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EFFECTS OF GLYCEROL ON COLD-HARDINESS IN INSECTS'
LAURITZ S#MME~ Entomology Section, Canada Agriculture Research Station, Lethbridge, Alberta
Received August 6, 1963
Abstract The effects of glycerol on cold-hardiness in insects and seasonal changes in
glycerol concentrations were investigated. The presence of this polyhydric alcohol was demonstrated in overwintering stages of 10 species. Larvae of one species also contained sorbitol, and eggs of two species another polyhydric alcohol, probably mannitol.
Evidence gathered from various diapausing speck showed that gIycero1 accumulated durina the fall. 7 his incrrasc in concentration was observed in eggs of one speciesat tcrnpcratures r a n ~ i r l ~ from -So to 20" C. KO decrease in gIycer01 content was observed in any species as long as it was in diapause. After diapnu*~vas brolien glyceml wits lost in all species, in some even a t temperatures clown to -5" C. Increase in concentration was never found in postdiapause insects. In two species, which do not have a diapause, the glycerol content in- creased below a certain temperature and decreased a t higher temperatures.
Three species were freezing-tolerant, although one contained less than 3% and another no glycerol, whereas eight species, most of which contained more than IS%, were killed by freezing. Thus glycerol alone cannot protect against freezing injuries.
In several species the cold-hardiness was increased by the presence of glycerol because supercooling points were depressed. These depressions were more than those of the corresponding melting points. The regression of amount of super- cooling on concentration of glycerol was linear in five species.
Introduction The presence of glycerol in insects was reported independently by different
authors a t about the same time. Wyatt and Kalf (1958) found glycerol in hernolymph of diapausing pupae of Hyalophora cecropia (L.) and Chino (1957) showed that glycogen is converted to glycerol and sorbitol during diapause in eggs of Bombyx mori (L.). After diapause was broken a reconversion to glycogen took place. Wyatt and Meyer (1959) also showed that glycerol accumulated slowly in H. cecropia during several months of diapause. During postdiapause development glycerol was lost, and the concentration approached zero a t the time of emergence of the adults.
Glycerol protects human red blood cells and bull spermatozoa from injuries caused by freezing. Several kinds of mammalian tissues may also recover after being frozen in the presence of glycerol (Smith 1961). Because of this protective action, Salt (1957, 1959) suggested that the natural occurrence of glycerol explains the ability of some insects to survive freezing. He found that freezing-tolerant, overwintering stages of Eurosta solidaginis (Fitch) , Antheraea polyphemus (Cram.), and Bracon cephi (Gahan) all contained glycerol, while with one exception no glycerol was detected in a number of freezing-susceptible species. Dubach et al. (1959) suggested that glycerol may play a role in the cold-hardiness of Camponotus pennsylvanicus (DeGeer). This was indicated
'This work was made possible through a postdoctoral fellowship from the National Research Council of Canada and the provision of facilities by the Canada Department of Agriculture a t the Research Station, Lethbridge.
2Present address: The Norwegian Plant Protection Institute, Division of Entomology, Vollebekk, Norway.
Canadian Journal of Zoology. volume 42 (1964)
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88 CANADIAN JOURNAL OF ZOOLOGY. VOL. 42, 1964
by an increase in glycerol contents in ants stored a t O0 to 5" C, and by the observation that ants collected outdoors in wintertime contained 10% glyc- erol.
Some protection by glycerol from the normally harmful effect of freezing was observed in isolated hearts of Popilius disjunctus (Illiger) by Wilbur and McMahan (1958). A small number of hearts frozen in the presence of glycerol survived more than 3 weeks a t -20°, but all were killed a t -70' C.
In B. cephi, Salt (1959) found that glycerol contents during the fall increased in concentration up to 5 molal. Larvae tested in late fall were able t o survive freezing. Parallel to the increase in glycerol content a lowering of supercooling points took place, some being as low as -47' C. During the spring glycerol was lost, while supercooling points rose. With the presence of glycerol a depression of the melting points of the hemolymph was also observed. This depression was slightly larger than in corresponding solutions of glycerol in water, and for concentrations of 15% to 20y0 the difference was about 1" to 1.5" C. Melting point depressions of a homogenate of H. cecropia pupae with added glycerol were the same as in water solutions of glycerol. In B. cephi the supercooling points were lowered more than the melting points, so that the amount of supercooling increased proportionally to the melting point depression. I t was concluded that glycerol makes the larvae more cold-hardy both by protecting against freezing injuries and by lowering the supercooling points. Later, freezing-tolerant larvae of Eurytoma gigantea Walsh and Mordel- listena sp. were found to contain amounts of glycerol similar t o those of B. cephi (Salt 1961).
