Mitochondria, Temperature, and the Pace of Life

Mitochondria, Temperature, and the Pace of Life Abstract Life history strategies, physiological traits, and behavior are thought to covary along a “pace of life” axis, with organisms at the fast end of this continuum having higher fecundity, shorter lifespan, and more rapid development, growth, and metabolic rates. Countergradient variation represents a special case of pace of life variation, in which high-latitude organisms occupy the fast end of the continuum relative to low-latitude conspecifics when compared at a common temperature. Here, we use Atlantic killifish (Fundulus heteroclitus) to explore the role of mitochondrial properties as a mechanism underlying countergradient variation, and thus variation in the pace of life. This species is found along the Atlantic coast of North America, through a steep latitudinal thermal gradient. The northern subspecies has faster development, more rapid growth, higher routine metabolic rate, and higher activity than the southern subspecies when compared at a common temperature. The northern subspecies also has greater mitochondrial respiratory capacity in the liver, although these differences are not evident in other tissues. The increased respiratory capacity of liver mitochondria in northern fish is associated with increases in the activity of multiple electron transport complexes, which largely reflects an increase in the amount of inner mitochondrial membrane per mitochondrion in the northern fish. There are also differences in the lipid composition of liver mitochondrial membranes, including differences in cardiolipin species, which could also influence respiratory capacity. These data suggest that variation in mitochondrial properties could, at least in part, underlie variation in the pace of life in Atlantic killifish. Introduction Species and populations within species exhibit substantial variation in life history traits such as fecundity, hatching success, developmental rate, growth rate, and size at maturity (Roff 1992). However, variation in these traits does not occur in all possible combinations in nature (Stearns 1976, 1992). Instead, it has been proposed to occur along a slow-to-fast continuum, with species or populations with low reproductive rate, slow development, and long lifespan at one end of the continuum, and those with faster reproductive rates and development, but shorter lifespan at the other (Ricklefs and Wikelski 2002). As such, the pace of life hypothesis has its foundation in the classic concept of “r” and “K” selection (MacArthur and Wilson 1967; Pianka 1970). The pace of life concept has been extended to include a variety of physiological and behavioral traits (Reale et al. 2010), suggesting the existence of a complex syndrome that links multiple traits across levels of biological organization to variation in life history strategies. These observed differences in the apparent pace of life and the associated physiological and behavioral traits (Reale et al. 2010) represent different allocation choices among investments in reproduction, growth, and survivorship (Stearns 1976, 1992) resulting from trade-offs due to the allocation of limited resources among different functions (Stearns 1992; Zera and Harshman 2001). The pace of life concept has inspired substantial research effort and has been the subject of multiple recent syntheses (see e.g., Dammhahn et al. 2018), but its validity and underlying mechanistic basis are still a matter of vigorous debate (Royauté et al. 2018). The purpose of the present review is to examine this concept in the context of thermal physiology, and specifically to explore the potential role of variation in mitochondrial function as a mechanism underlying variation in the pace of life as an adaptation to environmental temperature. Variation in metabolic rate is thought to play an important role as either a cause or consequence of variation in the pace of life (Ricklefs and Wikelski 2002; Wiersma et al. 2007; Williams et al. 2010; Glazier 2015), as individuals on the fast end of the pace of life spectrum tend to have higher resting metabolic rates, even after differences in body size have been accounted for (Ricklefs and Wikelski 2002). Indeed, in an elegant study with Trinidadian guppies, Auer et al. (2018) showed that standard metabolic rate had a strong positive correlation with a suite of traits associated with the pace of life (such as male age and mass at maturity, female age and mass at first parturition, inter-litter interval, and reproductive allotment) in natural populations exposed to differing predation pressure, and that evolutionary change in the pace of life following experimental transplant to sites with different predation pressure was accompanied by changes in metabolic rate. These studies demonstrate the potential for a tight association between variation in metabolic rate and the pace of life, and they highlight the evolutionary importance of these associations. However, the underlying physiological and biochemical mechanisms that cause variation in metabolic rate, and thus potentially in the pace of life, are not fully known and may include processes acting at many levels of biological organization (Versteegh et al. 2012; Konarzewski and Książek 2013; Wone et al. 2013; Pettersen et al. 2018). One cellular process that could play a key role in influencing the pace of life is mitochondrial oxidative phosphorylation (Fig. 1). Mitochondria are the principle source of ATP generation in animal cells, and thus are intimately involved in nutrient and energy metabolism. Aerobic ATP production in the mitochondrion occurs via oxidative phosphorylation, which is accomplished by the proteins of the mitochondrial electron transport system (ETS) and the ATP synthase (Complex V), which are a series of membrane-spanning or membrane-associated proteins located on the inner mitochondrial membrane. The proteins of the ETS accept electrons from NADH and FADH2 at complexes I and II of the ETS, respectively. These electrons are then transported through the ETS, and this electron transport process results in the translocation of protons (H+) by complexes I, III, and IV from the mitochondrial matrix into the intermembrane space. The resulting proton gradient is harnessed by the ATP synthase to produce ATP. Note that this process is not perfectly efficient, as protons can also move across the inner mitochondrial membrane through a variety of pathways such as the uncoupling proteins and the adenine nucleotide translocase, or even through the membrane itself (Jastroch et al. 2007), bypassing the ATP synthase, which results in the dissipation of the proton gradient without the synthesis of ATP. Fig. 1 View largeDownload slide Mitochondrial electron transport and ATP synthesis. The electron transport system (ETS) consists of a series of membrane associated protein complexes (Complexes I–IV) embedded in the inner mitochondrial membrane that separates the mitochondrial matrix and inter-membrane space (IMS). Electron carriers transfer electrons primarily to complexes I and II, which then passes them to coenzyme Q, a fat-soluble molecule that is present within the lipid phase of the membrane. Coenzyme Q then passes the electrons to Complex III, and they are ultimately transferred to oxygen through the action of Complex IV (cytochrome c oxidase). The transfer of electrons through the ETS is associated with the translocation of protons (H+) into the IMS. The resulting proton (ΔP) and electrical (Ψm) gradient is used by Complex V (ATP synthase) to synthesize ATP. Protons can also pass through the membrane through a variety of leak pathways such as uncoupling proteins (UCP) and the adenine nucleotide translocator (ANT) that do not result in ATP synthesis. Fig. 1 View largeDownload slide Mitochondrial electron transport and ATP synthesis. The electron transport system (ETS) consists of a series of membrane associated protein complexes (Complexes I–IV) embedded in the inner mitochondrial membrane that separates the mitochondrial matrix and inter-membrane space (IMS). Electron carriers transfer electrons primarily to complexes I and II, which then passes them to coenzyme Q, a fat-soluble molecule that is present within the lipid phase of the membrane. Coenzyme Q then passes the electrons to Complex III, and they are ultimately transferred to oxygen through the action of Complex IV (cytochrome c oxidase). The transfer of electrons through the ETS is associated with the translocation of protons (H+) into the IMS. The resulting proton (ΔP) and electrical (Ψm) gradient is used by Complex V (ATP synthase) to synthesize ATP. Protons can also pass through the membrane through a variety of leak pathways such as uncoupling proteins (UCP) and the adenine nucleotide translocator (ANT) that do not result in ATP synthesis. Oxygen is the final electron acceptor along the ETS and is thus critical for the continued functioning of the ETS and oxidative phosphorylation. However, under certain circumstances (Murphy 2009) oxygen can also interact with electron carriers at various steps along the ETS, forming superoxide, a type of reactive oxygen species (ROS). ROS production by mitochondria can result in oxidative damage to mitochondrial membranes, proteins, and DNA. Damage is not restricted to the mitochondrion, as these ROS can also be released into the cell and cause widespread damage, which is thought to be a major determinant of cellular aging, and thus of lifespan (Selman et al. 2012). However, ROS production is not the only determinant of the extent of oxidative damage. Cells have multiple efficient mechanisms to buffer ROS production through a variety of non-enzymatic and enzymatic antioxidants (Birben et al. 2012). The extent of oxidative stress experienced by an organism, and thus the potential for oxidative damage, will reflect the balance between the production of ROS and the extent of the antioxidant mechanisms. Thus, it is likely that this balance between ROS production and antioxidant defenses is an important component of the tradeoffs that are thought to underlie variation in the pace of life (Monaghan et al. 2009). The fact that the mitochondrion plays a key role both in the mechanisms underlying aerobic metabolic rate and in ROS production suggests that this organelle may be a critical nexus of life-history evolution that could play a role in establishing the trade-offs that underlie the pace of life hypothesis (Speakman et al. 2015; Janssens and Stoks 2018). Killifish and the pace of life A number of observations suggest that the Atlantic killifish, Fundulus heteroclitus, is an interesting system in which to investigate the role of the mitochondria in variation in the pace of life. Populations of Atlantic killifish are found in marshes and estuaries along the east coast of North America from as far north as the Bay of Islands in Newfoundland (Dickinson and Threlfall 1975) to just south of Jacksonville, Florida, where this species is replaced by the closely related congener, Fundulusgrandis (Gonzalez et al. 2009). There are two recognized subspecies of F. heteroclitus, which have been designated F. heteroclitus macrolepidotus in the northern part of the range, and F. heteroclitus heteroclitus in the southern part of the range (Morin and Able 1983; Jordan and Everman 1896). One of the striking environmental features of the habitat of