Life style and biochemical adaptation in Antarctic fishes

Journal of Marine Systems 27 Ž2000. 253–265 www.elsevier.nlrlocaterjmarsys

Life style and biochemical adaptation in Antarctic fishes Guido di Prisco ) Institute of Protein Biochemistry and Enzymology, C.N.R., Via Marconi 10, I-80125, Naples, Italy Received 10 February 1999; accepted 12 October 1999

Abstract Respiration and metabolism are under investigation in Antarctic fish, in an effort to understand the interplay between ecology and biochemical and physiological processes. Fish of the dominant suborder Notothenioidei are red-blooded, except Channichthyidae Žthe most phyletically derived family., whose genomes retain transcriptionally inactive DNA sequences closely related to the a-globin gene of red-blooded notothenioids and have lost the b-globin locus. Our structurerfunction studies on 38 of the 80 red-blooded species are aimed at correlating sequence, multiplicity and oxygen binding with ecological constraints and at obtaining phylogenetic information on evolution. For comparative purposes, this work has been extended to non-Antarctic notothenioids. All sluggish bottom dwellers have a single major hemoglobin ŽHb. and often a minor, functionally similar one. Three species of the family Nototheniidae have different life styles. They have uniquely specialised oxygen-transport systems, adjusted to the mode of life of each species. Artedidraconidae have a single Hb, lacking oxygen-binding cooperativity, similar to the ancestral hemoproteins of primitive organisms. The amino acid sequences are currently used in the molecular modelling approach. The study of several enzymes with key roles in metabolism Že.g. glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase, phosphorylase b, carbonic anhydrase. indicate that some aspects of the molecular structure Že.g. molecular mass, number of subunits, amino acid sequence, temperature of irreversible heat inactivation. have been conserved during development of cold adaptation. However, high catalytic efficiency, possibly due to subtle molecular changes, is observed at low temperature. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Antarctica; cold adaptation; fish; Notothenioidei; hemoglobin; enzyme

1. Oxygen transport and metabolism of the Antarctic fish fauna In our studies on the molecular basis of cold adaptation, we are finally beginning to reach a less fragmentary understanding of the complex interplay among ecology, biochemical and physiological processes, and adaptive evolution of Antarctic fish. This )

Tel.: q39-081-725-7242; fax: q39-081-593-6689. E-mail address: [email protected] ŽG. di Prisco..

article reviews our recent studies on the structure and function of hemoglobin ŽHb. and of some enzymes having a key role in metabolism Ždi Prisco and Giardina, 1996; di Prisco, 1997; di Prisco et al., 1998.. Fishes of the perciform suborder Notothenioidei, mostly confined within Antarctic and sub-Antarctic waters, are the dominant component of the Southern Ocean fauna. They comprise 95 of the 174 benthic species of the continental shelf and upper continental slope, and 120 Žincluding non-Antarctic notothe-

0924-7963r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 4 - 7 9 6 3 Ž 0 0 . 0 0 0 7 1 - 3

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nioids. of the 274 Southern Ocean species described to date ŽGon and Heemstra, 1990; Eastman, 1993.. No fossil record of Notothenioidei is available, leaving a void of 38 million years from the Eocene to the present. There is lack of information on their site of origin, existence of a transition fauna and time of their radiation in the Antarctic. Indirect indications suggest that notothenioids appeared in the early Tertiary ŽEastman, 1993. and began to diversify in isolation on the Antarctic continental shelf in the middle Tertiary, gradually adapting to progressive cooling. According to a recently revised classification ŽBalushkin, 1992; Pisano et al., 1998., Bovichtidae, monotypic Pseudaphritidae and Eleginopidae, Nototheniidae, Harpagiferidae, Artedidraconidae, Bathydraconidae and Channichthyidae are the families of the suborder ŽTable 1.. Notothenioids are red-blooded except Channichthyidae, the only known adult vertebrates whose blood is devoid of Hb. During 20–30 million years of increasing isolation south of the Antarctic Polar Front Ža barrier to migration in both directions and thus a key factor for fish evolution. cold adaptation was developed. Fish have now physiological and biochemical — often unique — specialisations. For the study of temperature adaptations, Antarctica — more than any other habitat on earth — is indeed a unique natural laboratory. Oxygen carriers are one of the most interesting systems for studying the interrelationships between environmental conditions and molecular evolution. Hb, being a direct link between the exterior and body requirements and fulfilling its primary function under extremely vari-

Table 1 The families of the suborder Notothenioidei ŽGon and Heemstra, 1990; Eastman, 1993; Pisano et al., 1998. Family

Antarctic species

Non-Antarctic species

Total

Bovichtidae Pseudaphritidae Eleginopidae Nototheniidae Harpagiferidae Artedidraconidae Bathydraconidae Channichthyidae

1 0 0 34 6 24 15 15 95

9 1 1 14 0 0 0 0 25

10 1 1 48 6 24 15 15 120

able conditions, has experienced a major evolutionary pressure to adapt and modify its functional features, while largely retaining its molecular structure. Thermodynamic analysis is a tool of choice in view of the role of temperature in modulating the oxygenation–deoxygenation cycle in tissues. The adaptive reduction in Hb contentrmultiplicity and erythrocyte count in the blood of Antarctic fish counterbalances the increase in blood viscosity produced by the subzero seawater temperature ŽWells et al., 1990. with potentially negative physiological effects Ži.e. higher demand of energy needed for circulation.. At the same time, low temperatures reduce the overall metabolic demand for oxygen, while increasing its solubility in the plasma, so that more oxygen can be carried in physical solution, and less needs to be bound to Hb. Nevertheless, only one notothenioid family has completely forsaken Hb as oxygen carrier. Why have Channichthyidae alone taken such a radical course, leaving the other families with only partial reductions in Hb? The physiological role of Hb in oxygen transport in temperate and tropical fish is undisputed; however does Hb remain absolutely vital for adequate oxygen transport in the Antarctic red-blooded families, or is it a vestigial relict which may be redundant under stress-free conditions? We sought to test the dependence of the nototheniid Trematomus bernacchii on Hb by complexing Hb with carbon monoxide to block oxygen binding, using two alternative experimental protocols Ždi Prisco et al., 1992. in which specimens bearing a caudal venous cannula were used ŽTable 2.. In the first one Žwhich took less than 3 min., approximately half of the total volume of whole heparinised blood was removed, saturated with carbon monoxide by gentle bubbling, and reinjected into the caudal vein. Aliquots of blood were taken periodically and analysed specrophotometrically for carbomonoxy–Hb. Ten minutes after reinjecting the blood, the latter derivative accounted for 56% of the total Hb. Carbomonoxide–Hb was thereafter reconverted slowly but totally to oxyHb within 48 h. In the second protocol, water of a sealed aquarium was equilibrated with 7% carbon monoxide in air. The aquarium contained cannulated specimens of T. bernacchii, as well as control specimens of Hb-less Chionodraco hamatus. After 5 h, over 95% of T. bernacchii Hb was converted to the carbomonoxy

