Insect Cold Hardiness: Freezing and Supercooling-An Ecophysiological Perspective

REVIEW

INSECT COLD HARDINESS: FREEZING AND SUPERCOOLING-AN ECOPHYSIOLOGICAL PERSPECTIVE

J. S. BALE Department of Pure and Applied Zoology, University of Leeds, Leeds LS2 9JT, England

(Received 22 October 1986; revised 10 March 1987)

INTRODUCTION hardiness, Salt (1961) states, “Insects hibernating in An earlier review in this journal (Baust and Rojas, cold regions are generally able to withstand fairly low 1985) encouraged investigators to “critically reas- temperatures for long periods of time. Under natural conditions, the only mortality directly attributable to sess” much of the “generally accepted dogma” which temperature is from freezing.” From this founding characterises research on insect cold hardiness and to hypothesis has arisen the classification of insects as undertake an assessment of the “founding hypoth- freezing tolerant or intolerant, the concept that super- eses” of the subject. In their contribution Baust and cooling is the only protection against freezing for Rojas considered the factors which may influence freezing-intolerant species, and the assumption, that the classification of a species as freezing tolerant since freezing occurs at the limit of supercooling, the or intolerant (supercooling point, optimal cooling/ supercooling point is a measure of the lethal limit of warming rates, state of adaptation and methods of low temperature. This review considers the meth- determining survival) and summarized the obser- odologies of research on freezing-intolerant insects vations which challenge the consensus view which and examines the theories and conclusions which identifies the gut as the probable prime site for -ice currently prevail. Can the cold hardiness of insects be nucleation in freezing-intolerant species. adequately described in terms of two strategies both The likelihood of death for an individual insect of which are concerned only with the formation of from the effects of cold depends on (i) the cold ice? Is freezing, which is avoided by supercooling, hardiness of the specimen and (ii) the temperatures universally the most damaging effect of low tem- and periods of exposure experienced in the over- perature? How effective is supercooling in protecting wintering site. The interaction between these two insects in winter? Do insects survive in nature as long factors will determine the proportion of a population as their supercooling ability exceeds the coldest days that lives or dies. it is important to recognise that the of winter? Is chill-injury fatal only after prolonged term cold hardiness refers to the combined attributes exposure? In short, where does the theory end and the required by an insect to overcome the various delete- reality begin? rious effects of low temperature. Viewed from an ecological perspective insect cryobiology is therefore concerned with all the events and processes governed STRATEGIES OF INSECT COLD HARDINESS by low temperature which influence and ultimately determine survival or mortality in the natural envi- It is evident from a comprehensive bibliography of ronment. In practise, research over 50 years has reviews and research papers on low temperature and concentrated on the physiological and biochemical insects (Baust et al., 1982; Lee et al., 1986) that the mechanisms of surviving or avoiding freezing while terms cold hardiness and cold tolerance refer only to largely disregarding the possibility that for some or mechanisms associated with the survival or avoidance many species (studied or unstudied) other injurious of freezing; furthermore, cold hardiness and super- effects of cold may be a more important threat to life. cooling ability are virtually synonomous when applied Additionally much of this work on cold hardiness has to freezing-intolerant species. Within these restrictive been based on laboratory temperature regimes which definitions (which are rarely acknowledged by au- take no account of ecological aspects such as behav- thors) the strategies of insect cold hardiness can be iour, overwintering site microclimate and the inter- summarized in this form: insects are either freezing action of mortality factors in nature (Danks, 1978). tolerant or intolerant depending on their ability to The aim of this review is to focus attention on one survive the formation of extracellular (and possibly of the fundamental principles of insect cold hardiness intracellular) ice (Salt, 1936, 1961). Freezing-tolerant which has largely determined the pattern of research species often contain ice-nucleating agents (proteins for the last half-century, namely that freezing is the or peptides) which are normally only present in most important lethal effect of low temperature and winter and ensure protective extracellular freezing at must be tolerated or avoided to ensure survival of the high sub-zero temperatures (Zachariassen and Ham- species. In a review of the principles of insect cold mel, 1976; Zachariassen, 1980, 1982; Duman, 1980), I.P.33,11--A 899 900 J. S. BALE

