a histological assessment of the effects of elevated
TRANSCRIPT
The University of Maine The University of Maine
DigitalCommons@UMaine DigitalCommons@UMaine
Honors College
Spring 5-2018
A Histological Assessment of the Effects of Elevated Temperature A Histological Assessment of the Effects of Elevated Temperature
and Nitrogen on the Symbiodinium of Palythoa Toxica (Walsh and and Nitrogen on the Symbiodinium of Palythoa Toxica (Walsh and
Bowers 1971) Bowers 1971)
Molly Westbrook University of Maine
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Recommended Citation Recommended Citation Westbrook, Molly, "A Histological Assessment of the Effects of Elevated Temperature and Nitrogen on the Symbiodinium of Palythoa Toxica (Walsh and Bowers 1971)" (2018). Honors College. 360. https://digitalcommons.library.umaine.edu/honors/360
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A HISTOLOGICAL ASSESSMENT OF THE EFFECTS OF ELEVATED
TEMPERATURE AND NITROGEN ON THE SYMBIODINIUM OF PALYTHOA
TOXICA (WALSH AND BOWERS 1971)
by
Molly Westbrook
A Thesis Submitted in Partial Fulfillment of the Requirements for a Degree with Honors
(Marine Science)
The Honors College
University of Maine
May 2018
Advisory Committee:
Dr. Ian Bricknell, Professor of Aquaculture Dr. Rhian Waller, Associate Professor of Marine Sciences Dr. Seth Tyler, Professor of Zoology, Cooperating Professor of Marine Sciences Dr. Heather Hamlin, Associate Professor of Aquaculture Dr. François Amar, Dean of the University of Maine Honors College, Professor of
Chemistry
ABSTRACT
Coral reefs around the world are suffering mass bleaching events caused by a
combination of stressors, including rising ocean temperatures, acidity, pollution,
increased suspended sediments, and increasing nitrogen levels. Corals harbor a complex
microbial ecosystem consisting of bacteria, and algal symbionts known as Symbiodinium.
This study examines the effects of elevated temperature, a known cause of bleaching, and
elevated nitrogen, an increasingly important potential stressor for reefs, on the
Symbiodinium of a zoanthid coral, Palythoa toxica. A total of 65 polyps were subjected to
5 different water treatments of 2 levels of elevated nitrogen, and 2 levels of elevated
temperature in populations of 12. Samples were removed every four days and at the
experiment’s termination. The samples were fixed for histological examination.
Symbiodinium size was analyzed using Feret diameter. The results of this experiment
confirm the effect of elevated temperature as a bleaching mechanism for P. toxica and
provide evidence that elevated nitrate is a potential bleaching trigger in prolonged
exposure. Furthermore, there is evidence that combined elevated nitrate and temperature
compound bleaching mechanisms, with the highest amount of bleaching occurring in the
most stressed treatments. When exposed to low levels of nitrate and low levels of
temperature elevation, there is a trend of Symbiodinium Feret diameter increase. With
time, however, there is an eventual collapse of the system, resulting in Symbiodinium
Feret diameter decrease and an increase of algal mass potentially from cell lysis within
bleached Symbiodinium colonies remaining between tentacles and within the coelenteron.
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ACKNOWLEDGEMENTS
Thank you to Dawna Beane for histological fixation of slides, Robert Harrington
for system design and construction help, Scarlett Tudor for statistical analysis and results
guidance, Jeremy Rich for microscope use, Corinne Mooring and the Bricknell laboratory
team of summer 2017 for system care, and Ashley Rossin for methodology of size-
frequency analysis. The author would also like to graciously thank Rhian Waller, Seth
Tyler, Heather Hamlin and François Amar for their knowledge, resources, and invaluable
input to this project. Finally, the author would like to express their endless gratitude to
Ian Bricknell for helping to make this project a reality, and providing guidance and
enthusiasm at every step. Thank you.
