Effects of Low Salinity and High Temperature on Anemone Locomotion

Kaitlyn Mitchell and Bridgette Wathen

Abstract

Climate change poses significant threats for many marine organisms due to projected
increases in sea surface temperatures and increased storm frequency and intensities that result in
highly variable salinity. Mass bleaching events have been observed where Anthozoans, corals
and anemones expel symbiotic algae from gastrodermal cells in response to environmental stress
induced from climate change. Aiptasia, a class of Anthozoans, is a symbiotic anemone found in
variable tropical ecosystems rendering them a favorable model for the limitations of other
Anthozoans in tropical reef systems. In this study, Aiptasia were subjected to low salinity and
high temperature treatments. During treatment, locomotor ability was assessed based on time to
open after stimulus. After two weeks, half of the low salinity treated Aiptaisa were returned to
control conditions to determine resiliency to stressful conditions. Locomotor ability did not
significantly differ from Aiptasia in control conditions in either the low salinity or high
temperature treatments. Yet there were a significantly larger number of closed or dead Aiptasia
in low salinity compared to control conditions. Aiptasia were able to return to normal locomotor
capability after return to control conditions. These findings suggest that Aiptasia may be
equipped to handle the current high temperatures and acute periods of low salinity induced by
climate change better than other tropical Anthozoans. However, if climate change were to induce
significantly lowered salinity for increased durations, all Anthozoan species may be at extreme
risk of increased mortality.

Introduction

Anthropogenic carbon emissions have continued to increase since the Industrial Age,
negatively impacting atmospheric and oceanic processes. Side effects of increased carbon
emissions include warming oceans, increased sea ice melt in the Arctic, and increased storm
frequency and intensity (Harley et al 2006; IPCC, 2007). These effects alter ocean salinity, pH
and temperature presenting new challenges to marine organisms. Sessile organisms tend to be
more phenotypically plastic due to their inability to relocate despite environmental challenges. In
other words, sessile organisms are better able to change biochemical, physiological, anatomical
or morphological characteristics in response to environmental changes. Anthozoans, a class of
cnidarians, have already presented evidence of limited phenotypic plasticity in changing oceans.
A process referred to as bleaching occurs in anthozoans when their symbiotes are either
exocytosed from the host cells or apoptosis of host cells occurs altogether (Mayfield and Gates
2007). Host organisms, unable to maintain necessary body functions, perish soon afterwards.
This eliminates the highly ecologically productive reef systems that many Anthozoans support.
Consequently, a better understanding of the physiological responses and limitations of
anthozoans to changes in environmental conditions will allow us to better predict the future
ecological demographics of our ocean.

The coastal waters surrounding Florida are naturally subjected to seasonal variations in
salinity and temperature, creating an environment inhabited by resilient marine organisms. Water
salinity can be greatly affected by tropical storms and hurricanes, which are relatively common
off the coast of Florida (Johns and Lee). After Hurricane Irene in 1999, the salinity of Florida
Bay dropped from 1.025 sg to 1.019 sg, negatively impacting marine life in the area (Kelble et
al, 2007). Anthozoans in the region can provide models of phenotypic plasticity in the face of
global climate change due to known resiliency coupled with previously understood physiological
limitations demonstrated by large bleaching events (Fitt et al. 2001). Actinaria, an order of
anthozoans colloquially called anemones, provide critical habitat for many reef dwelling
organisms (Stella et al. 2011) and are therefore favorable models for all anthozoan species
including corals. Aiptasia spp. are a representative of dinoflagellate symbiote systems for
tropical anthozoans. Aiptasia is a common sea anemone in the tropics with one primary symbiote
species, zooxanthellae. In addition, the use of Aiptasia allows for non-invasive, relatively
straightforward physiological assays such as measuring mesentery filament contraction.

Changes in temperature can alter resource partitioning between zooxanthellae and

Aiptasia (Clark and Jensen 1982), limiting growth and performance of Aiptasia. Suharsono et al.
(1993) noted that symbiote presence could be considered disadvantageous at higher temperatures
after heat-shocking Anemonia viridis and observing destabilization of lysosomes in the
mesentery filaments, which may have been caused by substances produced by zooxanthellae
when stressed. Later, Perez et al. (2004) observed high symbiote expulsion at increased
temperatures of Aiptasia pallida. Other research has suggested that intracellular protein
phosphorylation in high temperature environments may promote bleaching (Sawyer and
Muscatine 2001). Therefore, significant increases in temperature might limit the productivity and
reactivity of anemones due to limited resources changes in protein phosphorylation, or mesentery
filament degradation from decreased lysosome integrity facilitated by zooxanthellae.

