Biol. RLV. (1992). 67, pp.

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

(2) A Current Synthesis . . . . . . . . . . . . 3

11. Anhydrobiosis in Tardigrades . . . . . . . . . . . 7
( I ) Primary Dehydration . . . . . . . . . . . . 7
(2) Mobitisation of Membrane Protectants . . . . . . . . . I0
(3) Attainment of the Ametabolic State . . . . . . . . . 11
(4) Recovery from Anhydrobiosis . . . . . . . . . . I1

(5) Ecological Considerations . . . . . . . . . . . 13

111. Osmobiosis . . . . . . . . . . . . . . . 13
( I ) Primary IIyperosmosis . . . . . . . . . . . . '3
(2) Mobilisation of Membrane Protectants . . . . . . . . . 14
(3) Attainment of the Ametabolic State . . . . . . . . . IS
(4) Recovery from Osmobioeis . . . . . . . . . . . 15
( 5 ) Ecological Considerations . . . . . . . . . . . IS
IV. Cryobiosie . . . . . . . . . . . . . . . 16
( I ) Primary Cooling . . . . . . . . . . . . . 17
(2) Cryoprotectants and Ice-nucleation . . . . . . . . . 18
(3) Attainment of the Ametabolic State . . . . . . . . . 20
(4) Recovery from Cryobiosis . . . . . . . . . . . 20
(5) Ecological Considerations . . . . . . . . . . . 20
V. Anoxybiosis . . . . . . . . . . . . . . . 21
VI. Conclusions . . . . . . . . . . . . . . . 21

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.

