survivorship, development, and dna damage in echinoderm embryos and larvae exposed to ultraviolet...

www.elsevier.com/locate/jembe

Journal of Experimental Marine Biology and Ecology

292 (2003) 75–91

Survivorship, development, and DNA damage

in echinoderm embryos and larvae exposed to

ultraviolet radiation (290–400 nm)

Michael P. Lesser*, Thomas M. Barry

Department of Zoology and Center for Marine Biology, University of New Hampshire, Durham, NH 03824, USA

Received 11 September 2002; received in revised form 12 March 2003; accepted 14 March 2003

Abstract

Laboratory experiments utilizing ecologically relevant irradiances of ultraviolet radiation (UVR)

known to occur in shallow Gulf of Maine waters were conducted on the planktonic embryos and

larvae of two common benthic echinoids; the green sea urchin Strongylocentrotus droebachiensis

and the sand dollar Echinarachnius parma. Significant decreases in survivorship were observed in

freshly fertilized embryos of both species with greater mortality in E. parma that was associated with

the absence of UVR-absorbing compounds, the mycosporine-like amino acids. Experiments on

blastula, gastrula, and prism larval stages of S. droebachiensis also showed significant decreases in

survivorship, delays in development, and abnormal embryos and larvae associated with exposure to

UVR. Additionally, all developmental stages of S. droebachiensis experimentally exposed to UVR

resulted in significant increases in DNA damage, measured as cyclobutane pyrimidine dimer

photoproducts. The observed delays in early cleavage and subsequent developmental stages for S.

droebachiensis are correlated with DNA damage. It is postulated that cell cycle arrest at critical

checkpoints after DNA damage, mediated by a suite of cell cycle genes, is a component of the

observed UVR induced developmental delays.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: DNA damage; Echinoids; Embryos; Larvae; Ultraviolet radiation

1. Introduction

The effects of exposure to ultraviolet radiation (UVR: 290–400 nm) have become a

major concern in the marine environment. In particular, the decrease of stratospheric

0022-0981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0022-0981(03)00141-2

* Corresponding author. Tel.: +1-603-862-3442; fax: +1-603-862-3784.

E-mail address: [email protected] (M.P. Lesser).

M.P. Lesser, T.M. Barry / J. Exp. Mar. Biol. Ecol. 292 (2003) 75–9176

ozone from anthropogenic inputs of chlorinated fluorocarbons has resulted in an increase

in the amount of biologically damaging ultraviolet-B radiation (UV-B: 290–320 nm)

reaching the sea surface. Attention has largely been focused on the Antarctic, and more

recently the Arctic, where the autocatalytic destruction of stratospheric ozone (‘‘the ozone

hole’’) leads to enhanced fluxes of UV-B (Smith and Baker, 1989; Madronich et al.,

1998) and a decrease in primary productivity (Smith et al., 1992). Recent global

assessments of the depletion of stratospheric ozone have shown increases of unweighted

UV-B radiation of up to 130% in the Antarctic, 22% in the Arctic, and 4–7% at temperate

latitudes (Madronich et al., 1998). The temperate latitudes include coastal areas that

contain many productive estuarine, intertidal, and shallow subtidal habitats such as those

found in the Gulf of Maine. Current predictions suggest that higher irradiances of UV-B

radiation will affect the marine environment for at least the next 25 years, if not longer

(Madronich et al., 1998). Of particular concern at any latitude is that ozone depletion

results in an increase in damaging UV-B wavelengths without a proportional increase in

longer UV-A and blue wavelengths involved in photoreactivation and photorepair (Smith,

1989; Halliwell and Gutteridge, 1999). In the Gulf of Maine, UVR can penetrate to

depths as deep as 7–12 m depending on the optical properties of the water column

(Banaszak et al., 1998; Adams and Shick, 2001; Lesser et al., 2001). The transmission of

UVR in the Gulf of Maine varies with changes in the stability and optical properties of

the water column due to seasonal variation in solar irradiation, phytoplankton blooms, or

increases in dissolved organic matter (Kirk, 1994). These changes in the attenuation of

UVR could have significant biological effects on the planktonic life-history phases of

invertebrates, fish, and macrophytes. Ultraviolet radiation is known to have a detrimental

effect on phytoplankton, bacteria, macrophytes, and zooxanthellate corals (Jokiel and

York, 1984; Cullen et al., 1992; Gleason and Wellington, 1993; Herndl et al., 1993;

Lesser et al., 1996; Lesser and Lewis, 1996; Shick et al., 1996; Franklin and Forester,

1997), and the harmful effects of UVR may involve damage to DNA and proteins,

oxidation of membrane lipids, and photooxidation of chlorophyll or damage to photo-

system II (Halliwell and Gutteridge, 1999). Structural damage to DNA can be caused

directly by UV-B and indirectly by UV-A (320–400 nm) wavelengths. The cyclobutane

pyrimidine dimer (CPD) is the predominant photoproduct of direct UV-B exposures,

while UV-A wavelengths cause damage indirectly to DNA through the photodynamic

production of hydroxyl radicals causing strand breaks and DNA cross-links (Peak and

Peak, 1990). The amount of photodamage to DNA is dependent on the equilibrium

between damage, which is dependent on the absorbed dose of UVR, rates of repair, and

rates of cell division.

Additionally, there are significant synergistic effects of solar UVR with oxidative stress

in marine organisms. Reactive oxygen species (ROS) are formed by the univalent

reduction of molecular O2, yielding superoxide radicals (O2�), and further reduction of

superoxide yields hydrogen peroxide (H2O2) and hydroxyl radicals (HO�) via the iron-

catalyzed Haber–Weiss reaction (Fridovich, 1986). These reactions are known to occur

normally in many metabolic processes in the cytosol, mitochondria, and chloroplasts

(Asada and Takahashi, 1987; Halliwell and Gutteridge, 1999) but concentrations of these

radicals increase under stressful conditions (Fridovich, 1986; Asada and Takahashi, 1987).

