Global Change Biology (2004) 10, 408–416, doi: 10.1111/j.1529-8817.2003.00750.x
Molecular response to climate change: temperature
dependence of UV-induced DNA damage and repair
in the freshwater crustacean Daphnia pulicaria
E M I L Y J . M A C F A D Y E N *, C R A I G E . W I L L I A M S O N * , G A B R I E L L A G R A D *,
M E G A N L O W E R Y w, W A D E H . J E F F R E Y z and D AV I D L . M I T C H E L L w
*Department of Earth and Environmental Sciences, Lehigh University, Bethlehem, PA 18015, USA, wDepartment of Carcinogenesis,
University of Texas, M.D. Anderson Cancer Center, Science Park, Research Division, Smithville, TX 78957, USA,
zCenter for Environmental Diagnostics and Bioremediation, University of West Florida, Bldg 58, 11000 University Parkway,
Pensacola, FL 32514, USA
Abstract
In temperate lakes, asynchronous cycles in surface water temperatures and incident
ultraviolet (UV) radiation expose aquatic organisms to damaging UV radiation at
different temperatures. The enzyme systems that repair UV-induced DNA damage are
temperature dependent, and thus potentially less effective at repairing DNA damage at
lower temperatures. This hypothesis was tested by examining the levels of UV-induced
DNA damage in the freshwater crustacean Daphnia pulicaria in the presence and
absence of longer-wavelength photoreactivating radiation (PRR) that induces photoenzymatic repair (PER) of DNA damage. By exposing both live and dead (freeze-killed)
Daphnia as well as raw DNA to UV-B in the presence and absence of PRR, we were able
to estimate the relative importance and temperature dependence of PER (light repair),
nucleotide excision repair (NER, dark repair), and photoprotection (PP). Total DNA
damage increased with increasing temperature. However, the even greater increase in
DNA repair rates at higher temperatures led net DNA damage (total DNA damage minus
repair) to be greater at lower temperatures. Photoprotection accounted for a much greater
proportion of the reduction in DNA damage than did repair. Experiments that looked at
survival rates following UV exposure demonstrated that PER increased survival rates.
The important implication is that aquatic organisms that depend heavily on DNA repair
processes may be less able to survive high UV exposure in low temperature
environments. Photoprotection may be more effective under the low temperature, high
UV conditions such as are found in early spring or at high elevations.
Key words: DNA damage, DNA repair, photoreactivation, temperature, ultraviolet radiation,
zooplankton
Received 13 June 2003; revised version received 6 November 2003 and accepted 7 November 2003
Introduction
As climate change is raising global surface temperatures (IPCC WGII, 2001), depletion of the stratospheric
ozone layer is increasing levels of ultraviolet radiation
(UV) reaching the Earth’s surface (Madronich et al.,
1998), with potential impacts on terrestrial and aquatic
organisms. The interaction of temperature and UV is
likely to alter the impacts of UV and climate change on
Correspondence: Craig E. Williamson, fax 610-758-3677,
e-mail: [email protected]
408
aquatic ecosystems. For lakes at north temperate
latitudes there is evidence for a seasonal lag in peak
epilimnetic (surface) water temperatures relative to
peak levels of incident UV (Williamson et al., 2002).
Epilimnetic water temperature peaks in July and
remains high ( 20–25 1C) through September. In contrast, incident UV irradiance peaks at summer solstice
in late June. Thus exposure to high UV occurs at lower
temperature before summer solstice than after.
UV causes the formation of detrimental photoproducts in the DNA molecule (Giese et al., 1957; Mitchell
& Karentz, 1993). For example, two adjacent pyrimidine
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nucleotide bases become linked to form a dimer that
bends the phosphate backbone of the DNA molecule.
This disrupts the activity of DNA polymerase, interferes with gene transcription, and can result in
mutation and cell death. Two types of dimers are
commonly induced, cyclobutane pyrimidine dimers
(CPDs) and pyrimidine (6–4) pyrimidone photoproducts ((6–4)PDs), both of which are examined in this
study. While CPDs generally account for the majority of
photoproducts formed (80–90%), (6–4)PDs can be up to
300 times more effective in blocking DNA polymerase,
and therefore more cytotoxic than CPDs (Mitchell &
Nairn, 1989).
The focus of this study is on the temperature
dependence of the molecular processes by which UVdamaged DNA is repaired. The two primary repair
mechanisms are nucleotide excision repair (NER) and
photoenzymatic repair (PER). NER is a complex, multiprotein, multi-step pathway that is powered by metabolic energy (ATP). NER is found in all taxa but is not
specific to UV-induced DNA damage (Mitchell &
Karentz, 1993; Sancar, 1994a; Sinha& Häder, 2002;
Buma et al., 2003). In contrast, PER is a single-enzyme
repair process that is driven by UVA and visible light
energy. It is thus not as energetically costly to the cell as
is NER. PER is specific for UV-induced DNA damage
but is not present in all taxa (Sancar, 1994b). The
enzyme involved in PER, photolyase, has been identified in a number of organisms as diverse as archebacteria and marsupials, but is lacking in many other
species including some diatoms, a couple of angiosperms, and humans (Mitchell & Karentz, 1993).