Further examples of freezing-tolerant insects containing glycerol were reported by Takehara and Asahina (1960), who found concentrations of 4% and 2y0 in larvae of Pyrausta nubilalis (Hbn.) and prepupae of Papilio mach- aon Felder, respectively. Glycerol contents increased in prepupae of Afonema jlavescens Walker stored outdoors and a t 10" C in the laboratory during the fall but not a t 0" and 20' C (Takehara and Asahina 1961). When diapause was broken, glycerol was rapidly lost a t lo0, while in prepupae stored outdoors a t lower temperatures the concentration remained a t 3 to 4% for several months. A maximum degree of freezing-tolerance was observed in prepupae that con- tained more than 2y0 glycerol.
An exception to the expectation that insects containing glycerol are freezing- tolerant was pointed out by Salt (1957). In spite of concentrations as high as 4.3y0, larvae of Loxostege sticticalis (L.) did not survive freezing. Takehara and Asahina (1960) found that workers of C. obscuripes with 270 glycerol did not survive freezing, and non-overwintering stages of M . Jlavescens were not made freezing-tolerant by injection of glycerol. For this reason they believed that glycerol is only effective in increasing freezing-tolerance in insects where certain protoplasmic changes have probably occurred. As demonstrated by Aoki (1962), eggs of B. mori are also freezing-susceptible, although the ones tested by him contained about 3% glycerol. Tanno (1962) found that workers of C. obscuripes contained up to 4ajo glycerol, but died within 3 days after being frozen a t -10" C. On the other hand, larvae of Sasakia charonda and Hestina japonica survived freezing a t -15" C for a full day without any glycerol (Takehara and Asahina 1960).
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SDMME: GLYCEROL AND COLD-HARDINESS IN INSECTS 89
Although the data available suggest a strong relation to cold-hardiness, the role of glycerol is not clear. Quantitative data on relation to freezing- tolerance have not been worked out, and the general picture is obscured by the findings of glycerol-containing species that are killed by freezing. The objective of the present investigation was to resolve some of these questions.
Studies of seasonal changes in glycerol content are essential in this con- nection. In species that survive freezing it is of interest to know if the change from freezing-susceptible to freezing-tolerant in the fall is related to increase in glycerol concentration. T o classify an overwintering insect as freezing- susceptible i t is important t o show that it is killed by freezing even when the concentration of glycerol is a t its highest level.
Another relation of glycerol to cold-hardiness that calls for a closer study is the effect on supercooling. Studies to see if an increase in amount of super- cooling with increasing glycerol concentrations, as found by Salt (1959) in B. cephi, is a general rule were included in the present investigation.
Materials and Methods Insects used in the present study were collected during the winter of 1962-
63. Carpenter ants, Camponotus herculeanus (L.), were found in fallen logs near Pincher Creek, Alberta, and larvae of the mountain pine beetle, Dendro- ctonus monticolae Hopkins, under the bark of lodgepole pine from Invermere, British Columbia. All other insects used were collected in the vicinity of Leth- bridge, Alberta. Eggs of the black willow aphid, Pterocomma smithia (Monell), were common on willow, and eggs of the fall cankerworm, Alsophila pometaria (Harris), appeared in clusters on twigs of Manitoba maple. Larvae of a gall midge, Rhabdophaga globosa Felt, and of a gallfly, Euura nodus Walsh, were found in galls on willow. Galls of the rose root gallfly, Diplolepis radicum (Osten Sacken), were collected from roots of roses, and an unidentified species of Diplolepis was taken from twig galls. Larvae of the goldenrod gallfly, Eurostu solidaginis (Fitch), were available from galls on goldenrod, and a parasitic wasp, Eurytoma gigantea Walsh, was frequently found in the same galls. A leaf-cutter bee, Megachile rotundata (Fabr.), was reared outdoors at this laboratory, and larvae used in the present study had been stored a t 5' C.