Defenses against ROS include various non-enzymatic antioxidants and quenchers (e.g.,

M.P. Lesser, T.M. Barry / J. Exp. Mar. Biol. Ecol. 292 (2003) 75–91 77

ascorbate, tocopherols, carotenoids), and enzymes such as superoxide dismutase which

removes O2� but in so doing produces H2O2, catalase which then removes H2O2, and

ascorbate and glutathione peroxidases which remove H2O2 and other peroxides (Asada

and Takahashi, 1987; Halliwell and Gutteridge, 1999).

Reactive oxygen species also have multiple adverse effects on marine organisms as

described for UVR, including damage to DNA/RNA, inhibition of photosynthesis, and

bleaching of zooxanthellate corals (Dykens et al., 1992; Shick et al., 1995, 1996; Lesser,

1996, 1997; Lesser et al., 2001). Additionally, oxidative stress is known to play a role in

apoptosis or programmed cell death through several cell cycle genes such as p53. The

principal function of p53 is to promote the repair or deletion of cells exposed to agents that

cause DNA damage, like hypoxia, UVR, ROS, or mutagens (Graeber et al., 1996; Renzing

et al., 1996; Griffiths et al., 1997).

Defenses against solar UVR include blocking potentially harmful wavelengths by using

UV-absorbing compounds. Many of these compounds have been identified as mycospor-

ine-like amino acids (MAAs), and are found in a wide variety of marine organisms

(Karentz et al., 1991; Shick and Dunlap, 2002). A protective role of these compounds has

been inferred from their UVR absorbing properties and their decrease in concentration

with increasing depth or after experimental shielding from UVR (Shick and Dunlap,

2002). Eggs, embryos, and larvae from temperate sea urchins contain MAAs (Banaszak et

al., 1998; Carroll and Shick, 1996; Adams and Shick, 1996, 2001), as do sea star and

holothuroid eggs and larvae (Lesser unpublished).

Several adverse effects of UVR have already been documented for marine organisms,

including species of crustacean zooplankton that are normally found in near-surface waters

where high fluxes of UVR are present (Karansas et al., 1979; Damkaer and Dey, 1983;

Dey et al., 1988). Recent data have shown a significant increase in DNA damage,

measured as an increase in CPD formation, in ice fish and krill collected from shallow

waters in Antarctica during the occurrence of the ozone hole despite active DNA repair

capabilities (Malloy et al., 1997). DNA damage and up-regulation of p53 has also been

reported in laboratory experiments on the embryos of cod fish (Gadus morhua) in

response to UVR irradiances similar to exposures where developing embryos would be

found in the Gulf of Maine (Lesser et al., 2001). Near-surface waters also contain the

gametes and larvae of many species of benthic invertebrates. Larvae of the sand dollar

Dendraster excentricus exposed to UVR resulted in negative phototaxis and significant

mortality (Pennington and Emlet, 1986). Gleason and Wellington (1995) described

significant depth-dependent mortality of planula larvae from the coral, Agaricia agaricites

by UV-B radiation that was associated with differences in the MAA concentrations that

larvae obtained from their parent colonies. Urchin larvae also obtain their MAAs through

the eggs from their parents and the concentration of these compounds can be manipulated

by pre-conditioning adults with algal sources of these compounds (Adams and Shick,

1996; Carroll and Shick, 1996). Exposure of gametes and larvae to high doses of UVR

could potentially affect fertilization success, the timing of cleavage, and development time

for embryos that survive as observed for the temperate sea urchin, Strongylocentrotus

droebachiensis (Adams and Shick, 1996, 2001). Many of these effects on gametes and

larvae might be ameliorated if sufficient MAAs can be translocated from adults to gametes

(Adams and Shick, 1996).


The present study is the first of a series from laboratory and field experiments on the

effects of UVR on survival, development, and the biochemical and molecular response to

UVR in echinoderm larvae from the Gulf of Maine. Laboratory experiments examining the

effects of UVR on survivorship, development, and DNA damage on the embryos and

larvae from the green sea urchin S. droebachiensis and the sand dollar, Echinarachnius

parma are presented here.

2. Materials and methods

2.1. Experimental animals

Adult green sea urchins, S. droebachiensis (Muller), and sand dollars, E. parma

(Lamarck), were collected from around the Isles of Shoals, Gulf of Maine (42j59.29VN,70j37.01VW) at a depth of 10 m in June of 1999 for sand dollars and March of 2000 for sea

urchins. All animals were brought back to the University of New Hampshire Coastal

Marine Laboratory where they were maintained at 4–5 jC for sea urchins and 10 jC for

sand dollars. Animals were not feed and all were used within 2 weeks of collection.

Reproductively mature animals were brought into the laboratory and induced to spawn by

intracoelomic injection of 0.5 M KCl at 4 jC. Eggs from at least five females were

combined and washed three times with 0.22-Am filtered seawater and collected using a

100-Am Nitex filter after each wash. Sperm were left ‘‘dry’’ until used and the sperm of at

least three males combined and diluted in 0.22 Am filtered seawater at f 1:10,000 to

fertilize eggs at a density of 500 individuals ml� 1 in 2 l sterile plastic containers (Corning).

