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Protection against lethal freezing temperatures by mucus in an antarctic limpet
(:RYOBlO1.o(:Y IO, x31-:3:37
Protection
( 1973 )
Against
Lethal Freezing Temperatures in an Antarctic Limpet1
“Anchor kc:” and its effects on the distriImtion of some rpibenthic, antarctic animals have been rcportc~d (2, 12). Intertidal zone temperature is often substantially less than seawater temperature in polar regions (8). SCUBA diving observations of the limpet Patinigera polaris during the antarctic winter of 1970 showed that these limpets we’re naturally frozen into either anchor ice or intertidal SC& iccl (Fig. 1). Furthermore, sometimes they were e\~n csposed to air temperatures considerably bc1ow -2” C during low-tide periods. Icccvvcred scawatcr temperatures near P&WI Station, Antarctica ranged frown - 1.80” C daily air tcmpcrato - 1.95” C and meaii tures ranged from -3.5” C to - 11.0” C. Received
May 18, 1973.
n Presently, American I Irart Associntion Rrsearch Fellow, lkpartmcnt of Rioengineering, School of Medicine, University of Californin. ~:ur Diego, La Jolla. California 920:37.
by Mucus
In all casc~s in-frozen limpets were surrounded b,- small ice-free pockets filled with a high viscosity mucus. These ice-free regions were l-11 mm thick around 11 animals observed between June and August. ,4fter the anchor ice lifted or thr high tide released the limpets, the animals cshibitcd normal behavior. These observations seemed to indicate that mucus, secrctcsd by the limpets frozen in these pock&s, was in sornc’ way prevrnting freeze damnq~. \l.STERIALS
AND
METHODS
In o&r to ascertain the protective value of mucus in these molluscs, a group of 270 limpets ( Patinigera polaris) were collected in earl>- December 1970. These individuals were collected from a depth of 12 m and from the intertidal zone. This group was divided into three sets of NH)limpets which were exposed to a series of air temperatures ranging from -3” C to -22” C in a top-opening industrial freezer capable of maintaining the desired temperaturc to :-50.5” C. The exposure period at each temperature was 2 hr. In the first set of 90 animals a freezing environment was crcaatcaclto conform closely to natural, inter-
332
HARGENS
AND
SHABICA
FK. 1. An antarctic limpet Pat~nigera polaris is scale ice. Air temperature ranged from -16.5” C exposure to the freezing, above-water milieu. Most facilitate photography. An ensuing high tide released Scale is in centimeters.
tidal, freezing conditions. To facilitate these conditions each limpet was individually sealed within a water-impermeable plastic bag (TOPCO Associates, Inc. ) such that a 4-mm space surrounded the animal. The water-impermeable bag portrayed intertidal scale ice which formed as the ebbing tide exposed the intertidal region. A small vent hole was punctured in each bag to allow air passage. A second set of 90 limpets was exposed to freezing air temperatures without the plastic wrap to retain the protective mucus. In the third, and control set, 90 limpets were individually sealed in the bags, but all secreted mucus was withdrawn via a hypodermic needle which penetrated the bag and was externally connected to a 20-ml glass syringe. Heat was conducted from the limpets dur-
trapped above water level in intertidal to -20.0” C during the limpet’s 4.3-hr of the surrounding ice was removed to the limpet which survived the experience.
ing low tides by both the air and the rocky intertidal substrate. To ensure that the latter property was observed, all experimental subjects were placed on granite rocks similar to those of the intertidal zone. Supercooling of the limpets was prevented by placing a small piece of ice into each experimental bag (sets 1 and 3) or in contact with the animal (set 2). Consequently, ice formation began near -2” C and increased as the run proceeded. The core temperatures of the limpets were determined by a thin, stainless steel thermistor probe ( Yellow Springs Instruments ) inserted into the visceral cavity between the foot and the gonad. Following each exposure to the experimental temperature, the subjects were placed in continuously flowing seawater tanks at approximately
PROTECTION
AGi\ISST
FREEZIS(:
BY ;Z LIMPET
hlI:CUS
333
Fro. 2. Apparatus used for obscrviug ice crystal behavior The insulated copper box (A) provides good temperature control and sa~nplrs are held adjacent to thermometer (T) in the lift-out holder ( B ). The rotatable contrast rod ( C.R. ) is stationed between the sample capillary and the heating wire (H.W.). A cross-sectional view of the box (C) shows i\~o chnnibers: the adjusting bath (A.B.) and the constant temperature bath (C.B.) with circulating bath inlet (B.I.) and outlet (B.O.). ICC is observed microscopically through the illunrinated viewing window (V.W.). The contrast cylinder (CC.) is aligned behind the ice crystal under study (see D). A close-up of the capillary tube vvith stirring rod inside (see E) shows monoclinic ice spears propagating front a bottom frozen layer.
