ABSTRACT The magnitude of many biological traits relates strongly and regularly to body size. Consequently, a major goal of comparative biology is to understand and apply these ‘size-scaling’ relationships, traditionally quantified by using linear regression analyses based on log-transformed data. However, recently some investigators have questioned this traditional method, arguing that linear or non-linear regression based on untransformed arithmetic data may provide better statistical fits than log-linear analyses. Furthermore, they advocate the replacement of the traditional method by alternative specific methods on a case-by-case basis, based simply on best-fit criteria. Here, I argue that the use of logarithms in scaling analyses presents multiple valuable advantages, both statistical and conceptual. Most importantly, log-transformation allows biologically meaningful, properly scaled (scale-independent) comparisons of organisms of different size, whereas non-scaled (scale-dependent) analyses based on untransformed arithmetic data do not. Additionally, log-based analyses can readily reveal biologically and theoretically relevant discontinuities in scale invariance during developmental or evolutionary increases in body size that are not shown by linear or non-linear arithmetic analyses. In this way, log-transformation advances our understanding of biological scaling conceptually, not just statistically. I hope that my Commentary helps students, non-specialists and other interested readers to understand the general benefits of using log-transformed data in size-scaling analyses, and stimulates advocates of arithmetic analyses to show how they may improve our understanding of scaling conceptually, not just statistically.

ABSTRACT There are two types of polyunsaturated fatty acids (i.e. fats that contain multiple carbon-carbon double bonds) – omega-6 and omega-3. They are not interconvertible, and they contribute ‘double-bonded carbons’ to different depths in bilayer membranes, with different effects on membrane processes. This Commentary emphasises the importance of these fats for biological membrane function and examines their evolution and biochemistry. Omega-6 and omega-3 fatty acids are separately essential in the diet of animals, and they pass up the food chain largely from plants, with ‘seeds’ being a prevalent source of omega-6, and ‘leaves’ a prevalent source of omega-3. The dietary balance between these fatty acids has a strong influence on membrane composition. Although this aspect of diet has been little investigated outside of the biomedical field, emerging evidence shows it can alter important physiological capacities of animals (e.g. exercise endurance and adiposity), which has implications for activities such as avian migration and hibernation and torpor, as well as significant implications for human health. This Commentary will focus on the separate effects of omega-3 and omega-6 on membrane properties and will emphasise the importance of the balance between these two fatty acids in determining the function of biological membranes; I hope to convince the reader that fats should be considered first and foremost as the basic unit of biological membranes, and secondarily as a means of energy storage.

ABSTRACT Physiologists often assume that mitochondria are the main producers of reactive oxygen species (ROS) in cells. Consequently, in biomedicine, mitochondria are considered as important targets for therapeutic treatments, and in evolutionary biology, they are considered as mediators of life-history tradeoffs. Surprisingly, data supporting such an assumption are lacking, at least partially due to the technical difficulties in accurately measuring the level of ROS produced by different subcellular compartments in intact cells. In this Commentary, we first review three potential reasons underlying the misassumption of mitochondrial dominance in the production of cellular ROS. We then introduce some other major sites/enzymes responsible for cellular ROS production. With the use of a recently developed cell-based assay, we further discuss the contribution of mitochondria to the total rate of ROS release in cell lines and primary cells of different species. In these cells, the contribution of mitochondria varies between cell types but mitochondria are never the main source of cellular ROS. This indicates that although mitochondria are one of the significant sources of cellular ROS, they are not necessarily the main contributor under normal conditions. Intriguingly, similar findings were also observed in cells under a variety of stressors, life-history strategies and pathological stages, in which the rates of cellular ROS production were significantly enhanced. Finally, we make recommendations for designing future studies. We hope this paper will encourage investigators to carefully consider non-mitochondrial sources of cellular ROS in their study systems or models.