In addition to coping with temperature changes, Aiptasia must also cope with changes in
salinity. Aiptasia are euryhalines, meaning they can tolerate a wide range of salinities. This
occurs through the initiation of regulatory volume decrease (RVD) using sodium potassium
pumps and altering the number of osmolytes in cells (Marino et al. 2010; Mayfield et al. 2007).
Symbiotic anemones regulate compatible organic osmolytes (COOs) composed of polyols, or
waste and glycerol produced by zooxanthellae in gastrodermal cells (Mayfield et al. 2007). This
requires no additional energy expenditure to change intracellular osmolarity in less variable
salinity. However, as salinity more drastically changes, the synthesis of COOs requires more
energy, limiting energy expenditure in other areas such as locomotion.

This study attempts to reveal how Aiptasia performance, measured by locomotor

responses, is affected by changes in both salinity and temperature of their seawater environment.
Due to the additional energetic use associated with osmoregulation paired with the effects of
high temperatures on protein and organ function, we hypothesize that increased temperature and
decreased salinity will affect locomotion of Aiptasia anemones. To test this hypothesis, we
attempted to stress Aiptasia at either high temperature or low salinity conditions. Due to the
physiology of Aiptasia discussed previously, and the principle of allocation of resources, we
predicted a decrease in locomotor activity in all experimental conditions compared to the control.
Aiptasia are relatively resilient organisms, so we also predicted that Aiptasia would be able to
recover normal locomotor activity when moved from stressful to favorable conditions. Because
of this resiliency, it is also reasonable to imagine that these same changes in environment could
have more extreme effects on less adaptable species of subtropical marine anemones.

Methods

Selection of Experimental Conditions

Aiptasia are naturally found in Florida’s coastal waters where the average salinity is
1.025 sg and the average sea surface temperature is 25°C (NOAA). However, Aiptasia can be
found deeper in the water column indicating a slightly lower temperature. We recreated the
salinity of 1.019 sg in Florida’s coastal waters following Hurricane Irene to test the effects of
changes in salinity from naturally occurring meteorological events (Kelbe et al. 2007). Perez et
al. (2001) reported that Aiptasia showed signs of stress at 32°C based on the expulsion of
symbiotic zooxanthellae. While the average temperature off Florida’s coast is 25°C, waters have
been recorded up to 30°C with projections to increase up to 2°C by the end of the century.
Therefore, we also tested Aiptasia’s locomotor function at high, naturally occurring temperatures
of 30°C. These environmental conditions reveal how Aiptasia is being physiologically impacted
in Florida when extreme environmental conditions occur.

Experimental Setup

Twenty Aiptasia were ordered from Carolina Biosciences. Upon arrival to the University
of Washington, the Aiptasia were divided into 3 groups which each had a separate tank. The
Aiptasia were allowed two weeks to acclimatize in control conditions of 1.025 sg and 22°C.
Each tank was provided an individual aerator hose. Stock water samples were adjusted to
appropriate salinity levels using tap water and a urine hydrometer, while freshwater baths
monitored with a thermometer controlled individual tank temperatures. After acclimatization,
two groups of Aiptasia were moved into experimental conditions of either 30°C or 1.019 sg.
Measurements of locomotor rates were taken twice a week with at least two days in between
each assay. Following testing, water was changed and Aiptasia were fed brine shrimp eggs. After
two weeks, three Aiptasia were removed from low salinity conditions (1.019 sg) and placed into
control conditions (1.025 sg). The Aiptasia returned to control conditions were in a separate tank
and subsequently tested using the same locomotor assay for an additional two weeks.

Locomotor Assay

The effects of temperature and salinity modification on Aiptasia were examined based on
changes in locomotor ability. A finger was moved toward the oral disk and in one circle around
the body column, which stimulated Aiptasia to contract their mesentery filaments and tentacles.
This also resulted in ejection of the nematocysts. Aiptasia were determined to be closed when all
tentacles were fully contracted toward the oral disk. Once closed, a timer was started and then
stopped when the oral disk reappeared. Visual appearance was also recorded throughout the
experiment.

