Hymenopteran-specific TRPA channel from the Texas leaf cutter ant (Atta texana) is heat and cold activated and expression correlates with environmental temperature.

Leaf cutting ants of the genus Atta cultivate fungal gardens, carefully modifying environmental conditions to maintain optimal temperature for fungal growth. Antennal nerves from Atta are highly temperature sensitive, but the underlying molecular sensor is unknown. Here, we utilize Atta texana (Texas leaf cutter ant) to investigate the molecular basis of ant temperature sensation and how it might have evolved as the range expanded northeast across Texas from ancestral populations in Mexico. We focus on transient receptor potential (TRP) channel genes, the best characterized temperature sensor proteins in animals. Atta texana antennae express 6 of 13 Hymenopteran TRP channel genes and sequences are under a mix of relaxed and intensified selection. In a behavioral assay, we find A. texana workers prefer 24 °C (range 21-26 °C) for fungal growth. There was no evidence of regulatory evolution across a temperature transect in Texas, but instead Hymenoptera-specific TRPA (HsTRPA) expression highly correlated with ambient temperature. When expressed in vitro, HsTRPA from A. texana is temperature activated with Q10 values exceeding 100 on initial exposure to temperatures above 33 °C. Surprisingly, HsTRPA also appears to be activated by cooling, and therefore to our knowledge, the first non-TRPA1 ortholog to be described with dual heat/cold activation and the first in any invertebrate.

Temperature activated transient receptor potential ion channels from Antarctic fishes.

Antarctic notothenioid fishes (cryonotothenioids) live in waters that range between -1.86°C and an extreme maximum +4°C. Evidence suggests these fish sense temperature peripherally, but the molecular mechanism of temperature sensation in unknown. Previous work identified transient receptor potential (TRP) channels TRPA1b, TRPM4 and TRPV1a as the top candidates for temperature sensors. Here, cryonotothenioid TRPA1b and TRPV1a are characterized using Xenopus oocyte electrophysiology. TRPA1b and TRPV1a showed heat-evoked currents with Q10s of 11.1 ± 2.2 and 20.5 ± 2.4, respectively. Unexpectedly, heat activation occurred at a threshold of 22.9 ± 1.3°C for TRPA1b and 32.1 ± 0.6°C for TRPV1a. These fish have not experienced such temperatures for at least 15 Myr. Either (1) another molecular mechanism underlies temperature sensation, (2) these fishes do not sense temperatures below these thresholds despite having lethal limits as low as 5°C, or (3) native cellular conditions modify the TRP channels to function at relevant temperatures. The effects of osmolytes, pH, oxidation, phosphorylation, lipids and accessory proteins were tested. No conditions shifted the activity range of TRPV1a. Oxidation in combination with reduced cholesterol significantly dropped activation threshold of TRPA1b to 11.3 ± 2.3°C, it is hypothesized the effect may be due to lipid raft disruption.

A potential cost of evolving epibatidine resistance in poison frogs.

BACKGROUND: Some dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor ß2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4ß2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration-response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the ß2 S108C into the α4ß2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs. RESULTS: The introduction of S108C into the α4ß2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4ß2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity. CONCLUSIONS: The most parsimonious explanation of our current and previous work is that the S108C substitution renders the ß2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while ß2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 receptor function.

A potential cost of evolving epibatidine resistance in poison frogs.

Background: Some poison arrow frogs sequester the toxin epibatidine as a defense against predators. We previously identified a single amino acid substitution (S108C) at a highly conserved site in a neuronal nicotinic acetylcholine receptor (nAChR) ß2 subunit that prevents epibatidine from binding to this receptor. When placed in a homologous mammalian nAChR this substitution minimized epibatidine binding but also perturbed acetylcholine binding, a clear cost. However, in the nAChRs of poison arrow frogs, this substitution appeared to have no detrimental effect on acetylcholine binding and, thus, appeared cost-free. Results: The introduction of S108C into the α4ß2 nAChRs of non-dendrobatid frogs also does not affect ACh sensitivity, when these receptors are expressed in Xenopus laevis oocytes. However, α4ß2 nAChRs with C108 had a decreased magnitude of neurotransmitter-induced currents in all species tested ( Epipedobates anthonyi , non-dendrobatid frogs, as well as human), compared with α4ß2 nAChRs with the conserved S108. Immunolabeling of frog or human α4ß2 nAChRs in the plasma membrane using radiolabeled antibody against the ß2 nAChR subunit shows that C108 significantly decreased the number of cell-surface α4ß2 nAChRs, compared with S108. Conclusions: While S108C protects these species against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 nAChR function. These results may explain the high conservation of a serine at this site in vertebrates, as well as provide an example of a tradeoff between beneficial and deleterious effects of an evolutionary change. They also provide important clues for future work on assembly and trafficking of this important neurotransmitter receptor.