After 5 min, freshly fertilized embryos (FFE) were washed to remove excess sperm and

maintained at a density of 20–30 individuals ml� 1 until used for experiments. Both S.

droebachiensis and E. parma FFE were used within 15 min of fertilization in experimental

exposures to UVR, and samples taken at this time showed that fertilization success was

over 95% (three subsamples of 5 ml and counting 100 embryos in each sample). For

experiments on other stages of development in S. droebachiensis, embryos were allowed to

develop to blastula, gastrula, and prism stages and then exposed to UVR, as described

below. Embryos were aerated and gently stirred using magnetic stir bars. Larvae were fed

with phytoplankton (Isochrysis galbana) grown in nutrient replete f/2 medium as the prism

stage was reached. No larvae were fed during the experiments and all experiments were

conducted at 10 jC for E. parma and 4–5 jC for S. droebachiensis. A temperature-

controlled water bath was used to maintain (F 0.5 jC) these seawater temperatures.

2.2. Embryo and larval experiments

Freshly fertilized embryos, blastula, gastrula, and prism stage larvae were exposed to

artificial visible and UVR using four UV-340 lamps (Q-Panel) and four F40 Sun lamps

(General Electric) suspended f 20 cm from the top of the filters (see below) to provide a

downwelling mixed field (visible and UVR) exposure. Glass beakers (400 ml) containing

f 5 individuals ml� 1 were used for testing the effects of UVR on embryos and larvae.

Three treatments were used to partition the effects of UV-B from UV-A and visible


radiation with three replicates per treatment. Schott WG and GG long pass filters (6� 6W)with nominal cutoffs (50% T) at 280, 320, and 400 nm were used to cover the beakers

containing 400 ml of embryos or larvae. Treatments were designated as UVO (GG 400

filter), UVA (WG 320 filter), and UVT (WG 280 filter). All experiments on embryos and

larvae tested were conducted for 3 days on a 12:12-h light/dark cycle. Using this design,

successive replicate experiments were carried out using embryos and larvae at different

developmental stages for S. droebachiensis, while only FFE were analyzed experimentally

for E. parma. During the 3-day experiments, samples were taken at T0, 30, 60, 180 min; 6,

12, 24, 48, and 72 h after fertilization for S. droebachiensis and E. parma to assess percent

survivorship under each treatment. Subsamples (10 ml) were fixed in 3% glutaraldehyde

and filtered (0.22 Am) seawater and counts of normally developed stages of development

were recorded as survivors using light microscopy. Percent survivorship for all treatments

was determined by comparing the counts of embryos or larvae for each sampling period

against the T0 counts. In addition, the developmental stage at the end of the experiment in

each treatment, and whether embryos and larvae were developing normally was recorded.

Digital light micrographs of S. droebachiensis were taken for each developmental stage

and for some stages during exposure to UVR. At the end of 3 days, all remaining embryos

or larvae were collected for additional analyses, as described below.

A duplicate set of experiments was run to determine the effects of UVR on delays in

cleavage for the FFE of S. droebachiensis and E. parma as described above with the

following exceptions. Preliminary experiments indicated that S. droebachiensis and E.

parma take from 2.5–3.0 h and 30–60 min, respectively, to begin their first mitotic

division, as indicated by the start of a cleavage furrow, at the experimental temperatures

described above. To assess the effects of UVR on delays in cleavage, samples of FFE were

taken every 15 min beginning at 105 min after fertilization for S. droebachiensis and every

10 min for E. parma immediately after fertilization until all FFE had cleaved and fixed to

determine the percent cleaved as a function of time as described by Adams and Shick

(1996). From 50 to 100 embryos were counted for each sample and the time at which FFE

for each species reached 50% cleavage was determined by a nonlinear regression fit of the

data using a logistic (exponential) equation.

2.3. Measurements of ultraviolet radiation

For laboratory experiments, UVR (UV-B and UV-A) and photosynthetically active

radiation (PAR: 400–700 nm) were measured using a wavelength and radiometrically

calibrated (NIST traceable standards) CCD spectrometer and fiber optics (Ocean Optics).

Three scans were taken and the mean reported was in units of W m� 2 nm� 1. Integrated

values of unweighted UVR (W m� 2) were calculated for each treatment and biologically

weighted irradiances (W m� 2) were obtained by multiplying the unweighted irradiance

with the DNA weighting function of Setlow (1974).

2.4. UV absorbing compounds (mycosporine-like amino acids)

Samples of embryos and larvae were analyzed for MAAs using the methods described

in Shick et al. (1995). Individual MAAs were separated with reverse-phase isocratic high


performance liquid chromatography (HPLC) on a Brownlee RP-8 column (Spheri-5: 4.6

mm ID� 25 cm) protected with an RP-8 guard column (Spheri-5: 4.6 mm ID� 3 cm)

using an aqueous mobile phase of 40–55% methanol (v/v) and 0.1% acetic acid at a flow

rate of 0.6 ml min� 1. Detection of peaks is by UVabsorbance at 313 and 340 nm. Identity

of peaks is accomplished using co-chromatography with known secondary standards

prepared from primary standards provided by Dr. Walt Dunlap via Dr. Deneb Karentz.

Quantification of individual peaks is accomplished using response factors calculated from

known amounts of standard. Soluble protein composition was assessed using Coomassie

brilliant blue, and bovine serum albumin as a standard (Bio-Rad Laboratories) to

normalize data.

