THE RESPONSE OF SYMBIOTIC ZOOXANTHELLAE (
SYMBIODINIUM
 
SPP.) 
DIVERSITY AND GENE EXPRESSION TO STRESS IN GEOGRAPHICALLY 
DISTINCT REEFS
 
 
By
 
 
Briana Patricia Hauff Salas
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
A DISSERTATION
 
 
Submitted to
 
Michigan State University
 
i
n
 
partial fulfillment of the requirements 
 
f
or the degree of 
 
 
Zoology
 
-
 
Doctor of Philosop
h
y
 
Ecology, Evolutionary Biology and Behavior
 
-
 
Dual Major
 
 
2015
 
 
ABSTRACT
 
 
THE RESPONSE OF SYMBIOTIC ZOOXANTHELLAE (
SYMBIODINIUM
 
SPP.) 
DIVERSITY AND GENE EXPRESSION TO STRESS IN GEOGRAPHICALLY 
DISTINCT REEFS
 
 
By
 
 
Briana Patricia Hauff Salas
 
 
The persistence of coral reefs in the Florida Keys reef tract is of concern as coral 
bleaching, due to increased ocean temperatures, and human
-
linked disease outbreaks 
have led to 
a reduction in 
coral 
cover
 
of
 
40% since
 

E
vidence suggests a 
vari
ation in stress susceptibility of conspecific coral from inshore and offshore reefs in 
the Florida Keys. However, the mechanism behind the disparity in stress susceptibility is 

Symbiod
inium 
spp.; referred to as zooxanthellae) has been proposed as a mechanism to withstand stress. 
As such, I investigated zooxanthellae composition of 
Porites astreoides
 
and 
Montastraea 
cavernosa 
from an inshore and offshore reef in the Lower Florida Keys to
 
determine 
links to stress susceptibility. Additionally, I investigated variation in the expression of 
metabolically related genes in zooxanthellae of 
P. astreoides
 
reciprocally transplanted 
between reefs, as well as exposed to elevated temperatures and di
sease. 
 
 
Chapter one examines changes in the dominant zooxanthellae subclade type in 
P. 
astreoides 
and 
M. cavernosa
 
throughout a two
-
year reciprocal transplant study. The goal 
of this study was to determine if zooxanthellae subclade typ
e could explain higher rates 
of bleaching in offshore reef coral, as well as assessing the possibility of acclimatization 
to different environments. Increased complexity and diversity was seen in the 
composition of zooxanthellae subclade types from coral c
ollected at offshore reefs, 
compared to inshore reefs. As a result, offshore reef zooxanthellae displayed less stability, 
possibly explaining higher bleaching susceptibility. Additionally, zooxanthellae composition 
patterns were retained throughout the re
ciprocal transplant, demonstrating a lack of 
acclimatization. 
 
 
Chapter two examined site
-
specific variation in the expression of metabolically related 
genes in zooxanthellae from 
P. astreoides
 
following the reciprocal transplant. Symbionts from 
offshore c
orals experienced significantly increased expression in 
PCNA
, 
SCP2
, 
G3PDH
, 
PCP
 
and 
psaE
 
(p<0.05) compared to inshore symbionts, a pattern consistent with increased bleaching 
susceptibility. Significant differences in gene expression between zooxanthellae
 
from inshore 
and offshore reef indicate functional variability and are likely a result of localized adaptation. 
Similar to results in chapter one, gene expression patterns from site of origin were retained 
throughout the reciprocal transplant, suggesting 
no acclimatization. 
 
 
Chapter three investigated variation in the response of the same zooxanthellae genes 
when 
P. astreoides
 
was exposed to extreme temperatures and disease. Here disease was 
mimicked 
by the application of
 
lipopolysaccharide from 
Serratia 
marcescens
, the causative 
agent of acroporid serratosis. Gene expression did not differ in zooxanthellae from inshore and 
offshore reefs, nor as a consequence of extreme temperature or disease. Several factors may 
explain the lack of variation including zo
oxanthellae response to acute versus moderate stress, 
host protection and targeting of symbionts by 
S. 
marcescens
. 
These
 
results suggest that 
symbionts of 
P. astreoides
 
may be locally adapted to chronic moderate stress, but respond 
simila
r
ly to acute extre
me conditions.
 
 
 
 
iv
 
ACKNOWLEDGEMENTS
 
 
Although m
y name alone
 
appears on this dissertation as author, this work would not have 
been possible without the work of many. In particular, I wish to extend my thanks to the 
following people:  
 
My co
-
chairs Kevin 
Strychar and 
Peggy
 
Ostrom for taking me through this proces
s with 
your guidance and wisdom
 
and always believing in my success.
 

s advisor, current collaborator
 
and above all, friend, James Cervino. Without 
any doubt, had our paths not crossed at S
t. Francis Prep, this dissertation would never have 
happened. Your passion for marine science and making the world a better place has inspired 
many, including myself. 
 
Josh
ua Haslun, your friendship and passion for science no doubt brought me through 
many 
parts of the last five years. How else would I have been able to collect any coral if not for 

 
My fellow graduate students Karen Drumhiller, Rachel Williams, Knute Gundersen, 
Kateri Salk, Sam Rossman and Kaycee Mora who showed m
e the importance of 
taking off my 
headphones
 
in lab
,
 
and 
having fun.
 
Finally
, I would like to thank the countless others who have given their time, knowledge 
and advice that led to making this work possible: Nathaniel Ostrom, Bopi Biddanda, Brian 
Mauer, 
Jeff Landgraf, Diana Bello
-
DeOcampo, Ari Grode, 
Erich Bartels and the entire staff at 
Mote Marin
e Tropical Research Laboratory
, Coastal Preservation Network for research funding 
support and the College of Natural Science at Michigan State University for te
aching assistant 
funding and support
.
 
 
v
 
On a personal note:
 
My mom and dad, Christine and John Hauff, for always believing in my 
wild aspirations
.
 
My best friend and sister 
Lexie
 
Mannix whose love, support and friendship is 
unparalleled.
 
My friend and person
al editor 
Sam
 
Kerath, who 
has 
taught me the art of prose since high 
school. 
 
And 
Dan
, whose unrelenting lov
e, support and belief in my ability 
was at many times the 
only thing moving me forward.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
vi
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
A n
ote on 
authorship:
 
 
I have had the pleasure of collaborating with a multitude of bril
liant scientists to publish the
 
work
 
you are about to read
. The first chapter of my dissertation will be published in The 
International Journal of Biology in January 2016, with c
oauthors: Joshua Haslun, Kevin 
Strychar, Peggy Ostr
om and James Cervino. Chapters two and three
 
will be submitted for 
publication with the same coauthors.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
vii
 
TABLE OF CONTENTS
 
 
 
 
LIST OF TABLES



ix
 
 
LIST 
OF FIGURES



..
x
 
 
CHAPTER 1
 
SYMBIONT DIVERSITY OF ZOOXANTHELLAE (
SYMBIODINIUM 
SPP.) IN 
PORITES 
ASTREOIDES
 
AND 
MONTASTRAEA CAVERNOSA 
FROM A RECIPROCAL TRANSPLANT 
IN THE LOWER FLORIDA KEYS


...

 
 
ABSTRACT




 
 
INTRODUCTION

.



 
 
METHODS

.


..4
 
 
Study Sites



 
4
 
 
Coral Fragment 
Collection



 
4
 
 
Reciprocal 
Transplant


.

.



5
 
 
Sample Collection for DNA Analysis

.
.


............
..
 
7
 
 
DNA Extraction and Sequencing

..




8
 
 
Data Analysis





...9
 
 
RESULTS


...10
 
 
Porites 
astreoides collected from Birthday Reef (PBB and PBA
)



 
10
 
 
Porites astreoides
 
collected from Acer24 Reef (PAA and P
A
B
)


16
 
 
Montastraea cavernosa collected from Birthday Reef (MBB and MBA)

.
.17
 
 
Montastraea cavernosa collecte
d from Acer24 
Reef (MAA and MAB)


.
 
22
 
 
DISCUSSION



 
 
Porites astreoides



23
 
 
Montastraea cavernosa



 
2
5
 
 
 
LITERATURE CITED



 
 
CHAPTER 2
 
SITE
-
SPECIFIC VARIATION IN 
GENE EXPRESSION FROM 
SYMBIODINIUM 
SPP. 
ASSOCIATED WITH OFFSHORE AND INSHORE 
PORITES ASTREOIDES 
IN THE LOWER 
FLORIDA KEYS



 
35
 
ABSTRACT


..
 
35
 
 
INTRODUCTION

.



 
 
METHODS

.



 
 
RNA extraction and cDNA preparation



 
 
qRT
-
PCR



 
 
Data analysis

.



 
 
RESULTS



 
42
 
 
DISCUSSION




 
47
 
 
LITERATURE CITED



 
54
 
 
CHAPTER 3
 
 
viii
 
FUNTIONALLY VARIABLE 
SYMBIODINIUM 
SPP. DISPLAY SIMILAR REPSONSES TO 
BLEACHING AND DISEASE STRESS IN THE LOWER FLORIDA 
KEYS



 
59
 
ABSTRACT



 
59
 
 
INTRODUCTION

.



 
 
METHODS

.


 
 
Coral collection and preparation


.

 
 
Temperature and bacteria experiments

.




62
 
 
RNA extraction, 
qRT
-
PCR and data analysis






64
 
 
RESULTS

.




65
 
 
DISCUSSION


.



68
 
 
LITERATURE CITED


.
74
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ix
 
LIST OF TABLES
 
 
 
 
Table 1. Genes used in this study, 
abbreviations
, function, primer sequences, melting 
temperatures and amplicon length for primers used for qRT
-




.40
 
 
Table 2. 
Output
 
for two
-


as 
fixed 
factors under the MCMC.qpr package for zooxanthellae gene expression from 
sub
samples 
collected at Birthday reef and Acer24 reef. 
Post means are reported as well as lower and upper 
credible intervals, effective sample size and p
-
values.  

presents non
-
transplanted 
sub
-


represents transplanted sub
-
samples that were collected 
at Acer24 reef and transplanted 

represents non
-
transplanted sub
-
sample
s that 
originated at Birthday reef, wh
ereas 

transplanted sub
-
samples 
that were collected at Birthday reef and 
transp
lanted to Acer24 reef. Baseline 
comparison, or reference factor, included non
-
transplanted 
sub
-
samples 
from Acer24
 
reef 
(i.e. AcerAcer) at the winter sampling time. Asterisk denotes a p
-
value <0.05




 
43
 
 
Table 3. 
Output for two
-

MCMC.qpr package for zooxanthellae
 
gene expression from sub
-
samples collected at Birthday 
reef and Acer24 reef. 
Post means are reported as well as lower and upper credible intervals, 
effec
tive sample size and p
-
values. 

-
transplanted sub
-
samples that 
originated at A

-
samples that were 

-
transplanted sub
-

 
represents 
transplanted sub
-
samples that were collected at Birthday reef and transplanted to Acer24 reef. 
Baseline comparison, or reference factor, included non
-
transplanted sub
-
samples from Birthday 
reef (i.e. BirthdayBirthday) at the winter sampling tim
e. Asterisk denotes a p
-
value 
<0.05



.

.
4
6
 
 
Table 4. Two
-
way model for an experiment testing the effects of reef, heat and heat+LPS on the 
gene expression of 
Cox
, 
G3PDH
, 
PCP
, 
psaE
, 
SCP2
 
and 
PCNA 
under the MCMC.qpr
 
package. 
Post means are reported as well as lower and upper credible intervals, effective sample size and 
p
-

PCNA 
as a housekeeping gene and run with 25,000 
iterations, with the first 4,000 discarded as burn
-
in. Cora
l colony individual was used as a 
random factor. Reef fragments were from Acer24 or Birthday reef and exposed to 28°C. Heat 
treatments were fragments from both reefs exposed to 32°C, and heat+LPS treatments were 
fragments from both reefs exposed to 32°C+LP
S from 
Serratia marcescens
. Asterisk denotes a 
p
-
value <0.05




.66
 
 
 
 
 
 
 
 
 
x
 
LIST OF FIGURES
 
 
 
 
Figure 1.
 
Experimental Design
. 
Graphical representation of 
my
 
reciprocal transplant design for a 
single coral species at one experimental level. A fragment of one species was collected at each of 
the two sites. Solid rectangles represent coral from Acer24 Reef and hollow rectangles represent 
coral from Birthday Reef
. Those fragments were sectioned into two fragments. Each fragment 
was then placed back out on to a reef with one fragment half remaining at the site of origin while 
the other fragment half was transplanted to 
the companion site. 
The fragments colored in r
ed 
were placed on to Acer24 Reef while the fragments colored in blue
 
were placed at Birthday 
Reef. 
Dashed arrows represent transplanted fragments. For the experiment, the above was 
repeated ten times for each coral species resulting in n=80 fragments







..6
 
 
 
Figure 2
.
 
Symbiodinium
 
subclade type frequencies for 
Porites astreoides
 
samples. Graphs are 
labeled with acronyms describing the (1) coral species, (2) field site of original collection (i.e. 
Acer24 or Birthday Reefs) and (3) site of transplan
t (i.e. Acer24 or Birthday Reefs). For 
example, PBA are samples from 
Porites astreoides
 
fragments collected at Birthday Reef and 
transplanted to Acer24 Reef.   Graphs in the left
-
hand column represent non
-
transplanted coral 
while graphs on the right repres
ent transplanted coral. Although each condition began at n=10, 
note variation in grou
p sizes due to sequencing error


.



.
 
12
 
 
Figure 3
.
 
Porites astreoides 
zooxanthellae transitions. Transitions in zooxanthellae subclade
 
type 
occurring within the same coral fragment between sampling times for all fragments of 
Porites 
astreoides 
zooxanthellae. 
Pie
 
charts represent the relative frequencies of individual transitions of 
clade subtypes through each field season.  Graphs are la
beled with acronyms describing the (1) 
coral species, (2) field site of original collection (i.e. Acer24 or Birthday Reefs) and (3) site of 
transplant (i.e. Acer24 or Birthday Reefs). For example, PBA represents samples from 
P. 
astreoides 
fragments
 
collect
ed at Birthday Reef and transplanted to Acer24 Reef. Color scales 
are determined by the outcome of the transition. For example, slices represented in shades of red 
describe transitions that resulted in A4 zooxanthellae, transitions to A4.1 in blue, A4.2 in
 
green 
and A4.3 in orange. Transitions that stayed the same are represented by a gray scale and outlined 

Summer. Years correspond to 2012 or 2013




.
.14
 
 
 
Figure 4.
 
Symbiodinium
 
subclade frequencies for 
Montastraea cavernosa 
samples.
 
Graphs are 
labeled with acronyms describing the (1) coral species, (2) field site of original collection (i.e. 
Acer24 or Birthday Reefs) and (3) site of transplant (i.e. Acer24 or Birthday Reefs).  For 
example, MBA represents samples from 
Montastraea caver
nosa 
fragments
 
collected at Birthday 
Reef and transplanted to Acer24 Reef. Graphs in the left
-
hand column represent non
-
transplanted 
coral while graphs on the right represent transplanted coral. Although each condition began at 
n=10, note variation i
n grou
p sizes due to sequencing 
error.



 
18
 
 
Figure
 
5
.
  
Montastraea cavernosa 
zooxanthellae t
ransitions. Transitions in zooxanthellae 
subclade type occurring within the same coral fragment between sampling times for all 
fragments of 
Montastraea 
cavernosa 
zooxanthellae. Pie charts represent the relative frequencies 
 
xi
 
of individual transitions of clade subtypes through each field season. Graphs are labeled with 
acronyms describing the (1) coral species, (2) field site of original collection (i.e. Ace
r24 or 
Birthday reefs) and (3) site of transplant (i.e. Acer24 or Birthday reefs). For example, MBA 
represents samples from 
M. cavernosa 
fragments
 
collected at Birthday reef and transplanted to 
Acer24 reef.  Color scales are determined by the outcome of th
e transition
. 
. For example, slices 
represented in shades of red describe transitions that resulted in subtypes belonging to clade A 
zooxanthellae, transitions to C in blue, D in green. Transitions that stayed the same are 
represented 
by
 
gray
.
 
All transiti
ons resulting in the same subtype are represented by the same 
color, regardless of the original zooxanthellae subtype. Field seasons are abbreviated above each 

ears correspond to 2012 or 




21
 
 
 
Figure 6.
 
Temperatures at Birthday and Acer24 reefs. Temperature data for inshore (Birthday) 

placed on each reef site.




.28
 
 
Figure 7
. By
-
gene plot of transcript abundance for zooxanthellae genes obtained from sub
-
samples of 
Porites 
astreoides taken from Birthday reef and Acer24 reef during winter and 

-
transplanted sub
-
sa
mples that originated at 

-
samples that were collected at 

-
transplanted 
sub
-
samples that originated at Birthday re

sub
-
samples that were collected at Birthday reef and transplanted to Acer24 reef. Sub
-
samples 
collected in summer months are represented in orange, while sub
-
samples collected in winter 
months are represen
ted in blue. Whiskers denote 95% credible intervals


..
 
44
 
 
Figure 8.
 
By
-
gene plot of normalized log
2
-
transformed expression values (±SEM) of 
experimental genes from all samples. 
Orange
 
lines represent fragments exposed to control 
treatments (28°C
), 
green
 
lines represent fragments exposed to heat treatments (32°C) and 
blue
 
lines represent fragments exposed to heat+LPS treatments (32°C+LPS from 
Serratia marcescens
 
). Whiskers denote 95% credible intervals



.
.67
 
 
 
 
1
 
CHAPTER 1
 
 
SYMBIONT DIVERSITY OF ZOOXANTHELLAE (
SYMBIODINIUM 
SPP.) IN 
PORITES 
ASTREOIDES
 
AND 
MONTASTRAEA CAVERNOSA 
FROM A RECIPROCAL TRANSPLANT 
IN THE LOWER FLORIDA KEYS
 
 
ABSTRACT 
 
 
In recent years coral reefs worldwide have suffered high
 
mortality rates due to coral 
bleaching, a phenomenon contributing to a 40% decrease in coral cover in the Florida Keys since 
the 1997/98 El Niño event. In the Florida Keys, coral from inshore reefs are known to be more 
thermotolerant than their conspecifi
cs from offshore reefs, but the mechanism behind this 
difference is unclear. In this study we conducted a two
-
year, reciprocal transplant of 
Porites 
astreoides
 
and 
Montastraea cavernosa
 
from an inshore and offshore reef in the lower Florida 
Keys to determi
ne if changes in the dominant symbiotic algae (
Symbiodinium 
spp.) could explain 
variation in holobiont tolerance as well as to assess the possibility of acclimatization to a 
changing stress regime.  Increased complexity and diversity was demonstrated in th
e 
composition of 
Symbiodinium 
spp. from both coral species collected at the offshore reef when 
compared to conspecifics collected inshore. As a result of this complexity, the offshore reef 
samples displayed higher numbers of transitions of zooxanthellae su
bclade types between 
seasons, while inshore fragments demonstrated more stability and
 
this observation
 
may explain 
previously measured thermotolerance. Additionally, the known thermotolerant subclade type D1 
was associated with one 
M. cavernosa 
fragment fr
om the inshore reef. When fragments were 
transplanted, compositional patterns of 
Symbiodinium 
spp. were retained from site of collection, 
indicating a lack of acclimatization to a new environment over the lengthy two
-
year experiment. 
These results demonstr
ate variability in the dominant 
Symbiodinium
 
spp. of 
P. astreoides 
and 
M. 
cavernosa
 
conspecifics from inshore and offshore reefs in the lower Florida Keys and point to 
 
2
 
possible patterns in holobiont thermotolerance. 
Dominant symbiont
 
variability may 
contri
bute
 
to 
the continued persistence of these species in the face of climate change, but future studies are 
needed to determine the mechanisms and range in which these subclade types are able to 
withstand thermal stress.  
 
