J. Phycol. 37, 200–208 (2001)
DEPTH DISTRIBUTIONS OF DNA DAMAGE IN ANTARCTIC MARINE PHYTOAND BACTERIOPLANKTON EXPOSED TO SUMMERTIME UV RADIATION 1
Anita G. J. Buma,2 M. Karin de Boer, and Peter Boelen
Department of Marine Biology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
During a survey from January to March 1998, the
occurrence of UV-B radiation (UVBR)-induced DNA
damage in Antarctic marine phytoplankton and bacterioplankton was investigated. Sampling was done
in Ryder Bay, off the British base Rothera Station,
67⬚S, 68⬚W (British Antarctic Survey). Samples were
taken regularly during the survey period at fixed
depths, after which DNA damage was measured in
various plankton size fractions (⬎10, 2–10, and 0.2–
2 ␮m). Incident solar radiation was measured using
spectroradiometry, whereas attenuation of biologically effective UVBR was studied using a DNA dosimeter. A diatom bloom was found in the bay during the research period, judging from microscopic
observations and HPLC analyses of taxon-specific
pigments. The high phytoplankton biomass likely
caused strong attenuation of DNA effective UVBR
(Kbd-eff). Kbd-eff values ranged from 0.83⭈m⫺1 at the
peak of the bloom to 0.47⭈m⫺1 at the end of the season. UVBR-mediated DNA damage, as measured by
cyclobutane pyrimidine dimer (CPD) abundance,
was detected in all plankton size fractions. Highest
levels were found in the smallest size fraction,
mainly consisting of heterotrophic bacteria. Clear
CPD depth profiles were found during mid-summer
(January, beginning of February) with surface levels
exceeding 100 CPDs per million nucleotides in the
bacterioplankton fraction. At that time, melting of
the continuously present shelf ice caused strong salinity gradients in the upper meters, thereby stimulating water column stabilization. At the end of February and beginning of March, this phenomenon was
less pronounced or absent. At that time, DNA damage was homogeneously distributed over the first 10 m,
ranging between 20 and 30 CPDs per million nucleotides for the smallest size fraction.
There is unequivocal evidence that ultraviolet radiation (UVR: 280–400 nm) negatively affects marine
Antarctic organisms, in particular the primary producers (Voytek 1990). Antarctic phytoplankton and
ice algae form the first level of Antarctic marine
trophic structures by virtue of their capacity to use solar energy for photosynthesis. Because these organisms require visible light for growth, they will commonly be confronted with other potentially harmful
components of the solar spectrum: UV-A radiation
(UVAR: 315–400 nm) and UV-B radiation (UVBR:
280–315 nm). As a result of springtime ozone reduction in Antarctic regions and the resultant increases
in UVBR, there has been overwhelming interest in the
possible effects of (increased) UVBR on Antarctic marine primary productivity. Field experiments have
shown that even unaffected levels of UVBR lower the
performance of phytoplankton communities. Almost
all studies carried out so far, using short-term 14C
incorporation experiments, reveal that both UVBR
and UVAR reduce phytoplankton primary production
(Smith et al. 1992, Helbling et al. 1994, Neale et al. 1994,
Prezelin et al. 1994, 1998, Boucher and Prezelin 1996, McMinn et al. 1999). On top of naturally occurring UVR
stress, enhanced UVBR as a result of springtime ozone
reduction is estimated to cause an additional inhibition
of water column productivity between 6.7% in September and 2.4% in December (Helbling et al. 1994).
There has been considerable discussion in the literature as to the molecular target sites primarily affected
by UVR. It has been demonstrated for Antarctic ice algae that PSII efficiency (Kroon et al. 1994, Schofield
et al. 1995) or the RUBISCO pool (Lesser et al. 1996)
may be modified. A reduction in the performance of
both targets will decrease the ability of a cell to photosynthesize, thereby hindering the carboxylation process. Also the possibility of UVBR-induced DNA damage has been suggested (Karentz et al. 1991, Karentz
1994). A typical effect of UVBR is the dimerization of
certain DNA bases, leading to the formation of cyclobutane pyrimidine dimers (CPDs), such as TT, CC,
and TC dimers. The most abundant type of CPD as a
result of UVBR exposure is cyclobutane thymine
dimers (TT). CPDs may arrest the cell cycle in the
DNA synthesis phase unless the damage is quickly repaired. Meanwhile, damage will obstruct de novo synthesis of cellular components and substances required
for growth and cell maintenance by replication inhibition. There are several ways in which DNA damage can
be repaired, one of which is photoreactivation. This repair is known to be controlled by light in the UVARPAR region (330–450 nm) (Sancar and Sancar 1988).
Key index words: Antarctic; bacterioplankton; CPDs;
DNA damage; phytoplankton; solar radiation; UVAR;
UVBR; UV radiation
Abbreviations: CPD, cyclobutane pyrimidine dimer;
UVBR, UV-B radiation (280–315 nm); UVR, UV radiation (280–400 nm); UVAR, UV-A radiation (315–
400 nm); Kbd-eff, attenuation coefficient for DNA effective UVBR; TT, thymine dimer
1 Received
2 Author
18 January 2000. Accepted 15 January 2001.
for correspondence: e-mail [email protected].
200
201
DNA DAMAGE IN ANTARCTIC MICROPLANKTON
Photoreactivation has been demonstrated to occur in
marine viruses (Weinbauer et al. 1997, Wilhelm et al.
1998), whereas additional dark repair was found in marine bacteria (Jeffrey et al. 1996a, P. Boelen, M. J. W.
