BioSystems, 24 (1990) 1 7 - 2 4 17

Elsevier Scientific Publishers Ireland Ltd.

Towards a generalized model of encystment

(cryptobiosis) in ciliates: a review and a hypothesis

Juan C. Guti~rrez, A. Martin-Gonzalez and T. Matsusaka a

Departamento de Microbiologia-I, Facultad de Biologto, Universidad Cornplutense (UCM), ~8040 Madrid (Spain/and
•Department of Biology, Faculty of Science, Kumamoto University, Kumamoto (Japan/
(Received October 20th, 1989)
(Revised January 4th, 1990)

In this work we propose a generalized model to explain encystment (cryptobiosis) in cillates. The model is elaborated
from both structural and physiological studies previously reported, and some hypothetical considerations. The main
features of this model are the following: (a) starvation is considered as the most important inducer of ciliate encystment,
and it constitutes the Krst trigger of this cell differentiation process; (b) the "switch on" of encystment genes involves the
appearance of several new transcripts; it) starvation involves cell autophagy and protein turnover, which provides mate-
rlal to synthesize the new cystic proteins; (d) cytoplasmic dehydration has an important role during the resting cyst forma-
tion; iv) a gradual loss of intraeellular water can inactivate cell metabolism, leading to an ametabolic state (cryptobiosis);
if) as a late precystic event, the encysting cell forms a barrier (cyst wall), that isolates the cell from the hostile environ-
ment.

Keywords: Cryptobiosis; Ciliate encystment; Generalized model.

1. Introduction and other microbial cryptobiotic forms, inde-

Keilin {1959) introduced the term "crypto-
biosis" to define the state of an organism
when it shows no visible signs of life and
when its metabolic activity becomes hardly
measurable. Cryptobiosis is not limited to any
special living system. We can find numerous
and good examples in both prokaryotic and
eukaryotic microorganisms, plants and ani-
mals (Halvorson, 1961). Among microorgan-
isms, bacteria (bacterial spores), fungi (conidia
and spores) and protozoa (resting cysts) repre-
sent cryptobiotic forms. In protozoa, resting
cysts are perhaps the best example of a cryp-
tobiotic state, because while the cell remains
in cystic form, there is virtually no metabo-
lism going on, and the cell can remain stable
for long periods of time in that very particu-
lar state. Also, we can find numerous
similarities between resting cysts of protozoa
Correspondence to: J.C. Guti~rrez.
pendently of their phylogenetical distance
(Henis et al., 1987). This is also a good reason
to use a unified terminology for these living
states which exhibit a reduced metabolism.
We suggest the term "cryptobiosis" as a good
candidate for this generalized process.
In ciliates, the cell differentiation process
that involves resting cyst formation is partic-
ularly called "encystment" and the antagonis-
tic process, the emergence from the
cryptobiotic state (resting cyst) is named "ex-
cystment'. Both processes constitute a cycle
- the E-E (encystment-excystment) cycle -
with two stable differentiated states, the veg-
etative and the cystic or cryptobiotic one.
This facultative cycle is connected to the
growth-division cycle of the ciliate, which can
"jump" from one cycle to another when the
inducer conditions appear. This "leap" involves
the "switch on" of the genetic program
involved in the macromolecular biosynthesis
for the cryptobiotic state formation.

