ABSTRACT 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.

ABSTRACT 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 O 2 sensing and uptake were largely unaffected by water–air fluctuations during early life, but we found marked developmental changes in traits related to O 2 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 O 2 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.

ABSTRACT 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 ( P O 2 ) within artificial cavities excavated in wet clay soil, which simulated C. alboguttata underground aestivation chambers, and recorded hypoxic conditions ( P O 2 as low as 8.9 kPa). Using custom-built tunnels that maintained a longitudinal P O 2 gradient (hypoxic to normoxic), we then examined the P O 2 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 P O 2 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 O 2 consumption after 7 weeks compared with active frogs in normoxia, whereas frogs exposed to normoxia reduced O 2 consumption by only 51%. Overall, our findings indicate that some animals may seek microhabitats to maximally depress metabolic rate during dormancy, and that microhabitat O 2 availability can have significant implications for energy metabolism.

ABSTRACT 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 O 2 gradient (hypoxic to normoxic), we assessed the O 2 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 O 2 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 O 2 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.

ABSTRACT 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.

ABSTRACT 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.

ABSTRACT 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 O 2 cascade. Aerial O 2 consumption rate was measured over a step-wise decrease in O 2 levels. There were significant differences between strains, but time out of water had the largest impact on measured parameters. K ryptolebias marmoratus had improved respiratory performance [lower aerial critical oxygen tension ( P crit ), 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 P crit 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 O 2 uptake and O 2 carrying capacity.

ABSTRACT 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 O 2 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 O 2 levels and capitalizes on O 2 -rich opportunities to enhance O 2 utilization by skeletal muscle.

ABSTRACT Nitrogen metabolism in fishes has been a focus of comparative physiologists for nearly a century. In this Review, we focus specifically on early life stages of fishes, which have received considerable attention in more recent work. Nitrogen metabolism and excretion in early life differs fundamentally from that of juvenile and adult fishes because of (1) the presence of a chorion capsule in embryos that imposes a limitation on effective ammonia excretion, (2) an amino acid-based metabolism that generates a substantial ammonia load, and (3) the lack of a functional gill, which is the primary site of nitrogen excretion in juvenile and adult fishes. Recent findings have shed considerable light on the mechanisms by which these constraints are overcome in early life. Perhaps most importantly, the discovery of Rhesus (Rh) glycoproteins as ammonia transporters and their expression in ion-transporting cells on the skin of larval fishes has transformed our understanding of ammonia excretion by fishes in general. The emergence of larval zebrafish as a model species, together with genetic knockdown techniques, has similarly advanced our understanding of ammonia and urea metabolism and excretion by larval fishes. It has also now been demonstrated that ammonia excretion is one of the primary functions of the developing gill in rainbow trout larvae, leading to new hypotheses regarding the physiological demands driving gill development in larval fishes. Here, we highlight and discuss the dramatic changes in nitrogen handling that occur over early life development in fishes.

ABSTRACT Terrestrial animals must support their bodies against gravity, while aquatic animals are effectively weightless because of buoyant support from water. Given this evolutionary history of minimal gravitational loading of fishes in water, it has been hypothesized that weight-responsive musculoskeletal systems evolved during the tetrapod invasion of land and are thus absent in fishes. Amphibious fishes, however, experience increased effective weight when out of water – are these fishes responsive to gravitational loading? Contrary to the tetrapod-origin hypothesis, we found that terrestrial acclimation reversibly increased gill arch stiffness (∼60% increase) in the amphibious fish Kryptolebias marmoratus when loaded normally by gravity, but not under simulated microgravity. Quantitative proteomics analysis revealed that this change in mechanical properties occurred via increased abundance of proteins responsible for bone mineralization in other fishes as well as in tetrapods. Type X collagen, associated with endochondral bone growth, increased in abundance almost ninefold after terrestrial acclimation. Collagen isoforms known to promote extracellular matrix cross-linking and cause tissue stiffening, such as types IX and XII collagen, also increased in abundance. Finally, more densely packed collagen fibrils in both gill arches and filaments were observed microscopically in terrestrially acclimated fish. Our results demonstrate that the mechanical properties of the fish musculoskeletal system can be fine-tuned in response to changes in effective body weight using biochemical pathways similar to those in mammals, suggesting that weight sensing is an ancestral vertebrate trait rather than a tetrapod innovation.

