Reprint of: The ins and outs of water dynamics in cold tolerant soil invertebrates

Journal of Thermal Biology 54 (2015) 30–36

Contents lists available at ScienceDirect

Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio

Review

Reprint of: The ins and outs of water dynamics in cold tolerant soil invertebrates$ Martin Holmstrup n Department of Bioscience, Aarhus University, Vejlsøvej 25, DK-8600Silkeborg

art ic l e i nf o

a b s t r a c t

Available online 12 November 2015

Many soil invertebrates have physiological characteristics in common with freshwater animals and represent an evolutionary transition from aquatic to terrestrial life forms. Their high cuticular permeability and ability to tolerate large modifications of internal osmolality are of particular importance for their cold tolerance. A number of cold region species that spend some or most of their life-time in soil are in more or less intimate contact with soil ice during overwintering. Unless such species have effective barriers against cuticular water-transport, they have only two options for survival: tolerate internal freezing or dehydrate. The risk of internal ice formation may be substantial due to inoculative freezing and many species rely on freeze-tolerance for overwintering. If freezing does not occur, the desiccating power of external ice will cause the animal to dehydrate until vapor pressure equilibrium between body fluids and external ice has been reached. This cold tolerance mechanism is termed cryoprotective dehydration (CPD) and requires that the animal must be able to tolerate substantial dehydration. Even though CPD is essentially a freeze-avoidance strategy the associated physiological traits are more or less the same as those found in freeze tolerant species. The most well-known are accumulation of compatible osmolytes and molecular chaperones reducing or protecting against the stress caused by cellular dehydration. Environmental moisture levels of the habitat are important for which type of cold tolerance is employed, not only in an evolutionary context, but also within a single population. Some species use CPD under relatively dry conditions, but freeze tolerance when soil moisture is high. & 2015 Published by Elsevier Ltd.

Keywords: Arctic environments Cryoprotective dehydration Compatible osmolytes Freeze tolerance Soil invertebrates

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The soil environment and organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Protective dehydration strategy – a different kind of cold tolerance mechanism . . . . . . . . 4. Why desiccation in frozen soil? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Freezing-induced redistribution of water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Only two options for cold tolerance in permeable soil invertebrates: Freeze or dehydrate 7. How many species use CPD? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Adaptive traits for cryoprotective dehydration and freeze tolerance . . . . . . . . . . . . . . . . . . 9. Cross-tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ☆

This article belongs to the Special Issue dedicated to Prof. Ken Bowler for ongoing, long-term contributions to thermal biology : What sets the limit? how thermal limits, performance and preference in ectotherms are influenced by water and energy balance. n Tel.: þ 45 3018 3152. E-mail address: [email protected] http://dx.doi.org/10.1016/j.jtherbio.2015.10.006 0306-4565/& 2015 Published by Elsevier Ltd.

Animals of temperate regions living permanently in the soil or overwintering there may never or rarely experience temperatures low enough to produce a risk of freezing of their body fluids because thermal conditions in the soil are often much less extreme

M. Holmstrup / Journal of Thermal Biology 54 (2015) 30–36

than air temperatures. However, in cold regions of the world, soil will freeze every winter, and if insulating snow cover is low, or if permafrost prevails, soil animals cannot evade the low temperatures and must rely on physiological adaptations for survival. Cold hardy ectothermic animals have evolved two major strategies for survival of sub-zero temperatures. The freeze avoiding species, for which freezing of body fluids is lethal, depend on extensive supercooling of their body fluids, whereas a second strategy is deployed by the freeze tolerant species that are able to tolerate freezing of their extracellular body fluids. Intracellular freezing is generally considered to be lethal (Asahina, 1969; Zachariassen, 1985) although a few examples of tolerance of intracellular freezing have been shown in insects and nematodes (Lee et al., 1993; Wharton and Ferns, 1995). A comprehensive outline of general cold hardiness strategies of ectothermic animals will not be given here since several excellent reviews are available (Block, 1990; Denlinger and Lee, 2010; Ramløv, 2000; Storey and Storey, 1996; Zachariassen, 1985). This paper will focus on a third cold hardiness strategy termed cryoprotective dehydration (CPD), which is relevant for those smaller soil invertebrates that have only little cuticular resistance to desiccating conditions (Holmstrup et al., 2002). Since the first mechanistic description of CPD (Holmstrup and Westh, 1994), this cold tolerance strategy has been found in a growing number of species, and several previous observations support the (likely) widespread occurrence of CPD in soil invertebrates. The paper will discuss the physical conditions prevailing in soil, and the distinct relationships these have with water balance of soil invertebrates. Further, I will discuss which physiological traits are of importance for CPD, and show the similarities to adaptations making animals freeze tolerant. Lastly, I will present an inventory of species presently known to use CPD and from that attempt to predict characteristics of typical species employing this cold tolerance mechanism.

