The relationship between membrane fatty acid content and mitochondrial efficiency differs within- and between- omega-3 dietary treatments

Highlights

• Marine fish performance rely on dietary long chain omega-3 fatty acids (n-3), which are predicted to reduce in a near future.

• We examine the consequence of n-3 deficient diet on mitochondrial metabolism.

• Mitochondrial ability to make ATP increased in fish fed on lower n-3 diet.

• Surprisingly, individuals that have mitochondria with lower ability to make ATP had lower membrane n-3 content.

• Mitochondrial metabolism may provide new insights into the mechanisms underlying fish performance under n-3 deficiency.

Abstract

An important, but underappreciated, consequence of climate change is the reduction in crucial nutrient production at the base of the marine food chain: the long-chain omega-3 highly unsaturated fatty acids (n-3 HUFA). This can have dramatic consequences on consumers, such as fish as they have limited capacity to synthesise n-3 HUFA de novo. The n-3 HUFA, such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), are critical for the structure and function of all biological membranes. There is increasing evidence that fish will be badly affected by reductions in n-3 HUFA dietary availability, however the underlying mechanisms remain obscure. Hypotheses for how mitochondrial function should change with dietary n-3 HUFA availability have generally ignored ATP production, despite its importance to a cell's total energetics capacity, and in turn, whole-animal performance. Here we (i) quantified individual variation in mitochondrial efficiency (ATP/O ratio) of muscle and (ii) examined its relationship with content in EPA and DHA in muscle membrane of a primary consumer fish, the golden grey mullet Chelon auratus, receiving either a high or low n-3 HUFA diet. Mitochondria of fish fed on the low n-3 HUFA diet had higher ATP/O ratio than those of fish maintained on the high n-3 HUFA diet. Yet, mitochondrial efficiency varied up about 2-fold among individuals on the same dietary treatment, resulting in some fish consuming half the oxygen and energy substrate to produce the similar amount of ATP than conspecific on similar diet. This variation in mitochondrial efficiency among individuals from the same diet treatment was related to individual differences in fatty acid composition of the membranes: a high ATP/O ratio was associated with a high content in EPA and DHA in biological membranes. Our results highlight the existence of interindividual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variation in response to food chain changes.

Introduction

Fish provide critical sustenance for millions of people worldwide and have far reaching impacts on the productivity of ecosystems (McIntyre et al., 2016). Yet, ongoing and future climate change threatens the persistence of fish populations globally (Pörtner and Knust, 2007). An important, but underappreciated, consequence of climate change is the reduction in production at the base of the food chain of essential nutrient: the long-chain omega-3 highly unsaturated fatty acids (n-3 HUFA) (da Motta Pacheco et al., 2014; Galloway and Winder, 2015; Hixson and Arts, 2016). Water warming (Hixson and Arts, 2016), acidification (Bermudez et al., 2015), or UV irradiation (Kang, 2011), all affect n-3 HUFA primary producers physiology and community assemblages, leading to a dominance of n-3 HUFA-impoverished taxa (Galloway and Winder, 2015). The n-3 HUFA such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are essential to the structure and function of all biological membranes and are thus considered to be important drivers of organism performance (Hulbert et al., 2005; Ishizaki et al., 2001; Mazorra et al., 2003; Vagner et al., 2015). Endogenous biosynthesis of n-3 HUFA from precursors is limited in most vertebrates, including marine fish (Alimuddin et al., 2005; Arts and Kohler, 2009; Oboh et al., 2017). Changes in the n-3 HUFA availability in the fish diet causes strongly correlated changes in the fatty acid composition of their biological membranes (Guderley et al., 2008; Nogueira et al., 2001; Ramsey et al., 2005). Fish on n-3 HUFA-deficient diets can perform badly: reductions in EPA and DHA dietary content reduced growth (Norambuena et al., 2015; Vagner et al., 2014) and thermal tolerance (Vagner et al., 2015), and altered whole-organism metabolic traits (Vagner et al., 2014, 2015). Determining what causes animal performance to vary with n-3 HUFA diet constitutes a fundamental step in predicting the impacts of projected oceanic changes on fish resilience (Kang, 2011).

