Temporal resolution of cold acclimation and de-acclimation in the Antarctic collembolan, Cryptopygus antarcticus

Physiological Entomology (2007) 32, 233–239 DOI: 10.1111/j.1365-3032.2007.00569.x

© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society 233

Introduction

Cold hardiness in insects is a modal phenomenon. At sea-sonal temporal scales, this is expressed as a state of hardi-ness that is acquired in preparation for winter temperatures or lost in tandem with summer temperatures. For the small, but important, class of arthropods that are ‘chill tolerant’ ( sensu Bale, 1993, 1996 ), but for which the temperature of crystallization is a reliable proxy for lower lethal tempera-ture, this modality can be observed in the distribution of their supercooling points (SCPs). In some of these species, modal-ity in their SCPs is not just a seasonal, but also a diurnal phe-nomenon ( Worland & Convey, 2001 ; Sinclair et al ., 2003).

The Antarctic collembolan, Cryptopygus antarcticus (Willem), has in many ways become a model species for investigations into the cold hardiness of freeze-avoiding Antarctic invertebrates. Bimodality in its supercooling points (reflecting ‘summer’ and ‘winter’ modes of cold acclimatiza-tion/acclimation) has reached ‘textbook status’ ( Hopkin, 1997; Chown & Nicholson, 2004 ). Although the cues gov-

erning the shifts between these modes at seasonal timescales have been studied extensively ( Sømme & Block, 1982 ; Cannon, 1983; Cannon, 1987; Worland & Luke š ová, 2000 ) , recent investigations into its summer cryoprotection levels have highlighted its ability to change its cold tolerance at diurnal time-scales ( Worland & Convey, 2001 ). However, unlike most other Antarctic arthropods (Hawes et al ., 2006a), C. antarcticus shows little obvious evidence of a ‘transitional’/‘semicold-hardy’ state between its modes of cold hardiness; individuals appear to ‘switch’ rather than ‘flow’ from one group to the other. The present study sets out to characterize the temporal component of this apparently digital response.

Temporal scaling of cold hardening, particularly in polar arthropods, offers a means for testing the flexibility of their low temperature adaptations (i.e. their resilience in the face of climatic perturbation; Hawes et al ., 2006b). Although high Arctic summer habitat temperatures probably negate the necessity for short-term flexibility in cold hardening (Hawes et al ., 2006b), the frequency of subzero summer habitat tem-peratures in Maritime Antarctica means that C. antarcticus must retain the ability to lower its freezing points throughout polar summers ( Worland & Convey, 2001 ). This requirement, coupled with the need to attend to feeding, growth and repro-duction in the short temporal window (approximately 2 – 3

Temporal resolution of cold acclimation and de-acclimation in the Antarctic collembolan, Cryptopygus antarcticus

M . R . W O R L A N D 2 , T . C . H A W E S 1 and J . S . B A L E 1 1 University of Birmingham, School of Biosciences, Edgbaston, Birmingham, U.K. and 2 British Antarctic Survey, National

Environment Research Council, High Cross, Cambridge, U.K.

Abstract . The Antarctic collembolan, Cryptopygus antarcticus (Willem), can switch its supercooling point (SCP) between ‘winter’ and ‘summer’ modes of cold hardiness over a matter of hours. High resolution temporal scaling of the acquisition and loss of cold hardiness is undertaken by assaying changes in the proportion of animals freezing below – 15 °C in response to cooling rate, acclimation temperature, and access to food and moisture. Rapid de-acclimation to the ‘summer’ modal state is readily achieved after 1 – 6 h in response to warming and access to food; however, rapid acclimation to the ‘winter’ modal state is only evident in response to slow cooling and narrow ranges of temperature (0 – 5 °C). The rapid loss of cold tolerance at higher temperatures with access to food, in particular, emphasizes this species’ opportunistic responses to resource availability in the short polar summers. Cold hardiness is apparently more readily traded off against nutrient acquisition than vice versa in this maritime Antarctic species.

Key words . Collembola , flexibility , maritime Antarctic , rapid cold hardening , supercooling point , temporal scaling .