2. The soil environment and organisms Soil invertebrates are important components of soil ecosystems worldwide, with the exception of deserts and land permanently covered by ice. This diverse group of animals covers a range of taxa, the most important being protozoans, tardigrades, nematodes, oligochaete worms (earthworms and enchytraeids), mites, springtails (Collembola), millipedes, centipedes, and a range of insects (mostly belonging to Diptera and Coleoptera) whose larval stages complete their development in the soil. Many of these organisms such as tardigrades, nematodes, oligochaetes and soildwelling springtails have several characteristics that distinguish them from surface living forms, in particular with respect to water balance. These characteristics include small size, epidermal respiration, and high integumental permeability to water resembling aquatic animals more than truly terrestrial. These characters seem to match the soil environment, where the pore humidity is normally very close to 100% relative humidity (RH), and it could be argued that the soil has provided a habitat for an evolutionary transition from aquatic to terrestrial life forms (Ghilarov, 1958). Extreme winter (and summer) temperatures are also buffered due to insulation from snow, vegetation and dead plant litter, and from deeper soil layers that provide either a sink or a source of heat during warming and cooling from the air, respectively (Berry, 1981; Isard et al., 2007). This results in cooling rates that are mostly much slower than found aboveground, except in permafrost soils with little insulation by snow. In that case cooling rates in soil may be similar to those in air once all water in the soil has frozen (Coulson et al., 1995).

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3. Protective dehydration strategy – a different kind of cold tolerance mechanism Permeable invertebrates that are trapped in frozen soil or sediment may become dehydrated. Scholander et al. (1953) and Danks (1971) observed that chironomid larvae found in frozen sediment were wrinkled and appeared to have become dehydrated, and later Gehrken and Sømme (1987) reported that overwintering eggs of stoneflies became severely desiccated when kept in frozen creek water. Other organisms like Collembola and earthworm egg capsules (“cocoons”) also became severely desiccated if subjected to sub-zero temperatures in small vials where the air humidity was defined by ice, i.e. simulating the conditions in frozen soil (Holmstrup, 1992; Worland, 1996). In these early reports it was suggested that dehydration of the organism enhanced the supercooling capacity as a primary cold tolerance strategy. Later, using earthworm cocoons as models, Holmstrup and Westh (1994) showed that supercooling was of minor importance whereas the dehydration-induced lowering of body fluid melting point played a central role in the cold hardiness of permeable soil invertebrates. The difference in vapor pressure between ice and supercooled body fluids drives an outflux of water vapor. The force of this vapor pressure difference is so large (see following section) that even a few degrees of supercooling will result in substantial water loss, continuing until the vapor pressure of body fluids equals that of the surrounding ice (Holmstrup et al., 2002; Holmstrup and Westh, 1994). At this stage, the risk of tissue ice formation has been eliminated, and subzero survival is ensured. Thus, winter survival of permeable soil invertebrates is less dependent on supercooling than in most insects, and is instead based on dehydration and equilibration of body-fluid melting point with the ambient temperature of the surrounding ice. Since cryoprotective dehydration can sometimes be a slow process, some supercooling capacity is needed in the initial phases of dehydration (Elnitsky et al., 2008a, 2008b; Sørensen and Holmstrup, 2011). However, under field-relevant cooling rates the organism is rarely supercooled more than a few °C meaning that high supercooling capacity is not needed for winter survival (see Fig. 1 for a graphic outline of CPD). In some small sized organisms with high permeability this equilibration process is so rapid that melting points is practically keeping pace even with the extreme cooling rates seen in polar soils (Holmstrup et al., 2002). However, in other species less prone to dehydration the vapor pressure equilibrium may take much longer meaning that supercooling has a higher significance. The choice of overwintering strategy among soil organisms (supercooling versus CPD) should rather be seen as a continuum between these two strategies, than as either one or the other strategy.

