Selection on dispersal drives evolution of metabolic capacities for energy production in female wing-polymorphic sand field crickets, Gryllus firmus.

Life history and metabolism covary, but the mechanisms and individual traits responsible for these linkages remain unresolved. Dispersal capability is a critical component of life history that is constrained by metabolic capacities for energy production. Conflicting relationships between metabolism and life histories may be explained by accounting for variation in dispersal and maximal metabolic rates. We used female wing-polymorphic sand field crickets, Gryllus firmus, selected either for long wings (LW, flight-capable) or short wings (SW, flightless) to test the hypothesis that selection on dispersal capability drives the evolution of metabolic capacities. While resting metabolic rates were similar, long-winged crickets reached higher maximal metabolic rates than short-winged crickets, resulting in improved running performance. We further provided insight into the mechanisms responsible for covariation between life history and metabolism by comparing mitochondrial content of tissues involved in powering locomotion and assessing the function of mitochondria isolated from long- and short-winged crickets. Our results demonstrated that larger metabolic capacities in long-winged crickets were underpinned by increases in mitochondrial content of dorsoventral flight muscle and enhanced bioenergetic capacities of mitochondria within the fat body, a tissue responsible for fuel storage and mobilization. Thus, selection on flight capability correlates with increases in maximal, but not resting metabolic rates, through modifications of tissues powering locomotion at the cellular and organelle levels. This allows organisms to meet high energetic demands of activity for life history. Dispersal capability should therefore explicitly be considered as a potential factor driving the evolution of metabolic capacities.

Metabolic design in a mammalian model of extreme metabolism, the North American least shrew (Cryptotis parva).

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused ion beam electron microscopy imaging and tandem mass tag mass spectrometry proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart, permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogeneous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism. KEY POINTS: Least shrews were studied to explore the relationship between metabolic function, mitochondrial morphology and protein content in different tissues. Liver and kidney mitochondrial content and enzymatic activity approaches that of the heart, indicating similar metabolic demand among tissues that contribute to basal and maximum metabolism. This allows an examination of mitochondrial structure and composition in tissues with similar maximum metabolic demands. Mitochondrial networks only occur in striated muscle. In contrast, the liver and kidney maintain individual mitochondria with limited reticulation. Muscle mitochondrial reticulation is the result of dense ATPase activity and cell-spanning myofibrils which require networking for adequate metabolic support. In contrast, liver and kidney ATPase activity is localized to the endoplasmic reticulum and basolateral membrane, respectively, generating a locally balanced energy conversion and utilization. Mitochondrial morphology is not driven by maximum metabolic demand, but by the cytosolic distribution of energy-utilizing systems set by the functions of the tissue.

Mitochondria and the thermal limits of ectotherms.

Temperature is a critical abiotic factor shaping the distribution and abundance of species, but the mechanisms that underpin organismal thermal limits remain poorly understood. One possible mechanism underlying these limits is the failure of mitochondrial processes, as mitochondria play a crucial role in animals as the primary site of ATP production. Conventional measures of mitochondrial performance suggest that these organelles can function at temperatures much higher than those that limit whole-organism function, suggesting that they are unlikely to set organismal thermal limits. However, this conclusion is challenged by recent data connecting sequence variation in mitochondrial genes to whole-organism thermal tolerance. Here, we review the current state of knowledge of mitochondrial responses to thermal extremes and ask whether they are consistent with a role for mitochondrial function in shaping whole-organism thermal limits. The available data are fragmentary, but it is possible to draw some conclusions. There is little evidence that failure of maximal mitochondrial oxidative capacity as assessed in vitro sets thermal limits, but there is some evidence to suggest that temperature effects on ATP synthetic capacity may be important. Several studies suggest that loss of mitochondrial coupling is associated with the thermal limits for organismal growth, although this needs to be rigorously tested. Most studies have utilized isolated mitochondrial preparations to assess the effects of temperature on these organelles, and there remain many untapped opportunities to address these questions using preparations that retain more of their biological context to better connect these subcellular processes with whole-organism thermal limits.

Mitochondrial DNA segregation and replication restrict the transmission of detrimental mutation.