2.5. Detection of DNA photoproducts using an enzyme-linked immunoabsorbent assay

Cyclobutane pyrimidine dimer (CPD) formation was measured using the procedures

and monoclonal antibody (TDM-2) of Mori et al. (1991). Embryo or larval genomic DNA

was isolated using commercially available kits (Easy-DNA, Invitrogen) and quality and

concentration determined spectrophotometrically using 260:280 nm ratios. Subsequently,

50 ng of DNA from each sample was used in an enzyme-linked immunoabsorbent assay

(ELISA) technique with TDM-2 as the primary antibody. An affinity purified goat anti-

mouse IgG secondary antibody conjugated with horseradish peroxidase was used and the

final color development read in flat-bottomed 96-well microtiter plates using a plate

reader (Bio-Rad) at 490 nm as described by Mori et al. (1991). As in Mori et al. (1991),

we present the absorbance results from the ELISA. Although differences between

treatments can be explored within an experiment, differences in DNA damage between

developmental stage experiments are confounded by differences in total DNA content,

cell shading in different stages, difference in rates of cell division between stages, as well

as differences in maternal versus zygotic transcript expression that are likely to affect rates

of photorepair.

2.6. Statistical analysis

Survivorship, cleavage delay, and CPD formation were statistically analyzed using a

one-way ANOVA at a significance level of 5%. No unequal variances were detected using

the Fmax test, and individual treatment differences were assessed using the Student–

Newman–Keuls (SNK) multiple comparison test. Where appropriate, ratios and percen-

tages were arcsine or log transformed for analysis and back transformed for presentation.

3. Results

Table 1 shows the UVR irradiances for each treatment along with the total dose for the

experiments on both the embryos and larvae of S. droebachiensis and E. parma. Year to

year differences were less than 3% and for all treatments, the irradiance of PAR was f 70

Amol quanta m� 2 s� 1. Additionally, the biologically effective irradiance (Table 1) for

each treatment was calculated using the DNA damage weighting function of Setlow

Table 1

Unweighted experimental irradiances (Wm� 2) and total dose (kJm� 2) for each treatment group and DNA

weighted irradiances and dose using the DNA damage biological weighting function of Setlow (1974)

UVR

(Wm� 2)

Dose

(kJm� 2)

UV-A

(Wm� 2)

Dose

(kJm� 2)

UV-B

(Wm� 2)

Dose

(kJm� 2)

Unweighted treatments

UVT 3.08 399 2.78 360 0.29 38

UVA 2.88 373 2.73 354 0.14 18

UVO 0.05 6.5 0.05 6.5 0.00003 0.004

Weighted treatments

UVT 6.934� 10� 7 9� 10� 5 6.898� 10� 9 1�10� 6 6.865� 10� 7 8.9� 10� 5

UVA 1.511�10� 7 2� 10� 5 5.735� 10� 9 1�10� 6 1.454� 10� 7 1.9� 10� 5

UVO 1.006� 10� 10 1.3� 10� 7 0.00 0.00 1.006� 10� 10 1.3� 10� 7

Visible radiation (400–700 nm) was 70 Amol quanta m� 2 s� 1 for all treatments.


(1974). The maximum unweighted irradiance of UV-B is equivalent to that measured

around the Isles of Shoals in the Gulf of Maine at a depth of 1–3 m in the spring.

The percent survival of sand dollar FFE in the UVA and UVT treatments rapidly

declined compared to UVO treatments (Fig. 1A). Both time and treatment were significant

(two-way ANOVA: P= 0.0001 on log transformed data) factors contributing to lower

survival. Multiple comparison testing showed that all treatment groups were significantly

different from one another (SNK: P < 0.05, Fig. 1A) and a majority of pairwise comparisons

for the sampling periods (29 of 36) were significantly different (SNK: P < 0.05) from one

another. Additionally, there was a significant interaction term between time and treatment

(ANOVA: P= 0.0001), indicating a dose dependence for sand dollar embryo survival when

exposed to UVR. On the final day of the experiment, embryos in the UVO treatment had

reached the prism stage of development, while the UVA treatment was in the early prism

stage and UVT treatments had just completed gastrulation. These delays in development

during exposure to UVR occurred despite the lack of any significant treatment effect on

time to 50% cleavage (ANOVA: P= 0.066; UVO: 48.3 minF 2.89 [SD], UVA: 52.3F 2.52

[SD], UVT: 54.3F 2.08 [SD]). It should be remembered that these delays in cleavage (Fig.

2A) are always measured on the survivors. Embryos exposed to UVR exhibit significant

mortality and the survivors are likely more resistant to the lethal effects of UVR. For the

FFE of S. droebachiensis, the percent survival of embryos in the UVA and UVT treatments

also declined rapidly compared to UVO treatments (Fig. 1B) but not as high as observed in

E. parma (Fig. 1A). Additionally, S. droebachiensis embryos did not exhibit as great a

sensitivity to UVA radiation as observed for E. parma. Both time and treatment were

significant (two-way ANOVA: P= 0.0001 on log transformed data) factors contributing to

lower survival, and multiple comparison testing showed that all treatment groups were

significantly different from one another (SNK: P < 0.05, Fig. 1B) and that a majority of

pairwise comparisons for the sampling periods (35 of 36) were significantly different

(SNK: P < 0.05) from one another. There was a significant interaction term between time

and treatment (ANOVA: P= 0.0001) indicating a dose dependence for urchin embryo

survival when exposed to UVR. On the final day of the experiment, embryos in the UVO

treatment were at the hatched blastula stage of development while the UVA treatment was

Fig. 1. (A) Mean survival (F S.D.) of sand dollar (E. parma) freshly fertilized embryos exposed to UVR in the

three treatment groups. (B) Mean survival (F S.D.) of sea urchin (S. droebachiensis) freshly fertilized embryos

exposed to UVR in the three treatment groups. Each curve represents the percent survivors after exposure to the

UVR irradiances in Table 1 over the 3-day duration of the experiment.


in the blastula stage and UVT treatments were in the early blastula stage. These delays in

development during exposure to UVR, unlike the delays observed in E. parma, were

accompanied by significant treatment effects on time to 50% cleavage (ANOVA:

P < 0.0001; UVO: 193.7 minF 3.22 [SD], UVA: 202.3F 2.52 [SD], UVT: 261.0F 3.61

[SD]).