(2) A current synthesis

Before considering the specific case of Tardigrada, a brief review is given of recent
advances in cryptobiosis. We may then consider such questions as how the tardigrades
differ in specific details of the process, possible adaptive consequences of such
differences, and whether tardigrades support or refute physiological distinctions
between different classes of cryptobiosis.
Crowe ( 1 971, 1975)and Womersley ( I 98 I a) divide anhydrobiotic organisms into two
major groups. The first comprises examples confined to early ontogenetic stages : plant
seeds (Vegis, 1964), bacterial and fungal spores (Sussmann & Halvorsen, 1966), eggs of
certain Crustacea (Clegg, I 967 ; I 978) and Turbellaria (Wright, I 99 I), and larvae of
certain chironomid Diptera (Hinton, 1960; Grodhaus, 1979). The second group
constitutes those organisms capable of anhydrobiosis at any stage in their life cycle :
ciliate Protozoa, bdelloid and (occasional) monogonodont rotifers, tardigrades and
nematodes (Schmidt, 1948; Van Gundy, 1965;Crowe & Clegg, 1973; Crowe & Madin,
1974; Wright, 1991). This basic division can probably be applied to cryptobiotes in
general.
On induction of tissue dehydration by desiccation, hyperosmotic media or slow
freezing, most anhydrobiotes undergo physical/structural changes to reduce surface
area and so retard water-loss (Broca, I 860 ; Davis, I 873 ; Crowe, 197I). Tardigrades and
rotifers display an active, antero-posterior contraction to form the tun (Baumann, 1922)
and certain nematodes exhibit tight spiral coiling (Womersley, I 987). These
conformations are often critical to survival (Baumann, 1922 ; Ellenby, 1968a, b; Crowe
tk Madin, 1974, 1975; Wright, 1 9 8 9 ~ )and probably preserve structural integrity,
preventing physical disruption during bulk transfer of fluid (Crowe, 1971, 1975).
Further to this point, it may be noted that all known anhydrobiotes possess Some form
of exoskeletal cuticle or pellicle affording resistance to deformation. The necessity for
slow drying in many groups suggests a preparatory phase (Madin & Crowe, 1975;
Wright, 1 9 8 9 ~ Westh
; & Ramlev, 1991),yet several nematodes and the larva of the
chironomid Polypedilum wanderplanki Hinton appear to withstand rapid drying without
an intervening hydrated period (Hinton, 1960; Womersley, 1980); it is probably
significant that the species in question do not display active conformational changes
during drying.
The early demonstrations of prolonged viability of dried bdelloids and tardigrades in
a vacuum firmly established Doyhre’s belief that metabolism could cease during
cryptobiosis. This was essentially confirmed by Pigon & Weglarska ( 1 9 5 5 U , b) who
showed a logarithmic decline in CO, production with external humidity in desiccated
tardigrades, metabolism in the lowest humidities (25-48 %) being virtually un-
measurable. Clegg (1967, 1976a, 1986) has further established the existence of the
‘ametabolic state’ with his extensive work on Artemiu cysts and his series of papers
collectively entitled ‘ Interrelationships between water and cellular metabolism in
Artemia cysts ’ remains the most extensive study on metabolic consequences of
hydration to date. Below a water content of 0 3 g/g dry mass, cysts show no long-term
4 JONATHAN c. WRIGHT, PETER WESTH AND HANSRAMLOV
change in accumulated metabolites, as well unmeasurable accumulation of ‘‘C0,.
Survival of tardigrades and bdelloids cooled to -253 “C (Rahm, 1921)and -272.95 “C
(Bequerel, I 950) clearly illustrates the potential viability of ametabolic states since
chemical reactions at such temperatures, if possible in a solid cryobiotic system, would
be retarded several billion-fold.
All organisms can tolerate a degree of water-loss. The freely exchangeable water
(intra-or extracellular) must be distinguished from the ‘vicinal ’ water (often referred to
as ‘bound ’ or ‘unfreezable ’ water) which is intimately associated with such molecules
as membrane phospholipids, proteins and nucleic acids and forms an integral, though
dynamic, component of macromolecular tertiary structure (Cope, I 967 ; Drost-Hansen,
1971; Franks, 1986). Quantitative estimates of vicinal water differ, depending on
techniques used (calorimetric, NMR, gravimetric), but it is important to realise that
with standardised methodology, estimates are remarkably uniform for given molecular
species, Clearly, however, the wide variability in methods as well as definitions makes
a discrete concept of the vicinal water potentially misleading. Typically, water
molecules will be only transiently associated with polar groups, maintaining a dynamic
interchange with the ‘bulk’ or free water (Franks, 1985). The term ‘vicinal’ water was
introduced by Drost-Hansen ( I97 I ) and Etzler & Drost-Hansen ( I 979) who have
shown that water adjacent to membrane systems differs structurally from free water.
Clegg (1978, 1979) subsequently suggested that reduced metabolism in partially
hydrated Artemia cysts results from restricted transfer of metabolites in the vicinal
water, with the effective cessation of metabolism below 0.3 g H,O/g dry mass
attributable to the absence of an aqueous phase in which biochemical pathways could
operate. A growing body of evidence (see for example Somero & Hand, 1990) indicates
that changes in electrolyte concentration and perhaps disruption of the vicinal water
may induce abrupt metabolic transitions by altering assembly states of multiple sub-
unit proteins and disrupting the compartmentation and spatial proximity of enzyme