G. di Priscor Journal of Marine Systems 27 (2000) 253–265 Table 2 Reversible functional incapacitation of T. bernacchii Hb, by reversible conversion in vivo to the carbomonoxy derivative Ždi Prisco et al., 1992.. See text for experimental details of first Ž1; one specimen. and second Ž2; four specimens. protocols Hours

0 0.2 1 2 3 5 12 24 36 48 54

Carbomonoxy-Hb Ž%. 1

2

0 56 54 51

0

36 8 2 1

72"11 95"8 90"9 43"13 12"9

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ing this experimental challenge to physiologists, we are investigating the structure and function of coldadapted enzymes. High catalytic rates at low temperature may be due to higher intracellular enzyme concentration andror higher inherent catalytic activity per active site. In the first mechanism, an increased number of catalytic sites compensates for the temperature-induced lower rate per site; in the second, higher activity is achieved by means of fewer molecules of a more efficient enzyme. The effect of temperature on the enzyme stability and activity again calls for thermodynamic analysis.

2. Hemoglobin

2"2

2.1. Molecular modelling and site-directed mutagenesis derivative, and hence was unable to carry oxygen. Normal aeration and water circulation were then established, and analysis of blood showed that carbon monoxide had been completely removed from Hb within the next 48 h. During both regimes, fish showed no signs of distress, at least in the absence of metabolic challenge. The survival of red-blooded T. bernacchii, in spite of the functional incapacitation of Hb, leaves little doubt that routine oxygen delivery is still possible with virtually no functional Hb in the cold, stable environment of the Antarctic seas. In keeping with these experiments, gradual reduction of the hematocrit from 8–15% to less than 1% in cannulated T. bernacchii specimens ŽWells et al., 1990. has no obvious ill effect, even during bouts of enforced exercise. Similar to channichthyids, redblooded fish can thus carry routinely needed oxygen dissolved in the plasma. Our findings on globin genes in Hb-less species Žsee Section 2.5., suggesting that the loss of gene expression is a primitive character Žestablished in the ancestral channichthyid about 25 million years ago, prior to diversification within the clade., open promising pathways for evolutionary studies. The relationship between cold adaptation and enzymatic activity is directly andror indirectly related to the concept of Ametabolic cold adaptationB. Experiments on oxygen consumption and routine metabolic rate have led to controversial conclusions ŽClarke, 1991; Somero et al., 1998.. Although leav-

As it will be discussed below, some Antarctic fish Hbs show large differences in functional characteristics, despite high identity in primary structure. This identity indeed makes Antarctic Hbs a greatly simplified — therefore ideal — system, and molecular modelling becomes very helpful to approach the relationship between structural and biological features Žsee also Sections 2.2 and 2.3.. For instance, the major Hbs of T. bernacchii and Trematomus newnesi only differ by four residues in the a chain and 10 in the b ŽTable 3.. Despite this high identity, the Hbs are functionally very different, e.g. T. newnesi Hb 1 has no Root effect and a weak Bohr effect ŽD’Avino et al., 1994., whereas both effects are very large in T. bernacchii Hb 1. These Hbs are therefore an exceptional model for the study of Hb structure– function relationship and in particular, for the study of the molecular basis of the Root effect. A model of T. newnesi Hb 1 has been built Ždi Prisco et al., 1997. on the basis of the crystallographic coordinates of T. bernacchii Hb 1 in R and T state ŽCamardella et al., 1992; Ito et al., 1995. as template. The analysis of the two structures did not evidentiate substantial structural differences in the R state, as also established by crystallographic studies of T. newnesi Hb 1 in the R state ŽMazzarella et al., 1999.. A substitution at the a 1 b 2 interface Žthe crucial point in the R–T transition. in the T state, where bulkier Ile a41 in T. newnesi replaces Thr in

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Table 3 Differences in amino acid sequence between Hb 1 of T. bernacchii and T. newnesi Ždi Prisco et al., 1991. Position

T. bernacchii

T. newnesi

a chain 21 41 55 97

Ala Thr His Ala

Ser Ile Asn Ser

b chain 33 41 53 55 56 75 83 84 86 136

Ile His Ala Ile Gly Val Ala Thr Ala Val

Val Tyr Gly Met Ser Met Asp Ala Thr Ala

T. bernacchii, causes conformational modifications which may justify T-state destabilisation and loss of Root effect Ždi Prisco et al., 1997.. In fact the shift of helix G in T. newnesi affects the interaction between Asp b101 and Asp a95, considered responsible for T-state stabilisation in T. bernacchii ŽIto et al., 1995.. It is worth noting that Antarctic Žbut not temperate. fishes lacking the Root effect have this replacement in a41. In summary, the modelling approach is providing hints for conclusions of general bearing on a very important functional feature of fish Hb such as the Root effect. Previous investigations have suggested that several amino acid residues have a key role in the Root and Bohr effects ŽPerutz and Brunori, 1982.. However, often our Antarctic Hb sequences do not follow the predictions of the stereochemical model Žsee for instance Ser b93, Lys b82, Gln b144; di Prisco et al., 1991.. In general, caution is recommended in linking a Hb functional feature to a single or a few residues, since replacements in other domains may induce changes in the molecular configuration which may act in combination with, or substitute for, the role that specific residues have in many cases. If structural modifications driven by coldadaptation are indeed responsible for functional diversification in Antarctic fish Hbs, then it is not

surprising that the primary-structure characteristics correlated to absence or presence of the Root effect in Antarctic fish Hb can differ from those of temperate fish, exposed to profoundly different evolutionary constraints. The simple system of two functionally distinct Hbs, which differ by only 14 out of 288 residues, is ideal for approaching these questions. In fact, modelling-dictated site-directed mutagenesis is currently under way in order to understand the molecular basis of a number of unique features of Antarctic Hbs. 2.2. Antarctic Notothenioidei In general, Notothenioidei are by far the most thoroughly characterised group of fish in the world. Our studies on their oxygen-transport system have so far been addressed to 38 out of a total of 80 redblooded Antarctic species, encompassing all major families. Two species of non-Antarctic notothenioids have also been investigated. The theme of adaptive strategies is a central one in Antarctic biology, and we are currently engaged in attempting to correlate sequence, multiplicity and oxygen-binding features Žthermodynamics in particular. of fish Hb with ecological constraints. In the first stages of our research, the Antarctic fish fauna seemed a rather uniform and simplified community — even somewhat dull. The fish Ždi Prisco et al., 1991, 1998; di Prisco and Giardina, 1996; di Prisco, 1997, 1998. had a single major Hb ŽHb 1. and often a minor one ŽHb 2, generally having the b chain in common. both displaying — with some exceptions — the Bohr ŽRiggs, 1988. and Root ŽBrittain, 1987. effects ŽTable 4.. All these fish are bottom dwellers. Another trend was found in the amino acid sequences of Antarctic Hbs. The sequences of major and minor Hbs cluster in two groups; in each group, identity is high Ž73–99% and 84–100%, respectively.. However, the identity between major and minor Hbs is lower, ranging between 61% and 73%. The sequences and the similar functional features of major and minor Hbs in a given species led us to conclude that minor Hbs are vestigial Žperhaps larval. remnants Žsee also Section 2.4., devoid of physiological significance Ždi Prisco et al., 1991.. However, the life style of three nototheniids, T. newnesi, Pagothenia borchgreÕinki Žtwo active cry-

