oroboros Instruments, Innsbruck, Austria). Mitochondrial function was
209 assessed at four assay temperatures: 12°C, 15°C, 18°C, and 21°C. The 18°C condition corresponded to
210 the mean rearing temperature at the time of sampling, while 12°C and 15°C represented cooler
211 temperatures encountered during sardines' vertical migrations in the north Atlantic⁴⁷. The 21°C
212 condition simulated particularly high sea surface temperatures occurring during acute heat increase,
213 such as marine heatwaves. Each sardine was tested at two temperatures per experimental run, with
214 a second run performed later in the day to complete the four-temperature protocol.

215 OXPHOS respiration (complexes I+II) and ATP production rates were measured after ADP
216 addition (See ESM for full details). LEAK respiration was assessed by inhibiting ATP synthesis with
217 carboxyatractyloside, and residual oxygen consumption was determined after complex III inhibition
218 with antimycin A. The OxCE index (1 - LEAK/OXPHOS) was used to estimate mitochondrial respiratory
219 coupling efficiency²⁰, while the ATP/O ratio was calculated as the ATP production rate divided by
220 twice the OXPHOS respiration rate. To correct for latency effects observed between runs¹⁷,³⁶, an
221 algebraic correction factor was applied to second-run data at each temperature. Statistical analyses
222 were performed with and without this correction, yielding the same conclusions. Respiration rates
223 were expressed as pmol O₂·s⁻¹·mg⁻¹ of heart tissue, and ATP production rates as pmol ATP·s⁻¹·mg⁻¹ of
224 heart tissue.

225 Over the 144 mitochondrial measurements (18 sardines at 4 temperatures in duplicates), one
226 LEAK measurement failed (due to a missing addition of antimycin), and 15 ATP production
227 measurements (and thus ATP/O calculations) did not work due to a technical issue with a
228 fluorometric sensor. We estimated the repeatability of the measurements using intraclass correlation
229 coefficients (ICC⁵⁰). All ICC were highly significant (p < 0.001) and their values were very high for
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OXPHOS respiration and LEAK respiration (ICC r = 0.873 and 0.845, respectively), while ATP production had good repeatability (ICC r = 0.774).

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We averaged the duplicates for each measurement when possible, otherwise we used the single available value. As a result, rates of OXPHOS and LEAK respiration had no missing value, whereas rates of ATP production were missing for one single temperature for four sardines. To avoid gaps in the dataset for statistical analysis, we imputed the average ATP production value (calculated across all individuals in the study for each temperature) to these 4 missing values. The imputation of the overall average is conservative, i.e. minimizing any over-estimate of the dietary treatment effect due to this imputation.

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## 2.4. Fatty acids analyses

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The excess heart homogenised solution without BSA were centrifuged at 9.000 G for 10 min at 4°C. The supernatant was discarded, and heart homogenates were recovered and mixed in 6 mL chloroform/methanol (2/1, v/v, modified from Folch et al., 1957⁵¹) in a glass tube. Glass tubes were weighed before and after addition of heart samples to determine the tissue mass (47 ± 18 mg). The experimental pellets (4 samples/pellet) were ground with a ball mill, and similarly to the hearts, 10 mg were placed in 6 mL of chloroform/methanol (2/1, v/v). Pellet and heart samples were flushed with nitrogen gas, shaken, placed in an ultrasonic bath for 10 min, agitated for 1 hour, then stored at -20°C until FA analyses were performed.

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We analysed the FA in the heart of the 18 sardines sampled and in the pellets from the experiment according to the protocol described in Péron et al⁵² (See Electronic Supplementary Material ESM for full details). Briefly, samples were shaken 20 min, centrifuged for 15 min at 3.000 rpm and 1.5 mL of lipid heart extract or 1 mL for pellets was evaporated to dryness (Genevac centrifugal evaporator). Dry heart extracts were re-suspended thrice with 500 µL of chloroform/methanol (98/2; v/v) and gently deposited on top of a silica micro-column⁵³. Neutral lipids (i.e. reserve lipids⁵⁴) were eluted with 10 mL of chloroform/methanol (98/2; v/v) and polar lipids (i.e. membrane lipids⁵⁴) with 20 mL of methanol. Pellets were analysed as total lipids TL. After the addition of 20 µL of C23:0 as internal standard, lipid fractions were processed by saponification and transmethylation to obtain FA methyl ester (FAME⁵⁵). FAME were recovered in hexane, then processed by gas chromatography coupled with a flame ionisation detector (GC-FID, Varian CP8400).

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Their identification was carried out by comparing their retention times with those of commercial standards (Supelco 37 components FAME mix, PUFA1 and PUFA3, Sigma, MO, USA) on the software Galaxie Chromatography Data System (v.1.9.3.2). Results were expressed as
concentration (µg.mg⁻¹ of wet weight) and as percentage of the total FA (see formulas in Bertrand et al., 2022⁵³). The unsaturation index (UI) was calculated according to Hulbert et al. (2007)⁵⁶.

