Resistance to freezing by antarctic fauna: Supercooling and osmoregulation

Comp, Blochem, Physiol., 1976, Vol. 54A, pp. 291 to 300. Pergamon Pr¢,ss. Printed in Great Britain

RESISTANCE TO F R E E Z I N G BY A N T A R C T I C F A U N A : SUPERCOOLING AND OSMOREGULATION* S. RAKUSA-SuszczEWSKI t AND M. A. MCWHINNIE 2 INencki Institute of Experimental Biology Warsaw, Poland and 2De Paul University Chicago, IL 60614 U.S.A. (Received 4 November 1975)

Abstract--1. Body fluids of six antarctic invertebrates are hyperosmotic to their normal environmental sea water during the late austral summer and winter. Pelagic and benthic species respond as osmoconformers to changes in salinity but regulate slightly hyperosmotically when returned to normal sea water. Adjustment of the amphipod Orchomene plebs Hurley to a concentration gradient requires a small but significant increase in energy output. 2. Tolerance to supercooling and the extent of hyperosmotic regulation is greater jn pelagic than in benthic species. 3. Body fluids of antarctic invertebrates show equilibrium freezing points similar to ideal solutions while blood serum of the fish Trematomus bernacchi shows marked thermal hysteresis. The role of these resistance adaptations of antarctic marine fauna is discussed. INTRODUCTION

A n i m a l adaptations to all habitats, characterized by varying intensities of physical variables, are generally accomplished by magnification of particular mechanisms c o m m o n to all species, which change only in m a g n i t u d e reflecting the e n v i r o n m e n t a l demand. Temperature, one of the more a p p a r e n t variables to which organisms must acclimate or adapt, influences metabolic rates which reflect their over all response to, for example, seasonal variation. South circumpolar m a r i n e environments, however, are u n i q u e in the constancy of sea water temperature at - 1.86° to - 1.9°C where a n n u a l changes rarely exceed more than + 0 . 2 ° C at the highest southern m a r i n e latifudes yet sampled; ca. 78°S (Littlepage, 1965). Animals adapted to this e n v i r o n m e n t are therefbre not confronted with requirements to exploit capacity a d a p t a t i o n s with respect to temperature but must adapt to survive in constantly near-freezing conditions. I n this stenothermal e n v i r o n m e n t , a rich a n d moderately diverse fauna exists ( D a y t o n et al, i1969; Bushnell & Hedgpeth, 1969; Balech et al, 1968; Brown et al, 1974). With respect to sublittoral invertebrates it appears t h a t survival at low temperatures is generally achieved if the c o n c e n t r a t i o n of body fluids equals that of their e n v i r o n m e n t or, less c o m m o n l y , if they axe at least slightly hyperosmotic (Potts & Parry, 1964). As a supplement to this protection against freezing, tolerance to s u p e r c o o l i n g would add an advantage for survival. Tolerance to actual freezing, however, is also k n o w n , e.g. intertidal sessile or b u r r o w i n g clams have been found to survive ice formation t h r o u g h o u t their tissues (Kanwisher, 1966). H e d g p e t h (1969) suggested that shore a n d a n c h o r ice may be a more significant factor in limiting intertidal a n d shallow littoral fauna t h a n temperature. G r u z o v & P u s h k i n (1970) reported '*This w o r k was conducted at McMurdo Station, Antarctica Under a National Science Foundation grant GV39912 for which' we express our gratitude. 291

that in Antai:ctica, the upper 7-8 m of the littoral zone is lifeless at E n d e r b y L a n d due to a n c h o r ice, extending to 15 m along the coast of the Davis Sea, while in the South Shetland Islands, the lifeless zone was only 2-3 m. At M c M u r d o Station o n Ross Island, D a y t o n et al (1970) found that the d o m i n a t i n g influence of ice extends to 30 m. Sub-littoral i n h a b i t a n t s would n o t e n c o u n t e r temperatures as low as air and, with freedom to migrate to waters n o t subject to freezing, can survive if isomotic to sea water and tolerant to superc6oling. This study was u n d e r t a k e n to provide quantitative evidence for mechanisms of freezing resistance by selected antarctic m a r i n e invertebrates a n d to contrast these with an endemic benthic fish. MATERIALS AND METHODS Animals

Animals were collected from McMurdo Sound, Antarctica, in the vicinity of 77°51'S 166°42'E. The collection sites, location and depth are shown in Fig. I. Holes were cut through the sea-ice cover which ranged from 0.6 to 1.6 m in thickness between April and August, 1974. This study was conducted between February and July. At the end of summer and before the sea-ice developed to a thickness sufficient for vehicle travel, animals were collected with traps set along shore (8-10m). Benthic sp~.'ies were collected with 1.2 × 0.5 m cylindrical traps constructed of 1 cm hardware cloth; these were baited with seal meat and set for 24--48 hr. The species used in this study include the ubiquitous amphipod, Orchomene plebs Hurley, a predacious and necrophagous species; the common asteroid, Odontaster validus Koehler, an echinoid, Stereehinus sp.; the giant isopod,~ Glypfonotus antarcticus Eights; a pycnogonid, Colossendeis sp., and the fish,~Trematomus bernacchi. A l-m plankton net provided with a 200 nm mesh net was used for zoo-plankton species which included the copepod, Rhincalanus gigas Gesbrecht, the pteropod, Limacina sp, and the under-the-ice euphausiid, Euphausia crystalloropbias Holt. Throughout the winter months net samples were often filled with ice-platelets as was the sea water used for transport to the laboratory; the temperature ranged from --1.8°C to --2.0~C or :tenths of a degree lower. Under such conditions animals were :not injured

