Stress response in two Antarctic teleosts (Notothenia coriiceps Richardson andChaenocephalus aceratus Lönnberg) following capture and surgery

Blood chemistry and haematological parameters have been determined in two Antarctic teleosts,Notothenia coriiceps Richardson andChaenocephalus aceratus Lönnberg, held at around 1°C.Notothenia coriiceps has a low haemoglobin content compared to tem-perate-zone species, whereasC. aceratus apparently lacks respiratory pigments. Blood samples were obtained by cardiac puncture following landing or using chronically implanted post-branchial arterial cannulae. Although both species showed a similar acidosis on capture (arterial pH as low as 7.5 versus the final recovery value of around 7.9),C. aceratus took 48 h to reestablish baseline values whileN. coriiceps recovered within 12 h, despite initially showing a greater degree of hypercapnic hypoxia. Surgery led to a more severe disturbance of acid-base regulation inN. coriiceps thanC. aceratus (arterial pH of 7.5 versus 7.8) but needed only half as long for recovery. A progressive decrease in arterial oxygen tension and increase in arterial carbon dioxide tension (both more pronounced inN. coriiceps) with level of acidosis was observed down to arteria pH 7.2 InC. aceratus this was accompanied by a rise in blood lactate (up to 10 mmol·1^-1 in some individuals), whileN. coriiceps showed only a modest and transient lactacidosis. Stress inN. coriiceps therefore induces primarily a respiratory, rather than a metabolic acidosis, whereas inC. aceratus both components are present. A differential response to stress is also indicated by an elevated, though low noradrenaline titre inN. coriiceps following surgery and capture, whileC. aceratus was little affected by surgery. However, both species show an unusually weak catecholamine response to induced stress.Abbreviations pH/T °C thermal sensitivity of pH - Ad adrenaline - bw body weight - C.CO₂ total carbon dioxide content - C.O₂ total oxygen content - ED ₅₀ Median effective dose - EDTA ethylenediaminetertra-acetic acid - Hb haemoglobin - Hct haematocrit - HPLC high-performance liquid chromatography - lac lactate - MCH mean corpuscular haemoglobin content - MCHC mean corpuscular haemoglobin concentration - MCV mean cell volume - MS222 tricaine methane sulphonate - NAd noradrenaline - P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen tension - pHa arterial blood pH - RBCC red blood cell count - SW sea water - T _a ambient air temperature - VO₂ oxygen consumption     

Regulation of splenic contraction persists as a vestigial trait in white-blooded Antarctic fishes

In fishes, the spleen can function as an important reservoir for red blood cells (RBCs), which, following splenic contraction, may be released into the circulation to increase haematocrit during energy-demanding activities. This trait is particularly pronounced in red-blooded Antarctic fishes in which the spleen can sequester a large proportion of RBCs during rest, thereby reducing blood viscosity, which may serve as an adaptation to life in cold environments. In one species, Pagothenia borchgrevinki, it has previously been shown that splenic contraction primarily depends on cholinergic stimulation. The aim of the present study was to investigate the regulation of splenic contraction in five other Antarctic fish species, three red-blooded notothenioids (Dissostichus mawsoni Norman, 1937, Gobionotothen gibberifrons Lönnberg, 1905, Notothenia coriiceps Richardson 1844) and two white-blooded “icefish” (Chaenocephalus aceratus Lönnberg, 1906 and Champsocephalus gunnari Lönnberg, 1905), which lack haemoglobin and RBCs, but nevertheless possess a large spleen. In all species, splenic strips constricted in response to both cholinergic (carbachol) and adrenergic (adrenaline) agonists. Surprisingly, in the two species of icefish, the spleen responded with similar sensitivity to red-blooded species, despite contraction being of little obvious benefit for releasing RBCs into the circulation. Although the icefish lineage lost functional haemoglobin before diversifying over the past 7.8–4.8 millions of years, they retain the capacity to contract the spleen, likely as a vestige inherited from their red-blooded ancestors.     

Characterization of the intestinal microbiota of two Antarctic notothenioid fish species

