Aquatic Toxicology 103 (2011) 222–224
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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
Immune suppression of the echinoderm Asterias rubens (L.) following long-term
ocean acidification
Bodil Hernroth a,b,∗ , Susanne Baden c , Mike Thorndyke a , Sam Dupont c
a
b
c
The Royal Swedish Academy of Sciences, The Sven Lovén Centre for Marine Sciences, Kristineberg 566, 450 34 Fiskebäckskil, Sweden
Department of Biomedicine, Kristianstad University, 291 88 Kristianstad, Sweden
Department of Marine Ecology, University of Gothenburg, The Sven Lovén Centre for Marine Sciences, Kristineberg 566, 450 34 Fiskebäckskil, Sweden
a r t i c l e
i n f o
Article history:
Received 26 February 2011
Accepted 1 March 2011
Keywords:
Ocean acidification
CO2
Immunity
Stress indicators
Marine invertebrates
a b s t r a c t
We compared effects of exposure to predict near-future (2100) ocean acidification (OA; pH 7.7) and normal seawater (Control; pH 8.1) on immune and stress responses in the adult sea star Asterias rubens.
Analyses were made after one week and after six months of continuous exposure. Following one week
exposure to acidified water, the pH of coelomic fluid was significantly reduced. Levels of the chaperon
Hsp70 were elevated while key cellular players in immunity, coelomocytes, were reduced by approximately 50%. Following long-term exposure (six months) levels of Hsp70 returned to control values,
whereas immunity was further impaired, evidenced by the reduced phagocytic capacity of coelomocytes
and inhibited activation of p38 MAP-kinase. Such impacts of reduced seawater pH may have serious
consequences for resistance to pathogens in a future acidified ocean.
© 2011 Elsevier B.V. All rights reserved.
The rise in global atmospheric levels of CO2 not only warms the
Earth and destabilizes weather systems, but also dissolves easily
into the oceans changing the chemistry of seawater. From the preindustrial era until 2005, the average pH of ocean surface water
decreased by 0.1 pH units, representing an increase in the concentration of hydrogen ions of about 30%. A further decrease in
pH of about 0.4 units is expected by 2100 based on realistic scenarios for future CO2 emissions (Royal Society, 2005). Calcification
impairment of marine biota has been suggested as a potential consequence (Hofmann et al., 2010). However, there is a lack of long
term studies on the effects of OA on other vital processes of marine
organisms (Dupont et al., 2010) and virtually nothing is known
about immune and stress response.
In this study we compared immune response of the echinoderm Asterias rubens after long-term (six months) and short-term
(one week) exposure to seawater at pH 7.7 [predicted ocean acidification (OA) by the year 2100] with that of sea stars exposed
to seawater at pH 8.1 (Controls). Sea stars (n = 6) were kept in
400 L basins (flow-through system, temperature 12 ◦ C, salinity
32‰) and pH was maintained by bubbling CO2 which was regulated and controlled by a computerized system (AquaMedic;
Control: pH 8.1, pCO2 = 330.8 ppm, ca = 3.42, ar = 2.17; OA: pH
∗ Corresponding author at: The Royal Swedish Academy of Sciences, SLC
Kristineberg 566, 450 34 Fiskebäckskil, Sweden. Tel.: +46 523 18513;
fax: +46 523 18502.
E-mail addresses: [email protected], [email protected]
(B. Hernroth).
0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2011.03.001
7.7, pCO2 = 921.6 ppm, ca = 1.51, ar = 0.96). Animals were fed
with blue mussels (Mytilus edulis) twice a week until one week of
exposure remained, at which point the short-term experiment was
initiated with another batch of sea stars (n = 6).
At the end of the experiment an arm tip of each sea star was
cut and coelomic fluid collected to measure pH (NBS calibrated
Methrom pH-meter) and for microscopical determination of the
total coelomocyte count. Coelomocytes were harvested through
centrifugation and their phagocytic capacity was analyzed (Oweson
et al., 2008). Lyzed coelomocytes were prepared both for ELISAanalysis of heat shock protein Hsp-70 (Brun et al., 2008 with the
antibody as used by Holm et al., 2008) and for, phosphorylation
of mitogen activated protein kinase (MAPK) p38 after challenging coelomocytes with lipopolysaccharide. Data were analyzed
using Two-way ANOVA and the post hoc Student–Newman–Keuls
method.
