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.     

Mode of locomotion places selective pressures on Antarctic and temperate labriform swimming fish

The physiological responses to exercise and stress of the Antarctic labriform swimmer Pagothenia borchgrevinki were compared to the temperate labriform swimmers Notolabrus celidotus and Notolabrus fucicola. Basic swimming characteristics were very similar amongst the three species with P. borchgrevinki showing a reduced capacity for exercise. P. borchgrevinki showed large increases in haematocrit (Hct) following exercise that were not seen in the temperate species. Lactate dehydrogenase (LDH) activities were high in the white myotomal muscle from the Antarctic fish, with a distinct indication of metabolic cold adaptation in this enzyme. Nevertheless, although the temperate fish showed elevated muscle lactate concentrations following either exercise or electrical stimulation the Antarctic fish did not. The data suggest that poor anaerobic performance of white muscle is associated with the labriform mode of locomotion.     

Metabolic cold adaptation of polar fish based on measurements of aerobic oxygen consumption: fact or artefact? Artefact!

Whether metabolic cold adaptation in polar fish, based on measurements of aerobic standard metabolic rate, is a fact or an artefact has been a dispute since Holeton asked the question in 1974. So far polar fish had been considered to be metabolically cold adapted because they were reported to have a considerably elevated resting oxygen consumption, or standard metabolic rate, compared with oxygen consumption values of tropical or temperate fish extrapolated to similar low polar temperatures. Recent experiments on arctic and Antarctic fish, however, do not show elevated resting aerobic oxygen consumption values, or standard metabolic rate, and hence it is concluded that that metabolic cold adaptation in the traditional sense is an artefact.     

Expression of 70 kDa heat shock proteins in antarctic and New Zealand notothenioid fish

The cold and constant water temperature of the Southern Ocean surrounding Antarctica provides a natural laboratory to address questions of temperature adaptation in marine organisms. In this study, endogenous levels and the number of isoforms of the 70 kDa heat shock protein multigene family (hsp70) of Antarctic and cold temperate notothenioid fishes were determined by SDS-polyacrylamide gel electrophoresis and Western blotting. Tissues from three Antarctic Trematomus congeners had significantly lower levels of 70 kDa Hsp isoforms than their temperate confamilial from New Zealand waters. However, these two thermally disparate sets of fish did not differ in number or pattern of 70 kDa Hsp isoforms expressed under normal physiological conditions. Additionally, levels of 70 kDa Hsp isoforms in specimens of one Antarctic species, Trematomus bernacchii, acclimated to 4 degrees C were significantly higher than non-acclimated conspecifics, indicating a direct effect of temperature on Hsp expression in this species. This study shows that constitutive expression of some members of the 70 kDa Hsp multigene family have been maintained, despite the absence of environmental heat stress for at least 2.5 million years.     

Some like it hot, some like it cold: the heat shock response is found in New Zealand but not Antarctic notothenioid fishes

Previous research on Antarctic notothenioids has demonstrated that cells of cold-adapted Antarctic notothenioids lack a common cellular defense mechanism called the heat shock response (HSR), the induction of a family of heat shock proteins (Hsps) in response to elevated temperatures. The goal of this study was to address how widespread the loss of the HSR is within the Notothenioidei suborder and, specifically, to ask whether cold temperate non-Antarctic notothenioids possess the HSR. In general, Antarctic fish have provided an important opportunity for physiologists to examine responses to selection in the environment and to ask whether traits of the notothenioids represent cold adaptation, or whether the traits are related to history and are characteristics of the notothenioid lineage. Using in vivo metabolic labeling, results indicate that one of the two New Zealand notothenioids possess an HSR. The thornfish, Bovichtus variegatus Richardson, 1846, expressed heat shock proteins (Hsp) in response to heat stress, whereas the black cod, Notothenia angustata Hutton, 1875, did not display robust stress-inducible Hsp synthesis at the protein-level. However, further analysis using Northern blotting clearly demonstrated that mRNA for a common Hsp gene, hsp70, was present in cells of both New Zealand species following exposure to elevated temperatures. Overall, combined evidence on the HSR in notothenioid fishes from temperate New Zealand waters indicate that the loss of the HSR in Antarctic notothenioid fishes occurred after the separation of Bovichtidae from the other Antarctic notothenioid families, and that the HSR was most likely lost during evolution at cold and constant environmental temperatures.     

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0°C and after long-term warming to 5°C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0°C for the polar and 6°C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.     

