Effects of warm acclimation on serum osmolality,cortisol and hematocrit levels in the Antarctic fish, <Emphasis Type="Italic">Trematomus bernacchii</Emphasis>

Antarctic fish, such as the Trematomus bernacchii, living at −1.9°C maintain a serum osmolality of around 600 mOsm kg⁻¹, nearly twice that of temperate fish. Upon warm acclimation, Antarctic fish significantly lower their serum osmolality. It has been suggested that this response to warm acclimation is due to stress. The purpose of this study was to determine, whether upon warm acclimation there was a change in the levels of the stress hormone cortisol and hematocrit associated with the decrease in serum osmolality. T. bernacchii were warm acclimated up to 4 weeks and serum osmolality, cortisol and hematocrit were measured. Upon warm acclimation to +1.6 and +3.8°C over the course of 4 weeks, T. bernacchii significantly lowered their serum osmolality (from 547 ± 4 mOsm kg⁻¹ to 494 ± 6 and 489 ± 4 mOsm kg⁻¹, respectively), yet did not alter their serum cortisol (29 ± 6 nl ml⁻¹) or hematocrit (22 ± 1%) levels. These results suggest that warm acclimation does not induce a stress response in T. bernacchii.     

Freezing avoidance of the Antarctic icefishes (Channichthyidae) across thermal gradients in the Southern Ocean

Biogeographic studies separate the Antarctic Notothenioid fish fauna into high- and low-latitude species. Past studies indicate that some species found in the high-latitude freezing waters of the High-Antarctic Zone have low-serum hysteresis freezing points, while other species restricted to the low-latitude seasonal pack ice zone have higher serum hysteresis freezing points above the freezing point of seawater (−1.9°C), but the relationship has not been systematically investigated. Freeze avoidance was quantified in 11 species of Antarctic icefishes by determining the hysteresis freezing points of their blood serum, in addition, the freezing point depression from serum osmolytes, the antifreeze activity from serum antifreeze glycoproteins (AFGPs), and the antifreeze activity from serum antifreeze potentiating protein were measured for each species. Serum hysteresis freezing point, a proxy for organismal freeze avoidance, decreased as species were distributed at increasing latitude (linear regression r ² 0.66, slope −0.046°C °latitude⁻¹), which appeared largely independent of phylogenetic influences. Greater freeze avoidance at high latitudes was largely a result of higher levels of antifreeze activity from serum AFGPs relative to those in species inhabiting the low-latitude waters. The icefish fauna could be separated into a circum High-Antarctic Group of eight species that maintained serum hysteresis freezing points below −1.9°C even when sampled from less severe habitats. The remaining three species with low-latitude ranges restricted to the waters of the northern part of the west Antarctic Peninsula and Scotia Arc Islands had serum hysteresis freezing points at or above −1.9°C due to significantly lower combined activity from all of their serum antifreeze proteins than found in the High-Antarctic Zone icefish.     

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.     

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     

Thin-blooded Antarctic fishes: a rheological comparison of the haemoglobin-free icefishes Chionodraco kathleenae and Cryodraco antarcticus with a red-blooded nototheniid, Pagothenia bernacchii

The icefishes (family Channichthyidae) comprise a unique group of teleost fishes endemic to Antarctic and sub-antarctic seas. All members of the family totally lack haemoglobin. Haematological parameters and viscosity were determined for blood from 11 specimens of two channichthyid species (Chionodraco kathleenae Regan, 1914; Cryodraco antarcticus Dollo, 1900), and 14 specimens of a red-blood Antarctic nototheniid species (Pagothenia bernacchii (Boulenger, 1902)), captured near the Italian research station at Terra Nova Bay, Ross Sea, Antarctica. Channichthyid blood contained only a small number of non-pigmented cells (10 000-40 000 cells μI^?1, depending on species) in contrast to nototheniid blood (360 000-450 000 cells μI^?1 in unstressed specimens). Blood viscosity was measured by cone plate viscometry over a range of shear rates (11.3-450s ^?1), at six temperatures between – 1.8°C and + 15°C. At the ambient Antarctic seawater temperature of – 1.8° C, and at low shear rate (22.5 s^?1), the viscosity of channichthyid blood was relatively low (3.99 ± 0.40 cP) compared with blood taken from unstressed P. bernacchii, which was about 25% more viscous (4.91 ± 0.59 cP). The viscosity of channichthyid blood was almost independent of shear rate, approximating an ideal Newtonian fluid, while the viscosity of nototheniid blood was much more dependent upon both shear rate and temperature, increasing sharply at low shear rates and low temperatures. Viscosity of nototheniid blood varied with haematocrit, which was in turn strongly influenced by stress. Blood samples taken from P. bernacchii under moderate stress induced by handling during acute caudal venepuncture had haematocrit values in the range 15–20% and viscosities of 8-l0cP, while undisturbed specimens sampled through a venous cannula yielded haematocrits of 8–10%. The viscosity of nototheniid plasma did not differ significantly from that of channichthyid whole blood or channichthyid plasma. The higher viscosity of nototheniid blood is attributable to cell content, and in stressed specimens possibly also to adrenergic swelling of erythrocytes. The absence of erythrocytes in channichthyid blood avoids the great increases in viscosity which are induced in corpusculate blood by sub-zero seawater temperatures.     

