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The great majority of free-living eukaryotic organisms
cannot survive the absence of molecular oxygen (anoxia) for
more than a day, and even those species well-adapted to resist
anoxia die within a month or so (for books and reviews, see
Hochachka and Guppy, 1987; Bryant, 1991; Hochachka et al.
1993; Grieshaber et al. 1994). In the latter cases, anoxic
survival depends on an overall reduction in metabolic rate to
conserve substrates and reduce the accumulation of toxic end-
products, a phenomenon commonly referred to as ‘metabolic
rate depression’ or MRD (reviewed in the references cited
above and by Storey and Storey, 1990; Guppy et al. 1994;
Hand and Hardewig, 1996). With one possible exception,
MRD is not complete since the anoxic rate is easily measured,
amounting to approximately 1% or more of the aerobic rate.
The potential exception is the encysted embryo of the brine
shrimp Artemia franciscana, which survives years of anoxia
and reduces its metabolism under anoxia to the extent that its
very detection becomes a major experimental problem,
documented in the present paper. Hand and colleagues (for
example, Carpenter and Hand, 1986; Hand and Gnaiger, 1988;
Hand, 1990, 1995) have produced substantial data indicating
the existence of a continuing anoxic metabolism in these
embryos during 6 days of anoxia, although the possibility that
metabolism ceases under prolonged anoxia was recognized.
Our results on this system provide no direct evidence for the
presence of a continuing metabolism after a day or so of anoxia
(see Clegg and Jackson, 1989; Clegg, 1992, 1993, 1994), a
position also taken by Hontario et al. (1993). This matter
seemed worthy of further study since there are important
qualitative differences between the complete absence of
metabolism and a greatly reduced one.
Materials and methods
Sources of Artemia franciscana
Unless stated otherwise, the encysted embryos of Artemia
franciscana (Kellogg) were from the South San Francisco Bay
salterns, purchased from San Francisco Bay Brand, Hayward,
467
The Journal of Experimental Biology 200, 467–475 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JEB0602
Encysted gastrula embryos of the crustacean Artemia
franciscana have acquired an array of adaptations that
enable them to survive a wide variety of environmental
extremes.The present paper shows that at least 60%
survive 4 years of continuous anoxia at physiological
temperatures (20–23°C) when fully hydrated. Although
these embryos appear to carry on a metabolism during the
first day of anoxia, no evidence for a continuing metabolism
throughout the subsequent 4 years was obtained. During
this period, there were no measurable changes in the levels
of their stored, mobilizable carbohydrates (trehalose,
glycogen, glycerol). Calculations indicate that, if these
carbohydrates are being utilized at all during anoxia, the
rate is at the least 50000 times lower than the aerobic rate
(lower limit of detection). Indications of proteolysis during
prolonged anoxia were sought but not found. Under
starvation conditions, the life span of larvae produced from
embryos that had undergone 4 years of anoxia was not
significantly different from that of larvae produced by
embryos that had not experienced anoxia. Thus, all
substrates and other metabolites required to support
embryonic development to the nauplius, as well as
endogenous (unfed) larval growth and molting, are
retained during 4 years of anoxia. It is not possible to prove
experimentally the absence of a metabolic rate in anoxic
embryos under physiological conditions of hydration and
temperature. Nevertheless, on the basis of the results
presented here, I will make the case that the anoxic embryo
brings its metabolism to a reversible standstill. Such a
conclusion requires that these embryos maintain their
structural integrity in the absence of measurable
biosynthesis and free energy flow and are thus an exception
to a major biological generality. Potential mechanisms
involved in their stability are discussed.
Key words: Artemia franciscana, brine shrimp, embryo, anoxia,
metabolic rate depression, trehalose, protein stability in vivo, stress
proteins, bioenergetics.
Summary
Introduction
EMBRYOS OF ARTEMIA FRANCISCANA SURVIVE FOUR YEARS OF CONTINUOUS
ANOXIA: THE CASE FOR COMPLETE METABOLIC RATE DEPRESSION
JAMES S. CLEGG*
Bodega Marine Laboratory, University of California (Davis), Bodega Bay, CA 94923, USA
Accepted 23 October 1996
*e-mail: jsclegg@ucdavis.edu.