ABSTRACT Temperature is an important environmental factor governing the ability of organisms to grow, survive and reproduce. Thermal performance curves (TPCs), with some caveats, are useful for charting the relationship between body temperature and some measure of performance in ectotherms, and provide a standardized set of characteristics for interspecific comparisons. Endotherms, however, have a more complicated relationship with environmental temperature, as endothermy leads to a decoupling of body temperature from external temperature through use of metabolic heat production, large changes in insulation and variable rates of evaporative heat loss. This has impeded our ability to model endothermic performance in relation to environmental temperature as well as to readily compare performance between species. In this Commentary, we compare the strengths and weaknesses of potential TPC analogues (including other useful proxies for linking performance to temperature) in endotherms and suggest several ways forward in the comparative ecophysiology of endotherms. Our goal is to provide a common language with which ecologists and physiologists can evaluate the effects of temperature on performance. Key directions for improving our understanding of endotherm thermoregulatory physiology include a comparative approach to the study of the level and precision of body temperature, measuring performance directly over a range of body temperatures and building comprehensive mechanistic models of endotherm responses to environmental temperatures. We believe the answer to the question posed in the title could be ‘yes’, but only if ‘performance’ is well defined and understood in relation to body temperature variation, and the costs and benefits of endothermy are specifically modelled.

ABSTRACT Temperature influences many physiological processes that govern life as a result of the thermal sensitivity of chemical reactions. The repeated evolution of endothermy and widespread behavioral thermoregulation in animals highlight the importance of elevating tissue temperature to increase the rate of chemical processes. Yet, movement performance that is robust to changes in body temperature has been observed in numerous species. This thermally robust performance appears exceptional in light of the well-documented effects of temperature on muscle contractile properties, including shortening velocity, force, power and work. Here, we propose that the thermal robustness of movements in which mechanical processes replace or augment chemical processes is a general feature of any organismal system, spanning kingdoms. The use of recoiling elastic structures to power movement in place of direct muscle shortening is one of the most thoroughly studied mechanical processes; using these studies as a basis, we outline an analytical framework for detecting thermal robustness, relying on the comparison of temperature coefficients ( Q 10 values) between chemical and mechanical processes. We then highlight other biomechanical systems in which thermally robust performance that arises from mechanical processes may be identified using this framework. Studying diverse movements in the context of temperature will both reveal mechanisms underlying performance and allow the prediction of changes in performance in response to a changing thermal environment, thus deepening our understanding of the thermal ecology of many organisms.

‘Omics’ methods, such as transcriptomics, proteomics, lipidomics or metabolomics, yield simultaneous measurements of many related molecules in a sample. These approaches have opened new opportunities to generate and test hypotheses about the mechanisms underlying biochemical and physiological phenotypes. In this Commentary, we discuss general approaches and considerations for successfully integrating omics into comparative physiology. The choice of omics approach will be guided by the availability of existing resources and the time scale of the process being studied. We discuss the use of whole-organism extracts (common in omics experiments on small invertebrates) because such an approach may mask underlying physiological mechanisms, and we consider the advantages and disadvantages of pooling samples within biological replicates. These methods can bring analytical challenges, so we describe the most easily analyzed omics experimental designs. We address the propensity of omics studies to digress into ‘fishing expeditions’ and show how omics can be used within the hypothetico-deductive framework. With this Commentary, we hope to provide a roadmap that will help newcomers approach omics in comparative physiology while avoiding some of the potential pitfalls, which include ambiguous experiments, long lists of candidate molecules and vague conclusions.

ABSTRACT The avian pectoralis muscle demonstrates incredible plasticity. This muscle is the sole thermogenic organ of small passerine birds, and many temperate small passerines increase pectoralis mass in winter, potentially to increase heat production. Similarly, this organ can double in size prior to migration in migratory birds. In this Commentary, following the August Krogh principle, I argue that the avian pectoralis is the perfect tissue to reveal general features of muscle physiology. For example, in both mammals and birds, skeletal muscle fiber diameter is generally accepted to be within 10–100 µm. This size constraint is assumed to include reaction-diffusion limitations, coupled with metabolic cost savings associated with fiber geometry. However, avian muscle fiber structure has been largely ignored in this field, and the extensive remodeling of the avian pectoralis provides a system with which to investigate this. In addition, fiber diameter has been linked to whole-animal metabolic rates, although this has only been addressed in a handful of bird studies, some of which demonstrate previously unreported levels of plasticity and flexibility. Similarly, myonuclei, which are responsible for protein turnover within the fiber, have been forgotten in the avian literature. The few studies that have addressed myonuclear domain (MND) changes in avian muscle have found rates of change not previously seen in mammals. Both fiber diameter and MND have strong implications for aging rates; most aging mammals demonstrate muscular atrophy (a decrease in fiber diameter) and changes in MND. As I discuss here, these features are likely to differ in birds.

