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Supercooling and thermal hysteresis in the adult bark beetle, Ips acuminatus Gyll
J. Insect Physiol. Vol. 35, No. 4, pp. 347-352, 1989 Printed in Great Britain
0022-1910/89 $3.04 + 0.00 Pergamon Press plc
SUPERCOOLING AND THERMAL HYSTERESIS IN THE ADULT BARK BEETLE, IPS ACUMINATUS GYLL. Division of Zoology, Department
UNN GEHRKEN of Biology, University of Oslo, P.O. Box 1050, Blindem N-0316. Oslo 3, Norway
(Received 28 June 1988; revised 19 Seprember 1988) Abstract-The adult bark beetle Ips acurninarus relies on supercooling for overwintering success. The beetle produces ethylene glycol in concentration up to 2-3 mol/kg wet wt and antifreeze proteins leading to thermal hysteresis of about 24°C. The supercooling capacity is always higher in the presence of antifreeze proteins. After a complete depletion of ethylene glycol, the loss of protein antifreezes results in an elevation of supercooling points from about -20 to - 18°C. For equivalent levels of ethylene glycol, the breakdown of antifreeze proteins results in a substantial rise in supercooling points. In beetles exposed to temperatures above their mean supercooling point, survival decreases both with exposure time and decreasing temperature. Irrespective of the presence of ethylene glycol, lower survival of beetles is always seen following depletion of antifreeze proteins. A correlation between survival for 1 week and supercooling capacity is only evident in groups exhibiting thermal hysteresis. Freezing appears to be the cause of death in beetles with ethylene glycol in their body fluids, whereas pre-freeze injury occurs frequently in individuals depleted of this antifreeze. However, the beetles with antifreeze proteins are able to recover from pre-freeze injury in the course of 3 days at 3°C. In the absence of these proteins, beetles are fatally injured, and pre-freeze injury seems to be the main cause of death. The reduced supercooling capacity, higher variance of supercooling point measurements and lower survival in I. acwninatus depleted of antifreeze proteins suggests that these proteins offer protection against freezing over the entire supercooling range, and thus ensure winter survival at low subzero temperatures. Key Word Index: Adult, bark beetle, cold hardiness, supercooling, thermal hysteresis, survival/mortality
INTRODUCTION To improve winter survival, a number of arthropods rely on supercooling to avoid freezing. An enhanced
ability to supercool is generally ascribed to the accumulation of low molecular weight antifreezes (Somme, 1982). Antifreeze proteins which produce thermal hysteresis (a difference between melting and freezing points) have been found in a number of insect species (Zachariassen, 1985). These proteins do not alter the melting point of plasma but prevent growth of ice crystal (Raymond and DeVries, 1977). Zachariassen and Husby (1982) suggested that antifreeze proteins may stabilize the supercooled state over the entire supercooling range. However, more data are needed on the antifreeze effect of these proteins in insects. The present study evaluates the effect of antifreeze proteins on the cold tolerance of the adult bark beetle, Ips ncuminatus. The beetle produces antifreeze proteins corresponding to a thermal hysteresis of 3-4”C and also accumulates the polyhydric alcohol, ethylene glycol in a concentration of about 2-3 mol/kg wet wt (Gehrken, 1984). Experiments were performed in order to study survival near the supercooling point (the temperature at which spontaneous freezing occurs). Comparisons were made on supercooling and survival between beetles which had been treated so as to retain or remove the antifreeze proteins. These experiments were performed on beetles in which ethylene glycol also was present or absent according to the temperature regime in which the beetles were reared.
MATERIALS AND METHODS
Logs of Scats pine (Pinus sihestris) infested with I. acuminatus were collected near Kong&erg (59”40’), SE Norway and stored out of doors at Blindern, University of Oslo. The procedure in the following experiments was based on previous findings (Gehrken, 1984, 1985). After initiation by November, the beetles continue to accumulate ethylene glycol at subzero temperature throughout the winter. High temperatures, however, cause degradation of ethylene glycol, and a complete loss proved to be irreversible at subzero temperatures. Furthermore, continuous light causes depletion of antifreeze proteins within 24 h at 21°C. On each occasion in December, January and March, samples of about 700 beetles were obtained from the outdoor supply and sorted into 4 experimental groups (Fig. 1). Two groups of beetles were kept at 21°C for 6 days to achieve complete depletion of ethylene glycol. Group I was kept in total darkness for the entire experimental period so as to retain antifreeze proteins. Group II was kept during continuous light after the fifth day in order to deplete the antifreeze proteins. The remaining two groups were treated so as to retain ethylene glycol. One group (Group III) was kept at 0°C for 2 days in total darkness, while the other group (Group IV) was kept in continuous light. Survival was thereafter studied in beetles from each group exposed to a range of temperatures above their mean supercooling point. Beetles depleted of ethylene glycol, with (Group I) and without (Group II) antifreeze proteins were kept 347
348
UNN GEHRKEN
12 measurements. Melting and freezing points were measured on haemolymph obtained from individual beetles and the mean values were based on measurements from 3 specimens. Quantification of ethylene glycol was carried out on 3 extracts each from 3 beetles. Each determination of water content represented the mean of 5 measurements. Survival was determined on the basis of 20 beetles from each group after 1, 2, 3 and 7 days of exposure to subzero temperatures. Measures of survival were restricted to observations of coordinated walking after 3 days at 3°C.
outdoor sample
DDfP6
DD18d Groupie
Group1
-16%
GrouplV
Groupill
/l--T-l
-14%
LLt2d
-17.5%
-25%
-22%
-26%
RESULTS
Fig. 1. Acclimation schedule used to study the effect of antifreeze proteins on supercooling and survival of Ips acumina~ exposed to temperatures above their mean supercooling points. The beetles were either kept in complete darkness (DD) or continuous light (LL) for the time periods indicated for each acclimation temperature.
at - 14, - 16 or - 17.5”C. Similarly, the beetles containing ethylene glycol, with (Group III) and without (Group IV) antifreeze proteins were kept at -22, -25 or -26°C. Experiments were run to check whether freezing was the sole cause of death. In December, samples of 8 beetles were connected to thermocouples and kept for 24 h at 2-3°C above their mean supercooling points. Beetles containing antifreeze proteins but no ethylene glycol (Group I) were kept at - 17.5”C, while those depleted of both antifreezes (Group II) were held at - 16°C. Beetles from Group IV which contained ethylene glycol but no antifreeze proteins were kept at -25°C. The supercooling, melting and freezing points and thermal hysteresis were determined in beetles from Group I and Group II after 6 days at 21°C and after 7 days at - 14, and - 16°C. Similar measurements were obtained from individuals in Group III and Group IV after 2 days at 0°C and after 7 days at - 22 and - 25°C. Ethylene glycol concentration and water content were only recorded after subzero temperature exposure. The techniques for measuring supercooling point, melting and freezing points, ethylene glycol concentration and water content have been described earlier (Gehrken, 1984). Supercooling points are presented as the mean of
Table 1 shows the supercooling, melting and freezing points and thermal hysteresis measurements for I. acuminatus collected from the outdoor supply in December, January or March and depleted of ethylene glycol at 21°C for 6 days. For beetles kept at 21°C in complete darkness (Group I), there was a significant (t-test, P < 0.001) difference between the melting and freezing points, indicating that thermal hysteresis-producing antifreeze proteins were present. The magnitude of this difference, which is dependent on the concentration, ranged from 1.9 to 3.5”C. The stabilization of supercooling and melting points at about -20 and - 1.2”C (Table 1) corresponded to earlier measurements of beetles depleted of ethylene glycol (Gehrken, 1984). No correlation was found between mean supercooling point and mean concentration of antifreeze proteins (r2 = 0.1). For warm acclimated beetles exposed to continuous light after the fifth day (Group II), no significant difference was found between melting and freezing points, indicating that antifreeze proteins were absent. The loss of thermal hysteresis corresponded to a significant (t-test, P < 0.01) rise in supercooling points from about -20 to - 18°C. Evidence that this rise is not attributable to differences in solute concentration was provided by the melting points which were similar to those of beetles displaying thermal hysteresis. No substantial changes in mean supercooling, melting and freezing points of beetles from Group I and II were found after exposure to - 14 and - 16°C for 7 days in December, January or March (Table 2). No ethylene glycol was detected in extracts of beetles from these two groups, and their water content was above 60%. This high water content agrees with
Table I. Mean ( f SE) supercooling, melting depleted of ethylene glycol during
Month
Dec.