Determination of Supercooling Points T o measure supercooling points, each insect was placed in contact with a
24- or 40-gauge copper-constantan thermocouple, which was connected to a recording potentiometer. The thermocouple was kept in a fixed position inside a glass tube, which was closed a t the end with cotton after the insect was inserted. T o slow down the rate of cooling to lo to 3O C per minute the glass tube was placed inside one or two larger tubes and cooled in a freezer. Larger insects were cooled more slowly than smaller ones to assure that the temper- ature measured on the outside did not differ significantly from the inner temper- ature. T o keep them in contact with the thermocouple, the small-sized eggs of A. pometaria and P. smithia, and larvae of D. monticolae were attached to i t with vaseline.
Normally 10 to 20 specimens were used to determine the average supercool- ing point of a group of insects. However, 5 t o 10 larvae of E . solidaginis, workers of C. herculeanus, and 20 to 30 eggs of A. pornetaria were used.
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90 CANADIAN JOURNAL OF ZOOLOGY. VOL. 42, 1964
Glycerol Analysis Samples of insects were prepared for glycerol analysis by a method de-
scribed by Salt (1959). Single insects or groups of specimens were ground up in 70% ethanol and 400-mesh carborundum powder. The mixture was centri- fuged, the supernatant removed, and the residue washed with more ethanol and centrifuged twice more. The three ethanol extractions were combined and dried a t 45' C. The resulting residue was taken up in a known volume of water, varying from 0.1 to 1.0 ml.
Usually five or six samples were prepared from each group of insects to be analyzed, but according to size of specimens a varying number were used in each sample. For this reason i t was impossible to obtain paired data on super- cooling and glycerol content for individual insects in most of the species.
Glycerol contents were estimated by a chromatographic technique, slightly modified from a method described by Perkins and Aronoff (1959). Ascending chromatograms were run on Whatman No. 1 paper (20x20 cm) with n- butanol - acetic acid - water (12 : 3 : 5) as the solvent. After drying, the chroma- tograms were sprayed with 0.01 M aqueous potassium periodate solution, dried again, and oversprayed with a solution of the following composition: 35% saturated sodium tetraborate, 0.8% potassium iodide, 0.9% boric acid, and 3% soluble starch. By this method glycerol and other polyhydric alcohols appeared as distinct white spots on a blue background.
Three samples of a standard solution containing 0.1% glycerol and 0.06% sorbitol were run on each chromatogram. Within a range of 5 to 15 pg of glycerol and 3 to 9 pg of sorbitol a linear relationship was found between amount of polyol applied and the size of the resulting spot. At least two parallel tests were run of each extract.
The area of each spot was accurately measured with a planimeter, and the size of the standard solution spots were plotted on a graph against the con- centration of glycerol or sorbitol. A straight line was fitted to these data, and the amounts of glycerol or sorbitol in the unknown samples were calculated from this line. For the purpose of the present investigation this method gave sufficient accuracy, and procedures to determine more exactly the amount of glycerol and sorbitol were not undertaken.
In the present investigation glycerol concentrations are given as percent- ages of the sum of the weights of moisture content plus glycerol. Moisture contents were determined by drying the insects to constant weight a t 45', or in some cases a t 75' C. Glycerol is not lost by these treatments, but the lower temperature was preferred when the same specimens were to be used later for glycerol analysis.
Results The Identification of Glycerol and Other Polyhydric Alcohols
On ascending chromatograms periodate-reducing substances from extracts of insects left spots with the same Rr value as glycerol. Large quantities of a substance suspected to be sorbitol were found in larvae of E. solidaginis, and small amounts of other periodate-reducing substances were present in most of the other insects. C
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S0MME: GLYCEROL AND COLD-HARDINESS IN INSECTS 91
T o identify some of these substances, and to verify the identification of glycerol, comparisons were made on descending chromatograms with four different solvent systems. As recommended by Smith (1958) the solvents were run off the paper, and glucose was used as a reference. The resulting R, values for several sugars and polyhydric alcohols are given in Table I together with corresponding values from extracts of E. solidaginis, A . pometaria, and P. smith&.
All extracts gave one spot that clearly corresponds to glycerol, and there can be no doubt that this is the substance found in the different species. The other polyhydric alcohols are more difficult to separate, but the R, values strongly suggest that the substance found in larvae of E. solidaginis is sorbitol. A substance present in eggs of A . pometaria and P. smithia gave R, values with a strong resemblance to that of mannitol. No evidence was found tha t sorbitol, mannitol, or similar substances in any of the species are of importance to cold-hardiness. For this reason further identifications were not carried out.
Glycerol and Freezing-tolerance As mentioned in the introduction, glycerol has previously been found in
most freezing-tolerant insects but only in a few freezing-susceptible species. In the present study several species of insects that overwinter above snow level on the bark of trees or in galls were examined. Glycerol was present in most of them and also in some species that overwinter in more protected places.
