Unger: Photoprotection in Ascidia
ABSTRACT
Background: A photoprotective role has been ascribed to the ultraviolet radiation-absorbing
mycosporine-like amino acids (MAAs) present in the follicle cells surrounding eggs of the
tunicate Ascidia ceratodes.
Approach: Studies were performed using natural sunlight and a variety of wavelength filters to
expose intact Ascidia eggs and eggs in which the MAA-containing follicle cells had been
removed to various types of ultraviolet radiation. The kinetics of development were examined
to ascertain whether the follicle cells were acting to protect the eggs from UV damage.
Results: This series of studies demonstrated that at ambient solar radiation levels there are no
observable differences in rates of early development between Ascidia embryos where follicle
cells were removed and embryos with an intact follicle cell layer.
Conclusions: This study suggests that the tunicate embryo has a variety of defenses against UV
damage, and the defensive role played by mycosporine-like amino acids may not be of primary
importance.
INTRODUCTION
The bulk of the ultraviolet radiation that penetrates the Earth's atmosphere is
traditionally classified into UVA, with wavelengths between 400 and 320 nanometers, and
UVB, with wavelengths between 320 and 280 nm (Chrispeels 1996). The biological relevance
of such radiation to marine organisms was first recognized in the middle of this century (Koller
168). At this time, it was also discovered that UV radiation can penetrate into seawater to
depths of 20 meters or more (Jerlov 1950).
It is in this UV-exposed water column that eggs of the solitary ascidian Ascidia
ceratodes are fertilized and undergo their early development. Ascidian eggs do not
immediately sink out of the range of UV-irradiation; they are buoyed by their surrounding layer
of follicle cells (Lambert and Lambert, 1978). These follicle cells are also thought to be
Unger: Photoprotection in Ascidia
responsible for shielding the egg from potentially damaging wavelengths of UV radiation
through the use of UV-absorbing sunscreen molecules known as mycosporine-like amino acids,
or MAAs (Hemela 1993)
The mycosporine compounds present in the follicle cells of A. ceratodes are found in
other marine taxa ranging from the Antarctic to temperate and tropical waters (Karentz 1991;
Shick et al 1992) including the eggs of marine invertebrates (Chioccara et al 1986). It is
believed that most marine taxa cannot synthesize these mycosporine-like amino acids (Karentz
et al) and so it is generally assumed that mycosporines are either accumulated dietarily as in
green sea urchin eggs (Carroll and Shick 1996), or else they are obtained from algal or fungal
symbionts. As noted, these MAAs might play a UV-protective role, a theory that gained
support in 1995 when Shick et al noted a pattern of increasing UV sensitivity and decreasing
concentrations of mycosporine-like amino acids with increasing depth of occurrence in the
zooxanthellate coral Acropora microphthalma.
Removal of A. ceratodes follicle cells results in a noticeable cleavage delay and delay to
tadpole hatching when embryos are irradiated with a UV lamp in the laboratory (Hemela 1993)
Adams and Shick in 1996 described a similar trend in the green sea urchin: a cleavage delay
was observed in UV-irradiated urchin eggs with few dietarily-supplied mycosporines when
compared to urchin eggs from a female fed a diet abundant in MAAS.
A problem with the above two studies is that both were performed in the absence of
visible light, which prevented any DNA repair by the action of the photorepair enzyme
photolyase. This enzyme, first noticed in Arabacia eggs in 1949 by Marshak, absorbs radiation
in the range of visible blue light and uses this energy for the photorepair of pyrimidine dimers
formed when DNA is irradiated with UVB (Todo et al 1996).
The research described here addresses how applicable the previous research on UV-
induced developmental delays is to organisms living in their natural setting, i.e. where they
experience only the levels of radiation present in natural sunlight. The studies done by Adams
Unger: Photoprotection in Ascidia
and Shick and by Hemela both used UV lamps as a source of ultraviolet radiation, which
provide a higher dose of radiation than would typically be experienced by an animal living in
the sunshine. More importantly, the embryos in their studies were exposed only to ultraviolet
radiation so as to accentuate any damage; no visible light was present, which would have
activated DNA repair by the photolyase system.
The goal of this research was to compare delays in time to early cleavage as well as
time to development to the tadpole hatching stage between embryos that retained the protection
afforded by the MAAs present in their follicle cells and in embryos where the MAA-containing
follicle cells had been removed. The embryos were exposed only to the radiation provided by
the midday spring sun.
