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Frost resistance in adult insects
_Y,InsectPhysiol.,1972, Vol. 18, pp. 267 to 282. PergamonPress. Printedin
FROST
RESISTANCE
YOSHIKUNI
IN ADULT
OHYAMA
and I%60
G&at
Britain
INSECTS ASAHINA
Zoological Institute and The Institute of Low Temperature University, Sapporo, Japan
Science, Hokkaido
(Received 20 &ly 1971) Abstract-Some adult Coleoptera and Hymenoptera collected in winter in Hokkaido, Japan, proved to be frost-resistant, The limit of low temperatures at which they could tolerate prolonged freezing without any post-thawing injury was only - 10°C regardless of their glycerol content. Field observations suggested that these frost-resistant adult insects were rarely exposed to dangerous temperatures below -10°C in their natural habitats. A remarkable seasonal change in glycerol content, which rose to about 10 per cent of fresh body weight in midwinter, was observed in a wasp, Chasmias sp. The supercooling point and the degree of frost resistance in this wasp were, however, found to be nearly constant throughout the overwintering period. In the following spring when glycerol entirely disappeared in the wasps, a remarkable decrease in the capacity of supercooling and of frost resistance was observed. A characteristic freezing curve with two supercooling points, the first at - 8°C and the second at a lower temperature, was obtained from overwintering carpenter ants. The process of ice formation in an individual ant was therefore examined by use of both differential thermal analysis and freeze-sectioning. The results obtained strongly suggested that in a slowly cooled ant ice tist formed spontaneously in the foregut but the other tissues, even other parts of the alimentary tract, remained unfrozen until the insect was cooled down to the second supercooling point at which temperature the entire insect froze solid. Ice formation within the foregut was observed to be innocuous, whereas freezing throughout the entire tissue was fatal to the carpenter ant. INTRODUCTION
IT HAS long been known that the majority of hibernating insects in temperate and cold regions pass the winter in a supercooled state, whereas some can survive in a frozen state. The former are, as a rule, susceptible and the latter are resistant to freezing. Almost all of the frost-resistant insects so far known have been in the larval or pupal stage (ASAHINA,1969). Adult insects, even those which are very active in winter and can move under supercooled conditions, are freezing: susceptible (ASAHINA,1957; SGMMEand OSTBYE, 1969). Evidence indicating frost resistance of overwintering adult insects is beginning to accumulate. Some ichneumonid female wasps were found to survive freezing at relatively high subzero temperatures for many days (ASAHINA and TANNO, 1968 ; OHYAMA and ASAHINA,1970). A tiny carabid beetle in Alaska was shown to be frost-resistant at temperatures as low as - 87°C (MILLER, 1969). Even large carabid and silphid beetles
commonly
found
in temperate
regions
were
found
to be frost-resistant
(ASAHINAand OHYAMA, 1969). Workers of the carpenter ant, Camponotus obscuripes, which was believed to be freezing-susceptible was found to be resistant 267
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YOSHIKUNIOHYAMA ANDEIZO ASAHINA
to freezing, if the ice formation was limited to the alimentary tract (OHYAMAand ASAHINA, 1969). The present paper deals with the nature of frost resistance in some of the adult insects in relation to environmental temperatures. A detailed observation of the freezing process in the entire body of a carpenter ant is also described.
MATERIALS
AND METHODS
Insects used in the present study were collected in two winter seasons of both 1969-70 and 1970-71. Carabid beetles, Damaster blaptoides rugipennis Mots. and Pterostichus orientalis Mots., and carrion beetles, Phosphuga atrata L., were collected from decayed wood in Shibecha, eastern Hokkaido, which is one of the coldest places in Japan. The first named is one of the largest ground beetles in Japan having a body weight of more than 1 g and a body length of about 40 mm. Carpenter ants, Camponotus obscuripes Mayr, were collected from partly decayed stumps in Shibecha. Ichneumonid wasps, Hoplismenus pica japonica Uchida, Pterocormus molitorius L., and Chasmias sp., were collected from decayed wood in both Shibecha and Sapporo. The temperature conditions in the habitat were determined at the time of collection of these insects with a slender thermistor with a tip of 1 mm in dia. The collected insects were transferred to a laboratory in Sapporo where they were kept at - 5°C in a cold box until they were used for the experiment. To determine the frost resistance of the insects, dried Petri dishes containing usually more than 2 to 5 individuals were separately cooled slowly in freezers at - 10, - 15, and - 20°C. They were kept at a constant low temperature for 24 hr from the time of spontaneous freezing of the insect bodies. After thawing in air at room temperatures, recoveries of their normal activity such as walking, feeding, and avoidance response, were observed for several days. Supercooling points were determined from the freezing curves of individual insects using more than two for large beetles and up to ten for wasps and ants. Body temperatures were measured with a O-2 mm copper-constantan thermocouple connected to an electronic recorder. Insects were wrapped in dry cottonwool into which the tip of a thermocouple was inserted in such a way that the tip was in direct contact with the insect body. Carpenter ants were fixed to the tip of the thermocouple with a small dab of Vaseline. These two methods gave the same results in determining the supercooling point of the cooled insect. Insects with a thermocouple were cooled in a polystyrene tube in a freezer at about - 30°C. The rate of cooling was regulated to 1 to l*S”C!/min on the tangents along the freezing curves at about 0°C by inserting the insect in double or triple tubes in a freezer. To examine the process of freezing occurring in various parts of an ant body, differential thermal analysis (D.T.A.) was used. A tip of the thermocouple was in direct contact with the thorax and another tip with the abdomen (Fig. 1). In this manner the thermal difference between the thorax and abdomen were estimated simultaneously with the recording of the freezing curve of the insect (Fig. 4).
FROST RESISTANCE IN ADULT
INSECTS
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To determine the loci where ice forms within an ant’s body, frozen sections were used. The insects were separately frozen at various cooling rates as will be described later. In a cooled room at -20°C frozen insects were fixed to wooden blocks with a small amount of aniline and the freeze-sectioning was made in
m
..-. a AEJ
c
.:($ :: .. . . . .:. :..c;;:.:.....
:”
..
..: .
FIG. 1. Tips of thermocouples in contact with the ant’s body. Tip A is for the recording of body temperature. Tips B and C are for D.T.A. between the thorax and the abdomen.
parallel with the median plane of the insect by use of a microtome (ASAHINA and TANNO, 1967). The frozen sections obtained were observed under a microscope immediately after sectioning in the same cold room at -20°C. The cut surface of the remaining body, from which the frozen sections were separated, was also observed in the cold room. Since the knife marks on the cut surface, which usually occurred immediately after cutting, obstructed a clear observation of the cut surface, ice crystals on the surface were slightly sublimated by exposing to air for 1 day at -20°C before observation. To obtain a cut surface similar in appearance to that of an unfrozen control insect, insects frozen very rapidly by immersion in liquid nitrogen were used. On the cut surface of such rapidly frozen insects no large ice crystals were observed (Fig. 6), and the appearance of the internal tissues was almost the same as unfrozen ones except for the midgut, in which relatively large ice crystals were sometimes observed. Insects, usually 1 large beetle or 3 to 5 wasps and ants, were ground in 80% ethanol and the extract and residue were separated by centrifugation. The glycerol content in the extract was estimated by use of paper chromatographic analysis described by TAKEHARA (1966). The spot of glycerol was detected by Trevelyan’s alkaline silver nitrate method (TREVELYAN et al.,1950). The appropriate area was cut from the unsprayed paper chromatogram strip, and the glycerol on the strip was eluted with 20 ml of deionized water. The glycerol
YOSHIKUNI OHYAMA ANDEIZO ASAHINA
270
content was determined calorimetrically by periodic acid oxidation and subsequent chromotropic acid-formaldehyde reaction (BURTON, 1957). Glycogen was detected from the precipitated residue remaining after extraction of the soluble substances with 80% ethanol. The residue was suspended in a 5% trichloroacetic acid solution and the glycogen was extracted by heating at 100°C for 15 min. The amount of glycogen was calorimetrically determined as sugar with anthrone reagent. RESULTS
Frost resistance and environmental temperature In a variety of adult insects collected, three wasps, and a species of ant were observed to these insects, glycerol contents, supercooling resistance are indicated in Table 1. In spite TABLE ~-GLYCEROL IN
ALL
species of beetles, three species of be frost-resistant. The names of points, and the degree of frost of the relatively large content of
CONTENT, SUPERCOOLING POINTS, AND FROST RESISTANCE THE
FROST-RESISTANT
Glycerol content * (mg/g fresh wt.)
