Heat exposure limits pentose phosphate pathway activity in bumblebees.

Bumblebee populations across the globe are experiencing substantial declines due to climate change, with major consequences for pollination services in both natural and agricultural settings. Using an economically important species, Bombus impatiens, we explored the physiological mechanisms that may cause susceptibility to extreme heat events. We tested the hypothesis that heat exposure limits the activity of the pentose phosphate pathway (PPP)-a parallel pathway to glycolysis that can use nectar sugar to generate antioxidant potential and combat oxidative stress. Using isotopically labelled glucose, we tracked PPP activity in B. impatiens at rest, during exercise and during a post-exercise recovery period under two different temperature regimes (22°C and 32°C). We found that the PPP is routinely used by B. impatiens at moderate temperatures, but that its activity is markedly reduced when ATP demands are high, such as during periods of exercise and heat exposure. We also exposed B. impatiens to either 22°C or 32°C for 5 hours and assessed levels of oxidative damage (lipid peroxidation, protein carbonyls) and antioxidant potential [reduced (GSH) and oxidized (GSSG) glutathione concentrations]. Interestingly, bees exhibited little oxidative damage after the thermal exposure, but we found a lower GSH:GSSG ratio in 32°C-exposed bees, reflecting lower antioxidant potential. Overall, our study demonstrates that acute heat stress severely limits PPP activity and may constrain antioxidant potential in B. impatiens. The repeated attenuation of this pathway in a warming climate may have more severe physiological consequences for this species, with potential implications for pollination services across North America.

Cortisol enhances aerobic metabolism and locomotor performance during the transition to land in an amphibious fish.

Amphibious fishes on land encounter higher oxygen (O2) availability and novel energetic demands, which impacts metabolism. Previous work on the amphibious mangrove killifish (Kryptolebias marmoratus) has shown that cortisol becomes elevated in response to air exposure, suggesting a possible role in regulating metabolism as fish move into terrestrial environments. We tested the hypothesis that cortisol is the mechanism by which oxidative processes are upregulated during the transition to land in amphibious fishes. We used two groups of fish, treated fish (+metyrapone, a cortisol synthesis inhibitor) and control (-metyrapone), to determine the impact of cortisol during air exposure (0 and 1 h, 7 days) on O2 consumption, terrestrial locomotion, the phenotype of red skeletal muscle, and muscle lipid concentration. Metyrapone-treated fish had an attenuated elevation in O2 consumption rate during the water to air transition and an immediate reduction in terrestrial exercise performance relative to control fish. In contrast, we found no short- (0 h) or long-term (7 days) differences between treatments in the oxidative phenotype of red muscles, nor in muscle lipid concentrations. Our results suggest that cortisol stimulates the necessary increase in aerobic metabolism needed to fuel the physiological changes that amphibious fishes undergo during the acclimation to air, although further studies are required to determine specific mechanisms of cortisol regulation.

Leptin Resistance Does Not Facilitate Migratory Fattening in Ruby-Throated Hummingbirds (Archilochus Colubris).

In mammals, leptin is an important energy homeostasis hormone produced by adipose tissue. Circulating leptin concentrations correlate positively with fat mass and act in a negative feedback fashion to inhibit food intake and increase energy expenditure, thereby preventing fat gain. For some species, leptin resistance is advantageous during times of year where fat gain is necessary (e.g., prior to hibernation). While the function of leptin in birds remains controversial, seasonal leptin resistance may similarly benefit migratory species. Here, we used the ruby-throated hummingbird (Archilochus colubris) to test the hypothesis that leptin resistance promotes fattening prior to migration. We predicted that during the migratory fattening period, leptin levels should correlate positively with fat mass but should not inhibit food intake or increase energy expenditure, resulting in fattening. We tracked the body (fat) mass, the concentration of leptin-like protein in the urine, and the food intake of 12 captive hummingbirds from August 2021 to January 2022. In a subset of hummingbirds, we also quantified voluntary physical activity as a proxy for energy expenditure. We found remarkable age-related variation in fattening strategies, with juveniles doubling their body fat by mid-September and adults exhibiting only a 50% increase. Changes in fat mass were strongly associated with increased food intake and reduced voluntary activity. However, we found no correlation between leptin-like protein concentration and fat mass, food intake, or voluntary activity. Since increased torpor use has been shown to accelerate migratory fattening in ruby-throated hummingbirds, we also hypothesized that leptin is a mediator of torpor use. In an experimental manipulation of circulating leptin, however, we found no change in torpor use, body fat, or food intake. Overall, our findings suggest that leptin may not act as an adipostat in hummingbirds, nor does leptin resistance regulate how hummingbirds fatten prior to migration.

