Plasma Antifreeze Research Articles

American eel (Anguilla rostrata) substrate selection for daytime refuge and winter thermal sanctuary

We addressed hypotheses that anguillid eels use mud as a substrate refuge only in the absence of substrate cavities, and that the winter distribution of eels in coastal bay and estuarine habitat is limited to waters warmer than the freezing point of fish tissue (~–0.7°C). In the seasonally ice-covered southern Gulf of St Lawrence, Canada, locations of summer fyke and winter spear fisheries indicate that American eels (Anguilla rostrata) are widely distributed in both summer and winter in shallow soft-bottomed bay and estuarine habitat. Captive eels in fresh water preferred mud substrates during summer, during pre-winter cooling and during post-winter warming periods, but in winter chose mud and cobble substrates at approximately equal frequencies. Plasma antifreeze was not detected in blood sampled from eels speared in mud under winter ice. Winter bottom water temperatures in an eel wintering site were below the approximate freezing point of fish tissue 29.9% of the time. Mud of eel wintering grounds is warmer than overlying water and appears to serve as a thermal sanctuary that allows eels to safely overwinter under ice-covered waters. American eels in the southern Gulf of St Lawrence spend ~67% of their annual cycle within the substrate.

A re-evaluation of the role of type IV antifreeze protein

A lipoprotein-like antifreeze protein (type IV AFP) has previously been isolated only from the blood plasma of the longhorn sculpin. However, the plasma antifreeze activity in all individuals of this species tested from Newfoundland and New Brunswick waters ranges from low to undetectable. A close relative of the longhorn sculpin, the shorthorn sculpin, does have appreciable antifreeze activity in its blood but this is virtually all accounted for by the α-helical, alanine-rich type I AFP, other isoforms of which are also present in the skin of both fishes. We have characterized a putative ortholog of type IV AFP in shorthorn sculpin by cDNA cloning. This 12.2-kDa Gln-rich protein is 87% identical to the longhorn sculpin’s type IV AFP. Recombinant versions of both orthologs were produced in bacteria and shown to have antifreeze activity. Immunoblotting with antibodies raised to type IV AFP shows this protein present in longhorn sculpin plasma at levels of less than 100 μg/mL, which are far too low to protect the blood from freezing at the temperature of icy seawater. This confirms the results of direct antifreeze assays on the plasmas. It appears that type IV AFP has the potential to develop as a functional antifreeze in these fishes but may not have been selected for this role because of the presence of type I AFP. Consistent with this hypothesis is the observation that the type IV AFP gene has not been amplified the way functional antifreeze protein genes have in all other species examined.

Seasonal modulation of plasma antifreeze protein levels in Atlantic ( Anarhichas lupus) and spotted wolffish ( A. minor)

The Atlantic and spotted wolffish ( Anarhichas lupus and A. minor, respectively) inhabit the cold waters of the northeast Atlantic Ocean. Although both species experience subzero water temperatures during winter, the Atlantic wolffish, which occupies shallower waters than the spotted wolffish, faces the greater threat of coming into contact with ice and freezing. This laboratory study was designed to determine whether these species differed in their abilities to resist freezing by examining the seasonal changes in blood plasma freezing points, antifreeze protein (AFP) activity and Na + and Cl − concentrations when exposed to seasonally cycling water temperatures and photoperiod. The plasma of both species showed distinct seasonal cycles in all parameters with the highest values occurring during the winter. However, of the two species, only the Atlantic wolffish produced sufficient AFP to protect the fish down to the freezing point of seawater (− 1.80 °C). The levels of AFP in the spotted wolffish were too low to impart any significant improvement in their resistance to freezing (approximately − 0.8 °C). When wolffish were maintained in warm water under a seasonally changing photoperiod, the amplitude of the seasonal cycle in AFP activity was greatly reduced, indicating that low water temperatures are necessary to maximize plasma AFP levels. However, despite being maintained in warm water, plasma levels of AFP activity began to increase over summer values at the same time of year as did the fish exposed to seasonally changing water temperatures. This suggests that photoperiod plays a major role in the timing of the annual AFP cycle.