Correspondence: Professor J. S. Bale, School of Biosciences, University of Birmingham, Edgbaston Birmingham B15 2TT, U.K. Tel.: (0) 121 414 5908; fax: (0) 121 414 5925; e-mail: [email protected]

234 M. R. Worland, T. C. Hawes and J. S. Bale

© 2007 The Authors Journal compilation © 2007 The Royal Entomological Society, Physiological Entomology, 32, 233–239

months) available to them in the Antarctic summer ( Burn, 1981, 1984; Convey, 1996 ), means that C. antarcticus must regularly ‘switch’ from one mode (i.e. ‘summer’ or ‘winter’) of cold hardiness to the other over rapid time scales.

The primary cues for switching animals between modes of cold hardiness (i.e. feeding, moisture and temperature) are well known ( Cannon, 1986; Cannon & Block, 1988 ). However, to date, no attempt has been made to characterize this ‘switch’ in detail or, indeed, to determine whether it is truly a ‘switch’ or whether it has only been observed previ-ously with insufficient temporal resolution. Given the ability of these animals to acclimate over diurnal timescales, the present study, using a fine-scale temporal discrimination of acclimatory changes, aims to identify the cues, manner and speed with which cold hardiness changes in this species.

Materials and methods

Sample collection

Cryptopygus antarcticus is extremely common in vegeta-tion (moss and algae) close to the British Antarctic Survey’s research station at Rothera Point, Adelaide Island, on the west coast of the Antarctic Peninsula (67°34 ¢ S, 66°08 ¢ W). Samples for this study were collected during January to February 2005 from Lagoon Island in Ryder Bay, approxi-mately 3 – 4 km from the research station. Samples were extracted from wet moss ( Sanionia uncinata ) by gently teas-ing it apart in a large sieve (300 × 2.0 mm mesh) over a col-lecting tray. Extracted cultures were kept in plastic boxes containing a base of moist plaster of Paris and some vegeta-tion (sieved moss) and maintained in controlled environment cabinets at 0, 5 and 10 ± 1 °C (18 h of light), for at least 7 days before experimental treatment.

Measurement of cold tolerance

Two methods were used to assess cold tolerance: (i) quan-titative determination of the number of springtails that sur-vived cooling to – 15 °C (the traditional ‘break-point’ between winter and summer modes; Worland & Convey, 2001 ) and (ii) measurement of individual SCPs using differential scan-ning calorimetry (Worland & Luke š ová, 2001). For the former, animals were placed in microcentrifuge tubes (2.5 mL) enclosed with a wad of moist paper (held above the ani-mals to prevent inoculative freezing) and placed in test tubes in an alcohol bath (Thermo Haake Phoenix P2 Circulator; Thermo Haake International, Germany). Sample tubes for animals from 0 °C were kept on ice during sorting until placed in the bath. The bath was programmed to either cool or warm at a set rate over a predetermined range (see below). After the treatment period, all samples were held at the final temperature for 15 min, then cooled at 0.5 °C min – 1 to – 15 °C, held for a further 15 min at this temperature and then warmed at 0.5 °C min – 1 to 5 °C. Survival was immediately assessed after removal from the bath. Groups of springtails were then placed in small (15 mL) screw top vials containing

a base of moist plaster of Paris and survival (capable of coor-dinated locomotion) was re-assessed after a 24-h recovery period. The treatment temperature was constantly monitored using a dummy sample tube containing a fine thermocouple connected to an electronic thermometer.

The effect of cooling rate on acclimation and de-acclimation

Springtails were cultured at 0 and 5 °C for 14 days to pro-duce sample populations that were predominantly in either the low or high supercooling groups, respectively. The modal state of these sample populations was characterized by deter-mining the SCP distributions of 100 springtails (four samples of 25 animals) from each culture. Sample populations were then cooled and warmed in an alcohol bath (see above) at different rates to determine their relative responses to differ-ent rates of temperature change ( Table 1); ten replicate groups of ten were used for each treatment.