Fig. 2. Developmental stages of the green sea urchin (S. droebachiensis) (A) first cleavage (f 175 Am) (B)

blastulae (f 200 Am), (C) gastrula (f 300 Am), (D) prism stage larvae (325–350 Am), (E) four arm pluteus larva

(f 550 Am), and (F) packed blastula (f 125 Am).



Only trace amounts of the MAA mycosporine-glycine were detected in the FFE of

embryos from E. parma which were too low and variable to be quantified accurately. In

the FFE of S. droebachiensis, large amounts of shinorine and porphyra-334 were observed

and trace amounts of mycosporine-glycine and palythine. Only shinorine and porphyra-

334 were quantified and analyzed. Neither shinorine (UVO; 82.2 nmol mg

protein� 1F1.05 [SD], UVA: 75.8F 6.61 [SD], UVT 86.7F 7.68 [SD]) nor porphyra-

334 (UVO: 562.6 nmol mg protein� 1F 56.80 [SD], UVA: 535.1F 21.32 [SD], UVT:

671.5F 121.69 [SD]) concentrations were significantly different (ANOVA: P>0.05) in

time zero samples of FFE.

Three additional survivorship experiments were conducted on the blastula, gastrula, and

prism stages of the green sea urchin S. droebachiensis. For the experiment on blastula (Fig.

2B), both time and treatment were significant (two-way ANOVA: P= 0.0001 on log-

transformed data) factors contributing to lower survival and multiple comparison testing

showed that all treatment groups were significantly different from one another (SNK:

P < 0.05, Fig. 3A) and that all pairwise comparisons for the sampling periods (36 of 36)

were significantly different (SNK: P < 0.05) from one another. There was a significant

interaction term between time and treatment (ANOVA: P= 0.0001) indicating a dose

dependence for blastula survival when exposed to UVR. Overall mortality in each

treatment group was less than that observed in the FFE experiment. On the final day of

the experiment, blastula in the UVO and UVA treatments was at the early gastrula stage

(Fig. 2C) of development while the UVT treatment were in the hatched blastula stage.

Survivorship results for the experiment on gastrula (Fig. 2C) were similar to those

observed on blastula. Both time and treatment were significant (two-way ANOVA:

P= 0.0001 on log transformed data) factors contributing to lower survival, and multiple

comparison testing showed that all treatment groups were significantly different from one

another (SNK: P < 0.05, Fig. 3B) and that all pairwise comparisons for the sampling

periods (36 of 36) were significantly different (SNK: P < 0.05) from one another. There

was a significant interaction term between time and treatment (ANOVA: P= 0.0001),

indicating a dose dependence for gastrula survival when exposed to UVR. Again, overall

mortality in each treatment group was less than that observed in the FFE experiment and

similar to the blastula experiment. On the final day of the experiment, gastrula in the UVO

and UVA treatments were at the late gastrula and early prism stage of development while

the UVT treatment was in the late gastrula stage. Lastly, the survivorship results for the

experiment on prism stage larvae (Fig. 2D) were again similar to those observed on

blastula and gastrula. Both time and treatment were significant (two-way ANOVA;

P= 0.0001 on log transformed data), and multiple comparison testing showed that all

treatment groups were significantly different from one another (SNK: P < 0.05, Fig. 3C)

and that all pairwise comparisons for the sampling periods (36 of 36) were significantly

different (SNK: P < 0.05) from one another. There was a significant interaction term

between time and treatment (ANOVA: P= 0.0001) indicating a dose dependence for prism

larvae survival when exposed to UVR. Overall mortality in each treatment group was less

than that observed in the FFE experiment and similar to experiments on other devel-

opmental stages. On the final day of the experiment, prism larvae in the UVO and UVA

treatments were at the four-arm pluteus stage (Fig. 2E) of development while the UVT

treatment was in the late prism, early four-arm plutei stage.

Fig. 3. Mean survival (F S.D.) of the green sea urchin (S. droebachiensis) (A) blastulae, (B) gastrula, and (C)

prism stage larvae exposed to UVR in the three treatment groups. Each curve represents the percent survivors

after exposure to the UVR irradiances in Table 1 over the 3-day duration of the experiment.


Fig. 4. DNA damage (meanF S.D.), measured as cyclobutane pyrimidine dimmers (CPDs) of green sea urchin

(S. droebachiensis) embryos and larvae exposed to UVR in the three treatment groups. Treatment groups with

common superscripts are not significantly different from one another using multiple comparisons testing (SNK)

at a significance level of 0.05%.


At the end of each experiment, CPD photoproducts were measured, as described above.

All experiments exhibited significant (ANOVA: P < 0.05 on log-transformed values)

treatment effects on CPD formation (Fig. 4). For FFE, blastula, and gastrula, multiple

comparison testing of CPD formation revealed that all treatment groups were significantly

different from one another (SNK: P < 0.05, Fig. 4) and that UVT treatments in all cases

exhibited higher concentrations of CPDs followed by UVA and UVO treatments. For the

prism stage experiments, the UVO and UVA treatments were indistinguishable from each

other (SNK: P>0.05) but significantly different (SNK: P < 0.05) than the UVT treatment

(Fig. 4) that exhibited the highest concentration of CPDs.