It has long been realized that most enzyme-catalyzed
reactions are temperature dependent (Arrhenius, 1889;
Keeton et al., 1993), and DNA repair processes are no
different. For example, PER has been demonstrated to
be temperature dependent in cell-free extracts (Langenbacher et al., 1997; Harm, 1980) and in mold spores
(Coohill & Deering, 1969). NER has been demonstrated
to increase with temperature between 5 1C and 28 1C in
yeast (Giese et al., 1957). Few studies, however, have
addressed the question of temperature dependence of
DNA repair in aquatic organisms. In Antarctic zooplankton, the rate of PER increased with temperature
(Malloy et al., 1997). A study of a marine red alga
provided evidence that the temperature optimum for
repair by PER is different for CPDs and (6–4)PDs, with
the temperature optimum for CPD photolyase closer to
12 1C and that of 6–4 photolyase closer to 25 1C (Pakker
et al., 2000).
In this study, the common water flea Daphnia pulicaria
is used as a model organism for studying the
temperature dependence of UV-induced DNA damage
and repair. Daphnia is an appropriate model organism
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here for four reasons: (1) planktonic crustaceans in the
genus Daphnia are perhaps the most widespread and
abundant crustacean zooplankton in lakes in the
northern hemisphere, and some of the dominant
primary consumers in freshwater lakes worldwide
(Zagarese et al., 1994); (2) Daphnia exhibits sensitivity
to UV under typical ambient surface water conditions
(Williamson et al., 1994; Zagarese et al., 1994); (3)
survival under UV stress increases with temperature
from 10 1C to 25 1C (Williamson et al., 2002); and (4) PER
accounts for a significant proportion of its UV tolerance
(Grad et al., 2001).
Approach
The primary objective of the current study was to
quantify the primary components and temperature
dependence of Daphnia’s physiological tolerance to UV
including PER, NER, and photoprotection. The general
approach involved exposing Daphnia to damaging
levels of UV-B in the presence and absence of PERstimulating radiation (photoreactivating radiation or
PRR), at a range of temperatures (5 1C, 15 1C, 25 1C) for
a 12 h period. This range of temperatures is within the
range that Daphnia populations experience in north
temperate lakes. Several different exposure regimes
were used to separate out the primary DNA damage
and repair processes (Fig. 1). Live Daphnia were
exposed to no UV (dark controls) or to UV-B in the
presence ( 1 PRR) or absence (PRR) of photoreactivating radiation to separate out net DNA damage and
PER. The damage accumulated by the live Daphnia
1 PRR treatment minus the damage accumulated by
live Daphnia incubated in the dark (dark controls) is
termed ‘net damage’. This value represents the damage
response of Daphnia to the exposure conditions when
all defense mechanisms (photoprotection, NER, and
PER) are available. PER is estimated by the difference
between the live Daphnia in the 1 PRR and –PRR
treatments since PRR is necessary for photoreactivation.
Dead (freeze-killed just before start of experiment)
Daphnia treatments were used to block all DNA repair
processes. This permitted NER to be estimated as the
difference between the –PRR live and –PRR dead
treatments. The PRR had a small amount of damaging
UV, which was factored out of the 1 PRR treatments as
a ‘lamp difference’. Solutions of raw DNA (DNA
dosimeters) were exposed to the same UV treatments
as the Daphnia at each temperature. The difference
between the damage measured in the raw DNA and
that measured in the dead Daphnia provides an estimate
of photoprotection broadly defined. This includes any
and all compounds that might reduce UV damage to
DNA, whether they are more specifically UV-absorbing
410 E . J . M A C F A D Y E N et al.
Fig. 1 Conceptual model of molecular responses (CPDs or (6–4)PDs) of live and freeze-killed (dead) Daphnia and DNA dosimeter to
UV-B radiation with and without concurrent exposure to photoreactivating radiation (PRR). Photoprotection consisted of all processes
and compounds leading to the difference in DNA damage in dead Daphnia vs. the DNA dosimeters, PER, photoenzymatic repair, NER,
nucleotide excision repair.
(such as mycosporine-like amino acids, MAAs) or any
of the diverse array of biochemical compounds, such as
proteins, that play a role in cell structure and function
but also happen to absorb UV. An estimate of the ‘total
damage’ sustained by the Daphnia throughout the
course of the experiment, prior to repair, is determined
by adding the damage repaired by both NER and PER
to the measured net DNA damage (Fig. 1).