SPECIES
COLLECTED
Supercooling t points
Freezing temperature tolerated for 24 hr (“C)
Coleoptera D. 6. rugipennis P. orientalis P. strata
0.9 2.5 31.6
-6.3 -7.5 -8.3
-10 -10 -10
Hymenoptera C. obscuripes (worker) H. pica P. molitovius Chasmias sp.
34.6 +I 38.8 96-l
-85 -9.1 -6.0 -6.5
-10 -10 -10 -10
* Maximum value in samples measured. t Mean value for 2 to 10 individuals. 1 Only qualitatively identified.
glycerol, all insects showed supercooling points invariably higher than -10°C. The limit of low temperatures at which they can survive prolonged freezing were found to be about - 10°C regardless of the amount of glycerol possession. All the insects examined had only one supercooling point (Fig. 2) except the carpenter ants which had two supercooling points (Fig. 4). This indicates that in the former insects when ice forms in a readily freezable part of the insect body, it immediately seeds the other tissue and freezing throughout the entire body results, whereas in the latter insects this is not so, as will be described in detail later.
FROST
RESISTANCE
IN
ADULT
271
INSECTS
Almost all of these insects were collected from big trunks (more than 40 cm dia.) of partly decayed wood appearing above snow level. They were most frequently found in holes or chinks in the xylem. The temperatures in these habitats, which were determined at the time of collection, ranged from - 5.7 to - 8.7”C. IO
YZ
0
-IO
-2c I
20 FIG. 2. A
t
40 min
freezing curve of a carabid beetle, P. orientalis.
The temperature fluctuation in the xylem of these wood samples was small except for the surface layer, where the temperature was nearly the same as that of the outside air. Even on a morning when atmospheric temperature decreased to - 25”C, the temperature in the central xylem was observed to be - 9°C. An overnight determination indicated that the central xylem temperature in a wood sample remained between - 7 and - 9°C from the evening to the following morning when a minimum atmospheric temperature of - 15°C was recorded. SAKAI and WADA (1963) observed the temperature fluctuations of various parts in living trees during winter. They found that the temperature in the center of big trunks changed very little. In case of fallen wood with a snow cover, the wood was observed to be more insulated from the cold than those exposed. These results suggest that overwintering insects collected may actually freeze in their habitat, but they are rarely exposed to dangerous temperatures at which they would be fatally injured. In fact, some of the large beetles, which regained their normal activity after thawing, were found under completely frozen conditions in their habitats. Glycerol and frost resirtame Seasonal variations in glycerol content, supercooling points, and degree of frost resistance in relation to environmental temperature were studied in an ichneumonid wasp, Chamzias sp. collected in Sapporo during the winter of 1969-70
272
YOSHIKUNI
OHYAMA AND firzo ASAHINA
(Fig. 3). The wasps had a glycerol content of about 1% of fresh body weight at the beginning of November when the daily mean atmospheric temperature still remained above 5°C. At the end of November the mean air temperature decreased to 0°C and was maintained below 0°C till the end of March. During the overwintering period wasps were collected several times to determine the respective
1969
1970
Seasonal variations in glycerol (0) and glycogen (0) content, supercooling point ( o), and atmospheric temperature. From 6 April insects were kept at 2°C FIG. 3.
for 2 weeks after which glycerol (9) and glycogen ( 0) content were estimated. Insects were then exposed to room air maintained at 20°C and kept there for 1 week after which glycerol ( 0) and glycogen (0) content, and the supercooling point (A) were measured.
glycerol content from a big fallen log with a thick snow cover in which a habitat temperature of around 0°C was usually recorded. The glycerol content in this wasp showed a remarkable increase during December, a maximum value of 10% was measured at the beginning of February and thereafter decreased to about 4% at the beginning of April, when the mean air temperature again increased to above 0°C. The effect of environmental t_emperature on the glycerol content was examined in the period when both glycerol increases and decreases in the insects. On 20 December wasps were exposed to - 10°C at which temperature they invariably froze and became hard. After being kept at - 10°C for 40 days they were thawed. All of them regained their normal activity. The glycerol content in these immediately after thawing was 10%. This indicates that in frozen insects at - lO”C, glycerol may increase remarkably to the same level as in the control insect overwintering in its habitat at about 0°C. At the beginning of April when the outdoor temperature was increasing daily, insects were kept at 2°C. This resulted in the maintenance of a constant glycerol level for at least 2 weeks (Fig. 3). Apparently no glycerol decrease was seen in the insects at 2°C in this period. These kept at 2°C were then exposed to room temperature (over ZO’C). A complete loss
FROST RESISTANCE
IN ADULT
INSECTS
273
of glycerol content was observed within 1 week (Fig. 3). The amount of glycogen decreased in reverse relation to the glycerol increase only at the beginning of winter. During the 4 months from the end of December to the following spring, the glycogen content remained at a low level below 0.5%. Even in insects reared at room temperature at the end of April, when a remarkably rapid decrease of glycerol took place, no glycogen was observed to increase (Fig. 3). The supercooling point of the insect showed no remarkable change, giving an average value of about -6”C, throughout the 5 months from the beginning of November, regardless of the glycerol content in the insect. At the end of April when the insects began to move actively their capacity to supercool decreased remarkably. Insects overwintering outdoors could survive freezing at - 10°C for 1 day or more without any post-thawing injury at the beginning of November, when they have a glycerol content of only 1%. The degree of frost resistance in this wasp was observed to be nearly the same throughout the full overwintering period. In midwinter freezing at - 10°C was innocuous even for many days, but freezing at - 15°C caused some injury and freezing at - 20°C resulted in a serious injury which became fatal within 1 month. When the insects were reared at room temperatures at the end of April, they completely lost their ability to survive freezing. Process of ice formation within an insect body The carpenter ant, Camponotus obscuripes, was assumed to be a freezingsusceptible form, since it could not survive freezing at - lO”C, when it was previously seeded by ice and became hard and brittle (TANNO, 1962). However, a thermal determination of cooling ants suggested that ice actually formed within the insect body before the insects became hard and brittle. Ice propagation within the body was, therefore, studied by using overwintering carpenter ants. Since most of the carpenter ants in winter nests were observed to be workers, workers were exclusively used. A definite form of freezing curve with two supercooling points was obtained from the carpenter ants (Fig. 4). The first supercooling points, which were observed to be remarkably stable, were at - 8.5 & 0*3”C, whereas the second supercooling points ranged from - 10.3 to - 25*6”C and were at - 20 + 4*7”C on average. The heat release at the former temperature was much less than at the latter. The insects being cooled were still soft and flexible even after they were cooled down to the first supercooling point. When the second supercooling point was reached on the freezing curve, the cooled insects became hard and brittle. If the insects were rewarmed during the cooling process before the second supercooling point was attained, they invariably regained their normal activity. This was also the case in the insects kept for 1 day at a constant temperature of - 10°C in a cold box, in which the first supercooling point appeared on the freezing curve of the insect within about 1 hr after transference from room temperatures. None survived freezing even for a short time at temperatures below the second supercooling point.
