Tolerance to freezing of hydrated and partially hydrated larvae of Polypedilum (Chironomidae)

J. Ins. Physic& 1962, Vol. 8, pp. 155 to 163. Pergamon Press Ltd. Printed in Great Britain

TOLERANCE PARTIALLY

TO FREEZING OF HYDRATED

HYDRATED

AND

LARVAE OF POLYPEDILUM

KHIRONOMIDAE) J. P. LEADER Department

of Zoology, University of Bristol

(Received 19 October 1961)

Abstract-It has been shown that when fully hydrated larvae of Polypedilum vandmplankiare frozen they recover temporarily but fail to complete their development. Histologicaj examination of larvae fixed immediately after thawing reveals the destruction of the fat body. A similar breakdown of the fat body occurs in larvae dried over P,OS. When larvae are partially dehydrated before freezing they do not show such signs of damage and many complete their development. The relevance of this to current theories of freezing damage is discuss&d.

INTRODUCTION

ALTHOUGH the tolerance of sub-zero temperatures

by insects has been studied for many years, their ability to withstand internal freezing has received comparatively little attention. ROBINSON (1927) put forward the first theory of freezing tolerance in insects by invoking the concept of ‘bound’ water, but the investigations of DITMANet al. (1943) showed that “bound’ water bore no relation to freezing tolerance. The study of the mechanical changes taking place on freezing, and of the physical conditions necessary for freezing, notably by LWET, SALT,and others, has led to a clearer idea of the nature of the freezing process and has given rise to the theory that it is the mechanical disruption of cells by intracellular ice formation which hills them. Unfortunately the considerable variety both of cell types and of the mechanics of ice formation *makes it diEcult to define conditions or effects quantitatively. The view that cells are hilled by intracellular ice formation is not held by botanists. SACHS(1873) accepted the idea that ice formation was normally extra-

cellular in plants, a concept supported by many observations (see reference in LEVITT, 1956), and he suggested that it was the rise in electrolyte concentration caused by crystallization of water as ice which hilled cells on freezing, death being caused by the denaturation of proteins. However, MAXIMOV (1912) and AKERMAN (1927) rendered this theory untenable by showing that protection from freezing injury could be gained by treatment with non-penetrating non-electrolytes, and that this protection was related to the degree of plasmolysis induced by the 155

156

J. P.

bADER

treatment. A similar theory of electrolyte damage in animal tissues on freezing has been put forward by LOVELOCK (1953,1954) on the basis of his work on red blood corpuscles, although the implied relation between freezing and dehydration had been demonstrated earlier in frog muscle by MORAN(1930). LOVELOCK (1954) has also claimed that this theory can be used to explain the protective action of glycerol, although, using his data, some of his conclusions have been convincingly attacked by LEVITT (1958). This theory does, however, have the merit that it is open to quantitative investigation. More successful in explaining the phenomena of freezing injury in plant tissues is ILJIN’S ‘mechanical’ theory (1933), which supposes that it is the stresses and strains associated with dehydration on extracellular freezing which kills protoplasm by disrupting its organization. LEVI~~ (1960) has shown that the latter theory is sufficient to explain all known features of freezing damage in plants, and it will also explain the demonstrated fact that frost resistance, drought resistance, and plasmolysis resistance of plants are correlated. LEVITT claims that freezing damage can cause disruption of protoplasmic bonds in one of three ways: (1) by pressure of extracellular ice, particularly in liquid cultures of micro-organisms; (2) by the formation of relatively large crystals of intracellular ice, as opposed to the microcrystals formed in ultra-rapid freezing, e.g. LWET (1938) and LUYET and GEEIENIO (1940) particularly in small cells; or (3) by the collapse and expansion of large cells on freezing and thawing. He also suggests that freezing injury in animal cells is essentially similar, since their protoplasmic structure is not apparently widely different, but this idea has received little support from zoologists, for, as SALT (1961a) points out, animal cells lack rigid cell walls, and the stresses and strains to which the protoplasm will be subjected will be correspondingly less. It does receive some support from ASAHINA(1959a, b, c, 1961), ASAHINAet al. (1953), and &AHINA and AOKI (1958a, b), who claim that in the animals studied by them only extracellular freezing is tolerated, although they give no suggestion as to the possible nature of damage by internal freezing. CHAMBERSand HALE (1932) also showed that cells could not tolerate internal freezing. The demonstration by SALT (1959), however, that the fat body cells of Eurosta solidaginis will withstand intracellular freezing seems to show that this theory is at least open to exceptions. More recently, SALT (1961b) claims to have shown that in Cephus cinctus the differences in injury and activity level after freezing at - 15 and -20°C between animals frozen intra- or extra-cellularly were insufficient to justify the use of a freezing site as a basis for a theory of freezing injury. Since, however, neither group tolerated this treatment successfully, few surviving more than 5 days, this argument cannot be said to be decisive. Polypedilum vanderplanki Hinton is a Chironomid, the larva of which can successfully tolerate complete dehydration (HINTON, 1951, 1960a, b). It was therefore considered that subjection of the hydrated larva to freezing temperatures might help to throw some light on the nature of freezing damage, since it can apparently tolerate an extreme electrolyte concentration when drying at normal temperatures.