468
California. In some cases, embryos from the Great Salt Lake,
Utah, were used (generously supplied by Professor Patrick
Sorgeloos, Artemia Reference Center, Ghent, Belgium).
Encysted embryos were stored dry, under nitrogen gas, at
approximately
−10°C before use. In both cases, approximately
90% of the embryos developed into swimming larvae when
incubated in aerobic sea water at 23–25°C for 72h.
Anoxic incubations
In some cases, the dried embryos were hydrated under
anoxic conditions by placing approximately 75mg into an 8ml
screw-capped glass vial to which was added 6ml of 0.4moll
−1
NaCl buffered to pH7.2 with 0.1moll
−1
sodium/potassium
phosphate and degassed for 5h with nitrogen gas. The 2ml air
space was replaced with 100% N
2
, and the cap was screwed
tight and wrapped with several layers of Parafilm to prevent
loosening. The vial was oscillated (50revsmin
−1
) for the first
24h to allow the embryos to become uniformly hydrated, and
thereafter stored on its side under ambient laboratory
conditions (see Clegg, 1994, for additional details, including
evidence that these conditions lead to anoxia). For some
studies, the embryos were first hydrated in the same solution
(aerobic) at 2°C, and then given a 4h incubation under aerobic
conditions at 23°C. These embryos were collected on cloth
filters, blotted to remove interstitial solution and transferred to
8ml vials (approximately 180mg wet mass, equivalent to
approximately 75mg dry mass). Anoxic incubation medium
was then added and the vials treated as described above.
Biochemical assays
After anoxic incubation, the embryos were collected on
cloth filters, washed rapidly with ice-cold distilled water,
blotted ‘dry’ and measured wet weights were transferred to
homogenizers. Some samples were placed in tared aluminum
cups for determination of dry mass. For trehalose and glycerol
determinations, embryos were homogenized in 75% ethanol
(approximately 100mgwetmassml
−1
) and the supernatant
obtained by centrifugation (2000g, 5min). Samples were
analyzed using a Waters high-performance liquid
chromatography (HPLC) system (BioRad Aminex HPX-87H
column) with a differential refractometer as detector and
authentic compounds to determine retention times. Glycogen
was extracted from the 75% ethanol-insoluble pellet using
50% KOH (95°C, 1.5h) and reprecipitated with ethanol (66%
final concentration at 2°C for 24h). The glycogen was
dissolved with water and determined by a colorimetric method
(Dubois et al. 1956) after chitin fragments had been removed
by centrifugation at 2000g for 5min. Results are expressed as
µgcompoundmg
−1
drymass.
For analysis of organic acids, embryos were homogenized
in 6% perchloric acid (PCA) at 0°C. Samples of the
supernatant (2000g, 5min) were applied to the Aminex
column described above and the effluent was monitored at
210nm using a Kratos ultraviolet detector. Flow rate was
0.6mlmin
−1
at 41°C, and the mobile phase was 4mequivl
−1
.
Retention times were determined using authentic compounds
which were also used, separately, to ‘spike’ PCA extracts.
Results are expressed as µgorganicacidmg
−1
drymass.
Incorporation of NaH[
14
C]O
3
and electrophoresis
Encysted embryos are impermeable to nonvolatile solutes
(De Chaffoy et al. 1978) but
14
CO
2
(from H[
14
C]O
3
−
) is
incorporated into a variety of amino acids (and other
metabolites), enabling examination of protein synthesis
(Clegg, 1976, 1977). In the present study, embryos hydrated at
2°C were incubated at 23°C under aerobic conditions for 4h
in 0.4moll
−1
NaCl buffered to pH7.2 with 0.1moll
−1
sodium/potassium phosphate and containing 1.11GBqml
−1
NaH[
14
C]O
3
(1.94GBqmmol
−1
from Amersham). After
incubation, the embryos were collected on cloth filters, washed
rapidly with ice-cold distilled water and either assayed
immediately (see below) or transferred to 8ml vials for anoxic
incubation as described above.