Scaling cold de-acclimation

To quantify the rate of loss of cold tolerance cold tolerant springtails (SCP < – 15 °C) were selected and cultured under conditions that were likely to raise their SCP. To separate high group from low group springtails, approximately 500 animals from a mass culture maintained at 0 °C were placed in large tubes and cooled slowly in an alcohol bath (0.5 °C min – 1 ) from 5 to – 15 °C and held at this temperature for 15 min before being re-warmed at the same rate to 5 °C. The animals were then tipped onto a sorting tray placed on an ice bath, and the survivors removed. This provided a sample of springtails with SCPs solely in the low group (< – 15 °C). The standard DSC (Differential Scanning Calorimetry) protocol was used to confirm this by measuring the SCP frequency distribution of 100 animals (5 × 20 individuals).

Replicate culture vials (15 mL) with a base of plaster of Paris, containing approximately 100 springtails, either floating on 5 mL of distilled water, with moist food (finely sieved moss) or with only damp filter paper, were placed in an environmen-tal cabinet at 5 and 10 °C. Initially, the SCP frequency distribu-tion of springtails was monitored at time intervals of 12 – 72 h. However, because the majority of springtails given access to food had increased their SCP after 12 h, a second experiment was conducted using shorter time periods of 1, 3 and 6 h to fur-ther define the timescale for the effect of feeding.

Scaling cold-acclimation

To investigate the rate at which springtails gain cold tolerance, animals that had been kept at relatively high tem-peratures (5 and 10 °C), and which might be expected to have

Table 1. Experimental treatment.

Treatment Cool Warm

Range (°C) 5 to 0 10 to 0 0 to 5 0 to 10 Rate (°C h – 1 ) 2, 1, 0.5, 0.25 4, 2, 1, 0.5 2, 1, 0.5, 0.25 4, 2, 1, 0.5

Cold acclimation and de-acclimation in the Antarctic collembolan 235

© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society, Physiological Entomology, 32, 233–239

predominantly high SCPs (high group animals), were trans-ferred to a lower temperature (0 °C) for different periods of time.

To provide baseline data, the SCP frequency distribution of 100 springtails taken from both cultures was measured using the standard DSC protocol before the experimental treatment. Replicate vials (15 mL) containing approximately 100 springtails floating on 5 mL of distilled water), with moist food (finely sieved moss) or with only damp filter paper, were placed in an environmental cabinet at 0 °C. Two samples of 25 animals were removed at time intervals from 1 – 72 h and the SCP frequency distribution (2 × 25 individu-als) measured using the DSC protocol.

Switch or flow?

As noted above, the shift in SCPs between animals in ‘summer’ and ‘winter’ modes of cold hardiness appears, at low temporal resolution, to be a digital response. To iden-tify whether animals demonstrated evidence of a transition between cold hardy states at the higher temporal resolutions used in this study, SCP distributions from experiments that showed significant, rapid changes in cold hardiness (i.e. the switch from ‘low’ to ‘high’ group in animals transferred to higher temperatures with food) were re-analysed to dis-criminate ‘low’ and ‘high’ group animals from ‘transitional’ individuals. The rationale for the division of these three modalities is described in Hawes et al . (2006b). In brief, summer cold-hardy animals were classified as animals with SCPs > – 10 °C; transitional animals had SCPs between – 10 and – 20 °C; and winter cold-hardy animals had SCPs < – 20 °C.

Results

Effect of cooling rate on cold acclimation and de-acclimation

Acclimating springtails at different temperatures (0 and 5 °C) resulted in populations of animals with significantly different levels of cold tolerance. Of the springtails cultured at 0 °C, 59.9 ± 4.6% (mean ± SE of four samples of 25) sur-vived cooling to – 15 °C whereas only 19.1 ± 5.6% of those kept at 5 °C survived the same treatment. The two cultures were significantly different (Students t -test, P < 0.001, d.f. = 189) and provided populations of animals with SCPs predominantly in the high or low group. These cultures were subsequently used to determine the effect of warming and cooling on cold tolerance.

Springtails with a low level of cold tolerance from the 5 °C culture demonstrated a further loss in cold hardiness when taken to 10 °C and cooled to 0 °C, with levels below untreated controls at all rates of cooling ( Fig. 1). However, cooling samples from the same population but from 5 to 0 °C resulted in a significant increase ( Table 1 ) in cold tolerance, particu-larly with slow cooling rates (0.5 and 0.25 °C h – 1 ).