4. Discussion

The sea urchin, S. droebachiensis, and the sand dollar, E. parma, are common

echinoderms found in the Gulf of Maine. These benthic echinoderms inhabit hard and

soft-bottom environments from shallow to deep-water in the GOM. The depth distribution of

these species in the shallow subtidal is relevant to the studies reported here. UVR penetration

to depths of 7–12 m has been reported in the GOM. Both of the target species have

significant populations at these depths where their principal food sources, macrophytes and

phytoplankton, are available. It is not unreasonable to hypothesize that the buoyant,

swimming, or vertically advected planktonic phases of these benthic echinoderms are

exposed to UVR during the spring and early summer in the GOM, the amount of which will


be greatly affected by their depth of occurrence, and the stability and optical properties of the

water column. In this regard, an important result of the experiments described above is the

dose dependency of the decrease in survivorship with time. This has important consequences

for embryos and larvae in the plankton whose residence time within the upper portion of the

water column (< 10 m) is controlled by physical processes. An important component of

assessing the effects of a longer residence time in the upper portion of the water column is

whether reciprocity holds, that is, does a short-term exposure to high irradiances to UVR

have the same effect as a longer-term, lower irradiance exposure where the total dose is

equivalent.

Previous work on echinoid embryos and larvae has shown significant effects of UVR

on cell division, development, morphology, and DNA damage (Rustad, 1960; Eima et al.,

1984; Akimoto and Shiroya, 1987a,b). However, all of these studies utilized artificial light

sources containing germicidal UV-C (254 nm) radiation that is not ecologically relevant, as

UVR below 290 nm does not reach sea level. Recent studies by Adams and Shick (1996,

2001) have confirmed the occurrence of some of these effects on urchin embryos when

exposed to environmentally relevant wavelengths of UV-A and UV-B. Additionally, the

work reported here also confirms the adverse effects of environmentally relevant UVR on

echinoid survivorship, development, morphology, and DNA damage. Freshly fertilized

embryos of S. droebachiensis and E. parma, which have markedly different concentrations

of MAAs, exhibit clear species-specific differences in survivorship. Surprisingly, there

were no significant effects of UVR on cleavage delay in E. parma. One interpretation of

this result is that cleavage delay was measured on the low number of highly resistant

survivors in the experiment. Action spectra for the effect of UVR on cleavage delay in a

conspecific of the green sea urchin, Strongylocentrotus purpuratus, showed that UVR of

302 nm caused greater delays in cell division than the UVR of either 313 nm or 366 nm

(Geise, 1939). These results are similar to the results reported here for UVR effects on

cleavage delay in S. droebachiensis. On a daily basis, the unweighted and weighted

irradiances of UV-B used in this study were greater than that reported in Adams and Shick

(2001) but the proportional amounts of visible and UV-A, which are important in the

photorepair of DNA damage (Smith, 1989), are higher. The exposures described here also

simulate irradiances of UVR measured in the shallow (1–3 m) waters of the Gulf of Maine

in the spring which these embryos and larvae are likely to be exposed to.

For the subsequent developmental stages, blastula, gastrula, and prism, exposure to

UVR also resulted in significant decreases in survivorship but not as high as observed for

FFE and most of which can be explained by exposure to UV-B versus UV-A radiation. The

two dominant MAAs in all of the experiments were shinorine and porphyra-334 that

absorb maximally at 334 nm. Even though MAAs have broad absorption spectra

extending into the UV-B portion of the spectrum, and very high molar extinction

coefficients, the effects of UV-B in these experiments are significant for all developmental

stages. Previous work has shown that MAAs are invariant during development up to the

pluteus stage (Adams and Shick, 1996) or when exposed to UVR (Adams and Shick,

2001), and can have has high as an 86% efficiency of self-shading at 334 nm with high

MAA concentrations, as reported above. This suggests that the embryos and larvae in the

experiments reported here, up to the prism stage, have relatively stable MAA concen-

trations and should provide some protection from UVR if present.


During the experiments reported above only normally developing embryos and larvae

were counted as survivors because of the assumption that abnormal embryos and larvae

would not complete their developmental program. Beyond any delay in early cleavage,

there were also delays in the remainder of the developmental program when exposed to

UVR. Qualitative observations of the abnormal embryos showed similar types of abnormal

embryos as described by Adams and Shick (2001) including ‘‘packed blastula’’ (Fig. 2F),

exogastrula, and abnormal larvae. In contrast, the percentage of abnormal embryos or

larvae never exceeded 20% of the survivors at the end of the experiment, while Adams and

Shick (2001) report as high as 50% abnormal development in embryos with low

concentrations of MAAs and 10–15% abnormal development in embryos with high

concentrations of MAAs.

The DNA damage, measured as CPD photoproducts, observed in these experiments is

the direct result of exposure to UVR. DNA damage was not linearly related to survivorship

in any developmental stage. That is, in early developmental stages, it appears that small

amounts of DNA damage had greater effects on survivorship compared to the larger

amounts of DNA damage observed in later developmental stages. One reason for this

could be that differences in repair capacity exist as a result of differing amounts of

maternally derived transcripts of important repair enzymes such as photolyase compared to

the available zygotic transcripts in later developmental stages. Additionally, later stages of

development may be more tolerant of accumulating DNA damage than early stages (Epel

et al., 1999). Also, DNA damage may have also been caused indirectly by exposure to

UVR through the photodynamic production of ROS to a greater degree in early embryos

that resulted in decreased survivorship (Imlay and Linn, 1988). Either type of DNA

damage can lead to apoptosis if repair is not possible or delays in cell division while repair

is taking place (Paulovich et al., 1997). p53 can initiate cell cycle delays through either the

G1/S or G2/M checkpoints (Geyer et al., 2000; Innocente et al., 1999). Cleavage delays in

the FFE of S. droebachiensis and E. parma are the result of exposure to UVR. The

evidence presented here supports DNA damage as a precursor to those delays in cell

division and suggests the involvement of cell cycle genes in the observed delays. Delays in

development in the experiments on other developmental stages could be the result of the

time required to repair UVR-induced DNA damage, or the result of direct damage to

essential genes in the developmental program that are not repaired (Epel et al., 1999).

