Vol. 280, Issue 1, R123-R131, January 2001
Survival of water stress in annual fish embryos: dehydration
avoidance and egg envelope amyloid fibers
Jason E.
Podrabsky1,
John F.
Carpenter2, and
Steven C.
Hand1
1 Section of Integrative Physiology and Neurobiology,
Department of Environmental, Population, and Organismic Biology,
University of Colorado, Boulder 80309 - 0334; and
2 Department of Pharmaceutical Sciences, University of Colorado
Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
Diapausing embryos of
Austrofundulus limnaeus survive desiccating conditions by
reducing evaporative water loss. Over 40% of diapause II embryos
survive 113 days of exposure to 75.5% relative humidity. An early loss
of water from the perivitelline space occurs during days
1-2, but thereafter, rates of water loss are reduced to near
zero. No dehydration of the embryonic tissue is indicated based on
microscopic observations and the retention of bulk (freezable) water in
embryos as judged by differential scanning calorimetry. Such high
resistance to desiccation is unprecedented among aquatic vertebrates.
Infrared spectroscopy indicates frequent intermolecular contacts via
-sheet (14%) in hydrated egg envelopes (chorions). These -sheet
contacts increase to 36% on dehydration of the egg envelope.
Interestingly, the egg envelope is composed of protein fibrils with
characteristics of amyloid fibrils usually associated with human
disease. These features include a high proportion of intermolecular
-sheet, positive staining and green birefringence with Congo red,
and detection of long, unbranched fibrils with a diameter of 4-6
nm. The high resistance of diapause II embryos to water stress is not
correlated with ontogenetic changes in the egg envelope.
Austrofundulus limnaeus; diapause; chorion
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INTRODUCTION |
POPULATIONS OF THE ANNUAL
KILLIFISH Austrofundulus limnaeus persist in ephemeral
pond habitats in coastal desert and inland savanna regions of Venezuela
(32). Survival of desiccating conditions is accomplished
not by the adult or juvenile forms but by diapausing embryos that are
presumably drought tolerant (37). Survival of
environmental water stress in embryos of annual killifish has received
little attention, despite the obvious requirement and biological
significance to the survival of these species. In this paper, we
investigated the survival and hydration state of A. limnaeus
embryos exposed to varying degrees of dehydration stress, the role of
the egg envelope (chorion) to survival under xeric conditions, and the
structure of the egg envelope proteins.
Fertilized embryos of annual killifish may spend the majority of their
lives (from several months to years) in a state of diapause (29,
37). In embryos of A. limnaeus, diapause is a state
of developmental and metabolic arrest (26) that is part of
the natural developmental program (37). Continuous
development in annual killifish may be interrupted at three distinct
developmental stages, termed diapause I, II, and III (37).
Entry into diapause precedes environmental insult, and as a result,
embryos will enter diapause even under conditions that appear optimal
(i.e., normoxic and fully hydrated) for development. Although there are
no data describing the distribution of A. limnaeus embryos
in pond sediments, these embryos are likely buried within a few
centimeters of the soil surface, too shallow to access moisture
resources often found deep in the soil. Diapausing embryos of A. limnaeus are likely exposed to a highly variable and unpredictable
environment including intense dehydration pressures for extended
periods of time while encased in the dry mud. Ponds inhabited by
A. limnaeus may experience several cycles of innundation and
drying during a single annual cycle or remain dry for years at a time
(J. Thomerson, personal communication). The life history of
A. limnaeus and the nature of their ephemeral pond habitat
suggest that embryos must either enter a state of anhydrobiosis
(5, 6) or else dramatically reduce evaporative water loss
to survive desiccating conditions.
One mechanism for survival of water stress is to allow the removal of
virtually all cellular water and enter a state of anhydrobiosis (5, 6). Anhydrobiotes include invertebrates such as
nematodes, tardigrades, and encysted embryos of the brine shrimp
Artemia franciscana. Survival in the dried state is
dependent on the stabilization of macromolecules by trehalose, which
can in effect replace water associated with cellular structures
(15). A slow transition (on the order of days) from wet to
dry is often required for survival of anhydrobiosis and is thought to
allow organisms time to make the biochemical adjustment (e.g.,
accumulation of trehalose) necessary for survival (36).
