Thermostability of haemoglobins from Antarctic fish

Summary Proteins from Antarctic fish are less stable at high temperatures than those from fish from lower latitudes. Investigations into the thermostability of haemoglobins from a range of Antarctic teleosts have been carried out for comparison with data from temperate species. Haemoglobin concentrations following periods of heating at 50°C were analysed spectrophotometrically and the time taken for 50% denaturation (t_50%) determined. The effects of pH and salt concentrations were also examined. With the exception of that of Rhigophila dearborni, the haemoglobins were found to be relatively unstable with t_50% values ranging from 7.7 to 29.9 min at pH 7. All haemoglobins became less stable on addition of KCl but the effect of pH was variable. Freezing had no effect on the stability of haemoglobin from Dissostichus mawsoni. The thermostability of haemoglobin from a temperate nototheniid, Notothenia angustata, was within the range displayed by its antarctic relatives and it would seem that in general the differences between genera are as great as those between Antarctic and temperate species as a whole.     

Comparative study of some haematological and biochemical blood parameters in fishes from the Skagerrak

Marine fishes caught in the Skagerrak, 27 different species representing various groups of fishes (Cyclostomi, Holocephali, Elasmobranchii and Teleostei), were examined for the following haematological and biochemical blood parameters: haematocrit, haemoglobin, mean corpuscle haemoglobin concentration, total plasma protein, blood glucose and blood lactate. Interspecies variations as well as variations within some species were observed. The haemoglobin values for all species showed a positive correlation to the corresponding haematocrit values. Relatively low values for haematocrit and haemoglobin were found in cyclostomes, holocephaleans and elasmobranchii compared to the majority of teleosts. Within the teleost group, the haematocrit and haemoglobin levels were positively correlated with the activity of the fish species. The cyclostome Myxine glutimsa L. had a total plasma protein content in the same range as most teleosts, whereas holocephaleans, elasmobranchii and the deep-water teleost Coryphaenoides rupestris Gunnerus showed comparatively low values. Among teleosts some relationship seemed to exist between the total plasma protein level and the activity of the fish species. In addition, a correlation between plasma protein content and levels of blood lipids were noted. Values for blood glucose and blood lactate were found to be lower in cyclostomes, holocephaleans and elasmobranchii than in most teleosts. Higher blood glucose levels were observed in the more active teleost species.     

Variability in brain ganglioside composition: A further molecular mechanism beside serum antifreeze-glykoproteins for adaptation to cold in Antarctic and Arctic-boreal fishes

Summary Gangliosides and sialoglycoproteins from brain, liver and muscle have been isolated from 6 Antarctic fish species from the suborder Notothenoids and from 4 Arctic-boreal fish species. In addition freezing and melting points from serum of both groups were examined in order to determine the presence of protein antifreezes. In comparison with eurythermic fishes of temperate climates in both groups the phylogenetical adaptation to cold is correlated with a significantly higher concentration of gangliosides in the brain. The ganglioside concentration of liver in Antarctic fish, but not in Arctic species, is 3 to 5fold higher than in mammals (rat); in muscle the ganglioside content is increased only in red-blooded Antarctic fish as compared with mammals. The concentration of neuronal sialo-glycoproteins generally is lower in Antarctic fish than in other marine teleosts; in muscles the content is 2 to 3fold higher than in mammals. The molecular composition of brain gangliosides is characterized by an extreme high polarity which is due to an equipment with highly sialylated fractions (40 to 50% higher sialylated than tetrasialogangliosides). There are distinct differences between the freezing and melting point of blood serum, especially in the Antarctic species in favour of the existence of protein antifreezes. The results are discussed with regard to the fact that the extremely high polarity of brain gangliosides reflects a very efficient mechanism on molecular level to keep the neuronal membrane functional under low temperature conditions.     

Plasma osmolarity, glucose concentration and erythrocyte responses of two Antarctic nototheniid fishes to acute and chronic thermal change

Haematocrit, haemoglobin concentration, plasma osmolarity and plasma glucose concentration of the Antarctic nototheniid fishes Pagothenia borchgrevinki and Trematomus bernacchii were monitored during 24 h periods of exposure to 3 and 6° C. The same haematological variables were also measured in P. borchgrevinki following a 5–6 week period of 4° C acclimation. The first plasma glucose measurements in acutely thermally‐stressed Antarctic nototheniids revealed a delayed hyperglycaemia which related well to the relatively slow stress‐related elevation of plasma cortisol in these species. Plasma osmolarity of both species was unchanged by acute 3° C exposure, but exhibited a delayed and transient increase during acute exposure to 6° C. Haematocrit was unaltered in T. bernacchii during the acute temperature increases but was elevated in the relatively active P. borchgrevinki . Following 5–6 weeks of warm‐acclimation (4° C) the plasma glucose concentration, haematocrit and haemoglobin concentration of P. borchgrevinki were not significantly different from fish at −1° C, but plasma osmolarity decreased toward the level found in temperate‐water teleosts.     