ABSTRACT Natural habitats are rapidly declining due to urbanisation, with a concomitant decline in biodiversity in highly urbanised areas. Yet thousands of different species have colonised urban environments. These organisms are exposed to novel urban conditions, which are sometimes beneficial, but most often challenging, such as increased ambient temperature, chemicals, noise and light pollution, dietary alterations and disturbance by humans. Given the fundamental role of physiological responses in coping with such conditions, certain physiological systems such as the redox system, metabolism and hormones are thought to specifically influence organisms’ ability to persist and cope with urbanisation. However, these physiological systems often show mixed responses to urbanisation. Does this mean that some individuals, populations or species are resilient to the urban environmental challenges? Or is something missing from our analyses, leading us to erroneous conclusions regarding the impact of urbanisation? To understand the impact of urbanisation, I argue that a more integrated mechanistic and ecological approach is needed, along with experiments, in order to fully understand the physiological responses; without knowledge of their ecological and evolutionary context, physiological measures alone can be misinterpreted. Furthermore, we need to further investigate the causes of and capacity for individual plasticity in order to understand not only the impact of urbanisation, but also species resilience. I argue that abiotic and biotic urban factors can interact (e.g. pollution with micro- and macronutrients) to either constrain or relax individual physiological responses – and, thereby, plasticity – on a temporal and/or spatial scale, which can lead to erroneous conclusions regarding the impact of urbanisation.

ABSTRACT Magnetoreception is the ability to sense the Earth's magnetic field, which is used for orientation and navigation. Behavioural experiments have shown that it is employed by many species across all vertebrate classes; however, our understanding of how magnetic information is processed and integrated within the central nervous system is limited. In this Commentary, we review the progress in birds and rodents, highlighting the role of the vestibular and trigeminal systems as well as that of the hippocampus. We reflect on the strengths and weaknesses of the methodologies currently at our disposal, the utility of emerging technologies and identify questions that we feel are critical for the advancement of the field. We expect that magnetic circuits are likely to share anatomical motifs with other senses, which culminates in the formation of spatial maps in telencephalic areas of the brain. Specifically, we predict the existence of spatial cells that encode defined components of the Earth's magnetic field.

ABSTRACT An animal's maneuverability will determine the outcome of many of its most important interactions. A common approach to studying maneuverability is to force the animal to perform a specific maneuver or to try to elicit maximal performance. Recently, the availability of wider-field tracking technology has allowed for high-throughput measurements of voluntary behavior, an approach that produces large volumes of data. Here, we show how these data allow for measures of inter-individual variation that are necessary to evaluate how performance depends on other traits, both within and among species. We use simulated data to illustrate best practices when sampling a large number of voluntary maneuvers. Our results show how the sample average can be the best measure of inter-individual variation, whereas the sample maximum is neither repeatable nor a useful metric of the true variation among individuals. Our studies with flying hummingbirds reveal that their maneuvers fall into three major categories: simple translations, simple rotations and complex turns. Simple maneuvers are largely governed by distinct morphological and/or physiological traits. Complex turns involve both translations and rotations, and are more subject to inter-individual differences that are not explained by morphology. This three-part framework suggests that different wingbeat kinematics can be used to maximize specific aspects of maneuverability. Thus, a broad explanatory framework has emerged for interpreting hummingbird maneuverability. This framework is general enough to be applied to other types of locomotion, and informative enough to explain mechanisms of maneuverability that could be applied to both animals and bio-inspired robots.