Jan. Jan. Mar. Mar.
Treatment
Group
2lYXd/DD 2I “C-Sd/DD and 21°C~ld/LL 21“C-6d/DD 2l”C-Sd/DD 2I”C-ld/LL
SCP (“C) n = 12
KG n=5
(E) If=5
THP (“C) (n =5 1.9kO.1
- I .2 f 0.03
-3.1
- 18.4 f 0.4 -20.3+0.1
-1.2&O.] -1.2+0.1
-1.4*0.1 -4.1 f 0.3
3.5 f 0.2
II I
- 18.6 + 0.3 -20.3kO.l
- I .2 * 0.04 - I .2 f 0.03
-1.4+0.1 -4.0 f 0.3
2.8 k 0.2
II
- 18.0 k 0.3
-1.2*0.1
-1.4*0.1
I
-20.1
II I
* 0.1
50.2
and
21°C-6d/DD Zl”C-WDD and 21”C-ld/LL
and freezing points and thermal hysteresis of fps acuminofw 6 days at 21°C. Group l-total darkness (DD); Group II-continuous light from dav 5 to dav 6 (LL)
Supercooling and thermal hysteresis
349
Table 2. Mean(*SE) supercooling, melting and freezing points, thermal hysteresisand watercontentof &s acuminarus depleted of ethylene glycol and exposed to - 14 or - 16°C for 7 days. Both beetles with (Group I) and without antifreeze proteins (Group II) are included
Month
Exposure temperature (“C) -16 -14 -16 -14 -16 -14 -16 -14 -16 -14 - 16/7d - 14/7d
Dec.
Jan.
Mar.
Group
SCP (“C) n = 12
I I II II I I II II I I II II
-20.1 +0.1 -20.4 + 0.2 - 18.5 * 0.4 -l&3+0.3 -20.0 f 0.1 -20.5 f 0.2 -18.1 +0.3 - 18.0 + 0.4 - 20.0 rt 0.2 -20.3 k 0.1 - 17.9 * 0.4 - 18.3 + 0.4
earlier findings from warm acclimated I. accuminatus depleted of ethylene glycol (Gehrken, 1984). Table 3 shows the survival rates at - 14, - 16 and - 17.5”C of I. acuminatus depleted of ethylene glycol. Throughout the three experimental periods, the survival decreased with exposure time and decreasing temperature. Changes in the ability of beetles to survive were correlated to the temperature but not the season. Pre-freeze injuries (uncoordinated walking) were frequently seen in animals from both groups. However, a higher incidence of mortality was always seen in the groups depleted of antifreeze proteins (Group II). In beetles showing thermal hysteresis (Group I), 30% survived for 1 week at - 175°C in December, and 75% survived for 1 day. These beetles were pre-freeze injured but recovered during 3 days at 3°C. Throughout the three experimental periods, about 50% of the beetles survived for 1 week at - 16°C. After 1 day of exposure, the mortality ranged from 15 to 25%, and the injurious effects of cold in the remaining beetles were reversed at 3°C. Furthermore, more than two-thirds of the sample survived for 1 week at -14°C. Altogether, 1 out of 60 beetles died during 1 day of exposure, and those with pre-freeze injury (3540%) recovered at 3°C. In contrast, all beetles depleted of antifreeze proteins (Group II) died during 1 day at - 17.5”C in
(y; fl=5
(Z) n=.5
- I .2 + 0.03 -1.3*0.1 -1.2+0.04 -1.2kO.l - 1.3+ 0.03 -1.3*0.1 - I .2 + 0.03 -1.2 * 0.04 -1.2*0.1 - I .2 * 0.03 -1.3*0.1 -1.2*0.1
-2.9 f0.1 -3.2kO.2 -1.4*0.1 - I .4 f 0. I -4.8 f 0.2 -4.5 * 0.1 -1.4+0.1 -1.5+0.1 -3.8 + 0.3 -3.6 k 0.2 -1.5*0.1 -1.4*0.1
Water content (%) n=5
THP (“C) n=5 1.7 f0.1 1.9*0.1
60.9 + 0.4 62.4 + 0.5 65.2 L-0.6 62.7 f 0.5 63.5 rt 0.5 63.1 f 0.5 60.5 k 0.4 60.6 f 0.5 61.3 k 0.4 62.1 + 0.4 62.9 f 0.5 60.7 + 0.4
3.5 * 0.2 3.2 +O.l
2.6 +_0.2 2.4 k 0.2
December. Throughout the three experimental periods, all individuals had “died” after 1 day at - 16°C; between 40-50% of the specimens had died and the remainder were fatally injured. No beetles from Group II survived for 1 week at - 14°C. The survival after 1 day, however, ranged from 45 to 55%. About one-third of the beetles were dead and the remainder were fatally injured. Apparently, complete recovery occurred only in the presence of antifreeze proteins. Table 4 shows the supercooling, melting and freezing points as well as thermal hysteresis of outdoor I. acuminatus kept at 0°C in darkness for 2 days in December, January and March. Similar data obtained from beetles subjected to continuous light are also included. The significant difference (t-test, P < 0.001) between the melting and freezing points indicated the presence of antifreeze proteins in beetles kept in complete darkness (Group III). Accordingly, the insignificant difference between the melting and freezing points suggested the absence of antifreeze proteins in those kept in continuous light (Group IV). In Group III beetles, the seasonal changes in thermal hysteresis activity agree with those found for warm acclimated beetles (Table 1) although the melting and freezing points are lower in the cold treated beetles (Group III). The mean supercooling points changed
Table 3. Survival rates after 1, 2, 3 and 7 days at - 14. - 16 and - 17.5”C for Ips acuminafus depleted of ethylene glycol, with (Group I) or without (Group II) antifreeze proteins. Degree above mean SCP refers to the difference between exposure temperature and the mean supercooling points of beetles after the treatment at 21°C (Table I)
Month Dec.
Jan.
Mar.
