Hypoxia tolerance of New Zealand triplefin fish brains (Tripterygiidae): an enzymatic and metabolomic investigation

Abstract

The human brain is extremely sensitive to lack of oxygen failing within minutes of hypoxia. Hypoxia promotes a shift from aerobic to anaerobic metabolism which causes lactate accumulation, redox imbalance, glutamate accumulation leading to excitotoxicity, and altered calcium homeostasis which all lead to potential cell death. In comparison, aquatic animals are effective at routinely surviving levels of less than 3% atmospheric oxygen. Triplefins, especially intertidal rock pool species, repeatedly undergo periods of hypoxia or anoxia whilst still maintaining brain function. Therefore, investigations into mechanisms enhancing survival of the aquatic brain may be critical for understanding how humans could increase tolerance to cope with the vast array of conditions under which hypoxia arises. Five New Zealand triplefin species ranging from intertidal to subtidal zones were compared each with different hypoxia tolerances. Two key mechanisms thought to be impacting hypoxia tolerance were investigated. Firstly, the activity of key enzymes was assessed to determine their contribution to overall metabolism. The second mechanism to compliment this is analysis of abundance in metabolites between species subjected to a ~35 minute hypoxic event. Enzymatic activity was determined spectrophotometrically, whilst Gas Chromatography-Mass spectrometry (GC-MS) was used to determine concentrations of key metabolites (sugars, organic acids, fatty acids, lipids and proteins). Enzyme analysis showed significant differences in Adenylate kinase, Lactate dehydrogenase, Citrate Synthase, Glutamate dehydrogenase, Malate dehydrogenase and Pyruvate kinase (p≤0.05). Lactate dehydrogenase (LDH) activity was lower in hypoxia tolerant species than hypoxia sensitive species (420±27 & 520±20 umol/min/g respectively) with Pyruvate kinase (PK) following a similar trend. Additionally, hypoxia tolerant species had ~ 22% greater Creatine kinase (CK) activity compared to their relatives. Results from GC-MS show that hypoxia influences metabolite abundance. Citric acid cycle and glycolysis intermediates along with antioxidants, neurotransmitters and fatty acids showed variations between normoxia and hypoxia that are indicative of hypoxia tolerance. Additionally analysis of both enzyme activity and metabolite abundance showed strong rank correlations against Pcrit. Overall these results show hypoxia tolerant species have reduced metabolism under hypoxia and an ability to maintain energy stores in order to sustain extreme hypoxic events.