Mitochondrial physiology and responses to elevated hydrogen sulphide in two isogenic lineages of an amphibious mangrove fish.

Hydrogen sulphide (H2S) is toxic and can act as a selective pressure on aquatic organisms, facilitating a wide range of adaptations for life in sulphidic environments. Mangrove rivulus (Kryptolebias marmoratus) inhabit mangrove swamps and have developed high tolerance to environmental H2S. They are hermaphroditic and can self-fertilize, producing distinct isogenic lineages with different sensitivity to H2S. Here, we tested the hypothesis that observed differences in responses to H2S are the result of differences in mitochondrial functions. For this purpose, we performed two experimental series, testing (1) the overall mitochondrial oxidizing capacities and (2) the kinetics of apparent H2S mitochondrial oxidation and inhibition in two distinct lineages of mangrove rivulus, originally collected from Belize and Honduras. We used permeabilized livers from both lineages, measured mitochondrial oxidation, and monitored changes during gradual increases of sulphide. Ultimately, we determined that each lineage has a distinct strategy for coping with elevated H2S, indicating divergences in mitochondrial function and metabolism. The Honduras lineage has higher anaerobic capacity substantiated by higher lactate dehydrogenase activity and higher apparent H2S oxidation rates, likely enabling them to tolerate H2S by escaping aquatic H2S in a terrestrial environment. However, Belize fish have increased cytochrome c oxidase and citrate synthase activities as well as increased succinate contribution to mitochondrial respiration, allowing them to tolerate higher levels of aquatic H2S without inhibition of mitochondrial oxygen consumption. Our study reveals distinct physiological strategies in genetic lineages of a single species, indicating possible genetic and/or functional adaptations to sulphidic environments at the mitochondrial level.

Hydrogen sulphide sensitivity and tolerance in genetically distinct lineages of a selfing mangrove fish (Kryptolebias marmoratus).

Mangroves are critical marine habitats. High hydrogen sulphide (H2S) is a feature of these important ecosystems and its toxicity creates a challenge for mangrove inhabitants. The mangrove rivulus (Kryptolebias marmoratus) is a selfing, hermaphroditic, amphibious fish that can survive exposure to 1116 µM H2S in the wild. These fish rely on cutaneous respiration for gas and ion exchange when emerged. We hypothesized that the skin surface is fundamentally important in H2S tolerance in these mangrove fish by limiting H2S permeability. To test our hypothesis, we first disrupted the skin surface in one isogenic lineage and measured H2S tolerance and sensitivity. We increased water H2S concentration until emersion as a measure of the ability to sense and react to H2S, which we refer to as sensitivity. We then determined H2S tolerance by preventing emersion and increasing H2S until loss of equilibrium (LOE). The H2S concentration at emersion and LOE were significantly affected by disrupting the skin surface, providing support that the skin is involved in limiting H2S permeability. Capitalizing on their unique reproductive strategy, we used three distinct isogenic lineages to test the hypothesis that there would be genetic differences in H2S sensitivity and tolerance. We found significant differences in emersion concentration only among lineages, suggesting a genetic component to H2S sensitivity but not tolerance. Our study also demonstrated that external skin modifications and avoidance behaviours are two distinct strategies used to tolerate ecologically relevant H2S concentrations and likely facilitate survival in challenging mangrove habitats.

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.

Skin ionocyte remodeling in the amphibious mangrove rivulus fish (Kryptolebias marmoratus).

Amphibious fishes have evolved a variety of physiological modifications allowing them to survive in water and air. In air, the amphibious mangrove rivulus, Kryptolebias marmoratus, uses its skin as a site of ionoregulation. Skin ionocytes actively transport ions into/out of the body; however, it is unclear if there are specific morphological or functional changes occurring in skin ionocytes during air exposure. We used two microscopy techniques to describe skin ionocyte morphology and to investigate their plasticity after salinity challenges and air exposure. Immunohistochemical staining in air-exposed fish revealed ionocytes with Na + /K + ATPase (NKA), Na + /H + exchanger (NHE3b) and cystic fibrosis transmembrane conductance regulator (CFTR) immunoreactivity, whereas ionocytes from aquatic fish had only NKA (freshwater) or NKA and CFTR (brackish and hypersaline water). Following salinity challenges, we noted increases in the number and area of ionocyte apical surfaces, indicating that skin ionocyte activity increased in high salinity environments compared with control conditions. Furthermore, we show increased ionocyte area during air exposure suggesting increased ionocyte activity in all salinity conditions. Using energy dispersive X-ray spectroscopy to analyze the skin surface, we report decreases in magnesium, phosphorous, and sulfur after 7 days in air compared with fish in water, suggesting ionic movement in the skin surface during air exposure. Our study highlights morphological and functional features of skin ionocytes that are involved in ionoregulation in an air-exposed amphibious fish.