1967
The Journal of Experimental Biology 213, 1967-1975
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jeb.039990
Sea ice protects the embryos of the Antarctic sea urchin Sterechinus neumayeri from
oxidative damage due to naturally enhanced levels of UV-B radiation
Kathryn N. Lister1, Miles D. Lamare1 and David J. Burritt2,*
1
Department of Marine Science, 410 Castle Street, University of Otago, 9016 Dunedin, New Zealand and 2Department of Botany,
464 Great King Street, University of Otago, 9016 Dunedin, New Zealand
*Author for correspondence ([email protected])
Accepted 19 February 2010
SUMMARY
The ‘ozone hole’ has caused an increase in ultraviolet B radiation (UV-B, 280–320nm) penetrating Antarctic coastal marine
ecosystems, however the direct effect of this enhanced UV-B on pelagic organisms remains unclear. Oxidative stress, the in vivo
production of reactive oxygen species to levels high enough to overcome anti-oxidant defences, is a key outcome of exposure to
solar radiation, yet to date few studies have examined this physiological response in Antarctic marine species in situ or in direct
relation to the ozone hole. To assess the biological effects of UV-B, in situ experiments were conducted at Cape Armitage in
McMurdo Sound, Antarctica (77.06°S, 164.42°E) on the common Antarctic sea urchin Sterechinus neumayeri Meissner
(Echinoidea) over two consecutive 4-day periods in the spring of 2008 (26–30 October and 1–5 November). The presence of the
ozone hole, and a corresponding increase in UV-B exposure, resulted in unequivocal increases in oxidative damage to lipids and
proteins, and developmental abnormality in embryos of S. neumayeri growing in open waters. Results also indicate that embryos
have only a limited capacity to increase the activities of protective antioxidant enzymes, but not to levels sufficient to prevent
severe oxidative damage from occurring. Importantly, results show that the effect of the ozone hole is largely mitigated by sea ice
coverage. The present findings suggest that the coincidence of reduced stratospheric ozone and a reduction in sea ice coverage
may produce a situation in which significant damage to Antarctic marine ecosystems may occur.
Key words: Antarctica, oxidative damage, ozone hole, sea ice, sea urchin, ultraviolet radiation.
INTRODUCTION
In the past, the Antarctic marine environment has been exposed to
relatively low levels of ultraviolet-B radiation (UV-B) due to its
polar location, seasonal sea ice coverage and high atmospheric ozone
(O3) concentrations (McKenzie et al., 2003; Karentz et al., 2004;
Lesser et al., 2004; Lamare et al., 2007). However, in recent years
two relatively rapid changes to the global climate have occurred
that are causing biologically important changes to the Antarctic
environment.
Firstly, a reduction in stratospheric ozone has resulted in increased
levels of UV-B reaching the Earth’s surface (Madronich et al., 1998).
The greatest loss of stratospheric O3 has occurred over the Antarctic
continent and the surrounding Southern Ocean, where mean austral
springtime O3 concentrations are now 40–50% lower than they were
in the late 1970s (Malloy et al., 1997; Balis et al., 2009). This
reduction in springtime O3 concentrations, to less than 220 Dobson
units, has resulted in the formation of the Antarctic ‘ozone hole’
(Madronich et al., 1998), which results in large transient increases
in the levels of UV-B as the ozone hole moves across the Antarctic
continent (McKenzie et al., 2003; Karentz et al., 2004; Lesser et
al., 2004; Lamare et al., 2007). Although research suggests that
global ozone levels are stabilizing (Häder et al., 2003), full recovery
of the ozone layer is not expected for many decades (Newman et
al., 2006; McKenzie et al., 2007) and hence UV-B levels are
expected to remain high for the immediate future. For example, in
2008 the fifth largest ever ozone hole was recorded over Antarctica
(http://www.nasa.gov/topics/earth/features/ozonemax_ 2008.html).
Secondly, increased greenhouse gas emissions have resulted in
an increase in global temperatures and as a consequence a rapid
reduction in Arctic sea ice coverage (Turner and Overland, 2009).
Although, at present, the impact of global warming on Antarctic
sea ice has not been as significant as that seen in the Arctic,
researchers have shown accelerated melting of the West Antarctic
ice sheet (Velicogna and Wahr, 2006; Shepherd and Wingham,
2007) and it is likely that increasing temperatures will eventually
lead to an even greater loss of Antarctic sea ice.
Although it has been demonstrated that UV-B can, to a limited
extent, penetrate sea ice and cause damage to animals living under
the ice (Lesser et al., 2004), studies have also shown that sea ice,
which is relatively opaque and highly reflective especially when
covered with snow, reduces the UV-B exposure of aquatic organisms
living beneath it to a few percent of that above the ice (Trodahl and
Buckley, 1989; Perovich, 1993; Karentz et al., 2004). Therefore sea
ice can act as a protective filter greatly reducing the potential for
severe UV-B-induced damage in organisms living under it. Most
Antarctic marine organisms have highly specialised adaptations to
survive at low temperatures and have evolved under relatively low
UV-B levels (Karentz, 1991; Karentz et al., 2004; Lesser et al.,
2004). Studies have already shown that increased UV-B can reduce
primary productivity (Bidigare, 1989; Smith et al., 1992; Arrigo,
1994; Cullen and Neale, 1997) and affect reproduction and
development in Antarctic marine organisms (Karentz et al., 2004;
Lesser et al., 2004) and hence the combination of elevated UV-B
and a reduction in sea ice could have significant ecological
consequences (Häder et al., 2007).
At the level of the individual organism UV-B induces cellular
damage directly, as it is absorbed by macromolecules such as DNA
and proteins, and indirectly by increasing the formation of reactive
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1968 K. N. Lister, M. D. Lamare and D. J. Burritt
oxygen species (ROS) such as the superoxide anion (O2•), hydrogen
peroxide (H2O2), and the extremely reactive hydroxyl radical (•OH)
(Lesser et al., 2001; Lesser and Barry, 2003). Direct absorption of
UV-B by DNA results in the formation of cyclobutane-pyrimidine
dimers (CPDs) and 6-4 photoproducts, and causes increased
mutation rates in phytoplankton, macroalgae, and in the eggs and
larval stages of fish and other aquatic animals (Arrigo, 1994; Malloy
et al., 1997; Lesser et al., 2001; Lesser et al., 2003; Lamare et al.,
2006; Tedetti and Sempéré, 2006; Lamare et al., 2007). ROS are
produced continuously in living cells, largely as a normal by-product
of respiration in animals, however, changes in environmental
conditions that result in stress can lead to the over production of
ROS (Halliwell and Gutteridge, 1999). When ROS are produced at
high enough levels to overcome the antioxidant defences that
normally keep an organism’s ROS levels in check, oxidation of
DNA, proteins and membrane fatty acids occurs, the later resulting
in lipid peroxidation and a loss of membrane function (Halliwell
and Gutteridge, 1999). Such damage is commonly referred to as
oxidative stress and is considered to be a very sensitive biomarker
of many important environmental stressors, including UV-B (Lesser,
2006; Burritt and MacKenzie, 2003; Burritt, 2008). Although CPD
formation has been extensively studied, comparatively little is known
about the impact of UV-B exposure on oxidative damage and
prevention in Antarctic marine invertebrates, especially under field
conditions.
