Marshall D. McCue

¹³ C breath testing is increasingly used in physiology and ecology research because of what it reveals about the different fuels that animals oxidize to meet their energetic demands. Here I review the practice of ¹³ C breath testing in humans and other animals and describe the impact that contamination by ambient/background CO ₂ in the air can have on the accuracy of ¹³ C breath measurements. I briefly discuss physical methods to avoid sample contamination as well as the Keeling plot approach that researchers have been using for the past two decades to estimate δ ¹³ C from breath samples mixed with ambient CO ₂ . Unfortunately, Keeling plots are not suited for ¹³ C breath testing in common situations where (1) a subject's VCO ₂ is dynamic, (2) ambient [CO ₂ ] may change, (3) a subject is sensitive to hypercapnia, or (4) in any flow‐through indirect calorimetry system. As such, I present a mathematical solution that addresses these issues by using information about the instantaneous [CO ₂ ] and the δ ¹³ CO ₂ of ambient air as well as the diluted breath sample to back‐calculate the δ ¹³ CO ₂ in the CO ₂ exhaled by the animal. I validate this approach by titrating a sample of ¹³ C‐enriched gas into an air stream and demonstrate its ability to provide accurate values across a wide range of breath and air mixtures. This approach allows researchers to instantaneously calculate the δ ¹³ C of exhaled gas of humans or other animals in real time without having to scrub ambient CO ₂ or rely on estimated values.

¹³ C-Breath testing is increasingly used in physiology and ecology research because of what it reveals about the different fuels that animals oxidize to meet their energetic demands. Here we review the practice of ¹³ C-breath testing in humans and other animals and describe the impact that contamination that ambient/background CO ₂ in the air can have on the accuracy of ¹³ C-breath measurements. We briefly discuss physical methods to avoid sample contamination as well as the Keeling plot approach that researchers have been using for the past two decades to estimate δ ¹³ C from breath samples mixed with ambient CO ₂ . Unfortunately, Keeling plots are not suited for ¹³ C breath testing in common situations where 1) a subject’s VCO ₂ is dynamic 2) ambient [CO ₂ ] may change, 3) a subject is sensitive to hypercapnia, or 4) in any flow-through indirect calorimetry system. As such, we present a mathematical solution that addresses these issues by using information about the instantaneous [CO ₂ ] and the δ ¹³ CO ₂ of ambient air as well as the diluted breath sample to back-calculate the δ ¹³ CO ₂ in the CO ₂ exhaled by the animal. We validate this approach by titrating a sample of ¹³ C-enriched gas into an air stream and demonstrate its ability to provide accurate values across a wide range of breath and air mixtures. Researchers can now instantaneously calculate the δ ¹³ C of alveolar gas of humans or animals in real time without having to scrub ambient CO ₂ or rely on estimated values.

Seasonal migration is a physiologically demanding endeavour that animals prepare for by finding and storing energy. Species and populations vary in their tendencies to migrate and the energetic demands of migration likely determine the degree and type of preparation that different animals undergo. Birds fuel their migratory flight using stored fat. Previous work has suggested that in addition to being energetically dense, certain lipids like polyunsaturated fatty acids (PUFAs) may be particularly efficient fuels that are preferentially mobilized for flight or serve may additional functions, such as modulating membrane fluidity or stimulating gene expression as ligands, during long‐distance migratory flight. Using a series of behavioural and metabolic experiments in a partial transplant aviary setting of Common Blackbirds (Turdus merula) populations that range from being fully migratory to partially migratory to sedentary, we asked how migratory restlessness (Zugunruhe), weight gain and oxidation of stearic acid (18:0), oleic acid (18:1n‐9), linoleic acid (18:2n‐6) and alpha linolenic acid (18:3n‐3; ALA) varied with migratory mode and season. We found no population‐level differences in the timing of Zugunruhe. However, before migration, a greater proportion of individuals from fully migratory populations exhibited weight gain and migratory restlessness, as well as increased oxidation of PUFAs. After Zugunruhe, only individuals from migratory populations showed reduced PUFA oxidation. All populations showed increased oxidation of saturated fat after Zugunruhe. Our results demonstrate that certain migratory traits, like the timing of Zugunruhe are expressed similarly across populations in response to local environmental conditions, while other traits, like lipid oxidation, can vary among populations. Read the free Plain Language Summary for this article on the Journal blog.

