Organization of neuropeptide tyrosine-like immunoreactive system in the brain of the Antarctic fish, Trematomus bernacchii

Antarctic notothenioids have developed unique freezing-resistance adaptations, including brain diversification, to survive in the subzero waters of the Southern Ocean surrounding Antarctica. In this study we have investigated the anatomical distribution of neuropeptide tyrosine (NPY)-like immunoreactive elements in the brain of the Antarctic fish Trematomus bernacchii, by using an antiserum raised against porcine NPY. Perikarya exhibiting NPY-like immunoreactivity were observed in distinct regions of the brain. The most rostral group of immunoreactive perikarya was found in the telencephalon, within the entopeduncular nucleus. In the diencephalon, three groups of NPY-like immunoreactive perikarya were found in the hypothalamus. Two groups of positive cell bodies were found in distinct populations of the preoptic nucleus, whereas the other group was found in the nucleus of the lateral recess. More caudally, NPY immunoreactivity was detected in large neurons located in the subependymal layers of the dorsal tegmentum of the mesencephalon, medially to the torus semicircularis. NPY-like immunoreactive nerve fibres were more widely distributed throughout the telencephalon to the rhombencephalon. High densities of nerve fibres and terminals were observed in several regions of the telencephalon, olfactory bulbs, hypothalamus, tectum of the mesencephalon and in the ventral tegmentum of the rhombencephalon. The distribution of NPY-like immunoreactive structures suggests that, in Trematomus, this peptide may be involved in the control of several brain functions, including olfactory activity, feeding behaviour, and somatosensory and visual information. In comparison with other neuropeptides previously described in the brain of Antarctic fish, NPY is more widely distributed. Our data also indicate the existence of differences in the brain distribution of NPY between Trematomus and other teleosts. In contrast with previous results reported in other fish, Trematomus contains positive fibres in the olfactory bulbs and immunoreactive perikarya in the nucleus of the lateral recess, whereas NPY-immunopositive cell bodies are absent in the thalamus and rhombencephalon, and no NPY immunoreactivity is present in the pituitary. These differences could be related to the Antarctic ecological diversity of notothenioids living at subzero temperatures.     

Olfactory search tracks in the Antarctic fish Trematomus bernacchii

Video observations were made of benthic fish approaching the source of an olfactory plume. Fish (identified as Trematomus bernacchii from their “saddleback” markings) all approached the source from downstream, with relatively straight approach tracks. When the plume was made visible it showed a distinct concentration of the stimulus at the point of release, and a plume which extended down-stream with a rapidly decreasing stimulus gradient. Computer simulations were made that could generate search strategies based on chemical gradient information, and current (rheosensory) information alone or in combination. Only the combination of chemosensory and rheosensory information provided an adequate match to the observed approach tracks. Accepted: 27 August 1998     

Effects of temperature on mitochondrial function in the Antarctic fish Trematomus bernacchii

Effects of temperature on O₂ consumption by mitochondria of the Antarctic fish Trematomus bernacchii were compared with effects obtained with mitochondria from tropical (Sarotheridon mossambica) and temperate zone fishes (Sebastes carnatus and Sebastes mystinus). Arrhenius plots of O₂ consumption versus temperature exhibited slope discontinuities (“breaks”) at temperatures (Arrhenius break temperatures: ABTs) reflective of the species' adaptation temperatures. The ABT for mitochondria of T. bernacchii is the lowest reported for any animal and is ∼12 °C below the value predicted by a regression equation based on ABT data for several invertebrates and fishes. The temperature at which the acceptor control ratio (ACR), an index of efficiency of coupling of electron transport to synthesis of ATP, began to decrease with rising temperature also reflected adaptation temperature. The decrease in ACR with rising temperature began at ∼18 °C for mitochondria of T. bernacchii, in contrast to ∼35 °C for mitochondria of Sarotheridon mossambica. Maintaining T. bernacchii at 4 °C for 2 weeks led to no changes in ABT or in the response of ACR to temperature. The thermal sensitivities of mitochondria of T. bernacchii reflect the high level of cold adaptation and stenothermy that is characteristic of Antarctic Notothenioid fishes. Accepted: 5 January 1998     

Dermal and epidermal chromatophores of the Antarctic teleost Trematomus bernacchii