Materials and methods
Experiments were conducted using adult female (eggbearers or equivalent size) D. pulicaria collected from
Dutch Springs, a spring-fed quarry in Bethlehem, PA,
USA. Daphnia were collected on March 8, 11, and 15,
2002 by taking several vertical tows of the water
column from 0–20 m with a 202 mm mesh net. Samples
were filtered through a 363 mm mesh to isolate larger
adult Daphnia. The isolated adults were pre-incubated
for 3 days at experimental temperatures in 4 L aquaria
filled with 0.2 mm-filtered surface water from Dutch
Springs with ad libitum cultured Ankistrodesmus sp.
(green alga) as food.
DNA damage experiments were conducted in the
lamp phototron apparatus (Williamson et al., 2001). This
apparatus permits the isolation of the effects of longer
wavelength photoreactivating radiation (PRR) from
damaging UV-B radiation. The intensity of UV-B is
manipulated in the presence and absence of the PRR in
order to assess the importance of photoenzymatic
repair (PER). In all experiments, the intensity of the
PRR (89 kJ m2 280–400 nm, 93% of which is 320–
400 nm UV-A) was held constant, while the intensity
of the damaging radiation from the UV-B lamp was
manipulated to adjust the sensitivity of the experiment
for the survival response. In addition to the experiments that measured DNA damage, a comparable set of
experiments that examined survival of Daphnia at the
same experimental exposure levels was carried out in
the lamp phototron. Both DNA damage and survival
experiments were performed at three temperatures:
5 1C, 15 1C, and 25 1C.
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The lamp phototron apparatus was located inside a
temperature- and light-controlled growth chamber set
to a specified temperature and kept in the dark. The
phototron apparatus consisted of a horizontal black
acrylic plankton wheel that rotated (2 rpm) above a
light-tight box. A UV-B lamp (Spectronics XX15B;
Spectronics Corporation, Westbury, NY, USA) was
covered with new cellulose acetate before each experiment and suspended 24 cm above the plankton wheel.
Full exposure to the UV-B lamp (52 kJ m2 of 280–
400 nm UV over 12 h) approximates the ambient UV
exposure in the surface waters of a lake at 401N latitude
on a sunny day around summer solstice when
weighted for the spectral sensitivity of D. pulicaria
(Williamson et al., 2001). The box below the plankton
wheel contained four fluorescent bulbs (2, 40 W cool
white bulbs and 2, 40 W Q-panel 340 bulbs) situated
32 cm from the bottom of the dishes. Stainless-steel
mesh screens were placed on top of the quartz lids of
the dishes and used to manipulate the intensity of UVB. Control dishes were placed in the incubator adjacent
to the plankton wheel and were kept in the dark
throughout the duration of the experiment. DNA
dosimeters consisted of salmon testes DNA (SigmaAldrich Co., St Louis, MO, USA) dissolved in sterile
1 SSC buffer solution at a concentration of
0.1 mg mL1 (Jeffrey et al., 1996).
On the morning of the experiment, Daphnia were
filtered through a 363-mm mesh to isolate larger adults.
Approximately half of the Daphnia were then killed by
isolation on a dry 202 mm mesh cup, and placed in the
freezer (20 1C) for approximately 45 min. The remaining Daphnia were distributed among the quartz dishes
for the ‘live’ treatments with 30–40 healthy adults per
dish. Freeze-killed Daphnia were rinsed with deionized
water and similarly distributed among dishes for ‘dead’
treatments. Live and dead Daphnia and dosimeters
were exposed in the phototron for 12 h. Twenty dishes
were exposed to 25 kJ m2 of UV from the UV-B lamp in
the absence of PRR, and 20 were exposed to 52 kJ m2 in
the presence of PRR. The lower exposure levels were
necessary for the –PRR treatments to assure survival of
adequate numbers of animals at the end of the
experiments since UV-B tolerance is reduced in the
absence of PRR. An additional 20 dishes were incubated in the dark alongside the phototron as controls.
Two of the 20 dishes contained DNA dosimeters, nine
contained live Daphnia, and nine contained killed
Daphnia. At the end of the 12 h exposure period, a
1 mL sample was taken from each DNA dosimeter dish
and frozen. For the Daphnia, groups of three dishes
were combined to provide adequate material for DNA
analysis. This resulted in three replicates of about 100
Daphnia each. Only live Daphnia were selected for the
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DNA analyses to ensure that DNA repair processes
were active for the duration of the experiment. The
samples were immediately frozen at –20 1C to preserve
the DNA.
A series of survival experiments was also carried out
to assess the effects of temperature on Daphnia survival
at the same experimental temperatures. For each
survival experiment Daphnia were collected at the same
time and location as for the DNA damage experiments.