 
INTRODUCTION
 

(Glynn 
et al.
, 2001; Gardner 
et al.
, 2003)
 
due to bleaching, disease and poor water quality 
(Dustan, 1977; In 
et al.
, 2007)
. Coral bleaching is the result of the expulsion of symbiotic, 
single
-
celled dinoflagellate algae known as zooxanthellae (
Symbiodinium 
spp.)
 
from the tissues 
of coral animals
, and may be caused by many stressors including, but not limited to, 
elevated
 
sea 
surface temperatures and increased irradiance 
(Fitt 
et al.
, 2001; Lesser & Farrell, 2004)
.  
Bleaching can be fatal depending on the intensity and duration of a bleaching event 
(Brown, 
1997)
, as many coral derive up to 95% of their daily metabolic needs from the byproducts of 
zooxanthellae 
(Muscatine, 1990)
. However, through genetic variability of their in
-
hospite 
zooxanthellae
, coral
-
algal assemblages vary in their ability to tolerate bleaching conditions 
(Sampayo 
et al.
, 2008)
.
 
Defining how specific reef systems and their resident symbionts respond 
to increases in bleach
ing
 
inducing stressors is important for delineating coral survivability and 
determining appropriate target populations for conservation efforts.
 
Symbiodinium 
spp
. are classified into clades A
-
I
 
and 
are 
further divided into sub
-
clade 
types, with hermatypic coral known to form symbioses with members of clades A
-
D 
(Baker, 
2003)
. In response to stress, coral may shuffle or increase the abundance of a more tolerant type 
(Baker, 2001; Rowan, 2004)
.  The ability to increase the relative frequency of stress tolerant 
 
3
 
symbiont subclade types may enhance the survival of corals experiencing long or short
-
term 
deterioration of en
vironmental quality such as sea surface temperatures
 
above mean averages
 
or 
irradiance. 
 
Previous investigations demonstrated that stress tolerance varies between clades and even 
subclades 
(Rowan & Knowlton, 1995; Fitt 
et al.
, 2001; Berkelmans & van Oppen, 2006; Hauff 
et al.
, 2014)
, the latter demonstrated by Sampayo et al. 
(
2008)
. They showed that while variation 
in susceptibility to bleaching was attributed to specific thermotolerant subclade types in 
Stylophora pistillata
, zooxanthellae tolerance was 
also assemblage specific. Thus, to relate 
survival to the composition of symbionts, we need to learn more about how stress tolerance is 
related to subclade type and assemblage specificity.
 
Inshore and offshore reefs of the Florida Keys display distinct env
ironmental parameters, 
which may result in locally adapted coral populations 
(Kenkel 
et al.
, 2013)
. There is significant 
population genetic subdivision between host coral populations originating from offshore and 
insho
re reefs, with inshore coral demonstrating higher thermotolerance 
(Kenkel 
et al.
, 2013)
. 
Additionally, recent work documented changes in holobiont health and photosynthetic efficiency 
of zooxanthellae in response to re
ciprocal transplants between ins
hore and offshore reefs (Haslun 
et al. 
In Review
). These studies, however, did not describe how zooxanthellae subclade type 
diversity between inshore and offshore coral responded to stress. 
 
To expand 
my
 
knowledge of how 
subclades vary among coral host conspecifics and how 
these a
ssemblages respond to stress, I
 
investigated patterns in the dominant populations of 
zooxanthellae from an inshore and offshore reef in the Low
er Florida Keys. In addition, I
 
investigated changes 
in the dominant populations of zooxanthellae from these coral in response 
to a two
-
year reciprocal transplant to assess the capacity of symbiont shuffling, and therefore 
 
4
 
acclimatization, to an altered stress regime. 
Porites astreoides
 
and 
Montastraea caver
nosa
 
were 
used as they commonly occur in 
both inshore and offshore 
patch reefs of the Florida Keys and 
are likely targets of conservation and repopulation efforts. These species also possess distinct life 
strategies and modes of symbiont acquisition and th
erefore, are likely to alter their zooxanthellae 
subclade types differently in response to stress. Fragments were sampled biannually and the 
predominant zooxanthellae were identified down to the subclade type by direct sequencing of the 
ITS2 region 
(LaJeunesse 
et al.
, 2003; Thornhill 
et al.
, 2009
;
 
T. LaJeunesse pers. comm.).  
 
 
METHODS
 
 
Study
 
sites
 
Two reefs off the coast of Summerland Key, Florida near Mote Marine Tropical 
Laboratory (MMTL) were used for collection of coral samples for genetic analyses and 
reciprocal transplant experiments. Bir
thday reef (inshore, 24.57917N, 
81.49693W) and Ac
er24 
reef (offshore, 24.55268N, 
81.43741W) are patch reefs separated by Hawk Channel. These two 
reefs experience notably distinct temperature and turbidity regimes, with Birthday reef 
experiencing higher turbidity and higher annual average temperatures (~1
°C) than Acer24 reef 
(Erich Bartels, pers.com.), but are otherwise similar (i.e. depth, 
coral and fish 
spec
ies diversity
). 
 
 
Coral fragment collection
 
In September 2011, fragments of 
Montastraea cavernosa 
and 
Porites astreoides
 
were 
collected from Acer 24 
and Birthday reef. At each site, ten 16x16 cm coral fragments of 
P. 
astreoides
, and ten fragments of 
M. cavernosa
,
 
were obtained (Permit # FKNMS
-
2011
-
107, 
n=40) using a hammer and chisel and stored in coolers with site
-
derived seawater during 
 
5
 
transport to 
MMTL where they were sectioned into two pieces (16x8 cm) using a table saw. The 
fragments were allowed to recover for 24 hours in a flow through water table shaded from direct 
sunlight.  Following this recovery period, fragments were attached to labeled co
ncrete pucks (1 
part concrete: 3 parts sand) using a two
-
part epoxy (All Fix Epoxy®, AFP; Philadelphia, PA 
USA) and allowed to recover for an additional 72 hours under the same conditions described 
above. These fragments were then used for the reciprocal t
ransplant experiment.
 
 
Reciprocal Transplant
 
A reciprocal transplant experiment was designed such that half of the original sample 
fragment was placed back at the site of origin and the other replicate fragment was placed at the 
transplant site (i.e. the o
ther reef site, Figure 1). 
 
 
6
 
 
Figure 1.
 
Experimental Design
. 
Graphical representation of 
my
 
reciprocal transplant design for a 
single coral species at one experimental level.
 
A fragment of one species was collected at each of 
the two sites. Solid rectangles represent coral from Acer24 Reef and hollow rectangles represent 
coral from Birthday Reef. Those fragments were sectioned into two fragments. Each fragment 
was then placed 
back out on to a reef with one fragment half remaining at the site of origin while 
the other fragment half was transplanted to 
the companion site. 
The fragments colored in red 
were placed on to Acer24 Reef while the fragments colored in blue
 
were placed at
 
Birthday 
 
7
 

Reef. 
Dashed arrows represent transplanted fragments. For the experiment, the 
above was repeated ten times for each coral species resulting in n=80 fragments
.
 
 
Transplants 
were established at each reef using six concrete cinder
 
blocks placed on 
a 
level benthic substrate in the shape of a hexagon. Each cinder block was attached to the substrate 
using AFP.  A total of 40 coral were placed at each site, ten fragments of 
P. astreoides 
from 
Acer24 reef and ten fragments of 
P. astreoi
des
 
from Birthday reef, as well as ten fragments of 
M. 
cavernosa 
from Acer24 reef and ten fragments of 
M. cavernosa
 
from Birthday reef.  This design 
yielded a total of 80 coral fragments.
 
Fragment halves were arranged on cinder blocks as follows. Due to un
equal division of 
samples to cinder blocks, six or seven coral replicate fragment halves were attached to each 
cinder block using AFP. Individual cinder blocks contained one species of coral (i.e. either 
P. 
astreoides
 
or 
M. cavernosa
), with neighboring blo
cks containing the other species. Within each 
cinder block, replicate fragment halves were placed randomly.  
This experimental design was 
conducted to assure that a
ll cinder blocks received equal exposure to 
homogenous 
environmental 
conditions. Temperature
 

 
 
Sample Collection for DNA Analysis 
 
After transplant, coral fragments remained at their placement site for two years (i.e. 
August 2011 through August 2013). 
Sampling for DNA analysis was
 
conducted biannually 
(February and August, 2012
-
2013, n=4) to reflect both conditions in which temperature stress is 
high (summer/August) and low (winter/February). These events will be referenced as sampling 
times throughout the manuscript. Sampling was 
achieved by chipping off a 1x1 cm piece of coral 
from fragments using a hammer and chisel. 
Porites astreoides
 
pieces were stored in 70% ethanol 
(EtOH), while 
Montastraea 
cavernosa pieces were flash
-
frozen in liquid nitrogen and stored at 
-
 
8
 
80°C (n=80/field 
season) within 15 minutes of initial collection.  
The distinction
 
in storage 
technique resulted from 
an
 
inability to extract viable DNA from 
M. cavernosa
 
fragments stored 
in EtOH. 
 
 
DNA Extraction and Sequencing
 
Tissues of 
P. astreoides
 
were collected from coral fragments stored in 70% EtOH. Tissue 
was removed from the skeleton using a razorblade in 10 mL of zooxanthellae isolation buffer 
(ZIB, 
Rowan & Powers, 1991)
. The homogenate was poured into centrifuge
 
tubes and pelleted 
(500 x g for 10 minutes), and the supernatant decanted, washed in 5 mL of ZIB, and pelleted 
(500 x g for 10 minutes) a second time. Upon collection of the tissue pellet, DNA was extracted 
using a plant DNA extraction kit (MoBio Laborato
ries) according to manufacturers instructions. 
 
Samples of 
M. cavernosa,
 
stored at 
-
80°C were macerated into a fine powder using a 
mortar and pestle pre
-
chilled in liquid nitrogen.  DNA was extracted (Extract
-
n
-
amp, Sigma 
Aldrich) from 0.05 g of powder fol

 
For polymerase chain reactions (PCR) of the internal transcriber 2 region (ITS2), 1 µL of DNA 
was added to 10uL of GoTaq (Promega), 7 µL of nuclease free H
2
O and 1 µL each of primers 
ITSintfor2 (GAATTGCAGAACTCCGTG) 
and ITS2
-
reverse 
(GGGATCCATATGCTTAAGTTCAGCGGGT)
(Lajeunesse & Trench, 2000)
 
using a 
touchdown PCR method outlined in LaJeunesse et al. 
(2003)
.  Amplification was verified using 5 
µL of PCR product examined on a 2% agarose gel (60 minutes and 110 volts).  PCR products 
were purified (GeneElute PCR purification kit, Sigma Aldrich) and quantified. 
Po
rites astreoides 
and 
Montastraea cavernosa
 
samples were subsequently sequenced using a capillary ABI 3130xl 
platform at the DNA sequencing facilities located at the Annis Water Resources Institute, Grand 
 
9
 
Valley State University and at the Research Technolo
gy Support Facility (RTSF) at Michigan 
State University, respectively. Both sequencing reactions used the forward or reverse ITS2 
primers listed above. 
 
 
Data Analysis
 
Chromatograms were visually inspected and aligned using BioEdit
 
(v7.2.5; Hall, 1999).  
All statistical analyses were conducted in R (Version 3.1.3; R Core Team, 2015). Sequences 
from each coral species were divided into four groups based upon fragment origin and 
transplantation (see Figure 2). For example, sequences o
f samples taken from 
Porites astreoides
 
fragments were categorized into two main categories, from Acer 24 reef or Birthday reef, with 
their site of relocation determining their further classification (i.e. four groups). Sequences 

 
of 
P. astreoides
 
fragments originating from Acer 24 reef that were 

P. astreoides
 
fragments originating from Acer 24 reef that were placed back on Birthday reef. An analogous 
sample ide
ntification scheme was used for sequences obtained from 
Montastraea cavernosa
 
samples. 
 
A multinomial model was used to analyze differences in population frequencies of clade 
subtypes within and between sampling periods.  Overall, four models were run, one
 
for each 
coral species and one for each collection site.  For example, one model was run comparing 
P. 
astreoides 
native to Acer24 reef with coral native to Acer24 but transplanted to Birthday reef. 
 
Within each model
,
 
frequency of zooxanthellae subclade t
ypes were used as a response 
variable, with sampling season and treatment (i.e. transplant 
vs
. non
-
transplant) as predictors. 
Model outputs, termed coefficients, represent probabilities of individual response variables. 
 
10
 
Residual deviances of models with an
d without the interaction term (sampling time:treatment) 
were compared to determine any significance of interaction, while AIC values of full models and 
individual response variables were compared to chose best fit models for further analysis. In 
order to 
perform t
-
tests on individual subclade type abundance comparisons, fitted values and 
standard errors were calculated from coefficients. 
 
 
 
RESULTS
 
 
A total of 139 zooxanthellae sequences were obtained from samples of 
Porites astreoides
 
fragments and 111 zo
oxanthellae sequences obtained from samples of 
Montastraea cavernosa
 
fragments.  To identify these samples, 
Symbiodinium 
sp
p
. sequences or subclade types were 
categorized into codes describing the (1) species (P or M for 
P. astreoides 
or 
M. cavernosa
, 
resp
ectively), (2) collection site (A or B for Acer24 reef or Birthday reef, respectively) and (3) 
transplant site (A or B for Acer24 reef or Birthday reef, respectively).  For example, sequences 
or subclade types obtained from fragments collected and replaced
 
at Birthday reef will be 


 
 
Porites astreoides collected from Birthday Reef (PBB and PBA)
 
A total of three subclade types were recovered 
from transplant and non
-
transplant 
Porites 
astreoides
 
originating from Birthday reef. These types included members of subclades A4.1, 
A4.2 and A4.3. Subclade type A4.1 was the predominant type between PBB and PBA samples 
(Figure 2A).  Subclade type A4.2 wa
s absent in PBB samples in Summer 2012 as well as PBA 
samples in Winter 2012 and Winter 2013. Subtype A4.3 was absent only in PBA samples from 
 
11
 
Summer 2012, but reemerged in the follow two field seasons.
 
In a multinomial model, the interaction 
term between experimental treatment and field 
season was not significant (p>0.25). Based upon AIC values of the full model (zooxanthellae 
subclade types explained by both experimental treatment and sampling time) and those of the 
individual independent var
iables, the model containing experimental treatment only (transplant 
vs
. non
-
transplant) fit the data best and was used for further analysis. T
-
tests of individual 
subclade types between experimental treatments (i.e. subclade type A4.1 in PBB 
vs
. PBA 
sampl
es) were insignificant (p>0.05).  In PBB samples, the probability of subclade type A4.1 
was significantly higher than A4.2 (p=0.000001) and A4.3 (p=0.0005). Additionally, the 
probability of subclade type A4.3 was significantly higher than A4.2 in PBB sampl
es (p=0.01). 
In PBA samples, the probability of subclade type A4.1 was significantly higher than A4.2 
(p=0.001) 
and A4.3 (p=0.008) (Figure 2A).
 
12
 
 
 
Figure 2
.
 
Symbiodinium
 
subclade type frequencies for 
Porites astreoides
 
samples.
 
Graphs are labeled with acronyms describing the
 
 
 
13
 

(1) coral species, (2) field site of original collection (i.e. Acer24 or Birthday 
Reefs) and (3) site of transplant (i.e. Acer24 or Birthday Reefs). For example, PBA ar
e samples 
from 
Porites astreoides
 
fragments collected at Birthday Reef and transplanted to Acer24 Reef.   
Graphs in the left
-
hand column represent non
-
transplanted coral while graphs on the right 
represent transplanted coral. Although each condition began 
at n=10, note variation in group 
sizes due to sequencing error.
 
 
Figure 3 outlines the specific changes in zooxanthellae subclade type observed within 
individual sample fragments between sampling times (i.e. subclade type change from Summer 
20
12 to Winter 2012), here referred to as transitions.  Greater than 50% of subclade types present 
in PBB and PBA samples did not change throughout the field seasons (Figure 3A). Additionally, 
with the exception of PBA in Winter 2013 to Summer 2013, more tha
n 50% of the samples were 
of subclade type A4.1. Not all A4.1 samples transitioned, however.  Many A4.1 samples from 
PBA transitioned to A4.3, specifically in the Summer 2012 to Winter 2013 transition. There 
were also high transitions to A4.2 in the 2013
 
W
inter to Summer field season.
 
14
 
 
Figure 3
.
 
Porites astreoides 
zooxanthellae transitions. Transitions in zooxanthellae subclade type occurring within the same coral
 
 
15
 
 
Figure
 

Porites astreoides 
zooxanthellae. Pie charts represent the
 
16
 

 
relative frequencies of individual transitions of clade subtypes through each 
field season.  Graphs are labeled with acronyms describing the (1) coral species, (2) field site of 
original collection (i.e. Acer24 or Birthday Reefs) and (3) site of transplant (i.e. Acer24 or 
Birthday Reefs). For example, PBA represents samples from 
P. astreoides 
fragments
 
collected at 
Birthday Reef and transplanted to Acer24 Reef. Color scales are de
termined by the outcome of 
the transition. For example, slices represented in shades of red describe transitions that resulted 
in A4 zooxanthellae, transitions to A4.1 in blue, A4.2 in green and A4.3 in orange. Transitions 
that stayed the same are represen
ted by a gray scale and outlined in black. Sampling times are 

Y
ears correspond 
to 
2012 or 2013. 
 
 
 
Porites astreoides collected from Acer24 Reef (PAA and PAB)
 
A total of four subclade
 
types were observed from sequences originating from transplant 
and non
-
transplant 
Porites astreoides
 
collected from Acer24 reef, including members of 
subclade types A4, A4.1, A4.2 and A4.3. Subclade type A4 was present only during the Summer 
2012 field se
ason in PAA and PAB samples (Figure 2B). Additionally, subclade type A4.2 was 
present during each sampling time except Summer 2012 for PAA and PAB samples. The other 
three subclade types were present at all other sampling times in both PAA and PAB samples.
 
Experimental treatment (transplant 
vs.
 
non
-
 
transplant) did not have a significant effect 
on sequence frequencies (p>0.05). Additionally, the interaction term between experimental 
treatment and sampling time was not significant (p>0.25). According to AIC 
values of individual 
models, the model containing only sampling time fit the data best and was used for further 
analysis. 
 
The probability of having subclade type A4 was significantly higher in Summer 2012 
than the other sampling times (p<0.03). Although t
he probability of subclade type A4.1 was 
higher in both Winter sampling times compared to Summer sampling times, these increases were 
not significant (p>0.3) (Figure 2B).  
 
A higher number of transitions to different subclade
 
types were seen in PAA samples 
than in PAB samples (Figure 3B). Overall, ~78% of the zooxanthellae subclade types from PAA 
 
17
 
changed between field seasons, where as 52% changed in PAB samples (i.e. the difference 
between the total number of transitions and 
those in grey). Notable transitions are those of PAA 
samples from Winter 2012 to Summer 2012 (Figure 3B).  Approximately 50% of the transitions 
resulted in the presence of the A4 subtype, a subtype that was only observed again in the same 
transition time p
oint in PAB samples, but in fewer numbers than in PAA samples. Overall, PAB 
samples were dominated by transitions to A4.3, particularly from summer to winter, where as 
PAA had a high number of transitions to A4.1.  
 