Out of water in the dark: Plasticity in visual structures and function in an amphibious fish.

Many fishes encounter periods of prolonged darkness within their lifetime, yet the consequences for the visual system are poorly understood. We used an amphibious fish (Kryptolebias marmoratus) that occupies dark terrestrial environments during seasonal droughts to test whether exposure to prolonged darkness diminishes visual performance owing to reduced optic tectum (OT) size and/or neurogenesis. We performed a 3-week acclimation with a 2 ×$\times $ 2 factorial design, in which fish were either acclimated to a 12 h:12 h or 0 h:24 h light:dark photoperiod in water or in air. We found that water-exposed fish had poorer visual acuity when acclimated to the dark, while air-acclimated fish had poorer visual acuity regardless of photoperiod. The ability of K. marmoratus to capture aerial prey from water followed a similar trend, suggesting that good vision is important for hunting effectively. Changes in visual acuity did not result from changes in OT size, but air-acclimated fish had 37% fewer proliferating cells in the OT than water-acclimated fish. As K. marmoratus are unable to eat on land, reducing cell proliferation in the OT may serve as a mechanism to reduce maintenance costs associated with the visual system. Overall, we suggest that prolonged darkness and air exposure can impair vision in K. marmoratus, and that changes in visual performance may be mediated, in part, by OT neurogenesis. More broadly, we show that plastic changes to the visual system of fishes can have potential consequences for organismal performance and fitness.

Physiological and behavioural strategies of aquatic animals living in fluctuating environments.

Shallow or near-shore environments, such as ponds, estuaries and intertidal zones, are among the most physiologically challenging of all aquatic settings. Animals inhabiting these environments experience conditions that fluctuate markedly over relatively short temporal and spatial scales. Living in these habitats requires the ability to tolerate the physiological disturbances incurred by these environmental fluctuations. This tolerance is achieved through a suite of physiological and behavioural responses that allow animals to maintain homeostasis, including the ability to dynamically modulate their physiology through reversible phenotypic plasticity. However, maintaining the plasticity to adjust to some stresses in a dynamic environment may trade off with the capacity to deal with other stressors. This paper will explore studies on select fishes and invertebrates exposed to fluctuations in dissolved oxygen, salinity and pH. We assess the physiological mechanisms these species employ to achieve homeostasis, with a focus on the plasticity of their responses, and consider the resulting physiological trade-offs in function. Finally, we discuss additional factors that may influence organismal responses to fluctuating environments, such as the presence of multiple stressors, including parasites. We echo recent calls from experimental biologists to consider physiological responses to life in naturally fluctuating environments, not only because they are interesting in their own right but also because they can reveal mechanisms that may be crucial for living with increasing environmental instability as a consequence of climate change.

Context-dependent relationships between swimming, terrestrial jumping and body composition in the amphibious fish Kryptolebias marmoratus.

Understanding the mechanisms that create phenotypic variation within and among populations is a major goal of physiological ecology. Variation may be a consequence of functional trade-offs (i.e. improvement in one trait comes at the expense of another trait) or alternatively may reflect the intrinsic quality of an organism (i.e. some individuals are simply better overall performers than others). There is evidence for both ideas in the literature, suggesting that environmental context may mediate whether variation results from trade-offs or differences in individual quality. We tested this overarching 'context dependence' hypothesis by comparing the aquatic and terrestrial athletic performance of the amphibious fish Kryptolebias marmoratus captured from two contrasting habitats, a large pond and small burrows. Overall, pond fish were superior terrestrial athletes but burrow fish were better burst swimmers, suggestive of a performance trade-off at the population level. Within each population, however, there was no evidence of a performance trade-off. In burrow fish, athletic performance was positively correlated with muscle content and body condition, consistent with the individual quality hypothesis. In pond fish, there was only a relationship between glycolytic white muscle and aquatic burst performance. Notably, pond fish were in better body condition, which may mask relationships between condition and athletic performance. Overall, our data highlight that population-level trends are insufficient evidence for the existence of phenotypic trade-offs in the absence of similar within-population patterns. Furthermore, we only found evidence for the individual quality hypothesis in one population, suggesting that patterns of phenotypic covariance are context dependent.