The embryonic and early larval stages of many marine
invertebrates are ecologically extremely important and have been
shown to be especially sensitive to increased levels of UV-B as they
have less effective avoidance strategies than adults, because of their
small size, rapid cell division, low metabolic activity, potentially
limited energy reserves and distribution near the ocean surface
(Hoegh-Guldberg et al., 1991; Johnsen and Widder, 2001; Karentz
et al., 2004). Although many embryonic and larval marine
invertebrates do contain UV-B screening molecules in the form of
mycosporine-like amino acids (MAAs), provided by the maternal
parent, the levels of these molecules can vary greatly depending on
the diet of the parent and are often not present at sufficiently high
enough levels to provide complete protection from UV-B induced
damage (Dunlap et al., 2000; Adams and Shick, 2001; Shick and
Dunlap, 2002; Lesser and Barry, 2003; Lamare et al., 2007). This
lack of complete protection by screening molecules is compounded
by the fact that springtime phytoplankton blooms and many
invertebrate spawning times coincide with the development of the
ozone hole, making it highly likely that they will be exposed to
maximal UV-B levels (Smith et al., 1992).
Sea urchins (Echinodermata: Echinoidea) are a diverse class of
benthic invertebrates that are present in relatively high abundance
across most marine biomes. Most sea urchins have a free-swimming
larval stage that can be in the plankton for days to months
depending on the species. Several species of sea urchins are
important broadcast spawners in the benthic communities of
Antarctica, with their embryos and larvae found throughout the
water column for up to four months in spring and summer (Pearse
et al., 1991; Lamare et al., 2006). The Antarctic sea urchin,
Sterechinus neumayeri Meissner is one of the most common
animals in the shallow subtidal benthos surrounding Antarctica
(Brey, 1991; Brey et al., 1995) and so any climatic changes that
negatively influence S. neumayeri populations could have a
detrimental impact on the whole Antarctic marine ecosystem. S.
neumayeri embryos and larvae when compared with those of other
echinoids have relatively low metabolic rates and require 20 days
of development after fertilisation before they are capably of
feeding, during which time they are dependent on the reserves
deposited in what is a relatively small egg (Bosch et al., 1987;
Shilling and Manahan, 1994). Previous work has shown that despite
the presence of UV-B-absorbing MAAs, the embryos and larvae
of S. neumayeri are very sensitive to changes in UV-B levels,
possibly as a result of their evolution in a low UV-B environment
and their slow physiological adaptation rates in the cold, food-poor
Antarctic waters (Lesser et al., 2004; Lamare et al., 2007).
The present study was designed to test the potential impact of
naturally increased UV-B doses, due to seasonal ozone depletion,
combined with an artificial reduction in sea ice cover on S.
neumayeri embryos. It has been suggested, but not experimentally
demonstrated in the field, that the embryos and larvae of Antarctic
invertebrates such as S. neumayeri could be predisposed to oxidative
damage because of adaptations required for life at low temperatures
(Lesser, 2006). For this reason we chose to use oxidative damage
to proteins and lipids as two sensitive biomarkers of oxidative stress,
in addition to monitoring abnormal embryo development and
aspects of enzymatic antioxidant defence. We discuss our results
in the context of life history trade-offs and the potential impact of
climate change on Antarctic marine communities.
MATERIALS AND METHODS
Species and study sites
In situ experiments were conducted at Cape Armitage in McMurdo
Sound, Antarctica (77.06°S, 164.42°E) on the embryos of
Sterechinus neumayeri Meissner (Echinidae) over two consecutive
four-day periods in the spring of 2008 (26–30 October and 1–5
November 2008). Ambient sea temperature during the experimental
period was –1.9°C. Adult urchins were collected by SCUBA divers
from the seafloor in the immediate vicinity of the study site, and
were placed in indoor flowing-seawater tanks plumbed directly with
seawater from McMurdo Sound.
Spawning and larval rearing
Reproductively mature sea urchins were induced to spawn within
days of collection by intracoelomic injection of potassium chloride
(0.5moll–1). Injected animals were inverted over 200ml beakers
containing filtered seawater in order to collect gametes. After the
gamete flow had stopped, animals were removed and the gametes
washed by serial partial water changes. In order to gain sufficient
material for each experiment, eggs from 13 females were combined
and fertilised by adding several drops of dilute sperm from three
ripe males. Fertilisation was determined by the appearance of a
fertilisation envelope, and only batches of eggs with a fertilisation
rate >95% were used in experiments. After 5min freshly fertilised
eggs were washed, to remove excess sperm, and stored at a density
of approximately 25 individuals per ml, in 3litre sterile plastic
containers, until they were used for experiments. Temperature was
maintained at the environmental ambient sea temperature of –1.9°C.
Embryos were used within 12h of fertilisation and remained in a
non-feeding stage throughout the experiments.
In situ experiments
In situ exposures of embryos to ambient solar radiation were
undertaken using an adaptation of the method of Lesser et al. (Lesser
et al., 2004). Briefly, embryos were sealed in one litre UVtransparent polyethelene bags (Whirlpak, Nasco–USA) and
suspended in the sea at depths of 1 and 4m by attaching them to
polyvinyl chloride racks (31cm⫻26cm horizontal dimensions), with
Vexar netting, moored to a rope with appropriate anchors and
flotation devices.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Sea ice protects sea urchins from UV-B
At each depth, the bags of embryos were subjected to one of three
radiation treatments: (1) photosynthetically active radiation (PAR)
but no UV (‘UV-O’ treatment); (2) PAR and UV-A, but no UV-B
(‘UV-A’ treatment); and (3) PAR and both UV-A and UV-B (‘UV-T’
treatment). The three UV treatments were achieved using Plexiglas
filters (50cm⫻50cm) attached by cable ties directly above the bags
of embryos, thus exposing the embryos to electromagnetic radiation
passing through the filters. The UV-T treatment consisted of UV
transparent Plexiglas that transmitted PAR (84.5%), UV-A (84.6%)
and UV-B (80.6%); the UV-A treatment consisted of UV transparent
Plexiglas that transmitted PAR (77.9%) and UV-A (46.5%) but
minimal UV-B (0.1%) and the controls (UV-O treatment) consisted
of UV opaque Plexiglas that transmitted PAR (81.0%) but minimal
UV-A (5.2%) and UV-B (0.0%; Fig.1). For each treatment at each
depth, four replicate bags of embryos were used. Nine moorings (three
per treatment) were deployed under sea ice (approximately 1.8m deep)
and three moorings (one per treatment) were deployed in an ice-free
area (approximately 2m in diameter) for 90h. This entire set-up was
repeated a second time with different larvae and conducted for 92h.
At the termination of each experiment the embryos were removed
from the racks and immediately placed in a dark, thermally insulated
container containing ambient temperature seawater until processing.
Care was taken to avoid exposure to direct sunlight during removal
of the bags. Processing of each replicate involved taking three, 1ml
random sub-samples from each bag for later analysis of abnormality,
with the remaining embryos concentrated and stored frozen at –80°C,
in 1.5ml Eppendorf tubes, for later biochemical analyses.
Ozone and radiation levels
Transmission (%)
Daily images of column ozone concentrations during the study period
were obtained from the US National Aeronautics and Space
Administration (NASA) web site and were accessed routinely during
the field season (see http://jwocky.gsfc.nasa.gov). Daily values for
stratospheric ozone concentrations over the study area were provided
from the Total Ozone Mapping Spectrometer (TOMS) mounted on
the Earth Probe Satellite. Hourly data on ambient UV-B (280–320nm),
UV-A (320–400nm) and the 400–600nm subset of visible radiation
(VIS) were obtained from the National Science Foundation UVR
Monitoring Program using a SUV-100 spectroradiometer
(Biospherical Instruments Inc.). Data are available at the Biospherical
Instruments, Inc. website (http://www.biospherical.com).