YOSHIKLJNI OHYAMAAND &zo
274
ASAHINA
On the D.T.A. curve shown in Fig, 4, the part below the zero level indicates a larger heat release in the abdomen than in the thorax, and the part above zero level indicates the reverse. A simultaneous recording of both freezing and D.T.A. curves appeared to suggest that the first freezing, i.e. the freezing initiated at the first
I 0
40
20
60
min
FIG. 4. Freezing curve (A) and D.T.A. curve (B) of a worker of overwintering carpenter ant. The first (1) and the second (2) supercooling points appeared on the freezing curve. In D.T.A. curve (B), the part below the zero level indicates a larger heat release in the abdomen than in the thorax, and the part above zero level indicates the reverse.
supercooling point, mainly took place in the abdomen and the second freezing, i.e. the freezing initiated at the second supercooling point, took place in both the abdomen and the thorax (Fig. 4). Figures of D.T.A. curves for the first freezing were distinctly uniform in many cases of the freezing ants, whereas those for the second freezing were not uniform. Thermal difference between the anterior and posterior ends of the abdomen at the time of the first freezing is indicated in a D.T.A. curve presented in Fig. 5. The curve suggested that the first freezing might occur mainly at the anterior part of the abdomen. From these observations it was assumed that in a cooling body ice first formed in the anterior part of the abdomen and remained there without any further growth to other parts for a period of time until the insect was cooled down to the second supercooling point when ice formed throughout the entire body. This assumption led the authors to make direct observations of freeze-sectioned carpenter ants, using insects frozen by the following three different methods: (1) insects were very rapidly frozen by direct immersion into liquid nitrogen from room temperatures; (2) insects were cooled in air in a cold room at - 30°C for 2 hr during which period they became invariably hard and brittle ; (3) insects were
275
FIG. 6. The cut surfaces of frozen ants. (a) Insect cooled very rapidly by direct immersion into liquid nitrogen. (b) Insect frozen hard at -30°C. r (c) Insect cooled at - 10°C for 15 hr and then rapidly immersed in liquid nitrogen.
276
FIG. 7. Frozen cross-sections of the alimentary tract in ants frozen in three different manners, showing a part of the gut wall and ice crystals in the lumen. Crop (a), stomach (b), and rectum (c) of the insect cooled very rapidly by direct immersion into liquid nitrogen. Crop (d), stomach (e), and rectum (f) of the insect frozen hard at -30°C. Crop (g), stomach (h), and rectum (i) of the insect cooled at - 10°C for 15 hr and then rapidly immersed in liquid nitrogen.
FROST RESISTANCE IN ADULT INSECTS
277
cooled in air in a cold room at - 10°C for 15 hr, during which period they remained soft and flexible. They were then rapidly immersed in liquid nitrogen.
“C
6 0
-
A
A
-10 t 0
\ 30 min
FIG. 5. Freezing curve (A) and D.T.A. curve (B) for the first freezing in an ant. In D.T.A. curve (B) the part above zero level indicates a larger heat release in the anterior than in the posterior part of the abdomen.
Following these freezing treatments, median sections were made to observe the cut surfaces. As already mentioned the cut surface of insects frozen by the first treatment appeared intact as in the unfrozen control (Fig. 6A). On the cut surface of the insects frozen by the second treatment, large ice crystals were observed in various parts of alimentary tract. In addition, the muscle in the thorax showed a dehydrated and contracted appearance (Fig. 6B) and between muscle tissues ice crystals were actually observed. This suggests that in the frozen insect muscle tissues are dehydrated as a result of extracellular freezing. In the insects frozen by the third treatment, on the other hand, large ice crystals were observed only in the oesophagus, crop, and at times in the stomach (Fig. 6C). Other parts of the frozen insect appeared to be the same as in the rapidly frozen one shown in Fig. 6(A). By observing the frozen sections a detailed comparison of the size of ice crystals in the lumen of the alimentary tract of insects frozen in the above-mentioned three ways was made (Fig. 7). The sections from the insects frozen by the first treatment showed that only fine ice crystals occurred in various parts of the alimentary tract (Fig. 7A-C). This was also so in the insects frozen by the third treatment except in the oesophagus and the crop, where onIy large ice crystals were observed (Fig. 7G-I). In the insects frozen by the second treatment large ice crystals were invariably observed throughout the lumen of the crop, the stomach, and the rectum (Fig. 7D-F). These results seem to indicate that by cooling the
278
YOSHIKUNIOHYAMA AND&zo ASAHINA
insects at - 10°C for 15 hr, ice only forms within the foregut. On the cut surface of the insect frozen by the third treatment, as to why the stomach at times contained large ice grains was uncertain. Even in the insects frozen very rapidly by the first treatment, large ice crystals were at times observed in the stomach on the cut surface. However, immediately after sectioning no large ice crystals were observed in the stomach in the sections from the insect frozen by the third treatment (Fig. 7H). Since the freeze-cut surface of the insect was exposed to air at -20°C for 1 day before observation, the fine ice crystals initially formed in the stomach may have grown to large ones, if there were some factors to accelerate the ready process of migratory recrystallization, for instance a high solute concentration in the stomach. All observations described in the present paper seem to suggest that in the freezing process of the carpenter ant, ice spontaneously forms first in the crop at the first supercooling point, but that the propagation of ice crystals is limited only to the foregut until the insect is cooled down to the second supercooling point at which time ice forms throughout the entire tissue of the insect. DISCUSSION Spontaneous freezing in insects has been assumed to originate in either the haemolymph or the gut content (SALT, 1966; ASAHINA, 1969). As discussed extensively by Salt, it seems almost certain that freezing in insects commences from ice crystal nucleators or motes (DORSEY, 1948) introduced from the outside into the alimentary tract at least in feeding larvae (SALT, 1953, 1958a, 1966). When the larvae ceased to feed with the approach of the time for spinning cocoons a remarkable increase in supercooling ability was observed (SALT, 1936). This may suggest a decreased activity of the gut content as nucleators and an increased probability of spontaneous freezing of the haemolymph in the body cavity. During overwintering periods, many larvae and pupae have supercooling points around -20°C even without any glycerol or alternative agents. In these insects a remarkable correlation between glycerol content and supercooling ability has frequently been reported (SALT, 1958b; SBMME, 1964, 1965). S ome of them, possessing more than 10% glycerol, were very readily supercooled to - 30°C or below. These observations seem to suggest that in these insects the onset of freezing takes place in the haemolymph. Many insects, which pass winter in the adult stage are known to be freezingsusceptible (ASAHINA, 1969). They have, as a rule, high supercooling ability and can withstand cold by means of supercooling. Frost-resistant adult insects, on the other hand, were so far observed to have a relatively poor supercooling ability regardless of the amount of glycerol possession. The supercooling points of the adult insects examined in the present work invariably fell between a narrow temperature range of - 6 to - 1O’C. According to a detailed study by BAUST and MILLER (1970), an Alaskan carabid beetle, Pterostichus brevicornti, was observed to have a high supercooling point of about - 11 “C in spite of having a large amount of glycerol estimated to be more than 20% in winter. In this insect a clear seasonal
FROST
RESISTANCE
IN
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279