Haematological studies on Aethotaxis mitopteryx DeWitt,a high-Antarctic fish with a single haemoglobin

Summary The haematological parameters (haematocrit, erythrocyte number, haemoglobin concentration, MCHC, MCH, oxygen-carrying capacity, pH, p_1/2, , pCO₂, pO₂) of the Antarctic fish Aethotaxis mitopteryx DeWitt are reported. The erythrocyte number (0.39 × 10¹²/1) and the haemoglobin concentration (27.8 g/l) were found to be among the lowest values known for red-blooded Antarctic fishes. Among the species of the family Nototheniidae, this is the only one found so far to have a single haemoglobin in its blood. The results have been analysed in comparison with those of other Antarctic species, in an effort to establish correlations between the physiology of this pelagic-benthopelagic fish and its ecology. From the haematological parameters in this study and the functional properties of haemoglobin outlined in the following paper, it is suggested that A. mitopteryx has an extremely sluggish mode of life.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Branchial morphology and ion–water transport proteins in Antarctic teleosts with different modes of life

In the Antarctic Ocean salt concentration differs from the bottom to the surface owing to the seasonal forming and melting of sea ice. Antarctic teleosts present different lifestyle from benthic to pelagic. While benthic animals face a constant seawater salinity, benthic–pelagic animals have to face different salt concentration. Branchial morphology and ion–water transport proteins were compared in animals with different lifestyle. The ultrastructure of the gills was investigated by scanning electron microscopy (SEM). Na⁺/K⁺/ATPase, Na⁺/K⁺/Cl⁻ cotransport protein NKCC₁ and Aquaporin 3 (AQP3), were investigated by immunohistochemistry. The immunoreactivity for the ion transporter proteins were more intense in the active benthic–pelagic animals and in the icefishes than in the sluggish benthic ones. Conversely, AQP immunoreactivity was stronger in the animals with sedentary lifestyles. The SEM showed the secondary lamellae in the benthic–pelagic animals more densely packed with the exception of the haemoglobin free teleosts.     

Polar cod, Boreogadus saida (Gadidae), show an intermediate stress response between Antarctic and temperate fishes

To examine whether the attenuated stress response observed in Antarctic notothenioid fishes is a specialism for life in sub-zero waters, the polar cod, Boreogadus saida, and the temperate shorthorned sculpin, Myoxocephalus scorpius, were subjected to various stress treatments. Activity stress in both species had no effect on plasma catecholamine and cortisol levels, splenic mass, and on the haematological variables in B. saida. In contrast, heat stress caused a significant rise in circulating noradrenaline and adrenaline levels in B. saida, accompanied by a significant increase in haematocrit and haemoglobin concentrations, at constant plasma cortisol levels, red blood cell count and splenic mass. A concomitant rise in blood lactate concentrations indicated that heat-stressed B. saida were hypoxaemic. The capacity to synthesise catecholamines in B. saida was 38% of the value in M. scorpius, but similar to the values for Antarctic notothenioids. The lack of any adrenergic response to activity stress suggests that dominance of cholinergic control of the cardiovascular system may not be restricted to Antarctic notothenioids. Rather, the stress response in B. saida appears to be intermediate between Antarctic and temperate teleosts, in keeping with their relatively recent occupation of cold Arctic waters.     

Identification and distribution of nitric oxide synthase in the brain of adult Antarctic teleosts

We investigated the presence of nitric oxide synthase (NOS) in brain of adult Antarctic teleosts by indirect immunofluorescence technique using a synthetic rat neuronal NOS (nNOS) antibody. The following species were examined: Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni (haemoglobin-rich), Chionodraco hamatus and Pagetopsis macropterus (haemoglobin-free). Immunoreactive cell bodies were localized in dorsal telencephalon, in hypothalamus, in optic tectum of the mesencephalon as well as in Purkinje cells of the cerebellum. No differences were observed in the localization of the nNOS immunopositivity in the Antarctic teleosts brains examined and NOS distribution was similar to that described in other teleosts, suggesting that nitric oxide (NO) may also function as a neurotransmitter in the brain of Antarctic teleosts. A strong immunopositivity was observed in the cerebral blood vessels of the icefishes suggesting that NO may play a pivotal role in the regulation of the cerebral blood flow especially in these haemoglobin-free species.     

