Grigg GC

1. Fishes in Antarctic seas live at a temperature lower than that at which most other poikilotherms remain active. 2. Hematocrit, absolute oxygen capacity and oxygen equilibrium curves were determined for whole blood from each of four related species of Antarctic fishes. These parameters were related to the habits and activity of each species. 3. Temperature increase was found to have a marked effect on the affinity of the blood for oxygen. This effect was compared with data from other fishes and found to be extreme in the Antarctic species. 4. These fishes are known to be stenothermal and geographically restricted in distribution. It seems likely that the sensitivity of their oxygen transport systems to temperature increase could provide at least some of the physiological basis for this. INTRODUCTION The Low temperature of the Antarctic seas imposes on animals the necessity for certain adaptations, particularly in their metabolism and resistance to freezing. Some aspects of metabolism of nototheniid fishes have been studied, cf. Wohlschlag (1964). His work indicates that the Antarctic fishes are very stenothermal and that their metabolic rates are higher than would be expected at such low temperatures. He therefore considers them to be “cold adapted”. In his various papers Wohlschlag has commented extensively on the nature of the environment in McMurdo Sound where the fishes in the present study were caught. Physicochemical conditions are more stable than in lower latitudes and data from Littlepage (1965) illustrate this. The mean water temperature is about –1.9C, with a seasonal range from –1.4 to –2.0°C. Oxygen content is near saturation (about 8.0 ml/l at this temperature) and salinity is constant within narrow limits. For most of the year McMurdo Sound lies under an ice cover several feet in thickness and this results in reduced light penetration in summer when the sun is above the horizon. Antarctic fishes live at temperatures lower than those at which most other poikilotherms remain active. In general, low temperatures increase the oxygen affinity of hemoglobin, an effect first investigated in fishes by Krogh & Leitch (1919). Subsequent studies on fishes by Kawamoto (1929), Dill et al. (1932) and Irving et al. (1941) indicate that the hemoglobin characteristic of the species is functional in the temperature range for that species. However, no studies appear to have been done at Antarctic temperatures, and since some Antarctic fishes have no respiratory pigment (Ruud, 1954, 1959), one object of the present study was to see if nototheniid hemoglobin is functional. The oxygen-hemoglobin equilibrium was investigated at environmental temperatures, and again at higher temperatures for comparison. General comparisons between respiratory characteristics of blood and modes of life in different fishes have been made by many authors, notably Willmer (1934), Black (1940), Irving et al. (1941), Fish (1956) and Dubale (1959). In most cases these comparisons were made between fishes from different taxonomic groups, different environments, and having different habits. Apart from work on Salmonids, this is the first study of this nature to be made comparing several closely related fishes. The four species of Trematomus in the present study live under very similar environmental conditions and an attempt was made to relate differences in blood respiratory properties to different habits. MATERIALS AND METHODS The fishes used in this study were caught in 75-350 ft of water in McMurdo Sound (77° 51' S., 166° 38'E.), from October through December 1966. At this season the water temperature is about –1.8°C. Trematomus bernacchii (50-440g), T. centronotus (116210 g) and T. hansoni (90-200 g) were trapped on the bottom using methods described by Wohlschlag (1964). Specimens of T. borchgrevinki (40-112 g) were caught by hand line through holes in the ice. Specimens were identified from descriptions by Norman (1938). After collection, the fish were removed to the McMurdo Biological Laboratory where they were maintained in thermally controlled tanks until required. Blood was removed from the common cardinal vein of smaller fish and by cannulation of the caudal artery in larger specimens. In several cases, a large nematode living in the dorsal aorta of T. bernacchii made this latter procedure difficult by obstructing the cannula. Solid heparin was used to prevent clotting. Attempts to store the chilled blood were unsuccessful because the plasma became opaque