Cryptobiosis: in Tardigrada
Cryptobiosis: in Tardigrada
Cryptobiosis: in Tardigrada
1-29 I
Printed in Great Britain
CRYPTOBIOSIS IN TARDIGRADA
BY c. WRIGHT*,PETERWESTHt and HANSR A M L 0 V f
JONATHAN
Zoological Museum, University of Copenhagen, I 5 Universitetsparken, DK-2 I 00
Copenhagen 0, Denmark
(Received 3 May 1991, revised 23 September 1991 accepted 25 October 1991)
CONTENTS
I. Introduction . . . . . . . . . . . . . . I
( I ) Historical background . . . . . . . . . . . . 2
VII. Summary . . . . . . . . . . . . . . . 22
VIII. Acknowledgements . . . . . . . . . . . . . 23
IX. References . . . . . . . . . . . . . . . 23
I. I N T R O D U C T I O N
A common mechanism by which organisms adapt to physiological stress is to reduce
metabolism and enter a state of quiescence (Cooper & Van Gundy, 19-71; Evans &
Perry, I 976) associated with retarded senescence. Smaller life forms with high surface
area :volume ratios are prone to very rapid changes in their internal milieu following
environmental perturbations (temperature, hydration, osmolality), and adaptation is
correspondingly critical, Many such forms display quiescence where metabolism is
retarded to undetectable levels, a state termed cryptobiosis (Keilin, 1959). This should
be distinguished from diapause, the hormonal suppression of development, which
Department of Zoology, University of Toronto, 25 IIarbord Street, Toronto, Ontario, Canada Sl5S I A I .
t Carlsberg Foundation Fellow, Department of Chemistry, H.C. Orsted Institute, University of Copenhagen,
5 Universitetsparken, DH-2 loo Copenhagen, Denmark.
f Department of Physiology, Medical School, University of Otago, Dunedin, New Zealand.
2 JONATHAN c. WRIGHT, PETER WESTH AND HANSR A M L ~ V
persists largely independent of short-term environmental cues (Womersley, 198I a).
Although cryptobiosis has been the subject of several excellent reviews (Schmidt, 1948 ;
Keilin, 1959;Crowe, 1971, 1975; Womersley, 1981a),the Tardigrada have only been
specifically reviewed in this context in one recent work (Crowe, 1975). The substantial
body of literature accumulated over the past 15 years now permits a more complete
understanding of the physiological basis and adaptive significance of cryptobiotic
processes in this phylum. A synthesis of current information is therefore timely and
should facilitate comparisons between the different cryptobiotic states in tardigrades
and other taxa, as well as pointing to fruitful avenues for future research.
( I ) Historical background
Knowledge of cryptobiosis dates back to 1702 when the pioneer Dutch microscopist
Anton van Leeuwenhoek described the revival of ' animalcules ' (bdelloid rotifers) from
re-wetted gutter sediments. Although Leeuwenhoek ( I702) did not assume internal
organs of the rotifers to be dehydrated, he observed the longevity of the dried animals
and speculated that the phenomenon was probably widespread. T h e remarkable
observations prompted little scientific enquiry until Needham ( I743) reported that
blighted wheat grains contained 'white fibres' which 'took life' on wetting. Some of
these nematodes where sent to the distinguish microscopist Henry Baker who observed
.
that they remained viable for 4 years and wrote '. . life may be suspended and seemingly
.
destroyed., .(yet) may begin anew., by replenishing the organs and vessels with a fresh
supply of fluid.' Despite repudiation from Spallanzani in I 769, these early observations
on rotifers and nematodes were soon substantiated and significantly extended by
Roffredi ( I775-76) and Fontana ( I776).
