1. Environmental Effects on Population Density and Carbonic Anhydrase Activity
in Cultured Zooxanthellae (Symbiodinium sp.)
Roni Devassy, Amy Parekh, Erin Graham, and Dr. Robert Sanders
Temple University, Philadelphia, PA
Introduction
Tropical marine waters are generally nutrient poor. In order to fulfill daily nutrient
requirements, many marine invertebrates have developed a symbiotic relationship
with zooxanthellae, photosynthetic dinoflagellates of the genus Symbiodinium.
Zooxanthellae use dissolved inorganic carbon (DIC) and sunlight to perform
photosynthesis and translocate photosynthetic product to their hosts. In exchange
for the fixed carbon (sugars) produced by zooxanthellae, the invertebrate host
provides zooxanthellae protection and nutrients such as nitrogen and phosphorus
(Muscatine et al., 1984).
Zooxanthellae fix carbon dioxide (CO2) via the Calvin Cycle, yet CO2 composes only
0.5% of the total DIC supply in seawater. To acquire sufficient CO2 for zooxanthellae
photosynthesis, the invertebrate host takes up seawater bicarbonate (HCO3-) and
converts it to CO2 via the enzyme carbonic anhydrase (CA). Free-living Symbiodinium
may acquire CO2 directly from seawater by passive diffusion or active uptake using a
CO2 transporter, however, evidence shows that independent Symbiodinium rely as
well on uptake of HCO3-, the dominant species in seawater (Leggat et al., 1999), and
Symbiodinium have both internal and external CA enzyme (Al-Moghrabi et al., 1996).
In addition, recent evidence suggests that the primary method of CO2 acquisition by
Symbiodinium may be phylotype-dependent (Brading et al., 2011). Biotic factors such
as pCO2 and temperature may further affect DIC uptake. In this study, we investigated
and compared the varying degrees of tolerance to thermal stress seen in cultured
Symbiodinium representing clades A, B, D, and F.
References
Acknowledgements
Methods
Results
Summary of Results and Conclusions
Special thanks to Dr. Scott A. Fay and Dr. Mary Alice Coffroth for providing Symbiodinium
cultures. Thanks are also in order to Sarah DeVaul, Zaid McKie-Krisberg, and Grier Sellers for
support in various capacities throughout the year.
Figure 1 (top left): Zooxanthellae use carbonic anhydrase to obtain sufficient CO2 for
photosynthesis. In the animal, carbonic anhydrase is found in all three germ layers, essential for
maximum DIC uptake(Photo credit: Furla 2005). Figure 2 (top right): Plexaura kuna, an octocoral
species, and associated Symbiodinium comprise the model system used by Dr. Mary Alice Coffroth
to study the origin of cnidarian-algal symbioses (Photo Credit: Smithsonian Tropical Research
Institute). Figure 3 (bottom right): Porites divaricata is a coral species typically found in shallow
tropical waters. P. divaricata has a characteristic wide branching arrangement in its structure
(Photo Credit: Coralpedia).
Cultures of Symbiodinium clade A isolated from Cassiopeia xamachana (Cx) and an unknown invertebrate
species (Symbio A), clade B isolated from Plexaura kuna (13), clade D isolated from Porites divaricata
(Pdiv), and clade F isolated from Montipora verrucosa (Mv), were obtained from BURR Cultures (Buffalo,
NY). Cell cultures lines were established by combining 500µl aliquots from parent cultures with 25ml
artificial seawater (ASW-32) with F/2 culture medium. After zooxanthellae underwent a two week growth
period, cultures were secured in three glass aquaria containing deionized water heated to 25˚C, 29˚C, and
33˚C, respectively, using aquarium heaters. Cultures were grown for two months under cool fluorescent
lighting on a 12:12 light:dark cycle prior to sampling.
Samples were prepared by withdrawing 6ml of culture into a centrifuge tube, centrifuging the sample for 8
minutes at 4000rpm, removing the supernatant through decanting, and resuspension of cells in 3.5ml DI
water. After glass beads were added to each tube, each sample was vortexed. Sonication on ice for five-
30 second periods was performed to break open the Symbiodinium cells and release carbonic anhydrase.
After sonication, 1500µl of the mixture was set aside for assays. The remaining culture extraction was
boiled for 5 minutes at 101˚C to denature the enzyme. Chilled veronal buffer was added in an equivalent
amount to both the active and boiled enzyme fractions. Fractions were vortexed and divided into three
vials (1 mL buffered mixture each). Each vial was then diluted with an additional 1mL chilled veronal
buffer. Samples were placed on ice until the assay.
