The energetic cost of mating in a promiscuous cephalopod

Costs that individuals incur through mating can play an important role in understanding the evolution of life histories and senescence, particularly in promiscuous species. Copulation costs, ranging from energy expenditure to reduced longevity, are widely studied in insects but have received substantially less attention in other taxa. One cost of mating, the energetic cost, is poorly studied across all taxa despite its potential importance for the many species where copulation is physically demanding and/or frequent. Here, we investigated the energetic cost of mating in both male and female dumpling squid (Euprymna tasmanica). In this species, copulation can last up to 3 h and requires that the male physically restrains the female. We report that the act of copulation halves the swimming endurance of both sexes, and that they take up to 30 min to recover. Such a reduction in post-copulatory performance may have important implications for predator avoidance, foraging ability and energy allocation. Therefore, quantifying this cost is essential to understand the evolution of reproductive strategies and behaviours such as female receptivity and male and female mating frequency.     

The energetic cost of NNT-dependent ROS removal

Under conditions of high nutrient availability and low ATP synthesis, mitochondria generate reactive oxygen species (ROS) that must be removed to avoid cell injury. Among the enzymes involved in this scavenging process, peroxidases play a crucial role, using NADPH provided mostly by nicotinamide nucleotide transhydrogenase (NNT). However, scarce information is available on how and to what extent ROS formation is linked to mitochondrial oxygen consumption. A new study by Smith et al. shows that NNT activity maintains low ROS levels by means of a fine modulation of mitochondrial oxygen utilization.