S. RAKUSA-SuszczEwsKi AND M. A. McWH1NNIE

292

Fig. 1. Locations o f sampling holes cut through the sea-ice cover on McMurdo Sound, Antarctica. The location and depth of the water column at each site wa~: A, 750m; Nr. I, 100m; (77°51'31.3"S 166°42'3.2"E); Nr. 2, 7 6 m ; (77°51'19"S 166°38'30.6"E); Nr. 3, 5 6 0 m ; (77°53'5"S 166°33"34.7"E), Ice holes A and Nr. 3 would appear to have been through the permanent Ross Ice Shelf. However, that position marked its northei'n extent in 1965. The Ice Barrier has receded with the calving of icebergs since that time and these sampling sites were through sea-ice.

and within 1-3 hr after collection they reached the laboratory and were maintained in aerated sea water aquaria at approx - 1.8°C. In M c M u r d o Sound of the RoSs Sea, water temPerature is essentially vertically isothermal in the winter at -1.91 ° _ 0.1?C (Littlepag¢, !965). Throughout this season ice platelets occur in the upper 30 m and smal ! ice needles appear at increasing depth. The freezing point of sea water from 100 to 500 m declines to approximately --1.98 ° t o - - 2 . 2 8 ° C as a consequence of hydrostatic pressure providing a supercooled environment.

Supercooling Animals were supercooled in approx 4 I. of filtered sea w a t e r in a deep-froze chest (--27°C). The assembly was protected from direct contact with m e t a l t o reduce thermal gradients. Cooling rate varied from approx 2.0°C hr7 t initially to 0.5°C' hr " t as: the e x p o s u r e period continued. Throughout a series of measurements the lowest temperature reached was -: 8.0°C; each w a s voluntarily terminated and often achieved within, 4 hours without spontaneous crystallization. Careful movement of the assembly from the chest generally, permitted t h e temperature to return :to abo~e the freezing point without i c e formation, *Cryoscope designed and constructed by Dr. A. L Dc,Vries who kindly permitted .our use of it.

Osmoreoulation Studies of organismic adaptation to changing salinities were conducted by measurement of freezing points of body fluids using a cryoseope assembled with a circulating ethanol bath (Brinkman ,Instruments) and provided with a regulator and proportional temperature controller (Yellow' Springs Instrument Co,, Ohio) to an accuracy of O.01°C. * Ice-crystal growth and melt of the sample being measured in a capillary ( < 1 m m din), were monitored optically. Hernolymph, coelomic fluid and blood samples were collected through a number of time and acclimation sCh¢= dules. Normal values for freezing and melting points of body fluids were measured with samples taken immediately after the a n i m a l s arrived in the laboratory a n d : a g a i n within a few days after acclimation to l a b o r a t o r y conditionS. H e m o l y m p h and e o e l o m i c fluid of invertebrates .have melting and freezing: points which do n o t differ by more than O.01?C, Freezing points of t h e body: fluid s (Afp) were therefore t a k e n as the point of melting ( A m ) o f a pre-frozen sample which was melted t0 a point where a,diserete icecrystal remained, S u b s e q u e n t growth Or decrease Of the crystal Was used t o establish freezing arid melting p0ints. For comparison ~with invertebrates, the freezing point depression,of blood serum of T. bernacchi was measured. Blood. was :collected b y c a r d i a c punciure land l~rmitted to coagulate normally at 40C,: Serum-was :separated i n a

Freezing resistance: antarctic fauna hematocrit centrifuge for measurement of Alp and Am. In some cases aliquots of serum, in a sealed capillary, were held at 100°C for 30--60see to effect protein denaturation. A protein-free filtrat.e of serum was then obtained by eentrifugation. Osmoregulatory responses to changing salinities were studied with groups of O. plebs (22), G. amarcticus (14) and O. t,alidus (10) which were transferred from laboratory sea water to hyperosmotic (43%0, Alp = 2.3°C), normal (35~, Alp = 1.91°C), and hypoossmotic (27,°~, A f p !.44°C) filtered sea water maintained at -- 1.8°C. Sea water was concentrated by evaporation or diluted with distilled water to reach these conk:entrations which were established cryoscopically. The thermometer was calibrated with use of boiled distilled water. To determine the extent and rate of change of internal osmoconcentration" with changes in external salinity, hemolymph and coelomic fluid samples were taken at intervals of 1/2-2 hr. After measurement of body fluids through 8-13 hr in hyper- and hypoosmotic media, animals were transferred to normal sea water and again body fluid samples were collected at intervals.

