heterogeneity and functional properties of hemoglobins from south brazilian freshwater fish

Corap. Biochem. Physiol. Vol. 69B, pp. 463 to 470, 1981 0305-0491/81/070463-08102.00/0 Printed in Great Britain. All rights reserved Copyright {31981 Pergamon Press Ltd

HETEROGENEITY AND FUNCTIONAL PROPERTIES OF HEMOGLOBINS FROM SOUTH

BRAZILIAN FRESHWATER FISH

DANILO WILHELM F ° and EVALDO REISCHL* Laborat6rio de Biofisica Molecular, Departamento de Fisiologia, Farmacologia e Biofisica,

Instituto de Bioci6ncias, Universidade Federal do Rio Grande do Sul, Caixa Postal, 1953, 90.000 Porto Alegre, RS, Brazil

(Received 16 September 1980)

Abstract--1. The hemoglobin heterogeneity of twelve freshwater fish species from southern Brazil was characterized by starch-gel electrophoresis; the species from the super-order Acanthopterygii displayed a higher Hb heterogeneity than those from the super-order Ostariophysi; 2 species from different genera of the Cichlidae family displayed the same eleetrophoretic pattern in all their 9 components, resolved in two different buffer systems.

2. The hemolysates from 13 species were examined for the presence of a Root effect, in 10 of them the effect was positive.

3. Oxygen equilibria of the total hemolysates from 9 species, and of stripped hemolysates with and without ATP 1 mM from three catfish species were performed at pH 7.3.

4. Some aspects of the fish's biology are reviewed, and possible relations with their hemoglobin properties are discussed.

INTRODUCTION

Hemoglobin electrophoretic heterogeneity is generally high in fish, when compared with that of terrestrial vertebrates (Riggs, 1970; Sharp, 1973; Fyhn et al., 1979). The hemoglobin multiplicity probably rep- resents qualitative adaptations that can attend the physical and chemical variations of the organism's environment (Powers, 1974; Sullivan, 1977; Fyhn et al., 1979). The degree of polymorphism found in natural populations seems to be related to latitudinal variations (reviewed by Johnson, 1977).

Similar electrophoretic patterns may occur among different species (Sick et al., 1962; Sindermann & Honey, 1963; Yamanaka et al., 1965) or even among different genera (Sindermann & Honey, 1963; Fyhn et al., 1979), revealing that, although some phenotype features had been established in related species, another features can remain unchanged, like the hemoglobin pattern.

Blood from numerous teleost fish shows great de- crease in 02 capacity as the blood pH becomes acidic, making impossible the complete saturation of their hemoglobins, even under atmospheric pO2; this great dependence of oxygen saturation on pH is called the Root effect (Root, 1931). This effect has been impli- cated in the swimbladder and the choroid fete mirabile functioning (Fang6, 1966; Wittemberg & Wittem- berg, 1974; Farmer et al., 1979a). It was proposed (Black, 1958) that high Root effect hemoglobins can contribute to asphyxiate fish when the blood pH de- creases significantly in cases of intense exercise.

Obligate water breathing fish living in hypoxic and acidic waters must first guarantee maximal oxygen uptake from the environment, through relatively

* To whom reprint requests should be addressed.

high 02 affinity Hbs, when compared witl~ fish that live in better oxygenated waters (Krogh & Leitch, 1919; Riggs, 1970; Powers, 1974; Powers et al., 1979a). 02 affinities are also related to the metabolic rate of the organism (Riggs, 1970).

In the present work we described the electrophoretic heterogeneity of Hb hemolysates from 12 freshwater fish species from southern Brazil, as well as, the presence of a Root effect in ten of them, and oxygen equilibria studies in 11 species, one of them being a facultative air-breather.

MATERIALS AND METHODS

Biological material

Most of the specimens proceeded from the Jacui delta, situated near Porto Alegre, capital of the southermost state of Brazil; the remaining (Callichthys callichthys and the cichlids) were caught in ponds, brooks or sluices at the vicinity of this city. The former were kept, at least, 1 hr outside water by the fishermen, period elapsed since they were fished until sold at the dock; despite this, more than 70% recovered later in laboratory aquariums.