Evolution of Transient Receptor Potential (TRP) Ion Channels in Antarctic Fishes (Cryonotothenioidea) and Identification of Putative Thermosensors.

Animals rely on their sensory systems to inform them of ecologically relevant environmental variation. In the Southern Ocean, the thermal environment has remained between -1.9 and 5 °C for 15 Myr, yet we have no knowledge of how an Antarctic marine organism might sense their thermal habitat as we have yet to discover a thermosensitive ion channel that gates (opens/closes) below 10 °C. Here, we investigate the evolutionary dynamics of transient receptor potential (TRP) channels, which are the primary thermosensors in animals, within cryonotothenioid fishes-the dominant fish fauna of the Southern Ocean. We found cryonotothenioids have a similar complement of TRP channels as other teleosts (∼28 genes). Previous work has shown that thermosensitive gating in a given channel is species specific, and multiple channels act together to sense the thermal environment. Therefore, we combined evidence of changes in selective pressure, gene gain/loss dynamics, and the first sensory ganglion transcriptome in this clade to identify the best candidate TRP channels that might have a functional dynamic range relevant for frigid Antarctic temperatures. We concluded that TRPV1a, TRPA1b, and TRPM4 are the likeliest putative thermosensors, and found evidence of diversifying selection at sites across these proteins. We also put forward hypotheses for molecular mechanisms of other cryonotothenioid adaptations, such as reduced skeletal calcium deposition, sensing oxidative stress, and unusual magnesium homeostasis. By completing a comprehensive and unbiased survey of these genes, we lay the groundwork for functional characterization and answering long-standing thermodynamic questions of thermosensitive gating and protein adaptation to low temperatures.

A systems change framework for evaluating academic equity and inclusion in an Ecology and Evolution Graduate Program.

While academia is moving forward in terms of diversifying recruitment of undergraduate and graduate students, diverse representation is still not found across the academic hierarchy. At the graduate level, new discussions are emerging around efforts to improve the experiences of women and underrepresented minorities through inclusive graduate programming. Inclusive graduate programs are that which actively center and prioritize support for diverse experiences, identities, career goals, and perspectives, from recruitment through graduation. Establishing regular and rigorous evaluation of equity and inclusion efforts and needs is a critical component of this work. This is recognized by funding agencies that increasingly require reporting on inclusion efforts; here, we suggest use of a systems change framework for these evaluations. A systems change approach emphasizes three levels: explicit change (e.g., policies), semi-explicit change (e.g., power dynamics), and implicit change (e.g., biases). We use the Ecology, Evolution, and Behavior (EEB) PhD Program at the University of Texas at Austin in an exercise to (a) identify areas of concern regarding inclusive programming voiced by graduate students, (b) categorize efforts to address these concerns, and (c) integrating and evaluating which areas of the systems change framework show the greatest progress or potential for progress. We argue this framework is particularly useful for academic systems as they are complex, composed of variable individuals, and must address diverse stakeholder needs.

A morphometric analysis of the lungs of high-altitude ducks and geese.

