Arruda, Bucknamand Montes

Bleaching, Recovery, and Growth of Glass Anemone, Aiptasia pallida, as a Result of
a Heat Stress.
Marina Arruda, Keatyn Bucknam, and Esmeralda Montes
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Photosynthetic dinoflagellates, Symbiodinium spp. not only have a symbiotic
relationship with glass anemones, but also provide energy and food. Both glass
anemones and photosynthetic dinoflagellate require precise environmental
parameters to survive, therefore unfavorable conditions would cause anemones to
stress and undergo a process called bleaching by releasing their endosymbionts.
Therefor it was predicted that A. pallida that had previously recovered from a
bleaching event would bleach and recover more at a different rate than anemones
that had not experienced any stresses. Two 2.5 gallon tanks each containing varying
sizes of anemones were divided to house two different groups: those growing in ideal
environmental conditions, and those having recovered from a previous bleaching
event. The temperature in the experimental tank was gradually increased to 32°C
for 48 hours, and then reduced to normal temperature. The color value change for
bleaching was then obtained by subtracting the initial value from the maximum
color value, indicating a significance of bleaching color change (ANOVA, p=0.0079),
that occurred between the Experimental Normal group, Control Normal and
Control Recovered groups (Post Hoc-Bonferroni Correction, p<0.05). The
Experimental Normal group revealed a color change during recovery that was
statistically different than that of the other groups, including the experimental
group, having previously recovered from a stress and being exposed to the same
condition, showing that there was a significant difference between recovered and
normal anemone bleaching and recovery as a result of a heat stress.
pallida) have a symbiotic relationship
with a photosynthetic dinoflagellate,
Symbiodinium spp. (Muller-Parker &
Davy, 2001 and Weis, 2008), which
provides additional energy and food to
the anemones and gives the anemones
their color. A. pallida and Symbiodinium
parameters to survive which include
water temperature between 24.0-26.0°C,
specific gravity of 1.024-1.027, pH 8.09.0, levels of ammonia between 0.01.0ppm, nitrate levels of 0.0-40.0ppm,
nitrite levels of 0.0-5.0ppm, and
phosphate levels of 0.0-5.0ppm;
unfavorable conditions will cause the
anemones to stress and release their
endosymbionts (a process called
bleaching). If adequate food is not
available, they perish quickly without
the nutrients necessary to survive
(Wissmann, 2003).
Multiple stresses have been
shown to cause bleaching in A. pallida.
For instance, a study by Arruda et al.
(2013) demonstrated that anemones
placed in complete darkness bleach
endosymbionts are unable to undergo
metabolism without the presence of
light. Another avenue of bleaching was
demonstrated in a study by Dunn et al.
(2007), which showed that a temperature
increase between 33-34°C over a period
of 30 hours caused A. pallida anemones
to bleach. This method of bleaching is
comparable to coral bleaching induced
by a heat stress, since the most pressing
cause of coral bleaching is due to
climate change (Hughes, et al.).
The present project will study the
color change due to bleaching and
recovery of A. pallida after they have
been exposed to a heat stress (increase in
temperature) for a short period of time.
The purpose of this experiment is to
compare the bleaching and recovery of
anemones that have bleached and
recovered prior to the experiment
(recovered) and anemones that have
grown in ideal conditions (normal) in
order to determine if there is a
correlation between recovery and
previous bleaching events. It is proposed
that there will be a significant difference
between recovered anemone and normal
anemone bleaching and recovery. This
could provide further insight into the
loss of coral reef habitats due to coral
Materials and Methods
A total of 124 Aiptasia Pallida
anemones maintained from a previous
study (Arruda et al., 2013) previously
obtained from Berghia Breeder (Laguna
Niguel) were monitored over a period of
7.5 weeks. The anemones were
separated into four groups and kept in
two 2.5-gallon glass tanks (Petco, Aliso
Viejo). Groups included anemones
recovered from a previous bleaching
event in the experimental tank
(experimental recovered), anemones that
have grown in ideal conditions in the
normal), anemones recovered from a
previous bleaching event in the control
tank (control recovered), and anemones
that have grown in ideal conditions in
the control tank (control normal). One
2.5-gallon tank, placed on top of an egg
crate (Home Depot, Laguna Niguel)
within a 30-gallon glass tank obtained
from a Saddleback College student
experimental group of anemones. The
other 2.5-gallon tank housed the control
group and was separate from the 30gallon tank. Each 2.5-gallon tank housed
six large (>2.5cm), six medium (12.5cm), and fifty small (<1cm)
anemones. A porous ceramic cube
(Reefapalloza, Anaheim) was placed in
the corner of each 2.5-gallon tank. Tank
dividers were constructed from clear
plastic canvas (Michael’s, Aliso Viejo)
and plastic slide locks cut to fit between
each individual 2.5-gallon tank to
separate the recovered from the normal
anemones in each tank. These were
soaked in reverse osmosis deionized
water, RODI, (Sand Bar Pet Shop,
Mission Viejo) prior to installation. The
dividers were placed in the middle of
each tank and did not restrict the flow of
food, heat or water throughout the tanks,
but prevented recovered and normal
anemones from mixing. On the bottom
each tank, 1×2 cm ceramic tiles (Home
Depot, Laguna Niguel) were placed on
top of 10×5 cm ceramic tiles (Home
Depot, Laguna Niguel) to allow easier
measurements and pictures. The 2.5
gallon tanks were filled approximately
1.5 cm from the top with saltwater (Sand
Bar Pet Shop, Mission Viejo), and the 30
gallon tank was filled up to 2.5 cm from
the smaller tanks with tap water (no
water circulation between the 30 gallon
and 2.5 gallon tanks). All the materials
that were placed into the tank or came in
contact with the tanks were rinsed with
RODI prior to installment.