 
Montastraea cavernosa collected from Bi
rthday Reef (MBB and MBA)
 
A total of 59 sequences were recovered from transplant and non
-
transplant 
Montastraea 
cavernosa
 
originating from Birthday reef. These sequences were representative of seven 
different subclade types, including A4.1, A4
.2, A4.3, C1, C3 and D1. A large range of sample 
sizes between field sites in 
M. cavernosa
 
samples resulted.  Although the exact subclade types 
are detailed here, for analytical purposes and in corresponding figures, subclade types were 
binned by clade typ
e (clade type A or clade type C) due to low replicate numbers (Figure 4A). 
Clade C subclade types were the dominant clade represented in MBB and MBA samples. Some 
subclade types were only represented in one sampling time, such as A4.3 in Summer 2012 
(MBB),
 
A4.2 in Winter 2012 (MBA) and D1 in Summer 2013 (MBA). The presence of subclade 
type D1 in MBA samples in the Summer of 2013 marks the only appearance of a D subclade 
type in all samples 
tested.
 
18
 
 
 
Figure 4.
 
Symbiodinium
 
subclade frequencies for 
Montastra
ea cavernosa 
samples.
 
Graphs are
 
labe
led with acronyms describing the
 
 
 
 
19
 

(1) coral species, (2) field site of original collection (i.e. Acer24 or Birthday 
Reefs) and (3) site of transplant (i.e. Acer24 or Birthday Reefs
).  For example, MBA represents 
samples from 
Montastraea cavernosa 
fragments
 
collected at Birthday Reef and transplanted to 
Acer24 Reef. Graphs in the left
-
hand column represent non
-
transplanted coral while graphs on 
the right represent transplanted coral. Although each condition began at n=10, note variation in 
group sizes due to
 
sequencing error
.
 
 
 
A comparison of MBB and MBA samples described an insignificant 
interaction between 
experimental treatment and field season (p>0.25). Additionally, when comparing AIC values 
between the full model and those of the individual independent
 
variables, the model containing 
only the experimental treatment provided the best fit to the data and was used for further 
analysis. 
 
 
A comparison of
 
fitted values of the probability of clades A,
 
C and D in MBB and MBA 
samples displayed
 
no significant di
fference in the probability of any clade type between 
experimental treatments. For example, there was no significant difference in the presence of 
clade A in MBB 
vs.
 
MBA samples. However, there was a significant difference in the probability 
of clade A and
 
C within MBB samples (p=0.0000003), and within MBA samples (p=0.0000002), 
with probabilities of clade C being higher than clade A in both cases (Figure 4A).  
 
The majority of transitions seen in MBB samples resulted in clade C types (Figure 5
A).  
Interestingly, a few transitions resulted in changes from clade A to C or vice versa. The single 
appearance of clade D results from a transition from clade C in MBA samples during the Winter 
2013 to Summer 2013 seasons. 
 
20
 
 
 
Figure
 
5
.
  
Montastraea 
cavernosa 
zooxanthellae Transitions. Transitions in zooxanthellae subclade type occurring within the same
 
 
21
 
 
Figure
 
5
 

coral fragment between sampling times for all fragments of 
Montastraea cavernosa 
zooxanthellae. Pie charts 
 
22
 

 
represent the relative
 
frequencies of individual transitions of clade subtypes 
through each field season. Graphs are labeled with acronyms describing the (1) coral species, (2) 
field site of original collection (i.e. Acer24 or Birthday r
eefs) and (3) site of transplant (i.e. 
Acer24 or Birthday reefs). For example, MBA represents samples from 
M. cavernosa 
fragments
 
collected at Birthday reef and transplanted to Acer24 reef.  Color scales are determined by the 
outcome of the transition
. 
For
 
example, slices represented in shades of red describe transitions 
that resulted in subtypes belonging to clade A zooxanthellae, transitions to C in blue, D in green. 
Transitions that stayed the same are represented 
by
 
gray
.
 
All transitions resulting in th
e same 
subtype are represented by the same color, regardless of the original zooxanthellae subtype. Field 

correspond to 2012 or 2013
.
 
 
Montastraea cavernosa
 
collected from Acer24 Reef (MAA and MAB)
 
A total of 52 sequences were recovered from transplant and non
-
transplant 
Montastraea 
cavernosa
 
originating from Acer24 reef. These sequences were representative of seven different 
subclade types, including A3, A4,
 
A4.1, A4.2, A4.3, C1 and C3.  Again, although detailed here, 
subclade types were binned by overall clade for analysis and outlined by clade in corresponding 
figures (Figure 4B). 
 
 
According to the mutinomial model frequencies of clade types in MAA and MAB
 
samples, experimental treatment (transplant 
vs
. non
-
 
transplant) did not have a significant effect 
on clade frequencies (p>0.05).  In addition, the interaction term between experimental treatment 
and sampling time was not significant (p>0.25). When compar
ing AIC values between the full 
model and those of the individual independent variables, the model containing only sampling 
time provided the best fit to the data and was used for further analysis. Across all samples, the 
Winter 2013 sampling time was asso
ciated with a significant increase in the probability of clade 
type C (p=0.003) (Figure 4B).  
 
All but one MAA sample transitioned to a different subclade type over the sampling 
times (Figure 5B). All but two MAB samples transitioned to a different subclad
e type over the 
sampling times. Interestingly, there were multiple occurrences of A to C, or C to A subclade type 
 
23
 
shifts in both MAA and MAB samples. 
 
 
DISCUSSION
 
Since the 1997/98 El Niño event coral cover on offshore reefs in 
the Florida Keys Reef 
Tract 
have 
remained at low levels 

(Ruzicka 
et al.
, 2013)
. 
Prior
 
studies have demonstrated 
differences in the ability of certain cora
l assemblages to 
endure
 
stress. Kenkel et al. 
(2013)
 
found 
that coral assemblages from inshore reefs 
exhibited
 
higher thermotolerance than their offshore 
conspecifics. Additionally, Haslu
n 
et al.
 
(In R
eview) found lower incidences of chronic 
bleaching
 
inshore
 
relative
 
to assemblages from offshore. Zooxanthellae subclade type may be an 
important driver of holobiont stress tolerance 
(Sampayo 
et al.
, 2008)
. In this study, I
 
investigated 
whethe
r observable patterns of change
 
in the
 
abundances of
 
dominant zooxanthellae subclade 
type
s
 
from fragmen
ts of 
Porites astreoides
 
and 
Montastraea cavernosa 
provide information 
regarding inshore and offshore reef coral stress tolerance. 
 
 
Porites astreoides
 
Subclade type A4.1 was the predominant subclade type among fragments collected at 
Birthday Reef (Figure 
2A). Haslun 
et al.
 
(In R
eview) proposed that due to moderate 
temperatures and low irradiance, coral inhabiting the inshore (Birthday) reef experience less 
stress than their conspecifics at 
my
 
offshore (Acer24) reef. Temperatures at Birthday reef during 
sum
mer months were within the mean summer maximum for this 
reef
. These similar 
temperatures associated with 
presumed 
low
-
level irradiance are likely responsible for the 
relatively stable associations at Birthday reef compared to 
assemblages from Acer24 reef t
hat
 
demonstrated fewer transitions. Additionally, associations
 
at Birthday reef
 
consisted mostly of 
 
24
 
subclade type A4.1 and A4.3. Further studies will be needed to determine if the prominence of 
subclade type A4.1 found at the inshore site compared to the o
ffshore site is related to 
a
 
lower 
stress environment. 
 
According to Haslun 
et al.
 
(In R
eview), coral 
offshore 
experience
 
higher bleaching than 
coral at the inshore site 
presumably 
due to higher irradiance. This implies that coral fragments 
transplanted from Birthday Reef to the offshore Acer24 reef (PBA) may experience more stress 
than coral fragments that remained inshore (PBB). As a consequence, 
I
 
expect
ed
 
a transition in 
subclade
 
type
s in PBA fragments
 
to more stress tolerant subclades. Yet there is no significant 
difference in the probabilities of 
occurrence
 
for 
individual subclade types between transplanted 
and non
-
transplanted 
P. astreoides 
fragments from Birthday reef (p>0.05)
. In fact, composition 
of zooxanthellae from PBB and PBA samples are very similar, indicating a lack of 
acclimatization to a presumed higher stress environment. 
The lack of a transition in subclade 
abundance
 
may 
indicate that
 
Porites astreoides 
fragments o
riginating from the inshore site do 
not possess a 
Symbiodinium 
spp.
 
subclade type adapted to higher irradiance.
 
P. astreoides
 
fragments collected at the offshore site, 
I
 
observed zooxanthellae subclade 
type A4 in fragments of 
P. astreoides 
solely during th
e Summer 
of 
2012 sampling time (Figure 
2B).  Further, subclade type A4 was never observed in 
P. 
astreoides collected at the inshore site, 
indicating that this subclade type may originate from and confer a specific advantage to 
environmental parameters expe
rienced offshore. Summer 2012 was the warmer of the two 
summer sampling times
 
(Figure 6)
. 
The combination of
 
high
er
 
irradiance levels characteristic of 
summer
 
and
 
higher temperatures may have 
favored 
the higher 
relative abundance
 
of subclade 
type A4 during
 
the s
ummer 
of 
2012 relative to 
the s
ummer 
of 
2013
 
offshore
. 
 
 
25
 
Porites astreoides
 
is known to contain multiple subclade types 
in addition to
 
members of 
A4 
(LaJeunesse, 2002)
. However, little variability 
in subclade type abundance 
was found in this 
study as conspecifics of 
P. astreoides
 
displayed 
subclade types A4, A4.1, A4.2 and A4.3.  This 
lack of variability in zooxanthellae subclade type is common in corals that reproduce 
via
 
brooding as their zooxanthellae are acquired 
via
 
vertical transmission 
(Van Oppen, 2004)
.  T
he 
long
-
term coevolution of the
 
mutualism 
between the coral host and zooxanthellae 
likely leads to 
a tight symbiosis, with little flexibility, and m
ay explain the 
lack of marked changes in subclade 
type abundances
 
observed in this study 
(Thornhill 
et al.
, 2006)
. 
 
The altering of zooxanthellae subclade types
, here demonstrated 
via
 
transitions, is one 
mechanism that can alter the relative abundance of subclade types 
(Kinzie 
et al.
, 2001; Rowan, 
2004)
. 
Less than 50% of z
ooxanthellae subclade types 
in fragments 
originating from the inshore 
reef 
displayed
 
transitions (Figure 3A), demon
strating a relatively stable association even at the 
higher stress reef. In contrast, transitions between subclade types were much more frequent for 
coral fragments originating from the offshore 
reef
 
(Figure 3B).  Retention of zooxanthellae 
composition and
 
diversity
 
even though transferred to new sites indicates
 
a lack of acclimation
 
to 
changing stress regimes, 
particularly notable 
after a lengthy transplant
 
period of two years
. 
 
 
Montastraea cavernosa
 
Fragments of 
M. cavernosa 
collected from the inshore reef contained zooxanthellae 
subclade types representative of clades A, C and D (Figure 4A). To 
my
 
knowledge this is the 
first time that subclade types of clade A have been found in association with 
M. cavernosa
 
in this 
region. H
owever, members of clade A are 
frequnetly
 
isolated from species within the genus 
 
26
 
Montastraea 
(Garren 
et al.
, 2006)
.
 
C
lade A 
is also
 
a common zooxanthellae type in this region, 
and is common in
 
P. astreoides 
for example
. 
 
Subclade type C3 is commonly associated with 
M. cavernos
a
 
(LaJeunesse, 2002; 
Banaszak, 2007; Serra
no 
et al.
, 2014)
 
and has been described as a pandemic host generalist 
commonly associated with 
M. cavernosa 
in times of low stress 
(Lajeunesse, 2005)
. The 
probability of 
finding 
clade type C 
at the inshore reef was 
significantly higher 
than offshore and
 
likely 
reflects the
 
low
er
 
stress environmental conditions
 
inshore
 
and the generalist nature of 
clade type C.  A notable
 
exception in the subclade type abundances
 
found in MBA samples 
(Figure 4A) 
was
 
the presence of a D1 subclade type in the Summer
 
of
 
2013. Although this 
subclade type was only present in one fragment and at one sampling time, clade D has many 
implications f
or holobiont thermotolerance. For instance, the dominance of clade D subclade 
types 
relative to other subclade types 
during stress events has been demonstrated repeatedly in 
the literature 
(Baker, 2001; Thornhill 
et al.
, 2006; Silverstein 
et al.
, 2015)
, leading to the belief 
that clade D subclade types confer a specific advantage to holobionts during times of stress. 
Ul
strup and V
an
 
Oppen 
(
2003)
 
found that subclade types of clade D may be present in and 
acquir
ed from the surrounding environment, but may also exist at low concentrations within the 
host. Subclade types of clade D become more prevalent when environmental stress increases due 
to elevated temperatures or changes in turbidity. This confers resistance
 
to stressful conditions 
(Berkelmans & van Oppen, 2006)
 
a
nd may 
explain
 
why inshore coral have been found to be 
more thermotolerant than offshore conspecifics. 
 
In contrast to the brooder 
Porites 
astreoides
, 
Montastraea cavernosa
 
is a broadcast 
spawner, whose 
gametes 
lack
 
zooxanthellae. Thus, they acquire 
symbionts 
via
 
horizontal 
transmission, from the surrounding environment 
(Van Oppen, 2004)
, as their eggs are void of 
 
27
 
sym
biont cells 
(Szmant, 1991)
. This ability to acquire zooxanthellae from the surrounding 
environment may also explain the presence of clade type A in 
M. cavernosa
 
samples. Compared 
to brooders, broadcast spawners are thought to rely more heavily 
on zooxanthellae 
as a 
mechanism for surviving temperature stress
 
as they possess the ability to switch and shuffle their 
zooxanthellae 
(Silverstein 
et al.
, 2015)
. While 
I
 
did not attempt to identify the 
origin
 
of 
zooxanthellae clade types, the presence of D1 in MBA samples from Summer 2013 suggest 
stressful conditions during this sampling time, likely due to increases in irradiance demonstrated 
by Haslun 
et al.
 
(In R
eview) 
and 
as 
a means 
to counteract this
 
stress
 
b
y
 
the holobiont.
 
Fragments of 
Montastraea cavernosa
 
collected from the offshore site contained 
zooxanthellae subclade types representative of clades A and C. Similarly to fragments of 
Porites 
astreoides
, subclade
 
type A4 was isolated only from 
M. cavernosa
 
fragments from the offshore 
site, supporting the notion that subclade type A4 may be endemic to offshore sites. Symbionts 
from clade C, however, were the predominant clade type 
observed
 
in 
M. 
cavernosa fragments
. 
The w
inter 
of 
2013 was warm
er, and therefore milder, than w
inter
 
of
 
2012 (Figure 6). This mild 
winter, combined with the generalist nature of subtype C3 mentioned earlier, may explain the 
significant increase in clade C displayed by 
M. cavernosa
 
fragment
s collected from the offshore 
reef
 
at that time
. 
 
28
 
 
 
Figure 6.
 
Temperatures at Birthday and Acer24 reefs. Temperature data for inshore (Birthday) and offshore (Acer24) reefs. 

 
 
 
29
 
 
Transitions between zooxanthellae clade types, rather than subclade types, are significant 
as individual clades can differ drastically 
physiologically
. We observed multiple instances of 
transitions between subclade types A and C in 
M.
 
cavernosa
 
fragments collected at the inshore 
and offshore reef (Figure 5). Transitions between clade types are commonly reported in 
M. 
cavernosa
 
(Ulstrup & Van Oppen, 2003; Thornhill 
et al.
, 2006; Silverstein 
et al.
, 2015)
 
and are 
likely due to their reproductive strategy and ability to acquire symbionts from the surrounding 
environment. Similar to trends observed in 
P. astreoides
, fragments of 
M. cavernosa
 
collected 
inshore displayed fewer transitions, and therefore more stability, compared to fragments of 
M. 
cavernosa 
collected offshore. Additionally, similar transition frequencies, as well as 
zooxanthellae compositi
on, within samples from the same collection site suggest a lack of 
acclimatization to 
the 
new 
environmental conditions present at the transplant site
.  
 
In 
response to
 
global climate change, many 
researchers
 
are focused on finding particular 
coral assembla
ges that are better adept at 
surviving
 
a myriad of environmental stresses
 
that 
climate warming will impose
. 
My
 
study found significant differences in subclade type 
frequencies between inshore and offshore 
P. astreoides
 
and 
M. cavernosa
, suggesting a 
mechan
istic role of zooxanthellae subclade type in their thermotolerance variation. It also 
showed a site
-
dependent
 
effect of seasonal zooxanthellae compositional changes suggesting a 
lack of acclimatization
 
by zooxanthellae to the
 
new environment. 
 
This observa
tion has
 
important 
implications for the choice of which coral populations to target
 
for conservation or 
repopulation efforts. While these findings are the first step in identifying specific holobiont 
populations best equipped to survive stressors associate
d with global climate change, future 
studies will be required to determine differences in these subclade types at the functional genetic 
 
30
 
level, as well as the response of these subclade types to bleaching temperatures and other local 
synergistic stress.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
31
 
 
 
 
 
 
 
 
 
 
 
 
 
LITERATURE CITED
 
 
 
 
 
 
 
 
 
 
 
 
32
 
LITERATURE CITED
 
 
Baker AC (2001) Reef corals bleach to survive change. 
Nature
, 
411
, 765

766.
 
Baker C (2003) FLEXIBILITY AND SPECIFICITY IN CORAL
-

Diversity , Ecology , and Biogeography of Symbiodinium. 
34
, 661

689.
 
Banaszak AT (2007) Optimization of DNA extraction from a scleractinian coral for the detection 
of thymine dimers by i
mmunoassay. 
Photochemistry and photobiology
, 
83
, 833

8.
 
Berkelmans R, van Oppen MJH (2006) The role of zooxanthellae in the thermal tolerance of 

Proceedings. 
Biological sciences / The 
Royal Society
, 
273
, 2305

12.
 
Brown BE (1997) Coral bleaching: causes and consequences. 
Coral Reefs
, 
16
, S129

S138.
 
Dustan P (1977) Vitality of reef coral populations off Key Largo, Florida: Recruitment and 
mortality. 
Environmental Geology
, 
2
, 51

58.
 
Fitt W
, Brown B, Warner M, Dunne R (2001) Coral bleaching: interpretation of thermal 
tolerance limits and thermal thresholds in tropical corals. 
Coral Reefs
, 
20
, 51

65.
 
Gardner T a, Côté IM, Gill J a, Grant A, Watkinson AR (2003) Long
-
term region
-
wide declines 
i
n Caribbean corals. 
Science (New York, N.Y.)
, 
301
, 958

960.
 
Garren M, Walsh SM, Caccone A, Knowlton N (2006) Patterns of association between 
Symbiodinium and members of the Montastraea annularis species complex on spatial scales 
ranging from within colonies to between geographic regions. 
Coral Reefs
, 
25
, 503

512.
 
Glynn PW, Maté JL, Baker AC, Calderon MO (2001) Coral bleaching and mortality in Panama 
and Ecuador during the 1997
-
1998 el nino
-
southern oscillation event: spatial/temporal 
patterns and comparisons with the 1982
-
1983 event. 
Bulletin of Marine Science
, 
69
,
 
79

109.
 
Hall TA (1999)
 
BioEdit: A user friendly biological sequence alignment editor and analysis 
program for Windows95/98/NT. 
Nucelic Acid Symposium Series.
 
Oxford University 
Press, 
41
, 95
-
98.
 
 
Haslun JA, Hauff B
, 
Strychar K,
 
Cer
vino JM
 
(In Review) Decre
ased turbidity decouples 
seasonal relationship between seawater temperature and symbiont density resulting in 
chronic bleaching of 
Montastraea cavernosa 
 
and 
Porites astreoides
 
inhabiting the 
Florida Keys. 
International Journal of Marine Biology
. 
 