Exposure temperature (“C) -17.5 -16 -14 - 17.5 -16 -I4 -I6 -14 -16 -14 -16 -14 -16 -14
“C above mean SCP
Group
n =20
2.6 4.1 6.1 0.9 2.4 4.4 4.3 6.3 2.6 4.6 4.3 6.3 2.0 4.0
I I I II II II I I II II I I II II
75 85 100 0 0 50 75 95 0 55 75 100 0 45
I
Survival (%) 2 3 n = 20 n = 20 55 60 85 0 0 30 65 100 0 25 55 90 0 25
40 60 70 0 0 5 50 75 0 15 50 75 0 10
7 n = 20 30 55 70 0 0 0 50 75 0 0 55 70 0 0
350
UNN GEHRKEN Table 4. Mean (k SE) supercooling, melting and freezing points and thermal hysteresis in Ipsacuminatus kept at 0°C for 2 days in either total darkness (DD) (Group III) or continuous light (LL) (Group IV) SCP
THP
( Cl Month Dec. Jan. Mar.
0 C-Zd/DD O‘C-Zd/LL O”C-Zd/DD O”C-Zd/LL 0 ‘C-2d/DD O’C-2d/LL
P-3
n = 12
Group
Treatment
III IV 111 IV III IV
- 29.6 - 27.4 -31.5 -28.3 -28.9 -24.9
+ 0.2 f 0.4 kO.2 t 0.5 k 0.4 + 0.5
Dec.
Jan.
Mar.
-25 -22 -25 -22 -25 -22 -25 -22 -25 -22 -25 -22
-7.4 i 0.2 -5.3*0.1 < -9.5 -6.5 + 0.2 - 7.0 + 0.3 -4.5 kO.1
2.1 * 0.2 >3.1 2.5 f 0.2
melting and freezing points, thermal hysteresis. water content and ethylene glycol concentration of Ips for 7 days. Both animals with (Group III) and without antifreeze proteins (Group IV) are included
Table 5. Mean ( ? SE) supercooling, acuminatus kept at - 22 or -25°C
Month
-5.3 f 0.3 -5.1 f0.3 -6.4 k 0.3 -6.3 + 0.3 -4.5 * 0.3 -4.4 + 0.3
substantially lower (l-test, P < 0.001) than for beetles depleted of ethlylene glycol (Table 2). Table 6 shows the survival rate at -22, -25 and -26°C of 1. acuminates in the presence of ethylene glycol. Since no movements were observed in dead specimens, it was assumed that they died from freezing. Thus, pre-freeze injured beetles were never observed in Groups III and IV. Irrespective of the ethylene glycol concentration (Table 5), survival was always lower in beetles depleted of their antifreeze proteins (Group IV). Thus, while only 25% of the beetles showing thermal hysteresis (Group III) survived for 1 week at -26°C in December, 12 out of 20 beetles in this group survived after 1 day. In contrast, none survived after 1 day at -26°C when depleted of their antifreeze proteins. Furthermore, the seasonal difference in survival at - 25 and - 22°C
markedly during the season and between the two groups. In contrast, the mean melting point differed only during the season but not between the groups. In December, January and March, the substantially higher (r-test, P -c0.01)supercooling points of beetles in Group IV corresponded with the depletion of their antifreeze proteins. No substantial changes in mean supercooling, melting and freezing points of beetles from Groups III and IV were observed after 7 days of exposure to -22 and -25°C (Table 5). Thus, the cold hardiness of beetles had not been enhanced during exposure to these temperatures. The melting points of beetles from each group varied directly with seasonal changes in ethylene glycol concentration (Table 5), in agreement with earlier findings (Gehrken, 1984). The mean water content of about 50% (Table 5) was
Exposure temperature (“C)
n=5
Group
-29.4 + 0.5 -29.1 +0.2 - 21.6 ? 0.8 - 27.1 + 0.6 -31.2+0.3 - 30.9 I 0.2 -28.6 k 0.5 -28.5 f 0.5 - 28.8 * 0.3 -28.2 k 0.3 -24.9 i 0.5 -25.5 i 0.4
III III IV
IV III III IV IV 111 III IV IV
-5.1 -4.9 -4.9 - 5.0 -6.3 -6.2 -6.1 -5.9 -4.3 -4.5 -4.5 -4.2
Ethylene glyml (mmolikg) n=3
THP
MP (“0 II=5
SCP (“C) n = 12
(::, II=5
f0.3 f 0.2 * 0.3 i_ 0.3 + 0.3 * 0.3 Ifr 0.2 f 0.3 + 0.2 f 0.3 * 0.3 + 0.3
(“C) n=5
- 7.0 + 0.2 -7.1 kO.2 - 5.0 * 0.2 -5.2 ? 0.3 < -9.5 < -9.5 -6.3 k 0.2 -6.1 f 0.2 -6.8 + 0.3 -6.7 f 0.2 -4.3 * 0.4 -4.5 * 0.3
1.9 f0.1 2.2 f 0.2
> 3.2 >3.3
2.5 f 0.2 2.2 f 0.2
1752+70 1899It65 1821 f 99 1731 + 72 2385 f 88 2113 k98 2229 + 107 2205 f 102 1554 * 120 1609+90 1579+ 116 1487 k 139
Table 6. Survival rates after 1,2, 3 and 7 days at -22, -25 and -26’C for Ipsocuminafus displaying ethylene glycol, with (Group III) or without antifreeze proteins (Group IV). Degree above mean SCP refers to the difference between exposure temperature and the mean supercooling point of beetles followinn 2 davs at 0°C (Table 5)
Month
Dec.
Jan
Mar.