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TABLE OF CONTENTS
Table of Figures v Introduction 1
Anthropogenic Effects on Coral Reefs 1 Symbiodinium and the Adaptive Bleaching Hypothesis 2 The Interaction Between Nitrogen, Coral, and Symbiodinium 5 Palythoa toxica Overview and Study Objectives 6
Materials and Methods 8 System Design and Sampling 8 Symbiodinium Cell Size Analysis 10 Statistical Analysis 11 Results 12 Experiment One 12 Effects of Elevated Nitrogen 14 Effects of Elevated Temperature 17 Variance Among Treatments 20 Experiment Two 21 Effects of Combined Elevated Nitrogen and Temperature 22 Variance Among Treatments 27 Discussion 28 Nitrogen Effects on P. toxica 28 Temperature Effects on P. toxica 30 Combined Stress Effects 31 Conclusions 33 References 35 Appendices 40 Appendix 1: ImageJ Feret Diameter Calculations 41 Appendix 2: R Script for Statistical Analysis 45 Author’s Biography 49
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TABLE OF FIGURES
Figure 1. Image of P. toxica 6 Figure 2. Image of care system 8 Figure 3. Image of experimental system 9 Table 1. Treatment water quality parameters of experiment 1 and 2 9 Figure 4. Images of histological slide s (1) 12 Figure 5. Images of histological slides (2) 12 Figure 6. Images of histological slides (3) 14 Figure 7. Images of histological slides (4) 14 Figure 8. Images of histological slides (5) 15 Figure 9. Box plot of Symbidoinium size comparison (1) 16 Figure 10. Images of histological slides (6) 17 Figure 11. Images of histological slides (7) 17 Figure 12. Images of histological slides (8) 18 Figure 13. Box plot of Symbidoinium size comparison (2) 19 Figure 14. Box plot of Symbiodinium size variance (1) 20 Figure 15. Images of histological slides (9) 21 Figure 16. Images of histological slides (10) 22 Figure 17. Images of histological slides (11) 22 Figure 18. Images of histological slides (12) 23 Figure 19. Images of histological slides (13) 24 Figure 20. Images of histological slides (14) 25 Figure 21. Box plot of Symbidoinium size comparison (3) 25 Figure 22. Box plot of Symbiodinium size variance (2) 27
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INTRODUCTION
Anthropogenic Effects on Coral Reefs
It is abundantly clear that anthropogenic effects are changing the nature of coral
reefs (Hughes et al 2003, Bellwood et al 2004), and that as humans continue to impact the
global climate, the negative effects of these changes on coral reefs will increase (Hughes
et al 2003, Hughes et al 2017). There are a number of different factors that can influence
these effects, including stress effects from sedimentation and pollution (ISRS 2004),
temperature (Glynn 1990, Winter et al 1998), high levels of solar radiation (Lesser et al
1990, Gleason and Wellington 1993, Brown 1997), ocean acidification (Anthony et al
2008), and nitrogen enrichment (Wiedenmann et al 2012, Vega-Thurber et al 2014).
One of the most well-known coral stress indicators is that of coral bleaching, or
the expulsion of symbiotic dinoflagellate algae known as Symbiodinium normally
maintained in coral polyp tissues (Brown 1997). This loss of Symbiodinium results in the
coral tissue becoming pale or transparent, causing the skeleton to become visible and
resulting in a white color. The first observed and published mass bleaching event of a
wide range of coral reefs simultaneously occurred in Gulf of Chiriquí, Panama in 1982
during the El Niño temperature anomaly (Glynn 1984). Since this event, there have been
numerous local and widespread mass bleaching events worldwide, including three
widespread mass bleaching events in 1998, 2002, and from 2016 to 2017, the first back-
to-back mass bleaching event (Hughes et al 2017) on the Indo-Pacific Archipelago. Other
major bleaching events include the 2005 Caribbean reef bleaching event (Eakin et al
2010), and the 1982 Eastern Pacific bleaching event (Harriot 1985, Glynn 1990). The
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result is clear: bleaching events are becoming increasingly common, and understanding
these events is essential for reef protection and recovery (Hughes et al 2003).
The bleaching mechanism lies in the symbiotic relationship between
Symbiodinium and the coral animal, and understanding this relationship is essential to the
coral bleaching mechanism. With a more complete understanding of this mechanism,
human conservation policy can be accurately shaped to protect reefs from anthropogenic-
caused stress.
Symbiodinium and the Adaptive Bleaching Hypothesis
Symbiosis, or “the living together of unlike organisms” (Douglas 1994) has been
a long-observed relationship in biology. First used to describe the relationships among
lichens in the late 1800’s (Trappe 2005), there are several required factors for a
relationship to be classified as symbiosis. First, at least one organism must play a
significant role in the life cycle of the second, and second, the organisms are in sustained
and close contact throughout their lifetimes (Lewis 1973).
Symbiodinium are a genus of dinoflagellates found maintaining symbiotic
relationships with several marine invertebrate phyla including Cnidaria, Mollusca,
Platyhelminthes and Porifera (Stat et al 2006). The most well-known relationship of these
groups is that between Symbiodinium and the Cnidarian Anthozoa.
The relationship between coral animals and Symbiodinium, known colloquially as
zooxanthellae, is an essential relationship within the coral animal life cycle.