ABSTRACT A powerful way to evaluate scientific explanations (hypotheses) is to test the predictions that they make. In this way, predictions serve as an important bridge between abstract hypotheses and concrete experiments. Experimental biologists, however, generally receive little guidance on how to generate quality predictions. Here, we identify two important components of good predictions – criticality and persuasiveness – which relate to the ability of a prediction (and the experiment it implies) to disprove a hypothesis or to convince a skeptic that the hypothesis has merit. Using a detailed example, we demonstrate how striving for predictions that are both critical and persuasive can speed scientific progress by leading us to more powerful experiments. Finally, we provide a quality control checklist to assist students and researchers as they navigate the hypothetico-deductive method from puzzling observations to experimental tests.

ABSTRACT Diverse organisms use Earth's magnetic field as a cue in orientation and navigation. Nevertheless, eliciting magnetic orientation responses reliably, either in laboratory or natural settings, is often difficult. Many species appear to preferentially exploit non-magnetic cues if they are available, suggesting that the magnetic sense often serves as a redundant or ‘backup’ source of information. This raises an interesting paradox: Earth's magnetic field appears to be more pervasive and reliable than almost any other navigational cue. Why then do animals not rely almost exclusively on the geomagnetic field, while ignoring or downplaying other cues? Here, we explore a possible explanation: that the magnetic sense of animals is ‘noisy’, in that the magnetic signal is small relative to thermal and receptor noise. Magnetic receptors are thus unable to instantaneously acquire magnetic information that is highly precise or accurate. We speculate that extensive time-averaging and/or other higher-order neural processing of magnetic information is required, rendering the magnetic sense inefficient relative to alternative cues that can be detected faster and with less effort. This interpretation is consistent with experimental results suggesting a long time course for magnetic compass and map responses in some animals. Despite possible limitations, magnetoreception may be maintained by natural selection because the geomagnetic field is sometimes the only source of directional and/or positional information available.

ABSTRACT The epigenome determines heritable patterns of gene expression in the absence of changes in DNA sequence. The result is programming of different cellular-, tissue- and organ-specific phenotypes from a single organismic genome. Epigenetic marks that comprise the epigenome (e.g. methylation) are placed upon or removed from chromatin (histones and DNA) to direct the activity of effectors that regulate gene expression and chromatin structure. Recently, the cytoskeleton has been identified as a second target for the cell's epigenetic machinery. Several epigenetic ‘readers, writers and erasers’ that remodel chromatin have been discovered to also remodel the cytoskeleton, regulating structure and function of microtubules and actin filaments. This points to an emerging paradigm for dual-function remodelers with ‘chromatocytoskeletal’ activity that can integrate cytoplasmic and nuclear functions. For example, the SET domain-containing 2 methyltransferase (SETD2) has chromatocytoskeletal activity, methylating both histones and microtubules. The SETD2 methyl mark on chromatin is required for efficient DNA repair, and its microtubule methyl mark is required for proper chromosome segregation during mitosis. This unexpected convergence of SETD2 activity on histones and microtubules to maintain genomic stability suggests the intriguing possibility of an expanded role in the cell for chromatocytoskeletal proteins that read, write and erase methyl marks on the cytoskeleton as well as chromatin. Coordinated use of methyl marks to remodel both the epigenome and the (epi)cytoskeleton opens the possibility for integrated regulation (which we refer to as ‘epiregulation’) of other higher-level functions, such as muscle contraction or learning and memory, and could even have evolutionary implications.