systems comprising metabolic pathways. Osmotic dehydration of Artemia cysts in 5 M-
NaCl induces a reversible inhibition of trehalase, hexokinase and phosphofructokinase
reactions (Glasheen & Hand, I 988). Anoxia induces similar enzyme-based arrest of
carbohydrate catabolism via intracellular acidosis (Carpenter & Hand, I 986 ; Hofmann
& Hand, 1990) though maintenance of high intracellular pH by ammonium perfusion
of anhydrobiotic cysts does not restore enzyme activity, indicating that dehydration
exerts an independent inhibitory effect (Glasheen & Hand, 1988).
Cytological studies of anhydrobiotic tissues (Hickernell, 1917;May, 1949) reveal the
cytoplasm to be strongly basophilic and condensed into a fused ‘gelified’ mass, but
anhydrous TEM fixation shows that the basic structure of organelles is preserved
(Crowe, 1975 ; Greven & Greven, 1979; Walz, 1979;Wright, 1 9 8 8 ~ )Cellular
. integrity
must therefore be maintained following reduction or loss of bulk water and probably
much of vicinal water fractions. Falk et al. (1962, 1963) showed that DNA loses
structural integrity in humidities below 70 yo and similar critical hydration levels have
been demonstrated for the stability of proteins (Kuntz & Kauzmann, 1974; Crowe
et al., 1987),and phospholipid bilayers (Crowe & Crowe, 1984,1986;Crowe et al., 1983,
I 987). Depression of water activity by elevated osmolality induces similar structural
transitions to desiccation (Parsegian et al., 1986). The ‘Water Replacement Hypothesis ’
advanced by Webb, ( 1 964, 1965) proposed that certain polyhydroxy derivatives
 Cryptobiosis in tardigr ada 5
maintain structural integrity in dry biological materials, substituting for the vicinal
water fractions. Protective properties of glycerol had been demonstrated previously
(Wells tk Zapposodi, I 948) but the substantial protection afforded by certain hexacyclic
sugars and alcohols like inositol suggested the importance of steric factors (Bather et al.,
1964, 1965; Webb, 1965). More recently, the protective role of trehalose has been
clearly established (Clegg et al., 1982; Crowe et id.,1984~1, b ; 1987). Warner (1965)
performed molecular model analyses, showing that steric conformations of common
protective compounds allow them to mimic polar associations of the vicinal water
closely. Use of such compounds by cryptobiotes requires that substantial quantities are
available on dehydration, and synthesis/mobilization and subsequent catabolism can
proceed a t sufficient rates to confer adequate protection and avoid toxicity.
Trehalose and glycerol constitute the major protective compounds in anhydrobiotes
studied to date. Both are accumulated in substantial quantities in dried Arternia cysts
(Clegg, 1964, 1965) and in the anhydrobiotic nematode Aphefenchus avenue (Madin &
Crowe, 1975), although other nematodes do not appear to utilise glycerol (Womersley
& Smith, I 981). Several studies, demonstrating preferential storage of trehalose,
indicate production in direct response to dehydration (Womersley & Smith, 1981)and
Loomis et al. ( I 980) have demonstrated a decrease in trehalase activity on induction of
anhydrobiosis in A . avenue, suggesting a mechanism by which regulation could occur.
Using NMR,Clegg ( I 978) has provided strong supportive evidence that these sugars
replace bound water and also facilitate its removal by H-bonding. Being a non-
reducing sugar, trehalose will not participate in a ' browning reaction between reduced
sugars and free amino groups (Crowe & Clegg, 1973) and has been shown to inhibit
other browning reactions (Loomis et al., 1980).
The peculiar tolerances of anhydrobiotes to environmental extremes, including high
temperatures, rapid freezing to temperatures approaching o K, shock doses of UV and
X-rays, and immersion in saturated brine and organic solvents (see Becquerel, 1950;
Keilin, 1959; Iwasaki, 1973) are probably consequences of metabolic cessation and
absence of an aqueous phase in which osmotic stress or oxidative reactions could
operate (Clegg, I 978). Nevertheless, prolonged dehydration or repeated dehydration
and rehydration leads to reduced viability (Clegg, 1967; Perry, 1977). This can be
partly attributed to exhaustion of lipid and glycogen reserves (Crowe, 1975), especially
if anhydrobiotes are maintained in atmospheres of high water activity where metabolism
persists at significant rates (Pigon & Weglarska (1955a, b). In addition, several
oxidative reactions produce free radicals such as peroxide and superoxide (Oh) (Mead,
1976; Heckley, 1978; Senaratna et al., 1986), highly destructive in dried tissues where
scavenging reactions are suppressed. Superoxide dismutase plays a vital role in
removing 0;in hydrated tissues. Crowe et al. ( I 983) found a decline in ATPase activity
and Ca2+transport in sarcoplasmic reticulum protected with trehalose and stored in air ;
no such decline was noted in nitrogen-stored material. Addition of glycerol suppressed
this decline in enzyme activity, apparently acting as an antioxidant. Anhydrobiotes may
utilize several antioxidants to compensate for the reduced activity of superoxide
dismutase (SOD) and other scavengers. Nitrogen-stored anhydrobiotes show seemingly
indefinite longevity (Clegg, 1967; Crowe, 1975).
Cryptobiotes typically recover rapidly on return of favourable conditions (Crowe,,
1971 ; Wright, 1 9 8 9 ~ Westh
; Lk Ramlov, 1991), withstanding mechanical stresses
6 JONATHAN PETERWESTHAND HANSR A M L ~ V
C. WRIGHT,
(81