In the corner of each 2.5-gallon
tank, a Hydor centrifugal water pump
was installed (Amazon, LLC). An
aquarium 2-way gang valve and a fusion
air pump were installed in order to direct
airflow within the tanks (Amazon, LLC).
Both tanks received light via a light
hood 3.48cm away (Danielle Breski). An
Aquatop GH Series 150 watt
Submersible Heater (Amazon, LLC) was
placed inside the 30-gallon tank, while a
Hydor 25-watt submersible aquarium
heater (Amazon, LLC) was placed into
the Control 2.5-gallon so that its
temperature would remain independent
of that within the experimental tank.
Water parameters within each
2.5-gallon tank were measured and
tested in the afternoon or early evening
each day. In order to measure the salinity
of the controlled and experimental tanks,
a salinity refractometer (Ade Advanced
Optics, Amazon, LLC) was used and
calibrated with RODI water. API tests
were used to measure ammonia, nitrate,
nitrite and phosphate levels (Amazon,
LLC) by collecting samples from each
tank with their corresponding plastic
graduated pipettes (Lake Charles
Manufacturing, Amazon) and placing
the water samples into separate glass
vials or test tubes (Amazon, NPS), then
conducting the appropriate tests. The
water from each tank was also tested for
pH using a high range pH test kit (The
Paws, Amazon, LLC). All water
parameters, except temperature in the
experimental tank, remained consistent
at ideal levels in both aquariums
(Temperature 24.0-26.0 °C, pH 8-9,
Ammonia 0-1 ppm, Nitrate 0-40 ppm,
Nitrite 0-5 ppm, Phosphate 0-5 ppm,
Specific Gravity 4.1-4.8 %, Alkalinity
180-300 ppm).
Both heaters were initially set to
maintain a water temperature of 24.026.0°C. Preliminary photographs and
diameter measurements were taken for
each anemone before the experiment
began. Once the experiment began, the
control aquarium was maintained at the
ideal temperature (24.0-26.0°C), while
the experimental aquarium was stressed
to a higher temperature of 32.0-33.0°C
for a period of 48 consecutive hours and
then returned to ideal temperature for the
rest of the experiment. Temperature
within each tank was measured using
coral life digital thermometer (Sand Bar
Pet Shop, Mission Viejo). Before
starting the photograph and diameter
measurement procedures, the specimens
were fed with 0.05g of Cyclop-Eeze
freeze-dried Mysis Shrimp (Sand Bar Pet
Shop, Mission Viejo). Once the
anemones finished eating, they were
transferred on their tiles into a glass
10×13 inch rectangular dish, owned by
Saddleback College student Marina
Arruda, that was filled with water that
had previously been siphoned out from
their own tanks with aquarium airlines
(Amazon, LLC). Anemones were
transferred and photographed one tank at
a time, and with recovered and normal
groups kept separate. After allowing the
anemones to acclimate, photographs
were taken with a Panasonic DMC-TS20
camera (BestBuy, Laguna Niguel) at
approximately 6 cm way from each
anemone and each diameter was
measured. Once the measurements were
recorded, the anemones were placed
back into their appropriate tank and the
water from the dish was siphoned back
into that tank through a filter (to remove
excess algae). This process was repeated
for the second tank. This procedure was
done before the heat stress, 24 hours
after the experimental tank was brought
back to ideal temperature, every 7 days
for two weeks, and every 14 days for the
remaining four weeks.