Some aquatic vertebrates and soil-dwelling arthropods have developed
mechanisms for preventing water loss when faced with dehydration
stress. Examples include desert-dwelling amphibians such as spadefoot
toads (24, 35), the African lungfish Protopterus aethiopicus (12), and soil-dwelling Collembola
(1). Avoidance of dehydration is usually achieved by
burrowing deep into the soil where water resources are available and
water loss is less severe. Additionally, many species produce cocoons
or secrete cutaneous lipids that substantially reduce water loss
(33). Mechanisms for the prevention of water loss are
typically only effective at mild or moderate dehydration pressures
[>98% relative humidity (RH), water potentials >
2.8 MPa].
In this study, we exposed embryos of A. limnaeus to a slow
dehydration regime to evaluate how these embryos survive water stress.
This study addresses four issues concerning survival of annual
killifish embryos under xeric conditions: 1) the stages of
development and diapause that are tolerant to water deprivation, 2) the mechanism, water loss or retention, that allows
embryos to survive, 3) the role of the egg envelope in
survival of water stress, and 4) the structure of the
proteins comprising the egg envelope.
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MATERIALS AND METHODS |
Developmental stages investigated.
Three developmental stages of fertilized embryos are compared in this
study: dispersion/reaggregation (D/R) stage, diapause II, and diapause
III embryos. D/R stage embryos are the earliest stage investigated.
This stage occurs between 6 and 8 days postfertilization (DPF) before
the formation of the embryonic axis. Diapause II is the next stage
investigated and occurs at ~24-25 DPF (25). Diapause II is accompanied by a substantial metabolic depression that
may last for several months or more (25). Diapause II
embryos have 38-40 pairs of somites, the foundations of the
central nervous system, and a functional tubular heart (25,
36). Diapause III embryos are the latest stages of development
investigated. Metabolic depression was also observed in these embryos.
Diapause III embryos are fully developed and ready to hatch given the
appropriate stimuli (36). Embryos of A. limnaeus enter diapause III ~23-25 days postdiapause II.
Evaporative water loss and survival.
Embryos were collected and maintained before experiments, as described
previously (25, 26). Embryos were exposed to atmospheres of controlled RH for various durations of time in glass desiccators (volume 2-5 liters) at 25°C. Groups of 30 embryos were removed from their aqueous rearing medium (embryo medium: 10 mM NaCl, 2.15 mM
MgCl2, 0.8 mM CaCl2, 0.14 mM KCl, 1.3 µM
MgSO4, and 10 mg/l gentamycin sulfate) and placed in Petri
dishes (diameter 50 mm) that held a sterilized filter pad (Fisher
Scientific) soaked in 2 ml of embryo medium (25). Two
milliliters of embryo medium were chosen because it allowed for a slow
initial dehydration. Petri dishes were then introduced into glass
desiccators to expose embryos to one of three RHs: 85, 75.5, or 50%.
These RHs in the air phase were controlled with saturated solutions of
KH2PO4, NaCl, and
Ca(NO3)2, respectively (34). The
solutions were continuously stirred with a Teflon-coated magnetic stir
bar. At 50% RH, the filter pads lost most of their water after 2 days,
whereas it took 4 days at 75.5% RH and ~8 days at 85% RH (data not
shown). At various time intervals, Petri dishes containing 30 embryos were removed from the desiccators, and mortality of embryos was recorded. Embryos were considered dead when they failed to retain water
and were physically shriveled. Embryos that experience a severe loss of
tissue water never recover. Dead embryos were not used in
determinations of water content unless mortality rates were high, in
which case the embryos were processed for comparative purposes. Water
content (g H2O/g dry mass) was determined for 10 embryos
randomly selected from each pad by subtracting dry mass from wet mass.
Dry mass was obtained by drying the embryos at 60°C to constant mass.
Differential scanning calorimetry.