Cloning and characterization of cytoplasmic carbonic anhydrase from gills of four Antarctic fish: insights into the evolution of fish carbonic anhydrase and cold adaptation

Although carbonic anhydrase is a ubiquitous enzyme involved in a variety of physiological processes, the information on its evolution and cold adaptation among Antarctic fish is still limited: the only Antarctic fish carbonic anhydrase characterized up-to-date is from Chionodraco hamatus, a member of the Channichthyidae family. In this work, we characterized orthologous genes within two other fish families: Nototheniidae (Trematomus eulepidotus, Trematomus lepidorhinus, Trematomus bernacchii) and Bathydraconidae (Cygnodraco mawsoni). The cDNAs of epithelial gill carbonic anhydrases were cloned and sequenced. Both coding and deduced amino acid sequences were used in phylogenetic analyses. The group of enzymes preferentially expressed in fish erythrocytes (CAIIb) represented the most conserved variant. This result suggests that, although the two variants derived from the same ancestor, CAIIc genes have a more complex evolutionary history than CAIIb. The peculiar distribution of Antarctic CAs among fish CAIIcs suggests that the CAIIc gene appeared at different times through independent duplication events, even after the speciation that led to the differentiation of Antarctic fish families. Using the new CA sequences, we built homology models to trace the expected consequences of sequence variability at the protein structure level. From these analyses, we inferred that sequence variability in Antarctic fish CAs affect important physicochemical properties of these proteins and consequentially influence their reactivity. Furthermore, we searched and tested the validity of various potential molecular trademarks for cold adaptation: significant features that can be related to cold adaptation in fish CAs include reduction of positively charged solvent accessible surfaces and an increased flexibility of N-terminal and C-terminal regions.     

The complete mitochondrial genome of the Antarctic sea spider Ammothea carolinensis (Chelicerata; Pycnogonida)

Mitochondria are responsible for the oxidative phosphorylation process. Accordingly, putatively adaptive changes in their genomic features have been variously associated with major eco-physiological shifts in animal evolution, including increased metabolic rates and heat adaptation. Antarctic pycnogonids offer an interesting system to test whether the selective pressure for heat production and increased aerobic metabolism may be driving genomic changes like: (a) unusual compositional biases at the nucleotide and amino acid level, possibly related to cold adaptation; (b) an accelerated rate of mutations/genomic rearrangements, possibly related to the mutagenic effects of oxygen intermediates. The complete mitochondrial genome (mtDNA) of the Antarctic sea spider Ammothea carolinensis Leach, 1814 (Arthropoda: Pycnogonida), the type species for the genus Ammothea, has been determined and is here compared to known genomes from Antarctic and temperate species. We describe a marked heterogeneity in base composition skewness parameters as well as a strong signature of purifying selection toward an increase in thymines at second codon positions, possibly associated with an increased stability of hydrophobic inter-membrane domains. We further observe a fairly high rate of genomic changes, including a possible hot spot of recombination at the level of tRNA-Q. Nevertheless, these features do not seem to be restricted to the two Antarctic pycnogonids analyzed, as to suggest a causal relationship between cold adaptation and genomic changes, and are better interpreted as basal features shared by the entire group. The relevance of the newly determined sequence for the phylogeny of pycnogonids, including its base composition and genomic rearrangements, is further discussed.     

南极鱼类多样性和适应性进化研究进展

南极地区是地球上唯一未被人类活动大量影响的地区, 其极端寒冷的环境为南极生物的进化提供了“温床”。过去三千万年间, 南极鱼亚目鱼类在南极海洋逐渐变冷的过程中快速进化, 从一个温暖海域的底栖祖先分化成南极海域最为多样化的鱼类类群。由于其在南极圈内和南极圈外的各种温度区间都有分布, 因而成为研究鱼类适应性进化和耐寒机制的良好生物模型。本文综述了有关南极海域鱼类区系组成与物种多样性现状, 南极鱼亚目鱼类适应低温的一系列特化的生物学性状及其关键的遗传进化机制。现有研究表明: 南极鱼类在几千万年零度以下低温环境的进化中发生了大量基因的大规模扩增和基因表达的改变, 如铁调素、卵壳蛋白和逆转座子等118个基因发生了显著的扩增。另外, 有些从南极鱼中获得的抗寒基因已经用于提高动植物低温抗性的研究并取得了良好的效果。在今后的几年中, 将会有多个南极鱼物种的全基因组得到破译, 在低温适应相关基因的功能和进化方面的研究也会更加深入, 这些研究将深入揭示低温压力下基因组的进化规律以及鱼类低温适应的分子机制。     

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.     

A comparison of plasma vitamin C and E levels in two Antarctic and two temperate water fish species

Antarctic fish have a high polyunsaturated lipid content and their muscle cells have a high mitochondria density suggesting that Antarctic fish are under greater oxidative stress than temperate water fish. To test this hypothesis, the plasma concentrations of the antioxidant vitamins E and C were measured in two Antarctic fish species, Pagothenia borchgrevinki and Trematomus bernacchii, and compared with the plasma concentrations of these vitamins in two New Zealand temperate water fish species, blue cod (Parapercis colias) and banded wrasse (Notolabrus fucicola). Neither vitamin is known to be synthesised in fish and so must be obtained from the diet. The plasma from both Antarctic fish species had vitamin E concentrations five to six times higher than those found in the two temperate water fish species. However, significantly higher levels of vitamin C were only found in the plasma of T. bernacchii, a benthic Antarctic fish. The average level of vitamin C in the plasma of the cryopelagic P. borchgrevinki was approximately one-third that of T. bernacchii. The T. bernacchii plasma yielded a high range of vitamin C values, possibly reflecting differences in nutritional status among the animals captured. No beta-carotene was found in any of the fish plasma samples studied. The data suggest that even though Antarctic fish live at -1.5 degrees C they may be exposed to greater metabolic stress from free radical mediated oxidation than temperate water species.     