and polyhydroxy alcohols which function to limit mid-winter sample suggests that the response is even freeze damage (Duman and Horwath, 1983). Freez- more rapid with all changes occurring within about ing is lethal to freezing-intolerant species; this event 7 days (Bale and Smith, 1981). is avoided by supercooling in which the body tissues Cryoprotectant analyses indicated higher levels of and fluids are maintained in the liquid state below glycerol and trehalose in weevils collected in winter their equilibrium freezing point (Salt, 1936). Seasonal or acclimated in the laboratory than in samples increases occur in the concentration of one or more maintained at higher temperatures. When adults were polyols which extend the inherent ability to supercool collected in March and tested in a wet and dry (Baust, 1981, Somme, 19X2), and the activity of condition, surface moisture reduced supercooling by antifreeze proteins (Duman, 1977) which lower the more than 13” from - 23.6 to - 9°C and all individ- freezing point of the haemolymph relative to its uals were equally affected. Feeding in spring begins as melting point and may act to stabilise the supercooled soon as the new growth of beech leaves becomes state (Zachariassen and Husby, 1982). Similar cryo- available and this has a marked and immediate effect protective antifreezes and antifreeze proteins are on supercooling, which is reduced by about 6-7” from found in both freezing-tolerant and intolerant insects; the end of wintet level (from - 22.6 to - 16.8”C). So, the characteristic difference between the strategies is in terms of acclimatisation, acclimation, cryo- the winter loss or masking of the nucleators in protectants, surface moisture and food effects, this supercooling-dependent species and the synthesis or weevil exhibits all of the typical features of a freezing- unmasking of nucleating agents in freezing-tolerant intolerant insect. species (Duman, 1982; Baust and Rojas, 1985). The introduction to the first publication on cold hardiness by Salt, “Studies on the freezing process in insects” (1936) described the effects of low tem- FREEZING INTOLERANCE perature on the distribution and abundance of insects “Ability to supercool is the only protection against in an applied context. Preparations for the control of freezing that most hibernating insects possess; it is a pest species could be made more intelligently if direct measure of their cold hardiness. Supercooling, information was available on the ability of the insect therefore, is the dominant factor in the winter sur- to resist winter temperatures. vival of such insects, and anything that influences it Routine monitoring in Britain of a number of becomes of importance” (Salt, 1958). Many possible aphid species of economic importance, which reduce influences on supercooling have been investigated by crop yields by feeding damage or transmission of Salt, including time (1950, 1966), feeding (1953) viral diseases, has shown a clear link between winter moisture content and temperature (1956), glycerol temperatures, survival of active stages (adults and (1957), cooling rate (1966a) and the action of nucle- nymphs) and the timing of spring migrations @Vat- ators (1966b, 1968). The general characteristics of son et al., 1975). The cold hardiness of a number of freezing-intolerant insects based on these pioneering aphid species has been assessed by a range of super- studies and many similar reports have been reviewed cooling experiments similar to those adopted for by %xnme (1982). It should be noted that these the beech weevil. No aphid survived cooIing below features of cold hardiness are expressed only in terms the supercooling point. The mean supercooling point of an ability to supercool: of adults and nymphs reared under favourable laboratory conditions, or collected from host plants in the 1. There is a seasonal variation in cold hardiness field for much of the year, showed a consistent which is at a maximum in winter. pattern of supercooling with little variation between 2. Increased cold hardiness is related to the accu- or within age groups (Table 1). When maintained at mulation of cryoprotective substances. 5°C for up to 4 weeks there was no shift in the mean 3. Production of cryoprotectant antifreezes may be supercooling points of the aphids, Myzus persicae or induced by low temperature. Sitobion avenae @‘Doherty and Bale, 1985; Knight 4. Contact with surface moisture can reduce super- and Bale, 1986). Few aphids, adult or nymphal were cooling by inoculative ice formation through the killed when surface moisture froze on the cuticle, but cuticle. mortality increased with repeat toolings (Knight and 5. Feeding reduces supercooling through the action Bale, 1986). The most counterintuitive result was of gut nucleators. found in supercooling profiles of mid to late winter If this albeit simplified account is accepted by an samples when a proportion of the population showed investigator and experiments on previously unstudied a marked reduction in supercooling with individual species are designed according to the same methods, supercooling points as high as - 5°C. Such individu- then the results obtained can indeed “reiterate and als would be at risk of an instantaneous freezing intensify” (Baust and Rojas, 1985) the established death but only 520% of the population fell into this view. For instance there are clear changes in the cold category and the mean supercooling point of the hardiness of the beech leaf mining weevil Rhyn- remainder was consistently below -20°C (O’Doherty chaenus fagi with mean supercooling points of - 15.4 i 0.6”C on emergence in July, - 19.3 5 0.7”C in autumn and -23.1 + 0.4”C in mid-winter (Bale, Table 1. Supercooling points (mean + SE) of aphid species 1980). The seasonal azmatisation which occurs in Species First instar Adult nature can be induced in the laboratory. An early Myzus persicoe - 21.4 + 0.2 -26.0 f 0.2 winter sample of adults shows an acclimation re- Aphis fabae -26.3 + 0.6 -25.3 + 0.2 sponse of 5-6” from -19.5 to -25.3”C which is Sitobion avenue -26.9 + 0.2 -25.5 It 0.2 -23.0 + 0.3 complete within 2 weeks. An experiment with a Breuicoryne brassicae -26.9 + 0.3 Review 901 and Bale, 1985). So, when studied in terms of super- ration of “field studies” is usually only a sampling cooling responses, the features of aphid cold hardi- exercise in which insects are taken back to the ness can be summarized in these terms: laboratory for supercooling or cryoprotectant analyses. As Storey (1984) observed with reference to 1. There is no seasonal increase in cold hardiness the dominant areas of cold-hardiness research, and some aphids lose supercooling ability in winter. physiological ecologists have examined hundreds of 2. There is no acclimation response at low tem- species determining freeze tolerance/intolerance, perature. supercooling/freezing/melting points of body fluids, 3. Surface moisture inoculates a variable propor- and type/amount of cryoprotectant, while biochem- tion of a population. ists and biophysicists have concentrated on problems 4. Feeding (on sap) does not reduce supercooling. such as the structure of water in cells, cryo- These features largely contrast the “typical” char- enzymology and the protein chemistry of antifreeze acteristics of other so-called “freezing-intolerant” molecules. Immediately apparent from Storey’s accu- insects and while it is of scientific interest to identify rate summary is the absence of comparative studies a group of insects which are apparently atypical in which relate laboratory estimates of cold hardiness their cold hardiness it also suggests that some caution (e.g. supercooling) to environmental temperatures is required in the use of supercooling date to predict and some measure of survival/mortality during the survival or death at low temperature, particularly winter season; and unless this is done there is no basis under field conditions. for assessing the requirement or efficacy of the pro- posed cold-hardiness strategy in nature, or making the transition from hypothesis to fact. Paradoxically ECOLOGICAL VALIDITY OF SUPERCOOLING many studies by population ecologists have identified MEASUREMENTS winter cold stress as an important density- The primary use of supercooling points is to pro- independent regulating mechanism of insects, with- vide a comparative index of cold hardiness between out investigating the freezing-tolerant/intolerant na- different stages of the life cycle, species or seasons. ture of the species or considering the value of indices Somrne (1982) has compiled a detailed list of the of cold hardiness such as the supercooling point in lowest mean supercooling points for freezing- predictive models. Against this background a more intolerant eggs, larvae, pupae and adults in different comprehensive research protocol is required to inte- orders of insects and Block (1982) provides similar grate knowledge on the physiology, biochemistry and data for invertebrate poikilotherms across many ecology of insects at low temperature, allow labora- phyla. In theory all of these “freezing intolerant” tory assumptions and hypotheses to be examined and species will die from the effects of freezing following tested in the field, and expand our conception of a momentary exposure below their supercooling insect cold hardiness beyond the process and effects point. However, the established view that the super- of freezing. cooling point is a direct measurement of the lower lethal limit (instantaneous low temperature death INTEGRATED STUDIES ON COLD HARDINESS point) is only tenable for those species (or individuals) which (i) die from the effects of freezing, and (ii) An attempt is made here to describe a research remain alive (and capable of recovery) down to the protocol which combines field and laboratory meth- temperature at which freezing occurs. These are ods with particular reference to the determination of critical requirements which must be satisfied to vali- the freezing status of insects and the ecological date the use of the supercooling point as a direct interpretation of supercooling data in apparently measure of cold hardiness and of the lower lethal freezing-intolerant species. The areas of study out- limit of freezing-intolerant insects. For most species lined below have been applied to the grain aphid currently class$ed as freezing intolerant, survival in Sitobion avenae and appropriate results are included the supercooled state is an untested assumption both in the relevant sections. during the time-course of a supercooling experiment and in longer-term exposures; and far from being the Determination of freezing status starting point of an integrated study of cold hardi- Cooling of specimens at 1°C min-’ to the super- ness, supercooling is often the only aspect that is cooling point followed by an assessment of survival investigated. It is therefore not surprising that. the after warming (or thawing) will provide a preliminary literature provides few answers to these basic ques- indication of the freezing status of the species and tions, even for “model” species which have been allow comparisons with much of the existing litera- studied over many years by different investigators: ture. The influence of optimal and sub-optimal cool- What temperatures do insects experience in over- ing and warming rates on the designation of a species wintering sites? What proportion of the population as freezing tolerant or intolerant together with the dies in winter from the effects of cold? How does low difficulty of selecting an appropriate time interval temperature kill insects? Is the supercooling point the after exposure to assess survival has been discussed true lower lethal limit? Clearly from an ecological by Baust and Rojas (1985). In an ecological context viewpoint these questions are of fundamental im- survival per se is unimportant unless the individual portance. The lack of information in key areas arises can develop normally, and as an adult, reproduce and because insect cryobiology is primarily a laboratory so contribute to the next generation; although indi- science which utilises physiological and biochemical viduals which recover but are incapable of re- methods to investigate an ecological relationship production may still contribute to the predator-prey between insects and low temperature. The incorpo- balance in the ecosystem. 902 J. S. BALE