0303-2647/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

Published and Printed in Ireland
18
In the present paper, we have tried, after
revision of the data published to date, to elab-
orate a model that might be useful for study-
ing, at molecular level, the mechanism by
which ciliate cryptobiosis takes place. The
model is based on both cytological-structural
and physiological-molecular results. Naturally
it is possible that this model can be revised or
modified in the future in order to reflect new
data.
2. Structural and cytological basis of the
model
Although many inducers for ciliate encyst-
ment are known, for example, desiccation or
crowding, the most universal exogenous indu-
cer of ciliate encystment is starvation or the
deficiency of an essential nutrient in the
medium (Corliss and Esser, 1974; Wagten-
donk, 1955). When the ciliated protozoon
receives the initial trigger of cryptobiosis, the
cell undergoes progressive morphological,
biochemical and physiological changes which
conclude with the formation of a resting cyst
(Gutierrez and Walker, 1983). One of the most
remarkable changes is a drastic decrease of
cellular volume. In some ciliates, this volume
loss represents 60-80O/o, and it is a conse-
quence of the loss of intracellular water (Ricci
et al., 1985; Walker and Maugel, 1980; Walker
et al., 1980). This cell water loss results in a
cellular density change, manifested by a dif-
ferent sedimentation (or migration) of resting
cysts and vegetative cells on a discontinuous
density gradient of Percoll.
Perhaps, some peculiar changes in cell
organelles of the encysting ciliate are a conse-
quence of the cytoplasmic water loss, e.g. the
so-called mitochondrial clustering (Gutierrez
and Perez-Silva, 1983; Matsusaka, 1976;
Walker and Hoffman, 1985; Walker and Mau-
gel, 1980; Walker et al., 1975, 1980), which
may represent a decrease of respiratory activ-
ity (Giese, 1973; Pigon and Edstrom, 1961). A
high macronuclear chromatinic condensation
is also produced in the majority of encysting
ciliates, with or without fusion of macronu-
clear masses (many ciliates have more than
one macronuclear mass in the vegetative
state). Generally, during encystment, these
macronuclear masses fuse into a single cystic
macronuclear mass (Grimes, 1973; Gutierrez,
1985; Gutierrez and Perez-Silva, 1983; Gutier-
rez et al., 1981; Matsusaka, 1976; Matsusaka
and Kimura, 1981; Tikhonenko et al., 1984;
Verni et al., 1984, Walker and Hoffman, 1985;
Walker and Maugel, 1980; Walker et al., 1980).
This nuclear condensation may involve the
transcriptional inactivation of the ciliate
genetic system in the cryptobiotic state and
probably decreases the mutagenic effects
induced by desiccation (Webb, 1967).
Ribosomes, vacuoles and lysosomes are also
involved in the cytoplasmic changes during
encystment (Corliss and Esser, 1974; Delgado
et al., 1987; Garnjobst, 1937; Giese, 1973;
Grimes, 1973; Gutierrez and Perez-Silva, 1983;
Gutierrez and Walker, 1983; Kay, 1945; Mat-
susaka, 1976; Rios et al., 1985; Rosati et al.,
1984; Verni et al., 1984; Walker and Hoffman,
1985; Walker and Maugel, 1980; Walker et al.,
1980).
One of the cytoplasmic changes which prob-
ably has more physiological consequences is
the early appearance of high autophagic activ-
ity (Gutierrez and Perez-Silva, 1983; Matsu-
saka, 1976; Walker et al., 1980).
In some ciliates the cryptobiotic state
shows a characteristic pigmentation. Hypo-
trichs grown on unicellular green algae as
food usually form yellowish resting cysts. The
pigment responsible for this coloration
showed characteristics of a-carotene or its
derivative. The authors (Gutierrez et al., 1982)
suggest that this cystic pigment may be
obtained from the algae, which are the habit-
ual food of these protozoa, and then
accumulation occurs in the encysting cell. The
presence of pigmentation in ciliate resting
cysts appears to be a protective mechanism
against the photooxidation process, which
increases as the medium desiccates.
A fundamental characteristic of the major-

