A. Pisani
Salinity Stress in A. elegantissima
ABSTRACT
The cnidarian Anthopleura elegantissima is subject to a wide
variety of external stresses. The effects of decreased salinity were
examined in the field and in a laboratory environment. Animals
were experimentally subjected to decreased salinities of 10 ppt, 17
ppt, and 25 ppt over a period of 4 days. Laboratory results suggest
that salinity stress induces algal cell loss, however, cell loss was not
observed in the field. Mechanisms of release active in bleaching
under salinity stress appear to result in the release of free algae.
Salinity Stress in A. elegantissima 9
A. Pisani
INTRODUCTION
Symbiotic relationships between photosynthetic unicellular
algae and cnidarians are common. The host supplies phosphorus and
nitrogen to the algae, as well as carbon dioxide for photosynthesis.
In return, the symbiotic algae generally referred to as zooxanthellae
reduce nitrogen and supply the host with soluble photoassimilates
(Shick 1991). The algae are housed in membrane-bound vacuoles of
the endodermal cells in the tentacles and oral disc of the host givin,
a greenish-brown color to the animal. The sea anemone Anthopleura
elegantissima has such a relationship with the dinoflagellate
Symbiodinuim californium.
The symbiotic relationship between zooxanthellae and some
tropical cnidarian hosts has been shown to break down with various
environmental stresses including changes in water temperature
(Buchsbaum 1968, Steen and Muscatine 1987, Muscatine let al 1991).
intense levels of ultraviolet radiation in sunlight (Lesser and Shick
1989), and changes in salinity (Reimer 1971). The breakdown of the
association, referred to as bleaching, is accompanied by a reduction
in chlorophyll a within the algae (Szmant and Gassman 1990) or by
a loss of the algae themselves (Fisk and Done 1985). During
bleaching the host expels the algal symbiont by gathering the algae
in the coelenteron and egesting them upon contraction of its body
wall. There are five possible mechanisms of algal cell release in
bleaching: a) exocytosis; b) apoptosis; c) necrosis; d) pinching off of
the host cell containing the symbiont: and e) host cell detachment
(Gates et al. 1992). Although host cell detachment has been
A. Pisani
Salinity Stress in A. elegantissima 3
documented in tropical cnidarians as a result of heat stress, no
investigations have been undertaken to determine the mechanism of
release due to other environmental stresses.
Anemones such as A. elegantissima have adapted to thrive in
harsh conditions. They are found on the West coast of North America
at varied vertical intertidal heights over a 2m range (Hand 1955).
The environment A. elegantissima inhabits subjects these animals to
many stresses. Included among these stresses is patchy, periodic
exposure to low salinity, occurring due to fresh water runoff during
rains and floods. Despite these disturbances, these anemones are
able to maintain a stable relationship with their algal symbionts.
Heavy rains in March caused flooding in the Carmel area.
Samples were taken to detect a possible bleaching event due to the
influx of fresh water. Experimental salinities were also tested in a
controlled laboratory environment in efforts to reproduce such an
event. The mechanism active in bleaching due to salinity stress was
investigated.
MATERIALS AND METHODS
Collection and Maintenance of the animals:
To investigate bleaching in the field, on April 19th tentacles
from animals were collected at Carmel Point and Carmel River. The
tentacles were frozen at -20°C. Carmel Point animals were used as a
control for the samples taken at Carmel River where heavy flooding
had occurred.
Experimental animals were collected in the mid intertidal from
Aggassiz beach at Hopkins Marine Station. The individuals were
A. Pisani
Salinity Stress in A. elegantissima 4
removed from the rocks using a small spatula and placed in
containers of seawater for immediate transportation to a running
seawater table. Three clones were sampled to ensure genetic
variation. Two clones were from vertical rock faces, the third from a
horizontal surface. The animals were identified by threading tags
through the column signifying clone and treatment. Experimental
animals were kept in aerated seawater at ambient salinity (35 ppt)
and temperature (13.5°C) in a 12:12 (L:D)h photoperiod. The animals
were not fed during the experiment.
Experimental Treatment:
Baseline assessments of zooxanthellae populations were
obtained as described below. Seawater was diluted using deionized
water to achieve the experimental salinities of 10 ppt, 17 ppt, and 25
ppt as measured with a conductivity meter. Control salinity was 35
ppt. Two 2 liter containers were prepared for each salinity
treatment and the control. The containers rested in a water bath to
maintain ambient seawater temperatures throughout the stress, and
the containers were aerated with an aquarium bubbler. Evaporation
guards were placed over the containers to maintain the salinity
Three animals, one from each clone, were placed in each container,
The treatment period lasted four days. Salinity was monitored
throughout the treatment period. On Day 2 of the stress one
container of 17 ppt had an elevated salinity of 20 ppt for an
estimated period of 4 hours. It was adjusted. After the stress all
animals were returned to running seawater.
