First published online June 29, 2007
Journal of Experimental Biology 210, 2419-2429 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.002568
Rhesus glycoprotein gene expression in the mangrove killifish Kryptolebias marmoratus exposed to elevated environmental ammonia levels and air
C. Y. C. Hung1,2,
K. N. T. Tsui2,
J. M. Wilson3,
C. M. Nawata2,
C. M. Wood2 and
P. A. Wright1,*
1 Department of Integrative Biology, University of Guelph, Guelph, Ontario,
N1G 2W1, Canada
2 Department of Biology, McMaster University, 1280 Main Street West,
Hamilton, Ontario, L8S 4K1, Canada
3 Ecofisiologia CIMAR Rua dos Bragas 289, 4050-123, Porto,
Portugal
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Fig. 1. Amino acid sequence alignment of Kryptolebias marmoratus Rh with
other Rh sequences. Conserved amino acids are shaded in black and similar
amino acids are shaded in grey. Potential N-glycosylation sites of K.
marmoratus RhCGs are underlined in red. (A) Amino acid sequence alignment
of Kryptolebias marmoratus RhBG with other RhBG sequences. Accession
numbers of sequences: Takifugu rubripes AAM48577; Danio
rerio AAQ09527; Homo sapiens NP_065140; Rattus
norvegicus AAH79365 and Mus musculus NP_067350. (B) Amino acid
sequence alignment of K. marmoratus RhCGs with other RhCG sequences.
Accession numbers of sequences: Takifugu rubripes RhCG1 AAM48578;
Takifugu rubripes RhCG2 AAM48579; Danio rerio AAM90586;
Homo sapiens AAH30965; Rattus norvegicus NP_898876 and
Mus musculus NP_062773.
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Fig. 2. Protein characteristics of K. marmoratus RhBG, RhCG1 and RhCG2.
Amino acid compositions indicate that all three Rh proteins consist of
hydrophobic and polar amino acids, and all three proteins are negatively
charged at physiological pH. Hydropathy profiles (Kyte–Doolittle scale)
indicate that high hydrophobicity regions are dispersed along all three
sequences and these high hydrophobicity regions correspond to the predicted
transmembrane domains (red blocks). All three Rh proteins have intracellular
N- and C-terminals.
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Fig. 3. Phylogenetic relationships of K. marmoratus RhBG and
RhCGs and other homologues. Two major clusters are identified:
Cluster I (RhCG), Cluster II (RhBG). Cluster I is subdivided into Ia
(fish/amphibian/mammals/aves) and Ib (invertebrates). Cluster II is also
further divided into IIa (fish) and IIb (mammals/amphibian/aves). Sequences
are obtained from GenBank with accession numbers indicated.
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Fig. 4. Whole-body ammonia content of K. marmoratus after 5 days of 0 mmol
l–1, 1 mmol l–1 and 2 mmol
l–1 ammonia exposure. Asterisk indicates that tissue ammonia
was significantly higher at 2 mmol l–1 ammonia compared to 0
mmol l–1 and 1 mmol l–1 ammonia. Values are
means ± s.e.m., N=6 (one-way ANOVA, P<0.05).
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Fig. 5. RT-PCR mRNA expression of RhBG, RhCG1 and RhCG2 in
control (0 mmol l–1 ammonia) and ammonia-exposed (2 mmol
l–1 ammonia) K. marmaratus. For each gene, the
following tissues are shown from left to right: brain, eye, gill, gonad, gut,
kidney, liver, skeletal muscle and skin. Each gel represents one individual
(control N=3, ammonia-exposed N=3). Note RhBG,
RhCG1 and RhCG2 are expressed strongly in gill tissue.
RhBG expression is low in control tissues except in gill and skin,
but higher in many tissues in ammonia-exposed fish. RhCG1 expression
in skin is induced with ammonia exposure and RhCG2 expression remains
restricted to gill tissues.
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Fig. 6. Relative mRNA expression levels of gill Rh genes in excretory
organs (gills and skin) of K. marmaratus exposed to 0 mmol
l–1 (control) or 1 and 2 mmol l–1
NH4HCO3. (A) Gill RhBG, RhCG1 and
RhCG2 relative to 18S rRNA. Asterisk indicates that RhCG2
mRNA levels were significantly higher than in fish exposed to 2 mmol
l–1 ammonia compared to 0 or 1 mmol l–1
ammonia. RhBG and RhCG1 were not significantly different
between control and ammonia-exposed fish. Values are means ± s.e.m.; 0
and 2 mmol l–1 ammonia, N=7, 1 mmol
l–1 ammonia, N=8; one-way ANOVA,
P<0.05). (B) Skin RhBG, RhCG1 and RhCG2 relative
to EF1a. Letters (a,b,c) indicate that RhCG1 mRNA levels
were significantly higher in fish exposed to 1 mmol l–1
relative to 0 mmol l–1 ammonia, and significantly higher in 2
mmol l–1 relative to 0 and 1 mmol l–1
ammonia. Asterisk indicates that RhCG2 was significantly higher in 1
mmol l–1 compared to 0 and 2 mmol l–1
ammonia. RhBG was not significantly different between control and
ammonia-exposed fish. Values are means ± s.e.m.; N=6 (one-way
ANOVA, P<0.05).
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Fig. 7. Relative mRNA expression levels of RhBG to EF1a in
internal organs of K. marmaratus exposed to 0 mmol
l–1 (control) or 1 and 2 mmol l–1
NH4HCO3. In brain, no significant difference was
observed between control and ammonia-exposed fish. In liver, RhBG was
significantly higher in liver of fish exposed to 2 mmol l–1
(b) compared to 0 mmol l–1 (a), but there was no significant
difference between 0 (a) and 1 mmol l–1 ammonia (a,b), as
well as between 1 and 2 mmol l–1 ammonia-exposed fish. In
muscle, RhBG in muscle was significantly higher in fish exposed to 2
mmol l–1 (asterisk) than 0 and 1 mmol l–1
ammonia. Values are means ± s.e.m.; N=6 (one-way ANOVA,
P<0.05).
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Fig. 8. Relative mRNA expression levels of RhBG, RhCG1 and RhCG2
to EF1a in the skin of control (immersed) or air-exposed K.
marmoratus. Asterisks indicate both RhCG1 and RhCG2
were significantly higher in skin of air-exposed relative to control fish.
Values are means ± s.e.m.; N=6; air-exposed: N=4
(t-test, P<0.05).
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© The Company of Biologists Ltd 2007