Veldhuis, and A. G. J. Buma, unpublished data).
Over the past years much information has become
available on UVBR-related DNA damage accumulation in marine organisms. CPDs have been detected
in situ in tropical marine bacteria (Jeffrey et al. 1996a,b,
Visser et al. 1999, Boelen et al. 2000), marine tropical
picophytoplankton (Boelen et al. 2000, P. Boelen,
M. J. W. Veldhuis, and A. G. J. Buma, unpublished
data ), viruses (Weinbauer et al. 1999), and fish eggs
and larvae (Vetter et al. 1999). In the Antarctic, CPDs
are shown to be induced in situ in ice algae and phytoplankton (Prezelin et al. 1998) and in several developmental stages of ice fish (Malloy et al. 1997). Whenever
the damage formation rate exceeds the damage removal rate, damage accumulates. It is striking that,
also in (sub)tropical planktonic organisms, DNA damage accumulates in the surface over the day (Jeffrey et
al. 1996a,b, Weinbauer et al. 1999), indicating that repair mechanisms are not sufficient to undo the damage during UVBR exposure hours. This is despite the
expected adjustment of these organisms to the high
ambient UVR levels. Divergence between damage induction rate and repair rate would particularly be an
issue in Antarctic organisms. Enzyme-based mechanisms are generally temperature dependent, whereas
damage induction is not. Therefore, it can be hypothesized that DNA damage accumulates in Antarctic organisms, because slow repair cannot keep up with the
accumulation of damage.
The aim of the present study was to collect baseline
information on DNA damage levels in various size
classes of Antarctic marine phyto- and bacterioplankton in order to study the possibility of DNA as a UVBR
target site in situ. Also, investigating the various size
components of the plankton would allow us to increase our understanding of how UVBR influences
trophic dynamics and food web composition in these
Antarctic waters. To this end, depth profiles of DNA
damage were measured in several size fractions over the
course of nearly 3 months. Simultaneously, phytoplankton species composition was monitored, as well as some
physical and chemical parameters. Incident irradiances
of the various wavelength bands were studied using
spectroradiometry, and attenuation of DNA effective
UVBR was measured using a DNA dosimeter (Boelen
et al. 1999).
Fig. 1.
The Rothera research area.
202
ANITA G. J. BUMA ET AL.
Table 1. Sampling dates for the depth profiles, meteorological
conditions, and surface salinity (‰).
Date (Julian day)
January 20 (20)
January 28 (28)
January 31 (31)
February 1 (32)
February 27 (58)
March 4 (63)
Weather
Salinity
(‰)
Wind speed
(m⭈s⫺1)
Air
temperature
(⬚ C)
Few clouds
Cloudy
Sunny
Sunny
Cloudy
Few clouds
30.6
31.7
31.1
30.2
32.4
32.5
4.5
4.0
2.4
2.8
9.5
10.7
5.9
2.5
3.5
3.2
⫺0.3
⫺0.1
Weather, cloud conditions during morning hours; Wind speed,
average daily wind speed (m·s⫺1); Air temperature, maximum
daily temperature (⬚ C).
materials and methods
The survey was carried out in Ryder Bay, 67 ⬚.34⬘S, 68⬚.08⬘W,
near the British base Rothera Station (British Antarctic Survey,
BAS, Fig. 1) located on Adelaide Island, Antarctica, between
January 17 and March 4, 1998. Air temperature and wind speed
were recorded at 6-h intervals by a Modular Automatic Weather
Station (Meteorology Section, BAS) at Rothera Point. To study
incident UVBR, UVAR, and PAR, a spectroradiometer (Bentham
Instruments Ltd., Reading, UK) was positioned on the roof of
the Rothera Base laboratory. Spectra were recorded at 1-nm intervals on an hourly basis during daytime. Due to technical
problems, the spectroradiometer was not operative between Jan-
uary 20 and February 8, 1998. Calibration of the spectroradiometer was done using a mercury lamp for wavelength calibration
and a 1-kW FEL lamp for irradiance calibration.
Attenuation of biologically effective UVBR was studied following Boelen et al. (1999) on five occasions during the research
period. Quartz tubes filled with DNA solution (1 ␮g⭈mL⫺1) were
incubated in situ at 0, 2.5, 5, and 10 m and harvested after 24 h
to be analyzed for DNA damage (Boelen et al. 1999). Biologically effective attenuation coefficients (K bd-eff) were calculated
from linear regressions of natural logarithmic CPDs per million
nucleotides against depth using the log-linear part of the curve.
Depth profiles were taken six times during the research period on sunny and cloudy days (Table 1). Water samples were
taken in the bay around noon at 0, 5, and 10 m and transported
directly to the laboratory facilities. Surface samples were taken
in duplicate, whereas 5 m and 10 m were single samplings. Between 1 and 3 L of water was size fractionated over Poretics
membrane filters (Osmonics, Livermore, CA) of various pore
sizes (0.2, 2, and 10 ␮m). Filtrations were done in the dark at
4⬚ C. Then filters were frozen at ⫺20⬚ C until further analysis in
the home laboratory (see below). Samples for phytoplankton
species composition (50 mL) were fixed with formalin (0.5% final concentration), stored at 4⬚ C, and analyzed at home.
