Thermoacclimatory Changes in the Ionic Microenvironment of Haemoglobin in the Stenothermal Rainbow Trout (Salmo Gairdneri) and Eurythermal Carp (Cyprinus Carpio)

Increases in environmental temperature confront teleost fishes with the problem of satisfying elevated oxygen requirements in the fact of diminished oxygen availability. Enhancement of oxygen-carrying capacity through increase in total haemoglobin content does not appear to play either a general or a major role in this, although such changes may be of supplementary importance in some species (Houston, Mearow & Smeda, 1976). More subtle forms of haematological response are, however, possible. The individual components of the typically polymorphic haemoglobin systems of these animals often exhibit significant differences in oxygen affinity and sensitivity to erythrocytic affinity modulators (Iuchi, 1973; Brunori, 1975; Peterson & Poluhowich, 1976; Weber, Lykkeboe & Johansen, 1976; Reischl, 1977). Furthermore, changes in the abundance of specific haemoglobins are a common feature of the thermoacclimatory process (Falkner & Houston, 1966; Grigg, 1969; Houston & Cyr, 1974; Houston et al. 1976; Weber, Wood & Lomholt, 1976), and in some species at least can occur rapidly enough to be of physiological significance (Houston & Rupert, 1977). Thus, adaptively useful modifications in oxygen transport might be achieved by coupling selective adjustments in haemoglobin complement with appropriate alterations in red cell modulator levels.

Evaluation of this hypothesis will, however, require definition of the transport characteristics of specific haemoglobins under physiologically-realistic conditions and, at present, there is little information concerning the effects of acclimation upon the microenvironment in which haemoglobin actually functions. This is particularly true in the case of inorganic modulators of haemoglobin-oxygen affinity. Numerous studies upon vertebrates have suggested or demonstrated that these play a central role in affinity regulation (Bunn, Ransil & Chao, 1971; Wood, Johansen & Weber, 1972; Poluhowich, 1972; de Bruin et al. 1974; Benesch & Benesch, 1974; Brunori, 1975; Rollema et al. 1975; Lykkeboe, Johansen & Maloiy, 1975; Kaloustian & Poluhowich, 1976; Peterson & Poluhowich, 1976; Weber, Lykkeboe & Johansen, 1976). Relatively few studies upon the ionic composition of teleostean erythrocytes have, however, been reported (Munroe & Poluhowich, 1974; Fugelli & Zachariassen 1976; Börjeson, 1976; Calla, 1977). Little information is available with respect to divalent cation levels, and in only two instances (Grigg, 1969; Catlett &: Millich, 1976) have the consequences of thermal acclimation been evaluated.

In the present study we have considered haematological status and red cell electro lyte content in thermally-acclimated rainbow trout (Salmo gairdneri), and carp (Cyprinus carpio); species selected on the basis of major differences in thermal tolerance Black, 1953), metabolic activity (Flörke, Keiz & Wangorsch, 1954; Beamish, 1964) and haemoglobin system complexity (Houston & Cyr, 1974; Houston et al. 1976).

Yearling trout ranging in weight from 30·0–420·0 g (219·0 ± 12·1 g) were obtained from a commercial supplier (Goossens Trout Farm, Otterville, Ontario), and maintained in the laboratory under conditions similar to those described earlier (Houston & McCarty, 1977). All groups were acclimated for at least 4 weeks prior to sampling on a 12 h light: 12 h darkness photoperiod regime to 2, 10 or 18 °C. In view of reported seasonal variations in the haematological and ion-regulatory characteristics of this species (DeWilde & Houston, 1967; Houston et al. 1968; Murphy & Houston, 1977) summer (July-August) and early winter (October-November) populations were sampled. Carp (135·8 ± 26·4 g, 17·3 ± 444·8 g) were seined from a local drainage during June and July, and acclimated under similar conditions to 2, 16 and 30 °C. It was not possible to secure adequate stocks of winter animals.