The effect of freezing on the various species was examined shortly after diapause was broken. At this time the glycerol content was a t its highest level as will be shown later. After freezing, the insects were stored a t room temperature and 75% R. H., and compared to unfrozen controls over a period of several days or weeks.
TABLE I R, values from chromatograms of extracts of three different insects and of various sugars and
polyhydric alcohols
Solvent*
IsoPr BuPy EtAcPy IsoPrBu
Eurosta solidaginis
Alsophila pornetaria 113 98 99 112 173 135 173 182
Pterocomma smithia 113 97 98 110 171 135 176 181
Glucose 100 100 -
100 - 100 Galactose 93 - L
90 Fructose 114 - Mannose - - 117 - Sucrose 85 - - - Dulcitol 108 93 92 108 Mannitol 112 98 98 110 Sorbitol 106 94 90 102 Glycerol 173 134 175 -
*IsoPr: iso-propanol. water (4: 1); BuPy: n-butanol. pyridine, water (2:2: 1); EtAcPy: ethyl acetate, pyridine. water (12:5:4): IsoPrBu: iso-propanol. n-butanol. water (7:1:2).
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92 CANADIAN JOURNAL O F ZOOLOGY. VOL. 42. 1964
The maximum concentrations of glycerol found in postdiapause insects and the corresponding supercooling points are given in Table 11. Data are also given for D. monticolae and C. herculeanus that do not have a diapause. Although most species contained large amounts of glycerol, only a few sur- vived freezing. All insects listed as freezing-susceptible in Table I1 were killed b y 1 or 2 minutes' freezing a t their supercooling points. Complete mortality from freezing was also always observed in specimens of A. pometaria, P. smithia, D. monticolae, D. radicum, and C. herculeanus with higher super- cooling points and corresponding lower glycerol contents. No further tests were carried out with the rest of the s~ecies.
Freezing of insects a t temperatures above the supercooling points may be accomplished by inoculation. In this way all prepupae of D. radicum exposed at -15" C on moist filter paper for 20 hours froze and died. Workers of C. herculeanus were killed by freezing for a few minutes a t - 10' to - 15" C.
Freezing-tolerant larvae of E. solidaginis supercool only to about - 10" to - 1.5" C , but all groups of 10 larvae frozen a t -30" C for 16 and 32 days devel-
oped to adults with normal appearance, and after 64 days of exposure 90% survived. In postdiapause larvae transferred to 20' C the formation of puparia started within a few days. Larvae frozen for 1 hour a t - 30' C, when puparium formation had just started, did not survive although they contained about 1.8% glycerol. This concentration is only slightly lower than that found in larvae outdoors in the middle of the winter (Fig. 1).
Larvae of E. gigantea developed to normal adults after being frozen for a few minutes a t temperatures corresponding to their supercooling points. Salt (unpublished) found that these larvae will develop normally after being dropped into liquid nitrogen. Larvae of E. gigantea stored a t 20' C for 12 days lost all their glycerol, had an average supercooling point of -28.2" C , and were killed by freezing for a few minutes.
Larvae of E. nodus collected outdoors in February contained no detectable amounts of glycerol or other polyhydric alcohols. Their average supercooling
TABLE I1 Highest average concentrations of glycerol and the corresponding supercooling points in
nine postdiapause and two non-diapausing species
Supercooling point Species and stage % glycerol (" c)
I. Freezing-susceptible Alsophila pometaria, eggs 15.1 +0.83 -44.6+0.38 Pterocomma smithia, eggs 15.5k 0.78 -41.9k1.17 Megachile rotundata, larvae 2.2k0.18 -27.7k0.25 Rhabdophaga globosa, larvae 32.452.22 -49.1k1.22 Dendroctonus monticolae, larvae* 23.4k1.20 -34.0k0.70 Diplolepis radicum, prepupae 6.4k0.42 -32.7k0.33 Diplolepis sp., prepupae 17.6 -39.5k1.33 Camponotus herculeanus, workers* 5.8k0.85 -28.7k0.81
11. Freezing-tolerant Eurosta solidaginis, larvae 2.92 0.09 - 9.6-tO.16 Eurytoma gigantea, larvae 23.4f 0.75 -49.2k0.58 Euura nodus, larvae None - 7.1k0.17
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SBMME: GLYCEROL AND COLD-HARDINESS I N INSECTS 93
point was as high as -7.1" C. Ten larvae frozen a t -60' C for 2 hours sur- vived and later developed to normal-appearing adults. Because of their high supercooling points, the same specimens must have been frozen in their galls during most of January when temperatures were mainly below - 7" C.