1.0” C, observed for 24 hr, and counted for survivors. In order to characterize more fully the freeze-resistant properties of limpet blood and mucus, 21 Patinigera polaris limpets were collected near the end of the antarctic summer ( mid-February). These animals were collected from tide pools and at a depth of 12 m. Blood samples wcrc taken by heart puncture after the animals were carefully removed from their shells 1~~ scalpel dissection. Mucus samples were obtained by external recovery of the secreted
substance. Very fine drawn-out, l-mm glass capillaries were employed as collecting vessels for both blood and mucus. Blood capillary tubes were cut and the end was carefully occluded by a microalcohol flame. Then the blood was centrifuged and the plasma separated. Thus, freezing experiments were conducted in the same fine capillary tubes in which the samples were collected. Plasma melting and freezing equilibria were investigated using an apparatus accurate to 0.01” C (Fig. 2). Actual observation
3x4
HARGENS
Air wmperatwe (“Cl
Set 1
(with plastic U-i-rtp)
set 2
AND
Set 3
(without plas- (with plastic tir wrap) m-&p, muCUY withdrawn)
- :i -. i -8 -10 -12 -15 -18 -20 -22
3 1 0
of the minute crystals precluded any confusion between the melting-freezing equilibrium point and the temperature of initial ice propagation. Protein solutions, espccially those having glycoproteins, often have substantia1 disparities between the equihbrium freezing point and the temperature of initial ice propagation (3). The ice-equilibria apparatus ( Fig. 2)) modified considerably from its original design ( ll), was a two-chambered, copper box surrounded by 3-cm Styrofoam insulation. All copper walls were 2-mm thick. The 300-ml adjusting bath (A.B.) was filled with filtered, 10% ethylene glycol and held the sample tube, contrast rod (C.R. ), - 10” to +5” C precision thermometer (T) (A.S.T.M. 32 C, Butadiene, B.P.R., 162-mm long), fine heating wire ( H.W. ) (0.3-mm diam. lacquered, copper wire ) , and stirring propeller. Fogging on the viewing window (V.W.) was avoided by a few crystals of desiccant in the airtight observation port or by a heating wire along the port’s inside periphery. Physically separated but in thermal contact with the adjusting bath was a 150-ml, -4” C constant temperature bath (C.B.) connected by an inlet (B.I.) and outlet (B.O.) to a thermostatic, circulating bath (model K-2/R, Lauda, Inc. ). The circulating fluid was 20% ethylene glycol.
SHABICA
The holder (B) placed over the adjusting bath was Styrofoam except for a Lucite block backing the thermometer and sample tube. The heating wires connected externally to a variable transformer (Superior Elect. Co., Bristol, Ct.) which was calibrated to yield constant A.B. tcmpcratures at various settings. The sample tube passed through the holder (B) at an an& such that the 5-1n111” sample was positioned adjacent to the thermometer (see D, Fig. 2). The contrast cylinder ( C.C. ) could be rotated to facilitate ice crystal observation through a 15 X or 30X microscope. Illumination through the viewing window was provided bp a lamp positioned at the microscope’s side. The sample tube was a very thin walled, glass capillary in which a fine glass stirring rod was placed for sample mixing. A4 5-ni1ii~~ samplc~ was frozen with “SpraFreeze” ( Laboratory Supplies Co. ) and ice-cr)-stal behavior was investigated b\ observing the last small upward-floating ice crystal. The melting point of the sample was the temperature at which the last small upward-floating crystal began to blur. The freezing point was the temperature at which the edges of this last crystal sharpc,ned. The temperature of initial ice propagation was the point at which the smal1 crystal began to grow. All three points could bc determined to within 0.01” C. Rates of ice propagation at constant temperature were measured by timing the linear progress of ice needles emanating from a thin ln\,ctr of frozen fluid at the bottom of the: snmplc tube (see E, Fig. 1). A lo-mm microscope reticle (100 clivisions) apposed the sample t&e. RESULTS The results of survival studies on the three sets of experimental limpets are summarized in Table 1. During 2-hr exposures to air temperatures from -3” C to -22” C, each limpet’s core temperature was ambient k1.0” C. As freezing progressed, limpets secreted a profuse mucus which in