Hemoglobin hemolysates The fishes were bled by decapitation or by tail sectioning

after decerebration, according to their design and our con- venience. Blood was collected in a saline and anticoagulant solution (NaCl and Na-Citrate) always kept around 0°C on broken ice. The erythrocytes were washed three times in isotonic saline and later lysed in three volumes of 0.5 mM Tris--HCl buffer also 0.01 mM in EDTA at pH 8.0 at 5°C during 2-3 hr.

Electrophoresis The electrophoretic analysis were performed on starch-

gel with Tris-EDTA-Borate buffer at pH 7,9, and 0.3 M borate at pH 9.0 was used in the electrode vessels (Huehns, 1968). Samples runned for 7-9 hr at room temperature and the heating of the gel was prevented by ventilators and

463

464 DANILO WILHELM F ° a n d EVALDO REISCHL

evaporation of a water film kept over the gel. The electric field strength was 6-7V/cm. Staining was made with Amido Black and Benzidine. All the samples had more than 24 hr of storage at -15°C before being used. The oxy or cyanmet forms were used; neutralized KCN was added to samples (Yamanaka et al., 1965a). The Hb concentration varied between 2-5% (20-50 mg/ml), and 10-15 #1 of these solutions were applied in each slot.

Root effect One of the methods used to detect the presence of a

Root effect was that of Farmer et al. (1979a). Samples were diluted in 0.05 M citrate buffer at pH 5.5 in a 10ram cuvette, the Hb concentration (tetramer) ranged 18 to 28/*M. Recordings in A 576, A 560 and A 540 nm were

performed in a Perkin-Elmer Coleman 46 BCD spectro- photometer under atmospheric pO2. Solid buffer salt was added to the solution to raise the pH to ca. 8.0-8.2, thus saturating Hb at atmospheric pO2, and recordings were again performed. Finally recordings were made on deoxygenated samples, obtained with the addition of dithionite. A deoxygenation index of 0.67 related to the ratio of ab- sorbance values at A 580/A 560 nm was always obtained (Benesch & Benesch, 1965). All the samples examined for the presence of a Root effect were used no more than 10 hr after bleeding and were kept at 5°C until analysis. The experiments were performed at 20°C. Some samples were also studied in Tris-HCl 0.025 M, 0.025 M in NaCI and 0.5 mM in EDTA, being deoxygenated with a vacuum pump in tonometers.

A B C D1 D2 E F

!iii~iiiiliiiiiii!i~j~iiiiiiiii!iiiiill

G H I J K L M

Fig. 1. Synoptic electrophoretic pattern of hemoglobin hemolysates in starch-gel with Tris-EDTA- Borate buffer at pH 7.9 of 12 species. Start is indicated by an arrow; relative mobility of normal human Hb A is signalized at right of each electropherogram, because the present figure is composed from different electropherograms. Anode at the bottom. Number of specimens analysed is given after the respective scientific names: (A) Curimata gilberti (3); (B) Oligosarcus robustus (3); (C) Callichthys callichthys (3); (D1; D2) Plecostomus commersonii phenotype 1 and 2 (5); (E) Loricariichthys anus (2); (F) Rhamdia sp (5); (G) Synbranchus marmoratus (36); (H) Crenicichla lacustris (5); (I) C. lepidota (2); (J) Geophagus brasiliensis (4); (K) Aequidens portalegrensis (4); (L) Geophagus brachiurus (2); (M) normal

Human Hb.

Hb from Brazilian fish 465

Oxygen equilibria Curimata gilberti showed four bands with similar The procedures for oxygen equilibria were simitar to ~ H b distxibtttion:, (Fig. AA}; Oli#osarcus robustus

those described by Riggs (1951) and Benesch et al. (1965). (Fig. 1B) exhibited an interesting pattern of alternated Tris--HC10.025 M buffer at pH 7.3, 0.025 M in NaC1 and 0.5 mM in EDTA have been used for total hemolysates. The deoxygenation was performed in tonometers with the aid of a high vacuum pump; 1 or 2 ml of distilled water was added to samples to compensate evaporation during evacuation. Equilibration time was 7 min at 20°C. Least square method was employed in the determination of n and Pso from Hill plots; only values between 20-80% of oxygenation were considered.

Stripping The hemolysates were chromatographed in a

1.4 x 75 cm column of Sephadex G-100 at 6°C with flow rate of 0.3 ml/min. Tris-HCl 0.05 M buffer at pH 7.3 with NaCI 0.1 M and EDTA 1 mM was used. One mililiter of the diluted fraction after chromatography was employed in stripped hemolysates analysis. Concentration ranged from 70-170/aM for Hb tetramer, either in total and stripped hemolysates.