## 2.5. Statistical analyses

The concentration of each lipid fraction (reserve and membrane) between the two dietary groups were compared using ANOVA, while the FA percentages in each lipid fraction were compared using t-tests. Normality (Shapiro-Wilk test) and homoscedasticity (Levene's test) were assessed on residuals. Since concentration of reserve and membrane lipid fraction did not meet the assumptions of parametric ANOVA, we performed a rank-based ANOVA⁵⁷ using the ARTool package⁵⁸. A principal component analysis (PCA) was performed to quantify the overall variation in membrane FA proportions between the two experimental groups. We included only FA representing more than 1% of the total FA composition and/or relevant as precursors or intermediates of n-3, n-6 or n-9 PUFA in the analysis (n=44 FA in total). The first two principal components (PC1 and PC2) of the PCA, summarizing the inter-individual variation in the membrane FA composition, were extracted for further analysis. Additionally, a hierarchical clustering on principal components was performed, using Ward's method and Euclidean distance as the dissimilarity metric, to classify individuals based on their FA composition, providing further insights into the grouping patterns of individuals from the two dietary treatments.

The effects of dietary treatment and mitochondrial assay temperature on mitochondrial function were tested using linear mixed-effects models with individual identity as a random effect to account for repeated measures. Post-hoc Tukey tests were conducted when significant effects were found. Model assumptions were verified by visual inspection of residual plots, Shapiro-Wilk tests, and Levene's test. All mitochondrial variables met the assumptions required for these analyses, except for LEAK respiration and OxCE. LEAK respiration data were log-transformed to improve residual normality and homogeneity of variances, as indicated by diagnostic tests. OxCE, being a bounded ratio and showing residual deviations from normality, was arcsin square-root transformed prior to analyses, a common transformation for proportions and bounded ratios⁵⁹.

To determine the relationships between membrane FA composition and the mitochondrial function, we used linear models with two-way interaction terms for dietary treatment with PC1 and PC2. Because fatty acid composition was measured in the heart of each individual after sacrifice, only one measurement per fish was available. We therefore correlated FA composition with mitochondrial performance measured at 18°C, which reflected the acclimation temperature during the last month of the experiment. For this reason, linear models rather than mixed models were used. The best model was selected through stepwise-backward selection. We proceeded as follows: starting from the
complete model, we removed the least non-significant effect at each step, beginning with the interactions. All statistical analyses were conducted using R v.4.2.2<sup>60</sup> and the package tidyverse<sup>61</sup>, dplyr<sup>62</sup>, ARTool<sup>58</sup>, factoextra<sup>63</sup>, FactoMineR<sup>64</sup>, nlme<sup>65</sup>, emmeans<sup>66</sup>, ggplot2<sup>67</sup>, and ggpubr<sup>68</sup>. FA values are presented as mean ± SD, and statistical tests were considered significant at p < 0.05.

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## 3. Results

Sardines did not exhibit any significant differences in body weight between the two dietary conditions, neither at the beginning of the experiment (50.1 ± 4.3 g for control vs. 49.9 ± 12.0 g for DHA-deficient; t = -0.03, df = 10.01, p = 0.98) nor after 6 months of dietary conditioning (57.8 ± 12.4 g for control vs. 50.9 ± 13.5 g for DHA-deficient; t = -1.12, df = 15.89, p = 0.28). The same was true for total length, with an initial length of 19.0 ± 0.9 cm and 18.6 ± 1.4 cm for control and DHA-deficient sardines, respectively, and a final length of 19.6 ± 0.8 cm and 19.1 ± 1.3 cm, respectively. The sex ratio (F:M) per dietary treatment was unbalanced with 4:3 (2 not identified) for control sardines and 7:1 (1 not identified) for the DHA deficient group. All sardines were adults but sexually immature at the time of sampling. Testing the effect of sex on heart FA composition and mitochondrial performance revealed no significant differences, justifying its exclusion from final analyses.

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### 3.1. Fatty acid composition

The dietary treatment did not impact the concentrations of total lipids (TL), nor the concentrations and percentages of reserve and membrane lipid fractions in fish heart (Table S3). However, it significantly modified the proportion of several FA in both reserve and membrane lipid fractions (Fig.SX, Table S4), modifying most FA categories (Table 1). The DHA-depleted sardines presented a significantly lower DHA content in their membrane (-5.5% of total FA), leading to a reduced total PUFA, total n-3 PUFA, and n-3/n-6 FA ratio. In addition, they showed a higher n-6 PUFA percentage, leading to a lower n-3/n-6 FA ratio. Notably, linoleic acid (LA, 18:2n-6) and arachidonic acid (ARA, 20:4n-6) were 1.2-fold and 1.7-fold higher in DHA-depleted sardines than in control fish (Table S4), respectively. In contrast, EPA percentage was higher in the DHA-deficient group (+1.5% of total FA), leading to a 1.4-fold higher EPA/DHA ratio compared to the control group. The overall reduction in DHA and total n-3 PUFA resulted in a lower unsaturation index (UI) of membrane lipids in DHA-depleted sardines (Table 1).