For each assay, buffered lysed algal cells were equipped with a calibrated, temperature-sensitive pH meter
and magnetic stir bar. Once the temperature of the sample reached 13.5-14˚C, an initial pH value was
recorded and 1 mL of CO2-enriched water (pH 3.5) was injected into the sample. PH values were recorded
every 10 seconds for two minutes. The mean ΔpH of the three denatured samples was subtracted from
the mean ΔpH of the three active samples to obtain carbonic anhydrase activity, which was normalized to
algal cell population density.
To determine Symbiodinium population density, 200µl of culture sample and 1200µl ASW were placed into
a Phycotech Settling Chamber observed at 1000X magnification. Cell population density was calculated
using the average number of cells per approximately 80 grids for each sample and applying the
appropriate dilution factor.
Al-Moghrabi, S., C. G Oiran, D. Allemand, N. Speziale, and J. Jaubert. 1996. Inorganic carbon uptake for photosynthesis
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Brading, P., M. E. Warner, P. Davey, D. J. Smith, E. P. Achterberg, and D. J. Suggetta. 2011. Differential effects of ocean acidification on growth
and photosynthesis among phylotypes of Symbiodinium (Dinophyceae). Limnol. Oceanogr., 56(3): 927–938.
Furla, P., D. Allemand, J.M. Shick, C. Ferrier-Pages, S. Richier, A. Plantivaux, P.L. Merle, and S. Tambutte. 2005. The symbiotic anthozoan: A
physiological chimera between alga and animal. Integr. Comp. Biol. 45: 595-604.
Leggat, W., M. Badger, and D. Yellowlees. 1999. Evidence for an inorganic carbon-concentrating mechanism in the symbiotic dinoflagellate
Symbiodinium sp. Plant Physiol 121: 1247–1255.
Muscatine, L., Falkowski, P., J. Porter and Z. Dubinsky, Z. 1984. Fate of photosynthetically fixed carbon in light and shade adapted colonies of the
symbiotic coral Stylophora pistillata. Proceedings of the Royal Society of London Ser B. 222: 181-202.
Plexaura kuna. Smithsonian Tropical Research Institute. 12 March 2008 <http://biogeodb.stri.si.edu/bioinformatics>.
Porites divaricata. Coralpedia. 25 April 2012 <http://coralpedia.bio.warwick.ac.uk/en/corals/porites_divaricata.html>.
Carbonic Anhydrase Activity
•Carbonic anhydrase activity increased in clade D (Pdiv) as temperature increased. This suggests
that clade D Symbiodinium are well adapted to take up DIC in a warmer environment.
•Carbonic anhydrase activity greatly decreases at 33˚ C in clade A (Symbio A) and was
completely eliminated in clades B (13) and the Cx culture (clade A), indicating that these
Symbiodinium clades have a thermal tolerance limit below 33˚ C.
•CA activity for clade F (Mv) dropped significantly from the room temperature treatment, yet
CA activity was still able to be detected at the highest temperature.
•Clade D (Pdiv) relies more on CA for DIC uptake than the other clades investigated in this
study.
Symbiodinium Population Density
•Both clade F (Mv) and clade B (13) increased in population density across all temperature
treatments. The greatest increase was at 29˚ C, suggesting that this is their optimal
temperature.
•Clade D (Pdiv) experienced the same decline in population density across all temperature
treatments, suggesting that an increase in water temperatures has a minimal effect on clade D
(Pdiv). The population decline must have been due to the nutrient composition of the culture
media or overpopulation of the cultures.
•Different responses among clade A cultures (Cx and Symbio A) make it difficult to generalize a
clade response. Both cultures had large decreases in population at 29˚ C and 33˚ C, which
shows that clade A is more sensitive to high temperatures than the other clades studied.
Our results show that different clades of free-living Symbiodinium have varied tolerance to thermal
stress. Future studies will look into the extent to which different Symbiodinium species can maintain
essential functioning in response to rising aquatic pCO2 levels, which is a component of global
warming.
Future Directions
Figure 4 (top right graph): Carbonic
anhydrase activity per cell in four
Symbiodinium clades at different
temperatures. Bars represent means of five
replicates with standard error. Figures 5 and
6 (bottom graphs): Percent change in
population density for four clades of
Symbiodinium at different temperatures
grouped by temperature (left) and clade
(right). Bars represent means of five
replicates.
Note: No living cells were present in 13 and CX
cultures after 2 months in treatment.
Figure 7 (left):
Experimental set-up
consisted of culture
flasks maintained at
each temperature
treatment. Cells
were exposed to
the treatments for 2
months.
Figure 8 (right):
Typical
Symbiodinium cell
diameter appeared
to range between 5
to 10 µm (1000X).