A. Pisani
Salinity Stress in A. elegantissima 5
Tentacle samples were taken 0, 1, 7, 12, 16, and 20 days after
initiation of treatment. The samples were placed in filter sterilized
seawater (FSW), frozen at -20°C, and subsequently analyzed for
zooxanthellae content as described below. If the animals were
tightly closed magnesium chloride was used as a sedative to facilitate
sampling.
Estimation of Zooxanthellae populations:
To quantify the number of algae in the tissue, previously
frozen tentacle clips were thawed and homogenized in a 1.5 ml tube
with a micro pestle. The homogenate was centrifuged for 5 minutes
at 5000 rpm in a microfuge to separate the algae from the animal
supernatant. The supernatant was removed and frozen at -20°C for
subsequent protein assays. The pellet was then resuspended in FSW,
vortexed and spun again at 5000 rpm for 5 minutes. The resulting
pellet was resuspended in a known volume of FSW. The
zooxanthellae were counted using a hemacytometer and protein
concentrations of the animal supernatant were determined
colorimetrically (Sedmark and Grossberg 1977). Algal cell numbers
were normalized to mg animal protein.
Data Analysis:
Data were analyzed using a 3 factor ANOVA with Salinity and
Time as orthogonal fixed factors and Containers nested within
Salinity and orthogonal to Time (Table 1).
A. Pisani
Salinity Stress in A. elegantissima 6
Staining and epifluorescence microscopy:
In order to assess the state of algal cells released upon salinity
stress, released cells were collected, prepared, and viewed using
pifluorescence microscopy. Coelenteron contents released after
salinity stress at 25 ppt were collected by placing poly-L-lysine (1%
in distilled water) coated coverslips at the base of an animal in FSW,
The animal was agitated causing contraction of its oral disk and
expulsion of coelenteron contents. For 10 minutes the cells were
allowed to settle onto the coverslips. The coverslips were then
removed and treated with fluorescein diacetate, a vital stain showing
general cytoplasmic esterase activity (stock solution 15 mg/ml in
acetone; working solution 0.004 ml in 10 ml 0.1 M Tris buffer, 3%
sodium chloride, 0.004% calcium chloride, pH 7.4) according to the
methods of Gates et al., (1992). The coverslips were washed in the
Tris buffer, mounted on slides and viewed under epifluorescence
with an Olympus microscope. Additional coverslips were treated
with Hoescht 33258 which stains nuclei blue by binding to
chromatids (stock solution 5 mg/ml; working solution 0.004 ml in 10
ml 0.1 M Tris buffer, 3% sodium chloride, 0.004% calcium chloride,
pH 7.4) for 30 minutes. The coverslips were washed with the Tris
buffer, mounted, and viewed under epifluorescence The fluorescein
diacetate was viewed with a blue filter, wavelength 480 um, the
Hoescht used ultra violet light, wavelength 380 um.
RESULTS
Zooxanthellae populations after treatments:
A. Pisani
Salinity Stress in A. elegantissima
There was very little change in color through the stress and
sampling periods. However, significant changes in algal populations
of experimental anemones were detected with cell counts. Control
animals had an average population of 3.8 X 106 cells/ mg protein.
Despite some variation, this number remained fairly constant
throughout the sampling regime.
A. elegantissima placed in 10 ppt salinity did not survive
longer than three days post-stress. During the initial three days, the
animals did exhibit some loss of algal cells. This treatment was
excluded from statistical analysis, since no data could be obtained
after Day 3.
Animals placed in 17 ppt and 25 ppt salinity showed a steady
decrease in cell number during the 20 day sampling period
compared to control animals, and both exhibited similar rates of
decline (Fig. 1). Algal counts declined more quickly in 25 ppt, but
significant differences between groups were not significant until Day
16. The populations in 17 ppt and 25 ppt decreased to an average
symbiont density of 1.5 x 10° cells/mg protein after 20 days post-
stress. The zooxanthellae populations in these treatments both were
significantly different from control populations by Day 16 and Day
20 (p««0.001).
There was also a significant difference between the 2
containers in the 17 ppt treatments (pe«0.001). This difference may
be explained by an increase in the salinity in one container for a 4
hour time period due to accidental removal of the splash guard. This
change in salinity in the container was detected and corrected, but it
may have caused the 2 containers to differ from each other.