It was when Spallanzani returned to the field with revised wisdom that he gave the
first description of tardigrades in 1776 as one of the groups which 'enjoy the advantage
of real resurrection after death '. He showed tardigrades, nematodes and bdelloids to
withstand freezing, although freeze-tolerance in nematodes had been recognized over
a century earlier by Power (1663-64). Spallanzani was also the first to recognize the
enhanced environmental resistance of dried forms, noting that whilst dehydrated
rotifers could withstand 73 OC,hydrated animals were killed at temperatures above
45 "C, although he made the significant observation that both would withstand cooling
to -24 "C. Similar conclusions were drawn by Doykre (1842) who showed dried
tardigrades (Macrobiotus hufelandi) to survive for a few minutes at 120-125 "C. He
made the penetrating comparison with the stability of hydrated and dried albumin,
assuming the living tissues were truly dehydrated but their molecular composition
remained intact. Doykre further believed that the dried animal sustained complete
desiccation and metabolic arrest, ideas largely substantiated by an appointed
commission of the Biological Society of France (Broca, 1860).
The extreme drought-tolerance, together with similar resistance induced by cooling
below o "C (Broca, 1860), was named anabiosis or 'return to life' (Preyer, 1872) and
abiosis (Schmidt, 1948).Keilin (1959) proposed the term cryptobiosis (latent life), which
is usually adopted today. Among conditions inducing cryptobiosis (Keilin lists : loss of
water, lowering of temperature, elevated solute concentrations and reduction of oxygen
levels ; for which he proposed the respective terms anhydrobiosis (Giard, I 894)'
cryobiosis, osmobiosis and anoxybiosis (Keilin, 1959). There is still speculation as to
Cryptobiosis in tardigrada 3
whether these represent homologous adaptive states since, excepting anoxybiosis, all
involve loss of free (‘bulk ’) water with consequent suspension of dependent metabolic
processes.
Fig. I . Scanning electron micrographs (scale bars = 25 pm) of tardigrades during different stages of tun
formation. ( a )hlacrobiotus richfersi in a fully extended condition. ( b ) Hypsibius nberhcuseri showing partial
retraction of the anterior segments and limbs; the pronounced cuticular depressions are points of muscle
insertion. (c) ffypsibius oberhaeuseri in the completed tun state. Limbs and intersegment cuticle are
inflexed and regular wax extrusions adorn the cuticle surface.
8 JONATHAN c. WRIGHT, PETER WESTH AND HANSh M L 0 V
1983; Greven 81 Peters, 1986) but concurs essentially with the arthropod scheme. T h e
endocuticle is divisible into a distal, lipid-rich intracuticle, and proximal procuticle
(Baccetti & Rosati, 1971; Wright, 1988~). Morphometric analysis of the cuticles of four
species (Wright, 1988b)shows the intersegmental regions to be approximately half the
thickness of intrasegmental cuticle, with maximum thinning in the (by inference) stiff
procuticle, suggesting adaptations for flexibility. Minimum thinning occurs in the
intracuticle where the permeability-reducing role of lipids would represent a premium
(see below). In the heterotardigrade Echiniscus testudo the inner epicuticle is
dramatically modified as thickened dorsal plates containing an apparently air-filled
lacunar system (Greven, I 97 I a, b ; I 972; Wright, I 988a). These apparently impose
severe restrictions on surface-area reduction during tun formation, and hence on
transpiration reduction, but this may be offset by the long diffusion path of water
traversing the lacunae (Wright, 198bb).
Gravimetric studies in controlled water activities (Crowe, I 972; Wright, 1989 a)
reveal consistent transpiration profiles during primary desiccation (Fig. 2 a). During
tun formation, transpiration declines linearly (exponential mass-loss) as surface area is
reduced, in contrast to the transpiration of dead animals (killed by heat-shock) which
remains almost constant. Comparison of half-times of mass-loss between living animals
following tun formation, and dead animals at a comparable stage, shows transpiration
to be approximately halved by tun formation, depending on species. This agrees closely
with micrometric measurements of surface-area reduction in tuns (Wright, I 989a)
indicating that selective invagination of more permeable cuticle regions, and hence
reduction of mean cuticle permeability, is of minor significance. At this stage, the
cuticle is apparently freely permeable, transpiration following the rate parameters of the
preceding free water surface.