Stress response in the Antarctic teleosts (Notothenia neglecta Nybelin and N. rossii Richardson)

Blood chemistry and haematological parameters have been determined in the Antarctic teleosts, Notothenia neglecta Nybelin and Notothenia rossii Richardson at 2° C. Samples were taken using chronically implanted dorsal aortic cannulae following a minimum of 24–36 h recovery. Broadly similar results were obtained for the two species. In N. neglecta , routinely active specimens had high values of arterial pH (7.81) and PO ₂ (9.26 kPa), and modest haemoglobin levels (5.6 g dl⁻¹) relative to temperate species. Following 3 min strenuous activity there was a decrease in arterial pH (7.63) and a small rise in lactate from 0.41 to 0.68 mm, but no significant change in the calculated net metabolic acid load (δH_m+). PaO₂ and PaCO₂ varied inversely during exercise, and oxygen content declined by 22%. pHa and most other haematological parameters returned to routine values between 1–3 h post-exercise. The results suggest that the major effect of strenuous activity in Notothenia spp. is a respiratory, rather than a metabolic acidosis.     

Hydrogen ion binding properties of tuna haemoglobins.

Tunas are very active fish with a high aerobic capacity, but they also regularly perform burst swimming with massive production of lactic acid. The present study examines whether H(+) buffering by tuna haemoglobin (Hb) is elevated to cope with metabolic acidoses (by analogy with the high buffer capacity of tuna white musculature) or whether the Hb-H(+) binding properties resemble other teleosts that have low buffer values and high Haldane effects. H(+) titration of oxygenated and deoxygenated composite Hb from yellowfin tuna, skipjack tuna and bigeye tuna in 0.1 M KCl revealed low Hb-specific buffer values in all three tunas. Values at physiological pH were comparable to those reported in less active species such as carp and eel. The fixed acid Haldane effect was large (maximal uptakes of close to 4 mol H(+) per mol Hb tetramer upon deoxygenation). Thus, the Hb-H(+) binding properties of very active tuna species correspond to other teleosts. Low Hb buffer values may be a pre-requisite for the regulation of red blood cell pH via Na(+)/H(+) exchange. Approximately nine "neutral" groups were titratable in tuna Hbs, suggesting that two alpha-amino groups and seven histidine residues are titrated within each tetramer.     

Haematology of juvenile Acipenser oxyrinchus and Acipenser brevirostrum at rest and following forced activity

In vivo experiments were conducted to examine the haematology of juveniles from two relic bony fishes, Atlantic sturgeon Acipenser oxyrhinchus and shortnose sturgeon Acipenser brevirostrum . Oxygen transport characteristics (haematocrit, haemoglobin and mean erythrocytic haemoglobin concentration), ionic composition (Na⁺, Cl⁻, K⁺ and osmolality), metabolite concentration (lactate, cortisol and glucose) and protein content in blood were measured or calculated at rest and during recovery from forced activity. Under resting conditions, plasma osmolality and concentrations of Na⁺, Cl⁻, lactate, cortisol and total protein were significantly different between Atlantic and shortnose sturgeon. All other resting variables were not different between species. Following forced activity, plasma lactate levels were significantly higher in both species than at rest. Plasma cortisol levels in both species were only significantly higher 1 h following forced activity compared to resting values. Plasma lactate levels were significantly higher in Atlantic sturgeon than in shortnose sturgeon, but these levels returned to resting levels by 1 h in both species. Cortisol increases were greater in shortnose sturgeon than in Atlantic sturgeon. In general, oxygen transport characteristics, blood glucose, plasma protein and plasma osmolality were not altered by forced activity in either sturgeon species. Overall, both species had reduced responses ( i.e . the magnitude of changes in measured variables) to forced activity compared with teleosts.     

Brain gangliosides and cold-adaptation in high-antarctic fish

The concentration and composition of gangliosides from the brain of eight species of Antarctic Notothenioid fishes belonging to the class of perciformes and two species of boreal fishes (tropic cichlid fish Oreochromis mossambicus; Codfish Gadus morhua) were investigated. The concentration of whole brain gangliosides in Notothenioid fishes (between 1622 and 2183 μg NeuAc/g dry wt.) was slightly lower than that in the brains of fish species, which live in warm, temperate habitats (2483 μg NeuAc/g dry wt.). The composition of brain gangliosides was completely different from that of warm adapted fish species (e.g. the tropic cichlid fish Oreochromis mossambicus). The relative concentration of polysialogangliosides (GT1b-GH) is strongly increased in all the investigated Antarctic species. They were found to have the most complex and most polar brain ganglioside pattern (high degree of sialylation and alkali-lability) within the teleosts. This may be one of the mechanisms, beside antifreeze proteins, to keep the neuronal membranes functional even below the freezing point.     