 
Hauff B, Cervino JM, Haslun J a et al. (2014) Genetically divergent Symbiodinium sp. display 
distinct molecular responses to pathogenic Vibrio and thermal stress. 
Diseases of aquatic 
 
33
 
organisms
, 
112
, 149

59.
 
In F, Approaches G, Precht WF, Miller SL (2007) 9
 
. Ecological Shifts along the Florida Reef 


312.
 
Kenkel CD, Meyer E, Matz M V. (2013) Gene expression under chronic heat stress in 
populations of the mustard hill coral ( 
Porites astreoides
 
) from different ther
mal 
environments. 
Molecular Ecology
, 
22
, 4322

4334.
 
Kenkel CD, Sheridan C, Leal MC et al. (2014) Diagnostic gene expression biomarkers of coral 
thermal stress. 
Molecular Ecology Resources
, 
14
, 667

678.
 
Kinzie R a, Takayama M, Santos SR, Coffroth M a (2001)
 
The adaptive bleaching hypothesis: 
experimental tests of critical assumptions. 
The Biological bulletin
, 
200
, 51

8.
 
LaJeunesse T (2002) Diversity and community structure of symbiotic dinoflagellates from 
Caribbean coral reefs. 
Marine Biology
, 
141
, 387

400.
 

-
Pacific since the Miocene
-
Pliocene transition. 
Molecular biology and evolution
, 
22
, 570

81.
 
LaJeunesse TC, Loh WKW, van Woesik R, Hoegh
-
Guldberg O, Schmidt GW,
 
Fitt WK (2003) 
Low symbiont diversity in southern Great Barrier Reef corals relative to those of the 
Caribbean. 
Limnology and Oceanography
, 
48
, 2046

2054.
 
Lesser MP, Farrell JH (2004) Exposure to solar radiation increases damage to both host tissues 
and a
lgal symbionts of corals during thermal stress. 
Coral Reefs
, 
23
, 367

377.
 
Muscatine L (1990) The role of symbiotic algae in carbon and energy flux in reef corals. 
Ecosystems of the world
, 
25
, 75

87.
 
R Core Team (2015)
 
R: A language and environment for statistical computing. R Foundation for 
Statistical Computing, Vienna, Austria. URL http://www.R
-
project.org/.
 
 
Rowan R (2004) brief communications Thermal adaptation in. 
Nature
, 
430
, 742.
 
Rowan R, Knowlton N (1995) Intra
specific diversity and ecological zonation in coral
-
algal 
symbiosis. 
Proceedings of the National Academy of Sciences of the United States of 
America
, 
92
, 2850

3.
 
Rowan R, Powers D a (1991) A molecular genetic classification of zooxanthellae and the 
evoluti
on of animal
-
algal symbioses. 
Science (New York, N.Y.)
, 
251
, 1348

51.
 
Ruzicka RR, Colella M a., Porter JW et al. (2013) Temporal changes in benthic assemblages on 
Florida Keys reefs 11 years after the 1997/1998 El Niño. 
Marine Ecology Progress Series
, 
489
,
 
125

141.
 
 
34
 
Sampayo EM, Ridgway T, Bongaerts P, Hoegh
-
Guldberg O (2008) Bleaching susceptibility and 
mortality of corals are determined by fine
-
scale differences in symbiont type. 
Proceedings 
of the National Academy of Sciences of the United States of Americ
a
, 
105
, 10444

9.
 

the Caribbean coral Montastraea cavernosa despite high levels of horizontal connectivity at 
shallow depths. 
Molecular Ecology
, 4226

4240.
 
Silv
erstein RN, Cunning R, Baker AC (2015) Change in algal symbiont communities after 
bleaching, not prior heat exposure, increases heat tolerance of reef corals. 
Global Change 
Biology
, 
21
, 236

249.
 
Szmant  A
 
M (1991) Sexual reproduction by the Caribbean reef 
corals Montastrea annularis and 
M. cavernosa. 
Marine Ecology Progress Series
, 
74
, 13

25.
 
Tarze  a, Deniaud A, Le Bras M et al. (2007) GAPDH, a novel regulator of the pro
-
apoptotic 
mitochondrial membrane permeabilization. 
Oncogene
, 
26
, 2606

2620.
 
Thornhill DJ, Fitt WK, Schmidt GW (2006) Highly stable symbioses among western Atlantic 
brooding corals. 
Coral Reefs
, 
25
, 515

519.
 
Thornhill DJ, Kemp DW, Sampayo EM, Schmidt GW (2009) Comparative analyses of amplicon 
migration behavior in differing denatur
ing gradient gel electrophoresis (DGGE) systems. 
Coral Reefs
, 
29
, 83

91.
 
Ulstrup KE, Van Oppen MJH (2003) Geographic and habitat partitioning of genetically distinct 
zooxanthellae (Symbiodinium) in Acropora corals on the Great Barrier Reef. 
Molecular 
Ecolo
gy
, 
12
, 3477

3484.
 
 
 
 
 
 
 
 
 
 
 
35
 
CHAPTER 2
 
 
SITE
-
SPECIFIC VARIATION IN GENE EXPRESSION FROM 
SYMBIODINIUM 
SPP. 
ASSOCIATED WITH OFFSHORE AND INSHORE 
PORITES ASTREOIDES
 
IN THE LOWER 
FLORIDA KEYS
 
 
ABSTRACT 
 
Scleractinian
 
coral are experiencing unprecedented rates of mortality due to increases in 
sea surface temperatures 
in response to
 
global climate change. Some coral species however, 
survive high temperature events due to a reduced susceptibility to bleaching. 
I
 
investig
ated the 
relationship between bleaching susceptibility and expression of five metabolically related genes 
of 
Symbiodinium
 
spp. from the coral 
Porites astreoides 
originating from an inshore and offshore 
reef in the Florida Keys. 
The a
cclimatization potentia
l of 
Symbiodinium
 
spp. to changing 
temperature regimes was also measured 
via 
a two
-
year reciprocal transplant between the sites. 
Offshore 
coral fragments
 
displayed significantly higher expression in 
Symbiodinium
 
spp. genes 
PCNA
, 
SCP2
, 
G3PDH
, 
PCP
 
and 
psaE
 
t
han their inshore counterparts (p<0.05), a pattern 
consistent with increased bleaching susceptibility
 
in offshore corals
. Additionally, gene 
expression patterns
 
in
 
Symbiodinium
 
spp. 
from site of origin were conserved throughout the two
-
year reciprocal tran
splant, indicating acclimatization did not occur. Gene expression reported 
here indicates functional variation in populations of 
Symbiodinium
 
spp. associated with 
P. 
astreoides 
in the Florida Keys, and is likely a result of localized adaptation. 
 
 
INTRODUC
TION
 
Over the last 30
-
40 years, a decline in global coral populations as high as 50% has been 
recorded and linked to coral bleaching 
(Bruno 
et al.

et al.
, 2012; Jackson 
et al.
, 
2014)
. Bleaching
 
i
s a response to 
elevated
 
seawater temperatures and high irradiance, 
and 
results 
 
36
 
when the density of symbiotic algae (
Symbiodinium
 
spp. commonly referred to as zooxanthellae) 
declines severely in the host coral 
allowing the white skeleton below to show 
(Gates 
et al.
, 
1992)
. Considering 
that 
coral hosts derive up to 95% of their daily metabolic needs from 
zooxanthellae 
(Mu
scatine 
et al.
, 1989)
, the loss of symbiont
s
 
compromises the host during a 
bleaching event. Depending on the extent and duration of a bleaching event, loss of 
zooxanthellae may lead to coral host mortality 
(Brown, 1997)
. Given the importance of the host
-
zooxanthellae symbiosis, plasticity in 
algal response to stress is an important area of coral 
research 
(Baker, 2003)
.
 
Coral susceptibility to bleaching is influe
nced by the 
Symbiodinium
 
spp. clade type 
harbored by the host 
(Rowan, 2004)
. For example, Sampayo 
et al.
 
(
2008)
 
demonstrated
 
that
 
bleaching susceptibility of 
Stylophora 
pistillata
 
colonies 
was 
determined by 
compositional 
differences at the subclade leve
l within 
Symbiodinium
 
spp. of clade C. In the Florida Keys, 
several studies 
found
 
variation in bleaching susceptibility between inshore and offshore 
communities of 
P. astreoides
 
(Kenkel 
et al.
, 2013, 2015
; Haslun 
et al.
, I
n Review
)
. The 
observation of variation in subclades between proximate inshore and offshore reefs prompts 
questions of the role of genetically associated functional variation in zooxanthellae to bleaching 
susceptibility (Hauff 
et al.
 
In Press). However,
 
the role of zooxanthellae in bleaching 
susceptibility variation in these coral is unknown 
(Kenkel 
et al.
, 2015)
.
 
 
Changes in metabolic processes as markers of bleaching susceptibility have a long
-
standing history in the study of zooxanthellae
 
response to stress 
(Szmant & Gassman, 1990; 
Jones & Hoegh
-
Guldberg, 2001; Rodrigues & Grottoli, 2007)
. Changes in metabolism are 
commonly assessed 
vi
a
 
gene expression analysis, which targets genes related to the production 
of metabolically important proteins. Common examples include, proliferating cell nuclear 
 
37
 
antigen (
PCNA
), a protein involved in DNA synthesis and replication 
(Prelich 
et al.
, 1987)
, non
-
specific lipid
-
transfer protein (
SCP2
), which mediat
es the transfer of all common lipids 
(Thoma 
& Kaneko, 1991)
, Glyceraldehyde
-
3
-
phosphate dehydrogena
se (
G3PDH
), an enzyme involved 
in glycolysis 
(Sirover, 1997)
, peridinin chlorophyll alpha binding protein I chloroplastic (
PCP
) 
and photosystem I reaction center subunit IV (
psaE
). 
PCP
 
is invol
ved 
in
 
the capture of solar 
energy 
(Norris & Miller, 1994)
 
and 
psaE
 
stabilizes interactions with
in
 
the photosystem 1 
complex 
(Bengis & Nelson, 1977)
. Variation in the expression of these metabolic genes affords 
functional variability to zooxanthellae and
 
be involved in
 
acclimatization to bleaching conditions 
(Rosic 
et
 
al.
, 2010, 2011a; Leggat 
et al.
, 2011)
.
 
We studied the role of zooxanthellae in bleaching susceptibility of 
P. 
astreoides
 
by 
evaluating symbiont gene expression in reciprocal transplants of 
P. 
astreoides
 
in the Florida 
Keys. The expression of 
zooxanthellae genes 
PCNA
, 
SCP2
, 
G3PDH
, 
PCP
 
and 
psaE
 
was 
measured in coral sub
-
samples from inshore and offshore reefs differing in thermal and 
irradiance regimes. The offshore Acer24 reef experiences lower temperatures and higher 
irradiance than the inshor
e counterpart, Birthday reef. 
Differences in
 
patterns of gene expression 
in the reciprocal transplant allowed us to 
evaluate
 
the role of 
these genes in 
acclimatization 
to 
environmental stress
.
 
 
METHODS
 
In this study, 
Symbiodinium
 
spp. from 
Porites 
astreoid
es
 
reciprocally transplanted from 
an inshore and offshore site in the lower Florida Keys were analyzed for changes in gene 
expression over two years. 
Study
 
sites, coral collection, reciprocal transplant design and sample 
collection for analyses were comple
ted as outlined in Hauff 
et al.
 
(In Press). Briefly, in 
 
38
 
September 2011, ten 16 cm
2
 
fragments of 
Porites astreoides
 
were collected from Birthday reef 
(inshore:
 
24.57917N 
-
81.49693W) and 
an additional ten 16 cm
2
 
fragments were obtained from 
Acer24 reef (offshore:
 
24.55268N 
-
81.43741W)
. Birthday reef and Acer24 reef
 
are patch reefs 
separated by Hawk Channel
 
and have
 
notably distinct temperature and turbidity regimes,
 
but are 
otherwise similar (i.e. depth, species 
diversity
)
.
 
Relative to Acer24 reef,
 
Birthday reef 
has
 
higher 
turbidity
, lower light
 
and higher annual average temperatures (~1°C) (
Haslun 
et al.
, In Review, 
Erich Bartels, pers.com.). 
 
The 20 
Porites astreoides
 
fragments taken from Birthday reef and Acer2
4 reef were 
sectioned into nearly equal halves. These small fragments were approximately 8 cm
2
 
each. One 
small fragment was placed at the site of origin and the other transplanted to the alternate site (i.e. 
fragments from offshore transplanted to inshore 
site) (n=40). Small coral fragments remained at 
each site for a total of two years, but were sub
-
sampled biannually (i.e. February and August of 
2012 and 2013) to capture gene expression during winter and summer conditions. At each sub
-
sampling time, 1 cm
2
 
sub
-
samples were taken with hammer and chisel and flash frozen in liquid 
nitrogen within 15 minutes of initial collection and stored at 
-
80°C.  
 
 
Sub
-
samples from six of the small coral fragments from each treatment within the 
reciprocal transplant experi
ment were randomly chosen for qRT
-
PCR analysis. For example, six 
small fragments of 
P. astreoides
 
collected and replaced at Birthday Reef and sub
-
sampled over 
the four sampling times were analyzed and six small fragments of 
P. astreoides
 
collected at 
Birth
day Reef, transplanted to Acer24 reef, and sampled over the four sampling times, were 
analyzed. Sample analysis for sub
-
samples originating from Acer24 reef was the same. This 
sampling design yielded a total of n=96 sub
-
samples. 
 
 
39
 
RNA extraction and cDNA pr
eparation
 
 
 
Sub
-
samples of 
P. astreoides 
were
 
stored at 
-
80°C and crushed to a fine powder in liquid 
nitrogen using a pre
-
chilled mortar and pestle. 
Between samples, mortar and pestles were 
cleaned with liquid detergent and RNase AWAY
®
 
(Sigma
-
Aldrich, St. Louis MO, USA) and 
rinsed in ultrapure water. 
 
TRI Reagent® (Sigma
-
Aldrich, 
St. Louis MO, USA
) was used to extract RNA from 0.1 g 

two
-

ples were stored at 
-
80°C. Prior to reverse transcription, samples of RNA were 

-
extracted if 

DNase I (Life 
Technologies, San Antonio TX, USA) and reverse transcribed into cDNA following 
manufacturer instructions using the SuperScript
®
 
III First
-
Strand Synthesis SuperMix for qRT
-
PCR (Life Technologies, San Antonio TX, USA). 
 
qRT
-
PCR
 
Fu
lly transcribed cDNA was diluted 1:16 for qRT
-
PCR. Reaction volumes for qRT
-
PCR 
were 10 

L and as follows: 5 

L Power SYBR
®
 
Green PCR Master Mix, 3.5 

L H
2
O, 1

L 
cDNA, 0.5

L primer. Cycling conditions for all reactions were: 50°C
-
2 minutes (1x), 95°C
-
10 
mi
nutes (1x), 95°C
-
15 seconds and 60°C
-
1 minute (40x). Primer information for all
 
genes can be 
found in Table 1.
 
40
 
 
 
Table 1. Genes used in this study, 
abbreviations
, function, primer sequences, melting temperatures and amplicon
 
length for primers 
used for qRT
-
PCR
.
 
41
 
In this study, six zooxanthellae
-
specific genes were analyzed across all sub
-
samples, and 
include one housekeeping gene and five experimental genes (Table 1). The housekeeping gene, 
cytochrome oxidase subunit 1 (
Cox
) h
as previously been identified as a viable housekeeping 
gene in 
Symbiodinium
 
spp. 
(Rosic 
et al.
, 2011b)
. Technical replicates were run in duplicate and 
negative controls were run to confirm no genomic DNA contaminati
on for a total of n=1,152 
reactions. All analyses were completed at the Research Technology Support Facility at Michigan 
State University on an Applied Biosystems® Prism 7900HT (Thermo Scientific, New York, NY 
USA).
 
Data Analysis  
 
All analyses were perfor
med in R (v3.1.3) using the specialized package 
MCMC.qpcr
 
(Matz 
et al.
, 2013
; R Core Team 2014). In this analysis, raw qRT
-
PCR data (i.e. Ct values) is 
represented as molecule counts and described under a Poisson
-
logn
ormal error using generalized 
linear mixed models. There are numerous benefits to this approach. It is fully flexible for all 
levels of random and fixed effects, enables evaluation of unlimited interactions, increases power 
via
 
simultaneous analysis of all
 
genes in one model, accounts for low amplification targets by 
using molecule counts, and eliminates the need for control genes 
(Matz 
et al.
, 2013)
. For this 
study, however, a control gene was used to balance conservatism and the risk of bias, as 
suggested by 
Matz 
et al.
 
(
2013)
.  
 
For analysis, a two
-

-


lted in a model describing the individual effect of each factor, as well 
as the effect of their interaction (i.e. the treatment dependent effect of sampling time). Two 
models were run in order to report all relevant comparisons. The first model used fragme
nts 
 
42
 
originating from, and replaced at, Acer24 reef as a baseline comparison, with the second using 
fragments originating and replaced at Birthday reef for baseline comparison. Additionally, the 

-
PCR data generated here from on
e previously established 
control gene (
Cox
, Table 1, 
Rosic 
et al.
, 2011b)
. Diagnostic plots using the function 
diagnostic.mcmc() were analyzed to confirm linear modeling
 
as an appropriate application for 
this data set. 
 
 
RESULTS
 
There were no significant effects 
in gene expression between winter and summer 
observed, however, significant differences in zooxanthellae
 
gene expression were found between 
sub
-
samples from Acer24 and Birthday reefs (p>0.05). Overall, gene expression from 
zooxanthellae was higher
 
in algae
 
from Acer24 reef than 
those present at 
Birthday reef. Gene 
expression patterns 
observed in zooxanthella
e 
from the original site were retained in the 
symbiont throughout the transplant 
period
 
for both collection reefs. For example, 
Symbiodinium 
spp.
 
from Acer24 reef that were transplanted to Birthday reef displayed gene expression patterns 
similar to 
zooxant
hellae 
that were from, and held, at Acer24 reef.
 
Several genes from zooxanthellae harbored in 
P. 
astreoides
 
sub
-
samples originating from 
Birthday reef exhibited a significant down regulation relative to symbionts originating from 
Acer24 reef. These include
d 
PCP 
(p=0.002), 
PCNA
 
(p=<0.001) and 
psaE
 
(p=0.044) and 
G3PDH
 
(p=0.052) (Figure 7
, Table 2). Relative to zooxanthellae from sub
-
samples originating at Acer24 
reef, 
Symbiodinium 
spp.
 
originating from Birthday reef and transplanted to Acer24 reef exhibited 
significant down
-
regulation of the zooxanthellar genes 
PCP
 
(p<0.001), 
PCNA
 
(p<0.001), 
psaE
 
(p=0.018), and 
G3PDH 
(p=0.024)(Figure 7
, Table 2). 
Relative
 
to zooxanthellae originating at 
 
43
 
Birthday reef, 
Symbiodinium
 
spp. originating at Acer24 reef and transplanted to Birthday reef 
exhibited significant up
-
regulation of zooxanthellar genes 
PCP 
(p<0.001), 
PCNA 
(p<0.001), 
psaE 
(p=0.002), 
G3PDH 
(p<0.001) and 
SCP2 
(p=0.028)(Figure 7
, Table 3). 
 