Seasonal and Temperature-induced Variations i n Glycerol Content In eggs of A. pometaria and P. smithia collected outdoors an increase in
glycerol content was observed in the fall (Fig. 1). When the maximum level was reached, i t remained about the same for 2 to 3 months in both species. From the beginning of February, which was mild in 1963, the concentration rapidly decreased. An increase in fall and decrease in spring was also observed in prepupae of D. radicum. Larvae of E. solidaginis contained less glycerol than the other s~ecies examined, and the concentration reached in October was retained all winter. By the beginning of April more than two-thirds of the glycerol had disappeared.
The glycerol content did not decrease in any species as long as i t was in diapause. Diapause was already broken in eggs of A. pometaria collected out- doors in mid-January. At this time some of the eggs of P. smithia were able to continue their development, and all were out of diapause by the middle of February. In larvae of E. solidaginis and prepupae of D. radicum diapause was broken early in January.
Similar observations were made on eggs of A . pometaria and P. smithia collected in November and stored a t various temperatures in the laboratory (Fig. 2). In the aphid eggs the concentration of glycerol increased during the first week a t -5" , 0°, 5", and 20" C and in cankerworm eggs a t 0" and 5". This increase terminated about the time d i a~ause was broken. From then on a gradual decrease was observed a t all temperatures. As in P. smithia glycerol
x Pterocomma smlthia Alsophilo pometaria
A Diplolepis radicum
Eurosta solidaginis
U
OCT NOV DEC JAN FEB MAR APR
FIG. 1. Seasonal changes in glycerol contents in four species.
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94 CANADIAN JOURNAL OF ZOOLOGY. VOL. 42. 1964
was lost even a t -5' C in eggs of A . pometaria. In postdiapause eggs of -4. pometaria collected in February the concentration of glycerol decreased from 13.9 to 2.9y0 during 12 weeks' storage a t -5' C.
In P. smithia it was demonstrated that all glycerol is produced by the eggs themselves. No detectable amounts of glycerol were found in mature eggs removed from oviparous females in the fall or in eggs examined 1 day after they were deposited in the laboratory. In the few eggs obtained in this way, traces of glycerol appeared in 2 to 3 days. In about 1 week the concentration was 5.5% in eggs stored a t 20' and 1.2% in eggs stored at 5' C.
As mentioned previously larvae of E. solidaginis contain fairly large amounts of sorbitol. In larvae stored a t -5' C only small changes in glycerol content were observed, while the concentration of sorbitol increased slightly (Table 111). At 5" C diapausing larvae accumulated glycerol slowly, while sorbitol was gradually lost. Sorbitol also disappeared a t 20" C, while no changes in glycerol content were observed as long as the larvae were in diapause. Thus the concentrations of glycerol and sorbitol varied independently of each other.
In postdiapause insects glycerol was lost rapidly a t 20' C (Table IV). In eggs of A . pometaria the concentration decreased much faster a t 20' C than a t -5", 0°, and C (see text and Fig. 2). Practically all glycerol disappeared in 3 to 6 days in prepupae of D. radicum a t 20°, while a t 5' C the concentration decreased only from 5.7 to 5.0% in 9 weeks. The rate a t which glycerol is lost is therefore dependent on the temperature. This is also seen from Fig. 2, where the glycerol content in eggs of P. smithia decreased fastest a t the highest temperature. No increase in cbhcentration was observed in any species after diapause was broken.
FIG. 2. Changes in glycerol contents in eggs of Pterocomma smithia and Alsophila pometaria collected in the fall and stored a t various constant temperatures.
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SBMME: GLYCEROL AND COLD-HARDINESS IN INSECTS 95
Glycerol was also found in the overwintering stages of two species that do not have a diapause., In workers of C. herculeanus collected outdoors in the fall and stored a t 0' C it was found that the glycerol content increased grad- ually (Table V). After 4 weeks a t O0 C some of the ants were transferred to So and some to 20' C. Glycerol was lost a t both temperatures but faster a t 20° C.
Larvae of D. monticolae contained large amounts of glycerol when collected outdoors in December (Table VI). The larvae were in their first to third instar, but no differences in glycerol content were apparent among instars.