These factors, and our interpretation of experimental results, require us to put the effects of

UVR on DNA damage and subsequent delays in cell division in the context of the

equilibrium between damage and repair. Many cells of a developing embryo may require

the repair of all DNA damage or the repair of just enough DNA damage to proceed with

the developmental program (Epel et al., 1999). For fast developing embryos, such as sea

urchins, many cells may incur DNA damage that cannot be repaired but instead leads to

apoptosis and the removal of those cells. Sea urchin embryos may be able to tolerate the

loss of these cells and still complete their developmental program (Epel et al., 1999).

If DNA damage is the principal insult to developing embryos exposed to ecologically

relevant UVR, we should develop biological weighting functions to better compare our

experimental exposures to UVR, and to determine whether the irradiances of UVR in very

dynamic temperate waters will have similar effects on developing embryos in situ.

Weighting functions will also tell us something about the efficacy of MAAs in preventing


DNA damage. Lastly, an examination of the genes involved in repair of DNA damage

(e.g., photolyase) and the differential expression of cell cycle genes will provide critical

insights into the effects of UVR on the cell cycle in developing echinoids.

Acknowledgements

Technical assistance from Mary Maravic is gratefully acknowledged, and a special

thanks to Dr. Toshio Mori for generously supplying the monoclonal antibodies to CPD.

This project was supported by the National Science Foundation (Biological Oceanography

Program, OCE-9818918). [SS]

References

Adams, N., 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., 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.

Akimoto, A., Shiroya, T., 1987a. Photoreversability of UV-induced thymine dimmers and abnormal morpho-

genesis in sea-urchin embryos. Photochem. Photobiol. 45, 403–406.

Akimoto, A., Shiroya, T., 1987b. Photoreversal of abnormal morphogenesis in sea-urchin embryos caused by

UV-radiation. Photochem. Photobiol. 45, 407–412.

Asada, K., Takahashi, M., 1987. Production and scavenging of active oxygen in photosynthesis. In: Kyle, D.J.,

Osmond, C.B., Arntzen, C.J. (Eds.), Photoinhibition. Elsevier, Amsterdam, pp. 228–287.

Banaszak, A.T., Lesser, M.P., Kuffner, I.B., Ondrusek, M., 1998. Relationship between ultraviolet (UV) radiation

and mycosporine-like amino acids (MAAS) in marine organisms. Bull. Mar. Sci. 63, 617–628.

Carroll, A.K., Shick, J.M., 1996. Dietary accumulation of UV-absorbing mycosporine-like amino acids (MAAs)

by the green sea urchin (Strongylocentrotus droebachiensis). Mar. Biol. 124, 561–569.

Cullen, J.J., Neale, P.J., Lesser, M.P., 1992. Biological weighting function for the inhibition of phytoplankton

photosynthesis by ultraviolet radiation. Science 258, 646–651.

Damkaer, D.M., Dey, D.B., 1983. UV damage and photoreactivation potentials of larval shrimp, Pandalus

platyceros, and adult euphausiids Thysanoessa raschii. Oecologia 60, 169–175.

Dey, D.B., Damakaer, D.M., Heron, G.A., 1988. Dose/dose rate responses of seasonally abundant copepod of

Puget Sound. Oecologia 76, 321–329.

Dykens, J., Shick, J.M., Benoit, C., Buettner, G.R., Winston, G.W., 1992. Oxygen radical production in the sea

anemone Anthopleura elegantissima and its endosymbiotic algae. J. Exp. Biol. 168, 219–241.

Eima, Y., Ikenaga, M., Shiroya, T., 1984. Action spectrum for photoreactivation of ultraviolet-induced morpho-

logical abnormality in sea urchin eggs. Photochem. Photobiol. 40, 461–464.

Epel, D., Hemela, K., Shick, M., Patton, C., 1999. Development in the floating world: defenses of eggs and

embryos against damage from UV radiation. Am. Zool. 39, 271–278.

Franklin, L.A., Forester, R.M., 1997. The changing irradiance environment: consequences for marine macrophyte

physiology, productivity and ecology. Eur. J. Phycol. 32, 207–232.

Fridovich, I., 1986. Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247, 1–11.

Geise, A.C., 1939. The effects of ultra-violet radiations of various wave-lengths upon cleavage of sea urchins.

Biol. Bull. 75, 238–247.

Geyer, R.K., Nagasawa, H., Little, J.B., Maki, C.G., 2000. Role and regulation of p53 during ultraviolet

radiation-induced G1 cell cycle arrest. Cell Growth Differ. 11, 149–156.

Gleason, D.F., Wellington, G.M., 1993. Ultraviolet radiation and coral bleaching. Nature 365, 836–838.

Gleason, D.F., Wellington, G.M., 1995. Variation in UV-B sensitivity of planula larvae of the coral Agaricia

agaricites along a depth gradient. Mar. Biol. 123, 693–703.


Graeber, A.G., Osmanian, C., Jack, T., Housman, D.E., Koch, C.J., Lowe, S.W., Graccia, A.J., 1996. Hypoxia-

mediated selection of cells with diminished apoptotic potential in solid tumors. Nature 379, 88–91.

Griffiths, S.D., Clarke, A.R., Healy, L.E., Ross, G., Ford, A.M., Wyllie, A.H., Greaves, M., 1997. Absence of p53

permits propagation of mutant cells following genotoxic damage. Oncogene 14, 523–531.

Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford Science Publications,

London. 936 pp.