Supercooling data is normally presented as the -20°C) and the remaining six winters were “aver- mean value f the standard error. The range of age” with the coldest days between - 10°C and supercooling points is also relevant information since - 15°C. Descriptions of winters as “mild”, “average” a proportion of a population may be at risk of a and “very cold” are relative and will vary in usage freezing death even though the mean supercooling with different investigators and climates; additionally point exceeds the lowest winter temperatures the most deleterious effect of cold on one species may (Somme, 1982); additionally the distribution of su- be the frequency of exposure in the supercooled state percooling points may be skewed or bimodal (Block, (total number of,frosts) whereas for others the critical 1982a). factor may be the lowest temperature experienced The mean supercooling point of laboratory popu- throughout the winter. In the nine year records there lations of 5’. auenae varied from -27.0 f 0.2”C in was considerable variation in both these variables first instars, to -24.2 f 0.3”C as adults. There was although on no occasion did the temperature fall no acclimation in supercooling at 0 or 5°C and the below the mean supercooling point of the over- mean supercooling point of the majority of a field wintering population of Sitobion avenue. population remained below -20°C throughout the Microclimate of overwintering sites. Winter habitats winter (Knight and Bale, 1986). There were no sur- of insects are often described in terms of some single vivors below the supercooling point and S. auenae in standard measurement of air temperature whereas common with other aphids studied so far was the microclimate temperatures of the overwintering classified as freezing intolerant. site may be markedly different, above or below the air value. For instance beech weevils (Rhynchaenus Annual, seasonal and microclimate temperatures fagi) overwinter in the leaf litter of the forest floor The winter season imposes physiological stress on and the aerial canopy of conifers (Bale, 1981); the insects but the level of this effect varies temporally grain aphid overwinters on young wheat and barley Y-1 f plants O-10 cm above the soil surface. The vertical and spatially at any one moment in time. Variation; stratification of temperature on the coldest night in are seen in both the total number of frost days and the years 1975-1984 in northern England and on a the minimum temperatures of winter. normal cold night that would occur many times in Annual variations in frost days and minimal tem- most winters is shown in Fig. 1. On both nights the peratures. In nine successive winters (1975-76 to grass minimal temperatures were 3-5” colder than 1983-84) in northern England the total number of would occur at the top of a 1.5 m high tree, although air-frost days per winter varied from 50 to 100 with a frost of - 18.4”C at ground level did not penetrate a mean of 67 Ifr 6. Equivalent figures for ground level 1Ocm into the soil. How relevant are the air tem- frosts (grass minimal temperatures) ranged from 110 peratures of - 13.1 and - 1.5”C to the level of cold to 160 with a mean of 140 k 5. When compared on experienced by the weevil under the leaf litter or the the basis of lowest temperatures, one winter aphid on a cereal seedling? This meso- or microscale (1975-76) could be described as “mild” when there inversion of temperature such that the ground is was no air or ground frost below - 10°C two winters colder than the air above it, is caused by nocturnal 11978-79 and 1981-82) were “verv cold” with 6 davs radiant cooling and accompanying cold-air drainage with a ground-level frost below ‘- 15°C (but above (Wellington and Trimble, 1984). In hillside forests,