Although mitochondrial DNA (mtDNA) is prone to accumulate mutations and lacks conventional DNA repair mechanisms, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations is restricted through the maternal lineage is debated. Here, we demonstrate that mitochondrial fission, together with the lack of mtDNA replication, segregate mtDNA into individual organelles in the Drosophila early germarium. After mtDNA segregation, mtDNA transcription begins, which activates respiration. Mitochondria harboring wild-type genomes have functional electron transport chains and propagate more vigorously than mitochondria containing deleterious mutations in hetreoplasmic cells. Therefore, mtDNA expression acts as a stress test for the integrity of mitochondrial genomes and sets the stage for replication competition. Our observations support selective inheritance at the organelle level through a series of developmentally orchestrated mitochondrial processes. We also show that the Balbiani body has a minor role in mtDNA selective inheritance by supplying healthy mitochondria to the pole plasm. These two mechanisms may act synergistically to secure the transmission of functional mtDNA through Drosophila oogenesis.

Mitochondrial Ecophysiology: Assessing the Evolutionary Forces That Shape Mitochondrial Variation.

The mitonuclear species concept hypothesizes that incompatibilities between interacting gene products of the nuclear and mitochondrial genomes are a major factor establishing and maintaining species boundaries. However, most of the data available to test this concept come from studies of genetic variation in mitochondrial DNA, and clines in the mitochondrial genome across contact zones can be produced by a variety of forces. Here, we show that using a combination of population genomic analyses of the nuclear and mitochondrial genomes and studies of mitochondrial function can provide insight into the relative roles of neutral processes, adaptive evolution, and mitonuclear incompatibility in establishing and maintaining mitochondrial clines, using Atlantic killifish (Fundulus heteroclitus) as a case study. There is strong evidence for a role of secondary contact following the last glaciation in shaping a steep mitochondrial cline across a contact zone between northern and southern subspecies of killifish, but there is also evidence for a role of adaptive evolution in driving differentiation between the subspecies in a variety of traits from the level of the whole organism to the level of mitochondrial function. In addition, studies are beginning to address the potential for mitonuclear incompatibilities in admixed populations. However, population genomic studies have failed to detect evidence for a strong and pervasive influence of mitonuclear incompatibilities, and we suggest that polygenic selection may be responsible for the complex patterns observed. This case study demonstrates that multiple forces can act together in shaping mitochondrial clines, and illustrates the challenge of disentangling their relative roles.

Subspecies differences in thermal acclimation of mitochondrial function and the role of uncoupling proteins in killifish.

Thermal effects on mitochondrial efficiency and ATP production can influence whole-animal thermal tolerance and performance. Thus, organisms may have the capacity to alter mitochondrial processes through acclimation or adaptation to mitigate these effects. One possible mechanism is through the action of uncoupling proteins (UCPs), which can decrease the proton-motive force independent of the production of ATP. To test this hypothesis, we examined the mRNA expression patterns of UCP isoforms and characterized the effects of thermal acclimation and putative local thermal adaptation on mitochondrial capacity, proton leak and P/O ratios in two subspecies of Atlantic killifish (Fundulus heteroclitus). Ucp1 was the dominant isoform in liver and was more highly expressed in northern killifish. We found that cold acclimation increased mitochondrial capacity (state III and maximum substrate oxidation capacity), state II membrane potential, proton leak and P/O ratios in northern, but not southern, killifish liver mitochondria. Palmitate-induced mitochondrial uncoupling was detected in northern, but not southern, killifish liver mitochondria, consistent with the differences in Ucp mRNA expression between the subspecies. Taken together, our data suggest that mitochondrial function is more plastic in response to thermal acclimation in northern killifish than in southern killifish and that UCP1 may play a role in regulating the proton-motive force in northern, but not southern, killifish in response to thermal acclimation. These data demonstrate the potential for adaptive variation in mitochondrial plasticity in response to cold.

Mitochondria, Temperature, and the Pace of Life.

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.

Patterns of mitochondrial membrane remodeling parallel functional adaptations to thermal stress.