Ten dishes of Daphnia were exposed in the lamp
phototron for 12 h to each of three UV-B lamp exposure
levels: 4, 25 and 52 kJ m2 (total UV 280–400 nm). Five
replicate dishes with 10 Daphnia each were exposed to
1 PRR, PRR and dark control conditions as with the
DNA damage experiments. Immediately following the
end of the 12 h exposure period, all dishes were
removed from the wheel and survival was scored by
visual examination with a dissecting microscope. An
individual was scored as ‘live’ if a heartbeat was
observed during 10 sec of observation at 30 magnification. Any dead individuals were removed. Survival
was scored daily for 5 days (25 1C), 10 days (15 1C) or 20
days (5 1C) to provide physiologically similar time
periods for response at the different temperatures.
Concentrations of both CPD and (6–4)PD photoproducts of DNA damage were quantified in the Daphnia
and dosimeter samples using a radioimmunoassay
(RIA) (Mitchell, 1996). Prior to data analysis, dimer
data were adjusted by subtracting the average background level of photoproducts measured in the dark
control (unexposed) samples. Due to the differences in
the exposure levels and spectral composition of the UVB and PRR lamps, all irradiance values were weighted
with the Setlow action spectrum for DNA damage (286–
339 nm) normalized to 1 at 300 nm (Setlow, 1974) and
exposure levels expressed per Setlow-weighted kJ m2
over this wavelength range.
Two-way analysis of variance (ANOVA) with replication on the photoproducts (both CPDs and (6–4)PDs)
was used to test for the presence of significant NER,
PER, and net DNA damage according to our conceptual
model (Fig. 1). Statistically significant effects of temperature on NER, PER, and net DNA damage were
determined from the interaction terms. PER and
temperature effects on PER were tested with a twoway ANOVA on photoproducts in the live Daphnia
treatments where temperature and PRR ( 1 /) were
the two factors. NER and temperature effects on NER
were tested with a two-way ANOVA on photoproducts
in the treatments lacking PRR where temperature and
live/dead were the two factors. Net DNA damage and
temperature effects on net DNA damage were tested
with a two-way ANOVA on photoproducts in the live
Daphnia treatments where temperature and UVB&PRR
412 E . J . M A C F A D Y E N et al.
( 1 /) exposure were the two factors. This latter test
compared the raw data for Daphnia in the dark controls
with those in the 1 PRR live treatment at the three
different temperatures. For the survival experiments,
corrected daily percent survival data were arcsine
transformed and used in two-way ANOVA with replication to determine if temperature had a significant effect
on survival.
Results
Total DNA damage to Daphnia was substantial in all
treatments with the levels of CPD damage generally
about an order of magnitude higher than the levels of
(6–4)PD damage (Fig. 2). Both PER and NER contributed significantly to reducing this damage at all
temperatures (Fig. 2, Table 1). Of the total DNA damage
sustained by Daphnia, 57–71% was repaired by NER
and 7–38% was repaired by PER for both CPD and (6–
4)PDs (Table 2). This led to net DNA damage levels
after repair that were only 6–27% of the total DNA
damage to Daphnia (Fig. 2, Table 2). In spite of the
effectiveness of these repair processes significant net
DNA damage still remained after repair at all temperatures. (Fig. 2, Table 1).
For CPDs temperature had a significant effect on PER
and on net DNA damage but not on NER (Fig. 2, Table
1). Though the temperature effects on NER were not
significant, both NER and PER repaired greater
numbers of CPDs at higher temperatures, leading to a
Table 1 Results of two-way ANOVA to test for presence and
temperature dependence of PER, NER and net DNA damage
in Daphnia
CPDs
Test group and factors
Live Daphnia
Temperature
PRR ( 1 /)
Interaction (PER)
PRR Daphnia
Temperature
Live/dead
Interaction (NER)
Live Daphnia
Temperature
1 PRR/dark control
Interaction
(net DNA damage)
F stat
(6–4)PDs
P-value
F stat
P-value
5.8
28.7
10.1
o0.05
o0.01
o0.01
0
24.3
2.6
1
o0.01
0.11
36.3
113.9
3.14
o0.01
o0.01
0.08
9.4
122.8
4
o0.01
o0.01
o0.05
10.5
127.7
4.6
o0.01
o0.01
o0.05
31.6
160.8
11.7
o0.01
o0.01
o0.01
The interaction term indicates whether there were significant
effects of temperature on PER, NER, and net DNA damage as
indicated.
PER, photoenzymatic repair; NER, nucleotide excision repair;
CPDs, cyclobutane pyrimidine dimers; (6–4)PDs, pyrimidine
(6–4) pyrimidone photoproducts; PRR, photoreactvating
radiation.