Differential scanning calorimetry (DSC) was performed using a Perkin
Elmer model DSC 7 calorimeter (Norwalk, CT). Five embryos that had been
incubated for 8 days at 75.5% RH were sealed in an aluminum sample pan
for DSC analysis. Temperatures were scanned from 40°C to 25°C at
a scan rate of 10°C/min.
Isolation and solubilization of egg envelopes.
Egg envelopes were isolated manually with fine-tipped forceps.
Unfertilized egg envelopes were isolated in a saline solution containing 10 mM EDTA, 131 mM NaCl, 2.5 mM KCl, and 10 mM
Tris · HCl (pH 7.3) to help prevent spontaneous hardening
(31). Isolation of egg envelopes from fertilized stages
was performed in embryo medium. All isolated egg envelopes were washed
three times in 20 ml of reagent-grade water before use.
The dry mass of isolated egg envelopes and the solubility of egg
envelope proteins were determined on the same samples. Isolated envelopes were dried to constant mass at 60°C before determination of
protein solubility. Solubilization of egg envelopes was achieved by
homogenizing 10-30 envelopes in 100-400 µl of 8 M guanidine HCl containing 50 mM Tris · HCl, pH 8 (31).
Homogenates were boiled for 30 min in the presence or absence of 5%
-mercaptoethanol to aid solubilization. Solubilized proteins from
egg envelopes of unfertilized eggs were then dialyzed against 10 mM
sodium phosphate buffer (pH 7) overnight at 4°C. Dialysis against the
dilute phosphate buffer caused some of the protein to precipitate from
solution. Precipitated protein was isolated by centrifugation (10,000 g for 10 min) and resuspended in 10 mM sodium phosphate
buffer, pH 7. The protein secondary structure in the precipitated
protein was investigated using infrared (IR) spectroscopy. The
secondary structure of solubilized proteins in the supernatant fraction was analyzed using far-ultraviolet (UV)-circular dichroism (CD) spectroscopy and SDS-PAGE electrophoresis.
IR spectroscopy.
IR spectra were acquired using a Bomem MB-104 spectrometer (Quebec,
Canada). Solutions were prepared immediately before use by
homogenization of 10 egg envelopes in 50 µl of 10 mM sodium phosphate
(pH 7) using a ground glass tissue homogenizer. Homogenates were placed
in a liquid sampling cell with CaF2 windows and a path
length of 6 µm. Background spectra were collected under the same
conditions using only sample buffer (10 mM sodium phosphate). For
collection of IR spectra from dried egg envelopes, envelopes were
isolated as described above and dried over P2O5
for 12-24 h. Dried egg envelopes were crushed and mixed with
IR-grade potassium bromide (KBr). A disc of the material was prepared
by subjecting the powder to 5.4 metric tons of pressure. An
interferogram (256 scans) was generated for each sample in single-beam
mode with a 4 cm
1 resolution. For aqueous
samples, the contributions of liquid and gaseous water were subtracted
using previously established criteria (8). Protein spectra
in the amide 1 region (1,600-1,700/cm) were transformed to the
second derivative and smoothed by a seven-point Savitsky-Golay smooth
function to remove noise. Spectra were then baseline adjusted and area
normalized (18) using Grams v.4.04 software (Bomem).
Far-UV-CD spectroscopy.
Far-UV-CD spectra were obtained at 25°C with an Aviv 62DS CD
spectrometer and thermoelectric temperature-control unit (Lakewood, NJ). Samples were placed in a quartz cell with a 1-mm path length. CD
spectra were obtained for two samples of solubilized egg envelopes isolated from unfertilized eggs. Protein concentration in the samples
was 0.35-0.50 mg/ml in 10 mM sodium phosphate (pH 7). Background
spectra were recorded under identical conditions and subtracted from
the protein spectrum.
Congo red staining.
Congo red staining was performed on egg envelope aggregates formed in
vitro (see MATERIALS AND METHODS) and on
homogenized/sonicated egg envelopes isolated from unfertilized eggs and
fertilized embryos (11 h postfertilization). Samples were applied to a
glass microscope slide, allowed to air dry, and stained with Congo red
(27). The slides were incubated at room temperature for 20 min in a saturated solution of NaCl in 80% ethanol and 0.1% NaOH.
