Delayed onset of adult antifreeze activity in juveniles of the Antarctic icefish <Emphasis Type="Italic">Chaenocephalus aceratus</Emphasis>

Many Antarctic notothenioid species endemic to the Seasonal Pack-ice Zone have converged on adult blood serum freezing points that are several tenths of a degree above the freezing point of seawater. While these fishes share high adult serum freezing points, the development of their freeze avoidance during ontogeny has not been studied. We investigated this in wild caught juveniles of one such species, Chaenocephalus aceratus (family Channichthyidae), using blood serum antifreeze activity as a proxy for their freeze avoidance. Juvenile serum antifreeze activity was significantly below that of adults through the oldest year 2+ specimens collected. This increased at an estimated rate of 0.368 × 10⁻³ ± 0.405 × 10⁻⁴°C day⁻¹ which, if sustained, would leave C. aceratus below their adult serum antifreeze activity levels of 0.57 ± 0.08°C until 4.2 years after hatching. Underlying the 2.7-fold increase in their serum antifreeze activity from late year 0+ juveniles to adults was an even greater 10.4-fold increase in the concentration of their serum antifreeze glycopeptides, which increased proportionally across all of their serum AFGP size isoforms. With insufficient antifreeze activity to avoid freezing in the ice-laden surface waters, both adult and juvenile C. aceratus are most likely restricted to the year round ice-free waters where a metastable supercooled state can be maintained.     

Drinking in Antarctic fishes

Drinking rates have never been measured in Antarctic fish. Drinking rates were measured in four species of notothenioid fish, including a hemoglobinless icefish, found in the near-freezing waters of the Ross Sea of the Southern Ocean. All of the fish, with the exception of the icefish, had low drinking rates and high serum osmolalities relative to temperate seawater fish. The icefish had significantly higher drinking rates and serum osmolalities relative to the Antarctic fishes containing hemoglobin, including Trematomus bernacchii. Warm acclimation of T. bernacchii, from −1.5°C to +4°C for 4 weeks, significantly increased their drinking rates 4.6-fold, significantly decreased their serum and intestinal osmolality by 11% and 12%, respectively, relative to cold-acclimated fish. These results indicate that increased drinking rates in Antarctic fish at elevated temperatures are involved in maintaining a lower serum osmolality.     

Cold hardiness in Entomobrya nivalis (Collembola, Entomobryidae): annual cycle of polyols and antifreeze proteins, and antifreeze triggering by temperature and photoperiod

In the Swiss Prealps Entomobrya nivalis hibernates in an inactive state, hidden under bark flakes on spruce. For freeze avoidance it relies on thermal hysteresis proteins (THPs) and polyols (mainly ribitol, with small amounts arabitol and threitol). Polyols are present only during the inactive state, THPs additionally protect during the transition phase in spring and autumn, when animals are still active but frosts may occur. Peak values were recorded in February/March for THPs (3.5 °C hysteresis between melting and freezing point) and for polyols (26 μg mg⁻¹ FW; hemolymph osmolality 680 mosmol l⁻¹). E. nivalis is able to control its hemolymph osmolality independently of body water content. Mean osmolality in summer was 350– 440 mosmol l⁻¹, in winter it was elevated to 650 mosmol l⁻¹, due to a synthesis mainly of ribitol. Body water content varied between 1.8 and 3.3 mg H₂O mg⁻¹ DW, depending on humidity conditions. Experiments on triggering of antifreeze synthesis showed the action of temperature and photoperiod as cues, but there was also evidence for an endogenous rhythm. No clear correlation between antifreeze concentration and supercooling ability could be established, suggesting that gut content or other parameters also play an inportant role. Accepted: 18 November 1995     

Environmental influences on regulation of blood plasma/serum components in teleost fishes: a review