Specimens were stunned and blood drawn from the caudal vessel into ammonium heparin-treated syringes. Whole blood subsamples were taken for immediate haematological assessment and chilled on ice before use. The remainder of each sample was then centrifuged (5000 g, 5-0 min, 20–23 °C), and plasma drawn off by micropipette. Any plasma remaining in immediate contact with the packed cell column was removed by absorption to filter paper. Plasma and packed cell samples were stored at – 80 °C in individual sealed plastic containers prior to analysis. No significant changes in water or ion content could be detected after storage periods of up to 1 year.

Analyses carried out on packed cells were found to provide more consistent estimates of erythrocytic electrolyte levels than did those performed on whole blood. However, plasma retained in the interstices of cell columns may lead to errors in estimation of cell ion levels. A ‘trapped plasma’ correction factor was therefore calculated by addition of [¹⁴C]PEG-4000 (polyethylene glycol, MW4000) to whole blood samples prior to centrifugation. The value obtained, 2·810-15% of packed cell volume, was independent of haematocrit and mean erythrocytic volume and compared well with that (3%) reported by Catlett & Millich (1976) for goldfish (Carassius auratus), blood using [¹⁴C]inulin. Excellent correspondence was observed in preliminary trials between whole blood analyses of sodium, potassium, calcium, magnesium and chloride, and the sums of plasma and packed cell electrolytes adjusted for haematocrit and ‘trapped plasma’.

Haematological values were estimated using methods previously outlined (DeWiide & Houston, 1967; Houston & DeWilde, 1968) except that the alkaline haematin Ipethod of haemoglobin determination was substituted for the pyridine haemochro-Tnagen technique previously employed.

A Unicam SP-90 AAS was used in the emission mode for sodium and potassium analyses, and in the absorption mode for calcium and magnesium. Chloride deter-, minations were carried out with the Buchler-Cotlove chloridometer. Water content was estimated by dehydration at 103 °C for 24 (plasma) or 72 h (packed cells).

Single classification analysis of variance was used in data evaluations. Value were routinely subjected to base-10 logarithmic or arc-sin transformation, and significance normally attributed to differences at the 0·05 level or better. Differences significant at the 0·10, but not quite at the 0·05 level were sometimes encountered in comparisons of summer and winter trout held at common temperatures. These have been designated as ‘approaching significance’ (→ P < 0·05). In several cases correlation analyses were carried out. In each instance linear (Y = aX + b), power (Y = aX^b), exponential ((Y = ae^bx) and logarithmic (V = a + b In X) curves and coefficients of determination were calculated. Decisions on ‘best fit’ were based upon the relative magnitudes of the correlation coefficients obtained.

The haematological characteristics of rainbow trout were not greatly altered by acclimation (Table 1). Consistent with earlier findings (DeWilde & Houston, 1967) warm-acclimated summer animals had somewhat larger numbers of smaller erythrocytes of slightly reduced haemoglobin content. In the winter groups red cell numbers, haematocrit and haemoglobin levels tended to decline at higher temperatures. while maximum values for mean erythrocytic volume and haemoglobin content occurred at the mid-range temperature (10 °C). No differences were encountered between winter fish at 2 °C and summer trout at 18 °C, i.e. at what might be regarded as ‘normal’ seasonal temperatures. In general, these observations support the contention that adaptive changes at the haematological level cannot be a major factor in resolution of temperature-oxygen demand problems by rainbow trout (Houston et al. 1976).

Over a corresponding temperature range (2–16 °C) carp also displayed little haematological change (Table 1). More obvious variations were, however, seen at 30 °C. Mean erythrocytic volume declined significantly, and there were near-significant increases in erythrocyte abundance and haemoglobin concentration as well. Mean erythrocytic haemoglobin content was again reduced. These observations compare favourably with those of earlier studies on this species (Houston & DeWilde, 1968; Houston et al. 1976). The occurrence of somewhat more pronounced haematological responses to increased temperature in this metabolically sluggish species than is the case in the more active trout appears paradoxical. It should be recalled, however, that the increase in oxygen consumption which takes place in the carp between its lower and upper incipient lethal temperatures (ca. x 6, Beamish, 1964) is proportionally twice that effected by the more stenothermal trout (ca. 3, Flörke et al. 1954).