This step was followed by a 50-min incubation in a saturated solution of Congo red and NaCl prepared in 80% ethanol and 0.1% NaOH. The slides were rinsed with three changes of 100% ethanol, air dried, and
the stained tissue was mounted under a glass coverslip with acrylic
mounting medium. Stained slides were observed using a light microscope
with crossed polarization filters at 630× magnification. Red staining
under bright-field optics and green birefringence under crossed
polarizers were considered diagnostic for amyloid protein structure
(2, 27).
Electron microscopy.
Protein aggregates formed in vitro and homogenates of egg envelopes (as
prepared above for Congo red staining) were applied to formvar and
carbon-coated grids (400 mesh). After 1 min, the grids were negatively
stained with 2% uranyl acetate for 1 min and allowed to air dry.
Samples were observed at 80 kV on a JEOL transmission electron
microscope (model 100C, Peabody, MA) at magnifications of
×50,000-130,000.
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RESULTS |
Evaporative water loss and survival.
Embryos of A. limnaeus survive desiccating conditions by
substantially reducing water loss and retaining a large portion of their total water (Fig. 1). Diapause II
embryos are the most resistant developmental stage to water loss and
retain ~50% of their initial water even after 32 days of exposure to
dehydrating conditions in 50% RH. Embryos in the dispersion and
reaggregation (DR) stage of development and diapause III embryos are
almost completely dehydrated after only 2 days of exposure to 50% RH
(Fig. 1). At 75.5% RH (Fig. 1), diapause II embryos display a similar
pattern of water retention to that seen at 50% RH. Diapause III
embryos are essentially dehydrated after 8 days, and DR-stage embryos have an intermediate ability to resist water loss. The latter embryos
retain ~50% of their water during the first 8 days of exposure and
are not completely dehydrated until day 16. Finally, at 85%
RH, both DR-stage and diapause II embryos retain ~60% of their
water, whereas diapause III embryos retain 65% of their water during
37 days of exposure. It is appropriate to note that the water retained
by diapause II embryos exposed for 8 days to 75.5% RH is freezable
and, therefore, likely to be bulk water. DSC indicates a large
heat-flow signal near 0°C during warming of samples previously cooled
to 40°C, which indicates the melting of water that froze during
cooling (Fig. 2).
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Fig. 1.
Water content of Austrofundulus limnaeus
embryos exposed to 50, 75.5, and 85% relative humidity (RH) for 113 days. Symbols are means ± SE (n = 3).
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Fig. 2.
Differential scanning calorimetry indicates a large
endothermic event near 0°C indicative of melting water in diapause II
embryos of Austrofundulus limnaeus subjected to 8 days of
dehydration stress at 75.5% RH.
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The rate of water loss is initially high in diapause II embryos exposed
to all levels of RH (Fig. 3). Maximal
rates ranged from 0.018 to 0.035 g
H2O · g1 dry
mass · h1. After the initial 4 days of exposure,
water loss is reduced to extremely low but nonzero values
(t-test, using 0 as the parametric value, df = 2, P < 0.05). After 32 days of exposure, water loss is
indistinguishable from zero (t-test as above). The less
dehydration-resistant embryos (DR stage and diapause III) continue to
lose water at a significant rate until dehydrated (data not shown). The
initial and rapid loss of water in diapause II embryos is thought to
come primarily from the perivitelline fluid. This suggestion is
substantiated by the observation that the volume of the embryonic
tissues and yolk do not change appreciably during the initial phase of
water loss (Table 1), whereas the
perivitelline space, as observed microscopically, is greatly reduced
(Fig. 4). Calculations indicate that loss
of water from the perivitelline space can account fully for the initial
water loss on exposure to desiccating conditions (see
DISCUSSION).
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Fig. 3.