Consideration of mitochondrial capacity to make ATP may improve our understanding of the links between dietary n-3 HUFA availability and whole-animal performance. Mitochondrial ATP is produced via oxidative phosphorylation, a process through which energy substrates are oxidized to generate a protonmotive force that drives the phosphorylation of ADP to ATP. Hypotheses for how mitochondrial function should change with dietary n-3 HUFA availability have focused on variation in respiratory capacities and have generally ignored variation in ATP production (Kraffe et al., 2007; Ramsey et al., 2005; but see Herbst, 2014; Vagner et al., 2015; Vagner et al., 2014). Although ATP production depends on the rate of substrate oxidation, the number of ATP molecules produced for each atom of oxygen consumed by the mitochondria during substrate oxidation (ATP/O ratio) can vary (Brand, 2005; Salin et al., 2015). A fraction of the protonmotive force that is generated from substrate oxidation is dissipated through proton leak across mitochondrial inner membranes and this leakage can decrease the protonmotive force available to produce ATP (Brand, 2005; Kadenbach, 2003). Thus, the greater the mitochondria leak, the less efficiently an animal converts its metabolic substrates into ATP, and the lower the ATP/O ratio. The proportion of energy dissipated in the proton leak and the efficiency to make ATP vary among conspecific (Bottje and Carstens, 2009; Robert and Bronikowski, 2010; Salin et al., 2016b) and can be influenced by environmental factors including diet (Fontaine et al., 1996; Salin et al., 2018). A number of studies have found positive links between intraspecific heterogeneity in efficiency to produce mitochondrial ATP and the whole-organism performance, such as locomotory performance (Coen et al., 2012; Distefano et al., 2018; but see Jahn and Seebacher, 2019), developmental rate (Salin et al., 2012), growth efficiency (Bottje and Carstens, 2009; Salin et al., 2019) and reproductive output (Robert and Bronikowski, 2010), suggesting that it might be a trait of ecological relevance.

Recent research has recognized the importance of accounting for individual heterogeneity in predicting responses to global changes (Hamel et al., 2018). Because some individuals perform much better than others within the same environment, individual heterogeneity is likely to directly influence the potential for species to evolve adaptations for a reduced n-3 HUFA availability. A number of studies have found positive links between n-3 HUFA content in membranes and mitochondrial proton leak when comparing among species (Brand et al., 1994; Brookes et al., 1998), but contradictory results were found when comparing treatment groups with studies reporting positive (Martin et al., 2013), negative (Fontaine et al., 1996; Guderley et al., 2008) or no relationships (Guderley et al., 2008) between content in n-3 HUFA in mitochondrial membranes and leak respiration. N-3 HUFA are thought to have important effects on the mitochondrial capacity to make ATP; but until now, there has, to our knowledge, been no assessment of whether membrane n-3 HUFA content could explain variation in mitochondrial metabolism among individuals.

The present experiment integrates measurements of mitochondrial efficiency and mitochondrial proton leak to determine whether reductions in EPA and DHA availability in food led to changes in energy metabolism of a primary consumer fish. We first examined the effect of dietary n-3 HUFA content on mitochondrial function, in particular on the efficiency to produce ATP (ATP/O ratio) and the respiratory capacities to offset the proton leak (LEAK respiration). Secondly, we tested whether differences between individuals in mitochondrial efficiency and mitochondrial LEAK respiration vary with membrane n-3 HUFA content. To address this, we experimentally manipulated the quantity of n-3 HUFA in food for wild-caught juvenile golden grey mullet (Chelon auratus). Fish were fed either a high n-3 HUFA or low n-3 HUFA diet, and their membrane fatty acid composition and mitochondrial functioning were determined in skeletal muscle. We choose juvenile golden grey mullet as our study organism because they are likely to be the first levels of the food chain to face a decline in availability of dietary n-3 HUFA, as this fish fed mainly on primary producers (Lebreton et al., 2011; Mourente and Tocher, 1993). We analysed mitochondrial properties in the skeletal muscle because the mitochondrial function of this tissue is known to influence whole-animal performance (Coen et al., 2012; Salin et al., 2016a), and that fatty acid dietary content influences muscle membrane fatty acid composition (McKenzie et al., 1998; Vagner et al., 2015).