Plasma electrolyte levels in summer trout were not significantly affected by the acclimatory process (Table 2). In the winter series of fish, on the other hand, significant increases in sodium and potassium concentrations accompanied acclimation to higher temperatures. This was also true of the sum of all plasma electrolytes (an approximation of plasma osmolarity) and the sum of cations. ‘Anion deficit’ (Σ cations)-(Cl⁻) also increased under these conditions, suggesting the likelihood of increases in bicarbonate and/or phosphate. Several seasonal differences were present. Sodium levels in summer fish consistently exceeded those of the winter groups, and notably so in cold-acclimated specimens. The converse was true of chloride, calcium, the sums of all electrolytes and of all cations and ‘anion deficit’. Thus, the observations made in this study compare well with those of earlier reports on rainbow trout (Houston et al. 1968; Murphy & Houston, 1977).

Carp differed from trout in several respects (Table 3). Sodium and chloride levels, for example, were well below those of the trout. Sodium decreased significantly with increasing acclimation temperature, whereas chloride levels were elevated. Potassium was maintained at somewhat higher concentrations than are characteristic of salmonids but did not vary significantly with temperature. By contrast, calcium levels were lower than those of the trout, and rose with increases in temperature. The opposite was true of magnesium. The sum of electrolytes, the sum of cations and ‘anion deficit’ decreased at higher temperatures. Significant differences were encountered in the case of the latter two parameters, and contrast with the situation observed in winter, but not summer rainbow trout. Again, the findings of the present study were in reasonably good agreement with those of earlier reports on carp (Houston, Madden & DeWilde, 1970).

Changes in packed cell electrolyte levels with temperature, corrected for ‘trapped plasma’, are summarized in Table 4. In both summer and winter trout sodium levels declined with rising temperature. Chloride, however, increased and this was true of potassium as well. Calcium and magnesium concentrations in the summer groups tended to decline at higher temperatures, although some evidence of an intermediate (10 °C) maximum was encountered in each case. Among winter animals calcium reached minimum values at 10 °C. Magnesium content, however, increased steadily at higher temperatures. Red cell water content tended to decline in these animals as acclimation temperature increased. In the summer groups, however, water content was highest at 10 °C. Several seasonal differences were apparent. Potassium and magnesium levels in winter fish consistently exceeded those of summer animals. Chloride, and more particularly calcium, concentrations were lower. In general, temperature-related variations in packed cell electrolyte content were qualitatively similar to those seen in plasma. There were, however, some exceptions. For example, in the winter groups of trout plasma sodium was increased at higher temperatures, whereas packed cell sodium declined under these circumstances. Similarly, the 10 °C maximum in plasma calcium content was associated with minimum values for packed cell calcium.

In the carp packed cell sodium increased markedly with temperature, contrasting sharply with changes in the plasma concentration of this ion. Chloride and potassium levels also rose, mirroring in a general way concomittant variations in plasma. Calcium levels were little influenced by temperature, and this was true of water content as well. In the case of calcium this again contrasted with variations in plasma concentration. Finally, magnesium content dropped by about 30% between 16 and 30 °C.

The estimation of red cell ion concentrations has been complicated by uncertainties regarding the proportion of cell water actually available for the solution of electrolytes. Early studies on the aberrant osmotic behaviour of erythrocytes suggested that a substantial fraction of total cell water was bound to haemoglobin (Perutz, 1946; Drabkin, 1950; Savitz, Sidel & Solomon, 1946), and thus might not be available for solvent purposes. Gary-Bobo & Solomon (1968) have, however, provided convicing evidence that there is little, if any, exclusion of sodium, potassium and chloride from haemoglobin-associated water at physiologically-realistic haemoglobin concentrations. Accordingly, cellular ion concentrations were calculated on the assumption that all cell water is available to electrolytes. It is appreciated that this represents an approximation and furthermore, that the values obtained are means for the entire cellular phase and give no indication of intracellular compartmentalization, specific binding or the consequences of non-specific interionic attraction upon activity.