Habitat choice promotes and constrains phenotypic plasticity.

Habitat choice can either speed up or slow rates of phenotypic evolution, depending on which trait is measured. We suggest that habitat choice plays an analogous, and generally overlooked, role in shaping patterns of phenotypic plasticity. Using our work with an amphibious fish, we discuss two case studies that demonstrate how habitat choice can both promote and constrain expression of plasticity. First, habitat choice during the dry season accentuates adaptive metabolic plasticity and minimizes maladaptive changes to muscle, ultimately increasing survival time out of water. Second, a trade-off between water- and air-breathing drives matching habitat choice, resulting in positive feedback that reinforces respiratory specialization and environmental preference. Overall, these case studies demonstrate that we must consider the interactions between plasticity and habitat choice to fully understand how animals survive in the face of environmental change. Without considering both processes simultaneously, the performance of animals in challenging conditions can be either under- or over-estimated. Finally, because habitat choice shapes the frequency and predictability of environmental changes that animals experience, feedback between habitat choice and expressions of phenotypic plasticity may be an important factor that influences how plasticity evolves.

Seeing in the swamp: hydrogen sulfide inhibits eye metabolism and visual acuity in a sulfide-tolerant fish.

In fish, vision may be impaired when eye tissue is in direct contact with environmental conditions that limit aerobic ATP production. We hypothesized that the visual acuity of fishes exposed to hydrogen sulfide (H2S)-rich water would be altered owing to changes in cytochrome c oxidase (COX) activity. Using the H2S-tolerant mangrove rivulus (Kryptolebias marmoratus), we showed that a 10 min exposure to greater than or equal to 200 µM of H2S impaired visual acuity and COX activity in the eye. Visual acuity and COX activity were restored in fish allowed to recover in H2S-free water for up to 1 h. Since K. marmoratus are found in mangrove pools with H2S concentrations exceeding 1000 µM, visual impairment may impact predator avoidance, navigation and foraging behaviour in the wild.

More than Breathing Air: Evolutionary Drivers and Physiological Implications of an Amphibious Lifestyle in Fishes.

Amphibious and aquatic air-breathing fishes both exchange respiratory gasses with the atmosphere, but these fishes differ in physiology, ecology, and possibly evolutionary origins. We introduce a scoring system to characterize interspecific variation in amphibiousness and use this system to highlight important unanswered questions about the evolutionary physiology of amphibious fishes.

Does leaving water make fish smarter? Terrestrial exposure and exercise improve spatial learning in an amphibious fish.

Amphibious fishes transition between aquatic and terrestrial habitats, and must therefore learn to navigate two dramatically different environments. We used the amphibious killifish Kryptolebias marmoratus to test the hypothesis that the spatial learning ability of amphibious fishes would be altered by exposure to terrestrial environments because of neural plasticity in the brain region linked to spatial cognition (dorsolateral pallium). We subjected fish to eight weeks of fluctuating air-water conditions or terrestrial exercise before assessing spatial learning using a bifurcating T-maze, and neurogenesis in the dorsolateral pallium by immunostaining for proliferating cell nuclear antigen. In support of our hypothesis, we found that air-water fluctuations and terrestrial exercise improved some markers of spatial learning. Moreover, air-water and exercised fish had 39% and 46% more proliferating cells in their dorsolateral pallium relative to control fish, respectively. Overall, our findings suggest that fish with more terrestrial tendencies may have a cognitive advantage over those that remain in water, which ultimately may influence their fitness in both aquatic and terrestrial settings. More broadly, understanding the factors that promote neural and behavioural plasticity in extant amphibious fishes may provide insights into how ancestral fishes successfully colonized novel terrestrial environments before giving rise to land-dwelling tetrapods.