RESULTS

Heterogeneity

Results concerning electrophoretic heterogeneity from the Hb hemolysates are shown in Fig. 1 (see Table 1 for taxonomic informations).

All species showed the presence of multiple hemoglobins ranging from 4--10 components.

high and low relative concentrations among their 4 major and 3 minor components; Callichthys callichthys (Fig. 1C) presented five bands, two of them being cathodal; Plecostomus commersonii showed two phenotypes, both presenting one major component with more than 70% of the total hemoglobin; phenotype D1 presented more 4 minor components, and phenotype D2 only more 3 minor components; the major component found in Plecostomus commersonii resembles the major one revealed for phenotype II, IV, V and V var. of Hypostomus sp (= Plecostomus) from Amazonia (Fyhn et al., 1979); Loricariichthys anus (Fig. IE) showed among its eight components the fastest here verified, with 1.9 of relative mobility, referred to the normal human Hb A. Rhamdia sp (Fig. IF) showed three major components among a total of five which are present in other previous analysis (Reischl & Tondo, 1974a; Fyhn et al., 1979) in species also referred only as Rhamdia sp. The hemolysates from the air-breather Synbranchus marmoratus (Fig. 1G) presented three major groups of bands, in a total of 10 components, two of them being cathodal; 36 specimens, even some young ones with less than 10 cm of length, caught and analysed during the year, didn't display polymorphism, while seven specimens from Amazonia showed 3 phenotypes (Fyhn et al., 1979). All the cichlids showed high degree of hetero-

Table 1. Presence and intensity of Root effect in arbitrary ranges studied in thirteen species in Citrate 0.05 M and some in Tris-HCl 0.025 M buffer system, both at pH 5.5 and 20°C

Root effect intensity Citrate buffer Tris-HC1 buffer

Super-order Ostariophysi Order Cypriniformes

Sub-order Characoidei Family Curimatidae

Curimata gilberti (bird) Family Cynodontidae

Oligosarcus robustus (tambic6) Sub-order Siluroidei

Family Callichthyidae Callichthys callichthys (tamboath)

Family Loricariidae Loricariichthys anus (viola) Plecostoraus commersonii (cascudo)

Family Pimelodidae Rhanulia sp (jundi~i)

Super-order Acanthopterygii Order Perciformes

Sub-order Percoidei Family Cichlidae

Crenicichla lacustris (michola) Crenicichla lepidota (michola) Geophagus brasiliensis (carfi) Geophagus brachiurus (car/t) Geophaous gymnogenis (carfi) Cichlaurus facetus (carfi) Aequidens portalegrensis (car/0

- - 0

- - 0

- - 0

m

m

The Root effect notation refers to the degree of hemolysate deoxygenation: 0 means less than 10% (absence); *, 11-20% (weak); **, 21-40% (moderate); ***, 41-60% (strong) and ****, more than 61% (very strong); ranges similar to those used by Farmer et al. (1979a). (--) means not examined.

466 DANILO WILHELM F ° and EVALDO REISCHL

geneity. Two species from different genera, Crenicichla lacustris (Fig. 1H) and Geophagus brasiliensis (Fig. 1J) showed both the same electrophoretic pattern over their nine components, resolved in two different buffer systems (Tris-EDTA-Borate at pH 7.9 and Citrate at pH 8.65); two cathodal bands appeared when analysed in pH 7.9. Crenicichla lepidota (Fig. 1I) showed a pattern that resembles the two former cichlids, with seven evenly separated bands in a more pronounced anodal migration. Aequidens portaleorensis (Fig. 1K) displayed nine components, two of them being cathodal, and with the two anodal minor bands exhibiting a very low Hb concentration. Geophagus brachiurus (Fig. 1L) showed eight bands with three major and five minor components of very low Hb concentration; one major band was cathodal and one minor stayed at the origin.

Root effect

Results concerning the presence of a Root effect are presented in Table 1, together with other pertinent data about the species studied here.

The same arbitrary ranks used by Farmer et al. (1979a) have been adopted: 0 means less than 10~o of deoxygenated Hb; *, 11-20~o; **, 21-40; ***, 41-60~o and ****, more than 60~o of deoxygenated Hb at pH 5.5 and atmospheric pO2.