Height or depth (m) 1.5 -

0.57 +2.0° +3.7O

Fig. 1. Vertical stratification of temperature in winter (“C). Review 903

30 A population 1 A decline nymphs mature and reproduce 20 . % plants with aphids

L September October November December January

Fig. 2. Population sequences of Sitobion avenm on winter barley in winter 1984-85. Aphid density declined from 30% to 1% of plants infested (1 m to 30,000 ha-‘) from A to C when the lowest grass minimal temperature was -8.I”C between A and B and -9.7”C between B and C. The mean supercooling point was < - 20°C (from Knight et al., 1986). the temperature variation between the vertical strata Mortality in relation to supercooling ability can be so large that the spring abundance and Integration of the information on S. avenue damage potential of some pest species varies at showed that (i) the mean supercooling point through- different elevations according to the differential mor- out the winter was below -2O”C, (ii) temperatures tality of the overwintering stages (Tenow, 1975). close to -20°C were extremely rare in the cool Clearly, the vertical distribution of overwintering temperate winters of Britain and did not occur during sites together with the insulating properties of host the study period, but (iii) aphid mortality was very plants and leaf cover at the soil surface are important high in the field at temperatures much above the considerations when assessing the level of cold experi- known supercooling point. When the field mortality enced by insects in winter. of a species in winter is much higher than would be expected from a knowledge of supercooling capacity and environmental temperatures, the influence of low Population dynamics of insects in winter temperature can be examined in more detail under The most important requirement for the study of experimental conditions. By varying the cooling rate, population changes of insects in winter is an accurate minimal temperature and periods of exposure and sampling method taking into consideration the div- prior acclimation, in the absence of interactions with ersity of overwintering sites occupied by the species wind or precipitation and with the exclusion of and the distribution of the insect (random or aggre- natural enemies, it is possible to estimate the direct gated) in each site. A detailed study of the over- effect of low temperature on the species and separate wintering biology of the grain aphid S. avenue (Mc- time-dependent deaths by freezing above the super- Grath and Bale, in Knight et al., 1986) monitored an cooling point from pre-freeze mortality. aphid population on 100 randomly selected plants of When large samples of S. avenae taken from a 20°C winter barley, from the early stages of colonisation of maintained laboratory population were cooled at 1°C the crop by alates (winged migrants) in autumn, rein-’ to a range of minimal temperatures and mor- through the phases of population increase, peak, and tality assessed at daily intervals after exposure it was decline (Fig. 2). The peak density equivalent to found that (i) mortality commenced at -5°C and l,OOO,OOOaphids ha-’ occurred in mid-winter and there were no survivors at - 15”C, (ii) most aphids declined to less than 30,000 ha-‘, a 97% mortality were fatally injured rather than killed instantaneously over 2 weeks, when the lowest grass minimal tem- and died progressively within 24h of exposure, (iii) perature was - 8.1”C in the first week (approx 50% aphids which died up, to 96 h after exposure neither kill) and -9.7”C in the second week. The mean moulted nor reproduced before death, and (iv) the supercooling point of the same field population was adult L’T,, 24 h after exp’osure was - 8.1 “C whereas below -20°C. A similar study in an adjacent field the mean sunercoolina I I.noint was -24.2”C (Knight et indicated an abrupt increase in mortality when ‘the al., 1986). In similar experiments with Myzuspekxe temperature decreased to -8°C (Knight and Bale, (mean supercooling point -25”Q the LT, of adult 1987). In nature, most aphids are killed in winter at aphids maintained at 20,lO and 5% throughout their temperatures above their inherent ability to super- nymphal development and examined 3 days after cool. Extensive supercooling alone is not necessarily exposure was -6.9, - 11,l and - 11.6”C respectively an indicator of a cold-hardy insect, nor a guarantee (Bale et al., 1987 and unpublished data). Acclimation of winter survival. therefore depresses the LT,, although in all cases 904 J.S. BALE

there was 100% mortality above the mean super- From this information it appears that under nor- cooling point. mal conditions the phloem food source of aphids in However, the relationship between the pre-freeze cold-hardy plants remains unfrozen throughout win- mortality of aphids observed in the laboratory and ter. If inoculative freezing by a “phloem bridge” is the decline in winter of field populations of the same important in aphids it would seem to require the species at temperatures above their known super- phloem sap to freeze (or be nucleated) within a cooling point, do not necessarily have a common phloem cell or in the stylets, at an elevated tem- origin. Supercooling points and LT5,, values are de- perature compared to that at which it freezes in the rived from aphids which may have recently fed (on aphid gut, since feeding aphids removed from plant plant sap) but are removed from their host plants for tissue consistently supercool to below -20°C. Inocu- the experiments. In nature, unlike most overwintering lation may occur from intercellular ice formation insects, aphids normally remain in feeding contact around the stylet bundle since the stylets of aphids are with their host plants throughout winter, but may thought to penetrate the plant mainly via intercellular have to move to new leaves or plants a number of routes in order to reach individual phloem cells [sieve times to survive (Harrington and Cheng, 1984); addi- tubes] (Pollard, 1973). However the intercellular path tionally, the imbibition of plant sap is not a con- is lined by a secreted salivary sheath and this may act tinuous process although the stylets are not usually as a barrier to inoculative freezing. withdrawn between periods of feeding at the same To investigate the potential for inoculative freezing site. Powell (1974) observed that green spruce aphids from feeding, a culture of M. persicae was reared at (Elatobium abietinum) in feeding contact with their 10°C throughout their nymphal development to pro- host, were killed at temperatures above their inherent duce an acclimated population. Newly moulted supercooling level when the spruce needles froze, adults were allowed to establish feeding sites on leaf whereas detached aphids survived when cooled to the discs of oil seed rape (a winter host plant) at the same same temperature. It was suggested that during feed- temperature. When cooled at 1°C min-’ the leaf discs ing, a “bridge” of phloem sap is formed from the froze consistently between -5 and -6°C. The LT,, plant vascular tissues via the stylets to the aphid gut values for adult aphids cooled during feeding on leaf resulting in inoculative freezing of the insect. This discs were -16.6, -13.6 and -12.5”C, when as- phenomenon is a possible alternative explanation for sessed 24, 48 and 72 h after exposure; the equivalent the high mortality in winter populations of S. avenae values for acclimated aphids cooled in isolation from (Knight et al., 1986) and M. persicae (Harrington and plant tissue were - 15.6, - 11.4 and - 1l.l”C re- Cheng, 1984), but would have to operate at tem- spectively (Bale, Harrington and Clough, un- peratures above the pre-freeze event to have any published data). In combination with previous ex- importance under field conditions. periments, these results (i) confirm the occurrence of Crop plants on which aphids feed in winter must pre-freeze mortality processes in aphids, including be sufficiently cold hardy to survive at low tem- acclimated populations (ii) indicate that inoculative perature and continue their growth in spring; such freezing from feeding does not increase mortality plants would freeze many times in a normal tem- above that resulting from the direct effect of cold and perate winter. The process of extracellular freezing (iii) suggests a largely artefactual irrelevance for the tolerance in plants has been reviewed by Levitt supercooling ability of aphids in nature. (1980). Ice normally crystallizes first at a few nucleation sites in the main xylem vessels because of their Life and death at low temperature: physiology and large diameter and dilute sap. Ice from the vessels biochemistry spreads throughout the plant via the intercellular The inclusion of physiological and biochemical spaces but is prevented from inoculating the contents analyses at this stage in a study reflects the opinion of living cells by the lipid plasma membrane. If the of the author that until the causes of low-temperature temperature continues to fall at a gradual rate, the death @e-freezing, freezing or post-freezing) and intercellular ice masses increase in size as cell water their relative importance have been established, it is diffuses progressively through the semipermeable not possible to plan experiments on a rational basis lipid plasma membrane resulting in a “freeze concen- to identify mechanisms and strategies of survival. tration” of the cell sap including the phloem. Intra- Intuitively most investigators would screen insects for cellular freezing of the phloem is lethal. cryoprotectants, antifreeze and nucleator proteins, or The extent of ice formation in a plant and the bound-water content, whereas for some species, par- likelihood of intracellular freezing is governed largely ticularly those which suffer pre-freeze injury, it would by the rate of cooling relative to the rate of diffusion be more informative to target research at the mem- of water from within the cell to ice loci in the brane, enzyme or mitochondrial level. Storey (1984) intercellular spaces which is limited by the perme- recognises this distinction as adaptations for low- ability of the cell membrane. In nature, the freeze is temperature preservation and low-temperature func- likely to be so slow that the ice front will not spread tion and gives a comprehensive account of appropri- throughout the plant body. At the upper limit of ate methodologies including new technologies such as “slow” cooling, the ice front spreads throughout the nuclear magnetic resonance. It is important to recog- plant and contacts all the cells; at faster rates the nise that death can occur through a failure in any one diffusion of cell water to extracellular ice cannot process and that species inhabiting very cold climates occur with sufficient speed to increase the concen- have probably evolved a complex array of adapta- tration of the cell contents as the temperature falls so tions for both function and preservation, only one of that eventually there will be spontaneous intracellular which, the tolerance and avoidance of freezing, has freezing. yet been studied in any detail. Review 905