My research demonstrated that there are no differences in either cleavage times or time
to tadpole hatching between intact or defolliculated eggs when natural sunlight is the UV
source. Two trials examining cleavage delay failed to turn up any significant mycosporine
effects, and of three tadpole hatching trials only the first demonstrated any effects of the follicle
cells, which were not repeatable in two subsequent studies. These results indicate that the
mycosporines in Ascidia follicle cells are not the only defense against ultraviolet radiation that
the embryos possess, and that these alternate defenses can compensate for the loss of the
putative follicle cell protection.
MATERIALS AND METHODS
Collection
Specimens of Ascidia ceratodes were collected from approximately the mean low-low
tide level of the pilings supporting Fisherman's Wharf in Monterey, California. The animals
were stored in an aquarium continuously supplied with filtered aerated seawater from Monterey
Bay for a maximum of ten days.
Unger: Photoprotection in Ascidia
To obtain gametes, an incision was made along the tunic on the animal's ventral side
through which the animal was removed from its tunic. The red oviduct was then punctured
with a 25 gauge syringe needle and the eggs transferred with a glass pipet directly into seawater
adjusted to pH 6.0 with 1.0 and 0.1 MHCl. The eggs were incubated at pH 6.0 for one hour at
15°C and then pelleted in a hand centrifuge and washed three times with filtered seawater.
Eggs not used immediately were stored in filtered seawater for a maximum of twelve hours at
15°C. Sperm were collected undiluted with seawater by puncture of the white sperm duct and
transfer to a 1.5 mL Eppendorf tube with a glass pipet. Sperm were stored for a maximum of
24 hours at 4°
Defolliculation
Eggs were typically harvested from several individuals in the late afternoon or early
evening and left overnight at 15°C. Between 9 and 10:30 AM the following morning the
settled eggs were thoroughly resuspended and divided between two 12 mL conical glass
centrifuge tubes. They were then spun down in a hand centrifuge, and the resulting supernatant
was aspirated away to leave a slurry of approximately 2 to 3 mL egg suspension in each
aliquot. One aliquot of eggs was left intact, while the other was transferred to a 30 mL beaker
Next the follicle cells were removed by slowly running the slurry through a 22-gauge syringe
10 to 15 times. The eggs were monitored under a microscope during the defolliculation
process to assay levels of damage and follicle cell removal. The process was continued until
approximately 70% of the follicle cells had been removed. The eggs were combined with 10-
12 mL seawater in a glass centrifuge tube and both the defolliculated and intact eggs were spun
down, aspirated, and washed with filtered seawater three times. Finally the eggs were diluted
in seawater to a final concentration of approximately 1 to 3%.
UV Treatments
A water bath cooled with circulating seawater at ambient seasonal water temperature of
15-18°C was divided into three sections and used as an outdoor chamber for UV exposure.
Unger: Photoprotection in Ascidia
Each section was covered with a different filter: what shall subsequently be referred to as
receiving no UV was covered with a sheet of a transparent plastic known as OP3, which does
not transmit wavelengths below 400 nm. The UVAB portion of the chamber was filtered with
a similar material, UVT, which is completely UV transparent. The covering for the UVA
section consisted of a sheet of UVT and a thin sheet of mylar, which only transmitted
wavelengths above 320 nm. Eggs were placed in the chamber on clear to partly cloudy days
between 10 and 11 AM and left for varying lengths of time as indicated. The chamber was
oriented with the long axis of the water bath running east and west. The partitions were
oriented in a north to south direction so that each section received comparable amounts of
direct sunlight from sunrise until approximately 1:30 P.M., at which time the chamber was
blocked from direct sunlight by a nearby building.