Fig. 1. For legend see opposite.

 Cryptobiosis in tardigrada 7
imposed by rapid rehydration. Protective conformations such as the tun may play a
further role here in limiting rates of water uptake. Crowe et al. (1979) showed that
anhydrobiotic A. avenue (tightly coiled) leaked inorganic ions and primary amines
during rehydration at only the rate of quick-dried (killed) specimens. However, in
both samples, permeability to ions declined sharply when the nematodes attained
20-30 % rehydration, water contents shown to be critical for the structural stability of
phospholipid bilayers (Luzzatti 8z Husnon, 1962). We interpret these results to indicate
that substitution of the vicinal water with protective sugars increases membrane
permeability, a finding which could have further implications for viability during
rehydration.
11. ANIIYDAOBIOSIS IN TARDIGRADES
A capacity for cryptobiosis is apparently restricted to the ‘terrestrial ’ or ‘bryophilous ’
tardigrades, which form a major meiofaunal component in the interstitial water of
cryptogams and humic soils (Ramazzotti 8z Maucci, 1983; Wright, 1991),and to the
littoral species Echiniscoides sigismundi, E. travei and Archechiniscus marchi (Drs C . I.
Morgan & R. M. Kristensen, personal communication). T h e substantial fraction of
lentic, psammolittoral and oceanic species is thus excluded.
Most of the recent advances in cryptobiosis in tardigrades have been made in
anhydrobiosis which thus merits special consideration, As with osmobiosis and
cryobiosis, entry into an anhydrobiotic state will be treated in three stages: primary
dehydration and structural adaptations ; retarded dehydration and mobilization of
protectant compounds ; and (in low water activities) attainment of the ametabolic state.
Brief attention will then be given to revival during rehydration, and to ecological
considerations.
( I ) Primary Dehydration
Depending on the rate of drying, anhydrobiotic tardigrades contract into a compact
tun by active longitudinal contraction and infolding of intersegmental cuticle (Crowe,
1975 ; Wright, 1989a), (Fig. I ) . T h e anterior (cephalic) segments and lobopodial limbs
are fully invaginated and lipid extrusions, observable with cryo-SEM, appear from
putative pore canals (Wright 1 9 8 8 ~ ) These
. may serve to reduce transpiration or may
afford anti-fungal protection (Kiyoaki, I 957), tardigrades being susceptible to attack
from predaceous phycomycetes (Marcus, 1929; Morgan, 1977 ; Wright, 1987). T u n
formation is a vital prerequisite to anhydrobiosis. It may protect internal organs from
mechanical disruption during dehydration/rehydration (Crowe, I 975) in addition to
retarding transpiration. Animals dried at excessive rates, or under anoxia, collapse into
an irregular flattened form and do not revive; they adopt a bloated appearance on
rehydration, attributable to osmoregulatory failure.
T h e tardigrade cuticle demonstrates several structural adaptations which facilitate
tun formation. Cuticle ultrastructure varies substantially among species (Crowe et al.,
1970, 1971; Baccetti & Rosati, 1971 ; Bussers 8z Jeuniaux, 1973a, b; Greven, 1975,

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

1.2 I
0
L
1000
, I
2000
-2
0 1000 2000

Time (s) Time (s)

Fig. 2. (a)Semi-logarithmic plot of mass-loss in 1CZucrobiotw richtern' dehydrated in 80 ?' A r.h. The plot
portrays mean animal masses from five trials, each using samples of 25-30 individuals. At approximately
30 % initial mass (Xd), transpiration is abruptly reduced by the permeability slump, with subsequent
losses declining exponentially. (b) Residual plot of (a),the mass after 30 min dehydration subtracted from
preceding masses and depicted semi-logarithmically. The permeability slump shows a 2-stage reduction
in water conductivity, the second derivatives (DIand D2 - decreases in transpiration rate) showing
different half-times.

slump (Wright, 19896). Prolonged solvent extraction of lipids with chloroform :