Statistical Methods
 The significance of change in locomotor times was evaluated for the first two weeks
using an ANOVA followed by a Tukey test. The recovery ability of Aiptasia after low salinity
conditions and the observed data on the number of Aiptasia that were closed or severely swollen
in each condition were also evaluated using ANOVA corrected with a Tukey test. Graphs were
generated in Excel and R.

Results

Locomotor Ability

Locomotor ability, characterized by the time for Aiptasia to reopen after closure, was measured
over the course of two weeks in control, high temperature, and low salinity treatments. An
ANOVA followed by a post-hoc Tukey test revealed that there was no significant difference in
locomotor ability of Aiptasia between the control and high temperature or low salinity conditions
(Tukey HSD, p > 0.1).

300.0

250.0

200.0
Time.to.Open.(s)

150.0

100.0

50.0

0.0
0 2 4 6 8 10 12 14 16 18 20
Day

Control High.Temp Low.Salinity

Fig.  1.  Locomotor  ability,  m easured  as  time  to  open  after  closing,  of  Aiptasia  in  control  (1.025  sg  and  22°C),  low  
salinity  (1.019  sg),  and  high  temperature  (30°C)  conditions.  Aiptasia  did  not  display  any  significant  change  in  
locomotor  ability  between  low  salinity  (Tukey  HSD,  p  =  0.15)  or  high  temperature  (Tukey  HSD,  p  =  0.18)  and  control  
conditions.  

To test the resiliency of Aiptasia in stressful conditions, four of the seven anemones in
the low salinity treatment were moved into control conditions after two weeks. The time to open
for the returned Aiptasia was similar to that of the Aiptasia in control conditions (Tukey HSD, p
= 0.993). Despite insignificant differences between Aiptasia returned to the control treatment and
the original control group, Fig. 2 shows that returned anemones more closely resemble the
locomotor ability of control anemones than those in low salinity after two weeks in control
conditions.
 Time  to  Open  (s)  

Control   Low  Salinity     Returned  

     
 
Fig.  2.  Locomotor  ability  of  Aiptasia  in  low  salinity  conditions  was  not  significantly  slower  than  Aiptasia  in  control  
conditions  (Tukey  HSD,  p  =  0.196).  Aiptasia  returned  to  control  conditions  after  two  weeks  also  did  not  show  a  
significantly  different  locomotor  ability  from  those  in  low  salinity  treatment  (Tukey  HSD,  p  =  0.226),  and  approached  
locomotor  times  of  the  control  group  (Tukey  HSD,  p  =  0.993).    

Change in Appearance or Death

Although the locomotor assay did not show significant differences in locomotion of
Aiptasia between conditions, drastic changes in physical appearance were observed in low
salinity treatments where anemones often presented as swollen and closed, or even dead.
Statistical analysis via ANOVA and Tukey test confirmed that a significantly larger number of
Aiptasia were closed or dead in low salinity group treatment compared to the control group (p =
0.034). However, there was an insignificant change in number of closed Aiptasia between the
control condition and the high temperature treatment (p = 0.67) (Fig. 3). A post-hoc Tukey test
revealed that there was no significant difference in the number of closed anemones during the
second half of the experiment between any treatments (p > 0.05). Additionally, a number of
polyps formed when Aiptasia were in control conditions for three weeks. Young moved to a high
temperature treatment were able to survive and grow.

 
 Number  of  Closed  Aiptasia  

Control   High  Temp   Low  Salinity  

     
 
Figure  3  Comparison  of  number  of  Aiptaisa  that  appeared  as  closed  or  dead  in  control,  high  temperature,  and  low  
salinity  conditions.  There  were  significantly  more  closed  Aiptasia  in  the  low  salinity  vs.  control  conditions  (Tukey  
HSD,  p  =  0.034).  There  was  no  significant  difference  between  control  and  high  temperature  conditions  (Tukey  HSD,  
p  =  0.67)  

Discussion

Aiptasia subjected to a low salinity treatment more significantly presented as closed or

dead upon testing in the first two weeks, however Aiptasia in a high temperature treatment
exhibited insignificant periods of closure and no deaths. All Aiptasia in control conditions for at
least three weeks reproduced, and young polyps survived in the high temperature treatment.
Analysis showed that locomotor rates in the high temperature group, low salinity group and
control group were not significantly different following two weeks of treatment. Interestingly,
Aiptasia in low salinity treatments presented concerning appearances throughout the first two
weeks of experiments as well. Often Aiptasia in the low salinity treatment were intensely
swollen in both their body column and many or all of their tentacles after only two weeks.