More than 50% of the variation of membrane FA percentages was explained by the first two components of the PCA (Fig. 1). The first principal component (PC1) explained 32.3% of the variance in FA proportions. It was primarily positively correlated to DHA (22:6n-3) and negatively to the 24:0
330 (Fig. 1, Table 2). Three LC PUFA (DHA, 21:5n-3 and 22:5n-6) were positively correlated, and three LC PUFA (ARA and EPA) were negatively correlated (with|r| > 0.75, Table 2). The second principal component (PC2) explained 21.9% of the variance (Fig. 1, Table 2) and was related to increasing proportions of three saturated fatty acids SFA (15:0, 20:0, 17:0) and decreasing proportions of two PUFA (20:2n-9, 22:2n-9). Overall, PC1 represented well the effect of the dietary treatment on membrane lipid composition, as all sardines fed the control diet had positive PC1 values, whereas all but one sardine fed the DHA-deficient diet had negative PC1 values (Fig. 1). On PC2, the two dietary treatments were not separated but we observed a strong negative correlation with the sardine body mass (Fig. 1 and S1, body mass = 54.3 - 3.2 * PC2, R² = 0.62, p<0.001). The hierarchical clustering analysis confirmed the presence of two groups and classified sardines of our dietary treatments with 100% accuracy within these clusters.

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342 **3.2. Mitochondrial performance**

343 Assay temperature and dietary treatment significantly impacted all mitochondrial variables 344 measured (LEAK and OXPHOS respirations, ATP production, and ATP/O, Table 3, Fig. 2), but no 345 interaction between the two factors occurred. Regardless the assay temperature, mitochondria of 346 DHA-depleted fish displayed a higher LEAK respiration and a lower OXPHOS respiration (+ 16.8% and 347 - 23.2%, respectively, Fig. 2A & B) than the control fish. Consequently, they had a lower respiration 348 coupling efficiency OE (- 3.4%, Fig. S2). They also had a lower rate of ATP production (- 29.8%, Fig. 349 2C) and required 10.7% more oxygen to produce a molecule of ATP than sardine of control group 350 (Fig. 2D).

351 For both dietary conditions, as the assay temperature increased, LEAK respiration, OXPHOS 352 respiration and ATP production increased, while the ATP/O ratio progressively decreased (Table 3, 353 Fig. 2). Specifically, LEAK respiration was the lowest at 12 and 15°C, then increased at 18 and 21°C 354 (Fig. 2A). OXPHOS respiration exhibited a linear increase from 12 to 21°C with each temperature 355 different from the other (Fig. 2B). The effect of temperature on ATP production was less pronounced, 356 with a significant increase observed only at 21°C compared to the other temperatures (Fig. 2C). The 357 ATP/O ratio decreased from 12 to 18°C, with the highest efficiency observed at 12°C and the lowest 358 at 18 and 21°C (Fig. 2D). However, ATP/O remained similar between 12°C and 15°C, and between 359 15°C and 18°C, indicating a gradual decline in efficiency with increasing temperature.

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361 **3.3. Relationship between fatty acid composition and mitochondrial performance among**
362 **individuals**
Total LEAK respiration ranged from 6.6 to 16.7 pmol O₂.sec⁻¹.mg⁻¹ of fresh heart tissue and varied up to 2.5-fold among individuals on the same dietary treatment. No significant effect of FA percentage (PC1 and PC2) and diet was found to explain this variation (Table S5). Overall OXPHOS respiration ranged from 81.6 to 203.4 pmol O₂.sec⁻¹.mg⁻¹ of fresh heart tissue, differing by up to 1.9-fold among individuals of the same dietary treatment. Individual variation in OXPHOS was clearly affected by dietary treatments but was not significantly related to membrane FA composition (Table S5). The rate of ATP production ranged from 293.2 to 1111.7 pmol ATP.sec⁻¹ mg⁻¹ of heart and varied up to 2.5-fold among individuals on the same dietary treatment. Neither dietary treatment nor FA composition had a significant effect on ATP production (Table S5). The overall ATP/O ratio varied from 1.6 to 3.1, varied up to 2-fold among individuals within a treatment group and was positively related to PC1 (β = 0.12 ± 0.05 (standard error), *P* = 0.04; Fig. 3, Table S5).

## 4. Discussion

This study aimed to investigate the impact of a dietary DHA deficiency on the cardiac mitochondrial performance of the European sardine. Our findings revealed that a dietary DHA deficiency led to substantial reductions in mitochondrial efficiency, with increased proton leakage, decreased oxidative phosphorylation capacity, and lower ATP production across all assay temperatures. These impairments were associated with a marked reduction in DHA content in cardiac cell membranes, suggesting a role of membrane DHA levels in regulating mitochondrial efficiency.