Fluctuating environments during early development can limit adult phenotypic flexibility: insights from an amphibious fish.

The interaction between developmental plasticity and the capacity for reversible acclimation (phenotypic flexibility) is poorly understood, particularly in organisms exposed to fluctuating environments. We used an amphibious killifish (Kryptolebias marmoratus) to test the hypotheses that organisms reared in fluctuating environments (i) will make no developmental changes to suit any one environment because fixing traits to suit one environment could be maladaptive for another, and (ii) will be highly phenotypically flexible as adults because their early life experiences predict high environmental variability in the future. We reared fish under constant (water) or fluctuating (water-air) environments until adulthood and assessed a suite of traits along the oxygen cascade (e.g. neuroepithelial cell density and size, cutaneous capillarity, gill morphology, ventricle size, red muscle morphometrics, terrestrial locomotor performance). To evaluate the capacity for phenotypic flexibility, a subset of adult fish from each rearing condition was then air-exposed for 14 days before the same traits were measured. In support of the developmental plasticity hypothesis, traits involved with O2 sensing and uptake were largely unaffected by water-air fluctuations during early life, but we found marked developmental changes in traits related to O2 transport, utilization and locomotor performance. In contrast, we found no evidence supporting the phenotypic flexibility hypothesis. Adult fish from both rearing conditions exhibited the same degree of phenotypic flexibility in various O2 sensing- and uptake-related traits. In other cases, water-air fluctuations attenuated adult phenotypic flexibility despite the fact that phenotypic flexibility is hypothesized to be favoured when environments fluctuate. Overall, we conclude that exposure to environmental fluctuations during development in K. marmoratus can dramatically alter the constitutive adult phenotype, as well as diminish the scope for phenotypic flexibility in later life.

Hypoxia-seeking behavior, metabolic depression and skeletal muscle function in an amphibious fish out of water.

Several animals enter a state of dormancy to survive harsh environmental conditions. During dormancy, metabolic depression can be critical for economizing on limited endogenous energy reserves. We used two isogenic strains (strain 1 and strain 2) of a self-fertilizing amphibious fish (Kryptolebias marmoratus) to test the hypothesis that animals seek hypoxic microhabitats that, in turn, accentuate metabolic depression during dormancy. Using custom-built tunnels that maintained a longitudinal O2 gradient (hypoxic to normoxic), we assessed the O2 preference of K. marmoratus during prolonged air exposure. In support of our hypothesis, we found that one isogenic strain (strain 2) spent more time in hypoxia compared with normoxia after 21 days in air. Prolonged air exposure in both strains resulted in lower O2 consumption rates compared with active fish (35% depression), which was accentuated (51% depression) when fish were exposed to aerial hypoxia acutely. We then tested the hypothesis that chronic aerial hypoxia acclimation would protect endogenous energy reserves and skeletal muscle integrity, thereby maintaining locomotor performance, possibly owing to hypoxic hypometabolism. We found that air-acclimated fish from both strains were in poorer body condition relative to fish acclimated to aerial hypoxia. Furthermore, aerial hypoxia acclimation minimized glycogen usage (strain 1), lipid catabolism (strain 2) and white muscle atrophy (strain 2), as well as preserved terrestrial locomotor performance compared with fish in air (strain 2). Overall, our findings suggest that some K. marmoratus strains seek microhabitats that accentuate metabolic depression during dormancy, and that microhabitat O2 availability may have significant implications for energy metabolism, and the structure and function of skeletal muscle. Furthermore, the differential responses between isogenic strains suggests that genetic factors also contribute to phenotypic differences in the emersion behavior and physiology of this species.

Frogs seek hypoxic microhabitats that accentuate metabolic depression during dormancy.