The role of adrenaline as a modulator of cardiac performance in two Antarctic fishes

The present work was performed to test the hypothesis that Antarctic teleosts rely mostly on cholinergic inhibition for autonomic modulation of the heart. The effects of adrenaline on the inotropic properties on paced, isometrically contracting muscle strips were examined in two distinct Antarctic teleosts, the haemoglobinless icefish Chaenocephalus aceratus and the red-blooded Notothenia coriiceps. All tissues examined revealed a negative force-frequency relationship. Under baseline conditions C. aceratus contracted with a force twice as great as that of N. coriiceps. While the degree to which ventricular tissues responded to adrenaline varied between species, adrenergic stimulation significantly increases myocyte contraction force in this group of fishes. Contraction and relaxation times were not significantly affected by adrenaline concentration while absolute rates of contraction were. Adrenergic stimulation does not enable tissues to achieve higher contraction frequencies, but is shown to be a potent modulator of contraction force.     

Molecular evolution of haemoglobins of polar fishes

The Arctic and the Antarctic differ by age and isolation of the respective marine faunas. Antarctic fish are highly stenothermal, in response to stable water temperatures, whereas the Arctic ones are exposed to seasonal and latitudinal temperature variations. The knowledge of the mechanisms of phenotypic response to cold exposure in species of both polar habitats offers fundamental insights into the nature of environmental adaptation. In the process of cold adaptation, the evolutionary trend of Antarctic fish has led to unique specialisations, including modification of haematological characteristics, e.g. decreased amounts and multiplicity of haemoglobins.Unlike Antarctic Notothenioidei, Arctic teleosts have high haemoglobin multiplicity. Although the presence of functionally and structurally distinct haemoglobins is a plesiomorphic condition for many perciform-like fishes, it seems that the oxygen-transport system of teleost fish in the Arctic region has been adjusted to temperature differences and fluctuations of Arctic waters, much larger than in the Antarctic. The amino-acid sequences used to gain insight into the evolution history of α and β globins of polar fish have clearly shown that Antarctic and Arctic globins have different phylogenies, leading to the hypothesis that the selective pressure of environment stability allows the phylogenetic signal to be maintained in the Antarctic sequences, whereas environmental variability would tend to disrupt this signal in Arctic sequences.     

Antarctic teleost immunoglobulins: more extreme, more interesting

We have investigated the immunoglobulin molecule and the genes encoding it in teleosts living in the Antarctic seas at the constant temperature of −1.86 °C. The majority of Antarctic teleosts belong to the suborder Notothenioidei (Perciformes), which includes only a few non-Antarctic species. Twenty-one Antarctic and two non-Antarctic Notothenioid species were included in our studies. We sequenced immunoglobulin light chains in two species and μ heavy chains, partially or totally, in twenty species. In the case of heavy chain, genomic DNA and the cDNA encoding the secreted and the membrane form were analyzed. From one species, Trematomus bernacchii, a spleen cDNA library was constructed to evaluate the diversity of VH gene segments. T. bernacchii IgM, purified from the serum and bile, was characterized. Homology Modelling and Molecular Dynamics were used to determine the molecular structure of T. bernacchii and Chionodraco hamatus immunoglobulin domains. This paper sums up the previous results and broadens them with the addition of unpublished data.     

Functional characterization of parvalbumin from the Arctic cod (Boreogadus saida): similarity in calcium affinity among parvalbumins from polar teleosts

Calcium dissociation constants (KD) were measured as a function of temperature for parvalbumin, a small acidic protein expressed abundantly in fast-twitch muscle, from the Arctic cod (Boreogadus saida) and compared to values previously determined for Antarctic and temperate zone teleosts. Estimates of KD were derived independently from fluorometric titrations and calorimetry. In addition, the primary structure of B. saida parvalbumin was determined. Calcium KDs for parvalbumin from B. saida were fundamentally similar to those for parvalbumins from Antarctic species (6.68+/-0.59 nM and 7.77+/-0.72 nM at 5 degrees C, respectively), but significantly different from temperate zone species (1.35+/-0.28 nM at 5 degrees C). However, estimates of KD for B. saida parvalbumin at 5 degrees C closely matched values for temperate zone fish at 25 degrees C (6.54+/-0.56 nM), recapitulating the prior observation that calcium affinity of parvalbumin is conserved at the native temperature of teleost fish. Full sequence of B. saida parvalbumin was generated using reverse-phase HPLC and RACE-PCR. The Arctic parvalbumin showed 83% homology to a carp parvalbumin. None of the 16 total substitutions between the two parvalbumins resided in the cation binding sites of the protein, indicating that the structural locus of the thermal sensitivity of function lies outside the active regions.     