Exposure temperature (“C)
:C above mean SCP
-26 -25 -22 -26 -25 -22 -25 -22 -25 -22 -25 -22 -25 -22
3.6 4.6 8.6 1.4 2.4 5.4 6.5 9.5 3.3 6.3 3.9 6.9 0.0 3.0
I Group III 111 111 IV IV IV III 111 IV IV III III IV IV
n = 20 60 15 100 0 25 50 95 100 45 70 65 100 0 35
Survival (%) 2 3 n = 20 n = 20 45 50 100 0 5 40 100 100 20 55 55 100 0 5
30 40 100 0 0 25 90 100 0 40 50 95 0 0
I n = 20 25 40 100 0 0 0 85 100 0 0 35 80 0 0
Water content (%) n=5 49.7 f 0.3 50.1 * 0.4 50.3 * 0.3 51.5 2 0.3 51.450.3 50.2 5 0.3 50.6 f 0.3 50.9 * 0.3 51.3 f 0.3 50.3 f 0.3 51.7kO.4 51.1 f0.3
Supercooling and thermal hysteresis
351
at 2-3°C above their mean supercooling points. The survival in these groups agreed with corresponding data from December given in Tables 3 and 6. In the absence of ethylene glycol and antifreeze proteins (Group II), however, the majority of beetles died due to pre-freeze injuries. Only 3 out of 8 individuals froze at - 16°C and the remainder were fatally injured. DISCUSSION
In overwintering adult bark beetles I. acuminatus, freeze avoidance is potentiated by the accumulation of high concentrations of ethylene glycol. At temperatures above the mean supercooling point of the beetles, percentage survival decreased with exposure time and decreaging temperature. However, the antifreeze proteins had a marked effect on survival rates, both in the presence and in the absence of ethylene _i _; 4 4 -b 4 glycol (Tables 4, 5 and 6 and 1, 2 and 3, respectively). Mean meltlng point,“C At temperatures ranging up to 6°C above their mean Fig. 2. Mean supercooling points of Ips acuminatus as a supercooling point, no beetles depleted of antifreeze function of the corresponding mean melting points. The proteins survived 1 week, whereas 70% survival solid line represents d&a from beetles diplaying thermal or more was found in those displaying thermal hysteresis (Y = - 17.5 + 2.3X, r2 = 0.99 and P < O.OOl), hysteresis. Evidence that this change in survival was while the dashed line represents similar measurements for not related to changes in the concentration of low those depleted of these antifreeze proteins (Y = - 15.7+2.2X, molecular weight substances was provided by the r* = 0.99 and P c 0.001). melting points. In I. acuminatus with and without antifreeze proreflected changes in the supercooling points (Table 5). teins, low molecular weight substances lower superIn January, the supercooling points were well below cooling points 2.2-2.3”C for each degree of melting those obtained from beetles in December and March. point depression (Fig. 2), in agreement with earlier Accordingly, survival was higher after 1 week at findings (Gehrken, 1984). If supercooling capacity -25°C in January than in December and March in along determined survival, one might expect a groups displaying thermal hysteresis. No beetles correlation between percentage survival after 1 week depleted of these antifreeze proteins survived for 1 and “C above mean supercooling points, even for week at - 25 or -22”C, and differences in survival I. acuminatus depleted of thermal hysteresis proteins, related to changes in the supercooling points were however, the reverse was true. Thus, the overall only found after 1 day of exposure. change in survival rates arises from the breakdown of thermal hysteresis proteins. However, injurious The data from Group I-IV revealed that irreeffects of cold other than freezing also influenced the spective of the presence of ethylene glycol, the supercooling points were always substantially lower in mortality. Freezing seemed to be the cause of death in animals which retained ethylene glycol and in those beetles displaying thermal hysteresis. Thus, the mean supercooling points were linearly related to the which retained antifreeze proteins after a complete loss of the solute antifreeze. In contrast, the majority mean melting points in the presence and absence of antifreeze proteins (Fig. 2). Also survival of of beetles died from pre-freeze injury in the absence beetles was markedly improved in the presence of of both these antifreezes. That cold can lead to death in the absence of freezing has been observed in several antifreeze proteins. Using the data on survival after species which experience subzero winter temperatures 1 week (Tables 3 and 6), a correlation between (Turnock et al., 1983; Lee and Denlinger, 1985; survival and “C above mean supercooling point was Knight et a1.,1986; Bale et al., 1987). found for animals displaying thermal hysteresis The causes of pre-freeze injury are still a matter of (Y = 0.83 + 0,08X, r* = 0.90, P < O.OOl), but not for those depleted of their antifreeze proteins (r* = 0.1). speculation. In I. acuminatus, pre-freeze injury was Experiments using I. acuminates connected to only found in groups depleted of ethylene glycol, and might arise from the high water content and the lack thermocouples for 24 h in December showed that of low molecular weight substances. In the presence the mortality was caused by freezing and pre-freeze injury. In the presence of ethylene glycol but no of antifreeze proteins, however, beetles were capable of recovery, at least after 1 day of exposure to thermal hysteresis (Group IV), 5 out of 8 beetles froze temperatures near the supercooling point. This sugat -25”C, and the remainder showed no injurious gests that these antifreezes may also help to alleviate effects of cold. In the absence of ethylene glycol but presence of thermal hysteresis proteins (Group I), 2 pre-freeze damage. It has been suggested that thermal hysteresisout of 8 beetles froze at -17.5”C. The remaining 6 producing proteins are responsible for supercooling suffered pre-freeze injury, but recovered in the course of 3 days at 3°C. Accordingly, freezing was the sole point depression in several species (Duman et al., 1982). A correlation between increasing thermal hyscause of death, while 38% of animals from Group IV teresis activity and decreasing supercooling points and 75% of those from Group I survived after 1 day
352
UNN
was demonstrated in larvae of Merachanta contracta (Duman, 1977). In contrast, no such correlation was found in I. acuminatus depleted of ethylene glycol. However, depletion of antifreeze proteins resulted in an overall reduced capacity to supercool (Fig. 2). Since these proteins can inhibit growth of ice crystals, it is logical to assume that they also prevent embryo crystals from reaching the critical size necessary to induce freezing in a supercooled system. The lower supercooling capacity, greater variance of supercooling point measurements and lower survival of I. acuminatus depleted of thermal hysteresis, suggests that these proteins reduce the probability that the beetles will freeze. Thus, the present results support the theory of Zachariasscn and Husby (1982) and suggest that antifreeze proteins are necessary to stabilize the supercooled state of insects down to the observed supercooling points predicted by the levels of low molecular weight substances. Seasonal changes in day length may be of particular importance in controlling the level of thermal hysteresis. In larvae of the darkling beetle Merachantha contracta held at 22°C a long photoperiod leads to a loss of thermal hysteresis-producing proteins (Duman, 1977). In larvae of the Phyrochroid beetle Dendroides canadensis, long photoperiod cause depletion of antifreeze proteins at both 10 and 23°C (Duman, 1980). In the spider, Philodromus, no photoperiodic effect was observed at low (4°C) or high (22°C) temperatures (Duman, 1979). The fact that exposure to constant light results in a complete loss of thermal hysteresis activity, even in I. acuminatus kept at 0°C suggests that the primary environmental parameter responsible for triggering depletion is photoperiodic. High temperature is of less importance for depletion of antifreeze proteins in I. acuminatus, and these antifreezes might therefore provide a failsafe system to ensure supercooling to about - 20°C in the absence of ethylene glycol. Since the overwintering site is generally above the snow (Bakke, 1968), specimens are subjected to low and high ambient air temperatures. In fact, Bakke (1968) reported that the temperature in the microhabitat might increase by 20°C above the air temperature due to sun radiation. The beetles enter a facultative diapause during autumn which is terminated by mid-winter (Gehrken, 1985). While in diapause, the beetles remain underneath the bark of Scot’s pine (Gehrken, unpublished data). High ambient temperatures from mid-winter onwards might therefore be fatal to I. acuminatus if
GEHRKEN
the beetles leave the logs, and risk exposure to full daylight, leading to a complete breakdown of the antifreeze proteins. Acknowledgement-I
am most grateful to Dr L. Somme for valuable advice during the preparation of the manuscript. REFERENCES
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Bale J. S., Harrington R. and Clough M. S. (1987) Effect of low temperature on the survival of the peach-potato aphid Myzus persica. Proc. Third European Congress of Entomology, Part 2, PP. 243-246. Amsterdam. Duman J. G-(1977) Variation in macromolecular antifreeze levels in larvae of the darkling beetle, Merachantha contracta. J. exp. Zool. 201, 85-92.
Duman J. G. (1979) Subzero temperature tolerance in spiders: the role of thermal hysteresis factor. J. camp. Physiol. 131, 347-352. J. exp. Zool. 201. 85-92. Duman J. G. (1980) Factors involved in the overwintering survival of the freeze tolerant beetle, Dendroides canadensis. J. camp. Physiol. 136, 347-352. Duman J. G., Horwarth K., Tomchaney A. and Patterson J. L. (1982) Antifreeze agents of terrestrial arthropods, Comp. Biochem. Physiol. 73A, 545-555.
