Abstracts: CryoLetters 21 (2), 2000

CryoLetters is a bimonthly, international journal for low temperature science and technology

CryoLetters 21, (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU


17 September 1999, Department of Geography,  University of Cambridge

The Symposium explored the diversity of adaptations to cold in a wide range of organisms from microbes to vertebrate animals and highlighted the characteristics of the many and varied adaptations to cold at ecological, physiological, biochemical and molecular levels. Emphasis was placed on the environmental context to aid understanding of the cryobiology. Discussion of the various strategies involved in nature focussed on the potential to apply particular features of natural systems for Man’s benefit.

A summary of the oral and poster presentations made at the Symposium is given below followed by selected abstracts and three papers (*) arising from the meeting.

William Block
Scientific Programme Organiser
Guest Editor


Oral presentations

*How does cold constrain life cycles of terrestrial plants and animals? Peter Convey (Biological Sciences Division, British Antarctic Survey, Cambridge)

Life in the cold and dark - Antarctic lake plankton Johanna Laybourn-Parry (School of Biological Sciences, University of Nottingham)

*Natural cryoprotection - plant and invertebrate strategies Pedro Montiel (Biological Sciences Division, British Antarctic Survey, Cambridge)

Cold adaptation in crop and wild plant species - molecular aspects Roger Pearce (Department of Biological & Nutritional Sciences, University of Newcastle upon Tyne)

The role of water in insect cold tolerance Roger Worland & William Block (Biological Sciences Division, British Antarctic Survey, Cambridge)

*Hibernation and non-freezing cold adaptations in vertebrates Colin Green (Northwick Park Institute for Medical Research, Harrow)

Physiological water stress [SLTB Special Lecture] Felix Franks (BioUpdate Foundation, Cambridge)


Poster presentations

Antarctic collembolan response to climate: a sensitive bioindicator of change William Block and Peter Convey (British Antarctic Survey, Natural Environment Research Council, Cambridge)

A potential invertebrate indicator of climate change on sub-Antarctic South Georgia Peter Convey and Rodney J. Arnold (British Antarctic Survey, Natural Environment Research Council, Cambridge)

The kinetics of glycerolisation of skin grafts Q. Huang and D.E. Pegg (Medical Cryobiology Unit, Biology Department, University of York, York)

Simultaneous cryopreservation of orchid seed and it's endophytic fungal symbiont C. B. Wood, H. W. Pritchard and A. P. Miller (Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly)

Temperature gradients in intact/undisturbed soil cores - an experimental system for use in freeze-thaw studies M. Roger Worland (British Antarctic Survey, Natural Environment Research Council, Cambridge)

Water and dimethyl sulphoxide permeability of porcine vascular endothelial and smooth muscle cells M. C. Wusteman and D.E. Pegg (Medical Cryobiology Unit, Biology Department, University of York, York)



CryoLetters 21, 73-82 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

How does cold constrain life cycles of terrestrial plants and animals?


British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET


Short summer seasons with low thermal energy input characterise polar ecosystems. Climatic variables and physical isolation of terrestrial habitats act as selective filters which must be passed to allow colonisation , establishment and survival in these extreme environments. Life history studies of the terrestrial biota of such ecosystems give little evidence of adaptive responses to low temperatures having evolved in situ, even though behavioural, ecophysiological and biochemical features allowing tolerance of the likely extremes are well-developed. Observed life history strategies are often consistent with the general predictions of “adversity” (A) or “stress” (S) selection. However, biota successful in these extreme environments may be better-grouped by the lack of particular life history features, rather than common possession.

Keywords. Cold, ecophysiological, life history, polar regions, seasonality.



CryoLetters 21, 83-90 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Soluble carbohydrates (trehalose in particular) and cryoprotection in polar biota.

P.O. Montiel

British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET


Data are presented here on low molecular weight carbohydrates contents (assessed from samples collected in situ) of selected, representative terrestrial species from the maritime Antarctic. Significant seasonal variation for most species were observed. Increased levels overwinter in arthropods contrasted with cryptogams where levels were higher in spring and summer samples. The occurrence and seasonal variation of trehalose in particular is demonstrated for invertebrate and lichen species. The data are discussed in relation to published results from experimental manipulation of growth temperatures on Antarctic fungi and an Arctic springtail providing further evidence for the protective role of trehalose. The widespread presence of trehalose in polar biota is documented and discussed regarding acclimatisation to both low temperature and partial dehydration.