Warming springtails with high levels of cold tolerance from the 0 °C culture resulted in a loss of cold tolerance over both temperature ranges. All treatments resulted in a lower level of survival to – 15 °C compared with the original untreated population ( Table 2).

The effect of the treatments was analysed using analysis of variance and the results are presented in Table 3. Because not all the samples were cooled at all five rates, only 2, 1 and 0.5 °C min – 1 treatments were used in the statistical analysis. Logistic regression analysis with two treatments versus the experimental factors (rate and range) showed a significant difference between the effect of rate of change in tempera-ture and the cooling and warming treatments ( P = 0.027, F = 3.74; for analysis results, see Table 2 ). The effect of range also varied between cooling and warming treatments ( P < 0.01, F = 6.96). The highly significant difference between the cooling and warming treatments is the result of the animals being from different cultures (0 and 5 °C).

rate of change of temperature (°C min-1)

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Fig. 1. The effect of different cooling and warming rates on the cold tolerance of springtails cultured at two different temperatures. Grey filled symbols show the percentage of animals with supercooling points (SCPs) below – 15 °C taken directly from the cultures at 5 ( ) and 0 °C ( ). Samples from the 5 °C culture were used in cooling regimes and samples from the 0 °C culture in warming regimes. (a) Showing the effect of treating animals over a 5 °C range ( � ), co ol-ing from 5 to 0 °C; (�) warm from 0 to 5 °C; (b) showing similar results for samples treated over a 10 °C range ( � ), cooled from 10 to 0 °C and (�) warmed from 0 to 10 °C.



Scaling cold de-acclimation

The process of identifying low group springtails by cooling bulk samples to – 15 °C and selecting only the survivors, resulted in a success rate of 96% ± 1.9 (mean ± SE of four groups of 25) when a sample of the population was tested using the standard DSC technique. In the initial experiment, the first sample was taken after 12 h of treatment (with food, moisture or on water). However, after this period, springtails with access to food already showed a decrease in the number of cold tolerant animals (from 96% to 48% at 5 °C and to 42% at 10 °C). There was no significant change over the same period for those without food at either temperature ( Fig. 2a,b). Consequently, it was necessary to conduct a further experiment with sampling periods of 1, 3 and 6 h to determine exactly how fast the springtails started to lose cold tolerance. In this refined experiment, only two treatments were used; with food or with

moisture. At 10 °C, there was an almost immediate effect after feeding with a decrease from 96% to 84% after 1 h and a fur-ther reduction to 66% after 3 h (Fig. 2a), The effect was only slightly slower at 5 °C, with 86% in the low group after 3 h and only 56% remaining after 6 h (Fig. 2b). After 48 h, cold tolerance was fairly constant at both temperatures, with approximately 32% remaining in the low group. Springtails without food but with moisture, or floating on water, showed no significant change in SCP at either temperature and remained unchanged over the entire treatment period of 72 days.

Because there was no significant difference between simi-lar treatments at different temperatures ( P = 0.233), the data for samples at 5 and 10 °C with food were combined to show the highly significant relationship between time and loss of cold tolerance (logistic regression, P < 0.001) ( Fig. 3a ). Log 10 time against the percentage of animals in the low group produced a linear relationship ( Fig. 3b , inset) (regression sta-tistics, r 2 = 0.898, intercept = 0.849, slope = – 0.3058, P < 0.0001, n = 14).

Scaling cold acclimation

Of the springtails cultured at 10 °C, 64 ± 5.6% had low group SCPs (mean ± SE of four groups of 25 springtails). When transferred to 0 °C with food, the percentage of cold tolerant animals declined to 34% after 24 h and then increased to 40% after 72 h. The group without food followed an oppo-site trend, increasing in cold tolerance to 92% after 24 h and then falling back to 82% after 72 h. This group showed the maximal cold tolerance of any of the treatments in this experiment.