Respiration The metabolic response of O. plebs to varying salinity was determined by study of oxygen consumption of individual animals throughout acclimation to the same concentrations of sea water as were used in the study of osmoregulation. A Gilson differential respirometer was operated at -1.8°C and an oscillation frequency of 96 m i n - L Respiration flasks were provided with glass wool for animal attachment; filtered sea water (Millipore, HA, <0.45 nm pores) was used as the medium and 30~o KOH was the CO, absorbent. At the conclusion of the study animals were rinsed free of sea water and dried to constant weight at 60°C. Data are expressed as /zl O2 a n i m a l - l h r - l and plotted as a function of dry weight. Glass wool was used since it was observed that this species exercises continuous rapid swimming in search for a solid or food substrate. Respiration values were highly variable as a consequence of motor activity until substrate was provided and to which the animals usually adhered. Initially the proportionality between body size and oxygen consumption was measured using amphipods ranging from ca. 1.8 to 51 mg dry wt. Individual animals were maintained in normal, hyperosmotic .and hypoosmotic sea water and were measured respirometrically for 2-2.5 hr. The relationship between animal size and oxygen consumption was determined with the relation, R = aPVb, where, R = ,ul O , consumed hr- ~; W" = body wt in mg dry; a and b are constants, e.g. the intercept (or weightindependent metabolism) and slope, respectively. Regression computations employed the least squares method and confidence intervals were determined. To determine the time course of metabolic response to differing salinities, O2 consumption of individual amphipods was measured at 2, 4 and 6 hr of acclimation. The number of individuals and the mean body weights used in this study are given below. These animals were selected to avoid variation in oxygen consumption due to weight differences. Between measurement periods (4 a n d 6 hr) the respirometric system was opened for gas equilibration and subsequently closed for

Salinity 9/oo 27 35 43

(N)

2 hr Mean, dry wt (rag)

(13) (12) (10)

21.02 + 3.62 21.53 + 3 . o 4 22,76 ± 4.10

293

the following measurement. This study was conducted during mid-winter (18-25 June). RESULTS

Supercooling Survival of m a r i n e invertebrates below freezing temperatures has been k n o w n from a n u m b e r of studies. T o estimate the extent o f tolerance to the supercooled state, nine antarctic invertebrate and o n e fish species were supercooled to sub-freezing temperatures and held at low values for extended periods of time. Variously, ice-platelet formation occurred a p p a r e n t l y spontaneously or, as in the case of the isopod G. antarcticus, platelet formation occurred predictably o n the gill surfaces as these c o n t i n u e d in rhythmic respiratory m o t i o n (Fig. 2). This activity increased with depth of supercooling, While platelet size ot~en increased, the a n i m a l showed little distress t h r o u g h as much as o n e hour. The large antarctic pycnogonid, Colossendeis sp., the nemertean L. corruoatus a n d an octopus also increased m o t o r activity at sub-freezing temperatures which resembled strong escape reactions. Permitting w a r m i n g to above - 2 ° C removed the platclets a n d n o r m a l b e h a v i o r ensued. Consecutive return to the supercooled state 3-5 times was met with n o apparent persistent injury and animals were frequently supercooled o n subsequent or alternate days with n o mortality. A s u m m a r y of the temperatures reached in s u p e r c o o l i n g a n d the s u m m e d d u r a t i o n these species spent below - - 2 " C is given in T a b l e 1. Characteristic of all species so cooled was a m a r k e d increase in m o t o r activity as the temperature dropped below the freezing point of sea water. As temperatuce c o n t i n u e d to fall, before ice-platelet formation occurred, m o t o r activity declined. Tolerance to supercooling was lowest in the. fish. The octopus was the least tolerant a m o n g the invertebrates while the pelagic species showed the greatest tolerance. R. g@as was m a i n t a i n e d a m o n g d e n s e ice-platelets in sea water at --2.1°C for two days in late July without evident effect. T h e a m p h i p e d O. plebs, cosmopolitan in its distribution from epipelagic to benthic, and similarly highly t o l e r a n t of supercooling, did s h o w a decrease (ca. 59/o) in the o s m o c o n c e n t r a t i o n of h e m o l y m p h (Table 2A) one day after being supercooled.