In order to analyze the
photographs, the histogram feature of
Photoshop Elements 6.0 was used. For
large and medium anemones, three
0.5x0.5mm areas of separate tentacles
were analyzed at 100% zoom and
averaged to get a color value for each
anemone. For small anemones, one
0.5x0.5mm area was used, which
encompassed most of the anemone
tentacles. Small anemone color values
were averaged for each tank. Larger
color values indicated a lighter color
(greater amount of bleaching). Data was
placed into MS Excel (Microsoft,
Redmond, WA) and comprised into a
line graph of color value as a function of
date measured. Bleaching color value
change was obtained by subtracting the
initial color value from the maximum
color value for each group and groups
were analyzed against each other using
an ANOVA with Post Hoc test
(Bonferroni correction). Recovery color
value change was obtained by
subtracting the final color value from the
maximum color value for each group
and analyzed accordingly. Diameter
measurements were graphed in a line
graph as the average diameter of the
small anemones in each tank as a
function of day measured to show a
general growth trend.
Color Value
Date Measured
Figure 1. Bleaching and recovery rates for Experimental Recovered (ER, N=7), Experimental Normal (EN,
N=7), Control Recovered (CR, N=7), and Control Normal (CN, N=7). There is a significance between
bleaching color change (ANOVA, p=0.0079). Represents groups that exhibit a significant difference of
bleaching color change against the Experimental Normal group (Post Hoc- Bonferroni Correction, p<0.05).
There is a significant difference between recovery color changes (ANOVA, p=0.0001). * Represents
groups that exhibit a significant difference of recovery color change against the Experimental Normal
group (Post Hoc- Bonferroni Correction, p<0.05).
Average Diameter (cm)
Control Normal
Control Recovered
Experimental Normal
Experimental Recovered
Day Measured
Figure 2. Average diameter of small anemones as a function of day measured showing a general growth
rate for Control Normal, Control Recovered, Experimental Normal, and Experimental Recovered groups.
The four groups of anemones
were analyzed to determine their color
value change for bleaching and recovery.
Each group had three large, three
anemones present. Figure 1 indicates the
average of color values (three large,
three medium, and one small were added
together and averaged; the small color
values were averaged, giving one value)
for each day they were measured. The
color value change for bleaching was
obtained by subtracting the initial value
from the maximum color value (the
lightest color the anemones reached,
therefore their maximum bleach color).
There was a significant color difference
(ANOVA, p=0.0079) between the
Experimental Normal group (color
lightened) and the Control Normal and
the Control Recovered groups, which
showed no significant change (Post HocBonferroni Correction, p<0.05). The
color value change for recovery was
obtained from subtracting the final value
from the maximum value. There was a
significant difference of recovery color
change (ANOVA, p=0.0001) between
the Experimental Normal group and the
Normal, and Control Recovered groups
(Post Hoc- Bonferroni Correction,
Figure 2 represents the general
growth trends for each group of
anemones. During the initial four weeks,
the control groups indicate a higher
growth margin than the experimental
groups. Between day one and two of
measurements (before and after the heat
treatment), some of the anemones in the
experimental tank died, indicated by the
drop in average size. After week four,
the control groups exhibit a drop in size
because new polyps started to grow,
reducing the average diameter values. In
the experimental groups, there were no
new polyps, so the average diameter
values continued to increase.
Water parameters remained ideal
for each tank during the experiment. The
control tank temperature ranged between
24.0 and 25.6 °C. During the
temperature stress, the experimental tank
temperature reached 32.0 °C and was
brought back down to between 24.0 and
26.0 °C to allow for recovery. The
control tank parameters were: alkalinity
180-300ppm, salinity 4.2-4.5 %, pH 88.4, nitrate 0-20ppm, nitrite 0-0.45ppm,
ammonia 0-0.6ppm, and phosphate
0ppm. The experimental tank parameters
were: alkalinity 180-300ppm, salinity
4.1-4.8 %, pH 8-8.3, nitrate 0-20ppm,
nitrite 0-0.45ppm, ammonia 0-0.6ppm,
and phosphate 0ppm.
The higher color value indicates
that the anemones became lighter in
color (the higher the value, the closer the
color is to white). The color of A. pallida
is derived from their endosymbiotic
dinoflagellates Symbiodinium, so the
lightening indicates that some of these
dinoflagellates have been released into
the water column or have perished (the
experimental normal group showed a
color change during recovery that was
statistically different than all of the other
groups, including the experimental
group that had previously recovered
from a stress and was exposed to the
same conditions. As a result, the null
hypothesis can be rejected, and the
statistical analysis shows that there is a
significant difference between recovered
and normal anemone bleaching and
recovery as a result of a heat stress.
The bleaching of normal
anemones in this experiment supports
past studies concerning the bleaching of
A. pallida as a result of a heat treatment
at 32-33°C for a period of 24-48 hours
(Ferrier-Pages et al., 2007). The results
of this experiment suggest that
recovered from a bleaching event may
be more resilient to a future bleaching
event, since the experimental recovered
anemones did not show a significant
change in color when compared to the
control groups. This warrants further
study on the subject, which could be
useful to testing the Adaptive Bleaching
Hypothesis (Kinzie, Robert A, et al.,
Figure 1 indicates a slight
fluctuation in the color values for the
control anemone groups (recovered and
normal) and the experimental recovered.