Atlantic killifish is that mean environmental temperature varies steeply along the Atlantic coast (Fig. 2), such that the extreme northern populations of F. heteroclitus experience a mean monthly temperature approximately 10°C lower than do the extreme southern populations. There is also substantial seasonal variation in temperature, such that in any given location, winter temperatures are typically about 10°C lower than summer temperatures. However, these changes occur largely in parallel along the coast (Schulte 2007), and as a result the difference in temperatures between habitats at the extreme northern and southern end of the species range for F. heteroclitus is present throughout the year. Another major difference between the northern and southern extremes of the species range is in the length of the reproductive and growing season. In general, the growing season at high latitudes is shorter than the growing season at low latitudes, which limits the time available for fish to reach the minimum size required for over-wintering. Consistent with this pattern, the northern subspecies breeds multiple times beginning in early–mid June through the end of July (McMullin et al. 2009), while the southern subspecies breeds over a substantially longer time period—from the beginning of February through the end of September, with an apparent semi-lunar periodicity (Kneib 1986). Fig 2 View largeDownload slide Latitudinal temperature gradient along the Atlantic Coast of North America in relation to the distribution of killifish subspecies. The northern subspecies, F. h. macrolepidotus, is found from southern Canada to northern New Jersey (blue). The southern subspecies F. h. heteroclitus is found from southern New Jersey to northern Florida (red), with a hybrid zone in central New Jersey (purple). Colors in the Atlantic Ocean represent mean sea surface temperature for the week of July 3–9, 2017. Data downloaded from http://www.cpc.ncep.noaa.gov/products/GIS/GIS_DATA/sst_oiv2/index.php. Fig 2 View largeDownload slide Latitudinal temperature gradient along the Atlantic Coast of North America in relation to the distribution of killifish subspecies. The northern subspecies, F. h. macrolepidotus, is found from southern Canada to northern New Jersey (blue). The southern subspecies F. h. heteroclitus is found from southern New Jersey to northern Florida (red), with a hybrid zone in central New Jersey (purple). Colors in the Atlantic Ocean represent mean sea surface temperature for the week of July 3–9, 2017. Data downloaded from http://www.cpc.ncep.noaa.gov/products/GIS/GIS_DATA/sst_oiv2/index.php. Temperature has a profound effect on the physiology of ecotherms, as biochemical and physiological processes are expected to slow by approximately two-fold for every 10°C decrease in temperature (Hochachka and Somero 2001). Thus, the direct effects of temperature would be expected to result in lower metabolic rates in organisms at high latitudes compared with those at low latitudes. However, compensatory adaptations to offset these negative effects of low temperature (Cossins and Bowler 1987) might be expected to result in faster growth and higher metabolic rate in cold-adapted northern forms when they are compared with warm-adapted forms at a common temperature. Thus, natural selection that counters the effects of temperature would result in a pattern where the observed phenotype under common temperatures in the laboratory is in the opposite direction to that predicted for the direct effect of temperature in natural environments—a phenomenon that has been termed countergradient variation (Conover and Schultz 1995). Countergradient variation in phenotypes such as growth rate has been detected in multiple species of fish along the Atlantic coast (Conover and Present 1990; Conover and Schultz 1995; Conover et al. 2009), suggesting that this may be a widespread phenomenon in this habitat. In addition to the direct effects of temperature on metabolic rates, the shorter reproductive and growing season at northern latitudes would also be expected to select for faster growth, faster developmental rate, and higher metabolic rate in northern populations compared with southern populations, reinforcing the action of direct selection for thermal compensation. Together, selection due to the lower temperature and shorter growing season in northern compared with southern habitats thus likely work together to shape the observed patterns of countergradient variation. Because these phenotypes are all key life history traits that involve rate processes, countergradient variation represents a specific case of life history evolution that affects the pace of life. The observed patterns of countergradient variation also imply that rapid growth and development are not favored under the environmental conditions present at low latitudes, suggesting the presence of costs of rapid development, or tradeoffs with other traits that are selected when temperatures are high and growing seasons are long. Thus, the idea of countergradient variation and the pace of life hypothesis are both implicitly built on a framework that stems from a perspective of tradeoffs among different life history strategies. Given the clear similarities between patterns of countergradient variation and the variation considered by the pace of life hypothesis, it is surprising that there has been little consideration of the potential inter-relationships between thermal adaptation, patterns of countergradient variation, and pace of life syndromes (although see Goulet et al. 2017). There are several features of the biology of Atlantic killifish which suggest that they have undergone local adaptation in response to the differences in temperature and seasonality along the Atlantic coast, and thus may be an interesting system in which to investigate the relationships among thermal physiology, countergradient variation, and the pace of life hypothesis. Atlantic killifish are the numerically and ecologically dominant fish species in Atlantic coast marshes in North America, such that many thousands of fish can easily be captured at a single marsh location (Sweeney et al. 1998). These large population sizes means that genetic drift is likely to play a relatively small role in shaping the genetics of these populations, and that even relatively weak selection has the potential to drive adaptive variation (Lanfear et al. 2014). Although Atlantic killifish can move fairly long distances up and down tidal creeks within a marsh with the movement of the tides as they feed (Sweeney et al. 1998; Teo and Able 2003; McMahon et al. 2005) they have also been shown to have small home ranges and high site fidelity in terms of breeding sites (Lotrich 1975; Skinner et al. 2005; Able et al. 2012). In addition, although there may be substantial movement of fish within a single marsh, there is likely only modest dispersal of fish among marshes along the coast as Atlantic killifish lay adherent eggs, and once hatched, the larval fish are unlikely to disperse in large numbers from marsh to marsh (Morin and Able 1983). As a result, there is modest but significant genetic differentiation among populations even for marshes located within a few tens of kilometers of each other along the coast (McKenzie et al. 2016; Wagner et al. 2017). At a larger geographic scale, there are latitudinal clines in many loci (Strand et al. 2012; McKenzie et al. 2016), including a very steep cline in mitochondrial genotype and a subset of nuclear genes (Gonzalez-Vilasenor and Powers 1990; McKenzie et al. 2016). For example, the mitochondrial genome transitions from a fixed northern genotype to a fixed southern genotype across about 100 km along the coast of New Jersey, and there are similar clines up the rivers of the Chesapeake Bay (Whitehead et al. 2012). The steep clines in nuclear and mitochondrial allele frequency, as well as consistent differences in egg and adult morphology (Morin and Able 1983; Able and Felley 1986; Bernardi et al. 1993) along the coast and in the rivers of the Chesapeake Bay support the designation of the northern and southern forms as subspecies that hybridize across a contact zone along the coast of New Jersey and in the Chesapeake Bay. There is substantial evidence to suggest that northern and southern Atlantic killifish exhibit countergradient variation in a variety of life history traits that are consistent with potentially adaptive variation in the pace of life (Fig. 3). Northern killifish appear to pursue a strategy in which they lay smaller (Marteinsdottir and Able 1988, 1992) but more numerous eggs (Fig. 3A; Bosker et al. 2013) compared with the southern killifish, which lay larger eggs but produce smaller clutches. In addition, time to hatch varies between northern and southern killifish (Fig. 3B), with northern fish developing faster than southern killifish when compared at a common temperature (DiMichele and Westerman 1997; McKenzie et al. 2017). These differences in developmental rate are likely associated with differences in embryonic metabolic rate, as embryos from intermediate latitudes that differ in allozyme genotypes differ in metabolic rate, with individuals having the northern genotype exhibiting higher metabolic rates than individuals having the southern genotype when compared at a common temperature (DiMichele and Powers 1991; DiMichele et al. 1991; Paynter et al. 1991). Similarly, growth rate after hatching also differs between northern and southern F. heteroclitus (Schultz et al. 1996), with fish from northern populations growing approximately 10–20% faster than fish from southern populations when compared at a common temperature. In adults, growth rate is strongly dependent on temperature (Healy and Schulte 2012), and the two subspecies have differing thermal optima for adult growth, but the patterns of changes in growth rate are such that northern and southern fish have similar growth rates when compared at their respective thermal optima, as would be predicted for a pattern of countergradient variation. Associated with these differences in growth, are differences in routine metabolic rate between the populations (Fig. 3C), with northern fish having higher metabolic rate than southern fish across a wide range of acclimation temperatures (Healy and Schulte 2012). There are similar differences in maximum capacities between the subspecies such that, compared with southern fish, northern fish have higher maximum metabolic rate (Healy and Schulte 2012) and higher maximum aerobic swimming speed (Fangue et al. 2008) measured as critical swimming velocity, Ucrit (Fig. 3D) across a range of acclimation temperatures. Taken together, these data demonstrate that northern killifish occupy the “fast” end of the pace of life continuum, relative to their southern counterparts. This difference in the intrinsic pace of life between the subspecies is predicted to have evolved to offset the negative effects of lower temperature and shorter growing season in the northern habitats. Indeed, modeling of development rates of killifish populations along the coast suggests that there may have been strong selection on this trait (Williamson and DiMichele 1997). Fig. 3 View largeDownload slide Variation in life history traits between killifish subspecies. (A) Clutch size (number of eggs per female). Data are from crosses of a group of five females from each subspecies