For assay, the embryos were collected as usual and
homogenized in a solution containing 150mmoll
−1
sorbitol, 70
mmoll
−1
potassium gluconate, 5mmoll
−1
MgCl
2
, 5mmoll
−1
NaH
2
PO
4
, 40mmoll
−1
Hepes at pH7.9. Samples of these
homogenates were added to equal volumes of 2× ‘sample
buffer’ (Laemmli, 1970) heated at 100°C for 5min and
electrophoresed on 12% SDS–polyacrylamide gels. The gels
were stained for proteins with Coomassie Blue, dried and
exposed to X-ray film for autoradiography (see Clegg et al.
1994).
To quantify the amount of
14
C present in the proteins of
embryos after the 4h aerobic incubation with NaH[
14
C]O
3
and
after prolonged anoxia, samples of these same homogenates
were processed as described in detail previously (Clegg et al.
1996). Briefly, homogenates were prepared in 5%
trichloroacetic acid (TCA) and the insoluble fraction was
washed twice with 5% TCA, then heated in 5% TCA at 95°C
for 1h. The hot-TCA-insoluble fraction was washed twice with
5% TCA, resuspended in 88% formic acid and samples were
taken for scintillation counting. Previous work (Clegg, 1977)
has shown that this treatment produces radioactive proteins and
that little else is labeled by aerobic incubation with
NaH[
14
C]O
3
.
Results
Anoxic longevity and post-anoxic developmental rate
Fig. 1 shows hatching levels for embryos that had not
experienced anoxia (zero time controls) compared with those
that had. Data for embryos undergoing 3 and 4 years of anoxia
were obtained in the present study, the continuation of an
earlier work (Clegg, 1994). Results from that study (0, 1 and
2 years of previous anoxia) are given here for comparison with
the present findings.
In the previous study (Clegg, 1994), it was shown that
drying embryos after anoxia increased the hatching levels
when they were rehydrated in aerobic sea water. This
suggested that anoxia caused these embryos to re-enter
diapause since this treatment is known to terminate diapause
J. S. CLEGG
469Artemia embryos survive 4 years of anoxia
in embryos from San Francisco Bay (i.e. those used in Fig. 1).
However, drying embryos that had undergone 4 years of
anoxia did not increase the proportion that hatched in aerobic
sea water (data not shown).
Fig. 2 describes the onset and rate of hatching by embryos
that had previously undergone anoxia, compared with those of
controls. As the duration of anoxia increased, the onset of post-
anoxic hatching was markedly delayed, amounting to a 10-fold
difference between controls and the 4 year anoxic sample. The
rate of hatching was also slowed in relation to the duration of
the anoxic period. Thus, the time required for 50% of the final
hatching level to be achieved can be used as an overall measure
of developmental rate: these times are approximately 22h for
control animals, 81h after 2 years of anoxia, 106h after 3 years
of anoxia and 167h after 4 years of anoxia.
Hatching in the latter two samples did not appear to be
complete when the assay was terminated (Fig. 2) and,
therefore, longer hatching assays were carried out for the 4 year
anoxic sample. Typical results (Fig. 3) showed clearly that
hatching was still continuing in this sample, albeit at a very
slow rate, even when the study was ended after 30 days of post-
anoxic, aerobic incubation. Therefore, the results shown in
Figs 1 and 2 represent minimal hatching levels for post-anoxic
embryos. Fig. 3 also raises the curious problem of identifying
‘dead’ embryos in anoxic populations.
Longevity of nauplii obtained from controls and previously
anoxic embryos
It has long been established that embryonic trehalose, a
disaccharide of glucose stored in very large amounts, provides
the energy and carbon source needed to support embryonic
development to the nauplius (see Clegg and Conte, 1980;
Slegers, 1991). However, the nauplius still contains substantial
amounts of glycogen, glycerol and yolk that enable it to molt,
undergo further morphogenesis and survive for 3–4 days in the
absence of food (see references cited above). If a metabolism
occurs in encysted embryos during such prolonged anoxia, one
might expect these and/or any other vital endogenous
substrates to be utilized, thus reducing the endogenous
longevity of nauplii derived from them. Such an effect should
be most marked in nauplii after 4 years of anoxia. However,
the longevity of nauplii hatched from control embryos (3.6±0.7
days, mean ± S.D., N=33) did not differ from that of nauplii
obtained from embryos that had previously undergone 4 years
of continuous anoxia (3.8±0.7 days, mean ± S.D., N=36).