DNA dosimeter incubations were accompanied by sampling
for phytoplankton pigment composition (HPLC) and yellow
substance (colored dissolved organic matter [DOM]) measurements. Surface samples (10 L) were filtered onto GF/F Whatman
filters (Whatman, Clifton, NJ) and stored frozen until analysis in
the home laboratory. From the filtrate, 50 mL was stored at 4 ⬚ C
until spectrophotometric analysis of yellow substance content was
performed. Salinity was measured in the samples for phytoplank-
Fig. 2. (A) Incident irradiance conditions and
(B) mean wind speed (m⭈s⫺1) and surface salinity during the research period. PAR (400–700 nm), UVAR
(315–400 nm), and UVBR (280–315 nm) were maximum noontime values, as measured with the Bentham
spectroradiometer.
DNA DAMAGE IN ANTARCTIC MICROPLANKTON
ton floristic analysis, using a ProfiLab Conductivity meter (model
LF 597-S).
DNA was extracted from the filters as described before (Boelen
et al. 1999). In brief, filters were incubated at 60⬚ C for 30 min with
cetyltrimethylammonium bromide (CTAB) isolation buffer (2%
[w/vol] CTAB [Sigma], 1.4 M NaCl, 0.2% [vol/vol] ␤-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0). After chloroform/isoamylalcohol purification, the DNA was precipitated
with isopropanol and subsequently washed with 80% ice-cold
ethanol. The DNA was dried under vacuum and resuspended in
TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). To remove
RNA, the extracts were incubated for 1 h with 75 ␮g⭈mL⫺1
RNAse (Boehringer Mannheim) at room temperature. The
amount of DNA was determined fluorometrically using Picogreen dsDNA quantitation reagent (dilution 1:400, Molecular
Probes, Eugene, OR) on a 1420 Victor multilabel counter (EG ␥
Wallac, excitation 485 nm, emission 535 nm) using a standard
calibration series of calf thymus DNA (Sigma).
The amount of CPDs in DNA was determined using the H3
antibody (Roza et al. 1988) applied in an immuno-dot blot procedure slightly modified after Boelen et al. (1999). The H3 antibody was raised against cyclobutane thymine dimers (TT) but
also had a high affinity for 5⬘TC dimers. Heat-denaturated
DNA samples (100 ng) were slot-blotted (Minifold I SRC96D
slotblot apparatus; Schleicher & Schuell, Keene, NH) onto nitrocellulose filters (Protran BA79, pore size 0.1 ␮m, Schleicher
& Schuell). To immobilize the DNA, the filters were baked at
80⬚ C for 1 h, after which they were incubated in 5% (w/vol)
skimmed-milk powder in PBS-T (PBS [137 mM NaCl, 2.7 mM
KCl, 4.3 mM Na2HPO4.7H2O, 1.4 mM KH2PO4] ⫹ 0.1% [vol/
vol] Tween 20 [Sigma]) for 30 min at room temperature. During the whole procedure, filters were shaken gently and washed
three times (10 min) with PBS-T between incubation steps. The
filters were incubated over night at 4 ⬚ C with the H3 antibody
(0.12 ␮g⭈mL⫺1 in 0.5% milk powder–PBS-T) followed by incubation with the secondary antibody rabbit-anti-mouse-horseradish peroxidase (Dako Corp., Carpinteria, CA) (diluted
1:2000 in 0.5% milk powder–PBS-T) for 2 h at room temperature. Then the filters were transferred to 15 mL ECL detection reagents (Amersham) for 1 min and sealed in Photogene development folders (GIBCO BRL, Basel, Switzerland). Photographic
films (Amersham Hyperfilm ECL) were exposed to the filters
for 1 to 30 min. The films were developed, scanned, and analyzed as described before (Boelen et al. 1999). CPDs were quantified by comparing sample DNA with a dilution series of damaged standard DNA. The amount of CPDs in the standard DNA
was determined by calibrating it against DNA isolated from irradiated HeLa cells, kindly provided by Dr. Len Roza. The
amount of CPDs in this DNA was determined by means of
HPLC (Roza et al. 1988). The detection limit of the CPD assay
was ⬍1 CPD⭈10⫺6 nucleotides⫺1. All measurements were done
in duplicate.
Pigments were measured with HPLC. In short, pellets were
extracted overnight in 1–5 mL acetone (90%). Extracts were filtered over Whatman GF/F filters, after which 20–100 ␮L was injected into a Kratos HPLC system equipped with an RPC18 column. Expansion of peaks was done using a reversed-phase
gradient elution method as described in Buma et al. (1996b).
Detection of peaks was done at 436 nm with an LKB 2141 detector after which peaks were integrated using Nelson software
(Perkin Elmer, Nelson Systems, Inc.). Floristic composition of
the phytoplankton was followed using an inverted microscope
after additional fixing/coloring by modified Lugol’s solution.
Yellow substance was measured in a Varian spectrophotometer (Cary, 3E, UV/vis, Varian Instruments, San Francisco, CA)
in a 10-cm quartz cuvette after which absorbance at 440 nm was
measured. Statistical data analysis used paired two-tailed t-tests
for means (P ⬍ 0.05).
results
Maximum midday levels of UVBR, UVAR, and PAR
decreased during the research period. On cloudy days,
203
Fig. 3. Daily courses of PAR (400–700 nm), UVAR (315–
400 nm), and UVBR (280–315 nm) as measured on two sunny
days during the survey period. 䊉, February 8; 䊐, March 3.
levels of all three wavelength bands were strongly reduced (Fig. 2A). Highest UVBR levels were found in
January and the beginning of February, reaching dose
rates of around 1 W⭈m⫺2. As the season progressed,
roughly a 50% decrease in irradiance (PAR, UVAR,
UVBR) was observed. During the course of the season,
maximum daytime air temperatures dropped from 4
to 6⬚ C in January to temperatures close to zero in
March (Table 1). Generally, average wind speeds were
lower in January than in February and March (Fig. 2B,
Table 1). The weather in the bay area was further
characterized by a high daily variation in mean daily
wind speed during the whole research period (Fig.
