QUANTIFICATION OF MICROSCOPIC ZOOXANTHELLAE ALGAE EXPULSION
AFTER INCREASED TEMPERATURE BY CANDY CANE CORAL, Caulastrea furcata
by
Nathalie Danielle Staiger
Spring 2015
A thesis
submitted in partial fulfillment
of the requirements
for a baccalaureate degree
in Biology
in cursu honorum
Reviewed and approved by:
___________________________________________
Dr. Frances S. Raleigh
Thesis Supervisor
Submitted to
the Honors Program, Saint Peter’s University
May 11, 2015
Acknowledgements
I give my sincere thanks to several individuals who helped me complete this research
project. First, I would like to thank Dr. Raleigh, who not only made countless edits, but also
helped me with changes to the design of the research when roadblocks were met. You motivated
me to continue, even at the lowest points of this project.
I would also like to thank Dr. Twersky, who helped me to hit the ground running and
create a successful grant proposal, which was accepted by the TriBeta Biological Research
Foundation.
My thanks are also thereby given to the TriBeta Biological Research Foundation, who
generously supplied a grant to support this research.
My thanks also go to Dr. Wifall and the Honors Department, for providing additional
funding and for encouraging the unyielding pursuit of an even higher level of education,
maturity, and self-growth.
Additional thanks are given to Professor Ruppert, who built the shelf for the corals,
devised a proper running water system, and overall provided great instructional advice over the
past two years as my boss and friend in the Biology Work-Study program.
I wish to thank Kristian Gutierrez for his everlasting support, in both the forms of
encouragement to accomplish anything to which I set my mind, as well as physical support for
lending a hand whenever I am in need.
Last, but certainly not least, I give my love and thanks to my parents, Philip and Monika
Staiger, for without whom I would not be the person I am today.
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ABSTRACT
Since the early 1980s, intense coral bleaching events have yielded increasingly expansive
coral reef ecosystems devoid of recovery. Commonly distinguished as the “rainforest of the sea,”
the biodiversity of coral reef ecosystems is unparalleled by any other marine ecosystem. Recent
global changes—primarily increased sea surface temperature (SST)—have threatened the
continued prosperity of corals, thereby threatening the innumerable lives of organisms depending
on reefs. Efforts to protect coral reefs have been initiated, yet questions still remain: exactly how
much of the mutualistic dinoflagellate algae dwelling within coral tissue are expelled from the
first encounter of environmental stress? This study focuses on imposing stress-inducing
temperature change to candy cane corals, Caulastrea furcata, from an ideal water temperature of
27ºC to elevated levels of 29 ºC and 30 ºC, in the span of two days. After the corals experienced
these higher temperatures, they were immediately reverted to ideal conditions. The corals were
monitored over the course of several weeks to determine the degree to which recovery was made.
The change in zooxanthellae density was determined from multiple extraction periods and coral
color was categorized via the Siebeck et al. Color Reference Card. Results indicated that the
corals experiencing 29 ºC had better recovery than 30 ºC, and that more than half of the algae
were expelled in immediate response to this thermal stress.
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Table of Contents
Table of Contents…………………………………………………………..………………….……………….…4
Introduction: Diversity…………………………..…………………………………………………………….….5
Location……………………....…………………………………………………………….…………...5
Human Benefits………………………………………………………………………...……………….6
Ideal Conditions………………………………………………………………………….....................7
Coral Reproduction and Budding…………………………………………………………………….....8
Fringing, Barrier, and Atoll Reefs………………………………………………………......................9
Coral Bleaching…………………………………………………………………………......................9
Anthropogenic Factors…………………………………………………………………......................10
Recorded Bleaching Events…………………………………………………………….......................12
Caulastrea furcata: Candy Cane Coral………………………………………………………………....13
Coral Adaptability: Symbiont Shuffling………………………………………………………………..13
Research Objectives……………………………………………………………………......................14
Materials and Methods: Invertebrate Organisms………………………………………………………………....14
Figure 1: The Test Corals…………………………………………………………..…........................14
Acclimation……………………………………………………………………………………………..15
Figure 2: The Corals Upon Arrival……………………………………………………………………..15
Figure 3: The Acclimation Process………...…………………………………………………………..16
Proper Water Flow…………………………………………………………………………………...…16
Figure 4: The Closed Tap Water System………………………………………………......................17
Figure 5: Blue Food Dye………………...……………………………………………………………..17
Figure 6: Siphoned Connecting Tubes……………………………………………..….......................18
Figure 7: Temporary Housing……...………………………………………………………………….18
Running Water System………………………………………………………………………………...18
Figure 8: The Complete System……………………………………………………………………….19
Lighting………………………………………………………………………………..……………….20
Feeding………………………………………………………………………………….....................20
Figure 9: Brine Shrimp Hatching Kit………………………………………………………………….20
Heating………………………………………………………………………………….....................21
Water Quality Stability………………………………………………………………………………...21
Figure 10: Salt Water Comparison Chart……………………………………………………………...22
Figure 11: Hydrometer………………………………………………………………………………...22
Figure 12: Calcium Test Kit…………………………………………………………………………...22
Figure 13: Ammonia Test Kit………………………………………………………………………….22
Quantifying Bleaching………………………………………………………………….....................23
Figure 14: Siebeck et al. Colour Reference Card…..…………………………………......................23
Figure 15: Zooxanthellae Extraction Process………………………………………...………………..25
Photography……………………………………………………………………………......................25
Results: Water Quality……………………………………………………………………………..….………....26
Figure 16: Main Tank Water Factor Quality Levels…………………………………………………...27
Figure 17: Control Tank Water Factor Quality Levels………………………………………………...27
Figure 18: +2ºC Tank Water Factor Quality Levels…………………………………………………...28
Figure 19: +3ºC Tank Water Factor Quality Levels………………………………………………..….28
Temperature Changes……………………………………………………………………….………….29
Figure 20: Temperature Changes for All Tanks………………………………………........................29
Zooxanthellae Extractions…………………………………………………………………..………….29
Table 1: Baseline Count of Zooxanthellae Before Heat Stress…………………..………….…………30
Table 2: Count of Zooxanthellae After Heat Stress…………………………………...........................31
Table 3: Average Count of Zooxanthellae Before and After Heat Stress…………..………………….31
Coral Color Changes………………………………………………………………………….………..31
Figure 21: Control Coral Color……………………………………………………………….………..31