An increase in the proportion of nauplii from anoxic
embryos that exhibited obvious developmental defects was
observed in this study. In a sample of 368 control nauplii, 4
were abnormal (1%); of 401 nauplii from the 4 year anoxic
embryos, 16 showed abnormalities (4%). The most common
defects involved stunted appendages and abnormal abdomens.
Abnormal nauplii were not used in the longevity study.
Stored energy sources in control and anoxic embryos
Although these longevity studies suggested that endogenous
100
80
60
40
20
0
% Hatching
0 1 2 3 4
Time previously anoxic (years)
100
80
60
40
20
0
% Hatching
0 50 100 150 250200 300 350
No anoxia
2 years
3 years
4 years
Aerobic incubation (post-anoxia) time (h)
Fig. 1. Effect of previous anoxic incubation of encysted embryos on
their subsequent production of nauplius larvae (hatching) when
returned to aerobic conditions and incubated for 10 days. Bars are
standard deviations for 3 groups of at least 200 embryos for each data
point (mean).
Fig. 2. Production of nauplius larvae (hatching) as a function of
aerobic incubation time after different periods of embryonic anoxia,
as indicated. The data points represent means of at least 200 embryos.
70
60
40
20
10
50
30
0
% Hatching
0 5 10 15 2520 30
Aerobic incubation (post-anoxia) time (days)
Fig. 3. Long-term aerobic incubation of encysted embryos previously
made anoxic for 4 years. The data points are means for 367 embryos
and represent cumulative hatching.
470
stores were not being used during embryonic anoxia, I compared
the concentrations of trehalose, glycerol and glycogen in
embryos after 4 years of anoxia with levels in those not made
anoxic (Fig. 4). Results for each compound were subjected to a
one-way analysis of variance (ANOVA) followed by pairwise
multiple comparisons (Student–Newman–Keuls test) where
appropriate. The results of the analysis indicate that levels of
trehalose and glycerol did not change significantly with time
under anoxia (P=0.58 for trehalose and P=0.52 for glycerol).
Glycogen levels, however, dropped significantly (P=0.002)
during the first month of anoxia, but did not change significantly
from that level over the subsequent 4 years of anoxia.
Fig. 5 plots the initial decrease in glycogen concentration
during shorter exposures to anoxia. Evidently, this decrease
occurred only during the first day of anoxia. Also shown in
Fig. 5 are concentrations of metabolites considered to be
probable end-products of an anoxic energy metabolism. Levels
of all these metabolites either decreased or did not change
during anoxia. Since propionate has been reported to
accumulate as an end-product during the first 5 days of anoxia
(Hand, 1990), I looked for this compound. A peak was detected
by HPLC that was close to that of authentic propionate, but
since its retention time was not identical and the peaks
separated when co-applied to the HPLC column, further study
was not carried out.
Although the utilization of glycogen occurs during the first
day of anoxia, indicating an anoxic metabolism, its metabolic
fate remains to be determined. The decrease in glycogen
concentration is not due to a simple hydrolysis since no
significant increase in free glucose concentration was observed
(data not shown).
Effects of previous aerobic incubation on carbohydrate
levels during subsequent anoxia
The preceding results were obtained from dried embryos that
were hydrated from the start under anoxic conditions. To
enable comparison with other work on anoxia in these
embryos, the effects of an initial aerobic incubation on
carbohydrate levels during anoxia (Fig. 6) were also examined.
As mentioned, previous studies have shown that, during
subsequent aerobic incubations, trehalose concentrations fall
while glycogen and glycerol concentrations increase (reviewed
by Clegg and Conte, 1980; Slegers, 1991), a result confirmed
J. S. CLEGG
180
170
150
130
120
160
140
110
100
Trehalose concentration (µgmg
−1
drymass)
0 20 30 50
10 40
Glycogen
Trehalose
Glycerol
Period of anoxia (months)
Glycerol and glycogen concentration
(µgmg
−1
drymass)
80
70
60
50
40
30
20
10
0
Fig. 4. Concentrations of mobilizable carbohydrates in dried encysted
embryos that were hydrated and incubated under anoxic conditions
for the times shown. Data points are means ±
S.D. (N=3 independent
samples). The only significant change is the decrease in glycogen
concentration during the first month of anoxia (see text for statistical
analysis).