Concentrations of both inorganic and organic blood plasma/serum components of teleost fishes were reviewed in seven habitat/life-history categories. These were: freshwater; inland saline; estuarine and nearshore marine; pelagic and deep-sea; diadromous; southern cold-water; and northern cold-water. Plasma/serum osmolalities were compared among groups acclimated to/living in fresh and in salt water. Contributions of inorganic ions and colligative and non-colligative organic molecules were evaluated including with respect to melting and freezing points, and “antifreeze activity” of plasma/serum in species from cold marine waters. Possible roles of TMAO in deep-water fishes were reviewed. Discussion also included influences of ambient salinity and temperature on concentrations of plasma/serum components. Seasonal cycles of blood plasma/serum components were discussed, along with antifreeze concentrations in other body fluids and tissues of cold-water fishes. Regulatory patterns of plasma/serum osmolalities were compared among the most euryhaline of teleosts evaluated here. Highest mean values of plasma/serum osmolalities in sea water were seen in southern cold-water and in pelagic and deep-sea fishes. The southern cold-water group also had the lowest plasma/serum freezing points among these groups. Comparisons of mean plasma/serum Na⁺ and Cl⁻ concentrations among fishes from fresh waters did not differ significantly among groups, but species from cold marine waters showed higher levels than did other groups in marine waters. Plasma/serum osmotic, Na⁺ and Cl⁻ concentrations of these seven groups of teleosts were compared with those of other fish-like vertebrate groups. Possible impacts of global warming on regulatory responses of plasma/serum components were discussed.     

The effect of habitat temperature on serum antifreeze glycoprotein (AFGP) activity in Notothenia rossii (Pisces: Nototheniidae) in the Southern Ocean

The marble notothen, Notothenia rossii, is widely distributed around the waters of sub-Antarctic islands in the Southern Ocean and is exposed to different temperatures that range from ?1.5 to 8 °C. This study investigates whether the different environmental conditions experienced by N. rossii at different latitudes in the Southern Ocean affect the levels of its blood serum antifreeze glycoprotein (AFGP). N. rossii specimens were collected from four localities, including the Ob’ Seamount in the Indian Ocean sector, and South Georgia Island, South Shetland Islands and Dallman Bay in the Atlantic Ocean sector. Serum AFGP activity was determined in terms of thermal hysteresis, i.e. the difference between the equilibrium melting and non-equilibrium freezing points (f.p.s.). Among the four populations, the Ob’ Seamount specimen had the lowest serum AFGP activity (0.44 °C) and concentration (4.88 mg/mL), and the highest non-equilibrium f.p. (?1.39 °C). These results are consistent with the warmer, ice-free waters around the Ob’ Seamount. The other three higher latitude populations have 2–3 times greater serum AFGP activity and concentration, and much lower non-equilibrium f.p.s. In contrast, the physiological profiles of serum AFGP size isoforms revealed that all N. rossii populations, including the Ob’ Seamount specimen, possess an extensive complements of AFGP proteins. Isoform variation was observed, especially in the large size isoforms (AFGPs 1–5), when compared to AFGP of the high Antarctic Dissostichus mawsoni. The lower levels of AFGP and the absence of some of the large isoforms are likely responsible for higher non-equilibrium f.p.s. of the Ob’ seamount specimen.     

Chlorophyll/nutrient characteristics in the water masses to the north of South Georgia, Southern Ocean