FREEZING OF LARVAE OF POLYPEDILUM

157

(CHIRONOMIIMI~)

1. Freezing tolerance of fully hydrated larvae In the first series of experiments, the animals were placed in water in small polythene tubes and left in a refrigerator previously equilibrated to the desired temperature. The rate of cooling of the tubes and animals to -40°C was approximately 1°C per minute. After a known time, the tubes were removed, thawed rapidly in the hand or in a bath of water at 3O”C, and recovery observed. Results of these experiments are shown in Table 1. TABLE

&--RECOVERY

OF HYJJRATED LARVAE EXPOSED TO FRERZING TRMPERATURES FOR VARIOUS TIMES

No. of larvae 10 10 10 5 10 5 5 5 5 6 10 10 10 5

r’emp. (“C) -10 -10 -10 -12 -12 -12 -30 -30 -30 -30 -40 -40 -40 -40

Time (W

13

6 24 1-k 2 1 ” :t 2% 2 6 26 1

No. recovered 10 10 10 5 In G : 2 10 9 10 5

Max. life (dw)

24-t 4

J+ _. 24+ : 3 2 1 3 1 I8

It was considered that the animals showed some degree of recovery if they showed pulsation of the dorsal organ, pumping movements of the pharynx, or when contraction of the body wall musculature occurred either spontaneously or could be elicited by a light touch with a glassneedle. Although nearly all the animals used in these experiments recovered they cannot be said to survive the treatment unless subsequent development is completed. It may be seen that the only treatment which did not result in the death of the animals within a few days was a short exposure to - 10 or - 12°C. In all other cases the animals, almost without exception, failed to recommence feeding and quickly became immobile, although death in most did not occur for some days. In view of the tolerance of Polypedilum to a short exposure to - lO”C, it was decided to test the effect of exposing larvae to - 4O”C, after pretreatmentat - 10°C. These results are shown in Table 2. It may be seen that such pretreatment does have a slight effect on the ability of the larvae to tolerate -4O”C, but the treatment still usually results in the death of the animal.

158

J. P. LEADER

Experiments in which the larvae were exposed to liquid air temperatures after a variety of pretreatments were also tried. Only one animal lived long enough to pupate, so it seems reasonable to conclude that under these conditions Polypedilum cannot tolerate liquid air temperatures, although it is probable that the gut, some TABLE 2--RECOvERY OF HYDRATED LARVAFa EXPOSED TO -40°C PRETREATMENT AT - 10°C

No.

of larvae

Hours at - 10°C

5 5

1 1Q

5 5

1 2

Hours at - 40°C

Al?TBR

Max.

No. recovered

life

(days) 15 2 adults emerged 4 9

sense organs, the nervous system, and musculature are unharmed, since the normal behaviour patterns are apparently unchanged when the animal is observed immediately after thawing. The results of these experiments are shown in Table 3. TABLE 3--RECOVERY

OF HYDRATED

LARVAE EXPOSED TO -

190%

AFTER

VARIOUS PRETREATMENTS

No. of larvae

Houra at - 10°C

10

26

10 5 4 5

-

5 5 5 5” 5

-

Houra at - 30°C -

Hours at -40°C

Hours at -190°C

No

recovesed

*

M&x. life

(days)

-

6

2

1

6 2 2 2+

10 4 4 3

2 2 2

5 4 3

: 3 3

2 2

5 S 4

: 0

T :,

1

-

2”

