Increase of internal ion concentration triggers trehalose synthesis associated with cryptobiosis in larvae of Polypedilum vanderplanki
National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan
* Author for correspondence (e-mail: oku{at}affrc.go.jp)
Accepted 31 March 2003
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Summary |
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Key words: Polypedilum vanderplanki, cryptobiosis, anhydrobiosis, trehalose, ion
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Introduction |
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Accumulation of disaccharides at an extremely high level is a common
physiological trait among cryptobiotic organisms. Trehalose is often found in
cryptobiotic fungi (Sussman and Lingappa,
1959), Artemia cysts
(Clegg, 1965
), nematodes
(Madin and Crowe, 1975
;
Loomis et al., 1980
), yeast
(Panek et al., 1986
) and
bacteria (Payen, 1949
). The
mechanism of induction for trehalose production may differ in the wide array
of organisms; for example, Saccharomyces cerevisiae accumulates
trehalose in response to various external conditions, such as starvation, heat
shock and osmotic stress (e.g. Lillie and
Pringle, 1980
; Ribeiro et al.,
1994
). Intracellular trehalose has an important role to enable
high desiccation tolerance in human cells
(Guo et al., 2000
). This
compound provides the most effective protection against desiccation among
sugars and polyols because of its high ability for water replacement and
vitrification (Crowe et al.,
1987
,
1998
;
Green and Angell, 1989
;
Sano et al., 1999
). On the
other hand, trehalose is rarely used in higher plants, where sucrose, together
with other sugars, seems to substitute for trehalose
(Ingram and Bartels,
1996
).
An African chironomid, Polypedilum vanderplanki, is the highest
and largest multicellular animal with cryptobiotic ability. The cryptobiotic
larva can tolerate not only exposure to extremely high (106°C) and low
(270°C) temperatures but also submersion in pure ethanol (Hinton,
1960a,
b
,
1968
). Recently, we reported
that this chironomid also accumulates a large amount of trehalose
(approximately 20% of the dry body mass) in the state of cryptobiosis, and
that cerebral regulation is not involved in the process for induction of
cryptobiosis (Watanabe et al.,
2002
). However, the factor(s) that triggers explosive synthesis of
trehalose during desiccation remains unknown. In this study, we demonstrate
that elevation of internal ion concentration is an important factor for
triggering trehalose synthesis associated with cryptobiosis in larvae of
P. vanderplanki.
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Materials and methods |
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Procedures for desiccation and incubation in solutions
Groups of 57 final-instar larvae (approximately 1 mg wet body mass)
were placed on pieces of filter paper with 0.44 ml or 1.5 ml of distilled
water in a glass Petri dish (diameter, 65 mm; height, 20 mm). These dishes
were immediately transferred to a desiccator (<5% relative humidity) at
room temperature (2426°C), where they were gradually dehydrated
over 2 days (0.44 ml distilled water) or 7 days (1.5 ml distilled water).
In a separate experiment, groups of 520 larvae were submerged in approximately 20 ml of various kinds of solution in a glass Petri dish and incubated for 324 h at room temperature. Solutions of NaCl, mannitol, glycerol and dimethyl sulfoxide (DMSO) were applied at various osmotic pressures (205547 mosmol l-1 NaCl, 164412 mosmol l-1 mannitol, 2171628 mosmol l-1 glycerol and 128768 mosmol l-1 DMSO). In addition, eight different salt solutions (KCl, NaNO3, KNO3, Na2SO4, K2SO4, NaH2PO4, KH2PO4 and CaCl2) were prepared at 342 mosmol l-1 equivalents to a 1% NaCl solution. Larval activity was checked just after 1 day of treatment with each solution and classified into four categories as follows: category 3, all or most larvae moved actively in the same way as untreated larvae; category 2, most larvae moved their bodies slowly only when they were stimulated by tweezers; category 1, most larvae were moribund; category 0, all larvae were dead.
Measurements for water content
Larvae desiccated for 8 h, 16 h, 24 h, 32 h or 48 h in the 2-day
desiccation treatment or for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or
7 days in the 7-day desiccation treatment were individually heated at
100°C for 1 day. Water content was calculated from the difference in mass
before and after the heat treatment.
Pre-treatment with 1% NaCl solution before desiccation
1020 larvae were incubated for 3 h, 6 h or 9 h in 20 ml of 1% NaCl
solution in a glass Petri dish at room temperature and were then desiccated
with 0.1 ml or 0.44 ml of distilled water. The completely desiccated larvae
were used for determining trehalose content and for examining recovery after
rehydration with distilled water.