methanol destroys the phenomenon and gas chromatography-mass spectrometry
analysis of extracted lipids reveals a predominance of free fatty acids (C12-C18).Shorter
extractions fail to extract lipids and have no pronounced effect on the permeability
slump. Being amphiphilic lipids, fatty acids would be potential candidates for
hydration-dependent (lyotropic) phase transitions, well established in, for example,
phospholipids (Chapmann et a f . , 1967; Caffrey, 1986) which could account for
permeability changes. Substantiating this, the lipid-rich intracuticle has been shown,
using tracer studies, to constitute a hydration-dependent transpiration barrier (Crowe,
19-75; Greven & Greven, 1987; Wright, 19896). A model for the mechanism of the
permeability slump, involving three hydration-dependent lipid phases of different
permeability, is proposed by Wright (1989b).
Anhydrobiosis in tardigrades is apparently only attainable following slow dehydration
(Pouchet, I 959 ; Crowe, I 972 ; Wright, I 989 a ) and studies of primary desiccation
tolerance in seven cosmopolitan species reveal substantial variation (Wright, I 989 a ) .
Thus the xerophilic species Hypsibius oberhaeuseri ( = Ramazzottius oberhaeuseri, Binda
& Pilato (1986)) tolerates drying in humidities down to 59 yo (LD,, estimate) whilst H.
dujurdini, a hygrophile, tolerates a minimum humidity of only 78%. Once the
permeability slump has been effected, all species can apparently withstand exposure to
low humidities. Comparisons of these species permit evaluation of factors which may
limit tolerance. Much lower primary tolerances (minimum > 97% r.h.) have been
shown for the soil nematode Rotyfenchufus reniformis (Womersley & Ching, 1989), and
may be more representative of soil tardigrades.
Xeric species show improved water-retention during drying which can be attributed
in part to more efficient tun formation: Eutardigrada show a significant positive
I0 JONATHAN c. WRIGHT, PETER WESTH AND HANSRAML0V
correlation between surface-area reduction in the tun and desiccation tolerance.
Probably more significant is that the permeability slump is effected more rapidly in
more tolerant species, evident in both living and dead animals. Morphometric analysis
reveals corresponding cuticular adaptations : tolerant species show greater thinning of
inter-segmental cuticle, and possess the thickest intracuticle ; acceleration of the
permeability slump by a thicker lipid layer is in accordance with the proposed model
(Wright, 1988b, 1989b). Again, if a critical degree of water-retention is essential for
survival, the earlier initiation of the permeability slump in more tolerant species would
allow them to withstand more rapid transpiration and hence lower humidities.
(2) Mobilization of membrane protectants
The first study of biochemical changes in anhydrobiotic tardigrades was made by
Crowe ( I 975) who reported that paper chromatography analysis of ethanol extracts
from Macrobiotus areolatus showed more intense trehalose spots in anhydrobiotic
specimens, T h e first quantitative study is that of Westh & Ramlev (1991) who studied
accumulation of trehalose in Adorybiotus coronifer during exposure to decreasing water
potentials. Animals were progressively dehydrated in wet sand using an air stream at
50% r.h. Water potential within the sand equilibrated with the air stream
(-90000 kPa) after 3 h. Tardigrades hydrated for 24 h (after several months as
anhydrobiotes in dry moss) contained 0 1yo dry mass (d.m.) trehalose. Trehalose
concentration showed a pronounced increase during dehydration, stabilising after 5-7 h
at 2-3 yo d.m., a 23-fold increase (Fig. 3). Following 72 h drying by this treatment,
animals showed no significant reduction in viability (recovery = 92 5 4 %). Clearly,
trehalose synthesis continues after tun formation and the permeability slump, both of
which would be necessarily complete within 3 h. Water-retention thus permits
prolonged metabolic synthesis of trehalose in this species.
Although substantial, the accumulation of trehalose in A. coronifer is modest
compared to levels observed in anhydrobiotes from other phyla (Clegg, 1964;
Carpenter & Hand, 1986; Madin & Crowe, 1975; Womersley & Smith, 1981;
Womersley, I 98 I b, I 988). Possible roles of glycerol and other membrane protectants in
this species and other tardigrades await investigation.

( 3 ) Attainment of the ametabolic state

T o date, the studies of Pigon & Weglarska (1955a, b) remain the only quantitative
estimates of metabolism in anhydrobiotic tardigrades. Oxygen uptake in anhydrobiotic
M. hufelandi declined logarithmically with falling humidity, and was not measurable
below 48 % r.h. The corresponding water activity above an aqueous solution requires
an osmolality of over 60 osmoles. Assuming a normal haemolymph osmolality of CQ.
300 mOsm. for bryophilous tardigrades (Crowe, 1972), this corresponds to a 200-fold
dehydration (0.5 yo water-retention) in the absence of osmolyte sequestration and with
retention of bulk water. Polar interactions reduce water potential, so a vicinal water
model (Clegg, 1979) would be compatible with higher water contents. Nevertheless, the
ability of anhydrobiotes to tolerate vacuum-drying and high temperatures (see Keilin,
1959) can only be explained by a reduction of internal water activity to negligible
values.
We may conclude, extrapolating these results to other species, that tardigrades dried
 Cryptobiosis in tardigrada I1

J
1

-0.001 - 3.0

- 2.5 2
.-0
E
- 2.0 $
u-
0
g
- 1.5 p)

-I0mn
r
- 1.0 E
- 0.5
I 1 1 I 1 I I I I 1 1 I 1 1
1 2 3 4 5 6 7
Time (h)
8
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.

(4) Recovery from anhydrobiosis

On re-immersion in water, anhydrobiotic tardigrades remain contracted as a tun for
several minutes before gradual extension and resumption of movement (Wright,
1 9 8 9 ~ )Revival
. following desiccation for I h in high humidities typically takes from
10-20 min, and 1-2 h for prolonged drying in low humidities (Wright, 1 9 8 9 ~Westh;
& Ramlov, 1991).However, animals dried at near-lethal rates frequently require several
hours for complete recovery.
Revival demands that the intracuticular permeability barrier is negated for
resumption of free water and solute exchange. This could operate in several ways.
Crowe (1972) showed preferential penetration of dyes into the mouth and rectum of
newly hydrated M. areolatus although this may succeed the major rehydration. Oral or
rectal uptake would require that the permeability barrier be quashed from within,
which seems unlikely since it is stable with high internal water contents ( > 30%
hydrated mass) and hence high water activities, It could, however, be disrupted by
I2 JONATHAN c. WRIGHT, PETER WESTH AND HANSRAMLPIV

