Effects of temperature and nutrient variation on bleached and unbleached

Astrangia poculata.

Lucero Flores

Northwestern University

December 18, 2017

1

Abstract

Corals have a symbiotic relationship with the unicellular algae zooxanthellae. The coral

hosts zooxanthellae in its tissue and the zooxanthellae transfers up to 95% of its photosynthetic

material to the coral. However, coral bleaching, characterized by a loss in zooxanthellae, is

occurring more often as global temperatures and nutrient pollution increases. This weakens coral

health and ability to grow.

This experiment explores the effects of two temperature treatments (12oC and 23

oC) and

two nutrient treatments (ambient ammonium levels in seawater and 20uM ammonium levels in

seawater) on bleached and unbleached Astrangia poculata. Six bleached and six unbleached

corals were set in water baths in two seperate growth chambers at 12oC and 23

oC . Three of the

six bleached and unbleached corals were treated with 20uM of ammonium. This treatment

continued for 16 days. A Zeiss Axio Zoom v16 and the Fiji ImageJ application was used to

examine zooxanthellae densities (zooxanthellae cells / mm2) before and after the treatment. Coral

were fed and water was exchanged every third day. On the second water exchange, subsamples

of the corals’ seawater were collected at four intervals during a 60 minute period to examine

ammonium uptake and release with Solorzano (1969) method. At the end of the experiment

chlorophyll-a (ug) / dry weight coral (g) were examined with a modified version of the Lorenzen

(1967) and Erickson (2009) methods.

After the 16 day treatment there was no statistically significant change in zooxanthellae

except for unbleached coral at 23oC with added ammonium where there is a definite decrease

from 59.934 to 28.154 cells*mm-2

*102

. Average chlorophyll-a concentrations for bleached coral

were lower than concentrations for unbleached coral in all treatments. Results show that

bleached corals experience a net excretion of ammonium while unbleached corals experience a

net uptake of ammonium at both temperature treatments.

Key Words: Symbiodinium symbiosis, nitrogen pollution, coral bleaching

Introduction

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Corals, a type of marine invertebrate usually live in colonies of individual polyps (Figure

1). Coral have a symbiotic, or mutually beneficial relationship with Symbiodinium, a unicellular

algae also known as zooxanthellae. The coral hosts zooxanthellae in its tissue and the

zooxanthellae transfers up to 95% of its photosynthetic material to the coral (Figure 2). The coral

uses this material as energy to respire, grow, reproduce and to accumulate carbon calcium

(CaCO3) for its skeleton (Moynihan 2017).

Coral reefs are a valuable part of the world’s ecosystem because more than 25% of

world’s fish biodiversity are dependent on them (Spaulding et al. 2001). Corals have been used

to find treatments for cancer, asthma, arthritis, and inflammatory disorders. Corals also serve as

storm buffers that take energy out of waves reaching shores. Unfortunately, many studies show

that as global temperature and pollution has increased over time corals have experiences major

bleaching events, characterized by a loss in zooxanthellae, which weakens corals’ health and

ability to grow (Donner 2005). One specific type of pollution that affects coral health and

bleaching is nutrient overloading. Marine environments are usually nitrogen limited; a shift from

nitrogen limitation where coral live results in a phosphate limitation which increases corals’

vulnerability to heat and promotes bleaching (Radecker et al 2015).

As such, this project studies coral colonies of bleached and unbleached Astrangia

poculata coral colonies under two temperature and two nutrient levels. A. poculata commonly

called the Northern Star Coral is a temperate coral native to the western Atlantic Ocean and can

be found in Woods Hole, Massachusetts. A. poculata can tolerate temperatures from -1o C to 25

o

C (Dimond and Carrington 2007). Maximum photosynthesis for zooxanthellae in a variation of

astrangia coral occurs at about 400 umoles photons/ m2 / s (Jacques and Pilson 1983). A.

poculata is also one of the only coral that naturally occurs in bleached and unbleached forms so

it serves as a good way to study corals’ relationship to zooxanthellae. This experiment explores

how zooxanthellae and levels of chlorophyll-a in A. Poculata are impacted at a higher (23oC) and

a lower (12oC) temperature and in ambient and above ambient (20uM) ammonium

concentrations in seawater.