Soon after completion of tun formation, transpiration dynamics are abruptly altered
by a rapid decline in cuticle permeability (Crowe, 1972; Wright, 1989~).This
' permeability slump ' reduces transpiration by a factor of 20-60 within a few minutes
and is evident in living and dead animals (Wright, 1 9 8 9 ~ ) .It is not, therefore, a
metabolic phenomenon. Subsequent transpiration continues to decline slowly to
scarcely measurable levels. If residual masses are subtracted from the mass-loss curve,
and plotted semi-logarithmically, a biphasic curve results (Fig. 2b) with the
permeability slump and subsequent further decline in transpiration showing different
half- times.
Owing to minimal residual permeability, the permeability slump permits animals to
retain a substantial fraction of their bulk water for extended periods of drying.
Calorimetric estimates of vicinal water content (water not revealing an endothermic
phase transition close to o "C) give 2-3 % of the hydrated mass (Wright, 1989b). Taking
this into account, the species studied retain between 5 and 1 5 % of bulk water when
dried in 80 Yo r.h. However, on more rapid drying the onset of the permeability slump
is delayed, with only 105-3 yo of bulk water retained by Mucrobiotus richtersi dried in
25-30% r.h. (Wright, 1 9 8 9 ~ ) If
. a critical proportion of the bulk water must be
retained for sustained metabolism, in accordance with the vicinal network model, the
rate-dependent timing of the permeability slump could explain why slow drying is
essential for survival.
Substantial evidence has been amassed to indicate a lipid basis for the permeability
Cryptobiosis in tardigrada 9
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Fig. 3. Mean (+sE) trehalose levels for Adorybiotus coronifer dried embedded in sand; typical
morphological stages of the animals are illustrated below the abscissa. Zero hours represents active
animals (hydrated for 24 h). The dashed line shows the sand water potential (data points omitted).
in situ regularly suffer effective metabolic arrest since mosses and lichens rapidly
dehydrate to stable masses and thus attain equilibrium water activities with the
surrounding air (Wright, 1991). However, it is also clear that animals dried in
significantly higher humidities will sustain appreciable metabolism, displaying
correspondingly reduced viability.
1 2 3 4 5 6 24 25 26 27
Time (hours after hydration)
Fig. 4. Trehalose content and [3H]leucine incorporation in rehydrating Adorybiotus coronifer collected dry
from mosses in Oeland (Sweden). Solid squares : trehalose level when rehydrated in distilled water. Open
squares : trehalose level when rehydrated in C0,-perfused medium. Open circles: [SH]leucine
incorporation (['H]lleucine added I 5 min, after rehydration); solid triangles : ['H]leucine incorporation
(['€I]leucine added 24 h after rehydration); both under normal gassing conditions.
111. OSMOBIOSIS
Following Keilin (I 959), osmobiosis is defined as cryptobiosis induced by osmotic
extremes and it remains the most poorly studied and least understood of cryptobiotic
processes. T h e only directed studies concerning Tardigrada have been those of Collin
& May (1950) and Wright (1987),which only warrant brief coverage. All terrestrial
species thus far investigated survive prolonged immersion in distilled water and hyper-
regulate efficiently. This also applies to the littoral Echiniscoides sigismundi which can
withstand freshwater, although abrupt salinity changes cause temporary immobility.
Osmobiosis is thus induced chiefly, perhaps exclusively, by hyperosmotic media.
( I ) Primary hyperosmosis
On immersion in saline solutions above ca. 300 mOsm kg-', the approximate
haemolymph osmolality (Crowe, I 972),bryophilous tardigrades contract rapidly into a
tun (Collin & May, 1950;Wright, 1987). This suggests that the primary response is to
dehydration. Unlike anhydrobiosis, tun formation is not indispensable since active and
asphyxiated animals show comparable survival in high salinities (Collin & May, 1950).
