The nature of hematological response in fish : Studies on rainbow troutOncorhynchus mykiss exposed to simulated winter, spring and summer conditions.

Hematological status was examined in rainbow trout,Oncorhynchus mykiss, held for 3-4 weeks under temperature, photoperiod and PO 2 conditions approximating those of their winter, spring and summer habitats. The most striking change observed was in red cell population composition. In 'winter' fish mature cells were predominant; juvenile and developing erythrocytes characterized 'spring' and 'summer' animals. Hemoglobin, hematocrit and both mean erythrocytic volume and hemoglobin were modestly lower in 'spring' and 'summer' than in 'winter' fish. Red cell numbers were not significantly affected. These observations suggest that avoidance of viscosity-based increases in circulatory work cost is more advantageous than elevation of blood O2-carrying capacity. Although hemoglobin isomorph profiles were significantly altered, there is little evidence that such changes are of critical adaptive importance. Given presumed age-based reduction in gas transport effectiveness, the replacement of mature and senescent cells by more metabolically-competent juvenile cells appears to be the pivotal event in hematological response. Leucocyte counts were significantly elevated in 'spring' and 'summer' as compared to 'winter' fish. Lymphocyte/heterophil ratios declined from 8.27 in 'winter' fish to 3.13 in 'summer' trout. Thrombocyte, monocyte, eosinophil and basophil abundances were little changed.

Hematological response in fish: pronephric and splenic involvements in the goldfish,Carassius auratus L.

Pronephric and splenic involvements in erythropoiesis and in stress-induced hematological response by goldfish,Carassius auratus, were examined under conditions of minimal stress and following transient (3h) temperature-induced elevation of O2 demand and transient (3h) exposure to hypoxia. Although hemoglobin content and red cell numbers were little affected, the ontogenic composition of the circulating erythrocyte population was significantly altered by both types of respiratory challenge and also by the stresses associated with capture, air exposure during transfer and intraperitoneal administration of label. Juvenile cell numbers increased sharply while mature erythrocyte abundances declined. Consistent with this, [(3)H] methylthymidine-labeled cell numbers rose in both spleen and pronephros following imposition of stress. Splenic erythropoietic activity was modest relative to that of the pronephros. Our observations point to a readily triggered response involving: [1] release of cells reservoired in the spleen and pronephros, [2] increased erythropoiesis and [3] karyorrhexis. We suggest that while the magnitude of response may depend on the quality and magnitude of the imposed stress, its central features are essentially constant.

Variable versus constant temperature acclimation regimes: Effects on hemoglobin isomorph profile in goldfish,Carassius auratus.

Goldfish,Carassius auratus, were acclimated for 2 to 3.5 weeks to three temperature regimes: [1] temporally-constant (10, 20 and 30°C), [2] diurnally-cycling (20 ± 10°C) and [3] randomly-fluctuating (± 2°C at approximately 2h intervals between extremes of 10 and 30°C). No significant differences in hematocrit were evident. Hemoglobin levels in fish at constant 30°C and under randomly fluctuating temperature were significantly elevated. Of the three hemoglobin isomorphs observed, the two minor components (G1, G3) tended to decrease in relative abundance with increase in constant temperature, but increased under varying temperature regimes. The converse was true of the principal hemoglobin, G2. Extent of isomorph variation was correlated with extent of temperature variability. These observations confirm that temperature variability significantly effects thermoacclimatory response. The functional significance of changes in isomorph abundances during the acclimatory process is considered.

Maturation of the goldfish (Carassius auratus) erythrocyte.

1. Cohorts of [3H]thymidine-labelled erythrocytes were examined over a 42-day period in goldfish (Carassius auratus L.) recovering from phenylhydrazine HCl-induced anemia under normoxic conditions at 20 +/- 1 degree C and maintained with minimal disturbance on a high nutritional plane. 2. As judged by changes in primary and derived hematological variables, maturation required 16-20 days. 3. Similar estimates were obtained using cytomorphic variables obtained by image analysing methods. 4. These suggest that juvenile red cells in this species can be identified on the basis of the following characteristics: major axis less than 9.2 microM; one-sided surface area not greater than approximately 50 microns2; axis ratio greater than 0.774; form factor greater than 0.938. 5. Corresponding values for mature cells are: major axis greater than 11.2 microns; area greater than 68.5 microns2; axis ratio less than 0.716; form factor less than 0.912. 6. These criteria, with values for dividing and karyorrhectic cell numbers, offer a basis for more detailed and dynamic characterization of the erythron during response to environmental variation than has previously been possible.

Insulin affects ionic composition of rainbow trout erythrocytes.