The two members of sub-order Characoidei showed moderate and strong Root effects, Oligosarcus robustus and Curimata gilberti, respectively, analysed on Tris-HC! 0.05 M buffer. The Siluroidei fishes here analysed are devoid of Root effect, with the exception of the facultative air-breather Callichthys callichthys, which presented a moderate Root effect. The other facuitative air-breather, Synbranchus marmoratus, was not examined for this effect here, because previous results, which are available in the literature, indicate that it is devoid of a Root effect (Johansen, 1966; Powers et al., 1979a; Farmer et al., 1979a). All the cichlids displayed a strong or very strong Root effect, when examined in Citrate buffer, all of them with less than 50% of 02 saturation in the experimental conditions; different specimens of Cichlidae members, when analysed in the same buffer (Tris-HC1 or Citrate), showed very similar values for Root effect intensity.

Oxyyen equilibria

The hemolysates from nine species were studied without stripping: two Characoidei fish, Curimata oilberti and Oli#osarcus robustus; three Siluroidei species, Rhamdia sp, Plecostomus commersonii and Cal- lichthys callichthys; and four Percoidei species from the Cichlidae family, Crenicichla lacustris, C. lepidota, Cichlaurus facetus and Aequidens portalegrensis. Stripped hemolysates and stripped hemolysates with addition of ATP I mM were studied in the three cat- fishes from suborder Siluroidei mentioned above. The results concerning oxygen equilibria experiments are shown in Figs 2-4.

Curimata gilberti exhibited the highest 02 affinity (0.8 torr) and the lowest cooperativity (n = 1.1) and the other Characoidei, Oligosarcus robustus, showed the lowest affinity (5.9 torr), for non stripped hemolysates. The obligatory water breathing siluroid species here studied revealed total hemolysates of high O2

affinity. Rhamdia sp with Pso of 1.0 torr and Plecos- tomus commersonii with Pso of 1.7 torr. In Plecos- tomus commersonii ATP 1 mM lowered approx, four times the 02 affinity in the stripped hemolysate and no great change in cooperativity was revealed; Rham- dia sp showed the lowest Pso for stripped hemolysates (0.5 torr) and ATP 1 mM depressed the 02 affinity ca. three times. The facultative air-breather Callichthys callichthys showed a relatively high Pso (4.3 torr) for non-stripped hemolysate; the addition of ATP 1 mM didn't change the 02 affinity of the stripped hemolysate, both with Pso = 2.8 torr. All the hemolysates from the cichlids displayed relatively low values for Pso, with the exception of Crenicichla lacustris (3.2 torr); all the n values were low, only Crenicichla lepidota presented a relatively high value (n = 2.0).

0.5

0.0

0.5

I O0

/

I I 0.5 i.o log pO 2

Fig. 2. Oxygen equilibria of non-stripped hemolysates from two Characoidei and three Siluroidei freshwater fishes in Tris-HC10.025 M at pH 7.3 and 20°C; P~o and Hill coeffi- cient (n) are given after scientific names, respectively: (11) Curimata #ilberti (0.8 torr; n = 1.1); (e) Oligosarcus robustus (5.9 torr; n = 1.7); (A) Callichthys callichthys (4.3 torr; n = 1.4); (O) Plecostomus commersonii (1.7 torr;

n = 2.3); (A) Rhamdia sp (1.0 torr; n = 1.7).

_g 0.5

0.0

0.5

/!

o~ o15 ,.~ I,Q ,o~ Fig. 3. Oxygen equilibria of non-stripped hemolysates from four Percoidei (Cichlidae) species in Tris-HCl 0.025 M at pH 7.3 and 20°C. Pso and n are given after sciontific names, respectively: (0) Crenicichla lacustris (3.2 torr; n = 1.3); (O) C. lepidota (1.0 torr; n = 2.0); (A) Cichlaurus facetus (1.5 torr; n = 1.3); (A) Aequidens portaleorensis (0.6 torr;

n = 1.4).


~5

0.0

-0.5

Fig. 4. Oxygen equilibria of stripped hemolysate (open symbols) and stripped hemolysates plus ATP I mM (dosed symbols) from three siluroid species in Tris-HCl 0.05 M at pH 7.3 and 20°C. Pso and n are given after scientific names, according to the symbols order: ([~, II) Callichthys callichthys (2.8 torr; n = 2.5; 2.8 torr; n --- 2.1); (&, &) Ple- costomus commersonii (1.0 torr, n = 2.0; 4.3 torr; n = 2.2);

(0,0) Rhamdia sp (0.5 torr, n = 2.2; 1.4 torr; n = 2.4).