The rate of human energy expenditure fluctuates, increasing during periods of weight gain and decreasing during weight loss, to prevent large swings in body weight. Central to this ability are mitochondrial redox circuits responsible for nutrient oxidation and reactive oxygen species (ROS) generation. The redox circuits coupling the partial reduction of oxygen with ROS removal are linked with the major redox circuit represented by the complete reduction of oxygen into water at the level of the electron transport chain (ETC). This link enables cells to respond to changes in nutrient availability and energy demand. A better understanding of how these circuits are intertwined could lead to new therapeutic avenues for the treatment of metabolic disorders. New work by Smith et al. (1) reveals a mechanism by which mitochondria sense excess energy supply—particularly when energy demand is low—via ETC. This mechanism dependent on nicotinamide nucleotide transhydrogenase (NNT) couples the maintenance of low ROS levels with an increased oxygen consumption (i.e. energy expenditure).Within the inner mitochondrial membrane (IMM), the energetically favorable flow of electrons from NADH(H⁺) and FADH₂ to oxygen allows proton pumping from the matrix to the intermembrane space. The resulting proton gradient (Δp) is utilized for ATP synthesis, as well as for other mitochondrial processes, such as ion homeostasis, protein import, etc.A minor fraction of the electrons flowing through the ETC is diverted, causing the partial reduction of O₂ into superoxide, which is subsequently converted to H₂O₂ (2, 3). This electron detour is favored when flow is slowed down by a decrease in ATP demand. The carnitine-dependent β-oxidation of fatty acids exacerbates this situation, as it can increase NADH(H⁺) and FADH₂ availability, promoting mitochondrial ROS formation. This potentially deleterious process is counterbalanced by efficacious ROS removal systems. In particular, H₂O₂ reduction into water is catalyzed by peroxidases using reduced GSH and thioredoxin (Trx). The resulting oxidized forms of GSH and Trx are reduced by the reductases catalyzing NADPH(H⁺) oxidation. Various cytosolic and mitochondrial enzymes are involved in restoring the high NADPH(H⁺)/NADP⁺ ratio required not only for an optimal redox balance, but also for many anabolic processes. NNT, a ubiquitously expressed integral protein of the IMM (4), plays a major role among the enzymes contributing to NADP⁺ reduction.NNT functions as a redox-driven proton pump catalyzing the reversible reduction of NADP⁺ to NADPH(H⁺) at the expense of NADH(H⁺) oxidation into NAD⁺. Much of our current knowledge on the role of NNT derives from studies on the mouse strain C57BL/6J (B6J) displaying a markedly lower NNT protein expression as compared with the control B6N strain (5). The lack of NNT curtails NADPH(H⁺) availability and thus peroxidase activities leading to oxidative stress. A severe increase in mitochondrial ROS levels has been linked to various pathologies. On the other hand, a slight increase in mitochondrial ROS formation appears to contribute to endogenous defense mechanisms against cell injury (6). Because NNT is relevant for maintaining NADPH availability necessary for the peroxidase activities required for buffering H₂O₂, the following question arises: To what extent does NNT activity link mitochondrial ROS production resulting from excess substrate availability with mitochondrial oxygen consumption?Dr. Neufer''s group investigated whether an increase in H₂O₂ production due to high rates of substrate oxidation under resting conditions is counterbalanced by a corresponding increase in NNT-mediated oxygen consumption (1). Experiments in mitochondria isolated from hind limb skeletal muscle from B6N mice under conditions mimicking a resting state (i.e. in the absence of ADP) demonstrated that increasing carnitine from 25 μm to 5 mm to maximize palmitoyl-CoA oxidation resulted in a 3-fold increase in the rate of H₂O₂ emission. The combined inhibition of Trx reductase by auranofin (AF) and GSH reductase by carmustine (BCNU) increased H₂O₂ emission by almost 4-fold, demonstrating that the activity of mitochondrial peroxidases buffers >70% of H₂O₂ production driven by β-oxidation. In addition, H₂O₂ formation was shown to depend on a complete fatty acid oxidation, including acetyl-CoA buffering by carnitine acetyltransferase and/or acetyl CoA utilization by the TCA cycle. Notably, the highest rate of fatty acid oxidation obtained at 5 mm carnitine caused an increase in proton conductance that was prevented by AF/BCNU treatment. This interesting finding suggests that GSH and Trx reductases utilize NADPH(H⁺) produced by NNT, which in turn uses Δp generated by mitochondrial respiration. The authors validated this hypothesis comparing permeabilized fibers from B6J and B6N mice. Indeed, the increase in proton conductance was absent in B6J fibers that also were not affected by AF/BCNU addition. Notably, oxygen consumption was 18.6% lower in B6J samples. Therefore, a significant fraction of mitochondrial respiration supports NNT activity in mediating an optimal rate of ROS removal (Fig. 1).Open in a separate windowFigure 1.Schematic of the pathways involved in NNT coupling of mitochondrial ROS formation with oxygen consumption under resting conditions (i.e. no ATP synthesis). ROS removal requires NADPH(H⁺) provided by NNT in a process coupled with the utilization of the proton gradient generated by oxygen consumption. For the sake of simplicity, flavin nucleotides and thioredoxin are omitted, as well as the utilization of the proton gradient for ATP synthesis. FAO, fatty acid oxidation; GPX, GSH peroxidase; GR, GSH reductase; LCACoA, long-chain acyl-CoA; SOD, superoxide dismutase(s).The work conducted by Smith et al. suggests that NNT performs direct and indirect coupling activities that are tightly linked. NNT directly couples NADPH(H⁺) formation from NADH(H⁺) with mitochondrial proton uptake, as is well-established. Smith et al. (1) demonstrate that Δp is maintained by oxygen consumption, such that NNT-mediated ROS removal is physiologically coupled with mitochondrial respiration. However, it is worth noting that the current study does not include in situ or in vivo experiments, perhaps because the carnitine titration of β-oxidation along with the use of reductase and respiration inhibitors could not be applied to intact cells or organs. Thus, it will be important to extend this work to more intact models. Moreover, a word of caution should be mentioned for the use of B6N mice as control strain. A recent study demonstrated that B6N hearts are more prone to contractile failure because of the absence of MYLK3, a protein kinase required for actin assembly (7). However, this defect is unlikely to impact the findings of Smith et al. (1). Nevertheless, a proteomic analysis of BJ6 mitochondria is lacking. Future studies should investigate whether the lack of NNT is compensated by changes in mitochondrial proteins involved in substrate oxidation and ROS removal.