However, survival does not compare with anhydrobiosis for corresponding water
activities, M. hufefandi only surviving 6 h immersion in I IOO mOsm kg-' NaCl(,,,
(Collin & May, 1950),of equivalent water activity to a humidity of 98.0 %.
I4 JONATHAN c. WRIGHT, PETER WESTH AND HANSR A M L ~ v
Viability decreases with prolonged immersion in even modest salinities and higher
salinities ( > 3.0 Osm kg-' NaCl) are lethal within a few minutes (Wright, 1987);
durations of immersion in different salinities inducing 50 % mortality (LD,, estimates)
for M. richtersi and H . oberhaeuseri are listed in Table I . Other inorganic osmolytes
appear to induce similar effects, including KCl, CaCl,, and MgCl,, whilst glucose (a
non-electrolytic osmolyte) is tolerated in much higher (ca. x 10)concentrations (Collin
& May, 1950). Clearly, tardigrades suffer substantial influx of many species of inorganic
ion to which they display only modest tolerance. It may be noted that Artemia cysts,
which tolerate prolonged immersion in even saturating NaCl concentrations (Clegg,
1976b) possess an outer membrane completely impermeable to inorganic ions (ContC et
al., 1977). Lowered external water activities would be expected to induce a cutaneous
permeability decline in tardigrades, but dynamics in this process would be altered by
the increasing haemolymph activity. Since animals maintained for a few hours in sub-
lethal salinities do not subsequently survive much higher salinities (J. C. Wright,
unpublished observations), a substantial permeability decline may not occur. Survival
would thus be limited by an animal's tolerance of equilibration to external solute
activities. Tolerances of different solutes during the equilibration period would
therefore depend on diffusion coefficients through the cuticle and epidermis, as well as
specific effects on cellular physiology.
Across the spectrum of eukaryotes, inorganic ions are only tolerated over a narrow
range of intracellular concentrations, elevated levels (typically > zoo mOsm kg-' for K',
Na+, C1-) perturbing cellular reactions via effects on enzyme p K values and Michaelis
constants (&). Compatibility of ionic solutes is traditionally classified in the
Hofmeister series (Hofmeister, I 888). Deleterious effects are thought to result from
ligand binding to enzyme active sites and/or substrate molecules (see Yancey et al.,
1982; Somero, 1986). Many organic osmolytes are tolerated in much greater
concentrations which may relate to the absence of ionic charge or to entropic instability
of larger molecules interacting with the structured water of proteins (Somero, 1986;
Arakawa 8z Timasheff, 1985). Passive tolerance may account for the high concentrations
of glucose tolerated by tardigrades although the low diffusion coefficient of glucose
would also facilitate hyper-regulation, Acclimation to elevated osmolalities presumably
involves osmoconformity and the intracellular accumulation of compatible solutes
(certain amino acids, polyhydric alcohols, glycerol, betaine) as shown in many groups
(see Somero, 1986). By contrast, the superior tolerance of elevated external osmolyte
concentrations by anhydrobiotic animals probably depends on the permeability slump
and stabilization of proteins and lipid bilayers by protectants.
(2) Mobilisation of membrane protectants
There is currently no evidence for synthesis of trehalose or other possible membrane
protectants in osmobiotic tardigrades or any other osmobiotes. Since initial immersion
in aqueous sodium chloride solutions above 600 mOsm kg-' (only inducing dehydration
to ca. 50 Yo water-retention) is not tolerated for appreciable periods, further
dehydration, requiring protection, remains conjectural. Tolerance of higher salinities
probably depends on acclimation (see below) ; such animals display normal activity.