G. di Priscor Journal of Marine Systems 27 (2000) 253–265 Table 4 The oxygen-binding system of Notothenioidei Ždi Prisco, 1998. Family

No. of species examined

Major Hb components

Bohr and Root effectsa

Pseudaphritidae Nototheniidae Bathydraconidae Artedidraconidae Harpagiferidae

1 16 8 9 1

1 Ž95%. 1 Ž95–99%. 1 Ž90–99%. 1 Ž99%. 1 Ž99%.

strong strong b strong c weak Žnot investigated.

a Bohr and Root effects were measured according to Giardina and Amiconi Ž1981. and D’Avino and di Prisco Ž1989.. Bohr coefficients Ž Dlog P50 rD pH. higher than y0.6 and lower than y0.3 denote strong and weak Bohr effects, respectively. Strong and weak Root effects correspond to Hb oxygenation at atmospheric pressure, pH 6.0, lower than 40% and higher than 80%, respectively. b Except in Aethotaxis mitopteryx. c Except in Gymnodraco acuticeps.

opelagic species, especially the latter. and Pleuragramma antarcticum Ža pelagic, sluggish but migratory fish., differs from that of the sluggish benthic species. Each species has a unique oxygen-transport system ŽTables 5 and 6. and each system appears adjusted to the fish specific mode of life. Moreover, sluggish Artedidraconidae also have Hbs with unique features, suggesting that Hb has a reduced physiological role Žsee also Section 1.; in fact, Hb might merely act as an oxygen store when these fish undergo anoxic conditions. All these Hbs are highly suitable for model building and site-directed mutagenesis. The Hb system of T. newnesi ŽD’Avino et al., 1994. is made of Hb C, Hb 1 Žwith the a chain in common with Hb C. and Hb 2 Žwith the chain b in common with Hb 1.. This notothenioid is the only species having two major Hbs, only one of which ŽHb C, a mere trace in all other notothenioids. displays pH and organophosphate regulation. Temperature changes bring about a small effect on the oxygen affinity of the most abundant component, as shown by the small overall DH of oxygen binding, constant in the pH range 8.0–6.5. Thus, Hb 1 does not require significant amounts of energy during the oxygenation–deoxygenation cycle, and this may reflect molecular adaptation to the extreme conditions in the Antarctic. This Hb system can ensure oxygen

257

binding at the gills Žvia Hb 1. and controlled delivery to tissues Žvia Hb C. also when active behaviour produces acidosis. High levels of Hb C, conceivably redundant in other notothenioids Žwhich count on Root and Bohr-effect Hb., compensate for lack of protonreffector regulation of Hb 1 and Hb 2. Ple. antarcticum, a pelagic migratory species ŽHubold, 1985., is the most abundant and the only fully pelagic nototheniid of high-Antarctic shelf systems. It combines the more general adaptations of all notothenioids with specialisations for life in the water column, and has great biological importance in the circum-Antarctic pelagic system. It has three major Hbs, the highest multiplicity within notothenioids. Hb 1 has the a chain in common with Hb 2 and the b with Hb 3; Hb 2 and Hb 3 have no chain in common. The amino acid sequences of the four globins ŽTamburrini et al., 1996, 1997. show high identity between Hb 1 of Ple. antarcticum and of other species, and between the chains of Hb 2 and Hb 3 not shared by Hb 1 and those of minor Hbs. In terms of oxygen-binding regulation, the three Hbs are very similar; all display strong, effector-enhanced Root and Bohr effects. However, there are important Table 5 Antarctic notothenioid species Žfamily Nototheniidae. with higher Hb multiplicity and functionally distinct components Ždi Prisco, 1998. Species

Hb components

Bohr and Root effects No ATP

3 mM ATP

strong weak or absent weak or absent

enhanced not enhanced

Ple. antarcticum Ž3 major Hbs. Hb C Žtraces. Hb 1 Ž25–30%. Hb 2 Ž20–25%. Hb 3 Ž45–50%.

strong strong strong strong

enhanced enhanced enhanced enhanced

Pag. borchgreÕinki Ž1 major Hb. Hb C Žtraces. Hb 0 Ž5–10%. Hb 1 Ž70–80%. Hb 2 Ž5–10%. Hb 3 Ž5–10%.

strong strong weak weak weak

enhanced enhanced not enhanced slightly enhanced slightly enhanced

T. newnesi Ž2 major Hbs.

Hb C Ž20–25%. Hb 1 Ž70–75%. Hb 2 Ž3–5%.

not enhanced

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Table 6 Heat of oxygenation ŽD’Avino et al., 1994; di Prisco, 1998. Species

Hb

DH Žkcalrmol oxygen. a No effector

100 mM NaCl, 3 mM ATP

pH 7.0

pH 8.0

pH 7.0

pH 8.0

Hb C Hb 1 Hb 2

y6.0 y4.0 n.d.

y12.0 y4.0 n.d.

y3.0 y4.0 n.d.

y9.0 y4.0 n.d.

Hb 1 Hb 2 Hb 3

y12.8 y3.6 y0.1

y15.3 y6.4 y16.5

y8.6 y1.8 y4.1

y17.4 y8.1 y7.6

Hb C Hb 0 Hb 1 Hb 2 Hb 3

n.d. y7.2 y3.7 y3.2 y5.3

n.d. y14.7 y5.5 y5.9 y7.6

n.d. y5.1 y3.7 y8.0 n.d.

n.d. y4.1 y4.5 y7.2 n.d.