As is the case with the anucleate mammalian erythrocyte, ionic composition in anucleate red cells of rainbow trout, Salmo gairdneri, is insulin-sensitive. By comparison with erythrocytes cultured in insulin-free medium, those exposed to insulin concentrations of 20, 200 and 2000 microU/ml for 2 and 6 h exhibited dose-dependent increases in potassium and water content coupled with reductions in the levels of sodium, magnesium and chloride. These observations suggest that the membrane of this type of erythrocyte possesses insulin receptors.

Environmentally-related changes in red cell levels of ionic modulators of hemoglobin-O2 affinity in rainbow trout, Salmo gairdneri.

Hemoglobin content, plasma and red cell levels of chloride and magnesium and molar ion:hemoglobin ratios were examined in trout acclimatized to eight combinations of two treatment levels of temperature (5, 20 degrees C), O2 availability (less than or equal to 30%, greater than or equal to 75% saturation) and photoperiod (16L:8D, 8L:16D). Increases in hemoglobin content were associated with exposure to higher temperature, abbreviated daylength and hypoxia, with hypoxia greater than photoperiod greater than temperature. Under nominal "summer" conditions (20 degrees C, hypoxia, 16L:8D) photoperiodic influence was apparently masked by hypoxic and thermal effects. Temperature was the principal determinant of plasma and cellular chloride levels as well as [Cl:Hb]. O2 availability and photoperiod had little effect. Temperature was also the primary factor influencing magnesium, with hypoxia exerting a lesser influence. Photoperiod effects were negligible. With increased temperature and reduced O2 availability, plasma magnesium increased white cell magnesium levels and [Mg:Hb] declined. These observations suggest that with normal seasonal changes in environmental conditions, temperature-induced increases in the O2 requirements of summer trout are probably accompanied by increases in blood O2-carrying capacity and reductions in hemoglobin-O2 affinity with consequent increases in O2 delivery to tissues.

Rainbow trout red cells in vitro.

Washed rainbow trout erythrocytes incubated at 14 degrees C in Eagle's minimal essential medium and Cortland saline displayed sharp reductions in volume and water content, nucleoside triphosphate, K+ and Cl- concentrations. Mg2+ and, to a lesser extent, Na+ concentrations increased. Cellular to medium Cl- ratios were indicative of membrane hyperpolarization. Morphological irregularities were also observed. Oxygen consumption and hemoglobin system organization were not grossly affected. Supplementation with pyruvate stabilized nucleoside triphosphate concentrations for at least 24 hr, and reduced rates of volume and compositional change to some extent. Addition of norepinephrine at physiologically realistic levels led to stabilization of Cl- content and reductions in Mg2+ accumulation and water loss. Transient but modest increases in K+ and Ca2+ were coupled, under these circumstances, with some decrease in Na+ concentration. Factors which may contribute to the dysfunctional status of these cells in vitro are discussed.

Erythrocytic magnesium in freshwater fishes.

Erythrocytic magnesium in rainbow and speckled trout (Salmo gairdneri, Salvelinus fontinalis) carp (Cyprinus carpio) and goldfish (Carassius auratus) was examined by correlation analyses involving weight, hemoglobin content, plasma magnesium, erythrocytic sodium, potassium, calcium, chloride and unidentified anion concentration and chloride equilibrium potential, and in relation to possible diurnal and seasonal variation as well as with respect to constant, diurnally cycling and progressively increasing temperature conditions. Given that magnesium is not actively transported, cellular concentrations appear to reflect processes influencing membrane potential and may also be related to maintenance of cellular electroneutrality. No evidence of significant diurnal variation was encountered under either constant or diurnally cycling temperature conditions. Variations in relation to temperature, season and weight were, however, consistent with the suggestion that these animals have the ability to alter erythrocytic magnesium concentrations. It is hypothesized that by adjusting magnesium in relation to organophosphate, the availability of the latter to hemoglobin may be influenced and in this way contribute to adaptive regulation of hemoglobin-oxygen affinity.

Plasma and red cell ionic composition in rainbow trout exposed to progressive temperature increases.