Cryptobiosis in tardigrada 15
Table I . LD,, mortality estimates for Macrobiotus richtersi and Hypsibius oberhaeuseri,
transferred to distilled water following immersion in difj'erent concentrations of aqueous
N a C l for various durations ( t in minutes). Animals were hydrated for 24 h in the
habitat plant material prior to study; n = 16 for all trials
"aCI,,,I M . richtersi If. oberhaeuseri
I Yo (319 mOsm./kd 300 ( > 2000)
2 Yo (638 mOsm./kg) I 1 0 700
3 Yo (962 mOsm./kg) 70 140
4 Yo (1,295mOsm./kd 60 I20
5 Yo (1,638mOsm./kg) 50 I00
10% ( 3 , 5 9 2 mOsm./kg) 20 30
( 5 ) Ecological considerations
Although adaptations to hyperosmosis appear moderate compared to anhydrobiosis,
they are supplemented by an impressive capacity for acclimation. T h e cosmopolitan
bryophilous species H. oberhaeuseri, E. quadrispinosus and Milnesium tardigradum have
been reported from littoral habitats (Dr C. I. Morgan, personal communication ;
Renaud-Debyser, I 964). Probably the most euryhaline of tardigrades are the
mesolittoral heterotardigrades Echiniscoides sigismundi and Archechiniscus marci which
live on rocky shores, largely associated with Enteromorpha and barnacle tests (GrohC,
1976; Kristensen & Hallas, 1980), and survive tidal cycles of sea water and severe
desiccation, combined with huge fluctuations in osmolality during evaporation and
rainfall.
In typical bryophilous habitats, hyperosmotic conditions will be largely restricted to
periods of prolonged hydration on soluble substrates, and to late stages of habitat
dehydration. Tardigrades are often abundant in cryptograms subjected to salt-spray
which may necessitate more extreme resistance; these probably rely in part on
16 JONATHAN c. WRIGHT, PETER WESTH AND HANSRAML0V
acclimation, although periodic flushing of solutes by rainfall will necessitate sustained
tolerance of very low salinities also.
IV. CRYOBIOSIS
Cryobiosis is defined as cryptobiosis induced by freezing and should be distinguished
from the freeze-resistance of anhydrobiotes. Although Rahm (1921)showed fully
hydrated H. oberhaeuseri to survive rapid freezing to -253 "C, such extreme freeze-
tolerance is often misinterpreted as an exclusive property of the anhydrobiotic state
(Block, 1982;Storey & Storey, 1988).
Poikilothermic metazoans seem to have evolved two main mechanisms for tolerating
sub-zero temperatures (see Block, 1990,or Storey & Storey, 1988,for recent reviews).
In the first, freezing is always lethal and tolerance depends on 'freeze avoidance':
freezing-point depression of body fluids and/or a capacity for supercooling. Freezing-
point depression (FPD) involves the use of osmolytes such as glycerol to depress
freezing (and melting) points below o "C (Mazur, 1980; Baust & Rojas, 1985). Since
FPD is a direct function of osmolality, and molal FPD = 1.858 "C osmol-' kg, even
high concentrations of compatible solutes only induce modest FPD. Supercooling
relies largely on prevention of ice nucleation by so-called 'antifreeze proteins ' (which
also depress the freezing point) (see, for example, Miller, 1982; Ssmme, 1982;
Zachariassen, I 985), although osmolytes such as glycerol and other polyhydric alcohols
also exert a pronounced effect on supercooling capacity. Supercooling points of - 20 to
-45 "C are common in arthropods and values below - 70 "C are known (see S ~ m m e ,
1982).
The second adaptive mechanism, less common in invertebrates, is the ability to
tolerate extracellular ice-formation (Lee, 1989; Block, 1990).Such animals are termed
'freeze-tolerant ), regardless of the degree of ice accumulation withstood. Typical
adaptations again include the accumulation of osmolytes (frequently glycerol, inositol
and trehalose) and ice-nucleating proteins, the latter ensuring controlled ice-nucleation
at only a moderate level of supercooling (Storey 8z Storey, 1988).Some tardigrades (in
a hydrated state) tolerate temperatures where freeze-avoidance seems impossible. T h e
distinction of cryobiosis as a specialized form of freeze-tolerance depends on sustained
viability following low temperature-induced metabolic arrest. Few systematic bio-
chemical and physiological experiments on tardigrade cold-hardiness have been
performed, and it remains unclear whether the processes involved differ qualitatively
from those described in other freeze-tolerant animals. T h e distinction of cryobiotes
must thus be based on indirect studies of their metabolic status.