Figure 22: +2ºC Polyp Coral Color………………………………………………….………………...31
Figure 23: +3ºC Polyp Coral Color………………………………………………….………………...32
ANOVA…………………………………………………………………………….….……….………32
Table 4: Nested ANOVA: Analysis of Variance for Cell Number……………………........................32
Discussion…………………………..………………………………………………..………………………..…33
Literature Cited……………………………………………………………………………………..….………...35
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INTRODUCTION
Diversity
Coral reefs have existed on the planet for over 500 million years (Mondadori, 1999),
providing a source of both food and shelter to countless inhabitants of the ocean. Ranging from
simple organisms such as sponges, anemones, and algae to more complex organisms including—
but not limited to—worms, mollusks, crustaceans, and fish, coral reefs provide a home to a
widely diverse aggregation of species. Reefs are so intricately diverse with intra- and
interspecific relationships that combined with tropical rainforests, they represent the upper
boundaries of evolution of life on Earth (Woodward, 2003). Co-evolution has also led to
complex two-way interactions between species. Commensal relationships are abundant in coral
reefs, including that between large predatory fish and relatively much smaller and weaker cleaner
fish. The predator fish assumes a non-threatening posture while the cleaner fish grooms, by
nipping off ectoparasites from the gills and even the insides of the jaws (Thurman and Webber,
1984). Roughly 4,000 coral reef fish species exist worldwide, accounting for almost a fourth of
all marine fish (Spalding et al., 2001). Additionally, research performed in the Caribbean
concluded that 534 species from 27 phyla, with a further 30% of species not fully identified were
located in a 5 square meter reef microcosm (Spalding et al., 2001). Although researchers
disagree on the precise diversity of species associated with coral reefs, a broad estimation of
600,000 to 9 million species are interconnected with the fascinating underwater ecosystem
(Plaisance et al., 2011).
Location
Reefs are mainly prevalent in a latitudinal band between the Tropic of Cancer and the
Tropic of Capricorn that provides particular requirements for growth, such as light, temperature,
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salinity, waves, currents, tides, and sedimentation of organic and inorganic matter (Woodward,
2003). The world’s most expansive coral reef is found off the eastern coast of Australia, known
as the Great Barrier Reef. It is a string of 2,500 individual reefs that parallel the coast for
approximately 2,600 kilometers (Woodward, 2003). Reefs are also commonly found off the coast
of East Africa, south of Somalia, in the Indian Ocean (Woodward, 2003). According to Spalding
et al. (2001), coral reefs cover less than 1.2 percent of the world's continental shelf. In a research
article, Woodward (2003) confirmed the relatively tiny surface area coverage of coral reefs,
stating that in total, reefs cover approximately 2 million square kilometers, which is equivalent to
0.2 percent of the ocean’s surface area and 15 percent of the sea floor at depths of 0 to 30 meters.
Considering that reefs account for so little continental shelf area, the abundance of life dependent
on reefs is staggering (Spalding et al., 2001). Aside from the impressive role of supporting such
an overwhelming number of marine organisms, coral reefs also provide humans great support, as
well.
Human Benefits
Reefs play a critical role in coastal defense, food, tourist income, and medicinal
resources, generating billions of dollars of revenue (Carte, 1996). Over the course of several
centuries, corals build upon their predecessors and form great structures. These structures,
whether they are barrier, fringing, or atoll in form, protect coastal regions from crashing waves
(Spalding et al., 2001). The layout of many coastal lands is determined by underlying former
reefs. Many islands, even, are built on coral rock and sand (Spalding et al., 2001). Many coastal
and island communities are mainly surrounded by water, thereby making them nearly completely
dependent upon reefs as sources of food (Spalding et al., 2001). Coral reefs also provide great
sources of income for many small costal communities by advertising snorkeling and diving tours.
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For example, Birkeland (1997) cited a value of 1.6 billion dollars of revenue annually for the
Florida Keys alone.
Coral reefs are incredibly valuable for medicinal purposes, as well. Since 1985, coral
fragments have been implemented in bone graft surgeries. As documented in a journal by Roux,
et al. (1988), in a total of 167 coral grafts implanted, 150 coral “corks” filled burr holes, five
were used to repair skull defects, and 12 were used to reconstruct the floor of the anterior cranial
fossa. In an article by Costanza et al. (1997), total services and resources obtained from coral
reefs has been estimated at $375 billion per year. Indian medicinal practices also incorporate
corals for treating a multitude of diseases. The ash produced from incinerated coral has been
used to treat cough, phthisis, asthma, chronic bronchitis, fever, and urinary diseases among many
other diseases (Gopal, et al. 2008).
Ideal Conditions
The key component comprising a reef is the coral of Order Scleractinia, or hard coral
polyps that produce a strong calcium carbonate shell to protect their soft, fleshy polyp bodies
(Spalding et al., 2001). Although red coralline algae and green Halimeda algae aid in the
construction and maintenance of coral reefs by also secreting calcium carbonate, the limestone
backbone of the reef derives from these “reef-building” hermatypic corals that undergo budding
in ideal water conditions (Thurman and Webber, 1984). Coral reefs thrive in warm, clear,
nutrient-poor waters of gentle to moderate current, which provides a constant influx of warm
water. Clear, nutrient-poor water allows the slow-growing corals enough time to settle and
colonize on a sturdy foundation without having to compete for space against much fastergrowing organisms such as algae and porifera often found in nutrient rich waters. The clear water
also allows sunlight to travel to the corals uninhibited, so that symbiotic photosynthetic
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dinoflagellates known as zooxanthellae dwelling in the tentacles of coral polyps are able to
obtain proper nutrients to sustain themselves as well as the corals (Smith et al., 2004).
Coral Reproduction and Budding
The majority of coral species have evolved over the years to live in colonies and build up
a communal skeleton (Spalding et al., 2001). Once an individual coral polyp has reached full
maturity, there are two methods in which that polyp can contribute to the overall growth of a
reef. The first method is through sexual reproduction, in which coral sperm and egg from a preexisting colony unite and can begin the formation of a new reef.