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.

osmotic shock (Dr C. Dempsey, personal communication). Alternatively, the barrier

may be rehydrated externally; since the proposed lipid phase transitions depend on
reduced external water potential, re-exposure to high potentials is a feasible means of
negation.
Although metabolic rate has not been studied during revival from anhydrobiosis in
tardigrades, it probably relates closely to rehydration. Westh & Ramlov ( I 99 I ) studied
aerobic catabolism of trehalose in rehydrated A. coronifer, previously dried for several,
months in mosses. Trehalose concentrations declined exponentially from ca.
1'7% d.m., with a half-time (q)
of approximately I h (Fig. 4), the decline being
initiated within 1 2 min of re-immersion whilst animals were still in a tun state. Under
I Yo of the initial trehalose content of the animals was detected as leakage. More
appreciable leakage of smaller molecules on rehydration has been recorded by Crowe
et al. (1977, 1979) and Womersley (1981b). Aerobic catabolism is clearly indicated by
the lack of a significant decline in trehalose levels when animals were hydrated in
20 Yo CO, : I 7 yo 0,-equilibrated water, inducing immobility and asphyxia ; normal
activity and trehalose catabolism were readily resumed by perfusion with atmospheric
air (Westh & Ramlev, 1991).Activity of trehalase from Artemia is strongly dependent
on pH, showing a marked reduction due to enzyme polymerisation below pH 6 4 (Hand
& Carpenter, 1986). Similar pH-sensitivity of trehalase could explain reduced trehalose
catabolism during CO, perfusion. Incorporation of [3H]leucine in rehydrating A.
coronifer also indicated that protein synthesis is resumed much more slowly than
 Cryptobiosis in tardigrada I3
trehalose degradation ; incorporation was almost constant over the initial 4 hours but
had doubled after 24 h (Fig. 4).
( 5 ) Ecological considerations
The peculiar resistances of anhydrobiotes to environmental extremes are clearly of
adaptive significance, facilitating the role of tuns in airborne and vector-assisted
dispersal (Kristensen, r 987; Nelson & Higgins, 1990) and permitting prolonged
survival, at extreme temperatures, in the absence of liquid water. Nevertheless, the
limited tolerance of tardigrades to primary dehydration is likely to have pronounced
ecological implications, restricting animals to slowly desiccating habitats. Semi-
quantitative analysis of tardigrade habitats using four xeric parameters has revealed
xeric trends relating closely to desiccation tolerance (Wright, I 99 I ) . Substrate heat
capacity, drainage and exposure, as well as insolation and habitat plant species, may all
constitute limiting factors for tardigrade survival through effects on desiccation rate. Soil
tardigrades are subject to much slower desiccation regimes than bryophilous species,
and may display corresponding modest tolerances, although many anhydrobiotes fall
into both ecotypic categories.
Rapid synthesis of adequate membrane protectants is essential in anhydrobiotes
suffering rapid desiccation. A. coronifer is a xeric tardigrade, frequently inhabiting
exposed screes subject to rapid variations in temperature and humidity (Westh 8z
Ramlov, 1988;Dr R. M.Kristensen, personal communication). The ability of this
species to accumulate trehalose ten times faster, and survive anhydrobiosis with levels
at least five times lower, than reported for the soil-dwelling nematode A. avenue (Madin
& Crowe, 19-75) may represent adaptations to xeric biotopes. The possibility of
acclimation to xeric extremes, including dynamics of the permeability slump, tun
dimensions and trehalose accumulation, awaits investigation.

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

( 3 ) Attainment of the ametabolic state

Metabolic arrest in osmobiotic tardigrades would be predicted following appreciable
concentration of the intracellular osmolyte systems and corresponding reduction in the
vicinal water (Clegg, 1979) as well as reduction of enzyme activities and sub-unit or
enzyme :enzyme ('metabolon ') dissociation (see Hand, 1991).Studies of these aspects
and of metabolic rate parameters are awaited.

(4) Recovery from Osmobiosis

When returned to distilled water, osmobiotic tardigrades remain contracted in a tun
for variable periods before resuming activity (Wright, I 987). Recovery following
hyperosmotic exposures for which animals show high tolerance (low osmolalities
and/or short immersions) is typically rapid (8-1 5 min), but revival times increase
exponentially following more severe treatments (Wright, I 987). Lethal effects may
depend on osmotic damage sustained on return to freshwater, in which case graded
diminution in external solute activities could increase survival. However, animals
suffering lethal hyperosmosis show instant osmoregulatory failure on return to
freshwater, swelling and extending abruptly.