### 4.1 Impact of diet on cardiac membrane

In our study, sardines fed a DHA-deficient diet exhibited a significant reduction in DHA percentage in heart membranes. However, despite the fourfold decrease in dietary DHA, the decline in heart membrane DHA remained moderate, suggesting a selective retention of this fatty acid (REF). This retention is expected, as DHA is a major component of fish mitochondrial membranes, sometimes accounting for nearly half of total mitochondrial phospholipids³³⁻³⁵. Previous studies have shown that dietary-induced shifts in FA composition led to correlated changes in mitochondrial membrane lipids across various fish tissues, including liver and muscles³³⁻³⁵. Therefore, it is likely that DHA-deficient sardines also experienced a reduction in DHA levels within their inner mitochondrial membranes, which may have affected mitochondrial function.
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Interestingly, independently of the dietary effect and membrane DHA content, we observed that individuals with higher body mass exhibited higher proportions of 20:2n-9 and 22:2n-9 in their membrane lipids. These n-9 LC PUFA are synthesized from dietary 18:1n-9 through Δ6 desaturase activity, which first converts it to 18:2n-9 before elongation into 20:2n-9 and 22:2n-9⁶⁹. Notably, 18:1n-9 was the most abundant FA in both diets as well as in fish reserve lipids, suggesting that individuals with greater lipid assimilation capacity incorporated and metabolized more of this FA, potentially facilitating higher energy storage. This inter-individual variation in FA assimilation could stem from differences in feeding behavior within rearing tanks and/or intrinsic variability in nutrient absorption efficiency. Such metabolic flexibility, independent of dietary DHA levels, highlights the contribution of additional physiological mechanisms to individual differences in FA profiles. These variations could also influence membrane properties and, consequently, modulate mitochondrial function.

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## 4.2 Impact of diet on cardiac mitochondrial performance

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Our results showed that the reduction in DHA proportion measured in cardiac mitochondrial membranes of the fish fed the DHA-depleted diet led to an increase proton leakage across the inner mitochondrial membrane and a decrease in oxidative capacity (OXPHOS respiration). Consequently, mitochondria of DHA-depleted fish exhibited lower respiratory efficiency, with reduced oxygen uptake. A greater proportion of this oxygen was lost to compensate for proton leakage, ultimately decreasing ATP/O efficiency and overall ATP output. These findings are consistent with previous work on salmonids, including Guderley et al. (2008)³³ on rainbow trout (*Oncorhynchus mykiss*) and Závorka et al. (2021)³⁷ on Atlantic salmon (*Salmo salar*), who observed similar effects in red and white muscles following an eight-weeks dietary DHA and ten-weeks n-3 LC PUFA depletion, respectively. However, our results contrast with those of Martin et al. (2013)³⁵, who found that an eight-month DHA depletion in rainbow trout reduced LEAK respiration in red muscle mitochondria. Similarly, Salin et al. (2021)³⁶ reported that a two-month dietary deficiency in n-3 LC PUFA (EPA+DHA) had no effect on LEAK respiration in juvenile golden grey mullet white muscle but instead increased their ATP/O ratio, suggesting a potential improvement in mitochondrial efficiency. Taken together, these discrepancies suggest that the impact of dietary FA composition on mitochondrial function may be tissue-specific, influenced by treatment duration, the type of FA affected (DHA, EPA, or both), or even species-specific metabolic adaptations. DHA is known to remodel cardiolipin acyl chains, and cardiolipin content is tightly linked to respiratory supercomplex stability and proton conduction at the inner mitochondrial membrane [Sullivan 2017; Pfeiffer 2003; Schlame 2009; Haines 2001]. Then, a DHA dietary deficiency, by impacting the cardiolipin content, may have impacted the mitochondrial efficiency of heart cells.
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Our findings reveal a significant inter-individual variation in mitochondrial efficiency among sardines within the same dietary treatment. In particular, a higher ATP/O ratio was associated with a greater DHA percentage in cardiac membranes in both dietary groups, suggesting that individuals maintaining higher DHA levels preserved a more efficient ATP production. This relationship between mitochondrial efficiency and membrane FA composition has also been observed by Salin et al. in juveniles of golden grey mullet, in which higher n-3 LC PUFA (EPA + DHA) percentage in muscle was linked to improved ATP synthesis efficiency³⁶. The authors hypothesized that mitochondrial FA composition influences proton conductance across the inner mitochondrial membrane, thereby influencing oxygen consumption dedicated to offset proton leakage⁵⁶ and ultimately affecting ATP production efficiency¹⁹. However, in our study, no direct correlation was observed between LEAK respiration and membrane FA composition. This lack of correlation could indicate that membrane permeability and its ability to maintain the proton gradient do not depend solely on DHA content but rather on a complex and nonlinear combination of multiple membrane FA. Notably, several other key n-3 and n-6 LC PUFA—including EPA (+2.1%), ALA (+0.3%), ARA (+1.1%), and LA (+1.2%)—present in higher proportions in the DHA-depleted diet were also found in higher proportion in the heart mitochondrial membranes of DHA-deficient fish. These FA may have partially compensated for the reduction in DHA and helped limit excessive proton leakage. While most DHA-deficient sardines exhibited lower membrane DHA levels than control sardines, some individuals from the DHA-deficient group maintained mitochondrial functions and efficiency comparable to, or even exceeding, those of certain control-fed individuals. This highlights the complexity of the role of FA composition in shaping membrane properties and mitochondrial function, and suggests that DHA alone does not solely determine mitochondrial function.