2B). Due to the continuous presence of melting shelf
ice in the bay, surface salinity was highly variable. Lowest levels were found, however, in January and the beginning of February (Fig. 2B). Finally, daily courses of
PAR, UVBR, and UVAR, monitored on sunny days
204
ANITA G. J. BUMA ET AL.
(Fig. 3, i.e. February 8 and March 3), further demonstrated the strong reduction in incident irradiance
with the progression of the season. Highest dose rates
were found around 1300 h, when samples for the
depth profiles were taken.
Phytoplankton biomass in the bay was high throughout the season, with surface chl a levels between 1
␮g⭈L⫺1 and 15 ␮g⭈L⫺1 (Fig. 4). Occasionally, low biomass levels were observed, when sampling was done in
waters with low surface salinity (⬍30.5%0, day 30, Fig.
4). The phytoplankton were composed mainly of diatoms: the most abundant genera were Thalassiosira,
Fragillariopsis, Pseudonitzschia, and Eucampia. The dominance of diatoms was confirmed by the presence of
high levels of the diatom-specific pigments fucoxanthin
and diadino/diathoxanthin (Fig. 4). Taxon-specific
pigments of prasinophytes (prasinoxanthin, chl b),
prymnesiophytes (hexanoyloxyfucoxanthin, butanoyloxyfucoxanthin), and other phytoplankton classes were
present in traces only, indicating that these taxonomic groups contributed little to total phytoplankton biomass (Fig. 4). For example, Prasinophyceae would
constitute only 3% of total phytoplankton biomass in
terms of chl a, when assuming a mean chl b :chl a ratio of
0.75 as found for marine Antarctic Prasinophyceae
(Buma et al. 1992).
DNA effective radiation was rapidly attenuated in
the water column (Fig. 5). Attenuation coefficients
ranged from 0.47 m⫺1 in March to 0.83 m⫺1 in the beginning of February (Table 2). Due to this rapid attenuation, 1% depths ranged between 5.4 m and 9.6 m. At
the same time, yellow substance content was low with
absorption coefficients between 0.041 and 0.183 (m⫺1).
Kbd-eff correlated reasonably well with surface chl a concentrations (Table 2), resulting in the following (lin-
Fig. 4. Surface pigment distribution during the survey period, as measured with HPLC. Vertical lines, SD of the mean.
ear) relation: Kbd-eff ⫽ 0.025 ⫻ chl a ⫹ 0.393 (n ⫽ 5, R 2 ⫽
0.8796).
DNA damage was observed in all size fractions of
the (bacterio)plankton. CPD levels ranged from ⬍3
CPDs⭈10⫺6 nucleotides⫺1 (January 20, 10 m, all size
fractions) to ⬎120 CPDs⭈10⫺6 nucleotides⫺1 (January
31, February 1, surface, 0.2–2 ␮m size fraction). Except
for January 28 (Fig. 6B), which was a cloudy day, clear
depth profiles were found in the beginning of the research period, coinciding with strong salinity profiles
(Fig. 6, A–D). The input of melt water obviously promoted stabilization of the water column, thereby increasing the residence time of phytoplankton in the
very surface. This led to the highest damage levels recorded for the research period (Fig. 6, C and D). Results for all three depths were significantly different
from each other (P ⬍ 0.05, t-test, n ⫽ 12). Also, plankton biomass was not evenly distributed in the water
column at the end of January–beginning of February:
Cell counts of the main diatom species revealed significant (P ⬍ 0.05, t-test, n ⫽ 12) increases with depth
(Table 3). The observed effects were less pronounced
when the season proceeded; at the end of February
and the beginning of March, the salinity gradient was
weak or absent and DNA damage levels were not significantly different between depths. Also, phytoplankton cell counts did not show the large depth dependent changes, as observed earlier (Fig. 6, E and F;
Table 3).
CPD levels were generally highest in the smallest
size fraction: 0.2–2 ␮m. This fraction consisted mainly
Fig. 5. Depth profiles of mean CPD induction in dosimeter DNA during 24-h incubation periods at various water
depths. At each depth two tubes were incubated.
DNA DAMAGE IN ANTARCTIC MICROPLANKTON
205
of heterotrophic bacteria. The 2- to 10-␮m size fraction contained mainly flagellates (Prasinophyceae,
some Cryptophyceae), small diatoms, but probably
also Pseudonitzschia sp., which due to their needlelike
shape occasionally could pass through the 10-␮m filters. In this fraction intermediate levels of damage
were found. The largest diatom-containing fraction
(⬎10 ␮m) exhibited damage throughout, but levels
were almost always lower than those found for the
other two fractions. Significant differences between
all three fractions (P ⬍ 0.05, t-test, n ⫽ 18) were
found.
discussion
There is growing awareness that solar UVR is an environmental factor governing aquatic productivity,
similar to PAR, temperature, nutrients, and trace elements. It is evident from this study that solar summertime radiation causes DNA damage in Antarctic plankton organisms and therefore that UVR directly impairs
marine planktonic cells in the water column.