Fig. 5. Concentrations of glycogen and various potential end-products
of anoxic metabolism in dried encysted embryos hydrated and
incubated under anoxic conditions. Note the different scale for the
glycogen data (N=1).
15.0
10.0
20.0
0.6
0.4
0.2
0
Concentration (µgmg
−1
drymass)
0 5 10 15 20
Glycogen
Succinate
Lactate
α-Ketoglutarate
Malate
Fumarate
Pyruvate
Period of anoxia (days)
180
160
200
120
80
40
140
100
60
20
0
Concentration (µgmg
−1
drymass)
0 100 150 250
50 200
300
Glycogen
Begin anoxia
Aerobic incubation
Trehalose
Glycerol
Time (h)
Fig. 6. Concentrations of mobilizable carbohydrates in encysted
embryos first incubated under aerobic conditions for 4h (filled
symbols) followed by transfer to anoxic conditions (open symbols).
Data points are means ±
S.D., most of which are within the data point
(N=3 independent measurements).
471Artemia embryos survive 4 years of anoxia
in the present work (Fig. 6). When these aerobic embryos were
then transferred to anoxic conditions, results qualitatively
similar to those shown in Figs 4 and 5 were obtained: namely,
glycogen levels decreased initially, while no change was
observed in the levels of trehalose or glycerol (Fig. 6). Thus,
an initial period of aerobic metabolism had no qualitative effect
on the fate of these carbohydrates during subsequent
embryonic anoxia.
Comparison of embryos from Great Salt Lake (GSL) and San
Francisco Bay (SFB)
All our previous work on A. franciscana, anoxia has
involved embryos collected from SFB, while others have used
embryos from GSL with different results. Although these
animals are the same species, it was possible that they differed
in their response to embryonic anoxia. Therefore, a limited
study was carried out on GSL embryos (Fig. 7). In this case,
we examined changes in concentrations of glycogen and total
carbohydrates soluble in 75% ethanol. It is known that
trehalose makes up approximately 90% of the latter fraction
(Clegg and Jackson, 1989) so the use of this rapid colorimetric
assay provided a convenient estimate for the amount of
trehalose in these embryos. As for the studies on SFB embryos,
two cases were examined: embryos were either made anoxic
from the dried state or given an aerobic incubation prior to
anoxia. The results of this study on GSL embryos (Fig. 7) were
comparable to those obtained with SFB embryos, indicating
that geographic origin was not of importance, at least in terms
of the effects of anoxia on ‘trehalose’ and glycogen levels.
Proteins in control and anoxic embryos
Encysted embryos are impermeable to amino acids, and
probably to all non-volatile compounds (De Chaffoy et al.
1978; Clegg and Conte, 1980), so NaH[
14
C]O
3
([
14
C]O
2
) was
used because it is known to label a number of metabolites
including several amino acids (Clegg, 1976, 1977). Embryos
hydrated at 2°C for 24h were incubated under aerobic
conditions for 4h at 25°C with NaH[
14
C]O
3
as described in
Materials and methods. After aerobic incubation, embryos
were rapidly washed free of external radioactivity with ice-cold
distilled water and samples of known wet mass (approximately
100mg) were either frozen at once (−72±2°C) or transferred
to anoxic NaH[
14
C]O
3
-free incubation medium (phosphate-
buffered 0.4mmoll
−1
NaCl) for further incubation at 20–23°C
for periods of up to 1 year. All samples were analyzed at the
same time, as described in Materials and methods.
Fig. 8A is a Coomassie-stained gel (SDS–PAGE) of
proteins extracted from embryos after the 4h aerobic
incubation (‘0 anoxia’) and after subsequent anoxic
incubations. At this level of resolution, the profiles appear to
be the same, with no indication of protein loss or degradation.
Embryos containing
14
C-labeled proteins were studied in an
attempt to increase the sensitivity of detection of protein
hydrolysis. Fig. 8B is an autoradiogram of this gel after drying.
Once again, the profiles are all remarkably similar, with no
evidence of protein hydrolysis over an entire year of anoxia.