Rate of water loss in diapause II embryos exposed to
environmental dehydration stress. Rates are indistinguishable from 0 after 32 days of exposure (t-test, using 0 as the parametric
value, df = 2, P < 0.05). Symbols are means ± SE (n = 3).
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Fig. 4.
Photomicrographs of diapause II embryos of
Austrofundulus limnaeus incubated in aqueous embryo medium
(A) and after 5 days of dehydration in 75.5% RH
(B). Note the lack of the perivitelline space (PVS) in the
embryo exposed to desiccating conditions (B). E, embryo; O,
oil droplet; Y, large yolk mass. The outer edge of the egg envelope
(EV) and the PVS in A are indicated.
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For all stages of development, survival of water stress is highest in
85% RH, intermediate in 75.5% RH, and lowest in 50% RH (Fig.
5). Diapause II embryos have the highest
survivorship, and 40% of these embryos survive for over 100 days at
75.5% RH. Diapause III embryos show the least resistance and all die
after 8 days at 75.5% RH, whereas DR-stage embryos are all dead after 16 days of exposure. Embryos placed on filter pads preequilibrated to
the various RH conditions (but not moistened as in the above experiments) always died within 24-48 h of exposure (data not shown). Thus embryo contact with a wetted surface early in the dehydration experiments is required to obtain the survivorship data
reported here.
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Fig. 5.
Survival of embryos of Austrofundulus limnaeus
during exposure to dehydrating conditions for 113 days. Symbols are
means ± SE (n = 3).
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Protein structure of the egg envelope.
The dry mass of the egg envelope does not change as a result of
fertilization and hardening nor during subsequent development (ANOVA,
P > 0.05; Table 2).
Approximately 80% of the dry weight of an unfertilized egg envelope is
soluble protein in 8 M guanidine HCl (Table 2). As judged by SDS-PAGE,
two major proteins (34 and 24 kDa) are extracted in this manner (Fig.
6). Only ~10% of the egg envelope dry
weight is soluble protein after hardening, even in the presence of both
8M guanidine HCl and 5% -mercaptoethanol (Table 2). Furthermore,
the solubility does not differ among prediapause II, diapause II, and
diapause III embryos.
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Fig. 6.
SDS-PAGE gel of solubilized egg envelope proteins. Two
major protein bands are observed at 34 and 24 kDa. Stds, standards;
me, -mercaptoethanol.
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To investigate the secondary structure of the proteins in homogenates
of egg envelopes we chose IR spectroscopy, which can be used to
investigate proteins in any state, including those with large insoluble
particles (10). For egg envelopes isolated from
unfertilized eggs and hardened embryos (11 h postfertilization), the
second-derivative IR spectra in the conformationally sensitive amide I
region were very similar (Fig. 7). The
deconvoluted spectra indicate that the egg envelope proteins of
A. limnaeus contain mostly -sheet (47%) and turns (30%;
Fig. 8, Table
3) with a small fraction of 
-helix
(10%). Surprisingly, the spectra also exhibit bands at 1,613 and
1,690/cm, which indicate that the egg envelope contains 14%
intermolecular -sheet (10). This structure is often
found in nonnative aggregated states of proteins that are formed by
perturbing conditions in vitro [e.g., thermally induced precipitation,
(10)] or pathological processes in vivo causing amyloid
fibril formation (19, 22).
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Fig. 7.
Second-derivative infrared (IR) spectra in the amide-I
region of isolated egg envelopes from unfertilized eggs, fertilized
embryos (11 h postfertilization), and in vitro aggregates of egg
envelope proteins. dI2(dx2)1,
Second-derivative of infrared absorbance.
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Fig. 8.
Deconvoluted second-derivative IR spectra of hydrated
(top) and dehydrated (bottom) egg envelopes
isolated from fertilized embryos 11 h postfertilization
(posthardening).
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To determine the macroscopic structure associated with the
intermolecular -sheet, we investigated egg envelope homogenates with
transmission electron microscopy (TEM). The micrographs (Fig. 9) revealed bundles of unbranched
fibrillar structure with a diameter of ~4-6 nm and indeterminate
length. This structure is a hallmark of amyloid fibrils
(22). Another diagnostic test for these fibrils is green
birefringence of Congo red-stained fibrils under cross-polarized light
(22, 30). Remarkably, the fibrils from egg envelopes display this staining behavior (Fig.