Chlorophyll a and nutrient concentrations along with temperature and salinity values were measured at 22 CTD stations along a 735-km transect running to the northwest of the island of South Georgia, Southern Ocean. Measurements were repeated during five summer surveys (January and February 1994, January 1996, December 1996, January 1998) and one spring survey (October 1997). The transect sampled Sub-Antarctic Zone water in the north, Polar Frontal Zone water and Antarctic Zone water in the south. Chlorophyll a concentrations were lowest to the north of the transect and frequently high (up to 17 mg m⁻³) in the deep open ocean of the Antarctic Zone. Sub-surface peaks were measured in all zones and chlorophyll a was detectable to a depth of 150 m. There was a clear latitudinal temperature gradient in the near-surface waters (0–50 m), the warmest water occurring in the north (∼12 °C), and the coolest in the Antarctic Zone (∼2 °C). There was also a well-defined latitudinal gradient in summer near-surface silicate concentrations (∼2, 4, and 10 mmol m⁻³ in the Sub-Antarctic Zone, the Polar Frontal Zone and the Antarctic Zone, respectively), increasing to >20 mmol m⁻³ near South Georgia. Distinct differences in silicate concentrations were also evident in all three zones to a depth of 500 m. Near-surface nitrate and phosphate concentrations were relatively low to the north of the transect (∼14 and 1 mmol m⁻³, respectively) and higher in the Polar Frontal Zone and Antarctic Zone (∼18 and 1.4 mmol m⁻³, respectively). Ammonium and nitrite were restricted to the upper 200 m of the water column, and exhibited sub-surface concentration peaks, the lowest being in the Sub-Antarctic Zone (0.68 and 0.25 mmol m⁻³, respectively) and the highest in the Antarctic Zone (1.72 and 0.29 mmol m⁻³, respectively). Surface (∼6 m) spring nutrient measurements provided an indication of pre-bloom conditions; ammonium and nitrite concentrations were low (∼0.27 and 0.28 mmol m⁻³, respectively), while silicate, nitrate and phosphate concentrations were high and similar to previously measured winter values (e.g. ∼26, 23, 2 mmol m⁻³, respectively in the Antarctic Zone). Although the values measured were very variable, and there was some evidence of a seasonal growth progression, the chlorophyll a and nutrient distribution patterns were dominated by intercruise (interannual) factors. Approximate nutrient depletions (spring minus summer) appeared similar in the Polar Frontal Zone and Antarctic Zone for nitrate and phosphate, while silicate showed a marked latitudinal increase from north to south throughout the transect. Highest chlorophyll a concentrations coincided with the highest apparent silicate depletions over the deep ocean of the Antarctic Zone. In this area, relatively warm, easterly flowing Antarctic Circumpolar Current water meets cooler, westerly flowing water that is influenced by the Weddell-Scotia Confluence and is rich in nutrients, especially silicate. Accepted: 27 November 1999     

Simultaneous freeze tolerance and avoidance in individual fungus gnats, <Emphasis Type="Italic">Exechia nugatoria</Emphasis>

Freeze tolerance and freeze avoidance are typically described as mutually exclusive strategies for overwintering in animals. Here we show an insect species that combines both strategies. Individual fungus gnats, collected in Fairbanks, Alaska, display two freezing events when experimentally cooled and different rates of survival after each event (mean ± SEM: −31.5 ± 0.2°C, 70% survival and −50.7 ± 0.4°C, 0% survival). To determine which body compartments froze at each event, we dissected the abdomen from the head/thorax and cooled each part separately. There was a significant difference between temperature levels of abdominal freezing (−30.1 ± 1.1°C) and head/thorax freezing (−48.7 ± 1.3°C). We suggest that freezing is initially restricted to one body compartment by regional dehydration in the head/thorax that prevents inoculative freezing between the freeze-tolerant abdomen (71.0 ± 0.8% water) and the supercooled, freeze-sensitive head/thorax (46.6 ± 0.8% water).     

Freezing and desiccation injury resistance in the filamentous green alga Klebsormidium from the Antarctic, Arctic and Slovakia

The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aeroterrestrial, terrestrial, and hydro-terrestrial) from distinct geographical regions (Antarctic — South Shetlands, King George Island, Arctic — Ellesmere Island, Svalbard, Central Europe — Slovakia) were studied. Each strain was exposed to several freezing (−4°C, −40°C, −196°C) and desiccation (+4°C and + 20°C) regimes, simulating both natural and semi-natural freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity) was evaluated by chlorophyll a content, viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation injuries. Freezing down to −196°C was the most harmful regime for all studied strains. Freezing at −4°C did not influence the survival of studied strains. Further, freezing down to −40°C (at a speed of 4°C/min) was not fatal for most of the strains. RDA analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4°C; however, freezing at −40°C, as well as desiccation at +20°C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic, and Central Europe (Slovakia) survived desiccation at temperatures of +4°C, and freezing down to −40°C. It appears that species of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability prevail, are well adapted to freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor is the resilience only found in polar strains as it is also a feature of temperate strains. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia. This paper is dedicated to the memory of the late Dr. Bohuslav Fott (1908–1976), Professor of Botany at the Charles University in Prague, to mark the centenary of his birth.     