Fertilization can occur either
within the parent polyp or externally. Fertilization that occurs from within the bodies of the
polyps involves the immature larvae swimming out through the mouth opening of the parent
coral polyp. The planulae, or immature coral polyp, then searches for a new suitable home by
swimming for anywhere from several hours to a couple weeks (Johnson, 1984). The other form
of coral sexual reproduction involves a “Full Moon Mass Spawning Event,” where hundreds of
thousands of individual polyps mysteriously synchronize in the expulsion of sperm and eggs into
the water, where planulae subsequently develop (Johnson, 1984).
As explained by Johnson (1984), upon surviving the search for a new home, as soon as
planulae settle on a hard surface—no deeper than 45 meters below sea level—they immediately
begin producing their protective limestone chalice and permanent home.
The secondary method in which coral polyps contribute to the growth of a reef is through
asexual budding. There are also two methods of this form of reef production: intra-tentacular and
extra-tentacular budding. Intra-tentacular budding is when two daughter corallites are produced
from the splitting of the corallite wall of a coral colony parent polyp (“Intra-tentacular Budding”,
2015). It normally appears as an outgrowth on the body tissue of the original polyp (Johnson,
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1984). When a corallite algal member undergoes budding external to the wall, it is termed extratentacular budding (“Extra-tentacular Budding”, 2015). During this process, the new polyp is
formed on a thin layer of connective tissue that extends out from the original polyp body
(Johnson, 1984).
Fringing, Barrier, and Atoll Reefs
Due to the diverse species, shapes, and sizes of corals, the dispersion and formation of all
reefs vary. The most basic of reef formations is the fringing reef, which develop in an upward
growth from a limestone base plate (Spalding et al., 2001). They form a shallow shelving
platform which drops off abruptly, descending to the sea floor (Spalding et al., 2001). Farther out
from shore, fairly older structures, called barrier reefs, rise up from a deeper sea floor base and
are separated from the mainland by a lagoon. Atoll reefs, the most recluse of reef formations,
consist of a broad, circular formation enclosing a large lagoon (Spalding et al., 2001). These
structures may at first seem to be extremely peculiar, because there seemingly is no nearby
landmass available to create suitable shallow waters. However, atolls originate as the fringing
reefs of mid-ocean (mainly volcanic) islands, which over many years subside and sink below the
surface of the water (Spalding et al., 2001).
Coral Bleaching
Corals typically require water temperatures to be above 18°C, with 24°C as an average
optimal temperature (Fatherree, 1999). Though capable of withstanding slight temperature
differences above or below the average optimal temperature, water temperatures as little as one
degree Celsius above normal summer maxima pertaining specifically to the coral species in
question, lasting for at least two or three days, can be used as a predictor of coral bleaching
events (Goreau and Hayes, 1994). The act of coral bleaching occurs when conditions such as
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water temperature, salinity, alkalinity, turbidity, or a number of other factors create an
environment no longer conducive for the well-being of corals.
In such times of stress, corals expel their symbiotic zooxanthellae, which is significant
because a substantial ninety percent of nutrients derived from zooxanthellae’s photosynthetic
process, including glucose, amino acids, and glycerol, are used by the coral (“Corals”, 2008).
Without the nutrients provided from these dinoflagellates, the corals will die if conditions remain
unresolved and new symbionts do not step in to replace the former. Studies indicate that most
coral are likely to recover from bleaching if the temperature anomalies persist for less than a
month, but afterward the stress from sustained high temperatures can cause physiological
damage that may be irreversible (Wilkinson et al., 1999). Some opportunists, such as invasive
algae, take advantage of a dead reef, quickly growing over all available surfaces. This invasive
alga then overpowers the nutrient cycling in that ecosystem, creating murky water unsuitable for
other marine species (Spalding et al., 2001). Studies show that global climate change in the 21st
century presents coral reefs with further strife, as ocean waters are expected to increase 1.5-3°C
(Howells, et al., 2013).
Anthropogenic Factors
Warming Waters
There are multiple types of stress that can be induced on a coral reef, including global
climate change, pollution, sedimentation, and overfishing. Out of these four main factors,
however, climate change—and thereby temperature related stress—has been the most widely
reported (Spalding et al., 2001). A number of human actions are warming seawaters at a rate
faster than what is tolerable for a coastal coral community. Point source thermal pollution—
heated seawater that can be traced back to a specific location as the primary source of
disturbance—has been observed with electric power plants. The effluent of electric power plants
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can cause bleaching of a reef, as this runoff increases seawater temperatures beyond the
maximum range of corals for an extended period of time (Woodward, 2003). Changes in oceanic
circulation, upwelling, storm paths, annual precipitation amounts, and increased water
temperatures all relate to the effects of global climate change (Woodward, 2003).
Pollution
Not all sewage treatment systems around the world are created equal. Especially in some
poorer nations, urban sprawl has overridden the sewage system, so that copious amounts of
sewage is either dumped directly into the ocean, or piped through a drainage system to the ocean
(Spalding et al., 2001). The spike in nutrients provided by sewage dumping provides an
appropriate platform for the growth of phytoplankton, microscopic plant plankton. Although
phytoplankton are crucial in the marine web of life and play an essential role in removing
greenhouse gases from the atmosphere, along with being key members of the carbon, nitrogen,
and phosphorous cycles, (Przyborski, 2006) they are not welcome near a coral reef. As
mentioned earlier, reefs thrive in clear, nutrient poor water. The presence of phytoplankton
floating near the surface of the water skews the nutrient levels as well as blocks the full amount
of sunlight that would otherwise reach corals dwelling below. The damage produced by pollution
on coral reefs is also point sourced to oil spills and the detergents used to clean up these spills
(Woodward, 2003).
Sedimentation
Sedimentation is another anthropogenic factor that leads to coral bleaching; it is a direct
result of soil erosion from land. This ties in with the notion mentioned above, that corals require
clear waters in order to maximize their contact with sunlight. Sedimentation can affect corals in
multiple ways. The first is the most obvious: suspended sediment in the water blocks some
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sunlight from reaching corals. The second effect, however, happens when the sediments begin to
settle. Corals may become smothered by sediment, which truly blocks sunlight and can infringe
on a coral’s ability to extend its tentacles beyond its limestone cup (Spalding et al., 2001). The
corals then must secrete a mucus to slough off the sediment, an action which not only uses
energy and nutrients, but also weakens the coral overall, thereby affecting their growth rate,
reproductive potential, and competitiveness (Spalding et al., 2001).