During cooling of aphids at 1°C min-’ in a system Clearly the tolerance/intolerance categories are capable of resolving temperature changes to O.l”C ecologically meaningless for tropical insects which die (Bale et al., 1984) no “physiological” event was at temperatures above 0°C because they are unable to observed in the cooling curve in the mortality range adjust their metabolism to enter a dormant state, and from -5 to - 15°C only the supercooling point in nature never experience sub-zero temperatures. rebound at around -24°C. In the first use of a The classification is also inappropriate for species differential scanning calorimeter to study the cold from colder climates, if some or all of an over- hardiness of whole insects, Knight et al. (1986) wintering population are killed by cold, above the detected a pre-freeze exotherm occurring consistently supercooling point and without freezing. within the mortality range of S. auenae (usually The integrated, ecophysiological approach to the between - 7 and - 11“C), corresponding to less than study of insect cold hardiness recommended in this 1% of the heat released during freezing. The exo- review relates a laboratory determined index of cold therm appears to be of direct biological origin, tolerance (supercooling) to known winter micro- irreversible and non-repeatable, since there was no climate temperatures, and assessments of survival endotherm on warming and the exotherm was never (which ideally should include different combinations observed in repeat cooling of the same specimen. At of cooling/warming rates, exposure periods and min- this stage it is not known whether the exotherm is imal temperatures in the laboratory), and an accurate related to the cause of pre-freeze cold death in aphids sampling procedure to monitor population density in or is a coincidental event occurring at the same the field. By this approach it becomes apparent temperatures, although recently, individuals of M. whether (a) the species is genuinely freezing intolerant persicae have been observed to survive through the and survives to the supercooling point, or (b) a exotherm and moult and reproduce when returned to proportion of the population is killed or fatally normal culture conditions (Bale et al., 1987). There is injured before freezing, and (c) at what temperatures no doubt however that aphids die (or are fatally and under what conditions such mortality occurs and injured) before they freeze and the possible causes varies. Additionally, a comparison of laboratory and could include a thermotropic phase transition in a field survival, provides an estimate of the winter membrane, a cold inactivation of proteins, or the mortality which is attributable to factors other than decoupling of normal metabolic processes. cold. With this information it is also possible to plan a more rational sequence of physiological and biochemical experiments. For instance, is it logical to DISCUSSION analyse the spectrum and concentration of antifreezes The classification of insects as freeze tolerant or in an insect before assessing the ability of the species intolerant is not redundant; nor is it suggested that to survive above the supercooling point? many species currently regarded as freezing intolerant Same recent studies on insect cold hardiness have will be found by future research to die in large combined physiological and biochemical methods in numbers above their supercooling point. It is unlikely the laboratory with field ecology to assess cold toler- that alpine and polar insects could survive winter ance and mortality and the examples selected below microhabitat temperatures between - 20 and - 30°C exemplify the benefits of this interdisciplinary ap- without extensive and effective supercooling and the proach. The goldenrod gall moth Epiblema scud- absence of any significant pre-freeze mortality or deriana overwinters as mature larvae in stem galls on long-term chill injury. It is equally unlikely that the goldenrod plant inNorth America. Mean suP;er- species from such very cold regions should have cooling points of the larvae decreased from - 13.9 in evolved complex triggering mechanisms for the syn- the early autumn to stabilise between - 35 to -4Q”C thesis of ice-nucleating agents or antifreeze cryo- throughout the winter; the lowest winter temperature protectants, only then to die in large numbers was -26°C. There was a 90% successful pupation from thermal processes unrelated to the tolerance or and emergence of a field population in spring and avoidance of freezing. Nevertheless the tolerance/ 100% survival of winter larvae maintained at - 18°C intolerance classification, originally based on strate- for 197 days (Rickards et al, in press). Clearly this gies apparent in insects from extreme climates (for species survives at low temperatures by virtue of which it may be essentially correct) has gained a extensive supercooling which is accompanied by a general acceptance among investigators as a descrip- winter decrease in body water content and an increase tive code, and a basis for experimentation on insects in glycerol concentration to 18.7% of the fresh from many taxa which inhabit widely differing cli- weight. mates ranging from the cool temperate to the mar- The collembolan Cryptopygus antarcticus and, the itime Antarctic or the northern tundras. The hetero- cryptostigmatid mite Alaskozetes antarcticus are rela- geneity of form and function in insects combined with tively abundant species in the, very limited arthropod the diversity of their winter habitats predisposes any fauna of the Antarctic. The mean supercooling point common theory of cold hardiness to be oversimple of Cryptopygus decreases seasonally from -5 to and restrictive. The lethal effect of low temperature - 10°C in summer to about -25°C in winter, associ- on insects cannot be described in terms of a single ated with the evacuation of gut contents and its partly process, freezing, and to do so places a rigid concep- dehydrated condition in winter, and an increase in tual framework around experimental protocols and total potential cryoprotectant concentration. In a allows untested hypotheses and generalisations to standard test exposure to - 15°C for 24 h, the low prevail; none more so than the assumption of “sur- survival of summer field samples (<50%) increased vival by supercooling” in species known to be dead to more than 80% in winter populations (Block, when frozen. 