Yearling rainbow trout, Salmo gairdneri Richardson, were exposed to progressive increases in temperature from 10 to 26.1 degrees C, and variations in haemoglobin, haematocrit and plasma and erythrocytic concentrations of Na+, K+, Ca2+, Mg2+ and Cl- were compared with those of animals maintained at 10 degrees C. Despite the effects which increases in temperature are known to have on branchial ventilation, perfusion and effective exchange area, and consequently upon passive water and electrolyte fluxes, plasma ion concentrations were little affected at other than acutely stressful temperatures. Presumably this reflects the consequences of previously demonstrated changes in branchial, renal and erythrocytic (Na+/K+)- and (HCO3-)-activated ATPase and carbonic anhydrase activities. Haemoglobin and haematocrit were also little influenced by increases in temperature between 10 degrees C and about 25 degrees C. It is unclear whether the decreases seen at higher temperatures reflected accelerated ageing and breakdown of circulating red cells or were a consequence of changes in tissue water content and distribution with resulting haemodilution. Red cell levels of Cl- and K+ increased more or less steadily at temperatures exceeding 16-18 degrees C. So also did [Cl-]: [Hb] and [K+]: [Hb]. Sodium and [Na+]: [Hb] were essentially constant up to about 25 degrees C but thereafter increased sharply. Much the same was true of Ca2+. However, red cell Ca2+ concentrations were normally less than 0.15 mmol l-1 cell water, and may be physiologically insignificant in the context of the present study. A more complex pattern of change was encountered in the case of Mg2+. Concentrations of this ion and the [Mg2+]: [Hb] ratio declined between 10 degrees C and 20-22 degrees C and thereafter increased. A significant positive correlation existed between red cell levels of Cl- and K+; significant negative correlations between Cl- and Mg2+ and K+ and Na+. Negative, but insignificant correlations were also seen between Ca2+ and both Cl- and K+. Given the known direct and indirect effects of inorganic ions upon haemoglobin-oxygen affinity, the reductions in intraerythrocytic pH which accompany increases in temperature and the effects of temperature per se on affinity, the compositional changes observed in this study would be expected to prompt reductions in haemoglobin-oxygen affinity and increases in P50 values. Previous studies have, however, revealed little thermo-acclimatory variation in the P50 of this species. Thus, some as yet unidentified factor or factors may operate in opposition to these influences.

Water-electrolyte balance in goldfish Carassius auratus, under constant and diurnally cycling temperature conditions.

1. The effects of acclimation to constant and diurnally cycling temperatures upon water-electrolyte regulation were examined in goldfish held at 20, 25, 30 and 25 +/- 5 degrees C, and sampled at 03.00, 09.00 15.00 and 21.00 h. Plasma and epaxial muscle levels of Na, K, Ca, Mg, Cl and water were determined. Using Cl space as an indicator of extracellular phase volume, mean cellular cation concentrations were estimated. 2. Fish held at constant temperature exhibited significant diurnal variations in all ions except plasma magnesium and muscle potassium. With the exception of muscle choride, however, the occurrence of peak and/or minimum concentrations tended to be inconsistent in relation to specific sampling times. Somewhat more regularity was apparent in terms of dark or light periods. 3. Under constant temperature conditions plasma Cl increased with increasing temperature, while Na declined. Plasma magnesium was consistently higher at 25 degrees C than at either 20 or 30 degrees C. This was true of muscle Mg as well and, generally, also of Na, K and Cl. Water content tended to rise at higher temperatures in these animals, as did cellular phase volume, while extracellular phase volume was reduced. 4. Exposure to cycling temperatures was associated with a number of significant departures from the pattern seen at constant temperature. Ionic concentrations tended to be lower. By comparison with animals sampled at constant temperature and comparable times, diurnal stability was greater. In several instances (e.g. muscle Cl- and K+, cellular and extracellular phase volumes) variations with temperature were significantly different. This was also the case with ion pairs such as K and Na, and Ca and Mg. 5. These observations raise obvious questions regarding the validity of earlier descriptions of water-electrolyte status in species normally exposed to fluctuating temperatures. The variations seen under cycling temperature conditions, however, appeared to be adaptively appropriate. Reductions in plasma ion levels, for example, would tend to reduce costs of ionic regulation. The stability of plasma: cellular K concentrations should desensitize muscular excitability in relation to changing temperature conditions. This was also true of cellular levels of generally stimulating (Mg, K) and generally inhibitory ions (Ca, Na) known to influence metabolic processes.

Branchial and renal (Na+/K+) ATPase and carbonic anhydrase activities in a eurythermal freshwater teleost, Carassius auratus L.

1. Plasma sodium and chloride levels were determined in goldfish, Carassius auratus L., following acclimation to 5, 15, 25 and 35 degrees C. Carbonic anhydrase and (Na+/K+)-stimulated ATPase activities of gill and kidney were also assayed at both acclimation temperature and 41 degrees C. 2. Consistent with earlier findings this eurythermal species exhibits more variation in plasma composition with temperature than do the more stenothermal salmonids. Seasonal changes were also observed. 3. Despite differences in detail the overall pattern of transport enzyme activity change with acclimation was comparable to that previously observed in trout. The goldfish is, however, notable for high levels of renal carbonic anhydrase activity, and presumably employs this system more than does the trout to drive urinary recovery of ions by H+:Na+ and HCO3-:Cl- exchanges.