ABSTRACT In rainbow trout, the dominant site of Na + uptake ( J Na,in ) and ammonia excretion ( J amm ) shifts from the skin to the gills over development. Post-hatch (PH; 7 days post-hatch) larvae utilize the yolk sac skin for physiological exchange, whereas by complete yolk sac absorption (CYA; 30 days post-hatch), the gill is the dominant site. At the gills, J Na,in and J amm occur via loose Na + /NH 4 + exchange, but this exchange has not been examined in the skin of larval trout. Based on previous work, we hypothesized that, contrary to the gill model, J Na,in by the yolk sac skin of PH trout occurs independently of J amm . Following a 12 h exposure to high environmental ammonia (HEA; 0.5 mmol l −1 NH 4 HCO 3 ; 600 µmol l −1 Na + ; pH 8), J amm by the gills of CYA trout and the yolk sac skin of PH larvae, which were isolated using divided chambers, increased significantly. However, this was coupled to an increase in J Na,in across the gills only, supporting our hypothesis. Moreover, gene expression of proteins involved in J Na,in [Na + /H + -exchanger-2 (NHE2) and H + -ATPase] increased in response to HEA only in the CYA gills. We further identified expression of the apical Rhesus (Rh) proteins Rhcg2 in putative pavement cells and Rhcg1 (co-localized with apical NHE2 and NHE3b and Na + /K + -ATPase) in putative peanut lectin agglutinin-positive (PNA + ) ionocytes in gill sections. Similar Na + /K + -ATPase-positive cells expressing Rhcg1 and NHE3b, but not NHE2, were identified in the yolk sac epithelium. Overall, our findings suggest that the mechanisms of J Na,in and J amm by the dominant exchange epithelium at two distinct stages of early development are fundamentally different.

ABSTRACT Air and water differ dramatically in density and viscosity, posing different biomechanical challenges for animal locomotion. We asked how terrestrial acclimation influences locomotion in amphibious fish, specifically testing the hypothesis that terrestrial tail flip performance is improved by plastic changes in the skeletal muscle. Mangrove rivulus Kryptolebias marmoratus , which remain largely inactive out of water, were exposed to water or air for 14 days and a subgroup of air-exposed fish was also recovered in water. Tail flip jumping performance on land improved dramatically in air-acclimated fish, they had lower lactate levels compared with control fish, and these effects were mostly reversible. Muscle plasticity significantly increased oxidative muscle cross-sectional area and fibre size, as well as the number of capillaries per fibre. Our results show that reversible changes to the oxidative skeletal muscle of K. marmoratus out of water enhance terrestrial locomotory performance, even in the absence of exercise training.

ABSTRACT Amphibious fishes spend part of their life in terrestrial habitats. The ability to tolerate life on land has evolved independently many times, with more than 200 extant species of amphibious fishes spanning 17 orders now reported. Many adaptations for life out of water have been described in the literature, and adaptive phenotypic plasticity may play an equally important role in promoting favourable matches between the terrestrial habitat and behavioural, physiological, biochemical and morphological characteristics. Amphibious fishes living at the interface of two very different environments must respond to issues relating to buoyancy/gravity, hydration/desiccation, low/high O 2 availability, low/high CO 2 accumulation and high/low NH 3 solubility each time they traverse the air–water interface. Here, we review the literature for examples of plastic traits associated with the response to each of these challenges. Because there is evidence that phenotypic plasticity can facilitate the evolution of fixed traits in general, we summarize the types of investigations needed to more fully determine whether plasticity in extant amphibious fishes can provide indications of the strategies used during the evolution of terrestriality in tetrapods.

ABSTRACT Aquatic hypercapnia may have helped to drive ancestral vertebrate invasion of land. We tested the hypothesis that amphibious fishes sense and respond to elevated aquatic P CO 2 by behavioural avoidance mechanisms, and by morphological changes at the chemoreceptor level. Mangrove rivulus ( Kryptolebias marmoratus ) were exposed to 1 week of normocapnic control water (pH 8), air, hypercapnia (5% CO 2 , pH 6.8) or isocapnic acidosis (pH 6.8). We found that the density of CO 2 /H + chemoreceptive neuroepithelial cells (NECs) was increased in hypercapnia or isocapnic acidosis-exposed fish. Projection area (a measure of cell size) was unchanged. Acute exposure to progressive hypercapnia induced the fish to emerse (leave water) at water pH values ∼6.1, whereas addition of HCl to water caused a more variable response with a lower pH threshold (∼pH 5.5). These results support our hypothesis and suggest that aquatic hypercapnia provides an adequate stimulus for extant amphibious fishes to temporarily transition from aquatic to terrestrial habitats.