TABLE 111 Changes in glycerol and sorbitol contents in diapausing lanrae of
Eurosta solidaginis stored a t -So, So, and 20" C
Treatment
-S°C, 3 wk 8 wk
16 wk 32 wk*
5" C, 0 wk 1 wk 2 wk 7 wk 9 wk
% glycerol
1 .5k0 .18 2.0rt0.11 1.750.15 2.0f0.06 1.5kO.18 1 .5k0 .17 1.6k0.08 2.4k0.12 2.4k0.14
% sorbitol -
1 .6 f0 .14
20" C, 0 days 2.6L-0.24 2.5k0.31 8 days 2.5k0.19 <0.2
24 days 2 .7k0 .26 <O. 2 40 davs 2 .4 f0 .12 -
*Diapause broken.
TABLE IV Postdiapause changes in glycerol contents and supercooling points in four species stored
a t 20" C
Supercooling Species No. days a t 20' C OJo glycerol points ( O C)
Alsophila pornetaria
Eurosta solidaginis
Diplolepis radicum
Eurytoma gigantea
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96 CANADIAN JOURNAL OF ZOOLOGY. VOL. 42. 1964
When larvae of D. monticolae were stored in logs of pine a t O0 C the glycerol disappeared almost entirely in 7 weeks (Table VI). The logs were then trans- ferred to - So, a t which temperature the larvae rapidly increased their glycerol content during the first 2 weeks. After 10 more weeks' storage a t - So C, when no further increase was observed, the logs were transferred to So C, where most of the glycerol was lost in 3 days. When returned to --So the concentra- tion increased within a few days.
Thus the concentration of glycerol appears to be regulated by the temper- ature in non-diapausing species. However, the two species reacted differently. At 0" C glycerol was built up in workers of C. herculeanus, whereas it was lost in larvae of D. monticolae. At So C it was lost much faster in the beetle larvae than in the ants.
Glycerol and Amount of Sufiercooling In most species the supercooling points decreased when the concentration
of glycerol increased (Tables IV, V, and VI). A depression of melting points of the hemolymph was also expected. As mentioned previously, Salt (1959)
TABLE V Changes in glycerol contents and supercooling points in workers of
Camponotus herculeanus stored at 0°, So, and 20" C
Supercooling Treatment % glycerol point (" C)
0" C, 0 wk 0.4r t0 .11 -19.7f1.73 1 wk 2 . 0 f 0 . 3 6 -22.3f 0.51 2 wk 3 .5k0 .93 -26.lrt0.65 4 wk 4 . 4 f 0 . 5 0 -25.3f0.85 6 wk 5 . 7 k 0.37 -27.7f0.90 8 wk 5 .5 f0 .75 -28.3f 0.67
12 wk 5 . 8 f 0 . 8 5 -28.7f0.81
TABLE VI Changes in glycerol contents and supercooling points in larvae of
Dendroctonus monticolae stored a t --So, 0°, and 5" C
Supercooling Treatment % glycerol point ( O C)
0" C, 0 wk 7 wk
-5" C, 0 wk 2 wk 3 wk
12 wk 5" C, 0 days
3 days -So C, 0 days
3 days 6 days
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S0MME: GLYCEROL A N D COLD-HARDINESS IN INSECTS . 97
found that the melting point depression was the same in a homogenate of H. cecropia with added glycerol as in a solution of glycerol in water, but it was- slightly larger in hemolymph of B. cephi. Melting points were measured accord- ing to a method by Salt (1959) in two groups of larvae of E. gigantea. The average glycerol content in the first group was 25.1% and in the second 0.5%;. the difference in average melting points was 8.2' C, whereas the difference in melting points between solutions of the same concentrations of glycerol in. water is 7.4' C. Thus the melting point depression of insect hemolymph a t high concentrations of glycerol may be slightly more than in corresponding solutions of glycerol in water. For concentrations in the range found in the present study this difference is of little significance, and the amount of super- cooling in the various insects was estimated from the supercooling points by subtracting the melting point of a corresponding glycerol-water solution. One, more degree was subtracted to account for other solutes resent.
The sipercooling points were lowered more than t h i calculated melting points in all species except E. solidaginis. Thus the amount of supercooling increased with increasing melting point depression. The relation between the concentration of glycerol and amount of supercooling in five species is shown in Fig. 3. Since paired data on supercooling and glycerol from individual in- sects were not available, each point represents the average amount of super- cooling in a group of insects plotted against their average concentration of
Alsophilo pometorio
A Eurytomo gigonteo
Diplolepis rodicum
A Componotus herculeanus 0 Dendroctonus monticoloe
0 5 10 15 20 2 5 X GLYCEROL
FIG. 3. Relation between amounts of supercooling and concentrations of glycerol in five species (calculated regression lines).