Antifreeze proteins in the grubby sculpin,Myoxocephalus aenaeus and the tomcod,Microgadus tomcod: comparisons of seasonal cycles

Synopsis Antifreeze protein levels in the plasma of the grubby sculpin, Myoxocephalus aenaeus and the tomcod, Microgadus tomcod of Long Island coastal waters start to increase by November in anticipation of midwinter freezing conditions. Peak levels of antifreeze, as measured by the difference in plasma melting and freezing points, were detected in January for both species. The thermal hysteresis values reached 0.459°C in sculpin and 0.51°C in tomcod. Antifreeze peptides and glycopeptides start to disappear when water temperatures begin to rise and are at insignificant levels by late spring. Aspects of the seasonal cycle and the level of antifreeze activity were compared in three sympatric species (sculpin, tomcod, flounder); in two closely related but ecologically distinct gadids (tomcod, Atlantic cod); and within the genus Myoxocephalus.     

Expression of biologically active recombinant antifreeze protein His-MpAFP149 from the desert beetle (<Emphasis Type="Italic">Microdera punctipennis dzungarica</Emphasis>) in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An insect antifreeze protein gene Mpafp149 was cloned by the RT-PCR approach from the desert beetle Microdera punctipennis dzungarica. Sequence analysis revealed that this gene encoding a protein of 120 amid acids and this protein showed 65–76% homology with other insect antifreeze proteins, the deduced amino acid sequence displays very high similarities in those regions that contain tandem the 12-residue repeats (TCTxSxxCxxAx) domain and the TCT motif. Mpafp149 gene was cloned into pET-28a vector and expressed in Escherichia coli. A single-step purification based on specific binding of histidine residues was achieved. The purified His-MpAFP149 was SDS–PAGE analyzed, showing an atypical migration with molecular weight of about 24 kDa. The expression of His-MpAFP149 was confirmed by Western blot with specific binding to anti-GST-MpAFP149 antibody. The thermal hysteresis activity of the purified recombinant protein was 0.915°C at 0.09 mg/ml, and the supercooling point was −9.6°C at 0.03 mg/ml. In vitro antifreeze activity assay by measuring the survival rate of bacteria at −7 and −20°C respectively, with the protection of His-MpAFP149 showed that the His-MpAFP149 fusion protein was able to enhance the freeze resistance of bacteria.     

Osmotic and ionic regulation in response to salinity variations and cold resistance in the Arctic under-ice amphipod <Emphasis Type="Italic">Apherusa glacialis</Emphasis>

Amphipods living at the underside of Arctic sea ice are exposed to varying salinities due to freezing and melting, and have to cope with the resulting osmotic stress. Extracellular osmotic and ionic regulation at different salinities, thermal hysteresis, and supercooling points (SCPs) were studied in the under-ice amphipod Apherusa glacialis. The species is euryhaline, capable to regulate hyperosmotically at salinities S _R < 30 g/kg, and osmoconforms at salinities S _R ≥ 30 g/kg. Hyperosmotic regulation is an adaptation to thrive in low-salinity meltwater below the ice. Conforming to the ambient salinity during freezing reduces the risk of internal ice formation. Thermal hysteresis was not observed in the haemolymph of A. glacialis. The SCP of the species was −7.8 ± 1.9°C. Several ions were specifically downregulated ([Mg²⁺], [SO₄ ²⁻]), or upregulated ([K⁺], [Ca²⁺]) in comparison to the medium. Strong downregulation of [Mg²⁺], is probably necessary to avoid an anaesthetic effect at low temperatures.     

Maintenance of the supercooled state in overwintering pyrochroid beetle larvae, Dendroides canadensis : role of hemolymph ice nucleators and antifreeze proteins

Freeze-avoiding fire-colored beetle larvae, Dendroides canadensis, were monitored seasonally to explore the role of endogenous hemolymph ice nucleators and antifreeze proteins on the maintenance of supercooling. In preparation for overwintering, D. canadensis depressed hemolymph ice nucleator activity and increased thermal hysteresis activity [mean value circa 0. 5 °C (summer) versus circa 5 °C (midwinter)] resulting in decreased larval and hemolymph supercooling points [−7 °C (summer) versus −20 °C (midwinter)]. Results of gel filtration chromatography, flotation ultracentifugation and quantitative investigation of ice nucleator activity using hemolymph from summer and winter collected larvae strongly suggest that highly active protein and lipoprotein ice nucleators are removed in preparation for overwintering. Additions of either purified antifreeze proteins or midwinter hemolymph with high antifreeze protein activity to a mixture of protein or lipoprotein ice nucleators isolated from D. canadensis hemolymph inhibited the activity of these nucleators. This suggests that in addition to seasonal removal, inhibition of hemolymph ice nucleators by antifreeze proteins contributes to seasonal increases in hemolymph supercooling capacity. Accepted: 8 August 1996     

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.     