The fish fauna of the Southern Ocean is dominated by species of the perciform suborder Notothenioidei, which constitute 46% of fish species and 90% of biomass. Notothenioids have undergone rapid morphological and ecological diversification and developed physiological adaptations to a cold, highly oxygenated environment. Microbes inhabiting animal intestines include those that perform essential nutritional functions, but notothenioid gut microbial communities have not been investigated using cultivation-independent approaches. We analyzed bacterial 16S rRNA gene sequences obtained from the intestinal tract of Notothenia coriiceps and Chaenocephalus aceratus, which differ in their pelagic distribution and feeding strategies. Both samples showed dominance of Gammaproteobacteria (mostly Vibrionaceae), as has been reported for temperate teleost species. Both samples showed low diversity relative to that reported for other fish microbiota studies, with C. aceratus containing fewer OTUs than N. coriiceps. Despite the small sample size of this preliminary study, our findings suggest that Antarctic notothenioids carry a gut microbiota similar in composition to that of temperate fish, but exhibiting lower species-level diversity. The omnivorous N. coriiceps individual exhibited greater diversity than the exclusively carnivorous C. aceratus individual, which may indicate that increasing herbivory in fish leads to gut microbe diversification, as found in mammals. Lastly, we detected members of taxa containing known microbial pathogens, which have not been previously reported in Antarctic notothenioid fish.     

Triacylglycerol lipase activities in tissues of Antarctic fishes

. The activity of the lipolytic enzyme, triacylglycerol lipase (TAG lipase; E.C. 3.1.1.3) was measured in heart ventricle, liver, oxidative skeletal muscle, and adipose tissue of four species of Antarctic fishes (Chaenocephalus aceratus, Notothenia coriiceps, Trematomus newnesi, Gobionotothen gibberifrons). Estimates of weight-specific activities at 0°C in tissues of the Antarctic fishes were broadly comparable to those reported for temperate-zone fish species assayed at their physiological temperature. Within each species, liver tissue displayed the highest weight-specific activity of TAG lipase in C. aceratus, T. newnesi, and G. gibberifrons, while adipose tissue of N. coriiceps showed the highest activity within that species. Combining these estimates with carefully measured somatic indices of the tissues, permitted assessment of the relative importance of different tissues to overall lipolytic capacity on an organismal level. On this basis, liver tissue displays the greatest capacity of TAG lipase per 100 g body weight in each of the species studied.     

A comparison of the response to induced exercise in red- and white-blooded Antarctic fishes

The physiological responses to forced exercise were studied in yellowbelly and marbled rockcod (Notothenia coriiceps and N. rossii), and the haemoglobinless icefish (Chaenocephalus aceratus), from blood samples obtained via indwelling catheters. The maximal exertion tolerable by N. coriiceps was 3–5 min, although N. rossii was not fully exhausted by this effort, and it proved difficult to elicit sustained maximal activity in C. aceratus. Arterial O₂ tension reflected the relative degree of exhaustion, showing a significant fall in the case of N. coriiceps, little change in N. rossii, and even a rise in C. aceratus as a result of hyperventilation. Such changes in the red-blooded species were not caused by altered O₂ carrying capacity, as there was no change in haematocrit. In Notothenia spp. the decrease in arterial pH was better correlated with a rise in arterial CO₂ tension than with blood lactate concentration, which is reflected in a modest net metabolic acid load. In contrast, the icefish showed an attenuated hypercapnia and a more pronounced lactacidosis, but an insignificant net metabolic acid load. Disturbance in ionoregulation following exercise was limited to an elevated [Cl⁻] in Notothenia, while circulating catecholamine levels remained unusually low in all specimens. The response to stress appears to reflect lifestyle and/or endemic speciation, rather than specific adaptations to the stenothermal environment. Accepted: 9 August 1996     

The role of the rock cod Notothenia coriiceps Richardson, 1844 in the life-cycle of Antarctic parasites

Fifty specimens of Notothenia coriiceps caught in Potter Cove, King George Island, were examined for ecto- and endoparasites. Of the 22 parasite species found, 18 were helminths, 2 were hirudineans and 2 were crustaceans. The isopod Aega antarctica and an unidentified hirudinean are reported for the first time from this fish host. Dominant parasites were the adults of Aspersentis megarhynchus, the invasive stage of Corynosoma spp. (cystacanth) and the adults of Macvicaria pennelli, with respective prevalences of infestation of 94, 76 and 74%. The preferred sites of infestation were the pylorus and intestine, where five different larval (nematodes and cestodes) and eight adult (digeneans and acanthocephalans) parasite species were found. No adult nematodes and cestodes were found and no parasites could be isolated from the musculature. The results of the present study are related to previous findings on the parasite fauna of N. coriiceps. The comparison implies a high parasite diversity in this benthic Antarctic fish species. Most parasites found appear to have a wide range of distribution within Antarctic waters together with a low host specificity. Besides its role as final host for several species of trematodes and acanthocephalans, N. coriiceps serves as transmitter of parasite larvae to piscivorous birds and seals. It is concluded that the parasite fauna in Antarctic fish species provides important insights into the different habitat use and trophic relationship of their fish hosts. Received: 11 September 1997 / Accepted: 12 January 1998     

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

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

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.     