4. Why desiccation in frozen soil? The reason for the dehydration of the cocoons must be found in the physics of water and ice. Consider a closed system, held at subzero temperature, consisting of a volume of supercooled water surrounded by air and ice. In such a system, vapor pressure of the supercooled water is higher than vapor pressure of the ice (Lundheim and Zachariassen, 1993; Salt, 1963; Weast, 1989). This will cause a net transport of water (vapor) from the supercooled water to the ice, where water vapor is condensing onto the ice. The force of this desiccating effect of ice can be calculated as the relative humidity (RH) from:

RH (%) =

P VPice = ⋅100 P* VPwater

where VP is the vapour pressure at a given temperature. Since the

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M. Holmstrup / Journal of Thermal Biology 54 (2015) 30–36

Difference in osmotic pressure (bar)

-80 Body fluid MP = -0.8°C

-70

Body fluid MP = -2.0°C

-60

Body fluid MP = -4.0°C

-50 -40 -30 -20 -10 0

Sugars + polyols

0

Water content

-1

-2

-3

-4

-5

-6

-7

-8

Ambient temperature (°C) Osmotically inactive water Time

Temperature/melting point/SCP

Ambient temperature -0.8°C

Supercooling

Melting point of body fluids

-6°C

Supercooling point

-15°C Time -60°C

Fig. 1. A general model of cryoprotective dehydration. Decreasing soil temperature causes dehydration of the organism, which in turn induces accumulation of sugars and polyols (SP: upper panel). At low temperature (  15 to  20 °C) practically all osmotically active water is lost. Dehydration and SP accumulation bring about a lowering of melting point (MP), largely at the same rate as soil temperature decreases. Supercooling is therefore restricted to only a few degrees and only during a short initial period at relatively high subzero temperatures (lower panel). A temperature rise causes the animal to take up water, and increases the body fluid MP. Fully hydrated animals have supercooling points at about  6 °C. Dehydration result in a depression of the supercooling point. When dehydration approaches the level of osmotically inactive water, freezing cannot occur at environmental temperatures. The photos show fully hydrated cocoons of Dendrobaena octaedra and collembolan Megaphorura arctica (left), and animals dehydrated at  6 °C for 7 days (right).

RH(%) Vapour pressure of ice Vapour pressure of supercooled water

4

100 98 96

3

94 2

92 90

1

88

Relative humidity (%)

Vapour pressure (mm Hg)

5

86

0 -15

Fig. 3. Differences in water potential (WP) between the body fluids of a supercooled animal with a melting point of  0.8,  2.0 and  4.0 °C, respectively, and the surrounding ice at varying ambient temperature. Note that the more intense supercooling becomes, the larger becomes the WP difference. The water potential of body fluids at a given temperature was calculated using Van't Hoff's equation: ψ¼ Osm  R  T, where Osm is the osmolality of body fluids, R is the gas constant, and T is absolute temperature (°K). The melting point of body fluids (MP) was calculated by application of the osmolal melting point depression constant.

-10

-5

0

Temperature (°C) Fig. 2. Changes in the vapor pressure of supercooled water and ice with temperature. Note that at the same time there is an absolute pressure lowering of both supercooled water and ice with decreasing temperature, which will cause the relative humidity (RH) to steadily decline with the lowering of the temperature. Data from Weast (1989).

pressure difference between water and ice increases with decreasing temperature (at least down to  10 °C), and there is an absolute pressure lowering of both water and ice with decreasing

temperature, the RH will be steadily declining with the lowering of temperature (Fig. 2). The pressure difference between ice and the supercooled water can more conveniently be expressed in bar from:

ψ (bar ) = − 10.6Tlog (100/RH ) where ψ (psi) is the water potential or”osmotic pressure of ice”, and T is the absolute temperature (°K). These relationships make it possible to calculate the difference in water potential between a supercooled soil animal surrounded by ice in the soil. As it appears from Fig. 3, these differences in water potential are substantial, even in situations where there is only a slight difference between the water potential of the animal body fluids and the ice (corresponding to a difference between the body fluid MP and temperature of the ice). For example, we may consider the particular case with an animal body fluid MP of 2 °C surrounded by ice at  3 °C (“ambient MP”) ambient temperature. By transforming MP to osmolality using the osmolal MP depression constant of  1.86 °C Osmol  1, we find using Van't Hoff's equation that the difference in osmotic pressure between the animals body fluids and the surrounding ice, is 12 bar (see Fig. 3). Thus, even with a “MP inequality” of only 0.1 °C there is a difference in osmotic pressure of about 1.2 bar, which is sufficient to drive the dehydration at most natural cooling rates.

5. Freezing-induced redistribution of water When soils freeze, ice first forms in the larger pores. Water in very fine pores may still be warmer than its freezing point due to capillary forces. Because of differences in chemical potential, water from the finer pores moves to ice in larger pores and freezes on the interface. This process is termed “freezing-induced redistribution”, and is the mechanism by which ice lenses in arctic soils are formed (Miller, 1980). Freezing-induced re-distribution of water occurs at rates that are much too rapid to be explained by vapor transport. Rather, it must be based on bulk transport of liquid water from finer pores to the ice (Miller, 1980). Interestingly, there are some examples suggesting that water may also be transported from supercooled organisms to external ice by freezing-induced redistribution. Holmstrup and Zachariassen (1996) reported that

M. Holmstrup / Journal of Thermal Biology 54 (2015) 30–36

Water loss (% of fresh weight)

0

In frozen soil Over ice Over frozen soil

10 20 30 40 50 60 70 0

2

4

6

8

10

12

14

Exposure time (days) Fig. 4. Loss of water in supercooled earthworm cocoons (Dendrobaena octaedra) when surrounded by ice (at  3 °C). Dehydration rates were much faster when the cocoons were buried in frozen soil as compared to cocoons exposed in air, perhaps due to freeze-induced re-distribution of water. Data from Holmstrup and Zachariassen (1996).

earthworm cocoons dehydrated rapidly and reached vapor pressure equilibrium with external ice at 3 °C in less than 24 hours, whereas cocoons suspended over ice or frozen soil (but not in physical contact with ice) needed 4-6 days to reach equilibrium (Fig. 4). Wharton et al. (2003) studied CPD in the Antarctic nematode, Panagrolaimus davidi, and found that nematodes did not freeze, but dehydrated substantially if the substrate in which the nematodes were kept was inoculated at  1 °C and then cooled slowly to  5 °C. Both these examples suggest that the loss of water from the organism was not mediated by water vapor transport to surrounding ice, but rather by bulk flow similar to the formation of ice lenses in freezing soil. Salt (1963) proposed a similar framework in his theories about the mechanisms for delayed inoculative freezing in insects, and suggested that the water in sub-micron pores of the insect cuticle could be supercooled or have much lower melting points than bulk body fluids due to capillary forces reducing the vapor pressure. It follows that two outcomes could be the result if organisms are exposed in frozen soil: either the supercooled water would be drawn out of the organism along the chemical energy gradient, or the ice would grow and penetrate through pores in the cuticle (or through other openings) until it seeds bulk freezing of body fluids. The race between these two processes would then determine if an organism must use freeze tolerance or can use CPD as its cold tolerance strategy.

6. Only two options for cold tolerance in permeable soil invertebrates: Freeze or dehydrate Having established that supercooling is of minor importance in permeable soil invertebrates, it may be stated that such animals are left with only two sustainable strategies available for survival of sub-zero temperatures: freeze-tolerance or CPD. There is no doubt that inoculative freezing from external ice in the soil is a major issue. Thus, it is unlikely that larger animals living or hibernating in the soil, such as large enchytraeids, earthworms, slugs or frogs (macrofauna; diameter 42 mm), are able to avoid contact with ice crystals in frozen soil. There are numerous examples in the literature showing the importance of inoculative freezing for cold tolerance (Costanzo et al., 1999, 1997; Danks, 1971; Frisbie and Lee, 1997; Hoshikawa et al., 1988; Packard and Packard, 1993; Pedersen and Holmstrup, 2003; Salt, 1963; Slotsbo et al., 2012).