Osmoregulation and conformity Six arthropod a n d echinoderm species of antarctic invertebrates studied t h r o u g h o u t the late austral summer a n d winter, were slightly hyperosmotic to their e n v i r o n m e n t . This was evident w h e t h e r measured by freezing or melting p o i n t o f their h e m o l y m p h or coelomic fluid (Table 2A). These body fluids, rich in protein; approach a n ideal solution in their physical behavior showing essentially n o hysteresis a n d therefore

(N)

4 and 6 hr Mean, dry wt (rag)

9/o of wet wt

(14) (12) 04)

21.01 + 4.13 26.26 ± 3.50 23.05 +4.45

22.22 +__.3.24 22.i6 ± 1.85: 23.05 ± 3.29

294

S. R A K U S A - S u s z C Z E W S K i AND M . A . M c W H I N N I E

Fig. 2. Glyptonotus antarcticus, benthic isopod in normal sea water during spontaneous crystallization of ice after supercooling to --6.6°C. Ice platelets usually form on the gills first and subsequently at leg joints. 1 a b l e 1. M i n i m u m temperatures reached i n supercooling of antarctic fauna in f i l t e r e d s e a w a t e r (Ar~, 1.85-1.93°C) SPECIES

NEMERTZNEA Lineus corrugatus

HABITAT

SUPERC~OLING, °c.

Av. T~me, Hrs. below -2.0°C w i t h i n 24 hrs.

Benthic

-5.0

6

Strong movements ; withstands platelets

Pelagic

-7.8

2'

W i t h s ta~ds p l a t e lets, ice thickening

Nekton

- 4.5

3

V e r y d i s t u r b e d as platele~ form

-7,5

8

W i t h s t a n d s it h i c k e n -

MOLLuScA

Limacina

Spo

O c t o p u s sp.

C o m m e ~ ts

ARTHROPODA Orchomene plebs

Benthic/

Pelagic

_

i n g :~.ce

Rhlncalanus glgas

Pelagic

-7.6

Glyptonotua an t a r c t l c u s

Benthic

--6.9

lO ;

--3.9

7

Strong movement; w l t h s tandJ p l a t e lets

Benthic

-5.2

3

Active splne movements

Odontaster valldus Benthic

-4.5

3

Active ray movemen~

"--4.0.

2.5

PycNOGONIDAColossendela

ap.

~ Benthic

W ~ t h S t~unds p l a t e lets a n ~ t h i c k ice Platelets form on g i l ! s ; assoc, w i t h r e s p . movelnents

ECHINODE~ATA

Sterechinus

sp,

VERTEBRATA Tre~atomus hAnsoni*

Benthic

* A b o u t SOt ~]ULed a t / t h e s e o f i c e formation.

temperaturea

R e s p l r a t o r y ~dls~cess ~ p l a t e l e t s f0zID/oper=ul a , as a ~ r ~ e q u e n ~

Freezing resistance: antarctic fauna

295

Table 2A, Freezing of body fluids of antarctic invertebrates SPECIES

Pu~BITAT

BODY FLUID

~ 0

FREEZING, * Oc. + Z.d. ~fP

Orcho~mne

~ ' (Amph-~da)

Primarily benthic, also pelaglo

Hemolymph

(I0)** 1.93 + 0.171

De-proteinlzed (pooled, 15 anlmale) Supercooled,

2 hrs.

(16) 2.04 + 0.111

~m (16)

2.03 + 0.111 2.15

2.16 o -5

C ( 3} 1.91 + 0.023

( 3) 1 , 9 0 + 0 . 0 2 3

Benthic

Hemolymph

CI0}

1.93 + 0.I06

(13) 1.97 + 0.087

(13)

1.96 + 0.086

Pelagic

Hemolyml=h

(2)

2.00

(2)

2.05

(2)

2.04

Pelagic

Hemolymph

(i)

2.13

(1)

2.10

Coloezendelz rap. Benthic (Pycnogonida)

Hemolymph

( I} 2.15

( I} 2.14

Odontam tot validue (AS tero£dea)

Coelomlo

(10) 1.98 + 0..152

(i0) 1.97 ! 0.150

(measured 24 hrH. later) Ulyptonotum antarcticue {Ieopo~a) P,h i n c a l a n u s (Copepodz}

~:upheuela

cryetellorophias (EUpheumi~d)

* ~o,

Benthic

Alp)Am;

( 8}

1.95 + 0.164

freezing point of sea water; freezing and melting p o i n t 0f sample.

** Number of a ~ l e z .

Table 2B. Freezing and melting points (°C) of Trematomus bernacchi blood serum

Normal (N -' 3)

Z~ fp

4m

i.* 3.23 2, 3.17 3. 3.49

1.26 I. 14 1.24

Mean, + S.d.

3.30 + 0.17

De-proteinized I. (~ - 4) 2. 3.