Funding and additional information—This work was supported by Leducq Transatlantic Network of Excellence Grant 16CVD04 and COST Action EU-CARDIOPROTECTION Grant CA16225.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

ROS

reactive oxygen species

ETC

electron transport chain

IMM

inner mitochondrial membrane

NNT

nicotinamide nucleotide transhydrogenase

TCA

tricarboxylic acid cycle

AF

auranofin

BCNU

carmustine.

     

Effects of obesity and sex on the energetic cost and preferred speed of walking.

The metabolic energy cost of walking is determined, to a large degree, by body mass, but it is not clear how body composition and mass distribution influence this cost. We tested the hypothesis that walking would be most expensive for obese women compared with obese men and normal-weight women and men. Furthermore, we hypothesized that for all groups, preferred walking speed would correspond to the speed that minimized the gross energy cost per distance. We measured body composition, maximal oxygen consumption, and preferred walking speed of 39 (19 class II obese, 20 normal weight) women and men. We also measured oxygen consumption and carbon dioxide production while the subjects walked on a level treadmill at six speeds (0.50-1.75 m/s). Both obesity and sex affected the net metabolic rate (W/kg) of walking. Net metabolic rates of obese subjects were only approximately 10% greater (per kg) than for normal-weight subjects, and net metabolic rates for women were approximately 10% greater than for men. The increase in net metabolic rate at faster walking speeds was greatest in obese women compared with the other groups. Preferred walking speed was not different across groups (1.42 m/s) and was near the speed that minimized gross energy cost per distance. Surprisingly, mass distribution (thigh mass/body mass) was not related to net metabolic rate, but body composition (% fat) was (r2= 0.43). Detailed biomechanical studies of walking are needed to investigate whether obese individuals adopt novel energy saving mechanisms during walking.     

Muscle mechanical work requirements during normal walking: the energetic cost of raising the body's center-of-mass is significant

Inverted pendulum models of walking predict that little muscle work is required for the exchange of body potential and kinetic energy in single-limb support. External power during walking (product of the measured ground reaction force and body center-of-mass (COM) velocity) is often analyzed to deduce net work output or mechanical energetic cost by muscles. Based on external power analyses and inverted pendulum theory, it has been suggested that a primary mechanical energetic cost may be associated with the mechanical work required to redirect the COM motion at the step-to-step transition. However, these models do not capture the multi-muscle, multi-segmental properties of walking, co-excitation of muscles to coordinate segmental energetic flow, and simultaneous production of positive and negative muscle work. In this study, a muscle-actuated forward dynamic simulation of walking was used to assess whether: (1). potential and kinetic energy of the body are exchanged with little muscle work; (2). external mechanical power can estimate the mechanical energetic cost for muscles; and (3.) the net work output and the mechanical energetic cost for muscles occurs mostly in double support. We found that the net work output by muscles cannot be estimated from external power and was the highest when the COM moved upward in early single-limb support even though kinetic and potential energy were exchanged, and muscle mechanical (and most likely metabolic) energetic cost is dominated not only by the need to redirect the COM in double support but also by the need to raise the COM in single support.     

Metabolic cost of walking: equation and model

Twenty years of published experience with the Workman-Armstrong equation for predicting walking VO2 is reviewed. The equation is reexpressed in currently accepted terminology, and it is shown that the equation serves well as a basic model of normal walking. Employing this model to analyze VO2/step leads to the elaboration of a three-compartment model of the metabolic cost of walking. This three-compartment model provides a rational estimate of the fraction of walking's metabolic cost that powers the actual walking movement. Doubt is expressed that "comfortable speed of walking" is definable in energy terms. It is suggested that the requirements of maintaining balance while walking may determine both the comfortable speed of walking and the curvilinearity of the relationship between ground-speed and freely chosen step frequency of walking.     