across multiple spawning rounds (unpublished data from experiments presented in McKenzie et al. 2017). (B) Days to hatch at 21°C, 20 ppt salinity (McKenzie et al. 2017). (C) Routine metabolic rate at 15°C, 20 ppt salinity (Healy and Schulte 2012). (D) Critical swimming speed (Ucrit) in body lengths (BL)/second for fish acclimated to 15°C, 20 ppt salinity (Fangue et al. 2008). Fig. 3 View largeDownload slide Variation in life history traits between killifish subspecies. (A) Clutch size (number of eggs per female). Data are from crosses of a group of five females from each subspecies across multiple spawning rounds (unpublished data from experiments presented in McKenzie et al. 2017). (B) Days to hatch at 21°C, 20 ppt salinity (McKenzie et al. 2017). (C) Routine metabolic rate at 15°C, 20 ppt salinity (Healy and Schulte 2012). (D) Critical swimming speed (Ucrit) in body lengths (BL)/second for fish acclimated to 15°C, 20 ppt salinity (Fangue et al. 2008). The role of mitochondria Many of the differences between the northern and southern subspecies of Atlantic killifish are associated with differences in aerobic metabolic processes, suggesting that differences in mitochondrial function could play an important role in shaping the differences in the pace of life between these two subspecies. We have shown that the maximum capacity for oxidative phosphorylation of mitochondria isolated from liver is greater in northern killifish than in southern killifish (Fig. 4A, B) at both cold (A) and warm (B) acclimation temperatures (Fangue et al. 2009; Chung et al. 2018), which suggests that mitochondria may play a role in setting or supporting at least some of the observed differences in pace of life. However, this pattern is not evident when mitochondria from brain or heart tissue are compared, as we detect no difference between the populations in maximal mitochondrial respiration in these tissues (Chung et al. 2017a; Fig. 4C–F). Similarly others have detected few differences in mitochondrial respiration in heart tissue between the northern and southern subspecies at higher (28°C) acclimation temperatures (Baris et al. 2016), and these authors in fact detected significantly higher respiration in heart tissue from southern killifish than in northern killifish at an acclimation temperature of 12°C (Baris et al. 2016). However, it should be noted that our studies in liver utilized isolated mitochondria, whereas the studies in heart and brain have used a permeabilized tissue preparation, and these two types of methods can yield somewhat different results (Brand and Nicholls 2011; Mathers and Staples 2015). Fig. 4 View largeDownload slide Maximal ADP phosphorylating respiration rate in tissues of killifish acclimated to 5 (panels A, C, E) or 15°C (panels B, D, F). (A, B) Isolated mitochondria from liver. (C, D) Permeabilized tissue preparations from heart. (E, F) Permeabilized tissue preparations from brain. Gray bars = fish of the northern subspecies. Open bars = fish of the southern subspecies. ** indicates significant differences (P < 0.01) between the subspecies at a given acclimation temperature. All preparations provided with pyruvate, malate, and glutamate as substrates. All preparations were assayed at the acclimation temperature (Chung et al. 2017a, 2018). Fig. 4 View largeDownload slide Maximal ADP phosphorylating respiration rate in tissues of killifish acclimated to 5 (panels A, C, E) or 15°C (panels B, D, F). (A, B) Isolated mitochondria from liver. (C, D) Permeabilized tissue preparations from heart. (E, F) Permeabilized tissue preparations from brain. Gray bars = fish of the northern subspecies. Open bars = fish of the southern subspecies. ** indicates significant differences (P < 0.01) between the subspecies at a given acclimation temperature. All preparations provided with pyruvate, malate, and glutamate as substrates. All preparations were assayed at the acclimation temperature (Chung et al. 2017a, 2018). Ultimately, we observe clear subspecies differences in mitochondrial function in the liver, and there is little evidence for effects in other tissues. Thus, if mitochondrial function is playing a role in establishing at least some of the differences in the pace of life between northern and southern killifish, then these effects are most likely taking place in liver. The liver stores, mobilizes, and inter-converts energy reserves, and thus the metabolic activities of the liver are essential for providing fuel to other critical tissues such as the brain and muscle. Thus, the biochemical functions of the liver can be plausibly connected to differences in whole-organism standard metabolic rate in adult fish, and thus to other processes related to the pace of life such as growth rate. Variation in liver mitochondrial properties (specifically in cytochrome C oxidase activity and citrate synthase activity) has previously been shown to correlate with variation in standard metabolic rate and maximum metabolic rate in Brown Trout (Norin and Malte 2012), suggesting the importance of liver mitochondrial metabolism in setting metabolic rate across fish species. However, variation in mitochondrial properties in adult liver tissue is less likely to play a role in establishing differences in maximum aerobic swimming performance or early developmental rates, suggesting that the variation that we observe provides only a partial explanation for the variation in the full suite of traits associated with variation in the pace of life in killifish. Mechanisms of variation in mitochondrial properties It is possible to interrogate the role of the different respiratory complexes in establishing the differences in mitochondrial capacity between northern and southern killifish by assessing flux using different substrates, and by testing function in the presence of inhibitors of various parts of the ETS (Pesta and Gnaiger 2012). Using these approaches (Chung et al. 2017b, 2018), we find that multiple, and perhaps all, components of the ETS have higher activity in liver mitochondria in northern than in southern killifish. The relatively parallel increases in the activities of the ETS components in northern compared with southern killifish could reflect a greater amount of inner mitochondrial membrane per mitochondrion with a concomitant increase in ETS components in northern killifish, or a greater density of ETS components per unit inner mitochondrial membrane. To begin to distinguish between these possibilities, we measured the amount of cardiolipin per milligram liver mitochondrial protein in both northern and southern killifish held under common conditions in the laboratory to ensure a common diet. Cardiolipin is an unusual phospholipid that contains four acyl chains attached to the head group. This phospholipid is found almost exclusively in the inner mitochondrial membrane in eukaryotic cells (Hoch 1992). As such, cardiolipin amount can provide an estimate of the amount of inner mitochondrial membrane per mitochondrion (Larsen et al. 2012), if the amount of cardiolipin per unit membrane is assumed to be constant. As can be seen from Fig. 5, northern killifish have a greater amount of cardiolipin per milligram mitochondrial protein than do southern killifish, suggesting that they may have a greater surface area of inner mitochondrial membrane per mitochondrion (or possibly a higher cardiolipin concentration per unit inner mitochondrial membrane), which could account for the largely parallel increases in the activities of the various respiratory complexes in northern killifish compared with southern killifish. Interestingly, similar patterns in cardiolipin amount have been detected in comparisons of tropical and temperate birds (Calhoon et al. 2014; Jimenez et al. 2014), with higher levels in the animals from temperate climates that are correlated with differences in cellular metabolic rate. These similarities across very diverse taxa suggest the potential importance of mitochondrial membrane amount or mitochondrial membrane properties as a correlate of variation in the pace of life. This observation has some parallels with the membrane pacemaker theory of aging (Hulbert 2010), which suggests that membrane composition may be an important correlate of aging and lifespan. However, the specific role of the mitochondrial membrane, and of cardiolipin content, in the membrane pacemaker theory of aging remains poorly understood (Valencak and Azzu 2014), and thus further research will be necessary before it is possible to unify these two somewhat distinct hypotheses. Fig. 5 View largeDownload slide Total cardiolipin content in mitochondria isolated from the liver of killifish acclimated to 5 or 15°C. Total cardiolipin is the sum of cardiolipin plus monolysocardiolipin expressed per milligram mitochondrial protein in isolated mitochondrial preparations from liver (Chung et al. 2018). Gray bars = fish from the northern subspecies, open bars = fish from the southern subspecies. * indicates a significant difference between the subspecies (P < 0.05) at a given acclimation temperature. Different letters indicate significant differences (P < 0.05) between acclimation temperatures within a subspecies. Fig. 5 View largeDownload slide Total cardiolipin content in mitochondria isolated from the liver of killifish acclimated to 5 or 15°C. Total cardiolipin is the sum of cardiolipin plus monolysocardiolipin expressed per milligram mitochondrial protein in isolated mitochondrial preparations from liver (Chung et al. 2018). Gray bars = fish from the northern subspecies, open bars = fish from the southern subspecies. * indicates a significant difference between the subspecies (P < 0.05) at a given acclimation temperature. Different letters indicate significant differences (P < 0.05) between acclimation temperatures within a subspecies. Figure 5 also suggests that northern killifish may increase inner mitochondrial membrane amount substantially with cold acclimation in liver, whereas only modest changes occur in southern killifish. This observation is consistent with previous work that used electron microscopy to estimate the surface area of the inner mitochondrial membrane in muscle tissues (Dhillon and Schulte 2011). This study demonstrated that mitochondrial volume density and cristae surface area in both red and white muscle increased with cold acclimation in northern but not southern killifish, although the functional consequences of this variation remain unknown. Using RNA-seq we have also detected significant differential regulation of genes involved in mitochondrial fission and fusion in cold-acclimated killifish muscle (Healy et al. 2017), suggesting that there may be changes in mitochondrial morphology in response to cold acclimation as well. Taken together these data suggest that there may be differences between killifish subspecies in the effects of cold on the regulation of mitochondrial structure and function in multiple tissues. In addition to differences between subspecies in inner mitochondrial membrane amount or cardiolipin content, the relative proportions of different membrane phospholipids are not identical between the subspecies (Chung et al. 2018). Changes in the phospholipid composition of biological membranes can alter their properties, and in particular may influence membrane fluidity (Hazel 1995). This may be particularly relevant in the context of thermal acclimation and