In the rainbow trout red cell only potassium was significantly altered during acclimation; concentrations at 18 °C being some 20% above those of cold-acclimated animals in both the winter and summer series of fish (Fig. 1). Modest increases in chloride, and in magnesium (winter series) were also encountered at higher temperatures. Sodium content tended to decrease under these conditions. Calcium levels were notable for their thermostability. Largely because of changes in potassium content the sums of all electrolytes and all cations, as well as ‘anion deficit’, tended to rise at higher temperatures (Table 5). Well-defined seasonal differences were again seen. Potassium and magnesium concentrations in winter animals exceeded those of summer specimens. Chloride, and particularly calcium values, were substantially less. Differences in the sums of all electrolytes and cations were present at 2 °C, but less obvious in warm-acclimated animals. In a more limited sense this was true of ‘anionic deficit ‘as well.

In the carp a somewhat different pattern of response was observed. Potassium levels were similar to those of trout, but did not change with temperature. Chloride, however, rose sharply. Concentrations at 30 °C were comparable to those seen in the trout, and some 40 m-equiv ⁻¹ in excess of the values encountered in cold-acclimated specimens. Sodium content was far below the levels characteristic of the trout red cell. There was, in fact, little evidence of sodium in the erythrocytes of carp acclimated to 2 °C. Detectable amounts (0·75 – 8-22 m-equiv 1 ⁻¹, cell water) were found in 16 °C specimens, and an approximately five-fold increase took place between 16 and 30 °C. The mean value at 30 °C was, however, only about one-quarter of that characterizing rainbow trout red cells. Magnesium levels at 2 and 16 °C were approximately twice those of trout. Between 16 and 30 °C, however, these dropped by one Third. Calcium concentrations were similar to those in trout, and were apparently unaffected by the thermoacclimatory process.

As in trout, the sums of all cellular electrolytes and of all cations varied directly with temperature (Table 5), and these differences were in some instances both substantial and significant. In contrast to the trout, however, such changes involved chloride and sodium rather than potassium. Furthermore, because of modifications in cell chloride content ‘anion deficit’ dropped significantly at higher temperatures, a situation which differed sharply from that seen in rainbow trout.

Table 6 includes recently reported estimates of plasma and erythrocytic sodium, potassium and chloride in a number of freshwater, or freshwater-adapted species. Although some obvious differences are apparent, the overall pattern of ionic composition is at least qualitatively similar to that of most higher vertebrates. Acclimatory changes in the red cell electrolyte levels of bullhead, Ictalurus nebulosus (Grigg, 1969) and goldfish, Carassius auratus (Catlett & Millich, 1976) were similar to those observed in the present study. In the bullhead, for example, sodium increased by approximately 80% and potassium by almost 50% between 9-10 and 24-25 °C. Corresponding values in goldfish exposed to temperatures between 1 and 21·5 °C were: potassium, + 20%; sodium, +25%; chloride, +14%. Calcium in the bullhead was low (ca. 1 m-equiv I⁻¹) and thermostable (Grigg, 1969). Thus, the overall pattern of thermo-acclimatory modification in red cell ionic composition is reasonably consistent in the four species which have so far been investigated.

Since both mean erythrocytic haemoglobin content and cell ion levels were changed in some groups following acclimation, ion: haemoglobin ratios were calculated (Table 7). Rainbow trout were notable for the constancy with which proportionalities between haemoglobin and chloride, calcium and magnesium were maintained. Only in the case of potassium did well-defined variations occur, the K+:Hb ratio rising significantly with increases in acclimation temperature. A barely significant decrease in Na+:Hb was also observed in winter, but not summer fish between 2 and 10 °C, but it is unlikely that changes of the magnitude observed could have any profound physiological effect. Seasonal variations were again present. Chloride: haemoglobin and Ca^2;Hb were typically higher in summer than in winter fish, with the converse being true of K⁺:Hb and Mg²⁺;Hb.

No significant changes in K⁺:Hb were observed in carp. However, Na⁺:Hb and Cl–:Hb rose sharply at higher temperatures. Magnesium: haemoglobin decreased significantly between 16 and 30 °C. Interestingly Cl⁻¹:Hb and Mg²⁺:Hb values in carp held at 30 °C were comparable to those characteristic of trout. Calcium: haemoglobin ratios were not modified as a consequence of acclimation, and were virtually identical to those of summer trout.

