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Student Project

The effect of heterotrophically sourced nutrition on the density of zooxanthellae in Aiptasia Pallida (Verrill).

Vanessa Clark 2016


Anthozoans form symbiotic relationships with zooxanthellae, through which both are involved in each other’s nutrient supply. Nutrients are obtained heterotrophically by the host and autotrophically by the symbiont. The level of partitioning between the host and the symbiont can be affected by many factors including temperature and the balance between heterotrophic and autotrophic nutrient input. Aiptasia pallida (Verrill) is a model organism for this symbiosis due to its hardiness and the ease at which it can be cultured in the lab. By subjecting A. pallida to two treatments, fed and unfed the density of zooxanthellae, as well as the A. pallida size was expected to increase in those that were fed compared to those that were not. Individuals were measured seven days post treatment, where size was determined by liquid displacement, and zooxanthellae density with a haemocytometer. There was a significant difference between the two treatment groups, with fed A. pallida having a zooxanthellae density less than half of those that were unfed. There was also a significant difference between the sizes of A. pallida in each treatment group, where those that were fed appeared to have grown more. However, as size was not measured prior to treatment it cannot be concluded whether this was a result of treatment. Studies such as this are important in assessing the Anthozoan-Zooxanthellae symbiotic relationship and can be important determining what can affect and change it. 


Anthozoan Cnidarians are known to form symbiotic relationships with unicellular photosynthetic dinoflagellates of the species Symbiodinum commonly called zooxanthellae. From this relationship the cnidarian host gains photosynthates,and the zooxanthellae is provided with a protected, light rich environment as well as waste products such as nitrogen and phosphorus (Davy and Cook 2001).


As a result of this symbiotic relationship zooxanthellae-Anthozoaare capable of obtaining nutrients heterotrophically, through the capture of zooplankton and marine snow, and autotrophically from the symbionts. These nutrients are used by both the Anthozoan and the zooxanthellae, but the ratio at which they are partitioned changes with environmental conditions (Rodriguez-Lanetty et al. 2003). Leal et al. (2014)through the use of DNA tissue analysis determined that prey assimilation occurred over roughly eight days. This rate was affected by prey species as well as the amount of prey captured. Suggesting that the capture of prey would affect the nutrient balance over the course of eight days and thus would affectthe way they were partitioned between the Anthozoan and the zooxanthellae. Many factors have been found to affect the retention and absorption of photosynthate from zooxanthellae by Anthozoan hosts.

When supplied with zooplankton for heterotrophic consumption Anthozoans still rely on photosynthate for immediate metabolism, while storing the rest as lipids (Farrant et al. 1987a, b). There have been observations of decreased (Clayton 1986, Cook et al. 1988)zooxanthellae density in cnidarians as a result of ‘starvation’ treatment, aswell as observations of increased density (Muller-Parker 1985). Decreased density was suggested to be as aresult of nitrogen limitation, while in studies that observed an increaseddensity was accounted for by an increase in the biomass of the host.

Bleaching occurs when stresses such as high temperatures, or low oxygen levels are experienced by zooxanthellae – Anthozoans. The bleaching effect is usually a result of the strain placed on the symbiont, preventing photosynthesis and leading to the expulsion of zooxanthellae (Fransolet et al. 2013). The effect of temperature on zooxanthellae density and photosynthate production has been widely investigated(Clark and Jensen 1982, Davy and Cook 2001). The Anthozoan host has been observed to draw from the zooxanthellae at a constant rate regardless of thez ooxanthellae density. However, it has been suggested that reduced symbiont density results in a greater amount of available carbon and thus a higher rate of photosynthesis in those remaining. 

Aiptasia pallida (Verrill) as a result of its hardiness and the ease at which it can be cultured in thelab has become a model organism for investigating the cnidarian – zooxanthellae symbiosis (Leal et al. 2013).A known aquarium pest, A. Pallida are naturally found throughout the tropical and subtropical oceans. Initially thoughtto be separate to Aiptasia pulchella the phylogeny is currently debated as to whether A. pallida and A. pulchella arespatially differentiated species or not (Thornhill et al. 2013, Voolstra 2013).