The presence of shelf ice caused highly variable
conditions in the bay, with changing stabilization of
the water column. Earlier in the season the higher elevation of the sun caused higher air and possibly water temperatures (the latter were not measured) and
likely thereby increased melting. Also, earlier in the
season lower mean daily wind speeds were recorded
(Fig. 2B), leading to lower forcing of vertical mixing.
For example, in a theoretically homogenous (temperature, salinity) water column, the wind mixed layer
would have been 11.3 m (January 20), 9.9 m (January
28), 6.0 m (January 31), 6.9 m (February 1), but 23 m
(February 27) and 26 m (March 4) at the end of the
season (calculated using wind data, following Veth
1991). However, as observed in the bay, only the last
two sampling dates showed homogenous distributions
of DNA damage and phytoplankton cell counts, as
well as the absence of a salinity gradient. It is conceivable that due to this regularly occurring stabilization
earlier in the season, the diatom bloom could develop. On the other hand, it seems that the input of
melt water initially diluted plankton biomass at the
very surface or, more likely, stimulated the removal of
diatoms from surface waters by sinking (Table 3).
Table 2. Attenuation of DNA effective UVBR, yellow substance,
and chl a in Ryder Bay.
Date ( Julian day)
Kbd-eff
(m⫺1)
1% depth
(m)
A440 nm
(m⫺1)
Chl a
(␮g⭈L⫺1)
January 27 (27)
February 5 (36)
February 17 (48)
February 25 (56)
March 3 (62)
0.71
0.83
0.66
0.64
0.47
6.5
5.4
6.9
7.1
9.6
0.183
0.046
0.071
0.047
0.041
12.8
15.1
11.5
12.3
2.62
Kbd-eff, attenuation coefficient of biologically effective UVBR, as
measured with the biodosimeter; 1% depth, estimated 1% depth
for DNA effective UVBR; A440 nm, absorbance at 440 nm, as
measured in filtered seawater.
Fig. 6. Depth distributions of CPDs in three phyto- and
bacterioplankton size fractions and salinity profiles over 6 days
during the research period. (A) January 20, (B) January 28, (C)
January 31, (D) February 1, (E) February 27, and (F) March 4.
Error bars, SD of the mean (n ⫽ 2–4).
206
Table 3.
ANITA G. J. BUMA ET AL.
Cell numbers (cells ⫻ 103·L⫺1) of the four main diatom genera (January 20 to March 4, 1998).
January 20
January 28
January 31
February 1
February 27
March 4
Depth (m)
Thalassiosira
Eucampia
Pseudonitzschia
Fragillariopsis
Total
0
5
10
0
5
10
0
5
10
0
5
10
0
5
10
0
5
10
11.1
26.2
17.2
2.7
26.2
114.7
2.3
5.1
42.7
0.4
14.8
44.1
34.1
41.9
48.8
18.4
20.0
17.0
0.8
3.2
3.8
1.1
5.9
6.4
0.3
1.3
12.8
0
1.1
17.9
29.8
26.1
31.4
10.2
13.8
15.4
58.6
68.8
84.1
231.9
331.3
649.9
173.0
130.0
583.6
89.2
298.2
688.1
45.9
76.5
40.8
56.0
50.9
63.7
7.1
42.8
18.8
0
3.8
44.1
0
0
13.8
0
0.3
28.2
6.8
1.0
12.8
6.8
1.0
1.3
77.6
141.0
264.9
235.7
367.2
1182.3
175.6
136.4
652.9
89.6
314.4
1092.7
116.6
145.5
133.8
91.4
85.7
97.4
Attenuation of DNA effective UVBR was very rapid
and seemed to be related to the persistent phytoplankton bloom. Stambler et al. (1997) found a
strong correlation between phytoplankton biomass
and attenuation of UVAR in Antarctic waters. Yellow
substance is also a major UVR-absorbing component
but was found in low concentrations in the Ryder Bay,
in keeping with open ocean levels given by Kirk
(1996). It should be noted that the pigment data (Table 2) were obtained from single samplings around
noon, whereas effective attenuation was measured
over 24-h periods. Therefore, the measured Kbd-eff values (Table 2) reflect attenuation averaged over whole
daily periods, leveling out variable chlorophyll concentrations over the day. Also, the calculated Kbd-eff
values may be less valid for the days when melt water
input caused the observed strong stabilization of the
water column. Proper Kbd-eff calculation using semilog
linear regression assumes constant attenuation over
depth. This was probably not the case here: phytoplankton was not evenly distributed over the water
column, because cell numbers were greatly reduced
in the surface. However, the resolution of the data
(Fig. 5) was too low to reveal deviation from linearity.
Highest DNA damage levels were found in the surface samples of stabilized water columns on sunny
days (i.e. January 31 and February 1). As found elsewhere (Jeffrey et al. 1996a,b) occasional sampling in
the late afternoon showed higher CPD levels than the
noontime levels presented in this study (A.G.J. Buma,
unpublished results). Therefore, the CPD levels presented here cannot be considered maximal. Despite
the high Kbd-eff values giving rise to shallow 1% depths
for DNA effective radiation, between 5.4 m and 9.6 m
(Table 2), damage could be detected in the 10-m samples. This can be explained first by the presence of residual damage from previous UVBR exposures. Damaged cells could have been transported to greater depths
by sinking or previous vertical mixing events. In addi-
tion, cells may have been damaged to an extent beyond their capability to perform repair. As shown in a
number of studies, residual DNA damage is often
found in natural plankton samples even when sampled
under low or absent UVBR irradiance conditions (Jeffrey et al. 1996a,b). Also, as shown under laboratory conditions, UVBR exposure causes loss of viability in marine diatoms (Karentz 1994, Buma et al. 1996a). The
CPD levels found at the end of the season in our study
seem to support these observations: significant levels
of CPDs were detected despite low UVBR incident irradiance levels combined with the deeply mixed water
column, further decreasing mean UVBR levels experienced by the cells. We postulate here two consequences of UVBR-induced DNA damage: retardation
of cell division and depression of overall cell metabolism until the damage is repaired and an unrepairable
fraction of the community no longer able to contribute to biomass accumulation due to viability loss.
