Transcription profiling of acute temperature stress in the Antarctic plunderfish Harpagifer antarcticus

Harpagifer antarcticus (the Antarctic plunderfish), a shallow-water benthic fish distributed around the Antarctic Peninsula, is a member of the notothenioid family, one of whose adaptations to the cold waters of Antarctica has been the loss of the classic heat shock response. In order to gain a more comprehensive understanding of the effects of temperature stress on H. antarcticus, we constructed a liver cDNA library and a 10,371 feature microarray. This was hybridized with material from a time course series of animals held at 6°C for 48h. The resulting expression profiles show that this fish displays the classical vertebrate acute inflammatory response. There was also a pronounced signal for increased energy requirements via up-regulation of genes involved in the β oxidation of fatty acids and also a strong signature of response to oxidative stress. Genes in the latter category did not include the "classic" antioxidants such as glutathione S-transferase, but genes involved in the production of reducing potential in the form of NADPH, peroxisome proliferation via peroxisomal acyl co-enzyme A oxidase 1 and genes known to be up-regulated by hypoxia-inducible factor 1 (HIF1). These identifications provide clear support for oxygen being the whole animal limiting factor at least in acute short-term temperature challenges. The classical heat shock proteins were not up-regulated during this trial, although numerous clones for each were present on the gene chip, confirming the lack of this response in this species. These data significantly increase our knowledge of the cellular stress response from animals in this unique environment.     

Digestion rate, gut passage time and absorption efficiency in the Antarctic spiny plunderfish

The absolute gut evacuation rate (GER) (g day⁻¹) of Harpagifer antarcticus increased with increasing ration mass, fish mass only influenced the absolute GER at a daily ration level of 0·3% wet fish mass (approximately a maintenance ration). The relative GER (% of meal fed day⁻¹) was also affected differently by fish and ration mass depending on the relative ration level being fed; at rations of 0·7% wet fish mass or above the relative GER decreased with increasing fish or ration mass (in such a way that the absolute GER remained constant and unaffected by fish mass). At maintenance (0·3% wet fish mass) rations the relative GER was not affected by fish size or ration mass. Thus, there appears to be a ration threshold above which the digestion physiology alters. Mass-specific GER (% g fish⁻¹ day⁻¹) decreased with increasing fish mass. Within a set relative ration level (% wet fish mass) an increase in fish mass decreased the mass-specific GER. At a fixed ration mass, an increase in fish mass (i.e. a reduction in the ration expressed as % fish mass) resulted in a decrease in mass-specific GER. Gut evaluation time (GET) decreased and absorption efficiency (A) increased with increasing absolute GER. The effect of ration and fish mass on the absolute and relative GER followed the same pattern irrespective of the diet, however the A and GER (% day⁻¹ and g day⁻¹) were higher and the GET shorter when the fish were fed shelled krill rather than amphipods.     

Nitrogenous excretion in the Antarctic plunderfish

The nitrogenous excretion rates (total ammonia nitrogen, urea, and primary amines) of plunderfish Harpagifer antarcticus were related significantly to length and to wet mass (mass exponents of 0·94, 1·01, 1·07 and 0·93 for total ammonia nitrogen, urea, primary amines, and total nitrogen, respectively). The routine total ammonia excretion rates [22·23 & 2·0 mg N kg⁻¹ day⁻¹ (mean±S.E.)] of plunderfish measured in Antarctica are 10–69% lower than those of comparable non-polar species. Plunderfish are ammonotelic, but the proportion of the total nitrogenous waste attributable to each category was variable between individuals. On average (ranges in parentheses), total ammonia nitrogen, urea, and primary amines accounted for c .82 (57–97), 13 (2–28), and 5 (0·6–22)%, respectively, of the total nitrogen excreted. Polar fish differ from their non-polar relatives only in the rate, and not the nature, of their nitrogenous waste excretion processes.     

Energy partitioning in the Antarctic collembolan Cryptopygus antarcticus

Abstract. 1. Consumption, production and assimilation rates were determined for two age groups of Crypropygus antarcticus to give an estimate of energy utilization, and to investigate low temperature adaptation in its energy partitioning.
2. Feeding selectivity shown in laboratory preference tests was supported by gut analysis of field animals from contrasting sites. Although moulting rate was not significantly affected by food type, rates of growth were slowest and mortality highest when fed on a non-preferred substrate.
3. Both a radio labelling and a more direct method for measuring dry weight consumed gave similar results for Cvpropygus feeding on algae. The consuniption rate for animals when feeding on algae was lower than that on moss peat. The assimilation efficiency for immature animals feeding on algae was 46% and for mature animals was 19%; the values when feeding on moss peat were 7% and lo%, respectively, The net production efficiency ranged from 35%(inimatures) to 13% (matures) and was similar on both substrates.
4. Food consumption exceeded assimilation over the range 2.5–10°C, but the two converged from 2.5 to 0°C. Immature Cryptopygus maintained a net positive energy balance over 0–10°C, whilst below 1S°C respiration exceeded assimilation for mature individuals.
5. An estimate of the annual dry matter consumption (7 g m^-1 y^-1) by Ctypropygus in a moss turf at Signy Island agrees with one based on respiration data alone (Davis, 1981). The consumption at an alga-dominated site was c . 26 g m^-2 y^-l, and Crypropygus may have a locally limiting effect on net priniary production at such sites.     