adaptation, as temperature alters the fluidity of biological membranes, and changes in membrane lipid composition can be used to compensate for these effects (Hazel and Prosser 1974; Hazel 1995). Using a combination of liquid chromatography (LC) with ultraviolet (UV) detection followed by gas-chromatography with flame ionization detection (GC-FID) and high-performance liquid chromatography (HPLC) prior to electro-spray ionization mass spectrometry (ESI/MS) we performed high resolution lipid analysis of the mitochondrial membranes (both inner and outer membranes combined). This produces a set of more than 60 inter-related variables expressing the percentage of various lipid head group and acyl chain combination in the total membrane fraction (Chung et al. 2018). Principle component analysis (PCA) of these data clearly indicates that there is an effect of acclimation temperature on mitochondrial membrane lipid profile, and that there are differences in membrane composition between the subspecies (Fig. 6). Fig. 6 View largeDownload slide Variation in mitochondrial membrane lipid composition between killifish subspecies. Principle component analysis of data from high-resolution lipid analysis of northern (blue) and southern (red) killifish acclimated to either 5 (circles) or 15°C (triangles). PC1 and PC3 are shown. PC2 separates based on inter-individual variation (Chung et al. 2018). Fig. 6 View largeDownload slide Variation in mitochondrial membrane lipid composition between killifish subspecies. Principle component analysis of data from high-resolution lipid analysis of northern (blue) and southern (red) killifish acclimated to either 5 (circles) or 15°C (triangles). PC1 and PC3 are shown. PC2 separates based on inter-individual variation (Chung et al. 2018). Membrane composition can affect mitochondrial properties in a number of ways. First, membrane composition can affect membrane fluidity, which can affect the activities of proteins embedded within the membrane (Hazel 1995), such as the proteins of the ETS. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) comprise the majority of the phospholipids present in mitochondrial membranes (Mejia and Hatch 2016). Changes in the relative proportions of these two phospholipids (the PC/PE ratio) have been demonstrated to be associated with changes in membrane fluidity (Cullis et al. 1996). However, there were no significant differences between the northern and southern subspecies in PC/PE, although there were some differences in the way this indicator changed with thermal acclimation between the two subspecies (Chung et al. 2018). Similarly, the number of double bonds in the acyl chains of phospholipids and their chain length can influence membrane fluidity (Cullis et al. 1996). However, there were no differences in the global unsaturation index or chain length between the mitochondrial membranes of northern and southern killifish, and these parameters did not change with acclimation temperature (Chung et al. 2018). Despite the fact that there were few differences between subspecies in the broad measures of membrane composition that correlate with membrane fluidity, there were many differences between subspecies in the amounts of specific phospholipids in the membrane (Fig. 6). Of particular interest are differences between the subspecies in cardiolipin composition, because cardiolipin plays a key functional role in a variety of mitochondrial processes. For example, cardiolipin has been shown to be necessary for the optimal functioning of complexes I, III, IV, and V (Paradies et al. 2014), and it also acts as a “proton trap” (Haines and Dencher 2002) that localizes pumped protons close to the inner mitochondrial membrane. There were differences in the relative proportions of several cardiolipin species between northern and southern killifish, which suggests that there could be effects of these changes in membrane composition on ETS function. However, the functional significance of changes in cardiolipin species in mitochondrial membranes remains enigmatic (Ye et al. 2016), and thus the effects of the observed changes in cardiolipin species between northern and southern killifish are not yet known. These data suggest that there are specific differences in the composition of liver mitochondrial membranes between killifish subspecies that could play a role in determining the functional properties of the mitochondria. In addition, the data on cardiolipin content suggest that there may be substantial differences in the amount of the inner mitochondrial membrane per mitochondrion, or of the amount of cardiolipin per unit membrane. These differences in liver inner mitochondrial membrane amount and respiratory chain activity are consistent with the greater adult standard metabolic rate in northern compared with southern killifish, and suggest a potential role for variation in liver mitochondrial properties in at least some aspects of variation in the pace of life between the subspecies. As discussed previously, this is consistent with previous work in Brown Trout that detected correlations between liver cytochrome C oxidase and both standard and maximum metabolic rate (Norin and Malte 2012). In addition, these measures are just an initial examination of the variety of mitochondrial properties that could contribute to variation in either standard or maximum metabolic rate, and to variation in the pace of life. In particular, little is currently known about the relative coupling efficiency, leak respiration, or ROS generation between killifish subspecies, although these parameters may be particularly critical in establishing the tradeoffs at the cellular level that could lead to the tradeoffs that shape the pace of life continuum (Janssens and Stoks 2018). Although we detect differences in mitochondrial properties between the subspecies in the livers of adult fish, these differences are not likely to account for the observed differences in developmental rate between F. heteroclitus subspecies. Nothing is known about mitochondrial function in the developing embryos of F. heteroclitus, so the role of the mitochondria in variation in rate processes in these fish prior to hatch remains. There are, however, some hints that mitochondrial processes could also be important in metabolic rate variation in killifish embryos, as embryos with a northern genotype at the enzyme malate dehydrogenase A (MDH-A) develop faster than those with the southern genotype at this enzyme (DiMichele and Powers 1991). MDH-A is the cytoplasmic form of the enzyme, which participates in the malate–aspartate shuttle that translocates electrons produced by glycolysis into the mitochondrion where they can be transferred to the ETS. Thus, the role of variation in mitochondrial properties in early developmental stages remains a fruitful and largely unexplored avenue that could yield insights into the biochemical mechanisms underlying variation in life history strategies. Conclusions and future directions The results from the studies presented here suggest that variation in mitochondrial properties could be associated with at least some aspects of variation in the pace of life in Atlantic killifish. More broadly, these findings emphasize the intimate link between thermal adaptation, metabolism, and life history variation in the context of the pace of life, and reveal a potential role for variation in mitochondrial membrane structure and function as a physiological mechanism underlying this variation. However, many important questions remain to be addressed. Although we present evidence that variation in liver mitochondrial function between killifish subspecies is in the direction expected based on observed differences in growth rate and metabolic rate, little is known about the potential for variation in mitochondrial function during early embryogenesis in this species, and whether it could account for observed differences in developmental rate. Similarly, the potential costs of increased mitochondrial metabolism in northern killifish are unknown. Studies in other systems suggest that a rapid pace of life may come at a cost in terms of increased oxidative stress (Janssens and Stoks 2018), but the relative levels of oxidative damage in northern and southern killifish have not been examined, although variation in a variety of oxidative stress parameters among killifish populations with differing levels of contaminant exposure has been detected (Bacanskas et al. 2004). Also, little is known about variation in metabolic rate and mitochondrial properties within populations of killifish, and whether the trends observed when comparing the northern and southern subspecies are also apparent when within-population variation is examined. Killifish populations from the center of the intergrade zone between the northern and southern subspecies might present a particularly interesting experimental system in which to test these ideas, and to examine the genetic basis of variation in mitochondrial properties, metabolic rate, and the pace of life. More broadly, the lessons learned from the killifish system point to a series of important questions that need to be answered when considering the role of mitochondrial processes in variation of the pace of life, regardless of the experimental system. First, it is important to recognize that there can be substantial tissue to tissue variation in mitochondrial properties, and thus the connection between mitochondrial function and whole organism metabolism and the pace of life is likely to be complex. The data presented here suggest that liver tissue may be important in establishing whole organism standard metabolic rate, but this observation needs to be repeated across multiple species to determine whether this represents a consistent pattern. Second, the pace of life hypothesis includes life history traits that are expressed at various life stages (e.g., development rate, growth rate, and longevity). At present, it is not known whether variation in mitochondrial properties in early development is correlated with variation in mitochondrial properties in adult organisms, or whether mitochondrial function changes across ontogeny. A better understanding of these patterns is critical in order to fully assess the role of variation in mitochondrial properties in life history variation and the pace of life. However, despite these remaining questions, the data presented here highlight the potential role of the mitochondria in thermal adaptation and in life history evolution in ectotherms, and demonstrate the commonalities among important patterns of life history variation such as countergradient variation and variation in the pace of life. Acknowledgment The authors thank Alex Cooper for the design and production of Fig. 1. Funding This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Discovery Grant [to P.M.S.] and a U.S. Department of Agriculture National Institute of Food and Agriculture and an American Heart Association grant [to A.J.C.] and via Michael Smith Travelling Fellowship [to D.J.C.]. References Able KW , Felley JD. 1986 . Geographical variation in Fundulus heteroclitus: tests for concordance between egg and adult morphologies . Integr Comp Biol 26 : 145 – 57 . Able KW , Vivian DN , Petruzzelli G , Hagan SM. 2012 . 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Abstract