Cleavage Delay
Intact and defolliculated eggs from 6 to 8 individuals were collected and each aliquot
was diluted to a 30 mL final volume. Sperm motility was activated by diluting 3 mL dry sperm
into 1.5 mL 0.003 M Tris seawater adjusted to pH 9.2 with NaÖH, and the resulting sperm
suspension was checked for motility under a dark field microscope at 100x magnification 5
minutes after activation. Intact and defolliculated eggs were each fertilized in one large batch
with 600 mL of sperm suspension a maximum of 20 minutes after sperm activation. The
embryos were then transferred to 5 mL petri dishes. Petri dishes were prepared to prevent
sticking with a rinse in a 0.1% gelatin and 0.1% formaldehyde solution followed by two rinses
with deionized water and one rinse with filtered seawater. 5 mL aliquots of fertilized egg
suspension were placed into each petri dish and 2 replicate dishes of each folliculation
treatment were exposed to each of the three treatments in the UV chamber. In the first
experimental trial the eggs were fixed in a 1% final concentration of formaldehyde at 1 hour
and 55 minutes after fertilization, while in the second trial the eggs were allowed to develop for
only 90 minutes before fixation. For the first trial, 500-800 embryos exposed to each treatment
Unger: Photoprotection in Ascidia
were scored for development to the 1-, 2-, 4-, and 8- cell stage, while in the second trial as
many as 2500 embryos per treatment were scored to compensate for an unusually low
percentage of developing embryos.
Total minutes of development reached by embryos exposed to each treatment were
calculated by multiplying the proportion of embryos at the 2-cell stage by 60 minutes, the
proportion at the 4-cell stage by 90 minutes, the proportion at the 8-cell stage by 120 minutes.
and then summing the three terms to calculate the total number of minutes of development
represented by each sample. Embryos at the 1-cell stage were not included in data analysis due
to the inability to distinguish whether these eggs experienced a developmental delay, or were
simply unfertilized.
Tadpole Hatching
For the first experimental trial, which included just one time point, intact and
defolliculated eggs were left for 24.5 hours in the UV chamber in petri dishes treated with
gelatin and formaldehyde as described above. Samples were then scored for percent of total
eggs that hatched into tadpoles within that time period. The second trial also included just one
time point, where the ascidians were fixed 23 hours and 40 minutes after fertilization. The
third trial included four time points: two replicate dishes of each treatment were scored at 21
hours and 45 minutes after fertilization, then replicate dishes were scored at 22 hours 15
minutes, 23 hours 15 minutes, and 24 hours and 45 minutes. Untreated petri dishes were used
for the second and third trials.
MAAs in Floaters and Sinkers
Methanol Extraction
Eggs were collected from 2 individuals for the first experimental trial and 6 individuals
for the second trial. Eggs from each individual were stored separately overnight in filtered
seawater at 15°C. (The pH 6.0 treatment was not used for this series of experiments.) The
eggs were spun down in conical glass centrifuge tubes and then allowed to settle for
Unger: Photoprotection in Ascidia
approximately one hour until all eggs had settled into the pellet. The seawater was then
aspirated away, leaving a final volume of eggs and water that totaled approximately 1.2 mL.
To separate floaters from sinkers, this slurry was carefully pipetted with a wide-
mouthed 10 mL glass pipet into 40 mL of filtered seawater in a 50 mL conical plastic
centrifuge tube. The eggs were allowed to settle for 5 minutes, after which time the water
above the 5 mL mark was collected into conical glass centrifuge tubes and classified as
"floaters," while the bottom 5 mL were transferred to another centrifuge tube and labeled as
"sinkers.
In the second experimental trial the procedure was followed as above with the
following additional steps. The sinkers from the first separation were added to another 40
mL of seawater and again allowed to settle; the floaters from this second settling were
pooled together with the floaters from the first settling
All samples of both the floaters and the sinkers were spun in a hand centrifuge and then
allowed to settle until all eggs had sunk. Next most of the seawater was aspirated from
each sample and the eggs were transferred to 1.5 mL Eppendorf tubes with a glass pipet.
The samples were again spun in a hand centrifuge and allowed to settle completely. As
much seawater as possible was aspirated from the eggs, and 1 mL methanol at 15°C was
added to each sample.
The eggs were allowed to extract in the methanol for one hour at 15°C, where each was
resuspended four times over the course of the hour by inversion of the tube. At the end of
the hour the samples were spun down in a hand centrifuge and 900 mL of the methanol
from each sample were transferred to Eppendorf tubes. Another 1 mL of methanol at 15°C
was added to each sample and the extraction process was repeated. For assaying protein in
the pellet, residual methanol was removed from each sample and discarded. 0.5 mL of hot
(50-60°C) IN NaÖH was added to each pellet, vortexed, and left overnight to dissolve for
use in the protein assay (see below).
Unger: Photoprotection in Ascidia
Spectrophotometry
Only the first methanol extract was scanned in the spectrophotometer. A wavelength
scan was performed on each sample using a background standard of 90% methanol.