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.     

Temperature preferences of the mite, Alaskozetes antarcticus, and the collembolan, Cryptopygus antarcticus from the maritime Antarctic

Abstract. The thermal preferences of Alaskozetes antarcticus (Acari, Cryptostigmata) and Cryptopygus antarcticus (Collembola, Isotomidae) were investigated over 6 h within a temperature gradient (?3 to +13 °C), under 100% relative humidity (RH) conditions. After 10 days of acclimation at ?2 or +11 °C, individual supercooling points (SCP) and thermopreferences were assessed, and compared with animals maintained for 10 days under fluctuating field conditions (?6 to +7 °C). Acclimation at ?2 °C lowered the mean SCP of both A. antarcticus (?24.2 ± 9.1) and C. antarcticus (?14.7 ± 7.7) compared to field samples (?19.0 ± 9.0 and ?10.7 ± 5.2, respectively). Acclimation at +11 °C increased A. antarcticus mean SCP values (?13.0 ± 8.5) relative to field samples, whereas those of C. antarcticus again decreased (?16.7 ± 9.1). Mites acclimated under field conditions or at +11 °C selected temperatures between ?3 and +1 °C. After acclimation at ?2 °C, both species preferred +1 to +5 °C. Cryptopygus antarcticus maintained under field conditions preferred +5 to +9 °C, whereas individuals acclimated at +11 °C selected +9 to +13 °C. For A. antarcticus, thermopreference was not influenced by its cold hardened state. The distribution of field specimens was further assessed within two combined temperature and humidity gradient systems: (i) 0–3 °C/12% RH, 3–6 °C/33% RH, 6–9 °C/75% RH and 9–12 °C/100% RH and (ii) 0–3 °C/100% RH, 3–6 °C/75% RH, 6–9 °C/33% RH and 9–12 °C/12% RH. In gradient (i), C. antarcticus distributed homogeneously, but, in gradient (ii), C. antarcticus preferred 0–3 °C/100% RH. Alaskozetes antarcticus selected temperatures between 0 and +6 °C regardless of RH conditions. Cryptopygus antarcticus appears better able than A. antarcticus to opportunistically utilize developmentally favourable thermal microclimates, when moisture availability is not restricted. The distribution of A. antarcticus appears more influenced by temperature, especially during regular freeze‐thaw transitions, when this species may select low temperature microhabitats to maintain a cold‐hardened state.     

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.     

Eco-morphological patterns of the sagitta of Antarctic fish

Morphology and morphometry of the sagittae otolith were studied in pelagic and mesopelagic fish. The shape, margins and rostrum of four groups of otoliths from several species were analyzed: group 1 (pelagic fish associated with the under ice cover N = 42), group 2 (pelagic fish associated with water offshore N = 9), group 3 (mesopelagic fish associated with extensive vertical migration N = 57) and group 4 (mesopelagic fish associated with short vertical migration N = 54). E (maximum width of the sagitta /maximum length of the sagitta %), R (rostrum length (RL)/maximum length of the sagitta %) and S (sulcus area (SS)/otolith area (OS) %) indexes were calculated for each species. Sagittae of pelagic groups (1 and 2) showed the smallest sagitta dimensions in relation to the total length of the fish, in this group the sagitta shape is variable. Sagittae of mesopelagic fish (groups 3 and 4) showed variable shape and edges. The shape in group 4 was polygonal and these species have more width than length. Statistical analysis showed significant differences in the E, R and S indexes. These results were compared with other 19 species, belonging to six families, taken from a publisher-edited literature. E, R and S-values could be used to characterize the sagittae of the Antarctic fish and could be considered as a useful tool for fish ecology studies.     

Sub-lethal heat stress causes apoptosis in an Antarctic fish that lacks an inducible heat shock response

The endemic fish fauna of the Southern Ocean are cold-adapted stenotherms and are acutely sensitive to elevated temperature. Many of these species lack a heat shock response and cannot increase the production of heat shock proteins in their tissues. However, some species retain the ability to induce other stress-responsive genes, some of which are involved in cell cycle arrest and apoptosis. Here, the effect of heat on cell cycle stage and its ability to induce apoptosis were tested in thermally stressed hepatocytes from a common Antarctic fish species from McMurdo Sound in the Ross Sea. Levels of proliferating cell nuclear antigen were also measured as a marker of progression through the cell cycle. The results of these studies demonstrate that even sub-lethal heat stress can have deleterious impacts at the cellular level on these environmentally sensitive species.     