Gehrken U. (1984) Winter survival of an adult bark beetle, Ips acuminatus Gyll. J. Insect Physioi. 30, 421429. Gehrken U. (1985) Physiology of diapause in the adult bark beetle, Ips acuminatus Gyll.: studied in relation to cold hardiness. J. Insect Physioi. 31, 909-916. Knight J. D., Bale J. S., Franks F., Mathias S. and Baust J. G. (1986) Insect cold hardiness: supercooling points and pre-freeze mortaility. Cryo-Lefters 7, 194203. Lee R. E. Jr and Denlinger D. L. (1985) Cold tolerance in diapausing and non-diapausing stages of the flesh fly, Sacrophaga crassipaipis. Physiol. Ent. 10, 309-315. Raymond J. A. and De Vries A. L. (1977) Absorption inhibition as a mechanism of freezing resistance in polar fishes. Proc. natl Acad. Sci. U.S.A. 74, 2589-2593.Somme L. (1982) Sunercoolina and winter survival in terrestrial ‘arthropods. Campy Biochem. Physiol. 73A, 519-543.
Tumock W. J., Lamb R. J. and Bodaryk R. P. (1983) Effects of cold stress during pupal diapause on the survival and development of Mamestra confiaurata (Lepidoptera: Noctuidae). Oecologia. 56, 185-192.Zachariassen K. E. (1985) Cold tolerance in insects. Phvsiol. , ’ Rev. 65, 800-832: Zachariassen K. E. and Husby J. A. (1982) Antifreeze effects of thermal hysteresis protects highly supercooled insects. Nature, Land. 298, 865-869.
0022-1910/89 $3.04 + 0.00 Pergamon Press plc
SUPERCOOLING AND THERMAL HYSTERESIS IN THE ADULT BARK BEETLE, IPS ACUMINATUS GYLL. Division of Zoology, Department
UNN GEHRKEN of Biology, University of Oslo, P.O. Box 1050, Blindem N-0316. Oslo 3, Norway
(Received 28 June 1988; revised 19 Seprember 1988) Abstract-The adult bark beetle Ips acurninarus relies on supercooling for overwintering success. The beetle produces ethylene glycol in concentration up to 2-3 mol/kg wet wt and antifreeze proteins leading to thermal hysteresis of about 24°C. The supercooling capacity is always higher in the presence of antifreeze proteins. After a complete depletion of ethylene glycol, the loss of protein antifreezes results in an elevation of supercooling points from about -20 to - 18°C. For equivalent levels of ethylene glycol, the breakdown of antifreeze proteins results in a substantial rise in supercooling points. In beetles exposed to temperatures above their mean supercooling point, survival decreases both with exposure time and decreasing temperature. Irrespective of the presence of ethylene glycol, lower survival of beetles is always seen following depletion of antifreeze proteins. A correlation between survival for 1 week and supercooling capacity is only evident in groups exhibiting thermal hysteresis. Freezing appears to be the cause of death in beetles with ethylene glycol in their body fluids, whereas pre-freeze injury occurs frequently in individuals depleted of this antifreeze. However, the beetles with antifreeze proteins are able to recover from pre-freeze injury in the course of 3 days at 3°C. In the absence of these proteins, beetles are fatally injured, and pre-freeze injury seems to be the main cause of death. The reduced supercooling capacity, higher variance of supercooling point measurements and lower survival in I. acwninatus depleted of antifreeze proteins suggests that these proteins offer protection against freezing over the entire supercooling range, and thus ensure winter survival at low subzero temperatures. Key Word Index: Adult, bark beetle, cold hardiness, supercooling, thermal hysteresis, survival/mortality
INTRODUCTION To improve winter survival, a number of arthropods rely on supercooling to avoid freezing. An enhanced
ability to supercool is generally ascribed to the accumulation of low molecular weight antifreezes (Somme, 1982). Antifreeze proteins which produce thermal hysteresis (a difference between melting and freezing points) have been found in a number of insect species (Zachariassen, 1985). These proteins do not alter the melting point of plasma but prevent growth of ice crystal (Raymond and DeVries, 1977). Zachariassen and Husby (1982) suggested that antifreeze proteins may stabilize the supercooled state over the entire supercooling range. However, more data are needed on the antifreeze effect of these proteins in insects. The present study evaluates the effect of antifreeze proteins on the cold tolerance of the adult bark beetle, Ips ncuminatus. The beetle produces antifreeze proteins corresponding to a thermal hysteresis of 3-4”C and also accumulates the polyhydric alcohol, ethylene glycol in a concentration of about 2-3 mol/kg wet wt (Gehrken, 1984). Experiments were performed in order to study survival near the supercooling point (the temperature at which spontaneous freezing occurs). Comparisons were made on supercooling and survival between beetles which had been treated so as to retain or remove the antifreeze proteins. These experiments were performed on beetles in which ethylene glycol also was present or absent according to the temperature regime in which the beetles were reared.
MATERIALS AND METHODS
Logs of Scats pine (Pinus sihestris) infested with I. acuminatus were collected near Kong&erg (59”40’), SE Norway and stored out of doors at Blindern, University of Oslo. The procedure in the following experiments was based on previous findings (Gehrken, 1984, 1985). After initiation by November, the beetles continue to accumulate ethylene glycol at subzero temperature throughout the winter. High temperatures, however, cause degradation of ethylene glycol, and a complete loss proved to be irreversible at subzero temperatures. Furthermore, continuous light causes depletion of antifreeze proteins within 24 h at 21°C. On each occasion in December, January and March, samples of about 700 beetles were obtained from the outdoor supply and sorted into 4 experimental groups (Fig. 1). Two groups of beetles were kept at 21°C for 6 days to achieve complete depletion of ethylene glycol. Group I was kept in total darkness for the entire experimental period so as to retain antifreeze proteins. Group II was kept during continuous light after the fifth day in order to deplete the antifreeze proteins. The remaining two groups were treated so as to retain ethylene glycol. One group (Group III) was kept at 0°C for 2 days in total darkness, while the other group (Group IV) was kept in continuous light. Survival was thereafter studied in beetles from each group exposed to a range of temperatures above their mean supercooling point. Beetles depleted of ethylene glycol, with (Group I) and without (Group II) antifreeze proteins were kept 347
348
UNN GEHRKEN
12 measurements. Melting and freezing points were measured on haemolymph obtained from individual beetles and the mean values were based on measurements from 3 specimens. Quantification of ethylene glycol was carried out on 3 extracts each from 3 beetles. Each determination of water content represented the mean of 5 measurements. Survival was determined on the basis of 20 beetles from each group after 1, 2, 3 and 7 days of exposure to subzero temperatures. Measures of survival were restricted to observations of coordinated walking after 3 days at 3°C.
outdoor sample
DDfP6
DD18d Groupie
Group1
-16%
GrouplV
Groupill
/l--T-l
-14%
LLt2d
-17.5%
-25%
-22%
-26%
RESULTS
Fig. 1. Acclimation schedule used to study the effect of antifreeze proteins on supercooling and survival of Ips acumina~ exposed to temperatures above their mean supercooling points. The beetles were either kept in complete darkness (DD) or continuous light (LL) for the time periods indicated for each acclimation temperature.