DISCUSSION

Heterogeneity

The mean number of hemoglobin bands from the species studied here was 7.0 _+ 0.33 (mean + SE). The mean number for the super-order Acanthopterygii species was 8.7 + 0.18, bigger than the mean number found for Ostariophysi, 5.4 +_ 0.37. These data, despite the small number of species analysed, are according to those of the recent Alpha Helix expedition to Amazonia (Fyhn et al., 1979); the mean numbers for both super-orders found there were lower (6.7 _ 0.38 and 3.3 + 0.15, for super-order Acanthopterygii and Ostariophysi respectively). Significant discrepancies in fish hemoglobin electrophoretic heterogeneity appear when the number of small components resolved is considered (compare data from some fish in Reischl & Tondo, 1974a with those in Fyhn et al., 1979). We suspect that the low Hb concentration used in polyacrylamide-gel relatively to starch-gel electrophoresis could be responsible for this differences, including the mean values mentioned before. Also, as noted by Sul- livan (1977), polyacrylamide gel electrophoresis falls to reveal the cathodai components, due to experimental conditions; he got an average number of 5.0 components for many freshwater fish Sullivan, 1977), and a similar value was revealed for almost 90 freshwater fish species from Amazonia (Fyhn et al., 1979).

The alternation, in the electrophoretic pattern, of the major and minor components found in Oligo- sarcus robustus could be a nongenetic heterogeneity, due to a chemical modification in vivo, such as a com- bination of part of the major components with some kind of intra-erythrocyte compound, e.g. gluthation (Tondo, 1971) or glicose (Bunn & McDonald, i978). The pimelodian fishes exhibited very similar patterns (Fyhn et al., 1979), showing two major components of slow mobility that are more prominent than the other (s) one (s), and this was also verified for Pimelodus maculatus (Reischl, 1977) and Rhamdia sp (Reischl &

Tondo, 1974a). Fyhn et al. (1979), in an Amazonian species also referred as Rharadia sp, resolved only the three components that correspond to the major and more anodal ones here verified, for two phenotypes (I and II) displayed by three specimens analysed there. Weber & Wood (1979) found five components in one phenotype (V) of Hypostomus sp (a loricariid from Amazonia also referred as Plecostomus) when examined in isolectric focusing, while disc polyacrylamide-gel electrophoresis revealed only the two major components. Synbranchus marmoratus didn't show polymorphism in 36 specimens studied, which were caught during winter and summer, including some young specimens of less than 10 cm of length. These data contrast with those found in Amazonia, where three different phenotypes were described for only seven specimens studied (Fyhn et al., 1979). The higher degree of polymorphism found in tropical fresh and seawater fish species (Fyhn et al., 1979; Somero & Soul6, 1974), and also for many other animal groups that inhabit lower latitudinal regions (reviewed by Johnson, 1977 and Valentine, 1977), probably reflect many interelated environmental features. These genetic strategies have proved difficult to interpret (reviewed by Pianka, 1966 and Valentine, 1977).

The identical patterns exhibited by two cichlids examined here, Crenicichla lacustris and Geophagus brasiliensis, in two different buffer systems, suggest that their Hb components seem to have remained unchanged in their primary structure. Some previous data of identical electrophoretic patterns in agar were described for fishes in the two components from two eel species (Sick et al., 1962) and among five clupeoid fish species (Sindermann & Honey, 1963), and similar patterns in four cichlid species from Amazonia were described for nine components of similar mobility but different Hb distribution (Fyhn et al., 1979). The two species studied here displayed a remarkable identical pattern, showing almost uniform Hb distribution among the six more cathodal bands in each electropherogram. This could imply that, although selective forces along their evolution leaded to great morpho- logical differences between them, these pressures didn't affect their gas transport proteins. The similarity of components doesn't mean necessarily a functional similarity in vivo, since their intra-erythrocytic levels of ailosteric effectors can be different.