Hypercapnia induced shifts in gill energy budgets of Antarctic notothenioids

Mechanisms responsive to hypercapnia (elevated CO₂ concentrations) and shaping branchial energy turnover were investigated in isolated perfused gills of two Antarctic Notothenioids (Gobionotothen gibberifrons, Notothenia coriiceps). Branchial oxygen consumption was measured under normo- versus hypercapnic conditions (10,000 ppm CO₂) at high extracellular pH values. The fractional costs of ion regulation, protein and RNA synthesis in the energy budgets were determined using specific inhibitors. Overall gill energy turnover was maintained under pH compensated hypercapnia in both Antarctic species as well as in a temperate zoarcid (Zoarces viviparus). However, fractional energy consumption by the examined processes rose drastically in G. gibberifrons (100–180%), and to a lesser extent in N. coriiceps gills (7–56%). In conclusion, high CO₂ concentrations under conditions of compensated acidosis induce cost increments in epithelial processes, however, at maintained overall rates of branchial energy turnover.     

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.     

Diversity, relative abundance, new locality records and population structure of Antarctic demersal fishes from the northern Scotia Arc islands and Bouvetøya

A primary objective of the ICEFISH 2004 cruise was to collect and study notothenioid fishes from remote localities in the Atlantic sector of the Southern Ocean. Nearly 1 month was devoted to bottom trawling for fishes on the shelf and upper slope (to 1,000 m) areas around Shag Rocks, South Georgia, South Sandwich Islands and Bouvetøya. The focus was on the latter two locations, because their faunas are more poorly known. Eight species were collected at Shag Rocks with Patagonotothen guntheri most abundant; 17 at South Georgia with Lepidonotothen nudifrons, L. larseni and Gobionotothen gibberifrons most abundant; 13 at the South Sandwich Islands with L. larseni, L. nudifrons and G. gibberifrons most abundant; and 11 at Bouvetøya with L. larseni, Macrourus holotrachys and L. squamifrons most abundant. Ten new locality records were established: Shag Rocks (1), South Georgia (1), South Sandwich Islands (5), South Sandwich Trench at 5,350 m (1) and Bouvetøya (2). Total known demersal fish diversity on the shelf and upper slope at Shag Rocks/South Georgia, South Sandwich Islands and Bouvetøya is 42, 31 and 17 species, respectively. To examine population structure in the four most abundant notothenioids at Bouvetøya (L. larseni, L. squamifrons, Notothenia coriiceps and Chaenocephalus aceratus), we examined the ND2 portion of mitochondrial DNA. Chaenocephalus aceratus, N. coriiceps and L. larseni exhibited no significant genetic differentiation in comparison with samples from localities in the Scotia Sea and the Antarctic Peninsula. However, L. squamifrons showed significant genetic differentiation between the South Shetlands and Bouvetøya populations (F _ST = 0.189, P = 0.015). Thus, these data combined with previous studies of two other notothenioids suggest that five of the six notothenioid species at Bouvetøya are not genetically differentiated from other localities in the Atlantic sector of the Southern Ocean. The location of Bouvetøya within the Antarctic Circumpolar Current and the long (1–2 years) pelagic stages of the notothenioids at Bouvetøya may be at least partly responsible for this genetic homogeneity.     

Cardiac force-interval relationship,adrenaline and sarcoplasmic reticulum in rainbow trout

The force-interval relationship was examined at 20 and 10 °C in electrically paced atrial and ventricular tissue of rainbow trout,Oncorhynchus mykiss, regarding dependence on the sarcoplasmic reticulum and influence of adrenaline. In both tissues, adrenaline (10^-6 mol·l^-1) doubled control force developed at 0.5 Hz. In atrial but not in ventricular tissue it also shortened the diastolic interval needed for recovery of a given fraction of the control force. In atrial tissue and in ventricular tissue at 20 °C, the fraction of force recovered in the presence of adrenaline was diminished by 10 mol·l^-1 of ryanodine, a specific inhibitor of the sarcoplasmic reticulum. In atrial tissue not exposed to adrenaline and in ventricular tissue at 10 °C irrespective of adrenaline, ryanodine did not affect recovery. In atrial but not in ventricular tissue it also diminished control force. In conclusion, the cardiac sarcoplasmic reticulum of trout seems to support force development during adrenaline dependent increases in heart rate, and in atrial tissue also the force at steady state.Abbreviations E-C coupling excitation-contraction coupling - P-R potential - SR sarcoplasmic reticulum - SE standard error of the mean     

Aspects of the foraging behaviour of the Antarctic Tern Sterna vittata gaini at Harmony Point, South Shetland Islands

During January and February of 2002 and 2003, we studied the diet of the Antarctic Tern Sterna vittata gaini at two colonies in Nelson Island, South Shetland Islands, by identifying the prey fed to chicks by breeders. The fish Notothenia coriiceps was the main prey in both seasons, followed by the myctophid Electrona antarctica, Antarctic krill Euphausia superba and gammarid amphipods. The contribution of fish to the diet increased as chicks grew older. Fish and amphipods were brought to chicks during the day, whereas adults brought Antarctic krill at sunrise and sunset. Both the duration of the feeding trips and the number of trips per foraging bout varied according to the type of prey caught. Preliminary information suggests that, among other causes, the foraging strategy is strongly influenced by the predation pressure of skuas on chicks. Results are compared with the only two previous study on the diet of the Antarctic Tern at the South Shetland Islands.     