T. newnesi

Ple. antarcticum

Pag. borchgreÕinki

a

The overall oxygenation enthalpy change D H Žkcalrmol; 1 kcal s 4.184 kJ., corrected for the heat of oxygen solubilisation Žy3 kcalrmol., was calculated by the integrated van’t Hoff equation: D H s y4.574 wŽT1 P T2 .rŽT1 y T2 .x log P50r1000 Ž P50 is the partial pressure of oxygen required to achieve Hb half saturation.. N.d., not determined.

differences in the thermodynamic behaviour. Hb 1 and Hb 3 show a very strong enthalpy change at pH 8.0, enhanced by chloride and organophosphates in the former, but drastically decreased in the latter; the heat of oxygenation of Hb 2, in the presence and absence of effectors, is much lower. A dramatic decrease is observed at lower pH in Hb 3 and Hb 2 Ž DH approaches zero.; in contrast, Hb 1 retains high oxygenation enthalpy, especially when effectors are absent. These observations indicate a stronger Bohr effect at physiological temperatures in Hb 1 Žin the presence of effectors. and Hb 3 Žin their absence.. In addition, the moderate effect of temperature on Hb 2 in the pH range 7.0–8.0 and on Hb 3 at pH 7.0 suggests that energy-saving mechanisms of oxygen loading and unloading may also be of advantage. From a thermodynamic standpoint, the oxygen-transport system of Ple. antarcticum is one of the most specialised ever found in fish. It appears designed to fit the unusual pelagic life style through molecular adaptation in the thermodynamics of each Hb. Although pelagic, this fish is very sluggish. Therefore, rather than having to respond to acidosis, this Hb system responds to the need to optimise oxygen

loading and unloading during seasonal migrations through water masses which can have different and fluctuating temperatures. Thus, Ple. antarcticum relies on three major Hbs, which differ functionally mainly in thermodynamic behaviour rather than pH and organophosphate regulation. Cryopelagic Pag. borchgreÕinki has a higher Hb amount than other notothenioids ŽMacdonald and Wells, 1991., which could be linked to the mode of life of this fish, the most active notothenioid. It has five Hbs Ždi Prisco, 1997., among which Hb C is in trace amounts and Hb 1 is 70–80% of the total. Hb 1, Hb 2 and Hb 3 are functionally similar, with weak Bohr and Root effects, not significantly influenced by organophosphates. Neglecting Hb C, Hb 0 is the only component with strong, effector-enhanced Root and Bohr effects. The heats of oxygenation are lower than those of Hbs of temperate fish, with different values in the absence and presence of the effectors, indicating that also in this species temperature variations may differentially affect the functional properties of each Hb, and that chloride and phosphates play an important role in the conformational change between the oxy and deoxy structures. The multiplic-

G. di Priscor Journal of Marine Systems 27 (2000) 253–265

ity of functionally distinct Hbs indicates that also this active, cryopelagic species has a very specialised Hb system. Artedidraconidae are benthic fish which, in the adult stage, have a single Hb. The amino acid sequences of Artedidraco orianae and Pogonophryne scotti Hbs have very high identity with the major Hbs of species of other families. The Hbs of several artedidraconids Ž Artedidraco shackletoni, Dolloidraco longedorsalis, Pogonophryne sp. 1, sp. 2 and sp. 3; di Prisco and Giardina, 1996; Tamburrini et al., 1998., show a modest Bohr effect and no Root effect. ATP enhances the Bohr effect and induces a weak Root effect ŽTable 7.. However, ATP was never found in the erythrocytes at saturating concentration, as demonstrated — in addition to quantitative analysis — by further induction of the Root effect upon addition of ATP to erythrocytes or unstripped hemolysate. The physiological role of organophosphates, therefore, appears critical in the pH regulation of Hbs of species of this family, and calls for further analysis. These Hbs display a remarkable and unique feature, i.e. absence of cooperativity of oxygen binding in the whole pH range. Consequently, the Hb–oxygen dissociation curve is hyperbolic rather than sigmoidal, not allowing large volumes of oxygen to be bound or released in response to small changes in the blood oxygen partial pressure. In a Triassic reptilian relict, Sphenodon punctatus ŽTetens et al., 1984., the exceptionally low resting metabolic rate and low temperature of this primitive vertebrate is considered consistent with reduced Bohr effect, absence of cooperative oxygen binding and high oxygen affinity of its Hbs. These features are shared by the Hbs of A. orianae and Pog. scotti. It is very surprising that Hbs of far-

Table 7 The oxygen-binding system of artedidraconids Ždi Prisco, 1998. Species

Bohr and Root effects; effect of ATP

A. orianae A. shackletoni Histiodraco Õelifer Pog. scotti D. longedorsalis Pogonophryne sp. 1, 2, 3

weak ŽRoot only with ATP. weak, only with ATP weak weak ŽRoot only with ATP. weak Žin hemolysate. weak ŽRoot only with ATP.

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from-primitive Artedidraconidae display functional properties typical of the multimeric Hbs of primitive vertebrates. If extreme conditions have driven evolution towards low metabolism and decreased dependence on Hb-mediated oxygen transport, this functional behaviour may well be consistent with the less critical physiological role of Hb. The lack of cooperative oxygen binding is even more striking in the light of the similarity of primary and quaternary structures of these Hbs Žto be consequently regarded as AmodernB . with the major Hbs of the other notothenioids — which do show cooperative interactions — and raises interesting questions on the evolution and mode of function of multi-subunit molecules. 2.3. Non-Antarctic Notothenioidei This research has been extended to non-Antarctic notothenioids, since comparison between coldadapted and non-cold-adapted species may help to understand their evolutionary history, as well as the molecular mechanisms of cold adaptation. A recent classification ŽBalushkin, 1992. separates the monotypic family Pseudaphritidae from Bovichtidae. These notothenioid families inhabit waters also north of the Antarctic and sub-Antarctic. Morphological ŽIwami, 1985; Balushkin, 1984. and karyological evidence ŽPrirodina, 1986. indicates that these are the most primitive notothenioids. We have studied Hbs of euryhaline Pseudaphritis urÕillii ŽD’Avino and di Prisco, 1997; di Prisco et al., 1998., very common in estuaries and lower portion of Australian rivers, considered a relict species. It has no antifreeze glycoproteins ŽEastman, 1993.. Like the majority of notothenioids, the Hb electrophoretic pattern shows a single major Hb 1 and a minor Hb 2. It has a strong Bohr effect in the presence and absence of ATP. The oxygen affinity is extremely high; in fact, log P50 in the absence of ATP stays below zero in the pH range 8.0–7.0. The Root effect is ATP-induced. Although the Hb multiplicity closely resembles that of sedentary Antarctic bottom dwellers, the exceptionally high affinity for oxygen of Hb 1 of this species Žmaybe due to the different habitat constraints. clearly differentiates it from the others notothenioids and is most likely reflected in substantial changes in the primary struc-