10) and appear similar to typical
pathological fibrils formed in disease processes such as Alzheimer's
disease (22) and systemic amyloidosis (30).
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Fig. 9.
Transmission electron micrographs (×100,000)
illustrating the fibrillar structure of egg envelope protein aggregates
formed in vitro (top) and in vivo (bottom). The
scale bar is equal to 100 nm. Individual fibers are ~4-6 nm in
diameter with indeterminate length.
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Fig. 10.
Light microscopy of egg envelope homogenates and in
vitro aggregates, both stained with Congo red. Photographs on the left
were obtained with bright-field optics, whereas the images on the right
were obtained with cross polarization. Protein aggregates formed in
vitro from precursors previously solubilized in 8 M guanidine HCl
(top). Egg envelopes isolated from unfertilized eggs
(middle). Egg envelopes isolated from fertilized (hardened)
embryos at 11 h postfertilization (bottom).
Magnification is ×630.
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To test whether amyloid fibrils from egg envelope proteins could form
in vitro, we solubilized isolated egg envelopes in 8 M guanidine HCl
and removed the denaturant by dialysis. This process often fosters
aggregation and precipitation of proteins during refolding procedures
(11). Over 80% of the egg envelope protein precipitated
during dialysis to remove the denaturant. The IR spectra of the
precipitates were similar to those of the original egg envelope
homogenates and again confirmed the presence of a substantial fraction
of intermolecular -sheet structure (Fig. 7). Furthermore, in these
in vitro precipitated preparations, both Congo red staining and TEM
showed the presence of amyloid fibrils that were virtually
indistinquishable from those in egg envelope homogenates (Figs. 9 and
10).
The secondary structure of the soluble protein (comprised of 2 major
proteins; Fig. 6) remaining after dialysis was studied with far-UV-CD
spectroscopy. This method was chosen because it can be used with
relatively low protein concentrations compared with the requirements of
IR spectroscopy. The far-UV-CD spectra (Fig.
11) have a prominent minimum
ellipticity at 205 nm, which indicates that the proteins are mostly
random coil (23).
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Fig. 11.
Circular dichroism spectra of soluble proteins from the
egg envelope indicate random coil structure. Protein concentration of
samples was 0.35-0.5 mg/ml. The soluble proteins are a mixture of
2 major and a few minor proteins as illustrated in Fig. 6.
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Considering the potential role of the egg envelope in desiccation
resistance of the embryos, we next investigated the effects of drying
over phosphorous pentoxide on the secondary structure of the envelope
proteins. The IR spectrum of the dried sample was grossly altered
relative to that for the hydrated proteins (Figs. 8 and
12). There is a large increase in bands
associated with intermolecular -sheet at 1,613 and 1,694/cm,
concomitant with reduction in absorbances for turn and intramolecular
-sheet (Table 3). Rehydration of the envelopes fully reversed these structural alterations (Fig. 12).
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Fig. 12.
Second-derivative IR spectra of hydrated and dehydrated
egg envelopes isolated from diapause II embryos. Changes in secondary
structure caused by dehydration are reversible on rehydration.
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DISCUSSION |
Survival of environmental dehydration.
Survival of A. limnaeus embryos under severely desiccating
conditions is achieved by a large reduction in evaporative water loss
(Fig. 1). A slow transition from wet to dry is required for survival
and may indicate that the embryos make biochemical or physiological
adjustments that foster water retention and survival. All developmental
stages of embryos of A. limnaeus investigated in this study
appear to possess a substantial ability to reduce water loss when faced
with dehydrating conditions of 85% RH. This suggests that short-term
or moderate drying of the soil may be tolerated by diapausing as well
as developing embryos. However, diapause II embryos are the most
resistant stage to water stress, and 40% survive for over 100 days at
75.5% RH. These data indicate that diapause II embryos are likely the
life history stage responsible for long-term survival of environmental
dehydration and the persistence of populations of A. limnaeus
in ephemeral habitats.