The energetic cost of maintaining lateral balance during human running

To quantify the energetic cost of maintaining lateral balance during human running, we provided external lateral stabilization (LS) while running with and without arm swing and measured changes in energetic cost and step width variability (indicator of lateral balance). We hypothesized that external LS would reduce energetic cost and step width variability of running (3.0 m/s), both with and without arm swing. We further hypothesized that the reduction in energetic cost and step width variability would be greater when running without arm swing compared with running with arm swing. We controlled for step width by having subjects run along a single line (zero target step width), which eliminated any interaction effects of step width and arm swing. We implemented a repeated-measures ANOVA with two within-subjects fixed factors (external LS and arm swing) to evaluate main and interaction effects. When provided with external LS (main effect), subjects reduced net metabolic power by 2.0% (P = 0.032) and step width variability by 12.3% (P = 0.005). Eliminating arm swing (main effect) increased net metabolic power by 7.6% (P < 0.001) but did not change step width variability (P = 0.975). We did not detect a significant interaction effect between external LS and arm swing. Thus, when comparing conditions of running with or without arm swing, external LS resulted in a similar reduction in net metabolic power and step width variability. We infer that the 2% reduction in the net energetic cost of running with external LS reflects the energetic cost of maintaining lateral balance. Furthermore, while eliminating arm swing increased the energetic cost of running overall, arm swing does not appear to assist with lateral balance. Our data suggest that humans use step width adjustments as the primary mechanism to maintain lateral balance during running.     

The energetic cost of various behaviors in the laboratory mouse

The continuous 12-hr observation of a mouse placed in a respiratory chamber has made it possible to record the succession of behaviors of the animal, as well as the associated variations of CO2 concentrations in the chamber. Ten behaviors have been considered; these included rest, locomotion, sniffing, feeding, drinking, nest building and various types of grooming. These data have been used for the estimation, by means of Kalman filtering, of the energetic cost of each behavior. Thus, at 20 degrees C, these costs vary between 1.51 ml CO2/g/hr (rest) and 4.90 ml CO2/g/hr (locomotion). These costs seem independent of any underlying biological rhythm; they yield basal metabolic estimates and average daily metabolic rates similar to those found in the literature. The low energetic cost of each behavioral bout, as compared to the daily energy intake of the mouse, leads one to believe that the behavioral transitions of this animal are not to be ascribed to energetical reasons. These results have been validated with data obtained from two other mice under similar conditions.     

The energetic cost of locomotion: humans and primates compared to generalized endotherms

A wide range of selective pressures have been advanced as possible causes for the adoption of bipedalism in the hominin lineage. One suggestion has been that because modern human walking is relatively efficient compared to that of a typical quadruped, the ancestral quadruped may have reaped an energetic advantage when it walked on two legs. While it has become clear that human walking is relatively efficient and human running inefficient compared to "generalized endotherms", workers differ in their opinion of how the cost of human bipedal locomotion compares to that of a generalized primate walking quadrupedally. One view is that human walking is particularly efficient in comparison to other primates. The present study addresses this by comparing the cost of human walking and running to that of the eight primate species for which data are available and by comparing cost in primates to that of a "generalized endotherm". There is no evidence that primate locomotion is more costly than that of a generalized endotherm, although more data on adult Old World monkeys and apes would be useful. Further, human locomotion does not appear to be particularly efficient relative to that of other primates.     

Buccal cell DNA extraction: yield, purity, and cost: a comparison of two methods

Simple and cost-effective methods are needed to extract DNA in order to use it in large-scale studies. Blood is an excellent DNA source; however, it is costly and invasive thus an alternative is needed. Several kits and chemical protocols using buccal cells have been proposed for DNA extraction. The objective of the study is to evaluate buccal NaOH chemical protocol and Nucleospin Tissue Kit (BD Biosciences, Macery-Nagel, Germany) for DNA extraction. DNA swab samples were collected from 300 voluntary participants. DNA yields and purity were measured by NaOH and Nucleospin Tissue Kit techniques; the cost and time consumption for DNA extraction per sample were assessed as well. Results have shown that DNA amount and purity extracted by NaOH procedure was compared to that of the kit (p = 0.164; p = 0.249, respectively). NaOH method was considered cheaper and less time consuming (0.06 versus 3.80 USD, and 1.33 versus 3.59 minutes per sample, p < 0.001). Buccal cell derived DNA extracted by NaOH protocol can be considered a feasible substitute for more expensive and time-consuming kits.     