It is clear that stabilization of the water column strongly
promotes CPD accumulation at the surface. This does
not, however, necessarily mean that stable water columns contain more damage or damaged cells as compared with wind mixed water columns. Neale et al.
(1998) calculated interactive effects of ozone depletion and vertical mixing on UVR-mediated inhibition
of water column productivity. They showed that UVBR
stress (14C incorporation) may be either diminished or
enhanced, depending on the mixing depth. Jeffrey et al.
(1996a) demonstrated high CPD abundance in surface
bacterioplankton on a calm day in the Gulf of Mexico,
whereas on the previous day strong winds caused deep
vertical mixing, resulting in much lower CPD abundances. Integration of damage over the first 10 m from
the “calm” day gave a total CPD amount almost twice
that of the day before (Jeffrey et al. 1996a). Our data
set does not allow for such a comparison, because the
time interval between the “stable” and “mixed” days
was more than a few weeks. During this time period
DNA DAMAGE IN ANTARCTIC MICROPLANKTON
many variables, such as irradiance, attenuation, and
biotic conditions (biomass, species composition),
were likely to have changed. Therefore, it remains unclear whether in the Ryder Bay water column integrated CPD levels were higher under stabilizing conditions, especially given the low phytoplankton cell
numbers in the very surface (Table 3).
It seems likely that UVR, but in particular UVBR,
stress is a multiple target phenomenon. First, photoinhibition is usually more reduced by natural UVAR levels than by natural UVBR levels, including Antarctic
systems. Second, DNA damage is induced by solar
UVBR (Visser et al. 1999), not by natural UVAR. Also,
biological weighting functions as measured with natural plankton populations, using polychromatic UVR
exposures, seem to reflect a composite of known action spectra for different target sites (Cullen et al.
1992, Behrenfeld et al. 1993), the latter measured using monochromatic light and isolated molecules or
cell components. It seems that especially in the UVBR
region of the solar spectrum, multiple targets are affected, as recently demonstrated in situ for phytoplankton in Patagonian waters (Helbling et al. 2001).
The smallest size fraction contained most of the damage. This 0.2- to 2-␮m fraction consists mainly of heterotrophic bacteria, because phototrophic bacteria and
eukaryotic picophototrophs are not abundant in Antarctic waters. It has been demonstrated numerous times
that bacteria are seriously affected by UVBR in marine
systems (Jeffrey et al. 1996a,b, Visser et al. 1999, Boelen
et al. 2000). Helbling et al. (1995) showed UVR-mediated
decreases in viability in natural bacterial assemblages
in the Antarctic. This study, however, also demonstrated
a strong UVAR effect. Because CPD formation is exclusively mediated by UVBR, this indicates that not only
CPD formation is governing UVR stress in marine Antarctic bacteria. The other size fractions also showed DNA
damage, revealing that also in these larger cells daytime repair processes were inadequate. The same depthdependent pattern of damage was found for the larger
fractions; however, CPD levels were lower throughout.
Consequently, the diatoms contained on the 2- to 10-␮m,
but mainly on the 10-␮m, filters were also UVBR stressed
via DNA damage induction.
Diatoms appear less vulnerable to UVBR as compared with other phytoplankton organisms. This may
be related with size or the presence of photoprotective
compounds (mycosporine-like amino acids [MAAs]).
It has been suggested that among other aspects, cell
size determines the vulnerability of species for DNA
damage accumulation (Karentz et al. 1991), due to
unfavorable surface area to volume ratios and the
small effectiveness of screening pigments in small cells.
Tropical phytoplankters fall in the picoplankton size
range. Here damage accumulates rapidly over the day
(Jeffrey et al. 1996a,b). As has been suggested and
demonstrated for isolated diatom species (Karentz et
al. 1991), large species exhibit lower vulnerability to
DNA damage induction as compared with smaller cells.
A long-term study conducted by Villafañe et al. (1995)
207
in the Antarctic showed higher diatom:flagellate ratios in UVBR-exposed phytoplankton assemblages compared with UVBR-excluded conditions. In general, diatoms suffer less from UVBR stress as compared with
representatives from other taxonomic groups and size
classes (Davidson and Marchant 1994). Yet, diatoms are
negatively affected by solar UVBR, as shown here with
respect to DNA damage induction. Also, Antarctic diatom-dominated communities exhibited decreased primary production rates as a result of UVBR (Boucher
and Prezelin 1996).
In conclusion, this study shows that DNA damage
occurs in summertime in a variety of Antarctic marine
microorganisms. Despite the lower vulnerability of diatoms for CPD induction, this important Antarctic floristic group experiences UVBR stress via DNA damage. Therefore, ozone reduction will strongly increase
the risk for CPD accumulation in the bulk of bacterioand phytoplankton, due to spectral shifts in the UVBR
wavelength band in favor of the shortest DNA effective wavelengths. Finally, increased CPD accumulation
will not only reduce community growth but will also increase the loss of biomass from the water column.