The HSP70 heat shock response in the Antarctic fish <Emphasis Type="Italic">Harpagifer antarcticus</Emphasis>

Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) genes, plus GRP78 (Glucose-regulated protein 78 kDa) were surveyed for expression levels via Q-PCR after both an acute 2-h heat shock experiment and a time course assay in the Antarctic plunderfish Harpagifer antarcticus. In general, down regulation of all genes was observed during the course of the heat shock experiments. This thermally induced down regulation was particularly acute for the GRP78 gene, which at one time point was more than 100-fold down regulated. These results demonstrate the loss of the heat shock response in H. antarcticus, a basal member of the Notothenioidei. This finding is discussed with reference to the survival of Notothenioids during observed ocean warming and also the reorganisation of cellular protein mechanisms of species living in extreme environments.     

Moulting reduces freeze susceptibility in the Antarctic mite Alaskozetes antarcticus (Michael)

Abstract.  The effect of moulting on the cold hardiness of the oribatid mite Alaskozetes antarcticus (Michael) is investigated. Non moulting animals show clear seasonal patterns of cold hardiness with high supercooling points (SCPs) at the peak of summer and an increasing proportion of low SCPs with declining environmental temperatures. By contrast, both field-fresh and laboratory acclimated (5 °C) mites in the moult state are consistently found to have low SCPs regardless of environmental temperature.     

Temporal resolution of cold acclimation and de-acclimation in the Antarctic collembolan, Cryptopygus antarcticus

Abstract.  The Antarctic collembolan, Cryptopygus antarcticus (Willem), can switch its supercooling point (SCP) between 'winter' and 'summer' modes of cold hardiness over a matter of hours. High resolution temporal scaling of the acquisition and loss of cold hardiness is undertaken by assaying changes in the proportion of animals freezing below −15 °C in response to cooling rate, acclimation temperature, and access to food and moisture. Rapid de-acclimation to the 'summer' modal state is readily achieved after 1–6 h in response to warming and access to food; however, rapid acclimation to the 'winter' modal state is only evident in response to slow cooling and narrow ranges of temperature (0–5 °C). The rapid loss of cold tolerance at higher temperatures with access to food, in particular, emphasizes this species' opportunistic responses to resource availability in the short polar summers. Cold hardiness is apparently more readily traded off against nutrient acquisition than vice versa in this maritime Antarctic species.     

The reproductive behaviour of Pogonophryne scotti confirms widespread egg-guarding parental care among Antarctic notothenioids

In this paper, the first documentation of egg-guarding behaviour in an artedidraconid species, Pogonophryne scotti, through in situ photographic imagery obtained during video transects is provided. The male specimen was observed closely guarding a well-defined multi-layered egg mass deposited on the sea floor at 240 m on the southern South Orkney Islands shelf in the northern Weddell Sea. Egg-guarding parental care is present in species that are distributed among all of the major lineages of Antarctic notothenioids; however, lack of information on egg-deposition behaviours in Bovichtidae and Pseudaphritis prevents assessment of whether parental care originated prior to the origin of the Antarctic notothenioid radiation.     

Electrophoretic analysis of Antarctic fish from South Georgia

White muscle samples of Antarctic fish ( Notothenia rossii, N., neglecta. N. gibbe-rifrons and Chaenocephalus (aceratus ) were subjected to electrophoretic tests. lsoelectric focusing of general muscle proteins provides a quick method of iden-tyfying species. Six muscle enzymes were resolved by horizontal starch gel clectrophoresis. These are presumed to be the products of ten independent gene loci. Coefficients of genetic distance were calculated and substantially agree with the conventional taxnomy. Levels of genetic variability are within the normal range for marine teleosts. Two polymorphic enzymes. GPI and PGM. might prove useful as 'genetic tags' for stock identification.     

Identification of a novel helitron transposon in the genome of Antarctic fish

Rolling-circle (RC) eukaryotic transposons, known as helitrons, are found in a wide range of organisms, from protist to mammals. Autonomous helitrons have a distinctive open reading frame (ORF) encoding a polypeptide that contains typical domains for RC replication (RCR): the Rep (RCR initiator) and the DNA helicase domains. These elements are believed to have an important role in the host genome evolution, owing to their frequent capture of host genes, some of which can evolve into novel genes or become essential for helitron transposition. We conducted a molecular analysis of the suborder Notothenioidei, a group of Perciformes that currently dominate the Antarctic waters by virtue of their remarkable cold-adaptation ability. A novel helitron from the genome of the icefish species Chionodraco hamatus, belonging to the Channichthyidae, the most derived Notothenioids family, was isolated, characterized and designated as HeliNoto (8.9 kb). Its ORF was compared to homologous sequences from different species in a comprehensive phylogenetic analysis. For the first time the putative functional domains of a helitron were subjected to a well accurate structural analysis including chromosomal localization. Finally, the distribution of HeliNoto among Notothenioids was investigated.     