change of glycerol content was observed in good correlation with the freezing point of the haemolymph, but the relation between the supercooling point and glycerol content appeared to be somewhat obscure. In the case of an ichneumonid wasp, Chasmias sp., practically no change in supercooling capacity was observed throughout the overwintering period regardless of the glycerol content (Fig. 3). When the wasps began to take food or exogenous water by mouth with the approach of the warm season, a remarkable loss of supercooling ability was seen. One of the possible explanations for these facts may be an enhanced occurrence of spontaneous freezing in the content of the alimentary tract rather than in the haemolymph in these adults. This was clarified at least in the carpenter ants as will be discussed later. Supercooling points higher than - 10°C were also reported in some larvae and pharate pupae with a very high ability to tolerate freezing (S~~MME, 1964; TANNO, 1970). An easy release from a supercooled state at a relatively high subfreezing temperature may reasonably be profitable for insects in surviving freezing by decreasing the opportunity of the occurrence of dangerous intracellular freezing. It was observed in a Chasmias wasp that the glycerol content increased from about 5 to lo?; within 40 days at a constant temperature of - lO”C, at which temperature they invariably froze solid. It may be worthy of note that even under frozen conditions at - 10°C glycerol accumulates in the insect to the same level as in the unfrozen control insect from the habitat where the temperature is usually maintained at about 0°C under a snow cover. CHINO (1957) f ound that in diapausing eggs of the silkworm, Bombyx mod, glycogen was converted to polyols and was later resynthesized from the polyols. A similar relation was also observed in an overwintering slug caterpillar by TAKEHARA (1966). In this insect glycerol increased remarkably in autumn and decreased in the following spring indicating a good inverse relation with the amount of glycogen. On the other hand, an’inverse relation between the amount of glycerol and glycogen was shown in a Chasmias wasp only at the beginning of winter. In the following spring, when glycerol decreased remarkably in the wasp, no increase in the glycogen content was observed (Fig. 3). D uring the period of glycerol decrease, the environmental temperature of the wasps remained around 0°C and therefore no remarkable consumption of carbohydrates was expected. These results seem to suggest an interconversion of glycerol to appropriate substance(s) other than glycogen. In spite of a large amount of glycerol, this wasp was found to be moderately frost-resistant. The degree of frost resistance was also almost constant throughout the 5 months of over-wintering period regardless of the glycerol content. In this period freezing at - 10°C was innocuous, but not at lower temperatures. In the Alaskan carabid beetle, too, the haemolymph glycerol was observed to decrease without a loss of frost resistance (BAUST and MILLER, 1970). In addition, it was revealed in a remarkably frost-resistant caterpillar, Monema flavescens, that after the glycerol content had reached about 2% of the fresh body weight, further glycerol increase was unnecessary for the insect to maintain its high resistance
280
YOSHIKUNI OHYAMA
ANDfi~zo ASAHINA
(ASAHINAand TAKEHARA,1964). An accumulation of a relatively small amount of glycerol may perhaps be enough to maintain the maximum degree of frost resistance in Umsmia-swasps. Overwintering carpenter ants from both Japan and Canada were reported to be freezing-susceptible, since they could not survive after solidification of their bodies by freezing with ice-seeding at temperatures below - 10°C (TANNO, 1962; %MME, 1964). In the present work, however, it was revealed that in the cooling process of a carpenter ant, freezing progressed in two steps, i.e. the first freezing occurred at - 8.5 t_ 0*3”C, the first supercooling point, and the second at - 20 +_4*7”C, the second supercooling point. The first freezing did not solidify the insect and was shown to be innocuous, whereas the second freezing made the insect hard and brittle, resulting in fatal injury. Since artificial ice-seeding from the body surface could solidify the insect even at - 10°C (TANNO, 1962), such a mode of initiation of freezing appears to result in the second freezing in an ant at a higher temperature than the second supercooling point. Observations of frozen sections and of freezecut surfaces of bodies strongly suggested that the first freezing corresponded to the ice formation limited only to the foregut and the second freezing to all the remaining parts of the tissues. D.T.A. curves obtained from frozen ants indicated that at the beginning of first freezing a remarkable ice formation took place at the anterior end of the abdomen. These results seems to suggest that when an ant is cooled gradually to temperatures below - lO”C, the ice first forms spontaneously in the crop and develops through the lumen of foregut, but the other tissues, even other parts of the alimentary tract, still remain unfrozen until the insect is cooled down to the second supercooling point. It is well known that the inside wall of the foregut of an ant, throughout the oesophagus, crop, and proventriculus, is completely covered with a well-developed cuticular layer. In addition the posterior narrow end of the proventriculus is assumed to function as a valve, since the walls of the proventriculus, especially at this narrow end, are provided with powerful transverse muscles (WHEELER, 1960). Such a ‘valve’ may prevent the penetration of ice through the alimentary tract from the foregut to the midgut, at least at the temperature of the first supercooling point. The cuticular layer in the foregut may also serve as a barrier to prevent ice-seeding through the gut wall to the body cavity. In the second freezing in ants, an easy interpretation of the freezing process appears difficult. Since the D.T.A. curves obtained from freezing ants were not always uniform for the second freezing, various processes of ice propagation may be assumed. As mentioned before, the second supercooling point, at which temperatures the second freezing commenced, were observed to occur over a range from - 10.3 to -25*6X An inoculation through the lumen to the midgut from the ice mass formed by the first freezing in the foregut may be responsible for the occurrence of the second freezing at a high temperature near the first supercooling point, for example at - 10.3”C. On the other hand, spontaneous freezing of the haemolymph within the body cavity may cause the second freezing at a low temperature, say -25.6”C. Many larval or pupal insects having a glycerol content comparable
FROSTRESISTANCE IN ADULTINSECTS
281
with that in the overwintering carpenter ant, were observed to have supercooling points ranging from - 20 to - 30°C. REFERENCES A~AHINAE. (1957) Motility of some animals and an excised tissue in supercooled state. Low Temp. Sci. (B) 15,45-50. ASAHINAE. (1969) Frost resistance in insects. A&. Insect. Physiol. 6, l-49. ASAHINAE. and OHYAMAY. (1969) Cold resistance in insects wintering in decayed wood. Low Temp. Sci. (B) 27, 143-152. ASAHINAE. and TAI~EHARA I. (1964) Supplementary notes on the frost resistance in a slug caterpillar, Monemaflavescens. Low Temp. Sci. (B) 22, 79-90. ASAHINAE. and TANNO K. (1967) Observations of ice crystals formed in frozen larvae of the European corn-borer by the use of freeze-sectioning. Low Temp. Sci. (B) 25, 105-111. ASAHINA E. and TANNO K. (1968) A frost-resistant adult insect, Pterocormus molitorius (Hymenoptera, Ichneumonidae). Low Temp. Sci (B) 26, 85-89. BAUSTJ. G. and MILLER L. K. (1970) Variations in glycerol content and its influence on cold hardiness in the Alaskan carabid beetle, Pterostichus brevicovnis. J. Insect Physiol. 16, 979-990. BURTONR. M. (1957) The determination of glycerol and dihydroxyacetone. In Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLAN N. 0.) 3,246-249. Academic Press, New York. CHINO H. (1957) Conversion of glycogen to sorbitol and glycerol in the diapause egg of the bombyx silkworm. Nature, Lond. 180, 606-607. DORSEY N. E. (1948) The freezing of supercooled water. Trans. Am. phil. Sot. 38, 247-328. MILLER L. K. (1969) Freezing tolerance in an adult insect. Science, Wash. 166, 105-106. OHYAMAY. and ASAHINAE. (1969) Process of spontaneous ice formation within the body of the carpenter ant. Low Temp. Sci. (B) 27, 153-160. OHYAMA Y. and ASAHINAE. (1970) Frost resistance and glycerol in overwintering adult wasp Chasmias sp. Low Temp. Sci. (B) 28, 79-85. SAKAI A. and WADA J. (1963) Temperature variation in over-wintering trees. Low Temp. Sci. (B) 21, 25-40. SALT R. W. (1936) Studies on the freezing process in insects. Tech. Bull. Minn. agric. Exp. Stn 116, l-41. SALT R. W. (1953) The influence of food on the cold-hardiness of insects. Can. Ent. 85, 261-269. SALT R. W. (1958a) Application of nucleation theory to the freezing of supercooled insects. J. Insect Physiol. 2, 178-188. SALT R. W. (1958b) Role of glycerol in producing abnormally low supercooling and freezing points in an insect, Bracon cephi (Gehan). Nature, Lond. 181, 1281. SALT R. W. (1966) Factors influencing nucleation in supercooled insects. Can. J. 2002. 44,117-133. SBMMEL. (1964) Effects of glycerol on cold-hardiness in insects. Can.J. Zool. 42,87-101. SBMME L. (1965) Further observations on glycerol and cold-hardiness in insects. Can. J. Zool. 43, 765-770. S~MME L. and &.TBYE E. (1969) Cold-hardiness in some winter active insects. Nor& ent. Tidsskr. 16, 45-48. TAKEHARAI. (1966) Natural occurrence of glycerol in the slug caterpillar, Monemaflavescens. Contr. Inst. Low Temp. Sci. Hokkaido Univ. (B) 14, l-34. TANNO K. (1962) Frost-resistance in a carpenter ant Camponotus obscuripes obscuripes-I. The relation of glycerol to frost resistance. Low Temp. Sci (B) 20, 25-34.
282
YOSHIKUNIOHYAMAAND I?IZO ASAHINA
TANNO K. (1970) Frost injury and resistance in the poplar sawfly, Trichiocampus pop& Okamoto. Corm. Inst. low Temp. Sci. Hokkaido Univ. (B) 16, 1-41. TRJZ~ELYAN W. E., PROCTERD. P., and HARRISONJ. S. (1950) Determination of sugar on paper chromatograms. Nature, Land. 166, 444-445. WHEELERW. M. (1960) Ants, Their Structure, Development and Behavior. Columbia University Press, New York.