Methods

Collection and Acclimation

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12 bleached and 12 unbleached coral were collected near the one of the Marine

Biological Laboratory docks in Woods Hole, Massachusetts (41°31'28.4"N 70°40'23.1"W). I

kept the coral at ambient temperature in a common garden for five days. Then I placed the each

coral in individual 9 oz. cups with 200 mL of filtered seawater. I distributed coral into four 33

cm x 23 cm x 7 cm white plastic containers (three bleached and three unbleached coral in each

container). I filled the container with filter seawater to serve as a bath that could keep water

temperature even throughout the six cups in each container. Next I acclimated the corals in two

growth chambers at 12oC and 23

oC. Each growth chamber housed two of the white containers

with corals. The coral were acclimated at the two temperatures for threes days before the

ammonium treated samples were stained with calcein to mark the start of the treatment. Then,

the coral started their 16 day treatment.

Treatment

After acclimating to the two treatment temperatures (12oC and 23

oC) for three days, I

designated one container in each growth chamber to hold above ambient level ammonium treated

corals. These corals (three bleached and three unbleached) were stained with calcein to mark the

start of the 16 day treatment. I made calcein in a 2500 mg/ L stock solution. I applied 0.8 mL of

calcein to 199.2 mL of filtered seawater for 15 hours (Holcomb et al 2012). I exchanged water

for 200mL of fresh seawater. The corals in the other container served as a control with no dye

and no added ammonium during the treatment (Figure 3).

To add ammonium I prepared a 10,000 uM stock solution of ammonium chloride

(NH4Cl). I started the treatment by giving 200mL of clean filtered seawater to the control

treatment corals. I gave 196 mL of clean filtered seawater and 4 mL of 10,000 uM NH4Cl to the

corals in the ammonium treated container for a 20 uM ammonium treatment. Corals were fed

artemia (brine shrimp) every third morning. I exchanged water for the corals every third night

after feeding. During the treatment, two carboys filled with filtered seawater were kept in the

growth chambers to make sure new water would be at the same temperature as the treatment.

Any macroalgae growing on the coral was also removed before clean water was added.

Lighting in the growth chambers was kept at 450 umoles photons * m-2

* s-1

on a 12:12

hour light:day schedule. After 16 days of treatment, I applied alizarin red to the ammonium

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treated corals to stain them and mark the end of the treatment. I made alizarin in a 125 mg /100

mL stock solution. 0.8mL of stock solution added to 199.2 mL of seawater for 15 hours

(Holcomb et al 2012).

Imaging for Zooxanthellae

On the second day of acclimation I briefly removed each 12oC coral to take images of

their zooxanthellae using a Zeiss Axio Zoom v16 microscope. On the third day of acclimation I

briefly removed each 23oC coral to take images of their zooxanthellae using the same

microscope. These images serve as the “before” treatment zooxanthellae images for the corals. I

took a macro shot of two polyps that represented the distribution and density of zooxanthellae for

that coral. Then I zoomed into each of the two polyps and took three photos of different areas on

each polyp that also had a good representation of zooxanthellae distribution and density.

I used the cell counter plugin on the Fiji Imagej application to count the zooxanthellae in

each zoomed image. I drew a box around an area of each zoomed image that represented

zooxanthellae distribution and density well. Each zooxanthellae in this boxed area was counted. I

used the amount of zooxanthellae cells in the image area (on the scale of um2) and scaled up to

have zooxanthellae cells / mm2 for each image from each polyp. These densities were averaged

to have a concentration of zooxanthellae / mm2 for each coral. I used the average concentrations

for each coral to get an average zooxanthellae concentration for each treatment. I took “after”

images of the coral 20 days later, after the initial calcein dying process, 16 day treatment period,

and alizarin red dying process was complete. The same method was used to determine the

average zooxanthellae cells / mm2 for bleached and unbleached corals after the treatment was

over.