Tissue carbonate buffers could help to maintain internal pH in
the short term, but in sea urchins such compensation has been
proven to be incomplete even under moderate environmental
hypercapnia (Miles et al., 2007). Accordingly, the present study
showed that after only one week of exposure to OA the pH of
sea star coelomic fluid was significantly reduced compared to that
of controls (Fig. 1A; Diff of mean 0.14; p = 0.008). Similar differences in pH levels were also recorded after six months (Diff of
mean 0.16; p = <0.001), indicating that the rate of proton-equivalent
ion exchange between the sea stars and ambient seawater was
maintained at the same level in both normal and acidified
conditions.
B. Hernroth et al. / Aquatic Toxicology 103 (2011) 222–224
223
Fig. 1. (A) pH of coelomic fluid was significantly lowered in A. rubens exposed to acidified seawater (OA) compared to that of Controls (C) both after one week (1wk) and six
months (6mo) of exposure. (B) Hsp70 increased in coelomocytes of OA treated sea stars after one week of exposure but not after six months compared to that of the controls.
(C) Activation of MAPKp38 in LPS challenged coelomocytes was significantly inhibited after six months of OA treatment compared to that after one week and the controls.
(D) OA treatment significantly reduced the total coelomocyte counts (TCC) and did so already after one week of exposure. (E) The phagocytic capacity of the coelomocytes
decreased in OA-treated sea stars and particularly so after six months of exposure. Letters indicate significant relationship between values.
During stress, energy is allocated to maintain protein integrity
via the chaperon Hsp70 (Mayer and Bukau, 2005), as well as
transferring peptides through the cell thus also affecting immune
defense (Moseley, 2000). In echinoderms, enhancement of Hsp70
has been recorded after short term exposure to environmental
stressors (Matranga et al., 2002; Oweson et al., 2008) and to physiological stress such as injury and tissue regeneration (Patruno et al.,
2001; Holm et al., 2008). In the current study Hsp70 was found to
be elevated after one week of OA exposure (Fig. 1B; Diff of means
79.6 abs units per mg protein; p < 0.001) but not after the longterm exposure. It has been suggested that it is too costly for sea
stars to over-express Hsp70 in the long-term (Clark et al., 2007)
and that lowered pH inhibits transcriptional and/or translational
levels (Langenbuch et al., 2006; Todgham and Hofmann, 2009).
Another activating pathway of immunity is that of the highly
conserved MAPK-p38. The short term exposure to OA did not
significantly affect the phosphorylation in LPS triggered coelomo-
cytes (Fig. 1C). However, after six months OA had a negative effect
with levels of this enzyme reduced by approximately 35% compared to controls (Diff of mean: 3.371, p = 0.049). Such suppression
of both Hsp70 and MAPK-p38 caused by long-term exposure to
OA may indicate a weakened activation of the immune response.
Furthermore, OA-exposed sea stars showed reduced numbers of
coelomocytes by approximately 50% after one week (Fig. 1D;
Diff of mean 6 × 103 cells per ␮l; p = 0.021) and this reduction
remained after six months. Not only was the number of coelomocytes affected, but their phagocytic capacity was also reduced to
approximately 30% after six months in acidified seawater (Fig. 1E;
Diff of means: 800 phagocytic units, p = 0.001). Thus, only 15% of
full phagocytic capacity remained. Such an impairment of phagocytosis after exposure to lowered pH for one month has previously
been reported in studies of M. edulis (Bibby et al., 2008).
In conclusion, we did not see any lethal effects but identified
the likely negative impact of near-future ocean acidification on
224
B. Hernroth et al. / Aquatic Toxicology 103 (2011) 222–224
immunity in the sea star A. rubens. This suggests potential serious consequences for resistance to pathogens in a future acidified
sea. Furthermore, our data illustrate the fact that ocean acidification is not solely a calcification issue and highlight the importance
of long-term experiments.
Acknowledgement
This work was supported by Gothenburg University Platform for
Integrative Physiology (GRIP; http://www.grip.science.gu.se/).
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