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Variations in membrane FA composition can influence the structure and fluidity of mitochondrial membranes, thereby modulating the function of key membrane-bound proteins⁹, including electron transport chain (ETC) complexes and ATP synthase. This may result in the variability observed in ATP/O ratios among individuals. Additionally, substrate utilization could explain differences in mitochondrial efficiency, as demonstrated in Atlantic salmon, in which variations in DHA and EPA contents in muscle were associated with differences in gene expression related to lipid and carbohydrate metabolism⁷⁰. In our study, while we observed a relationship between ATP/O ratio and membrane DHA proportion, no correlation was found between ATP/O ratio and OXPHOS respiration or ATP production rates, reinforcing the hypothesis that DHA alone does not linearly explain these mitochondrial performances. Beyond the compensatory role of alternative FA in DHA-depleted sardines, broader metabolic and physiological mechanisms may also contribute to
the observed variability in mitochondrial function. For example, the capacity of some individuals to
accumulate energy reserves—reflected in higher body mass and increased incorporation of n-9 LC
PUFA—suggests that differences in lipid assimilation and reserve synthesis could play a role in
shaping mitochondrial performance. These results highlight the complexity of mitochondrial
responses to dietary FA availability and suggest that multiple regulatory mechanisms contribute to
maintaining bioenergetic function under nutritional constraints, rather than being solely determined
by DHA availability.

Interestingly, our findings in sardine heart mitochondria challenge the commonly held
hypothesis that reduced unsaturation decreases membrane permeability, leading to lower proton
leakage and increased mitochondrial efficiency. Instead, the lower unsaturation level of cardiac
membranes in DHA-depleted sardines did not reinforce mitochondrial integrity but rather increased
proton leakage, reducing the mitochondria's ability to maintain the proton gradient. Moreover, the
decline in oxidative (OXPHOS respiration) and phosphorylative capacities (ATP production) suggests
an impairment of key proteins involved in the ETC and ATP synthase activity. These results confirm
that both the structural integrity and functional capacity of cardiac mitochondrial membranes were
compromised by reduced membrane DHA, emphasizing the essential role of this FA in maintaining
mitochondrial function under varying environmental conditions.

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480 **4.3 Dietary effects on in vitro cardiac mitochondrial thermal performance**

In vitro measurement of mitochondrial performance showed that a lower DHA proportion in
membrane impaired mitochondrial efficiency at all four assay temperatures (ranging from 12 to
21°C). This indicates that alternative FA and other regulatory mechanisms potentially involved were
not sufficient to stabilize membrane integrity and sustain mitochondrial efficiency under a thermal
gradient. In addition, our results highlight a clear effect of temperature on mitochondrial function in
both dietary groups, with LEAK respiration increasing exponentially and OXPHOS respiration rising
linearly from 12 to 21°C. Such thermodynamic increases in mitochondrial respiration have been
widely reported in ectotherms⁷¹,⁷², including fish, specifically in the liver⁷³ and heart²³,²⁴. Generally, as
temperature rises, proton LEAK increases exponentially due to higher membrane fluidity and
permeability⁷⁴, while OXPHOS respiration follows a classic enzymatic thermodynamic pattern, initially
increasing before plateauing and declining at suboptimal temperatures²⁴,⁷⁵. At 21°C, in both dietary
groups, we observed no significant respiratory decoupling (i.e., no imbalance in the proportion of
oxygen consumption dedicated to ATP synthesis relative to the oxygen required to offset proton
leakage, Fig. S2), as OXPHOS respiration continued to increase. This suggests that, in vitro, ETS
enzyme function remained operating, and cardiac mitochondria did not reach, or at least exceed, their thermal optimum. As a result, ATP production also increased with temperature (Fig. 2). These results contrast with those of Iftikar et al. (2013)²³, who found a decline in ATP production between 20 and 32°C in the heart of the spotty fish *Notolabrus celidotus*. Beyond species-specific differences in habitat and trophic ecology (small pelagic vs. reef-associated fish), this discrepancy may also stem from methodological differences in the protocol we used (e.g., homogenates vs. permeabilized fibers, Kd determination for each assay temperature; see ESM for details).

