Andy J. Turko

Amphibious fishes: evolution and phenotypic plasticity

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 O2 availability, low/high CO2 accumulation and high/low NH3 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. Summary: We review the use of phenotypic plasticity by amphibious fishes and ask whether studying plasticity in extant fishes can provide insight into evolutionary changes during the tetrapod invasion of land.

Evolution, ecology and physiology of amphibious killifishes (Cyprinodontiformes).

The order Cyprinodontiformes contains an exceptional diversity of amphibious taxa, including at least 34 species from six families. These cyprinodontiforms often inhabit intertidal or ephemeral habitats characterized by low dissolved oxygen or otherwise poor water quality, conditions that have been hypothesized to drive the evolution of terrestriality. Most of the amphibious species are found in the Rivulidae, Nothobranchiidae and Fundulidae. It is currently unclear whether the pattern of amphibiousness observed in the Cyprinodontiformes is the result of repeated, independent evolutions, or stems from an amphibious common ancestor. Amphibious cyprinodontiforms leave water for a variety of reasons: some species emerse only briefly, to escape predation or capture prey, while others occupy ephemeral habitats by living for months at a time out of water. Fishes able to tolerate months of emersion must maintain respiratory gas exchange, nitrogen excretion and water and salt balance, but to date knowledge of the mechanisms that facilitate homeostasis on land is largely restricted to model species. This review synthesizes the available literature describing amphibious lifestyles in cyprinodontiforms, compares the behavioural and physiological strategies used to exploit the terrestrial environment and suggests directions and ideas for future research.

The amphibious fish Kryptolebias marmoratus uses different strategies to maintain oxygen delivery during aquatic hypoxia and air exposure

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 O2 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 O2; 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 O2-carrying capacity of blood via a large (40%) increase in red blood cell density and a small increase in the affinity of hemoglobin for O2 (P50 decreased 11%). In contrast, air exposure increased the hemoglobin O2 affinity (decreased P50) by 25% without affecting the number of red blood cells. Acclimation to aerial hypoxia both increased the O2-carrying capacity and decreased the hemoglobin O2 affinity. These results suggest that O2 transport is regulated both by O2 availability and also, independently, by air exposure. The ability of the hematological system to respond to air exposure independent of O2 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.

Fish embryos on land: terrestrial embryo deposition lowers oxygen uptake without altering growth or survival in the amphibious fish Kryptolebias marmoratus.

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. Highlighted Article: Terrestrially reared embryos from the amphibious fish Kryptolebias marmoratus consume less oxygen and reach hatching competency with larger yolk reserves relative to embryos reared in water.

Hypercapnia and low pH induce neuroepithelial cell proliferation and emersion behaviour in the amphibious fish Kryptolebias marmoratus

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 PCO2 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% CO2, pH 6.8) or isocapnic acidosis (pH 6.8). We found that the density of CO2/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. Summary: Amphibious fish may use oxygen-sensing neuroepithelial cells to sense and avoid high CO2 or low pH in the water.

Out of the frying pan into the air—emersion behaviour and evaporative heat loss in an amphibious mangrove fish (Kryptolebias marmoratus)

Amphibious fishes often emerse (leave water) when faced with unfavourable water conditions. How amphibious fishes cope with the risks of rising water temperatures may depend, in part, on the plasticity of behavioural mechanisms such as emersion thresholds. We hypothesized that the emersion threshold is reversibly plastic and thus dependent on recent acclimation history rather than on conditions during early development. Kryptolebias marmoratus were reared for 1 year at 25 or 30°C and acclimated as adults (one week) to either 25 or 30°C before exposure to an acute increase in water temperature. The emersion threshold temperature and acute thermal tolerance were significantly increased in adult fish acclimated to 30°C, but rearing temperature had no significant effect. Using a thermal imaging camera, we also showed that emersed fish in a low humidity aerial environment (30°C) lost significantly more heat (3.3°C min−1) than those in a high humidity environment (1.6°C min−1). In the field, mean relative humidity was 84%. These results provide evidence of behavioural avoidance of high temperatures and the first quantification of evaporative cooling in an amphibious fish. Furthermore, the avoidance response was reversibly plastic, flexibility that may be important for tropical amphibious fishes under increasing pressures from climatic change.

Copper alters hypoxia sensitivity and the behavioural emersion response in the amphibious fish Kryptolebias marmoratus

Elevated levels of metals have been reported in mangrove ecosystems worldwide. Mangrove fishes also routinely experience severe environmental stressors, such as hypoxia. In the amphibious fish Kryptolebias marmoratus (mangrove rivulus), a key behavioural response to avoid aquatic stress is to leave water (emersion). We hypothesized that copper (Cu) exposure would increase the sensitivity of this behavioural hypoxia avoidance response due to histopathological effects of Cu on gill structure and function. K. marmoratus were exposed to either control (no added Cu) or Cu (300μg/L) for 96h. Following this period, fish were exposed to an acute hypoxic challenge (decline in dissolved oxygen to ∼0% over 15min), and the emersion response was recorded. Gills were examined for histological changes. Fish exposed to Cu emersed at a higher dissolved oxygen level (7.5±0.6%), relative to the control treatment group (5.8±0.4%). Histological analysis showed that the gill surface area increased and the interlamellar cell mass (ILCM) was reduced following Cu exposure, contrary to our prediction. Overall, these data indicate that Cu induces hypoxia-like changes to gill morphology and increases the sensitivity of the hypoxia emersion response.

Environmental calcium regulates gill remodeling in a euryhaline teleost fish

Some cyprindid and cyprinidontiform fishes undergo gill remodeling via the proliferation or regression of an interlamellar cell mass (ILCM), resulting in the modification of gill surface area in response to environmental hypoxia or ion levels. We hypothesized that ion-related gill remodeling is regulated by water hardness through the interactions of Ca2+ with tight junctions, predicting that gills will exhibit a lower ILCM and more surface area in a high Ca2+ environment than in a low Ca2+ environment. To test this hypothesis, we acclimated euryhaline mangrove rivulus (Kryptolebias marmoratus) to natural hard water ([Ca2+] = 2.77 mmol/L), low Ca2+ ([Ca2+] = 0.13 mmol/L) freshwater, or high Ca2+ water (5.88 mmol/L). Fish exposed to hard water had a significantly lower ILCM height than fish exposed to low Ca2+ water. The addition of Ca2+ to low Ca2+ water restored gill surface area. Plasma Ca2+ activity was not significantly different between groups. This study provides support for an influence of external Ca2+ on gill remodeling and represents the first evidence of an ionic trigger (Ca2+) for gill remodeling in teleost fishes.

Amphibious fish jump better on land after acclimation to a terrestrial environment

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. Summary: Reversible changes to the oxidative skeletal muscle of the amphibious fish Kryptolebias marmoratus out of water enhance terrestrial locomotory performance, even in the absence of exercise training.

Skeletal Stiffening in an Amphibious Fish Out of Water is a Response to Increased Body Weight

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. Highlighted Article: Did weight responsiveness evolve during the tetrapod invasion of land or is it ubiquitous among vertebrates? Skeletal stiffening in amphibious fish out of water supports the latter hypothesis.