Blood Lipids in Antarctic and in Temperate-Water Fish Species

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 ω-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, ω-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.     

Inducible heat tolerance in Antarctic notothenioid fishes

Significant increases in heat tolerance (time of survival at 14°C) were observed for some, but not all, species of notothenioid fishes collected from McMurdo Sound, Antarctica (77°51′S) following acclimation to 4°C. The increase in thermal tolerance was rapid in Trematomus bernacchii, developing within 1–2 days of acclimation to 4°C. Long-term (6–8 weeks) acclimation to 4°C led to greater heat tolerance in Trematomus pennellii than in T. bernacchii. Unlike its demersal congeners, the cryopelagic notothenioid Pagothenia borchgrevinki did not increase heat tolerance during warm acclimation. A deep-living zoarcid fish, Lycodichthys dearborni, also failed to increase heat tolerance, but survived significantly (> threefold) longer at 14°C than the notothenioids.     

Induction of mixed function oxidase (MFO) system in two species of antarctic fish from Terra Nova Bay (Ross Sea)

Summary The aim of this study was to investigate the detoxicant Mixed Function Oxidase system in two species of Antarctic fish, C. hamatus and P. bernacchii, collected during the Antarctic summer of 1989–1990 at the Italian Scientific Station at Terra Nova Bay (Ross Sea). Several specimens were induced by injection of phenobarbital (PB), 3-methylcholanthrene (3-MC) and PCBs in the caudal vein. The results show significant differences between the two species. In C. hamatus, basal activity of benzo(a)pyrene monooxygenase (BPMO) was among the lowest measured even in fish of temperate seas, and in P. bernacchii it was 20 times lower. The values of regenerating activities (NADPH-cytochrome c reductase (NADPH-CYTCRED), NADH-cytochrome c reductase (NADH-CYTCRED) and NADH-ferricyanide reductase (NADH-FERRIRED) suggest that the two species use different electron donor molecules. Injection of chemicals in the caudal vein did not provoke induction of MFO activity in P. bernacchii. In C. hamatus, phenobarbital and PB-type inducers did not cause induction but there was a statistically significant response to 3-MC.     

Qualitative and quantitative analysis of serum immunoglobulins of four Antarctic fish species

Immunoglobulins from the Antarctic fish species Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi, and Chionodraco hamatus were analysed in whole serum and after purification by affinity chromatography on protein A-sepharose. Using SDS-PAGE, the apparent masses of the heavy and light chains were, respectively, 83.5 kDa and 27.5 kDa for T. bernacchii, 83.5 kDa and 27 kDa for T. hansoni, 81 kDa and 27.5 kDa for T. newnesi, and 74.5 kDa and 30 kDa for C. hamatus. It was not possible to purify immunoglobulins from T. newnesi due to their low concentration in serum. Heterogeneity in mass of both heavy and light chains was observed in all species. By using a polyclonal antibody raised against sea bass immunoglobulins, cross-reactivity was observed with heavy and light chains of all species. With this antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and results showed the relative immunoglobulin concentration in sera of the Antarctic fish species considered, using as standard sea bass immunoglobulins. Received: 25 November 1996 / Accepted: 3 February 1997     