Survival and metabolism of <Emphasis Type="Italic">Rana arvali</Emphasis>s during freezing

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of −3°C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and −1°C). Lactate accumulation was only observed in the liver (4.1 ± 0.8 against 16.6 ± 2.4 μmol g⁻¹ fresh weight (FW) between 4 and −1°C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4 ± 0.7 μl O₂ g⁻¹ FW h⁻¹ at −4°C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.     

Cold tolerance and dehydration in Enchytraeidae from Svalbard

When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C. In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions. Accepted: 12 December 1997     

Physiological constraints on the life cycle and distribution of the Antarctic fairy shrimp <Emphasis Type="Italic">Branchinecta gaini</Emphasis>

Maturation to adulthood and successful reproduction in the Antarctic fairy shrimp, Branchinecta gaini, must be completed within a physiologically challenging temporal window of ca. 2.5 months in the southern Antarctic Peninsula. Although adults show considerable metabolic opportunism at positive temperatures, little is known of their tolerance of two physiological insults potentially typical to pool life in the maritime Antarctic: sub-zero temperatures and salinity. B. gaini are freeze-avoiding crustaceans with temperatures of crystallisation (T _cs) of −5°C. No antifreeze proteins were detected in the haemolymph. Adults osmoregulate in relation to temperature, but rapid mortality in saline solutions of even low concentration, indicate they cannot osmoregulate in relation to salinity. Survival of ice encasement at temperatures above their T _c was found to be pressure but not time dependent: at severe inoculative ice pressures, there was little immediate survival and none survived after 48 h below −2°C; at mild inoculative ice pressures, immediate survival was ca. 100% at −3°C, but <20% after 48 h. There was no significant difference in survival after 1 and 6 h encasement at −3°C. Observations of ventilation suggest that it is not low temperature per se, but ice that represents the primary cryo-stress, with ventilatory appendages physically handcuffed below the freezing point of pool water. Both sub-zero temperatures and salinity represent real physiological constraints on adult fairy shrimp.     

Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-tolerant and freeze-intolerant earthworms

In this paper we present the results of physiological responses to winter acclimation and tissue freezing in a freeze-tolerant Siberian earthworm, Eisenia nordenskioeldi, and two freeze-intolerant, temperate earthworm species, Lumbricus rubellus and Aporrectodea caliginosa. By analysing the physiological responses to freezing of both types we sought to identify some key factors promoting freeze tolerance in earthworms. Winter acclimation was followed by a significant increase in osmolality of body fluids in E. nordenskioeldi, from 197 mosmol kg⁻¹ in 10 °C-acclimated animals to 365 mosmol kg⁻¹ in animals acclimated to 0 °C. Cold acclimation did not cause any change in body fluid osmolality in the two freeze-intolerant species. As a response to ice formation in the body, the freeze-intolerant species produced copious amounts of slime and expulsion of coelomic fluids, and thereby lost 10–30% of their total water content. Contrary to this, the freeze-tolerant species did not lose water upon freezing. At temperatures down to −6.5 °C, the ice content in the freeze-tolerant E. nordenskioeldi was significantly lower than in L. rubellus. At lower temperatures there were no differences in ice content between the two species. Cold acclimated, but unfrozen, specimens of all three species had low levels of ammonia, urea, lactate, glycerol and glucose. As a response to ice formation, glucose levels significantly increased within the first 24 h of freezing. This was most pronounced in E. nordenskioeldi where a 153-fold increase of glucose was seen (94 mmol · l⁻¹). In L. rubellus and A. caliginosa a 19-fold and 17-fold increase in glucose was seen. This is the first study on physiological mechanisms promoting freeze tolerance in E. nordenskioeldi, or any other oligochaete. Our results suggest that the cryoprotective system of this species more closely resembles that of freeze-tolerant anurans, which synthesize cryoprotectants only after tissues begin to freeze, than that of cold-hardy invertebrates which exhibit a preparatory accumulation of cryoprotectants during seasonal exposure to low temperature. Accepted: 10 February 1999     