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Because of the high risk of inoculative freezing in frozen soils, natural selection should be expected to favor freeze-tolerance in larger soil animals. Small soil animals associated with the air-filled pores of the soil (mesofauna; 0.1 mm odiametero2 mm), e.g. collembolans, can in some cases also be vulnerable to inoculative freezing (Coulson et al., 2000; Sømme and Conradi-Larsen, 1977). However, the smaller soil arthropods may be able to avoid inoculative freezing by behavioural means, probably because ice crystals in the soil are discrete leaving opportunities for small organisms to avoid direct contact with ice (see also preceding section). Since ice almost exclusively forms as a pure water phase, the ice formed when soils freeze may be found segregated in quite large ice crystals. This is easily observed if one looks at frozen soil in a dissection microscope. Temperature will remain at 0 °C until all soil water is frozen (due to heat of fusion). Since melting points of fully hydrated microarthropods is typically  0.8 °C or lower there is no desiccation taking place until soil temperature goes below the animals' melting point. Studies have shown that polar microarthropods can remain active at temperatures from 0 °C to  3 °C (Block et al., 1994; Hayward et al., 2000, 2003) and it is therefore reasonable to speculate that microarthropods can actively avoid ice crystals in the soil. However, as discussed in the previous sections, if the organism is very permeable for water, the avoidance of freezing will inevitably lead to dehydration if temperature goes below the melting point of the organism's body fluids. The competition and balance between risk of inoculative freezing and ice-induced dehydration seems therefore to be highly influenced by thermal and hydric conditions in the soil. High cooling rates would be expected to increase the risk of inoculative freezing because depression of the melting point by dehydration cannot keep pace with ambient temperature, and similarly, high moisture content of the soil would result in an increased contact between the animal and the ice in the soil, and thereby a higher risk of inoculative freezing. Three examples of this being the case are known. Wharton et al. (2003) demonstrated that the freeze tolerant nematode P. davidi froze when subjected to high cooling rates, but switched to CPD as a survival strategy at lower cooling rates that allowed time for efficient dehydration. Pedersen and Holmstrup (2003) manipulated soil moisture content and observed that the enchytraeid, F. ratzeli, used freeze tolerance as a strategy when soil moisture was high, but shifted to a higher likelihood of CPD when soil moisture was decreased. Very similar results were shown for the Antarctic midge, Belgica antarctica, in two recent studies (Elnitsky et al., 2008b; Kawarasaki et al., 2014a). In common for these three species, they are able to survive sub-zero temperature almost equally well using either of these two strategies, showing that they are well adapted to very variable environmental conditions, and indicating that the physiological adaptations to freeze tolerance and CPD have much in common (see later sections). Most studies have investigated CPD under simulated field conditions in the laboratory, whereas very few studies have shown CPD to take place under natural field conditions. To do so, it is necessary to bring frozen blocks of soil or substrate to the laboratory and quickly thaw them in order to extract the studied organism and determine body fluid melting point and water contents. This is not without practical problems because desiccated soil invertebrates will rapidly (within hours) absorb water when the soil is thawed (Holmstrup, 1995; Sørensen et al., 2010). Kawarasaki et al. (2014b) sampled B. antarctica in the field and found that this midge primarily used freeze tolerance as a survival strategy, but that some individuals collected from dry substrates were considerably desiccated suggesting that they were cryoprotectively dehydrated. Since freeze tolerance has not been shown in any collembolan species, whereas several are capable of CDP (Sørensen and Holmstrup, 2011) and survive in deeply frozen

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M. Holmstrup / Journal of Thermal Biology 54 (2015) 30–36

Arctic soils, there can be no doubt that CPD is probably the predominant overwintering strategy of permeable collembolans in their natural habitat.