cDNA microarray analysis reveals the capacity of the cold-adapted Antarctic fish <Emphasis Type="Italic">Trematomus bernacchii</Emphasis> to alter gene expression in response to heat stress

During their many millions of years of evolution in the extreme and stable cold, Antarctic notothenioid fishes have acquired profoundly cold-adapted physiologies. Gene expression profiling via cDNA microarray was used to determine the extent to which one species of notothenioid, Trematomus bernacchii, has retained the ability to alter gene expression in response to heat stress. While an inability to up-regulate the expression of any size class of heat shock proteins (except for a 1.1-fold induction of the co-chaperone Hsp40) was observed, hundreds of additional genes, associated with a broad range of cellular processes, were responsive to heat. Many of these genes are associated with central aspects of the evolutionarily conserved cellular stress response (CSR), which plays a pivotal role in responding to physical and chemical stresses. The inability of T. bernacchii to mount a heat shock response underscores the potential susceptibility of this species to the effects of global warming.     

The ultrastructural characterization of mitochondria‐rich cells as a response to variations in salinity in two types of teleostean pseudobranch: milkfish (Chanos chanos) and Mozambique tilapia (Oreochromis mossambicus)

The pseudobranchs of two euryhaline teleost species, the milkfish (Chanos chanos ) and the Mozambique tilapia (Oreochromis mossambicus ), were studied after acclimization to different salinities using optical and electron microscopy. The milkfish pseudobranch was the lamellae‐free type, with separate lamellae along the filaments containing two groups of mitochondria (Mt)‐rich cells: chloride cells (CCs) and pseudobranch type cells (PSCs). Conversely, the tilapia pseudobranch was the embedded type, covered with connective tissues and with only one group of Mt‐rich PSCs. Chloride cells were identified according to the apical openings and branched tubular networks around randomly distributed and diversely shaped Mt. Pseudobranchs type cells, however, were characterized according to the orderly arrangement of parallel tubules around closely packed Mt; both the tubules and the Mt were distributed in the vascular side of the cell, but were absent from the apical region. Compared with those of seawater (SW)‐acclimated milkfish, the pseudobranchial lamellae of freshwater (FW) specimens were longer on average, and the Mt of the CCs had fewer cristae, were less electron‐dense, and were often vacuolated. The Mt in the PSCs of FW‐acclimated milkfish and tilapia were larger and more electron‐dense than those of their SW‐acclimated counterparts; in addition, more tubules were found to aggregately surround the Mt and basolateral membranes in the PSCs of fish from the hypo‐osmotic environment. Conversely, the PSCs of tilapia were periodic acid‐Schiff (PAS)‐positive, and Mt in PSCs were concentrated with more parallel arrays of the tubule system than those of milkfish. Therefore, salinity‐dependent changes in the ultrastructures of PSCs suggest their potential role in energy metabolism of both lamellae‐free and embedded pseudobranchs, whereas the PAS‐positive staining characteristics suggest a role in releasing or storaging polysaccharides in the embedded pseudobranch. J. Morphol. 278:390–402, 2017. © 2017 Wiley Periodicals, Inc.     

The effect of feeding on specific soil algae on the cold-hardiness of two Antarctic micro-arthropods (Alaskozetes antarcticus and Cryptopygus antarcticus)

The effect of consuming terrestrial algae on the cold tolerance of two Antarctic micro-arthropods was examined. From the results of preferential feeding experiments, seven species of Antarctic terrestrial micro-algae were chosen and fed to two common, freeze-avoiding Antarctic micro-arthropods: the springtail Cryptopygus antarcticus (Collembola: Isotomidae), and the mite Alaskozetes antarcticus (Acari: Oribatida). Mites were very selective in their choice of food whereas the springtails were less discriminating. The ice nucleating activity of each species of alga was measured using an ice nucleator spectrometer and a differential scanning calorimeter. Pure cultures of individual species of algae had characteristic supercooling points ranging from ca. −5 to −18 °C. The effect of eating a particular alga on the supercooling point of individual micro-arthropods cultured at two different temperatures (0 and 10 °C) was examined. Neither species showed a preference for algae with low ice-nucleating activity and there was no clear correlation between the supercooling point of food material and that of the whole animal. However, feeding on certain algae such as Prasiola crispa, which contained the most active ice nucleators, decreased the cold tolerance of both species of arthropods. Accepted: 6 May 2000