Influence of plasma antifreeze glycoproteins on temperature selection by Atlantic cod (Gadus morhua) in a thermal gradient

Atlantic cod (Gadus morhua) produce antifreeze glycoproteins when exposed to cold conditions in winter which increase their physiological resistance to extreme low temperatures. Two size categories of cod (23-31 and 40-60 cm), containing subgroups with and without antifreeze, were acclimated to 2 °C (November-February). A thermal gradient (-0.5 to 1°C approximately) was established in a compartmented trough and fish were introduced into the temperature gradient singly (experiments conducted between December and February). Frequency of occupation of the compartments and temperatures throughout the trough were recorded during hour-long observation periods. Mean gradient temperatures occupied by cod in the four groups (large and small, with and without antifreeze) were not correlated with plasma antifreeze levels. Cod with antifreeze did not show a preference for subzero water. Overall, large cod occupied a lower mean temperature than small cod. However, small cod with antifreeze occurred significantly more frequently in the cold and less in the warm and middle gradient areas than did small cod with no antifreeze. This effect was not seen in the two groups of large cod. Thus, the presence of plasma antifreeze increased the likelihood of small cod entering water at subzero temperatures, while having no such effect on large cod. Resume : Les morues franches (Gadus morhua) produisent des glycoproteines antigel lorsqu'elles sont exposees au froid durant l'hiver, ce qui augmente leur resistance physiologique aux temperatures extremement basses. Des morues divisees en deux categories de taille (23-31 cm et 40-60 cm), et subdivisees en groupes possedant ou non l'antigel, ont ete acclimatees a une temperature de 2°C (novembre a fevrier). Un gradient de temperature (de -0,5 a 1 °C, approximativement) a ete etabli a l'interieur d'une auge compartimentee; les poissons ont ete exposes a ce gradient de temperature un par un (experiences realisees de decembre a fevrier). La frequence d'occupation des differents compartiments, de meme que les temperatures, ont ete enregistrees a travers toute l'auge, au cours de periodes d'observation de 1 h. Les temperatures moyennes des compartiments occupes par les morues n'etaient pas en correlation avec les concentrations d'antigel dans le plasma, et ce, pour les quatre groupes (grandes et petites morues, avec et sans antigel). Les morues possedant l'antigel n'ont pas manifeste de preference pour les temperatures inferieures a zero. Dans l'ensemble, les grandes morues ont occupe des compartiments dont la temperature moyenne etait plus basse que celle des compartiments occupes par les petites morues. Cependant, les petites morues possedant l'antigel se retrouvaient, de facon significative, plus souvent dans les compartiments froids et moins souvent dans les parties chaudes ou moyennes du gradient que les petites morues depourvues d'antigel. Cet effet n'a pas ete observe dans les deux groupes de grandes morues. La presence d'antigel dans le plasma a donc augmente la probabilite pour les petites morues de penetrer dans de l'eau a des temperatures inferieures a zero, alors qu'elle n'avait pas cet effet chez les grandes morues. (Traduit par la Redaction)

Movements of farmed and wild Atlantic cod (Gadus morhua) released in Trinity Bay, Newfoundland

Fishery scientists and managers are investigating the feasibility of enhancing annual recruitment to the northern Atlantic cod (Gadus morhua L.) stock complex off Labrador and northeastern Newfoundland through the release of farmed fish back into the sea. Release of newly matured fish and adults with farm-advanced fecundity would increase the spawning biomass. Enhancement efforts might be measurably successful in major bays that are year-round habitats for cod. To determine if farmed cod would remain and spawn in Trinity Bay, 14 fish with surgically implanted transmitters were released in November 1992. Sonic tracking confirmed that farmed cod released on the western side of Trinity Bay overwintered within the bay, and integrated with wild cod approaching spawning condition in April 1993. Blood plasma antifreeze levels confirmed that these wild cod had overwintered inshore in subzero waters. A spawning aggregation was found in July 1993, providing evidence that northern cod reproduce in Newfoundland bays. These findings suggest that it may be possible to increase the number of cod spawning inshore through the release of farmed fish.

Overwintering of Adult Northern Atlantic Cod (Gadus morhua) in Cold Inshore Waters as Evidenced by Plasma Antifreeze Glycoprotein Levels

Adult Atlantic cod (Gadus morhua) are known to produce antifreeze glycoproteins in response to cold temperatures. Our laboratory studies demonstrated that blood plasma levels in adult cod were positively correlated with the number of days they spent in subzero water. Between April 1991 and June 1993, we monitored concentrations of antifreeze glycoproteins in the plasma of late juvenile and adult cod in Trinity Bay, Newfoundland, and used the results to estimate how long cod had been exposed to low water temperatures. A consideration of these data in conjunction with detailed temperature profiles of the area taken over the course of the study allowed us to deduce the distribution of cod in relation to the temperature field. This study provides evidence that (1) blood antifreeze glycoprotein levels can be used to deduce the recent thermal history of cod in the wild and (2) after their inshore summer feeding period, considerable numbers of adult cod overwintered inshore in Trinity Bay in subzero water, producing antifreeze glycoproteins as temperatures fell below 0 °C. From May onwards, "cold-adapted" cod moved into warming surface waters, where they became available to an early inshore trap fishery.