FROST
RESISTANCE
YOSHIKUNI
IN ADULT
OHYAMA
and I%60
G&at
Britain
INSECTS ASAHINA
Zoological Institute and The Institute of Low Temperature University, Sapporo, Japan
Science, Hokkaido
(Received 20 &ly 1971) Abstract-Some adult Coleoptera and Hymenoptera collected in winter in Hokkaido, Japan, proved to be frost-resistant, The limit of low temperatures at which they could tolerate prolonged freezing without any post-thawing injury was only - 10°C regardless of their glycerol content. Field observations suggested that these frost-resistant adult insects were rarely exposed to dangerous temperatures below -10°C in their natural habitats. A remarkable seasonal change in glycerol content, which rose to about 10 per cent of fresh body weight in midwinter, was observed in a wasp, Chasmias sp. The supercooling point and the degree of frost resistance in this wasp were, however, found to be nearly constant throughout the overwintering period. In the following spring when glycerol entirely disappeared in the wasps, a remarkable decrease in the capacity of supercooling and of frost resistance was observed. A characteristic freezing curve with two supercooling points, the first at - 8°C and the second at a lower temperature, was obtained from overwintering carpenter ants. The process of ice formation in an individual ant was therefore examined by use of both differential thermal analysis and freeze-sectioning. The results obtained strongly suggested that in a slowly cooled ant ice tist formed spontaneously in the foregut but the other tissues, even other parts of the alimentary tract, remained unfrozen until the insect was cooled down to the second supercooling point at which temperature the entire insect froze solid. Ice formation within the foregut was observed to be innocuous, whereas freezing throughout the entire tissue was fatal to the carpenter ant. INTRODUCTION
IT HAS long been known that the majority of hibernating insects in temperate and cold regions pass the winter in a supercooled state, whereas some can survive in a frozen state. The former are, as a rule, susceptible and the latter are resistant to freezing. Almost all of the frost-resistant insects so far known have been in the larval or pupal stage (ASAHINA,1969). Adult insects, even those which are very active in winter and can move under supercooled conditions, are freezing: susceptible (ASAHINA,1957; SGMMEand OSTBYE, 1969). Evidence indicating frost resistance of overwintering adult insects is beginning to accumulate. Some ichneumonid female wasps were found to survive freezing at relatively high subzero temperatures for many days (ASAHINA and TANNO, 1968 ; OHYAMA and ASAHINA,1970). A tiny carabid beetle in Alaska was shown to be frost-resistant at temperatures as low as - 87°C (MILLER, 1969). Even large carabid and silphid beetles
commonly
found
in temperate
regions
were
found
to be frost-resistant
(ASAHINAand OHYAMA, 1969). Workers of the carpenter ant, Camponotus obscuripes, which was believed to be freezing-susceptible was found to be resistant 267
268
YOSHIKUNIOHYAMA ANDEIZO ASAHINA
to freezing, if the ice formation was limited to the alimentary tract (OHYAMAand ASAHINA, 1969). The present paper deals with the nature of frost resistance in some of the adult insects in relation to environmental temperatures. A detailed observation of the freezing process in the entire body of a carpenter ant is also described.
MATERIALS
AND METHODS
Insects used in the present study were collected in two winter seasons of both 1969-70 and 1970-71. Carabid beetles, Damaster blaptoides rugipennis Mots. and Pterostichus orientalis Mots., and carrion beetles, Phosphuga atrata L., were collected from decayed wood in Shibecha, eastern Hokkaido, which is one of the coldest places in Japan. The first named is one of the largest ground beetles in Japan having a body weight of more than 1 g and a body length of about 40 mm. Carpenter ants, Camponotus obscuripes Mayr, were collected from partly decayed stumps in Shibecha. Ichneumonid wasps, Hoplismenus pica japonica Uchida, Pterocormus molitorius L., and Chasmias sp., were collected from decayed wood in both Shibecha and Sapporo. The temperature conditions in the habitat were determined at the time of collection of these insects with a slender thermistor with a tip of 1 mm in dia. The collected insects were transferred to a laboratory in Sapporo where they were kept at - 5°C in a cold box until they were used for the experiment. To determine the frost resistance of the insects, dried Petri dishes containing usually more than 2 to 5 individuals were separately cooled slowly in freezers at - 10, - 15, and - 20°C. They were kept at a constant low temperature for 24 hr from the time of spontaneous freezing of the insect bodies. After thawing in air at room temperatures, recoveries of their normal activity such as walking, feeding, and avoidance response, were observed for several days. Supercooling points were determined from the freezing curves of individual insects using more than two for large beetles and up to ten for wasps and ants. Body temperatures were measured with a O-2 mm copper-constantan thermocouple connected to an electronic recorder. Insects were wrapped in dry cottonwool into which the tip of a thermocouple was inserted in such a way that the tip was in direct contact with the insect body. Carpenter ants were fixed to the tip of the thermocouple with a small dab of Vaseline. These two methods gave the same results in determining the supercooling point of the cooled insect. Insects with a thermocouple were cooled in a polystyrene tube in a freezer at about - 30°C. The rate of cooling was regulated to 1 to l*S”C!/min on the tangents along the freezing curves at about 0°C by inserting the insect in double or triple tubes in a freezer. To examine the process of freezing occurring in various parts of an ant body, differential thermal analysis (D.T.A.) was used. A tip of the thermocouple was in direct contact with the thorax and another tip with the abdomen (Fig. 1). In this manner the thermal difference between the thorax and abdomen were estimated simultaneously with the recording of the freezing curve of the insect (Fig. 4).
FROST RESISTANCE IN ADULT
INSECTS
269
To determine the loci where ice forms within an ant’s body, frozen sections were used. The insects were separately frozen at various cooling rates as will be described later. In a cooled room at -20°C frozen insects were fixed to wooden blocks with a small amount of aniline and the freeze-sectioning was made in
m
..-. a AEJ
c
.:($ :: .. . . . .:. :..c;;:.:.....
:”
..
..: .
FIG. 1. Tips of thermocouples in contact with the ant’s body. Tip A is for the recording of body temperature. Tips B and C are for D.T.A. between the thorax and the abdomen.
parallel with the median plane of the insect by use of a microtome (ASAHINA and TANNO, 1967). The frozen sections obtained were observed under a microscope immediately after sectioning in the same cold room at -20°C. The cut surface of the remaining body, from which the frozen sections were separated, was also observed in the cold room. Since the knife marks on the cut surface, which usually occurred immediately after cutting, obstructed a clear observation of the cut surface, ice crystals on the surface were slightly sublimated by exposing to air for 1 day at -20°C before observation. To obtain a cut surface similar in appearance to that of an unfrozen control insect, insects frozen very rapidly by immersion in liquid nitrogen were used. On the cut surface of such rapidly frozen insects no large ice crystals were observed (Fig. 6), and the appearance of the internal tissues was almost the same as unfrozen ones except for the midgut, in which relatively large ice crystals were sometimes observed. Insects, usually 1 large beetle or 3 to 5 wasps and ants, were ground in 80% ethanol and the extract and residue were separated by centrifugation. The glycerol content in the extract was estimated by use of paper chromatographic analysis described by TAKEHARA (1966). The spot of glycerol was detected by Trevelyan’s alkaline silver nitrate method (TREVELYAN et al.,1950). The appropriate area was cut from the unsprayed paper chromatogram strip, and the glycerol on the strip was eluted with 20 ml of deionized water. The glycerol
YOSHIKUNI OHYAMA ANDEIZO ASAHINA
270
content was determined calorimetrically by periodic acid oxidation and subsequent chromotropic acid-formaldehyde reaction (BURTON, 1957). Glycogen was detected from the precipitated residue remaining after extraction of the soluble substances with 80% ethanol. The residue was suspended in a 5% trichloroacetic acid solution and the glycogen was extracted by heating at 100°C for 15 min. The amount of glycogen was calorimetrically determined as sugar with anthrone reagent. RESULTS
Frost resistance and environmental temperature In a variety of adult insects collected, three wasps, and a species of ant were observed to these insects, glycerol contents, supercooling resistance are indicated in Table 1. In spite TABLE ~-GLYCEROL IN
ALL
species of beetles, three species of be frost-resistant. The names of points, and the degree of frost of the relatively large content of
CONTENT, SUPERCOOLING POINTS, AND FROST RESISTANCE THE
FROST-RESISTANT
Glycerol content * (mg/g fresh wt.)
