Bacterial flora of newly caught Antarctic fish Notothenia neglecta

Summary The heterotrophic bacterial flora of Antarctic fish Notothenia neglecta was studied in Potter Cove (King George Island, South Shetland Islands). Quantitative and qualitative analysis of aerobic bacteria from sea water, skin, stomach and intestine were carried out. In addition, anaerobic flora of stomach and intestine was studied and compared. Pseudomonas was dominant in sea water samples but, neither skin nor digestive tract samples showed to be rich in this genus. Stomach flora showed variable results between samples. The gut flora was composed almost exclusively of Vibrio. Despite extreme environmental conditions this Antarctic fish show an intestinal indigenous microflora very similar to warmer waters fish. Several factors, that we discussed, could be determining the Vibrio dominance in the gut of marine teleosts.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

Karyotypes of two Antarctic fishes,Notothenia gibberifrons andNotothenia coriiceps neglecta

The chromosomes of two species of Antarctic fishes,Notothenia (Gobionotothen) gibberifrons andNotothenia (Notothenia) coriiceps neglecta, were prepared by the air-drying method at the Polish Antarctic Station “Henryk Arctowski” during the austral summer 1984–1985. ForN. (G.) gibberifrons the diploid number is2n = 46 consisting of 2 metacentric (m) pairs, 1 submetacentric (sm) pair and 20 telocentric (t) or subtelocentric (st) pairs. ForN. (N.) coriiceps neglecta the diploid number is 2n = 22 consisting of 9 m pairs, 1 sm pair and 1st pair. Some aspects of karyological evolution of these fishes are discussed.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 2. Amino acid sequence of the alpha chain of Hb1

The complete amino acid sequence of the alpha chain of the main hemoglobin of the Antarctic fish Notothenia coriiceps neglecta (family Nototheniidae) has been determined. It consists of 142 residues; an acetylated seryl residue is at the amino terminal. The molecular mass is 15,519 Da. In comparison with alpha-chain sequences of non-Antarctic poikilothermic fish hemoglobins, the homology appears to be significantly lower than that existing among the latter species. A higher homology has been found with the alpha-chain sequence of the non-poikilothermic bluefin tuna.     

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.     

The amino acid sequence of the alpha chain of HB 2 completes the primary structure of the hemoglobins of the Antarctic fish Notothenia coriiceps neglecta

1. The blood of Notothenia coriiceps neglecta (a cold-adapted notothenioid fish, widely distributed in Antarctic waters, and characterized by a relatively low content of erythrocytes and hemoglobin), contains two hemoglobin components, Hb 1 and Hb 2; the amino acid sequences of the beta chain of Hb 1 and Hb 2 are identical. 2. The amino acid sequence of the alpha chain of Hb 2 has been established, thus completing the elucidation of the primary structure of the two hemoglobins.     

Hemoglobin from the antarctic fish Notothenia coriiceps neglecta. Amino acid sequence of the beta chain

1. Notothenia coriiceps neglecta is a cold-adapted notothenioid teleost, widely distributed in the Antarctic waters. 2. In comparison with fishes from temperate waters, the blood of this teleost contains a reduced number of erythrocytes and concentration of hemoglobin; the erythrocytes contain two hemoglobins, Hb1 and Hb2, respectively accounting for approximately 90, and 5% of the total. 3. The two components differ by the alpha chain; the amino acid sequence of the beta chain in common to the two hemoglobins has been established, thus completing the elucidation of the primary structure of the major component Hb 1.     

Gill morphometrics of two Antarctic fish species Pleuragramma antarcticum and Notothenia gibberifrons

Summary Gill dimensions of 27 juvenile and adult Pleuragramma antarcticum from the southern Weddell Sea and of 28 juvenile and adult Notothenia gibberifrons from the South Orkney and South Shetland Islands were estimated. The unit gill area (UGA) of P. antarcticum ranged from 75 to 167 mm²/g (mean=105); gill area index (GAI) and water-blood distance (WBD) were found to be 1.38 cm² and 3.3 m, respectively. The exponent d_g in the relationship total gill area to weight was found to be 0.90. It is concluded that P. antarcticum belongs to sluggish species and, although pelagic, its routine energy costs tend to be low. However, the closely packed lamellae (NL/mm=21) indicate more active behaviour in comparison with N. gibberifrons. A preliminary estimation of growth parameters (P, k) is presented. The sluggish behaviour of N. gibberifrons, as expected from its benthic mode of life, is reflected by the gill parameters: the very low UGA ranged from 39 to 118 mm²/g; GAI and d_g were found to be 0.73 cm² and 0.98, respectively. Comparing gill dimensions and general respiration characteristics of fish from antarctic, temperate and tropical waters it is concluded that antarctic fish have increased their scope for activity.     