3.30 2.99]** 2.93~ 2.S6J 2.82

4. Mean, +_, end.

1.21 + 0.06

4fp _ 4 m 1.97 2.03 2.25 2.09 + 0.15

1.53 1.31

1.77 1.44

2.s~

1.13 1.22

1.69 1.31

2.87 + 0.32

1,30 + 0.17

1.57 + 0.21

* Fish number. ** Consecutive measurementS of 1 sample.

have equilibrium freezing points. The difference between freezing and melting points rarely exceeded 0.01°C. Thus, n o macromolecular cryoprotective agents are indicated. Deproteinized bemolymph of O. plebs showed n o decrease in freezing o r melting points. However, ,high molecular weight proteins have negligible osmotic effects. The increase in osmoconcentration o f protein-free hemolymph. (Alp, from 2.03/2.04 to 2.15/2.16) may represent release o f pr otein,bound ions upon thermal denaturation :or, more probably the environmental salinity history of the animals (15)from which the hemolymph was pooled for this measurement, Hypcrosmotic regulation is apparent in both pelagic and benthic species which-live in antarctic water continuously below O°C. Studies conducted to determine the adjustment or indelaendence~of these species t o changing ~salinities, demonstrated that t h e y are osmoconformers (Table 3; Fig..3). T h r o u g h 8-13 + hr of acclimatizati6n:to hyper- and hypoosmotic sea wateri(C/i. 42.2 a n d 26.6 ~oo)the internal concentrations of t h e am~hiood a n d

starfish changed relatively rapidly achieving an isoo r limited hypcrosmotic concentration within 8 to 9 hours. The giant isopod responded more slowly. The ubiquitous amphipod, O. plebs, occurring in the s u b - i ~ community when epipelaglc, encounters changing salinities with seasonal sea-ice formation and melting. A small portion of the population was in the surface layers throughout the austral summer and winter. Rapid osmoconformity, with limited but r~urring, hypertonicity would provide a physiological advantagelin support of a vagrant habit, and some protection against freezing in the winter near-surface ice-platelct-ladcn water. The rate o f response to achieve a hyperosmotic condition When returned to normal sea water from hyper- and hypoosmotic sea water affords freedom of movement between a changing pelagic and m o r e stable benthic habitat. The -amphipod-reached fits characteristic hyperosmotic concentration, w h e n transferred to normal sea water ( 3 5 ' ~ ) w i t h i n - 8 hr after removal from concentrated a n d dilute s e a water, .However, within 2 hr: 82-94~/o o f the final, osmotic concentration was reached. Con-

296

S, RAKOsx-SusZCZL'WSKIAND M. A. McWH]NNm

,,,.r-.

/

, . ~ , o ' ~ . : - . -", ~-

!,,-'_Y"

:. . . . . .

-"T---~--~

,,,

it

~ .

~,

4

.

i~

~

~o.m.-~

1.5 - - ~ , 1.49 -- ~

1 G. ~mt41rctlc~l

,-,-~" .....

~

[

Fig. 3. Changes in the osmoconcentration of hemolymph of the amphipod O. plebs and the isopod, G. antarcticus and, eoelomie fluid of the asteroid O. validus, when acclimated to hyperosmotic and hypoosmotic sea water a t -1.9°C. Normal sea water ranged from A¢~ = 1.87 to 1.97°(2. Arrows indicate A,, changes when introduced into experimental salinities and return to normal sea water. trariwise, the benthic starfish and isopod did not reach 80% of the total osmotic adjustment for 4 and 8 hr respectively. Conversion of equilibrium freezing points of the crustacean hernolymph and starfish coelomicfluid to milliosmolar concentrations showed that the arnphipod has a hyperosmotic concentration approx. 1.7-2.3 times that of the benthic isopod and starfish. The habitat of the latter generally escapes ice formation except in the shallow in-shore littoral zone characterized by anchor-ice. Hyperosmotic regulation of a pelagic species entering the sub-ice platelet layer is a eryo-protective mechanism in a sub-zero environment. While data are limited for copepods a n d euphausiids (Table 2A), the values obtained indicate

a higher osmotic differential between the body fluids of these pelagic species and their environment, than is found in benthic species. The largest collections of G, antarcticus a n d O. validus were made at depths of 100 m o r greater throughout the winter. The advantage of a lowered freezing point of sea water with depth, and a limited degree of hyperosmotic regulation c o m b i n e to protect these species from freezing. Tolerance for supercooling adds to survival. In contrast to the freezing and melting' points of invertebrate body fluids which show properties of ideal solutions, blood serum of the fish T. bernacchi shows a wide difference between freezing and melting points. The fl"eezing points given in Table 2B are generally lower (--3.30°C)than those reported in other studies (-- 1.87° to - 1.98°C)and may represent the slowness with which the lower temperatures were approached; these values probably indicate a degree .of supercooling. This is likely since mechanical shock of the samples at these temperatures induced spontaneous freezing. The melting point for normal sera for ,three animals was reasonably constant at ,1.22°C. The wide difference between melting and freezing points ofsera from these fish is an expression of thermal hysteresis. Capillary samples of normal and heat de-proteinized sera were quick frozen, melted back to a single small crystal, and placed into the cryostat. Observation of such a sample maintained at --1.64°C for three days showed neither crystal growth nor recession. Further slow decrease in temperature through two days to --2.25°C similarly showed no change in crystal size. However, mechanical shock at this temperature induced spontaneous freezing. Similar slow thermal changes with normal sera showed widely separated points of freezing and melting. While it is likely that these .low values resulted from supercooling they reflect a capacity for low temperature tolerance even in excess of that indicated by more conventional modes of measuring Alp, e.g. a more rapid decrease in temperature. Since these animals range in

Fable 3. Time course: changes in osmoconcentration of three antarctic invertebrate species when transferred to sea water of different salinities at -- 1.9°C From O¢'ehomene

.1,87

2:23

TO

~ - -

2,19.