Springtails cultured at 5 °C were predominantly in the high group (99 ± 0.25%) and showed only a slight increase in cold tolerance over time when transferred to 0 °C either with or without food (maximum of 14% in the low group when given food).

Figure 4 shows the proportion of animals in the low group for the four treatments.

Table 2. Significance of the difference ( P , Students t -test, d.f. = 9) between the original sample (control) and each treatment.

Rate (°C h – 1 )

Treatment Range (°C) 4 2 1 0.5 0.25

Warm 0 – 5 0.015 0.009 0.015 0.017 0 – 10 0.0067 0.03 0.002 0.002

Cool 5 – 0 0.56 0.95 0.01 0.02 10 – 0 0.0001 0.0002 0.0006 0.005

Table 3. Analysis of variance: proportion surviving – 15 °C vs. treatment (CW = cool/warm), rate of change of temperature and range of change.

Factor Type Levels Values

Treatment Fixed 2 Cool, warm Rate ( °C min – 1 ) Fixed 3 0.5, 1.0, 2.0 Range (°C) Fixed 2 5, 10

Source d.f. SS MS F P

CW 1 102.6 102.6 0.00 < 0.001 Rate 2 9.81 4.91 1.18 0.092 Range 1 42 42 2.29 < 0.001 CW × Range 1 14 7.25 0.00 0.01 CW × Rate 2 15.05 4.36 0.00 0.027 Rate × Range 2 8.72 0.51 1.19 0.12 CW × Rate × Range 2 1.02 2.01 0.00 0.777

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Fig. 2. (a) Effect on cold-tolerant (low group = supercooling point < – 15) springtails of culturing at 10 °C with food ( � ), moisture ( � ) and floating on water ( � ); (b) similar to above but cultured at 5 °C.



Switch or flow?

At high temporal resolution, it is evident that the ‘switch’ between ‘low’ and ‘high’ groups is not a completely digital response (Fig. 5a,b). The proportion of transitional animals in each sample is directly related to the proportions of animals in each modal group, reaching its maximum at the point when the decline in ‘low group’ animals is paralleled by a corresponding increase in ‘high’ group animals.

Discussion

Although the modal switch to ‘summer’ levels of cryoprotec-tion in C. antarcticus is achieved readily by feeding and warm-ing over short timescales, the switch to a ‘winter’ acclimatory state is less readily achieved. Thus, animals lose their cold tol-

erance after just hours of exposure to higher temperatures and access to food, whereas the increase in cold tolerance in animals cooled from 5 to 0 °C is qualitatively similar to pre-vious observations of rapid cold hardening in C. antarcticus

time (h)0 20 40 60 80

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transferred from:

Fig. 4. The effect on cold tolerance (proportion in the low group) of animals cultured at 10 and 5 °C and subsequently transferred to 0 °C; with food and moisture; ( � ) 10 – 0 °C ( � ) 5 – 0 °C and with moisture only (�) 5 – 0 °C (¦) 10 – 0 °C.

Fig. 3. (a) Combined results for samples cultured at 5 and 10 °C with food. (b) Linear relationship between log 10 time against per-centage in the low group.

(a) combined data

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Fig. 5. Loss of cold tolerance when given access to food at (a) 10 °C�and�(b)�5 °C�showing�high�group�(solid�bar,�SCP>�10 °C)�transitional�(grey� bar,� between��10 and �20 °C)� and� low� group� (open� bar,�<�20 °C).



( Worland & Convey, 2001 ). However, cooling over a wider temperature range ( Fig. 2 ) and transfers to lower temperatures) fails to induce a modal switch to greater cold hardiness.