Absorbence between -0.05 and 0.3 absorbence units was measured for the first trial; while
for the second, absorbence was measured between -0.05 and 0.5 units to account for the
increased absorbence due to pooling of the two collections of floaters. The scan was
performed between 200 and 600 nm. Samples were diluted 2-, 3-, or 5-fold with 100%
methanol if they exceeded this absorbence range. Integration of the area enclosed beneath
the absorbence curve between 300 and 400 nm was calculated by the spectrophotometer
computer program. This integration value was multiplied by a dilution factor if the sample
had been diluted and standardized to the amount of protein present in the original sample as
determined by a protein assay (see below).
Protein Assay
A BCA assay for protein content was performed on each of the methanol-extracted egg
samples as described by Brown et al (1989), with several modifications. The samples left
overnight in IN NaÖH were heated to 60°C in a water bath and vortexed extensively to
dissolve the pellet. 10 mL and 20 mL aliquots of each of the floater protein samples, and 5
mL and 10 mL aliquots of each sinker sample were combined with enough IN NaÖH to
bring the volume to 20 mL. 980 mL deionized water were then added to each sample.
BSA standards were prepared using 20 mL IN NaÖH and 0, 10, 25, 50, and 100 mL of a 10
mg/mL solution of BSA in water. Deionized water was added to achieve a final volume of
1 mL for each standard.
10 mL of each sample or standard were combined in Eppendorf tubes with 990 mL
deionized water and 0.1 mL 0.15% DOC. Samples were incubated for 10 minutes at room
temperature and then 0.1 mL 72% TCA was added to each sample or standard. The tubes
were vortexed and then centrifuged at 13,000 rpm for 15 minutes. The resulting
Unger: Photoprotection in Ascidia
supernatant was aspirated and each pellet was resuspended in 100 mL of a 5% SDS, O.1 N
NaOH solution.
200 mL of working reagent were added to an appropriate number of wells of a flat-
bottomed 96-well plate and 10 mL of each sample or standard were added to triplicate
wells and mixed with pipet tips. The plate was then incubated at 60°C for between 90
minutes and 2 hours, until a good linear trend developed for the standards when read at 550
nm by a microplate reader.
Data Analysis
The integration value for each sample was multiplied by the appropriate dilution factor and
the resulting quantity was divided by mg protein present in the original sample as
determined by the BCA protein assay.
Integration X Dilution factor
Relative absorbence per milligram protein -
—----.
—---
mg protein
RESULTS
Cleavage Delay
Two experimental trials measured the effect of exposure to various wavelengths of UV
on early cleavage times. Intact and defolliculated eggs were fertilized, immediately placed in
petri dishes and exposed to solar UVA or UVA and UVB. Controls were placed under a filter
that did not transmit any ultraviolet radiation. The embryos were allowed to develop until a
test dish indicated that embryos were beginning to undergo cleavage to the 8-cell stage, at
which time all embryos were fixed in formaldehyde. Samples were then scored to determine
the distribution of embryos that had developed to the 1-,2-, 4- and 8-cell stage. Embryos at the
1-cell stage were not included in the data analysis as unfertilized eggs could not be
distinguished from embryos that had been fertilized but had not developed beyond the 1-cell
stage.
Unger: Photoprotection in Ascidia
The time of fixation in the two experiments was based on a time of development assay
on a test sample performed previous to the UV-exposure trials. This test indicated that
embryos first began to cleave into 8 cells (third division) at 125 minutes after fertilization when
incubated at 15°C. It was important that the embryos be fixed just as this third cell division
was taking place, so that any delays in development would be easily observed.
For the first trial, embryos were fixed at 115 minutes after fertilization, but as the
temperature in the chamber unexpectedly to 18°C, they developed more rapidly than expected
as indicated by the test development assay. As seen in Figures 1-3, these embryos were mostly
in the 8-cell stage by the time they were fixed. Figure 3 also shows that embryos exposed to
both UVA and UVB were slightly more abundant at the 4-cell stage when compared to the
control embryos and the embryos exposed only to UVA: 7% of intact and 16% of
defolliculated embryos exposed to UVA and B had not yet undergone the third division.
whereas the embryos exposed to UVA and the control group were 5 to 10% lower.