While our results suggest that increasing temperature enhances ATP production, potentially supporting cellular processes such as growth, locomotion, and reproduction, this does not necessarily translate into a net energetic gain. Indeed, the additional ATP produced must also offset the higher basal maintenance costs associated with elevated temperatures (e.g., ion gradient homeostasis⁷⁶). Although mitochondrial respiratory fluxes do not always directly reflect ATP synthesis efficiency⁷⁷, our results show a clear trend. Regardless of dietary treatment and despite the absence of significant respiratory uncoupling, mitochondria became less efficient at producing ATP per unit of oxygen consumed as temperature increased. They had to compensate for this loss of efficiency by increasing oxygen consumption with rising temperatures. A similar trend was observed by Iftikar et al. (2013)²³, who attributed this decline in efficiency to greater proton leakage across the inner mitochondrial membrane and a reduced uncoupling capacity—suggesting a diminished ability of mitochondria to regulate energy dissipation and optimize ATP production under heat stress. While reactive oxygen species (ROS) production could also contribute to this phenomenon, significant increases in mitochondrial ROS generation tend to occur near critical temperatures in fish hearts²³,⁷⁸, which does not appear to have been reached in our study.

Interestingly, DHA deficiency did not alter the thermodynamic effects of temperature on mitochondrial function and efficiency *in vitro*. The temperature-dependent trends in mitochondrial function remained consistent across the thermal gradient, regardless of dietary treatment, despite the potential additive effects of temperature and membrane DHA content on mitochondrial inner membrane properties. A lower DHA content could have been detrimental at low temperatures by exacerbating membrane rigidity, while it could have been beneficial at high temperatures by counteracting the temperature-induced increase in membrane fluidity and permeability, thereby potentially limiting excessive proton leakage. However, these hypotheses were not supported by our results. Across the tested temperature range, despite a clear overall impairment of mitochondrial performance due to reduced membrane DHA levels, we observed no interactive effects between membrane DHA content and temperature. This could be due to the relatively narrow temperature range studied and/or the heart's ability to maintain a sufficient DHA level through strong selective
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retention. Additionally, compensation through the assimilation of other FA present in the DHA-deficient diet, such as EPA, could also have mitigated the effects of reduced membrane unsaturation in the heart.

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Our in vitro study suggests that, even at 21°C—a temperature considered as a high one for sardines in the Bay of Biscay—cardiac mitochondria from both dietary groups maintained their respiratory capacity without signs of failure. The absence of a major collapse in respiratory function and ATP production may indicate that sardine cardiac mitochondria may be able to respond to rapid temperature changes. However, impairment of the mitochondrial performance of DHA-deficient sardines at all assay temperatures suggests that these individuals may reach their functional limits at relatively lower temperatures compared to control sardines when attempting to meet rising energetic demands with increasing temperature. Finally, it is important to note that these results were obtained *in vitro* under conditions of saturating oxygen and substrate availability, which may not fully reflect what we may observe in a whole *in vivo* individual.

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## Conclusions

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This study highlights a critical link between dietary FA composition and mitochondrial function, providing insights into how changes in membrane FA composition can influence the efficiency of sardine cardiac mitochondria exposed at different temperatures. Our findings demonstrate that a 6-month dietary DHA deficiency led to substantial changes in the FA composition of sardine heart membranes, significantly reducing DHA levels and thus lowering overall membrane unsaturation. These changes resulted in a consistent impairment in mitochondrial efficiency in producing ATP over a large range of temperature assays which might reduce cardiac mitochondrial efficiency and hence reduce individuals' overall performance. This may have critical implications for the resilience of sardines exposed to a decreasing DHA dietary availability in the context of global changes. Our *in vitro* results indicate that DHA-deficient sardines exhibit a reduced mitochondrial capacity to meet cellular energy demands, particularly under conditions where metabolic requirements may increase, such as acute temperature changes. This reduced mitochondrial efficiency may, in turn, impair sustained energy supply for crucial physiological functions. In small pelagic fish like sardines, sustained swimming abilities are particularly reliant on efficient ATP production, as they are essential for schooling, predator evasion, and feeding—all critical for survival. The high interindividual variability observed also highlighted that DHA alone does not fully explain mitochondrial oxidative capacity. While DHA remains a key player in the functional efficiency of ATP synthesis per oxygen consumed in mitochondria, EPA and other FA likely play a role in maintaining oxidative capacities and mitigating the effects of DHA deficiencies. The contribution of these fatty acids to cardiolipid remodelling has to be explored. Another perspective is to explore potential
compensation from alternative pathways, such as lipid or ketone metabolisms because these
substrates can differentially affect mitochondrial efficiency and thermal response (Ref du referee).
Overall, our findings underscore the complexity of mitochondrial efficiency, suggesting that
additional mechanisms contribute to the observed inter-individual variability.