( 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
100 1 1 % Trehalose
90
80
70
5
- 60
.-?
t
50
$
v)
40
30
20
10

1 10 100 1000
Cooling rate ("C rnin.-ll
100 0.1% Trehalose

80
70
A

5
- 60
.- 50
$ 40
v)
30
20
10

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.

(I) Primary coding

Tun formation in response to freezing has been observed at slow cooling rates
(Crowe, 1975)but tardigrades can survive cooling rates which preclude even partial
contraction into a tun state (Ramlov & Westh, 1990).A possible adaptive role of tun
formation during cryobiosis remains unresolved.
Having quantified rates of trehalose catabolism in A. coronifer following prolonged
anhydrobiosis, Ramlsv & Westh (I 990)used animals of pre-determined trehalose levels
( I % and 0 1 % d.m.) to investigate a possible cryoprotective role of this carbohydrate
and tolerance of different cooling rates. Animals were cooled to - 196 O C at rates up to
1500 O C min-', and maintained at this temperature for 15 min before thawing in water
at 30 "C. They were then maintained at 5 O C and recovery, judged as the resumption
of locomotor activity, estimated after 2 h and 24 h. Survival with I yo d.m. trehalose
declined steadily with increasing cooling rates, with IOOyo mortality at I 500 OC min-'
(Fig. 5 ) . Animals containing 0.1% d.m. trehalose showed a similar survival at high
cooling rates ( > 10OC min-'), but survival at lower (ecologically more realistic)
18 JONATHAN c. WRIGHT, PETER WESTH AND HANSRAML0V
Poling

s .* .'
. v
-... ....
0
* .
'
5:
F

80-
. i.
(D

Time (s)
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).

Cryoprotectants and Ice-nucleation

(2)

Several cold-hardy insects synthesize trehalose when subjected to prolonged cooling