SPECIES
COLLECTED
Supercooling t points
Freezing temperature tolerated for 24 hr (“C)
Coleoptera D. 6. rugipennis P. orientalis P. strata
0.9 2.5 31.6
-6.3 -7.5 -8.3
-10 -10 -10
Hymenoptera C. obscuripes (worker) H. pica P. molitovius Chasmias sp.
34.6 +I 38.8 96-l
-85 -9.1 -6.0 -6.5
-10 -10 -10 -10
* Maximum value in samples measured. t Mean value for 2 to 10 individuals. 1 Only qualitatively identified.
glycerol, all insects showed supercooling points invariably higher than -10°C. The limit of low temperatures at which they can survive prolonged freezing were found to be about - 10°C regardless of the amount of glycerol possession. All the insects examined had only one supercooling point (Fig. 2) except the carpenter ants which had two supercooling points (Fig. 4). This indicates that in the former insects when ice forms in a readily freezable part of the insect body, it immediately seeds the other tissue and freezing throughout the entire body results, whereas in the latter insects this is not so, as will be described in detail later.
FROST
RESISTANCE
IN
ADULT
271
INSECTS
Almost all of these insects were collected from big trunks (more than 40 cm dia.) of partly decayed wood appearing above snow level. They were most frequently found in holes or chinks in the xylem. The temperatures in these habitats, which were determined at the time of collection, ranged from - 5.7 to - 8.7”C. IO
YZ
0
-IO
-2c I
20 FIG. 2. A
t
40 min
freezing curve of a carabid beetle, P. orientalis.
The temperature fluctuation in the xylem of these wood samples was small except for the surface layer, where the temperature was nearly the same as that of the outside air. Even on a morning when atmospheric temperature decreased to - 25”C, the temperature in the central xylem was observed to be - 9°C. An overnight determination indicated that the central xylem temperature in a wood sample remained between - 7 and - 9°C from the evening to the following morning when a minimum atmospheric temperature of - 15°C was recorded. SAKAI and WADA (1963) observed the temperature fluctuations of various parts in living trees during winter. They found that the temperature in the center of big trunks changed very little. In case of fallen wood with a snow cover, the wood was observed to be more insulated from the cold than those exposed. These results suggest that overwintering insects collected may actually freeze in their habitat, but they are rarely exposed to dangerous temperatures at which they would be fatally injured. In fact, some of the large beetles, which regained their normal activity after thawing, were found under completely frozen conditions in their habitats. Glycerol and frost resirtame Seasonal variations in glycerol content, supercooling points, and degree of frost resistance in relation to environmental temperature were studied in an ichneumonid wasp, Chamzias sp. collected in Sapporo during the winter of 1969-70
272
YOSHIKUNI
OHYAMA AND firzo ASAHINA
(Fig. 3). The wasps had a glycerol content of about 1% of fresh body weight at the beginning of November when the daily mean atmospheric temperature still remained above 5°C. At the end of November the mean air temperature decreased to 0°C and was maintained below 0°C till the end of March. During the overwintering period wasps were collected several times to determine the respective
1969
1970
Seasonal variations in glycerol (0) and glycogen (0) content, supercooling point ( o), and atmospheric temperature. From 6 April insects were kept at 2°C FIG. 3.
for 2 weeks after which glycerol (9) and glycogen ( 0) content were estimated. Insects were then exposed to room air maintained at 20°C and kept there for 1 week after which glycerol ( 0) and glycogen (0) content, and the supercooling point (A) were measured.
glycerol content from a big fallen log with a thick snow cover in which a habitat temperature of around 0°C was usually recorded. The glycerol content in this wasp showed a remarkable increase during December, a maximum value of 10% was measured at the beginning of February and thereafter decreased to about 4% at the beginning of April, when the mean air temperature again increased to above 0°C. The effect of environmental t_emperature on the glycerol content was examined in the period when both glycerol increases and decreases in the insects. On 20 December wasps were exposed to - 10°C at which temperature they invariably froze and became hard. After being kept at - 10°C for 40 days they were thawed. All of them regained their normal activity. The glycerol content in these immediately after thawing was 10%. This indicates that in frozen insects at - lO”C, glycerol may increase remarkably to the same level as in the control insect overwintering in its habitat at about 0°C. At the beginning of April when the outdoor temperature was increasing daily, insects were kept at 2°C. This resulted in the maintenance of a constant glycerol level for at least 2 weeks (Fig. 3). Apparently no glycerol decrease was seen in the insects at 2°C in this period. These kept at 2°C were then exposed to room temperature (over ZO’C). A complete loss
FROST RESISTANCE
IN ADULT
INSECTS
273
of glycerol content was observed within 1 week (Fig. 3). The amount of glycogen decreased in reverse relation to the glycerol increase only at the beginning of winter. During the 4 months from the end of December to the following spring, the glycogen content remained at a low level below 0.5%. Even in insects reared at room temperature at the end of April, when a remarkably rapid decrease of glycerol took place, no glycogen was observed to increase (Fig. 3). The supercooling point of the insect showed no remarkable change, giving an average value of about -6”C, throughout the 5 months from the beginning of November, regardless of the glycerol content in the insect. At the end of April when the insects began to move actively their capacity to supercool decreased remarkably. Insects overwintering outdoors could survive freezing at - 10°C for 1 day or more without any post-thawing injury at the beginning of November, when they have a glycerol content of only 1%. The degree of frost resistance in this wasp was observed to be nearly the same throughout the full overwintering period. In midwinter freezing at - 10°C was innocuous even for many days, but freezing at - 15°C caused some injury and freezing at - 20°C resulted in a serious injury which became fatal within 1 month. When the insects were reared at room temperatures at the end of April, they completely lost their ability to survive freezing. Process of ice formation within an insect body The carpenter ant, Camponotus obscuripes, was assumed to be a freezingsusceptible form, since it could not survive freezing at - lO”C, when it was previously seeded by ice and became hard and brittle (TANNO, 1962). However, a thermal determination of cooling ants suggested that ice actually formed within the insect body before the insects became hard and brittle. Ice propagation within the body was, therefore, studied by using overwintering carpenter ants. Since most of the carpenter ants in winter nests were observed to be workers, workers were exclusively used. A definite form of freezing curve with two supercooling points was obtained from the carpenter ants (Fig. 4). The first supercooling points, which were observed to be remarkably stable, were at - 8.5 & 0*3”C, whereas the second supercooling points ranged from - 10.3 to - 25*6”C and were at - 20 + 4*7”C on average. The heat release at the former temperature was much less than at the latter. The insects being cooled were still soft and flexible even after they were cooled down to the first supercooling point. When the second supercooling point was reached on the freezing curve, the cooled insects became hard and brittle. If the insects were rewarmed during the cooling process before the second supercooling point was attained, they invariably regained their normal activity. This was also the case in the insects kept for 1 day at a constant temperature of - 10°C in a cold box, in which the first supercooling point appeared on the freezing curve of the insect within about 1 hr after transference from room temperatures. None survived freezing even for a short time at temperatures below the second supercooling point.