We examined the morphology of the lungs of five species of high-altitude resident ducks from Lake Titicaca in the Peruvian Andes (yellow-billed pintail [Anas georgica], cinnamon teal [Anas cyanoptera orinomus], puna teal [Anas puna], speckled teal [Anas flavirostris oxyptera], and ruddy duck [Oxyura jamaicensis ferruginea]) and compared them with those of the high-altitude migratory bar-headed goose (Anser indicus) and the low-altitude migratory barnacle goose (Branta leucopsis). We then determined the relationship between mass-specific lung volume, the volume densities of the component parts of the lung, and previously reported hypoxia-induced increases in pulmonary O2 extraction. We found that the mass-specific lung volumes and the mass-specific volume of the exchange tissue were larger in the lungs of high-altitude resident birds. The bar-headed goose had a mass-specific lung volume that fell between those of the low-altitude species and the high-altitude residents, but a mass-specific volume of exchange tissue that was not significantly different than that of the high-altitude residents. The data suggest that the mass-specific volume of the lung may increase with evolutionary time spent at altitude. We found an inverse relationship between the percentage increase in pulmonary O2 extraction and the percentage increase in ventilation across species that was independent of the volume density of the exchange tissue, at least for the resident Andean birds.

Cardiovascular responses to progressive hypoxia in ducks native to high altitude in the Andes.

The cardiovascular system is critical for delivering O2 to tissues. Here, we examined the cardiovascular responses to progressive hypoxia in four high-altitude Andean duck species compared with four related low-altitude populations in North America, tested at their native altitude. Ducks were exposed to stepwise decreases in inspired partial pressure of O2 while we monitored heart rate, O2 consumption rate, blood O2 saturation, haematocrit (Hct) and blood haemoglobin (Hb) concentration. We calculated O2 pulse (the product of stroke volume and the arterial-venous O2 content difference), blood O2 concentration and heart rate variability. Regardless of altitude, all eight populations maintained O2 consumption rate with minimal change in heart rate or O2 pulse, indicating that O2 consumption was maintained by either a constant arterial-venous O2 content difference (an increase in the relative O2 extracted from arterial blood) or by a combination of changes in stroke volume and the arterial-venous O2 content difference. Three high-altitude taxa (yellow-billed pintails, cinnamon teal and speckled teal) had higher Hct and Hb concentration, increasing the O2 content of arterial blood, and potentially providing a greater reserve for enhancing O2 delivery during hypoxia. Hct and Hb concentration between low- and high-altitude populations of ruddy duck were similar, representing a potential adaptation to diving life. Heart rate variability was generally lower in high-altitude ducks, concurrent with similar or lower heart rates than low-altitude ducks, suggesting a reduction in vagal and sympathetic tone. These unique features of the Andean ducks differ from previous observations in both Andean geese and bar-headed geese, neither of which exhibit significant elevations in Hct or Hb concentration compared with their low-altitude relatives, revealing yet another avian strategy for coping with high altitude.

Cardiovascular responses to progressive hypoxia in ducks native to high altitude in the Andes.

The cardiovascular system is critical for delivering O2 to tissues. Here we examine the cardiovascular responses to progressive hypoxia in four high-altitude Andean duck species compared to four related low-altitude populations in North America, tested at their native altitude. Ducks were exposed to stepwise decreases in inspired partial pressure of O2 while we monitored heart rate, O2 consumption rate, blood O2 saturation, haematocrit (Hct), and blood haemoglobin concentration [Hb]. We calculated O2 pulse (the product of stroke volume and the arterial-venous O2 content difference), blood O2 concentration, and heart rate variability. Regardless of altitude, all eight populations maintained O2 consumption rate with minimal change in heart rate or O2 pulse, indicating that O2 consumption was maintained by either a constant arterial-venous O2 content difference (an increase in the relative O2 extracted from arterial blood) or by a combination of changes in stroke volume and the arterial-venous O2 content difference. Three high-altitude taxa (yellow-billed pintails, cinnamon teal, and speckled teal) had higher Hct and [Hb], increasing the O2 content of arterial blood, and potentially providing a greater reserve for enhancing O2 delivery during hypoxia. Hct and [Hb] between low- and high-altitude populations of ruddy duck were similar, representing a potential adaptation to diving life. Heart rate variability was generally lower in high-altitude ducks, concurrent with similar or lower heart rates than low-altitude ducks, suggesting a reduction in vagal and sympathetic tone. These unique features of the Andean ducks differ from previous observations in both Andean geese and bar-headed geese, neither of which exhibit significant elevations in Hct or [Hb] compared to their low-altitude relatives, revealing yet another avian strategy for coping with high altitude.

Reduced metabolism supports hypoxic flight in the high-flying bar-headed goose (Anser indicus).