Sample
 
Gene
 
Post.mean
 
l
-
95% CI
 
u
-
95% CI
 
E
ff.samp  
 
pMCMC
 
 
Acer
Birthday
 
G3PD 
 
0.75786
 
-
0.32954
 
1.81690   
 
1100.1
 
0.182    
 
 
Acer
Birthday
 
PCP
 
 
0.57277
 
-
0.65011  
 
1.58568   
 
1000.0  
 
0.334    
 
 
Acer
Birthday
 
PCNA
 
0.45110
 
-
0.85448  
 
1.74646   
 
1000.0  
 
0.472    
 
 
Acer
Birthday
 
PSAE
 
0.82805
 
-
0.24776  
 
1.92126   
 
1095.1  
 
0.146    
 
 
Acer
Birthday
 
SCP2
 
0.51517
 
-
0.62875  
 
1.57658   
 
1140.2  
 
0.368    
 
 
Acer
Birthday
 
Cox
 
0.29714
 
-
0.81822  
 
1.34287   
 
1000
.0
 
0.598    
 
 
BirthdayAcer
 
G3PD 
 
-
1.27444
 
-
2.32264
 
-
0.17412   
 
1000.0  
 
0.024
 
*
 
BirthdayAcer
 
PCP
 
 
-
1.99936
 
-
3.00025
 
-
0.71156
 
1000.0
 
<0.001
 
*
 
BirthdayAcer
 
PCNA
 
-
2.41345
 
-
3.72389
 
-
1.14470   
 
1000.0
 
<0.001
 
*
 
BirthdayAcer
 
PSAE
 
-
1.33718
 
-
2.43305
 
-
0.23223   
 
1000.0
 
0.018
 
*
 
BirthdayAcer
 
SCP2
 
-
1.04736
 
-
2.08630  
 
0.05666   
 
1000.0
 
0.072
 
 
BirthdayAcer
 
Cox
 
-
1.15290
 
-
2.32132
 
-
0.11005   
 
1000.0
 
0.056
 
 
BirthdayBirthday
 
G3PD 
 
-
1.03375
 
-
2.11422
 
-
0.03793   
 
1024.5  
 
0.052
 
 
BirthdayBirthday
 
PCP
 
 
-
1.79766
 
-
2.94654
 
-
0.74262   
 
1122.8  
 
0.002
 
*
 
BirthdayBirthday
 
PCNA
 
-
2.49322
 
-
3.63201
 
-
1.13299   
 
1000.0
 
<0.001
 
*
 
BirthdayBirthday
 
PSAE
 
-
1.05099
 
-
2.07456
 
-
0.01014   
 
1063.0  
 
0.044
 
*
 
BirthdayBirthday
 
SCP2
 
-
0.80628
 
-
1.87348
 
0.26407   
 
1046.7  
 
0.156    
 
 
BirthdayBirthday
 
Cox
 
-
0.86286
 
-
1.96599  
 
0.21116   
 
1034.0  
 
0.140
    
 
 
Summer
 
G3PD 
 
-
0.42471
 
-
1.44246  
 
0.57653   
 
1000.0
 
0.400    
 
 
Summer
 
PCP
 
 
-
0.96875
 
-
2.08771  
 
0.07127   
 
1000
.0
 
0.076
 
 
Summer
 
PCNA
 
-
0.90811
 
-
2.04225  
 
0.42268   
 
1000
.0
 
0.136    
 
 
Summer
 
PSAE
 
-
0.53284
 
-
1.49807  
 
0.53535   
 
1000
.0
 
0.298    
 
 
Summer
 
SCP2
 
-
0.50555
 
-
1.67745  
 
0.41615   
 
1000
.0
 
0.336    
 
 
Summer
 
Cox
 
-
0.66882
 
-
1.56163  
 
0.42765   
 
1000
.0
 
0.176
 
 
Acer
Birthday:Summer
 
G3PD 
 
-
1.10678
 
-
2.53604  
 
0.23038   
 
1000.0
 
0.146    
 
 
Acer
Birthday:Summer
 
PCP
 
 
-
0.71755
 
-
2.36609  
 
0.71800   
 
1000
.0
 
0.366    
 
 
Acer
Birthday:Summer
 
PCNA
 
-
0.42148
 
-
2.29178  
 
 
1.34468   
 
1102.2  
 
0.660    
 
 
Acer
Birthday:Summer
 
PSAE
 
-
0.88605
 
-
2.41830  
 
0.49638   
 
1000
.0
 
0.258    
 
 
Acer
Birthday:Summer
 
SCP2
 
-
0.73566
 
-
2.13112  
 
0.78400   
 
1000
.0
 
0.346    
 
 
Acer
Birthday
:Summer
 
Cox
 
-
0.15287
 
-
1.43671  
 
1.36536   
 
1000
.0
 
0.860
 
 
BirthdayAcer:Summer
 
G3PD 
 
0.48370
 
-
1.05469  
 
1.93171   
 
1000
.0
 
0.528    
 
 
BirthdayAcer:Summer
 
PCP
 
 
1.17241
 
-
0.36614  
 
2.84003   
 
1000
.0
 
0.162    
 
 
BirthdayAcer:Summer
 
PCNA
 
-
0.44314
 
-
2.25117  
 
1.56216   
 
1000
.0
 
0.648    
 
 
BirthdayAcer:Summer
 
PSAE
 
0.51274
 
-
1.06876  
 
2.00907   
 
1043.4  
 
0.506    
 
 
BirthdayAcer:Summer
 
SCP2
 
0.24851
 
-
1.14452  
 
1.85562   
 
1000
.0
 
0.748    
 
 
BirthdayAcer:Summer
 
Cox
 
0.35344
 
-
1.07449  
 
1.79065   
 
1121.9  
 
0.672    
 
 
BirthdayBirthday:Summer
 
G3PD 
 
-
0.11873
 
-
1.52551  
 
1.26020   
 
1000
.0
 
0.860
 
 
BirthdayBirthday:Summer
 
PCP
 
 
0.28191
 
-
1.21268  
 
1.80808   
 
1000
.0
 
0.704    
 
 
BirthdayBirthday:Summer
 
PCNA
 
0.17796
 
-
1.53111  
 
2.15363   
 
1000
.0
 
0.822    
 
 
BirthdayBirthday:Summer
 
PSAE
 
-
0.06285
 
-
1.50997  
 
1.28971   
 
1000
.0
 
0.940    
 
 
BirthdayBirthday:Summer
 
SCP2
 
-
0.08662
 
-
1.60147  
 
1.34879   
 
1000
.0
 
0.900    
 
 
BirthdayBirthday:Summer
 
Cox
 
-
0.16014
 
-
1.51131  
 
1.16904   
 
1000
.0
 
0.818
 
 
Table 2. 
Output
 
for two
-


as 
fixed 
factors under the MCMC.qpr package for zooxanthellae gene expression from 
sub
samples 
collected at Birthday reef and Acer24 reef. 
Post means are reported as well as lower and upper 
credible intervals, effective sample size and p
-
values.  

-
transplanted 
 
44
 

sub
-


represents 
transplanted 
sub
-
samples that were collected 
at Acer24 reef and transplanted 
to Birthday reef. 

-
transplanted sub
-
samples that 
originated at Birthday reef, 
wh

transplanted sub
-
samples 
that were collected at
 
Birthday reef 
and transp
lanted to Acer24 reef. Baseline 
comparison, or reference factor, included non
-
transplanted sub
-
samples 
from Acer24 reef 
(i.e. AcerAcer) at the winter sampling time. Asterisk 
denotes a p
-
value <0.05
.
 
 
 
Figure 7
. By
-
gene plot of tra
nscript abundance for zooxanthellae genes obtained from sub
-
samples of 
Porites 
astreoides taken from Birthday reef and Acer24 reef during winter and 

-
transplanted sub
-
samples that originated at 
Acer24 reef,
 

-
samples that were collected at 

-
transplanted 
sub
-

 
transplanted 
sub
-
samples that were collected at Birthday reef and transplanted to Acer24 reef. Sub
-
samples 
collected in summer months are represented in orange, while sub
-
samples collected in winter 
months are represented in blue. Whiskers denote 95% cred
ible intervals
.
 
 
 
45
 
In contrast to the cases above, no significant effect of site was found when comparing 
zooxanthellae gene expression from sub
-
samples originating at Acer24 reef and zooxanthellae 
originating from Acer24 reef sub
-
samples transplanted to Bir
thday reef (p>0.05, Table 2). 
Furthermore, gene expression did not vary significantly between zooxanthellae originating from 
Birthday reef sub
-
samples and zooxanthellae originating from Birthday reef sub
-
samples that 
were tran
splanted to Acer24 reef (Table
 
3
).
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
46
 
 
Sample
 
Gene
 
 
Post.mean
 
l
-
95% CI
 
u
-
95% CI
 
E
ff.samp  
 
pMCMC
 
 
AcerAcer
 
G3PD 
 
 
1.21755  
 
0.17023  
 
2.20626   
 
1000
.0
 
0.022
 
*
 
AcerAcer
 
PCP
 
 
 
1.99231  
 
0.96816  
 
3.19196   
 
1000.0  
 
<0.001
 
*
 
AcerAcer
 
PCNA
 
 
2.72957  
 
1.49594  
 
3.84646   
 
1000.0
 
<0.001
 
*
 
AcerAcer
 
PSAE
 
 
1.25442  
 
0.20186  
 
2.25162   
 
1000.0
 
0.022
 
*
 
AcerAcer
 
SCP2
 
 
1.00775
 
-
0.02516  
 
2.11803   
 
1000.0
 
0.080
 
 
AcerAcer
 
Cox
 
 
1.09524  
 
0.05914  
 
2.19882   
 
1000
.0
 
0.056
 
 
AcerBirthday
 
G3PD 
 
 
1.64635  
 
0.54220  
 
2.66718   
 
1000.0  
 
<0.001
 
*
 
AcerBirthday
 
PCP
 
 
 
2.22900  
 
1.03429  
 
3.28904   
 
1000.0
 
<0.001
 
*
 
AcerBirthday
 
PCNA
 
 
2.83415  
 
1.54539  
 
4.01819   
 
1000.0
 
<0.001
 
*
 
AcerBirthday
 
PSAE
 
 
1.74730  
 
0.65014  
 
2.81450   
 
1000.0
 
0.002
 
*
 
AcerBirthday
 
SCP2
 
 
1.18808  
 
0.16333  
 
2.30082   
 
1000.0
 
0.028
 
*
 
AcerBirthday
 
Cox
 
 
0.98921
 
-
0.13981  
 
2.08998    
 
909.6  
 
0
.094
 
 
BirthdayAcer
             
 
G3PD 
 
 
-
0.31557
 
-
1.38858  
 
0.74282   
 
1092.6  
 
0.580    
 
 
BirthdayAcer
 
PCP
 
 
 
-
0.27211
 
-
1.36499  
 
0.85086   
 
1000.0  
 
0.650    
 
 
BirthdayAcer
 
PCNA
 
 
0.04449
 
-
1.31269  
 
1.16807   
 
1136.6  
 
0.934    
 
 
BirthdayAcer
 
PSAE
 
 
-
0.35742
 
-
1.51305  
 
0.65777    
 
943.5  
 
0.518    
 
 
BirthdayAcer
 
SCP2
 
 
-
0.30484
 
-
1.42683  
 
0.71932   
 
1113.7  
  
 
0.600    
 
 
BirthdayAcer
 
Cox
 
 
-
0.41275
 
-
1.52753  
 
0.68653   
 
1098.6  
 
0.444    
 
 
Summer                          
 
G3PD 
 
 
-
0.42119
 
-
1.38320  
 
0.59371   
 
1000.0
 
0.410    
 
 
Summer                         
 
PCP
 
 
 
-
0.56929
 
-
1.60197  
 
0.51766   
 
1000
.0
 
0.300    
 
 
Summer
                         
 
PCNA
 
 
-
0.50935
 
-
1.64950  
 
0.90378   
 
1110.9
 
0.414    
 
 
Summer
 
PSAE
 
 
-
0.47024
 
-
1.42073  
 
0.61740   
 
1107.8  
 
0.370
 
 
Summer
 
SCP2
 
 
-
0.46287
 
-
1.44652  
 
0.61586   
 
1000
.0
 
0.398    
 
 
Summer
 
Cox
 
 
-
0.69225
 
-
1.64933  
 
0.33131   
 
1000
.0
 
0.166    
 
 
Acer
Acer:
Summer
    
 
G3PD 
 
 
-
0.27623
 
-
1.60932  
 
1.10719    
 
933.0  
 
0.716
 
 
Acer
Acer:
Summer    
 
PCP
 
 
 
-
0.67608
 
-
2.14837  
 
0.73583    
 
888.2  
 
0.388    
 
 
Acer
Acer:
Summer    
 
PCNA
 
 
-
0.68121
 
-
2.33178  
 
0.95846   
 
1000
.0
 
0.446    
 
 
Acer
Acer:
Summer    
 
PSAE
 
 
-
0.33763
 
-
1.68846  
 
 
1.13446    
 
921.6  
 
0.654    
 
 
Acer
Acer:
Summer    
 
SCP2
 
 
-
0.32461
 
-
1.65484  
 
1.17265    
 
887.0  
 
0.652    
 
 
Acer
Acer:
Summer    
 
Cox
 
 
-
0.31844
 
-
1.64613  
 
1.04231    
 
959.7  
 
0.658
 
 
AcerBirthday:
Summer    
 
G3PD 
 
 
-
0.98030
 
-
2.38653  
 
0.45556   
 
1000
.0
 
0.182    
 
 
AcerBirthday:
Summer    
 
PCP
 
 
 
-
0.97558
 
-
2.51798  
 
0.46522   
 
1000
.0
 
0.208    
 
 
AcerBirthday:
Summer    
 
PCNA
 
 
-
0.60709
 
-
2.36205  
 
1.04494   
 
1000
.0
 
0.502    
 
 
AcerBirthday:
Summer    
 
PSAE
 
 
-
0.81838
 
-
2.28514  
 
0.60443   
 
1000
.0
 
0.272    
 
 
AcerBirthday:
Summer    
 
SCP2
 
 
-
0.65107
 
-
2.10597  
 
0.78232   
 
1000
.0
 
0.390    
 
 
AcerBirthday:
Summer    
 
Cox
 
 
0.01370
 
-
1.42340  
 
1.33326   
 
1000
.0
 
0.986    
 
 
 
BirthdayAcer
:
Summer    
 
G3PD 
 
 
0.52894
 
-
0.79296  
 
1.88105   
 
1150.7  
 
0.456    
 
 
BirthdayAcer
:
Summer    
 
PCP
 
 
 
0.82061
 
-
0.72111  
 
2.21444   
 
1125.8  
 
0.278
 
 
BirthdayAcer:
Summer    
 
PCNA
 
 
-
0.75939
 
-
2.51604  
 
0.91120   
 
1146.2  
 
0.406    
 
 
BirthdayAcer:
Summer    
 
PSAE
 
 
0.51246
 
-
0.88945  
 
1.90230   
 
1126.0  
 
0.480    
 
 
BirthdayAcer:
Summer    
 
SCP2
 
 
0.25853
 
-
1.00056  
 
1.84046   
 
1000.0
 
0.738    
 
 
BirthdayAcer:
Summer    
 
Cox
 
 
0.46346
 
-
0.91224  
 
1.83777   
 
1125.6
 
0.506    
 
 
Table 3. Output for two
-

MCMC.qpr package for zooxanthellae
 
gene expression from sub
-
samples collected at Birthday 
reef and Acer24 reef. 
Post means are reported as well as lower and upper credible intervals, 
effective sample size and p
-
values.  

-
transplanted sub
-
samples that 
originated at 

-
samples that were 

-
transplanted sub
-


transplanted sub
-
samples that were collected at Birthday reef and transplanted to Acer24 reef. 
 
47
 

Baseline comparison, or reference factor, included non
-
transplanted sub
-
samples from Birthday reef (i.e. BirthdayBirthday) at the 
winter sampling time. Asterisk denotes 
a p
-
value <0.05
.
 
 
For all of the zooxanthellae genes tested throughout all experimental conditions, 
expression was higher in winter, compared to summer, but the effect of season was n
ot 
significant (p>0.07, Figure 7
, 
Tables 2 & 3). In zooxanthellae from Birthday reef sub
-
samples 
transplanted to Acer24 reef,
 
G3PDH
 
and 
PCP 
experienced slight decreased expression in winter 
compared to summer, while 
psaE
 
experienced no change in expression. Finally, the interaction 
between
 
time and transplant was not significant (p>0.1, Table 2 & 3).
 
 
DISCUSSION
 
Gene expression analysis is a powerful tool increasingly utilized to determine responses 
of closely related coral and zooxanthellae populations to stress 
(Rosic 
et al.
, 2010, 2011a; 
McGinley 
et al.
, 2012a; Kenkel 
et al.
, 2014)
. Traditional techniques 
used in the study of stress 
response
s
 
rely on phenotypic variation and disregard 
differences
 
at the genetic level 
(Berkelmans 
& van Oppen, 2006; Middlebrook 
et al.
, 2008; Downs 
et al.
, 2012)
. Molecular stress markers, 
such as differences in 
gene expression, provide unique advantages over
 
visual assessments
. For 
example, molecular markers 
measure
 
changes in stress response
 
prior to the advent of 
phenotypic change 
(DeSalvo 
et al.
, 2008; Rosic 
et al.
, 2011a; Kenkel 
et al.
, 2013)
. 
Coral host 
populations of 
Porites 
astreoides
 
exhibit differential gene expression that has been linked to 
thermal tolerance 
(Kenkel 
e
t al.
, 2013)
. To understand the role of zooxanthellae in bleaching 
susceptibility 
I
 
studied gene expression in 
Symbiodinium
 
spp. from sub
-
samples of 
P. 
astreoides
 
from an inshore and offshore reef, as well as a reciprocal transplant conducted at the sam
e reefs. 
Results demonstrating increased gene expression in zooxanthellae from the offshore reef
 
 
48
 
experiencing more bleaching
 
and no change in gene expression patterns in reciprocal transplants, 
allowed 
me
 
to make inferences regarding the roles of zooxanthe
llae and acclimatization in 
bleaching susceptibility. 
 
 
Differences in zooxanthellae gene expression between 
P. 
astreoides
 
sub
-
samples from 
inshore and offshore sites are
 
indicative of functional variability within zooxanthellae. Elevated 
gene response
s
 
and high
er
 
rate
s
 
of bleaching at Acer24 reef 
relative to inshore 
may be a 
consequence of higher irradiance (Haslun 
et al.
, In Review). Owing to the elevated coral
-
host 
stress response at Acer24 reef, 
I
 
interpreted patterns of zooxanthellae gene expression
 
exhibited 
in Acer24 reef sub
-
samples to reflect bleaching susceptibility at their cellular level. With the 
exception of 
SCP2
 
in zooxanthellae from sub
-
samples originating and retained at Acer24 reef, 
four symbiont genes (
PCNA,
 
G3PDH
,
 
PCP 
and
 
psaE
) display
ed significantly higher expression 
in all offshore Acer24 reef sub
-
samples compared to inshore Birthday reef sub
-
samples (Tables 2 
& 3). These zooxanthellae genes are associated with metabolic processes that increase with 
stress, such as DNA synthesis, lip
id transfer, metabolism and photosynthesis 
(Karako
-
Lampert 
et 
al.
, 2005; Leggat 
et al.
, 2011; McGinley 
et al.
, 2012b)
. Consequently, increased zooxanthellae
 
gene expression observed at Acer24 reef is likely a function of differential bleaching 
susceptibility experienced by the symbiont. 
 