90
80
70
60
50
40
30
UVA
UVO
20
UVT
10
0
250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
Fig.1. Transmission profiles of the light filters use to control exposure to
UV radiation. The UV-T treatment consisted of UV-transparent Plexiglas
that transmitted PAR (84.5%), UV-A (84.6%) and UV-B (80.6%); the UV-A
treatment consisted of UV-transparent Plexiglas that transmitted PAR
(77.9%) and UV-A (46.5%), but minimal UV-B (0.1%); the controls (UV-O
treatment) consisted of UV-opaque Plexiglas that transmitted PAR (81.0%)
but minimal UV-A (5.2%) and UV-B (0.0%).
1969
Embryo morphology
The number of dead or abnormal embryos was determined within
a few hours of the termination of an experiment by direct
microscopic observation at magnifications of ⫻100 and ⫻400. Three
replicate sub-samples containing 100 embryos from each sample
under each light treatment and depth were counted. Embryos were
identified as abnormal if they showed one or more of the following
developmental aberrations: pronounced thickening of the
blastodermis in combination with reduction of the blastocoel,
abnormal development of primary mesenchyme cells, occlusion of
the blastocoel by cellular debris, exogastrulation or other forms of
aberrant archenteron development (Lamare et al., 2007).
Abnormality was cross checked by comparison with control embryos
kept in the laboratory and not exposed to field conditions of UV
radiation.
Determination of oxidative damage and antioxidant enzyme
analysis
Total protein was extracted for analysis of protein carbonyls and
enzyme activities by mixing a frozen pellet of approximately
25,000 embryos with 300l of potassium phosphate buffer (pH 7.0),
containing 0.1mmoll–1 Na2 EDTA, 1% polyvinylpolypyrrolidone
(PVPP), 1nmoll–1 phenylmethylsulphonyl fluoride (PMSF) and 0.5%
Triton X-100, and mixing the sample by vortexing for approx 30s
before centrifugation (13,000g) for 5min at 4°C. Protein extracts
were stored at –80°C until the assays were conducted. The protein
contents were determined as per Fryer et al. (Fryer et al., 1986) with
minor modifications.
Lipids were extracted following protein extraction by mixing the
pellet containing the remains of 25,000 embryos with 600l of
methanol:chloroform (2:1) and leaving the suspension to stand for
1min at room temperature. 400l of chloroform was then added
and the sample mixed by vortexing for 30s. 400l of deionised
water was then added and the sample mixed by vortexing for 30s,
before placing the tubes at 4°C overnight to allow the phases to
separate. 50l of the chloroform phase was used for the lipid
hydroperoxide analysis.
Protein carbonyl levels in the protein extracts were determined
via reaction with 2,4-dinitrophenylhydrazine (DNPH) as described
by Reznick and Packer (Reznick and Packer, 1994). Lipid
hydroperoxide levels in the lipid extractions were quantified using
the ferric thiocyanate method described by Mihaljevic et al.
(Mihaljevic et al., 1996). Both assays were adapted for measurement
using a microplate reader, with glass microplates used for the lipid
hydroperoxide analysis. Superoxide dismutase (SOD; EC 1.15.1.1)
was assayed using the microplate assay described by Banowetz et
al. (Banowetz et al., 2004) with minor modifications. Catalase (CAT;
EC 1.11.1.6) was assayed using the chemiluminescent method of
Maral et al. (Maral et al., 1977), as modified by Janssens et al.
(Janssens et al., 2000). All assays were carried out using a
PerkinElmer (Wallac) 1420 multilabel counter (Perkin Elmer, San
Jose, California, USA), controlled by a PC, and fitted with a
temperature control cell and an auto-dispenser. Data were acquired
and processed using the WorkOut 2.0 software package (Perkin
Elmer, San Jose, California, USA).
Statistical analyses
Statistically significant differences in abnormality, lipid
hydroperoxides, protein carbonyls, and enzyme activities were
tested against depths and treatments using two-way ANOVAs. Any
significant treatment or depth effects were then analysed using
post-hoc Tukey’s HSD tests. Residual values were assessed to
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1970 K. N. Lister, M. D. Lamare and D. J. Burritt
Table 1. Atmospheric ozone concentrations (A) over McMurdo Sound (77°51.618’S, 166°40.660’E) from 26 to 30 October, and 1 to 5
November 2008 and (B) surface irradiances, mean daily dose and total dose of UV-B, UV-A and visible light over McMurdo Sound during
the two experimental periods
A
Atmospheric ozone concentrations
26–30 Oct
1–5 Nov
Max ozone (DU)
Min ozone (DU)
Mean ozone (DU)
312
269
233
166
283
210
DU, Dobson units.
B
Surface irradiances
Max. irradiance (Wm s )
Mean daily dose (kJm )
–2 –1
UV-B
UV-A
VIS
UV-B:PAR
–2
Total dose (kJm–2)
26–30 Oct
1–5 Nov
26–30 Oct
1–5 Nov
26–30 Oct
1–5 Nov
1.05
32.81
194.19
0.01
1.80
32.89
159.71
0.02
34.15
1104.72
5338.52
54.52
1281.62
5998.20
128.06
4142.70
20019.45
208.81
4908.60
22973.09


UV-A, ultraviolet-A radiation (320-400nm) ; UV-B, ultraviolet-B radiation (290-320nm); VIS, visible light (400-600nm); UV-B-PAR, ratio of ultraviolet-B radiation
to visible light.
ensure that the test assumptions, normality and homogeneity of
variances, were met. All analyses were performed using SigmaStat
2.03 (SPSS Inc.) and the level of significance was P≤0.05 for the
post-hoc tests.
RESULTS
Ozone and surface irradiance
The ozone hole was located south of the study site in McMurdo
Sound during the first experimental period (26–30 October 2008),
but was directly over the study site for most of the second
experimental period (1–5 November 2008). Ozone concentrations
over McMurdo Sound ranged between 233 and 312DU (with an
average of 283DU) during the first experimental period, decreasing
to between 166 and 269DU (with an average of 210DU) during
the second experimental period (Table1). Concurrent with changes
in ozone concentrations were changes in ambient irradiances, the
most noticeable being an increase in incident UV-B during the
second experimental period (Table1). Maximum UV-B irradiance
increased 1.7-fold, total UV-B dose increased 3.8-fold and the ratio
of UV-B to visible radiation (measured from 400 to 600nm) doubled
(Table1).
Water column irradiance
In the sea-ice-free site at a depth of 1m radiation levels were 24.8%
of surface UV-B, 28.4% of surface UV-A and 27% of surface PAR,
whereas at 4m radiation was 2.4% of surface UV-B, 4.7% of surface
UV-A and 5% of surface PAR. The extinction coefficients (Kdm–1)
for UV-B, UV-A and PAR in the sea ice-free site were 1.14, 0.84
and 0.73m–1, respectively.
Oxidative damage, abnormal development and antioxidant
enzyme activities in embryos exposed to UV-A radiation
In general, embryos exposed to UV-A but protected from UV-B
showed no significant increases in abnormality or oxidative damage,
when compared with control embryos protected from both UV-A
and UV-B (Figs2 and 3). The exception was for embryos exposed
to UV-A at 4m under sea ice during the low ambient UV-B (high
ozone concentration) experiment which showed increased
abnormality (Fig.3D). In addition, exposure to UV-A did not cause
a significant increase in the activities of antioxidant enzymes, when
compared with control embryos (Figs4 and 5).