YOSHIKLJNI OHYAMAAND &zo
274
ASAHINA
On the D.T.A. curve shown in Fig, 4, the part below the zero level indicates a larger heat release in the abdomen than in the thorax, and the part above zero level indicates the reverse. A simultaneous recording of both freezing and D.T.A. curves appeared to suggest that the first freezing, i.e. the freezing initiated at the first
I 0
40
20
60
min
FIG. 4. Freezing curve (A) and D.T.A. curve (B) of a worker of overwintering carpenter ant. The first (1) and the second (2) supercooling points appeared on the freezing curve. In D.T.A. curve (B), the part below the zero level indicates a larger heat release in the abdomen than in the thorax, and the part above zero level indicates the reverse.
supercooling point, mainly took place in the abdomen and the second freezing, i.e. the freezing initiated at the second supercooling point, took place in both the abdomen and the thorax (Fig. 4). Figures of D.T.A. curves for the first freezing were distinctly uniform in many cases of the freezing ants, whereas those for the second freezing were not uniform. Thermal difference between the anterior and posterior ends of the abdomen at the time of the first freezing is indicated in a D.T.A. curve presented in Fig. 5. The curve suggested that the first freezing might occur mainly at the anterior part of the abdomen. From these observations it was assumed that in a cooling body ice first formed in the anterior part of the abdomen and remained there without any further growth to other parts for a period of time until the insect was cooled down to the second supercooling point when ice formed throughout the entire body. This assumption led the authors to make direct observations of freeze-sectioned carpenter ants, using insects frozen by the following three different methods: (1) insects were very rapidly frozen by direct immersion into liquid nitrogen from room temperatures; (2) insects were cooled in air in a cold room at - 30°C for 2 hr during which period they became invariably hard and brittle ; (3) insects were
275
FIG. 6. The cut surfaces of frozen ants. (a) Insect cooled very rapidly by direct immersion into liquid nitrogen. (b) Insect frozen hard at -30°C. r (c) Insect cooled at - 10°C for 15 hr and then rapidly immersed in liquid nitrogen.
276
FIG. 7. Frozen cross-sections of the alimentary tract in ants frozen in three different manners, showing a part of the gut wall and ice crystals in the lumen. Crop (a), stomach (b), and rectum (c) of the insect cooled very rapidly by direct immersion into liquid nitrogen. Crop (d), stomach (e), and rectum (f) of the insect frozen hard at -30°C. Crop (g), stomach (h), and rectum (i) of the insect cooled at - 10°C for 15 hr and then rapidly immersed in liquid nitrogen.
FROST RESISTANCE IN ADULT INSECTS
277
cooled in air in a cold room at - 10°C for 15 hr, during which period they remained soft and flexible. They were then rapidly immersed in liquid nitrogen.
“C
6 0
-
A
A
-10 t 0
\ 30 min
FIG. 5. Freezing curve (A) and D.T.A. curve (B) for the first freezing in an ant. In D.T.A. curve (B) the part above zero level indicates a larger heat release in the anterior than in the posterior part of the abdomen.
Following these freezing treatments, median sections were made to observe the cut surfaces. As already mentioned the cut surface of insects frozen by the first treatment appeared intact as in the unfrozen control (Fig. 6A). On the cut surface of the insects frozen by the second treatment, large ice crystals were observed in various parts of alimentary tract. In addition, the muscle in the thorax showed a dehydrated and contracted appearance (Fig. 6B) and between muscle tissues ice crystals were actually observed. This suggests that in the frozen insect muscle tissues are dehydrated as a result of extracellular freezing. In the insects frozen by the third treatment, on the other hand, large ice crystals were observed only in the oesophagus, crop, and at times in the stomach (Fig. 6C). Other parts of the frozen insect appeared to be the same as in the rapidly frozen one shown in Fig. 6(A). By observing the frozen sections a detailed comparison of the size of ice crystals in the lumen of the alimentary tract of insects frozen in the above-mentioned three ways was made (Fig. 7). The sections from the insects frozen by the first treatment showed that only fine ice crystals occurred in various parts of the alimentary tract (Fig. 7A-C). This was also so in the insects frozen by the third treatment except in the oesophagus and the crop, where onIy large ice crystals were observed (Fig. 7G-I). In the insects frozen by the second treatment large ice crystals were invariably observed throughout the lumen of the crop, the stomach, and the rectum (Fig. 7D-F). These results seem to indicate that by cooling the
278
YOSHIKUNIOHYAMA AND&zo ASAHINA
insects at - 10°C for 15 hr, ice only forms within the foregut. On the cut surface of the insect frozen by the third treatment, as to why the stomach at times contained large ice grains was uncertain. Even in the insects frozen very rapidly by the first treatment, large ice crystals were at times observed in the stomach on the cut surface. However, immediately after sectioning no large ice crystals were observed in the stomach in the sections from the insect frozen by the third treatment (Fig. 7H). Since the freeze-cut surface of the insect was exposed to air at -20°C for 1 day before observation, the fine ice crystals initially formed in the stomach may have grown to large ones, if there were some factors to accelerate the ready process of migratory recrystallization, for instance a high solute concentration in the stomach. All observations described in the present paper seem to suggest that in the freezing process of the carpenter ant, ice spontaneously forms first in the crop at the first supercooling point, but that the propagation of ice crystals is limited only to the foregut until the insect is cooled down to the second supercooling point at which time ice forms throughout the entire tissue of the insect. DISCUSSION Spontaneous freezing in insects has been assumed to originate in either the haemolymph or the gut content (SALT, 1966; ASAHINA, 1969). As discussed extensively by Salt, it seems almost certain that freezing in insects commences from ice crystal nucleators or motes (DORSEY, 1948) introduced from the outside into the alimentary tract at least in feeding larvae (SALT, 1953, 1958a, 1966). When the larvae ceased to feed with the approach of the time for spinning cocoons a remarkable increase in supercooling ability was observed (SALT, 1936). This may suggest a decreased activity of the gut content as nucleators and an increased probability of spontaneous freezing of the haemolymph in the body cavity. During overwintering periods, many larvae and pupae have supercooling points around -20°C even without any glycerol or alternative agents. In these insects a remarkable correlation between glycerol content and supercooling ability has frequently been reported (SALT, 1958b; SBMME, 1964, 1965). S ome of them, possessing more than 10% glycerol, were very readily supercooled to - 30°C or below. These observations seem to suggest that in these insects the onset of freezing takes place in the haemolymph. Many insects, which pass winter in the adult stage are known to be freezingsusceptible (ASAHINA, 1969). They have, as a rule, high supercooling ability and can withstand cold by means of supercooling. Frost-resistant adult insects, on the other hand, were so far observed to have a relatively poor supercooling ability regardless of the amount of glycerol possession. The supercooling points of the adult insects examined in the present work invariably fell between a narrow temperature range of - 6 to - 1O’C. According to a detailed study by BAUST and MILLER (1970), an Alaskan carabid beetle, Pterostichus brevicornti, was observed to have a high supercooling point of about - 11 “C in spite of having a large amount of glycerol estimated to be more than 20% in winter. In this insect a clear seasonal
FROST
RESISTANCE
IN
ADULT
INSECTS
279