Since oxygen content varies inversely with water temperature exposure to higher temperatures is, in some respects, equivalent to the imposition of hypoxic stress. It has been suggested (e.g. Eaton, 1974) that fishes, unlike air-breathing vertebrates, respond to this by increasing haemoglobin-oxygen affinity and thereby facilitate branchial oxygen loading. Although some evidence supporting this hypothesis can be found in studies by Powers (1974) and others, its generality is open to question. Whole blood oxygen equilibrium curves frequently plateau at tensions well below those ordinarily regarded as hypoxic (Cameron, 1971, 1973, Eddy, 1973; Hayden, Cech & Bridges, 1975). In such cases no obvious advantage accrues from increasing affinity. Any increase under circumstances which also elevate oxygen demand could, in fact, be considered anti-adaptive, since oxygen release at the tissue level would necessarily be impeded. In addition, there is evidence from a variety of studies which suggests that haemoglobin-oxygen affinity is reduced rather than increased (Heath & Hughes, 1973; Wood, Johansen & Weber, 1975; Lykkeboe et al. 1975; Weber, Lykkeboe & Johansen, 1976; Weber, Wood & Lomholt, 1976). The observations of the present study are consistent with the latter view, and suggest that temperature-related changes in the red cell ionic microenvironment would favour decreases in haemoglobin-oxygen affinity at higher temperatures. This type of response would, of course, facilitate oxygen release to cells under circumstances in which their oxygen requirements have been increased. In addition, both species apparently have the ability to reduce mean erythrocytic volume when exposed to increases in environmental temperature. Since rate of oxygen combination with haemoglobin varies inversely with cell volume (Holland, 1970) both species appear to have resolved the problem of enhancing branchial oxygen uptake and tissue oxygen release under conditions which simultaneously increase oxygen demand and reduce oxygen availability.

As noted earlier the modulatory effects of inorganic ions upon haemoglobinoxygen affinity in mammalian systems have been well-documented. In the context of this study, chloride, magnesium and hydrogen ions are of particular interest.

Chloride reduces affinity in much the same fashion as ATP and 2,3-DPG, i.e. through direct interaction with the haemoglobin molecule (de Bruin et al. 1974; Rollema et al. 1975; Laver et al. 1977). Not unexpectedly chloride influence is pH-dependent, and at pH values similar to those postulated for the teleostean red cell (Steen & Turitzin, 1968) increases in chloride prompt near-exponential reductions in affinity. Temperature-related changes in the chloride content of carp erythrocytes fall well within the critical range. Consequently, in this species negative modulation of affinity by chloride ion may have significant impact upon oxygen transport by haemoglobins amenable to this form of modulation. The rainbow trout is also characterized by increases in chloride concentration at higher temperatures. However, these were less pronounced, and their physiological relevance is not as apparent.

Magnesium has little influence upon the oxygen affinity of stripped haemoglobin (Bunn et al. 1971). The modulatory effects of this ion stem primarily from competition with haemoglobin for organophosphate modulators. In the deoxygenated mammalian red cell approximately two-thirds of the ATP present is complexed with magnesium, and essentially unavailable for modulation. By contrast, only about 5% of total 2,3-DPG is in this form (Bunn et al. 1971). ATP is the most abundant organophosphate modulator of haemoglobin-oxygen affinity in many teleosts including the rainbow trout (Weber, Wood & Lomholt, 1976). Its utilization a modulatory role would presumably depend in large measure on the establishment of conditions maximizing its availability.

The observations made in the present study suggest that rainbow trout and card may employ different strategies in this regard. Neither the Mg²⁺:Hb ratios encountered in this study, nor the nucleoside triphosphate (NTP):Hb ratios reported by Weber, Wood & Lomholt (1976) for rainbow trout were significantly altered following acclimation. Magnesium: NTP relationships were presumably also stable, suggesting that effective NTP levels are not influenced by the acclimatory p rocess. However, red magnesium levels are low by comparison with those of muscle and liver in this species (Murphy & Houston, 1977) and similar to values cited for mairmalian erythrocytes. Consequently, trout may be adapted for effective ATP modulation of affinity over much of their thermal tolerance zone. Such an arrangement provides no obvious means for adaptive response to temperature-induced increases in oxygen demand, and it is increasingly apparent that, beyond some amplification of cell numbers and reduction in cell volume, mature trout do not rely heavily upon response at the haematological level (DeWilde & Houston, 1967; Houston & Cyr, 1974; Weber, Wood & Lomholt, 1976). This points to the probable adaptive importance of adjustments in branchial function, and in mechanisms for enhancing extraction efficiency at the tissue level. Observations upon cardiovascular-ventilatory responses to experimentally induced anaemia in this species (Cameron & Davis, 1970), and adjustments in arteriovenous oxygen differentials in trout exposed to temperature increases (Heath & Hughes, 1973) are generally consistent with this supposition.

