First published online June 15, 2007
Journal of Experimental Biology 210, 2253-2266 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005116
Extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus: insights from a metabolomics analysis
Jason E. Podrabsky1,*,
James P. Lopez2,
Teresa W. M. Fan3,
Richard Higashi3 and
George N. Somero4
1 Department of Biology, Portland State University, PO Box 751, Portland, OR
97207-0751, USA
2 Department of Neurobiology, Pharmacology, and Physiology, The University
of Chicago, Chicago, IL 60637, USA
3 Department of Chemistry, Belknap Research Building, 2210 S. Brook Street,
University of Louisville, Louisville, KY 40208, USA
4 Hopkins Marine Station, Stanford University, Oceanview Boulevard, Pacific
Grove, CA 93950, USA
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Fig. 1. Lethal time to 50% mortality (LT50) for embryos of A.
limnaeus exposed to anoxic conditions at 25°C. Filled bars represent
early development through diapause II (days post-fertilization; d.p.f.). Open
bars represent post-diapause II development (days post-diapause II; d.p.d.).
Values are means ± s.e.m. (N=3). Bars with different letters
are statistically different (Student–Neuman–Keul's
post-hoc test, P<0.05).
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Fig. 2. Aerobic recovery from anoxia in embryos of A. limnaeus exposed to
anoxia for (A) 7, (B) 30 and (C) 60 days. Filled and open pairs of bars
represent the number of embryos (out of a total of 20) initially surviving
anoxia (filled) and those that resumed normal development after 1 week of
aerobic recovery (open) from early development through diapause II (age of
embryos given in days post-fertilization). Hatched and open pairs of bars
represent the number of embryos initially surviving anoxia (hatched) and those
that resume normal development (open) after 1 week of aerobic recovery as
post-diapause II embryos. The stages of these embryos are given as days post
diapause II. Bars are means ± s.e.m. (N=3). Asterisks over the
open bars indicate a significant difference between initial survival of anoxia
and survival after 1 week of aerobic recovery (paired t-test,
P<0.05, one-tailed).
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Fig. 3. Anoxia induces quiescence in embryos of A. limnaeus. The number of
somite pairs does not increase during long-term exposure (ANOVA,
P 0.05), indicating a
state of anoxia-induced quiescence. Values are means ± s.e.m.
(N=3).
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Fig. 4. Concentration of glyceraldehyde 3-phosphate and lactate in embryos during
normoxic development and after exposure to anoxia in developing and diapause
II embryos. Open squares represent the normal pattern of metabolite
concentration during normoxic development. Colored symbols represent embryos
exposed to anoxia. 1 and 2 d.p.f. embryos were sampled at 0.5 and 1 day of
anoxia. 4 d.p.f. embryos were sampled after 7, and 21 days of anoxia. 8, 16
and 32 d.p.f. embryos were sampled after 21 and 60 days of anoxia. Values are
means ± s.e.m. (N=3).
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Fig. 5. The rate of lactate accumulation during anoxia is highly correlated with
survival in embryos of A. limnaeus. (r=–0.97,
P0.000001). Values are
means ± s.e.m. (N=3) for both rate of lactate accumulation and
LT50 values. Each symbol represents a single developmental
stage.
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Fig. 6. Concentration of succinate, malate and citrate in embryos of A.
limnaeus during normoxic development and after exposure to anoxia.
Symbols are the same as in Fig.
4; values are means ± s.e.m. (N=3).
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Fig. 7. Concentration of total free amino acids (FAA) in embryos of A.
limnaeus during normoxic development and after exposure to anoxia. Total
free amino acids increase substantially during normoxic development, but do
not change during exposure to anoxia in embryos with extreme anoxia tolerance.
Symbols are the same as in Fig.
4; values are means ± s.e.m., N=3.
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Fig. 8. The composition of the free amino acid pool changes as a result of normoxic
development and exposure to anoxia in embryos of A. limnaeus.
N=normoxic, A=21 days of anoxia for all developmental stages. The composition
of amino acids in lipovitellin heavy chain I (LvH1) was deduced from amino
acid sequences (see text for details).
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Fig. 9. Essential amino acids (isoleucine, leucine, methionine, phenylalanine,
threonine and valine) that increase during exposure to anoxia in embryos of
A. limnaeus. Symbols are the same as in
Fig. 4; values are means
± s.e.m., N=3.
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Fig. 10. Non-essential amino acids (alanine, glycine and proline) that increase
during exposure to anoxia in embryos of A. limnaeus. Symbols are the
same as in Fig. 4; values are
means ± s.e.m., N=3.
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Fig. 11. Four amino acids (glutamate, glutamine, aspartate and asparagine) decrease
in concentration during exposure to anoxia in embryos of A. limnaeus.
Symbols are the same as in Fig.
4; values are means ± s.e.m. (N=3).
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Fig. 12. Three amino acids (lysine, serine and tryosine) exhibit no change in
concentration during exposure to anoxia in embryos of A. limnaeus.
Symbols are the same as in Fig.
4; values are means ± s.e.m., N=3.
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Fig. 13. There is a substantial increase in the concentration of
 -aminobutyrate in embryos that can survive long-term anoxia. Symbols
are the same as in Fig. 4;
values are means ± s.e.m., N=3.
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© The Company of Biologists Ltd 2007