ABSTRACT Few teleost fishes incubate embryos out of water, but the oxygen-rich terrestrial environment could provide advantages for early growth and development. We tested the hypothesis that embryonic oxygen uptake is limited in aquatic environments relative to air using the self-fertilizing amphibious mangrove rivulus, Kryptolebias marmoratus , which typically inhabits hypoxic, water-filled crab burrows. We found that adult mangrove rivulus released twice as many embryos in terrestrial versus aquatic environments and that air-reared embryos had accelerated developmental rates. Surprisingly, air-reared embryos consumed 44% less oxygen and possessed larger yolk reserves, but attained the same mass, length and chorion thickness. Water-reared embryos moved their opercula ∼2.5 more times per minute compared with air-reared embryos at 7 days post-release, which probably contributed to the higher rates of oxygen uptake and yolk utilization we observed. Genetically identical air- and water-reared embryos from the same parent were raised to maturity, but the embryonic environment did not affect growth, reproduction or emersion ability in adults. Therefore, although aspects of early development were plastic, these early differences were not sustained into adulthood. Kryptolebias marmoratus embryos hatched out of water when exposed to aerial hypoxia. We conclude that exposure to a terrestrial environment reduces the energetic costs of development partly by reducing the necessity of embryonic movements to dispel stagnant boundary layers. Terrestrial incubation of young would be especially beneficial to amphibious fishes that occupy aquatic habitats of poor water quality, assuming low terrestrial predation and desiccation risks.

ABSTRACT Relative to the gills, the mechanisms by which the kidney contributes to ammonia and acid–base homeostasis in fish are poorly understood. Goldfish were exposed to a low pH environment (pH 4.0, 48 h), which induced a characteristic metabolic acidosis and an increase in total plasma [ammonia] but reduced plasma ammonia partial pressure ( P NH3 ). In the kidney tissue, total ammonia, lactate and intracellular pH remained unchanged. The urinary excretion rate of net base under control conditions changed to net acid excretion under low pH, with contributions from both the NH 4 + (∼30%) and titratable acidity minus bicarbonate (∼70%; TA–HCO 3 − ) components. Inorganic phosphate (P i ), urea and Na + excretion rates were also elevated while Cl − excretion rates were unchanged. Renal alanine aminotransferase activity increased under acidosis. The increase in renal ammonia excretion was due to significant increases in both the glomerular filtration and the tubular secretion rates of ammonia, with the latter accounting for ∼75% of the increase. There was also a 3.5-fold increase in the mRNA expression of renal Rhcg-b (Rhcg1) mRNA. There was no relationship between ammonia secretion and Na + reabsorption. These data indicate that increased renal ammonia secretion during acidosis is probably mediated through Rhesus (Rh) glycoproteins and occurs independently of Na + transport, in contrast to branchial and epidermal models of Na + -dependent ammonia transport in freshwater fish. Rather, we propose a model of parallel H + /NH 3 transport as the primary mechanism of renal tubular ammonia secretion that is dependent on renal amino acid catabolism.

Despite the abundance of oxygen in atmospheric air relative to water, the initial loss of respiratory surface area and accumulation of carbon dioxide in the blood of amphibious fishes during emersion may result in hypoxemia. Given that the ability to respond to low oxygen conditions predates the vertebrate invasion of land, we hypothesized that amphibious fishes maintain O 2 uptake and transport while emersed by mounting a co-opted hypoxia response. We acclimated the amphibious fish Kryptolebias marmoratus , which are able to remain active for weeks in both air and water, for 7 days to normoxic brackish water (15‰, ~21kPa O 2 ; control), aquatic hypoxia (~3.6kPa), normoxic air (~21 kPa) or aerial hypoxia (~13.6kPa). Angiogenesis in the skin and bucco-opercular chamber was pronounced in air- versus water-acclimated fish, but not in response to hypoxia. Aquatic hypoxia increased the O 2 -carrying capacity of blood via a large (40%) increase in red blood cell density and a small increase in the affinity of hemoglobin for O 2 ( P 50 decreased 11%). In contrast, air exposure increased the hemoglobin O 2 affinity (decreased P 50 ) by 25% without affecting the number of red blood cells. Acclimation to aerial hypoxia both increased the O 2 -carrying capacity and decreased the hemoglobin O 2 affinity . These results suggest that O 2 transport is regulated both by O 2 availability and also, independently, by air exposure. The ability of the hematological system to respond to air exposure independent of O 2 availability may allow extant amphibious fishes, and may also have allowed primitive tetrapods to cope with the complex challenges of aerial respiration during the invasion of land.