1

13 s 1

1

-

1 1

8

1 l& 2 2 2

2. E#ect of freezing on the fat body A. Histological examination. The impression was gamed, after observations of animals after freezing and thawing, that the fat body was tiected in some way by this treatment. To investigate this further, a group of five animals was exposed to a temperature of -40°C for 1 hr in the same way as in earlier tests. After rapid thawing, they were fixed in Bouin’s fluid, embedded in the usual way, and sections

FRREZING OF LARVAF. OF POLYPEDZLUM

159

(CHIRONOMIDAE)

were cut at 8 ,u. These were stained with Mallory’s triple stain, and compared with unfrozen controls. Examination of the test animals revealed that almost all the cells of the fat body were destroyed. All other tissues appeared normal, and it therefore seems that death in some larvae is related to the disruption of the fat body. B. Comparison with rapidZy dried animals. It has been shown (author, unpublished) that when Polypedilum larvae are exposed, fully hydrated, in dry still air they will only tolerate a reduction to about 55 per cent moisture content. If exposure to these conditions is continued the animals fail to recover completely. If the animal is to survive complete dehydration a much slower rate of drying must be used. When larvae were examined histologically after drying over phosphorus pentoxide at ZO”C, and subsequent rehydration, it was seen that these also showed complete destruction of the fat body, while other tissues appeared normal. These animals showed almost no sign of recovery on rehydration. Larvae dried at 20°C and 80 per cent r.h. on filter paper showed a temporary recovery, and in these animalsthere was merely partial destruction of the fat bady. 3. Freezing tolerance of pa&z& dehydrated Earerae Since Polypedakm will tolerate freezing temperatures with a moisture content of 8 per cent (HINTON, 196Oa), a series of experiments was carried out to test the tolerance of partially hydrated larvae to freezing. Using a glass fibre balance reading to approximately 0.02 mg, the percentage loss in weight in unit time was calculated for animalsimmersed in 1.17 M solution af sucrose. From these results it was found that the solutian was strong enough to reduce the moisture content to SO-60 per cent, equilibrium being reached in about 8 hr. Aniials were immersed in this solution for a known time and then cooled, with or without pretreatment at - 10X!, to -40°C. This method gave a much higher number of successful results, as shown in Table 4. TABLE~---RECOVERY

HYDRATRD LARVAE EXPOSED TO --4o”c

OF P -LY

WITH OR WITHOUT

No. of larvae

Hours in CyFyE,

PRRTRRATMENT

Approximate Hours at moisture content - 10°C (%I

5 5 5 5 10

4 4 6 6 4

65 65 60 60 65

10 10 10

4 6 6

65 60 60

* All died when incubator overheated.

