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.

Through the looking glass: attempting to predict future opportunities and challenges in experimental biology.

To celebrate its centenary year, Journal of Experimental Biology (JEB) commissioned a collection of articles examining the past, present and future of experimental biology. This Commentary closes the collection by considering the important research opportunities and challenges that await us in the future. We expect that researchers will harness the power of technological advances, such as '-omics' and gene editing, to probe resistance and resilience to environmental change as well as other organismal responses. The capacity to handle large data sets will allow high-resolution data to be collected for individual animals and to understand population, species and community responses. The availability of large data sets will also place greater emphasis on approaches such as modeling and simulations. Finally, the increasing sophistication of biologgers will allow more comprehensive data to be collected for individual animals in the wild. Collectively, these approaches will provide an unprecedented understanding of 'how animals work' as well as keys to safeguarding animals at a time when anthropogenic activities are degrading the natural environment.

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.

Positive feedback promotes terrestrial emergence behaviour in an amphibious fish.

Major ecological transitions such as the invasion of land by aquatic vertebrates may be facilitated by positive feedback between habitat choice and phenotypic plasticity. We used the amphibious fish Kryptolebias marmoratus to test the hypothesis that aquatic hypoxia, emergence behaviour and respiratory plasticity create this type of positive feedback loop that causes fish to spend increasing amounts of time on land. Terrestrially acclimated fish were more sensitive to aquatic hypoxia (emergence at higher PO2) and were less hypoxia tolerant (shorter time to loss of equilibrium) relative to water-acclimated fish, which are necessary conditions for positive feedback. Next, we tested the prediction that exposure to aquatic hypoxia causes fish to emerge frequently, reduce gill surface area, and become less hypoxia tolerant. Indeed, fish exposed to severe aquatic hypoxia spent almost 50% of the time out of water and coverage of the gill lamellae by an inter-lamellar cell mass almost doubled. Fish exposed to aquatic hypoxia that could emerge from water were also more sensitive to subsequent acute aquatic hypoxia and were less hypoxia tolerant than normoxia-exposed controls. These results are opposite those of fish that cannot escape from aquatic hypoxia and presumably arise owing to plastic changes that occur during air exposure. Together, these results indicate that emergence behaviour begets further emergence behaviour, driven by gill remodelling which reduces aquatic respiratory function. This type of positive feedback may explain how amphibious behaviour has repeatedly evolved in fishes that occupy hypoxic aquatic habitats despite the associated challenges of life on land.

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.

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.

Novel spikey ionocytes are regulated by cortisol in the skin of an amphibious fish.

Cortisol is a major osmoregulatory hormone in fishes. Cortisol acts upon the gills, the primary site of ionoregulation, through modifications to specialized ion-transporting cells called ionocytes. We tested the hypothesis that cortisol also acts as a major regulator of skin ionocyte remodelling in the amphibious mangrove rivulus (Kryptolebias marmoratus) when gill function ceases during the water-to-land transition. When out of water, K. marmoratus demonstrated a robust cortisol response, which was linked with the remodelling of skin ionocytes to increase cell cross-sectional area and Na+-K+-ATPase (NKA) content, but not when cortisol synthesis was chemically inhibited by metyrapone. Additionally, we discovered a novel morphology of skin-specific ionocyte that are spikey with multiple cell processes. Spikey ionocytes increased in density, cell cross-sectional area and NKA content during air exposure, but not in metyrapone-treated fish. Our findings demonstrate that skin ionocyte remodelling during the water-to-land transition in amphibious fish is regulated by cortisol, the same hormone that regulates gill ionocyte remodelling in salinity-challenged teleosts, suggesting conserved hormonal function across diverse environmental disturbances and organs in fishes.

Genomic and physiological mechanisms underlying skin plasticity during water to air transition in an amphibious fish.