Oxidative damage and abnormal development in embryos
exposed to UV-B radiation
The proportion of abnormal embryos and oxidative damage to both
lipids and proteins increased significantly in embryos exposed to
UV-B at a depth of 1m under open-water conditions, when
compared with embryos protected from UV-B by artificial filters
(Fig.2A–C). In addition, both the proportion of abnormal embryos
and the levels of oxidative damage were dependent on column ozone
concentration and thus ambient UV-B dose (Fig.2A–C). Embryos
exposed to the higher ambient UV-B levels during the second
experimental period, when the ozone hole was directly over the study
site, had a significantly higher proportion of abnormal embryos and
greater oxidative damage when compared with embryos sampled
during the first experimental period, when ambient UV-B levels
were lower (Fig.2A–C). Specifically, during the first experimental
period embryos exposed to UV-B showed 1.8-fold and 2.5-fold
increases in oxidized lipids and proteins respectively, and a 1.8fold increase in the proportion of abnormal embryos, compared with
those protected from UV-B by filters (Fig.2A–C). During the second
experimental period, with lower ozone concentrations and higher
ambient UV-B levels, embryos exposed to UV-B showed a 2.4-fold
increase in oxidized lipids, a 4.4-fold increase in oxidized proteins
and a 2.5-fold increase in the proportion of abnormal embryos,
compared with those protected from UV-B by filters (Fig.2A–C).
A significant UV treatment ⫻ ambient UV-B interaction effect was
also observed for both measures of oxidative damage (F2,1212.245,
P0.005; F2,127.118, P0.001).
In contrast to embryos exposed to UV-B at 1m under open water
conditions those held at 1m under the sea ice showed no consistently
significant increases in either oxidative damage or embryo
abnormality, when compared with embryos protected from UV-B
by filters (Fig.2D–F). An ambient UV-B effect on embryo
abnormality and a UV treatment effect for protein carbonyl levels
were observed when the data from the two experimental periods
were pooled and subjected to an ANOVA (F1,1213.031, P0.005;
F2,124.205, P0.05). However, multiple comparison testing
revealed there were no significant differences in embryo abnormality
or protein oxidation due to the UV treatments (Fig.2D,E).
For embryos held at 4m, despite the differences being smaller
and results more variable, the same general trends with respect to
oxidative damage as seen in embryos held at 1m were observed,
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Lipid hydroperoxides (nmol
per 25,000 embryos)
Protein carbonyls
(nmol mg–1 protein)
Embryo abnormality
Sea ice protects sea urchins from UV-B
3
2
Open water
A
High ozone (26–30 Oct)
Low ozone (1–5 Nov)
1
b
a
a a
Sea ice
D
c
a
a
a
a
a
a
a
a
a
a
0
8
E
c
B
6
b
4
a
2
a
a
a
a
a
a
1971
Fig.2. The influence of ambient UV-B levels and UV
treatment [UV-minus: (UVO), UV-B-minus (UVA) and
UV-transparent (UVT)] on oxidative damage and
abnormal embryo development in Sterechinus
neumayeri embryos held at a depth of 1m, under
open water conditions or under sea ice. Proportion of
abnormal development in open water (A), protein
carbonyl contents in open water (B), levels of lipid
hydroperoxides in open water (C), proportion of
abnormal development under sea ice (D), protein
carbonyl contents under sea ice (E), levels of lipid
hydroperoxides under sea ice (F). Values are means
± s.e.m. calculated from four (open water) or three
(under ice) replicates. Open water and sea ice
experiments were analysed separately using two-way
ANOVAs. Within each graph, bars with the same
letter are not significantly different (P≤0.05).
0
10
C
F
c
8
b
a
6
4
a
a
a
a
a
a
a
a
a
2
0
UVO
UVA
UVT
UVO
UVA
with oxidative damage to both proteins and lipids increasing
significantly in embryos exposed to UV-B under open water
conditions (Fig.3B,C). The amount of oxidative damage was also
dependent on column ozone concentration and thus ambient UV-B
dose (Fig.3B,C), but unlike embryos held at 1m no increase in the
proportion of abnormal embryos was observed (Fig.3A). Sea ice
once again protected embryos held at 4m from oxidative damage
caused by UV-B exposure under both high and low ozone conditions
and from UV-B-induced abnormality during the low ozone high
ambient UV-B period (Fig.3E,F). However, a significant increase
in embryo abnormality was observed in embryos exposed to both
UV-A and UV-B during the low ambient UV-B experimental period
(Fig.3D). As there was no increase in oxidative damage observed
under these treatments and as the trend was not repeated under high
ambient UV-B conditions, this result was assumed to be due to a
significant ‘batch effect’, whereby the batch of embryos used had
a greater propensity for abnormality or was influenced by some
unknown factor (i.e. poly-spermy), rather than as a result of UV
exposure.
Antioxidant enzyme activities
Under open water conditions, the activities of SOD and CAT
increased significantly in embryos maintained at 1m and exposed to
UV-B, when compared with embryos protected from UV-B
(Fig.4A,B). Furthermore, the activities of both SOD and CAT were
significantly higher during the second experimental period with higher
ambient UV-B levels (Fig.4A,B), indicating that the increases in
enzyme activities were UV-B dose-dependent. Under sea ice no
significant differences in the activities SOD or CAT, due to UV-B
treatment, were observed (Fig.4C,D). Moreover, under the sea ice
no significant differences in enzyme activities between the two
experimental periods differing in ambient UV-B levels were observed.
Under open water conditions the basal activities of SOD and CAT
in embryos held at 4m were similar to those found in embryos held
UVT
at 1m and the same trends with respect to the presence or absence
of UV-B were observed, but were less clearly defined, with
increases in enzyme activity being much smaller than those observed
in embryos held at 1m (Fig.5A,B). Some variability in enzyme
activities between the two experimental periods was observed. Under
the sea ice no significant differences in the activities of SOD or
CAT, directly attributable to UV-B, were observed (Fig.5C,D).
DISCUSSION
Even without the influence of climate change the Antarctic
environment is metabolically demanding, with organisms requiring
a range of local adaptations to survive (Guderly, 2004; Pace and
Manahan, 2006). Most Antarctic marine organisms are
stenothermal and have cellular and metabolic adaptations that
allow them to survive and complete their life cycles at a constant
seawater temperature of –1.86°C (Pörtner et al., 2007). These
adaptations may include increased mitochondrial abundance and/or
volume density to help compensate for decreases in O2
conductance at constantly low temperatures, a higher proportion
of unsaturated fatty acids in their membrane phospholipids to help
maintain membrane fluidity at low temperatures, the maintenance
of greater lipid stores, and changes in both the levels and specific
activities of enzymes involved in key metabolic processes
including respiration (Hazel, 1995; Guderly, 2004). Although these
adaptations allow aerobic organisms to survive and function at
constantly low temperatures, such adaptations can greatly increase
the susceptibility of these organisms to environmental stresses
which cause excess ROS production and hence oxidative damage,
when compared with organisms living in warmer and more
variable environments (Viarengo et al., 1998; Guderly, 2004;
Pörtner et al., 2007). In addition, Antarctic seawater is rich in
oxygen, further increasing the likelihood of excessive ROS
production under environmental conditions that induce stress
(Lesser, 2006).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1972 K. N. Lister, M. D. Lamare and D. J. Burritt
Embryo abnormality
3
Open water
A
Sea ice
D
High ozone (26–30 Oct)
Low ozone (1–5 Nov)
2
a
a
1
a
b
b
a
a
a
a
a
a
a
0
Protein carbonyls
(nmol mg–1 protein)
8
E
B
6
c
4
2
a
a
a
a
a
b
a
a
a
a
a
a
a
Fig.3. The influence of ambient UV-B levels and UV
treatment [UV-minus: (UVO), UVB-minus (UVA) and
UV-transparent (UVT)] on oxidative damage and
abnormal embryo development in Sterechinus
neumayeri embryos held at a depth of 4m, under
open water conditions or under sea ice. Proportion
of abnormal development in open water (A), protein
carbonyl contents in open water (B), levels of lipid
hydroperoxides in open water (C), proportion of
abnormal development under sea ice (D), protein
carbonyl contents under sea ice (E), levels of lipid
hydroperoxides under sea ice (F). Values are means
± s.e.m., calculated from four (open water) or three
replicates (under ice). ‘Open water’ and ‘sea ice’
experiments were analysed separately using twoway ANOVAs. Within each graph, bars with the
same letter are not significantly different (P≤0.05).
a
Lipid hydroperoxides (nmol
per 25,000 embryos)
0
10
C
F
8
c
6
4
a
a
b
a
a
a
a
a
2
0
UVO
UVA
UVT
UVO
UVA
UV-B can directly induce ROS formation either in water, through
UV-B absorption by dissolved organic carbon (Cooper et al., 1994;
Richard et al., 2007), or inside animals, where photosensitisers such
as flavins and reduced pyridine nucleotides absorb UV energy and
are elevated to an excited state (Halliwell and Gutteridge, 1999).