Ammonium concentrations

To examine the rate at which the ammonium treated corals excreted or took up

ammonium I ran an ammonium testing experiment. On the second water exchange cycle, I

collected a 10 mL sub sample from the each coral cup before added the coral to it. This served as

the initial (time = 0) concentration of ammonium. Then I collected subsamples at time = 15, 45,

5

and 60 minutes after the corals were places in their cups again. Each subsample was collected

with clean acid washed syringes, vials, swinexes, and ashed 25mm G/GF filters.

Ammonium concentrations were measured with a Cary 50 scan spectrophotometer

following methods described by Solarzano (1969) and by Zumdahl, Chemical Principles.

Chlorophyll-a concentrations

After completing the treatment and final zooxanthellae images I removed coral tissue and

zooxanthellae from the each skeleton using a modified version of the air-spraying technique

described by Conlan et al (2017). Any macroalgae growing on the coral was removed before

removing tissue. Tissue was removed inside a clean upside down plastic bottle with two holes

2.5 cm in diameter cut into the sides of the bottle 90o from each other. I held the coral with

forceps in one hole and held the opening to the air spray through the other hole. Filtered seawater

was connected to the air spraying gun and I removed organic material by shooting this water at

each coral until all tissue was removed. A 50 mL falcon tube was placed under the plastic bottle

to collect the organic matter. Organic material was kept on ice.

Each falcon tube was centrifuged at the highest speed for 10 minutes so the tissue wand

zooxanthellae formed a pellet at the bottom of the tube. The pellet was collected with a pipet and

transferred into a glass tissue grinder. Remaining seawater was discarded. The tissue was ground

up to remove zooxanthellae from tissue. This material was placed back into the falcon tube and

centrifuged at the highest speed for two minutes. The centrifuge separated the ground up tissue

from the zooxanthellae forming a new pellet at the bottom of the tube. This pellet was ground up

and centrifuged twice more to ensure all zooxanthellae was removed from tissue and collected.

After the final round of centrifuging, the zooxanthellae pellets were filtered to remove

excess seawater. This process uses a modified version of the SES Chlorophyll -a method by

Lorenzen (1967) acidification. G/GF filters with zooxanthellae were wrapped in foil, frozen for

two days, then places in clean falcon tubes with 30 mL of 90% buffered acetone for 9 hours.

After 9 hours I placed 7mL sub samples from each falcon tube into test tubes to examine

chlorophyll-a via fluorescence analysis with standard operating procedure for chlorophyll-a

(Erickson 2009).

I took a reading for fluorescence before and after adding 100 uL HCl. I calculated

chlorophyll-a concentration with the equation ((RB - RA) * FS * (r/(r-1)) * dilution factor = ug

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of Chl a in 7mL. Where RB = Reading before adding acid , RA = Reading after adding acid, r =

acid ratio: RB/RA of calibrating standard, and FS = calibrating standard concentration / reading

of standard. I performed dilutions for corals with more zooxanthellae as needed. Chlorophyll-a

concentrations where scaled up to total the amount (ug) in the falcon tube with 30 mL of acetone.

Chlorophyll-a (ug) was divided by the dry weight of each coral. Chlorophyll-a (ug) / dry weight

coral (g) was averaged for each treatment.

Results

On average, bleached corals show a net excretion of ammonium while unbleached corals

show a net uptake of ammonium at both temperature treatments. Bleached corals at 12oC go

from 18.8 uM ammonium in seawater to 21.3 uM ammonium in seawater (Figure 4). Unbleached

corals go from 18.08 uM ammonium in seawater to 17.8 uM ammonium in seawater.

Unbleached corals at 23oC go from 17.7 uM ammonium in seawater to 19.7 uM ammonium in

seawater. Unbleached corals go from 18.5 uM ammonium in seawater to 17.3 uM ammonium in

seawater (Figure 5).

Average before and after zooxanthellae density (cells*mm-2

*102) measurements

decrease in bleached corals at 23oC with added ammonium (2.087 to 1.085), in bleached corals at

23oC with no added ammonium (5.006 to 3.944), and in unbleached corals at 23

oC with added

ammonium (59.934 to 28.154). Average zooxanthellae density measurements increase for

unbleached corals at 23oC with no added ammonium (75.496 to 83.255) (Figure 6). However,

error bars for all conditions are so large that there is no sign of a real change in zooxanthellae

taking place except for unbleached coral at 23oC with added ammonium where there is a definite

decrease.