Many animals occupy microhabitats during dormancy where they may encounter hypoxic conditions (e.g. subterranean burrows). We used the green-striped burrowing frog (Cyclorana alboguttata) to test the hypothesis that animals seek hypoxic microhabitats that accentuate metabolic depression during dormancy. We first measured the partial pressure of oxygen (PO2 ) within artificial cavities excavated in wet clay soil, which simulated C. alboguttata underground aestivation chambers, and recorded hypoxic conditions (PO2  as low as 8.9 kPa). Using custom-built tunnels that maintained a longitudinal PO2  gradient (hypoxic to normoxic), we then examined the PO2  preference of C. alboguttata in response to drying habitat conditions. In support of our hypothesis, we found that C. alboguttata chose to spend a greater proportion of time at the hypoxic end of the PO2  gradient compared with the normoxic end. To determine whether hypoxia accentuates metabolic depression in C. alboguttata, we exposed frogs to normoxia (21.0 kPa) or hypoxia (10.5 kPa) for 7 weeks during the transition from an active to an aestivating state. We found that hypoxia exposure accelerated the onset of metabolic depression in C. alboguttata by 2 weeks. Furthermore, we found that frogs exposed to hypoxia exhibited a 66% reduction in O2 consumption after 7 weeks compared with active frogs in normoxia, whereas frogs exposed to normoxia reduced O2 consumption by only 51%. Overall, our findings indicate that some animals may seek microhabitats to maximally depress metabolic rate during dormancy, and that microhabitat O2 availability can have significant implications for energy metabolism.

Amphibious fish 'get a jump' on terrestrial locomotor performance after exercise training on land.

Many amphibious fishes rely on terrestrial locomotion to accomplish essential daily tasks, but it is unknown whether terrestrial exercise improves the locomotor performance of fishes on land. Thus, we tested the hypothesis that terrestrial exercise improves locomotion in amphibious fishes out of water as a result of skeletal muscle remodeling. We compared the jumping performance of Kryptolebias marmoratus before and after an exercise training regimen, and assessed the muscle phenotype of control and exercise-trained fish. We found that exercise-trained fish jumped 41% farther and 48% more times before reaching exhaustion. Furthermore, exercise training resulted in the hypertrophy of red muscle fibers, and an increase in red muscle capillarity and aerobic capacity. Lactate accumulation after jumping indicates that white muscle is also important in powering terrestrial jumps. Overall, skeletal muscle in K. marmoratus is highly responsive to terrestrial exercise, and muscle plasticity may assist in the effective exploitation of terrestrial habitats by amphibious fishes.

Ageing impacts phenotypic flexibility in an air-acclimated amphibious fish.

The ability to tolerate environmental change may decline as fishes age. We tested the hypothesis that ageing influences the scope for phenotypic flexibility in the mangrove rivulus (Kryptolebias marmoratus), an amphibious fish that transitions between two vastly different environments, water and land. We found that older fish (4-6 years old) exhibited marked signs of ageing; older fish were reproductively senescent, had reduced fin regenerative capacity and body condition, and exhibited atrophy of both oxidative and glycolytic muscle fibers relative to younger adult fish (1-2 years old). However, age did not affect routine O2 consumption. We then acclimated adult fish (1-6 years) to water (control) or air for 10 days to assess the scope for phenotypic flexibility in response to terrestrial exposure. In support of our hypothesis, we found that older air-acclimated fish had a diminished scope for gill remodeling relative to younger fish. We also found that older fish exhibited poorer terrestrial locomotor performance relative to younger adult fish, particularly when acclimated to air. Our results indicate that ageing diminishes skeletal muscle integrity and locomotor performance of amphibious fishes, and may, therefore, impair terrestrial foraging ability, predator avoidance, or dispersal across the terrestrial environment. Remarkably, older fish voluntarily left water to a similar degree as younger fish despite the age-related deterioration of traits important for terrestrial life.

Mangrove Fishes Rely on Emersion Behavior and Physiological Tolerance to Persist in Sulfidic Environments.