Although the maintenance of intracellular osmolarity is complicated by zooxanthellae in

gastrodermal cells, the insignificance of changes in locomotor functions during the first two
weeks indicate low salinity did not result in additional energy expenditure. Nevertheless,
Aiptasia clearly experienced significant complications from low salinity based on the number of
anemones who presented as closed or dead following treatment. We predicted that under osmotic
stress from highly variable salinity, Aiptasia would be forced to undergo extensive synthesis of
COOs which would increase energy expenditure and reduce locomotor ability. Instead we saw
no indication of increased energy expenditure based on the insignificant changes in locomotor
ability. Coupled with the diminished appearance of low salinity anemones, this may provide
evidence that Aiptasia synthesize life sustaining molecules and render them useless in order to
reduce osmolytes and achieve lower osmolarity. It appears that RVD may have the intended
effect of reducing intracellularly osmolarity, but life-sustaining glycerol and proteins may be
compromised in the process, leading to tentacle loss and eventual death.
 If climate change is mitigated so that storm severity does not evolve to further reduce
salinity, Aiptasia may be seen as the primary Anthozoan in tropical areas since it is often
considered a hardier and faster reproducer than its tropical relatives (Sunagawa et al. 2009) and
has demonstrated ability to survive for short periods of severe osmotic stress. Kerswell and Jones
(2003) collected coral from the Great Barrier Reef and observed coral bleaching at 1.021 sg
indicating an inability for tested species to survive at reduced salinity. Aiptasia were able to cope
for two weeks at an even further reduced salinity than tested by Kerswell, providing further
evidence for possible increases in Aiptasia populations relative to other tropical Anthozoans.
However, the high mortality and closure responses as well as the absence of reproduction during
low salinity treatment reveals physiological limits to the duration of low salinity osmotic stress.
Such a severe acute response to conditions that naturally occur during storms in tropical areas
provides reason for concern when considering that the frequency and intensity of these
conditions may only increase as climate change continues (IPCC 2007). As anthropogenic
emissions continue to increase and climate change further evolves, Aiptasia populations may
experience increasingly greater mortality due to increasingly lower salinity.

Alternatively, Aiptasia displayed high phenotypic plasticity through their resilience to a

high temperature treatment. Suharsono et al. (1993) found that another Anthozoan, Anemonia
viridis, experienced decreased lysosome integrity and destabilization of the mesentery filaments
following exposure to 26°C seawater. A. viridis are found in the Mediterranean Sea which has
comparable temperatures to tropical waters. Additionally, Downs et al. (2002) reported
significant coral bleaching in the Florida Keys where temperatures reached 30°C as well. Despite
this previous research, Aiptasia did not present signs of destabilized mesentery filaments through
compromised locomotor function or bleaching at 30°C. Furthermore, young polyps thrived in
high temperature conditions, but it is important to consider that no further reproduction occurred
following high temperature treatment. High temperatures do not appear to impact Aiptasia’s
fitness compared to osmotic stress or other tropical Anthozoans, which indicates a potential for
larger Aiptasia populations in the future.

The current conditions appear uncompromising for Aiptasia, but future climate
predictions may still pose a threat. High phenotypic plasticity allows Aiptasia to survive in high
temperature and low salinity conditions that pose significant threats to other Anthozoan species.
Nevertheless, changes in salinity and temperature can occur simultaneously as climate change
progresses. Investigating how Aiptasia react to multiple stressors will provide further insight into
the limitations of Aiptasia’s phenotypic plasticity. Because of the predicted increase in bleaching
events, characterized by mass expulsion of symbiotes, studying aposymbiotic anemones will
provide further insight into the anemone’s physiology, uncomplicated by the presence of a
symbiote. Hosts are responsible for removing the waste and toxins produced by symbiotes. This
may be complicating osmoregulatory abilities of Anthozoans when symbiotes are present.
Furthermore, an Anthozoan like Aiptasia without a symbiotic algae like zooxanthellae present
will be free to undergo higher levels of protein synthesis because zooxanthellae will not be
utilizing nitrogen found in free amino acids. Future research is clearly necessary to understand
the complicated future of Anthozoans in the face of climate change with Aiptasia being able to
represent a resilient model for symbiotic species.

 
 
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