Nuclear magnetic resonance (NMR) studies on larvae of the freeze-tolerant gallfly
Eurosta soliduginis have detected significant metabolism in the frozen state at
temperatures down to -30 "C (Storey et al. 1984),close to the temperature inducing
freeze-injury in this species (Lee & Lewis, 1985), thus indicating that freeze-tolerance
in this species does not conform to cryobiosis. Monitoring of metabolic rate in
cryobiotic tardigrades has not been performed, but the tolerance of extremely low
temperatures shown for certain species (Rahm, 1921; Ramlev & Westh, 1990)can only
be explained by a reversible ametabolic state,
Cryptobiosis in tardigrada 17
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100 0.1% Trehalose
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20
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1 10 100 1000
Cooling rate ("C min.-')
Fig. 5. Semi-logarithmic plots of survival of Adorybiotus coronifer following cooling from 20 "C to
- 196 "C at different rates. The two samples were rehydrated for different periods following prolonged
anhydrobiosis to provide pre-determined trehalose levels of ca. 1.0and 0-1yo d.m. for curves ( 0 ) and (b)
respectively. Differences over this range have no clear effects on freeze-tolerance.
s .* .'
. v
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5:
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(D
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Fig. 6. Survival of Adorybiotus coronifer cooled at cu. 30 "C min-' to the indicated temperatures before
rapid cooling to - I 96 "C at a rate of cu. I 500 "C min,-I. The survival curve (A) shows yo recovery ( & SE)
of 3-4 experimental trials. Precooling temperatures are indicated on B. For example, at point X,animals
were cooled to - 5 5 "C at a rate of 30 "C min.-', over the period indicated on the abscissa, before rapid
cooling to - 196"C. Slow cooling to temperatures below 5 O C confers substantial freeze-tolerance,
suggesting that lethal events of rapid cooling are associated with ice-formation.
temperatures was reduced ; again, there was no recovery after cooling at I 500 "Cmin-'
(Fig. 5 ) . Elevation of the trehalose content from 0 1 to 1.0%would thus seem to offer
some additional protection from freezing at low cooling rates. Exposure to 0.5 "C for
16 h prior to freezing did not induce increased recovery, indicating that increased
protection is not afforded by short-term pre-acclimation.
The same workers investigated the effect of slow freezing on conferring tolerance to
more rapid cooling rates. Pre-cooling at 30 "C min-' to temperatures between the
crystallization temperature of the sample (- 5 "C) and - 90 "C provided subsequent
resistance to shock-cooling (to - 196 "C)at the otherwise lethal rate of 1500 "Cmin-',
with no significant differences in survival for different pre-cooling temperatures (Fig.
6). The results indicate that lethal events in rapid cooling occur very close to o°C,
subsequent cooling exerting little or no effect on viability (Ramlov & Westh, 1990).
V. ANOXYBIOSIS
Like other meiofaunal Metazoa, bryophilous tardigrades tolerate considerable
periods of asphyxia, whether induced by 0,-shortage or excess aqueous CO, and H,S.
In this state, referred to by some workers as ‘anoxybiosis’ (Keilin, 1959;Crowe, 1975),
animals typically remain extended, turgid and immobile. Unlike other forms of
cryptobiosis, asphyxia involves osmoregulatory failure and water uptake rather than
dehydration. Measurements of CO, production suggest only a small reduction in
metabolism and animals only remain viable for 3-4 days (Crowe, 1975). Whilst
tolerance of asphyxia may be adaptive in large, static water-masses and at high
temperatures, as well as conferring temporary resistance to polysaprobic and anoxic
habitats, it presently seems inappropriate to regard this as a form of cryptobiosis.
However, as a caution, Kristensen & Hallas (1980) reported survival of Eclziniscoides
sigismundi and E. hoepneri for 6 months in a hermetically closed vial containing
decomposing barnacles. T h e adaptive basis of such long- term tolerance is unknown.
