Characterization of oxygen-dependent processes in the anoxia-tolerant crucian carp

Abstract

Most vertebrates die within minutes when exposed to anoxia (no oxygen). However, a few exceptions exist, and a champion among these is the crucian carp, which at low temperatures can survive several months in an active state in the complete absence of oxygen. This fish survives anoxia by combining metabolic depression with up-regulating glycolytic ATP production, and by converting the lactate formed in this process into ethanol, allowing it to avoid acidosis. In this way it can survive as the only vertebrate in small ponds that get covered by ice and snow in the winter, blocking oxygen diffusion from air, and light for photosynthesis. Its extreme anoxia tolerance has made this fish a well suited model for investigating adaptation to anoxia. Anoxia related diseases are major causes of death in the industrialized world, and this fish may provide us with insight into mechanisms that can effectively counteract the damage caused by anoxia and reoxygenation of tissues.

However, while the mechanisms responsible for maintaining ATP levels in anoxia have been well studied in crucian carp, few studies have looked at how it tackles the numerous other processes that need oxygen. Oxygen-dependent processes in vertebrates include nitric oxide synthesis, monoamine neurotransmitter synthesis by tryptophan and tyrosine hydroxylases and the synthesis of DNA bases by ribonucleotide reductase. Can the crucian carp do without these substances in anoxia or have it found ways around the oxygen dependence of these systems? In this thesis I have investigated the function of these systems using different experimental approaches. First, the systems were investigated on the genetic level by cloning the responsible genes from mRNA and comparing them to other vertebrates. Second, their expression was estimated by measuring their mRNA levels in hypoxia and anoxia. Finally, these systems were investigated on the protein level by looking for adaptations in the function of some of the proteins involved, and by studying how metabolite levels may be adjusted to accommodate the oxygen dependence of these processes.

The results indicate an array of adaptations in crucian carp, from storing nitric oxide in the form of nitrite at very high levels, particularly in heart tissue, to adjusting the stability of a radical involved in DNA synthesis. It also disclosed an apparent lack of adaptive change in the enzyme synthetizing serotonin, suggesting that the crucian carp needs to economize with this neurotransmitter until oxygen returns. Apparently as a consequence of a recent genome duplication, numerous previously undiscovered variants of genes involved in oxygen dependent processes were discovered. Of the genes studied, I did not find any dramatic deviations from previously known versions of these genes, but it is clear that these extra gene variants gives evolution additional material to work on for providing new functions, and an increased capacity to adapt to such a serious challenge as anoxia.