Bath
temperature
[*Cl
FE. 3. Equilibrium freezing points (F.P.‘s), temperatures ice propagation rates in antarctic limpet (Putinigera poln~is) and mucus sitlnples (triangular points )
set 1 encapsulated the animal. Hy the end of each experimental period all the limpets appcarcd to be completely frozen. In set 1, experimental temperatures greater than -5” C yielded a slushy-type ice within the bag, whereas temperatures less than -5” C imparted an adamant kc shell around the animal. In the first set of Table 1 (conforming to the natural milieu) fatalities appeared at -10” C and survivorship extended to -20” C. When limpets were not allowed to retain total mucus coverage (set 2), fatalities commenced at -8” C with a survival limit of -15” C. In order to separate out any protection afforded by the plastic wrap in set 1, a control group (set 3) was exposed to the freezing conditions but with mucus withdrawn. This control group (set 3) showed initial incidence of death at -8” C and a survival limit of -15” C. Statistical analyses indicate a significant diffcrcnce in survivorship due to the mucus protection. A concordance coefficient test indicates a confidence limit greater than 99%’ that the mucus-protected limpets (set 1) had significantly greater survivorship. Furthermore, comparing sets 2 and 3, no protection can bc ascribed to the plastic wrap. No significant survival difference was
of initial ice propagation, and blood samples (round points)
noted between intertidal limpets and those caught at 12 m depth. Extracellular ice was present in sets 2 and 3 at temperatures below -8” C following the 2-hr exposures. However, when limpets were enclosed within a mucus layer (set 1 ), extracellular ice was encountered only at temperatures below -10” C. EquiLbrium freezing points of all limpet blood and mucus samples were very near -1.8” C, the freezing point of the February seawater (Fig. 3). At this temperature, however, ice crystals would not grow. Ice propagation was not initiated until a mean bath temperature of -2.0” C was reached in 10 limpet blood samples. Moreover, the initiation of ice growth in eight superficially drawn mucus samples was retarded until a mean temperature of -2.35” C. Rates of ice propagation in limpet blood increased linearly at temperatures below -2” C. Compared to the blood, the mucus showed significantly slower propagation rates at the same temperatures (Fig. 3). DISCUSSION
The survival experiments in Table 1 indicate that the process of mucus secretion prevents extracellular ice formation down to -8” C. Indeed, no ice was present in
3x
HARGENS
AND
any tissues above this lower limit. Upon exposure to a freezing situation, the limpet secretes an isotonic mucus to lower interstitial fluid content, thereby avoiding extracellular ice propagation. A compressed cellular matrix effect ( 1, 7, 10) is probably the mechanism retarding ice propagation through limpet tissues. The increased survival between -8” C and -IO” C (set 1, TabIe 1) must be due to the cryoprotective nature of the mucus itself. Statistical analyses showed that there is significantly increased survival in the mucus-protected limpets and that the plastic wrap had no protective effect. Even a crude sampling of the mucus during the antarctic summer revealed that mucus retards ice propagation below its freezing point (Fig. 3 ). Such a cryoprotective mechanism occurs in antarctic fishes living in the same ecosystem ( 3). Like glycoproteins in polar fishes, mucus couId inhibit the growing surface of any ice crystal with which it comes into contact. These two mechanisms, a compressed tissue matrix and a cryoprotectivc mucus (protecting antarctic limpets between -2” C and -8” C and between -8” C and -10” C, respectively) differ considerably from the mechanism of survival of intertidal molluscs in the North Atlantic ecosystem. Those intertidal molluscs avoid freezing injury by reducing intracellular water as a consequence of extracellular ice formation (5, 6, X3-15), Freezing tolerance in Atlantic clams is thus limited by the animal’s tolerance to intracellular dehydration which is 64% in Myth edulis (15) and up to 75% in some other bivalves (5 ) I It is interesting to note that this is the normal range (60--750/o ) in which intracellular elements lose their aqueous milieu and begin to pack together (4, 9)The mucus which was sampled from outside the pallial cavity for the ice-crystal behavior studies probably was not as cryoprotective as that secreted naturally in a limpet captured by intertidal ice. Dilution