The terrestrial radiation of vertebrates required changes in skin that resolved the dual demands of maintaining a mechanical and physiological barrier while also facilitating ion and gas transport. Using the amphibious killifish Kryptolebias marmoratus, we found that transcriptional regulation of skin morphogenesis was quickly activated upon air exposure (1 h). Rapid regulation of cell-cell adhesion complexes and pathways that regulate stratum corneum formation was consistent with barrier function and mechanical reinforcement. Unique blood vessel architecture and regulation of angiogenesis likely supported cutaneous respiration. Differences in ionoregulatory transcripts and ionocyte morphology were correlated with differences in salinity acclimation and resilience to air exposure. Evolutionary analyses reinforced the adaptive importance of these mechanisms. We conclude that rapid plasticity of barrier, respiratory and ionoregulatory functions in skin evolved to support the amphibious lifestyle of K. marmoratus; similar processes may have facilitated the terrestrial radiation of other contemporary and ancient fishes.

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.

Cutaneous respiration and osmoregulation in amphibious fishes.

In his early career, August Krogh made fundamental discoveries of the properties of cutaneous respiration in fish, frogs and other vertebrates. Following Krogh's example, the study of amphibious fishes provides an excellent model to understand how the skin morphology and physiological mechanisms evolved to meet the dual challenges of aquatic and terrestrial environments. The skin of air-exposed fishes takes on many of the functions that are typically associated with the gills of fish in water: gas exchange, gas sensing, iono- and osmoregulation, and nitrogen excretion. The skin of amphibious fishes has capillaries close to the surface in the epidermis. Skin ionocytes or mitochondrial-rich cells (MRCs) in the epidermis are thought to be responsible for ion exchange, as well as ammonia excretion in the amphibious mangrove rivulus Kryptolebias marmoratus. Ammonia gas (NH3) moves down the partial pressure gradient from skin capillaries to the surface through ammonia transporters (e.g., Rhcg) and NH3 is volatilized from the mucus film on the skin. Future studies are needed on the skin of amphibious fishes from diverse habitats to understand more broadly the role of the skin as a multifunctional organ.

Acclimation to prolonged aquatic hypercarbia or air enhances hemoglobin­oxygen affinity in an amphibious fish.

When the amphibious mangrove rivulus (Kryptolebias marmoratus) leaves water for extended periods, hemoglobin-O2 binding affinity increases. We tested the hypothesis that the change in affinity was a consequence of hemoglobin isoform switching driven by exposure to environments associated with increased internal CO2 levels. We exposed K. marmoratus to either water (control, pH 8.1), air, aquatic hypercarbia (5.1 kPa CO2, pH 6.6-6.8), or aquatic acid (isocarbic control, pH 6.6-6.8), for 7 days, and measured hemoglobin-O2 affinity spectrophotometrically. We found that mangrove rivulus compensated for elevated CO2 and aquatic acid exposure by shifting hemoglobin-O2 affinity back to aquatic (control) levels when measured at an ecologically-relevant high CO2 level that would be experienced in vivo. Using proteomics, we found that the hemoglobin subunits present in the blood did not change between treatments, but air and aquatic acid exposure altered the abundance of cathodic hemoglobin subunits. We therefore conclude that hemoglobin isoform switching is not a primary strategy used by mangrove rivulus to adjust P50 under these conditions. Abundances of other RBC proteins also differed between treatment groups relative to control fish (e.g. Rhesus protein type A, band 3 anion exchanger). Overall, our data indicate that both aquatic hypercarbia and aquatic acidosis create similar changes in hemoglobin-O2 affinity as air exposure. However, the protein-level consequences differ between these groups, indicating that the red blood cell response of mangrove rivulus can be modulated depending on the environmental cue received.

Calcified gill filaments increase respiratory function in fishes.

The morphology of fish gills is closely linked to aerobic capacity and tolerance of environmental stressors such as hypoxia. The importance of gill surface area is well studied, but little is known about how the mechanical properties of gill tissues determine function. In some fishes, the bases of the gill filaments are surrounded by a calcified 'sheath' of unknown function. We tested two non-exclusive hypotheses: (i) calcified gill filaments enhance water flow through the gill basket, improving aquatic respiratory function, and (ii) in amphibious fishes, calcification provides support for gills out of water. In a survey of more than 100 species of killifishes and related orders, we found filament calcification was widespread and thus probably arose before the evolution of amphibious lifestyles in killifishes. Calcification also did not differ between amphibious and fully aquatic species, but terrestrial acclimation caused calcium deposition on the filaments of the killifish Kryptolebias marmoratus, suggesting a possible structural role when out of water. We found strong evidence supporting a role for filament calcification in enhancing aquatic respiratory function. First, acclimation to increased respiratory demands (hypoxia, elevated temperatures) induced calcium deposition on the filaments of K. marmoratus. Next, gentle removal of filament calcification decreased branchial resistance to water flow, indicating disruption of gill basket positioning. Thus, the mechanical properties of the gill filaments appear to play an important and previously unappreciated role in determining fish respiratory function.