Antifreeze Production, Freeze Resistance, and Overwintering of Juvenile Northern Atlantic Cod (Gadus morhua)

Four size groups of northern Atlantic cod (Gadus morhua) (juveniles < 15, 15–25, and 26–40 cm and adults > 50 cm) were investigated over a winter cycle for their ability to increase freeze resistance by producing plasma antifreeze glycoproteins. All juveniles had significantly higher plasma antifreeze levels than adults during the winter (January–March) under similar conditions of cycling temperature and ambient photoperiod. Highest winter plasma antifreeze levels and lowest plasma freezing temperatures were consistently found in the 15–25 cm group. Fish length and plasma antifreeze levels were inversely related from January to April, with the exception of the 0 + cod. All juvenile groups developed considerable freeze protection early in the winter cycle before the onset of freezing temperatures. However, the adults were not significantly protected until the end of January, when ambient temperatures had fallen below 0 °C. The 15–25 cm cod were held in three cycling temperature regimes. Induction temperatures for antifreeze production in this size group appeared to be between 2 and 3 °C, as compared with between 1 and 0 °C for the adults. We suggest that these patterns of antifreeze production may have direct bearing on habitat selection and overwintering behaviour of the northern Atlantic cod at different stages of development.

Hormonal regulation of antifreeze protein gene expression in winter flounder

The essential features of experiments carried out over the past fifteen years are brought together with new data to formulate a model describing the hormonal regulation of the annual plasma antifreeze polypeptide (AFP) cycle in winter flounder (Pseudopleuronectes americanus). The precise time of onset of antifreeze synthesis in the fall appears to be regulated by photoperiod acting through the central nervous system (CNS) on the pituitary gland. During the summer, growth hormone (GH) blocks transcription of the AFP genes. With the loss of long daylengths in the fall, the CNS inhibits the release of GH allowing AFP gene transcription in the liver to proceed. In the spring GH is again released from the pituitary and AFP gene transcription is blocked.

Antifreeze proteins in the urine of marine fish

Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6-33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each of these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.

Fish antifreeze proteins: physiology and evolutionary biology

Many marine teleosts have adapted to ice-laden seawater by evolving antifreeze proteins and glycoproteins. These proteins are synthesized in the liver for export to the blood where they circulate at levels of up to 20 mg/mL. There are at least four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence, and secondary structure. In addition to antifreeze structural diversity, fish species differ considerably with respect to mechanisms controlling seasonal regulation of plasma antifreeze concentrations. Some species synthesize antifreeze proteins immediately before the onset of freezing conditions, some synthesize them in response to such conditions, whereas others possess high concentrations all year. Endogenous rhythms, water temperature, photoperiod, and pituitary hormones have all been implicated as regulators of plasma antifreeze protein levels. The structural diversity of antifreeze proteins and their occurrence in a wide range of fish species suggest that they evolved separately and recently during Cenozoic glaciation. Invariably, the genes coding for these antifreeze proteins are amplified, sometimes as long tandem arrays, suggesting intense selective pressure to produce large amounts of protein. The distribution of antifreeze gene types among fish species suggests that they could serve as important tools for studying phylogenetic relationships.

Juvenile Atlantic Cod (Gadus morhua) Can Be More Freeze Resistant than Adults

Adult Atlantic cod (Gadus morhua) produce antifreeze glycopeptides during the winter which help to depress the freezing point of their blood plasma to approximately −1.2 °C. Since the temperature at which fish can freeze and die approximates the freezing point of their blood plasma, it is evident that adult cod do not have the capacity to survive in ice-laden waters when the water temperatures drop to −1.5 °C. Templeman and Fleming hypothesized that small cod are more adapted to cold water than adults. The present study documents the fact that the blood plasma freezing points of juvenile cod (mean length = 33 cm, 3-yr-olds) were significantly lower than those of adult cod (> 45 cm) (juvenile, −1.55 °C; adult, −1.23 °C). This difference was largely attributable to the doubling of plasma antifreeze glycopeptide levels in the juvenile cod. These results directly support Templeman and Fleming's hypothesis and indicate that juvenile cod, unlike adults, are capable of surviving the icy marine conditions which prevail along the northeast coast of Newfoundland during the winter.

