Understanding Coral Symbiosis and Bleaching: E�ects of Climate Change and Genetic Diversity on the Stability of Symbiosis

Eadaoin Harney, Eric Eberhardt, Jodi Schwarz Biochemistry Program, Vassar College

Conclusions

IntroductionThe coral reef ecosystem is entirely dependent upon a symbiosis between Symbio-dinium (a photosynthetic dino�agellate) and corals. However, this essential relation-ship is threatened by environmental stressors, such as global warming, which leads to the breakdown of symbiosis through a process known as coral bleaching.

Like coral reefs, the sea anemone species Aiptasia pallida can form a symbiotic rela-tionship with Symbiodinium. This relationship is non-speci�c, as both hosts are known to enter into symbiotic relationships with a wide range of Symbiodinium.

Symbiodinium can be divided into 11 genetically distinct clades. While little is known about phenotypic di�erences between the clades, the global distribution of Symbio-dinium clades hosted by Aiptasia pallida suggests that speci�c symbionts confer ad-vantages to hosts under di�ering conditions. Globally, Aiptasia pallida host clade B, except in the Florida Keys, where anemones typically host clade A. We are interested in understanding why anemones in this area tend to host clade A symbionts rather than clade B. This region has experienced environmental changes, including large-scale coral bleaching events, and we hypothesize that clade A symbionts may be more tolerant of the local environmental conditions than clade B.

This study attempts to determine whether Aiptasia pallida exhibit a preference for speci�c Symbiodinum clades, and the e�ects of temperature upon this selection. It ex-pands upon previous research from our lab, which indicates that Aiptaisa prefers to take up clade B symbionts. It also suggested that anemones may host multiple symbi-ont types, as trace levels of clade A symbionts were observed in conjunction with clade B symbionts in many anemones.

Methods

Exposure:

RFLP Analysis:

Sample Lyse Bind Wash Elute Ready-to-use DNA

DNA extraction-

Extracted DNA Region of Interest: 18S Heat Denature Anneal Primers Primer Extension Replicated DNA

RepeatPCR of 18S DNA-

Restriction Digest & Gel Electrophoresis-

Control A only B only A+B

25oC 3 3 3 8

29oC 3 3 3 8

32oC 3 3 3 8

Previous Data Collected In Our Lab

Our data from two independent experiments support the conclusion that Aipta-sia exhibits selective uptake of Symbiodinium. Uptake of clade B Symbiodinium was favored under non-stressed conditions, however, under heat stress (32oC) they are almost incapable of becoming infected. The only anemone to uptake symbionts at this temperature became infected with clade A. These results are consistent with the hypothesis that speci�c clades of Symbiodinium may confer advantages to their hosts under di�ering environmental conditions. In both ex-periments some anemones under non-stressed conditions that exhbited uptake of clade B symbionts demonstrated an additional uptake of low concentrations of clade A Symbiodinium. These results indicate that anemones can host mul-tiple clades of symbionts. Through uptake of multiple clades of symbionts, Aip-tasia pallida may be better able to withstand environmental changes.

Even though Aiptasia living in the Florida Keys are dominated by clade A symbi-onts, our data suggest that during the initial infection process, anemones are in-fected primarily by clade B symbionts. We propose that clade A symbionts may be better adapted for warmer temperatures, and may be out-competing clade B symbionts in the local environmental conditions of the Florida Keys

We examined whether water temperature in�uences the ability of Aiptasia to form a symbiosis with clade A vs clade B. We performed infection experiments, in which we exposed Aiptasia to a mixture of clade A and clade B, and then used RFLP analysis to determine which clade successfully infected the host. The anemones in these experi-ments were obtained from the Florida Keys, were experimentally bleached to remove their clade A symbionts, and maintained in an aposymbiotic state until used in these experiments

Acknowledgements Thank you to the Research Corporation for Science Ad-vancement for providing funding for this project through the Cottrell College Science Multi-investigator Award. Additional thanks go to Sam Seymour (Vassar College ‘11) for sharing the re-sults of his thesis, which this project expands upon.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