Sugar and polyol measurements
Larvae incubated in solutions and/or desiccated for various periods were
homogenized individually with 0.1 mg of sorbitol as an internal standard in
0.5 ml of 90% ethanol. After centrifugation (1500 g,
2030 min), the supernatant was desiccated completely by using a vacuum
concentrator and was stored at room temperature. The dried residue was
dissolved in approximately 500 µl of MilliQ water (Millipore, Bedford, MA,
USA). After filtration through a 0.45 µm membrane, the amount of
low-molecular-mass carbohydrates and polyols was measured as described by
Watanabe et al. (2002).
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Results |
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Effect of various solutions on trehalose synthesis and larval
activity
As the occurrence of rapid trehalose synthesis coincided with a decrease in
body water loss (Fig. 1),
changes of osmolarity in the body were thought to be a cue for trehalose
synthesis. It was therefore postulated that we might induce trehalose
synthesis by increasing internal ion concentration even without the
desiccation treatment. Trehalose content was thus compared among larvae
treated for 1 day in solutions of NaCl, mannitol, glycerol or DMSO at various
osmotic pressures. Larvae incubated for 1 day in NaCl solution at around 350
mosmol l-1 (342 mosmol l-1=1% NaCl solution) accumulated
quite a large amount of trehalose (approximately 35 µg
individual-1; equal to approximately 20% of the dry body mass;
Fig. 2A). Most of these larvae
were moribund or dead just after the treatment. All larvae died even when they
were transferred into distilled water or rehydrated after desiccation over 48
h. Larvae treated at less than 300 mosmol l-1 of NaCl solution
moved actively but did not accumulate as much trehalose. Larvae treated with
solutions of mannitol and DMSO increased trehalose content slightly
(approximately 5 µg individual-1) only when osmotic pressure of
the solution was extremely high (Fig.
2B,D). Thus, the level of trehalose accumulation depended upon the
kinds of solutes and was not a simple osmotic response.
|
Comparison of the rate of trehalose synthesis between larvae
desiccated and incubated in 1% NaCl
Larvae started accumulating trehalose significantly 3 h after both
desiccation and incubation in 1% NaCl (MannWhitney U-test;
P<0.05, N=46;
Fig. 3). The desiccating larvae
then gradually increased trehalose content for 48 h (at a rate of
approximately 0.7 µg h-1), whereas the 1% NaCl-treated larvae
accumulated trehalose rapidly between 6 h and 15 h after the beginning of the
treatment (at a rate of approximately 4 µg h-1). These results
suggest that the treatment with 1% NaCl solution stimulated trehalose
synthesis much more efficiently than did desiccation.
|
Comparison of trehalose content among larvae incubated in various
salt and carbohydrate solutions
Fig. 4 shows trehalose
content in larvae incubated for 1 day in various salt and carbohydrate
solutions at the same osmotic pressures (342 mosmol l-1). There was
no significant difference in trehalose content between untreated larvae
(control) and those treated in solutions of glycerol or DMSO
(MannWhitney U-test; P>0.05,
N=58). All of the salt solutions, with the exception of KCl
and K2SO4, were more effective in eliciting trehalose
accumulation than those of carbohydrates such as mannitol, glycerol and DMSO
(MannWhitney U-test; P<0.05,
N=58).
|
Salt molecules dissociate into cations and anions in solution. We also compared the effect of cations on trehalose content between molecules with the same kinds of anions. Na+ tended to be more effective in stimulating trehalose accumulation than did K+, especially with Cl- and NO3- (MannWhitney U-test; P<0.05, except for PO4-; Fig. 4). Ca2+ also appeared to trigger trehalose synthesis effectively.
Effect of pre-treatment with 1% NaCl solution prior to
desiccation
We examined how short-term treatment with 1% NaCl solution affects
trehalose synthesis and induction of cryptobiosis after the subsequent
desiccation treatment. Desiccation with 0.1 ml of distilled water caused
trehalose synthesis in larvae to some extent (mean, 23.2 µg) but rarely
induced cryptobiosis (Table 1).
Pre-treatment with an NaCl solution for 3 h or 6 h prior to desiccation with
0.1 ml of distilled water greatly accelerated trehalose synthesis (80.8 µg
for 3 h or 70.1 µg for 6 h) but did not enhance the success rate of
induction of cryptobiosis. Desiccation with 0.44 ml of distilled water
triggered trehalose synthesis (54.7 µg) more than that with 0.1 ml
distilled water (MannWhitney U-test; P<0.05).