Abstract Life history strategies, physiological traits, and behavior are thought to covary along a “pace of life” axis, with organisms at the fast end of this continuum having higher fecundity, shorter lifespan, and more rapid development, growth, and metabolic rates. Countergradient variation represents a special case of pace of life variation, in which high-latitude organisms occupy the fast end of the continuum relative to low-latitude conspecifics when compared at a common temperature. Here, we use Atlantic killifish (Fundulus heteroclitus) to explore the role of mitochondrial properties as a mechanism underlying countergradient variation, and thus variation in the pace of life. This species is found along the Atlantic coast of North America, through a steep latitudinal thermal gradient. The northern subspecies has faster development, more rapid growth, higher routine metabolic rate, and higher activity than the southern subspecies when compared at a common temperature. The northern subspecies also has greater mitochondrial respiratory capacity in the liver, although these differences are not evident in other tissues. The increased respiratory capacity of liver mitochondria in northern fish is associated with increases in the activity of multiple electron transport complexes, which largely reflects an increase in the amount of inner mitochondrial membrane per mitochondrion in the northern fish. There are also differences in the lipid composition of liver mitochondrial membranes, including differences in cardiolipin species, which could also influence respiratory capacity. These data suggest that variation in mitochondrial properties could, at least in part, underlie variation in the pace of life in Atlantic killifish. Introduction Species and populations within species exhibit substantial variation in life history traits such as fecundity, hatching success, developmental rate, growth rate, and size at maturity (Roff 1992). However, variation in these traits does not occur in all possible combinations in nature (Stearns 1976, 1992). Instead, it has been proposed to occur along a slow-to-fast continuum, with species or populations with low reproductive rate, slow development, and long lifespan at one end of the continuum, and those with faster reproductive rates and development, but shorter lifespan at the other (Ricklefs and Wikelski 2002). As such, the pace of life hypothesis has its foundation in the classic concept of “r” and “K” selection (MacArthur and Wilson 1967; Pianka 1970). The pace of life concept has been extended to include a variety of physiological and behavioral traits (Reale et al. 2010), suggesting the existence of a complex syndrome that links multiple traits across levels of biological organization to variation in life history strategies. These observed differences in the apparent pace of life and the associated physiological and behavioral traits (Reale et al. 2010) represent different allocation choices among investments in reproduction, growth, and survivorship (Stearns 1976, 1992) resulting from trade-offs due to the allocation of limited resources among different functions (Stearns 1992; Zera and Harshman 2001). The pace of life concept has inspired substantial research effort and has been the subject of multiple recent syntheses (see e.g., Dammhahn et al. 2018), but its validity and underlying mechanistic basis are still a matter of vigorous debate (Royauté et al. 2018). The purpose of the present review is to examine this concept in the context of thermal physiology, and specifically to explore the potential role of variation in mitochondrial function as a mechanism underlying variation in the pace of life as an adaptation to environmental temperature. Variation in metabolic rate is thought to play an important role as either a cause or consequence of variation in the pace of life (Ricklefs and Wikelski 2002; Wiersma et al. 2007; Williams et al. 2010; Glazier 2015), as individuals on the fast end of the pace of life spectrum tend to have higher resting metabolic rates, even after differences in body size have been accounted for (Ricklefs and Wikelski 2002). Indeed, in an elegant study with Trinidadian guppies, Auer et al. (2018) showed that standard metabolic rate had a strong positive correlation with a suite of traits associated with the pace of life (such as male age and mass at maturity, female age and mass at first parturition, inter-litter interval, and reproductive allotment) in natural populations exposed to differing predation pressure, and that evolutionary change in the pace of life following experimental transplant to sites with different predation pressure was accompanied by changes in metabolic rate. These studies demonstrate the potential for a tight association between variation in metabolic rate and the pace of life, and they highlight the evolutionary importance of these associations. However, the underlying physiological and biochemical mechanisms that cause variation in metabolic rate, and thus potentially in the pace of life, are not fully known and may include processes acting at many levels of biological organization (Versteegh et al. 2012; Konarzewski and Książek 2013; Wone et al. 2013; Pettersen et al. 2018). One cellular process that could play a key role in influencing the pace of life is mitochondrial oxidative phosphorylation (Fig. 1). Mitochondria are the principle source of ATP generation in animal cells, and thus are intimately involved in nutrient and energy metabolism. Aerobic ATP production in the mitochondrion occurs via oxidative phosphorylation, which is accomplished by the proteins of the mitochondrial electron transport system (ETS) and the ATP synthase (Complex V), which are a series of membrane-spanning or membrane-associated proteins located on the inner mitochondrial membrane. The proteins of the ETS accept electrons from NADH and FADH2 at complexes I and II of the ETS, respectively. These electrons are then transported through the ETS, and this electron transport process results in the translocation of protons (H+) by complexes I, III, and IV from the mitochondrial matrix into the intermembrane space. The resulting proton gradient is harnessed by the ATP synthase to produce ATP. Note that this process is not perfectly efficient, as protons can also move across the inner mitochondrial membrane through a variety of pathways such as the uncoupling proteins and the adenine nucleotide translocase, or even through the membrane itself (Jastroch et al. 2007), bypassing the ATP synthase, which results in the dissipation of the proton gradient without the synthesis of ATP. Fig. 1 View largeDownload slide Mitochondrial electron transport and ATP synthesis. The electron transport system (ETS) consists of a series of membrane associated protein complexes (Complexes I–IV) embedded in the inner mitochondrial membrane that separates the mitochondrial matrix and inter-membrane space (IMS). Electron carriers transfer electrons primarily to complexes I and II, which then passes them to coenzyme Q, a fat-soluble molecule that is present within the lipid phase of the membrane. Coenzyme Q then passes the electrons to Complex III, and they are ultimately transferred to oxygen through the action of Complex IV (cytochrome c oxidase). The transfer of electrons through the ETS is associated with the translocation of protons (H+) into the IMS. The resulting proton (ΔP) and electrical (Ψm) gradient is used by Complex V (ATP synthase) to synthesize ATP. Protons can also pass through the membrane through a variety of leak pathways such as uncoupling proteins (UCP) and the adenine nucleotide translocator (ANT) that do not result in ATP synthesis. Fig. 1 View largeDownload slide Mitochondrial electron transport and ATP synthesis. The electron transport system (ETS) consists of a series of membrane associated protein complexes (Complexes I–IV) embedded in the inner mitochondrial membrane that separates the mitochondrial matrix and inter-membrane space (IMS). Electron carriers transfer electrons primarily to complexes I and II, which then passes them to coenzyme Q, a fat-soluble molecule that is present within the lipid phase of the membrane. Coenzyme Q then passes the electrons to Complex III, and they are ultimately transferred to oxygen through the action of Complex IV (cytochrome c oxidase). The transfer of electrons through the ETS is associated with the translocation of protons (H+) into the IMS. The resulting proton (ΔP) and electrical (Ψm) gradient is used by Complex V (ATP synthase) to synthesize ATP. Protons can also pass through the membrane through a variety of leak pathways such as uncoupling proteins (UCP) and the adenine nucleotide translocator (ANT) that do not result in ATP synthesis. Oxygen is the final electron acceptor along the ETS and is thus critical for the continued functioning of the ETS and oxidative phosphorylation. However, under certain circumstances (Murphy 2009) oxygen can also interact with electron carriers at various steps along the ETS, forming superoxide, a type of reactive oxygen species (ROS). ROS production by mitochondria can result in oxidative damage to mitochondrial membranes, proteins, and DNA. Damage is not restricted to the mitochondrion, as these ROS can also be released into the cell and cause widespread damage, which is thought to be a major determinant of cellular aging, and thus of lifespan (Selman et al. 2012). However, ROS production is not the only determinant of the extent of oxidative damage. Cells have multiple efficient mechanisms to buffer ROS production through a variety of non-enzymatic and enzymatic antioxidants (Birben et al. 2012). The extent of oxidative stress experienced by an organism, and thus the potential for oxidative damage, will reflect the balance between the production of ROS and the extent of the antioxidant mechanisms. Thus, it is likely that this balance between ROS production and antioxidant defenses is an important component of the tradeoffs that are thought to underlie variation in the pace of life (Monaghan et al. 2009). The fact that the mitochondrion plays a key role both in the mechanisms underlying aerobic metabolic rate and in ROS production suggests that this organelle may be a critical nexus of life-history evolution that could play a role in establishing the trade-offs that underlie the pace of life hypothesis (Speakman et al. 2015; Janssens and Stoks 2018). Killifish and the pace of life A number of observations suggest that the Atlantic killifish, Fundulus heteroclitus, is an interesting system in which to investigate the role of the mitochondria in variation in the pace of life. Populations of Atlantic killifish are found in marshes and estuaries along the east coast of North America from as far north as the Bay of Islands in Newfoundland (Dickinson and Threlfall 1975) to just south of Jacksonville, Florida, where this species is replaced by the closely related congener, Fundulusgrandis (Gonzalez et al. 2009). There are two recognized subspecies of F. heteroclitus, which have been designated F. heteroclitus macrolepidotus in the northern part of the range, and F. heteroclitus heteroclitus in the southern part of the range (Morin and Able 1983; Jordan and Everman 1896). One of the striking environmental features of the habitat of Atlantic killifish is that mean environmental temperature varies steeply along the Atlantic coast (Fig. 2), such that the extreme northern populations