This fluctuation appears to be a natural
phenomenon, as it was observed in all
anemones (except the experimental
normal group, which showed significant
bleaching and recovery due to the heat
stress). It also did not have any
correlation with water parameters, which
were kept within the ideal ranges for the
species. The experimental recovered
anemones did appear to have some color
values within the experimental period
that were higher than those in the control
groups, but this is understandable since
the experimental group was exposed to
the heat stress. It is important to note
however, that though this difference is
visible in the figure, it was not
During this experiment the
diameters of the anemones were
measured and recorded in order to
determine the general trend in growth of
the smaller anemones in each group
during the experimental period. As seen
in Figure 2, the control group diameters
experimental period until new polyps
began to grow, which decreased the
average diameter values for the tank.
The experimental groups showed a
decrease in diameter after the heat
treatment, then an increase as recovery
took place. In the previous study
involving these anemones (Arruda et al.,
2013) growth appeared to be reduced in
anemones that bleached. This suggests
that the initial observed decrease in
diameter is a result of the stress on the
anemones, since more resources must be
devoted to capturing food if some of the
symbiotic dinoflagellates are absent due
to bleaching.
The results of this project may
shed light on the effects of bleaching in
coral reefs due to a temperature stress
brought on by climate change. The cause
of death from a bleaching event to the
anemone or coral is usually due to the
decrease in nutrient intake because of the
loss of endosymbionts, not because of
the actual stress (Wissmann, 2003).
During this study, anemones were
consistently fed with high nutrientcontent food, allowing them to survive
and recover from the loss of those extra
endosymbionts. Corals and anemones
possess a capability referred to as
organism to tolerate changing conditions
that lie within certain parameters
dependent on species (Gates and
Edmunds, 1999).
The temperature
increase therefore causes bleaching in A.
pallida, but the loss of their
endosymbiotic dinoflagellates (and
therefore their source of many essential
nutrients) causes death.
We would like to thank Professor
Teh for his assistance and guidance with
this study. We would also like to thank
Danielle Breski and Patrick O’Hara for
supplying some of our materials.
alphabetically by last name and does not
reflect individual contribution to the
Literature Cited
Arruda, M., Breski, D., Moghadasi, M.,
Fastuca, J. (2013). The Effect of
Darkness on Bleaching in Glass
Anemones, Aiptasia pallida.
Saddleback Journal of Biology.
Dunn, S.R., Schnitzler, C.E., and Weis,
V.M. (2007). Apoptosis and
Autophagy as Mechanisms of
Dinoflagellate Symbiont Release
during Cnidarian Bleaching: Every
Which Way You Lose. Molecular
and Cellular Proteomics, 30793085.
Ferrier-Pages, C., Richard, C., Forcioli,
D., Allemand, D., Pichon, M.,
Malcolm Schick, J (2007).
Effects of Temperature and UV
Radiation Increases on the
Photosynthetic Efficiency in
Four Scleractinian Coral Species.
Biological Bulletin, Vol. 213,
No. 1, 76-87.
Gates, Ruth D. and Edmunds, Peter J.
(1999). The Physiological
Mechanisms of Acclimitization in
Tropical Reef Corals. American
Zoologist, Vol. 39, No. 1, 30-43.
Hughes, T.P., Baird, A.H., Bellwood,
D.R., Card, M., Connolly, S.R.,
Folke, C., Grosberg, R., HoeghGuldberg, O., Jackson, J.B.C.,
Kleypas, J., Lough, J.M.,
Marshall, P., Nystrom, M.,
Palumbi, S.R., Pandolfi, J.M.,
Rosen, B., Roughgarden, J.
(2003). Climate Change, Human
Impacts, and the Resilience of
Coral Reefs. Science, Vol 301,
Kinzie, R., Takayama, M., Santos, S.,
Coffroth, M. (2001). The Adaptive
Bleaching Hypothesis:
Experimental Tests of Critical
Assumptions. Biological Bulletin,
Vol. 200, No. 1, 51-58.
Muller-Parker, G., and Davy, S.K. (2001).
Temperate and Tropical Algal-Sea
Anemone Symbioses. Invertebrate
Biology, Vol 120, No. 2, 104-123.
Weis, Virginia M (2008). Commentary:
Cellular Mechanisms of Cnidarian
Bleaching: Stress Causes the
Collapse of Symbiosis. The
Journal of Experimental Biology,
Vol 211, 3059-3066.
Wissmann, S. (2003). The Effects of
Elevated Ultraviolet B Radiation
and Elevated Water Temperature
on the Loss of Zooxanthellae from
Aiptasia pallida. Transactions of
the Kansas Academy of Science
(1903-). Vol. 106, No. 1/2, 92-98.