260

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ture. In the model, there are indeed two substitutions that may alter the geometry of the invariantly hydrophobic heme pocket: in a 87ŽF7. and a92ŽFG3. Glu and Met replace Leu. The substitution in a92 is uncommon and Glu is found only in this species. Interestingly, all Antarctic Notothenioidei have Gln, whose codon differs from that of Glu of Pse. urÕillii by a single base change at the first position. Although Pse. urÕillii has never developed cold adaptation, the amino acid sequences reveal high identity with the globins of the other notothenioids. Most of the residues, which differentiate Antarctic notothenioids from temperate fish are found in Pse. urÕillii Hb. In Hb 1, the identity with Antarctic notothenioids is higher Ž73–80% for the a and 75–82% for the b chains. than with any temperate fish Ž60–63% for the a and 60–66% for the b chains., following the general trend of notothenioids. In addition, the b chain of Hb 2 which is not in common with Hb 1 is highly similar Ž85–88%. to those of the minor Antarctic Hbs. However, the identity between Hb 1 of Pse. urÕillii and other Antarctic fish is close to, or lower than, the low extreme of the ensemble of values of Antarctic notothenioids Ž82–99% and 77–93% for the a and b chains, respectively.. This argues in favour of a common origin within notothenioids but also suggest that the major Hb has undergone modifications only to a limited extent. If sequence mutations in Antarctic fish are indeed related to the development of cold adaptation, this may imply divergence during the first stages of the cooling process, and in any case before the event which gave origin to antifreeze glycoproteins. We have initiated an investigation on the Hb system of the bovichtid Cottoperca gobio; electrophoretic analysis showed two Hbs in similar amounts. In BoÕichtus Õariegatus, we also have evidence of higher multiplicity. Although this is yet merely indicative, it is in keeping with Pse. urÕillii being a non-bovichtid notothenioid. Some hematological parameters of the nonAntarctic nototheniid Notothenia angustata, e.g. high hematocrit, erythrocite number and Hb content and cellular concentration ŽMCHC., typically favour oxygen transport in a temperate environment ŽMacdonald and Wells, 1991.; but Hb multiplicity and structural and functional features ŽFago et al., 1992.

closely resemble those of Antarctic notothenioids. In fact, the amino acid sequence identity between Hbs of a non-cold-adapted and a cold-adapted species such as N. coriiceps of the same family is the highest ever found among notothenioids. Thus, N. angustata is an ideal link between temperate and Antarctic habitats. The two nototheniids diverged evolutionarily well before the establishment of the Antarctic Polar Front. If N. angustata migrated before cooling and never became cold adapted, then cooling could not exert any evolutionary pressure in determining the Hb primary structure, and the strong sequence similarity would merely reflect the common phylogenetic origin of notothenioids. On the other hand, at the end of the Miocene Ž5 million years ago. and during the Pliocene, the Antarctic Polar Front moved northwards up to 398S, the latitude of Northern New Zealand, greatly facilitating migration of fish fauna. The finding that the genome of N. angustata contains antifreeze genes ŽCheng, personal communication. suggests that — unlike Pse. urÕilli — this fish may have migrated from Antarctic to temperate waters in a relatively recent time and was cold adapted prior to radiation. Thus, the sequence similarity may indeed be a direct consequence of cold adaptation. 2.4. Phylogenetic approach to notothenioid Hb Our amino acid sequences of the a and b chains of Antarctic fish and Pse. urÕillii Hbs, together with those of several non-Antarctic fish species, have been analysed using maximum parsimony to build notothenioid cladograms and phylogenetic trees ŽStam et al., 1997, 1998.. The trees are in agreement with those obtained by morphological analysis and sequence studies on mitochondrial RNA ŽRitchie et al., 1996. and give strong support to the monophyly of Antarctic notothenioids, with non-Antarctic Pse. urÕillii as their sister taxon. The chains of minor Hbs appear to have diverged from the major components prior to cold adaptation; they also are a monophyletic group and form an unresolved cluster with the chains of major Hbs Žwhich appear to have undergone major sequence modifications after the formation of the Antarctic Polar Front. and with Hbs of temperate fish, e.g. tuna. This analysis leads to the tentative hypothesis that at least two gene duplica-

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tions of an adjacent a–b Hb pair occurred in ancient teleosts and that a more recent duplication near the divergence of Perciformes from the other teleost orders gave rise to separate clusters of the major and minor chains of Notothenioidei. The trees also indicate that Pse. urÕillii Žtherefore also Bovichtidae, from which Pseudaphritidae have evolved. diverged before the formation of the Polar Front. The phylogenetic approach has indicated that Ži. Nototheniidae and Bathydraconidae are paraphyletic, whereas Artedidraconidae may be monophyletic; Žii. the separate genus Ž Pagothenia. assigned to T. bernacchii in previous studies ŽAndersen, 1984. is not justified; Žiii. Bovichtidae are not monophyletic, since the b chain of Cot. gobio Hb 1 groups with notothenioid minor Hbs; Živ. the grouping of nonAntarctic N. angustata Hb with the Antarctic nototheniids supports the hypothesis that this species was cold adapted prior to its recent radiation to temperate waters north of the Polar Front. 2.5. Globin genes in Channichthyidae Channichthyidae, the most phyletically derived notothenioid family, have Hb-less blood. These unique vertebrates carry oxygen in physical solution at approx. 10% of the carrying capacity of redblooded notothenioids. They developed physiological adaptations that maintain adequate tissue oxygenation, e.g. enhanced gas exchange by highly vascularised gills and skin, and increased cardiac output, circulatory volume and heart size. Using cDNAs that encode the a and b globins of Hb 1 from red-blooded N. coriiceps, we have compared the hybridisation patterns of genomic DNAs from three icefish species representing primitive and advanced genera Ž Chaenocephalus aceratus, Champsocephalus gunnari, and C. rastrospinosus. with those from four red-blooded notothenioids, N. coriiceps, Gobionotothen gibberifrons, Parachaenichthys charcoti ŽAntarctic. and N. angustata Žtemperate.. As expected, the genomes of the four red-blooded fish yield strong hybridisation signals for both aand b-globin cDNA probes. In contrast, the genomes of the channichthyids show hybridisation signals when probed with a-globin cDNA, but fail to hybridise the b-globin probe, suggesting that icefish genomes share retention of DNA sequences closely