A. Pisani
Salinity Stress in A. elegantissima 8
Epifluorescence microscopy:
Coelenteron contents collected from the animals in the 25 ppt
treatment and stained with fluorescein diacetate appeared as single
red spheres (Fig. 2A) due to autofluorescence of the algal chloroplast
with a green halo and green inside of the red spheres (Fig. 2B). The
presence of green stain indicated that these algae are viable. Product
stained with Hoescht again appeared as red spheres containing a
single blue nuclei within the cell (Fig. 20). Additional nuclei on the
outside of the cell were never detected suggesting that the released
products were single algal cells and not host cells containing algae.
Discussion
Bleaching, or the loss of algal symbionts, is not a phenomenon
unique to A. elegantissima. It occurs on a large scale in tropical coral
reefs due to various stresses. For example, corals are able to survive
at salinities 25-40 ppt. However, if salinity drops below 20 ppt for a
time period longer than 24 hrs it is lethal. One example of the fragile
nature of the coral-symbiont relationship is the bleachin
documented in Jamaica and Easter Island after decreases in salinity
(Goreau 1964, Egana and DiSalvo 1892). With global warming and
other environmental changes a large scale change in salinity is not
expected, however, there will be local changes in areas with
increased run-off patterns due to human land use, and changes in
weather patterns. Unlike corals, anemones are easily maintained in
the laboratory. Therefore, anemones are an excellent model for
A. Pisani
Salinity Stress in A. elegantissima 9
studying bleaching.
Understanding the effects of different
environmental stresses, such as decrease in ambient salinity, on
symbiotic relationships will help in the development of hypotheses
about future global change and its consequences.
Anthopleura elegantissima reacts to low salinity stress by
gradual, but significant release of its symbionts. This was found to
happen over a 20 day period in laboratory manipulations after a 4
day stress in 17 ppt and 25 ppt water. After 16 days post-stress the
numbers of algae in experimental animals were significantly lower
than those from unstressed animals. A salinity 10 ppt was too low
for survival.
Samples collected from the field did not show signs of a
bleaching event. This component of the experiment was parallel to
an investigation by Engebretson and Martin, 1994, examining
bleaching in A. elegantissima found in LA County, March 1992.
Bleaching occurred in the field, but only 5 months after the heavy
rains (Engebretson pers. com.). It would be interesting to return to
Carmel Point in mid August to further investigate the effects of the
March flooding. Perhaps by that time effect of the flooding, if any,
would be detectable.
Engebretson and Martin also experimented with salinity stress
in a controlled lab environment, however their treatments were less
environmentally applicable. They kept their animals immersed in
hyposaline water (8 ppt, 16 ppt, and 24 ppt) for 21 days which is
unlikely length of stress in the field. This strong treatment resulted
in a bleaching event. My treatments which were 1/5 the length of
this stress, still induced cell loss, but at a slower rate.
A. Pisani
Salinity Stress in A. elegantissima 10
The mechanism by which A. elegantissima releases its
symbiotic algae after salinity stress is not known. It has been shown
that algal cells are released in intact host cells under temperature
stress (Gates et al., 1992). Is this mechanism of release uniform
under all stresses, or is the animal reacting differently to different
stresses on a cellular level? The results presented here provide
evidence for the probable release of free algal cells. Under
epifluorescence microscopy the algal cells were viable and appeared
to be free of a host cell. Only the nucleus of the algal cell could be
detected. It is possible that host cells degraded prior to collection, or
that they were missed due to a gradual release. Baring experimental
or sampling error, however, there are 3 possible mechanisms for
release of free algae: exocytosis, apoptosis, or necrosis of the host cell.
Presently I have no evidence for which of the 3 is most likely
occurring.
Anemones are osmoconformers. They quickly equilibrate to
ambient salinities. Few species are naturally found in brackish water
[15-20 ppt.], normal sea water being at a salinity of 35 ppt.. The
coelenteron is ventilated facilitating the periodic flow of fresh sea
water into the coelenteric cavity. This mechanism causes the salinity
of the coelenteric fluid to reach that of the water in approx. 3-6
hours. Cells must regulate their volume so as not to burst and are
able to do so at a range of salinities (Shick 1991).
Cell volume regulation is achieved through the use of the
intercellular free amino acid (FAA) pool. As salinity decreases, the
free amino acid pool decreases. The pool size increases with
increases in salinity (Shick 1991). Recent work has shown that
A. Pisani
Salinity Stress in A. elegantissima 11
animals and algal symbionts may communicate through the use of
amino acid signaling (Gates et al., 1992). With a decrease in the free
amino acid pool this signaling may be disrupted. The host may not
be stimulating the algae to return it's photoassimilates to the host.
The host may then consider the algae to be parasitic or too much of a
drain on the host's metabolism to be kept. Therefore, the expulsion
of the algae may bean indirect response to salinity stress indicated
through the process of cell osmoregulation. Further work is needed
to confirm this hypothesis.