Table 2 Percentage of total damage repaired by NER and
PER processes and net damage remaining at the end of the
experiment as a function of temperature
Fig. 2 DNA damage (positive values) and repair (negative
values) in Daphnia pulicaria, at 5 1 C, 15 1 C and 25 1 C, following
12 h UV-B exposure with and without photoreactivation radiation. DNA damage was estimated as CPD (a) and (6–4)PD (b)
photoproducts per mb DNA per Setlow-weighted kJ m2. Note
the 10-fold difference in scaling of the Y-axes – CPDs are more
prevalent than (6–4)PD photoproducts. Mean values are
estimated from differences between two treatments. Thus for
error propagation the standard errors (SE) shown here were
calculated as the square root of the sum of the squared SE of the
individual treatments.
CPD
photoproducts
Damage and repair
(6–4)PD
photoproducts
5 1 C 15 1 C 25 1 C 5 1 C 15 1 C 25 1 C
NER repaired damage 69
PER repaired damage
7
Net damage remaining 24
71
18
12
57
38
6
60
14
27
70
18
12
64
30
6
PER, photoenzymatic repair; NER, nucleotide excision repair;
CPD, cyclobutane pyrimidine dimer; (6–4)PD, pyrimidine
(6–4) pyrimidone photoproduct.
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413
Discussion
Fig. 3 Percent survival of the Daphnia in the 1 PRR and PRR
treatments of the survival experiments at the three experimental
temperatures.
significantly lower level of net DNA damage at higher
temperatures. For the (6–4)PD photoproducts both PER
and NER also increased at higher temperatures,
although the temperature effects were statistically
significant only for NER and for net DNA damage
(Fig. 2, Table 1). As observed for CPDs, net DNA
damage for (6–4)PDs decreased significantly with
increasing temperature.
The DNA dosimeter data were variable and not
considered reliable for comparing temperature effects.
The damage levels in the unprotected DNA were
extremely high compared to those in the DNA of intact
Daphnia. In all cases, the levels of both CPDs and (6–
4)PDs in the dead Daphnia were only 1–3% (at 5 1C and
15 1C) to 3–6% (at 25 1C) of those observed in the DNA
dosimeters. This indicates that photoprotection as
broadly defined here accounted for 94–99% of the
reduction in DNA damage in Daphnia and is much
more important than both NER and PER combined.
In the survival experiments, Daphnia survival remained above 90% in all dark controls but was
significantly reduced in the treatment dishes. Survival
in the treatments exposed to 52 kJ m2 was low (0–4%)
for both the 1 PRR and –PRR treatments (Fig. 3). After
exposure to 25 kJ m2 survival remained at 65–92%
when PRR was present, but was reduced to 0–6% when
PRR was absent. This was indicative of the importance
of PER to survival in Daphnia at this exposure level (Fig.
3). At 4 kJ m2 survival remained high (77–98%) in both
1 PRR and PRR treatments. No significant effects of
temperature were observed for the survival data.
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Asynchronous changes in both seasonal and global
patterns of environmental UV and temperature lead
aquatic organisms to be exposed to damaging UV
radiation at different temperatures (Williamson et al.,
2002). The data presented here indicate that ectotherms
that depend on temperature-dependent enzyme repair
processes may be less able to repair DNA at lower
temperatures. Thus low temperatures will likely favor
photoprotection rather than repair of DNA damage.
This suggests that differences in the ability of certain
species to utilize photoprotection vs. photorepair may
lead to changes in community structure. For species
that are capable of utilizing both photoprotection and
photorepair, shifts between these two UV defenses may
have important ecological implications. For example,
pigmented photoprotective compounds may lead to
either greater vulnerability to visually feeding predators (Hairston, 1976; Luecke & O’Brien, 1981; Morgan &
Christy, 1996) or to thermal advantages in cold
environments (Byron, 1981, 1982).
Several previous studies demonstrated that exposure
to UV-A and visible light can increase the survival of
Daphnia exposed to potentially damaging UV-B radiation (Siebeck & Bohm, 1991; Grad et al., 2001; Williamson et al., 2002). While this light-induced repair was
assumed to be due to PER of DNA damage, evidence at
the molecular level has been lacking. The actual
measurements of DNA damage coupled with the
observed increases in survival in this study demonstrated that PER can enhance the repair of both CPDs
and (6–4)PDs and is likely responsible for increases in
survival following UV-B exposure. Interestingly, our
experiments revealed that animals with detectable
levels of DNA damage did not live for long periods
of time postexposure. This suggests that it may be
difficult to detect measurable levels of DNA damage in
freshwater zooplankton in nature even with our quite
sensitive assay. DNA damage has been successfully
measured in nature in marine zooplankton and fish
(Malloy et al., 1997; Vetter et al., 1999).