7. How many species use CPD? Cryoprotective dehydration is a relatively recent supplement to the understanding of soil invertebrates' cold tolerance. The main reason for this is probably that experimental protocols developed for the study of cold tolerance in insects have often been uncritically applied to soil invertebrates. As outlined in previous sections, water balance of soil invertebrates is fundamentally different from truly terrestrial insects, and the thermal and hydric conditions are often very different from aboveground conditions. This calls for methodologies that simulate environmental conditions relevant for the soil as overwintering habitat, and are able to perceive the important physiological responses. To give an example, it is of little relevance to measure supercooling points and survival of e.g. tardigrades subjected to rapid cooling rates in a differential scanning calorimeter. Even though tardigrades may survive freezing under such conditions, studies of this kind add little to our understanding of cold tolerance mechanisms operating under field conditions. The list of species using CPD is steadily growing, and CPD has now been demonstrated in phylogenetically distant species such as oligochaeta, nematodes, Collembola and insects (Table 1). At the moment, CPD has been experimentally documented in about 15 species, but a number of previous studies suggest that it is a widespread strategy. Sørensen and Holmstrup (2011) tested for CPD in Collembola from Spitzbergen and noted that four out of five species used CPD, whereas a beetle (Atheta graminicola) did not. The observations by Scholander et al. (1953) and Danks (1971) that midge larvae desiccated in frozen sediment is well in line with studies on B. antarctica suggesting that CPD is probably also widespread in this group of insects.

8. Adaptive traits for cryoprotective dehydration and freeze tolerance Adoption of CPD by invertebrates requires that water loss rates are sufficiently high to ensure vapor pressure equilibration within relatively short time. Otherwise, the probability of freezing of body fluids by inoculation may be high. It is therefore obvious that high

permeability for water and small body size is in favor of CPD, and the opposite for freeze tolerance. Animals using CPD must be able to tolerate extensive dehydration, which is indeed the case for all species listed in Table 1. Some of these species (Collembola and earthworm embryos) can tolerate the loss of practically all osmotically active water (Holmstrup et al., 2002; Holmstrup and Sømme, 1998) by using accumulation of compatible osmolytes as dehydration protectants (Petersen et al., 2008; Worland et al., 1998) in a manner bearing similarities to anhydrobiotic organisms (Crowe et al., 1992). At least in M. arctica the production of protective osmolytes during CPD seems to be adjusted to the thermal conditions of the habitat. Bahrndorff et al. (2007) and Sørensen and Holmstrup (2013) compared Arctic and subarctic populations and noted that the arctic M. arctica accumulated more trehalose than populations originating from warmer regions. When occurring in combination, dehydration and accumulation of compatible osmolytes (e.g. trehalose or glycerol) can ultimately result in vitrification of the remaining body water. In these cases the organism is completely secured against extreme, low temperatures and no freezing of body water takes place (Bennett et al., 2005; Holmstrup et al., 2002; Sformo et al., 2010). The severe cellular dehydration that occurs in CPD and freeze tolerance causes cellular stress in the form of oxidative stress which can denature proteins and destroy membrane lipids (Hermes-Lima, 2004; Joanisse and Storey, 1996). Further, the removal of cellular water and following increase in solute concentration can denature and precipitate proteins (Zachariassen, 1985). Molecular chaperones and antioxidative defence mechanisms are therefore important in both CPD and freeze tolerance. Indeed, a handful of studies have shown that several heat shock proteins are involved in arthropods using CPD (Clark et al., 2009; Sørensen et al., 2010; Sørensen and Holmstrup, 2013; Teets et al., 2012). Perhaps of importance for CPD it should also be mentioned that Late Embryogenesis Abundant proteins (LEA), which are associated with desiccation tolerance in plants and some invertebrates (Tunnacliffe and Wise, 2007) might also be important for CPD. Bahrndorff et al. (2009) have shown that desiccation induce LEA in some Collembola including M. arctica, whereas Teets et al. (2012) in their comprehensive genomic profiling of cryoprotectively dehydrated B. antarctica did not find LEA sequences. For further discussion of molecular responses involved in CPD, please consult the recent review by Teets and Denlinger (2014).