Antioxidant defences in marine fish—II. Elasmobranchs

1. Tissues from four ray species and two shark species were examined concerning Superoxide dismutase and catalase content, and malonaldehyde and tocopherol concentrations.2. Livers displayed the highest Superoxide dismutase and catalase content in all species examined. Sharks exhibited higher mean enzyme contents than rays (3.6 and 2.4 nmol SOD g⁻¹ wet tissue, 185 and 120 pmol catalase g⁻¹ wet tissue).3. Tocopherol was detected in liver from all species, in kidney and in ovary from one ray and one shark species, and in shark cristaline in a range of 0.1–4.8 nmol g⁻¹ wet tissue.4. The content of antioxidant enzymes in elasmobranchs was lower than that of teleosts, and seems to follow the overall metabolic oxygen consumption or activity level from each fish major taxonomic group.5. Liver peroxidation as measured as MDA/TBARS concentration, revealed very high values (range of 1.5–4.5 μmoles g⁻¹ wet tissue), approximately one order of magnitude higher when compared with mammals.     

Elevated levels of trimethylamine oxide in deep-sea fish: evidence for synthesis and intertissue physiological importance

Tissue levels of trimethylamine oxide (TMAO) were compared for seven teleost and two elasmobranch species captured from three depth ranges: shallow (<150 m), moderate (500-700 m), and deep (1,000-1,500 m). Within the teleosts, the deep-caught species had significantly greater TMAO content than shallow- or moderate-caught species. In all teleosts, muscle had substantially more TMAO than all other tissues. Kidney or, in some cases, liver had elevated trimethylamine (TMA) content, 2.20-9.65 mmol/kg, along with appreciable trimethylamine oxidase (TMAoxi) activity, suggesting active TMAO synthesis. No correlation was found between TMAoxi activity and TMAO content. The elasmobranchs in this study, Squalus acanthias and Centroscyllium fabricii from shallow and deep water, respectively, were both squaliform sharks. The deep-caught species had significantly more TMAO in all tissues than the shallow species. Furthermore, urea was significantly less in the deep species in all tissues except liver, while the urea:TMAO ratio was significantly less in all tissues. As with teleosts, the TMAO content of muscle was substantially higher for both elasmobranchs than in all other tissues. TMAoxi was below levels of detection in both elasmobranch species, suggesting that TMAO is obtained solely from the diet. This study expands the trend of increased muscle TMAO in deep-sea fish to a variety of other tissues. The accumulation of TMAO in various tissues in deep-sea teleosts and the accumulation of TMAO and concurrent urea decrease in a deep-sea elasmobranch in comparison to a shallow water species strongly support the contention that TMAO is of physiological importance in deep-sea fish.     

Kinetic Characterization of Myoglobins from Vertebrates with Vastly Different Body Temperatures

Fish myoglobins are structurally distinct from the previously characterized mammalian myoglobins. Teleost fishes express generally lower levels of myoglobin than those found in mammals. Although the oxygen binding affinity is essentially the same as mammalian myoglobins, oxygen dissociation rates and carbon monoxide combination rates of the teleost myoglobins studied are significantly faster. Thus, the kinetic parameters of myoglobin from two Antarctic teleost species, measured close to their body temperature of −1°C, are comparable to those of mammalian myoglobins with higher body temperatures. These data suggest myoglobins from Antarctic teleosts may function at extreme environmental temperatures.     

Some aspects of haematology of a common Indian field rat, Rattus rattus arborious (Linn.) in relation to sex and size.

In the present investigation the studies on erythrocyte and leucocyte counts, haemoglobin concentration, haematocrit values, blood biochemistry of protein, glucose and cholesterol, erythrocyte sedimentation rate, pH value, diameter of erythrocyte and clotting time were made in different sex and size (weight group) of an Indian field rat, Rattus rattus arborious. The number of erythrocyte, haemoglobin concentration, haematocrit values were found to be higher in females than the males, while the number of leucocyte was lower in females in comparison to males. In the biochemical estimation the value of protein in females was high while that of the glucose and cholesterol were lower than that of the males. All the above values showed an increasing tendency with the increase in body weight. pH value, diameter of erythrocyte and clotting time were found to be almost constant in rats of different weight groups and different sex.