Keywords: Antarctic, polar, cryoprotection, desiccation, trehalose, compatible solutes.



CryoLetters 21, 91-98 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Mammalian hibernation: Lessons for organ preparation?

C. Green

Northwick Park Institute for Medical Research, Northwick Park Hospital, Harrow, Middlesex, HA1 3UJ


The adaptations to low environmental temperatures exhibited in mammalian hibernation are many and varied, and involve molecular and cellular mechanisms as well as the systematic physiology of the whole organism. Natural torpidity is characterised by a profound reduction in body temperature and other functions lasting from a few hours to several weeks. Controlled reduction of heart rate, respiration and oxygen consumption is followed by the fall in body temperature. However, thermoregulation persists such that a decrease in ambient temperature below dangerous levels typically triggers arousal, and shivering and non-shivering thermogenesis from brown fat provide the heat to restore body temperature to normal levels.

Many of the cellular mechanisms for survival are similar to those brought into play during medium-term storage of organs destined for transplantation. For example maintenance of ionic regulation and membrane fluxes is fundamental to cell survival and function at low body temperatures. Differences between hibernating and non-hibernating species are marked by differences in Na+/K+ transport and Ca++pumps. These in turn are probably associated with alterations in the lipoproteins of the plasma membrane and inner mitochondrial membrane.

We have accordingly conducted a series of pilot studies in captured Richardson’s ground squirrels kept in laboratory conditions as a model for hypothermic organ preservation. Tissue function was compared during the summer (non-hibernating season) with that in the winter when the animals could be: (i) in deep hibernation in a cold chamber at 4oC; (ii) maintained in an ambient temperature of 4oC but active and awake; or (iii) active at an ambient temperature of 22oC. The studies involved: whole animal monitoring of standard physiological parameters; whole organ (kidney) storage and transplantation for viability assessment; storage and functional assessment on an ex vivo test circuit with capacity for perfusion at normothermic and hypothermic temperatures; measurement of thyroid function; measurements of total nucleotides (ATP, ADP and AMP)and ratios by standard techniques after freeze-clamping of organs; similar nucleotide and pH measurements using31P-NMR as a non-invasive whole animal technique; and measurement of O2 uptake and gluconeogenesis using isolated renal tubules and isolated hepatocytes. Marked differences in cold tolerance were demonstrated between organs taken from hibernating versus non-hibernating individuals. In particular kidneys transplanted from animals in deep hibernation were capable of withstanding up to 72 hours of cold storage as compared with up to 24 hours in non-hibernating squirrels or in comparable sized rats. Adaptations which might provide valuable clues in our attempts to better preserve human organs for transplantation are explored in some depth in this report.

Keywords: hibernation, circannual rhythms, torpor, arousal, euthermia.



CryoLetters 21, 99-106 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

DSC studies of freezing in terrestrial enchytraeids (Annelida: Oligochaeta).

W. Block*1 and R. Bauer2

1 British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK (email: )
2 Institute of Zoology, University of Agricultural Sciences, Vienna, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria


Specimens of six enchytraeid species from a wide range of terrestrial habitats were cooled to –25oC and rewarmed at 1oC min-1 and their cryobiological features measured by DSC. All were freezing intolerant, supercooling on average to c.-9oC. The quantity of frozen (osmotically active) water, calculated from the melt endotherm, varied considerably from 17 to 62% depending on the species and formed three groups. It is suggested that variation in the amount of water frozen in the worms reflects the acclimatisation of these species to the environmental conditions of their habitats.

Keywords: Enchytraeidae, Differential Scanning Calorimetry, supercooling, freezing intolerant, frozen (osmotically active) water, temperature acclimation.



CryoLetters 21, 107-116 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.)