The effect of temperature on mitochondrial performance is thought to be partly due to its effect on mitochondrial membranes. Numerous studies have shown that thermal acclimation and adaptation can alter the amount of inner-mitochondrial membrane (IMM), but little is known about the capacity of organisms to modulate mitochondrial membrane composition. Using northern and southern subspecies of Atlantic killifish (Fundulus heteroclitus) that are locally adapted to different environmental temperatures, we assessed whether thermal acclimation altered liver mitochondrial respiratory capacity or the composition and amount of IMM. We measured changes in phospholipid headgroups and headgroup-specific fatty acid (FA) remodeling, and used respirometry to assess mitochondrial respiratory capacity. Acclimation to 5°C and 33°C altered mitochondrial respiratory capacity in both subspecies. Northern F. heteroclitus exhibited greater mitochondrial respiratory capacity across acclimation temperatures, consistent with previously observed subspecies differences in whole-organism aerobic metabolism. Mitochondrial phospholipids were altered following thermal acclimation, and the direction of these changes was largely consistent between subspecies. These effects were primarily driven by remodeling of specific phospholipid classes and were associated with shifts in metabolic phenotypes. There were also differences in membrane composition between subspecies that were driven largely by differences in phospholipid classes. Changes in respiratory capacity between subspecies and with acclimation were largely but not completely accounted for by alterations in the amount of IMM. Taken together, these results support a role for changes in liver mitochondrial function in the ectothermic response to thermal stress during both acclimation and adaptation, and implicate lipid remodeling as a mechanism contributing to these changes.

Intraspecific variation and plasticity in mitochondrial oxygen binding affinity as a response to environmental temperature.

Mitochondrial function has been suggested to underlie constraints on whole-organism aerobic performance and associated hypoxia and thermal tolerance limits, but most studies have focused on measures of maximum mitochondrial capacity. Here we investigated whether variation in mitochondrial oxygen kinetics could contribute to local adaptation and plasticity in response to temperature using two subspecies of the Atlantic killifish (Fundulus heteroclitus) acclimated to a range of temperatures (5, 15, and 33 °C). The southern subspecies of F. heteroclitus, which has superior thermal and hypoxia tolerances compared to the northern subspecies, exhibited lower mitochondrial O2 P50 (higher O2 affinity). Acclimation to thermal extremes (5 or 33 °C) altered mitochondrial O2 P50 in both subspecies consistent with the effects of thermal acclimation on whole-organism thermal tolerance limits. We also examined differences between subspecies and thermal acclimation effects on whole-blood Hb O2-P50 to assess whether variation in oxygen delivery is involved in these responses. In contrast to the clear differences between subspecies in mitochondrial O2-P50 there were no differences in whole-blood Hb-O2 P50 between subspecies. Taken together these findings support a general role for mitochondrial oxygen kinetics in differentiating whole-organism aerobic performance and thus in influencing species responses to environmental change.

Metabolic and regulatory responses involved in cold acclimation in Atlantic killifish, Fundulus heteroclitus.

Ectotherms often respond to prolonged cold exposure by increasing mitochondrial capacity via elevated mitochondrial volume density [V V(mit,f)]. In fish, higher V V(mit,f) is typically associated with increased expression of nuclear respiratory factor 1 (Nrf1), a transcription factor that induces expression of nuclear-encoded respiratory genes. To examine if nrf1 expression or the expression of other genes that regulate mitochondrial biogenesis contribute to changes in whole-organism metabolic rate during cold acclimation, we examined the time course of changes in the expression of these genes and in metabolic rate in Atlantic killifish, Fundulus heteroclitus. Cold acclimation rapidly decreased metabolic rate, but increased the expression of nrf1 more gradually, with a time course that depended on how rapidly the fish were transitioned to low temperature. Cold-induced nrf1 expression was not associated with increases in biochemical indicators of mitochondrial respiratory capacity, suggesting that cold-induced mitochondrial biogenesis may occur without increases in oxidative capacity in this species. These observations imply that changes in nrf1 expression and metabolic rate due to cold acclimation occur through different physiological mechanisms, and that increases in V V(mit,f) are likely not directly related to changes in metabolic rate with cold acclimation in this species. However, nrf1 expression differed between northern and southern killifish subspecies regardless of acclimation temperature, consistent with observed differences in metabolic rate and V V(mit,f) at 5 °C between these subspecies. Taken together, these results reveal substantial complexity in the regulation of V V(mit,f) and mitochondrial capacity with temperature in fish and the relationship of these parameters to metabolic rate.

Mechanisms and costs of mitochondrial thermal acclimation in a eurythermal killifish (Fundulus heteroclitus).