2.22

~:I.99-~

G 1 : /"~ o n o t u s antardticus --

~

_(Isopoda)

,

1,94

1.4, odo~.~,,,,,~e,,. , 1 . , 2 -~,~0:

2 :

2,30

.:~.~7 : " Z . " ~ 4

1.94

• ,

~.j?:,

lA',L~h~.da)

2,25

t 0

ii9¢"

'

2,25' ~ ~ ": "" . . . . 2,25

i.I04

i,

1.98

~:.'~0~ ~ 1 . s 6 2.p i~7

6

2,26..

2,29

1,4S

/,1,06:

1.92"

:1.6~,

8-

1.97.,:i.,6

2°02 '. . . . 2,212,10

~":_:~.',8 2.:30~ ~ 2..10

di~e~t~.~ransfer

,

:~.SX" ' X . 4 s , '

'2;00 ,~:-i;i ~ 7 5

(Arrows '£ndi~atQ

4

:

1,B3

/

~0.o7

: 1.44,!"

~ 37.6

2,14 :

2,1B

"

"

2,08'

1.78

1.7.,

1,71

1.,3

an8 ~hltiat~on

1.'51'

~

III

',

2 . 2 s 2 . 2 9 ,;:30 ~ 2.02 ~.:2.02~:.2.00 ~ -1.63 " ~ i. S5

14 , ~ S m ~ l e s

~ - -

1.81

::

,- 12

. 2,30

1,09 '2,09

10

'

~ 0.04

~ 21.5

.

_<0.0:3 :I.46 '

of.new'reSponse)-

~ "i6.~

~-I

Freezing resistance: .antarctic fauna

//.//.

Fig. 4. Correlation coefficients for respiration and body wt of O. plebs acclimatized to different salinities for 2-2.5 hr at --1.8°(2. Confidence intervals are given; 3 5 ~ log R = log (0.0607 :t: 0.1550) + (0,57 + 0.16) log W; 27}',,., log R = log (--0.0128 ± 0.0997) + (0.60 ~ 0.18) log W; 43~/~, log R --- log (--0.1184,4- 0.0891)+ (0.66 ± 0.15) log W. depths of 100-300m they could be supercooled to the freezing point of sea water by 0.075-0.225°C lower than the same salinity at the surface. Respiration Rates. of oxygen consumption of O. plebs are dependent upon body size and the interdependence of these is expressed by the coefficient of correlation of W TM. While there was small variance in this coefficient when amphipods of the same size were maintained in normal, hyper- and hypoosmotic sea water there was no significant difference (P---- 0.95) between them and animals in normal sea water. However, the latter show a slightly higher 0 2 consumption when compared at two hours with animals in higher and lower salinities. The correlations are within the range of those reported for other amphipods (Fig. 4), e.g. Orchomenella chilensis 0.665 (Armitage, 1962), Paramoera walkeri, 0.63 at 2.0°C, 0.60 at 0°C and 0.50 a t - 1.9°C (Klekowski et al., 1973). Since such correlations are known to depend upon many factors (tem•perature, salinity, season, past physiological history, developmental stage, among others) aniq,ae biochemical adaptations to stable polar t~mperaturcs may also influence the relatively low metaboli¢~ coefficients obtained in this study (e.g. 0.57, 0.60 and 0.66)which are befd~t-h~-generalized-value given f o r crustaceans (e.g. Wo.~s, Sushchenya, 1972). Differences in enzyme kinetics (Somdro, 1969; Somero & Hochachka, 197I; Low & Somero/1974) membrane permeability (Caldwell & Vcrnberg, 1 9 7 0 ) a n d m e t a b o l i c . pathways (in preparation) and their temperature correlations, may set polar species into a different category :consequent to a long evolutionary history Of low, temperature a d a p t a t i o n and: survival.-:In the low temperature adapted Gammaracanthus-lacustris (normaLenvironment;: ~ 6:5°C), - Ivanoy(1972)reported the,coefficient //vo:63,~for t e m p e r a t u r e s 0 f 4-5°C. T h u s , low c.a.a. 54/3A--a

297

values for endemic low temperature adapted species may signal fundamental differences characteristic of such fauna. The study of oxygen consumption by this amphipod through six hours of acclimation to sea water of 27, 35 and 43 ~oosalinity shows no significant differonce through the first 4 hr. The decrease in Oz uptake with time (Fig. 5) is judged to result from a decline in motor activity. By 6 hr of acclimation, respiration values for animals in normal sea water were significantly lower than for animals i n hyperosmotic sea water. It was at this time when hemolymph osmoconcentration had increased 15% (Afp, 2.30°C) above its value in normal sea water as the amphipods became isoosmotic with the environment. T h e response to become hyperosmotic appears to be expressed i n metabolic work. A similar response was observed in animals maintained in hypoosmotic media to which they became isosmotic i n 6-8 hr, (Table 3) but the difference was not statistically significant. DISCUSSION