Average before and after zooxanthellae density (cells*mm-2

*102) measurements

decrease in bleached corals at 12oC with added ammonium (2.789 to 2.562), in bleached corals at

12oC with no added ammonium (8.257 to 3.173), and in unbleached corals at 12

oC with added

ammonium (40.953 to 39.097). Average zooxanthellae density measurements increase for

unbleached corals at 12oC with no added ammonium (35.958 to 37.268) (Figure 7). However,

error bars for all conditions are so large that there is no sign of a real change in zooxanthellae

taking place at 12oC.

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Average chlorophyll-a concentrations for bleached coral are lower than concentrations

for unbleached coral in all treatments. Bleached coral with added ammonium at 23oC have a

chlorophyll-a concentration of 35.652 ug / g dry weight coral; bleached coral with no added

ammonium at 23oC have a chlorophyll-a concentration of 24.062 ug / g dry weight coral;

unbleached coral with added ammonium at 23oC have a chlorophyll-a concentration of 630.611

ug / g dry weight coral; unbleached coral with no added ammonium at 23oC have a chlorophyll-a

concentration of 915.6 ug / g dry weight coral (Figure 8). Bleached coral with added ammonium

at 12oC have a chlorophyll-a concentration of 32.434 ug / g dry weight coral; bleached coral with

no added ammonium at 12oC have a chlorophyll-a concentration of 87.342 ug / g dry weight

coral; unbleached coral with added ammonium at 12oC have a chlorophyll-a concentration of

591.870 ug / g dry weight coral; unbleached coral with no added ammonium at 23oC have a

chlorophyll-a concentration of 378.7 ug / g dry weight coral (Figure 9).

Discussion

Ammonium uptake/excretion results show that algae do use ammonium, but this

ammonium use is regulated by the host coral and can eventually result in bleaching as indicated

by unbleached coral with added ammonium at 23oC.

Results show that only unbleached coral with added ammonium at 23oC experienced a

statistically significant change in zooxanthellae. Added ammonium makes the coral bleach. It is

not temperature that is affecting the A. poculata because this coral species tolerant of a wide

range of temperature and we do not see changes in the control corals with no added ammonium

significantly change their zooxanthellae densities. These results make sense given the Radecker

et al (2015) discussion that a shift from nitrogen limitation promotes bleaching (Radecker et al

2015).

The chlorophyll-a analysis show expected data where bleached corals have less

chlorophyll-a than unbleached corals. However, error bars in Figure 8 and 9 show that

chlorophyll-a concentrations for bleached and unbleached coral may be closer than anticipated.

This may be due to a loss of zooxanthellae for unbleached corals during the tissue extraction

process as tissue was being transferred multiple times between the falcon tube and the grinder.

This may also be due to macroalgae stuck to the coral tissue accidentally making its way into the

chlorophyll-a extraction.

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Because there were only noticeable changes in zooxanthellae in one treatment for

unbleached corals I believe it is necessary to run this experiment for more than 16 days. Results

with a lower range of error may also be reached by using more than three coral replicates per

treatment. Having a longer incubation period will also clarify if the initial zooxanthellae counts

are due to the temperature differences or ambient temperature conditions. Next steps for this

project include looking at the bands of calcein and alizarin red in the corals treated with

ammonium to examine their skeletal growth.

Acknowledgements

Thank you to those who made my work possible. The principal investigator of the lab I

worked in, Dr. Loretta Roberson, and her research assistant, Mayra Sanchez Garcia. Thank you

to Dr. Jim Tang for sharing his time and space in the growth chambers. Thank you to Louie Kerr

and Bonnie Kwiatkowski who helped with everything microscope and computer related. Thank

you to the SES teachers’ assistants Emily Stone, Jordan Stark, Alana Thurston, Richard

McHorney and classmates Alondra Soto, Caitlyn Linehan, and Luis Cartagena for help with

miscellaneous tasks throughout this project.