SHABICA
of these samples by seawater was possibly another systematic error. An actual glandular mucus sample would have avoided these difficulties. Nevertheless, the mucus did exhibit a significant ice retardation property which has not been previously reported. SUMMARY
The antarctic limpet, Patinigera polar& is sometimes caught in near-shore ice and exposed to temperatures substantially below -2” C. In-frozen animaIs always secrete an envelope of mucus which prevents extracellular ice propagation down to -10” C. Survival in limpets without mucus protection is significantly lower. Ice propagation through limpet mucus is retarded below its equihbrium freezing point in a manner similar to polar fishes. The capacity of mucus as a cryoprotectant has not previously been described. ACKNOWLEDGMENTS We thank Drs. P. F. Scholander, J. W. Hedgpeth, and Hans Theede for their interest and helpful discussions. Support is also acknowledged from the U. S. Naval Support Forces, Antarctica, for logistics during the field studies, and from the School of Oceanography of Oregon State University. Thanks are due to Dr. Edvard Hemmingsen, Dr. Walter F. Garey, and Captain Bob Haines of Scripps Institution of Oceanography for organizing the R/V ALPHA HELIX 1971 Antarctic Expedition, to Mr. maul Berkman of the National Oceanic and Atmospheric Administration for the reduction of tide marigrams, and to Dr. Charles B. Miller of Oregon State University for assistance with the statistical analyses. Special thanks to Mr. Michael Bergin of Marine Acoustical Services, Inc., and Messrs. Harvey High, Jay Klinck, and Dennis Pat.ton of the U. S. Navy Deep-Freeze 1970 winter-over crew. REFERENCES 1. Bloch, R., Walters, D. H., and Kuhn, W. Structurally caused freezing point depression of biological tissues. J. Gen. Physiol. 46, 605-615 ( 1963). 2. Dayton, P. K., Robilliard, G. A., and DeVries, A. L. Anchor ice formation in McMurdo Sound, Antarctica, and its biological effects. Science 163, 273-274 ( 1969).
PROTECTION
AGAINST
FREEZING
3. Hargens, A. R. Freezing resistance in polar fishes. Science 176, 184-186 (1972). 4. Hargens, A. R. Macromolecular osmotic pressures and interstitial fluid pressures in marine multicellular systems. Ph.D. Dissertation, Univ. of Calif., San Diego, pp. 30-5i;, 84-101, 1971. 5. Kanwisher, J. W. Freezing in intertidal animals. Biol. Bull. 109, 56-63 (1955). 6. Kanwisher, J. W. Freezing in intertidal animals. In “Cryobiology” (H. T. Meryman, Ed.), pp. 487-494. Academic Press, New York, 1966. 7. Litvan, G. G. Mechanism of cryoinjury in biological systems. Cryobiology 9, 182-191 (1972). 8. Newell, R. C. “The Biology of Intertidal Animals,” pp. 451-454, Logos Press Ltd., London, 1970. 9. Scholander, P. F., Hargens, A. R., and Miller, S. L. Negative pressure in the interstitial fluid of animals. Science 161, 321-328 (1968).
BY A LIMPET
MUCUS
x!l’i
10. Scholander, P. F., and Maggert, J. E. Supercooling and ice propagation in blood from arctic fishes. Cryobiology 8, 371-374 (1971). 11. Scholander, P. F., van Dam, L., Kanwisher, J. W., Hammel, H. T., and Gordon, nl. S. Supercooling and osmoregulation in arctic fish. .I. Cell. Comp. Physiol. 49, 5-24 (1957). 12. Shabica, S. V. Tidal zone ecology at Palmer Station. Antarct. 1. U. S. 7, 184-185 (1972). 13. Theede, H. Comparative ecophysiological studies on cellular cold resistance of marine invertebrates. Mar. Rid. 1.5, 160-191 (1972). 14. Theede, H., and Lassig, J. Comparative studies on cellular resistance of bivalves from marine and brackish waters. Helgolaentler Wiss. Mceresunters. 16, 119-129 ( 1967 ). 15. Williams, R. J. Freezing tolerance in Mytilus cdcllis. Camp Biochem. Physiol. 35, 145161 (1970).