Symbiodinium provide essential nutrients to the coral animal through photosynthesis for
cell nutrition, maintenance and reproduction (Muscatine, Porter, 1977), fixing up to four
times as much carbon as coral lacking Symbiodinium in their tissues (Muscatine et al
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1984). Maintaining symbiotic relationships with algae allow coral to function as primary
producers as well as primary and secondary consumers (Muscatine, Porter 1977),
resulting in the role of coral in reef ecosystems as a keystone species. The carbon fixed
by Symbiodinium plays an especially important role in the complex calcium carbonate
skeleton certain coral secrete that comprises the physical shape of the reef itself (Anthony
et al 2008). These calcifying coral not only function as animals within the reef ecosystem,
coral compose the shape of the reef itself literally through their skeletal structures
(Tambutté et al 2011). Furthermore, Symbiodinium play an important role in juvenile
coral growth, fixing carbon as the coral polyp settles on the reef (Cantin et al 2008).
Symbiodinium have only recently become the focus of understanding the adaptive
mechanisms of coral to stress.
Symbiodinium were first grouped into 22 different taxonomic groups of
indeterminate level using restriction fragment length polymorphisms from the small
ribosomal subunit RNA (Rowan and Powers 1991). Later these groups became the
modern clade taxonomic classification of these algae, composing nine clades (A-I), and a
number of subclades, with primary associations of clade C with coral. Symbiodinium are
acquired in coral tissue through two ways: horizontally from the environment, or
vertically from parental populations (Byler et al 2013). Originally thought to be specific
to the host, Symbiodinium transmission is flexible both between species of coral and
within species, with species maintaining multiple subclades of a single Symbiodinium
clade within their tissues, and some maintaining even multiple clades (Little et al 2004).
It has been shown that different Symbiodinium clades adjust the metabolism and stress
coping mechanisms for different aspects of the holobiont system. For example, hosts
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harboring clade C Symbiodinium have an increased growth rate (Little et al 2004),
whereas populations maintaining clade D have a higher tolerance of thermal stress
(Rowan 2004, Baker et al 2004).
Herein lies the key to coral bleaching and coral adaptation to stress conditions.
Individual clades of Symbiodinium have relatively narrow ranges of stress tolerance due
to their thylakoid lipid-membrane composition (Tchernov 2004), and thus it is very
difficult for populations of Symbiodinium within a single clade to survive alone with the
holobiont system in changing environments. However, there are two mechanisms that
allow corals to shift the dominant clades of Symbiodinium maintained within their tissues:
switching, or completely expelling the symbionts to replace with others taken up
horizontally, and shuffling, or rearranging existing populations within their tissues for a
different symbiont clade to influence the coral’s adaption to the environment (Reich et al
2017). Bleaching was first hypothesized in 1993 and later refined as an adaptive
mechanism through which coral could shuffle Symbiodinium within their tissues to
increase tolerance to local conditions known as the Adaptive Bleaching Hypothesis
(Buddemeier and Foutin, 1993; Buddemeier et al 2004). Further research into this
mechanism lead to the hope that corals would be able to adapt for the short term to
human-influenced changes, such as increasing ocean temperatures up to a point
(Berkelmans, Oppen 2004), though it is increasingly clear that the complicated nature of
Symbiodinium taxonomy and nuanced understanding of thermal and stress tolerance
requires reclassification from the current taxonomic clade system (Sampayo et al 2008).
Whether or not scientists can use this relationship to help protect and potentially recover
reefs in future anthropogenic climate fluctuations is unknown.
5
The Interaction Among Nitrogen, Coral, and Symbiodinium
The interaction between coral reefs and nitrogen has been a subject of study since
the 1970’s, when the observation was first made of the coral reef nutrient paradox. Reefs
are located in areas of relatively low nutrient levels and yet are able to maintain high
levels of productivity (Webb et al 1975), thus the mechanisms through which coral are
able to efficiently use nitrogen are of major concern. Nitrogen is the primary limiting
nutrient for marine systems within coral- reef systems (Thomas 1969), and it has been
shown that coral’s response to the nitrogen cycle and internal nitrogen cycle are closely
tied with their susceptibility to bleaching and disease (Weidenmann et al 2013, Vega-
Thurber et al 2014, Rädecker et al 2015).