This study is one of the first to examine the ecological
importance of the temperature dependence of different
DNA repair mechanisms for UV-induced DNA damage. There was a clear and significant temperature
dependence of net DNA damage for both photoproducts as a result of the generally greater (but not always
statistically significant) effectiveness of both NER and
PER at the higher temperatures tested (Fig. 2). This
provides molecular evidence for the previously demonstrated temperature dependence of light repair in
Daphnia (Williamson et al., 2002), and is consistent with
the temperature dependence of repair rates of CPDs in
414 E . J . M A C F A D Y E N et al.
marine killifish at temperatures ranging between 6 1C
and 25 1C (Malloy et al., 1997). However, we found that
dark repair (NER) in Daphnia was about twice as
effective as PER in repairing both CPDs and (6–4)PDs.
This is in contrast to those previous studies on marine
organisms that reported PER rates for CPDs to be
between 31 and 190 times greater than those of NER
(Malloy et al., 1997). The reason for the differences
between these studies may be related to the species
used or to differences in experimental techniques.
Malloy et al. (1997) exposed krill and three species of
fish to UV-B and examined CPD repair rates during the
postexposure period in the light and in the dark. In
contrast, our phototron apparatus permitted us to
examine relative rates of PER and NER during the
actual UV-B exposure period by comparing live and
dead animals. On the one hand, the exposure of
organisms to both damaging and repair wavelengths
simultaneously would seem to be more environmentally realistic. On the other hand, we do not know if
short-term biochemical changes in dead animals may
somehow modify UV absorption and thus alter DNA
damage in live and dead Daphnia. It is often thought
that NER is less prevalent because it requires ATP and
is thus more energy intensive than PER which uses
incident light energy.
Perhaps one of the most striking results in our study
is that photoprotection, broadly defined, accounted for
over 95% of the reduction in potential DNA damage.
NER and PER together accounted for only 1–4% of the
reduction in DNA damage by incident UV when
compared to damage in the DNA dosimeters. This
was not expected in such a highly transparent small
invertebrate such as Daphnia. The population of
Daphnia that we used is not melanic and HPLC analyses
of several species of Daphnia have revealed essentially
no MAAs or carotenoids (R. E. Moeller, unpublished
results). Thus the ‘photoprotection’ that we observed is
likely due to the fact that proteins, many lipids, and
numerous other biomolecules have chromophores that
absorb radiation in the UV-B range (Häder & Tevini,
1987). The fact that most of the DNA is not at the
surface of the organism and that it is more condensed in
organisms than in solution may also contribute to the
lower levels of DNA damage observed in Daphnia
compared to the DNA dosimeters. The exoskeleton
may also absorb or reflect UV, though we have not
tested this directly, and the high sensitivity of Daphnia
to UV compared to other zooplankton species that we
have tested makes this unlikely.
The findings of our study are not consistent with the
notion that DNA damage is independent of temperature (Wang, 1976; Cadet & Vigny, 1990). In contrast, we
found that total DNA damage in Daphnia increased at
higher temperatures (Fig. 2). The mechanism for this
response is not known, but may be related to increased
growth rates at higher temperatures. The physical
protection of the DNA molecule from UV damage
may become compromised when the DNA is unwound
during DNA replication, transcription, and cell division, making faster growing cells more vulnerable to
UV damage. This potential for greater induction of
DNA damage at higher temperatures is supported by
findings from the human genome, where induction of
(6–4)PDs occurs at significantly higher frequencies in
linker DNA compared to nucleosome core DNA in
chromatin (Mitchell et al., 1990).
The lack of significant temperature effects on the
survival of Daphnia in the current experiments was
most likely due to the range of exposure levels that
were selected to optimize detection of DNA damage.
All of the exposure levels chosen led to either very high
or very low survival, leaving little latitude for variation
in survival at the different temperatures. Higher
resolution experiments at exposure levels between 11
and 34 kJ m2 have clearly demonstrated the effects of
temperature on survival in D. catawba. (Williamson
et al., 2002).
Conclusions
In conclusion, large-scale ecosystem changes result in
part from the impacts of environmental variables on
small-scale molecular processes, the effects of which are
propagated through the organism, population, community, and ultimately the ecosystem. In the case of UV,
one of the most important determinants is the direct
damage to DNA. As this study has demonstrated,
direct damage to DNA in Daphnia has the potential to
be altered by the interactive effects of UV and
temperature. Therefore, a shift in the UV : temperature
ratio (UV : T) of temperate lakes could have a significant
impact on Daphnia populations. Increases in UV : T are
likely to have the greatest impact on Daphnia in shallow,
alpine systems where water temperatures are cold, iceout occurs very near the peak UV at summer solstice,
and no depth refuge exists for behavioral avoidance of
UV. Even in clear low-elevation lakes at temperate
latitudes, shifts in UV : T could potentially impact
Daphnia survival, especially in the spring when cold
surface waters coincide with high UV irradiance during
a period of rapidly changing temperature and incident
solar radiation. The clear-water phase induced by
Daphnia grazing increases water transparency in late
spring (Lampert et al., 1986). Climate change related to
the North Atlantic Oscillation can alter the timing of the
development of both the clear-water phase and the
spring peak in Daphnia populations by up to 1 month
r 2004 Blackwell Publishing Ltd, Global Change Biology, 10, 408–416
T E M P E R AT U R E - D E P E N D E N T D N A D A M A G E B Y U V
(Gerten & Adrian, 2000; Straile & Adrian, 2000). Other
elements of global change may also be important. For
example, maximum ozone depletion occurs in late
winter to early spring (Madronich et al., 1998). Changes
in the timing of ice-out, seasonal temperatures, and
thus vertical mixing during the late winter and spring
(Likens, 2000; Magnuson et al., 2000a, b) may further
alter these UV–temperature relationships. Understanding how the molecular-level processes involved in DNA
damage and repair respond to changes in environmental temperature and UV will better enable us to
predict how these responses translate into organism
and ecosystem-level responses to climate change.