Hydrogen sulfide (H 2 S) is a potent respiratory toxin that makes sulfidic environments tolerable to only a few organisms. We report the presence of fishes ( Kryptolebias marmoratus , Poecilia orri , Gambusia sp., and Dormitator maculatus ) in Belizean mangrove pools with extremely high H 2 S concentrations (up to 1,166 µM) that would be lethal for most fishes. Thus, we asked whether the three most prevalent species ( Kryptolebias , Poecilia , and Gambusia ) persist in sulfidic pools because they are exceptionally H 2 S tolerant and/or because they can leave water (emerse) and completely avoid H 2 S. We show that both physiological tolerance and emersion behavior are important. Kryptolebias demonstrated high H 2 S tolerance, as they lost equilibrium significantly later than Poecilia and Gambusia during H 2 S exposure ( 1,188±21 µM H 2 S). However, the fact that all species lost equilibrium at an ecologically relevant [H 2 S] suggests that physiological tolerance may suffice at moderate H 2 S concentrations but that another strategy is required to endure higher concentrations. In support of the avoidance behavior hypothesis, H 2 S elicited an emersion response in all species. Kryptolebias was most sensitive to H 2 S and emersed at H 2 S concentrations 52% and 34% lower than Poecilia and Gambusia , respectively. Moreover, H 2 S exposure caused Kryptolebias to emerse more frequently and spend more time out of water compared to control conditions. We suggest that physiological H 2 S tolerance and emersion behavior are complementary strategies. The superior H 2 S tolerance and amphibious capability of Kryptolebias may explain why this species was more prevalent in H 2 S-rich environments than other local fishes.

Hydrogen sulphide toxicity and the importance of amphibious behaviour in a mangrove fish inhabiting sulphide-rich habitats.

We investigated amphibious behaviour, hydrogen sulphide (H2S) tolerance, and the mechanism of H2S toxicity in the amphibious mangrove rivulus (Kryptolebias marmoratus). We found that fish emersed (left water) in response to acutely elevated [H2S] (~ 130-200 µmol l-1). The emersion response to H2S may be influenced by prior acclimation history due to acclimation-induced alterations in gill morphology and/or the density and size of neuroepithelial cells (NECs) on the gills and skin. Thus, we acclimated fish to water (control), H2S-rich water, or air and tested the hypotheses that acclimation history influences H2S sensitivity due to acclimation-induced changes in (i) gill surface area and/or (ii) NEC density and/or size. Air-acclimated fish emersed at significantly lower [H2S] relative to fish acclimated to control or H2S-rich water, but exhibited no change in gill surface area or in NEC density or size in the gills or skin. Despite possessing exceptional H2S tolerance, all fish lost equilibrium when unable to emerse from environments containing extremely elevated [H2S] (2272 ± 46 µmol l-1). Consequently, we tested the hypothesis that impaired blood oxygen transport (i.e., sulphemoglobin formation) causes H2S toxicity in amphibious fishes. In vitro exposure of red blood cells to physiologically relevant [H2S] did not cause a substantial increase in sulphemoglobin formation. We found evidence, however, for an alternative hypothesis that H2S toxicity is caused by impaired oxidative phosphorylation (i.e., cytochrome c oxidase inhibition). Collectively, our results show that amphibious behaviour is critical for the survival of K. marmoratus in H2S-rich environments as fish experience impaired oxidative phosphorylation when unable to emerse.

Oxygen drives skeletal muscle remodeling in an amphibious fish out of water.

Skeletal muscle remodeling in response to terrestrial acclimation improves the locomotor performance of some amphibious fishes on land, but the cue for this remodeling is unknown. We tested the hypothesis that muscle remodeling in the amphibious Kryptolebias marmoratus on land is driven by higher O2 availability in atmospheric air, and the alternative hypothesis that remodeling is induced by a different environmental or physiological condition that fish experience on land. Fish were acclimated to 28 days of air, or to aquatic hyperoxia, hypercapnia, hypoxia, elevated temperature or fasting conditions. Air, fasting and hyperoxic conditions increased (>25%) the size of oxidative fibers in K. marmoratus while hypoxia had the reverse effect (23% decrease). Surprisingly, hyperoxia acclimation also resulted in a transformation of the musculature to include large bands of oxidative-like muscle. Our results show that K. marmoratus is highly responsive to environmental O2 levels and capitalizes on O2-rich opportunities to enhance O2 utilization by skeletal muscle.