Protection
( 1973 )
Against
Lethal Freezing Temperatures in an Antarctic Limpet1
“Anchor kc:” and its effects on the distriImtion of some rpibenthic, antarctic animals have been rcportc~d (2, 12). Intertidal zone temperature is often substantially less than seawater temperature in polar regions (8). SCUBA diving observations of the limpet Patinigera polaris during the antarctic winter of 1970 showed that these limpets we’re naturally frozen into either anchor ice or intertidal SC& iccl (Fig. 1). Furthermore, sometimes they were e\~n csposed to air temperatures considerably bc1ow -2” C during low-tide periods. Icccvvcred scawatcr temperatures near P&WI Station, Antarctica ranged frown - 1.80” C daily air tcmpcrato - 1.95” C and meaii tures ranged from -3.5” C to - 11.0” C. Received
May 18, 1973.
n Presently, American I Irart Associntion Rrsearch Fellow, lkpartmcnt of Rioengineering, School of Medicine, University of Californin. ~:ur Diego, La Jolla. California 920:37.
by Mucus
In all casc~s in-frozen limpets were surrounded b,- small ice-free pockets filled with a high viscosity mucus. These ice-free regions were l-11 mm thick around 11 animals observed between June and August. ,4fter the anchor ice lifted or thr high tide released the limpets, the animals cshibitcd normal behavior. These observations seemed to indicate that mucus, secrctcsd by the limpets frozen in these pock&s, was in sornc’ way prevrnting freeze damnq~. \l.STERIALS
AND
METHODS
In o&r to ascertain the protective value of mucus in these molluscs, a group of 270 limpets ( Patinigera polaris) were collected in earl>- December 1970. These individuals were collected from a depth of 12 m and from the intertidal zone. This group was divided into three sets of NH)limpets which were exposed to a series of air temperatures ranging from -3” C to -22” C in a top-opening industrial freezer capable of maintaining the desired temperaturc to :-50.5” C. The exposure period at each temperature was 2 hr. In the first set of 90 animals a freezing environment was crcaatcaclto conform closely to natural, inter-
332
HARGENS
AND
SHABICA
FK. 1. An antarctic limpet Pat~nigera polaris is scale ice. Air temperature ranged from -16.5” C exposure to the freezing, above-water milieu. Most facilitate photography. An ensuing high tide released Scale is in centimeters.
tidal, freezing conditions. To facilitate these conditions each limpet was individually sealed within a water-impermeable plastic bag (TOPCO Associates, Inc. ) such that a 4-mm space surrounded the animal. The water-impermeable bag portrayed intertidal scale ice which formed as the ebbing tide exposed the intertidal region. A small vent hole was punctured in each bag to allow air passage. A second set of 90 limpets was exposed to freezing air temperatures without the plastic wrap to retain the protective mucus. In the third, and control set, 90 limpets were individually sealed in the bags, but all secreted mucus was withdrawn via a hypodermic needle which penetrated the bag and was externally connected to a 20-ml glass syringe. Heat was conducted from the limpets dur-
trapped above water level in intertidal to -20.0” C during the limpet’s 4.3-hr of the surrounding ice was removed to the limpet which survived the experience.
ing low tides by both the air and the rocky intertidal substrate. To ensure that the latter property was observed, all experimental subjects were placed on granite rocks similar to those of the intertidal zone. Supercooling of the limpets was prevented by placing a small piece of ice into each experimental bag (sets 1 and 3) or in contact with the animal (set 2). Consequently, ice formation began near -2” C and increased as the run proceeded. The core temperatures of the limpets were determined by a thin, stainless steel thermistor probe ( Yellow Springs Instruments ) inserted into the visceral cavity between the foot and the gonad. Following each exposure to the experimental temperature, the subjects were placed in continuously flowing seawater tanks at approximately
PROTECTION
AGi\ISST
FREEZIS(:
BY ;Z LIMPET
hlI:CUS
333
Fro. 2. Apparatus used for obscrviug ice crystal behavior The insulated copper box (A) provides good temperature control and sa~nplrs are held adjacent to thermometer (T) in the lift-out holder ( B ). The rotatable contrast rod ( C.R. ) is stationed between the sample capillary and the heating wire (H.W.). A cross-sectional view of the box (C) shows i\~o chnnibers: the adjusting bath (A.B.) and the constant temperature bath (C.B.) with circulating bath inlet (B.I.) and outlet (B.O.). ICC is observed microscopically through the illunrinated viewing window (V.W.). The contrast cylinder (CC.) is aligned behind the ice crystal under study (see D). A close-up of the capillary tube vvith stirring rod inside (see E) shows monoclinic ice spears propagating front a bottom frozen layer.