The energy cost of walking or running on sand

Oxygen uptake (VO2) at steady state, heart rate and perceived exertion were determined on nine subjects (six men and three women) while walking (3-7 km.h-1) or running (7-14 km.h-1) on sand or on a firm surface. The women performed the walking tests only. The energy cost of locomotion per unit of distance (C) was then calculated from the ratio of VO2 to speed and expressed in J.kg-1.m-1 assuming an energy equivalent of 20.9 J.ml O2-1. At the highest speeds C was adjusted for the measured lactate contribution (which ranged from approximately 2% to approximately 11% of the total). It was found that, when walking on sand, C increased linearly with speed from 3.1 J.kg-1.m-1 at 3 km.h-1 to 5.5 J.kg-1.m-1 at 7 km.h-1, whereas on a firm surface C attained a minimum of 2.3 J.kg-1.m-1 at 4.5 km.h-1 being greater at lower or higher speeds. On average, when walking at speeds greater than 3 km.h-1, C was about 1.8 times greater on sand than on compact terrain. When running on sand C was approximately independent of the speed, amounting to 5.3 J.kg-1.m-1, i.e. about 1.2 times greater than on compact terrain. These findings could be attributed to a reduced recovery of potential and kinetic energy at each stride when walking on sand (approximately 45% to be compared to approximately 65% on a firm surface) and to a reduced recovery of elastic energy when running on sand.     

Predicting metabolic cost of level walking

Energy expenditure in walking is usually expressed as a function of walking speed. However, this relationship applies only to freely adopted step length-step rate patterns. Both the step length and the step rate must be used to preduct the energy expenditure for any combination of step length and step rate. Evidence on seven subjects indicates that the energy demand for such a combination can be determined by conducting two experiments. In the first, the subject is allowed to freely choose his own walking pattern to achieve a set of prescribed speeds. In the second, the speed is kept constant but the subject is forced to adopt a range of prescribed step rates. The results of the two experiments combined yield enough data to make possible the determination of the energy equation of the pattern, encompassing both "free" and "forced" gaits. Results show that the freely chosen step rate requires the least oxygen consumption at any given speed. Any other forced step rate at the same speed increases the oxygen cost over that required for the "free" step rate.     

The energetic cost of reproductive conflicts in the ant Pachycondyla obscuricornis

In a variety of social animals, individuals can secure reproductive rights through aggressive dominance. Direct individual benefits of aggression are widely recognized, but underlying costs affecting group productivity, and thus indirect benefits, are less clear. Costs of aggressive regulation of reproduction are especially important in small social insect colonies, where individual workers could potentially dominate male production. We estimated the energetic costs associated with the regulation of worker reproduction in the ponerine ant Pachycondyla obscuricornis, using the total CO2 emission of a colony as a measure. The level of CO2 emission of 12 experimental colonies varied significantly during five periods with varying levels of aggression and egg-laying. Overall, CO2 emission increased with the degree of fighting in a colony, but was not associated with differences in egg-laying. Aggressive regulation of reproduction and the formation of a dominance hierarchy thus pose an energetic cost to the colony. Furthermore, workers reduce their work-activities immediately after experimental orphaning, giving a further cost to the colony. These costs might influence the outcome of conflicts over male production in ants. This paper presents the first quantification of energetic costs of aggressive behavior regulating reproduction in ants.     

The cost of walking downhill: Is the preferred gait energetically optimal?