The bar-headed goose is famed for migratory flight at extreme altitude. To better understand the physiology underlying this remarkable behavior, we imprinted and trained geese, collecting the first cardiorespiratory measurements of bar-headed geese flying at simulated altitude in a wind tunnel. Metabolic rate during flight increased 16-fold from rest, supported by an increase in the estimated amount of O2 transported per heartbeat and a modest increase in heart rate. The geese appear to have ample cardiac reserves, as heart rate during hypoxic flights was not higher than in normoxic flights. We conclude that flight in hypoxia is largely achieved via the reduction in metabolic rate compared to normoxia. Arterial [Formula: see text] was maintained throughout flights. Mixed venous PO2 decreased during the initial portion of flights in hypoxia, indicative of increased tissue O2 extraction. We also discovered that mixed venous temperature decreased during flight, which may significantly increase oxygen loading to hemoglobin.

Control of breathing and respiratory gas exchange in high-altitude ducks native to the Andes.

We examined the control of breathing and respiratory gas exchange in six species of high-altitude duck that independently colonized the high Andes. We compared ducks from high-altitude populations in Peru (Lake Titicaca at ∼3800 m above sea level; Chancay River at ∼3000-4100 m) with closely related populations or species from low altitude. Hypoxic ventilatory responses were measured shortly after capture at the native altitude. In general, ducks responded to acute hypoxia with robust increases in total ventilation and pulmonary O2 extraction. O2 consumption rates were maintained or increased slightly in acute hypoxia, despite ∼1-2°C reductions in body temperature in most species. Two high-altitude taxa - yellow-billed pintail and torrent duck - exhibited higher total ventilation than their low-altitude counterparts, and yellow-billed pintail exhibited greater increases in pulmonary O2 extraction in severe hypoxia. In contrast, three other high-altitude taxa - Andean ruddy duck, Andean cinnamon teal and speckled teal - had similar or slightly reduced total ventilation and pulmonary O2 extraction compared with low-altitude relatives. Arterial O2 saturation (SaO2 ) was elevated in yellow-billed pintails at moderate levels of hypoxia, but there were no differences in SaO2  in other high-altitude taxa compared with their close relatives. This finding suggests that improvements in SaO2  in hypoxia can require increases in both breathing and haemoglobin-O2 affinity, because the yellow-billed pintail was the only high-altitude duck with concurrent increases in both traits compared with its low-altitude relative. Overall, our results suggest that distinct physiological strategies for coping with hypoxia can exist across different high-altitude lineages, even among those inhabiting very similar high-altitude habitats.

Electrostatic Tuning of a Potassium Channel in Electric Fish.

Molecular variation contributes to the evolution of adaptive phenotypes, though it is often difficult to understand precisely how. The adaptively significant electric organ discharge behavior of weakly electric fish is the direct result of biophysical membrane properties set by ion channels. Here, we describe a voltage-gated potassium-channel gene in African electric fishes that is under positive selection and highly expressed in the electric organ. The channel produced by this gene shortens electric organ action potentials by activating quickly and at hyperpolarized membrane potentials. The source of these properties is a derived patch of negatively charged amino acids in an extracellular loop near the voltage sensor. We demonstrate that this negative patch acts by contributing to the global surface charge rather than by local interactions with specific amino acids in the channel's extracellular face. We suggest a more widespread role for this loop in the evolutionary tuning of voltage-dependent channels.

Validation of a Pulse Oximetry System for High-Altitude Waterfowl by Examining the Hypoxia Responses of the Andean Goose (Chloephaga melanoptera).