VI. CONCLUSIONS
It is clear from the foregoing sections that different forms of cryptobiosis in
Tardigrada justify separate classification and may not even represent homologous
processes at the biochemical level. Nevertheless, it should also be stressed that the
results are derived from studies on a limited number of species, Anhydrobiosis depends
on tun-formation and a cutaneous permeability decline for water-retention, permitting
sustained metabolism for the synthesis of trehalose and other possible protectants.
Osmobiosis is superficially similar but does not permit tolerance of extreme water-loss
or comparable changes in water activity. T u n formation is observed but is not essential
for survival. Metabolic changes and the possible role of membrane protectants during
osmobiosis remain unresolved, Cryobiosis, like anhydrobiosis, does confer tolerance to
loss of bulk liquid water, but tun formation only occurs at low cooling rates. The low
levels of trehalose required by cryobiotic A. coronifer suggest a limited role in
membrane protection although this sugar may serve other vital roles in preventing
intracellular freezing. Loss of bulk water in anhydrobiotes and cryobiotes, with
suspension of oxidative reactions, probably explains their impressive longevity and
resistance to environmental extremes.
In addition to cryptobiosis, certain bryophilous, freshwater and soil tardigrades
undergo seasonal encystation, a process involving three incomplete ecdyses with
extensive tanning of the outer (old) cuticle (Weglarska, 1957). Cysts are not resistant to
prolonged desiccation but are probably produced in response to saprobic factors
(Weglarska, 1957). Internal organs remain fully hydrated and metabolism is moderate ;
0,-consumption in cysts of the freshwater species M. dispar was 24% of measured
values for active animals (Pigon & Weglarska, 1955a, b).
We would thus recommend the inclusion of anhydrobiosis and cryobiosis within
Keilin’s term ‘cryptobiosis’, at least as far as Tardigrada are concerned. Both states
permit reversible metabolic arrest and loss of bulk water. As Keilin (1959) writes : ‘ T h e
concept of life as applied to an organism in the state of anabiosis (cryptobiosis) becomes
synonymous with that of the structure which supports all the components of its
catalytic systems. Only when the structure is damaged or destroyed does the organism
22 JONATHAN c. W R I G H T , PETER 'IYESTH AND HANSh M L 0 V
pass from the state of anabiosis or latent life to that of death'. Osmobiosis is doubtfully
included in this scheme, given the lack of clear evidence for enhanced viability and
ability to tolerate suspended metabolism.
If the term cryptobiosis is restricted to organisms surviving in an ametabolic state,
the definition excludes moderate levels of dehydration where significant metabolism is
maintained. Since the critical preparatory processes take place in the first few hours of
dehydration or freezing, such a definition would also exclude the basic adaptive
mechanisms. Rather, it seems preferable to define cryptobiosis as a collective term for
those quiescent states in which metabolism may be reversibly arrested. This allows the
complete preparatory process to be incorporated into definitions of anhydro- and
cryobiosis. Anoxybiosis and encystation would be excluded but referred to as other
dormant states.
VII. SUMMARY
I , Cryptobiosis, a quiescent state characterised by reversible suspension of
metabolism following loss of free (' bulk ') water, is apparently restricted to terrestrial
(' bryophilous ') tardigrades and the littoral Echiniscoides sigisrnundi and Archechinisms
marci. Depending on the mode of induction, cryptobiosis is classified as anhydrobiosis,
osmobiosis or cryobiosis.
2. Anhydrobiosis is induced by slow dehydration. During the primary drying,
tardigrades display active, longitudinal contraction and invaginate limbs and inter-
segmental cuticle to form the tun. Then follows an abrupt decline in cutaneous
permeability attributed to a hydration-dependent phase-change in amphiphilic,
intracuticular lipids. These processes serve in water-retention, permitting sustained
metabolism and biochemical preparations ; desiccation tolerances among species show
clear correlations with cuticle composition, tun dimensions and kinetics of the
permeability decline.