Key enzymes of the kallikrein-kinin system in Antarctic teleosts

In this study, some of the mammalian kallikrein-kinin system (KKS)-like components were identified in two species of Antarctic notothenioid [Chionodraco hamatus (Channichthydae) and Trematomus bernacchii (Nothothenidae)]. The kidney and heart were assayed for kallikrein-like activity using the synthetic substrate d-Val-Leu-Arg-paranitroanilide. Values expressed as nmol p-nitroanilide/mg proteins, were in C. hamatus 15.5±1.5 and 15.2±1.4 in kidney and heart, respectively, and 15.8±2.2 and 15.9±1 in kidney and heart of T. bernacchii. Kallikrein-like activity was inhibited bvy aprotinin and phenylmethylsulfonyl fluoride (PMSF). The assay was stable at 20°C. Kininase II-like activity was performed on kidney, gills and heart using the substrate hippuryl-lhistidyl-l-leucine. The activity was inhibited by captopril, and in kidney and gills by high temperatures (20°C and 37°C); in the heart the enzymatic activity was measurable also at 20°C. Bradykinin-like immunoreactive cells were localized by immunohistochemistry in the nephron, in the gills, and in the arterial walls of the heart. These results show that Antarctic teleosts possess elements comparable to those of the KKS, including kallikrein-like, and kininase II-like activities, and the end product of the enzymatic cascade, bradykinin. The enzymatic cascade appears to be fully active only at low temperatures. Received: 19 April 1996/Accepted: 30 June 1996     

Comparison of angiotensin converting enzyme-like activity in the Antarctic teleosts <Emphasis Type="Italic">Trematomus bernacchii</Emphasis> and <Emphasis Type="Italic">Chionodraco hamatus</Emphasis>

Biochemical parameters of the angiotensin converting enzyme-like activity (ACELA) in the gills of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii were characterized. Enzymatic activity was revealed following hydrolysis of a specific substrate of angiotensin-converting enzyme N-[3-(2-furyl)acryloyl]l-phenylalanyl-glycyl-glycine (FAPGG) and metabolites were separated by reverse phase HPLC analysis. The results showed similar Km values for the substrate FAPGG at 5°C for the two species with an increase of Km value for T. bernacchii at 25°C. The optimum pH value was 8.5 at 25°C and optimum chloride concentrations were about 300 mM. In T. bernacchii the optimum temperature for maximum enzyme activity was 50°C, while maximum activity in C. hamatus occurred at 35°C. Lisinopril was more efficient in inhibiting ACELA in C. hamatus with an I ₅₀ value of 16.83 ± 5.11 nM, compared to an I ₅₀ value of 30.66 ± 5.19 nM in T. bernacchii. In conclusion, it appears that some biochemical parameters of ACELA in C. hamatus differ from those in T. bernacchii, probably due to different ways that the enzyme adapts to the constantly cold temperatures of the animal’s environment.     

A comparison of myocardial β-adrenoreceptor density and ligand binding affinity among selected teleost fishes

This study quantified the cell surface β-adrenoreceptor density and ligand binding affinity in the ventricular tissue of seven teleost species; skipjack tuna (Katsowonus pelamis), yellowfin tuna (Thunnus albacares), Pacific mackerel (Scomber japonicus), mahimahi (dolphin fish; Coryphaena hippurus), sockeye salmon (Oncorhynchus nerka), rainbow trout (Oncorhynchus mykiss) and an Antarctic nototheniid (Trematomus bernacchii). β-Adrenoreceptor density varied by almost fourfold among these species, being highest for the athletic fish: sockeye salmon among the salmonids and skipjack tuna among the scombrids. β-Adrenoreceptor density was lowest for the Antarctic icefish. β-Adrenoreceptor binding affinity varied by almost threefold. We conclude that there is a significant species-specific variability in myocardial β-adrenoreceptor density and binding affinity and these interspecific differences cannot be attributed to temperature even though intraspecifically cold temperature can stimulate an increase in myocardial β-adrenoreceptor density. Instead, we suggest that interspecifically myocardial β-adrenoreceptor density is highest in fish that inhabit tropical water. Accepted: 12 July 2000     

Osmotic regulation in aqueous humor and blood in the euryhaline fish,Eugerres plumieri

• 1.1. The estuarine fish Eugerres plumieri was acclimated to sea-water concentrations ranging from 6 to 85% sea-water.
• 2.2. Serum and aqueous humor osmolalities were moderately well regulated over the range of concentrations studied.
• 3.3. Serum osmolality and aqueous humor osmolalities conformed to the following relations: serum osmolality = (319 ± 3) + (0.56 ± 0.03) (% sea-water); aqueous humor osmolality = (314 ± 4) + (0.35 ± 0.04) (% sea-water).
• 4.4. Aqueous humor osmolality was more strictly regulated than that of serum, indicating that the retina and nervous system of the fish, which are encased in inextensible structures, are well protected from variations in sea-water concentration in order to minimize osmotically induced changes in cell volume.