Hypoxia at high altitudes constrains O2 supply to support metabolism, thermoregulation in the cold, and exercise. High-altitude natives that somehow overcome this challenge-who live, reproduce, and sometimes perform impressive feats of exercise at high altitudes-are a powerful group in which to study the evolution of physiological systems underlying hypoxia resistance. Here, we sought to determine whether a common pulse oximetry system for rodents (MouseOx Plus) can be used reliably in studies of high-altitude birds by examining the hypoxia responses of the Andean goose. We compared concurrent measurements of heart rate obtained using pulse oximetry versus electrocardiography. We also compared our measurements of peripheral arterial O2 saturation (SaO2) in uncannulated birds with published data collected from blood samples in birds that were surgically implanted arterial cannulae. Responses to acute hypoxia were measured during stepwise reductions in inspired partial pressure of O2. Andean geese exhibited very modest breathing and heart rate responses to hypoxia but were nevertheless able to maintain normal O2 consumption rates during severe hypoxia exposure down to 5 kPa O2. There were some minor quantitative differences between uncannulated and cannulated birds, which suggest that surgery, cannulation, and/or other sources of variability between studies had modest effects on the hypoxic ventilatory response, heart rate, blood hemoglobin, and hematocrit. Nevertheless, measurements of heart rate and SaO2 by pulse oximetry had small standard errors and were generally concordant and well correlated with measurements using other techniques. We conclude that the MouseOx Plus pulse oximetry system can be a valuable tool for studying the cardiorespiratory physiology of waterfowl without the deleterious effects of surgery/cannulation.

Respiratory mechanics and morphology of Tibetan and Andean high-altitude geese with divergent life histories.

High-altitude bar-headed geese (Anser indicus) and Andean geese (Chloephaga melanoptera) have been shown to preferentially increase tidal volume over breathing frequency when increasing ventilation during exposure to hypoxia. Increasing tidal volume is a more effective breathing strategy but is also thought to be more mechanically and metabolically expensive. We asked whether there might be differences in the mechanics or morphology of the respiratory systems of high-altitude transient bar-headed geese and high-altitude resident Andean geese that could minimize the cost of breathing more deeply. We compared these two species with a low-altitude migratory species, the barnacle goose (Branta leucopsis). We ventilated anesthetized birds to measure mechanical properties of the respiratory system and used CT scans to quantify respiratory morphology. We found that the respiratory system of Andean geese was disproportionately larger than that of the other two species, allowing use of a deeper breathing strategy for the same energetic cost. The relative size of the respiratory system, especially the caudal air sacs, of bar-headed geese was also larger than that of barnacle geese. However, when normalized to respiratory system size, the mechanical cost of breathing did not differ significantly among these three species, indicating that deeper breathing is enabled by morphological but not mechanical differences between species. The metabolic cost of breathing was estimated to be <1% of basal metabolic rate at rest in normoxia. Because of differences in the magnitude of the ventilatory response, the cost of breathing was estimated to increase 7- to 10-fold in bar-headed and barnacle geese in severe hypoxia, but less than 1-fold in Andean geese exposed to the same low atmospheric PO2.

Respiratory mechanics of eleven avian species resident at high and low altitude.

The metabolic cost of breathing at rest has never been successfully measured in birds, but has been hypothesized to be higher than in mammals of a similar size because of the rocking motion of the avian sternum being encumbered by the pectoral flight muscles. To measure the cost and work of breathing, and to investigate whether species resident at high altitude exhibit morphological or mechanical changes that alter the work of breathing, we studied 11 species of waterfowl: five from high altitudes (>3000 m) in Perú, and six from low altitudes in Oregon, USA. Birds were anesthetized and mechanically ventilated in sternal recumbency with known tidal volumes and breathing frequencies. The work done by the ventilator was measured, and these values were applied to the combinations of tidal volumes and breathing frequencies used by the birds to breathe at rest. We found the respiratory system of high-altitude species to be of a similar size, but consistently more compliant than that of low-altitude sister taxa, although this did not translate to a significantly reduced work of breathing. The metabolic cost of breathing was estimated to be between 1 and 3% of basal metabolic rate, as low or lower than estimates for other groups of tetrapods.

Morphological and morphometric specializations of the lung of the Andean goose, Chloephaga melanoptera: A lifelong high-altitude resident.