ABSTRACT The air volume in the respiratory system of marine tetrapods provides a store of O 2 to fuel aerobic metabolism during dives; however, it can also be a liability, as the associated N 2 can increase the risk of decompression sickness. In order to more fully understand the physiological limitations of different air-breathing marine vertebrates, it is therefore important to be able to accurately estimate the air volume in the respiratory system during diving. One method that has been used to do so is to calculate the air volume from glide phases – periods of movement during which no thrust is produced by the animal – which many species conduct during ascent periods, when gases are expanding owing to decreasing hydrostatic pressure. This method assumes that there is conservation of mass in the respiratory system, with volume changes only driven by pressure. In this Commentary, we use previously published data to argue that both the respiratory quotient and differences in tissue and blood gas solubility potentially alter the mass balance in the respiratory system throughout a dive. Therefore, near the end of a dive, the measured volume of gas at a given pressure may be 12–50% less than from the start of the dive; the actual difference will depend on the length of the dive, the cardiac output, the pulmonary shunt and the metabolic rate. Novel methods and improved understanding of diving physiology will be required to verify the size of the effects described here and to more accurately estimate the volume of gas inhaled at the start of a dive.

ABSTRACT The evolution of wings has played a key role in the success of insect species, allowing them to diversify to fill many niches. Insect wings are complex multifunctional structures, which not only have to withstand aerodynamic forces but also need to resist excessive stresses caused by accidental collisions. This Commentary provides a summary of the literature on damage-reducing morphological adaptations in wings, covering natural causes of wing collisions, their impact on the structural integrity of wings and associated consequences for both insect flight performance and life expectancy. Data from the literature and our own observations suggest that insects have evolved strategies that (i) reduce the likelihood of wing damage and (ii) allow them to cope with damage when it occurs: damage-related fractures are minimized because wings evolved to be damage tolerant and, in the case of wing damage, insects compensate for the reduced aerodynamic efficiency with dedicated changes in flight kinematics.

ABSTRACT The lateral accessory lobes (LALs), paired structures that are homologous among all insect species, have been well studied for their role in pheromone tracking in silkmoths and phonotaxis in crickets, where their outputs have been shown to correlate with observed motor activity. Further studies have shown more generally that the LALs are crucial both for an insect's ability to steer correctly and for organising the outputs of the descending pathways towards the motor centres. In this context, we propose a framework by which the LALs may be generally involved in generating steering commands across a variety of insects and behaviours. Across different behaviours, we see that the LAL is involved in generating two kinds of steering: (1) search behaviours and (2) targeted steering driven by direct sensory information. Search behaviours are generated when the current behaviourally relevant cues are not available, and a well-described LAL subnetwork produces activity which increases sampling of the environment. We propose that, when behaviourally relevant cues are available, the LALs may integrate orientation information from several sensory modalities, thus leading to a collective output for steering driven by those cues. These steering commands are then sent to the motor centres, and an additional efference copy is sent back to the orientation-computing areas. In summary, we have taken known aspects of the neurophysiology and function of the insect LALs and generated a speculative framework that suggests how LALs might be involved in steering control for a variety of complex real-world behaviours in insects.

ABSTRACT The biological effects of oxidants, especially reactive oxygen species (ROS), include signaling functions (oxidative eustress), initiation of measures to reduce elevated ROS (oxidative stress), and a cascade of pathophysiological events that accompany excessive ROS (oxidative distress). Although these effects have long been studied in animal models with perturbed ROS, their actions under physiological conditions are less clear. I propose that some of the apparent uncertainty may be due to confusion of ROS with endogenously generated reactive sulfur species (RSS). ROS and RSS are chemically similar, but RSS are more reactive and versatile, and can be stored and reused. Both ROS and RSS signal via oxidation reactions with protein cysteine sulfur and they produce identical effector responses, but RSS appear to be more effective. RSS in the form of persulfidated cysteines (Cys-S-S) are produced endogenously and co-translationally introduced into proteins, and there is increasing evidence that many cellular proteins are persulfidated. A number of practical factors have contributed to confusion between ROS and RSS, and these are discussed herein. Furthermore, essentially all endogenous antioxidant enzymes appeared shortly after life began, some 3.8 billion years ago, when RSS metabolism dominated evolution. This was long before the rise in ROS, 600 million years ago, and I propose that these same enzymes, with only minor modifications, still effectively metabolize RSS in extant organisms. I am not suggesting that all ROS are RSS; however, I believe that the relative importance of ROS and RSS in biological systems needs further consideration.