These results indicate a slight developmental delay may have been experienced by
embryos exposed to UVA and B, suggesting that UV-induced damage may have slowed the
rate of cleavage. However, the observed developmental delay was not affected by the presence
or absence of the mycosporine-containing follicle cells. The developmental delay is not as
apparent when expressed as minutes of development. There are only slight differences in
average length of development in different sets of embryos and no trend is apparent (see Table
In the second experiment, the distributions of the division stages are centered around
the 4-cell stage (see Figures 4-6), indicating that embryos were fixed slightly earlier in the
course of their development. In the control group, 41% of intact and 37% of defolliculated
embryos had reached the 8-cell stage, whereas for the UVA-irradiated embryos, just 10% and
4% respectively had undergone the third round of cleavage. For the UVAB group, the
percentages were only 0.3 and 0.9 %, respectively.
Unger: Photoprotection in Ascidia
In this trial, a developmental delay was demonstrated by both UVA-exposed and
UVAB-exposed embryos. This shift is also evident in the calculated minutes of development,
where the embryos not exposed to UV were 10 to 15 minutes faster in their development than
the embryos exposed to UV, regardless of whether they were exposed to UVA only or to both
UVA and UVB (Table 2). It is important to note that once again, the developmental delay
observed is experienced by both intact and defolliculated eggs.
Tadpole Hatchin
Three experimental trials were performed to assay the effects of UV exposure on time
to hatching. Intact and defolliculated eggs were fertilized and placed in the UV exposure
chamber. A test dish of intact embryos not exposed to UV was monitored for the first
appearance of hatched tadpoles to indicate when hatching had begun. In the first two
experiments, this time was then used to fix the experimental embryos and tadpoles in
formaldehyde. Each of these trials thus measured only a single time point. In the third trial.
the rate of hatching was determined by fixing embryos approximately 30 minutes to an hour
after hatching began, and subsequent samples at 30 minutes, 90 minutes, and three hours after
the first set was fixed. The time to hatching in each of the three trials was approximately 23
hours after fertilization. The proportion of hatched tadpoles in each of the UV treatments was
standardized to that of the appropriate control.
In the first trial, embryos exposed to UVA achieved 72.1% of the control hatching rate
for intact eggs and only 37.8% of the control hatching rate for defolliculated eggs (Figure 7).
Embryos exposed to UVA and UVB illustrated this trend even more dramatically, with intact
eggs hatching at 103.3% of control rates and defolliculated eggs hatching at just 14.9% of
control rates. A clear UV-induced delay in time to hatching was observed, which varied with
presence or absence of follicle cells.
In the second trial this trend was not apparent, despite the fact that the embryos were
fixed slightly earlier (Figure 8). The UVA-irradiated embryos hatched at 89.6% of control for
Unger: Photoprotection in Ascidia
intact eggs and 72% of control for defolliculated eggs, while eggs irradiated with both UVA
and UVB hatched at 72.3% and 64.4% of control for intact and defolliculated eggs.
respectively. A slight delay in development dependent on UV exposure was observed, but
there was no evidence that delays differed with the presence or absence of follicle cells.
The third experiment assayed multiple time points in order to better assess the kinetics
of hatching. As seen in Figure 9, the percentage of eggs that hatched increased with time, but
the hatching leveled off after about 23 to 25 hours of development. There was no difference
between eggs not exposed to UV and eggs exposed to UVA and UVB. (Exposure to UVA only
was omitted as a treatment for this experiment.) Intact eggs hatched at about 48% of total at
21.75 hours, and leveled off within the next three hours to a hatching rate of about 56% of
total, regardless of whether they were exposed to UV or not. Similarly, defolliculated eggs
hatched at about 17% at the first time point and subsequently leveled off at about 20%. The
fact that by the time the first time point was taken, a significant number of tadpoles had already
hatched indicates that additional kinetic studies are in order for future experiments, particularly
one which documents in detail the progression of hatching from the point at which tadpoles
first begin to appear.
Floaters and Sinkers
During the course of the experiments studying developmental delay it became apparent
that the method of egg isolation used was systematically selecting against eggs of high
buoyancy. The more buoyant eggs were often lost in the supernatant when eggs were being
washed in preparation for fertilization. This was studied in more detail in order to confirm that
this egg selection bias would not alter the results of studies of developmental delay. An
experiment measuring the mycosporine content of methanol extracts of both more buoyant and
less buoyant eggs was performed and the results of the spectophotometric analysis of the
methanol extracts was standardized to original protein content using a BCA protein assay
Unger: Photoprotection in Ascidia
The first trial of this experiment was composed of individuals 1-1 and 1-2 (Figure 10).