ity of cryptobiotic states, including ciliates, is
the presence of partially permeable barriers
Cwalls, envelopes, cuticles and other rigid
structures protecting the cell). Many authors
have studied the fine structure of the differ-
ent cyst wall layers, using transmission and
scanning electron microscopy (Delgado et al.,
1987; Gutierrez et al., 1983a; Matsusaka, 1976;
Tibbs, 1968; Walker and Hoffman, 1985).
Little is known about the chemical composi-
tion of the different cyst wall layers of cil-
iates. In general, the main macromolecular
cyst wall components are proteins, glycopro-
teins and carbohydrates (Bussers and Jeu-
niaux, 1974; Calvo et al., 1983; Gutierrez et
al., 1984; Matsusaka and Hongo, 1984; Tibbs,
1966).
The distinct cyst wall layers are derived
from different precursors, which are formed
in the precystic cytoplasm {Grimes, 1973;
Gutierrez et al., 1983b; Holt and Chapman,
1971; Matsusaka, 1976; Matsusaka, 1979;
Verni et al., 1984; Walker and Maugel, 1980;
Walker et al., 1980}. There is cytochemical
evidence which supports the idea that the
origin of these cystic precursors is from both
rough endoplasmatic reticulum and/or Golgi
complex (Calvo et al., 1986). Some of these
precursors, like those forming ecto- and endo-
cysts of some hypotrich ciliates (Calvo et al.,
1986; Gutierrez et al., 1984; Matsusaka and
Hongo, 1984) might undergo glycosylation.
The significance of glycosylation in ciliate
encystment has recently been shown using
Tunicamycin Ca very specific inhibitor of pro-
tein glycosylation, which inhibits the initial
step of N-glycans formation). This inhibitor
blocks the encystment process (Benitez et al.,
1989).
The kinetics of cyst wall precursors forma-
tion during encystment has only been exam-
ined in one hypotrich ciliate (Gutierrez et al.,
1983b). In this work, the authors conclude
that the genesis of the cyst wall, in ciliates
with multilayered walls, is a sequential pro-
cess at two levels: Ca) successive appearance
of precursors in the precystic cytoplasm, pos-
19
sibly determined by a sequential genetic
expression; and (b) successive secretion or
exocytosis of the different precursors which
will form the cyst wall layers.
3. Physiological and molecular basis of the
model
In ciliates, the encystment depends on both
RNA and protein synthesis (Gutierrez et al.,
1981; Matsusaka, 1979; Matsusaka and
Kimura, 1981; Ruthmann and Kuck, 1985;
Tibbs and Marshall, 1970; Yonezawa, 1985).
Actinomycin D blocks the encystment process
(Benitez et al., 1989; Gutierrez et al., 1981;
Matsusaka and Kimura, 1981; Ruthmann and
Kuck, 1985; Yonezawa, 1985), suggesting that
the macronucleus is transcriptionally active,
at least in the first stages of encystment.
These results agree with those obtained by
using a radioactive precursor ([~H]uridine),
and later detection of the newly synthesized
transcripts by electrophoresis and fluorogra-
phy (Gutierrez, 1987). The new transcripts are
detected in the early stages of encystment,
but transcriptions are not detected during the
rest of encystment. Thus, transcriptional
activity gradually decreases during encyst-
ment and ceases in the last phases of
encystment.
Encystment is clearly a protein synthesis
dependent process (Gutierrez et al., 1981;
Matsusaka, 1979; Ruthmann and Kuck, 1985;
Yonezawa, 1985}. The newly synthesized pro-
teins are presumably involved in the crypto-
biotic state formation and also in its main
component, the cyst wall.
As in other morphogenetic processes
induced by starvation, cyst formation depends
completely upon endogenous materials,
because an exogenous energy source is
absent. These endogenous materials are used
extensively for synthesizing the new
macromolecules directly or indirectly involved
in the cyst formation.
Experiments using inhibitors of protein
synthesis (e.g., cycloheximide; Matsusaka,
20
1979; Sendo and Matsusaka, 1982), suggest