     

Mitochondrial function in Antarctic notothenioid fishes that differ in the expression of oxygen-binding proteins

State III respiration rates were measured in mitochondria isolated from hearts of Antarctic notothenioid fishes that differ in the expression of hemoglobin (Hb) and myoglobin (Mb). Respiration rates were measured at temperatures between 2 and 40°C in Gobionotothen gibberifrons (+Hb/+Mb), Chaenocephalus aceratus (–Hb/–Mb) and Chionodraco rastrospinosus (–Hb/+Mb). Blood osmolarity was measured in all three species and physiological buffers prepared for isolating mitochondria and measuring respiration rates. Respiration rates were higher in mitochondria from G. gibberifrons compared to those from C. aceratus at 2°C, but were similar among all species at temperatures between 10 and 26°C. Respiration rates were significantly lower in icefishes at 35 and 40°C compared to G. gibberifrons. The respiratory control ratio of isolated mitochondria was lower in C. aceratus compared to G. gibberifrons at all temperatures below 35°C. At 35 and 40°C, mitochondria were uncoupled in all species. The Arrhenius break temperature of state III respiration was similar among all three species (30.5 ± 0.9°C) and higher than values previously reported for Antarctic notothenioids, likely due to the higher osmolarity of buffers used in this study. These results suggest that differences in mitochondrial structure, correlated with the expression of oxygen-binding proteins, minimally impact mitochondrial function.     

Is cold the new hot? Elevated ubiquitin-conjugated protein levels in tissues of Antarctic fish as evidence for cold-denaturation of proteins in vivo

Levels of ubiquitin (Ub)-conjugated proteins, as an index of misfolded or damaged proteins, were measured in notothenioid fishes, with both Antarctic (Trematomus bernacchii, T. pennellii, Pagothenia borchgrevinki) and non-Antarctic (Notothenia angustata, Bovichtus variegatus) distributions, as well as non-notothenioid fish from the Antarctic (Lycodichthys dearborni, Family Zoarcidae) and New Zealand (Bellapiscis medius, Family Tripterygiidae), in an effort to better understand the effect that inhabiting a sub-zero environment has on maintaining the integrity of the cellular protein pool. Overall, levels of Ub-conjugated proteins in cold-adapted Antarctic fishes were significantly higher than New Zealand fishes in gill, liver, heart and spleen tissues suggesting that life at sub-zero temperatures impacts protein homeostasis. The highest tissue levels of ubiquitinated proteins were found in the spleen of all fish. Ub conjugate levels in the New Zealand N. angustata, more closely resembled levels measured in other Antarctic fishes than levels measured in other New Zealand species, likely reflecting their recent shared ancestry with Antarctic notothenioids.     

Effects of aging on thermoregulatory responses and hormonal changes in humans during the four seasons in Japan

Physiological functions are impaired in various organs in aged people, as manifest by, e.g., renal and cardiac dysfunction and muscle atrophy. The elderly are also at increased risk of both hypothermia and hyperthermia in extreme temperatures. The majority of those over 65 years old have elevated serum osmolality. Our hypothesis is that the elderly have suppressed osmolality control in different seasons compared to the young. Eight healthy young men and six healthy older men participated in this study. The experiments were performed during spring, summer, autumn and winter in Japan, with average atmospheric temperatures of 15–20°C in spring, 25–30°C in summer, 15–23°C in autumn and 5–10°C in winter. Each subject immersed his lower legs in warm water at 40°C for 30 min. Core (tympanic) temperature and sweat rate at chest were recorded continuously. Blood was taken pre-immersion to measure the concentrations of antidiuretic hormone, serum osmolality, plasma renin activity, angiotensin II, aldosterone, leptin, thyroid stimulating hormone, fT₃ and fT₄. The results suggested that the elderly have suppressed osmolality control compared to the young; osmolality was especially elevated in winter compared to the summer in elderly subjects. Therefore, particularly in the elderly, balancing fluid by drinking water should be encouraged to maintain euhydration status in winter.     

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.