In each of these individuals, the more buoyant eggs exhibited higher ratios of absorbence
between 300 and 400 nm, per milligram original protein content, ranging from a 8.9%
difference in individual 1-1 to a 16.3% difference in individual 1-2. In the second experimental
trial, there was no general pattern to which class of egg buoyancy had the higher ratio;
percentages ranged from floaters being 13.4% higher than sinkers (individual 2-5) to sinkers
being 74.3% higher than floaters (individual 2-6). An example of an absorbence spectrum is
shown in Figure 11. These results indicate there was no correlation between mycosporine
content and egg buoyancy.
DISCUSSION
Research done previous to these experiments implied that the sunscreen qualities
afforded by mycosporine-like amino acids present in follicle cells play an important role in
absorbing potentially damaging ultraviolet radiation. Carroll and Shick (1996) noted that
mycosporine-like amino acids in eggs might act as sunscreen molecules to protect embryos
from UV damage as they develop.
This suggestion was bolstered by the papers of Hemela (1993), and Adams and Shick
(1996), which indicated that mycosporines allowed embryos to proceed through early cell
divisions faster than embryos who lacked such photoprotection. However, the research in both
these studies used UV lamps to provide the damaging radiation and the embryos were kept in
the dark so that photorepair of any DNA damage by enzymes such as photolyase could not take
place. The trends they describe are intriguing but tell us little about what happens to embryos
developing outside of the laboratory.
The cleavage delay experiments described here illustrate that under conditions more
closely approximating those experienced by ascidian embryos in a natural setting, the
mycosporine-containing follicle cells are not necessary for cleavage to proceed at a normal
Unger: Photoprotection in Ascidia
pace: embryos without follicle cells that were exposed to UVA and B did not differ
significantly in pace of development from intact embryos of the same radiation exposure. The
distribution of embryos in trial +2 indicated that a radiation-linked delay to the 8-cell stage was
present (especially in embryos exposed to UVAB: see Figure 6), but this delay was not
dependent on the presence or absence of the egg's follicle cells. This suggests that although
mycosporines may play a role in UV defense, it is not a critical one. One hypothesis that might
explain the developmental delay observed is that UV damage is occurring to a significant
extent in both intact and defolliculated eggs, where the photolyase enzymatic repair system is
robust enough to repair any solar UV-induced damage regardless of whether the mycosporines
in the follicle cells are present or not.
It is also conceivable that the MAAs did screen out UV, but that differences in speed of
development would not be apparent until later in development. Three examinations of time to
tadpole hatching were conducted to test this hypothesis. The first trial indicated that the
hypothesis was indeed correct: there was a clear difference between the numbers of intact and
defolliculated eggs that had hatched in both samples exposed to UVAB as well as in samples
exposed to just UVA. Further trials failed to repeat this result, however, and a final test in
which a time series was constructed indicated that there was no observable difference in time to
hatching between embryos exposed to UV radiation and non-irradiated controls, regardless of
the presence of follicle cells (Figure 9). Unfortunately, the first time point in this study was
taken later than would have been ideal, and further kinetic studies including time points taken
earlier in the hatching process should be done in the future. It is critical to examine the
samples at just the point in time where undamaged embryos have completed hatching but
damaged embryos have not.
One final experiment was performed to confirm that these results were not an artifact of
the method of egg isolation, where repeated washings in which eggs are pelleted and the wash
water aspirated away resulted in the less buoyant tunicate eggs being preferentially collected.
Unger: Photoprotection in Ascidia
A preliminary study of mycosporine content of more buoyant eggs versus that of less buoyant
eggs indicated that the eggs with higher buoyancy had higher concentrations of mycosporines,
which seemed reasonable given that such buoyant eggs would spend a significantly longer
portion of their time of development at the top of the water column, where UV radiation levels
are highest. A second trial in which more individuals were assayed indicated that this trend
was not consistent, and concentration of mycosporines in eggs of varying buoyancies varied
unpredictably from individual to individual. This indicates that the data discussed above are
not artifacts of the method of egg preparation used in the described experiments.
