Jpn. J. Protozool. Vol. 37, No. 2. (2004)

119

Original
Effects of porphyrins on encystment and excystment in ciliated protozoan
Colpoda sp.
Saori TSUTSUMI,1 Tomoko WATOH,1 Koji KUMAMOTO,2 Hiyoshizo KOTSUKI2 andTatsuomi MATSUOKA1＊
1

Institute of Biological Science, Faculty of Science, Kochi University, Kochi 780-8520, JapanSUMMARY

INTRODUCTION

Excystment of Colpoda sp. was found to be
triggered by a brownish fraction obtained by highperformance liquid chromatography (HPLC) of
cereal infusion that was expected to contain porphyrins. A commercial porphyrin, chlorophyllin(coppered, sodium salt) induced excystment andprevented encystment, while protoporphyrin IX
without metal, protoporphyrin IX bound to zinc,and riboflavin did not have any excystmentinducing effect. These results suggest that theporphyrins contained in cereal leaves might trigger
excystment and prevent encystment. Greencolored chlorophyllin is expected to be useful as amolecular probe to visualize and isolate receptors
for triggering the encystment and excystment processes.

The processes of encystment and excystment in protozoans involves overall reconstruction
at the cellular level (Grimes, 1973; MartínGonzález et al., 1992; Delmonte et al., 1996;
Gutiérrez et al., 2001). These processes might becontrolled by unknown intracellular signaling
chains including gene expression and silencingthat are activated by certain receptor molecules.
One of the strategies for elucidating intracellularsignaling chains leading to the gene expression orsilencing required for encystment or excystment
involves determining environmental signals for
activating receptors so that these signals can be
employed as a molecular probe for isolation of thereceptors. The receptor-like protein isolated todate may be only a 40 kD surface protein(encystment stimulating protein, ESP) of soil
amoeba that was screened by using immunologicalmethods (Yang and Villemez, 1994).
It has previously been reported that vegetative cells of Colpoda sp., which had been isolated
from cysts adhered to dried fallen leaves, encyst in
response to an increase in the concentration of ionssuch as Ca2+, which may be a signal for forthcom-*Corresponding author
Tel: + 81 88 844 8315
Fax: + 81 88 844 8356
E-mail: tmatsuok@cc.kochi-u.ac.jp
Received: 28 April 2004; Accepted: 16 August 2004.

120

Effect of porphyrin on encystment and excystment

ing desiccation (Watoh et al., 2003; Yamaoka etal., 2004). In addition, encystment of the cells canalso be induced by increasing cell density up to
1000~3000 cells/ml (our unpublished data). On
the other hand, Ca2+-induced encystment is prevented by certain molecules released from bacteria(Enterobacter aerogenes) suspended in a surrounding medium (Yamasaki et al., 2004).
Excystment of Colpoda is induced by certain
components contained in hay extracts (Thimann
and Barker, 1934) or cereal infusion (Watoh et al.,2003). In the present study, we focused on environmental factors initiating the excystment process,analyzing in particular what kinds of components
contained in cereal leaves activate the signaling
chains leading to excystment, and suggested thatcertain kinds of porphyrins could trigger excystment.

MATERIALS AND METHODS
Colpoda sp. was isolated from cysts adhered
to dried fallen leaves on the soil in Kochi prefecture, and cultured in 0.1% (w/v) infusion of dried
cereal leaves (autoclaved for 20 min) inoculatedwith bacteria (Enterobacter aerogenes) at 23°C in
the dark. Bacteria were cultured on agar plates
containing 1.5% agar, 0.5% polypeptone, 1% meat
extract and 0.5% NaCl. The cysts employed in thepresent assays for excystment (Figs. 1-4) wereprepared as follows: vegetative cells were collected by centrifugation (1,000 g, 1 min) and kept
suspended at a cell density of 1,000~3,000 cells/ml
for more than 1 month in 0.1 mM Tris-HCl (pH7.2). For induction of excystment, the cysts wererinsed twice in 0.1 mM Tris-HCl (pH 7.2) containing tested components such as porphyrins, and50~100 cysts were subsequently suspended in 1 mlof each test solution by transferring them using athin glass pipette. The rate (%) of excystment orencystment was examined at 10 hr (Figs. 1b, 1c,