Characterization of the cytoplasmic chaperonin containing TCP-1 from the Antarctic fish Notothenia coriiceps

The cytoplasmic chaperonin containing TCP-1 (CCT) plays a critically important role in the folding and biogenesis of many cytoskeletal proteins, including tubulin and actin. For marine ectotherms, the chronically cold Southern Ocean (−2 to +2°C) poses energetic challenges to protein folding, both at the level of substrate proteins and with respect to the chaperonin/chaperone folding system. Here we report the partial functional and structural characterization of CCT from an Antarctic notothenioid fish, Notothenia coriiceps. We find that the mechanism of folding by the Antarctic fish CCT differed from that of mammalian CCT: (1) the former complex was able to bind denatured β-tubulin but (2) when reconstituted with rabbit Cofactor A, failed to release the protein to yield the tubulin/cofactor intermediate. Moreover, the amino acid sequences of the N. coriiceps CCT β and θ chains contained residue substitutions in the equatorial, apical, and intermediate domains that would be expected to increase the flexibility of the subunits, thus facilitating function of the chaperonin in an energy poor environment. Our work contributes to the growing realization that protein function in cold-adapted organisms reflects a delicate balance between the necessity of structural flexibility for catalytic activity and the concomitant hazard of cold-induced denaturation.     

Solution structure of MT_nc,a novel metallothionein from the Antarctic fish Notothenia coriiceps

The structure of [113Cd(7)]-metallothionein (MT_nc) of the Antarctic fish Notothenia coriiceps, the first three-dimensional structure of a fish metallothionein, was determined by homonuclear 1H NMR experiments and heteronuclear [1H, 113Cd]-correlation spectroscopy. MT_nc is composed of an N-terminal beta domain with 9 cysteines and 3 metal ions and a carboxy-terminal alpha-domain with 11 cysteines and 4 metal ions. The position of the ninth Cys of the alpha domain of MT_nc is different from the corresponding Cys of mammalian MTs. As a result, the last CXCC motif in the mammalian MT sequence becomes CXXXCC in the fish MT. This difference leads to a structural change of the alpha domain and, in turn, to a different charge distribution with respect to that observed in mammalian metallothioneins.     

Fat absorptive processes in the intestine of the Antarctic fish Notothenia coriiceps (Richardson, 1844)

Absorption of lipids by the enterocytes of Notothenia coriiceps, an omnivorous Antarctic and subAntarctic fish, was studied by light and electron microscopy. The lipids are absorbed by the anterior and middle intestine segments. They appear as fat droplets that measure from 0.5 to 7 μm of diameter and which accumulate in the apical cytoplasm within the first 24 h and seem to be the main fat storage form in the enterocytes. Fat is also observed as lipid particles with 60–300 nm inside the rough endoplasmic reticulum and cytoplasmic smooth vesicles. The epithelial intercellular space and the mucosal inner lamina contain lipid particles, which probably are the fat transport form. Our observations show that an intense lipid absorptive process takes place in N. coriiceps digestive system, due to the great extension of the intestine involved and due to the great lipid accumulation found in the epithelial compartment.     

Metal (Fe, Zn, Mn and Cu) levels in the Antarctic fish Notothenia coriiceps

During the 1994/1995 Antarctic summer 20 Antarctic cod (Notothenia coriiceps) of both sexes were collected at King George Island, South Shetland Islands, Antarctica, to determine the levels of Fe, Zn, Mn and Cu in muscle, liver, gonad and kidney. Metal levels were assessed using the inductively coupled plasma-atomic emission spectrometry (ICP-AES) technique. The levels of Fe, Zn, Mn and Cu observed in both sexes of N. coriiceps were significantly lower in muscle tissue than in liver, kidney and gonad; the livers of males and gonads of females had the highest levels. The differences between the sexes were significant for Zn and Mn in gonads and for Cu in liver. Despite the modest size-range of the specimens, the Cu levels in liver, muscle and kidney were significantly inversely correlated with female mass (P < 0.05) as was gonad Cu level and male body length. Fe mainly presented the highest concentrations and Cu and Mn the lowest ones. Mean muscle metal levels are similar to those of hake, cod, herring and mackerel; however, the level of Fe was slightly higher. Accumulation may occur for Fe, Zn and Mn in liver, kidney and gonad, which have higher levels than those of most prey. Received: 21 October 1997 / Accepted: 1 February 1998     

Specific immunity proteomic profile of the skin mucus of Antarctic fish Chionodraco hamatus and Notothenia coriiceps

