Skin ionocyte density of amphibious killifishes is shaped by phenotypic plasticity and constitutive interspecific differences.

When amphibious fishes are on land, gill function is reduced or eliminated and the skin is hypothesized to act as a surrogate site of ionoregulation. Skin ionocytes are present in many fishes, particularly those with amphibious life histories. We used nine closely related killifishes spanning a range of amphibiousness to first test the hypothesis that amphibious killifishes have evolved constitutively increased skin ionocyte density to promote ionoregulation on land. We found that skin ionocyte densities were constitutively higher in five of seven amphibious species examined relative to exclusively water-breathing species when fish were prevented from leaving water, strongly supporting our hypothesis. Next, to examine the scope for plasticity, we tested the hypothesis that skin ionocyte density in amphibious fishes would respond plastically to air-exposure to promote ionoregulation in terrestrial environments. We found that air-exposure induced plasticity in skin ionocyte density only in the two species classified as highly amphibious, but not in moderately amphibious species. Specifically, skin ionocyte density significantly increased in Anablepsoides hartii (168%) and Kryptolebias marmoratus (37%) following a continuous air-exposure, and only in K. marmoratus (43%) following fluctuating air-exposure. Collectively, our data suggest that highly amphibious killifishes have evolved both increased skin ionocyte density as well as skin that is more responsive to air-exposure compared to exclusively water-breathing and less amphibious species. Our findings are consistent with the idea that gaining the capacity for cutaneous ionoregulation is a key evolutionary step that enables amphibious fishes to survive on land.

Retention of larval skin traits in adult amphibious killifishes: a cross-species investigation.

The gills are the primary site of exchange in fishes. However, during early life-stages or in amphibious fishes, ionoregulation and gas-exchange may be primarily cutaneous. Given the similarities between larval and amphibious fishes, we hypothesized that cutaneous larval traits are continuously expressed in amphibious fishes across all life-stages to enable the skin to be a major site of exchange on land. Alternatively, we hypothesized that cutaneous larval traits disappear in juvenile stages and are re-expressed in amphibious species in later life-stages. We surveyed six species spanning a range of amphibiousness and characterized cutaneous ionocytes and neuroepithelial cells (NECs) as representative larval skin traits at up to five stages of development. We found that skin ionocyte density remained lower and constant in exclusively water-breathing, relative to amphibious species across development, whereas in amphibious species ionocyte density generally increased. Additionally, adults of the most amphibious species had the highest cutaneous ionocyte densities. Surprisingly, cutaneous NECs were only identified in the skin of one amphibious species (Kryptolebias marmoratus), suggesting that cutaneous NECs are not a ubiquitous larval or amphibious skin trait, at least among the species we studied. Our data broadly supports the continuous-expression hypothesis, as three of four amphibious experimental species expressed cutaneous ionocytes in all examined life-stages. Further, the increasing density of cutaneous ionocytes across development in amphibious species probably facilitates the prolonged occupation of terrestrial habitats.

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.

Striped catfish (Pangasianodon hypophthalmus) use air-breathing and aquatic surface respiration when exposed to severe aquatic hypercarbia.

We investigated the extent to which the facultative air-breathing fish, the striped catfish (Pangasianodon hypophthalmus), uses air-breathing to cope with aquatic hypercarbia, and how air-breathing is influenced by the experimental exposure protocol and level of hypercarbia. We exposed individuals to severe aquatic hypercarbia (up to Pw CO2 = 81 mmHg) using step-wise and progressive exposure protocols while measuring gill ventilation rate, heart rate, mean arterial blood pressure, and air-breathing frequency, as well as arterial blood pH and PCO2 . We confirm that P. hypophthalmus is tolerant of hypercarbia. Under both protocols gill ventilation rate, heart rate, and mean arterial blood pressure were maintained near control levels even at very high CO2 levels. We observed a marked amount of individual variation in the PwCO2 at which air-breathing was elicited, with some individuals not responding at all. The experimental protocol also influenced the onset of air-breathing. Air-breathing began at lower Pw CO2 in the step-wise protocol (23 ± 4.1 mmHg) compared with the progressive protocol (46 ± 7.8 mmHg). Air-breathing was often followed by aquatic surface respiration, at higher PCO2 (71 ± 5.2 mmHg) levels. On average, the blood PCO2 was approximately 43% lower (46 ± 2.5 mmHg) than water Pw CO2 (~81 mmHg) at our highest tested CO2 level. While this suggests that aerial CO2 elimination is an effective, and perhaps critical, respiratory strategy used by P. hypophthalmus to cope with severe hypercarbia, this observation may also be explained by a long lag time required for equilibration.

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.

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.

Corrigendum: Hypoxic Induced Decrease in Oxygen Consumption in Cuttlefish (Sepia officinalis) Is Associated with Minor Increases in Mantle Octopine but No Changes in Markers of Protein Turnover.