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related to the a-globin gene of red-blooded nototheniods and loss of the b-globin locus ŽCocca et al., 1995, 1997; Zhao et al., 1998.. The assessment of steady-state globin mRNA levels in hematopoietic and non-hematopoietic tissues Žincluding the cellular component of blood. of Cha. aceratus reveals neither a-globin nor b-globin transcripts. Thus, the a-globin-related icefish sequences are not trancriptionally active. Work on myoglobin expression ŽSidell and Wayda, 1998. is complementing this research. In the light of these results, the most plausible mechanism leading to the Hb-less phenotype might be deletion of the b-globin locus in the ancestral channichthyid; the a-globin genes, no longer under positive selection pressure, would have accumulated mutations which caused loss of gene expression without complete loss of sequence information. We have initiated the analysis of the globin cluster in N. coriiceps, in order to gain information on the gene organisation in notothenioids. Preliminary results Ždi Prisco et al., 1997. revealed two sets of linked a- and b-globin genes Žsee also Section 2.4. with the same orientation and spaced about 1-kb apart. Each set shows the a- and b-globin genes spaced about 0.8-kb apart and oriented 5X to 5X relative to each other, with the coding sequences located on opposite DNA strands. This is the first evidence of such an arrangement, and it confirms previous findings that a- and b-globin genes are adjacent in the genome of amphibians and fishes. In mammals and birds, the genes are arranged in distinct clusters, a-like and b-like, located on different chromosomes. In the one other fish genome investigated to date for globin genes ŽAtlantic salmon; Wagner et al., 1994., two distinct clusters of linked adult a- and b-globin genes have been found, oriented 3X to 3X relative to each other and transcribed from opposite DNA strands. In N. coriiceps, we have found the same orientation between genes belonging to different sets. Therefore, the regions analysed in salmon may be part of a cluster having the same organisation found in N. coriiceps. This arrangement may be typical of the globin gene cluster in fishes. The deduced amino acid sequences from the four globin genes found in N. coriiceps differ from those expressed in the adult and may refer to juveniles.

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Considering the organisation in N. coriiceps and assuming a series of sets of linked a- and b-globin genes, a deletion leaving terminal a-globin geners might be considered as a mutational event in the icefish genome. Comparison with the icefish genomic region containing the a remnants will shed light on this intriguing question.

3. Enzymes Important contributions to the understanding of cold adaptation are coming from fish metabolism. Among environmental factors, temperature has profound effects on the catalytic activity and on enzyme regulation and structure. We are currently investigating several enzymes of special metabolic significance from red-blooded and Hb-less species, e.g. L-glutamate dehydrogenase ŽGDH., glycogen phosphorylase b and carbonic anhydrase ŽCA.. Glucose6-phosphate dehydrogenase has been described in the blood of the channichthyid C. hamatus and of the red-blooded nototheniid Dissostichus mawsoni ŽCiardiello et al., 1995, 1997a,b,c.; this enzyme catalyses the first reaction of the hexose monophosphate shunt, whose main function is to generate NADPH Žessential for glucose reductase and catalase.. GDH has a key regulatory function in cellular metabolism. It catalyses the reversible oxidative deamination of L-glutamate to a-ketoglutarate and ammonia through reduction of NADq or NADPq. In vertebrate cells, GDH has an important function in the production of ammonia and in feeding amino acids into the urea cycle. It plays a critical role also in the brain, because glutamate is an important neurotransmitter. GDH was purified to homogeneity from the liver of the channichthyid Cha. aceratus and its structural and functional characterisation was carried out in comparison with vertebrate homologous enzymes ŽCiardiello et al., 1997a,c.. The molecular masses of the subunit and of native GDH are compatible with a hexameric quaternary structure and are similar to those of tuna, dogfish and other hexameric GDHs. Similar to other vertebrates, the Antarctic enzyme displays dual coenzyme specifity, but yields higher catalytic rates with NADŽH.. This coenzyme preference is shared with the tuna and

dogfish enzymes and is probably due to metabolic requirements. Cha. aceratus GDH displays apparent temperature optima at 308C and 258C for the forward ŽFig. 1. and reverse reactions, respectively, whereas bovine GDH shows maximal activity at 458C and 508C in the forward and reverse reactions, respectively. k cat of forward and reverse reactions of Antarctic GDH in the range 5–258C is higher than that of bovine GDH. Thus, at low temperature Cha. aceratus GDH, similar to Antarctic G6PDs, is more efficient than the homologous mesophilic enzymes. Above 458C Cha. aceratus and bovine GDHs are irreversibly inactivated. The half-inactivation temperatures after 10-min incubation, are similar Ž508C and 538C, respectively.. The enthalpy of activation of the inactivation process, calculated from Arrhenius plots of heat-inactivation constants of Antarctic and bovine GDHs, is higher in Antarctic GDH, indicating that complete inactivation occurs in a much more narrow temperature range than the bovine enzyme ŽFig. 2.. Thus, although heat exposure causes irreversible inactivation at temperatures similar to

Fig. 1. Activity of Cha. aceratus Žcircles. and bovine Žtriangles. GDHs Žforward reaction. as a function of temperature.

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Fig. 2. Time course of heat inactivation at 458C and 528C of Cha. aceratus Žcircles. and bovine Žtriangles. GDHs. The enzyme was incubated in 100 mM potassium phosphate buffer, pH 7.5.

those inactivating the bovine enzyme, the molecular structure of Antarctic GDH is much more sensitive to small variations in the high temperature range. Phosphorylase b, a key enzyme in the metabolism of glycogen, was purified close to homogeneity from the white muscle of the nototheniid T. bernacchii. The time course of heat inactivation shows a strong effect of temperature on the activity in a narrow range Ž45–508C.. The allosteric activator AMP protects the enzyme; the most dramatic effect occurs at 508C, where the enzyme retains almost 100% activity after 20-min incubation in the presence of the ligand, but is completely inactivated in its absence. The temperatures of half inactivation are high, 498C in the absence and 558C in the presence of AMP ŽCiardiello et al., 1997c.. CA catalyses the conversion of gaseous carbon dioxide to bicarbonate and has an important role in pH regulation, since bicarbonate is the most important physiological buffer. In vertebrates it is found in tissues and blood Žerythrocytes.. The blood of channichthyids Cha. aceratus and C. hamatus reveals absence of CA in the plasma and the few erythrocyte-like cells, indicating that conversion of carbon dioxide, transported by gas diffusion into the plasma — similar to oxygen — does not depend on CA Ždi Prisco et al., 1997; Acierno et al., 1997.. On the other hand, the CA content in icefish gills is higher

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than in red-blooded fish gills. Following purification from the gills of T. bernacchii, the molecular mass is slightly different from erythrocyte CA, indicating isoenzymes with different tissue localisation. Two CA forms from the erythrocytes of T. bernacchii can be separated by anion-exchange chromatography; one form has a molecular mass of 30,000 Da, similar to erythrocyte CAs of other vertebrates. The structural and functional properties are under investigation. In summary, molecular characterisation of these enzymes, fulfilling important metabolic roles, and comparison with mesophilic counterparts provide indications that at least some structural features have been conserved during development of cold adaptation, as shown by the similarities in molecular mass, number of subunits, amino acid sequence, and temperature of irreversible heat inactivation. However, analysis of the catalytic properties reveals a range of differences Že.g. higher catalytic efficiency, shift towards low temperatures of the apparent optimum activity., adjusting the catalytic mechanisms to low temperatures. In order to obtain a suitable metabolic flux, a cold-adapted organism needs to rely on high catalytic efficiency at low temperature. In enzymes, the modifications adopted to reach this goal seem to be associated with a rather flexible structure, which does not necessarily imply heat inactivation temperatures lower than those of the mesophilic enzymes, but may induce subtle alterations of the temperature sensitivity. Such alterations may, for instance, be reflected in the narrow temperature range in which irreversible inactivation occurs. At decreasing temperature the rates of enzyme-catalysed reactions are reduced by the low heat content of the cellular environment Ž Q10 effect. that affects the protein structure and the interactions with low-molecularweight ligands — including the formation of the E–S complex. However, the biochemical strategies developed during cold-adaptation allow species with low body temperature to obtain an adequate metabolic flux, offsetting the Q10 effect.