What makes some species successful invaders while others fail, and why some invaders have major impacts in invaded ecosystems are pivotal questions that are attracting major research effort. The increasing availability of high resolution, georeferenced stable isotope landscapes (‘isoscapes’), coupled with the commercialization of stable isotope‐enriched tracer molecules and the development of new analytical approaches, is facilitating novel applications of stable isotope techniques in ecology. We can now address ecological questions that were previously intractable. We review and discuss how stable isotope analysis (SIA) can complement fundamental research themes in the study of biological invasions, especially in answering questions relating to the physiological and ecological mechanisms underlying invasion processes and invader impacts. SIA was first used for simply describing the diet of invaders but, more recently, SIA‐informed metrics of population and community trophic structure have been advanced. These approaches now permit the comparison of diets across space and time and provide quantitative tools to compare food webs across different stages of invasion. SIA has also been pivotal in quantifying competition for resources between native and non‐native species (e.g. competition for food, water, or nutrient use). Specific questions related to modes of dispersal (e.g. origin and distance/direction travelled) and mechanisms of establishment can also be addressed using SIA in diverse taxa. An overarching goal is to highlight examples of recent studies that have used SIA in key areas of invasion ecology and use these to synthesize testable predictions where SIA could be applied to future studies. We conclude by highlighting several paths forward and describing how unresolved challenges in quantifying the rates, impacts, and mechanisms underlying invasions could potentially benefit from the use of SIA.

Starvation is common among wild animal populations, and many individuals experience repeated bouts of starvation over the course of their lives. Although much information has been gained through laboratory studies of acute starvation, little is known about how starvation affects an animal once food is again available (i.e. during the refeeding and recovery phases). Many animals exhibit a curious phenomenon – some seem to ‘get better’ at starving following exposure to one or more starvation events – by this we mean that they exhibit potentially adaptive responses, including reduced rates of mass loss, reduced metabolic rates, and lower costs of digestion. During subsequent refeedings they may also exhibit improved digestive efficiency and more rapid mass gain. Importantly, these responses can last until the next starvation bout or even be inherited and expressed in the subsequent generation. Currently, however, little is known about the molecular regulation and physiological mechanisms underlying these changes. Here, we identify areas of research that can fill in the most pressing knowledge gaps. In particular, we highlight how recently refined techniques (e.g. stable isotope tracers, quantitative magnetic resonance and thermal measurement) as well as next-generation sequencing approaches (e.g. RNA-seq, proteomics and holobiome sequencing) can address specific starvation-focused questions. We also describe outstanding unknowns ripe for future research regarding the timing and severity of starvation, and concerning the persistence of these responses and their interactions with other ecological stressors.

It was recently shown that fasting alters the composition of microbial communities residing in the distal intestinal tract of animals representing five classes of vertebrates [i.e., fishes (tilapia), amphibians (toads), reptiles (leopard geckos), birds (quail), and mammals (mice)]. In this study, we tested the hypothesis that the extent of tissue reorganization in the fasted distal intestine was correlated with the observed changes in enteric microbial diversity. Segments of intestine adjacent to those used for the microbiota study were examined histologically to quantify cross‐sectional and mucosal surface areas and thicknesses of mucosa, submucosa, and tunica muscularis. We found no fasting‐induced differences in the morphology of distal intestines of the mice (3 days), quail (7 days), or geckos (28 days). The toads, which exhibited a general increase in phylogenetic diversity of their enteric microbiota with fasting, also exhibited reduced mucosal circumference at 14 and 21 days of fasting. Tilapia showed increased phylogenetic diversity of their enteric microbiota, and showed a thickened tunica muscularis at 21 days of fasting; but this morphological change was not related to microbial diversity or absorptive surface area, and thus, is unlikely to functionally match the changes in their microbiome. Given that fasting caused significant increases and reductions in the enteric microbial diversity of mice and quail, respectively, but no detectable changes in distal intestine morphology, we conclude that reorganization is not the primary factor shaping changes in microbial diversity within the fasted colon, and the observed modest structural changes are more related to the fasted state. Anat Rec, 300:2208–2219, 2017.