[This corrects the article DOI: 10.3389/fphys.2017.00344.].

Hypoxic Induced Decrease in Oxygen Consumption in Cuttlefish (Sepia officinalis) Is Associated with Minor Increases in Mantle Octopine but No Changes in Markers of Protein Turnover.

The common cuttlefish (Sepia officinalis), a dominant species in the north-east Atlantic ocean and Mediterranean Sea, is potentially subject to hypoxic conditions due to eutrophication of coastal waters and intensive aquaculture. Here we initiate studies on the biochemical response to an anticipated level of hypoxia. Cuttlefish challenged for 1 h at an oxygen level of 50% dissolved oxygen saturation showed a decrease in oxygen consumption of 37% associated with an 85% increase in ventilation rate. Octopine levels were increased to a small but significant level in mantle, whereas there was no change in gill or heart. There were no changes in mantle free glucose or glycogen levels. Similarly, the hypoxic period did not result in changes in HSP70 or polyubiquinated protein levels in mantle, gill, or heart. As such, it appears that although there was a decrease in metabolic rate there was only a minor increase in anaerobic metabolism as evidenced by octopine accumulation and no biochemical changes that are hallmarks of alterations in protein trafficking. Experiments with isolated preparations of mantle, gill, and heart revealed that pharmacological inhibition of protein synthesis could decrease oxygen consumption by 32 to 42% or Na+/K+ ATPase activity by 24 to 54% dependent upon tissue type. We propose that the decrease in whole animal oxygen consumption was potentially the result of controlled decreases in the energy demanding processes of both protein synthesis and Na+/K+ ATPase activity.

Pass the salt: physiological consequences of ecologically relevant hyposmotic exposure in juvenile gummy sharks (Mustelus antarcticus) and school sharks (Galeorhinus galeus).

Estuarine habitats are frequently used as nurseries by elasmobranch species for their protection and abundant resources; however, global climate change is increasing the frequency and severity of environmental challenges in these estuaries that may negatively affect elasmobranch physiology. Hyposmotic events are particularly challenging for marine sharks that osmoconform, and species-specific tolerances are not well known. Therefore, we sought to determine the effects of an acute (48 h) ecologically relevant hyposmotic event (25.8 ppt) on the physiology of two juvenile shark species, namely the school shark (Galeorhinus galeus), listed by the Australian Environmental Protection and Biodiversity Conservation Act as 'conservation dependent', and the gummy shark (Mustelus antarcticus), from the Pittwater Estuary (Australia). In both species, we observed a decrease in plasma osmolality brought about by selective losses of NaCl, urea and trimethylamine N-oxide, as well as decreases in haemoglobin, haematocrit and routine oxygen consumption. Heat-shock protein levels varied between species during the exposure, but we found no evidence of protein damage in any of the tissues tested. Although both species seemed to be able to cope with this level of osmotic challenge, overall the school sharks exhibited higher gill Na+/K+-ATPase activity and ubiquitin concentrations in routine and experimental conditions, a larger heat-shock protein response and a smaller decrease in routine oxygen consumption during the hyposmotic exposure, suggesting that there are species-specific responses that could potentially affect their ability to withstand longer or more severe changes in salinity. Emerging evidence from acoustic monitoring of sharks has indicated variability in the species found in the Pittwater Estuary during hyposmotic events, and together, our data may help to predict species abundance and distribution in the face of future global climate change.

Metabolic Adjustments to Short-Term Diurnal Temperature Fluctuation in the Rainbow Trout (Oncorhynchus mykiss).

In rainbow trout, warmer temperatures increase metabolic rate, which can be energetically stressful. Diel fluctuations in water temperatures are common in rivers, raising the question of whether fish experience metabolic preconditioning with repeated heat stress. In this study, rainbow trout (Oncorhynchus mykiss Walbaum, 1792) were subjected to three temperature treatments consisting of either a constant exposure to 16°C, a single exposure to 24°C, or three cycles between 16° and 24°C. Metabolic responses were investigated, including patterns of regulation of adenosine monophosphate-activated protein kinase and its substrates, key metabolic enzymes, and several relevant metabolites. In liver and, to a lesser extent, in heart, patterns of signal transduction suggest an increasingly anabolic phenotype with successive heat cycles. Inhibition of Raptor in the heart suggests lowered gross protein synthesis after multiple heat cycles. Fish also showed recovery of glycogen stores and lipid synthesis after multiple thermal cycles, while they maintained baseline plasma glucose levels. The animals showed no evidence of hypoxemia, and our results suggest rainbow trout exposed to repeated thermal cycles were not at risk of metabolic substrate depletion. Collectively, our data indicate that, when exposed to fluctuating but noncritical thermal cycles, rainbow trout may adopt a new metabolic phenotype to sequester readily accessible metabolic substrates in the liver in preparation for more severe or sustained thermal exposures.