Unsustainable Fishing
The fourth most common human-made factor that induces coral bleaching is
unsustainable fishing. Blast and chemical fishing, along growth overfishing, can alter the well
being of a reef (Spalding et al., 2001). Growth overfishing is when the average size of the fish
species under surveillance is reduced to that of the juvenile growth stage (Spalding et al., 2001).
Overfishing has become so extensive that there are very few reefs in existence whose ecosystems
are not threatened (Spalding et al., 2001).
Recorded Bleaching Events
As observed by Liu, et al. (2003) and Berkelmans et al. (2004), the Great Barrier Reef
(GBR) surrounding Australia has experienced two great bleaching events in 1998 and 2002, as a
result of increased sea surface temperature (SST). Corals underwent a peak of thermal stress
around February 11th, 2002, when site-specific “HotSpots” dispersed throughout the GBR
reached levels of +2°C and +3°C (Liu et al., 2003). It has been observed that the 2002 coral
bleaching event had more severe damage on corals in terms of bleaching than that of 1998.
Approximately 12% more corals underwent bleaching in 2002 than 1998, and both time periods
experienced roughly 18% “strong” bleaching of their respective coral bleaching coverage
(Berkelmans, et al., 2004). Berkelmans, et al. (2004) created a comparative model of coral
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sensitivity to thermal stress in both 1998 and 2002 identifying a maximum change of +3°C SST.
The conclusions of their model stated that a +1°C change in SST increases the likelihood of
bleaching from 50% to 82%, a +2°C change increases the bleaching likelihood to 97%, and a
+3°C SST change yields a 100% likelihood in coral bleaching (Berkelmans, et al., 2004).
Caulastrea furcata: Candy Cane Coral
Caulastrea furcata is a scleractinian, large-polyp coral native to the Indo-West Pacific,
and ranges specifically in the southwest and northern Indian Ocean, Southeast Asia, the central
Indo-Pacific, eastern Australia, Japan and the East China Sea, oceanic West Pacific, Pitcairn,
Tuomotos, northwestern Australia, and Palau (Randall, 1995). Its natural location is ideal as a
subject for this experiment, as sea surface temperature changes experienced by this species of
coral are also experienced by the majority of all other coral species in existence. The water
temperature in the shallow, tropical reef slopes and lagoons where Caulastrea furcata is found
typically ranges from 24°C to 28°C (Borneman, 2004). Because Caulastrea furcata is a
relatively hardy coral, and is ranked as an easy to moderate care level coral, it is a common
genus targeted for aquarium trade. Additionally, Caulastrea was the second most heavily
exploited genus in Lampung (Hogdson, 2006). Since this species of coral is less likely to
undergo stress than some other more sensitive species of coral, it can be presumed that any
damage resulting from this research would have been more severe on a more delicate species.
The Candy Cane coral used in this experiment was aqua cultured, meaning that it was grown in a
facility, not extracted from the wild.
Coral Adaptability: Symbiont Shuffling
Researchers in recent studies have found that within certain species of coral, a certain
“shuffling” effect of symbionts has taken place as a mechanism of corals to adapt to increased
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water temperatures (Jones, 2010). Although this shuffling can potentially help some species of
coral adapt to an increase of 1-2ºC (Jones, 2010), there are difficulties that arise with this
shuffling action and no true solution has been found in the protection of all species of these
invaluable marine organisms. Since this shuffling does not completely prevent coral bleaching, it
is still uncertain if corals will be capable of withstanding these global climate changes.
Research Objectives
This study analyzes the effect of +2°C and +3°C over the course of two days on
Caulastrea furcata corals and determines the initial level of bleaching induced by this increased
temperature via a Reference Card and zooxanthellae density extraction procedure. Additionally,
it assesses whether corals undergoing thermal stress will be capable of fully recovering after a
three-week recovery period. It also focuses on the extent of which recovery is made, respective
to the intensity of stress placed on the corals.
MATERIALS AND METHODS
Invertebrate Organisms
Three separate Caulastrea furcata brain coral colony fragments were ordered from the
Salty Underground LLC, located in St. Louis, Missouri. The first colony had ten polyps, the
second colony had eight polyps, and the third colony had seven polyps. Each of these colonies
came from “Heads Green Candy Cane Coral Company”.
Figure 1: The Test Corals. Three separate colonies of Caulastrea furcata, Candy Cane Coral. From left to
right: The Control Coral (10-polyp), the +2 ºC Coral (8-polyp), and the +3 ºC Coral (7-polyp).
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Acclimation
Upon arrival, the corals were admitted to the animal room in dim lighting, in order to
ensure they would not experience light shock. They underwent the acclimation process, which in
total lasted slightly less than two hours. The corals arrived in small plastic bags, and were left to
float on the surface of the main tank water for half an hour, so that the temperature of their
transportation water could adjust to that of their new environment. Once this period was over,
100mL of the Main Tank water was poured into each bag and left alone for 15 minutes. This step
was repeated four more times. A majority of the water within the bag was then poured down the
sink drain, and then the corals were placed in their new environment. For the duration of one
week, the corals were placed on a submersed shelf in the Main Tank. After that week, the corals
were placed in their respective testing tanks.
Figure 2: The Corals Upon Arrival. Each of the three test corals arrived in their individual bag.
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Figure 3: The Acclimation Process. Here, the corals are submerged in the main tank water, so that they may adjust
to water temperature of its new environment.
Proper Water Flow
In order to provide the best possible environment for the corals, completeness of water
circulation also had to be considered. It was not enough to know water was flowing from one
tank to another, but also to know the rate at which all water from one tank would be cycled to the
following tank. Before the shelf tanks were synched to the salt water’s Main Tank, they were
synched to a bucket containing tap water, in a closed circulation system. During this time, the
corals were kept in the main salt-water tank. In order to assess this factor, eight drops of blue
food-coloring dye was added to the bucket containing tap water, and the dye’s travel time was
recorded. From the tap water bucket, it took 5 seconds for the initial signs of blue dye to appear
in the first tank. Blue dye appeared in the second tank after 30 seconds, and completely filled the
first tank after 45 seconds. The blue dye completely left the first tank after several minutes. This
flow rate was ideal, so the blue dye was diluted out of the system, and a stock of salt water
replaced it.