Root effect

Our data on the taxonomic distribution of the Root effect agree well with those from Farmer et al. (1979a), where many Amazonian species were analysed. The members from the sub-order Siluroidei studied there were devoid of, or showed only a slight Root effect for total hemolysates. From the four siluroid species studied by us, only the facultative air-breather Cal- lichthys callichthys showed a median Root effect, the other three, obligatory water-breathers, being devoid of it (see Table 1). The suggestion (Johansen et al., 1978a) that an increase in Hb concentration could compensate partially the coexistence of Root effect hemoglobins with high blood pCO2 in air-breathing fishes, seems to fit in Callichthys callichthys, where a high Hb amount is noted when the animal is bled; the same feature is present in the obligatory air-breather

468 DAN1LO WILHELM F ° and EVALDO REISCHL

and related callichthyid from Amazonia, Hoploster- num littorale (Willmer, 1934; Johansen et al., 1978a), which could be related to the hypoxic and acidic waters where this fish inhabits (Willmer, 1934; Gar- lick et al., 1979b). The real value of Root effect intensity of one component can be masked by other components which are devoid of this effect or have reverse Bohr effect (Farmer et al., 1979a), e.g. components I and II from H. littoralle (Garlick et al., 1979b). Spe- cies from the suborder Percoidei show, generally, strong Root effect hemolysates (Farmer et al., 1979a), and all the cichlids studied here showed less than 50Yo of saturation of their hemolysates at pH 5.5 and under atmospheric 02 tensions.

According to Wittemberg & Haedrich (1974), almost all members from a large taxonomic group, such as family, sub-order or even at the order level, seem to present in common the choroid rete mirabile and/or the presence of a functional swimbladder. The benthic or epibenthic life habitats, typical of the siluroids here studied, and their reduced swimbladder (Alexander, 1965), can be related to the absence of a Root effect (with the exception of the air-breather Callichthys callichthys), or, as Farmer had pointed out (Farmer et al., 1979a), the lack of choroid rete mirabile in siluroids could be correlated with this lack of Root effect.

The experiments carried on with Tris-HCl buffer 0.05 M always revealed lower values for the Root effect (see Table 1) than with Citrate buffer; the latter, mimicrying the phosphate allosteric effectors (Anto- nini et al., 1972), must be closer to the natural conditions and responsible for this difference.

Black (1958) had suggested that asphyxia associated to violent exercises in fishes could be related to strong Root effect hemoglobins. In this respect, it was interesting to observe that all the obligatory water-breathing siluroids here studied, and also Pimelodus maculatus (Reischl, 1977), are very resistant to hypoxia situ- ations (maintenance outside water for long periods), being found alive in the market several hours after fished. The other obligatory water-breathers here analysed from sub-order Characoidei and Percoidei that presented strong Root effects are less resistant.

Oxygen equilibria

New insights about fish adaptation to the environment were presented in the recent results from the Alpha Helix expedition to Amazonia (1976), showing that the oxygen affinity from obligate water-breathing fishes is well correlated with the 02 availability of the different micro-habitats of lentic and specially of lotic habitats (Powers et al., 1979a). Significant differences appeared within the obligate water-breathers when fishes living in the lentic or slow waters from lotic habitats were compared with those inhabiting the better oxygenated waters from river centers. On the other hand, it was shown that the 02 affinities of Amazonian air-breathers (obligate and facultative) were englobed by the range of obligate water- breathers, not supporting previous generalization (Johansen, 1970; Johansen & Lenfant, 1972; Johan- sen, 1976; Johansen et al., 1978) that air-breathers have, in general, lower 02 affinities than obligate water-breathers.

From the fishes here studied, only the predatory

and also very active characoid Oligosarcus robustus inhabits the better oxygenated surface waters from river centers (Fink & Fink, 1979) and exhibited the lowest 02 affinity hemolysate (5.9 torr) (Fig. 2). The others inhabit both lotic and lentic habitats, but only the large and predatory cichlid Crenicichla lacustris can also be sometimes found in the central and rapid waters of lotic habitats (personal and fishermen observations).