The total amount of radioactive protein in the same samples
used in Fig. 8 was also determined (Table 1). Analysis of
variance of these data revealed that no significant differences
exist between the proteins of embryos after aerobic incubation
and those undergoing anoxic incubation. These results confirm
the visual impression given in Fig. 8 and document the
extraordinary resistance of the primary structures of proteins
in anoxic embryos.
Discussion
It is remarkable that at least 60% of these embryos give rise
to viable nauplii after experiencing continuous anoxia for 4
years (Fig. 1). To my knowledge, no laboratory study has
demonstrated this level of anoxic survival for any free-living
eukaryote, while fully hydrated at temperatures of 20–23°C.
A
200
180
160
140
120
100
80
60
Concentration (µgmg
−1
drymass)
Concentration (µgmg
−1
drymass)
0 5 10 15 20 25 30
4h aerobic → anoxia
Anoxic from start
Ethanol-soluble carbohydrates
Period of anoxia (days)
B
0 5 10 15 20 25 30
35
4h aerobic → anoxia
Anoxic from start
Glycogen
70
60
50
40
30
20
10
0
Fig. 7. Concentrations of 75% ethanol-soluble carbohydrates (A) and glycogen (B) during anoxia of encysted embryos from the Great Salt
Lake. The dried embryos were either hydrated and incubated under anoxic conditions or first hydrated at 2°C, given a 4h aerobic incubation
and then transferred to anoxic conditions. All data points are means ±
S.D. (N=3 independent measurements). In some cases, the standard
deviations are contained within the data points.
472
Of interest, however, is the work of Marcus et al. (1994), who
isolated copepod embryos from 40-year-old anoxic marine
sediments that hatched into nauplii when placed under aerobic
conditions. Even more remarkable are the results of Hairston
et al. (1995), who found that viable freshwater copepod
embryos could be isolated from 332-year-old anoxic,
freshwater sediments. If the ages of these embryos are the same
as those of the sediments (an assumption not currently subject
to test), then these embryos must either certainly be ametabolic
or are permeable to compounds in the interstitial water of these
sediments that support an anoxic metabolism. The latter
possibility seems unlikely, but appears to be testable.
Nevertheless, these observations on copepod embryos suggest
that a reversible metabolic standstill under anoxia is not limited
to Artemia embryos.
An important question in the present study concerns those
embryos that do not hatch after anoxia is terminated. It has
generally been assumed that embryos are dead if they do not
hatch after a period of incubation comparable to controls;
however, that is a risky assumption in view of the results
shown in Figs 2 and 3, which demonstrate that some anoxic
embryos require at least a month to complete the hatching
process when returned to aerobic conditions. These results
raise the question of how to distinguish between anoxic
embryos that are dead and those that simply require an
extremely prolonged aerobic incubation to hatch. I am not
aware of a way to do that at present by direct inspection.
Why should prolonged anoxia cause such lengthy delays in
hatching for those embryos that do eventually hatch (Figs 2, 3)?
One explanation is the need to repair damage that might have
accumulated during anoxia. Another possibility could be the
time needed to reverse the regulatory mechanisms that bring
metabolism to a standstill during anoxia and/or that are involved
in protecting the anoxic embryo from damage. It is known that
overall metabolic rates of post-anoxic embryos are reduced
compared with those of controls (Clegg, 1993). Consequently,
the return to a complete metabolic and developmental program
after prolonged anoxia is not a simple reversal of the events
involved in the aerobic–anoxic transition which is, by
comparison, very rapid. Further study of this matter is needed.
No evidence was found for a continuing metabolism during
prolonged anoxia. Although a metabolic rate cannot be proved
experimentally to be zero at temperatures of 20–23°C, the
results from the present study provide strong support for that
possibility in anoxic embryos. If these embryos are
metabolizing under anoxia, the rate is truly minuscule. For
instance, under aerobic conditions, trehalose is metabolized at
a rate of about 15µgmg
−1
drymassh
−1
(Fig. 6) but no change
in the content of this sugar was detected during 4 years of
anoxia (Fig. 4). Since a decrease in trehalose content of
10µgmg
−1
drymass could easily be measured, this will be used
as the lower limit of detection: 10µgmg
−1
drymass4years
−1
is
equivalent to the loss of 0.3ngmg
−1
drymassh
−1
, which is at
least 50000 times slower than the aerobic rate.