Humans tend to prefer walking patterns that minimize energetic cost, but must also maintain stability to avoid falling over. The relative importance of these two goals in determining the preferred gait pattern is not currently clear. We investigated the relationship between energetic cost and stability during downhill walking, a context in which gravitational energy will assist propulsion but may also reduce stability. We hypothesized that humans will not minimize energetic cost when walking downhill, but will instead prefer a gait pattern that increases stability. Simulations of a dynamic walking model were used to determine whether stable downhill gaits could be achieved using a simple control strategy. Experimentally, twelve healthy subjects walked downhill at 1.25 m/s (0, 0.05, 0.10, and 0.15 gradients). For each slope, subjects performed normal and relaxed trials, in which they were instructed to reduce muscle activity and allow gravity to maximally assist their gait. We quantified energetic cost, stride timing, and leg muscle activity. In our model simulations, increase in slope reduced the required actuation but also decreased stability. Experimental subjects behaved more like the model when using the relaxed rather than the normal walking strategy; the relaxed strategy decreased energetic cost at the steeper slopes but increased stride period variability, an indicator of instability. These results indicate that subjects do not take optimal advantage of the propulsion provided by gravity to decrease energetic cost, but instead prefer a more stable and more costly gait pattern.     

The cost of sex: quantifying energetic investment in gamete production by males and females

The relative energetic investment in reproduction between the sexes forms the basis of sexual selection and life history theories in evolutionary biology. It is often assumed that males invest considerably less in gametes than females, but quantifying the energetic cost of gamete production in both sexes has remained a difficult challenge. For a broad diversity of species (invertebrates, reptiles, amphibians, fishes, birds, and mammals), we compared the cost of gamete production between the sexes in terms of the investment in gonad tissue and the rate of gamete biomass production. Investment in gonad biomass was nearly proportional to body mass in both sexes, but gamete biomass production rate was approximately two to four orders of magnitude higher in females. In both males and females, gamete biomass production rate increased with organism mass as a power law, much like individual metabolic rate. This suggests that whole-organism energetics may act as a primary constraint on gamete production among species. Residual variation in sperm production rate was positively correlated with relative testes size. Together, these results suggest that understanding the heterogeneity in rates of gamete production among species requires joint consideration of the effects of gonad mass and metabolism.     

Swimming by sea otters: adaptations for low energetic cost locomotion

The energetics and hydrodynamics of surface and submerged swimming were compared in the sea otter (Enhydra lutris). 1. Sea otters used two distinct speed ranges that varied with swimming mode. Sustained surface swimming was limited to speeds less than 0.80 m/s, while sustained submerged swimming occurred over the range of 0.60 to 1.39 m/s. 2. Rates of oxygen consumption (VO2) at the transition speed (0.80 m/s) were 41% lower for submerged swimming by sea otters in comparison to surface swimming. 3. Total cost of transport for surface swimming sea otters, 12.56 joules/kg.m, was more than 12 times the predicted value for a similarly-sized salmonid fish. Transport costs for submerged swimming at the same speed was only 7.33 times the predicted value. 4. The allometric relationship for minimum cost of transport in surface swimming birds and mammals was y = 23.87 chi -0.15 where y = cost of transport in joules/kg.m and x = body mass in kg. This regression loosely parallels the relationship for salmonid fish. 5. Correlations between aquatic behavior, morphological specialization, and swimming energetics indicate that the development of swimming in mustelids involved transitions from fore-paw to hind-paw propulsion, and from surface to submerged swimming.     

The relative cost of bent-hip bent-knee walking is reduced in water

The debate about how early hominids walked may be characterised as two competing hypotheses: They moved with a fully upright (FU) gait, like modern humans, or with a bent-hip, bent-knee (BK) gait, like apes. Both have assumed that this bipedalism was almost exclusively on land, in trees or a combination of the two. Recent findings favoured the FU hypothesis by showing that the BK gait is 50–60% more energetically costly than a FU human gait on land. We confirm these findings but show that in water this cost differential is markedly reduced, especially in deeper water, at slower speeds and with greater knee flexion. These data suggest that the controversy about australopithecine locomotion may be eased if it is assumed that wading was a component of their locomotor repertoire and supports the idea that shallow water might have been an environment favourable to the evolution of early forms of “non-optimal” hominid bipedalism.