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.

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.

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.

Phenotypic flexibility in respiratory traits is associated with improved aerial respiration in an amphibious fish out of water.

Amphibious fishes have evolved multiple adaptive strategies for respiring out of water, but there has been less focus on reversible plasticity. We tested the hypothesis that when amphibious fishes leave water, enhanced respiratory performance on land is the result of rapid functional phenotypic flexibility of respiratory traits. We acclimated four isogenic strains of Kryptolebias marmoratus to air for 0, 1, 3 or 7 days. We compared respiratory performance out of water with traits linked to the O2 cascade. Aerial O2 consumption rate was measured over a step-wise decrease in O2 levels. There were significant differences between strains, but time out of water had the largest impact on measured parameters. Kryptolebiasmarmoratus had improved respiratory performance [lower aerial critical oxygen tension (Pcrit), higher regulation index (RI)] after only 1 day of air exposure, and these changes were strongly associated with the change in hematocrit and dorsal cutaneous angiogenesis. Additionally, we found that 1 h of air exposure induced the expression of four angiogenesis-associated genes - vegfa, angpt2, pecam-1 and efna1 - in the skin. After 7 days in air, respiratory traits were not significantly linked to the variation in either aerial Pcrit or RI. Overall, our data indicate that there are two phases involved in the enhancement of aerial respiration: an initial rapid response (1 day) and a delayed response (7 days). We found evidence for the hypothesis that respiratory performance on land in amphibious fishes is the result of rapid flexibility in both O2 uptake and O2 carrying capacity.

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.

Prolonged survival out of water is linked to a slow pace of life in a self-fertilizing amphibious fish.

Metabolic rate and life-history traits vary widely both among and within species, reflecting trade-offs in energy allocation, but the proximate and ultimate causes of variation are not well understood. We tested the hypothesis that these trade-offs are mediated by environmental heterogeneity, using isogenic strains of the amphibious fish Kryptolebias marmoratus that vary in the amount of time each can survive out of water. Consistent with pace of life theory, the strain that survived air exposure the longest generally exhibited a 'slow' phenotype, including the lowest metabolic rate, largest scope for metabolic depression, slowest consumption of energy stores and least investment in reproduction under standard conditions. Growth rates were fastest in the otherwise slow strain, however. We then tested for fitness trade-offs between 'fast' and 'slow' strains using microcosms where fish were held either with constant water availability or under fluctuating conditions where water was absent for half of the experiment. Under both conditions the slow strain grew larger and was in better condition, and under fluctuating conditions the slow strain produced more embryos. However, the fast strain had larger adult population sizes under both conditions, indicating that fecundity is not the sole determinant of population size in this species. We conclude that genetically based differences in the pace of life of amphibious fish determine survival duration out of water. Relatively slow fish tended to perform better under conditions of limited water availability, but there was no detectable cost under control conditions. Thus, pace of life differences may reflect a conditionally neutral instead of antagonistic trade-off.

Factors affecting terrestrial movement in the amphibious mangrove rivulus (Kryptolebias marmoratus).

We hypothesised that the exploration tendency of the amphibious mangrove rivulus Kryptolebias marmoratus would be inhibited in the terrestrial environment because of constraints on terrestrial locomotion or orientation. Using a novel object test, we showed that the fish explored objects in the aquatic but not the terrestrial environment, supporting the existence of constraints on terrestrial exploration. In further tests of the effects of extrinsic factors on terrestrial movement between aquatic refuges, shallow water depth simulating desiccation risk and the presence of a conspecific simulating intraspecific competition increased emersion outside of refuges, while high water salinity had no effect. These extrinsic factors had little effect on terrestrial movement between different aquatic refuges, except possibly for the lowest water depth tested. A significant association observed between emersion activity and movement between aquatic refuges suggested that terrestrial movement in K. marmoratus might depend on the tendency of individuals to emerse.