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TABLE VII The effect of loss of one-third of the moisture content on glycerol concentration, melting
point, and amount of supercooling in Diplolepis radicum
Stage
Amount of Melting point supercooling
% glycerol (" C) (" C) - - - . . . .
Prepupae, fresh 5 . 1 k 0 . 4 3 -2 .3k0 .11 28.7+0.49* Prepupae, dried 8 . 0 k 0 . 6 1 -3 .3k0 .16 30.2f 0.48* Pupae, fresh Pupae, dried
*Value of t =2.175 (significant a t 5 % level).
glycerol. Data for A. #ometaria and D. radicum include results from specimens collected outdoors, as well as from those stored a t various temperatures in the laboratory. The lines for the three other species are based on data obtained from specimens stored under various laboratory conditions.
With the exce~tion of Dart of the data from D. monticolae there is a linear regression of amount of supercooling on concentration of glycerol in all species, a s is seen from the calculated regression lines. No significant differences were found between the regression coefficients of any of the lines. This tendency for the lines t o be parallel suggests a similar effect of glycerol in all species. In D. monticolae no changes in amount of supercooling were observed with concentrations of glycerol up to about 11%. From then on a linear regression parallel to the ones in the other species was found.
Further evidence to show that glycerol is responsible for the increase in amount of supercooling was obtained by causing an artificial increase in the glycerol concentration of D. radicum. Prepupae of this species dried a t 4S0 C for 1 to 2 hours lost about one-third of their moisture. Supercooling points and glycerol contents were determined in 10 prepupae treated in this way, while melting points were measured in an equal number of different speci- mens.
Whereas the concentration of glycerol in fresh prepupae of D. radicum was about 5yo, i t was 8% in the dried specimens (Table VII). Depressions of both melting points and supercooling points were observed after drying, but the amount of supercooling was significantly larger in the dried prepupae than in the fresh ones. Other prepupae transferred to 20' C pupated within 4 days and lost almost all of their glycerol a t the same time. Small depressions ,of melting points and supercooling points were found in pupae dried in the same way as above. The amount of supercooling, however, remained the same, a s would be expected since no glycerol was present.
Discussion The seasonal changes in glycerol content seem to follow a similar pattern
in all diapausing insects so far investigated. The observations made by the author agree with those of Chino (1957), Salt (1959), Wyatt and Meyer (1959), and Takehara and Asahina (1961), who all found that glycerol is accumulated during the fall. Apparently glycerol is never lost until diapause is broken, and from then on the decrease in concentration is dependent on the
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SOMME: GLYCEROL AND COLD-HARDINESS IN INSECTS 99
temperature. No increase has been observed in postdiapause insects. Non- diapausing species differ from the diapausing ones in that glycerol may be built up or lost, a t any time during the season. A repeated increase and de- crease in glycerol content as a result of changes in temperature was shown by Dubach et al. (1959) in C. pennsylvanicus and by the author in D. monticolae.
The freezing-tolerance of some of the glycerol-containing species listed by Salt (1961) may be doubted. Aoki (1962) found that eggs of B. mori are killed by freezing, and no data showing that C. pennsylvanicus and M. striata sur- vive freezing are available. Tanno (1962) found that workers of C. obscuripes are freezing-susceptible and so are eight other glycerol-containing species ex- amined in the present investigation. Eleven species containing glycerol have so far been found to be killed by freezing, although as many as nine other species containing glycerol are freezing-tolerant. Thus the relation between glycerol and freezing-tolerance is not clear. If glycerol does protect against freezing injuries, this is only true in some insects, whereas no protection is provided in others. Freezing-tolerance can also be acquired without the pres- ence of glycerol, as in larvae of E. nodus (Table 11), or with only small amounts, as in E. solidaginis.
Larvae of E. gigantea became freezing-susceptible in 12 days a t 20' C, and all glycerol was lost during the same period (Table IV). This may, however, be a coincidence since other changes as well took place during postdiapause development. In larvae of E. solidaginis no quantitative relation between glycerol and freezing-tolerance was apparent. I t also seems unlikely that E. solidaginis should survive freezing because it contains 2 to 3% glycerol, where- as several other species containing more than 15yo are freezing-susceptible (Table 11).