after half a day or so. Hence, all observations were made on freshly drawn blood. Duplicate samples for cell volume determination were collected in heparinized capillary tubes and centrifuged in a standard microhematocrit centrifuge. Hematocrit determinations did not agree with those made on the same species by Kooyman (1963). However, he gave no indication of the type of centrifugation used and it was found early in the present study that the ordinary laboratory centrifuges were unsatisfactory. Measurements of the oxygen content of blood were made by the method described by Roughton & Scholander (1943) incorporating the modification for fish blood by Scholander & van Dam (1956). Further procedural modifications were made following the advice of Mr. Everett Douglas (personal communication, 1966). Oxygen capacity determinations were made on blood equilibrated to air in a temperature-controlled tonometer similar to that described by Finley et al. (1960). The water bath was held at –1.5°C for these determinations. Duplicate analyses usually agreed within 0.1 vol. per cent. If disagreement was more than 0.2 vol. per cent the readings were disregarded. Oxygen equilibrium curves were obtained by the technique described by Lenfant & Johansen (1965) after Haab et al. (1960). Whole blood was used, and sometimes pooling from four to five fish was necessary, but this was kept to a minimum. The well-mixed sample was halved and one half equilibrated to air, the other half to helium in the tonometer at experimental temperature. This provided a sample of fully oxygenated and a sample of fully deoxygenated blood. Oxygen content in these samples was determined by the method described above. By mixing subsamples from each of these in different proportions, a series of mixtures was obtained over a range of known oxygen contents. The oxygen tensions (PO2) of these mixtures was then measured by a Beckman Spinco Gas Analyser and a Beckman Macroelectrode. The electrode was calibrated at the temperature of the tonometer bath. Duplicate or triplicate measurements on the same sample were within 2 mm in the upper range of the 0-160 scale and within 1 mm over the 0-60 scale. Precautions were taken during the whole operation to keep the syringes and blood chilled at all times. All measurements were converted to standard temperature and pressure. RESULTS General respiratory properties of the blood That the species of Trematomus in this study have a functional oxygen transporting pigment is beyond question. The dark venous blood was in sharp contrast to the brightly colored arterial blood. The oxygen affinities were seen to be well within the range of those from fishes in lower latitudes. Blood oxygen capacities and cell volumes are displayed in Table 1. It was found that a straight line relates these parameters in each species. From these items and a knowledge of the oxygen capacity of the plasma (0.8 vol. per cent), the oxygen capacity of 100 ml of cells was calculated and this is included in Table l. This allows comparison of the carrying capacity of the red cells themselves. These cell capacities are similar in the four species examined, but by comparison with data tabulated by Redfield (1933) and Prosser & Brown (1961) it is seen that these capacities are lower than those for fish in lower latitudes. This is in agreement with Kooyman's (1963) data for T. borchgrevinki, T. bernacchii and T. centronotus, in which the hemoglobin concentrations were lower than in temperate or tropical fishes, but similar to Arctic and some other Antarctic forms. The percentage of oxygen dissolved in the plasma at air saturation is also shown in Table 1. Because of the high oxygen solubility at low temperatures, the transport of oxygen by solution in the plasma cannot be ignored, and this may partly explain the observed lower hemoglobin concentrations in fishes from high latitudes. TABLE 1-HEMATOCRIT, OXYGEN CAPACITY (OF CELLS AND WHOLE BLOOD) AND PERCENTAGE OF OXYGEN DISSOLVED IN THE PLASMA FOR FOUR SPECIES OF Trematomus AT –1.5oC Volumes per cent oxygen capacity Species Hematocrit Blood Cells Plasma per cent contribution to absolute oxygen capacity T. bernacchii 20.5%(14) [17-261 5.3 (10) 4.5-6.3 22.7 12.1 T. centronotus 22.0%(7) [17-24] 5.2(7) 4.2-5.5 21.0 12.4 T. hansoni 31.0% (3) [29-331 7.7(3) 7.1-8.1 22.9 7.3 T. borchgrevinki 30.0% (13) [26-32] 6.6(11) 6.1-7.0 20.3 8.5 Mean figures are given with the number of determinations in parentheses and the total range of determinations in