CONCLUSIONS
Under midday springtime sunshine, the mycosporines in follicle cells are not the solo
line of defense in Ascidia embryos. The eggs do not exhibit observable delays during early
cleavage when irradiated with natural sunlight and allowed the opportunity to photorepair,
regardless of whether mycosporine-containing follicle cells are removed or are allowed to
remain surrounding the egg. It is more difficult to draw the same conclusions with regard to
tadpole hatching kinetics, which indicates that further experiments on the matter are in order.
These observations complement conclusions drawn by previous researchers, who observed a
developmental delay in both Ascidia ceratodes and Strongylocentrotus droebachiensis when
eggs were irradiated with UV lamps and photorepair was prevented (Hemela 1993; Adams and
Shick 1996). The results of this study, however, are more indicative of what is likely to happen
to embryos developing in a natural setting.
Unger: Photoprotection in Ascidia
LITERATURE CITED
Adams, N. L. and Shick, J. M. (1996). Mycosporine-like amino acids provide protection
against ultraviolet radiation in eggs of the green sea urchin Strongylocentrotus
droebachiensis. Photochem. and Photobiol. 64(1). 149-158.
Brown, R. E., Jarvis, K. L. and Hyland K. J. (1989). Protein measurement using bicinchoninic
acid: elimination of interfering substances. Analytical Biochemistry. 180(1). 136-139,
Carroll, A. K. and 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.
Chioccara, F. et al. (1986). Occurrence of mycosporine related compounds in sea urchin eggs.
Comp. Biochem. Physiol. 85B. 459-461.
Chrispeels, H. E. (1996). Effects of ultraviolet radiation on maize. Doctoral dissertation
submitted to the Department of Biological Sciences, Stanford University.
Hemela, K. J. (1993). Test cells provide photoprotection for Ascidia ceratodes. Senior honors
thesis submitted to the Department of Biological Sciences, Stanford University.
Jerlov, N. G. (1950). Ultraviolet radiation in the sea. Nature. 166. 111-112.
Karentz, D. et al. (1991). Survey of mycosporine-like compounds in Antarctic marine
organisms: potential protection from ultraviolet exposure. Mar. Biol. 108. 157-166.
Koller, L. R. Ultraviolet Radiation. New York: John Wiley & Sons, Inc., 1952.
Lambert, C. C. and Lambert, G. (1978). Tunicate eggs utilize ammonium ions for floatation.
Science. 200. 64-65.
Marshak, A. (1949). Recovery from ultraviolet light-induced delay in cleavage of Arabacia
eggs by irradiation with visible light. Biol. Bull. 97. 315-322.
Shick, J. M., et al. (1992). Survey of ultraviolet radiation-absorbing mycosporine-like amino
acids in organs in coral reef holothuroids. Mar. Ecol. Prog. Ser. 90. 139-148.
Unger: Photoprotection in Ascidia
Shick, J. M. et al. (1995). Depth-dependent responses to solar ultraviolet radiation and
oxidative stress in the zooxanthellate coral Acropora microphthalma. Mar. Biol. 122,
41-51.
Todo, T. et al. (1996). Similarity among the Drosophila (6-4)Photolyase, a human photolyase
homolog, and the DNA photolyase-blue-light photoreceptor family. Science.
272(5258). 109-112.
TABLES
No UV
UVA
—UVAB
Table 1:
No U
UVA
UVAB
Table 2:
Unger: Photoprotection in Ascidia
Intact
Defolliculated
114.8
119.2
118.6
117.7
1174
115.1
Calculated total minutes of development reached by embryos of each
experimental treatment for cleavage delay trial H1. There are no consistent
trends present across the different treatments in these data.
Intact
Defolliculated
97.9
98.9
86.2
85.6
75.9
82.5
Calculated total minutes of development reached by embryos of each
experimental treatment for cleavage delay trial +2. Again, there are no
consistent trends present across the different treatments in these data.
Unger: Photoprotection in Ascidia
FIGURE LEGENDS
Figure 1:
Trial H1: Distribution of embryos not exposed to UV. The control embryos,
incubated under a filter that did not transmit UV, developed almost completely
to the 8-cell stage. 98.0% of the intact and 88.8% of the defolliculated embryos
had undergone the third cell division by the time of fixation, which for this trial
was 115 minutes after fertilization. 1.5% of the intact embryos and 5% of the
defolliculated embryos were still at the 4-cell stage. A total of 3656 embryos
were scored for all three treatments in this trial, 51.8% of which developed
beyond the 1-cell stage.