Processes acting at the level of the mitochondria have been suggested to affect the thermal limits of organisms. To determine whether changes in mitochondrial properties could underlie shifts in thermal limits, we examined how mitochondrial properties are affected by thermal acclimation in the eurythermal killifish, Fundulus heteroclitus - a species with substantial plasticity in whole-organism thermal limits. We hypothesized that thermal acclimation would result in functional changes in the mitochondria that could result in trade-offs in function during acute thermal shifts. We measured the mitochondrial respiration rate (V̇O2 ) through multiple complexes of the electron transport system following thermal acclimation (to 5, 15, 33°C) and assessed maintenance of mitochondrial membrane potential (Δp) and rates of reactive oxygen species (ROS) production as an estimate of costs. Acclimation to 5°C resulted in a modest compensation of mitochondrial respiration at low temperatures, but these mitochondria were able to maintain Δp with acute exposure to high temperatures, and ROS production did not differ between acclimation groups, suggesting that these increases in mitochondrial capacity do not alter mitochondrial thermal sensitivity. Acclimation to 33°C suppressed mitochondrial respiration as a result of effects on NADH dehydrogenase (complex I). These high-temperature acclimated fish nonetheless maintained levels of Δp and ROS production similar to those of the other acclimation groups. This work demonstrates that killifish mitochondria can successfully acclimate to a wide range of temperatures without incurring major functional trade-offs during acute thermal shifts and that high-temperature acclimation results in a suppression of metabolism, consistent with patterns observed at the organismal level.

Changes in the mitochondrial phosphoproteome during mammalian hibernation.

Mammalian hibernation involves periods of substantial suppression of metabolic rate (torpor) allowing energy conservation during winter. In thirteen-lined ground squirrels (Ictidomys tridecemlineatus), suppression of liver mitochondrial respiration during entrance into torpor occurs rapidly (within 2 h) before core body temperature falls below 30°C, whereas reversal of this suppression occurs slowly during arousal from torpor. We hypothesized that this pattern of rapid suppression in entrance and slow reversal during arousal was related to changes in the phosphorylation state of mitochondrial enzymes during torpor catalyzed by temperature-dependent kinases and phosphatases. We compared mitochondrial protein phosphorylation among hibernation metabolic states using immunoblot analyses and assessed how phosphorylation related to mitochondrial respiration rates. No proteins showed torpor-specific changes in phosphorylation, nor did phosphorylation state correlate with mitochondrial respiration. However, several proteins showed seasonal (summer vs. winter) differences in phosphorylation of threonine or serine residues. Using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry, we identified three of these proteins: F1-ATPase α-chain, long chain-specific acyl-CoA dehydrogenase, and ornithine transcarbamylase. Therefore, we conclude that protein phosphorylation is likely a mechanism involved in bringing about seasonal changes in mitochondrial metabolism in hibernating ground squirrels, but it seems unlikely to play any role in acute suppression of mitochondrial metabolism during torpor.

Regulation of succinate-fuelled mitochondrial respiration in liver and skeletal muscle of hibernating thirteen-lined ground squirrels.

Hibernating ground squirrels (Ictidomys tridecemlineatus) alternate between two distinct metabolic states throughout winter: torpor, during which metabolic rate (MR) and body temperature (Tb) are considerably suppressed, and interbout euthermia (IBE), during which MR and Tb briefly return to euthermic levels. Previous studies showed suppression of succinate-fuelled respiration during torpor in liver and skeletal muscle mitochondria; however, these studies used only a single, saturating succinate concentration. Therefore, they could not address whether mitochondrial metabolic suppression occurs under physiological substrate concentrations or whether differences in the kinetics of mitochondrial responses to changing substrate concentration might also contribute to mitochondrial metabolic regulation during torpor. The present study confirmed that succinate oxidation is reduced during torpor in liver and skeletal muscle at 37 and 10°C over a 100-fold range of succinate concentrations. At 37°C, this suppression resulted from inhibition of succinate dehydrogenase (SDH), which had a greater affinity for oxaloacetate (an SDH inhibitor) during torpor. At 10°C, SDH was not inhibited, suggesting that SDH inhibition initiates but does not maintain mitochondrial suppression during torpor. Moreover, in both liver and skeletal muscle, mitochondria from torpid animals maintained relatively higher respiration rates at low succinate concentrations, which reduces the extent of energy savings that can be achieved during torpor, but may also maintain mitochondrial oxidative capacity above some lower critical threshold, thereby preventing cellular and/or mitochondrial injury during torpor and facilitating rapid recruitment of oxidative capacity during arousal.

Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels.

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.