3. During progressive desiccation, Adorybiotus coronifer shows a 23-fold accumu-
lation of trehalose, stabilising after 5 7 h. This disaccharide has been shown to replace
the vicinal water of dried phospholipid bilayers, hence stabilising membranes, and is
accumulated in many other cryptobiotes. Roles of glycerol and other likely membrane
protectants in anhydrobiotic tardigrades await investigation.
4. Oxygen uptake in anhydrobiotic tardigrades effectively ceases below a water
activity of 0.48. Regular reduction of water activities below this level in xeric habitats
indicates that metabolic arrest is routine. Such animals show extreme longevity and
remarkable tolerance of environmental extremes.
5 . On rehydration, tardigrades display rapid aerobic catabolism of trehalose, initiated
within 12 minutes, with a half-time of approximately r h. This could operate via pH-
dependent trehalase activity as demonstrated for Artemiu cysts. Early resumption of
metabolism suggests that the permeability barrier is negated by external hydration.
rapid synthesis and catabolism of trehalose may be further determinants of desiccation
tolerance. Xeric trends across seven tardigrade species in southern England relate
closely to measured desiccation tolerances.
6. Osmobiosis - cryptobiosis induced by hyperosmosis - awaits convincing dem-
onstration in Tardigrada, although anhydrobiotic animals tolerate saturation concen-
trations of many osmolytes. Animals form a tun in response to significant hyperosmosis,
Cryptobiosis in tardigrada 23
but do not tolerate equivalent activity deficits to those inducing anhydrobiosis. Survival
is apparently limited by inward diffusion of osmolytes ; as in other eukaryotes, elevated
concentrations of inorganic ions are deleterious and tolerance may conform to the
established Hofmeister series. There is not evidence for a cutaneous permeability
decline or synthesis of membrane protectants. Tardigrades probably tolerate osmotic
perturbations chiefly by a substantial capacity for acclimation,
7. Cryobiosis may be considered a special form of freeze-tolerance. T h e boreal
eutardigrade Adorybiotus coronijer tolerates cooling to - 196 O C at rates up to
1500 "C min-'. Freeze injuries from high cooling rates occur during the initial ice
formation; DSC data reveal that over 80 ?{) of body water freezes at about - 7 OC,with
no measurable increase during further cooling. T u n formation only occurs with slow
cooling and its significance is unclear.
8. T h e high ice-crystallization temperature is attributable to ice-nucleating agents
(INAs) comprising molecular species over 200 kDa ; thermal denaturation reduces the
freezing temperature of animals from -7 O C to - 1 6 "C. Heat stability, size and
concentration of the INAs are similar to those described for other freeze-tolerant
animals.
9. Roles of cryoprotectants in A. coronifer are unclear. Elevation of whole-animal
trehalose concentration from 0.1yo d.m. to I yo d.m. apparently confers some additional
tolerance to freezing at low cooling rates. High freezing rates tolerated would preclude
synthesis or mobilization of cryoprotectants. Pre-existing concentrations of trehalose or
other polyols may promote intracellular vitrification, unfreezability, or colligative
reduction in extracellular ice formation, limiting cellular dehydration. In the absence
of vitrification, cellular dehydration may also necessitate membrane protection.
10. In addition to cryptobiosis, several meiofaunal tardigrades show seasonal
encystation. Cysts remain viable for several months but maintain significant
metabolism. Tardigrades also tolerate brief periods of asphyxia, formerly termed
'anoxybiosis ', though again characterized by sustained high (anaerobic) metabolism.
Such states qualify for quiescence but not cryptobiosis. It is recommended that the
latter term is restricted to anhydrobiosis and cryobiosis, adaptively different processes
characterized by enhanced longevity and environmental resistance, and reversible
metabolic arrest.
V I I I . ACKNOWLEDGEMENTS
The authors gratefully acknowledge the support of a Carlsberg Fellowship (Grant 90-0642/20)awarded to Petcr
Westh for research contributing to this review.
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