When returning to the ground state, excited photosensitisers transfer
energy to ground-state oxygen producing 1O2, H2O2 and O2•, which
can in turn lead to the production of •OH. The •OH radical is
potentially very damaging to cells as it can initiate lipid peroxidation,
which once started can proceed via a free-radical-mediated chain
reaction mechanism which continues unabated unless terminated
(Halliwell and Gutteridge, 1999). Polyunsaturated fatty acids, which
contain multiple double bonds and highly reactive hydrogens, are
most sensitive to lipid peroxidation. Animals adapted to cold water
environments, such as the Antarctic oceans, often have a higher
proportion of unsaturated fatty acids in their membranes and higher
levels of lipid reserves, which are also able to undergo peroxidation,
and therefore may be particularly susceptible to lipid peroxidation.
Moreover, oxidative damage to membranes and other key cellular
macromolecules, such as proteins, can lead to mitochondrial damage.
It has been estimated that normal undamaged mitochondria convert
between 1 and 3% of their entire oxygen consumption to ROS (Sohal
and Weindruch, 1996) and so any disruption of mitochondrial
function, especially in animals whose mitochondria are adapted to
cold-water environments, could result in a large increase in ROS
production and therefore greatly increase the potential for oxidative
damage.
In the present study we showed that under open-water conditions,
at a depth of 1m, exposure of S. neumayeri embryos to naturally
elevated levels of UV-B as a result of the presence of the Antarctic
ozone hole over the study site, greatly increased levels of oxidative
damage to proteins and lipids, and also increased the proportion of
UVT
abnormal embryos observed. At a depth of 4m increases in oxidative
damage from UV-B exposure also occurred, but the increases were
small compared with those observed at 1m and no increase in embryo
abnormality, which could be directly attributed to UV-B exposure,
was observed. This reduction in oxidative damage at increased depth
was most likely due to the attenuation of UV-B by sea water, for at
1m below the surface UV-B levels were 24.8% of surface levels
whereas at 4m UV-B levels were only 2.4% of surface levels. These
results support in part the findings of Karentz et al. (Karentz et al.,
2004), who found that at depths greater than 3m reduced or no UVB-induced embryo abnormality was evident and that DNA damage
in the form of CPDs was minimal. However, unlike Karentz et al.
(Karentz et al., 2004) who found that compared with visible light and
UV-A, UV-B had a relatively small impact on embryo abnormality
at depths less than 3m, the results of the present study support the
findings of numerous other studies that have shown that UV-B can
have a considerable impact on the embryos of sea urchins and other
marine larvae (Lesser et al., 2001; Browman et al., 2003; Lesser and
Barry, 2003; Lesser et al., 2003; Lesser et al., 2004; Lamare et al.,
2007). As abnormality can be the end result of exposure of a
developing embryo to several stressors, is not surprising that under
highly variable field conditions, such as those found in Antarctica,
the accumulation of cellular damage to a level sufficient to cause
abnormalities could be achieved in different ways depending on the
combined impacts of many environmental factors. For example,
Karentz et al. (Karentz et al., 2004) showed that abnormal embryo
morphology could be caused by exposure to 80kJm–2 of UV-B or
by exposure to more than 2000kJm–2 of UV-A, suggesting that under
conditions of very high UV-A the effects of UV-B could easily be
masked, as abnormal embryos would be observed in the presence or
absence of UV-B. The UV-A levels to which the embryos were
exposed in the present study were lower than those detailed for most
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Catalase (µmol
H2O2 mg–1 protein min–1)
Superoxide dismutase
(i.u. mg–1 protein min–1)
Sea ice protects sea urchins from UV-B
300
Open water
A
200
c
b
250
a
a
a
High ozone (26–30 Oct)
Low ozone (1–5 Nov)
a
a
150
a
a
a
a
a
a
a
a
100
50
0
250
B
200
150
D
c
b
a
a
a
a
a
a
a
100
50
0
UVO
UVA
UVT
UVO
UVA
of the experiments presented by Karentz et al. (Karentz et al., 2004),
hence the UV-A dose in the present study may have been insufficient
to cause enough cellular damage in the absence of UV-B to induce
abnormal embryo development. This is supported by the failure of
UV-A alone to induce significant amounts of oxidative damage.
To cope with ROS generation and to help prevent oxidative
damage animals have a number of antioxidant defences including
ROS scavenging molecules such as ascorbate, glutathione and tocopherol, and ROS detoxifying enzymes such as SOD and CAT
(Halliwell and Gutteridge, 1999). Animals adapted to low
temperatures often have increased antioxidant defences to enable
them to cope with the potential for increased mitochondrial ROS
production. In the present study two commonly used enzymatic
markers of ROS scavenging capacity, SOD and CAT, were used to
determine if S. neumayeri embryos are capable of increasing their
ROS scavenging capacity to protect themselves from UV-B-induced
oxidative damage. Our results clearly demonstrate that although S.
neumayeri embryos contain moderate to high levels of both SOD
Superoxide dismutase
(i.u. mg–1 protein min–1)
Fig.4. The influence of ambient UV-B levels and UV
treatment [UV-minus: (UVO), UVB-minus (UVA) and
UV-transparent (UVT)] on antioxidant enzyme
activities in Sterechinus neumayeri embryos held at
a depth of 1m. (A)superoxide dismutase activities
and (B) catalase activities in embryos in open water,
(C) superoxide dismutase activities and (D) catalase
activities in embryos under sea ice. Values are
means ± s.e.m., calculated from four replicates
(open water) or three replicates (under ice). ‘Open
water’ and ‘sea ice’ experiments were analysed
separately using two-way ANOVAs. Within each
graph, bars with the same letter are not significantly
different (P≤0.05).
Sea ice
C
300
Open water
A
200
a
a
High ozone (26–30 Oct)
Low ozone (1–5 Nov)
b
a
and CAT, when compared with the developing eggs and larvae of
other marine animals (Buchner et al., 1996; Rudneva, 1999; Korkina
et al., 2000; Regoli et al., 2002), that exposure to UV-B induces a
small but significant dose-dependent increase in both SOD and CAT
activities. Lesser et al. (Lesser et al., 2003) also found an increase
in SOD activity when the embryos of the sea urchin
Strongylocentrotus droebachiensis were exposed to UV-B. In the
present study although significant increases in the activities of
antioxidant enzymes were observed, the increases were insufficient
to completely protect embryos from oxidative damage to proteins
and lipids, especially at a depth of 1m. It is highly likely that this
increase in oxidative damage to these key macromolecules
contributed significantly to the increase in abnormality observed in
embryos exposed to UV-B at this depth.