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Figure 4: The Closed Tap Water System. Here, the water pump is submersed in tap water, contained in the green
bucket. Water flows from the green hose through the rest of the hoses (connected via siphon), and then falls back
into the bucket: a complete cycle.
Figure 5: Blue Food Dye. The dye was introduced to the green bucket and has now traveled to the first tank. This
helps us assess the rate at which water is cycling throughout the tank system.
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Figure 6: Siphoned Connecting Tubes (left). This image shows the way in which water flowed from tank to tank,
through the tubes.
Figure 7: Temporary Housing (right). The corals were located on a temporary shelf within the Main Tank for the
duration of these water trials.
Running Water System
A 30-gallon saltwater tank (Main Tank) containing live rock, sea urchins, sea mushrooms,
and various other marine animals was the source of salt water for this experiment. The tank was
fully equipped with proper current, oxygenizing bubbles, 1/10 HP Corallife Aquarium Chiller
and Heater, and Instant Ocean SeaClone Protein Skimmer, suitable for tanks up to 100 gallons.
The Corallife Aquarium Chiller and Heater kept the Main Tank at a temperature of 27 ºC, with a
variability of +/- 0. 5 ºC.
A shelf was constructed directly above the Main Tank to provide room for the six other
smaller tanks incorporated in this running water system. An Aqua Supreme 70 gallon/hour
Submersible Pump channeled a fresh, steady, and constant flow of salt water to the first fivegallon tank (Control Tank), which contained the control corals, a colony consisting of ten polyps.
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From the Control Tank, water flowed through a tube into Heating Tank 1, which held a fully
submersed Finnex Deluxe 800-watt Titanium Digital Heater Controller. This heater raised the
water temperature by two degrees, to 29 ºC. Water then flowed through another tube into the next
5-gallon tank named Test Tank 1 (+2 ºC), which had a colony of corals with eight polyps. From
this tank, water flowed into Heating Tank 2, with another fully submersed Finnex Digital Heater.
This heater raised the water temperature to 30 ºC, which was a full three degrees above the
original ideal control temperature.
Water then flowed into another 5-gallon tank, named Test Tank 2 (+3 ºC), within which
was the third and final coral colony, with seven polyps. From this tank, the water flowed into a
final “cool-off” 5-gallon tank. From there, the water poured into a funnel tube, connected to the
main tank’s nitrate and nitrite filtering system, which then cycled through the protein skimmer,
and back into the main tank for a complete cycle. All of the above-tank tubes were siphoned with
the Python Squeeze Siphon Pump Starter Aquarium Adapter, to ensure a lack of air bubbles,
which would prevent a flow of water from one tank to the next.
Figure 8: The Complete System. Water flows from the water pump, located in bottom left of tank, through the
green hose and throughout all the tanks. The water travels down funnel tube on far right, into the filtration sump.
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Lighting
Two T5 High output aquarium lights from Home Depot were hung from the ceiling,
above all of the tanks. The lights were high performance, energy efficient lighting fixtures that
gave off minimal heat and produced just the right balance of light that was ideal for corals. The
lights were manually regulated on a 12-hour cycle, simulating the amount of hours to which
corals would naturally be exposed in the ocean.
Feeding
Brine shrimp were hatched and distributed to each coral colony, to provide additional
nutrients aside from what was given by the zooxanthellae via photosynthesis. Roughly a
teaspoon of dry brine shrimp eggs were mixed into saltwater, within the Shrimpery Brine Shrimp
Hatching Kit. The lid was placed on top of the container, and over the course of the next day,
newly hatched brine shrimp swam into the collection chamber, and roughly 25 brine shrimp were
added to each tank containing corals. This process was done every other day to ensure a steady
feeding regimen.
Figure 9: Brine Shrimp Hatching Kit. Dried brine shrimp eggs (left) are placed in Hatching Kit (right, black box),
which is filled with salt water. Hatched brine shrimp swim towards light, into the Collecting Cup (right, clear cup).
| P a g e 20
Heating
Once the acclimation process was complete and the corals were ready for the experiment,
the first Finnex Titanium Digital Heater was adjusted from the original 27ºC to 29ºC, and the
second Finnex Heater was adjusted from 27ºC to 30ºC. These temperatures were sustained for
two days. At the end of this heating period, the heater settings were reduced back down to the
ideal 27ºC and maintained at that temperature for the rest of the experiment. A Colour Reference
Card was used three weeks later to assess the amount of recovery made.
Water Quality Stability
Another key factor considered in this research was that increased temperature was truly
the only variable in the coral’s tank environment. In other words, other water quality factors such
as nitrate, nitrite, total alkalinity, pH, calcium, ammonia, specific gravity, and salinity had to be
kept constant. This was done through the use of water testing equipment. The first was a Tetra
test strip, which was dipped in water for one second, and then left alone for 30 seconds. After the
waiting period, identifier squares changed color to reveal the levels of nitrate, nitrite, total
alkalinity, and pH. Specific gravity and salinity levels were measured through the use of a
Coralife Deep Six Hydrometer, where the range indicated by a black stripe represents ideal water
quality for corals, and the clear stripe range represents the ideal range for a saltwater tank
without the presence of corals. Calcium and ammonia quality was observed with the API
Calcium Test Kit and API Ammonia Test Kit. Water quality was tested and recorded on a daily
basis.
| P a g e 21
Figure 10: Saltwater Comparison Chart (left). This reference chart was used when assessing Nitrate, Nitrite, Total
Alkalinity, and pH water quality factors.
Figure 11: Hydrometer (right). The hydrometer is submersed in the saltwater being tested, as the dial moves
according to the water quality factor. This tested for Specific Gravity and Salinity.
Figure 12: Calcium Test Kit (left). This kit tested for the Calcium water quality level. After completing the test,
the salt water turned to the appropriate blue color within the necessary amount of drops.
Figure 13: Ammonia Test Kit (right). This kit tested for the Ammonia water quality level. After completing the
test, the salt water turned bright yellow, indicative of a 0.0ppm “Safe” reading.
| P a g e 22
Quantifying Bleaching
Siebeck et al. Colour Reference Card
The Siebeck et al. (2006) Colour Reference Card was implemented to assess visual
change in coral pigmentation, as associated with bleaching. There are four hues on the card,
which represent the four most common coral pigments found in nature. On each strip, there is a
very dark hue, representing the healthiest, and most zooxanthellae-rich pigment, and on the
opposite end, there is a very pale square, which represents the unhealthiest, and most
zooxanthellae-poor pigment. The Reference Card identifies bleaching when a pigment shifts at
least two squares, toward a lighter hue.