Thermal tolerance limits in six weevil species (Coleoptera, Curculionidae) from sub-Antarctic Marion Island

Supercooling points, lower lethal temperatures, and the effect of short-term exposures to low temperatures were examined during both winter and summer in the adults of six weevil species from three different habitats on Marion Island. Upper lethal limits and the effects of short-term exposure to high temperatures were also examined in summer-acclimatized adult individuals of these species. Bothrometopus elongatus, B. parvulus, B. randi, Ectemnorhinus marioni, and E. similis were freeze tolerant, but had high lower lethal temperatures (−7 to −10°C). Seasonal variation in these parameters was not pronounced. Physical conditions of the habitat appeared to have little effect on cold hardiness parameters because the Ectemnorhinus species occur in very wet habitats, whereas the Bothrometopus species inhabit drier areas. The adults of these weevil species are similar to other high southern latitude insects in that they are freeze tolerant, but with high lower lethal temperatures. In contrast, Palirhoeus eatoni, a supra-littoral species, avoided freezing and had a mean supercooling point of −15.5 ± 0.94°C (SE) in winter and −11.8 ± 0.98°C in summer. Survival of a constant low temperature of −8°C also increased in this species from 6 h in summer to 27 h in winter. It is suggested that this strategy may be a consequence of the osmoregulatory requirements imposed on this species by its supra-littoral habitat. Upper lethal temperatures (31–34°C) corresponded closely with maximum microclimate temperatures in all of the species. This indicates that the pronounced warming, accompanied by the increased insolation that has been recorded at Marion Island, may reduce survival of these species. These effects may be compounded as a consequence of predation by feral house mice on the weevils. Received: 4 February 1997 / Accepted: 3 May 1997     

The presence and quantification of splenic ice in the McMurdo Sound Notothenioid fish, Pagothenia borchgrevinki (Boulenger, 1902)

Survival of some polar fishes is associated with high levels of circulating antifreeze glycoproteins (AFGPs). AFGP prevent ice growth giving rise to thermal hysteresis. The inhibiting action of AFGPs implies that polar fish contain ice to which AFGPs adsorb. Cryopelagic Pagothenia borchgrevinki, inhabiting the ice-laden waters of McMurdo Sound, Antarctica, were assayed for ice and ice was found on skin, gills, in the intestine, and in the spleen. Two methods used to assess the number of ice crystals in spleens gave comparable results (12.1 +/− 1.9 and 22 +/− 3.8 per spleen). Attempts were made to measure the rate of uptake of ice by P. borchgrevinki held in cages immediately beneath the sub-ice platelet layer in McMurdo Sound; uptake was sporadic. Introduction of ice into fish by spray freezing a small patch of the integument resulted in detection of splenic ice after 1 h, illustrating that a mechanism exists for ice transport from the periphery to the spleen. Splenic ice did not seem to be eliminated from fish held in ice-free water at − 1.6 °C for approximately two months. The relatively small number of splenic ice crystals and the slow rate of ice uptake suggest efficient ice barriers exist in P. borchgrevinki.     

Freeze tolerance in larvae of the winter-active Diamesa mendotae Muttkowski (Diptera: Chironomidae): a contrast to adult strategy for survival at low temperatures

The winter-active Diamesa mendotae Muttkowski (Diptera: Chironomidae) is freeze intolerant in the adult stage with a low mean supercooling point (SCP) of ~−20 °C. However, cold-hardiness strategies for immatures of this species are unknown. In this study, we measured SCP values for D. mendotae larvae, pupae and adults using surface-contact thermometry. In addition, the lower lethal temperature (LLT) was determined for the larval stage. The mean SCPs for larvae (−7.4 °C) and pupae (−9.1 °C) were relatively high compared to adults (−19.7 °C). Our results indicate that the larvae of D. mendotae are freeze tolerant with a LLT₉₉ (−25.4 °C), ~−10 °C lower than their minimum SCP (−15.6 °C). Freeze tolerance in these larvae may be a strategy to provide protection from short-term exposures to ice crystals or to permit diapause within frozen substrates. The change in cold-hardiness strategy from freeze tolerant to freeze intolerant between the larval and adult stages of this species is likely a result of the different habitats occupied by these two life stages.