Although studies have shown that the UV-A portion of the
spectrum can cause oxidative damage in animal cells (McMillan et
al., 2008), studies on marine animals have provided mixed results,
with some studies demonstrating negative effects (Adams and Schick,
Sea ice
C
b
250
UVT
a
b
150
a
a
b
a
b
100
50
0
Catalase (µmol
H2O2 mg–1 protein min–1)
1973
250
B
D
c
200
150
a
a
a
a
b
100
b
b
b
a
a
UVO
UVA
Fig.5. The influence of ambient UV-B levels and
UV treatment [UV-minus: (UVO), UVB-minus
(UVA) and UV-transparent (UVT)] on antioxidant
enzyme activities in Sterechinus neumayeri
embryos held at a depth of 4m (A) superoxide
dismutase activities and (B) catalase activities in
embryos in open water, (C) superoxide dismutase
activities and (D) catalase activities in embryos
under sea ice. Values are means ± s.e.m.,
calculated from four (open water) or three
replicates (under ice). ‘Open water’ and ‘sea ice’
experiments were analysed separately using twoway ANOVA’s. Within each graph, bars with the
same letter are not significantly different at a
significance level of P≤0.05.
a
50
0
UVO
UVA
UVT
UVT
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1974 K. N. Lister, M. D. Lamare and D. J. Burritt
1996; Lesser and Barry, 2003; Lesser et al., 2003; Karentz et al.,
2004) whereas others showed little effect at all (Béland et al., 1999;
Kouwenberg et al., 1999). In the present study no significant effects
due to exposure to UV-A radiation were observed. This could have
been due to the fact that the filters used to block UV-B also reduced
UV-A doses by over 50% and as UV-A levels were not significantly
influenced by the presence or absence of the ozone hole this treatment
effectively reduced ambient UV-A levels. Moreover, as S. neumayeri
embryos were shown in the present study to have moderate to high
basal levels of SOD and CAT activity and sea urchin embryos and
larvae have been shown in other studies to have antioxidant capacity
(Lesser et al., 2003; Lesser, 2006), it is highly likely that significant
levels of oxidative damage only occur in embryos when their basal
antioxidant capacity is greatly exceeded by ROS production, for
example when exposed to high levels of UV-B.
The results of the open water experiments detailed above clearly
demonstrate that naturally increased UV-B doses, due to seasonal
ozone depletion, cause oxidative damage in S. neumayeri embryos
under field conditions, despite a limited capacity of the embryos to
increase the activities of antioxidant enzymes. However, during the
Austral spring McMurdo Sound is normally covered with 2–3m of
annual sea ice and it is generally accepted that sea ice provides a
physical barrier to UV-B radiation reaching the underlying water
column (Perovich, 1993; Perovich, 2002). The results of the present
study suggest a protective role for sea ice by demonstrating that sea
urchin embryos held beneath almost 2m of sea ice were completely
protected from oxidative damage and UV-B-induced abnormality.
Moreover, no significant increases in SOD or CAT activities were
observed in animals protected by sea ice, indicating that no
upregulation of ROS scavenging capacity was required, or that ROS
levels did not increase sufficiently to trigger increases in SOD and
CAT activities. Although the upregulation of other antioxidant
mechanisms cannot be ruled out, changes in the activities of SOD or
CAT are commonly used as cellular markers of oxidative stress and
ROS scavenging capacity in most animals (Halliwell and Gutteridge,
1999) and have been shown to be upregulated in response to oxidative
stress in marine animals (Lesser, 2006). Despite the above results it
should be noted that Lesser et al. (Lesser et al., 2004) found UV-B
wavelengths reached a depth of over 6m after being transmitted
through approximately 3m of annual sea ice in McMurdo Sound.
Furthermore, they observed mortality and DNA damage in sea urchin
embryos exposed to UV radiation beneath the ice over two consecutive
years and that higher mortality and DNA damage were associated
with greater irradiances of UV-B. There are many possible reasons
for the difference between the Lesser et al. (Lesser et al., 2004) study
and the present study. For example, seasonal variations in the levels
of screening pigments in maternal parents, which are dependent on
food abundance and quality, could influence the levels of UV-B
screening pigments passed on to the embryos, fluctuations in ozone
concentrations and cloud cover during experiments, variations in ice
thickness and optical properties (reviewed by Perovich, 2002), the
thickness of snow cover and water transmission characteristics, which
are highly variable and constantly changing on both a daily and
seasonal basis, making direct comparisons between studies difficult.
Irrespective of the above, both studies did show that ice reduces the
negative effects of UV-B on S. neumayeri embryos and therefore plays
an important protective function.
As mentioned previously, most studies on the influences of UVB on S. neumayeri embryos have concentrated on direct DNA damage,
in the form of CPDs, as a measure of the influence of UV-B exposure
at the cellular level and microscopic observations to determine the
levels of embryo abnormality. Although CPDs are a sensitive marker
for exposure to UV-B, because DNA dimers such as CPDs are
produced at high levels in cells exposed to UV-B, most organisms
have evolved very effective mechanisms to repair them (Sinha and
Hader, 2002). Unlike DNA dimers, which can be reversed effectively
by photoreactivation, the overproduction of ROS caused by exposure
of an organism to UV-B can have far more wide ranging
consequences. Oxidative damage to DNA, proteins and lipids is both
energetically expensive to prevent and potentially very resource
intensive to repair (Halliwell and Gutteridge, 1999; Sinha and Hader,
2002).
The cost of prevention and/or repair of oxidative damage could be
a very important life history trade-off for organisms living in the
Antarctic marine ecosystem. Moreover, S. neumayeri embryos require
20 days of development after fertilisation before the resultant larvae
are capably of feeding (Bosch et al., 1987) during which time they
are totally dependent on stored reserves for growth and development.
Thus any relocation of resources from growth and development to
the prevention or repair of oxidative damage could significantly
influence larval fitness and/or survivability. In response to increased
ROS levels, developing embryos could be required to invest valuable
cellular resources to prevent oxidative damage, but this is likely to
have consequences for other important traits (Monaghan et al., 2009).
In conclusion, basal levels of oxidative damage and antioxidant
enzyme activities in S. neumayeri embryos were within the range
of values published for other marine invertebrates. In embryos
exposed to UV-B under open water conditions antioxidant enzyme
activities increased, but not enough to prevent a significant increase
in oxidative damage and developmental abnormalities. The amount
of oxidative damage was dependent upon ambient UV-B levels and
increased as the ozone hole moved over the study site. Embryos
protected from UV-B by sea ice showed little oxidative damage
with few developmental abnormalities, directly attributable to UVB. Our results suggest that normal development of sea urchin
embryos could require the presence of sea ice, which acts as a UVB screening filter, protecting the embryos from potentially damaging
increases in UV-B. As embryos represent a key life-history stage,
lower survival rates caused by increased UV-B as a result of the
ozone hole and significant reductions in protective sea ice coverage
brought about by global warming, could reduce the long-term
viability of affected populations and potentially have a significant
impact on the sensitive Antarctic marine ecosystem. Sea urchin
embryos could therefore be an ideal indicator species for the potential
impact of climate change on Antarctic marine invertebrates.
ACKNOWLEDGEMENTS
We especially thank Antarctica New Zealand for logistical support over two field
seasons at Scott Base and Kelly Tarlton’s Antarctic Encounter and Underwater
World for financial support via a postgraduate scholarship. Thanks are also
extended to staff at the Portobello Marine Laboratory for assistance during
research. Thank you to the members of K-068: Nickolas Isley, Dana Clark,
Michael Gonsior and Michelle Liddy (both in the 07/08 and 08/09 seasons) and K065: Dr Steve Wing, Rebecca Macleod, Brian Grant, Greg Funnell and Jim) for
help with field-work in Antarctica. Financial support to write this manuscript was
provided by a University of Otago Publishing Bursary.