The white-blooded Antarctic icefish is the only known vertebrate lacking oxygen-transporting haemoglobins. Fish skin mucus, as the first line of defence against pathogens, can reflect fish welfare. In this study, we analysed the skin mucus proteome profiles of the two Antarctic fish species, the white-blooded Antarctic icefish, Chionodraco hamatus, and the red-blooded Antarctic fish, Notothenia coriiceps, unfolding the different proteins by liquid chromatography coupled with tandem mass spectrometry isobaric tags for relative and absolute quantitation (iTRAQ) technology. Of the 4444 totally identified proteins, 227 differentially expressed proteins (DEPs) were found in the comparison between C. hamatus and N. coriiceps, of which 121 were upregulated and 106 were downregulated in the icefish. In the Kyoto Encyclopedia of Genes and Genomes pathway annotation, we found two pathways “Legionellosis” and “Complement and coagulation cascades” were significantly enriched, among of which innate immune candidate proteins such as C3, CASP1, ASC, F3 and C9 were significantly upregulated, suggesting their important roles in C. hamatus immune system. Additionally, the DEP protein–protein interaction network analysis and “Response to stress” GO category provided candidate biomarkers for deep understanding of the distinct immune response of the two Antarctic fish underlying the cold adaptation.     

Comparison of fatty acid composition in nine organs of the sympatric Antarctic teleost fish species Notothenia coriiceps and Notothenia rossii (Perciformes: Nototheniidae)

Fatty acid (FA) composition of nine organs from two closely related Antarctic fish species, Notothenia coriiceps and Notothenia rossii, was determined through gas chromatography with flame ionization detection. A data set for each species was obtained using major FA profiles from specimens caught in the sea waters of Admiralty Bay during the summer season. The FA profiles for both species are overall similar, but organ peculiarities have been found, which could reflect metabolic specificities and feeding habits between species. With the exception of liver, the most abundant FA in organs was the n?3 polyunsaturated FA. The total n?6 polyunsaturated FAs were minor components in all evaluated organs. Palmitic acid was identified as the major saturated FA, whereas oleic acid was the most represented of the monounsaturated FA in almost all assessed organs of both species. The n? 3/n? 6 ratios of all organs were higher than 3.5. Differences in individual FA and FA metabolic profiles of some organs observed between N. coriiceps and N. rossii suggest specific requirements in the mobilization, transport, incorporation, and/or catabolism of lipids that were reinforced by differences on some FA ratios expressing the activity coefficient of enzymes implicated on the FA pathway flux.     

Rhythmic structural and chemical patterns in otoliths of the Antarctic fish Notothenia larseni: Their application to age determination

Summary Sagittal otoliths from Notothenia larseni contain microincremental growth rings which are distinctly visible in otolith sections using Scanning Electron Microscopy. These microincrements are similar to those deposited daily in the otoliths of fishes from temperate and tropical waters. Microincrements were easily enumerated and fish length was related to increment number by a logarithmic curve. Otolith microstructure analysis appears to provide a technique to accurately determine age and growth rates in these fish. Sr/Ca ratios in otolith aragonite were analyzed along a radius from the outside edge to the core of an otolith section using an electron microprobe. The strontium/calcium (Sr/Ca) ratios varied with a cyclic periodicity apparently related to seasonal water temperature cycles and the number of cycles agreed closely with age estimated from daily microincrement counts. Sr/ca cycles can potentially be used to determine age, validate growth rates determined by other methods, and establish thermal conditions experienced during the life of a fish. Microstructural and chemical analyses of otoliths demonstrate great potential in helping to answer many questions about the growth processes and ecology of Antarctic fishes.     

The oxidation process of Antarctic fish hemoglobins.

Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation process, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectroscopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemoglobins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemichrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly transformed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquomet and the hemichrome. In order to assign the heme region state of the alpha and beta chains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an alpha(aquomet)-beta(bishistidyl-hemichrome) state. This demonstrates that the alpha and beta chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrome state [Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A. & Mazzarella, L. (2002) Proc. Natl Acad. Sci. USA99, 9801-9806], the quaternary structures of these alpha(aquomet)-beta(bishistidyl-hemichrome) forms are intermediate between the physiological R and T hemoglobin states. Together, these structures provide information on the general features of this intermediate state.     

The hemoglobins of Notothenia angustata, a temperate fish belonging to a family largely endemic to the Antarctic Ocean.

The blood of the teleost Notothenia angustata contains a major hemoglobin (Hb 1, over 95% of the total), accompanied by a minor component (Hb 2). The two hemoglobins have identical beta chains and differ in their alpha chains. The primary structure of both hemoglobins has been established through the elucidation of the complete amino acid sequence of the three chains. The study of the oxygen-binding properties shows that Hb 1 displays the Bohr and Root effects and has high affinity for organic phosphates. N. angustata belongs to the family Nototheniidae, suborder Notothenioidei. Unlike the vast majority of nototheniid species, which live in isolation in the Antarctic Ocean and have developed cold adaptation, N. angustata inhabits the waters of southern New Zealand and is not cold adapted. Although some hematological parameters typically favour oxygen transport in a temperate environment, the hemoglobin multiplicity and structural and functional features closely resemble those of the Antarctic species of the same family and suborder. Thus, N. angustata may be considered as a link between temperate and Antarctic habitats. The hypothetical separation history of N. angustata from the Antarctic species of the same family is discussed in the light of the present findings.