that these new proteins appear early in the
encystment process, and they are essential
for normal cyst development. Evidence for
protein turnover, by pulse-labelling experi-
ments using [35S]methionine, has also been
shown (Gutierrez, 1987). This process involves
amino acid recycling from vegetative proteins
to cystic proteins. Protein turnover has also
been described in other eukaryotic cells
undergoing differentiation induced by starva-
tion, e.g. sporulation in Saccharomyces
cerevisiae (Klar and Halvorson, 1975) and Dic-
tyostelium discoideum (Watts, 1984).
When cells are placed in non-nutrient
encystment medium, many cellular compo-
nents are degraded by autophagy, and this
process provides the materials for cyst prod-
ucts, and allows the removal of excess cyto-
plasmatic baggage during encystment. Thus,
the autophagosomic activity detected during
encystment may be related to the protein
turnover.
Cellular levels of RNA and proteins
decrease substantially during encystment (Pi-
gon, 1961; Sendo and Matsusaka, 1982; Tibbs
and Marshall, 1970). Lysosomal hydrolases
(such as a- and ~-glucosidases, amylase, f3-gal-
actosidase, proteases, phosphatases, RNase
and DNase) may be responsible for these
degradative processes.
In general, DNA synthesis is not involved
in ciliate encystment. Inhibitors of DNA syn-
thesis do not block the encystment process
(Gutierrez et al., 1981; Ruthmann and Kuck,
1985). However, the macronucleus undergoes
drastic changes during the cryptobiotic pro-
cess, such as macronuclear fusion, chromatinic
condensation, transcriptional inactivation and
sometimes a loss of DNA by forming extru-
sion bodies. This last feature has been
reported in some ciliates (Martin-Gonzalez et
al., 1989; Morat et al., 1981}, and it may be a
result of both the volume decrease and degra-
dative activities.
Even though a selective loss of macronu-
clear DNA (by extrusion bodies) occurs during
encystment in some ciliates, the complete
genome, characteristic of each ciliate species,
is maintained. The maintenance of at least a
single copy of the genome, during the E-E
cycle, is revealed by the mating type that the
vegetative cell exhibits in conjugation, which
is maintained after excystment (Afon'kin and
Skovorodkin, 1987).
4. The model
On the basis of what is known about ciliate
encystment as well as our working hypothe-
sis, we can elaborate a generalized model
about the main mechanism involved in ciliate
encystment (Fig. 1). In our model we have
taken into account the following elements.
Firstly, we think that starvation is the most
universal exogenous inducer of cryptobiosis in
free living and fresh water ciliates. We do not
discard the existence of other encystment
inducers in ciliates (Corliss and Esser, 1974;
Wagtendonk, 1955), but they should be
considerated in another different model.
The exogenous inducer (nutritional defi-
ciency) might alter the levels of various meta-
bolites (endogenous inducers) and as a result,
trigger the "turn off" of some vegetative
genes involved in the growth-division cycle
and the "turn on" of encystment genes. This
"switch on" of encystment genes involves the
appearance of some new transcripts, which
are necessary for synthesizing the cystic ele-
ments. This point is corroborated by the
action of transcriptional inhibitors which
block ciliate encystment (Benitez et al., 1989;
Gutierrez et al., 1981; Matsusaka and Kimura,
1981; Ruthmann and Kuck, 1985; Yonezawa,
1985), and also by the detection of newly syn-
thesized transcripts in early phases of encyst-
ment (Gutierrez, 1987). The information
contained in these transcripts should be struc-
tural and/or enzymatic proteins involved in
the encystment process, in particular cyst
wall proteins.
Ciliate encystment is also blocked by inbib-
itors of protein biosynthesis (Gutierrez et al.,
1981; Matsusaka, 1979; Ruthmann and Kuck,
1985; Tibbs and Marshall, 1970; Yonezawa,
 21

