Metabolic and biochemical consequences of niche specialization of Antarctic fish - heat stressed proteins, metabolite levels, nucleotide ratios, neurotransmitters, muscle buffering capacity, blood erythrocytes

Antarctic notothenioid fishes have radiated to fill a range of ecological niches, forming an assemblage of over a hundred closely related species. Some groups have developed extreme specializations while others remain generalized. The metabolic and biochemical consequences of niche specialization in notothenioids from McMurdo Sound was investigated and comparisons were made between specialists and generalists both between and within families.

Several aspects were investigated:

1) Characterization of the stress related compounds such as catecholamines, cortisol, ubiquitin and other heat-stress proteins in fish - Groups of five Pagothenia borchgrevinki were subjected to stress in the form of confinement and elevated temperature for time intervals of 0-48 hours. Fish were killed at each time interval and samples of blood and other tissues were analysed for stress induced concentrations.

2) Quantify metabolite levels and nucleotide ratios in muscle tissue and erythrocytes of common notothenioid species (Trematomus bernacchii (bottom dwelling) and P. borchgrevinki (semi-pelagic)) - Analysis of phosphorylated nucleotides (which reflect the energy status of the cell) was measured from muscle samples of resting and exercised fish. Samples of haemoglobin were collected for investigation of multiple haemoglobins. Heart, gills, muscle and liver samples collected to study lactate dehydrogenase isoenzymes and packed red blood cells for study of the Root effect. For a combined study of levels of adrenaline, related transmitter compounds and stress related steroid hormones were kept at +5°C. Muscle and other tissues were stored for biochemical analysis.

3) Record the effects of temperature, acetylcholine and adrenaline on cardiac rate in Antarctic fishes - In vitro effects of temperature and neurotransmitters (acetylcholine and adrenalin) on rhythmic electrical activity (contraction rate) of atria isolation from the hearts of P. borchgrevinki, T. bernacchii and the icefish Chionodraco hamatus, were recorded.

4) Measure the buffering capacity of muscle and other tissues in active and inactive fish ecotypes - a series of measurements of the capabilities of different tissues to resist changes in acidity (pH0 which would occur under conditions of heavy exercise.

5) Test the hypothesis that changes in enzyme activity are responsible for the apparent low activation energy reported for synaptic currents in P. borchgrevinki by determining the effect of temperature on the enzyme kinetics of acetylcholinesterase (an enzyme catalysing the breakdown of acetylcholine) - Fish brain tissue was homogenized and pH change was measured following the addition of acetylcholine. A total of 169 reaction curves were recorded in varying substrate concentration, temperature and pH.

6) Assess the degree of cold-adaptation in Antarctic fish muscle - Glycerine extracted muscle fibres of T. bernacchii and P. borchgrevinki were measured for force and velocity of contraction in single muscle cells.

7) To characterise the modulation of haemoglobin oxygen binding in intact living erythrocytes and to investigate the properties of intact living erythrocytes - A number of Antarctic fish species were collected from Antarctica and shipped to New Zealand and stored in the aquarium at Kelly Tarlton's Underwater World, Auckland, on display to the public and available for non-lethal research projects. Blood samples were taken from T. bernacchii for determination of concentration of blood trinucleotides (ATP & GTP). Blood was also taken from T. bernacchii and P. borchgrevinki and incubated with adrenaline to test the role of adrenaline in red cell swelling.