567 Acknowledgements

568 We thank Y. Le Lay, C. Kerlorch, and his crew for sardine collection, Oceanopolis for technical
569 support, J.L. Zambonino for diet development, Polaris (G. Breton and L. Larvol) for providing DHA-
570 deficient oil, INRAE Saint-Pée-sur-Nivelle for preparing experimental pellets, and the Ifremer-
571 PHYTNESS team for fish care, samplings, and dissections. Special thanks to the Lipidocean platform
572 for their assistance with lipid analyses.

573 Ethics

574 All fish handling complied with the guidelines of the Animal Care Committee of France (ACCF)
575 and took place at Ifremer's facilities in Plouzané (France, Authorization: B29-212-05) in accordance
576 with protocol #347492022011916415126-v6 approved by the French Ministry of Higher Education
577 and Research.

578 Fundings

579 This work is a contribution to the OMEGA project, supported by ISblue project,
580 Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant
581 from the French government under the program "Investissements d'Avenir" embedded in France
582 2030. L.G. was funded by ISblue.

583 CRediT (Contributor Roles Taxonomy)

G.L., M.V., M.B. conceived the ideas and designed methodology. G.L., M.B., M.V., V.S.,
584 collected the data. G.L., M.B., P.S., F.L.G., C.L., K.S., M.V. analysed the data. M.B., V.S. ensured animal
585 rearing. G.L. led the writing of the manuscript and all authors revised the manuscript and gave final
586 approval for publication. G.L., M.V., L.P. acquired the funds.

588 Declaration of competing interest - The authors report no declarations of interest.

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<caption>Table 1: Percentages of main classes of fatty acids (% of total FA) of heart membrane lipids of sardines fed with a control or a DHA-deficient diet (n = 9 per dietary treatment). Results are expressed as mean ± SD. PUFA: PUFA: polyunsaturated FA (16 to 22 carbons), UI: unsaturation index, EPA: Eicosapentaenoic acid, DHA: Docosahexaenoic acid</caption>

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<table><thead><tr><th rowspan="2">(%)</th><th colspan="2">Dietary treatment</th><th colspan="3">t-test</th></tr><tr><th>Control</th><th>DHA-deficient</th><th>Statistic</th><th>df</th><th>p-value</th></tr></thead><tbody><tr><td>Total PUFA</td><td>60.7 ± 0.8</td><td>59.4 ± 1.6</td><td>-2.31</td><td>11.55</td><td>0.04*</td></tr><tr><td>Total n-3 PUFA</td><td>49.6 ± 0.9</td><td>45.6 ± 1.9</td><td>-5.76</td><td>11.5</td><td>&lt;0.001***</td></tr><tr><td>Total n-6 PUFA</td><td>10.5 ± 1.1</td><td>13.2 ± 1.5</td><td>4.41</td><td>14.66</td><td>&lt;0.001***</td></tr><tr><td>n-3/n-6</td><td>4.8 ± 0.6</td><td>3.5 ± 0.5</td><td>-4.91</td><td>15.55</td><td>&lt;0.001***</td></tr><tr><td>EPA</td><td>7.1 ± 1.2</td><td>8.6 ± 1.6</td><td>2.41</td><td>14.73</td><td>0.03*</td></tr><tr><td>DHA</td><td>39.0 ± 2.2</td><td>33.5 ± 3.2</td><td>-4.33</td><td>14.26</td><td>&lt;0.001***</td></tr><tr><td>EPA/DHA</td><td>0.18 ± 0.04</td><td>0.26 ± 0.07</td><td>3.19</td><td>13.14</td><td>&lt;0.01**</td></tr><tr><td>UI</td><td>324 ± 5</td><td>307 ± 10</td><td>-4.48</td><td>11.42</td><td>&lt;0.001***</td></tr></tbody></table>

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<caption>Table 2: Ranking of fatty acids (FA) with $|r| > 0.75$ based on their correlation with PCA axes 1 and 2 (Figure 1). ALA: alpha linolenic acid; ARA: Arachidonic acid; DHA: Docosahexaenoic acid; EPA: Ecosapentaenoic acid; LA: Linoleic acid. FA in bold are key contributors to total n-6, total n-3, and the n-3/n-6 balance indices, and correspond to PUFA representing more than 1% of total FA.</caption>