Pre-treatment with an NaCl solution did not cause the further accumulation of
trehalose by desiccation with 0.44 ml but did decrease the rate of recovery
after rehydration depending on the time for the pre-treatment
(MannWhitney U-test; P<0.05).
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Discussion |
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Recently, several groups of researchers partly demonstrated the existence
of some components of a general regulatory network governing synthesis of
compatible solutes against the stresses of desiccation or high osmolarity. In
the yeast S. cerevisiae, exposure to high extracellular osmolarity
induced a two-component osmosensor (Sln1 and Sho1) to activate the high
osmolarity glycerol response (HOG) and mitogen-activated protein (MAP) kinase
cascades and finally caused accumulation of glycerol as compatible solutes
(Maeda et al., 1994,
1995
;
Posas et al., 1996
;
Posas and Saito, 1997
;
Raitt et al., 2000
). This
activation response was induced by high osmolarity regardless of the kinds of
solute (Maeda, 1999
). Also, in
the higher plant Arabidopsis thaliana, an osmosensor (ATHK1)
homologous to Sln1 has already been found
(Urano et al., 1999
), and
signal transduction cascades of the osmotic response similar to those in the
yeast have been shown to be genetically conserved and activated under
desiccation and high salinity stress
(Shinozaki and Yamaguchi-Shinozaki,
1997
; Mizoguchi et al.,
1998
; Miyata et al.,
1998
; Urano et al.,
1998
). In the case of P. vanderplanki larvae, exposure to
both desiccation and solutions containing salt or other substances at high
concentrations was able to cause an internal increase of salt and other solute
concentrations. The change is caused by gradual evaporation of water from the
body in the former and invasion of solutes into the body in the latter. These
treatments allowed larval tissues and cells to become exposed to high
osmolarity and high concentrations of various solutes. Unlike S.
cerevisiae and A. thaliana, rapid accumulation of trehalose was
not a simple osmotic response in P. vanderplanki larvae; i.e. the
explosive production occurred mainly in high concentration salt solutions and
depended on the kind of cation in solution. Thus, increase of internal ion
concentration triggers trehalose synthesis associated with cryptobiosis in
this species.
One-day incubation in high concentrations of salt solution triggered
effective synthesis of trehalose in larvae, but all of the treated larvae died
immediately or after the subsequent desiccation. Pre-treatment with NaCl
solution accelerated trehalose synthesis rapidly during the following
desiccation with 0.1 ml of distilled water, but such desiccated larvae also
failed to recover after rehydration. There appeared to be two possible reasons
why individuals containing a large amount of trehalose died in these cases.
One could be due to the stresses of osmotic shock and/or invasion of ions into
the body. Ion stress often provides detrimental effects on organisms by
confusing various physiological states and functions
(Hasegawa et al., 2000). This
hypothesis is supported by the marked depression of larval recovery rate by
treatment of NaCl solution prior to desiccation with 0.44 ml of distilled
water. A second explanation could be that the accumulation of trehalose is not
the only essential factor. Indeed, additional unknown critical factor(s) and
mechanism(s) are often needed for successful induction of cryptobiosis
(reviewed in Crowe, 2002
;
Oliver et al., 2002
). A
similar phenomenon was also found in this study: namely, larvae desiccated
with 0.1 ml of distilled water accumulated a relatively large amount of
trehalose (mean, 23.2 µg), although most of them did not recover from the
dehydration state. On the other hand, decapitated larvae that contained less
than 20 µg of trehalose succeeded in inducing cryptobiosis
(Watanabe et al., 2002
),
indicating that additional factor(s) beside trehalose are needed for
successful induction of cryptobiosis. The physiological basis for regulating
cryptobiosis remains to be elucidated.
The carbon source for compatible solutes to protect against desiccation and
osmotic stresses is uniformly distributed in unicellular organisms and plants;
i.e. mainly glucose in the former and sucrose in the latter
(Ingram and Bartels, 1996). By
contrast, in insects, glycogen is the main source of sugars and polyols and is
distributed locally in the fat body
(Storey and Storey, 1991
).
Indeed, the larvae of P. vanderplanki had a large amount of glycogen
in the fat body (M. Watanabe, T. Kikawada and T. Okuda, unpublished data). We
presume that proper distribution of newly synthesized trehalose into all
tissues or cells, probably by the fat body through the hemolymph, might be an
important task for desiccating larvae. This insect may have more complex
physiological and molecular mechanisms for induction of cryptobiosis than
those of the osmotic responses found in S. cerevisiae and A.
thaliana.
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Acknowledgments |
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