1.0” C, observed for 24 hr, and counted for survivors. In order to characterize more fully the freeze-resistant properties of limpet blood and mucus, 21 Patinigera polaris limpets were collected near the end of the antarctic summer ( mid-February). These animals were collected from tide pools and at a depth of 12 m. Blood samples wcrc taken by heart puncture after the animals were carefully removed from their shells 1~~ scalpel dissection. Mucus samples were obtained by external recovery of the secreted
substance. Very fine drawn-out, l-mm glass capillaries were employed as collecting vessels for both blood and mucus. Blood capillary tubes were cut and the end was carefully occluded by a microalcohol flame. Then the blood was centrifuged and the plasma separated. Thus, freezing experiments were conducted in the same fine capillary tubes in which the samples were collected. Plasma melting and freezing equilibria were investigated using an apparatus accurate to 0.01” C (Fig. 2). Actual observation
3x4
HARGENS
Air wmperatwe (“Cl
Set 1
(with plastic U-i-rtp)
set 2
AND
Set 3
(without plas- (with plastic tir wrap) m-&p, muCUY withdrawn)
- :i -. i -8 -10 -12 -15 -18 -20 -22
3 1 0
of the minute crystals precluded any confusion between the melting-freezing equilibrium point and the temperature of initial ice propagation. Protein solutions, espccially those having glycoproteins, often have substantia1 disparities between the equihbrium freezing point and the temperature of initial ice propagation (3). The ice-equilibria apparatus ( Fig. 2)) modified considerably from its original design ( ll), was a two-chambered, copper box surrounded by 3-cm Styrofoam insulation. All copper walls were 2-mm thick. The 300-ml adjusting bath (A.B.) was filled with filtered, 10% ethylene glycol and held the sample tube, contrast rod (C.R. ), - 10” to +5” C precision thermometer (T) (A.S.T.M. 32 C, Butadiene, B.P.R., 162-mm long), fine heating wire ( H.W. ) (0.3-mm diam. lacquered, copper wire ) , and stirring propeller. Fogging on the viewing window (V.W.) was avoided by a few crystals of desiccant in the airtight observation port or by a heating wire along the port’s inside periphery. Physically separated but in thermal contact with the adjusting bath was a 150-ml, -4” C constant temperature bath (C.B.) connected by an inlet (B.I.) and outlet (B.O.) to a thermostatic, circulating bath (model K-2/R, Lauda, Inc. ). The circulating fluid was 20% ethylene glycol.
SHABICA
The holder (B) placed over the adjusting bath was Styrofoam except for a Lucite block backing the thermometer and sample tube. The heating wires connected externally to a variable transformer (Superior Elect. Co., Bristol, Ct.) which was calibrated to yield constant A.B. tcmpcratures at various settings. The sample tube passed through the holder (B) at an an& such that the 5-1n111” sample was positioned adjacent to the thermometer (see D, Fig. 2). The contrast cylinder ( C.C. ) could be rotated to facilitate ice crystal observation through a 15 X or 30X microscope. Illumination through the viewing window was provided bp a lamp positioned at the microscope’s side. The sample tube was a very thin walled, glass capillary in which a fine glass stirring rod was placed for sample mixing. A4 5-ni1ii~~ samplc~ was frozen with “SpraFreeze” ( Laboratory Supplies Co. ) and ice-cr)-stal behavior was investigated b\ observing the last small upward-floating ice crystal. The melting point of the sample was the temperature at which the last small upward-floating crystal began to blur. The freezing point was the temperature at which the edges of this last crystal sharpc,ned. The temperature of initial ice propagation was the point at which the smal1 crystal began to grow. All three points could bc determined to within 0.01” C. Rates of ice propagation at constant temperature were measured by timing the linear progress of ice needles emanating from a thin ln\,ctr of frozen fluid at the bottom of the: snmplc tube (see E, Fig. 1). A lo-mm microscope reticle (100 clivisions) apposed the sample t&e. RESULTS The results of survival studies on the three sets of experimental limpets are summarized in Table 1. During 2-hr exposures to air temperatures from -3” C to -22” C, each limpet’s core temperature was ambient k1.0” C. As freezing progressed, limpets secreted a profuse mucus which in