(Block, 1982; Miller, I 982; Semme, 1982). Nevertheless, although A. coronifer
synthesizes trehalose during desiccation, the foregoing results suggest a minor
 Cryptobiosis in tardigrada 19
cryoprotective role of this sugar, protection being afforded only at low cooling rates and
elevated trehalose concentrations. Indeed, from the extreme cooling rates tolerated, it
is unlikely that appreciable quantities of any cryoprotectant could be synthesized or
mobilized by metabolic processes. Moreover, differential scanning calorimetry (DSC)
studies of whole-animal samples indicated that over 80 Yo of the body water crystallizes
close to - 7 "C in this species during cooling at 2 "C min-' (Westh & Hvidt, 1990),and
further cooling does not lead to a measurable increase in ice content. T h e melting point
of winter-acclimated animals (collected in Sweden in February, 1990)was -0.5 "C,
indicating a haemolymph osmolality of 250-300 mOsm kg-' (Westh & Hvidt, 1990);
this value remained unchanged in animals collected in August, 1990 (P. Westh,
unpublished results). Limited depression of the melting point, showing no seasonal
variation and high ice contents at modern sub-zero temperatures, support this view.
Indigenous molecular species may nevertheless play a vital role in freeze-tolerance at
pre-existing concentrations,
Studies of whole and homogenised A. coronijer using DSC indicate catalysis of ice-
nucleation by an ice-nucleating agent (INA) (Westh et al. 1990).T h e crystallization
temperature of whole animals cooled at 2 "C min-' was -6.7 OC, decreasing to - 16 OC
following heating to 90 "C over 2 min. Prior heating of whole animals to 63-67 "C
induces a complete loss of ice-nucleating activity, probably due to thermal inactivation
of one or a few active molecular nucleators. Gel filtration of body fluids and DSC
analysis further indicate that ice-nucleating activity is confined to molecules larger than
a 200 kDa protein. T h e heat stability, size and concentration (estimated as the
reduction in nucleating activity following dilution) of the ice nucleators indicate
analogous compounds to those described in other freeze-tolerant animals (see Ssmme,
1978;Zachariassen et al., 1982; Duman et al., 1985;Wolanczyk et al., 1990;Duman,
1990).
Pre-existing levels of trehalose and other polyols may play a role in intracehlar
vitrification. Wasylyk et al. (1988)reported that mixtures of polyol cryoprotectants
could give rise to partial glass formation (i,e. a mixture of crystalline ice and glass)
between - 25 and -40 "Cand suggested this as a likely mechanism of cryoprotection.
However, since slow cooling of A. coronifer to temperatures close to - 5 OC confers
tolerance to near-instantaneous cooling rates, partial glass formation would need to
occur at corresponding temperatures. Becquerel (I950) proposed a similar mechanism
to explain cryobiosis in rotifers, tardigrades and nematodes, although he found no
evidence of vitrification from X-ray absorption spectra (see Keilin, 1959). It seems
more likely that freeze-concentration of polyols and other solutes at - 5 "C can render
the intracellular water unfreezable, removing dangers of intracellular ice-formation
during more severe freeze-dehydration incurred at lower temperatures (see Franks,
1985,for a thorough discussion of these aspects).
An alternative role of polyols, documented for freeze-tolerant insects, is colligative
reduction of the amount of ice formed at a given sub-zero temperature (Zachariassen,
I 980). By sustaining a modest vapour pressure, this will reduce cellular dehydration.
Since the extent of cellular dehydration induced by freezing is typically lower than that
induced by desiccation (Crowe et al., 19901,further reduction in dehydration dependent
on polyols may not be necessary for A. coronifer in view of its profound desiccation
tolerance. However, in either case, prevention of intracellular freezing would be vital
20 JONATHAN c.W R I G H T , PETER W E S T H AND HANSR A M L 0 V
and levels of cellular dehydration sustained may still require membrane stabilization,
for which minimum concentrations of certain polyols would presumably be essential.
To draw a tentative conclusion, cryobiosis in tardigrades seems to involve controlled
extracellular ice-nucleation after moderate supercooling and (long-term) tolerance of
extracellular freezing. Tolerance may depend on cytoplasmic vitrification or freeze-
concentration rendering the intracellular water unfreezable. As well as the concomitant
cellular dehydration, both these processes will probably depend on permanent
minimum levels of intracellular polyols.
( 3 ) Attainment of the ametabolic state
Given the exponential decline in metabolism with falling temperature, there will
evidently be no appreciable metabolic activity of tardigrades at temperatures below
- I 90 O C . This would lead to correspondingly increased viability and animals cooled to
such temperatures would be expected to survive indefinitely.
The metabolic status at ' natural ' sub-zero temperatures has not been investigated
but the extensive ice-formation at -7 "C observed in A. coronifer might indicate a
significant reduction in metabolism due to the reduction of free water. Since fully
hydrated animals contain approximately 3 2 g H,O g d.m.-' (Westh & Hvidt, I990), the
residual amount of free water following freezing at -7 "C is 06 g H,O d.m.-'. Clegg
(1978)has suggested that hydration between 0 3 and 0 6 g H,O g d.m.-' is sufficient to
induce ' restricted ' metabolism in Artenziu cysts.
(4) Recovery from cryobiosis
A . coronifer revives from the cryobiotic state when warmed abruptly on transfer to
water at 30 OC, ( R a m l ~ v& Westh, 1990) but no systematic study on the effects of
different thawing procedures has been performed to date. Since tun formation is
precluded by rapid cooling, without an associated reduction in viability, it is clearly not
required for protection against physical disruption ensuing from ice-thawing and
osmotic rehydration. Extracellular ice may, in fact, confer mechanical support of organ
systems, substituting for this probable role of tun formation in anhydrobiotes. Hypo-
osmosis will probably occur before the ' Malpighian tubules ' resume hyper-regulation,
although this might be overcome by mechanisms to retard rehydration.
( 5 ) Ecological considerations
Cryobiosis in A. coronijer will permit animals to tolerate rapid cycles of freezing and
thawing, including rapid cooling to the lowest temperatures ever encountered in nature.
Essentially similar tolerances have been shown for other tardigrades (Rahm, 1921).An
extreme example is Echiniscoides sigismundi groenlandicus, living in mesolittoral Arctic
habitats, which tolerates freezing and thawing with the tidal cycle during spring and
autumn and survives frozen in the icefoot for 6-8 months of the year (Kristensen &
Hallas, I 980). During cryobiosis, senescence will be greatly retarded, providing
adaptation to Arctic/cold- temperate water bodies undergoing thawing on irregular or
seasonal bases. Several species have been recorded from Arctic and Antarctic habitats
(Dougherty & Harris, 1963; Sudzuki, 1964; Jennings, 1976; Kristensen, 1982;
Dastych, 1984).Only exceptional cooling rates will prevent active tun formation which
can thus serve a dispersive role as in anhydrobiosis.
 Cryptobiosis in tardigrada 21

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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