REFERENCES
Adams, N. L. and Shick, J. M. (1996). Mycosporine-like amino acids provide protection
against ultraviolet radiation in eggs of the green sea urchin, Strongylocentrotus
droebachiensis. Photochem. Photobiol. 64, 149-158.
Adams, N. L. and Shick, J. M. (2001). Mycosporine-like amino acids prevent UV-B
induced abnormalities during early development of the green sea urchin,
Strongylocentrotus droebachiensis. Mar. Biol. 138, 267-280.
Arrigo, K. R. (1994). Impact of ozone depletion on phytoplankton growth in the Southern
Ocean: large-scale spatial and temporal variability. Mar. Ecol. Prog. Ser. 114, 1-12.
Balis, D., Bojkov, R., Tourpali, K. and Zerefos, K. (2009). Characteristics of the ozone
decline over both hemispheres. Int. J. Remote Sens. 30, 3887-3895.
Banowetz, G. M., Dierksen, K. P., Azevedo, M. D. and Stout, R. (2004). Microplate
quantification of plant leaf superoxide dismutases. Anal. Biochem. 332, 314-320.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Sea ice protects sea urchins from UV-B
Béland, F., Browman, H. I., Rodriguez, C. A. and St-Pierre, J. F. (1999). Effect of
solar ultraviolet radiation (280-400nm) on the eggs and larvae of Atlantic cod (Gadus
morhua). Can. J. Fish. Aquat. Sci. 56, 1058-1067.
Bidigare, R. R. (1989). Potential effects of UV-B radiation on marine organisms of the
Southern Ocean: distributions of phytoplankton and krill during austral spring.
Photochem. Photobiol. 50, 469-477.
Bosch, I., Beauchamp, K. A., Steele, M. E. and Pearse, J. S. (1987). Development,
metamorphosis, and seasonal abundance of embryos and larvae of the Antarctic seaurchin Sterechinus neumayeri. Biol. Bull. 173, 126-135.
Browman, H. I., St-Pierre, J. F. and Kuhn, P. (2003). Dose and dose-rate dependency
in the mortality response of Calanus finmarchicus embryos exposed to ultraviolet
radiation. Mar. Ecol. Prog. Ser. 247, 297-302.
Brey, T. (1991). Population dynamics of Sterechinus antarcticus (Echinodermata:
Echinoidea) on the Weddell Sea shelf and slope, Antarctica. Antarc. Sci. 3, 251-256.
Brey, T., Pearse, J., Basch, L., McClintock, J. and Slattery, M. (1995). Growth and
production of Sterechinus neumayeri (Echinoidea: Echinodermata) in McMurdo sound,
Antarctica. Mar. Biol. 124, 279-292.
Buchner, T., Abele-Oeschger, D. and Theede, H. (1996). Aspects of antioxidant status
in the polychaete Arenicola marina: tissue and subcellular distribution, and reaction to
environmental hydrogen peroxide and elevated temperatures. Mar. Ecol. Prog. Ser.
143, 141-150.
Burritt, D. J. (2008). The polycyclic aromatic hydrocarbon phenanthrene causes
oxidative stress and alters polyamine metabolism in the aquatic liverwort Riccia fluitans
L. Plant Cell Environ. 31, 1416-1431.
Burritt, D. J. and MacKenzie, S. (2003). Antioxidant metabolism during the acclimation
of Begonia x erythrophylla to high-light. Ann. Bot. 91, 783-794.
Cooper, W. J., Shao, C. W, Lean, D. R. S., Gordon, A. S. and Scully, F. E. (1994).
Environmental chemistry of lakes and reservoirs. In Advances in Chemistry Series,
vol. 237 (ed. L. A. Baker), pp. 391-422. Washington, DC: ACS Publications.
Cullen, J. J. and Neale, P. J. (1997). Effects of ultraviolet radiation on short-term
photosynthesis of natural phytoplankton. Photochem. Photobiol. 65, 264-266.
Dunlap, W. C., Shick, J. M. and Yamamoto, Y. (2000). UV protection in marine
organisms. 1. Sunscreens, oxidative stress and antioxidants. In Free Radicals in
Chemistry, Biology and Medicine (ed. T. Yoshikawa, S. Toyokuni, Y. Yamamoto and
Y. Naito). London: OICA International.
Fryer, H. J. L., Davis, G. E., Manthorpe, M. and Varon, S. (1986). Lowry protein assay
using an automatic microtitre plate spectrophotometer. Anal. Biochem. 153, 262-266.
Guderley, H. (2004). Metabolic responses to low temperature in fish muscle. Biol. Rev.
79, 409-427.
Häder, D., Kumar, H. D., Smith, R. C. and Worrest, R. C. (2003). Aquatic ecosystems:
effects of solar ultraviolet radiation and interactions with other climatic change factors.
Photochem. Photobiol. Sci. 2, 39-50.
Häder, D., Kumar, H. D., Smith, R. C. and Worrest, R. C. (2007). Effects of solar
radiation on aquatic ecosystems and interactions with climate change. Photochem.
Photobiol. Sci. 6, 267-285.
Halliwell, B. and Gutteridge, J. M. C. (1999). Free Radicals in Medicine and Biology,
Edition 3. Oxford: Oxford University Press.
Hazel, J. R. (1995). Thermal adaptation in biological-membranes-is homeoviscous
adaptation the explanation. Annu. Rev. Physiol. 57, 19-42.
Hoegh-Guldberg, O. H., Welborn, J. R. and Manahan, D. T. (1991). Metabolic
requirements of Antarctic and temperate asteriod larvae. Antarct. J. 26, 163-165.
Janssens, B. J., Childress, J. J., Baguet, F. and Rees, J. F. (2000). Reduced
enzymatic antioxidative defense in deep-sea fish. J. Exp. Biol. 203, 3717-3725.
Johnsen, S. and Widder, E. A. (2001). Ultraviolet absorption in transparent zooplankton
and its implications for depth distribution and visual predation. Mar. Biol. 138, 717-730.
Karentz, D. (1991). Ecological considerations of Antarctic ozone depletion. Antarc. Sci.
3, 3-11.
Karentz, D., Bosch, I. and Mitchell, M. (2004). Limited effects of Antarctic ozone
depletion on sea urchin development. Mar. Biol. 145, 277-292.
Korkina, L. G., Deeva, I. B., De Biase, A., Iaccarino, M., Oral, R., Warnau, M. and
Pagano, G. (2000). Redox-dependent toxicity of diepoxybutane and mitomycin C in
sea urchin embryogenesis. Carcinogenesis 21, 213-220.
Kouwenberg, J. H. M., Browman, H. I., Runge, J. A., Cullen, J. J., Davis, R. F. and
St-Pierre, J. F. (1999). Biological weighting of ultraviolet (280-400nm) induced
mortality in marine zooplankton and fish. II. Calanus finmarchicus (Copepoda) eggs.
Mar. Biol. 134, 285-293.
Lamare, M. D., Barker, M. F., Lesser, M. P. and Marshall, C. (2006). DNA photorepair
in echinoid embryos: effects of temperature on repair rate in Antarctic and nonAntarctic species. J. Exp. Biol. 209, 5017-5028.
Lamare, M. D., Barker, M. F. and Lesser, M. P. (2007). In situ rates of DNA damage
and abnormal development in Antarctic and non-Antarctic sea urchin embryos. Aqua.