The physiological response and ultrastructure of the pigment cells of Trematomus bernacchii, an Antarctic teleost that lives under the sea ice north of the Ross Ice Shelf, were studied. In the integument, two types of epidermal chromatophores, melanophores and xanthophores, were found; in the dermis, typically three types of chromatophores--melanophores, xanthophores, and iridophores--were observed. The occurrence of epidermal xanthophore is reported for the first time in fish. Dermal melanophores and xanthophores have well-developed arrays of cytoplasmic microtubules. They responded rapidly to epinephrine and teleost melanin-concentrating hormone (MCH) with pigment aggregation and to theophylline with pigment dispersion. Total darkness elicited pigment aggregation in the majority of dermal xanthophores of isolated scales, whereas melanophores remained dispersed under both light and dark conditions. Pigment organelles of epidermal and dermal xanthophores that translocate during the pigmentary responses are carotenoid droplets of relatively large size. Dermal iridophores containing large reflecting platelets appeared to be immobile.     

Haematological changes in an Antarctic teleost,Trematomus bernacchii,following stress

The effect of an acute increase in temperature, exhaustive exercise and hypoxia on the haematology of the benthic Antarctic teleost, Trematomus bernacchii was investigated. High temperature and hypoxia caused the biggest changes to the blood, with increases in haematocrit, haemoglobin concentrations and plasma chloride levels. The spleen decreased in mass. Exercise produced the smallest changes. Changes were substantially less than reported for the more active cryopelagic species Pagothenia borchgrevinki. The magnitude of the haematocrit increase is discussed in relation to life-style of fish living in the Antarctic.     

Histological observations on lymphomyeloid organs of the Antarctic fish Trematomus bernacchii (Teleostei: Nototheniidae)

Lymphomyeloid organs of the Antarctic fish Trematomus bernacchii were studied with the aim of analysing some morphological aspects related to adaptation to low environmental temperature. The thymus of T. bernacchii was flattened, incompletely lobated and scarcely regionalised. It was filled by lymphoid elements intermingled with stromal elements. The head kidney appeared highly vascularised and mainly lymphopoietic. The spleen appeared mainly erythropoietic, with scarcely developed areas of white pulp. Received: 10 February 1997 / Accepted: 4 May 1997     

Characterization of serum immunoglobulin M of the Antarctic teleost Trematomus bernacchii

Trematomus bernacchii immunoglobulin M concentration was determined in the serum by ELISA; the mean concentration value was 2.7 mg/ml corresponding to 9.6% of the total serum proteins. Purified IgM was analyzed by SDS-polyacrylamide gel electrophoresis, isoelectrofocusing and 2D electrophoresis. The relative molecular mass of the polymeric form was 830 kDa; that of separated H and L chains was, respectively, 78 and 25 kDa. The isoelectric points of the entire molecule ranged from 4.4 to 6.5, that of isolated H chains was between 4.0 and 6.0. Separated H chains were shown to reaggregate in tetrameric form. The cleavage site of trypsin was at the end of the CH1 domain, as confirmed by the N-terminal amino acid sequence of one of the resultant peptides. Immunoblotting was used to detect carbohydrates in the H and L chains labeled with digoxigenin. Glycosyl residues were detected only in the H chain. The carbohydrate content was evaluated to be 12.8% of the entire chain. Purified Igs were hydrolyzed by N-glycosidase F at different conditions and at least four different hydrolytic sites were revealed by limited deglycosylation. T. bernacchii IgM was also compared to those of five other polar fish species.     

Specific features of immunoglobulin VH genes of the Antarctic teleost Trematomus bernacchii

The somatic recombination of different germline-encoded gene segments constitutes a principal source of antibody diversity. In order to investigate the diversity in recombined gene segments encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii, a VH library was constructed by 5'-RACE (rapid amplification of cDNA ends) using RNA isolated from the spleen of an individual specimen. Analysis of cDNA sequences of 45 rearranged VH/D/JH segments revealed specific features, such as: high number of repeats, up to 8 bp long, and palindromic sequences, especially in CDRs (complementary determining regions); occurrence of the RGYW consensus, known as mutational hot spot, higher than in other species. Sixty-four percent of single base substitutions was found within this motif. In addition, the usage of serine codons showed a clear bias for AGY in CDRs, particularly in CDR2, and for TCN in FRs (framework regions). In CDRs, the frequency of non-synonymous changes was higher than that of synonymous changes. Diversity generated by insertions/deletions occurred more often than in other species; inserted bases were often repeats of adjacent bases. In particular the CDR2 showed the highest length variability as compared to other species. Alignment of VH sequences indicated that also the gene conversion mechanism may contribute to generating diversity. These data indicate a CDR mutability higher than in other species and provide some insights into the hypermutational events that may also occur in teleosts.     