This study aims to determine the effect of increased heterotrophically obtained nutrients by the Anthozoan host on the zooxanthellae density of individuals. In this study this relationship is being specifically assessed inthe species Aiptasia pallida.

It was expected that increased heterotrophic input would result in a decreased zooxanthellae density in A. pallida. This is due to the decreased reliance on, and partitioning of nutrients to the zooxanthellae resulting in expulsion, as well as the expected increased biomass of fed compared to unfed A. pallida. 

Materials and Methods

Experimental Treatment

60 individual Aiptasia pallida were collected from the aquarium located at the University of Queensland. A. pallida were divided equally into two plastic containers to ensure separation of the two treatment groups. These were labelled as ‘well fed’ and ‘unfed’, and left to settle for 60 minutes. The ‘well fed’ A. pallida were then target fed to repletion with small pieces of fish. Both containers were placed into separate isolated tanks that were part of the same filtration system and left for seven days. The term unfed is used rather than starved as the A. pallida still had access to nutrients within the water system.

Density Measurements

17 representative A. pallida were removed from each treatment container and each placed in a 1ml aliquot tube. 0.45µl of Calcium- and magnesium-free artificial seawater (CMF-ASW), and 0.5 µl of trypsin was pipetted into each aliquot tube. The liquid displacement method was used to determine the size class of each individual, with the size being noted as the overall volume of the aliquot tube. A plastic tissue grinder was used to homogenise each sample. The concentration of zooxanthellae in the homogenate was determined using a haemocytometer (Bright-Line, Hausser Scientific). The final calculated concentration for each sample was multiplied by the size class of the individual in order to standardise for differences in size.


Statistical analysis

Four individuals were removed from the data set for the analysis on the basis of being outliers by falling more than 1.5 interquartile ranges outside the first and third quartiles. A one way ANOVA was applied to the data treating the size corrected zooxanthellae concentration as the explanatory variable and the treatment of ‘fed’ or ‘unfed’ as the categorical predictor. All analysis was done using the R studio software (R Development Core Team 2015)


Size Class

There were a greater number of small (0.25ml) A. pallida observed in the fed treatment than the unfed (Figure 1). On average the unfed A. pallida had a larger size class (0.38 ± 0.028) than the fed A. pallida (0.30 ± 0.020) seven days post treatment. The size class differed significantly between treatments (ANOVA, F (1, 28) = 5.223, P = <0.05).

Zooxanthellae Concentration

There was a significantly (ANOVA, F (1, 28) = 10.6, P = <0.005) different zooxanthellae concentration (cells/ml) between the two treatments (Figure 2). Unfed A. pallida (mean = 693250 ± 94279.6 cells/ml) had a mean zooxanthellae concentration more than double that of fed A. pallida (340475 ± 94279.6 cells/ml)

Figure 1
Figure 2


The hypothesis that zooxanthellae density would decrease in A. pallida with a greater heterotrophic input was supported by these results. The unfed Aiptasia on average had a significantly higher size class, supporting the suggestions made by (Muller-Parker 1985) that the increased biomass in fed anthozoans was involved in the process of zooxanthellae density decrease. The unfed A. pallida were still able to obtain nutrient from within the water system, however, because the focus was on the increase of heterotrophic input and was present in both treatments there may have been no impact. But this presents an avenue for future research.


As size class was not measured prior to treatment it cannot be determined whether size class increased as a result of treatment or simply unequal samples. The following will be discussed under the assumption the observed greater size class in fed than unfed A. pallida was a result of the treatment. An increase in size by A. pallida as a result of increased heterotrophic input can be explained by higher rate of lipid storage because of excess nutrients, due to photosynthates being used as the primary source of metabolism (Farrant et al. 1987a, b). As the A. pallida were not observed over the course of the seven days it cannot be determined if the decrease in density of zooxanthellae in fed A. pallida was solely a result of increased biomass or if the expulsion of zooxanthellae was involved.