Marine invertebrates in polar regions have been considered resistant t o freezing because of, (a) the concentration of their b o d y fluids, e.g. isosmotic or, less often, slightly hyperosmotic to sea water (Potts & Parry, 1964) and, (b) a high tolerance to supercooling. However, few environments have the sustained l o w temperature (--1.91 -I-0.1°C) which prevails in south circumpolar waters. These seas are near their freezing point and as the temperature declines 0.1-0.2°C with the Onset of winter, ice-needles and platelets form in the upper water c o l u m n as well as through the shallow in-shore regions. Under these conditions, isosmoticity Would fail to b e protective and pelagic and shallow be~lthic fauna would become

ti

t I

I

|

!

!

t

t

I

....

f "

I

Fig. 5. Oxygen consumption of O. plebs during acclimatioh to varying salinities of sea water at --L8 oC ; Standard deviations are indicated.

298

S. RAKIJSA-SUSZCZEWSKIAND M. A. McWmNN)~

susceptible to freezing. Combining tolerance for supercooling and limited hyperosmotic regulation would provide protection against spontaneous freezing. Benthic species showing similar resistance adaptations gain a greater horizontal range extending from several hundred meters to shallow in-shore regions. The benthic species studied were collected with inshore traps ( ~ 10m) as well as in depthg of 100 and 560 m from January to August. Dayton et al.', (1969) also found a wide distribution of these animals in McMurdo Sound and t h a t these forage among the anchor-ice The antarctic invertebrates and fish reported in this study show a considerable tolerance for supercooling. The lower limits of sub-zero temperatures reached in laboratory supercooling far exceeded any natural depressions which would occur in the environment. They do, however, indicate the extent o f tolerance which these species have for sub-freezing temperatures. Increased motor responses elicited through supercooling, allow for behavioral activities which could result in translocation to waters" of greater depth and therefore lower freezing points. However, repeated and long-term supercooling may result in some physiol0gicai damage since the amphipod, O. plebs showed loss of hyperosmotic,, regulation so long as one day after being held at --'5°C for 2 hr. Coupled with tolerance for supercooling, each species studied has been shown to be slightly hyperosmotic (by 16-37 tocsin) to the environment, though all a r e osmoconformers. The response to changing salinity was greatest in the amphip0d O. plebs which appears to move freely from ~a benthic to a pelagic habit. This species is subject therefore to salinity changes which do not occur at greater depths. Hyperosmotic regulation bY the antarctic amphipod pararaoera walkeri, studied in the Atlantic Sector, has been reported for hemolymph at Alp = 2.06°C (RakusaSuszczewski & Klekowski, 1973); that value is essentially the same as found for O. plebs in McMurdo Sound. Increase in hemolymph concentration via osmoconformity in concentrated sea water~ provides increasing freezing resistance. In a temperate mussel, Mytilus edulis, Williams (1970) showed an increase in freezing resistance (from - - 1 0 ° t o ,157C) on simI)le osmotic grounds, when the animals were maintained in 150% sea water. Each of the-three antarctic species, reported here became isosmotic t o dilute (779/o) and concentrated (123~/o) sea-water," within 8-13+ hr and returned to a hyperosmotic state in normal sea water. However, the benthic: isopod (G. antareticus) and starfish (O. validus) showed only 43--57~ of- the ~osmoconcentration reached b y the move vagram !amphipod. Among crustaceans,.isopods show considerable diversity in habitat.ranging from deep marine to inter-tidal and, fres)i water and terrestrial environments, and all species are hyperosmoti¢ to their medium, The antarctic isopod, G.: antarcticus is, however, 0nly 20 molsm more concentrated than normal sea water. Its slow response to chfihging external Salinities resem~bles that o f isopods: in "general,;-which show a low p e ~ e a b i l i t y t o salts (Croghan: &: Lockwood,.; 1968). Similar to the.0smoconformity0bserved with these