Acknowledgements This research is in the framework of the Italian National Programme for Antarctic Research. The contribution of L. Camardella, V. Carratore, M.A.

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Ciardiello, E. Cocca, A. Riccio and M. Tamburrini is gratefully acknowledged.

References Acierno, R., Maffia, M., Rollo, M., Storelli, C., 1997. Buffer capacity in the blood of the hemoglobinless Antarctic fish Chionodraco hamatus. Comp. Biochem. Physiol. A 118, 989– 992. Andersen, N.C., 1984. Genera and subfamilies of the family Nototheniidae ŽPisces, Perciformes. from the Antarctic and Subantarctic. Steenstrupia 10, 1–34. Balushkin, A.V., 1984. Morphological bases of the systematics and phylogeny of nototheniid fishes. Acad. Sci. USSR, Zool. Inst., 1–140. Balushkin, A.V., 1992. Classification, phylogenetic relationships, and origins of the families of the suborder Notothenioidei ŽPerciformes.. J. Ichthyol. 30, 132–147. Brittain, T., 1987. The Root effect. Comp. Biochem. Physiol. B 86, 473–481. Camardella, L., Caruso, C., D’Avino, R., di Prisco, G., Rutigliano, B., Tamburrini, M., Fermi, G., Perutz, M.F., 1992. Haemoglobin of the Antarctic fish Pagothenia bernacchii. Amino acid sequence, oxygen equilibria and cristal structure of its carbomonoxy derivative. J. Mol. Biol. 224, 449–460. Ciardiello, M.A., Camardella, L., di Prisco, G., 1995. Glucose-6phosphate dehydrogenase from the blood cells of two Antarctic teleosts: correlation with cold adaptation. Biochim. Biophys. Acta 1250, 76–82. Ciardiello, M.A., Camardella, L., Carratore, V., di Prisco, G., 1997a. Enzymes in Antarctic fish: glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Comp. Biochem. Physiol. 21 Ž4., 443–450. Ciardiello, M.A., Camardella, L., di Prisco, G., 1997b. Enzymes in cold-adapted Antarctic fish: glucose-6-phosphate dehydrogenase. In: Battaglia, B., Valencia, J., Walton, D.W.H. ŽEds.., Proc SCAR 6th Biol. Symp., Venice ŽAntarctic communities: species, structure and survival.. Cambridge Univ. Press, Cambridge, pp. 261–265. Ciardiello, M.A., Camardella, L., di Prisco, G., 1997c. Enzymes of Antarctic fishes: effect of temperature on catalysis. Cybium 21 Ž4., 443–450. Clarke, A., 1991. What is cold adaptation and how should we measure it? Am. Zool. 31, 81–92. Cocca, E., Ratnayake-Lecamwasam, M., Parker, S.K., Camardella, L., Ciaramella, M., di Prisco, G., Detrich, H.W., 1995. Genomic remnants of a-globin genes in the hemoglobinless antarctic icefishes. Proc. Natl. Acad. Sci. U. S. A. 92, 1817– 1821. Cocca, E., Ratnayake-Lecamwasam, M., Parker, S.K., Camardella, L., Ciaramella, M., di Prisco, G., Detrich, H.W., 1997. Do the hemoglobinless icefishes have globin genes? Comp. Biochem. Physiol. A 118, 1027–1030. D’Avino, R., Caruso, C., Tamburrini, M., Romano, M., Rutigliano,

B., Polverino de Laureto, P., Camardella, L., Carratore, V., di Prisco, G., 1994. Molecular characterization of the functionally distinct hemoglobins of the Antarctic fish Trematomus newnesi. J. Biol. Chem. 269, 9675–9681. D’Avino, R., di Prisco, G., 1989. Hemoglobin from the Antarctic Fish Notothenia coriiceps neglecta. 1. Purification and characterisation. Eur. J. Biochem. 179, 699–705. D’Avino, R., di Prisco, G., 1997. The hemoglobin system of Antarctic and non-Antarctic notothenioid fishes. Comp. Biochem. Physiol. A 118, 1045–1049. di Prisco, G., 1997. Physiological and biochemical adaptations in fish to a cold marine environment. In: Battaglia, B., Valencia, J., Walton, D.W.H. ŽEds.., Proc. SCAR 6th Biol Symp, Venice ŽAntarctic communities: species, structure and survival.. Cambridge Univ. Press, Cambridge, pp. 251–260. di Prisco, G., 1998. Molecular adaptations in Antarctic fish hemoglobins. In: di Prisco, G., Pisano, E., Clarke, A. ŽEds.., Fishes of Antarctica. A Biological Overview. Springer, Milano, pp. 339–353. di Prisco, G., Camardella, L., Carratore, V., Ciardiello, M.A., Cocca, E., D’Avino, R., Romano, M., Tamburrini, M., 1997. Structure and function of hemoglobins and enzymes from Antarctic organisms. In: di Prisco, G., Focardi, S., Luporini, P. ŽEds.., The search for correlations with adaptive evolution. Proc. 3rd Meet Biol in Antarctica, S. Margherita Ligure. Camerino Univ. Press, pp. 83–104. di Prisco, G., D’Avino, R., Caruso, C., Tamburrini, M., Camardella, L., Rutigliano, B., Carratore, V., Romano, M., 1991. The biochemistry of oxygen transport in red-blooded Antarctic fish. In: di Prisco, G., Maresca, B., Tota, B. ŽEds.., Biology of Antarctic fish. Springer-Verlag, Berlin, pp. 263–281. di Prisco, G., Giardina, B., 1996. Temperature adaptation: molecular aspects. In: Johnston, I.A., Bennett, A.F. ŽEds.., Animals and temperature. Phenotypic and evolutionary adaptation, Soc Exptl Biol, Seminar Series 59. Cambridge Univ. Press, Cambridge, pp. 23–51. di Prisco, G., Macdonald, J.A., Brunori, M., 1992. Antarctic fish survive exposure to carbon monoxide. Experientia 48, 473– 475. di Prisco, G., Tamburrini, M., D’Avino, R., 1998. Oxygen-transport systems in extreme environments: multiplicity and structure–function relationship in hemoglobins of Antarctic fish. In: Portner, H.O., Playle, R. ŽEds.., Cold ocean physiology. ¨ Soc Exptl Biol, Seminar Series 66. Cambridge Univ. Press, Cambridge, pp. 143–165. Eastman, J.T., 1993. Antarctic fish biology, Evolution in a unique environment. Academic Press, San Diego. Fago, A., D’Avino, R., di Prisco, G., 1992. The hemoglobins of Notothenia angustata, a temperate fish belonging to a family largely endemic to the Antarctic Ocean. Eur. J. Biochem. 210, 963–970. Giardina, B., Amiconi, G., 1981. Measurement of binding gaseous and non-gaseous ligands to hemoglobins by conventional spectrophotometric procedures. Methods Enzymol. 76, 417–427. Gon, O., Heemstra, P.C., 1990. Fishes of the Southern Ocean. JLB Smith Institute of Ichthyology, Grahamstown, South Africa.