25 28

Per

cent

age

of A

nem

ones

In

fect

ed (

%)

Temperature (oC)

No Uptake

A

B

A+B

Aposymbiotic anemones were divided into 3 temperature groups, 25oC, 29oC, and 32oC. 25oC represents non-stressed conditions, while 32oC induces heat stress within anemones. 8 anemones in each group were bathed in a mixture containing an equal misture of clade A and B Symbiodinium. Remaining anemones were exposed to either clade A, B, or no Symbiodinium to act as controls. After exposure, anemones were placed in their respective temperatures and incubated for 16 days. Water was changed daily, and the anemones were fed twice during the experiment

*Numbers refer to number of anemones in each condition

Restriction Fragment Length Polymorphism (RFLP) is an analytical technique which exploits the exis-tence of minor genetic di�erences between homologous strands of DNA. Using restriction enzymes, pieces of DNA, which have been ampli�ed via PCR, are cut at speci�c restriction sites. Digested strands of DNA are then run on agarose gels, separating the fragments by size. RFLP is useful when genetic di�erences occur at sites which can be cut by restriction enzymes. When a di�erence occurs, one allele will be cut by the restriction enzyme, causing it to form two shortened strands, while one will not. These di�erences can then be seen as di�erent size bands on an agarose gel.

In Symbiodinium, minor genetic di�erences exist in the 18S gene, encoding the small subunit of ribo-somes, of di�erent clades. These di�erences can be observed through RFLP using the restriction enzyme TaqαI, which cuts at the site TCGA.

RFLP analysis of Symbiodinium DNA results in di�erent banding patterns for clade A and B Symbiodinium.

1 2 3 4Lane 1 contains a DNA ladder of known lengths.Lane 2 contains digested DNA from Aiptasia hosting clade A Symbiodinium. Lane 3 contains digested DNA from Aiptasia hosting clade A and B Symbiodinium.Lane 4 contains digested DNA from Aiptasia hosting clade B Symbiodinium.

Figure 1. E�ect of Temperature on Symbiont Uptake. The frequency of symbiont uptake by clade is present for each temperature condition, 25oC, 29oC, and 32oC. Anemones in this sample were exposed to both clade A and B Symbiodinium. Anemo-nes in non-stress temperature conditions (25oC and 29oC) exhibited a preference for uptake of clade B symbionts. Anemones at 32oC exhibited little symbiont uptake. How-ever, 1 anemone was infected with clade A symbionts.

This data suggests that under normal temperature conditions, clade B was more successful than clade A at infecting Aiptasia, but as temperature increased this trend began to decline, and clade A became the dominant clade in a subset of anemones. It is important to note that in both experiments, some anemones that exhibited strong uptake of clade B symbionts showed an additional uptake of a low concentrations of clade A symbionts, suggesting that dual uptake had occurred. Anemones in the 32oC condition exhibited little uptake of symbionts. These decreased uptake levels are consistent with the process of bleaching, or loss of symbionts, which is commonly observed under elevated temperatures.

In the current data set, uptake of symbionts in 2 of the 9 control anemones was observed. This is likely due to water contamination during the experiment, rather than the use of already infected anemones, as 7 of the 9 control anemo-nes showed no symbiont uptake.

Figure 1. E�ect of Temperature on Symbiont Uptake. The frequency of symbiont uptake by clade is pre-sented for each temperature condition, 25oC and 28oC. Anemones in this sample were exposed to both clade A and B Symbiodinium. Anemones in each temperature exhibited a preference for uptake of clade B symbi-onts. Data courtesy of Sam Seymour.

Results

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

25 29 32

Per

cen

tag

e of

An

emon

es I

nfe

cted

(%

)

Temperature (oC)

No UptakeABA+B

Aposymbiotic Anemone Symbiodinium Clade Unknown Clade A Clade B Symbiotic Anemone

and/or