Understanding of this zooxanthellae – Anthozoan symbiosis is becoming increasingly important due to the bleaching events that are being observed globally in reef ecosystems. Global climate change is increasing the stresses being experienced by the zooxanthellae – Anthozoans symbiosis and resulting in mass bleaching through the expulsion of zooxanthellae. 


I would like to thank Bernie and Sandie Degnan for their guidance throughout this project as well as BIOL3211 tutors. 


Clark, K. B., and K. R. Jensen. 1982. Effects of temperature on carbon fixation and carbon budget partitioning in the zooxanthellal symbiosis of Aiptasia pallida (Verrill). Journal of Experimental Marine Biology and Ecology 64:215-230.

Clayton, J. W. 1986. Ingestion, Digestion and Assimilation Efficiency of the Sea Anemone Aiptasia pallida Fed Zooplankton. Internationale Revue der gesamten Hydrobiologie und Hydrographie 71:iv-iv.

Cook, C. B., C. B. Cook, C. F. D'Elia, and G. Muller-Parker. 1988. Host feeding and nutrient sufficiency for zooxanthellae in the sea anemone Aiptasia pallida. Marine Biology 98:253-262.

Davy, S., and C. Cook. 2001. The relationship between nutritional status and carbon flux in the zooxanthellate sea anemone Aiptasia pallida. Marine Biology 139:999-1005.

Farrant, P. A., M. A. Borowitzka, R. Hinde, and R. J. King. 1987a. Nutrition of the temperate Australian soft coral Capnella gaboensis - I. Photosynthesis and carbon fixation. Marine Biology 95:565-574.

Farrant, P. A., M. A. Borowitzka, R. Hinde, and R. J. King. 1987b. Nutrition of the temperate Australian soft coral Capnella gaboensis - II. The role of zooxanthellae and feeding. Marine Biology 95:575-581.

Fransolet, D., S. Roberty, A.-C. Herman, L. Tonk, O. Hoegh-Guldberg, and J.-C. Plumier. 2013. Increased cell proliferation and mucocyte density in the sea anemone Aiptasia pallida recovering from bleaching. PloS one 8:e65015.

Leal, M. C., J. C. Nejstgaard, R. Calado, M. E. Thompson, and M. E. Frischer. 2014. Molecular assessment of heterotrophy and prey digestion in zooxanthellate cnidarians. Molecular Ecology 23:3838-3848.

Leal, M. C., C. Nunes, S. Kempf, A. Reis, T. L. da Silva, J. Serôdio, D. F. R. Cleary, and R. Calado. 2013. Effect of light, temperature and diet on the fatty acid profile of the tropical sea anemone Aiptasia pallida. Aquaculture Nutrition 19:818-826.

Muller-Parker, G. 1985. Effect of feeding regime and irradiance on the photophysiology of the symbiotic sea anemone Aiptasia pulchella. Marine Biology 90:65-74.

R Development Core Team. 2015. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.

Rodriguez-Lanetty, M., S.-J. Chang, and J.-I. Song. 2003. Specificity of two temperate dinoflagellate–anthozoan associations from the north-western Pacific Ocean. Marine Biology 143:1193-1199.

Thornhill, D. J., Y. Xiang, D. T. Pettay, M. Zhong, and S. R. Santos. 2013. Population genetic data of a model symbiotic cnidarian system reveal remarkable symbiotic specificity and vectored introductions across ocean basins. Molecular Ecology 22:4499-4515.

Voolstra, C. R. 2013. A journey into the wild of the cnidarian model system Aiptasia and its symbionts. Molecular Ecology 22:4366-4368.