G. di Priscor Journal of Marine Systems 27 (2000) 253–265 Hubold, G., 1985. On the early life history of the high-Antarctic silverfish Pleuragramma antarcticum. In: Siegfried, W.R., Condy, P.R., Laws, R.M. ŽEds.., Proc 4th SCAR Biol Symp ŽAntarctic nutrient cycles and food webs.. Springer-Verlag, Berlin, pp. 445–451. Ito, N., Komiyama, N.H., Fermi, G., 1995. Structure of deoxyhaemoglobin of the Antarctic fish Pagothenia bernacchii with an analysis of the structural basis of the Root effect by comparison of the liganded and unliganded haemoglobin structures. J. Mol. Biol. 250, 648–658. Iwami, T., 1985. Osteology and relationships of the family Channichthyidae. Mem. Natl. Inst. Polar Res., Tokyo, Ser. E 36, 1–69. Macdonald, J.A., Wells, R.M.G., 1991. Viscosity of body fluids from Antarctic notothenioid fish. In: di Prisco, G., Maresca, B., Tota, B. ŽEds.., Biology of Antarctic fish. Springer-Verlag, Berlin, pp. 163–178. Mazzarella, L., D’Avino, R., di Prisco, G., Savino, C., Vitagliano, L., Moody, P.C.E., Zagari, A., 1999. Crystal structure of Trematomus newnesi haemoglobin re-opens the Root effect question. J. Mol. Biol. 287, 897–906. Perutz, M.F., Brunori, M., 1982. Stereochemistry of cooperative effects in fish and amphibian haemoglobins. Nature 299, 421–426. Pisano, E., Ozouf-Costaz, C., Prirodina, V., 1998. Chromosome diversification in Antarctic fish ŽNotothenioidei.. In: di Prisco, G., Pisano, E., Clarke, A. ŽEds.., Fishes of Antarctica. A Biological Overview. Springer, Milano, pp. 275–285. Prirodina, V.P., 1986. Karyotypes of Cottoperca gobio ŽBovichthyidae, Notothenioidei. as compared to karyotypes of other Notothenioidei. USSR Acad. Sci. Proc. Zool. Inst., Leningrad 153, 67–71. Riggs, A.F., 1988. The Bohr effect. Annu. Rev. Physiol. 50, 181–204. Ritchie, P.A., Bargelloni, L., Meyer, A., Taylor, J.A., Macdonald, J.A., Lambert, D.M., 1996. Mitochondrial phylogeny of trematomid fishes ŽNototheniidae, Perciformes. and the evolution of Antarctic fish. Mol. Phylogenet. Evol. 5, 383–390. Sidell, B.D., Wayda, M.E., 1998. Physiological and evolutionary aspects of myoglobin expression in the haemoglobinless Antarctic icefishes. In: Portner, H.O., Playle, R. ŽEds.., Cold ¨ ocean physiology. Soc Exptl Biol, Seminar Series 66. Cambridge Univ. Press, Cambridge, pp. 121–142. Somero, G.N., Fields, P.A., Hofmann, G.E., Kawall, R.B., 1998.

265

Cold adaptation and stenothermy in Antarctic notothenioid fishes: What has been gained and what has been lost? In: di Prisco, G., Pisano, E., Clarke, A. ŽEds.., Fishes of Antarctica. A Biological Overview. Springer, Milano, pp. 97–109. Stam, W.T., Beintema, J.J., D’Avino, R., Tamburrini, M., Cocca, E., di Prisco, G., 1998. Evolutionary studies on teleost hemoglobin sequences. In: di Prisco, G., Pisano, E., Clarke, A. ŽEds.., Fishes of Antarctica. A Biological Overview. Springer, Milano, pp. 355–359. Stam, W.T., Beintema, J.J., D’Avino, R., Tamburrini, M., di Prisco, G., 1997. Molecular evolution of hemoglobins of Antarctic fishes ŽNotothenioidei.. J. Mol. Evol. 45, 437–445. Tamburrini, M., D’Avino, R., Carratore, V., Kunzmann, A., di Prisco, G., 1997. The hemoglobin system of Pleuragramma antarcticum: correlation of hematological and biochemical adaptations with life style. Comp. Biochem. Physiol. A 118, 1037–1044. Tamburrini, M., D’Avino, R., Fago, A., Carratore, V., Kunzmann, A., di Prisco, G., 1996. The unique hemoglobin system of Pleuragramma antarcticum, an Antarctic migratory teleost. Structure and function of the three components. J. Biol. Chem. 271, 23780–23785. Tamburrini, M., Romano, M., Carratore, V., Kunzmann, A., Coletta, M., di Prisco, G., 1998. The hemoglobins of the Antarctic fishes Artedidraco orianae and Pogonophryne scotti. Amino acid sequence, lack of cooperativity and ligand binding properties. J. Biol. Chem. 273, 32452–32459. Tetens, V., Brittain, T., Christie, D.L., Robb, J., Wells, R.M.G., 1984. Characterization and function of isolated hemoglobins from the tuatara, Sphenodon punctatus ŽReptilia: O. Rhynchocephalia.. Comp. Biochem. Physiol. B 79, 119–123. Wagner, A., Deryckere, F., McMorrow, T., Gannon, F., 1994. Tail-to-tail orientation of the Atlantic salmon alpha- and betaglobin genes. J. Mol. Evol. 38, 28–35. Wells, R.M.G., Macdonald, J.A., di Prisco, G., 1990. Thin-blooded Antarctic fishes: a rheological comparison of the haemoglobin-free icefishes Chionodraco kathleenae and Cryodraco antarcticus with a red-blooded nototheniid, Pagothenia bernacchii. J. Fish. Biol. 36, 595–609. Zhao, Y., Ratnayake-Lecamwasam, M., Parker, S.K., Cocca, E., Camardella, L., di Prisco, G., Detrich, H.W. III, 1998. The major adult-globin gene of Antarctic teleosts and its remnants in the hemoglobinless icefishes. Calibration of the mutational clock for nuclear genes. J. Biol. Chem. 273, 14745–14752.