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<table><thead><tr><th></th><th>FA</th><th>Correlation</th></tr></thead><tbody><tr><td rowspan="2">PC1</td><td>22:6n-3 (DHA)</td><td>0.94</td></tr><tr><td>24:0</td><td>-0.92</td></tr></tbody></table>
<table><tbody><tr><td></td><td>22:2n-6</td><td>-0.92</td></tr><tr><td></td><td><strong>20:4n-6 (ARA)</strong></td><td>-0.89</td></tr><tr><td></td><td><strong>18:3n-3 (ALA)</strong></td><td>-0.88</td></tr><tr><td></td><td>18:3n-4</td><td>0.85</td></tr><tr><td></td><td><strong>20:5n-3 (EPA)</strong></td><td>-0.79</td></tr><tr><td></td><td>22:1n-11</td><td>-0.79</td></tr><tr><td></td><td>21:5n-3</td><td>0.76</td></tr><tr><td></td><td>22:5n-6</td><td>0.75</td></tr><tr><td></td><td>18:3n-6</td><td>-0.75</td></tr><tr><td></td><td><strong>18:2n-6 (LA)</strong></td><td>-0.75</td></tr><tr><td rowspan="5"><strong>PC2</strong></td><td>15:0</td><td>0.93</td></tr><tr><td>20:2n-9</td><td>-0.89</td></tr><tr><td>22:2n-9</td><td>-0.81</td></tr><tr><td>20:0</td><td>0.78</td></tr><tr><td>17:0</td><td>0.77</td></tr></tbody></table>

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<table><caption>Table 3: Parameters from mixed-effects linear models of cardiac mitochondrial performance as a function of temperature assay and dietary treatment (Diet) of sardines fed with control or a DHA-deficient diet (n = 9 per diet). LEAK respiration was log transformed prior analyses. For each model, we also report the proportion of variance explained by the random factor (individual identity), calculated as the variance of the random intercept divided by the total variance (random + residual).</caption><thead><tr><th>Dependant variables</th><th>Fixed factors</th><th>Chi²</th><th>Df</th><th>p</th><th>Random variance</th></tr></thead><tbody><tr><td rowspan="3">Log(LEAK respiration)</td><td>Temperatures</td><td>76.15</td><td>3</td><td>&lt;0.001***</td><td rowspan="3">35%</td></tr><tr><td>Condition</td><td>4.51</td><td>1</td><td>0.034*</td></tr><tr><td>Temperatures*Condition</td><td>5.37</td><td>3</td><td>0.15</td></tr><tr><td rowspan="3">OXPHOS respiration</td><td>Temperatures</td><td>167.85</td><td>3</td><td>&lt;0.001***</td><td rowspan="3">49%</td></tr><tr><td>Condition</td><td>9.87</td><td>1</td><td>&lt;0.01**</td></tr><tr><td>Temperatures*Condition</td><td>6.60</td><td>3</td><td>0.08</td></tr><tr><td rowspan="3">arcsinV(OXCE)</td><td>Temperatures</td><td>14.94</td><td>3</td><td>&lt;0.01**</td><td rowspan="3">59%</td></tr><tr><td>Condition</td><td>26.67</td><td>1</td><td>&lt;0.001***</td></tr><tr><td>Temperatures*Condition</td><td>4.80</td><td>3</td><td>0.19</td></tr><tr><td rowspan="3">ATP production</td><td>Temperatures</td><td>35.76</td><td>3</td><td>&lt;0.001***</td><td rowspan="3">7%</td></tr><tr><td>Condition</td><td>19.78</td><td>1</td><td>&lt;0.001***</td></tr><tr><td>Temperatures*Condition</td><td>3.36</td><td>3</td><td>0.34</td></tr></tbody></table>

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790
Temperatures 9.45 3 0.024*
Condition 4.98 1 0.026*
Temperatures*Condition 0.31 3 0.96
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<figure><img src="image_3.png" /><figcaption>Figure 1: Principal component analysis and associated ellipses (95% confidence interval) discriminating the dietary treatment, based on membrane (polar lipid fraction) fatty acids (FA) percentages of sardine hearts. In total, 44 FA were considered in the analysis (black arrows), and only those (17) with absolute correlations greater than 0.75 along either axis 1 or 2 were labelled. Deficient sardines are indicated by a red triangle (n = 9), and control sardines by a blue circle (n = 9). Sardine body mass vector (in green) was added as an additional non-explanatory variable in the PCA for illustration purposes. DHA is 22:6n-3; EPA is 20:5n-3.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2: Thermal performance curves for cardiac mitochondrial (A) LEAK respiration, (B) OXPHOS respiration, (C) ATP production and (D) ATP/O ratio. DHA-deficient sardines are indicated by a red triangle (n = 9), and control sardines by a blue circle (n = 9). The mitochondria performance of each individual, measured *in vitro* at four assay temperatures, has its points connected by a thin line. Thick lines represent the means estimated by the mixed-effects linear models (Table 3). Small letters represent the results of post-hoc tests regarding the assay temperature effect. Assay temperature points not sharing a common letter are significantly different.</figcaption></figure>

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<figure><img src="image_5.png" /><figcaption>Figure 3: Effects of the FA percentage of membrane lipids (PC1 axis) and ATP/O ratio in the hearts of sardines at the sampling temperature (~18°C). The FA percentage was summarized by extracted PC1 from the PCA. The significant effect of FA is indicated by a solid line (Table S5, ATP/O, PC1 effect, p-value = 0.042). DHA-deficient sardines are indicated by a red triangle (n = 9), and control sardines by a blue circle (n = 9).</figcaption></figure>