In brief, the nitrogen cycle begins in the atmosphere as N2, nitrogen is fixed by
archaeobacteria and eubacteria located in sediment in an anaerobic process called
denitrification to NO3- or nitrate, which is then taken up by organisms, usually plants, and
converted to ammonium (NH4+). Microbial processes convert NH4+ to NH3, then NO2-,
and finally to NO3-, and the cycle can repeat. These converted forms of nitrogen,
especially NO3= and NH4+, can be taken up by organisms and transformed back to NH4+
or urea, which then sink down into the sediment and are converted again. NH4+ can also
be nitrified back into N2, where it is returned in the atmosphere (Falkowski 1997).
However, human impact on the nitrogen cycle through artificial fertilizers in
modern agriculture and burning fossil fuels have added a second influx of NO3- into the
system, where it is washed into the ocean, most notably manifesting as algae blooms and
areas of anoxic or dead zones along the coast (Vitousek et al 1997, Canfield et al 2010).
There is clear evidence that these anoxic blooms are increasing with time, resulting in
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decreasing diversity along coastal dead zones including among seagrass beds and coral
reefs, lower fish and shellfish resources for human fisheries and aquaculture, increasing
dominance of nuisance algae and increasing occurrences of toxic dinoflagellate blooms
or “red tides” known to cause shellfish poisoning among humans (Vitousek et al 1997).
Tropical coral reefs are most often located in coastal or near-coast ecosystems and
are thus subject to these anthropogenic influxes of nitrogen into the system. Because
coral maintain symbiotic algae within their tissues, it is likely that this influx of nitrogen
into the ocean is causing changes to these communities. Very little research has been
done on the interactions between coral and the nitrogen cycle, though there is increasing
evidence that nitrogen plays an important role in the dynamic of the coral holobiont and
the bacterial and algal members of its symbiotic community (Siboni et al 2008,
Pogoureutz et al 2017)
Palythoa toxica and Study Objectives
Figure 1. A colony of Palythoa toxica (Walsh and Bowers 1971) from the University of Maine Aquaculture Research Collection used in this experiment. Photo credit to Allyson Redcay.
Palythoa toxica (Walsh and Bowers 1971) is a zoanthid hexacoral that produces
the infamous palytoxin, one of the most deadly toxins within the natural world. This
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species is not a reef-building sclearactinian coral. Instead these animals have relatively
large anemone-like polyps, but maintain similar symbiotic algal associations as
scleractinian coral. The species was first isolated in 1971 from Hawaii from a tropical
coral referred to as limu-make-o-Hana, and was the first organism discovered to produce
palytoxin (Walsh and Bowers, 1971). Since its discovery, palytoxin and its effects on
humans have become the focus of research on this group (Moore and Scheuer, 1971;
Deeds and Schwartz 2010). Currently there is very little research on histological changes
during prolonged stress periods, though ecological studies of Symbiodinium in zoanthid
coral have been conducted (Kemp and Cook 2006; Burnett 2002) and indicate zoanthid
coral exhibit similar bleaching mechanisms as scleractinians (Kemp and Cook, 2006).
Furthermore, P. toxica is relatively easy to obtain and culture, and was used for this
experiment due to its accessibility and lack of research focusing on this particular
symbiotic relationship.
The goal of this study is understanding the relationship between P. toxica and its
associated Symbiodinium when exposed to elevated levels of nitrogen and temperature
both separately and in combination using histological analysis. Symbiodinium were not
genetically analyzed to determine clade. The hypothesis of the study was based on
increased growth of dinoflagellates when exposed to elevated nutrient levels (Parkhill
and Cembella 1999), and tests for a direct relationship between the level of nitrogen
within the coral tissues and the size of the Symbiodinium cells. Temperature was added as
a second factor to understand stress effects on coral in combination with nitrate stress,
and to retest the effects of temperature on coral bleaching, especially for the relatively
understudied zoanthid clade.
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MATERIALS AND METHODS
System Design and Sampling
Palythoa toxica samples were acquired from a local aquarist in Orono, ME, and
moved to the University of Maine Aquaculture Research Center where they were
maintained in a 710 liter raceway (290 L raceway, 420 L bottom sump) under a Tek light
T5. The water was maintained around 26˚C, 32 ppt salinity, a pH of approximately 8, and
a water exchange rate of 35 L/sec.
Figure 2. The system in which colonies of P. toxica were maintained before the experiment.
The samples comprised three genetically identical colonies of various size. 125
polyps were cloned from these samples using an Ecotech Marine Coral Propagation Kit,
glued onto cement coral plugs using Ecotech coral glue, and maintained in the raceway
for two weeks prior to the experiment. The design of the experiment comprised one
control and four experimental treatments with one 4-L tank per treatment. 60 polyps were
used for each experiment, in groups of 12 polyps per treatment.