In contrast to trout, carp reduce red cell magnesium content as temperature and oxygen demand increase. Information upon concomitant changes in erythrocytic organophosphate levels.does not appear to have been published. Decrease in erythrocytic magnesium would nevertheless be expected to enhance effective modulator concentrations and to prompt adaptively useful reductions in haemoglobin-oxygen affinity. It is worth noting in this context that much of the support for the view that teleosts respond to increases in oxygen requirements and/or reductions in oxygen availability by enhancing affinity is based upon observed changes in nucleoside triphosphate: haemoglobin relationships. Powers (1974), for example, has reported that ATP:Hb in catostomid fishes drops from ca. 1·o to ca. 0·6 between 20 and 30 °C. However, if these relatively eurythermal animals also reduce red cell magnesium content to the extent seen in carp the effective ATP:Hb ratio might well change very little with temperature. If this were the case accompanying changes in pH would tend to prompt significant reductions in the oxygen affinity of the cathodal haemoglobins of this species (Powers, 1974).

Reduction in magnesium content may not, however, lead to enchancement of ATP availability. The stability constant for Mg²⁺.ATP, unlike that of Mg·.2,2-DPG, increases with temperature (Bunn et al. 1971). Between 2 and 16–18 °C such increases are of relatively modest magnitude, but more pronounced changes take place at higher temperatures. Thus, export of magnesium may serve only to limit reductions in organophosphate availability, and restrict increases in haemoglobin-oxygen affinity. However, this too might be regarded as an adaptive response.

Given the presumed effects of chloride and magnesium upon affinity, the most obviously utilitarian response of the teleost lies in coupling increases in the former with reductions id the latter. We examined this by means of correlation analysis (Fig. 2). In carp the relationship between magnesium and chloride was biphasic in nature and best-fitted by linear equations through the extremes. There was no evidence of correlation in cold-acclimated specimens (r = 0·072). Among animals held at higher temperatures, however, a highly significant, negative relationship was present (r = –0·796, P < 0·01). In rainbow trout no differences attributable to temperature or season were apparent. Chloride and magnesium values were, however, again characterized by a significant negative correlation (r = 0-316, P < 0·05). It will be apparent that the chloride-magnesium relationship of warm-acclimated carp converges upon that found in the rainbow trout over its entire temperature range.

The basis of this relationship is not apparent from the data obtained in the present investigation. Among several possibilities is the likelihood that such changes are essentially passive in nature and reflect the consequences of temperature-related changes in membrane potential. In the instance of magnesium, at least, this hypothesis is attractive for there is little evidence of active magnesium transport across the red cell membrane (Christensen, 1975). Membrane potential can be approximated by the chloride equilibrium potential (Dalmark, 1976). Highly significant decreases in this were encountered in carp following acclimation to increased temperatures (Table 8). There was also some suggestion of comparable changes in winter trout as well.

In both species red cell magnesium was significantly correlated with chloride equilibrium potential (Fig. 3). In the carp all values could be fitted by a simple logarithmic function. The correlation coefficient obtained (r = 0·725, P < 0·01) was not statistically distinguishable from that associated with a linear relationship for values from warm-acclimated animals only (r = 0·734, P < 0·01). In the case of Ipinbow trout, best fit was obtained with a linear function, and the correlation coefficient relating the two variables was again significant (r = 0·504, P < 0·01).

Thus, these relationships provide some support, particularly in the case of carp, for the suggestion that magnesium content varies in relation to the red cell membrane potential.