Low temperature regulation of antifreeze glycopeptide levels in Atlantic cod (Gadus morhua)

The influence of water temperature and photoperiod on the timing of the annual cycle of plasma antifreeze glycoproteins (AFGP) was examined in Atlantic cod. Long day lengths (18 h) or continuous light had no effect on the time of appearance or disappearance of AFGP from the plasma. Cold water (0 °C) advanced the time of AFGP appearance by as much as 100 days. Long day lengths had no effect on this early induction of AFGP production. AFGP was not detectable in the plasma of fish exposed to water temperatures greater than 1 °C. Although small amounts of AFGP did appear in the plasma of cod exposed to 1 °C, it immediately began to disappear while plasma levels in normal and 0 °C acclimated cod continued to rise. The biological half time of AFGP activity was very sensitive to temperature, ranging from 15.6 days at 5 °C to 99.4 days at 0 °C. The results of this study suggest that the appearance of AFGP in cod during the winter months is dependent on the cod's exposure to water temperatures at least as low as 1 °C. Although 1 °C appears to be capable of initiating production of AFGP, it is not low enough to allow normal protective levels to be built up in the plasma.

Antifreeze peptides confer freezing resistance to fish

It has been widely accepted that plasma antifreeze proteins are directly responsible for the ability of many marine teleosts to survive in ice-laden seawater. However, there appears to be no direct experimental evidence to indicate that this assumption is correct. In the present study winter flounder (Pseudopleuronectes americanus) showed seasonal changes in freezing resistance that were quantitatively the same as the seasonal changes in plasma antifreeze protein levels. Moreover, when winter flounder antifreeze proteins were injected into rainbow trout (Salmo gairdneri) (a species that does not normally possess antifreeze proteins) they increased the freezing resistance of the trout in direct proportion to plasma antifreeze protein levels attained. These studies indicate that antifreeze proteins are directly responsible for the ability of many marine teleosts to survive icy seawater at temperatures below the colligative freezing points of their blood. There appears to be no requirement for species-specific antifreeze protein receptors in the fish in order for them to act.

Year-round presence of high levels of plasma antifreeze peptides in a temperate fish, ocean pout (Macrozoarces americanus)

The annual cycle of plasma antifreeze activity and Na+ and Cl− concentrations were measured in Newfoundland and New Brunswick ocean pout (Macrozoarces americanus) maintained in the laboratory under Newfoundland conditions of water temperature and photoperiod. The Na+ and Cl− concentrations and antifreeze activity showed distinct seasonal cycles in both groups of fish, with the highest values occurring during the winter. Relatively high concentrations of antifreeze peptides were present in the Newfoundland ocean pout at all times of the year. Antifreeze peptides were also present in the New Brunswick fish throughout the year; however, the concentrations found were approximately one-tenth of those observed in Newfoundland fish. A comparison of the antifreeze peptides from summer and winter samples from Newfoundland ocean pout and winter samples from New Brunswick ocean pout using high performance liquid chromatography revealed essentially identical elution profiles.

Annual antifreeze cycles in Newfoundland, New Brunswick and Long Island winter flounder Pseudopleuronectes americanus

Annual cycles of plasma antifreeze peptide levels were compared in winter flounder Pseudopleuronectes arnericanus from 3 areas: Conception Bay. Newfoundland; Passamaquoddy Bay. New Brunswick; and Shinnecock Bay, Long Island, New York. Winter flounder from the most southerly population, Shinnecock Bay, initiated antifreeze peptide production at the same time of year (Nov) as did flounder from the most northern population (Newfoundland). Flounder from a geographically intermediate population. Passamaquoddy Bay, produced antifreeze peptides approximately 2 mo later (Jan). The differences observed did not correlate with differences in seawater temperatures. Antifreeze peptides disappeared from the plasma during March in the Shinnecock Bay flounder, April in the New Brunswick flounder, and May in the Newfoundland flounder. These differences correlated with the regional differences in the time at which the water temperature increased It is hypothesized that the time of antifreeze production is a genetically determined population characteristic which reflects the earliest date that antifreeze peptides would be essential to their survival.