3d, 4, 5) or at 24 hr (Fig. 2d) after the onset of
induction, and is expressed as a percentage of thetotal number of tested cells (50-100 cells). Points(columns) and attached bars correspond to themeans of four identical measurements (50-100
cells per measurement) and standard errors.
Protoporphyrin IX zinc (II) (Aldrich) was
dissolved in 0.1 mM Tris-HCl buffer (pH 7.2) assodium salt, because original compound was lesssoluble in water; that is, the functional group CH2CH2COOH was replaced by -CH2CH2COONa
by adding an excess of NaOH, and subsequently
neutralized with HCl. The excess Na+ and Clwere removed by dialyzing with 500 Da cut-off
membrane (Float-A-Lyzer, Spectrum Laboratories,
Inc., USA) for 24 hr in a cooled 0.1 mM Tris-HCl
buffer (pH 7.2) on ice. Other porphyrins and riboflavin (Aldrich) were simply dissolved in 0.1 mM
Tris-HCl buffer (pH 7.2).
Cereal infusions (0.5%) with and without
0.1% trifluoroacetic acid (TFA) were fractionatedby reverse-phase high performance liquid chromatography (HPLC) equipped with a column(Cosmosil 5C18 AR-300) having a 0-100% acetonitrile gradient with or without 0.1% TFA. Fractionated samples (1 volume) were dried at room temperature using a rotary evaporator (Rotavapor,
Sibata) and dissolved in 5 volumes of 0.1 mM
Tris-HCl buffer (pH 7.2) for excystment assays.
For the ninhydrin test, the fractions were concentrated by using a rotary evaporator and applied on
a TLC plate (Funacel SF, Funakoshi, Japan). The
plate was dried and immersed in 95% ethanol solution containing 0.2% ninhydrin.
The density of bacteria was spectroscopically determined in a diluted bacterial suspension
whose value had been calibrated by comparison
with the cell density obtained by directly counting
the colonies on plates with the optical density
value at 600 nm (OD600).

Jpn. J. Protozool. Vol. 37, No. 2. (2004)

121

Fig. 1. Effect of cyst age on excystment of Colpoda
induced by cereal infusion or on spontaneous excystment, and excystment-inducing effect by bacteria. (a)Time course of excystment induced by 0.1% cereal
infusion containing 0.1 mM Tris-HCl (pH 7.2). The
rate of excystment is expressed as a percentage of thetotal number of tested cells (50-100 cells). (b) The
relationship between cyst age and excystment rate(%) induced by cereal components (closed circles)
and the rate (%) of spontaneous excystment (opencircles). The cysts were transferred from 0.1% TrisHCl (pH 7.2) into a fresh 0.1 mM Tris-HCl (pH 7.2)
buffer containing 0.1% cereal infusion (closed circles) or into a fresh 0.1 mM Tris-HCl (open circles).
Inset, the effect of cyst-free conditioned medium on
excystment. Three-day-old cysts adhered to the bottom of a petri dish were scraped and transferred intocyst-free conditioned medium or a fresh 0.1 mM
Tris-HCl buffer (pH 7.2). The label “No operation”
indicates the spontaneous excystment rate of thecysts without mechanical touch (cysts remained adhered to the bottom of the petri dish); in this case, thecysts were randomly selected (50-100 cysts) in thefield of the dissecting microscope and vacant cysts
were counted. (c) Excystment induction by bacteria(Enterobacter aerogenes) suspended in the surrounding medium containing 0.1 mM Tris-HCl (pH 7.2).