Retention of cellular water during environmental dehydration.
Considering the severely dehydrating conditions to which the embryos of
A. limnaeus are likely exposed in nature, retention of water
was an unanticipated mechanism for survival. Surface soils (<0.5 m) in
desert regions can become severely dried. Soils from an ephemeral
stream bed in the Chihuahuan desert have been reported to be extremely
dry, with water potentials reaching 13 to 16 MPa after long periods
(mo) without rain (28). Many amphibians and fish that
inhabit xeric or ephemeral environments have mechanisms that reduce
water loss (see introduction), but these animals can only resist water
loss under relatively mild dehydration stresses. In contrast, diapause
II embryos of A. limnaeus can retain water under extremely
dehydrating conditions for >113 days at 25°C. Such high resistance
to desiccation is unprecedented among aquatic vertebrates.
Embryos lose ~50% of their water during the initial period of
dehydration (Fig. 1), but importantly, this water does not appear to be
associated with embryonic tissues or yolk. Using an average egg
diameter of 1.73 ± 0.026 mm and an average yolk diameter of 1.46 ± 0.022 mm (25), we estimated the volume of the
perivitelline space to be 1.082 µl. This value corresponds to ~0.76
mg of water when one considers that 70% of the perivitelline fluid is
water and the balance is solutes, such as proteins and salts for
example (14). At 75.5% RH, diapause II embryos have lost
on average ~0.76 mg of water/embryo after 16 days of exposure to
dehydrating conditions. This value represents 100% of the estimated
water in the perivitelline space. Therefore, it is probable that all of
the water lost during the initial phases of dehydration is from the
perivitelline space and not from the embryo or yolk. This
interpretation would explain why the water content of diapause II
embryos always approaches the same value over a large range of
dehydration pressures. The large amount of freezable water present in
embryos exposed to 8 days of dehydration at 75.5% RH (Fig. 2) also
suggests a hydrated embryonic compartment in diapause II embryos. At
113 days of exposure to 75.5% RH, only 75% of the water loss is
explained by the perivitelline space. Thus the smaller amounts of water
lost after 16 days must come from the embryonic/yolk compartment.
Mechanisms for water retention.
The unchanged structure of the egg envelope among DR-stage, diapause
II, and diapause III embryos suggests that mechanisms other than
changes in the egg envelope proteins must be involved in the reduced
water loss observed in diapause II embryos. Damage to the egg envelope
during water stress, either mechanically or by fungal infection, causes
rapid water loss and death of embryos (data not shown). Thus the egg
envelope is required for survival under desiccating environmental
conditions, but it does not appear to be solely responsible for
resistance to water loss. Dehydration greatly increased the
intermolecular -sheet content of the egg envelope proteins, a
process that was reversible on rehydration (Fig. 12). These -sheet
structures are indicative of increased intermolecular contacts and
interactions (2, 9, 11, 22). Dehydration-induced increases
in intermolecular interactions could serve to decrease the water
permeability of the egg envelope. Further investigation of the
permeability of isolated egg envelopes in hydrated and dehydrated
conditions may clarify the role of the structural changes in the egg
envelope during dehydration.