We thank officers and crew from the British Antarctic Survey at
Rothera Base for their assistence during field operations and
Jacqueline Zeedijk and Leen Villerius for technical assistance
in the lab. This project was carried out within the framework of
BRUVA (Biotic Responses to Ultraviolet radiation in Antarctica, coordinated by Ad Huiskes, NIOO, The Netherlands and
Ron Lewis Smith, BAS, UK) and funded by the Dutch Committee for Antarctic Research (Dutch Science Foundation, Project
no. 751-499-02).
Behrenfeld, M. J., Chapman, J. W., Hardy, J. T. & Lee II, H. 1993. Is
there a common response to ultraviolet-B radiation by marine
phytoplankton. Mar. Ecol. Progr. Ser. 102:59–68.
Boelen, P., de Boer, M. K., Kraay, G. W., Veldhuis, M. J. W. & Buma
A. G. J. 2000. UVBR induced DNA damage in natural marine
picoplankton assemblages in the tropical Atlantic ocean. Mar.
Ecol. Progr. Ser. 193:1–9.
Boelen, P., Obernosterer, I., Vink, A. A. & Buma, A. G. J. 1999. Attenuation of biologically effective UV radiation in tropical Atlantic waters measured with a biochemical DNA dosimeter.
Photochem. Photobiol. 69:34–40.
Boucher, N. P. & Prezelin, B. B. 1996. Spectral modeling of UV inhibition of in situ Antarctic primary production using a field
derived biological weighting function. Photochem. Photobiol. 64:
407–18.
Buma, A. G. J., Gieskes, W. W. C. & Thomsen, H. A. 1992. Abundance of Cryptophyceae and chlorophyll b-containing organisms in the Weddell-Scotia Confluence area in the spring of
1988. Mar. Ecol. Progr. Ser. 12:43–52.
Buma, A. G. J., van Hannen, E. J., Veldhuis, M. J. W. & Gieskes,
W. W. C. 1996a. UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp. Proc. of the Second
Algal Photobiology Workhop, Malaga, Okt. 1994. Sci. Mar. 60
(Suppl. 1):101–5.
Buma, A. G. J., Zemmelink, H. J., Sjollema, K. & Gieskes, W. W. C.
1996b. UVB radiation modifies protein and photosynthetic
pigment content, volume and ultrastructure of marine diatoms. Mar. Ecol. Progr. Ser. 142:47–54.
Cullen, J. J., Neale, P. J. & Lesser, M. P. 1992. Biological weighting
function for the inhibition of phytoplankton photosynthesis by
ultraviolet radiation. Science 258:646–50.
208
ANITA G. J. BUMA ET AL.
Davidson, A. T. & Marchant, H. J. 1994. The impact of ultraviolet
radiation on Phaeocystis and selected species of Antarctic marine diatoms. In Weiler, C. S. & Penhale, P. A. [Eds.] Ultraviolet
Radiation in Antarctica: Measurements and Biological Effects. Antarctic Res. Ser. 62. Am. Geophys. Union, Washington DC, pp.
187–205.
Helbling, E. W., Buma, A. G. J., de Boer, M. K. & Villafañe, V. E.
2001. In situ impact of solar ultraviolet radiation on photosynthesis and DNA damage in temperate marine phytoplankton.
Mar. Ecol. Progr. Ser. (in press).
Helbling, E. W., Marguet, E. R., Villafañe, V. E. & Holm-Hansen, O.
1995. Bacterioplankton viability in Antarctic waters as affected
by solar ultraviolet radiation. Mar. Ecol. Progr. Ser. 126:293–8.
Helbling, E. W., Villafañe, V. E. & Holm-Hansen, O. 1994. Effects of
ultraviolet radiation on Antarctic marine photosynthesis with
particular attention to the influence of mixing. In Weiler, C. S.
& Penhale, P. A. [Eds.] Ultraviolet Radiation in Antarctica: Measurements and Biological Effects. Antarctic Res. Ser. 62. Am. Geophys. Union, Washington DC, pp. 207–29.
Jeffrey, W. H., Aas, P., Lyons, M. M., Coffin, R. B., Pledger, R. J. & Mitchell, D. L. 1996a. Ambient solar radiation-induced photodamage in
marine bacterioplankton. Photochem. Photobiol. 64:419–27.
Jeffrey, W. H., Pledger, R. J., Aas, P., Hager, S., Coffin, R. B., Von
Haven, R. & Mitchell, D. L. 1996b. Diel and depth profiles of
DNA photodamage in bacterioplankton exposed to ambient
solar ultraviolet radiation. Mar. Ecol. Progr. Ser. 137:283–91.
Karentz, D. 1994. Ultraviolet tolerance mechanisms in Antarctic
marine organisms. In Weiler, C. S. & Penhale, P. A. [Eds.] Ultraviolet Radiation in Antarctica: Measurements and Biological Effects. Antarctic Res. Ser. 62. Am. Geophys. Union, Washington DC,
pp. 93–110.
Karentz, D., Cleaver, J. E. & Mitchell, D. L. 1991. Cell survival characteristics and molecular responses of Antarctic phytoplankton
to ultraviolet-B radiation. J. Phycol. 27:326–41.
Kirk, J. T. O. 1996. Light and Photosynthesis in Aquatic Ecosystems.
Cambridge University Press, Cambridge, pp. 46–85.