Although normally involved in glycolysis, 
G3PDH
 
expression has been associated
 
with 
apoptosis activation 
(Tarze 
et al.
, 2007)
. Apoptosis, also called programmed cell death, occurs 
during the beginning stages of host coral bleaching 
(Strychar 
et al.
, 2004a, 2004b)
. The 
possibility of increased zooxanthellae 
G3PDH
 
expression signaling symbiont apoptosis and 
subsequent bleaching in coral originating at Acer24 reef is consistent with significantly higher 
levels
 
of bleaching among host coral fragments originating from Acer24 reef relative to those 
 
49
 
originating from Birthday reef (Haslun 
et al.
, In review). As such, this is the first report to 
use
 
zooxanthellae gene expression (i.e 
G3PDH
) 
as a 
precursor of apoptosi
s
 
to document bleaching
; 
G3PDH 
may also be a better marker of
 
symbiont 
stress then 
apoptosis. Documenting 
zooxanthellae
 
gene expression is an important mechanism to better understand symbiosis and 
climate stress in coral. The ease of sampling and analysis broadens the scope of potential 
analyses, as well as our understanding of the role of zooxanthellae apoptosis in host b
leaching. It 
is important to note that as zooxanthellar 
G3PDH
 
expression increases, downstream activation of 
apoptotic processes are stimulated and expressed on the surface of the symbiont cells 
(Strychar 
et 
al.
, 2004b)


(Strychar 
et al.
, 2004a)
. If 
zooxanthellae are being actively expelled from the host 
via
 
phagocytic cells, or other removal 
mechanisms, as indicated by increases in 
G3PDH 
expression, an increase in symbiont replication 
rates to counteract symbiont lo
ss may explain increases in 
PCNA 
and 
SCP2
. 
 
Increased expression of 
PCNA
 
and
 
SCP2
 
in 
zooxanthellae from 
Acer 24 reef suggests 
higher rates of DNA synthesis and lipid transfer/synthesis, processes that occur during 
cell 
replication and division. 
My
 
observation of i
ncreased expression of 
PCNA 
and 
SCP2
 
suggests 
that rates of zooxanthellae replication and division increase
s
 
with elevated stress 
(Suharsono & 
Brown, 1992)
. During a bleaching event 
in which
 
str
ess results in host bleaching, it is plausible 
that the demands placed upon the symbiont 
via
 
the host results in increased zooxanthellar lipid 
metabolism. As a consequence, the symbiont increases lipid use not only as a mechanism to 
increase its own metabo
lism, but also to increase lipid transfer to the host 
(Luo 
et al.
, 2009)
. The 
drawback of increased expression of 
SCP2
, however, is that the symbionts may become deficient 
in peroxisomes, thereby creating 
a negative feedback loop.  Peroxisomes, found in most, if not 
 
50
 
all, eukaryotic cells, play a major role in catabolism of long chain fatty acids 
(Gabaldon, 2010)
. 
As increased zooxanthellae 
SCP2
 
expression is maintained, the symbiont will continue to utilize 
lipid stores for its own metabolism, as well as increased 
translocation 
that benefits the
 
survivorship of the host.  
 
Parallel to increases in 
SCP2
, 
PCNA
 
expression increased in zooxanthellae at Acer24 reef
 
that is indicative of a stress response
. Increased expression of 
PCNA
 
during stress, when DNA 
repair and chromatin remodeling would be most important for symbiont survivorship,
 
could be a 
driver of the development of
 
new zooxanthellae subclade types.  
PCNA
 
is involved in two 
processes of post replication repair 
(Lehman & Fuchs, 2006)
. The first, referred to as the 

via
 
DNA polymerases. The translesion pathway acts as a 
mechanism to incorporate damaged DNA bases into their active sites. In this pathway, 
polymerases bypass damage c

polymerases would stall during this process 
(Lehman & Fuchs, 2006)
. Incorporating damaged 
DNA bases may result in different gene sequences, which may contribute to
 
an increase in 
the
 
number of 
Symbiodinium
 
spp
.
 
subclade type reported 
(LaJeunesse 
et al.
, 2004; Sampayo 
et al.
, 
2008
; Hauff 
et al
. In 
Press
)

attempts to bypass DNA damage, but this process involves homologous machinery (Lehmann 
and Fuchs, 2006).  Regardless of which pathway is chosen, PCNA is a vital mechanism of 
pathway 
activation used by a cell.  
 
Rates of photosynthesis increase in response to moderate stress in cultured 
Symbiodinium 
spp. 
(Iglesias
-
Prieto 
et al.
, 1992)
. The increased expression of 
PCP
 
in Acer24 reef samples is 
consistent with this observation as 
PCP 
is related to light harvesting. The scarcity of carbon 
dioxide (CO
2
) in an aquatic environment results from slow diffusion 
rates coupled with CO
2
 
 
51
 
availability existing mostly as bicarbonate, as well as the non
-
specificity of RuBisCO for CO
2
 
over O
2
 
(Raven, 
1970; Eckardt, 2012)
. To augment RuBisCO efficiency, symbionts increase 
carbon
-
concentrating mechanisms (CCM) when experiencing survivorship problems in a host 
coral (CCMs; 
Price 
et al.
, 2013
, Oakley 
et al.
 
2014
)
. Found in nearly all 
terrestrial 
plants
 
(Jungnick 
et al
. 2014) and a variety of algae (Xu 
et al
. 2012)
, there are two general types of 
CCMs, C4 and Crassulacean
 
Acid Metabolism (CAM) pathways 
(Jungnick 
et al.
, 2014)
. 
Fundamentally, both processes attempt to increase uptake and fixation of CO
2
, concentrating 
carbon around RuBisCO 
(Price 
et al.
, 2013)
. Increased 
PCP 
expression yields increases in light 
energy transfer and therefore, increased carbon availability, boosting potential survivorship of 
the symbiont 
(Reynolds 
et al.
, 2008)
. Hence, it is plausible that 
my
 
observation of increased 
PCP
 
expression under stress is a mechanism used by zooxanthellae to increase CCM. 
 
In addition to increased 
PCP
, 
PsaE
, a protein involved in the stabilization of interactions 
within the photosystem 1 complex (PSI), elevated expression in zooxanthellae from Ac
er24 sub
-
samples
 
relative to those at Birthday reef
. 
PsaE
 
expression increases in the cyanobacterium 
Synechocystis
 
spp. during light stress 
(Jeanjean 
et al.
, 2008)
. 
The
 
increases in 
psaE
 
expressio
n 
counteracts the effects of reactive oxygen species (ROS), whose presence breaks down 
photosystem II machinery 
(Mayfield 
et al.
, 2011)
. ROS production is a common response
 
of
 
Symbiodinium 
spp. to bleaching conditions 
(Lesser, 2006)
. Increases in the
 
expression of 
stabilizing protein
 
psaE
 
in Acer24 reef samples may represent an initial attemp
t to counteract 
degradation of photosynthetic machinery repair as reported in McGinley 
et al.
 
(
2012a)
 
for 
Symbiodinium
 
spp. A13. Increases in the expression of 
psaE
 
also demonstrate the higher 
stressful environment offs
hore. 
 
 
52
 
 
The 
lack of an
 
effect of transplantation and the retention of zooxanthellae gene 
expression patterns from reef of origin, demonstrates that environmental change does not 
influence the expression patterns of zooxanthellae genes investigated in this 
study. If 
acclimatization were an important means of counteracting bleaching susceptibility, zooxanthellae 
gene expression patterns would have likely changed in the transplant experiment. Instead, gene 
expression may be genetically determined by
 
adaptation
, as was also proposed for the coral host 
(Kenkel 
et al.
, 2015)
. The lack of acclimatization demonstrated in gene expression parallels 
previous work demonstrating a lack of change in zooxanthellae subclade type populations 
associated with 
P. a
streoides 
from Acer24 and Birthday reefs in response to reciprocal transplant 
(Hauff 
et al.
, In Review). Differences in zooxanthellae subclade populations seen in Hauff 
et al.
 
(In Review) reflect variation in genetic material for natural selection to act u
pon, and may have 
resulted in locally adapted populations of zooxanthellae at Acer24 and Birthday reefs. 
 
 
To predict how coral reefs will change in response to bleaching induced stress, it is 
important to understand mechanisms involved in bleaching suscep
tibility of zooxanthellae 
associated with scleractinian corals. Although demonstrating a lack of acclimatization to 
environmental change, 
my
 
gene expression results suggest that zooxanthellae associated with 
Porites astreoides
 
in the Florida Keys are functionally variable and 
the lower expression of stress 
response genes relative to the offshore likely reflects better adaptation 
to bleaching susceptibility 
inshore. If zooxanthellae are given time to adapt, 
P. astreoides
 
may res
pond positively in a 
climate change scenario due to r
educed bleaching susceptibility
. 
Additionally, I demonstrated a 
link between 
G3PDH 
gene expression
 
and bleaching, which may be a good bioindicator gene of 
zooxanthellae apoptotic activation. 
F
uture studi
es 
regarding responses of 
P. astreoides 
to heat 
 
53
 
stress should focus on evaluating the response of
 
Symbiodinium
 
spp.
 
gene expression under
 
bleaching temperatures
 
and other synergistic stressors such as disease. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
54
 
 
 
 
 
 
 
 
 
 
 
 
 
LITERATURE 
CITED
 
 
 
 
 
 
 
 
 
 
 
 
55
 
LITERATURE CITED
 
 
Baker C (2003) FLEXIBILITY AND SPECIFICITY IN CORAL
-

Diversity , Ecology , and Biogeography of Symbiodinium. 
34
, 661

689.
 
Bengis C, Nelson N (1977) Subunit structure of chloroplast photosystem I reaction center. 
Journal of Biological Chemistry
, 
252
, 4564

4569.
 
Berkelmans R, van Oppen MJH (2006) The role of zooxanthellae in the thermal tolerance of 

or coral reefs in an era of climate change. 
Proceedings. 
Biological sciences / The Royal Society
, 
273
, 2305

12.
 
Brown BE (1997) Coral bleaching: causes and consequences. 
Coral Reefs
, 
16
, S129

S138.
 
Bruno JF, Selig ER, Casey KS et al. (2007) Thermal stress 
and coral cover as drivers of coral 
disease outbreaks. 
PLoS biology
, 
5
, e124.
 

-
year decline of coral cover on 
the Great Barrier Reef and its causes. 
Proceedings of the National Academy of Sciences
, 
109
, 17995

17999.
 
DeSalvo MK, Voolstra CR, Sunagawa S et al. (2008) Differential gene expression during 
thermal stress and bleaching in the Caribbean coral Montastraea faveolata. 
Molecular 
ecology
, 
17
, 3952

71.
 
Downs C a, Ostrander GK, Rougee L et al. (2
012) The use of cellular diagnostics for identifying 
sub
-
lethal stress in reef corals. 
Ecotoxicology (London, England)
, 
21
, 768

82.
 
Eckardt NA (2012) Gene Regulatory Networks of the Carbon
-
Concentrating Mechanism in 
Chlamydomonas reinhardtii. 
the Plant Cel
l Online
, 
24
, 1713.
 
Gabaldon T (2010) Peroxisome diversity and evolution. 
Philosophical Transactions of the Royal 
Society B: Biological Sciences
, 
365
, 765

773.
 
Gates RD, Baghdasarian G, Muscatine L (1992) Temperature stress causes host cell detachment 
in s
ymbiotic cnidarians: implications for coral bleaching. 
Biological Bulletin
, 
182
, 324

332.
 
Iglesias
-
Prieto R, Matta JL, Robins W a, Trench RK (1992) Photosynthetic response to elevated 
temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. 
Proceedings of the National Academy of Sciences of the United States of Ameri
ca
, 
89
, 
10302

10305.
 
Jackson JBC, Kleypas J, Lough JM et al. (2014) of. 
301
, 929

933.
 
Jeanjean R, Latifi A, Matthijs HCP, Havaux M (2008) The PsaE subunit of photosystem I 
 
56
 
prevents light
-
induced formation of reduced oxygen species in the cyanobacterium 
Syn
echocystis sp. PCC 6803. 
Biochimica et biophysica acta
, 
1777
, 308

16.
 
Jones RJ, Hoegh
-
Guldberg O (2001) Diurnal changes in the photochemical efficiency of the 
symbiotic dinoflagellates (Dinophyceae) of corals: Photoprotection, photoinactivation and 
the rel
ationship to coral bleaching. 
Plant, Cell and Environment
, 
24
, 89

99.
 
Jungnick N, Ma Y, Mukherjee B et al. (2014) The carbon concentrating mechanism in 
Chlamydomonas reinhardtii: Finding the missing pieces. 
Photosynthesis Research
, 
121
, 
159

173.
 
Karako
-
Lam
pert S, Katcoff DJ, Achituv Y, Dubinsky Z, Stambler N (2005) Responses of 
Symbiodinium microadriaticum clade B to different environmental conditions. 
Journal of 
Experimental Marine Biology and Ecology
, 
318
, 11

20.
 
Kenkel CD, Meyer E, Matz M V. (2013) Gene 
expression under chronic heat stress in 
populations of the mustard hill coral ( 
Porites astreoides
 
) from different thermal 
environments. 
Molecular Ecology
, 
22
, 4322

4334.
 
Kenkel CD, Sheridan C, Leal MC et al. (2014) Diagnostic gene expression biomarkers o
f coral 
thermal stress. 
Molecular Ecology Resources
, 
14
, 667

678.
 
Kenkel CD, Setta SP, Matz M V (2015) Heritable differences in fitness
-
related traits among 
populations of the mustard hill coral, Porites astreoides. 
Heredity
, 1

8.
 
LaJeunesse T, Thornhill D, Cox E, Stanton F, Fitt W, Schmidt G (2004) High diversity and host 
specificity observed among symbiotic dinoflagellates in reef coral communities from 
Hawaii. 
Coral Reefs
, 596

603.
 
Leggat W, Seneca F, Wasmund K, Ukani L, Yellowle
es D, Ainsworth TD (2011) Differential 
responses of the coral host and their algal symbiont to thermal stress. 
PloS one
, 
6
, e26687.
 
Lehman AR, Fuchs RP (2006) Gaps and forks in DNA replication: Rediscovering old models. 
DNA Repair
, 
5
, 1495

1498.
 
Lesser MP 
(2006) OXIDATIVE STRESS IN MARINE ENVIRONMENTS: Biochemistry and 
Physiological Ecology. 
Annual Review of Physiology
, 
68
, 253

278.
 
Luo Y
-
J, Wang L
-
H, Chen W
-
NU et al. (2009) Ratiometric imaging of gastrodermal lipid bodies 
in coral

dinoflagellate endosymbiosis. 
Coral Reefs
, 
28
, 289

301.
 
Matz M V, Wright RM, Scott JG (2013) No control genes required: Bayesian analysis of qRT
-
PCR data. 
PloS one
, 
8
, e71448.
 
Mayfield AB, Wang L
-
H, Tang P
-
C, Fan T
-
Y, Hsiao Y
-
Y, Tsai C
-
L, Chen C
-
S (2011) Assessing 
the impacts of experimentally elevated temperature on the biological composition and 
 
57
 
molecular chaperone gene expression of a reef coral. 
PloS one
, 
6
, e26529.
 
McGinley MP, Aschaffenburg MD, Pettay DT, Smith RT, LaJeunesse TC, Warner ME (2012a) 
Transcriptional response of two core photosystem genes in Symbiodinium spp. exposed to 
thermal stress. 
PloS one
, 
7
, e50439.
 
McGinley MP, Aschaffenburg MD, Pet
tay DT, Smith RT, LaJeunesse TC, Warner ME (2012b) 
Transcriptional Response of Two Core Photosystem Genes in Symbiodinium spp. Exposed 
to Thermal Stress. 
Plos One
, 
7
, e50439.
 
Middlebrook R, Hoegh
-
Guldberg O, Leggat W (2008) The effect of thermal history on
 
the 
susceptibility of reef
-
building corals to thermal stress. 
The Journal of experimental biology
, 
211
, 1050

6.
 
Muscatine L, Falkowski PG, Dubinsky Z, Cook P a., McCloskey LR (1989) The Effect of 
External Nutrient Resources on the Population Dynamics of Z
ooxanthellae in a Reef Coral. 
Proceedings of the Royal Society B: Biological Sciences
, 
236
, 311

324.
 
Norris BJ, Miller DJ (1994) Nucleotide sequence of a cDNA clone encoding the precursor of the 
peridinin
-
chlorophyll a
-
binding protein from the dinoflagella
te Symbiodinium sp. 
Plant 
molecular biology
, 
24
, 673

7.
 
Prelich G, Tan CK, Kostura M, Mathews MB, So  a G, Downey KM, Stillman B (1987) 
Functional identity of proliferating cell nuclear antigen and a DNA polymerase
-
delta 
auxiliary protein. 
Nature
, 
326
, 517

520.
 
Price GD, Pehgelly JJ, Forster B et al. (2013) The cyanobacterial CCM as a source of genes for 
improving photosynthetic CO2 fixation in crop species. 
Journal of Experimental Botany
, 
63
, 753

768.
 
Raven JA (1970) Exogenous inorganic carbon sources in p
lant photosynthesis. 
Biological 
Review
, 
45
, 167

221.
 
Reynolds JM, Bruns BU, Fitt WK, Schmidt GW (2008) Enhanced photoprotection pathways in 
symbiotic dinoflagellates of shallow
-
water corals and other cnidarians. 
Proceedings of the 
National Academy of Scien
ces of the United States of America
, 
105
, 13674

13678.
 
Rodrigues LJ, Grottoli AG (2007) Energy reserves and metabolism as indicators of coral 
recovery from bleaching. 
Limnology and Oceanography
, 
52
, 1874

1882.
 
Rosic NN, Pernice M, Dunn S, Dove S, Hoegh
-
Guldberg O (2010) Differential regulation by 
heat stress of novel cytochrome P450 genes from the dinoflagellate symbionts of reef
-
building corals. 
Applied and environmental microbiology
, 
76
, 2823

9.
 
Rosic NN, Pern
ice M, Dove S, Dunn S, Hoegh
-
Guldberg O (2011a) Gene expression profiles of 
cytosolic heat shock proteins Hsp70 and Hsp90 from symbiotic dinoflagellates in response 
 
58
 
to thermal stress: possible implications for coral bleaching. 
Cell stress & chaperones
, 
16
,
 
69

80.
 
Rosic NN, Pernice M, Rodriguez
-
Lanetty M, Hoegh
-
Guldberg O (2011b) Validation of 
housekeeping genes for gene expression studies in Symbiodinium exposed to thermal and 
light stress. 
Marine biotechnology (New York, N.Y.)
, 
13
, 355

65.
 
Rowan R (2004) b
rief communications Thermal adaptation in. 
Nature
, 
430
, 742.
 
Sampayo EM, Ridgway T, Bongaerts P, Hoegh
-
Guldberg O (2008) Bleaching susceptibility and 
mortality of corals are determined by fine
-
scale differences in symbiont type. 
Proceedings 
of the National
 
Academy of Sciences of the United States of America
, 
105
, 10444

9.
 
Sirover M a. (1997) Role of the glycolytic protein, glyceraldehyde
-
3
-
phosphate dehydrogenase, 
in normal cell function and in cell pathology. 
Journal of Cellular Biochemistry
, 
66
, 133

140.
 
Strychar KB, Coates M, Sammarco PW, Piva TJ (2004a) Bleaching as a pathogenic response in 
scleractinian corals, evidenced by high concentrations of apoptotic and necrotic 
zooxanthellae. 
Journal of Experimental Marine Biology and Ecology
, 
304
, 99

121.
 
Stryc
har KB, Sammarco PW, Piva TJ (2004b) Apoptotic and necrotic stages of Symbiodinium 
(Dinophyceae) cell death activity: bleaching of soft and scleractinian corals. 
Phycologia
, 
43
, 
768

777.
 
Suharsono, Brown BE (1992) Comparative Measurements of Mitotic Index 
in Zooxanthellae 
From a Symbiotic Cnidarian Subject To Temperature Increase. 
Journal of Experimental 
Marine Biology and Ecology
, 
158
, 179

188.
 

reproduction of the reef coral Montastrea annularis. 
Coral Reefs
, 
8
, 217

224.
 
Tarze  a, Deniaud A, Le Bras M et al. (2007) GAPDH, a novel regulator of the pro
-
apopt
otic 
mitochondrial membrane permeabilization. 
Oncogene
, 
26
, 2606

2620.
 