Herndl, G.J., Muller-Niklas, G., Frick, J., 1993. Major role of ultraviolet-B in controlling bacterioplankton

growth in the surface layer of the ocean. Nature 361, 717–719.

Imlay, J.A., Linn, S., 1988. DNA damage and oxygen radical toxicity. Science 240, 1302–1309.

Innocente, S.A., Abrahamson, J.L.A., Cogswell, J.P., Lee, J.M., 1999. p53 regulates a G2 checkpoint through

cyclin B1. Proc. Natl. Acad. Sci. U. S. A. 96, 2147–2152.

Jokiel, P.L., York Jr., R.H., 1984. Importance of ultraviolet radiation in photoinhibition of microalgal growth.

Limnol. Oceanogr. 29, 192–199.

Karansas, J.J., van Dyke, H., Worrest, R.C., 1979. Mid-ultraviolet (UV-B) sensitivity of Acartia clausii Gies-

brecht (Copepoda). Limnol. Ocenogr. 24, 1104–1116.

Karentz, D., McEuen, F.S., Land, M.C., Dunlap, W.C., 1991. Survey of mycosporine-like amino acid compounds

in Antarctic marine organisms: potential protection from ultraviolet exposure. Mar. Biol. 108, 157–166.

Kirk, J.T.O., 1994. Optics of UV-B radiation in natural waters. Arch. Hydrobiol. Ergeb. Limnol. 43, 1–16.

Lesser, M.P., 1996. Exposure of symbiotic dinoflagellates to elevated temperatures and ultraviolet radiation

causes oxidative stress and inhibits photosynthesis. Limnol. Oceanogr. 41, 271–283.

Lesser, M.P., 1997. Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs

16, 187–192.

Lesser, M.P., Lewis, S., 1996. Action spectra for the inhibition of photosynthesis by ultraviolet radiation in the

hermatypic coral, Pocillopora damicornis. Mar. Ecol. Prog. Ser. 134, 171–177.

Lesser, M.P., Neale, P.J., Cullen, J.J., 1996. Acclimation of Antarctic phytoplankton to ultraviolet radiation: UV

absorbing compounds and carbon fixation. Mol. Mar. Biol. Biotechnol. 5, 314–325.

Lesser, M.P., Farrell, J.H., Walker, C.W., 2001. Oxidative stress and p53 expression in the larvae of Atlantic cod

(Gadus morhua) exposed to ultraviolet (290–400 nm) radiation. J. Exp. Biol. 204, 157–164.

Madronich, S., McKenzie, R.L., Bjorn, L.O., Caldwell, M.M., 1998. Changes in biologically active ultraviolet

radiation reaching the Earth’s surface. Photochem. Photobiol., B. 46, 5–19.

Malloy, K.D., Holman, M.A., Mitchell, D., Detrich III, H.W., 1997. Solar UV-B-induced DNA damage and

photoenzymatic DNA repair in Antarctic zooplankton. Proc. Natl. Acad. Sci. U. S. A. 94, 1258–1263.

Mori, T., Nakane, M., Hattori, T., Matsunaga, T., Ihara, M., Nikaido, O., 1991. Simultaneous establishment of

monoclonal antibodies specific for either cyclobutane pyrimidine dimers or (6–4) photoproduct from the

same mouse immunized with ultraviolet radiated DNA. Photochem. Photobiol. 54, 225–232.

Paulovich, A.G., Toczyski, D.P., Hartwell, L.H., 1997. When checkpoints fail. Cell 88, 315–321.

Peak, M.J., Peak, J.G., 1990. Hydroxyl radical quenching agents protect against DNA breakage caused by both

365-nm and gamma radiation. Photochem. Photobiol. 51, 649–652.

Pennington, J.T., Emlet, R.B., 1986. Ontogenetic and diel vertical migration of a planktonic echinoid larva,

Dendraster excentricus: occurrence, cause, and probable consequences. J. Exp. Mar. Biol. Ecol. 104, 69–95.

Renzing, J., Hansen, S., Lane, D.P., 1996. Oxidative stress is involved in the UV activation of p53. J. Cell Sci.

109, 1105–1112.

Rustad, R.C., 1960. Changes in the sensitivity to ultraviolet-induced mitotic delay during the cell division cycle

of the sea urchin egg. Exp. Cell Res. 21, 596–602.

Setlow, R.B., 1974. The wavelengths in sunlight effective in producing skin cancer: a theoretical analysis. Proc.

Natl. Acad. Sci. U. S. A. 71, 3363–3366.

Shick, J.M., Dunlap, W.C., 2002. Mycosporine-like amino acids and related gadusols: biosynthesis, accumula-

tion, and UV-protective functions in aquatic organisms. Annu. Rev. Physiol. 64, 223–262.

Shick, J.M., Lesser, M.P., Dunlap, W.C., Stochaj, W.R., Chalker, B.E., Wu Won, J., 1995. Depth-dependent

responses to solar ultraviolet radiation and oxidative stress in the zooxanthellate coral Acropora microphthal-

ma. Mar. Biol. 122, 41–51.

Shick, J.M., Lesser, M.P., Jokiel, P.L., 1996. Effects of ultraviolet radiation on corals and other coral reef

organisms. Global Change Biol. 2, 527–545.


Smith, R.C., 1989. Ozone, middle ultraviolet radiation, and the aquatic environment. Photochem. Photobiol. 50,

459–468.

Smith, R.C., Baker, K.S., 1989. Stratospheric ozone, middle ultraviolet radiation and phytoplankton productivity.

Oceanogr. Mag. 2, 4–10.

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, 952–959.

survivorship, development, and dna damage in echinoderm embryos and larvae exposed to ultraviolet...

Documents