1987, in press). Similar and more consistent increases 906 J. S. BALE in supercooling were found in adult Alaskozetes, and 15-20”, high survival in short exposures to sub-zero winter collected mites with a mean supercooling point temperatures, but considerable mortality overwinter of -30°C showed a 52% survival after 250 days at in the field for reasons as yet unknown (Crypto- - 15°C and 73% survival after 100 days at -20°C pygus), (c) species in which the lower lethal limit is (Cannon, 1987). Again these studies provide evi- consistently above the supercooling point, increases dence for the adequacy of the supercooling strategy seasonally by only 2” and overlaps soil surface tem- even after prolonged exposures to low temperatures. peratures such that behavioural avoidance may be However, in the light of an 80% survival of Crypto- more important than physiological tolerance of cold pygus in the laboratory in a 24 h exposure to - 15°C as a mechanism for survival (Orchesella and Tom- it is interesting to note that Block (1982b) has ocerus), and (d) species in which there is no seasonal recorded overwintering population declines of this increase in supercooling, the lower lethal limit is species of approx 22% in a moss turf (where the markedly above the supercooling point, population density was higher) and 79% in a moss carpet. These declines overwinter of 90-99% are common, and substantial mortalities may be related to freezing in survival depends on a diversity of favourable over- the supercooled state, chill injury, action of preda- wintering sites (e.g. glasshouses) and an additional tors, or other physiological stresses which occur with and supposedly “more cold hardy” overwintering the changing season, and also show how population state, the egg (Sitobion and Myzus). Interestingly, density and mortality can vary in different over- although aphid eggs supercool to -35°C in winter wintering sites. (Somme, 1969; James and Luff, 1982), and are re- A contrasting pattern of cold hardiness and strat- garded as the most cold-hardy stage of the aphid life egy for survival has been found in the temperate cycle, Leather (1980, 1981) has recorded winter egg Collembola Tomocerus minor and Orchesella cincta. mortalities in the absence of predators, of 35-50%, The mean supercooling point of the two species in although temperatures were always l&25” above the winter was - 11 and - 14°C respectively; however in supercooling points. The variation seen between these a 24 h exposure to a range of minimal temperatures, species in seasonal increases in supercooling, minimal although there was a seasonal increase in “cold supercooling points, lower lethal limits, field and hardiness”, the LT,, of winter collected samples was laboratory mortalities, environments and over- only -6.5 and -8.7”C, or 4-5” above the mean wintering sites, argues strongly against the proposi- supercooling point of each species (van der Woude tion of any common theory of cold tolerance in and Verhoef, 1986). These differences have been insects. attributed to the time-dependent nature of freezing Studies on the temperate Collembola and aphids during extended exposure (24 h) in the supercooled should not be viewed in isolation on the grounds that state, but in common with many “time” experiments, the insects are not “typical cold-hardy species”. Typ- there is no evidence that freezing was the cause of ical and atypical are relative terms used to describe death. In such a short exposure it is possible that species which do or do not conform to particular pre-freeze processes acting at relatively high tem- criteria, such as the supercooling characteristics of peratures may be an important mortality factor in “freezing-intolerant” insects; if the criteria are these species. Their limited tolerance of cold com- changed so do the relative proportions of typical and pared to other temperate insects is in any case atypical species. And what is an acceptable definition compensated by the insulation afforded by snow and of a “cold-hardy” insect? Does a species have to leaf-litter cover in winter such that the overwintering undergo a process of cold hardening or acclimation mortalities of adults or juveniles of either species such as the seasonal increase in supercooling capacity sampled from the soil did not exceed 65%. and correlated increases in cryoprotectant concen- The peach potato aphid Myzus persicae shows a tration, to be classed as cold-hardy? If then, 90% of very similar pattern of cold hardiness and winter the overwintering population is killed, is the species decline to that described for Sitobion avenae. The still cold-hardy, or is it partially cold-hardy? In the mean supercooling point of the field population final analysis, only the survivors are suficiently cold remains below -20°C throughout the year hardy. Clearly, what is required at this time is a (O’Doherty and Bale, 1985). In successive winters critical reassessment of current theories and an inte- there were population declines of approx 99.9 and gration of knowledge from different disciplines, taxa 98% when lowest grass minimal temperatures were and climates. - 18.8 and - 11.1 “C respectively, and the under-leaf Temperate aphids and Antarctic Collembola share minimal temperatures of a preferred overwintering at least one common character of cold hardiness; in site (winter cabbage) were only -9.4 and -7.2”C. winter both groups supercool and therefore avoid ice The LT,, of a laboratory population maintained at formation in short exposures to -20°C. However, 20°C was -7.4”C whereas the mean supercooling exposure to temperatures between - 10 and - 15°C point was -25°C (Harrington and Cheng, 1984; Bale kills large numbers of aphids whereas most of the et al., 1987). There is a clear indication from these Collembola survive. Either the Collembola can avoid results of a large-scale pre-freeze mortality in such or tolerate the process which kills the aphids, or they species. are physiologically so different that the process does In order of description these studies reveal (a) a not affect them. Identification of the processes which species which undergoes a winter increase in super- kill less cold-hardy species, which may inhabit cool cooling capacity of more than 20” with a high sur- rather than cold climates, will focus attention on the vival after prolonged exposure in the supercooled adaptive mechanisms which allow species (though state in both the field and laboratory (Epiblema), (b) sometimes few individuals) to survive winter in the a species with a seasonal increase in supercooling of most extreme environments. Freezing-intolerant in- Review 907 sects do not survive at low temperature only by virtue tally important to the development of ecological of