Table 1 Overview of species presently known to use cryoprotective dehydration as overwintering strategy. Larger taxonomic group Species

Life-stage

Body size (length or fresh weight)

Reference

Nematoda

second-stage juveniles post-embryonic stages adults egg capsules egg capsules egg capsules egg capsules adults adults adults adult adult adult adult adult 4th instar larvae

0.4 mm 20–120 mm 0.8 mm 4 mg 4 mg 13 mg 11 mg 1-3 mg 1–3 mg 6 mg 750 mg 100 mg 70 mg 100 mg 30 mg 1.5 mg

(Forge and MacGuidwin, 1992) (Wharton et al., 2003) Holmstrup and Ramløv, unpublished (Holmstrup and Westh, 1994) (Holmstrup, 1994) (Holmstrup, 1994) (Holmstrup, 1994) (Sømme and Birkemoe, 1997) (Sømme and Birkemoe, 1997) (Pedersen and Holmstrup, 2003) (Holmstrup and Sømme, 1998; Worland et al., 1998) (Elnitsky et al., 2008a; Worland and Block, 2003) (Sørensen and Holmstrup, 2011) (Sørensen and Holmstrup, 2011) (Sørensen and Holmstrup, 2011) (Elnitsky et al., 2008b; Kawarasaki et al., 2014a; Teets et al., 2012)

Tardigrada Oligochaeta

Collembola

Insecta

Meloidogyne hapla Panagrolaimus davidi Richtersius coronifer Dendrobaena octaedra Dendrodrilus rubidus Aporrectodea caliginosa Allolobophora chlorotica Enchytraeus kincaidi Mesenchytraeus sp. Fridericia ratzeli Megaphorura arctica Cryptopygus antarcticus Oligaphorura groenlandica Hypogastrura viatica Folsomia quadrioculata Belgica antarctica

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9. Cross-tolerance CPD bears some resemblance to freeze-tolerance in which extracellular ice formation induces dehydration of cells (Zachariassen, 1985). Parallel to this, organisms using CPD are protected by the dehydrating effects of environmental ice, which dehydrates the whole organism and its cells and prevent lethal freezing of cellular water. In essence, both strategies therefore work in the same way. As a response to dehydration both groups of organisms accumulate compatible osmolytes both extra- and intracellularly, which may prevent harmful effects of the extensive cellular water loss coupled with water potential equilibration. For those organisms that use CPD (except for true insects) it is characteristic that no preparative accumulation of compatible osmolytes occurs (as opposed to freeze-avoiding and freeze-tolerant insects) before the moment where ice is present in the environment (Holmstrup, 1995; Holmstrup and Sømme, 1998; Pedersen and Holmstrup, 2003; Sørensen and Holmstrup, 2011). Due to the elevated osmotic pressure this would result in an increased influx of water into the body at above-zero temperatures, requiring increased energy consumption to avoid salt losses in connection with excretion, and causing problems with the organism's volume control. Deferring cryoprotectant synthesis to the moment of tissue dehydration obviates the energetic expense of mobilizing cryoprotectants, and the potential for loss of energy-rich compounds from the body, that would be unnecessary so long as body temperatures remain above the melting point. In line with these similarities between freeze tolerance and CPD (even though the latter in principle is a freeze avoiding strategy) we saw that some species are capable of using both freeze tolerance and CPD for winter survival, external environmental conditions determining which of the two become effective. Several studies and reviews have discussed the relationship existing between desiccation and cold tolerance in the sense that responses to these stressors share protective mechanisms as discussed in the previous sections (Block, 1996; Sinclair et al., 2013). In line with this several studies have shown that pre-acclimation to mild desiccation often improves cold tolerance most likely because accumulation of protective osmolytes and other molecular responses are induced by cellular dehydration (Bayley et al., 2001; Benoit et al., 2009; Hayward et al., 2007). As proposed by Ring and Danks (1994, 1998) it seems therefore that several physiological adaptations considered as important for survival of extreme cold originally evolved as adaptations to living with occasional drought stress in a terrestrial environment. For those soil invertebrates that are highly permeable for water this has the implication that limits for cold tolerance are in essence set by their limits for desiccation tolerance.

Acknowledgements The author thanks Society of Experimental Biology and Journal of Thermal Biology for supporting this paper, and the constructive criticism of reviewers.

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