Figure 2:
Trial H1: Distribution of embryos exposed to UVA. Embryos exposed to UVA
showed a distribution similar to that of the control embryos: 97.0% of the intact
and 93.5% of the defolliculated embryos developed to the 8-cell stage by the
time of fixation. 1.4% of the intact embryos and 5.3% of the defolliculated
embryos were still at the 4-cell stage.
Figure 3:
Trial H1: Distribution of embryos exposed to UVAB. Embryos exposed to both
UVA and UVB were distributed similarly to the control group and the UVA
group, with a slight increase in the percentage of embryos still at the 4-cell
stage. 6.8% of the intact and 15.7% of the defolliculated embryos had only
undergone cleavage to the four-cell stage.
Figure 4:
Trial +2: Distribution of embryos not exposed to UV. The distribution of these
control embryos was centered around the 4-cell stage, while 41.8% of the intact
and 37.3% of the defolliculated embryos had cleaved into 8 cells. Embryos in
this trial were fixed 93 minutes after fertilization, and just 15.7% of the 9366
embryos scored for the trial developed beyond the 1-cell stage.
Figure 5
Figure 6:
Figure 7
Figure 8:
Figure 9:
Figure 10:
Unger: Photoprotection in Ascidia
Trial H2: Distribution of embryos exposed to UVA. The distribution of UVA¬
irradiated embryos was also centered around the 4-cell stage, while fewer
embryos (10.5% of intact and 4.29% of defolliculated) had cleaved into 8 cells.
Trial f2: Distribution of embryos exposed to UVAB. UVAB-irradiated embryos
had generally not yet undergone the third round of cleavage: only 0.3% of intact
and 0.9% of defolliculated embryos had cleaved to 8 cells.
Trial H: UV inhibition of tadpole hatching at a single time point. Both UVA¬
and UVAB-irradiated embryos show a drop in the percentage of defolliculated
eggs that hatched into tadpoles when compared to irradiated, intact eggs after
standardization to the non-irradiated control. Samples were fixed 24.5 hours
after fertilization. 22.4% of all eggs scored in this trial hatched into tadpoles.
Trial 42: UV inhibition of tadpole hatching at a single time point. The trend
evident in Figure 7 is no longer obvious in this trial, in which embryos were
fixed slightly earlier, at 23 hours and 40 minutes after fertilization. Differences
between various UV treatments and follicle cell removal are minimal, the
largest difference being the 12% drop between intact and defolliculated eggs
exposed to UVA. 27.5% of the eggs scored in the trial hatched into tadpoles,
Kinetics of UV inhibition of tadpole hatching. This time series suggests that UV
treatment has no clear effect on tadpole hatching, regardless of time of
development. Intact eggs hatched at approximately 50-55% of total, while
defolliculated eggs hatched at approximately 17-20% of total. 35.9% of the
eggs scored in this trial developed into tadpoles.
Relative mycosporine concentrations in Ascidia eggs. Comparison of ratios of
absorbence per mg protein between floating and sinking eggs from each of
seven specimens of A. ceratodes. Neither category of eggs consistently exhibits
a higher absorbence ratio.
Figure 11:
Unger: Photoprotection in Ascidia
Wavelength scan of methanol extract of "sinker" eggs from a single ascidian.
Absorbence spectrum of methanol extraction from the less buoyant eggs from
individual 2.5. The broad peak from about 290 to 380 nm contains the bulk of
the mycosporine-like amino acids in the extract.
8


8
8
soque jo juee
Unger
Photoprotection in Ascidia
8
8
e jo juele
23
Unger: Photoprotection in Ascidia
- +

L
Unger: Photoprotection in Ascidia


8
8
soqu peej jo juele
24
3akakaakakakaa-
o juee
Unger: Photoprotection in Ascidia
2

8akakatatakakata-
sonquig peresjo jo jueblea
Unger: Photoprotection in Ascidia
E
S
L
88888
se o ue
Unger: Photoprotection in Ascidia
8
8 8
8
joljuoo jo juebied
Unger: Photoprotection in Ascidia
2
8
8
joljuos jo juebled
8
Unger: Photoprotection in Ascidia
-
5
Unger: Photoprotection in Ascidia
a.
223
eno
1
8
8
LO
uejoig ueiij led eoueglosqy
Unger: Photoprotection in Ascidia
3
.
Unger: Photoprotection in Ascidia