Loss of perivitelline-space water and retention of embryonic water
suggests that the plasma membranes of the enveloping cell layer may be
important in the retention of water during environmental dehydration in
embryos of A. limnaeus. The permeability of the plasma
membranes of early fish embryos to water is among the lowest reported
for aquatic animals (13). However, the low permeability to
water and ions in typical fish embryos is obviously not sufficient to
retard evaporative water loss to the degree we observe in A. limnaeus; otherwise, we would expect survival of desiccating
conditions to be more common among fish embryos. Secretion of
substances into the perivitelline space (either at fertilization or
during subsequent development) could play a role in the dehydration
resistance of diapause II embryos of A. limnaeus. The
perivitelline space is known to have colloidal properties
(21) that are thought to be responsible for the
accumulation of a substantial internal hydrostatic pressure in eggs of
Fundulus on fertilization (17). The
perivitelline space of fish embryos contains significant amounts of
protein (20% by weight) (14), lipid (5% by weight)
(14), polysaccharides and mucopolysaccharides
(21), and polysialoglycoproteins (20). It is
possible that certain constituents of the perivitelline space of
diapause II embryos prevent water loss by creating a hydrophobic
barrier on drying. It is also possible that the constituents of the
perivitelline space may vitrify (form a glass) (4) when severely desiccated, thus substantially slowing water loss from the
embryonic and yolk compartments. The later possibility is supported by
observations that the outer layers of the embryo (egg envelope and
perivitelline space) become very brittle and glasslike after the
initial dehydration period is complete (Fig. 4), whereas the internal
compartments (yolk and embryonic cells) appear to remain relatively
hydrated as demonstrated by DSC (Fig. 2).
Amyloid protein structure of the egg envelope.
An unexpected result of the current study was the observation that the
egg envelope of A. limnaeus is composed of proteins that
share many characteristics of amyloid fibrils that are associated with
numerous human diseases (3, 22). This conclusion is substantiated by three major pieces of evidence: 1) positive
staining and green birefringence observed with Congo red (Fig. 10),
2) the fibrillar structure of the aggregates demonstrated
with TEM (Fig. 11), and 3) the characteristic intermolecular
-sheet secondary structure (Figs. 5, 8, 9). To our knowledge this is
the first report of a nonpathological amyloid fibril. Sequence analyses and in vitro studies with synthetic peptide fragments suggest that the
OsmB protein from Escherichia coli has the potential to form
fibrils (16), although in vivo amyloidlike fibrils have not been observed. In A. limnaeus and other fish embryos,
this fibrillar structure appears to protect against mechanical
disruption as well as to serve as a barrier to polyspermy, microbes,
low molecular weight solutes, and, in the case of A. limnaeus, perhaps water. We have found that the soluble protein
from the egg envelope is predominantly composed of random coil.
Furthermore, on the basis of our in vitro results, the egg envelope
proteins show a propensity to form amyloid fibrils instead of amorphous
precipitates. Thus all that may be needed to foster fibril formation in
the maternal ovaries is a sufficient concentration of precursor protein molecules. It is well established for many protein-aggregation, precipitation, and fibril-forming processes that the rate and extent
correlates directly with protein concentration (11).
Diapause II embryos of A. limnaeus can survive under
extremely dehydrating conditions by reducing evaporative water loss and retaining cellular water. The protein secondary structure, dry mass,
and solubility of the egg envelope do not change during development and
therefore do not correlate with increased resistance to environmental
dehydration in diapause II embryos. It appears that additional
mechanisms such as vitrification of the perivitelline space must be
important for reduction of evaporative water loss during exposure to
water stress in diapause II embryos of A. limnaeus.
| |
NOTE ADDED IN PROOF |
Recently, amyloid fibrils have been reported to play a
protective role in the chorion of the silkmoth (Bombyx mori)
embryo (Iconomidou VA, Vriend G, and Hamodrakas SJ. Amyloids protect the silkmoth oocyte and embryo. FEBS Lett 479:
141-145).
| |
ACKNOWLEDGEMENTS |
We thank Yong-sung Kim for assistance with CD measurements and Dr.
Suchart Chonprasert for help with DSC. The efforts of Natalia Gomez and
Tom Giddings in preparing the TEM samples are greatly appreciated.
| |
FOOTNOTES |
This work was supported by National Science Foundation Grants
IBN-9723746 to S. C. Hand and BES-9816975 to J. F. Carpenter.
Present address for S. C. Hand: Department of Biological Sciences,
Louisiana State University, Baton Rouge, LA 70803.
Address for reprint requests and other correspondence:
J. E. Podrabsky, Hopkins Marine Station of Stanford Univ.,
Oceanview Blvd., Pacific Grove, CA 93950 (E-mail:
podrabsk{at}leland.stanford.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 February 2000; accepted in final form 10 August 2000.
| |
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