Figure 14: Siebeck et al. Colour Reference Card.
Isolating Fresh Zooxanthellae
A technique developed by Zamoum and Furla (2012) was used to freshly isolate
individual dinoflagellate members. The coral colonies were taken from their respective tanks and
| P a g e 23
placed in temporary transportation tubs, filled with the same water from their holding tanks. Due
to the unevenness of polyp count per colony (the control colony consisted of 10 polyps, +2ºC
colony consisted of eight polyps, and the +3ºC colony consisted of seven polyps) seven polyps
from each colony were viewed for the duration of this research. A stock solution of 200 mL 1M
NaOH was prepared for all of the extractions. Using a micropipette, 500uL was pipetted into 21
separate microfuge tubes. Once the corals were in their temporary tubs, 0.5 grams of coral flesh
was extracted with forceps from all of the selected 21 polyps. The flesh extractions were then
placed in microfuge tubes and vigorously shaken. Once placed in the 25ºC incubator, the tubes
were shaken every fifteen minutes for four hours. After this incubation period, the tubes were
removed and examined for Zooxanthellae density. A drop from each tube was individually
examined at 400x magnification on the light microscope, and algae were counted for that field of
view. This procedure was done once before any temperature stress occurred, as well as two days
after heat stress exposure.
| P a g e 24
Figure 15: Zooxanthellae Extraction Process. This figure was taken from a poster that was designed for a
presentation at the 60th Annual NJAS Conference. Disregard the “Figure 11” label, as that was its label on the poster.
This process shows isolation of corals, preparation of NaOH solution, microfuge tubes with 500uL NaOH + 0.5 g
coral flesh, and the end result: freshly isolated zooxanthellae at 400x magnification under light microscope.
Photography:
The development of bleaching was observed on a daily basis, and photographs were taken
every other day to provide a stable comparison. Photos were taken with a Motorola Moto X
camera with 10 MP RBGC “Clear Pixel” which offers excellent low-light performance, with a
resolution of 1280x720 on a 4.7 inch AMOLED display. The bleaching of these corals was
analyzed upon comparison to the Siebeck et al. (2006) Coral Colour Reference Card in order to
establish a universally recognizable stage of the bleaching identification process.
| P a g e 25
RESULTS
Water Quality
For the month-long duration of this research, the water quality factors including Nitrate,
Nitrite, Total Alkalinity, pH, Calcium, Ammonia, Specific Gravity, and Salinity remained
constant, within the acceptable quality range. There were different ranges in respect to the factor
in question. Nitrate, Total Alkalinity, and pH worked with the “Ideal” scale: Ideal > Okay >
Acceptable > Not Desired > Unsafe. Nitrite worked with the “Safe” scale: Safe > Caution >
Stress > Danger. Ammonia was required to stay at 0.0 ppm for the entire experiment in order to
be termed “Safe.” Calcium had a range from 400-500 ppm in order to be termed “Safe.” The
ranges for Salinity and Specific Gravity were between 32-35 ppt and 1.024-1.026, respectively
for a coral-friendly water environment. The “Ideal” range for Total Alkalinity spans from 180300 ppm, which explains the acceptable “jump” half-way through research.
The Control Tank, +2ºC Tank, and +3ºC Tank experienced fluctuation in factor quality
levels at the beginning of research, during the water acclimation process from fresh water to
saltwater. During this period, the corals were kept in the Main Tank, and were thereby unaffected
by this closed-system acclimation.
| P a g e 26
Figure 16: Main Tank Water Factor Quality Levels. Note the stability of all eight factors. The slight variations in
Calcium readings and Total Alkalinity readings are fine, because they are still within “Ideal” Range. Note that
Nitrite and Ammonia levels are not seen on graph because they were constantly at 0.00 ppm for the entire research.
Salinity readings are actually in ppt, not ppm.
Figure 17: Control Tank Water Factor Quality Levels. Note the stability of all eight factors, past Day 9.
Fluctuations in the previous days were due to the switch from tap water to salt water. During Days 1-8, all corals
were in the Main Tank. Control corals were placed in Control Tank on Day 9. The slight variations in Calcium
readings and Total Alkalinity readings are fine, because they are still within “Ideal” Range (refer to Tetra Test
Strips). Note that Nitrite and Ammonia levels are not seen on graph because they were constantly at 0.00 ppm for
the entire research. Salinity readings are actually in ppt, not ppm.
| P a g e 27
Figure 18: +2ºC Tank Water Factor Quality Levels. Note the stability of all eight factors, past Day 9. Fluctuations
in the previous days were due to the switch from tap water to salt water. During Days 1-8, all corals were in the
Main Tank. +2ºC corals were placed in +2ºC Tank on Day 9. The slight variations in Calcium readings and Total
Alkalinity readings are fine, because they are still within “Ideal” Range (refer to Tetra Test Strips). Note that Nitrite
and Ammonia levels are not seen on graph because they were constantly at 0.00 ppm for the entire research. Salinity
readings are actually in ppt, not ppm.
Figure 19: +3ºC Tank Water Factor Quality Levels. Note the stability of all eight factors, past Day 9. Fluctuations
in the previous days were due to the switch from tap water to salt water. During Days 1-8, all corals were in the
Main Tank. +3ºC corals were placed in +3ºC Tank on Day 9. The slight variations in Calcium readings and Total
Alkalinity readings are fine, because they are still within “Ideal” Range (refer to Tetra Test Strips). Note that Nitrite
and Ammonia levels are not seen on graph because they were constantly at 0.00 ppm for the entire research. Salinity
readings are actually in ppt, not ppm.
| P a g e 28
Temperature Changes
The Main Tank and Control Tank were successfully kept at 27ºC for the duration of this
experiment. The +2ºC Tank experienced an increase of water temperature of two degrees Celsius
(reaching 29ºC) on days ten and eleven of the experiment. The +3ºC Tank experienced an
increase of water temperature of three degrees Celsius (reaching 30ºC) on days ten and eleven of
the experiment. The water temperature in the Control Tank, +2ºC Tank, and +3ºC Tank changed
from 24ºC to 27ºC once the full salt water running system replaced the closed tap water system.