That cold tolerance can so rapidly atrophy in this species re-emphasizes the well known association between feeding and lowered SCP in these animals ( Cannon & Block, 1988 ). Previous evidence indicating that uptake of water alone can be sufficient to terminate cold tolerance ( Cannon, 1986 ) was not corroborated. However, the present study employed shorter timescales and summer animals acclimated to low temperatures rather than winter-acclimatized animals with their complete suite of cryoprotective measures (colligative antifreezes, evacuated guts, low water content, winter dor-mancy) in place ( Cannon, 1986 ). Physiologically, whatever changes are undergone to attain rapid cold hardening are readily negated by feeding. From an ecological perspective, the experimental manipulations carried out in the present study provide valuable insight into the behavioural responses of C. antarcticus to changes in temperature and resource availability. Burn (1984) found that C. antarcticus showed evidence of a less opportunistic life cycle strategy than the collembolan Parisotoma octooculata on Signy Island. However, although C. antarcticus may rely, relatively, more heavily on overlapping gen erations and extended life cycles than rapid energy assim ilation ( Burn, 1984; Convey, 1996 ), opportunism is nonetheless an important attribute of its life-history strategy. Indeed, on the evidence of these experi-ments, it is clear that a large proportion of animals will resume or begin feeding almost immediately after temperatures rise above 0 °C (i.e. ‘warm’ Antarctic summer temperatures promote compensatory resource acquisition in C. antarcticus ).

Given this ready return to the feeding state (and the attend-ant risks to low temperature survival), it is perhaps all the more surprising that C. antarcticus do not turn ‘on’ their cold hardiness so easily. Shifts to the ‘winter’ mode of cold toler-ance are less predictable and perhaps subject to a greater variety of interacting cues. The modal switch of animals cooled at slow rates over a narrow temperature range sug-gests that, although less necessary for the atrophy of cold hardiness, ‘ecologically realistic’ ( sensu Kelty & Lee, 1999; Worland, 2005 ) cooling may be an important component of the response. Slow cooling rates, rather than transfer and acclimation to low temperatures, were the only factors that induced significant short-term changes. In that the acquisi-tion of cold hardiness, at a minimum, requires the cessation of feeding, perhaps animals are reluctant to trade-off feeding and somatic development unless cues are sufficiently ‘realis-tic’ in their suggestion of risk.

A proportion of the animals exposed to food and moisture at higher temperatures did not lose their cold tolerance at all (approximately 32% after 48 h). A likely explanation is that this element of the sample population (or part of it) might have been in the moult state, with their cold tolerance acquired ‘accidentally’ by moulting itself or their arrival at a nonfeeding pre-ecdysial stage. Moulting significantly reduces the freeze susceptibility of both Collembola and Acari ( Worland, 2005 ; Worland et al ., 2006; Hawes et al ., 2007 ) .

During moulting, the midgut lining is shed, resulting in the complete evacuation of the gut contents. Termination of moult-acquired hardiness would only be expected to occur when such animals have completed their ecdysis and are able to resume feeding and somatic development. Although higher temperatures might shorten the transitional period between resting and ecdysis ( Burn, 1981 ), the loss of cold hardiness would not be accelerated by access to food per se , only by the termination of this part of their developmental pro-gramme. However, although moulting is known to interact with cold hardiness, other unknown endogenous factors might also contribute to low supercooling points.

Rapid changes in cold hardiness are not really a digital response because the transition between winter and summer modes is a discrete process involving a transitional state of ‘semicold hardiness’ ( sensu Hawes et al ., 2006a ). However, in this species, the switch between modes, when it occurs, is nonetheless very rapid; it is not evident at lower temporal resolutions, nor indeed do animals remain ‘transitional’ for any longer than it takes to acquire their new mode of cold hardiness (i.e. a few hours).

The modal switch in the cold tolerance of C. antarcticus is thus a subtle process, governed by a matrix of factors, both endogenous and exogenous. Fine-scale examination of the temporal component of the acquisition and loss of cold hardiness reveals the rapidity with which the switch to a summer modal state is achieved in response to warming and feeding. The reverse process (i.e. the switch to the winter modal state) is more problematic, but is probably con-nected, at least partially, to cooling rate. The balance between these states apparently is managed, temporally, by trade-offs between the acquisition of nutrients and cryoprotection.

Acknowledgements

T.C.H. is supported by a BBSRC studentship. The NERC Antarctic Funding Initiative (CGS6/13) funded fieldwork at Rothera Research Station.

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Accepted 21 December 2006First published online 6 June 2007

Documents

Temporal resolution of cold acclimation and de-acclimation in the Antarctic collembolan, Cryptopygus antarcticus