Kroon, B. M. A., Schofield, O. & Prezelin, B. B. 1994. Icecolors ‘93:
UV-B radiation specifically decreases photosystem II (PsII)
quantum yield in a field community of Antarctic ice algae exposed to natural daylight. EOS 75:200.
Lesser, M. P., Neale, P. J. & Cullen, J. J. 1996. Acclimation of Antarctic phytoplankton to ultraviolet radiation: ultravioletabsorbing compounds and carbon fixation. Mol. Mar. Biol. Biotechnol. 5:314–25.
Malloy, K. D., Holman, M. A., Mitchell, D., & William Dettrich III,
H. 1997. Solar UV-B induced DNA damage and photoenzymatic DNA repair in Antarctic zooplankton. Proc. Natl. Acad.
Sci, USA. 94:1258–63.
McMinn, A., Ashworth, C. & Ryan, K. 1999. Growth and productivity of Antarctic sea ice algae under PAR and UV irradiances.
Bot. Mar. 42:401–7.
Neale P. J., Davis, R. F. & Cullen J. J. 1998. Interactive effects of
ozone depletion and vertical mixing on photosynthesis of
Antartic phyoplankton. Nature 392:585–9.
Neale, P. J., Lesser, M. P. & Cullen, J. J. 1994. Effects of ultraviolet
radiation on the photosynthesis of phytoplankton in the vicinity of McMurdo Station, Antarctica. In Weiler, C. S. & Penhale,
P. A. [Eds.] Ultraviolet Radiation in Antarctica: Measurements and
Biological Effects. Antarctic Res. Ser. 62. Am. Geophys. Union,
Washington DC, pp. 125–42.
Prezelin, B. B., Boucher, N. P. & Smith, R. C. 1994. Marine primary
production under the influence of the Antarctic ozone hole: Icecolors ‘90. In Weiler, C. S. & Penhale, P. A. [Eds.] Ultraviolet Radiation in Antarctica: Measurements and Biological Effects. Antarctic Res.
Ser. 62. Am. Geophys. Union, Washington DC, pp. 159–86.
Prezelin, B. B., Moline, M. A. & Matlick, H. A. 1998. Icecolors ‘93:
spectral UV radiation effects on Antarctic frazil ice algae. Ant.
Res. Ser. 73:45–83.
Roza, L., van der Wulp, K. J. M., McFarlane, S. J., Lohman, P. H. M.
& Baan, R. A. 1988. Detection of cyclobutane thymine dimers
in DNA of human cells with monoclonal antibodies raised
against thymine dimer-containing tetranucleotide. Photochem.
Photobiol. 48:627–33.
Sancar, A. & Sancar, G. B. 1988. DNA repair enzymes. Annu. Rev.
Biochem. 57:29–67.
Schofield, O., Kroon, B. M. A. & Prezelin, B. B. 1995. Impact of ultraviolet-B radiation on photosystem II activity and its relationship to the inhibition of carbon fixation rates for Antarctic ice
algae communities. J. Phycol. 31:703–15.
Smith, R. C., Prezelin, B. B., Baker, K. S., Bidigare, R. R., Boucher,
N. P., Coley, T., Karentz, D., MacIntyre, S., Matlick, H. A., Menzies, D., Ondrusek, M., Wan, Z. & Waters, K. J. 1992. Ozone depletion: ultraviolet radiation and phytoplankton biology in
Antarctic waters. Science 255:952–9.
Stambler, N., Lovengreen, C. & Tilzer, M. M. 1997. The underwater
light field in the Bellingshausen and Amundsen Seas (Antarctica). Hydrobiologia 344:41–56.
Veth, C. 1991. The evolution of the upper water layer in the marginal ice
zone, austral spring 1988, Scotia-Weddell Sea. J. Mar. Syst. 2:451–64.
Vetter, R. D., Kurtzman, A. & Mori, T. 1999. Diel cycles of DNA
damage and repair in eggs and larvae of Northern anchovy,
Engraulis mordax, exposed to solar ultraviolet radiation. Photochem. Photobiol. 69:27–33.
Villafañe, V. E., Helbling, E. W., Holm-Hansen, O. & Chalker, B. E.
1995. Acclimatization of Antarctic natural phytoplankton assemblages when exposed to solar ultraviolet radiation. J. Plankton Res. 17:2295–306.
Visser, P. M., Snelder, E., Kop, A. J., Boelen, P., Buma, A. G. J. & van
Duyl, F. C. 1999. UV radiation causes inhibited protein synthesis and DNA photodamage in bacterioplankton in the Carribean Sea. Aquat. Microb. Ecol. 20:49–58.
Voytek, M. A. 1990. Addressing the biological effects of decreased
ozone on the Antarctic environment. Ambio 19:52–60.
Weinbauer, M. G., Wilhelm, S. W., Suttle, C. A. & Garza, D. R. 1997.
Photoreactivation compensates for UV damage and restores
infectivity to natural marine virus communities. Appl. Environ.
Microbiol. 63:2200–5.
Weinbauer, M. G., Wilhelm, S. W., Suttle, C. A., Pledger, R. J. & Mitchell, D. L. 1999. Sunlight-induced DNA damage and resistance in
natural viral communities. Aquat. Microb. Ecol. 17:111–20.
Wilhelm, S. W., Weinbauer, M. G., Suttle, C. A., Pledger, R. J. &
Mitchell, D. L. 1998. Measurement of DNA damage and photoreactivation imply that most viruses in marine surface waters
are infective. Aquat. Microb. Ecol. 14: 215–22.