Y. Chang1, R.E. Barker2 and B.M. Reed3

1 Department of Horticulture, Oregon State University, Corvallis, OR 97331.
2 USDA/ARS National Forage Seed Production Research Center, 3450 SW Campus Way, Corvallis, OR 97331.
3 USDA/ARS National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333.
*Corresponding author. Email:


Cold acclimation of Lolium L. and Zoysia Willd. Grass cultivars significantly increased regrowth of cryopreserved meristems. One wk of cold acclimation improved recovery following cryopreservation but extended acclimation (4-8 wk) resulted in the best regrowth. Cold acclimation also significantly increased the dehydration tolerance of both Zoysia and Lolium meristems. Lolium apices cold acclimated for 4 wk produced 60-100% regrowth following cryopreservation by slow freezing or encapsulation-dehydration. Cold-acclimated Zoysia had greater than 60% regrowth following encapsulation-dehydration when beads were dehydrated to less than 22% water content. Non-acclimated meristems of both genera had little or no regrowth. Thawed meristems grew quickly without callus formation and the plantlets produced were transplanted to pots in the greenhouse after 4 to 6 wk. Samples of each cultivar were stored in liquid nitrogen as part of the U.S. National Plant Germplasm System.

Keywords: cold acclimation, dehydration tolerance, ryegrass, genetic resources, liquid nitrogen, meristem, zoysiagrass.



CryoLetters 21, 117-124 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Yiping Li 1*, He Gong 1 and Ho-Yong Park 2

1 State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
2 Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305-600, Korea


The pine needle gall midge of Thecodiplosis japonensis is a serious forest pest and overwinters as a 3rd instar larva at soil surface in Korea. The time necessary for killing 50% of larvae at -15C is 160 min. During overwintering period, T. japonensis larvae accumulate relatively high content of trehalose as the main cryoprotectant. In this paper, the proteinaceous cryoprotectants were identified. Two thermal hysteresis proteins (THP-1S and 2S) were purified from overwintering larvae by ethanol fractionation, trichloroacetic acid precipitation, ion-exchange chromatography (DEAE-Sephadex A-25) and gel permeation chromatography (Sephadex G-100). Their molecular weights are 34.9 and 37.8 kD respectively. T. japonensis THPs cannot be stained by periodic acid-Schiffs’ reagent, suggesting no carbohydrate in them. The thermal hysteresis activity of THP-2 at the concentration of 50 mg/ml is 11.020.08C (meanSD, n=10), perhaps the highest active insect THP. It is the first report of purified T. japonensis THPs in Diptera.

Keywords: Thecodiplosis japonensis, thermal hysteresis proteins, antifreeze protein, freeze tolerance.



CryoLetters 21, 125-136 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Simultaneous preservation of orchid seed and its fungal symbiont using encapsulation-dehydration is dependent on moisture content and storage temperature.

C.B. Wood*, H.W. Pritchard and A.P.Miller

Seed Conservation Department, Royal Botanical Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK


Seeds of Dactylorhiza fuchsii (common spotted orchid) and Anacamptis morio (green-winged  orchid) were encapsulated in alginate beads with hyphae of the basidomycete fungus Ceratobasidium cornigerum. Pre-treatment of beads for 18 h with sucrose at an optimum concentration of 0.75 M decreased the desiccation rate in a flow of sterile air (c. 23oC, 30% RH) and increased seed and fungal survival after up to 16 h drying. Pre-treated and 16-h dried beads were transferred to cryo-vials and subsequently stored at a range of low temperatures for up to 30 d. Neither embryo growth of both orchids nor fungal development was detrimentally affected by 1 d storage at –196oC when the beads were pre-dried to c. 20% moisture content. Encapsulated D. fuchsii seed and compatible fungus had < 5% and < 45% viability when beads of the same moisture content were stored for 1 d at –20oC and –70oC respectively. In contrast, viability of the seed and the fungus remained unchanged during 30 days storage at –196oC but was progressively lost at 16oC over the same interval. The results indicate opportunities for the use of simultaneous cryopreservation as a conservation tool for diverse taxa.

Keywords: cryopreservation, encapsulation, drying rate, seed, orchid, fungus.

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