The four siluroid fishes are benthic or epibenthic, feeding mainly on aquatic insect larvae found in the bottom (Kn/~ppel, 1970; Fink & Fink, 1979); Plecos- tomus commersonii, however, is a continuous ilyopha- gous benthic species that shares an ecological niche similar to those of Curimata gilberti (Gneri & Ange- lescu, 1951) and both displayed high 02 affinity for total hemolysates (1.7 and 0.8 torr, respectively). Ana- lagous correlation were described for two pimelodian species, Rhamdia sp and Pimelodus maculatus (Reischl, 1977 thesis). Callichthys callichthys can live in the bottom of very hypoxic waters and feeds mainly on Trichoptera larvae (KniSppel, 1970) and is sometimes found moving on land (Von Ihering, 1940); it is a facultative air-breather owing to a highly vascularized intestine (Rauther, 1911; Von Ihering, 1940) in ana- logy to the related Amazonian species, Hoplosternum littorale (Willmer, 1934; Johansen, 1970). A relatively low 02 affinity was found for non-stripped hemolysate (4.3 torr) and no modification in Pso appeared when ATP 1 mM was added to the stripped hemolysate (Fig. 4), suggesting that GTP can be the main phosphate modulator in Callichthys callichthys, like in other air-breathing fishes (Weber & Wood, 1979), despite the results found in the related callichthyid Hop- losternum littorale, where ATP caused a reduction in oxygen affinity (Garlick et al., 1979b). On the other hand, the other two siluroid species, Plecostomus commersonii and Rhamdia sp, displayed sensibility to ATP.

The low Pso values verified for the obligatory water-breathing siluroids here studied seems to be related to their environment and to the their low activity level; it is presumed that the oxygen available in the bottom of slow waters of lotic habitats is less than the fast and superficial waters from river centers.

The cichlids showed relatively low Ps0 in non- stripped hemolysates, with the exception of the more active, large and predatory Crenicichla lacustris, which displayed a relatively high Ps0 (3.2 torr). The remaining cichlids are smaller, and slow swimmers that feed on insect larvae, plant materials, debris or mud, being typical bottom feeders (KniSppel, 1970; Fink & Fink, 1979); they all exhibited high 02 affinity hemolysates. Cichlids from Amazonia showed, in general, high 02 affinities for whole blood (at 30°C), and those that presented the highest affinities seem to prefer the bottom of lentic habitats, where low 02 tensions prevailed (Powers et al., 1979a). The smaller cichlids here studied are usually found in the slow zones of lotic habitats or in lentic ones (sluices), where are easily caught (personal observations). Few references about habitat and 02 consumption of South- american cichlids are available but indicate that they often move to better oxygenated upper waters, just below the surface, for respiratory purposes (Carter & Beadle, 1931; Sawaya, 1946).


Comparative interspecies studies of non-stripped hemolysates, where the amount of diluted modu la to r s such as organic phosphates ions is not precisely known, associated to the unknown blood 02 capacity and physiological pH, permit only rough inferences. Besides, few data are available in neotropical freshwater fish ecology. Despite this, it seems that the low affinity found for the only strong swimmer studied here, Oligosarcus robustus, and in minor extend, for the predatory Crenicichla lacustris, agrees well with the high 02 pressures needed by their tissues, relative to their activity level, and contrasts with the relatively high affinities found for the other fishes, which are slow or moderate swimmers and inhabit either the slow waters from lotic habitats or lentic habitats.

The results here obtained, although the reduced number of species analysed, and the restrictions pre- viously appointed, suggest their agreement with those of Amazonian fishes, where correlation between O2 affinity with their ecological niches was evident (Powers et al., 1979a), indicating that the reduced Pso found in obligate water-breathers that inhabit preva- lently or temporarily low oxygenated waters and, on the other hand, the relatively high Ps0 of those fishes that inhabit better oxygenated waters from river centers, is not a phenomenon restricted to Amazonia, but can reflect a broad evolutionary trend in fish adaptation to environment in a different way of the specialized anatomic devices developed by the air- breathing fishes.

Acknowledgements--This work was supported by the Concelho Nacional de Pesquisas (CNPq) (Proc. No. 2222, 1191/78), the Comiss~to de Aperfeiqoamento de Pessoal do Ensino Superior--CAPES (UFRGS) and by the Fundacgo de Amparo fi Pesquisa do Estado do Rio Grande do Sul (FAPERGS).

We thank Cleuder V. Sim6es for the technical help in the laboratory work; H. Britsky from the University of S~o Paulo and Paulo Buckup from the Departamento de Zoo- logia--UFRGS for systematic identification and Paulo Buckup and Carlos O. da C. Diefenbach from the Departa- mento de Fisiologia, Farmacologia e Biofisica--UFRGS for some specimens collected in nature.

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heterogeneity and functional properties of hemoglobins from south brazilian freshwater fish

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