RESULTS AND DISCUSSION
It has been reported that the rate of excystment of Histriculus is influenced by the age of thecysts (Nakamura and Matsusaka, 1991). The rateof excystment of Colpoda employed in the present
study was also influenced by cyst ages. As shownin Fig. 1a, the rate (%) of excystment reached aplateau within 6 hr after the onset of induction.
Therefore, the excystment rates at 8 hr after the

onset of induction were examined (Figs. 1b, 1c,
2d, 3d, 4). The young cysts (aged less than 20days) tended to excyst when they were transferred
into a fresh medium (0.1 mM Tris-HCl, pH 7.2)
without cereal components (Fig. 1b, open circles)
and the rate (%) of excystment tended to be lower
in comparison with 30-day-old cysts (Fig. 1b).
Therefore, in the present study, cysts aged morethan 30 days were used. Such a spontaneous excystment of younger cysts might be triggered by122

Effect of porphyrin on encystment and excystment

Fig. 2. Induction of excystment of Colpoda by cereal components. (a, b) Elution profiles of cereal infusion
containing trifluoroacetic acid (TFA) by reverse-phase HPLC equipped with a column (Cosmosil 5C18 AR300) having a 0-100% acetonitrile gradient containing 0.1% TFA; profiles were monitored by the absorbances
at 260 (Fig. 2a) and 400 nm (Fig. 2b); in the present case, the monitored sensitivity was different between
Figs. 2a and b. (c) Absorption spectrum of colored fraction (fraction C). (d) The rate of excystment induced byeach fractionated component dissolved in 0.1 mM Tris-HCl buffer (pH 7.2). Their concentrations correspondto those contained in 0.1 % cereal infusion.

replacing the conditioned surrounding medium(0.1 mM Tris-HCl, pH 7.2) in which the cells hadencysted and had possibly excreted certain components such as metabolic products, with a fresh medium; that is, it might be triggered by the removalof Colpoda-derived substances accumulated in theconditioned surrounding medium. When 3-dayold cysts were transferred into cyst-free conditioned medium using a glass pipette, the excystment rate was not significantly different from therate in a fresh medium (Fig. 1b, inset; p > 0.05,
Mann-Whitney test). On the other hand, the cyststhat were not transferred rarely excysted (Fig. 1b,
inset, labeled “No operation”); there was a significant difference between “No operation” and “Cystfree conditioned medium” (p < 0.05, MannWhitney test). These results indicate that mechanical stimulation by pipetting might induce
excystment in younger cysts. Presumably, younger
cysts are more likely to be influenced by a me-

Jpn. J. Protozool. Vol. 37, No. 2. (2004)

123

Fig.3. Induction of excystment of Colpoda by cereal components. (a, b) Elution profiles of cereal infusion
without TFA by reverse-phase HPLC equipped with a column (Cosmosil 5C18 AR-300) having a 0-100%
acetonitrile gradient without TFA; profiles were monitored by the absorbances at 260 (Fig. 3a) and 400 nm(Fig. 3b); note that the monitored sensitivity was different. (c) Absorption spectrum of colored fraction(fraction A). (d) The rates of excystment induced by fractionated components (their concentration corresponding to those contained in 0.1 % cereal infusion) that were dissolved in 0.1 mM Tris-HCl buffer (pH 7.2). The
column labeled “None” indicates the excystment rate of cysts suspended in 0.1 mM Tris-HCl buffer without
any components.

chanical touch because the cyst wall might be thinner than in older, mature cysts.
Excystment of Colpoda is triggered by certain molecules contained in cereal leaves (Watoh
et al., 2003; Figs. 1a, 1b). In order to determine
whether the excystment-inducing activity of cereal
infusion could be due to bacteria in the infusion,
the bacterial density in 0.1% cereal infusion containing cysts was measured spectroscopically. The
density of proliferated bacteria was less than 105

cells/ml at 18 hr after the cysts were suspended ina sterilized fresh cereal infusion, a value which isat the lower limit of spectroscopic detectability.
On the other hand, even a bacterial density of 109
cells/ml failed to show excystment-inducing activity (Fig. 1c). Therefore, the excystment-inducing
effect of cereal infusion cannot be attributed tobacterial contamination, but to certain cereal components.
Figures 2a and 2b show the elution profiles