Although the basis of the latter changes is not clear at present, it is tempting to speculate that variations in potential may be related to temperature-induced modifications in acid-base status. The inverse relationship between plasma pH and temperature in fishes has been well described (Reeves, 1977). Malan, Wilson & Reeves (1976) have provided evidence that cellular pH in poikilotherms is also reduced as temperature increases. Changes in ionic composition (see below) suggest that this takes place in the teleostean red cell as well. Buffering capacity within the erythrocyte greatly exceeds that of plasma, and masking of negative charges within the cell by hydrogen ion could lead to some reduction of membrane potential. In the mammalian system decreases in pH are accompanied by increases in erythrocytic chloride content (Dalmark, 1976), and there is some evidence that this also occurs in fish (Börjeson 1977). If such is, in fact, the case postulated adaptive reductions in haemoglobin-[oxygen affinity by adjustment in chloride and magnesium levels could be construed as a form of passive response. Moreover, since mammalian red cell chloride is altered within minutes after temperature and pH change (Dalmark, 1976) adaptation would be essentially instantaneous (Hazel & Prosser, 1974). Earlier studies have indicated that haemoglobin system reorganization in at least the goldfish also occurs rapidly following temperature change (Houston & Rupert, 1976). Thus, there is some likelihood that adjustments in both haemoglobin type and modulating conditions can take place quickly enough to have physiological significance.

Increases in calcium prompt small increases in the P₅₀ value for stripped haemoglobin. In the presence of either 2,3-DPG or ATP, however, calcium action mimics that of magnesium (Bunn et al. 1971). The absence of any change in red cell calcium content or Ca²⁺: Hb ratio suggests, however, that carp and rainbow trout do not exploit the modulatory potential of this ion in relation to oxygen transport. The actions of potassium and sodium are less certain. Nevertheless, studies by Rossi-Fanelli, Antonini & Caputo (1961) indicate that at equimolar concentrations KC1 reduces affinity more than does NaCl, and this has been confirmed by Bunn et al. (1971). Thus, the increases in red cell potassium observed in the trout may be of some adaptive significance.

In both species acclimation to increased temperature was accompanied by erythrocytic accumulation of univalent ions; potassium in the case of rainbow trout, sodium and chloride by carp. These are the most abundant of the red cell electrolytes, and changes in their concentrations account for most of the increase in the sums of all electrolytes encountered at higher temperatures. It was of interest that the ratio, Σ plasma electrolytes: Σ cellular electrolytes, decreased with increasing temperature, for this indicates that concentration changes within the cell exceed those of plasma. In the summer group of rainbow trout, for example, ratios at 2, 10 and 18 °C were 1·157, 1·091 and 1·052, respectively. Corresponding values in winter fish were 1·077,1·073 and 1·060. More marked changes took place in carp: 2 °C, 1·345; 16 °C, 1·220; 30 °C, 1·075. This suggests that the osmolarity of the red cell rises under warm -conditions and, given that cell and plasma are in osmotic equilibrium, increases in mean erythrocytic volume would be expected at higher temperatures. Such increases would, however, be anti-adaptive since rate of oxygen combination with haemoglobin is a negative function of cell volume (Holland, 1970). Consequently, oxygen loading at the gill would be impeded. In fact, some greater or lesser reduction in mean erythrocytic volume is one of the more consistent features of haematological response to increased temperature in both species (present study; DeWilde & Houston, 1967; Houston & DeWilde, 1968).

Considerable emphasis has been given to the role of electrolytes in cell volume regulation (MacKnight & Leaf, 1977). Accordingly, relationships between mean erythrocytic volume and plasma and cellular ion levels were examined by correlation analysis. Several significant correlations were obtained (cellular chloride, Σ cell electrolytes, S cell cations). No obvious differences attributable to temperature or season were apparent. Fig. 4 A depicts the relationship between mean erythrocytic volume and Σ cellular cations. In this case, as well as in all others in which significant correlations were obtained the relationship was negative; reductions in cell volume were associated with increases in cell electrolyte levels.