Bath
temperature
[*Cl
FE. 3. Equilibrium freezing points (F.P.‘s), temperatures ice propagation rates in antarctic limpet (Putinigera poln~is) and mucus sitlnples (triangular points )
set 1 encapsulated the animal. Hy the end of each experimental period all the limpets appcarcd to be completely frozen. In set 1, experimental temperatures greater than -5” C yielded a slushy-type ice within the bag, whereas temperatures less than -5” C imparted an adamant kc shell around the animal. In the first set of Table 1 (conforming to the natural milieu) fatalities appeared at -10” C and survivorship extended to -20” C. When limpets were not allowed to retain total mucus coverage (set 2), fatalities commenced at -8” C with a survival limit of -15” C. In order to separate out any protection afforded by the plastic wrap in set 1, a control group (set 3) was exposed to the freezing conditions but with mucus withdrawn. This control group (set 3) showed initial incidence of death at -8” C and a survival limit of -15” C. Statistical analyses indicate a significant diffcrcnce in survivorship due to the mucus protection. A concordance coefficient test indicates a confidence limit greater than 99%’ that the mucus-protected limpets (set 1) had significantly greater survivorship. Furthermore, comparing sets 2 and 3, no protection can bc ascribed to the plastic wrap. No significant survival difference was
of initial ice propagation, and blood samples (round points)
noted between intertidal limpets and those caught at 12 m depth. Extracellular ice was present in sets 2 and 3 at temperatures below -8” C following the 2-hr exposures. However, when limpets were enclosed within a mucus layer (set 1 ), extracellular ice was encountered only at temperatures below -10” C. EquiLbrium freezing points of all limpet blood and mucus samples were very near -1.8” C, the freezing point of the February seawater (Fig. 3). At this temperature, however, ice crystals would not grow. Ice propagation was not initiated until a mean bath temperature of -2.0” C was reached in 10 limpet blood samples. Moreover, the initiation of ice growth in eight superficially drawn mucus samples was retarded until a mean temperature of -2.35” C. Rates of ice propagation in limpet blood increased linearly at temperatures below -2” C. Compared to the blood, the mucus showed significantly slower propagation rates at the same temperatures (Fig. 3). DISCUSSION
The survival experiments in Table 1 indicate that the process of mucus secretion prevents extracellular ice formation down to -8” C. Indeed, no ice was present in
3x
HARGENS
AND
any tissues above this lower limit. Upon exposure to a freezing situation, the limpet secretes an isotonic mucus to lower interstitial fluid content, thereby avoiding extracellular ice propagation. A compressed cellular matrix effect ( 1, 7, 10) is probably the mechanism retarding ice propagation through limpet tissues. The increased survival between -8” C and -IO” C (set 1, TabIe 1) must be due to the cryoprotective nature of the mucus itself. Statistical analyses showed that there is significantly increased survival in the mucus-protected limpets and that the plastic wrap had no protective effect. Even a crude sampling of the mucus during the antarctic summer revealed that mucus retards ice propagation below its freezing point (Fig. 3 ). Such a cryoprotective mechanism occurs in antarctic fishes living in the same ecosystem ( 3). Like glycoproteins in polar fishes, mucus couId inhibit the growing surface of any ice crystal with which it comes into contact. These two mechanisms, a compressed tissue matrix and a cryoprotectivc mucus (protecting antarctic limpets between -2” C and -8” C and between -8” C and -10” C, respectively) differ considerably from the mechanism of survival of intertidal molluscs in the North Atlantic ecosystem. Those intertidal molluscs avoid freezing injury by reducing intracellular water as a consequence of extracellular ice formation (5, 6, X3-15), Freezing tolerance in Atlantic clams is thus limited by the animal’s tolerance to intracellular dehydration which is 64% in Myth edulis (15) and up to 75% in some other bivalves (5 ) I It is interesting to note that this is the normal range (60--750/o ) in which intracellular elements lose their aqueous milieu and begin to pack together (4, 9)The mucus which was sampled from outside the pallial cavity for the ice-crystal behavior studies probably was not as cryoprotective as that secreted naturally in a limpet captured by intertidal ice. Dilution