The existence of an anoxic metabolism not involving the use
of carbohydrate as substrate cannot be excluded, but this would
be a unique process since we know of no example of an anoxic
metabolism in any free-living organism that uses lipids or
amino acids without concomitant carbohydrate mobilization.
J. S. CLEGG
B
A
0 1d 1m 1y Anoxia
b
b
6
Table 1. Radioactive proteins in encysted embryos during
anoxia
Duration Radioactivity in protein
of anoxia (disintsmin
−1
mg
−1
wetmassembryo)
None 3408±135
1 day 3497±206
1 month 3658±196
1 year 3444±162
Embryos were incubated with NaH[
14
C]O
3
under aerobic
conditions for 4h at 23°C and then transferred to anoxic conditions,
as described in Materials and methods. The amount of
14
C in the total
protein fraction was determined after the periods of anoxia shown
above.
Results are means ± S.E.M. (N=3).
Fig. 8. Analysis of proteins in homogenates of encysted embryos by
denaturing polyacrylamide gel electrophoresis (SDS–PAGE).
Encysted embryos were hydrated at 2°C in sea water, then incubated
with NaH[
14
C]O
3
under aerobic conditions, as described, for 4h.
After removal of external NaH[
14
C]O
3
, the embryos were incubated
under anoxic conditions for the times shown. (A) Coomassie-stained
proteins; (B) autoradiogram of this same gel. The arrowhead points
to an abundant protein of molecular mass 26kDa referred to in the
text as p26. 1d, 1 day; 1m, 1 month; 1y, 1 year.
473Artemia embryos survive 4 years of anoxia
The conclusion that anoxia in excess of 1 day reduces
metabolism in these embryos to a reversible standstill should
be compared with the results of Hand and colleagues (cited in
the Introduction). They reported significant anoxic decreases
in levels of trehalose and glycogen, the accumulation of
propionate, lactate and other end-products, and the production
of small but easily measurable amounts of heat, that persisted
over at least 6 days of anoxia and were interpreted as a
continuing anoxic metabolism. However, these workers also
recognized that metabolism might come to a standstill during
prolonged anoxia. Hontario et al. (1993), also using
microcalorimetry, showed that heat production (and therefore
metabolism) reached undetectable levels within hours of
anoxic incubation. They proposed that the very small amounts
of heat measured by Hand and colleagues during the first 6
days of anoxia could be accounted for by a combination of
microbial contamination and instrumental problems. Hand
(1995) responded by controlling contamination and increasing
the sensitivity of the microcalorimetric technique employed,
and concluded that heat continues to be produced by anoxic
embryos. Most recently, Hand (1995) accepted the possibility
that at least some of this heat may not be coupled to metabolic
activity, but rather could be the result of non-metabolic
chemical (exothermic) reactions taking place in dead embryos
(Clegg and Jackson, 1989). This is relevant since Hand and
colleagues have utilized embryos from Great Salt Lake, Utah,
whose hatching levels were typically in the range 65–75%
(hatching levels were not reported in Hand, 1995). Pursuing
the possibility that at least some of the heat production
represented a continuing metabolism, Hand (1995) proposed
that if only 10% of the minuscule amount of heat measured in
anoxic embryos were coupled to the metabolism of trehalose
(or any carbohydrate) it would take 18 years of anoxia to utilize
this sugar completely. Assuming that these numbers are
correct, and knowing that encysted embryos contain
approximately 165µgtrehalosemg
−1
drymass (Fig. 4), the
amount of trehalose that would be utilized during 4 years of
this postulated anoxic metabolism can be calculated to be
35µgtrehalosemg
−1
drymass, a change that would easily be
detected if it took place. Similar results are obtained for glycogen
and glycerol, apparently the only other mobilizable carbohydrates
(Glasheen and Hand, 1989). Thus, I believe the preponderance
of evidence supports the view that metabolism in these embryos
is brought to a standstill after the first day of anoxia.
It is important to attempt to determine whether metabolism
comes to a reversible standstill in anoxic embryos since cells
are viewed as highly unstable systems operating far from
equilibrium. Thus, it is a general and important rule of biology
that living cells require a continuous and substantial flow of
free energy to maintain their integrity when fully hydrated at
physiological temperatures. Eukaryotic cells do indeed seem
to obey this rule, in general, but those in the anoxic Artemia
franciscana embryo may not. In the final section of this paper,
the significance of that result is examined further.