Biol. 1, 21-32.
Lesser, M. P. (2006). Oxidative stress in marine environments: biochemistry and
physiological ecology. Annu. Rev. Physiol. 68, 253-278.
Lesser, M. P. and Barry, T. M. (2003). Survivorship, development and DNA damage in
echinoderm embryos and larvae exposed to ultraviolet radiation (290-400nm). J. Exp.
Mar. Biol. Ecol. 292, 75-91.
Lesser, M. P., Farrell, J. H. and Walker, C. W. (2001). Oxidative stress, DNA damage
and p53 expression in the larvae of Atlantic Cod (Gadus morhua) exposed to
ultraviolet (290-400 nm) radiation. J. Exp. Biol. 204, 157-164.
Lesser, M. P., Kruse, V. A. and Barry, T. M. (2003). Exposure to ultraviolet radiation
causes apoptosis in developing sea urchin embryos. J. Exp. Biol. 206, 4097-4103.
1975
Lesser, M. P., Lamare, M. D. and Barker, M. F. (2004). Transmission of ultraviolet
radiation through biological effects on sea urchin embryos. Limnol. Oceanogr. 49,
1957-1963.
Madronich, S., McKenzie, R. L., Björn, L. O. and Caldwell, M. M. (1998). Changes in
biologically active radiation reaching the Earth’s surface. Photochem. Photobiol. 45, 519.
Malloy, K. D., Holman, M. A., Mitchell, D. and Detrich, W. H., III (1997). Solar UVBinduced DNA damage and photoenzymatic repair in Antarctic zooplankton. Proc. Natl.
Acad. Sci. USA 94, 1258-1263.
Maral, J., Puget, K. and Michelson, A. M. (1977). Comparative-study of superoxidedismutase, catalase and glutathione peroxidase levels in erythrocytes of different
animals. Biochem. Biophys. Res. Commun. 77, 1525-1535.
McKenzie, R. L., Bjorn, L. O., Bais, A. F. and Ilyas, M. (2003). Changes in
biologically active ultraviolet radiation reaching the Earth’s surface. Photochem.
Photobiol. Sci. 2, 5-15.
McKenzie, R. L., Aucamp, P. J., Bais, A. F., Björn, L. O. and Ilyas, M. (2007).
Changes in biologically active ultraviolet radiation reaching the Earth’s surface.
Photochem. Photobiol. Sci. 6, 218-231.
McMillan, T. J., Leatherman, E., Ridley, A., Shorrocks, J., Tobi, S. E. and Whiteside,
J. R. (2008). Cellular effects of long wavelength UV light (UVA) in mammalian cells. J.
Pharm. Pharmacol. 60, 969-976.
Mihaljevic, B., Katusin-Razem, B. and Razem, D. (1996). The re-evaluation of the
ferric thiocyanate assay for lipid hydroperoxides with special considerations of the
mechanistic aspects of the response. Free Radical Biol. Med. 21, 53-63.
Monaghan, P., Metcalfe, N. B. and Torres, R. (2009). Oxidative stress as a mediator of
life history trade-offs: mechanisms, measurements and interpretation. Ecol. Lett. 12,
75-92.
Newman, P. A., Nash, E. R., Kawa, S. R., Montzka, S. S. and Schauffler, S. M.
(2006). When will the Antarctic ozone hole recover? Geophys. Res. Lett. 33, LI2814.
Pace, D. A. and Manahan, D. T. (2006). Fixed metabolic costs for highly variable
rates of protein synthesis in sea urchin embryos and larvae. J. Exp. Biol. 209, 158170.
Pearse, J. S., McClintock, J. B. and Bosch, I. (1991). Reproduction of Antarctic benthic
marine invertebrates: tempos, modes and timing. Am. Zool. 31, 65-80.
Perovich, D. K. (1993). A theoretical model of ultraviolet light transmission through
Antarctic sea ice. J. Geophys. Res. 98, 22579-22587.
Perovich, D. K. (2002). Ultraviolet radiation and the optical properties of sea ice and
snow. In UV Radiation and Arctic Ecosystems, Ecological Studies, vol. 153 (ed. D.
Hessen), pp. 73-89. Berlin Heidelberg: Springer-Verlag.
Pörtner, H. O., Peck, L. and Somero, G. (2007). Thermal limits and adaptation in
marine Antarctic ectotherms: an integrative view. Philos. Trans. R. Soc. Lond. B. Biol.
Sci. 362, 2233-2258.
Regoli, F., Nigro, M., Chiantore, M. and Winston, G. M. (2002). Seasonal variations of
susceptibility to oxidative stress in Adamussium colbecki, a key bioindicator species for
the Antarctic marine environment. Sci. Total Environ. 289, 205-211.
Reznick, A. Z. and Packer, L. (1994). Oxidative damage to proteins –
spectrophotometric method for carbonyl assay. In Oxygen Radicals in Biological
Systems Part C, Methods in Enzymology, vol. 233 (ed. J. N. Abelson, M. I. Simon and
H. Sies) pp. 357-363. San Diego, CA: Elsevier Science and Technology Books.
Richard, L. E., Peake, B. M., Rusak, S. A., Cooper, W. J. and Burritt, D. J. (2007).
Production and decomposition dynamics of hydrogen peroxide in freshwater. Environ.
Chem. 4, 49-54.
Rudneva, I. I. (1999). Antioxidant system of Black Sea animals in early development.
Comp. Biochem. Physiol. 122C, 265-271.
Shepherd, A. and Wingham, D. (2007). Recent sea-level contributions of the Antarctic
and Greenland ice sheets. Science 315, 1529-1532.
Shick, J. M. and Dunlap, W. C. (2002). Mycosporine-like amino acids and related
gadusols: biosynthesis, accumulation, and UV-protective functions in aquatic
organisms. Annu. Rev. Physiol. 64, 223-262.
Shilling, F. M. and Manahan, D. T. (1994). Energy-metabolism and amino-acid-transport
during early development of Antarctic and temperate echinoderms, Biol. Bull. 187, 398407.
Sinha, R. P. and Hader, D. P. (2002). UV-induced DNA damage and repair: a review.
Photochem. Photobiol. Sci. 1, 225-236.
Smith, R. C., Prezelin, B., Baker, K. S., Bidigare, R. R., Boucher, N. P., Coley, T.,
Karentz, D., MacIntyre, S., Matlick, H. A., Menzies, D. et al. (1992). Ozone
depletion: ultraviolet radiation and phytoplankton biology in Antarctic waters. Science
255, 952-959.
Sohal, R. S. and Weindruch, R. (1996). Oxidative stress, caloric restriction, and aging.
Science 273, 59-63.
Tedetti, M. and Sempéré, R. (2006). Penetration of ultraviolet radiation in the marine
environment: a review. Photochem. Photobiol. 82, 389-397.
Trodahl, H. J. and Buckley, R. G. (1989). Ultraviolet levels under sea ice during the
Antarctic spring. Science 245, 194-195.
Turner, J. and Overland, J. (2009). Contrasting climate change in the two polar regions.
Polar Res. 28, 146-164.
Velicogna, I. and Wahr, J. (2006). Measurements of time-variable gravity show mass
loss in Antarctica. Science 31, 1754-1756.
Viarengo, A., Abele-Oeschger, D. and Burlando, B. (1998). Effects of low temperature
on prooxidant processes and antioxidant defense systems in marine organisms. In
Cold Ocean Physiology (ed. H. O. Pörtner and R. C. Playle), pp. 212-238.
Cambridge: Cambridge University Press.
THE JOURNAL OF EXPERIMENTAL BIOLOGY