change of glycerol content was observed in good correlation with the freezing point of the haemolymph, but the relation between the supercooling point and glycerol content appeared to be somewhat obscure. In the case of an ichneumonid wasp, Chasmias sp., practically no change in supercooling capacity was observed throughout the overwintering period regardless of the glycerol content (Fig. 3). When the wasps began to take food or exogenous water by mouth with the approach of the warm season, a remarkable loss of supercooling ability was seen. One of the possible explanations for these facts may be an enhanced occurrence of spontaneous freezing in the content of the alimentary tract rather than in the haemolymph in these adults. This was clarified at least in the carpenter ants as will be discussed later. Supercooling points higher than - 10°C were also reported in some larvae and pharate pupae with a very high ability to tolerate freezing (S~~MME, 1964; TANNO, 1970). An easy release from a supercooled state at a relatively high subfreezing temperature may reasonably be profitable for insects in surviving freezing by decreasing the opportunity of the occurrence of dangerous intracellular freezing. It was observed in a Chasmias wasp that the glycerol content increased from about 5 to lo?; within 40 days at a constant temperature of - lO”C, at which temperature they invariably froze solid. It may be worthy of note that even under frozen conditions at - 10°C glycerol accumulates in the insect to the same level as in the unfrozen control insect from the habitat where the temperature is usually maintained at about 0°C under a snow cover. CHINO (1957) f ound that in diapausing eggs of the silkworm, Bombyx mod, glycogen was converted to polyols and was later resynthesized from the polyols. A similar relation was also observed in an overwintering slug caterpillar by TAKEHARA (1966). In this insect glycerol increased remarkably in autumn and decreased in the following spring indicating a good inverse relation with the amount of glycogen. On the other hand, an’inverse relation between the amount of glycerol and glycogen was shown in a Chasmias wasp only at the beginning of winter. In the following spring, when glycerol decreased remarkably in the wasp, no increase in the glycogen content was observed (Fig. 3). D uring the period of glycerol decrease, the environmental temperature of the wasps remained around 0°C and therefore no remarkable consumption of carbohydrates was expected. These results seem to suggest an interconversion of glycerol to appropriate substance(s) other than glycogen. In spite of a large amount of glycerol, this wasp was found to be moderately frost-resistant. The degree of frost resistance was also almost constant throughout the 5 months of over-wintering period regardless of the glycerol content. In this period freezing at - 10°C was innocuous, but not at lower temperatures. In the Alaskan carabid beetle, too, the haemolymph glycerol was observed to decrease without a loss of frost resistance (BAUST and MILLER, 1970). In addition, it was revealed in a remarkably frost-resistant caterpillar, Monema flavescens, that after the glycerol content had reached about 2% of the fresh body weight, further glycerol increase was unnecessary for the insect to maintain its high resistance
280
YOSHIKUNI OHYAMA
ANDfi~zo ASAHINA
(ASAHINAand TAKEHARA,1964). An accumulation of a relatively small amount of glycerol may perhaps be enough to maintain the maximum degree of frost resistance in Umsmia-swasps. Overwintering carpenter ants from both Japan and Canada were reported to be freezing-susceptible, since they could not survive after solidification of their bodies by freezing with ice-seeding at temperatures below - 10°C (TANNO, 1962; %MME, 1964). In the present work, however, it was revealed that in the cooling process of a carpenter ant, freezing progressed in two steps, i.e. the first freezing occurred at - 8.5 t_ 0*3”C, the first supercooling point, and the second at - 20 +_4*7”C, the second supercooling point. The first freezing did not solidify the insect and was shown to be innocuous, whereas the second freezing made the insect hard and brittle, resulting in fatal injury. Since artificial ice-seeding from the body surface could solidify the insect even at - 10°C (TANNO, 1962), such a mode of initiation of freezing appears to result in the second freezing in an ant at a higher temperature than the second supercooling point. Observations of frozen sections and of freezecut surfaces of bodies strongly suggested that the first freezing corresponded to the ice formation limited only to the foregut and the second freezing to all the remaining parts of the tissues. D.T.A. curves obtained from frozen ants indicated that at the beginning of first freezing a remarkable ice formation took place at the anterior end of the abdomen. These results seems to suggest that when an ant is cooled gradually to temperatures below - lO”C, the ice first forms spontaneously in the crop and develops through the lumen of foregut, but the other tissues, even other parts of the alimentary tract, still remain unfrozen until the insect is cooled down to the second supercooling point. It is well known that the inside wall of the foregut of an ant, throughout the oesophagus, crop, and proventriculus, is completely covered with a well-developed cuticular layer. In addition the posterior narrow end of the proventriculus is assumed to function as a valve, since the walls of the proventriculus, especially at this narrow end, are provided with powerful transverse muscles (WHEELER, 1960). Such a ‘valve’ may prevent the penetration of ice through the alimentary tract from the foregut to the midgut, at least at the temperature of the first supercooling point. The cuticular layer in the foregut may also serve as a barrier to prevent ice-seeding through the gut wall to the body cavity. In the second freezing in ants, an easy interpretation of the freezing process appears difficult. Since the D.T.A. curves obtained from freezing ants were not always uniform for the second freezing, various processes of ice propagation may be assumed. As mentioned before, the second supercooling point, at which temperatures the second freezing commenced, were observed to occur over a range from - 10.3 to -25*6X An inoculation through the lumen to the midgut from the ice mass formed by the first freezing in the foregut may be responsible for the occurrence of the second freezing at a high temperature near the first supercooling point, for example at - 10.3”C. On the other hand, spontaneous freezing of the haemolymph within the body cavity may cause the second freezing at a low temperature, say -25.6”C. Many larval or pupal insects having a glycerol content comparable
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with that in the overwintering carpenter ant, were observed to have supercooling points ranging from - 20 to - 30°C. REFERENCES A~AHINAE. (1957) Motility of some animals and an excised tissue in supercooled state. Low Temp. Sci. (B) 15,45-50. ASAHINAE. (1969) Frost resistance in insects. A&. Insect. Physiol. 6, l-49. ASAHINAE. and OHYAMAY. (1969) Cold resistance in insects wintering in decayed wood. Low Temp. Sci. (B) 27, 143-152. ASAHINAE. and TAI~EHARA I. (1964) Supplementary notes on the frost resistance in a slug caterpillar, Monemaflavescens. Low Temp. Sci. (B) 22, 79-90. ASAHINAE. and TANNO K. (1967) Observations of ice crystals formed in frozen larvae of the European corn-borer by the use of freeze-sectioning. Low Temp. Sci. (B) 25, 105-111. ASAHINA E. and TANNO K. (1968) A frost-resistant adult insect, Pterocormus molitorius (Hymenoptera, Ichneumonidae). Low Temp. Sci (B) 26, 85-89. BAUSTJ. G. and MILLER L. K. (1970) Variations in glycerol content and its influence on cold hardiness in the Alaskan carabid beetle, Pterostichus brevicovnis. J. Insect Physiol. 16, 979-990. BURTONR. M. (1957) The determination of glycerol and dihydroxyacetone. In Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLAN N. 0.) 3,246-249. Academic Press, New York. CHINO H. (1957) Conversion of glycogen to sorbitol and glycerol in the diapause egg of the bombyx silkworm. Nature, Lond. 180, 606-607. DORSEY N. E. (1948) The freezing of supercooled water. Trans. Am. phil. Sot. 38, 247-328. MILLER L. K. (1969) Freezing tolerance in an adult insect. Science, Wash. 166, 105-106. OHYAMAY. and ASAHINAE. (1969) Process of spontaneous ice formation within the body of the carpenter ant. Low Temp. Sci. (B) 27, 153-160. OHYAMA Y. and ASAHINAE. (1970) Frost resistance and glycerol in overwintering adult wasp Chasmias sp. Low Temp. Sci. (B) 28, 79-85. SAKAI A. and WADA J. (1963) Temperature variation in over-wintering trees. Low Temp. Sci. (B) 21, 25-40. SALT R. W. (1936) Studies on the freezing process in insects. Tech. Bull. Minn. agric. Exp. Stn 116, l-41. SALT R. W. (1953) The influence of food on the cold-hardiness of insects. Can. Ent. 85, 261-269. SALT R. W. (1958a) Application of nucleation theory to the freezing of supercooled insects. J. Insect Physiol. 2, 178-188. SALT R. W. (1958b) Role of glycerol in producing abnormally low supercooling and freezing points in an insect, Bracon cephi (Gehan). Nature, Lond. 181, 1281. SALT R. W. (1966) Factors influencing nucleation in supercooled insects. Can. J. 2002. 44,117-133. SBMMEL. (1964) Effects of glycerol on cold-hardiness in insects. Can.J. Zool. 42,87-101. SBMME L. (1965) Further observations on glycerol and cold-hardiness in insects. Can. J. Zool. 43, 765-770. S~MME L. and &.TBYE E. (1969) Cold-hardiness in some winter active insects. Nor& ent. Tidsskr. 16, 45-48. TAKEHARAI. (1966) Natural occurrence of glycerol in the slug caterpillar, Monemaflavescens. Contr. Inst. Low Temp. Sci. Hokkaido Univ. (B) 14, l-34. TANNO K. (1962) Frost-resistance in a carpenter ant Camponotus obscuripes obscuripes-I. The relation of glycerol to frost resistance. Low Temp. Sci (B) 20, 25-34.
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YOSHIKUNIOHYAMAAND I?IZO ASAHINA
TANNO K. (1970) Frost injury and resistance in the poplar sawfly, Trichiocampus pop& Okamoto. Corm. Inst. low Temp. Sci. Hokkaido Univ. (B) 16, 1-41. TRJZ~ELYAN W. E., PROCTERD. P., and HARRISONJ. S. (1950) Determination of sugar on paper chromatograms. Nature, Land. 166, 444-445. WHEELERW. M. (1960) Ants, Their Structure, Development and Behavior. Columbia University Press, New York.