Seasonal and Phenotypic Variations in Plasma Protein Antifreeze Levels in a Population of Marine Fish, Sea Raven (Hemitripterus americanus)

The annual cycle of plasma antifreeze polypeptide levels and Na+and Cl−concentrations were measured in sea raven (Hemitripterus americanus) from a New Brunswick population. Both Na+and Cl−concentrations showed a distinct seasonal cycle, with the highest concentration of both ions occurring during the winter months. Mean plasma antifreeze levels also showed a significant seasonal cycle, with the highest values occurring during winter. Two features of the annual plasma antifreeze cycle differed considerably from the annual cycles observed in other temperate water fish: antifreeze polypeptides were present throughout the year and, during the winter, 40–50% of the fish had antifreeze levels that were the same as those observed during the summer. Thus, our results modify the generally accepted view that the presence of plasma antifreeze proteins correlates with the seasonal period of low water temperatures and that only fish inhabiting polar oceans maintain high levels of antifreeze peptides in their blood all year round.

How does the brain control the pituitary's release of antifreeze synthesis inhibitor?

Previous studies of winter flounder (Pseudopleuronectes americanus) demonstrated that the pituitary inhibits the synthesis of antifreeze proteins during the summer and that the inhibition is removed with the approach of winter. Assuming that the pituitary is under the control of the central nervous system, the question posed was, Does the central nervous system stimulate the release of the pituitary antifreeze inhibitory factor during the summer or inhibit its release during the winter? Two experiments were carried out. In the first, flounder were hypophysectomized and a number of them were given pituitary autotransplants prior to the spring loss of plasma antifreeze. During July, flounder containing functional autotransplants had lost the capacity to synthesize antifreeze proteins and their plasma antifreeze activity had disappeared. In the second experiment, hypophysectomy and pituitary transplantation was carried out in the fall prior to the winter onset of antifreeze biosynthesis. Flounder containing functional auto- or homo-transplants showed no evidence of plasma antifreeze activity, whereas intact controls and hypophysectomized flounder had levels typical of winter fish. These results indicate that the central nervous system normally inhibits the pituitary glands release of antifreeze inhibitor during the winter.

Comparison of antifreeze polypeptides from newfoundland, nova scotia, new brunswick and long island winter flounder

1. 1. Seven plasma antifreeze polypeptides (AFP) isolated from winter flounder collected from Nova Scotia, New Brunswick, Long Island (New York) and Newfoundland demonstrated nearly identical elution profiles when analyzed by reverse phase high performance liquid chromatography. 2. 2. AFP from the different flounder populations had similar amino acid compositions. 3. 3. One of the major AFP components isolated from New Brunswick flounder was different from the AFP samples from other populations in that it contained valine. This minor variation suggests that a limited amount of genetic polymorphism may be present in the winter flounder from New Brunswick. 4. 4. In general this study suggests that there is little variation in the structures of AFP from winter flounder inhabiting different locations.

The role of pituitary in regulating antifreeze protein synthesis in the winter flounder

The antifreeze proteins are a unique class of serum proteins found in some polar and cold water fish. These proteins prevent the fish from freezing and are essential for their survival [ 1,2] . At present, there are two different types of antifreeze proteins known; the glycoproteins isolated from some Antarctic [3] and Arctic fishes [4] and the polypeptide antifreeze from the winter flounder [5,6]. The glycoproteins consist of repeating tripeptide units of alanyl-alanylthreonine with a disaccharide linked to the threonyl residue. Flounder antifreeze protein, on the other hand, contains 8 amino acids and no carbohydrate moiety. The only similarity between these two types of antifreezes are the abundance of alanine in their amino acid composition. The occurrence of the antifreeze protein (AFP) in the winter flounder inhabiting Newfoundland seawater is seasonal [7]. It appears in the plasma during November and disappears during May of each year. Both temperature and photoperiod are involved in regulating this annual cycle [S] . We have recently shown that the pituitary is necessary for the seasonal disappearance of the antifreeze from the plasma [9]. However the detailed mechanisms whereby the pituitary regulates plasma antifreeze has yet to be investigated. Here we describe our findings on the effect of hypophysectomy on AFP synthesis using both in vivo and in vitro procedures. Our results show that while control animals lose the ability to synthesis AFP during the summer months, the hypophysectomized animals continue to synthesize the protein.