Consider the proteins of these embryos during prolonged
anoxia. Protein turnover utilizes a very large fraction of the
free energy budget of cells (Hand and Hardewig, 1996). Thus,
it can be expected that proteolytic activity must be turned off
in Artemia franciscana embryos during anoxia, and the results
shown in Fig. 8 and Table 1 support that expectation. Further
support comes from the study of Anchordoguy and Hand
(1994), who showed that the ubiquitin-dependent pathway of
proteolysis was profoundly reduced during anoxia in this
system (also see Anchordoguy and Hand, 1995). It appears
from the present results (Fig. 8; Table 1) that non-ubiquitin
pathways of proteolysis in these embryos (see Warner, 1987,
1989) must also be turned off during anoxia. It should be noted
that Anchordoguy et al. (1993) found that the rate of
degradation of cytochrome oxidase in anoxic embryos was
reduced, but still measurable, over 28 days of anoxia (half-life,
101 days). If this rate continued over 4 years of anoxia (Fig. 1)
little, if any, cytochrome oxidase would remain (10 half-lives)
to support the respiration that takes place immediately after
anoxic embryos are returned to aerobic conditions (Clegg,
1993).
A more difficult problem for the anoxic embryo, in my
opinion, concerns the inherent instability of proteins, notably
their tertiary and quaternary structures. When hydrated,
globular proteins will tend to denature/unfold and participate
in deleterious aggregation reactions (Dill, 1990; Somero,
1995). It does not seem likely that the Artemia franciscana
embryo has evolved proteins whose structures intrinsically
resist unfolding and aggregation since it has been pointed out
that globular proteins must be unstable at physiological
temperatures in order to be sufficiently flexible to function
(Somero, 1995; Shoichet et al. 1995). How is protein
conformational integrity maintained in these embryos over
years of anoxia, during which replacement by synthesis does
not occur?
One possibility could involve the participation of molecular
chaperones that reduce unfolding and/or renature unfolded
proteins (for recent reviews, see Ellis, 1994; Buchner, 1996;
Hartl, 1996). We have been studying an extremely abundant
protein in these embryos (p26, Fig. 8) that exhibits a number
of features characteristic of the small heat shock (stress)
protein family (Clegg et al. 1994, 1995, 1996; Jackson and
Clegg, 1996). This protein, whose native relative molecular
mass is approximately 500000 (subunits of 26000), makes up
10–15% of the total non-yolk protein, partially translocates
into the nucleus and other cellular compartments during anoxia
and other stresses, and is restricted to the encysted embryo
stage of the life cycle. It is noteworthy that the small stress
protein family does not require nucleoside triphosphate(s) to
perform chaperone functions in vitro (Parsell and Lindquist,
1993; Jakob et al. 1993; Waters et al. 1996). Further study of
the potential role of p26 during prolonged anoxia in these
embryos is needed.
A second possibility concerns the presence of large amounts
of trehalose and glycerol in these embryos (Fig. 4). These
compatible solutes are well-known stabilizers of proteins and
other cellular constituents (reviewed by Yancey et al. 1983;
Crowe et al. 1992; Winzor et al. 1992; Somero, 1995; also see
474
Hottiger et al. 1994). Consequently, these compounds might
generate an intracellular environment that stabilizes
membranes and macromolecules during prolonged anoxia
without the need for other participants. Of course, the
involvements of molecular chaperones and compatible solutes
are not mutually exclusive possibilities.
Although the mechanism(s) of stabilization remains to be
described, the results of this study suggest that the anoxic
embryos of Artemia franciscana provide an exception to one
of the most pervasive generalities of biology: the need for a
constant flow of metabolic free energy to maintain integrity
under physiological conditions of hydration and temperature.
The expert technical assistance of Susan A. Jackson is
recognized as an important part of this research. I appreciate
thoughtful critiques by two anonymous reviewers. Mary
Bowers is thanked for efficient manuscript preparation.
Supported in part by a grant from the National Science
Foundation (DCB 88-20347).
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