Literature Cited

Conlan, J. A., Rocker, M. M., & Francis, D. S.2017. A comparison of two common sample

preparation techniques for lipid and fatty acid analysis in three different coral

morphotypes reveals quantitative and qualitative differences. PeerJ, 5, e3645.

Dimond J., Carrington E. 2007. Temporal variation in the symbiosis and growth of the

temperate scleractinian coral Astrangia poculata. Marine Ecology Progress Series

348:161–172.

Donner, S. D., Skirving, W. J., Little, C. M., Oppenheimer, M. and Hoegh-Guldberg, O. 2005.

Global assessment of coral bleaching and required rates of adaptation under climate

change. Global Change Biology 11: 2251–2265.

Erickson, M. 2009. Standard Operating Procedure for Chlorophyll-a and Pheophytin-a (Turner

Designs Method). The Ecosystems Center, MBL.

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Finnerty R. J. “ The Northern Star Coral Astrangia poculata.” Marine Genomics, Boston

University, Lecture 3.

Holcomb M., Cohen, A.L., and McCorkle D.C. 2013 An evaluation of staining techniques for

marking daily growth in scleractinian corals. Elsevier Journal of Experimental Marine

Biology and Ecology 440:126-131

Jacques T.G., Marshall N., Pilson M.E.Q. 1983. Experimental ecology of the temperate

scleractinian Astrangia danae. II. Effect of temperature, light intensity and symbiosis

with zooxanthellae on metabolic rate and calcification. Marine Biology 76:135–148.

Moynihan A. M. 2017. Microbial Diversity of the northern star coral, Astrangia poculata.

Marine Biological Laboratory.

Lorenzen, C. (1967) Limnology and Oceanography 12:343-346.

SES Chlorophyll A Analysis Method by Lorenzen Acidification (2017)

Smithsonian Ocean Team. 2016. Zooxanthellae and Coral Bleaching. Ocean Portal Smithsonian,

Smithsonian's National Museum of Natural History.

Solorzano L.1969. Determination of ammonia in natural waters by the phenol hypochlorite

method. Limnology and Oceanography 14: 799-801.

Spalding, M.D., C. Ravilious, and E.P. Green. 2001. United Nations Environment Programme,

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California Press: Berkeley. 416.

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Figures

Figure 1 : Coral Polyp Anatomy

Source: Finnerty R. J.

Figure 2 : Coral symbiosis with zooxanthellae

Source: Smithsonian Ocean Team

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Figure 3 : Experimental design includes 4 bath containers that hold three bleached three

unbleached coral each. Each bath has a different combination temperature and ammonium

concentration in seawater.

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Figure 4 : Ammonium uptake or release for bleached and unbleached coral treated in 20 uM

ammonium seawater at 23oC.

Figure 5: Ammonium uptake or release for bleached and unbleached coral treated in 20 uM

ammonium seawater at 23oC.

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Figure 6: Zooxanthellae concentration (zooxanthellae cells / mm2) *10

2 for bleached and

unbleached coral before and after a 16 day treatment in 23oC.

B = Bleached, U = Unbleached, N = treated in 20uM ammonium filtered seawater, CTL =

control with no added ammonium.

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Figure 7: Zooxanthellae concentration (zooxanthellae cells / mm2) *10

2 for bleached and

unbleached coral before and after a 16 day treatment in 12oC.

B = Bleached, U = Unbleached, N = treated in 20uM ammonium filtered seawater, CTL =

control with no added ammonium.

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Figure 8: Chlorophyll-a (ug) / dry weight coral (g) for bleached and unbleached coral in a 23oC

treatment. B = Bleached, U = Unbleached, N = treated in 20uM ammonium filtered seawater,

CTL = control with no added ammonium.

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Figure 9: Chlorophyll-a (ug) / dry weight coral (g) for bleached and unbleached coral in a 12oC

treatment. B = Bleached, U = Unbleached, N = treated in 20uM ammonium filtered seawater,

CTL = control with no added ammonium.