Exogenous inducer
(Starvation)

IINDUCTIO'~ ~ Early Precystic events // :- Late Precystic events • IResting Cyst I

End~ogenOus~~ Oegradat ire Cell . . ~ V o lume loss

inducers (?) \ processes
le \e (Autophagy)~ De h y d r a t i o ~ L M e t a b o l i c ~ C e l l
~t~0 inactivation spherical
Macronuclear Genome ~ \ " " ~ O r g a nella rganelle~ ! ~ form
[ I\ elimination clustering ~
Cystic~enes----Vegetative genes i \~
i Expression
" Prbtein
turnover
/
Chromatinic ~ Macronuclear
New transcripts condensation genome
i~ Translation 4
IAmino
pool
acids t inactivation Cyst Wall
formation
Cystic polypept ides
7
DGlycoproteins i=Cyst Wall , m,Exocytosis
if precursors
Glycosilat ion

Fig. 1. Schematic representation of the ciliate encystment model. In this scheme we are summarizing the main features
involved in the ciliate encystment. ( + } Positive effect (turn on). ( - ) Negative effect (turn off). (-~) Steps of the propoeed
model that when they are inhibited, by using different metabolic inhibitors, the ciliate encystment is blocked (for more
details see the text).

1985). Moreover, newly synthesized polypep-
tides have been identified during ciliate
encystment (Gutierrez, 1987).
Some of these proteins could undergo gly-
cosylation, because glycoproteins have been
detected in the ciliate cyst wall (Bussers and
Jeuniaux, 1974; Calvo et al., 1983; Gutierrez
et al., 1984; Matsusaka and Hongo, 1984) and
inhibitors of protein glycosylation block the
encystment (Benitez et al., 1989).
This protein biosynthesis could take place
using the amino acids released from a selec-
tive proteolysis (protein turnover), that might
be united to the autophagosomic activity
which takes place during a large part of cil-
iate encystment. By means of this autolytic
process the starved cell maintains a basic
metabolism that helps to synthesize the nec-
essary macromolecules to form the cyst, and
to eliminate some cellular elements not
necessary in the differentiated resting state
(e.g. in the majority of ciliates the cortical
structures, involved in mobility and/or the
uptake of food, are totally or partially
destroyed, and only in some ciliates are these
structures maintained almost intact). The
newly synthesized proteins could be protected
against proteolysis by different cell mecha-
nisms, one of which might be glycosylation
(Olden et al., 1985).
In this way, the precystic cytoplasm under-
goes drastic changes which may be due to two
different and simultaneous processes, auto-
phagy and cytoplasmic dehydration. Probably
the latter has a crucial role in obtaining the
main characteristic of any cryptobiotic state;
the metabolic inactivation or a metabolism.
The gradual loss of intracellular water can
inactivate the main metabolic pathways,
increase the cell density and induce the orga-
nellar clustering. Also, genetic inactivation
can be obtained by a double mechanism, the
cytoplasmic dehydration and the biosynthesis
of basic nuclear proteins involved in the
chromatinic condensation (Gutierrez, 1985).
Logically, the necessary biosynthetic pro-
22
cesses to establish the cryptobiotic state must
be started before the dehydration level inacti-
vates the cellular metabolism. Thus, specific
cystic transcriptions and translations should
take place during the early phases of encyst-
ment.
To conclude, ciliate encystment morphoge-
nesis is essentially an endogenous or self-suf-
ficient system (Wright, 1967). Like most other
living systems undergoing morphogenesis, cil-
iate encystment is also a closed system, as it
depends completely upon endogenous materi-
als to form all elements of the resting cyst.
Closed systems are characterized by the
formation of barriers {cyst walls in ciliates),
which isolate the cell from the adverse enviro-
ment, and utilize an endogenous metabolism
which requires a high rate of breakdown of
macromolecular elements. Thus, cryptobiosis
in ciliates includes any cell capable of making
a protective cyst wall from endogenous mate-
rials under starvation conditions.
Certainly, some details of several ciliate
encystment do not coincide exactly with the
general model proposed here for ciliate
encystment. However, we think that it is not
very important, because we must consider
that any scientific model is basically an impor-
tant tool for a better understanding of the
biological process that we are trying to study.
This model may help to continue study of this
very complex cell differentiation process with
a higher dedication and enthusiasm.
Acknowledgements
This work was supported by a grant from
Direccibn General de InvestigaciSn Cientifica y
T~cnica (DGICYT). Project: PB87-0006.
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