their supercooling ability. models of insect populations, prediction of pest out- The observation that cold can lead to injury and breaks and storage of biomedical materials at low death in the absence of freezing is not new although temperature, whether supercooled, frozen or vitrified. some of the earlier reports described mortality at In conclusion it is hoped that this review will encour- temperatures well above 0°C (e.g. Blattella german- age investigators to consider that the study of low- ica, Colhoun, 1954) and may be more relevant to temperature mortality will provide a clearer under- lethal processes in tropical species than those standing of the mechanisms and strategies of survival, affecting insects below 0°C in cool and colder cli- that physiological and biochemical studies on indi- mates. More recently pre-freeze cold death has been viduals in the laboratory must be related to the observed in species and life cycle stages which experi- ecology and performance of populations in the field, ence sub-zero temperatures overwinter (e.g. Mum- and finally, that the tolerance or avoidance of freez- estra cor$gurata, Turnock et al., 1983; Sarcophaga ing by a frequently small number of individuals of a crassipalpis, Lee and Denlinger, 1985; Sitobion ave- relatively limited number of species inhabiting ex- nue, Knight et al., 1986; Myzus persicae, Bale et al., treme environments, is only one of the principles of 1987). The causes of pre-freeze mortality are still a insect cold hardiness. matter of speculation rather than a distillation of evidence from purposeful research. Prosser (1973) Acknowledgements-The research at Leeds University de- states that the causes of death at either high or low scribed in-this paper was funded in part by granti and temperature are “not well understood” and are “cer- studentships from AFRC, SERC and MAFF. I am grateful tainly multiple”, including increased cell membrane for the contributions of Rose O’Doherty, Jonathan Knight permeability, failure to maintain ionic gradients and and Martin Clough and for collaboration with John Baust, Felix Franks. Sheila Mathias and Richard Harm&on. Dr pumps, insufficient energy liberation, decoupling of W. Block, Dr R. J. C. Cannon, Professor K. B. St&ey and enzyme reactions and malfunction of the central Dr H. Verhoef kindly allowed “in press” manuscripts to be nervous system. Similar hypotheses have been ad- viewed. Finally I thank John Baust for his comments during vanced by Hochachka and Somero (1973) who also the preparation of this review. discussed biochemical adaptations to the effects of temperature changes on biological systems (1984), Danks (1978), and Knight et al. (1986), who sug- REFERENCES gested that the lethal cooling inury to aphids may be attributable to changes in membranes, proteins or Bale J. S. (1980) Seasonal variation in cold hardiness of the normal metabolic processes. Apparently, our knowl- adult beech leaf mining weevil Rhynchaenusfagi L. Cryo- edge and understanding of the basal causes of low- letters 1, 372-383. Bale J. S. (1981) Seasonal distribution and migratory behav- temperature death in insects has not advanced greatly iour of the beech leaf mining weevil Bhynchuenus fugi L. over the last quarter-century, although over the same Ecol. Em. 6, 109-118. time period large numbers of species have been added Bale J. S. and Smith G. C. (1981) The nature of cold to the categories of freezing tolerance and intoler- acclimatisation in the beech leaf mining weevil fiyn- ance, based on studies which have frequently mea- chaenus fagi L. Cryoietters 2, 325-336. sured nothing other than supercooling, and usually Bale J. S., O’Doherty R., Atkinson H. J. and Stevenson R. excluded any assessment of mortality, particularly (1984) An automatic thermoelectric cooling method and under field conditions. computer-based recording system for supercooling point In nature as the temperature decrease seasonally studies on small invertebrates. Cryobiology 21, 340-347. Bale J. S., Harrington R. and Clough M. S. (1987) Effect of and daily, insects may pass through a sequence of low temperature on the survhd of the peach-potato potentially injurious or lethal states. The proportion aphid Myl~us persicae. Proceedings Third European Con- of the population that is injured or killed in each state g&s of Entomology, Amsterdam, Part 2, pp.-243-246. will depend on factors such as inherent levels of cold Baust J. G. (198 I) Biochemical correlates to cold hardening hardiness, acclimatisation and rate-based effects. in insects.’ Cryobiology 18, 18&198. Eventually as temperature continues to fall, the only Baust J. G., Lee R. E. Jr and Ring R. A. (1982) The remaining threat to the survivors (which may be a physiology and biochemistry of low temperature toler- high or low proportion of the original population) ance in insects and other terrestrial arthropods: A bibli- will be the process of freezing. The full return on the ography. Cryoletters 3, 191-212. Baust J. G. and Rojas R. R. (1985) Review-Insect cold metabolic investment in supercooling will be realised hardiness: Facts and fancy. J.‘Insect Physiol. 31,755-759. only by individuals which have survived at higher Block W. (1982) Cold hardiness in invertebrate noi- temperatures, and only those individuals are in the kilotherms. Co&. Biochem. Physiol. 73A, 581-593. _ strictest sense, freezing intolerant. If a species with Block W. (1982a) Supercooling points of insects and mites these properties is cooled under laboratory condi- on the Antarctic Peninsula. Ecol. Em. 7, l-8. tions, mortality will be observed to increase with Block W. (1982b) The Signy island terrestrial reference sites: decreasing temperature and probably with increasing XIV Population studies on the Collembola. Br. Antarct. time of exposure at a constant temperature and this Sum. Bull. 55, 3349. pattern of results has been described for many Block W. (1987) Temperature and drought affects on Ant- “freezing-intolerant” insects. arctic land arthronods. Proceedinrrs. Third Eurooean Con- gress of Entomology, Amsterdam, Part 2, pp. 247-250. Identification of the range and complexity of low- Cannon R. J. C. (1987) Effects of low temnerature accli- temperature mortality processes is fundamental to mation on the s&ival and cold tolerance of an Antarctic the development of insect cryobiology, irrespective of mite J. Insect Physiol. 33, 509-521. whether the primary interest is biochemical, phys- Colhoun E. H. (1954) Temperature and acclimatization in iological or ecological. Such information is also vi- insects. Nature, Lond. 173, 582. 908 J. S. BALE