Responses of lateral line receptors to water flow in the Antarctic notothenioid, Trematomus bernacchii

The response properties of anterior lateral line afferent neurones in Trematomus bernacchii were recorded extracellularly while stimulating the fish with unidirectional water flows of varying velocity. Afferent neurone responses were either flow-sensitive or flow-insensitive. Flow-sensitive neurones showed linear increases in response magnitude with increasing flow rate and tonic non-adapting response properties. These findings indicate that flow-sensitive afferent neurones originate from lateral line receptors that detect absolute flow velocity. The likely explanation is that flow-sensitive afferent neurones innervate neuromasts located superficially on the skin and flow-insensitive neurones innervate neuromasts situated in sub-epidermal fluid-filled canals.     

Effects of temperature acclimation on cardiorespiratory performance of the Antarctic notothenioid Trematomus bernacchii

Notothenioid fishes of the Southern Ocean have evolved under cold and stable temperatures for millions of years. Due to rising temperatures in the Southern Ocean, investigating thermal limits and the capacities for inducing a temperature acclimation response in notothenioids has become of increasing interest. Here, we investigated effects of temperature acclimation on cardiorespiratory responses and cardiac and skeletal muscle energy metabolism in a benthic Antarctic notothenioid, Trematomus bernacchii. We acclimated specimens to ?1, 2 and 4.5 °C for 14 days and quantified heart rates and ventilation rates during an acute increase in temperature. Ventilation rates showed an effect of acclimation both at initial steady-state acclimation conditions and during an acute temperature increase, suggesting a partial thermal compensatory response. However, acclimation did not affect heart rates at steady-state acclimation conditions and the temperatures at which onset of cardiac arrhythmia occurred, suggesting lack of inducible thermal tolerance in cardiac performance. Citrate synthase (CS), lactate dehydrogenase (LDH) and 3-hydroxyacyl dehydrogenase activities in skeletal muscle tissues suggested acclimation-induced shifts in metabolic fuel preferences, and a marked increase in LDH activity with acclimation to 4.5 °C showed an increase in anaerobic metabolism. In heart tissue, CS and LDH activities decreased with acclimation to 4.5 °C, suggesting reduced cardiac ATP production. Overall, the data suggest a partial acclimatory response to temperature by T. bernacchii and support the hypothesis that reduced cardiac acclimatory capacity may play a role in limiting the thermal plasticity of T. bernacchii.     

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.     

Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.     

Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M-type homopolymer.

Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is composed of a single subunit that contains both the ferroxidase center residues, typical of mammalian H chains, and the carboxylate residues forming the micelle nucleation site, typical of mammalian L chains. Comparison of the amino-acid sequence with those available from lower vertebrates indicates that T. bernacchii ferritin can be classified as an M-type homopolymer. Interestingly, the T. bernacchii ferritin chain shows 85.7% identity with a cold-inducible ferritin chain of the rainbow trout Salmo gairdneri. The structural and functional properties indicate that cold acclimation and functional adaptation to low temperatures are achieved without significant modification of the protein stability. In fact, the stability of T. bernacchii ferritin to denaturation induced by acid or temperature closely resembles that of mesophilic mammalian ferritins. Moreover iron is taken up efficiently and the activation energy of the reaction is 74.9 kJ.mol(-1), a value slightly lower than that measured for the human recombinant H ferritin (80.8 kJ.mol(-1)).     

Protonation of histidine 55 affects the oxygen access to heme in the alpha chain of the hemoglobin from the Antarctic fish Trematomus bernacchii

The Root effect describes the drastic drop of oxygen affinity and loss of cooperativity at acidic pH expressed in the hemoglobins (Hb) of certain fish. The comparison between the deoxy structures of the Root effect Hb from the Antarctic fish Trematomus bernacchii (HbTb) at different pHs (pH = 6.2 and pH = 8.4) shows that the most significant differences are localized at the CDα region, where a salt bridge between Asp48 and His55 breaks during the low-to-high pH transition. In order to shed light on the relationship between pH, CDα loop structure and dynamics, and oxygen access to the active site in the alpha chain of HbTb, different computer simulation techniques were performed. Our results highlight the importance of the protonation of His55 in regulating oxygen access, underscoring its pivotal role in the structural and functional properties of HbTb. These data provide further support to the hypothesis that this residue might contribute to the release of Root protons in HbTb and underline the fact that an efficient transport of molecular oxygen in Hbs relies on a subtle balance of tertiary structure and protein conformational flexibility.     