124

Effect of porphyrin on encystment and excystment

Fig. 4. Effects of porphyrins andriboflavin on Colpoda excystment. The porphyrins (sodium
salts) or riboflavin was dissolved
in 0.1 mM Tris-HCl buffer (pH7.2). Inset, growth of bacteria(Enterobacter aerogenes) in a
0.1 mM Tris-HCl (pH 7.2) containing 10-4 M chlorophyllin.

of cereal infusion containing 0.1% TFA by reverse-phase HPLC monitored by the absorbances
at 260 nm and 400 nm, respectively. Of fractions
A-C, only fraction C (light brownish in color; absorption spectrum shown in Fig. 2c) showed excystment induction activity (Fig. 2d). In this case,the cells did not excyst within 10 hr after induction, but did excyst within 24 hr after induction. In
addition, the excystment rate was lower, despite
the fact that the concentration of components offraction C corresponded to those contained in
0.1% cereal infusion (ref. Fig. 3d). It is possiblethat a large amount of colored components waslost as precipitates produced by TFA treatment.
Fraction C was ninhydrin-positive, as was fraction
A, and had an absorption ranging from 200 to 450
nm (Fig. 2c).
Figures 3a and 3b show the elution profiles of
cereal infusion by reverse-phase HPLC analyses
using solvents without TFA. Of these fractions,fractions A (absorption spectrum shown in Fig. 3c)
and E were colored, and only fraction A showed amarked excystment-inducing effect (Fig. 3d). In
this case, only the rate of excystment induced byfraction A was significantly different from thecontrol (labeled “None”; excystment rate in 0.1 mM
Tris-HCl without components) (p < 0.05, Mann-

Whitney test). Fractions B-E were slightly ninhydrin-positive while fraction A was strongly positive,
suggesting that fraction A may contain compoundswith amino groups and certain colored components
such as porphyrins derived from chlorophyll or
riboflavin.
We examined the excystment-inducing activity of commercially obtained porphyrins(chlorophyllin, coppered, trisodium salt; protoporphyrin IX, disodium salt; protoporphyrin IX zinc)and riboflavin. Chlorophyllin was found to have amarked excystment-inducing effect, while protoporphyrin IX, protoporphyrin IX bound to zincand riboflavin had no effect (Fig. 4). The densityof bacteria (Enterobacter) increased up to only 1.5
times at 10 hr in a 0.1 mM Tris-HCl buffer (pH7.2) containing 10-4 M chlorophyllin (Fig. 4, inset).
However, the excystment-inducing effect bychlorophyllin is not attributed to bacteria proliferated in chlorophyllin solution because even highdensity of bacteria (106~109 cells/ml) hardly induces excystment (Fig. 1c). These results imply
that the three-dimensional porphyrin structure conformed by coordination to copper may be involvedin the activation of receptors triggering excystment
processes. It is also possible that the excystmentinducing activity of the chlorophyllin may be at-Jpn. J. Protozool. Vol. 37, No. 2. (2004)

125

Fig. 5. Supression of encystment of Colpoda by chlorophyllin or cereal components. Encystment was inducedby suspending the vegetative cells (50-100 cells) in 1 ml of saline solution (1 mM CaCl2, 1 mM KCl, 5 mM
Tris-HCl, pH 7.2) containing various concentrations of chlorophyllin or 0.1% cereal infusion. (a) Relationshipbetween the encystment rate and chlorophyllin concentrations. (b) Effect of the elimination of bacteria onencystment suppression by chlorophyllin or cereal components. The cysts were transferred into fresh(sterilized) standard saline solutions containing 10-4 M chlorophyllin or 0.1% cereal infusion every hour(labeled “chlorophyllin, washed every hour” or “0.1% cereal infusion, washed every hour”). “None”, saline
solution (1 mM CaCl2, 1 mM KCl, 5 mM Tris-HCl, pH 7.2).