I t must be concluded that glycerol alone does not protect against freezing injuries in insects. Since one species was found to survive freezing without any glycerol, its presence in freezing-tolerant species may be a mere coin- cidence. However, as long as it is not known what kind of injuries result from freezing, i t cannot be definitely demonstrated whether glycerol has a protective action or not.
Although the ability of glycerol to protect against freezing injuries is open to question, the cold-hardiness of insects is increased by its presence because the supercooling points are lowered. Even if the amount of supercooling re- mained constant, supercooling points would decrease with the melting point depression caused by glycerol. However, in most species tested the cold- hardiness was increased more than that because the supercooling points were lowered more than the corresponding melting points.
This increase in amount of supercooling could have been caused by other changes taking place in the insects a t the same time as the glycerol content was increased. Data given in the present investigation are based on insects collected outdoors a t different times during the winter or stored for different times a t various temperatures in the laboratory. For this reason there was always a time factor involved and in postdiapause insects also a develop- mental factor.
If factors other than glycerol are responsible for the observed changes in supercooling, they must be affected simultaneously by the same conditions
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100 CANADIAN JOURNAL OF ZOOLOGY. VOL. 42, 1964
as is glycerol. Since the insects were treated in several different ways this seems unlikely. The regularity of the data from different species strongly in- dicates that the relation between glycerol and amount of supercooling is not a coincidence. Further evidence was found in D. radicum when the glycerol concentration was increased artificially by desiccation. Whereas the amount of supercooling was changed in prepupae, which contained glycerol, no changes took place in the pupae, which contained no glycerol (Table VII).
In larvae of D. monticolae the amount of supercooling did not change a t concentrations of glycerol lower than 11% (Fig. 3). No satisfactory explanation can be offered for this, although it may be related to the fact that these larvae overwinter in several instars. For this reason their digestive tracts may not be empty, a situation which would influence their supercooling points (Salt 1953). This effect may not be overcome until the glycerol content has reached a certain level.
Even without any glycerol the amounts of supercooling differ in various species. Little is known about why some insects supercool 20" or 30" C, whereas others, like E. solidaginis and E. nodus, only supercool about 10" C. The question of what causes an insect to freeze and why some survive freezing is of fundamental importance in the field of cold-hardiness, but is far from solved.
Acknowledgments I am most grateful to Dr. R. W. Salt for valuable help and suggestions
throughout this work. I also wish to thank Dr. T. H. Anstey for advice re- garding statistical analysis and Dr. H. J. Perkins regarding chromatographic technique. Dr. G. A. Hobbs kindly supplied me with larvae of M. rotundata and Dr. R. W. Reid of the Forest Biology Laboratory, Calgary, with larvae of D. monticolae. Mr. C. D. F. Miller of the Entomology Research Institute, Ottawa, has identified specimens of C. herculeanus used in the present study. The conscientious technical assistance of Mr. W. L. Pelham is gratefully acknowledged.
References AOKI, K. 1962. Protective action of the polyols against freezing injury in the silkworm egg.
Sci. Rept. Tohoku Univ., Fourth Ser., 28, 29-36. CHINO, H. 1957. Conversion of glycogen to sorbitol and glycerol in the diapause egg of the
Bombyx silkworm. Nature, 180, 606-607. DUBACH, P., PRATT, D., SMITH, F., and STEWART, C. M. 1959. Possible role of glycerol in
the winter-hardiness of insects. Nature, 184, 288-289. PERKINS, H. J. and ARONOFF, S. 1959. A paper chromatographic method for the purification
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SALT, R. W. 1953. The influence of food on cold-hardiness of insects. Can. Entomologist, 85, 261-269.
1957. Natural occurrence of glycerol in insects and its relation to their ability to survive freezing. Can. Entomologist, 89, 491-494.
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TANNO, K. 1962. Frost-resistance in a carpenter ant Camponotus obscuripes obscuri es. I. The relation of glycerol to frost-resistance. (In Japanese, English summary3 Low Temp. Sci. Ser. B, 20, 25-34.
WILBUR, K. M. and MCMAHAN, E. 1958. Idow temperature studies on the isolated heart of the beetle, Popilius disjunctus (Illiger). Ann. Entomol. Soc. Am. 51, 27-32.
WYATT, G. R. and KALF, G. F. 1958. Organic compounds of insect hemolymph. Proc. 10th Intern. Congr. Entomol. Montreal, 2, 333.
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