at - 14, - 16 or - 17.5”C. Similarly, the beetles containing ethylene glycol, with (Group III) and without (Group IV) antifreeze proteins were kept at -22, -25 or -26°C. Experiments were run to check whether freezing was the sole cause of death. In December, samples of 8 beetles were connected to thermocouples and kept for 24 h at 2-3°C above their mean supercooling points. Beetles containing antifreeze proteins but no ethylene glycol (Group I) were kept at - 17.5”C, while those depleted of both antifreezes (Group II) were held at - 16°C. Beetles from Group IV which contained ethylene glycol but no antifreeze proteins were kept at -25°C. The supercooling, melting and freezing points and thermal hysteresis were determined in beetles from Group I and Group II after 6 days at 21°C and after 7 days at - 14, and - 16°C. Similar measurements were obtained from individuals in Group III and Group IV after 2 days at 0°C and after 7 days at - 22 and - 25°C. Ethylene glycol concentration and water content were only recorded after subzero temperature exposure. The techniques for measuring supercooling point, melting and freezing points, ethylene glycol concentration and water content have been described earlier (Gehrken, 1984). Supercooling points are presented as the mean of
Table 1 shows the supercooling, melting and freezing points and thermal hysteresis measurements for I. acuminatus collected from the outdoor supply in December, January or March and depleted of ethylene glycol at 21°C for 6 days. For beetles kept at 21°C in complete darkness (Group I), there was a significant (t-test, P < 0.001) difference between the melting and freezing points, indicating that thermal hysteresis-producing antifreeze proteins were present. The magnitude of this difference, which is dependent on the concentration, ranged from 1.9 to 3.5”C. The stabilization of supercooling and melting points at about -20 and - 1.2”C (Table 1) corresponded to earlier measurements of beetles depleted of ethylene glycol (Gehrken, 1984). No correlation was found between mean supercooling point and mean concentration of antifreeze proteins (r2 = 0.1). For warm acclimated beetles exposed to continuous light after the fifth day (Group II), no significant difference was found between melting and freezing points, indicating that antifreeze proteins were absent. The loss of thermal hysteresis corresponded to a significant (t-test, P < 0.01) rise in supercooling points from about -20 to - 18°C. Evidence that this rise is not attributable to differences in solute concentration was provided by the melting points which were similar to those of beetles displaying thermal hysteresis. No substantial changes in mean supercooling, melting and freezing points of beetles from Group I and II were found after exposure to - 14 and - 16°C for 7 days in December, January or March (Table 2). No ethylene glycol was detected in extracts of beetles from these two groups, and their water content was above 60%. This high water content agrees with
Table I. Mean ( f SE) supercooling, melting depleted of ethylene glycol during
Month
Dec.
Jan. Jan. Mar. Mar.
Treatment
Group
2lYXd/DD 2I “C-Sd/DD and 21°C~ld/LL 21“C-6d/DD 2l”C-Sd/DD 2I”C-ld/LL
SCP (“C) n = 12
KG n=5
(E) If=5
THP (“C) (n =5 1.9kO.1
- I .2 f 0.03
-3.1
- 18.4 f 0.4 -20.3+0.1
-1.2&O.] -1.2+0.1
-1.4*0.1 -4.1 f 0.3
3.5 f 0.2
II I
- 18.6 + 0.3 -20.3kO.l
- I .2 * 0.04 - I .2 f 0.03
-1.4+0.1 -4.0 f 0.3
2.8 k 0.2
II
- 18.0 k 0.3
-1.2*0.1
-1.4*0.1
I
-20.1
II I
* 0.1
50.2
and
21°C-6d/DD Zl”C-WDD and 21”C-ld/LL
and freezing points and thermal hysteresis of fps acuminofw 6 days at 21°C. Group l-total darkness (DD); Group II-continuous light from dav 5 to dav 6 (LL)
Supercooling and thermal hysteresis
349
Table 2. Mean(*SE) supercooling, melting and freezing points, thermal hysteresisand watercontentof &s acuminarus depleted of ethylene glycol and exposed to - 14 or - 16°C for 7 days. Both beetles with (Group I) and without antifreeze proteins (Group II) are included
Month
Exposure temperature (“C) -16 -14 -16 -14 -16 -14 -16 -14 -16 -14 - 16/7d - 14/7d
Dec.
Jan.
Mar.
Group
SCP (“C) n = 12
I I II II I I II II I I II II
-20.1 +0.1 -20.4 + 0.2 - 18.5 * 0.4 -l&3+0.3 -20.0 f 0.1 -20.5 f 0.2 -18.1 +0.3 - 18.0 + 0.4 - 20.0 rt 0.2 -20.3 k 0.1 - 17.9 * 0.4 - 18.3 + 0.4
earlier findings from warm acclimated I. accuminatus depleted of ethylene glycol (Gehrken, 1984). Table 3 shows the survival rates at - 14, - 16 and - 17.5”C of I. acuminatus depleted of ethylene glycol. Throughout the three experimental periods, the survival decreased with exposure time and decreasing temperature. Changes in the ability of beetles to survive were correlated to the temperature but not the season. Pre-freeze injuries (uncoordinated walking) were frequently seen in animals from both groups. However, a higher incidence of mortality was always seen in the groups depleted of antifreeze proteins (Group II). In beetles showing thermal hysteresis (Group I), 30% survived for 1 week at - 175°C in December, and 75% survived for 1 day. These beetles were pre-freeze injured but recovered during 3 days at 3°C. Throughout the three experimental periods, about 50% of the beetles survived for 1 week at - 16°C. After 1 day of exposure, the mortality ranged from 15 to 25%, and the injurious effects of cold in the remaining beetles were reversed at 3°C. Furthermore, more than two-thirds of the sample survived for 1 week at -14°C. Altogether, 1 out of 60 beetles died during 1 day of exposure, and those with pre-freeze injury (3540%) recovered at 3°C. In contrast, all beetles depleted of antifreeze proteins (Group II) died during 1 day at - 17.5”C in
(y; fl=5
(Z) n=.5
- I .2 + 0.03 -1.3*0.1 -1.2+0.04 -1.2kO.l - 1.3+ 0.03 -1.3*0.1 - I .2 + 0.03 -1.2 * 0.04 -1.2*0.1 - I .2 * 0.03 -1.3*0.1 -1.2*0.1
-2.9 f0.1 -3.2kO.2 -1.4*0.1 - I .4 f 0. I -4.8 f 0.2 -4.5 * 0.1 -1.4+0.1 -1.5+0.1 -3.8 + 0.3 -3.6 k 0.2 -1.5*0.1 -1.4*0.1
Water content (%) n=5
THP (“C) n=5 1.7 f0.1 1.9*0.1
60.9 + 0.4 62.4 + 0.5 65.2 L-0.6 62.7 f 0.5 63.5 rt 0.5 63.1 f 0.5 60.5 k 0.4 60.6 f 0.5 61.3 k 0.4 62.1 + 0.4 62.9 f 0.5 60.7 + 0.4
3.5 * 0.2 3.2 +O.l
2.6 +_0.2 2.4 k 0.2
December. Throughout the three experimental periods, all individuals had “died” after 1 day at - 16°C; between 40-50% of the specimens had died and the remainder were fatally injured. No beetles from Group II survived for 1 week at - 14°C. The survival after 1 day, however, ranged from 45 to 55%. About one-third of the beetles were dead and the remainder were fatally injured. Apparently, complete recovery occurred only in the presence of antifreeze proteins. Table 4 shows the supercooling, melting and freezing points as well as thermal hysteresis of outdoor I. acuminatus kept at 0°C in darkness for 2 days in December, January and March. Similar data obtained from beetles subjected to continuous light are also included. The significant difference (t-test, P < 0.001) between the melting and freezing points indicated the presence of antifreeze proteins in beetles kept in complete darkness (Group III). Accordingly, the insignificant difference between the melting and freezing points suggested the absence of antifreeze proteins in those kept in continuous light (Group IV). In Group III beetles, the seasonal changes in thermal hysteresis activity agree with those found for warm acclimated beetles (Table 1) although the melting and freezing points are lower in the cold treated beetles (Group III). The mean supercooling points changed
Table 3. Survival rates after 1, 2, 3 and 7 days at - 14. - 16 and - 17.5”C for Ips acuminafus depleted of ethylene glycol, with (Group I) or without (Group II) antifreeze proteins. Degree above mean SCP refers to the difference between exposure temperature and the mean supercooling points of beetles after the treatment at 21°C (Table I)
Month Dec.