tributable to other elements such as functionalgroups characterized by chlorophyllin. Chlorophyllin (bound to copper) does not occur in nature.
It is presumable, therefore, that excystmentinducing elements contained in cereal leaves may
be chlorophyll-derived water-soluble porphyrins
associated with magnesium.
Cereal infusion (0.1% ) and chlorophyllin
remarkably prevented Ca2+-induced encystment(Fig. 5a). Such a suppression effect still remains to
be attributed to bacteria contaminated and proliferated in these media. In the present experiments,
therefore, the cells were transferred into the fresh
media every hour. In this case, both cereal infusionand chlorophyllin strongly suppressed encystment(Fig. 5b).
The binding of antibody to the receptor-likeprotein (ESP) located on the cell surface of vegetative cells of soil amoeba induces encystment, while
the binding of ESP antibody located on the cysts

suppresses excystment (Yang and Villemez,1994). The results obtained by Yang and Villemez show that a common receptor is involved inboth the induction of encystment and the suppression of excystment. The fact that chlorophyllin
triggers the excystment processes of Colpoda andsuppresses its encystment processes likewise implies that chlorophyllin may activate a common
receptor located on the vegetative cells and cysts.
Chlorophyllin may be useful as a molecular
probe to visualize and isolate receptor proteins towhich chlorophyllin is bound and which activate
signaling pathways for excystment processes.
Further research is needed to isolate and characterize these receptors by using chlorophyllin.

REFERENCES
Delmonte Corrado, M. U., Chessa, M. G. andPelli, P. (1996) Ultrastructural survey of126

Effect of porphyrin on encystment and excystment

mucocysts throughout the life cycle of Colpoda cucullus (Ciliophora, Colpodea). Acta
Protozool., 35, 125-129.
Grimes, G. W. (1973) Differentiation during encystment and excystment in Oxytricha fallax. J. Protozool., 20, 92-104.
Gutiérrez, J. C., Callejas, S., Borniquel, S.,
Benítez, L. and Martín-González, A. (2001)
Ciliate crypto-biosis: a microbial strategy
against environmental starvation. Int. Microbiol., 4, 151-157.
Martín-González, A., Benítez, L. and Gutiérrez, J.
C. (1992) Ultrastruc-tural analysis of restingcysts and encystment in Colpoda inflata: II.
Encystment process and a review of ciliate
resting cyst classification. Cytobios, 72, 93106.
Nakamura, T. and Matsusaka, T. (1991) Effects ofcyst age on encystment of the ciliate Histriculus cavicola(Kahl, 1935) (Ciliophora:
Sticho-trichia) Europ. J. Protistol., 27, 375-

380.
Thimann, K. V. and Barker, H. A. (1934) Studies
on the excystment of Colpoda cucullus. J.
Exp. Zool., 69, 37-57.
Watoh, T., Yamaoka, M., Nagao, M., Oginuma, K.
and Matsuoka, T. (2003) Inducing factors
for encyst-ment and excystment in ciliatedprotozoan Colpoda sp. Jpn. J. Proto-zool.,
36, 105-111. (In Japanese)
Yamaoka, M., Watoh, T. and Matsuoka, T. (2004)
Effect of salts concentra-tion on encystmentof ciliated protozoan Colpoda sp. Acta Protozool., In press.
Yamasaki, C., Kida, A., Akematsu, T. and Matsuoka, T. (2004) Effect of components released from bacteria on encystment in ciliated protozoan Colpoda sp. Jpn. J. Protozool., 37, 111-117.
Yang, S. and Villemez, C. L. (1994) Cell surface
control of differentiation in Acanthamoeba. J.
Cell. Biochem., 56, 592-596.