Thermoacclimatory changes in the ionic microenvironment of haemoglobin in the stenothermal rainbow trout (Salmo gairdneri) and eurythermal carp (Cyprinus carpio).

1. Haematological characteristics (erythrocyte number, haematocrit, haemoglobin, mean erythrocyte volume and haemoglobin content) and plasma and packed red blood cell water and electrolyte (Na+, K+, Ca2+, Mg2+, Cl-) levels were determined in summer and winter populations of rainbow trout acclimated to 2, 10 and 18 degrees C, and for carp held at 2, 16 and 30 degrees C. Erythrocyte electrolyte concentrations and ion:haemoglobin ratios were calculated from these data. 2. Modest increases in red cell abundance and reductions in mean erythrocytic volume were the most obvious haematological changes accompanying acclimation to higher temperatures. Haemoglobin levels in carp also tended to increase with temperature. 3. In winter trout plasma sodium and potassium were elevated following acclimation to increased temperature. No significant cges in plasma composition were observed in summer fish. Carp held at higher temperatures were characterized by increases in plasma chloride and calcium and reductions in sodium and magnesium levels. 4. Red cell potassium and magnesium and K+:Hb and Mg2+:Hb ratios tended to be higher in winter than in summer trout, with the converse being true of chloride and calcium and Cl-:Hb and Ca2+:Hb. Only potassium and K+:Hb were significantly altered following acclimation; rising at higher temperatures. In carp, potassium and K+:Hb were relatively thermostable, but sodium and chloride and Na+:Hb and Cl-:Hb increased with temperature while magnesium and Mg2+:Hb decreased. Changes in the ionic composition of carp red cells support the suggestion that cellular pH is reduced in the warm-acclimated animal. 5. These variations may be of adaptive value. Increases in chloride and hydrogen ion commonly reduce haemoglobin=oxygen affinity, and should facilitate oxygen unloading at the tissue level. Reductions in cellular magnesium, by maximizing organophosphate modulator levels, should produce much the same effect. 6. In both species reductions in mean erythrocytic volume took place at higher temperatures despite increases in cellular ion content which exceeded those of plasma. It is probable that reductions in cellular volume, which should favour branchial oxygen loading, were achieved by export of some as yet unidentified solute or solutes.

Temperature-related changes in the erythrocytic carbonic anhydrase (acetazolamide-sensitive esterase) activity of goldfish, Carassius auratus.

1. Carbonic anhydrase activity in 'membrane' and 'cytosol' fractions of goldfish erythrocytes was assayed by the p-nitrophenyl acetate procedure following thermal acclimation. 2. The thermal sensitivity of "membrane"-associated activity was apparently unaltered by acclimation. "Cytosol" activity in warm-acclimated specimens was somewhat more thermosensitive than that animals maintained at low temperature. 3. Significant increases in specific activity, and activity per unit volume of packed cells and blood were observed at higher temperatures when assays were conducted at the temperatures at which the system actually functions in the fish. By contrast, when determinations were carried out at a standard temperature (41 degrees C) corresponding to the upper incipient lethal for this species, activity was either unaffected, or declined as acclimation temperatures increased. 4. Changes in carbonic anhydrase activity following acclimation are consistent with the hypothesis that this system is implicated in the maintenance of stable plasma chloride levels, and the suggestion that alternations in red cell chloride levels with temperature are, in part at least, attributable to concomitant variations in enzyme activity.

Carbonic anhydrase (acetazolamide-sensitive esterase) activity in the blood, gill and kidney of the thermally acclimated rainbow trout, Salmo gairdneri.

1. Gill, kidney and blood levels of acetazolamide-sensitive esterase (carbonic anhydrase) activity were estimated at acclimation temperature and at a common temperature (25 degrees C) in rainbow trout acclimated to 2, 10 and 18 degrees C. Plasma levels of sodium, potassium and chloride were also examined for possible acclimatory variations. 2. Plasma sodium and chloride levels, and the sodium:chloride ratio were unaffected by thermal acclimation; potassium concentrations were significantly elevated at 18 degrees C. 3. Significant, but modest changes in renal and branchial carbonic anhydrase activity were observed under physiologically realistic incubation temperature conditions. Blood carbonic anhydrase activity was sharply elevated at higher acclimation temperatures. 4. The data are discussed in relation to the hypothesis that carbonic anhydrase in this relatively stenothermal freshwater salmonid, through its intimate association with the coupled HCO-3/CL- and H+ +NH+4/Na+ exchange systems may provide for relatively thermostable basal rates of sodium and chloride uptake from the medium and recovery from urine. The renal, and more notably the branchial (Na+/K+)-stimulated ATPase systems, and erythrocytic carbonic anhydrase may then serve primarily as high-temperature amplifiers of sodium and chloride recruitment respectively.