Jan.
Mar.
Exposure temperature (“C) -17.5 -16 -14 - 17.5 -16 -I4 -I6 -14 -16 -14 -16 -14 -16 -14
“C above mean SCP
Group
n =20
2.6 4.1 6.1 0.9 2.4 4.4 4.3 6.3 2.6 4.6 4.3 6.3 2.0 4.0
I I I II II II I I II II I I II II
75 85 100 0 0 50 75 95 0 55 75 100 0 45
I
Survival (%) 2 3 n = 20 n = 20 55 60 85 0 0 30 65 100 0 25 55 90 0 25
40 60 70 0 0 5 50 75 0 15 50 75 0 10
7 n = 20 30 55 70 0 0 0 50 75 0 0 55 70 0 0
350
UNN GEHRKEN Table 4. Mean (k SE) supercooling, melting and freezing points and thermal hysteresis in Ipsacuminatus kept at 0°C for 2 days in either total darkness (DD) (Group III) or continuous light (LL) (Group IV) SCP
THP
( Cl Month Dec. Jan. Mar.
0 C-Zd/DD O‘C-Zd/LL O”C-Zd/DD O”C-Zd/LL 0 ‘C-2d/DD O’C-2d/LL
P-3
n = 12
Group
Treatment
III IV 111 IV III IV
- 29.6 - 27.4 -31.5 -28.3 -28.9 -24.9
+ 0.2 f 0.4 kO.2 t 0.5 k 0.4 + 0.5
Dec.
Jan.
Mar.
-25 -22 -25 -22 -25 -22 -25 -22 -25 -22 -25 -22
-7.4 i 0.2 -5.3*0.1 < -9.5 -6.5 + 0.2 - 7.0 + 0.3 -4.5 kO.1
2.1 * 0.2 >3.1 2.5 f 0.2
melting and freezing points, thermal hysteresis. water content and ethylene glycol concentration of Ips for 7 days. Both animals with (Group III) and without antifreeze proteins (Group IV) are included
Table 5. Mean ( ? SE) supercooling, acuminatus kept at - 22 or -25°C
Month
-5.3 f 0.3 -5.1 f0.3 -6.4 k 0.3 -6.3 + 0.3 -4.5 * 0.3 -4.4 + 0.3
substantially lower (l-test, P < 0.001) than for beetles depleted of ethlylene glycol (Table 2). Table 6 shows the survival rate at -22, -25 and -26°C of 1. acuminates in the presence of ethylene glycol. Since no movements were observed in dead specimens, it was assumed that they died from freezing. Thus, pre-freeze injured beetles were never observed in Groups III and IV. Irrespective of the ethylene glycol concentration (Table 5), survival was always lower in beetles depleted of their antifreeze proteins (Group IV). Thus, while only 25% of the beetles showing thermal hysteresis (Group III) survived for 1 week at -26°C in December, 12 out of 20 beetles in this group survived after 1 day. In contrast, none survived after 1 day at -26°C when depleted of their antifreeze proteins. Furthermore, the seasonal difference in survival at - 25 and - 22°C
markedly during the season and between the two groups. In contrast, the mean melting point differed only during the season but not between the groups. In December, January and March, the substantially higher (r-test, P -c0.01)supercooling points of beetles in Group IV corresponded with the depletion of their antifreeze proteins. No substantial changes in mean supercooling, melting and freezing points of beetles from Groups III and IV were observed after 7 days of exposure to -22 and -25°C (Table 5). Thus, the cold hardiness of beetles had not been enhanced during exposure to these temperatures. The melting points of beetles from each group varied directly with seasonal changes in ethylene glycol concentration (Table 5), in agreement with earlier findings (Gehrken, 1984). The mean water content of about 50% (Table 5) was
Exposure temperature (“C)
n=5
Group
-29.4 + 0.5 -29.1 +0.2 - 21.6 ? 0.8 - 27.1 + 0.6 -31.2+0.3 - 30.9 I 0.2 -28.6 k 0.5 -28.5 f 0.5 - 28.8 * 0.3 -28.2 k 0.3 -24.9 i 0.5 -25.5 i 0.4
III III IV
IV III III IV IV 111 III IV IV
-5.1 -4.9 -4.9 - 5.0 -6.3 -6.2 -6.1 -5.9 -4.3 -4.5 -4.5 -4.2
Ethylene glyml (mmolikg) n=3
THP
MP (“0 II=5
SCP (“C) n = 12
(::, II=5
f0.3 f 0.2 * 0.3 i_ 0.3 + 0.3 * 0.3 Ifr 0.2 f 0.3 + 0.2 f 0.3 * 0.3 + 0.3
(“C) n=5
- 7.0 + 0.2 -7.1 kO.2 - 5.0 * 0.2 -5.2 ? 0.3 < -9.5 < -9.5 -6.3 k 0.2 -6.1 f 0.2 -6.8 + 0.3 -6.7 f 0.2 -4.3 * 0.4 -4.5 * 0.3
1.9 f0.1 2.2 f 0.2
> 3.2 >3.3
2.5 f 0.2 2.2 f 0.2
1752+70 1899It65 1821 f 99 1731 + 72 2385 f 88 2113 k98 2229 + 107 2205 f 102 1554 * 120 1609+90 1579+ 116 1487 k 139
Table 6. Survival rates after 1,2, 3 and 7 days at -22, -25 and -26’C for Ipsocuminafus displaying ethylene glycol, with (Group III) or without antifreeze proteins (Group IV). Degree above mean SCP refers to the difference between exposure temperature and the mean supercooling point of beetles followinn 2 davs at 0°C (Table 5)
Month
Dec.
Jan
Mar.