ACKNOWLEDGMENTS
1 am grateful to Dr. Virginia Weiss for her support, guidance, and
commitment to this project. Additional thanks to Dr. Paul Levine for
the use of his lab and his probing questions, and to Dr. Jim
Wantanabe for his statistical genius and field expertise.
A. Pisani
Salinity Stress in A. elegantissima 19
LITERATURE CITED
Buchsbaum, V.M. 1968. Behavioral and physiological, responses to
light by Anthopleura elegantissima. Ph.D. diss,. Stanford
University, Stanford California :5.
Egana, A.C. and L.H. DiSalvo. 1982. Mass expulsion of zooxanthellae
by Easter Island corals. Pacific Science 36:61-63.
Engebretson, H. and K.L.M. Martin. 1994. The effects of decreased
salinity on expulsion of zooxanthellae in Anthopleura
elegantissima. Pacific Sci. 4: 446-457.
Fisk
T. A., and T. J. Done. 1985. Taxonomic and bathymetric patterns
of bleaching corals. Proc. Fifth Int. Coral Reef Cong. 6: 149-154.
Gates, et al. 1992. Temperature stress causes host cell detachment in
symbiotic cnidarians: implications for coral bleaching. 182:
324-332.
Gates et al. 1995. Free amino acids exhibit anthozoan "host factor
activity: they induce the release of photosynthate from
symbiotic dinoflaggellates in vitro. Bio. Sci. not in print.
Goreau, T.F. 1964. Mass expulsion of zooxanthellae from Jamaican
reef communities. Science (Washington, D.C.) 16:383-386.
Hand, C. 1955. The seas anemones of Central California. Wasmann.
Biology., 37.
Lesser, M.P., and J.M. Shick. 1989. Effects of irradiance and
ultraviolet radiation on photoadaption of Aiptasia pallida:
Primary production, photoinhibition, and enzymatic defenses
against oxygen toxicity. Mar. Bio. 102:243-255.
A. Pisani
Salinity Stress in A. elegantissima 13
Muscatine, L., D. Grossman, and J Doino. 1991. Release of symbiotic
algae by tropical anemones after cold shock. Mar. Ecol. Prog.
Ser. 77: 233-243.
Reimer, A.A. 1971. Observations of the relationships between
several species of tropical zoanthids and their zooxanthellae. J.
Exp. Mar. Bio. Ecol. 7: 207-214.
Sedmak, J.J. and S. E. Grossberg. 1977. A versatile protein assay
using Coomassie Protein assay Reagent G250. Anal. Biochem.
79: 544-552.
Shick, J.M. 1991. Osmoconformity and cellular volume regulation. A
Functional Biology of Sea Anemones: 179-191.
Szmant, A., and N.J. Gassman. 1990. The effects of prolonged
"bleaching“ on tissue biomass and reproduction of reef coral
Montastrea annularis. Coral Reefs 8: 217-214.
Steen, R.G and L. Muscatine. 1987. Low temperature evokes rapid
exocytosis of symbiotic algae by a sea anemone. Bio. Bull.
(Woods Hole) 172: 246-263.
A. Pisani
Salinity Stress in A. elegantissima 14
Table 1
Statistical Analysis
Analysis of Variance
df
Source
MS
14.987
Salinity
5.252
0.105
12.130 34.317
Time
0.000
2.971 8.405
Sainity*Time
0.000
10
2.852
7.837
0.000
Container (Salinity)
0.353
0.494
Time* Container (Salinity) 15
0.971
0.364
Error
67
Post Hoc Comparisons (Tukey HSD)
Pairwise mean differences
Day O
Control
17 ppt
-1.00 (p=.846)
17 ppt
25 ppt
119 (p=1.00)
—219 (p=1.00)
* No significant difference Time 1-Time 4
Day 16
Control
17 ppt
2.097 (p=0.000)
17 ppt
1.812 (p=0.000)
285(p=1.00)
25 ppt
—
Control
Day 20
17 ppt
3.270 (p=0.000)
17 ppt
3.086 (p=0.000)
O.185(p=1.00)
25 ppt
A. Pisani
Salinity Stress in A. elegantissima 15
List of Figures
Figure 1: Cell/mg animal protein as calculated with n=6 per
treatment over a 20 day period.
Figure 2: a) algae cells autofluoressing red; b) green staining of
esterase activity with fluorescein diacetate staining; c) blue staining
of the chromatids in the nucleus with Hoescht
1.0E+07-
1.OE+06
1.OE405.
1.OE+04-


Figure 1





10
Time (days)
15
20
25
—+— 10 p
—H 17 pr
--O---- 25 pr
— A— 35 pr
A. Pisani
Figure 2
a.
in A. elegantissima 16