Danks H. V. (1978) Modes of seasonal adaptation in insects. Salt R. W. (1936) Studies on the freezing process in insects. 1. Winter survival. Can. Em. 110, 1168-1205. Tech. Bull. no. 116. Univ. Minn. Aaric. Exn. Stn. Duman J. G. (1977) The role of macromolecular antifreeze Salt R. W. (1950) Time as a facto; in the freezing of in the darkling beetle, Meracantha contracta. J. camp. undercooled insects. Can. J. Res. D. 28, 285-291. Physiol. 115, 279-286. Salt R. W. (1953) The influence of food on cold hardiness Duman J. G. (1980) Factors involved in overwintering of insects. Can. Ent. 85, 261-269. survival of the freeze tolerant beetle, Dendroides cana- Salt R. W. (1956) Influence of moisture content and tem- densis. J. camp. Physiol. 136, 53-59. perature on cold hardiness of hibernating insects. Can. J. Duman, J. G. (1982) Insect antifreezes and ice nucleatine Zool. 34, 283-294. agents. Cryobiology 19, 613627. Salt R. W. (1957) Natural occurrence of glycerol in insects Duman J. G. and Horwath K. L. (1983) The role of and its relation to their ability to survive freezing. Can. haemolymph proteins in the cold tolerance of insects. A. Ent. 89, 491-494. Rev. Physiol. 45, 261-270. Salt R. W. (1958) Cold-hardiness of insects. Proc. 10th Znt. Harrington R. and Cheng X. N. (1984) Winter mortality, Congr. Ent. 2, 73-77. development and reproduction in a field population of Salt R. W. (1961) Principles of insect cold-hardiness. A. Rev. Mysus persicae (Sulz.) in England. Bull. ent. Res. 74, Em. 6, 55-74. 633-640. Salt R. W. (1966) Relation between time of freezing and Hochachka P. W. and Somero G. N. (1973) Strategies of temperature in supercooled larvae of Cephus cinctus Nort. Biochemical Adaptation. W. B. Saunders, Philadelphia.” Can. J. Zool. 44, 947-952. Hochachka P. W. and Somero G. N. (1984) Biochemical Salt R. W. (1966a) Effect of cooling rate on the freezing Adaptation. Princeton University Press. ’ temperature of supercooled insects. Can. J. Zool. 44, James B. D. and Luff M. L. (1982). Cold hardiness and 655359. development of eggs of Rhopalosiphum inserturn. Ecol. Salt R. W. (1966b) Factors influencing nucleation in super- Ent. 7, 277-282. cooled insects. Can. J. Zool. 44, 117-133. Knight J. D. and Bale J. S. (1986) Cold hardiness and Salt R. W. (1968) Location and quantitative aspects of ice overwintering of the grain aphid Sitobion avenae. Ecol. nucleators in insects. Can. J. Zool. 46, 329-333. Ent. 11, 189-197. Somme L. (1969) Mannitol and glycerol in overwintering Knight J. D., Bale J. S., Franks F., Mathias S. and Baust aphid eggs. Norw. J. Ent. 16, 107-111. J. G. (1986) Insect cold hardiness: Supercooling points Somme L. (1982) Supercooling and winter survival in and pm-freeze mortality. Cryoletters 7; 1942031 _ terrestrial arthropods. Comp. Biochem. Physiol. 73A, Kniaht J. D. and Bale J. S. (1987) The effects of low 519-543. tekperature on the development, reproduction and sur- Storey K. B. (1984) A metabolic approach to cold hardiness vival of the grain aphid Sitobion avenae. Proceedings, in animals. Cryoletters 5, 147-161. Third European Congress of Entomology, Amsterdam, Tenow 0. (1975) Topographical dependence of an outbreak Part 2, pp. 279-282. of Oporinia antumnata Bkh. (Lep., Geometridae) in a Leather S. R. (1980) Egg survival in the bird cherry-oat mountain birch forest in northern Sweden. Zoon. 3, aphid, Rhopalosiphum padi. Entomologia exp. appl. 27, 85110. 9697. Turnock W. J., Lamb R. J. and Bodnaryk R. P. (1983) Leather S. R. (1981) Factors affecting egg survival in the Effects of cold stress during pupal diapause on the bird cherry-oat aphid Rhopalosiphim padi. (L) Ento- survival and development of Mamestra confguraia (Lep- mologia exp. appl. 30, 197-199. idoptera: Noctuidae) Oecologia 56, 185-192. Lee R. E. Jr, Baust J. G. and Ring R. A. (1986) Low Watson M. A., Heathcote G. D., Lauckner F. B. and temperature tolerance in insects and other terrestrial Sowray P. A. (1975) The use of weather data and counts arthropods: Bibliograph II. Cryoletters 7, 113-126. of aphids in the field to predict the incidence of yellowing Lee R. E. Jr, and Denlinger D. L. (1985) Cold tolerance in viruses of sugar beet crops in England in relation to the diapausing and non-diapausing stages of the fresh fly, use of insecticides. Ann. Appl. Biol. 81, 181-198. Sarcophaga crassipalpis. Physiol. Ent. 10, 309-3 15. Wellington W. G. and Trimble R. M. (1984) ‘Weather’ in Levitt J. (1980) Responses of Plants to Environmental Ecological Entomology (Ed. by HutTaker C. B. and Rabb Stresses (Physiological Ecology). Vol. 1, Chilling, Freezing R. L.). John Wiley. and High Temperature Stresses. Academic Press, London. van de Woude H. A. and Verhoef H. A. (1986) A compara- G’Doherty R. and Bale J. S. (1985) Factors affecting the tive study of winter survival in two temperate Collembola. cold hardiness of the peach potato aphid Myzus persicae. Ecol. Ent. 11, 333-340. Ann. Appl. Biol. 106, 219-228. Zachariassen K. E. and Hammel H. T. (1976) Nucleating Pollard D. G. (1973) Plant penetration by feeding aphids agents in the haemolymph of insects tolerant to freezing. (H&~YY Aphidoidea): a review. Bull. em. Res. 62, Nature Lond. 262, 285-287. Zachariassen K. E. (1980) The role of polyols and nucle- Powell W. (1974) Supercooling and the low temperature ating agents in cold-hardy beetles. J. camp. Physiol. 140, survival of the green spruce aphid Elatobium abietinum. 227-234. Ann. Appl. Biol. 78, 27-37. Zachariassen K. E. (1982) Nucleating agents in cold-hardy Prosser C. L. (1973) Comparative Animal Physiology. W. B. insects. Comp. Biochem. Physiol. 73A, 557-562. Saunders, Philadelphia. Zachariassen K. E. and Husby J. A. (1982). Antifreeze effect Rickards J., Kelleher M. J. and Storey K. B. (1987) Strate- of thermal hysteresis agents protects highly supercooled gies of freeze avoidance in larvae of the goldenrod gall insects. Nature, Lond. 298, 865-867. moth, Epiblema scudderiana : Winter profiles of a natural population. J. Insect Physiol. 33