2 2 2

Hours at -40°C

-

-

(day4

26 24 26 24 24

5’ 10

;: 26

10 10 10

2

No recovered Max. life

5 5

3* 3* 3* 3* 6 adults emerged 2 adults 7 adults 4 adults

160

J. P. LEADER

DISCUSSION The fact that Polypedihm is able to recover most of its faculties after cooling when fully hydrated to a temperature of - 190°C suggests that at least the musculature of the gut and body wall and the nervous and sensory systems are able to tolerate this treatment. Death appears to result from the destruction of the fat body. Since, however, Polypedilum can tolerate total dehydration, and hence presumably an extreme electrolyte concentration in the l&er stages of this process, it cannot be assumed that this is the cause of freezing damage in these experiments. It is equally unlikely that the damage is caused by the mechanical effects of ice formation. SALT (1961a) appears to suppose that supercooling is an inalienable property of all insects. In the absence of any suitable nucleating sites, supercooling will occur, crystallizationbeing governed by a composite probability/time relationship which is strongly temperature-dependent (SALT, 1958). When crystallization does occur, a large proportion of the body water will freeze rapidly, as a single though ramifying crystal, apparently unimpeded by cell membranes, and such an event may well result in mechanical disruption of cells. SALT(1958) has shown that the fat body of Eurostu soZ~dugintican tolerate such treatment and has later suggested (SALT, 1961a) that such tolerance may be in some way related to the presence of glycerol. The possibility of supercooling, however, is decreased if the rate of cooling is slow and if nucleating sites, e.g. contact moisture or food in the gut, are present. These factors make it unlikely that under natural conditions all insects exposed to freezing temperatures will supercool to any great extent. SCHOLANDER et aZ. (1953) have published an account of a freezing-tolerant chironomid la&a, at present unidentified, which rarely supercools : at temperatures .beiow freezing the body water is in a temperature-dependent equilibrium with a fixed percentage of ice. A few measurementsmade on Polypedilum indicate that this too does not supercool, at least when cooled slowly in water. The presence of food in the gut appeared to make no difference: starved animalsbehaved similarly to feeding specimens. Observations made on larvae cooled slowly on a microscope stage showed a gradual formation of ice, although the point at which the fat body disrupted could not be seen visually. MERY~C~AN (1957, 1960) has described a sequence of events which occur during slow cooling in the absence of supercooling. Freezing would begin in the extracellular fluid, causing a rise in the osmotic pressure outside the cells that will be balanced by a flow of water from the intracellularcomponents. As the temperature drops more water will freeze and this process will continue. The cells will become dehydrated, and freezing will be confined to the intercellular spaces. As MAZUR (1960) has pointed out, this process will only continue satisfactorilyas long as the rate of diffusion of water from the cells is not exceeded by the rate of formation of ice, which is dependent on the rate of cooling. If this is too fast, then the possibility of internal freezing is increased. This, it has been claimed by ASAHINA (1959a), is lethal. In Polypedilum, however, the damage caused by freezing is apparently identical to that caused by too rapid drying. This suggests that water is being lost from the cells so rapidly that it is either causing the disruption of any

FREEZINGOF LARVAEOF POLYPEDILUM(CFJIR~NOMIDAE)

161

existing cytoplasmic organization or preventing the establishment of a stable dehydrated configuration rather than that the rate of diffusion is too slow. Such a situation is analogous to the process which LEVITT(1960) has claimed to occur in plants. Little is known about the state of dehydrated cytoplasm in those animalswhich can tolerate complete loss of water. HICKEFWELL (1917) has described cells of rotifers fixed at different times during rehydration, and MAY (1950, 1951) has described similarly treated cells of tardigrades, but it is doubtful if these observations are of much value since they provide little information about the actual state of the dehydrated cellular constituents, and they used aqueous fixatives, which must influence the result to some extent. As &ILIN (1959) has stated, the electron microscope might be expected to provide information of value on this point. It does seem likely that when water is removed gradually from an animal capable of cryptobiosis, the cytoplasm will retain some kind of organized state, rather than become an amorphous mass. HINTON(1960a) has shown that dried PoZypediEzlm larvae will withstand immersion in absolute alcohol for several hours, even an animal damaged so that cell membranes were exposed showed a temporary recovery when wetted. In addition, the impression has been gained both by the writer and by H~NTON(1960a) that within the range of drying speeds permitting complete recovery, the slower the rate of drying, the more rapid was the subsequent uptake of water. 30th these observations seem to suggest that the dehydrated cytoplasm has some degree of organization, and the second that this is influenced by the rate of removal of water-* It would appear, therefore, that the results obtained tend to agree with the concept of freezing damage advanced by LEVITT(1960), namely that freezing damage is the result of disruption of protoplasmic bonds caused by stresses set up by removal of water. Protection against freezing damage is provided by partial removal of water, as is already known for plants (AKERMAN, 1927). It is interesting to note that A&NINAand AOKI(1958b) found that the fat body cells of Cnidocampa fluvesca were sometimes damaged by freezing and it seems that these cells may be peculiarly susceptible to freezing damage. This may be related to the large size of these cells. Pulypedthn is not, therefore, freezing tolerant when fully hydrated, and this does not favour LOVELOCK’s hypothesis that it is the rise in electrolyte concentration on freezing which kills cells. However, Po2yped&m vanderplanki is unique among insects in that its larva is capable of cryptobiosis, and it is possible that LEVITT’Stheory of freezing damage is only applicable to those animals which are capable of withstanding the adverse effects of a rise in electrolyte concentration in their extracellular fluid. Acknawledgements--I

wish to expressmy gratitudeto Dr. H. E.

arranging a supply of animals from Dr. M. W. assistance in the preparation of this paper.

HINTON both for SEZRVICE and for his helpful criticism and

162

J. P. LEAIXIR REFERENCES

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OF

POLYPEDILUM

(CHIRONOMIDAE)

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