of F. heteroclitus experience a mean monthly temperature approximately 10°C lower than do the extreme southern populations. There is also substantial seasonal variation in temperature, such that in any given location, winter temperatures are typically about 10°C lower than summer temperatures. However, these changes occur largely in parallel along the coast (Schulte 2007), and as a result the difference in temperatures between habitats at the extreme northern and southern end of the species range for F. heteroclitus is present throughout the year. Another major difference between the northern and southern extremes of the species range is in the length of the reproductive and growing season. In general, the growing season at high latitudes is shorter than the growing season at low latitudes, which limits the time available for fish to reach the minimum size required for over-wintering. Consistent with this pattern, the northern subspecies breeds multiple times beginning in early–mid June through the end of July (McMullin et al. 2009), while the southern subspecies breeds over a substantially longer time period—from the beginning of February through the end of September, with an apparent semi-lunar periodicity (Kneib 1986). Fig 2 View largeDownload slide Latitudinal temperature gradient along the Atlantic Coast of North America in relation to the distribution of killifish subspecies. The northern subspecies, F. h. macrolepidotus, is found from southern Canada to northern New Jersey (blue). The southern subspecies F. h. heteroclitus is found from southern New Jersey to northern Florida (red), with a hybrid zone in central New Jersey (purple). Colors in the Atlantic Ocean represent mean sea surface temperature for the week of July 3–9, 2017. Data downloaded from http://www.cpc.ncep.noaa.gov/products/GIS/GIS_DATA/sst_oiv2/index.php. Fig 2 View largeDownload slide Latitudinal temperature gradient along the Atlantic Coast of North America in relation to the distribution of killifish subspecies. The northern subspecies, F. h. macrolepidotus, is found from southern Canada to northern New Jersey (blue). The southern subspecies F. h. heteroclitus is found from southern New Jersey to northern Florida (red), with a hybrid zone in central New Jersey (purple). Colors in the Atlantic Ocean represent mean sea surface temperature for the week of July 3–9, 2017. Data downloaded from http://www.cpc.ncep.noaa.gov/products/GIS/GIS_DATA/sst_oiv2/index.php. Temperature has a profound effect on the physiology of ecotherms, as biochemical and physiological processes are expected to slow by approximately two-fold for every 10°C decrease in temperature (Hochachka and Somero 2001). Thus, the direct effects of temperature would be expected to result in lower metabolic rates in organisms at high latitudes compared with those at low latitudes. However, compensatory adaptations to offset these negative effects of low temperature (Cossins and Bowler 1987) might be expected to result in faster growth and higher metabolic rate in cold-adapted northern forms when they are compared with warm-adapted forms at a common temperature. Thus, natural selection that counters the effects of temperature would result in a pattern where the observed phenotype under common temperatures in the laboratory is in the opposite direction to that predicted for the direct effect of temperature in natural environments—a phenomenon that has been termed countergradient variation (Conover and Schultz 1995). Countergradient variation in phenotypes such as growth rate has been detected in multiple species of fish along the Atlantic coast (Conover and Present 1990; Conover and Schultz 1995; Conover et al. 2009), suggesting that this may be a widespread phenomenon in this habitat. In addition to the direct effects of temperature on metabolic rates, the shorter reproductive and growing season at northern latitudes would also be expected to select for faster growth, faster developmental rate, and higher metabolic rate in northern populations compared with southern populations, reinforcing the action of direct selection for thermal compensation. Together, selection due to the lower temperature and shorter growing season in northern compared with southern habitats thus likely work together to shape the observed patterns of countergradient variation. Because these phenotypes are all key life history traits that involve rate processes, countergradient variation represents a specific case of life history evolution that affects the pace of life. The observed patterns of countergradient variation also imply that rapid growth and development are not favored under the environmental conditions present at low latitudes, suggesting the presence of costs of rapid development, or tradeoffs with other traits that are selected when temperatures are high and growing seasons are long. Thus, the idea of countergradient variation and the pace of life hypothesis are both implicitly built on a framework that stems from a perspective of tradeoffs among different life history strategies. Given the clear similarities between patterns of countergradient variation and the variation considered by the pace of life hypothesis, it is surprising that there has been little consideration of the potential inter-relationships between thermal adaptation, patterns of countergradient variation, and pace of life syndromes (although see Goulet et al. 2017). There are several features of the biology of Atlantic killifish which suggest that they have undergone local adaptation in response to the differences in temperature and seasonality along the Atlantic coast, and thus may be an interesting system in which to investigate the relationships among thermal physiology, countergradient variation, and the pace of life hypothesis. Atlantic killifish are the numerically and ecologically dominant fish species in Atlantic coast marshes in North America, such that many thousands of fish can easily be captured at a single marsh location (Sweeney et al. 1998). These large population sizes means that genetic drift is likely to play a relatively small role in shaping the genetics of these populations, and that even relatively weak selection has the potential to drive adaptive variation (Lanfear et al. 2014). Although Atlantic killifish can move fairly long distances up and down tidal creeks within a marsh with the movement of the tides as they feed (Sweeney et al. 1998; Teo and Able 2003; McMahon et al. 2005) they have also been shown to have small home ranges and high site fidelity in terms of breeding sites (Lotrich 1975; Skinner et al. 2005; Able et al. 2012). In addition, although there may be substantial movement of fish within a single marsh, there is likely only modest dispersal of fish among marshes along the coast as Atlantic killifish lay adherent eggs, and once hatched, the larval fish are unlikely to disperse in large numbers from marsh to marsh (Morin and Able 1983). As a result, there is modest but significant genetic differentiation among populations even for marshes located within a few tens of kilometers of each other along the coast (McKenzie et al. 2016; Wagner et al. 2017). At a larger geographic scale, there are latitudinal clines in many loci (Strand et al. 2012; McKenzie et al. 2016), including a very steep cline in mitochondrial genotype and a subset of nuclear genes (Gonzalez-Vilasenor and Powers 1990; McKenzie et al. 2016). For example, the mitochondrial genome transitions from a fixed northern genotype to a fixed southern genotype across about 100 km along the coast of New Jersey, and there are similar clines up the rivers of the Chesapeake Bay (Whitehead et al. 2012). The steep clines in nuclear and mitochondrial allele frequency, as well as consistent differences in egg and adult morphology (Morin and Able 1983; Able and Felley 1986; Bernardi et al. 1993) along the coast and in the rivers of the Chesapeake Bay support the designation of the northern and southern forms as subspecies that hybridize across a contact zone along the coast of New Jersey and in the Chesapeake Bay. There is substantial evidence to suggest that northern and southern Atlantic killifish exhibit countergradient variation in a variety of life history traits that are consistent with potentially adaptive variation in the pace of life (Fig. 3). Northern killifish appear to pursue a strategy in which they lay smaller (Marteinsdottir and Able 1988, 1992) but more numerous eggs (Fig. 3A; Bosker et al. 2013) compared with the southern killifish, which lay larger eggs but produce smaller clutches. In addition, time to hatch varies between northern and southern killifish (Fig. 3B), with northern fish developing faster than southern killifish when compared at a common temperature (DiMichele and Westerman 1997; McKenzie et al. 2017). These differences in developmental rate are likely associated with differences in embryonic metabolic rate, as embryos from intermediate latitudes that differ in allozyme genotypes differ in metabolic rate, with individuals having the northern genotype exhibiting higher metabolic rates than individuals having the southern genotype when compared at a common temperature (DiMichele and Powers 1991; DiMichele et al. 1991; Paynter et al. 1991). Similarly, growth rate after hatching also differs between northern and southern F. heteroclitus (Schultz et al. 1996), with fish from northern populations growing approximately 10–20% faster than fish from southern populations when compared at a common temperature. In adults, growth rate is strongly dependent on temperature (Healy and Schulte 2012), and the two subspecies have differing thermal optima for adult growth, but the patterns of changes in growth rate are such that northern and southern fish have similar growth rates when compared at their respective thermal optima, as would be predicted for a pattern of countergradient variation. Associated with these differences in growth, are differences in routine metabolic rate between the populations (Fig. 3C), with northern fish having higher metabolic rate than southern fish across a wide range of acclimation temperatures (Healy and Schulte 2012). There are similar differences in maximum capacities between the subspecies such that, compared with southern fish, northern fish have higher maximum metabolic rate (Healy and Schulte 2012) and higher maximum aerobic swimming speed (Fangue et al. 2008) measured as critical swimming velocity, Ucrit (Fig. 3D) across a range of acclimation temperatures. Taken together, these data demonstrate that northern killifish occupy the “fast” end of the pace of life continuum, relative to their southern counterparts. This difference in the intrinsic pace of life between the subspecies is predicted to have evolved to offset the negative effects of lower temperature and shorter growing season in the northern habitats. Indeed, modeling of development rates of killifish populations along the coast suggests that there may have been strong selection on this trait (Williamson and DiMichele 1997). Fig. 3 View largeDownload slide Variation in life history traits between killifish subspecies. (A) Clutch size (number of eggs per female). Data are from crosses of a group of five females from each subspecies across multiple spawning rounds (unpublished data from experiments presented in McKenzie et al. 2017). (B) Days to hatch at 21°C, 20 ppt salinity (McKenzie et al. 2017). (C) Routine metabolic rate at 15°C, 20 ppt salinity (Healy and Schulte 2012). (D) Critical swimming speed (Ucrit) in body lengths (BL)/second for fish acclimated to 15°C, 20 ppt salinity (Fangue et al. 2008). Fig. 3 View largeDownload slide Variation in life history traits between killifish subspecies. (A) Clutch size (number of eggs per female). Data are from crosses of a group of five females from each subspecies across multiple spawning rounds (unpublished data from experiments presented in McKenzie et al. 2017). (B) Days to hatch at 21°C, 20 ppt salinity (McKenzie et al. 2017). (C) Routine metabolic rate at 15°C, 20 ppt salinity (Healy and Schulte 2012). (D) Critical swimming speed (Ucrit) in body lengths (BL)/second for fish acclimated to 15°C, 20 ppt salinity (Fangue et al. 2008). The role of mitochondria Many of the differences between the northern and southern subspecies of Atlantic killifish are associated with differences in aerobic metabolic processes, suggesting that differences in mitochondrial function could play an important role in shaping the differences in the pace of life between these two subspecies. We have shown that the maximum capacity for oxidative phosphorylation of mitochondria isolated from liver is greater in northern killifish than in southern killifish (Fig. 4A, B) at both cold (A) and warm (B) acclimation temperatures (Fangue et al. 2009; Chung et al. 2018), which suggests that mitochondria may play a role in setting or supporting at least some of the observed differences in pace of life. However, this pattern is not evident when mitochondria from brain or heart tissue are compared, as we detect no difference between the populations in maximal mitochondrial respiration in these tissues (Chung et al. 2017a; Fig. 4C–F). Similarly others have detected few differences in mitochondrial respiration in heart tissue between the northern and southern subspecies at higher (28°C) acclimation temperatures (Baris et al. 2016), and these authors in fact detected significantly higher respiration in heart tissue from southern killifish than in northern killifish at an acclimation temperature of 12°C (Baris et al. 2016). However, it should be noted that our studies in liver utilized isolated mitochondria, whereas the studies in heart and brain have used a permeabilized tissue preparation, and these two types of methods can yield somewhat different results (Brand and Nicholls 2011; Mathers and Staples 2015). Fig. 4 View largeDownload slide Maximal ADP phosphorylating respiration rate in tissues of killifish acclimated to 5 (panels A, C, E) or 15°C (panels B, D, F). (A, B) Isolated mitochondria from liver. (C, D) Permeabilized tissue preparations from heart. (E, F) Permeabilized tissue preparations from brain. Gray bars = fish of the northern subspecies. Open bars = fish of the southern subspecies. ** indicates significant differences (P < 0.01) between the subspecies at a given acclimation temperature. All preparations provided with pyruvate, malate, and glutamate as substrates. All preparations were assayed at the acclimation temperature (Chung et al. 2017a, 2018). Fig. 4 View largeDownload slide Maximal ADP phosphorylating respiration rate in tissues of killifish acclimated to 5 (panels A, C, E) or 15°C (panels B, D, F). (A, B) Isolated mitochondria from liver. (C, D) Permeabilized tissue preparations from heart. (E, F) Permeabilized tissue preparations from brain. Gray bars = fish of the northern subspecies. Open bars = fish of the southern subspecies. ** indicates significant differences (P < 0.01) between the subspecies at a given acclimation temperature. All preparations provided with pyruvate, malate, and glutamate as substrates. All preparations were assayed at the acclimation temperature (Chung et al. 2017a, 2018). Ultimately, we observe clear subspecies differences in mitochondrial function in the liver, and there is little evidence for effects in other tissues. Thus, if mitochondrial function is playing a role in establishing at least some of the differences in the pace of life between northern and southern killifish, then these effects are most likely taking place in liver. The liver stores, mobilizes, and inter-converts energy reserves, and thus the metabolic activities of the liver are essential for providing fuel to other critical tissues such as the brain and muscle. Thus, the biochemical functions of the liver can be plausibly connected to differences in whole-organism standard metabolic rate in adult fish, and thus to other processes related to the pace of life such as growth rate. Variation in liver mitochondrial properties (specifically in cytochrome C oxidase activity and citrate synthase activity) has previously been shown to correlate with variation in standard metabolic rate and maximum metabolic rate in Brown Trout (Norin and Malte 2012), suggesting the importance of liver mitochondrial metabolism in setting metabolic rate across fish species. However, variation in mitochondrial properties in adult liver tissue is less likely to play a role in establishing differences in maximum aerobic swimming performance or early developmental rates, suggesting that the variation that we observe provides only a partial explanation for the variation in the full suite of traits associated with variation in the pace of life in killifish. Mechanisms of variation in mitochondrial properties It is possible to interrogate the role of the different respiratory complexes in establishing the differences in mitochondrial capacity between northern and southern killifish by assessing flux using different substrates, and by testing function in the presence of inhibitors of various parts of the ETS (Pesta and Gnaiger 2012). Using these approaches (Chung et al. 2017b, 2018), we find that multiple, and perhaps all, components of the ETS have higher activity in liver mitochondria in northern than in southern killifish. The relatively parallel increases in the activities of the ETS components in northern compared with southern killifish could reflect a greater amount of inner mitochondrial membrane per mitochondrion with a concomitant increase in ETS components in northern killifish, or a greater density of ETS components per unit inner mitochondrial membrane. To begin to distinguish between these possibilities, we measured the amount of cardiolipin per milligram liver mitochondrial protein in both northern and southern killifish held under common conditions in the laboratory to ensure a common diet. Cardiolipin is an unusual phospholipid that contains four acyl chains attached to the head group. This phospholipid is found almost exclusively in the inner mitochondrial membrane in eukaryotic cells (Hoch 1992). As such, cardiolipin amount can provide an estimate of the amount of inner mitochondrial membrane per mitochondrion (Larsen et al. 2012), if the amount of cardiolipin per unit membrane is assumed to be constant. As can be seen from Fig. 5, northern killifish have a greater amount of cardiolipin per milligram mitochondrial protein than do southern killifish, suggesting that they may have a greater surface area of inner mitochondrial membrane per mitochondrion (or possibly a higher cardiolipin concentration per unit inner mitochondrial membrane), which could account for the largely parallel increases in the activities of the various respiratory complexes in northern killifish compared with southern killifish. Interestingly, similar patterns in cardiolipin amount have been detected in comparisons of tropical and temperate birds (Calhoon et al. 2014; Jimenez et al. 2014), with higher levels in the animals from temperate climates that are correlated with differences in cellular metabolic rate. These similarities across very diverse taxa suggest the potential importance of mitochondrial membrane amount or mitochondrial membrane properties as a correlate of variation in the pace of life. This observation has some parallels with the membrane pacemaker theory of aging (Hulbert 2010), which suggests that membrane composition may be an important correlate of aging and lifespan. However, the specific role of the mitochondrial membrane, and of cardiolipin content, in the membrane pacemaker theory of aging remains poorly understood (Valencak and Azzu 2014), and thus further research will be necessary before it is possible to unify these two somewhat distinct hypotheses. Fig. 5 View largeDownload slide Total cardiolipin content in mitochondria isolated from the liver of killifish acclimated to 5 or 15°C. Total cardiolipin is the sum of cardiolipin plus monolysocardiolipin expressed per milligram mitochondrial protein in isolated mitochondrial preparations from liver (Chung et al. 2018). Gray bars = fish from the northern subspecies, open bars = fish from the southern subspecies. * indicates a significant difference between the subspecies (P < 0.05) at a given acclimation temperature. Different letters indicate significant differences (P < 0.05) between acclimation temperatures within a subspecies. Fig. 5 View largeDownload slide Total cardiolipin content in mitochondria isolated from the liver of killifish acclimated to 5 or 15°C. Total cardiolipin is the sum of cardiolipin plus monolysocardiolipin expressed per milligram mitochondrial protein in isolated mitochondrial preparations from liver (Chung et al. 2018). Gray bars = fish from the northern subspecies, open bars = fish from the southern subspecies. * indicates a significant difference between the subspecies (P < 0.05) at a given acclimation temperature. Different letters indicate significant differences (P < 0.05) between acclimation temperatures within a subspecies. Figure 5 also suggests that northern killifish may increase inner mitochondrial membrane amount substantially with cold acclimation in liver, whereas only modest changes occur in southern killifish. This observation is consistent with previous work that used electron microscopy to estimate the surface area of the inner mitochondrial membrane in muscle tissues (Dhillon and Schulte 2011). This study demonstrated that mitochondrial volume density and cristae surface area in both red and white muscle increased with cold acclimation in northern but not southern killifish, although the functional consequences of this variation remain unknown. Using RNA-seq we have also detected significant differential regulation of genes involved in mitochondrial fission and fusion in cold-acclimated killifish muscle (Healy et al. 2017), suggesting that there may be changes in mitochondrial morphology in response to cold acclimation as well. Taken together these data suggest that there may be differences between killifish subspecies in the effects of cold on the regulation of mitochondrial structure and function in multiple tissues. In addition to differences between subspecies in inner mitochondrial membrane amount or cardiolipin content, the relative proportions of different membrane phospholipids are not identical between the subspecies (Chung et al. 2018). Changes in the phospholipid composition of biological membranes can alter their properties, and in particular may influence membrane fluidity (Hazel 1995). This may be particularly relevant in the context of thermal acclimation and adaptation, as temperature alters the fluidity of biological membranes, and changes in membrane lipid composition can be used to compensate for these effects (Hazel and Prosser 1974; Hazel 1995). Using a combination of liquid chromatography (LC) with ultraviolet (UV) detection followed by gas-chromatography with flame ionization detection (GC-FID) and high-performance liquid chromatography (HPLC) prior to electro-spray ionization mass spectrometry (ESI/MS) we performed high resolution lipid analysis of the mitochondrial membranes (both inner and outer membranes combined). This produces a set of more than 60 inter-related variables expressing the percentage of various lipid head group and acyl chain combination in the total membrane fraction (Chung et al. 2018). Principle component analysis (PCA) of these data clearly indicates that there is an effect of acclimation temperature on mitochondrial membrane lipid profile, and that there are differences in membrane composition between the subspecies (Fig. 6). Fig. 6 View largeDownload slide Variation in mitochondrial membrane lipid composition between killifish subspecies. Principle component analysis of data from high-resolution lipid analysis of northern (blue) and southern (red) killifish acclimated to either 5 (circles) or 15°C (triangles). PC1 and PC3 are shown. PC2 separates based on inter-individual variation (Chung et al. 2018). Fig. 6 View largeDownload slide Variation in mitochondrial membrane lipid composition between killifish subspecies. Principle component analysis of data from high-resolution lipid analysis of northern (blue) and southern (red) killifish acclimated to either 5 (circles) or 15°C (triangles). PC1 and PC3 are shown. PC2 separates based on inter-individual variation (Chung et al. 2018). Membrane composition can affect mitochondrial properties in a number of ways. First, membrane composition can affect membrane fluidity, which can affect the activities of proteins embedded within the membrane (Hazel 1995), such as the proteins of the ETS. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) comprise the majority of the phospholipids present in mitochondrial membranes (Mejia and Hatch 2016). Changes in the relative proportions of these two phospholipids (the PC/PE ratio) have been demonstrated to be associated with changes in membrane fluidity (Cullis et al. 1996). However, there were no significant differences between the northern and southern subspecies in PC/PE, although there were some differences in the way this indicator changed with thermal acclimation between the two subspecies (Chung et al. 2018). Similarly, the number of double bonds in the acyl chains of phospholipids and their chain length can influence membrane fluidity (Cullis et al. 1996). However, there were no differences in the global unsaturation index or chain length between the mitochondrial membranes of northern and southern killifish, and these parameters did not change with acclimation temperature (Chung et al. 2018). Despite the fact that there were few differences between subspecies in the broad measures of membrane composition that correlate with membrane fluidity, there were many differences between subspecies in the amounts of specific phospholipids in the membrane (Fig. 6). Of particular interest are differences between the subspecies in cardiolipin composition, because cardiolipin plays a key functional role in a variety of mitochondrial processes. For example, cardiolipin has been shown to be necessary for the optimal functioning of complexes I, III, IV, and V (Paradies et al. 2014), and it also acts as a “proton trap” (Haines and Dencher 2002) that localizes pumped protons close to the inner mitochondrial membrane. There were differences in the relative proportions of several cardiolipin species between northern and southern killifish, which suggests that there could be effects of these changes in membrane composition on ETS function. However, the functional significance of changes in cardiolipin species in mitochondrial membranes remains enigmatic (Ye et al. 2016), and thus the effects of the observed changes in cardiolipin species between northern and southern killifish are not yet known. These data suggest that there are specific differences in the composition of liver mitochondrial membranes between killifish subspecies that could play a role in determining the functional properties of the mitochondria. In addition, the data on cardiolipin content suggest that there may be substantial differences in the amount of the inner mitochondrial membrane per mitochondrion, or of the amount of cardiolipin per unit membrane. These differences in liver inner mitochondrial membrane amount and respiratory chain activity are consistent with the greater adult standard metabolic rate in northern compared with southern killifish, and suggest a potential role for variation in liver mitochondrial properties in at least some aspects of variation in the pace of life between the subspecies. As discussed previously, this is consistent with previous work in Brown Trout that detected correlations between liver cytochrome C oxidase and both standard and maximum metabolic rate (Norin and Malte 2012). In addition, these measures are just an initial examination of the variety of mitochondrial properties that could contribute to variation in either standard or maximum metabolic rate, and to variation in the pace of life. In particular, little is currently known about the relative coupling efficiency, leak respiration, or ROS generation between killifish subspecies, although these parameters may be particularly critical in establishing the tradeoffs at the cellular level that could lead to the tradeoffs that shape the pace of life continuum (Janssens and Stoks 2018). Although we detect differences in mitochondrial properties between the subspecies in the livers of adult fish, these differences are not likely to account for the observed differences in developmental rate between F. heteroclitus subspecies. Nothing is known about mitochondrial function in the developing embryos of F. heteroclitus, so the role of the mitochondria in variation in rate processes in these fish prior to hatch remains. There are, however, some hints that mitochondrial processes could also be important in metabolic rate variation in killifish embryos, as embryos with a northern genotype at the enzyme malate dehydrogenase A (MDH-A) develop faster than those with the southern genotype at this enzyme (DiMichele and Powers 1991). MDH-A is the cytoplasmic form of the enzyme, which participates in the malate–aspartate shuttle that translocates electrons produced by glycolysis into the mitochondrion where they can be transferred to the ETS. Thus, the role of variation in mitochondrial properties in early developmental stages remains a fruitful and largely unexplored avenue that could yield insights into the biochemical mechanisms underlying variation in life history strategies. Conclusions and future directions The results from the studies presented here suggest that variation in mitochondrial properties could be associated with at least some aspects of variation in the pace of life in Atlantic killifish. More broadly, these findings emphasize the intimate link between thermal adaptation, metabolism, and life history variation in the context of the pace of life, and reveal a potential role for variation in mitochondrial membrane structure and function as a physiological mechanism underlying this variation. However, many important questions remain to be addressed. Although we present evidence that variation in liver mitochondrial function between killifish subspecies is in the direction expected based on observed differences in growth rate and metabolic rate, little is known about the potential for variation in mitochondrial function during early embryogenesis in this species, and whether it could account for observed differences in developmental rate. Similarly, the potential costs of increased mitochondrial metabolism in northern killifish are unknown. Studies in other systems suggest that a rapid pace of life may come at a cost in terms of increased oxidative stress (Janssens and Stoks 2018), but the relative levels of oxidative damage in northern and southern killifish have not been examined, although variation in a variety of oxidative stress parameters among killifish populations with differing levels of contaminant exposure has been detected (Bacanskas et al. 2004). Also, little is known about variation in metabolic rate and mitochondrial properties within populations of killifish, and whether the trends observed when comparing the northern and southern subspecies are also apparent when within-population variation is examined. Killifish populations from the center of the intergrade zone between the northern and southern subspecies might present a particularly interesting experimental system in which to test these ideas, and to examine the genetic basis of variation in mitochondrial properties, metabolic rate, and the pace of life. More broadly, the lessons learned from the killifish system point to a series of important questions that need to be answered when considering the role of mitochondrial processes in variation of the pace of life, regardless of the experimental system. First, it is important to recognize that there can be substantial tissue to tissue variation in mitochondrial properties, and thus the connection between mitochondrial function and whole organism metabolism and the pace of life is likely to be complex. The data presented here suggest that liver tissue may be important in establishing whole organism standard metabolic rate, but this observation needs to be repeated across multiple species to determine whether this represents a consistent pattern. Second, the pace of life hypothesis includes life history traits that are expressed at various life stages (e.g., development rate, growth rate, and longevity). At present, it is not known whether variation in mitochondrial properties in early development is correlated with variation in mitochondrial properties in adult organisms, or whether mitochondrial function changes across ontogeny. A better understanding of these patterns is critical in order to fully assess the role of variation in mitochondrial properties in life history variation and the pace of life. However, despite these remaining questions, the data presented here highlight the potential role of the mitochondria in thermal adaptation and in life history evolution in ectotherms, and demonstrate the commonalities among important patterns of life history variation such as countergradient variation and variation in the pace of life. Acknowledgment The authors thank Alex Cooper for the design and production of Fig. 1. Funding This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Discovery Grant [to P.M.S.] and a U.S. Department of Agriculture National Institute of Food and Agriculture and an American Heart Association grant [to A.J.C.] and via Michael Smith Travelling Fellowship [to D.J.C.]. References Able KW , Felley JD. 1986 . Geographical variation in Fundulus heteroclitus: tests for concordance between egg and adult morphologies . Integr Comp Biol 26 : 145 – 57 . Able KW , Vivian DN , Petruzzelli G , Hagan SM. 2012 . 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For permissions please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Integrative and Comparative BiologyOxford University Press

Published: Sep 1, 2018

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