Water mould exposure induces enzymatic antioxidant defences in embryos of the Two-colored Oval Frog (Elachistocleis bicolor) (Anura: Microhylidae)

Water moulds are pathogens of amphibian eggs and embryos. However, little is known about oxidant or antioxidant status of amphibians in response to stress caused by water moulds. We exposed embryo stages of Two-colored Oval Frogs (Elachistocleis bicolor (Guérin-Méneville, 1838)) to a Saprolegnia-like species of water mould to explore homeostatic adjustments by the shifting of oxidative stress markers. We also tested whether water mould infection affected survivorship, hatching time, and morphology of hatching embryos. We found that the Saprolegnia-like species is a genuine stressor and substantially altered the physiological state of E. bicolor embryos. Among antioxidant defences, superoxide dismutase (SOD) and glutathione S-transferase (GST) activities increased in embryos exposed to the Saprolegnia-like species. However, no difference in lipid peroxidation levels was found between treatments, which might indicate that SOD and GST activations could be enough to prevent oxidative damage. Finally, we found higher mortality and number of malformations in the water mould treatment. We showed the stressful effect of water moulds on amphibian embryos, evidenced by the activation of their antioxidant system, and reveal the importance of considering physiological stress markers as key information when studying the potential consequences of disease outbreaks in the ecology and conservation of amphibians.

Amino Acid Transport in a Water-mould: The Possible Regulatory Roles of Calcium and N6-(Δ2-isopentenyl)adenine

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different transport systems elucidated are: (i) methionine, (ii) cysteine, (iii) proline, (iv) serine–threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine–tyrosine–tryptophan and leucine–isoleucine–valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.Calcium played more than a secondary role in the transport of the amino acids. When bound to the membrane-localized glycopeptide, it permits concentrative transport to take place. However, excess calcium can inhibit transport which can be overcome by chelating with citrate. Calculations show that the concentration of free citrate is most important. At low citrate concentrations (less than 1 mM) in the absence of exogenously supplied calcium, enhancement of amino acid transport occurs. At high concentrations (greater than 5 mM), citrate inhibits but this effect can be reversed by titrating with calcium. Evidently, the glycopeptide acts as a calcium sink to regulate the concentration of calcium made available to the cell for its membrane activities.N6-(Δ2-isopentenyl) adenine (a plant growth 'hormone') and analogues mimic the inhibitory effect of citrate and bind to the glycopeptide as well. Replot data for citrate and N6-(Δ2-isopentyl) adenine inhibition indicate that both agents have no more than one binding constant. These results implicate calcium, glycopeptide, and energy-dependent transport of solutes in some, as yet undefinable, way.