Thoma S, Kaneko Y (1991) A non
-
specific lipid transfer protein from. 
Plant Journal
, 
3
, 427

436.
 
 
 
 
 
 
 
59
 
CHAPTER 3
 
 
FUNCTIONALLY VARIABLE 
SYMBIODINIUM 
SPP. DISPLAY SIMILAR REPONSES TO 
BLEACHING AND DISEASE STRESS IN THE LOWER FLORIDA KEYS
 
 
ABSTRACT 
 

bleaching and disease. Recent variation in susceptibility to moderate bleaching stress has been 
rep
orted for symbiotic algae (
Symbiodinium
 
spp.) of the coral 
Porties astreoides
 
between inshore 
and offshore reefs in the Florida Keys. Continued survival of 
P. astreoides
 
depends on the ability 
of the coral to survive extreme bleaching events and synergisti
c disease stress.  
I
 
conducted a 
laboratory experiment to investigate the influence of acute high temperature (32°C for eight 
hours) and disease (lipopolysaccharide of 
Serratia marcescens
) on five metabolically related 
genes of 
Symbiodinium
 
spp. from insho
re and offshore 
P. 
astreoides
. Gene expression did not 
differ between reefs, or as a consequence of acute exposure to heat or heat and disease. This 
contrasts to results from a separate field study that showed differences in expression of several of 
the same genes between the same ins
hore and offshore reefs. Several factors may account for the 
lack of variation in gene expression such as zooxanthellae response to moderate chronic versus 
acute stress, host protection and/or the targeting of zooxanthellae by 
S.
 
marcescens
. Patterns 
repor
ted here imply that functional variation in zooxanthellae observed under conditions of 
chronic moderate stress is lost under
 
the
 
acute extreme conditions studied here. 
 
 
INTRODUCTION
 
Scleractinian coral have recently experienced up to 80% mortality in the 
Florida Keys 
(Ruzicka 
et al.
, 2013)
. Coral bleaching and disease are the leading stressors threatening the 
mortality of coral reef
s 
(Harvell 
et al.
, 2002)
. Recent studies show that coral symbiont clade 
 
60
 
types (
Symbiodinium
 
spp.), also known as zooxanthellae, vary in susceptibility to bleaching and 
disease stress 
(Sampayo 
et al.
, 2008; Hauff 
et al.
, 2014
;
 
Hauff 
et al.
,
 
In Review). However, these 
studies assessed stress responses of zooxanthellae in culture, or under conditions of chronic 
moderate stress. Ch
ronic moderate stress refers to elevated levels of a stressor experienced over a 
lengthy period of time (i.e. weeks or months). Chronic moderate stress will produce a response 
such as bleaching, but may not result in death
 
(Dikou & van Woesik, 2006)
.
 
Acute, extreme 
stress is often associated with short periods of drastically elevated temperatures, but may also be 
related to extremes in irradiance, disease, or the synergistic effect of two or more environmental 
variables 
(Lirman 
et al.
, 2011
)
.
 
Herein, 
I
 
differentiate chronic and acute stress on the basis of 
time. Importantly, the continued success of coral reefs is likely dependent on the ability of 
zooxanthellae to survive acute extreme stress and the synergistic effects of bleaching and 
disease 
(Hauff 
et al
.
, 2014; Palumbi 
et al.
, 2014)
. 
 
Coral bleaching has contributed to 
considerable 
declines in coral cover in the Florida 
Keys 
(Ruzi
cka 
et al.
, 2013)
. Bleaching is modestly defined as the expulsion of zooxanthellae 
from the host gastrodermal cavity 
resulting in the exposure of the white coral skeleton 
(Gates 
et 
al.
, 1992)
. It can result in mortality 
and i
s tightly linked to elevated
 
seawater temperatures 
(Goreau & Hayes, 1994)
, but can also be promoted by other stressors such as irradiance and 
sedimentation 
(Brown, 1997)
.
 
In addition to bleaching, disease 
plays
 
a large role in the mortality 
of coral in t
he Florida Keys. 
Increased
 
disease related mortality is a function of both the number 
of coral species with diseases and the frequency of diseased reefs 
(Porter 
et al.
, 2001)
.
 
A
croporid 
serratosis
 
refers to 
White Pox Disease 
specifically 
caused by 
Serratia marcescens
 
(Patterson 
et 
al.
, 2002)
. 
S.
 
marcescens 
is 
derived from 
the
 
gut
 
of animals, includin
g humans,
 
and
 
was 
 
61
 
identified
 
in wastewater
 
of the Florida Keys
 
(Sutherland 
et al.
, 2011)
. Acroporid se
rratosis
 
has 
caused
 
a 70% loss of 
Acropora palmata
 
species in the Florida Keys 
(Patterson 
et al.
, 2002)
. 
 
 
In the Florida Keys, higher rates of bleaching have been observed in offshore 
Porites 
astreoides
 
relative to their inshore counterparts (Haslun 
et al.
, In Review). The expression of 
metabolically related zooxanthellae genes was also higher in the offshore reef (Hauff 
et al.
, 
Chapter 2
). Both of these results likely reflect a response to chronic moderate stress. 
To my
 
knowledge, the effects of 
disease
 
on
 
the expression of zooxanthellae genes have not been 
described
 
previously
. 
 
In order to determine the synergistic effects of 
heat
 
and disease on functionally variant 
populations of zooxanthellae, 
I
 
measured the expression of 
five
 
metabolically related 
Symbiodinium 
spp. 
genes, 
Cox
, 
G3PDH
, 
SCP2
, 
PCP
 
and 
psaE
 
to bleaching temperatures and 
the 
combination of bleaching temperatures and 
lipopolysaccharide (LPS) from 
S. 
marcescens
. 
Lipopolysaccharide are large 
molecules found on the outer membrane of gram
-
nega
tive bacteria 
(Raetz & Whitfield, 2002)
. When present, LPS
 
elicit
s a strong immune response
 
in infected 
organisms 
(Maverakis 
et al.
, 2015)
. 
Conducting a laboratory study to determine 
the effects of 
acute (8
-
hour) heat and disease stress 
allowed 
me
 
to 
assess the response
 
of 
zooxanthellae
 
to
 
acute
 
bleaching and dise
ase.
  
Variation in the response 
of zooxanthellae 
to acute stress 
in the 
laboratory 
compared to 
the 
chronic moderate stress experienced in the field allowed us to make 
inferences of the role of the coral host in response to acute stress. 
 
 
 
METHODS
 
 
Coral 
Collection and Preparation
 
 
62
 
 
My
 
laboratory experiment involved fragments of 
Porites astreoides
 
associated with a 
two
-
year field experiment conducted at 
off
shore
 
Acer24 reef (
24.55268 N 
-
81.43741 W) and 
inshore Birthday reef (24.57917
 
N 
-
81.49693
 
W)
 
(Permit 
# FKNMS
-
2011
-
107)
 
(Haslun 
et al.
, In 
Review; Hauff 
et al.,
 
In Review)
.
 
The two reefs experience distinct temperature and turbidity 
regimes. Birthday reef experiences higher mean average annual temperatures (~1°C) and 
turbidity than Acer24 reef. However, re
lative to Birthday reef, there is more bleaching at Acer24 
reef, likely due to 
greater levels of 
irradiance (Haslun 
et al.,
 
In Review). For field experiments, 
16x16 cm
2
 
fragments of 
P. astreoides
 
were sampled at each reef and sectioned into equal halves. 
Each half was either replaced at the reef of origin or transplanted to the counterpart reef. At the 
end of the field experiment, 
P. astreoides
 
fragments originating and replaced at Birthday (n=6) 
and Acer24 (n=6) reef were brought back to Mote Marine Tropical Laboratory (MMTL). Upon 
arrival, all 12 fragments were sectioned again and allowed to recover for 72 hours in a shaded 
flow through water table prior to use in the laboratory experiment discus
sed herein. 
 
 
For the current experiment, 
P. astreoides 
fragments were sectioned into nine 2.5 cm
2
 
fragments for a total of 108 samples (54 per site). The nine pieces resulting from each of the 12 
fragments were used to accommodate a laboratory design cons
isting of three fragments for 
controls and three fragments for each of two treatment levels described below.
 
 
Temperature and Bacteria Experiments
 
 
Two treatment levels and one control were used in experiments: a 
temperature 
level 
known to cause bleaching
 

in which
 
both 
the 
bleaching 
temper
ature
 
and 
S. mars
escens
 
lipopolysaccharide 
were applied 

(Sigma Aldrich, St. Louis MO, USA)
; the latter application was induced 
to mimic the onset of 
 
63
 
disease. Purified 
LPS is a common reagent used to elicit immune responses in human and animal 
studies 
(Maverakis 
et al.
, 2015)
. 
I
 
chose to use purified LPS instead of 
S. mars
escens
 
colonies to 
minimize 
potential 
cross contamination
 
of 
S. marsescens 
into the environment
, as MMTL is 
located directly adjacent to a public waterway. Additionally, LPS was not added to flow
-
through 
sections of the experimental design, see below. Control fr
agments were maintained in 
experimental tanks at ambient temperature (28°C) without exposure to LPS. 
 
Experiments were conducted by placing coral fragments in custom made acrylic boxes 
(2.5 x 7.5 x 7.5 cm) set within 38
-
liter fish tanks. The acrylic boxes 
included three distinct 
subdivisions each of which housed a single coral fragment. Because each subdivision was 
isolated from one another, individual coral fragments were entirely independent. Six 38
-
liter 
tanks containing five acrylic boxes each were used
 
in the experiment. Because each acrylic box 
contained three subdivisions, this resulted in 90 experimental units. The experimental designs for 
the six tanks included two controls with a total of 30 experimental units and two tanks for each 
of the two trea
tments, with 30 experimental units for each. The six 38
-
liter tanks were filled with 
artificial sterile seawater (ASSW, Instant Ocean

) and placed in flow 
through water tables. Water tables were 680 liter, fiberglass troughs with seawat
er pumped 
through 
via
 
the adjacent 
waterway to aid in temperature maintenance
.
   
 
Coral fragments were rinsed in ASSW prior to placement in acrylic box subsections. 
ASSW held within the tanks was circulated 
via 
pumps inside the tanks but external to acryli
c 
boxes. Heaters placed external to the acrylic box maintained temperatures characteristic of 
bleaching (32
°
C
±
0.5) and were monitored with HOBO

Bourne MA, USA)
. For the disease response treatment, 
LPS from 
S. mars
escens
 
(1 mL, 5 

g/mL) 
was added using disposable plastic pipettes. Additionally, 1 mL of ASSW was added to 
 
64
 
the heat treatment and control tanks for consistency.
 
 
Coral fragments were exposed to treatments for eight hours.  Within that period of time, 
individual
 
subdivisions were aerated each hour 
via 
air infusion using disposable pipettes. Water 
was aerated (C. Page MMTL, per. obs.) taking care not to disturb the fragments. At the end of 
each experiment, coral fragments were removed from their individual tanks u
sing sterile tongs, 
wrapped in combusted foil and placed in freezer bags. Coral fragments were then immediately 
flash frozen in liquid nitrogen and stored at 
-
80
°C until processing. 
 
 
RNA Extraction, qRT
-
PCR and Data Analysis
 
 
RNA extractions, cDNA prepara
tions and qRT
-
PCR were conducted as outlined in Hauff 
et al.
, (In Review). Gene expression of zooxanthellae genes 
Cox
, 
PCNA
, 
SCP2
, 
G3PDH
, 
PCP
 
and 
psaE
 
were evaluated in each control and treatment coral fragment. 
The
se were the same 
genes used in 
zooxanthellae gene expression studies by Hauff 
et al.
,
 
(In Review). Gene and 
primer information may be found in Hauff 
et al.,
 
(In Review)
 
(Table 1). Technical replicates 
were analyzed in duplicate and negative controls confirmed the absence of genomic DNA 
contamination. This yielded a total of n=1,296 analyses.
 
 
Statistical analyses were performed in R (v3.1.3) using the specialized package 
MCMC.qpcr
 
(Matz 
et al.
, 2013
; R Core Team 2014).  Within this package, raw qRT
-
PCR 
data is 
converted to molecule counts. Counts are then described under a Poisson
-
lognormal error using 
generalized linear mixed models.
 
This approach was used as it allows any number of random and 
fixed effects, accounts for low amplification, increases
 
model power by analyzing all genes 
simultaneously in one model, and allows for analysis without the use of housekeeping genes 
 
65
 
(Matz 
et al.
, 2013)
.  The addition of a housekeeping gene 
PCNA 
however, decreases the risk of 
bias, and therefore, was used here. 
 
A 
two
-


This resulted in a model describing the individual effects of each fac
tor, in addition to the 
interaction effect (i.e. the site dependent effect of treatment). 
I
 
ran three Markov Chain Monte 
Carlo chains for 25,000 iterations with the first 4,000 discarded as burn
-
in. The model was 

-
PCR data generated her
e from one previously established housekeeping 
gene (
PCNA
, 
McGinley 
et al.
, 2012)
. Diagnostic plots using the function diagnostic.mcmc(
) 
were analyzed to confirm linear modeling as an appropriate application for this data set. 
 
 
RESULTS
 
No significant effect of site or treatment was found on the expression of 
Cox
, 
G3PDH
, 
SCP2
, 
PCP
 
or 
psaE
 
between Acer24 and Birthday reef zooxanthellae
 
associated with the 
coral 
fragments (p>0.05, Table 4
). For example, gene expression of 
Cox
 
was not different between 
Acer24 and Birthday reef fragments or between control and experimental treatments from the 
same site. The interaction of site and treatmen
t also did not have a significant effect on 
zooxanthellae expression (p>0.05, Table1). For instance, the gene expression of 
Cox
 
in heat
-
stressed 
Symbiodinium
 
spp. from Acer24 reef was not different from the expression of 
Cox
 
heat
-
stressed zooxanthellae fro
m Birthday reef. Pair
-
wise comparisons showed no significant 
differences in gene expressio
n among treatments and controls. 
The expression of 
Cox
 
in 
zooxanthellae harbored in control fragments from Birthday reef was not significantly different 
 
66
 
from the expr
ession of 
Cox
 
in zooxanthellae harbored in fragments from Birthday reef exposed 
to heat or heat+LPS treatments (p>0.05). 
 
 
 
Treatment
 
 
Gene
 
Post.mean
 
l
-
95% CI
 
u
-
95% CI
 
Eff.samp
 
pMCMC
 
Reef    
 
 
Cox 
 
2.187638
 
-
0.139690  
 
4.479140   
 
1000.0  
 
0.068
 
Reef    
 
 
G3PDH
 
0.504446
 
-
0.644153  
 
1.417098   
 
1000.0  
 
0.334    
 
Reef    
 
 
PCP
 
0.245573
 
-
0.812485  
 
1.259310   
 
1000.0  
 
0.636    
 
Reef    
 
 
PSAE    
 
0.038782
 
-
0.910168  
 
1.093089   
 
1000.0  
 
0.914    
 
Reef    
 
 
SCP2
 
1.174183
 
-
0.081910  
 
2.613642   
 
1000.0  
 
0.102    
 
Reef    
 
 
PCNA   
 
0.353228
 
-
0.635999  
 
1.392324   
 
1000.0  
 
0.538    
 
Heat
 
 
Cox 
 
0.631882
 
-
1.126961  
 
2.276879   
 
1000.0  
 
0.452    
 
Heat
 
 
G3PDH
 
0.370865
 
-
0.632919  
 
1.328137   
 
1000.0  
 
0.466    
 
Heat
 
 
PCP
 
0.410617
 
-
0.656092  
 
1.311776   
 
1000.0  
 
0.430    
 
Heat
 
 
PSAE    
 
0.418968
 
-
0.651622  
 
1.390965   
 
1000.0  
 
0.414    
 
Heat
 
 
SCP2
 
0.603772
 
-
0.547622  
 
1.572087   
 
1000.0  
 
0.300    
 
Heat
 
 
PCNA   
 
0.024704
 
-
0.753097  
 
0.877509   
 
1000.0  
 
0.980    
 
LPS
 
 
Cox 
 
0.007293
 
-
1.872295  
 
1.749692    
 
898.7  
 
0.986    
 
LPS
 
 
G3PDH
 
0.345562
 
-
0.654212  
 
1.385999   
 
1000.0  
 
0.520    
 
LPS
 
 
PCP
 
0.613552
 
-
0.434824  
 
1.643339    
 
798.5  
 
0.262    
 
LPS
 
 
PSAE    
 
0.556251
 
-
0.492400  
 
1.607295    
 
799.0  
 
0.314    
 
LPS
 
 
SCP2
 
0.223214
 
-
0.903428  
 
1.304645   
 
1000.0  
 
0.686    
 
LPS
 
 
PCNA   
 
-
0.001164
 
-
0.869460  
 
0.914153   
 
1000.0  
 
0.982    
 
Reef+Heat
 
 
Cox 
 
-
1.832442
 
-
4.065119  
 
0.415856    
 
872.3  
 
0.094
 
Reef+Heat
 
 
G3PDH
 
-
0.088287
 
-
1.412416  
 
1.204037   
 
1000.0  
 
0.904    
 
Reef+Heat
 
 
PCP
 
-
0.061582
 
-
1.457569  
 
1.138234    
 
709.4  
 
0.916    
 
Reef+Heat
 
 
PSAE    
 
0.009594
 
-
1.246905  
 
1.411503    
 
732.4  
 
0.994    
 
Reef+Heat
 
 
SCP2
 
-
0.163576
 
-
1.403211  
 
1.327639   
 
1000.0  
 
0.816    
 
Reef+Heat
 
 
PCNA   
 
-
0.145029
 
-
1.095284  
 
0.821031    
 
867.0  
 
0.768    
 
Reef+LPS
 
 
Cox 
 
-
0.438337
 
-
2.817670  
 
1.779693   
 
1000.0  
 
0.712    
 
Reef+LPS
 
 
G3PDH
 
-
0.110682
 
-
1.423377  
 
1.182371   
 
1000.0  
 
0.848    
 
Reef+LPS
 
 
PCP
 
-
0.414284
 
-
1.808073  
 
0.857795   
 
1000.0  
 
0.526    
 
Reef+LPS
 
 
PSAE    
 
-
0.086955
 
-
1.474356  
 
1.181806   
 
1000.0  
 
0.886    
 
Reef+LPS
 
 
SCP2
 
0.069792
 
-
1.298356  
 
1.489236   
 
1000.0  
 
0.918    
 
Reef+LPS
 
 
PCNA   
 
-
0.126965
 
-
0.963841  
 
0.885971   
 
1121.8  
 
0.770    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 4. Two
-
way model for an experiment testing the effects of reef, heat and heat+LPS on the 
gene expression of 
Cox
, 
G3PDH
, 
PCP
, 
psaE
, 
SCP2
 
and 
PCNA 
under the MCMC.qpr package. 
Post means are reported as well as lower and upper credible intervals, effect
ive sample size and 
p
-

PCNA 
as a housekeeping gene and run with 25,000 
iterations, with the first 4,000 discarded as burn
-
in. Coral colony individual was used as a 
random factor. Reef fragments were from Acer24 or Bir
thday reef and exposed to 28°C. Heat 
treatments were fragments from both reefs exposed to 32°C, and heat+LPS treatments were 
fragments from both reefs exposed to 32°C+LPS from 
Serratia marcescens
. Asterisk denotes a 
p
-
value <0.05
.
 
 
 
67
 
Although not significant
, t
he 
cumulative
 
effect of heat stress on the expression of each of 
these genes generally resulted in an increase in 
gene expression
 
through time.  For some genes 
(e.g. 
Cox
), an increase in temperature resulted in a decrease in expression (Figure
 
8
).  The only 
other time a decrease in gene expression was observed was when 
PCP
 
and 
PSAE
 
were exposed 
to the heat+LPS treatment.  In addition, depending upon treatment, the stress resulted in a 2
-
fold 
increase in expression
 
relative to the control
 
(e.g. 
Co
x
, 
SCP2
; Figure
 
8
).
 