Physiological responses to hypersalinity correspond to nursery ground usage in two inshore shark species (Mustelus antarcticus and Galeorhinus galeus).

Shark nurseries are susceptible to environmental fluctuations in salinity because of their shallow, coastal nature; however, the physiological impacts on resident elasmobranchs are largely unknown. Gummy sharks (Mustelus antarcticus) and school sharks (Galeorhinus galeus) use the same Tasmanian estuary as a nursery ground; however, each species has distinct distribution patterns that are coincident with changes in local environmental conditions, such as increases in salinity. We hypothesized that these differences were directly related to differential physiological tolerances to high salinity. To test this hypothesis, we exposed wild, juvenile school and gummy sharks to an environmentally relevant hypersaline (120% SW) event for 48 h. Metabolic rate decreased 20-35% in both species, and gill Na(+)/K(+)-ATPase activity was maintained in gummy sharks but decreased 37% in school sharks. We measured plasma ions (Na(+), K(+), Cl(-)) and osmolytes [urea and trimethylamine oxide (TMAO)], and observed a 33% increase in plasma Na(+) in gummy sharks with hyperosmotic exposure, while school sharks displayed a typical ureosmotic increase in plasma urea (∼20%). With elevated salinity, gill TMAO concentration increased by 42% in school sharks and by 30% in gummy sharks. Indicators of cellular stress (heat shock proteins HSP70, 90 and 110, and ubiquitin) significantly increased in gill and white muscle in both a species- and a tissue-specific manner. Overall, gummy sharks exhibited greater osmotic perturbation and ionic dysregulation and a larger cellular stress response compared with school sharks. Our findings provide physiological correlates to the observed distribution and movement of these shark species in their critical nursery grounds.

Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogfish shark (Squalus acanthias).

Salinity decreases are experienced by many marine elasmobranchs. To understand how these fishes cope with hyposmotic stress on a cellular level, we used the spiny dogfish shark (Squalus acanthias) as a model to test whether a reciprocal relationship exists between the cell's two primary protein protection mechanisms, the chemical (e.g., trimethylamine oxide, TMAO) and molecular (e.g., heat shock protein 70, HSP70) chaperone systems. This relationship is interesting given that many elasmobranchs are expected to gain water and lose osmolytes, chemical chaperones, and ions as they osmoconform to new, lowered salinity. Dogfish were cannulated for repeated blood sampling and exposed to 70% seawater (SW) for 48 h. These hyposmotic conditions had no effect on red blood cell (RBC) and white muscle TMAO concentrations, and did not result in HSP70 induction or signs of protein damage (i.e., increased ubiquitin), suggesting that TMAO levels were sufficiently protective in these tissues. However, in the gill, we observed a significant decrease in TMAO concentration and a significant induction of HSP70 as well as signs of protein damage. In the face of this cellular stress response, gill Na(+)/K(+)-ATPase (NKA) activity significantly increased during hyposmotic conditions, as expected. We suggest that this functional preservation in the gill is partly the result of HSP70 induction with lowered salinity. We conclude a reciprocal relationship between TMAO and HSP70 in the gills of dogfish as a result of in vivo hyposmotic stress. When osmotically induced protein damage surpasses the protective capacity of remaining TMAO, HSP70 is induced to preserve tissue and organismal function.

Cardiorespiratory toxicity of environmentally relevant zinc oxide nanoparticles in the freshwater fish Catostomus commersonii.

The inhalation of zinc oxide engineered nanomaterials (ENMs) has been linked to cardiorespiratory dysfunction in mammalian models but the effects of aquatic ENM exposure on fish have not been fully investigated. Nano-zinc oxide (nZnO) is widely used in consumer products such as sunscreens and can make its way into aquatic ecosystems from domestic and commercial wastewater. This study examined the impact of an environmentally relevant nZnO formulation on cardiorespiratory function and energy metabolism in the white sucker (Catostomus commersonii), a freshwater teleost fish. Evidence of oxidative and cellular stress was present in gill tissue, including increases in malondialdehyde levels, heat shock protein (HSP) expression, and caspase 3/7 activity. Gill Na(+)/K(+)-ATPase activity was also higher by approximately three-fold in nZnO-treated fish, likely in response to increased epithelial permeability or structural remodeling. Despite evidence of toxicity in gill, plasma cortisol and lactate levels did not change in animals exposed to 1.0 mg L(-1) nZnO. White suckers also exhibited a 35% decrease in heart rate during nZnO exposure, with no significant changes in resting oxygen consumption or tissue energy stores. Our results suggest that tissue damage or cellular stress resulting from nZnO exposure activates gill neuroepithelial cells, triggering a whole-animal hypoxic response. An increase in parasympathetic nervous signaling will decrease heart rate and may reduce energy demand, even in the face of an environmental toxicant. We have shown that acute exposure to nZnO is toxic to white suckers and that ENMs have the potential to negatively impact cardiorespiratory function in adult fish.