All corals were housed in the Main Tank during this time and were thereby unaffected by this
change in temperature.
Figure 20: Temperature Changes for All Tanks. The Main Tank remained at 27ºC for all 31 days. The Control
Tank, +2ºC Tank, and +3ºC tank adjusted to 27ºC during the switch from tap water to salt water (Days 1-8). From
that point on, each tank adjusted to its respective designated temperature.
Zooxanthellae Extractions
Primary zooxanthellae extractions were conducted on all three coral colonies. Due to the
uneven count of polyps to coral colony (one colony had seven polyps, the next had eight polyps,
and the third had ten polyps), seven polyps were selected from each colony and any excess
polyps were not included in any of the research data. The primary extraction numbers (Table 1)
signify how many individual zooxanthellae members were counted in a field of view under the
| P a g e 29
light microscope at 400x magnification, from one uniform drop of incubated dissolved coral
flesh in NaOH solution before any heat stress was induced on the coral. All three coral colonies
had roughly the same amount of algal members present.
The secondary extraction numbers (Table 2) signify how many individual zooxanthellae
members were counted in a field of view under the light microscope at 400x magnification, from
one uniform drop of incubated dissolved coral flesh in NaOH solution after respective heat stress
took place. There was a noticably smaller amount of zooxanthellae counted in the two test tanks
after heat stress. The Control Tank maintained fairly constant cell count data.
The average density (Table 3) was taken of all seven samples from all three coral colonies
before and after heat stress. There is a noticable decline in cell count after heat stress occurred.
It is to be noted that during the second week of recovery, technical malfunctions occurred,
which resulted in the corals being partially exposed to air for several hours. The zooxanthellae
density results were obtained before this incident took place, however, the exposure to air may
have affected the overall recovery time.
Zooxanthellae
Counted Per Polyp
Primary Extraction
Control Tank
+2ºC Tank
7
8
7
5
5
5
4
4
7
6
6
7
8
9
+3ºC Tank
7
12
10
4
8
4
10
Table 1: Baseline Count of Zooxanthellae Before Heat Stress. These numbers represent how many algae members
were counted in the field of view at 400x magnification on the light microscope, from one uniform drop of
NaOH/Coral flesh solution per polyp.
| P a g e 30
Secondary Extraction
Control Tank
+2ºC Tank
2
6
5
3
10
3
11
3
4
1
6
2
6
1
Zooxanthellae
Counted Per Polyp
+3ºC Tank
2
3
4
2
2
2
1
Table 2: Count of Zooxanthellae After Heat Stress. These numbers represent how many algae members were
counted in the field of view at 400x magnification on the light microscope, from one uniform drop of NaOH/Coral
flesh solution per polyp.
Coral Colony
Control
+2ºC Tank
+3ºC Tank
Average Density Before and After Heat Stress
Primary Extraction:
Secondary Extraction:
Average Algae Density
Average Algae Density
6.3
6.3
6.3
2.7
7.8
2.3
Table 3: Average Count of Zooxanthellae Before and After Heat Stress. These numbers represent on average how
many algae members were counted in the field of view at 400x magnification on the light microscope, from one
uniform drop of NaOH/Coral flesh solution per polyp.
Coral Color Changes
Below are four images of each coral colony taken throughout the span of the research, to
visually show the change in pigment of the stressed corals. There is clearly a stark difference
between the original color status and the end color status.
10 Polyp Control Coral



Figure 21: Control Coral Color. Note that essentially no color change has occurred. The only damage is in the
final picture, which was taken after accidental air exposure had taken place.
8 Polyp (+2ºC) Coral



Figure 22: +2ºC Polyp Coral Color. Note the drastic color change (bleaching).
| P a g e 31
7 Polyp (+3ºC) Coral



Figure 23: +3ºC Polyp Coral Color. Note the drastic color change (bleaching).
ANOVA
A commonly used statistical test called ANOVA (Analysis of Variance) was implemented
to assess the statistical significance of temperature on the corals. As stated by Lane (2015),
ANOVA is a tool used to separate the observed variance in a certain variable into components
attributable to different sources of variation. ANOVA analyzed the zooxanthellae density change
from Time 0 to Time 1. Time 0 represents when all three coral colonies had ideal water
temperature and Time 1 represents when the coral colonies underwent their respective
temperature changes. Via a Nested ANOVA trial, the variable of “Time” had a p-value of 0.090.
With the same Nested ANOVA trial, the variable of “Temperature” had a p-value of 0.007.
Nested ANOVA: Cell Number VS Time, Temperature
Source
DF
SS
MS
F
P-Value
Time
1
97.5283
97.5238
4.935
0.090
Temperature
4
79.0476
19.7619
4.136
0.007
Error
36
172.0000
4.7778
Total
41
348.5714
Table 4: Nested ANOVA: Analysis of Variance for Cell Number. Note that the P-Value for Temperature is 0.007
and the P-Value for Time is 0.090.
| P a g e 32
DISCUSSION
The averages of zooxanthellae differences before and after heat stress for all three
colonies were calculated (Table 3). It was confirmed that 57.1% of the zooxanthellae algal
members were expelled after two days of heat stress from the coral colony experiencing +2°C
water temperature change. 70.5% zooxanthellae algal members were expelled after two days of
heat stress with +3°C water temperature change.
Through the use of the Reference Card, it was confirmed that hue changed by three
levels, starting at the strongest, healthiest hue and declining to a paler square three segments
lower. This indicates that significant bleaching occurred. After a span of three weeks, the corals
did not regain health to full recovery. Corals regained to the second strongest hue. The coral
polyps associated with +2°C reached the second strongest hue 4 days sooner than the coral
polyps associated with +3°C. Although the +2°C corals recovered sooner chronologically, the
overall recovery rate was similar for both the +2°C and +3°C coral, as the +3°C experienced a
greater degree of stress.