antarctic species, Ltnce (1965) reported conformity in the temperate, planktonic copepod Acartia tonsa which however, remains hyperosmotic to all external concentrations. It was concluded that the hyperoSmotic state was due to the Donnan effect rather than active regulation of ion concentrations. In the antarctic amphipod O. plebs, hyperosmotic regulation occurred only in normal sea water and respiration increased when animals were maintained in dilute or concentrated sea water: Study of respiration of O. plebs through 6 hr exposure to different salinities showed that oxygen consumption was significantly higher in !23% sea water than in normal sea water. Moreover:, oxygen consumption of animals in 77% sea water was also above that in normal sea water, but the difference was not significant. When the antarctic amphipod Paramoera walkeri was exposed to 15~/oo(489/o) sea water, its respiration increased .by 50~, (Rakusa-Suszczewski & Klekowski, 1973). Conversion of the six hour respiration values for O. plebs to energy requirements for animals in salinities of 27, 35 and 43~oo indicates that approx 19.2, 15,5 and 23 x 10 -a g c a l h r - t are produced respectively, by amphipods with an average dry weight of 23 m g (mean body wt, ca. 100 rag). These increases in energy requirements, associated with ionic inbalance and subsequent osmo-adjustment, are 20-30% greater than the energy production of O. plebs in normal sea water. It may be this magnitude of energy requirement which renders amphipods stenohaline. Volume regulation was not studied and thus changes in osmo-concentration cannot be directly assigned to ion or water regulation. However, the respiratory responses and attainment of isosmoticity resulting from changing salinities suggest ion regulation rather than a Donnan effect, and it may also be required as a consequence o f a limiting exoskeleton. Kinne (1966) reported that some euryhalin¢ crustaceans (Oeypoda and Palaemonetes) increase oxygen consumption in both dilute a n d concentrated sea w a t e r and the increased metabolic rate was re!ated to ,ion balance. Summarily, :antarctic invertebrates, while characteristically stenohaline, show varying degrees of hyperoStactic regulation and Considerable tolerance to supercooling which :combine to permit survival in a constantlynear-freezing environment. Theirate of change in:osm6conformity t o :different external salinities differs arnong species, being greater in' an intermittent pelagic species ,which forages at the ice-sea water intcrfac~as well as at'the seabott0m, than in 'a benthic isopod and starfish; : The freezing characteristics o f b!0od of antarctic fish are considerably 'different from: those of invertebrates: The freezing point o f b l o o d sera of several ant~irctic fish species :was~¢ported: to be. a fraction Of adegre¢~ below~the!'inci~ent :.loWer lethal>temperature: for: the fish (DeCries; & S0mero,'~-1970,=DeVries, :1974)i:Furtheri:'a marked,thermd'l difference between freezing ! a n d 'melting ~:points
Freezing resistance: antarctic fauna rate of temperature decline achieved in the present study, and spontaneous freezing with mechanical shock (Afp, 2.53 ° to 3.40°C) it is evident that the low freezing points recorded here (2.99" + 0.31) represent the state of supercooling. D u m a n & DeVries (1972) used slow cooling rates (0.02°C r a i n - t to 2.01 ° below Alp) to control the rate of ice propagation. Similarly, R a y m o n d & DeVries (1972) reported that the freezing point of purified serum glycoproteins is a function of the rate with which freezing o c c u r s and that Alp is greater with a slow decline in temperature. In the pi'esent study low freezing points were a c h i e v e d by lowering the bath temperature slowly. A fish serum sample held at - 1 . 6 4 ° C (between Am and Alp) for three days failed to show a change in ice-crystal size.. DeVries (1971) maintained a fish serum sample containing a small ice-crystal at --0.75°C for 24 hr without crystal g r o w t h ; Afp o f t h e sample was 0.80°C. Variability in the freezing points observed arises from both the rate of freezing and the character of the icecrystal surface. In contrast, melting points do not show the Same variability ( L i n e t al., 1972; Scholander et al., 1957; Scholander & Maggert, 1971; Table 2B). The chemical basis for freezing characteristics of the blood of polar fish is, at least in part, due to salts and low molecular weight organic solutes (Gordon et al., 1962; Umminger, 1969; Smith, 1970). In addition, substantial evidence for conjugated proteins responsible for freezing-resistance in antarctic fish blood is rapidly accumulating. Eight molecular species of glycoproteins have been isolated and characterized (DeVries et al., 1970; L i n e t al., 1972). Purified solutions of these glycoproteins show freezing and melting characteristi::~ which mimic those of sera from polar fish. Moreover, neither trichloracetic acid precipitation, nor thermal denatui'ation of blood protein alters the freezing p o i n t d e p r e s s a n t - a c t i o n of fish sera (Table 2B; DeVries, 1968; DeVries et hi., 1970; R a y m o n d & DeVries, 1972), while molecular modification of the purified glycoproteins has resulted in the loss of their freezing characteristics. These features confirm this molecular class as the chemical basis for freezing resistance of antarctic fish (DeVries et a l . , 1970; Shier et al., 1972). Thus, survival :~; antarctic marine fauna i f f a n environment consistently at --1.8°C or lower, is achieved by high tolerance to supercooling and by maintaining a n internal freezing point lower than that of sea water. Invertebrates achieve this by hyperosrnotic regulation, w h e t h e r pelagic or benthic i n habit, while fish use conjugated glycoproteins whose solution Characteristics deviate substantially from true solutions.

Acknowledgements--We gratefully acknowledge the techni-

cal assistance Of St. M. O. Cahoon who helped in many of these measurements and of Stephen Grabacki and Dennis Schenborn who conducted the field work through~u~ the summer and winter of 1974. We are also grateful to the U.S. Navy Detachment Alpha who maintained wocking ice-holes as they continuously froze-in throughout t h e winter, Miss Elizabeth Obrebski contributed greatly bytranslation of early drafts in the Polish language for one of us (MAM).

299 REFERENCES

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