Acknowledgements
We thank Patrick Neale for his comments on the manuscript and
for providing the data for the Setlow action spectrum for DNA,
and Stuart Schooley for access to Dutch Springs to collect
Daphnia. This work was supported by NSF grants DEB 9973938
and DEB-IRCEB-0210972.
References
Arrhenius S (1889) On the reaction velocity of the inversion of
cane sugar by acids. Zeitschrift für Physikalische Chemie, 4,
226. Reproduced in Selected Readings in Chemical Kinetics. 1967
(eds Back MH, Laidler KJ), Pergamon, Oxford.
Buma AGJ, Boelen P, Jeffrey WH (2003) UVR-induced DNA
damage in aquatic organisms. In UV effects in aquatic
organisms and ecosystems. In: Comprehensive Series in Photochemical & Photobiological Sciences (eds Helbling EW, Zagarese
HE), pp. 291–327. The Royal Society of Chemistry, Cambridge.
Byron ER (1981) Metabolic stimulation by light in a pigmented
freshwater invertebrate. Proceedings of the National Academy of
Sciences, 78, 1765–1767.
Byron ER (1982) The adaptive significance of calanoid copepod
pigmentation: a comparative and experimental analysis.
Ecology, 63, 1871–1886.
Cadet J, Vigny P (1990) The photochemistry of nucleic acids. In:
Bioorganic Photochemistry, Photochemistry of the Nucleic Acids,
Vol. 1 (ed. Morrison H), John Wiley, New York.
Coohill TP, Deering RA (1969) Ultraviolet light inactivation and
photoreactivation of Bastocladiella emersonii. Radiation Research,
39, 374–385.
Gerten D, Adrian R (2000) Climate-driven changes in spring
plankton dynamics and the sensitivity of shallow polymictic
lakes to the North Atlantic Oscillation. Limnology and
Oceanography, 45, 1058–1066.
Giese AC, Iverson RM, Sanders RT (1957) Effect of nutrient state
and other conditions on the ultraviolet resisitance and
photoreactivation in yeast. Journal of Bacteriology, 74, 271–279.
Grad G, Williamson CE, Karapelou DM (2001) Zooplankton
survival and reproduction responses to damaging UV radiation: A test of reciprocity and photoenzymatic repair.
Limnology and Oceanography, 46, 584–591.
r 2004 Blackwell Publishing Ltd, Global Change Biology, 10, 408–416
415
Häder D-P, Tevini M (1987) General Photobiology. Pergamon Press,
New York.
Hairston NG (1976) Photoprotection by carotenoid pigments in
the copepod Diaptomus nevadensis. Proceedings of the National
Academy of Sciences, 73, 971–974.
Harm W (1980) Biological effects of Ultraviolet Radiation. Cambridge University Press, New York.
Intergovernmental Panel on Climate Change, Working Group 2
(2001) Summary for Policymakers: Climate Change 2001: Impacts,
Adaptation, and Vulnerability. www.grida.no/climate/ipcc_tar/
wg2/index.htm
Jeffrey WH, Pledger RJ, Aas P et al. (1996) Diel and depth
profiles of DNA photodamage in bacterioplankton exposed to
ambient solar ultraviolet radiation. Marine Ecology Progress
Series, 137, 283–291.
Keeton WT, Gould JL, Gould CG (1973) Biological Science, 5th
edn., pp. 80–85. WW Norton and Company, New York.
Lampert W, Fleckner W, Rai H et al. (1986) Phytoplankton
control by grazing zooplankton: A study on the spring clearwater phase. Limnology and Oceanography, 31, 478–490.
Langenbacher T, Zhao X, Bieser G et al. (1997) Substrate and
temperature dependence of DNA-photolyase repair activity
examined with ultrafast spectroscopy. Journal of the American
Chemical Society, 119, 10532–10536.
Likens GE (2000) A long-term record of ice cover for Mirror
Lake, New Hampshire: effects of global warming. Verhandlungen Internationale Vereinigung fur Theoretische und Angewandte
Limnologie, 27, 2765–2769.