Exposure temperature (“C)
:C above mean SCP
-26 -25 -22 -26 -25 -22 -25 -22 -25 -22 -25 -22 -25 -22
3.6 4.6 8.6 1.4 2.4 5.4 6.5 9.5 3.3 6.3 3.9 6.9 0.0 3.0
I Group III 111 111 IV IV IV III 111 IV IV III III IV IV
n = 20 60 15 100 0 25 50 95 100 45 70 65 100 0 35
Survival (%) 2 3 n = 20 n = 20 45 50 100 0 5 40 100 100 20 55 55 100 0 5
30 40 100 0 0 25 90 100 0 40 50 95 0 0
I n = 20 25 40 100 0 0 0 85 100 0 0 35 80 0 0
Water content (%) n=5 49.7 f 0.3 50.1 * 0.4 50.3 * 0.3 51.5 2 0.3 51.450.3 50.2 5 0.3 50.6 f 0.3 50.9 * 0.3 51.3 f 0.3 50.3 f 0.3 51.7kO.4 51.1 f0.3
Supercooling and thermal hysteresis
351
at 2-3°C above their mean supercooling points. The survival in these groups agreed with corresponding data from December given in Tables 3 and 6. In the absence of ethylene glycol and antifreeze proteins (Group II), however, the majority of beetles died due to pre-freeze injuries. Only 3 out of 8 individuals froze at - 16°C and the remainder were fatally injured. DISCUSSION
In overwintering adult bark beetles I. acuminatus, freeze avoidance is potentiated by the accumulation of high concentrations of ethylene glycol. At temperatures above the mean supercooling point of the beetles, percentage survival decreased with exposure time and decreaging temperature. However, the antifreeze proteins had a marked effect on survival rates, both in the presence and in the absence of ethylene _i _; 4 4 -b 4 glycol (Tables 4, 5 and 6 and 1, 2 and 3, respectively). Mean meltlng point,“C At temperatures ranging up to 6°C above their mean Fig. 2. Mean supercooling points of Ips acuminatus as a supercooling point, no beetles depleted of antifreeze function of the corresponding mean melting points. The proteins survived 1 week, whereas 70% survival solid line represents d&a from beetles diplaying thermal or more was found in those displaying thermal hysteresis (Y = - 17.5 + 2.3X, r2 = 0.99 and P < O.OOl), hysteresis. Evidence that this change in survival was while the dashed line represents similar measurements for not related to changes in the concentration of low those depleted of these antifreeze proteins (Y = - 15.7+2.2X, molecular weight substances was provided by the r* = 0.99 and P c 0.001). melting points. In I. acuminatus with and without antifreeze proreflected changes in the supercooling points (Table 5). teins, low molecular weight substances lower superIn January, the supercooling points were well below cooling points 2.2-2.3”C for each degree of melting those obtained from beetles in December and March. point depression (Fig. 2), in agreement with earlier Accordingly, survival was higher after 1 week at findings (Gehrken, 1984). If supercooling capacity -25°C in January than in December and March in along determined survival, one might expect a groups displaying thermal hysteresis. No beetles correlation between percentage survival after 1 week depleted of these antifreeze proteins survived for 1 and “C above mean supercooling points, even for week at - 25 or -22”C, and differences in survival I. acuminatus depleted of thermal hysteresis proteins, related to changes in the supercooling points were however, the reverse was true. Thus, the overall only found after 1 day of exposure. change in survival rates arises from the breakdown of thermal hysteresis proteins. However, injurious The data from Group I-IV revealed that irreeffects of cold other than freezing also influenced the spective of the presence of ethylene glycol, the supercooling points were always substantially lower in mortality. Freezing seemed to be the cause of death in animals which retained ethylene glycol and in those beetles displaying thermal hysteresis. Thus, the mean supercooling points were linearly related to the which retained antifreeze proteins after a complete loss of the solute antifreeze. In contrast, the majority mean melting points in the presence and absence of antifreeze proteins (Fig. 2). Also survival of of beetles died from pre-freeze injury in the absence beetles was markedly improved in the presence of of both these antifreezes. That cold can lead to death in the absence of freezing has been observed in several antifreeze proteins. Using the data on survival after species which experience subzero winter temperatures 1 week (Tables 3 and 6), a correlation between (Turnock et al., 1983; Lee and Denlinger, 1985; survival and “C above mean supercooling point was Knight et a1.,1986; Bale et al., 1987). found for animals displaying thermal hysteresis The causes of pre-freeze injury are still a matter of (Y = 0.83 + 0,08X, r* = 0.90, P < O.OOl), but not for those depleted of their antifreeze proteins (r* = 0.1). speculation. In I. acuminatus, pre-freeze injury was Experiments using I. acuminates connected to only found in groups depleted of ethylene glycol, and might arise from the high water content and the lack thermocouples for 24 h in December showed that of low molecular weight substances. In the presence the mortality was caused by freezing and pre-freeze injury. In the presence of ethylene glycol but no of antifreeze proteins, however, beetles were capable of recovery, at least after 1 day of exposure to thermal hysteresis (Group IV), 5 out of 8 beetles froze temperatures near the supercooling point. This sugat -25”C, and the remainder showed no injurious gests that these antifreezes may also help to alleviate effects of cold. In the absence of ethylene glycol but presence of thermal hysteresis proteins (Group I), 2 pre-freeze damage. It has been suggested that thermal hysteresisout of 8 beetles froze at -17.5”C. The remaining 6 producing proteins are responsible for supercooling suffered pre-freeze injury, but recovered in the course of 3 days at 3°C. Accordingly, freezing was the sole point depression in several species (Duman et al., 1982). A correlation between increasing thermal hyscause of death, while 38% of animals from Group IV teresis activity and decreasing supercooling points and 75% of those from Group I survived after 1 day
352
UNN
was demonstrated in larvae of Merachanta contracta (Duman, 1977). In contrast, no such correlation was found in I. acuminatus depleted of ethylene glycol. However, depletion of antifreeze proteins resulted in an overall reduced capacity to supercool (Fig. 2). Since these proteins can inhibit growth of ice crystals, it is logical to assume that they also prevent embryo crystals from reaching the critical size necessary to induce freezing in a supercooled system. The lower supercooling capacity, greater variance of supercooling point measurements and lower survival of I. acuminatus depleted of thermal hysteresis, suggests that these proteins reduce the probability that the beetles will freeze. Thus, the present results support the theory of Zachariasscn and Husby (1982) and suggest that antifreeze proteins are necessary to stabilize the supercooled state of insects down to the observed supercooling points predicted by the levels of low molecular weight substances. Seasonal changes in day length may be of particular importance in controlling the level of thermal hysteresis. In larvae of the darkling beetle Merachantha contracta held at 22°C a long photoperiod leads to a loss of thermal hysteresis-producing proteins (Duman, 1977). In larvae of the Phyrochroid beetle Dendroides canadensis, long photoperiod cause depletion of antifreeze proteins at both 10 and 23°C (Duman, 1980). In the spider, Philodromus, no photoperiodic effect was observed at low (4°C) or high (22°C) temperatures (Duman, 1979). The fact that exposure to constant light results in a complete loss of thermal hysteresis activity, even in I. acuminatus kept at 0°C suggests that the primary environmental parameter responsible for triggering depletion is photoperiodic. High temperature is of less importance for depletion of antifreeze proteins in I. acuminatus, and these antifreezes might therefore provide a failsafe system to ensure supercooling to about - 20°C in the absence of ethylene glycol. Since the overwintering site is generally above the snow (Bakke, 1968), specimens are subjected to low and high ambient air temperatures. In fact, Bakke (1968) reported that the temperature in the microhabitat might increase by 20°C above the air temperature due to sun radiation. The beetles enter a facultative diapause during autumn which is terminated by mid-winter (Gehrken, 1985). While in diapause, the beetles remain underneath the bark of Scot’s pine (Gehrken, unpublished data). High ambient temperatures from mid-winter onwards might therefore be fatal to I. acuminatus if
GEHRKEN
the beetles leave the logs, and risk exposure to full daylight, leading to a complete breakdown of the antifreeze proteins. Acknowledgement-I
am most grateful to Dr L. Somme for valuable advice during the preparation of the manuscript. REFERENCES
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