ABSTRACT Serotonin (5-HT) has largely been accepted to be inhibitory to vertebrate aggression, whereas an opposing stimulatory role has been proposed for invertebrates. Herein, we argue that critical gaps in our understanding of the nuanced role of 5-HT in invertebrate systems drove this conclusion prematurely, and that emerging data suggest a previously unrecognized level of phylogenetic conservation with respect to neurochemical mechanisms regulating the expression of aggressive behaviors. This is especially apparent when considering the interplay among factors governing 5-HT activity, many of which share functional homology across taxa. We discuss recent findings using insect models, with an emphasis on the stalk-eyed fly, to demonstrate how particular 5-HT receptor subtypes mediate the intensity of aggression with respect to discrete stages of the interaction (initiation, escalation and termination), which mirrors the complex behavioral regulation currently recognized in vertebrates. Further similarities emerge when considering the contribution of neuropeptides, which interact with 5-HT to ultimately determine contest progression and outcome. Relative to knowledge in vertebrates, much less is known about the function of 5-HT receptors and neuropeptides in invertebrate aggression, particularly with respect to sex, species and context, prompting the need for further studies. Our Commentary highlights the need to consider multiple factors when determining potential taxonomic differences, and raises the possibility of more similarities than differences between vertebrates and invertebrates with regard to the modulatory effect of 5-HT on aggression.

ABSTRACT The critical O 2 tension ( P crit ) is the lowest P O 2 at which an animal can maintain some benchmark rate of O 2 uptake ( Ṁ O 2 ). This P O 2 has long served as a comparator of hypoxia tolerance in fishes and aquatic invertebrates, but its usefulness in this role, particularly when applied to fishes, has recently been questioned. We believe that P crit remains a useful comparator of hypoxia tolerance provided it is determined using the proper methods and hypoxia tolerance is clearly defined. Here, we review the available methods for each of the three steps of P crit determination: (1) measuring the most appropriate benchmark Ṁ O 2 state for P crit determination ( Ṁ O 2 ,std , the Ṁ O 2 required to support standard metabolic rate); (2) reducing water P O 2 ; and (3) calculating P crit from the Ṁ O 2 versus P O 2 curve. We make suggestions on best practices for each step and for how to report P crit results to maximize their comparative value. We also discuss the concept of hypoxia tolerance and how P crit relates to a fish's overall hypoxia tolerance. When appropriate methods are used, P crit provides useful comparative physiological and ecological information about the aerobic contributions to a fish's hypoxic survival. When paired with other hypoxia-related physiological measurements (e.g. lactate accumulation, calorimetry-based measurements of metabolic depression, loss-of-equilibrium experiments), P crit contributes to a comprehensive understanding of how a fish combines aerobic metabolism, anaerobic metabolism and metabolic depression in an overall strategy for hypoxia tolerance.

ABSTRACT Closed kinematic chains (CKCs), links connected to form one or more closed loops, are used as simple models of musculoskeletal systems (e.g. the four-bar linkage). Previous applications of CKCs have primarily focused on biomechanical systems with rigid links and permanently closed chains, which results in constant mobility (the total degrees of freedom of a system). However, systems with non-rigid elements (e.g. ligaments and muscles) and that alternate between open and closed chains (e.g. standing on one foot versus two) can also be treated as CKCs with changing mobility. Given that, in general, systems that have fewer degrees of freedom are easier to control, what implications might such dynamic changes in mobility have for motor control? Here, I propose a CKC classification to explain the different ways in which mobility of musculoskeletal systems can change dynamically during behavior. This classification is based on the mobility formula, taking into account the number of loops in the CKC and the nature of the constituent joint mobilities. I apply this mobility-based classification to five biomechanical systems: the human lower limbs, the operculum–lower jaw mechanism of fishes, the upper beak rotation mechanism of birds, antagonistic muscles at the human ankle joint and the human jaw processing a food item. I discuss the implications of this classification, including that mobility itself may be dynamically manipulated to simplify motor control. The principal aim of this Commentary is to provide a framework for quantifying mobility across diverse musculoskeletal systems to evaluate its potentially key role in motor control.