SHABICA
of these samples by seawater was possibly another systematic error. An actual glandular mucus sample would have avoided these difficulties. Nevertheless, the mucus did exhibit a significant ice retardation property which has not been previously reported. SUMMARY
The antarctic limpet, Patinigera polar& is sometimes caught in near-shore ice and exposed to temperatures substantially below -2” C. In-frozen animaIs always secrete an envelope of mucus which prevents extracellular ice propagation down to -10” C. Survival in limpets without mucus protection is significantly lower. Ice propagation through limpet mucus is retarded below its equihbrium freezing point in a manner similar to polar fishes. The capacity of mucus as a cryoprotectant has not previously been described. ACKNOWLEDGMENTS We thank Drs. P. F. Scholander, J. W. Hedgpeth, and Hans Theede for their interest and helpful discussions. Support is also acknowledged from the U. S. Naval Support Forces, Antarctica, for logistics during the field studies, and from the School of Oceanography of Oregon State University. Thanks are due to Dr. Edvard Hemmingsen, Dr. Walter F. Garey, and Captain Bob Haines of Scripps Institution of Oceanography for organizing the R/V ALPHA HELIX 1971 Antarctic Expedition, to Mr. maul Berkman of the National Oceanic and Atmospheric Administration for the reduction of tide marigrams, and to Dr. Charles B. Miller of Oregon State University for assistance with the statistical analyses. Special thanks to Mr. Michael Bergin of Marine Acoustical Services, Inc., and Messrs. Harvey High, Jay Klinck, and Dennis Pat.ton of the U. S. Navy Deep-Freeze 1970 winter-over crew. REFERENCES 1. Bloch, R., Walters, D. H., and Kuhn, W. Structurally caused freezing point depression of biological tissues. J. Gen. Physiol. 46, 605-615 ( 1963). 2. Dayton, P. K., Robilliard, G. A., and DeVries, A. L. Anchor ice formation in McMurdo Sound, Antarctica, and its biological effects. Science 163, 273-274 ( 1969).
PROTECTION
AGAINST
FREEZING
3. Hargens, A. R. Freezing resistance in polar fishes. Science 176, 184-186 (1972). 4. Hargens, A. R. Macromolecular osmotic pressures and interstitial fluid pressures in marine multicellular systems. Ph.D. Dissertation, Univ. of Calif., San Diego, pp. 30-5i;, 84-101, 1971. 5. Kanwisher, J. W. Freezing in intertidal animals. Biol. Bull. 109, 56-63 (1955). 6. Kanwisher, J. W. Freezing in intertidal animals. In “Cryobiology” (H. T. Meryman, Ed.), pp. 487-494. Academic Press, New York, 1966. 7. Litvan, G. G. Mechanism of cryoinjury in biological systems. Cryobiology 9, 182-191 (1972). 8. Newell, R. C. “The Biology of Intertidal Animals,” pp. 451-454, Logos Press Ltd., London, 1970. 9. Scholander, P. F., Hargens, A. R., and Miller, S. L. Negative pressure in the interstitial fluid of animals. Science 161, 321-328 (1968).
BY A LIMPET
MUCUS
x!l’i
10. Scholander, P. F., and Maggert, J. E. Supercooling and ice propagation in blood from arctic fishes. Cryobiology 8, 371-374 (1971). 11. Scholander, P. F., van Dam, L., Kanwisher, J. W., Hammel, H. T., and Gordon, nl. S. Supercooling and osmoregulation in arctic fish. .I. Cell. Comp. Physiol. 49, 5-24 (1957). 12. Shabica, S. V. Tidal zone ecology at Palmer Station. Antarct. 1. U. S. 7, 184-185 (1972). 13. Theede, H. Comparative ecophysiological studies on cellular cold resistance of marine invertebrates. Mar. Rid. 1.5, 160-191 (1972). 14. Theede, H., and Lassig, J. Comparative studies on cellular resistance of bivalves from marine and brackish waters. Helgolaentler Wiss. Mceresunters. 16, 119-129 ( 1967 ). 15. Williams, R. J. Freezing tolerance in Mytilus cdcllis. Camp Biochem. Physiol. 35, 145161 (1970).