Isolation,characterization and nucleotide sequence of the muscle isoforms of creatine kinase from the Antarctic teleost Chaenocephalus aceratus

Creatine kinase (CK) was isolated from the white muscle of the Antarctic icefish Chaenocephalus aceratus, which is deficient in glycolytic capacity. C. aceratus white myotomal creatine kinase (MMCK) displayed an apparent K_m at 0.5 °C of 0.06 mM for ADP and 17 mM for Phosphocreatine. These K_m values are similar to those reported for other vertebrate MMCKs at their physiologically relevant body temperatures. C. aceratus MMCK exhibited optimal activity at pH of 7.6–7.7 at 0.5 °C, in contrast to rabbit MMCK which had optimum activity at pH 6.2 at 30 °C. The apparent V_max of C. aceratus MMCK at 0.5 °C is 94±4 S.D. (n=9) μmol ATP/min/mg (i.e. U/mg), which is comparable to rabbit MMCK assayed at 20 °C and 8-fold greater than rabbit MMCK measured at 0.5 °C. DEAE chromatography of C. aceratus white muscle CK resolved two distinct activity peaks. Cloning and sequencing of C. aceratus CK cDNAs confirmed that two muscle-specific isoforms of CK were expressed that were distinct from the mitochondrial and brain isoforms. Icefish MMCK was sensitive to transient temperature elevation, and the DEAE-fractionated forms were highly unstable. These results indicate that C. aceratus MMCK displays significant activity at physiological temperature and intracellular pH of icefish muscle that could contribute to sustaining energy charge during burst-swimming.     

Analysis of trace metals in the Antarctic host-parasite system Notothenia coriiceps and Aspersentis megarhynchus (Acanthocephala) caught at King George Island,South Shetland Islands

Concentrations of the elements Al, Ag, As, Ba, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb and Sr were analysed by high-performance quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS) in the acanthocephalan Aspersentis megarhynchus and in different tissues of its final host, Notothenia coriiceps. Infected fish were sampled at King George Island, South Shetland Islands, Antarctica. Most of the elements were found at significantly higher concentrations in the acanthocephalan than in muscle, liver and intestine of its host. Only Fe was concentrated in fish liver to a significantly higher level than in the parasite. Compared with the host tissues, the highest accumulation rates in A. megarhynchus were found for Pb, Cd, Ag, Ni and Cu. The acanthocephalans showed very high Ag and Pb levels, whereas the concentrations in the fish tissues were close to the detection limit. This study is the first proof that the enormous heavy-metal accumulation capacity reported for acanthocephalans from freshwater fish also occurs in acanthocephalans parasitizing marine fish. Consequently, acanthocephalans can be used to assess the occurrence and availability of even the lowest metal concentrations in all kinds of aquatic habitats, including remote areas such as the Antarctic.     

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     

Microsatellite analysis reveals genetic differentiation between year-classes in the icefish <Emphasis Type="Italic">Chaenocephalus aceratus</Emphasis> at South Shetlands and Elephant Island

Chaenocephalus aceratus is one of the most abundant Antarctic icefish species in the Atlantic sector and has been a by-catch species in the fishery for mackerel icefish, Champsocephalus gunnari, between the mid-1970s and mid-1980s at South Georgia, South Orkney, and South Shetland Islands. The species became the target of the fishery in particular seasons, such as at South Georgia in 1977/78. In our paper, we report results on genetic differentiation for 11 microsatellite loci in C. aceratus samples collected at the South Shetlands and Elephant Island. This study represents the first report on microsatellite variability of an icefish species. Our results support the evidence from previous studies on differences in infestation patterns of parasites that a single panmictic population of C. aceratus exists, spanning the two sampling sites separated by about 100 km. Moreover, our study indicates the presence of a significant genetic differentiation between individual year-classes pointing out the existence of dynamic processes acting at the population genetic level, according to recent results for broadly distributed marine species. Both small effective population size and immigration from unsampled differentiated stocks may be at the base of the differentiation found in C. aceratus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.