Luecke C, O’Brien WJ (1981) Photoxicity and fish predation:
selective factors in color morphs in Heterocope. Limnology and
Oceanography, 26, 454–460.
Madronich S, McKenzie RL, Bjorn LO et al. (1998) Changes in
biologically active ultraviolet radiation reaching the Earth’s
surface. Journal of Photochemistry and Photobiology B: Biology, 46,
5–19.
Magnuson JJ, Robertson DM, Benson BJ et al. (2000a) Historical
trends in lake and river ice cover in the Northern Hemisphere.
Science, 289, 1743–1746.
Magnuson JJ, Wynne RH, Benson BJ et al. (2000b) Lake and river
ice as a powerful indicator of past and present climates.
Verhandlungen Internationale Vereinigung fur Theoretische und
Angewandte Limnologie, 27, 2749–2756.
Malloy KD, Holman MA, Mitchell D et al. (1997) Solar UVBinduced DNA damage and photoenzymatic DNA repair in
antarctic zooplankton. Proceedings of the National Academy of
Sciences, 94, 1258–1263.
Mitchell DL (1996) Radioimmunoassay of DNA damaged by
ultraviolet light. In: Technologies for Detection of DNA Damage
and Mutations (ed. Pfeifer GP), pp. 73–85. Plenum Press, New
York.
Mitchell DL, Karentz D (1993) The induction and repair of DNA
photodamage in the environment. In: Environmental UV
Photobiology (eds Young AR, Bjorn LO, Moan J, Nultsch W),
pp. 345–377. Plenum, New York.
Mitchell DL, Nairn RS (1989) The biology of the (6–4)
PD photoproduct. Photochemistry and Photobiology, 49, 805–819.
Mitchell DL, Nguyen TD, Cleaver JE (1990) Nonrandom
induction of pyrimidine-pyrimidone (6–4) photoproducts in
416 E . J . M A C F A D Y E N et al.
ultraviolet-irradiated human chromatin. Journal of Biological
Chemistry, 256, 5353–5356.
Morgan SG, Christy JH (1996) Survival of marine larvae under
the countervailing selective pressures of photodamage and
predation. Limnology and Oceanography, 41, 498–504.
Pakker HS, Martins RST, Boelen P et al. (2000) Effects of
temperature on the photoreactivation of ultraviolet-B-induced
DNA damage in Palmaria palmata (Rhodophyta). Journal of
Phycology, 36, 334–341.
Sancar A (1994a) Mechanisms of DNA excision repair. Science,
266, 1954–1956.
Sancar A (1994b) Structure and Function of DNA photolyase.
Biochemistry, 33, 2–9.
Setlow RB (1974) The wavelengths in sunlight effective in
producing skin cancer: a theoretical analysis. Proceedings of the
National Academy of Sciences, 71, 3363–3366.
Siebeck O, Böhm U (1991) UV-B effects on aquatic animals.
Verhandlungen Internationale Vereinigung fur Theoretische und
Angewandte Limnologie, 24, 2773–2777.
Sinha RP, Häder D-P (2002) UV-induced DNA damage and
repair: a review. Photochemical and Photobiological Sciences, 1,
225–236.
Straile D, Adrian R (2000) The North Atlantic Oscillation and
plankton dynamics in two European lakes – two variations on
a general theme. Global Change Biology, 6, 663–670.
Vetter RD, Kurtzman AL, Mori T (1999) Diel cycles of DNA
damage and repair in eggs and larvae of Northern anchovy,
Engraulis mordax, exposed to solar ultraviolet radiation.
Photochemistry and Photobiology, 69, 27–33.
Wang SY (1976) Pyrimidine biomolecular photoproducts. In:
Photochemistry and Photobiology of Nucleic Acids, Vol. I (ed.
Wang SY), pp. 326–356. Academic Press, New York.
Williamson CE, Grad G, De Lange HJ et al. (2002) Temperature-dependent ultraviolet responses in zooplankton: implications of climate change. Limnology and Oceanography, 47,
1844–1848.
Williamson CE, Neale PJ, Grad G et al. (2001) Beneficial
and detrimental effects of UV radiation: implications of
variation in the spectral composition of environmental
radiation for aquatic organisms. Ecological Applications, 11,
1843–1857.
Williamson CE, Zagarese HE, Schulze PC et al. (1994) The impact
of short-term exposure to UV-B radiation on zooplankton
communities in north temperate lakes. Journal of Plankton
Research, 16, 205–218.
Zagarese HE, Williamson CE, Mislivets M et al. (1994) The
vulnerability of Daphnia to UV-B radiation in the Northeastern
United States. Archiv für Hyrobiologie Beihefte Ergebnisse der
Limnologie, 43, 207–216.
r 2004 Blackwell Publishing Ltd, Global Change Biology, 10, 408–416