This apparent paradox points to the likelihood that carp and trout regulate red cell volume by means comparable to those used by flounder, Pleuronectes flesus, following exposure to salinity change, i.e. adjustment in cellular levels of organic solutes (Fugelli, 1967; Fugelli & Zachariassen, 1976). Some idea of the magnitude of the changes involved can be obtained if it is assumed that, (1) red cell and plasma are in osmotic equilibrium and (2) that the difference value, Σ plasma electrolytes mmol-Σ cell electrolytes mmol, is indicative of osmotically active cellular solute. It is appreciated that difference values obtained in this way are at best approximations. In addition, they give no indication of whether the solute(s) involved is inorganic (e.g. HCO₃⁻,P_i or organic in nature. These limitations notwithstanding, if carp and trout regulate cell volume in this way difference values should decrease as temperature increases. In addition, a positive relationship between mean erythrocytic volume and difference would be anticipated.

Both expectations are largely met. Mean difference values at 2, 10 and 18 °C were 46·8 ±3·93, 26·313·97 and 19·013·44 mmol in summer trout, and 25·012·70, 19·613·28 and 18·914·23 mmol in the winter series of animals. Much the same was true of carp: 2 °C, 67·71 3·85, 16 °C, 48·714·62; 30 °C, 18-714-86 mmol. In both species mean erythrocytic volume was positively, and significantly correlated with Σ plasma electrolytes (mmol)-Σ cellular electrolytes (mmol) (Fig. 4B). These observations support the view that both species may have the ability to enhance red cell electrolyte levels in a manner appropriate for reduction of haemoglobin-oxygen affinity, while simultaneously reducing mean erythrocytic volume through export of some as yet unidentified solute and thereby facilitating branchial oxygen loading.

The differences seen between the carp and trout in terms of red cell ionic compo-Bsition are of interest in relation to the relative complexities of their haemoglobin Systems and the extent to which these are altered during acclimation. Those of salmonids are among the most complex which have been described in teleosts (Riggs. 1970). The rainbow trout, for example, possesses nine electrophoretically distinguishable components (Houston & Cyr, 1974; Braman et al. 1977). Because these animals are also among the most metabolically active of fishes, it has been suggested that complexity may be related to oxygen demand (Riggs, 1970). However, the bullhead and sucker, Catostomus commersoni, which have complex systems of seven and eight haemoglobins respectively (Grigg, 1969; Houston et al. 1976) are not noted for unusually high oxygen requirements. Moreover, it is difficult to reconcile this suggestion with the inverse relationship which exists between haemoglobin system complexity and thermal tolerance (Houston et al. 1976), since eurythermal species must frequently satisfy proportionally larger increases in oxygen demand over their more extended thermal tolerance zones than is the case with the relatively stenothermal salmonids. In addition, species such as the rainbow trout, bullhead and sucker do not exhibit very marked changes in the abundance of specific haemoglobins, or even groups of haemoglobins (Grigg, 1969; Houston & Cyr, 1974; Houston et al. 1976; Weber, Wood & Lomholt, 1976). Accordingly, it is difficult to see how any major adaptive advantage could be gained by adjusting the erythrocytic milieu in relation to relatively minor changes in the amounts of particular haemoglobins. The more appropriate strategy in this instance presumably lies in maintenance oí: a high chloride-low magnesium ionic microenvironment conducive to reduced haemo-globin-oxygen affinity over the entire range of tolerated temperatures.

In contrast to the Salmonidae, goldfish and carp are among the most eurythermal of freshwater teleosts. Each possesses a relatively simple two-or three-cor.iponent haemoglobin system and exhibits substantial changes in fractional abundance following acclimation (Houston & Cyr, 1974; Houston et al. 1976; Houston & Rupert, 1977). The coupling of significant changes in haemoglobin type with variations in erythrocytic composition of the kinds observed in the carp would, a priori, seem to offer more potential for significant adjustment in transport function than is the case in the trout. This, of course, assumes that the haemoglobins predominant at higher temperatures possess characteristics which can be adaptively exploited and, as yet, this has not been conclusively demonstrated. In any event, from the viewpoint of temperature-related variation in oxygen demand, adaptive advantage may be associated with a restricted form of polymorphism which allows for significant modification in the abundance of particular haemoglobins. If this is the case the complexities of the salmonid haemoglobin system may reflect a form of neutral rather than adaptive polymorphism.