Increased renal ammonia excretion in response to metabolic acidosis is thought to be a conserved response in vertebrates. We tested the hypothesis that Rhesus (Rh) glycoproteins in the kidney of the freshwater common carp, Cyprinus carpio , play a crucial role in regulating renal ammonia excretion during chronic metabolic acidosis. Exposure to water pH 4.0 (72 h) resulted in a classic metabolic acidosis with reduced plasma arterial pH and [HCO 3 − ], no change in P CO 2 and large changes in renal function. Urine [NH 4 + ] as well as [titratable acidity-HCO 3 − ] rose significantly over the acid exposure, but the profound reduction (fivefold) in urine flow rates eliminated the expected elevations in renal ammonia excretion. Low urine flow rates may be a primary strategy to conserve ions, as urinary excretion rates of Na + , Cl − and Ca 2+ were significantly lower during the acid exposure relative to the control period. Interestingly, renal Rhcg1 mRNA and protein levels were elevated in acid-exposed relative to control groups, along with mRNA levels of several ion transporters, including the Na + /H + exchanger, H + -ATPase and Na + /K + -ATPase . Immunofluorescence microscopy showed a strong apical Rhcg1 signal in distal tubules. Taken together, these data show that renal Rh glycoproteins and associated ion transporters are responsive to metabolic acidosis, but conservation of ions through reduced urine flow rates takes primacy over renal acid–base regulation in the freshwater C. carpio . We propose that an ‘acid/base–ion balance’ compromise explains the variable renal responses to metabolic acidosis in freshwater teleosts.

Serotonin containing neuroepithelial cells (NECs) are putative oxygen sensing cells found in different locations within the gills of fish. In this study we wished to determine the effect of sustained internal (blood) hypoxaemia versus external (aquatic) hypoxia on the size and density of NECs in the first gill arch of bowfin ( Amia calva ), a facultative air breather. We identified five different populations of serotonergic NECs in this species (Types I–V) based on location, presence of synaptic vesicles (SV) that stain for the antibody SV2, innervation and labelling with the neural crest marker HNK-1. Cell Types I–III were innervated, and these cells, which participate in central O 2 chemoreflexes, were studied further. Although there was no change in the density of any cell type in bowfin after exposure to sustained hypoxia (6.0 kPa for 7 days) without access to air, all three of these cell types increased in size. In contrast, only Type II and III cells increased in size in bowfin exposed to sustained hypoxia with access to air. These data support the suggestion that NECs are putative oxygen-sensing cells, that they occur in several locations, and that Type I cells monitor only hypoxaemia, whereas both other cell types monitor hypoxia and hypoxaemia.

SUMMARY In rainbow trout development, a switch occurs from high-affinity embryonic hemoglobin (Hb) and round, embryonic erythrocytes to lower-affinity adult Hb and oval, adult erythrocytes. Our study investigated the early ontogeny of rainbow trout blood properties and the hypoxia response. We hypothesized that hypoxia exposure would delay the ontogenetic turnover of Hb and erythrocytes because retention of high-affinity embryonic Hb would facilitate oxygen loading. To test this hypothesis we developed a method of efficiently extracting blood from individual embryos and larvae and optimized several techniques for measuring hematological parameters on microliter (0.5–2.0 μl) blood samples. In chronic hypoxia (30% of oxygen saturation), stage-matched embryos and larvae possessed half the Hb concentration, erythrocyte counts and hematocrit observed in normoxia. Hypoxia-reared larvae also had threefold to sixfold higher mRNA expression of the embryonic Hb α-1, β-1 and β-2 subunits relative to stage-matched normoxia-reared larvae. Furthermore, in hypoxia, the round embryonic erythrocytic shape persisted into later developmental stages. Despite these differences, Hb–oxygen affinity ( P 50 ), cooperativity and the Root effect were unaltered in hypoxia-reared O. mykiss . The data support our hypothesis that chronic hypoxia delays the ontogenetic turnover of Hb and erythrocytes, but without the predicted functional consequences (i.e. higher than expected P 50 ). These results also suggest that the Hb–oxygen affinity is protected during development in chronic hypoxia to favor oxygen unloading at the tissues. We conclude that in early trout development, the blood–oxygen transport system responds very differently to chronic hypoxia relative to adults, possibly because respiration depends relatively more on oxygen diffusion than convection.