The effect of temperature on catalytic and regulatory functions of pyruvate kinases of the rainbow trout and the Antarctic fish Trematomus bernacchii

1. The effects of temperature on the catalytic and regulatory properties of pyruvate kinases from the temperate-zone rainbow trout and the Antarctic fish Trematomus bernacchii were examined. 2. The K(m) value of pyruvate kinase for one of its two substrates, phosphoenolpyruvate, is temperature-dependent, and is lowest at temperatures that closely coincide with the habitat temperatures of the two fishes. 3. Two regulatory functions of pyruvate kinase, feedforward activation by fructose diphosphate and feedback inhibition by ATP, are temperature-independent. Enzyme-ADP interaction is also temperature-independent. 4. It is concluded that enzyme-substrate and enzyme-modulator interactions are important factors in short-term and in evolutionary adaptations by poikilotherms to changes in temperature. Though the K(m) for substrate may vary in apparently adaptive manners, the regulatory functions of an enzyme appear to be unchanged over the range of temperatures experienced by the organism in Nature.     

Analysis of a cDNA sequence encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii

A spleen cDNA library was constructed from the Antarctic teleost Trematomus bernacchii and immunoscreened with rabbit IgG specific for T. bernacchii Ig heavy chain. Eleven cDNA clones, varying in size and encoding the entire heavy chain or parts of it, were isolated. Here the complete nucleotide and deduced amino acid sequences of clone 2C2 encoding the secretory IgH chain form are reported. Comparison of the amino acid sequence of the entire constant region of the T. bernacchii Ig heavy chain with those from other teleosts and two holostean fish showed percent identity ranging 53.6-60.6%, with the highest values found for Salmoniformes. The multiple sequence alignment revealed the presence of two remarkable insertions: one at the VH-CH1 boundary and a second one, not found in any other IgM heavy chain, localised at the CH2-CH3 boundary. The latter occurred in the region proposed to act as a 'hinge', and resulted in a CH2-CH3 hinge peptide longer than any other IgM hinge. Differences were also found in the number and position of putative N-glycosylation sites of the compared sequences. It is suggested that the unusual features found in the T. bernacchii Ig heavy chain might contribute to the flexibility of the Ig molecule and help understand more about the adaptation of Ig molecules to the polar sea environment.     

Ion transport systems in the kidney and urinary bladder of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii

Na⁺-K⁺-Cl^- cotransport and Na⁺/K⁺ATPase were studied by immunohistochemistry in the kidney and urinary bladder of Trematomus bernacchii and Chionodraco hamatus. The activity was correlated to the density of mitochondria. The first segment of the renal proximal tubule was more active than the second one. In T. bernacchii and the temperate marine teleost Pagellus bogaraveo, the immunoreactivity for the antibody to cotransporters and to the !-subunit of the sodium pump was stronger than in the icefish. This difference indicates in the kidney of the icefish, a weaker secretory activity, a consequent lower osmolarity in the lumen and lower water loss, which correlates well with the need for a greater blood volume in the icefish. The epithelium of the urinary bladder in T. bernacchii, where intense immunostaining was observed, was composed of columnar cells. In C. hamatus the columnar cells, where the immunostaining was weaker, lined only a portion of the urinary bladder, the other region being composed of cuboidal cells.     

Immunocytochemical observations on pineal organ and retina of the Antarctic teleosts Pagothenia borchgrevinki and Trematomus bernacchii

In spite of the unique conditions they have to operate under, the pineal organs of Antarctic fishes have not previously been examined. We determined immunohistochemically that in the end-vesicles and the pineal stalks of Pagothenia borchgrevinki (a species found directly beneath the sea-ice) as well as Trematomus bernacchii (a species preferring somewhat deeper water than the former) at least two populations of physiologically-different cells occurred that displayed reactions indicative of typical vertebrate photoreceptors. Comparisons with immunocytochemically treated retinal sections from the eyes of the same two species showed that anti-opsin reactivity, characteristic of rods, was particularly strong in the lumina of the pineal stalks of both species. Anti-visinin reactions stained cones in the retinal sections of both fishes and occurred throughout the pineal organs, but in particular in the end vesicles of the pineals of both species. The difference in preferred habitat depth between the two species appears to have had very little influence on both retinal and pineal immunocytochemistry. It is concluded that the pineal organs of both species, at least during the austral summer, exhibit signs of being directly photo-sensitive.