CHILL-COMA TOLERANCE, A MAJOR CLIMATIC ADAPTATION AMONG DROSOPHILA SPECIES

Abstract.— Most drosophilid species can be classified either as temperate or tropical. Adults of species were submitted to a cold treatment (0°C) and then brought back to ambient temperature. They generally exhibited a chill coma and the time needed to recover was measured. We found in a set of 26 temperate species that recovery was rapid (average 1.8 min, range 0.15–4.9). In contrast, a long recovery time (average 56 min, range 24–120) was observed for 48 tropical species. A few species, like Drosophila melanogaster, are cosmopolitan and can proliferate under temperate and tropical climates. In 9 of 10 such species, slight genetic differences were found: a shorter recovery in temperate than in tropical populations. Comparing physiological data to phylogeny suggests that chill‐coma tolerance has been a recurrent adaptation that is selected for in cold climates but tends to disappear under a permanently warm environment. This major climatic adaptation, evidenced in drosophilids, seems to occur in other insect groups also.     

The heart of the Antarctic icefish as paradigm of cold adaptation

The stenothermal Antarctic fishes, particularly the hemoglobinless icefish, have developed biochemical, metabolic and morpho-functional features of cardiac performance that can help to decipher some mechanisms underlying cardiac cold adaptation. Examples taken from different levels of cardiac biology in Antarctic fish as a paradigm of cold adaptation include: the function of myoglobin in the icefish species that either express or do not express this pigment; the metabolic and ultrastructural reshaping of the myocardiocytes; and the intrinsic mechanical characteristics of the icefish heart ventricle as a low rate, low pressure and high volume pump.     

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.     

Hemoglobin of the Antarctic fishes Trematomus bernacchii and Trematomus newnesi: structural basis for the increased stability of the liganded tetramer relative to human hemoglobin

Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of representative hemoglobins from two Antarctic fishes, Trematomus newnesi (Hb1Tn) and Trematomus bernacchii (Hb1Tb), were determined by analytical ultracentrifugation as a function of pH in the range 6.0-8.6 and compared to HbA. HbA is more dissociated than fish hemoglobins at all pH values and in particular at pH 6.0. In contrast, both fish hemoglobins are mostly tetrameric over the whole pH range studied. The extent of hydrophobic surface area buried at the alpha(1)beta(2) interface upon association of dimers into tetramers and the number of hydrogen bonds formed are currently thought to play a major role in the stabilization of the hemoglobin tetramer. These contributions were derived from the X-ray structures of the three hemoglobins under study and found to be in good agreement with the experimentally determined L(2,4) values. pH affects oxygen binding of T. bernacchii and T. newnesi hemoglobins in a different fashion. The lack of a pH effect on the dissociation of the liganded proteins supports the proposal that the structural basis of such effects resides in the T (unliganded) structure rather than in the R (liganded) one.     

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.     

Hydrocarbons of antarctic midwater organisms

Summary Hydrocarbons have been isolated from most major biomass species of zooplankton and fish in an Antarctic mesopelagic community. Unlike the odd-carbon preference of aliphatic hydrocarbons which typifies terrestrial plants and temperate marine organisms, even carbon chain-length paraffins predominate in 80% of the Antarctic species analyzed. Although the ultimate source of these even carbon n-alkanes cannot be determined from our study, the dominance of these compounds suggests an unusual biochemical pathway may be responsible for their synthesis in this ecosystem.     

Cold-adapted tubulins in the glacier ice worm, Mesenchytraeus solifugus

Glacier ice worms, Mesenchytraeus solifugus and related species, are the only known annelids that survive obligately in glacier ice and snow. One fundamental component of cold temperature adaptation is the ability to polymerize tubulin, which typically depolymerizes at low physiological temperatures (e.g., <10 degrees C) in most temperate species. In this study, we isolated two alpha-tubulin (Msalpha1, Msalpha2) and two beta-tubulin (Msbeta1, Msbeta2) subunits from an ice worm cDNA library, and compared their predicted amino acid sequences with homologues from other cold-adapted organisms (e.g., Antarctic fish, ciliate) in an effort to identify species-specific amino acid substitutions that contribute to cold temperature-dependent tubulin polymerization. Our comparisons and predicted protein structures suggest that ice worm-specific amino acid substitutions stabilize lateral contact associations, particularly between beta-tubulin protofilaments, but these substitutions occur at different positions in comparison with other cold-adapted tubulins. The ice worm tubulin gene family appears relatively small, comprising one primary alpha- and one primary beta-tubulin monomers, though minor isoforms and psuedogenes were identified. Our analyses suggest that variation occurs in the strategies (i.e., species-specific amino acid substitutions, gene number) by which cold-adapted taxa have evolved the ability to polymerize tubulin at low physiological temperatures.