During fasting, animals rely on a mixture of fats, carbohydrates, and proteins that are derived solely from endogenous sources. The relative contributions of these metabolic fuels chiefly depend on the duration of the fast, but other factors including previous diet, environmental temperature, and activity level can modulate the fuel mixture. It has long been held that endogenous proteins are spared from catabolism until the final stages of prolonged fasting and contribute a significant proportion of energy once the other metabolic fuels have been depleted. However, evidence is mounting that protein is catabolized supplemental to fat metabolism under some circumstances. This has been shown in migratory birds that exhibit dramatic reductions in lean mass during flights. One hypothesis to explain this seemingly maladaptive metabolic strategy is that the catabolism and oxidation of protein in situ yields five times more metabolic water than that generated through fat oxidation alone. Recent support for this hypothesis is that birds benefit from such a strategy due to their uricotelic nature. However, it remains unclear whether ureotelic mammals would also employ this strategy. Here we test the protein-for-water hypothesis in resting laboratory mice subjected to water deprivation during fasting while we tracked rates of protein and lipid catabolism using endogenously incorporated ¹³C-leucine and ¹³C-palmitic acid. We found no differences in instantaneous leucine oxidation; however, cumulative differences in instantaneous leucine oxidation ultimately resulted in a higher total leucine oxidation after 72 h of fasting in water-deprived animals. We also found that lipid oxidation was 8% higher in the hydrated mice, but the difference was not significant presumably because of a concomitant reduction in metabolic rates of the water-deprived mice. Our results indicate that mammals do increase rates of protein catabolism during dehydration but to a lesser degree than birds. The ability of mammals to produce highly concentrated urine and their lower inherent rates of protein turnover apparently preclude mammals from taking full advantage of the protein-for-water strategy during fasting under dehydrating conditions.

The risk of food limitation and, ultimately, starvation dates back to the dawn of heterotrophy in animals, yet starvation remains a major factor in the regulation of modern animal populations. Researchers studying starvation more than a century ago suggested that animals subjected to sublethal periods of food limitation are somehow more tolerant of subsequent starvation events. This possibility has received little attention over the past decades, yet it is highly relevant to modern science for two reasons. First, animals in natural populations are likely to be exposed to bouts of food limitation once or more before they face prolonged starvation, during which the risk of mortality becomes imminent. Second, our current approach to studying starvation physiology in the laboratory focuses on nourished animals with no previous exposure to nutritional stress. We examined the relationship between previous exposure to food limitation and potentially adaptive physiological responses to starvation in adult rats and found several significant differences. On two occasions, rats were fasted until they lost 20% of their body mass maintained lower body temperatures, and had presumably lower energy requirements when subjected to prolonged starvation than their naive cohort that never experienced food limitation. These rats that were trained in starvation also had lower plasma glucose set -points and reduced their reliance on endogenous lipid oxidation. These findings underscore (1) the need for biologists to revisit the classic hypothesis that animals can become habituated to starvation, using a modern set of research tools; and (2) the need to design controlled experiments of starvation physiology that more closely resemble the dynamic nature of food availability.

The energetics of processing a meal is crucial for understanding energy budgets of animals in the wild. Given that digestion and its associated costs may be dependent on environmental conditions, it is crucial to obtain a better understanding of these costs under diverse conditions and identify resulting behavioural or physiological trade-offs. This study examines the speed and metabolic costs - in cumulative, absolute, and relative energetic terms - of processing a bloodmeal for a major zoonotic disease vector, the tsetse fly Glossina brevipalpis, across a range of ecologically-relevant temperatures (25°C, 30°C & 35°C). Respirometry showed that flies used less energy digesting meals faster at higher temperatures but that their starvation tolerance was reduced supporting the prediction that warmer temperatures are optimal for bloodmeal digestion while cooler temperatures should be preferred for unfed or post-absorptive flies. 13C-Breath testing revealed that the flies oxidized dietary glucose and amino acids within the first couple of hours of feeding and overall oxidized more dietary nutrients at the cooler temperatures supporting the premise that warmer digestion temperatures are preferred because they maximise speed and minimise costs. An independent test of these predictions using a thermal gradient confirmed that recently fed flies selected warmer temperatures and then selected cooler temperatures as they became postabsorptive, presumably to maximize starvation resistance. Collectively these results suggest there are at least two thermal optima in a given population at any time and flies switch dynamically between optima throughout feeding cycles.