 
Figur
e 8
: By
-
gene plot of normalized log
2
-
transformed expression values (±SEM) of 
experimental genes from all samples. 
Orange
 
lines represent fragments exposed to control 
treatments (28°C), 
green
 
lines represent fragments exposed to h
eat treatments (32°C) and 
blue 
lines
 
represent fragments exposed to heat+LPS treatments (32°C+LPS from 
Serratia marcescens
 
). Whiskers denote 95% credible intervals.
 
 
 
68
 
DISCUSSION
 
The continued success of coral reefs will likely be determined by the ability of 
coral
-
zooxanthellae 
association
 
to survive exposure to chronic moderate stress and acute extreme 
bleaching events 
(Sampayo 
et al.
, 2008; Palumbi 
et al.
, 2014)
. While chronic moderate stress is 
commonly experience
d by zooxanthellae on reefs in the Florida Keys 
(Ruzicka 
et al.
, 2013
; 
Haslun 
et al.
, In Review) acute stress is a more transient phenomenon poorly described in the 
literature. Most studies report 
a
 
response of the coral host and imply 
that 
such effects
 
also impact
 
the
 
metabolism of the
 
symbionts 
(Barshis 
et al.
, 2013; Kenkel 
et al.
, 2015)
. In a complimentary 
field study, 
I
 
showed bleaching and elevated expression of zooxanthellar metabolic genes in 
response to chronic moderate stress at offshore Acer24 reef (Hauff 
et al.,
 
In Review). To 
evaluate the influence of acute stress on zooxanthellae gene response 
I
 
exposed coral from 
Acer24 and Birthday reef to a high temperature (32°C) stress, and high temperature combined 
with LPS from 
S. mars
escens
; a treatment that represents
 
a primary stress coupled 
with
 
a 
secondary stress (i.e. disease).
 
 
 
Although 
my
 
data was
 
not statistically significant, cumulative trends demonstrate 
increased expression of five of the six zooxanthellar genes 
relative to the experimental control 
in 

-

 
reaction 
in which
 
cumulative gene expression increases one or two
-
fold, increasing 
photosynthetic capacity (i.e. 
PCP
 
and 
psaE
 
increases
, Figure 8
). Up regulation of photosynthetic 
genes is likely a mechanism to amplify zooxanthellae metabolism (i.e. incre
ased 
G3PDH
, Figure 
8
).  At a cellular level, the consequence of increased metabolism is likely an increase in symbiont 
DNA synthesis, as well as DNA repair due to mutations 
(Rui
z
-
J
ones & Palumbi, 2015)
. 
Increased DNA synthesis was observed in this study 
via
 
an overall trend of increased 
PCNA
 
 
69
 
concentration
 
(Figure 8
). As metabolic activity increases, 
Symbiodinium
 
spp. also augment
 
their 
ability to transfer lipids (i.e. the increase in lipid
-
transfer protein 
SCP2
, Figure
 
8
).  
My 
expectation is
 
that as photosynthetic machinery ramps
-
up its production of photosynthate (i.e. 
lipids), lipid products need to be used or transferred to the
 
host. 
SCP2
 
is likely a mechanism to 
increase lipid transport to the host coral.  
 
Although 
I
 
observed a general cumulative increase in metabolic activity in zooxanthellae, 
it is possible that the algae are less stressed (i.e. non
-
significant gene expressi
on) than the host in 
laboratory conditions experienced here.  This result is perplexing as significant changes in algal 
gene expression were measured in the companion field study, even though summer temperatures 
and irradiance in the field study were not o
utside the norm for these reefs (Hauff 
et al.
, In 
Review, Haslun 
et al.,
 
In Review). As elevated temperatures and irradiance persisted for several 
weeks in the field, perhaps these environmental conditions manifested themselves as a chronic 
stress. 
I
 
recom
mend that future studies pay close attention to subtle changes in temperature, even 
over a short duration. 
I
 
suspect that zooxanthellae genes respond differently under acute and 
chronic stress, with this difference contributing to dissimilarities reported 
in the current and field 
experiment. As such, the implications to the dynamics of symbiosis may be considerable.  
 
Contrary to chronic stress and variable gene expression observed from samples collected 
in the field in Hauff 
et al.
 
(In Review), there were 
no significant differences in zooxanthellae 
gene expression between Acer24 and Birthday reef fragments exposed to high temperature as the 
primary stressor, and disease as the secondary stressor.  However, 
I
 
did observe a cumulative 
increase in expression o
f zooxanthellae genes. For instance, the only symbiont genes showing 
decreased expression with the addition of disease were 
PCP
 
and 
PSAE
 
-
 
which are both related 
to photosynthesis.  
I
 
viewed this decrease as peculiar as it would seem natural to increase 
 
70
 
me
tabolism as a mechanism to counteract disease.  Instead, however, the opposite occurred.  
I
 

 
The evolution of plant and pathogen is a dynamic interaction where
by
 
pathogens evolve 
to maximize nutrient intake, ensuring their future success 
(Berger 
et al.
, 2007)
. In this context, 
the synthesis of an energy source 

 
photosynthate 

 
and its availability for use, is a competition 
between organisms 
(Selvaraj & Fofana, 2012)
, 
in which
 
a pathogen will often reprogram a plants 
metabolism to suit its own needs 
(Biemelt & Sonnewald, 2006)
. In 
this
 
case, 
the application of
 
LPS 
mimicked
 
a pathogen
 
attack
, 
I
 
believe that the zooxanthellae responded by undergoing a 

(i.e. 
Symbiodinium
 
spp.) down
-
regulated 
photosynthesis as a mechanism to starve the pathogen 
(Selvaraj & Fofana, 2012)
. This is a 
common occurrence in 
terrestrial plants 
(Kocal 
et al.
, 2008)
, but poorly described in aquatic 
habitats. Although not shown in this study Cervino 
et al.
 
(2012)
 
demonstrated decreased 
photosystem II quantum yields under disease stress, supporting 
my
 
speculations.  
 
Garavaglia 
et al.
 
(
2010)
 
describes rep
rogramming of a hosts metabolism as a first step in 
a pathogens attempt to control its host. As the host attempts to starve the pathogen, the microbe 

-

sensitivi
ty genes, resulting in localized programmed cell death (PCD) and/or hypersensitive 
responses restricting pathogen growth but preventing its elimination. In terrestrial plants the 
phenotypic response of this stress yields irregular spots that are visualized
 
as small dark brown 
spots with a tan/yellow border 
(Kim 
et al.
, 2010)
.  In coral reef ecology, the aforementioned 
phenotype displays resemblance to coral diseases such as Yellow Band Disease 
(Cervino 
et al.
, 
2001)
 
and dark spot disease 
(Gil
-
Agudelo & Garzón
-
Ferreira, 2001)
.  
 
 
7
1
 
I
 
speculate that the differences observed here over a short time period (8 hours) may have 
been insufficient when compared to the chronic expression obs
erved in an earlier study (Hauff 
et 
al
. In Review). It is also plausible that this coral species is well adapted to short acute responses 
of temperature and/or temperature and disease as observed by the increased concentration of 
particular genes (i.e. G3P
DH = metabolism) despite the lack of statistical significance.  This is 
plausible as 
P. astreoides 
is considered a dominant coral on reefs of the Florida Keys
 
in a region 
known to experience elevated temperatures and disease
 
(Green 
et al.
, 2008)
.
 
In addition to insufficient exposure time
 
as a cause of the lack of a gene response
, the 
coral host may influence zooxanthellae gene expression by providing protection to 
zooxanthellae. This possibility is suggested by the observation that 
in hospite
 
zooxanthellae 
experience less photosynthetic d
egradation than cultured zooxanthellae when exposed to short
-
term bleaching conditions 
(Bhagooli 
et al.
, 2010)
. Because the viability of zooxanthellae 
metabolism is not independ
ent of the coral host, understanding the response of the coral host to 
heat and disease conditions is relevant to the current study. Using the same samples as this study, 
Haslun 
et al
. (In Review B) evaluated the genetic response of the coral host. Coral h
ost genes 
associated with bacterial recognition increased under acute heat and disease stress. This suggests 
that coral, rather than zooxanthellae, are the first responders to acute stress. Cultured 
zooxanthellae provide a unique way to further understand 
the role of host protection, as they are 
completely void of host tissues. In order to determine whether the host is indeed providing 
protection, gene expression of cultured zooxanthellae exposed to
 
the same levels of
 
heat and 
disease stress should be compa
red to responses demonstrated here. Deciphering
 
which
 
symbiotic 
partner 
experiences the lions
-
share of acute stress  
(Sammarco & Strychar, 2009)
 
will be key in 
determining the range at 
which
 
coral holobionts can resist acute extreme episodes of stress.  
 
 
72
 
 
The response of zooxanthellae to heat and disease appears to vary among clades. Hauff 
et 
al.
 
(2014) reported higher rates of apoptosis in 
Symbiodinium
 
spp. clade C1 synergistically 
affected by heat and Yellow Band Disease (YBD) pathogens. The same synergistic effect was 
not demonstrated in 
Symbiodinium
 
spp. clade B2. While YBD specifically target
s zooxanthellae 
(Cervino 
et al.
, 2004)
, 
S.
 
marcescens
 
does not. 
If 
S.
 
marcescens
 
targeted host coral cells as 
shown in Haslun 
et al.
, (In ReviewB), rather than zooxanthell
ae, this could explain the absence 
of zooxanthellae gene expression in the current experiment. Owing to differences in variability 
among clades discussed above, and paucity of studies at different temperatures and disease 
regimes, the synergistic effect of
 
elevated temperature and disease
 
on zooxanthellae
 
remains 
unresolved. Investigations into the impacts of 
S.
 
marcescens
 
specifically are a particularly 
important line of investigation. I
ncreased virulence of 
S. marcescens
 
is
 
linked to elevated water 
temperatures and bleaching, yielding 
host 
tissue loss rates of up to 2.5 cm
2
 
per day 
(Patterson 
et 
al.
, 2002)
. 
 
While 
S.
 
marcescens
 
virulence is enhanced with elevated temperature 
(Patterson 
et al.
, 
2002)
, some pathogens are not able to e
stablish themselves under extreme temperatures. Cervino 
et al.
 
(
2004)
 
demonstrated increased spreading rates of YBD lesions with elevated temperatures, 
but only after 
infectivity of YBD 
at ambient temperatures. If exposure to ambient temperatures is 
similarly required for the 
S.
 
marcescens
 
pathogens, this could be another fa
ctor accounting for 
the absence of differential gene expression observed herein. 
 
Short
-
term perturbations such as acute stress are difficult to capture in the field. As a 
counterpart to field studies, laboratory studies are important as they allow for man
ipulation and 
the capture of acute stress perturbations. 
My
 
study demonstrated an absence of statistically 
significant functional variability to acute stress, even though functional variation in 
 
73
 
zooxanthellae populations from 
P. astreoides
 
as a consequence
 
of chronic stress was established
 
under similar or less stressful conditions
 
in the field. The role of the host as a protective 
mechanism may explain the apparent lack of variability reported. Alternatively, the short time 
period may have also skewed the 
results
, indicating longer sampling intervals are required
 
to 
induce a significant stress response in zooxanthellae
.  The ecological implications of the field 
and lab findings suggest that the response of the coral holobiont to stress is dynamic. As such, 
the future persistence of 
P. astreoides 
is dependent on a myriad of environmental and biological 

knowledge, this study marks the first study of zooxanthellae gene exp
ression in response to 
synergistic temperature as a primary stress coupled to disease as a secondary stress. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
74
 
 
 
 
 
 
 
 
 
 
 
 
LITERATURE CITED
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
75
 
LITERATURE CITED
 
 
 
 
Barshis DJ, Ladner JT, Oliver T a, Seneca FO, Traylor
-
Knowles N, Palumbi SR (2013) Genomic 
basis for coral resilience to climate change. 
Proceedings of the National Academy of 
Sciences of the United States of America
, 
110
, 1387

92.
 
Berger S, Sinha AK, Roit
sch T (2007) Plant physiology meets phytopathology: plant primary 
metabolism and plant pathogen interactions. 
Journal of Experimental Botany
, 
58
, 4019

4026.
 
Bhagooli R, Baird AH, Ralph PJ (2010) Does the coral host protect its algal symbionts from heat 
and
 
light stresses? 
The 11th International Coral Reef Symposium
, 113

117.
 
Biemelt S, Sonnewald U (2006) Plant

microbe interactions to probe regulation of plant carbon 
metabolism. 
Journal of Plant Physiology
, 
163
, 307

318.
 
Brown BE (1997) Coral bleaching: caus
es and consequences. 
Coral Reefs
, 
16
, S129

S138.
 
Cervino J, Goreau TJ, Nagelkerken I, Smith GW, Hayes R (2001) Yellow band and dark spot 
syndromes in Caribbean corals: Distribution, rate of spread, cytology, and effects on 
abundance and division rate of zo
oxanthellae. 
Hydrobiologia
, 
460
, 53

63.
 
Cervino JM, Hayes RL, Polson SW, Polson SC, Goreau TJ, Martinez RJ, Smith GW (2004) 
Relationship of Vibrio Species Infection and Elevated Temperatures to Yellow Blotch / 
Band Disease in Caribbean Corals Relationship 
of Vibrio Species Infection and Elevated 
Temperatures to Yellow Blotch / Band Disease in Caribbean Corals.
 
Cervino J, Hauff B, Haslun J et al. (2012) Ulcerated yellow spot syndrome: implications of 
aquaculture
-
related pathogens associated with soft coral S
arcophyton ehrenbergi tissue 
lesions. 
Diseases of Aquatic Organisms
.
 
Dikou A, van Woesik R (2006) Survival under chronic stress from sediment load: Spatial 
patterns of hard coral communities in the southern islands of Singapore. 
Marine Pollution 
Bulletin
, 
52
, 1340

1354.
 
Garavaglia BS, Thomas L, Gottig N, Zimaro T, Garofalo CG, Gehring C, Ottado J (2010) 
Shedding light on the role of photosynthesis in pathogen colonization and host defense. 
Communicative & integrative biology
, 
3
, 382

4.
 
Gates RD, Baghdasaria
n G, Muscatine L (1992) Temperature stress causes host cell detachment 
in symbiotic cnidarians: implications for coral bleaching. 
Biological Bulletin
, 
182
, 324

332.
 
Gil
-
Agudelo DL, Garzón
-
Ferreira J (2001) Spatial and seasonal variation of dark spots disea
se in 
coral communities of the Santa Marta area (Colombian Caribbean). 
Bulletin of Marine 
 
76
 
Science
, 
69
, 619

629.
 

Ambio
, 
23
, 176

180.
 
Green DH, Edmunds PJ, Carpenter RC (2008) Increasing relative abundance of Porites 
astreoides on Caribbean reefs mediated by an overall decline in coral cover. 
Marine 
Ecology Progress Series
, 
359
, 1

10.
 
Harvell CD, Mitchell CE, Ward JR, Altizer S, Dobson A
P, Ostfeld RS, Samuel MD (2002) 
Climate warming and disease risks for terrestrial and marine biota. 
Science (New York, 
N.Y.)
, 
296
, 2158

62.
 
Hauff B, Cervino JM, Haslun J a et al. (2014) Genetically divergent Symbiodinium sp. display 
distinct molecular resp
onses to pathogenic Vibrio and thermal stress. 
Diseases of aquatic 
organisms
, 
112
, 149

59.
 
Kenkel CD, Setta SP, Matz M V (2015) Heritable differences in fitness
-
related traits among 
populations of the mustard hill coral, Porites astreoides. 
Heredity
, 1

8.
 
Kim YM, Bouras N, Kav NN V, Strelkov SE (2010) Inhibition of photosynthesis and 
modification of the wheat leaf proteome by Ptr ToxB: A host
-
specific toxin from the fungal 
pathogen Pyrenophora tritici
-
repentis. 
Proteomics
, 
10
, 2911

2926.
 
Kocal N, Sonnewald 
U, Sonnewald S (2008) Cell Wall
-
Bound Invertase Limits Sucrose Export 
and Is Involved in Symptom Development and Inhibition of Photosynthesis during 
Compatible Interaction between Tomato and Xanthomonas campestris pv vesicatoria. 
Plant 
Physiology
, 
148
, 152
3

1536.
 
Lirman D, Schopmeyer S, Manzello D et al. (2011) Severe 2010 Cold
-
Water Event Caused 
Unprecedented Mortality to Corals of the Florida Reef Tract and Reversed Previous 
Survivorship Patterns. 
PLoS ONE
, 
6
, e23047.
 
Matz M V, Wright RM, Scott JG (2013) No control genes required: Bayesian analysis of qRT
-
PCR data. 
PloS one
, 
8
, e71448.
 
Maverakis E, Kim K, Shimoda M et al. (2015) Glycans in the immune system and The Altered 
Glycan Theory of Autoimmunity: a critical revie
w. 
Journal of autoimmunity
, 
57
, 1

13.
 
McGinley MP, Aschaffenburg MD, Pettay DT, Smith RT, LaJeunesse TC, Warner ME (2012) 
Transcriptional Response of Two Core Photosystem Genes in Symbiodinium spp. Exposed 
to Thermal Stress. 
Plos One
, 
7
, e50439.
 
Palumbi SR
, Barshis DJ, Bay R a (2014) To Future Climate Change. 
344
, 895

898.
 
Patterson KL, Porter JW, Ritchie KB et al. (2002) The etiology of white pox, a lethal disease of 
the Caribbean elkhorn coral, Acropora palmata. 
Proceedings of the National Academy of 
 
77
 
Scie
nces of the United States of America
, 
99
, 8725

30.
 
Porter JW, Dustan P, Jaap WC et al. (2001) Patterns of spread of coral disease in the Florida 
Keys. 
Hydrobiologia
, 
460
, 1

24.
 
Raetz CRH, Whitfield C (2002) L <scp>IPOPOLYSACCHARIDE</scp> E 
<scp>NDOTOXINS<
/scp>. 
Annual Review of Biochemistry
, 
71
, 635

700.
 
Ruiz
-
jones LJ, Palumbi SR (2015) Transcriptome
-
wide Changes in Coral Gene Expression at 
Noon and Midnight Under Field Conditions. 
Biological Bulletin
, 
228
, 227

241.
 
Ruzicka RR, Colella M a., Porter JW et a
l. (2013) Temporal changes in benthic assemblages on 
Florida Keys reefs 11 years after the 1997/1998 El Niño. 
Marine Ecology Progress Series
, 
489
, 125

141.
 
Sammarco PW, Strychar KB (2009) Effects of Climate Change/Global Warming on Coral Reefs: 
Adaptation/
Exaptation in Corals, Evolution in Zooxanthellae, and Biogeographic Shifts. 
Environmental Bioindicators
, 
4
, 9

45.
 
Sampayo EM, Ridgway T, Bongaerts P, Hoegh
-
Guldberg O (2008) Bleaching susceptibility and 
mortality of corals are determined by fine
-
scale diff
erences in symbiont type. 
Proceedings 
of the National Academy of Sciences of the United States of America
, 
105
, 10444

9.
 
Selvaraj K, Fofana B (2012) An Overview of Plant Photosynthesis Modulation by Pathogen 
Attacks. 
Advances in Photosynthesis 
-
 
Fundamenta
l Aspects
, 465

486.
 
Sutherland KP, Shaban S, Joyner JL, Porter JW, Lipp EK (2011) Human pathogen shown to 
cause disease in the threatened eklhorn coral Acropora palmata. 
PloS one
, 
6
, e23468.