High altitude flight in rarefied, extremely cold and hypoxic air is a very challenging activity. Only a few species of birds can achieve it. Hitherto, the structure of the lungs of such birds has not been studied. This is because of the rarity of such species and the challenges of preparing well-fixed lung tissue. Here, it was posited that in addition to the now proven physiological adaptations, high altitude flying birds will also have acquired pulmonary structural adaptations that enable them to obtain the large amounts of oxygen (O2) needed for flight at high elevation, an environment where O2 levels are very low. The Andean goose (Chloephaga melanoptera) normally resides at altitudes above 3000 meters and flies to elevations as high as 6000 meters where O2 becomes limiting. In this study, its lung was morphologically- and morphometrically investigated. It was found that structurally the lungs are exceptionally specialized for gas exchange. Atypically, the infundibulae are well-vascularized. The mass-specific volume of the lung (42.8 cm3.kg-1), the mass-specific respiratory surface area of the blood-gas (tissue) barrier (96.5 cm2.g-1) and the mass-specific volume of the pulmonary capillary blood (7.44 cm3.kg-1) were some of the highest values so far reported in birds. The pulmonary structural specializations have generated a mass-specific total (overall) pulmonary morphometric diffusing capacity of the lung for oxygen (DLo2) of 0.119 mlO2.sec-1.mbar-1.kg-1, a value that is among some of the highest ones in birds that have been studied. The adaptations of the lung of the Andean goose possibly produce the high O2 conductance needed to live and fly at high altitude.

Mitochondrial physiology in the skeletal and cardiac muscles is altered in torrent ducks, Merganetta armata, from high altitudes in the Andes.

Torrent ducks inhabit fast-flowing rivers in the Andes from sea level to altitudes up to 4500 m. We examined the mitochondrial physiology that facilitates performance over this altitudinal cline by comparing the respiratory capacities of permeabilized fibers, the activities of 16 key metabolic enzymes and the myoglobin content in muscles between high- and low-altitude populations of this species. Mitochondrial respiratory capacities (assessed using substrates of mitochondrial complexes I, II and/or IV) were higher in highland ducks in the gastrocnemius muscle - the primary muscle used to support swimming and diving - but were similar between populations in the pectoralis muscle and the left ventricle. The heightened respiratory capacity in the gastrocnemius of highland ducks was associated with elevated activities of cytochrome oxidase, phosphofructokinase, pyruvate kinase and malate dehydrogenase (MDH). Although respiratory capacities were similar between populations in the other muscles, highland ducks had elevated activities of ATP synthase, lactate dehydrogenase, MDH, hydroxyacyl CoA dehydrogenase and creatine kinase in the left ventricle, and elevated MDH activity and myoglobin content in the pectoralis. Thus, although there was a significant increase in the oxidative capacity of the gastrocnemius in highland ducks, which correlates with improved performance at high altitudes, the variation in metabolic enzyme activities in other muscles not correlated to respiratory capacity, such as the consistent upregulation of MDH activity, may serve other functions that contribute to success at high altitudes.

Oxygen in demand: How oxygen has shaped vertebrate physiology.

In response to varying environmental and physiological challenges, vertebrates have evolved complex and often overlapping systems. These systems detect changes in environmental oxygen availability and respond by increasing oxygen supply to the tissues and/or by decreasing oxygen demand at the cellular level. This suite of responses is termed the oxygen transport cascade and is comprised of several components. These components include 1) chemosensory detectors that sense changes in oxygen, carbon dioxide, and pH in the blood, and initiate changes in 2) ventilation and 3) cardiac work, thereby altering the rate of oxygen delivery to, and carbon dioxide clearance from, the tissues. In addition, changes in 4) cellular and systemic metabolism alters tissue-level metabolic demand. Thus the need for oxygen can be managed locally when increasing oxygen supply is not sufficient or possible. Together, these mechanisms provide a spectrum of responses that facilitate the maintenance of systemic oxygen homeostasis in the face of environmental hypoxia or physiological oxygen depletion (i.e. due to exercise or disease). Bill Milsom has dedicated his career to the study of these responses across phylogenies, repeatedly demonstrating the power of applying the comparative approach to physiological questions. The focus of this review is to discuss the anatomy, signalling pathways, and mechanics of each step of the oxygen transport cascade from the perspective of a Milsomite. That is, by taking into account the developmental, physiological, and evolutionary components of questions related to oxygen transport. We also highlight examples of some of the remarkable species that have captured Bill's attention through their unique adaptations in multiple components of the oxygen transport cascade, which allow them to achieve astounding physiological feats. Bill's research examining the oxygen transport cascade has provided important insight and leadership to the study of the diverse suite of adaptations that maintain cellular oxygen content across vertebrate taxa, which underscores the value of the comparative approach to the study of physiological systems.