The p-value for Temperature (p = 0.007) from the Nested ANOVA concludes that
temperature had a statistically significant effect on the count of algal cells per field of view. The
p-value for Time (p = 0.090) from the Nested ANOVA concludes that time did not have a
statistically significant effect on the count of algal cells counted per field of view. Interpretation
of these results means that temperature truly was the only factor working against the coral
colonies, and thereby affirms that the bleaching of the corals was due to temperature, and
temperature alone.
Coral reefs provide a home, as well as source of food, for countless marine species. The
diversity of species found at corals reefs is staggering, as many species still have yet to be
| P a g e 33
identified. Human beings also rely on reefs in terms of food source, revenue from tourism,
prevention of coastline degradation, and medicinal purposes. However, humans contribute in
many ways to the decline of coral reef health. The bleaching of coral reefs occurs in a time of
coral stress, when mutualistic zooxanthellae (which account for providing 90% of a coral polyp’s
nutrition) are expelled from coral tissue. If undesirable conditions persist and corals are unable to
regain zooxanthellae, the reef will die, thereby uprooting the lives of innumerable marine
species. Heightened awareness and active efforts by everyone to fish sustainably, minimize
pollution, and reduce carbon emissions will make a postive impact on coral recovery.
| P a g e 34
LITERATURE CITED
Berkelmans, R., De'ath, G., Kininmonth, S., Skirving, W.J. 2004. A comparison of the 1998 and
2002 coral bleaching events on the Great Barrier Reef: spatial correlation, patterns, and
predictions. Coral Reefs. 23(1): 74-83.
Birkeland, C. (ed) 1997. Life and Death of Coral Reefs. Chapman and Hall, New York, New
York, USA.
Borneman, E. 2004. Aquarium Corals: Selection Husbandry and Natural History. T.F.H.
Publications.
Carte, B. K. 1996. Biomedical potential of marine natural products. BioScience 46: 271-286.
Corals. (2008, March 25). Retrieved May 8, 2015, from http://oceanservice.noaa.gov/education/
kits/corals/coral02_zooxanthellae.html
Costanza, R., dArge, R., deGroot, R., Farber, S., Grasso, M., Hannon, B., et al. 1997. The value
of the world’s ecosystem services and natural capital. Nature 387: 253-260.
Extra-tentacular budding. 2015. Retrieved May 8, 2015, from http://www.coralhub.info/terms/
extra-tentacular-budding/
Fatherree, J. 1999. Soft Corals: Selecting and Maintaining Soft Corals, Feeding and Algal
Symbiosis, Lighting and Water Clarity. T.F.H. Publications, Inc, Neptune City, NJ, USA.
Gopal, R., Vijayakumaran, M., Venkatesan, R., Kathiroli, S. 2008. Marine organisms in Indian
medicine and their future prospects. Natural Product Radiance 7(2): 139-145.
Goreau, T.J. and Hayes, R.M. 1994. Coral bleaching and ocean 'hot spots'. Ambio 23: 176-180.
Hodgson, G. and Ochavillo, D. 2006. MAQTRAC Marine aquarium trade coral reef monitoring
protocol field manual. Reef Check Foundation. Pacific Palisades, California USA.
Howells, E. J., Berkelmans, R.., van Oppen, M.J.H., Willis, B.L., Bay, L.K. 2013. Historical
thermal regimes define limits to coral acclimatization. Ecology: 94: 1078-1088.
Intra-tentacular budding. 2015. Retrieved May 8, 2015, from http://www.coralhub.info/terms/
extra-tentacular-budding/
Johnson, S. A. (ed.) 1984. Coral Reefs. Lerner Publications Company, Minneapolis, Minnesota,
USA.
Jones, A. 2010. Double jeopardy for corals. Australasian Science. 31(8): 30-32.
| P a g e 35
Lane, David M. "Introduction to Analysis of Variance." Introduction to Analysis of Variance.
National Science Foundation, Rice University, University of Houston Clear Lake , and
Tufts University, n.d. Web. 11 May 2015.
Liu, G., Strong, A. E., Skirving, W. 2003. Remote sensing of sea surface temperatures during
2002 Barrier Reef coral bleaching. Eos Trans. AGU. 84(15): 137-141.
Mondadori, A. 1999. Reef Life. Firefly Books Ltd. 2000-2001.
Plaisance, L., Caley M. J., Brainard R. E., Knowlton, N. 2011. The diversity of coral reefs: what
are we missing? PLoS ONE 6(10): e25026. doi:10.1371/journal.pone.0025026
Przyborski, P. (2006, December 7). Warming Ocean Slows Phytoplankton Growth: Image of the
Day. Retrieved May 9, 2015, from http://www.earthobservatory.nasa.gov/
IOTD/view.php?id=7187
Randall, R. H. 1995. Biogeography of reef-building corals in the Mariana and Palau islands in
relation to back-arc rifting and the formation of the eastern Philippine Sea. Nat. Hist.
Res. 3: 193-210.
Roux, F., Brasnu, D., Loty, B., George, B., and Guillemin, G. 1998. Madreporic coral: a new
bone graft substitute for cranial surgery. Journal of Neurosurgery 69(4): 510-513.
Siebeck, U. E., Marshall, N.J., Klüter, A., Hoegh-Guldberg, O. 2006. Monitoring coral bleaching
using a colour reference card. Coral Reefs. 25: 453-460.
Smith, D.J., Suggett, D.J., Baker, N.R. 2004. Is photoinhibition of zooxanthellae photosynthesis
the primary cause of thermal bleaching in corals? Global Change Biology, 11: 1-12.
Spalding, M.D., Ravilious, C., and Green, E.P. 2001. World Atlas of Coral Reefs. UNEP World
Conservation Monitoring Centre. University of California Press, Berkeley, USA.
Thurman, H.V. and Webber, H. H. 1984. Marine Biology. Bell and Howell Company, Ohio,
USA.
Wilkinson, C., Linden O., Cesar, H., Hodgson, G., Rubens, J., Strong, A. E.1999. Ecological and
socioeconomic impacts of 1998 coral mortality in the Indian Ocean: An ENSO impact
and a warning of future change? Ambio 28(2): 188-196.
Woodward, S.L. 2003. Biomes of Earth: Terrestrial, Aquatic, and Human- Dominated.
Greenwood Press. Westport, CT.
Zamoum, T. and Furla, P. 2012. Symbiodinium isolation by NaOH treatment. J. Exp. Biol. 22: 4.
| P a g e 36