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Figure 3. A reconstruction of the experimental system setup.
Two experiments were run over the course of this project, both lasting 14 days, of
two levels of elevated nitrate and two levels of elevated temperature (Table 1). Nitrate
levels were elevated by adding powdered NaNO3. After completion of experiment one
tanks were drained and cleaned using a water and bleach rinse before being randomly
rearranged for experiment two. Experiment two consisted of the same experimental
setup. Nitrate levels were readjusted for the second experiment, and the same temperature
levels were used.
Table 1. Treatment temperature and nitrate levels for experiment 1 and experiment 2.
Treatment Experiment 1 Experiment 2 1 26˚C, 0 ppm 26˚C, 0 ppm 2 26˚C, 7 ppm 30˚C, 10 ppm 3 26˚C, 15 ppm 30˚C, 20 ppm 4 30˚C, 0 ppm 35˚C, 10 ppm 5 35˚C, 0 ppm 35˚C, 20 ppm
10
All tanks contained a fish tank heater of 50 or 100 watts to maintain temperature,
and were maintained beneath two 50 watt lights, on a light schedule of 12 hours on, 12
hours off (Figure 3).
For both experiments two polyps were sampled from the control treatment on day
one, and then from every treatment every four days. On day 14 all remaining polyps were
sampled from each treatment. Histology fixation was conducted using a standardized
procedure as follows: samples were placed in 10% neutral buffered formalin, placed in a
standard tissue processor for 16.5 h, then embedded in paraffin. Five-µm thick sections
were cut and stained with hematoxylin and eosin. Sampling protocol was designed using
IACUC Power Study protocol (University of Maine). Nitrogen-enrichment levels were
based on two Australian reefs with higher levels of nitrate: Yongola Reef in the mid-
Great Barrier (Vridaka) as the low-enriched level, and Rottnest Island, in Western
Australia as the high-enrichment level (Thomson et al 2011). Nitrogen levels were tested
every day for the first four days of each experiment using a LaMotte water quality testing
kit and then every other day for the duration of the experiment. When nitrogen levels
were lower than the required level, approximately 0.01 grams of NaNO3 was added to the
tanks (increasing NO3- by approximately 2 ppm).
Symbiodinium Cell Size Analysis
Feret diameter was used as a quantitative measurement of the change of size in
Symbiodinium (See Appendix 1). Slides were photographed using an Olympus CX31
microscope with Infinity Capture software. Photographs were analyzed using ImageJ
version 1.51s and Java1.8.0_151. Analysis was calibrated for 40x images using a Motis
calibration for 40x, 100x and 400x magnification. The perimeters of 100 randomly
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selected Symbiodinium were traced using the freehand trace function and measured for
each polyp for a total of 200 Feret diameters for each treatment barring the samples from
Trial 1 Treatment 1 Day 1 (3 polyps), Treatment 3 Day 12 (3 polyps), Treatment 4 Day 8
(1 polyp), Treatment 5 Day 8 (3 polyps), and Trial 2 Treatment 1 Day 1 (1 polyp), as well
as samples from the 14th day of the experiments (3-4 polyps). Due to stress, the polyps
from the 12th day of Trial 2 for treatments 4 and 5 were too degraded for histology, and
thus there are no data for those samples. Symbiodinium were treated as the individual
(N=1) for this process instead of polyps within the treatment.
Statistical Analysis
All statistical analysis was conducted in RStudio version 3.3.3 on a basis of 95%
confidence. Data were organized by Experiment (1 or 2), treatment (control, high/low
nitrate, high/low temperature), time of sampling (Day 4, 8, 12, or 14) and polyp. Each
Symbiodinium Feret measurement was assigned a number, and average Feret diameter
was calculated by averaging the Feret maximum and minimum. One-way analysis of
variance (ANOVA) was used to compare mean Feret of treatments (See Appendix 2).
Outliers were removed from each dataset and assumptions of the ANOVA were met by
each set (Levene’s test for symmetry, p>0.05, Shapiro-Wilk test for normality, p>0.05).
Two ANOVA analyses were conducted. The first ANOVA used treatments as the
explanatory variable, and the second used day of sampling as the explanatory variable. A
Tukey HSD post-hoc was used for both ANOVA results for individual mean comparison.
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RESULTS
Experiment One
Figure 4. Control Day 4, from left to right, top to bottom: 400x, 100x and 40x images of Polyp II.
Figure 5. Images of control polyps throughout different sampling times of the experiment. Green arrows indicate healthy Symbiodinium cells. To establish a concept of a ‘healthy’ polyp as a basis of comparison for the
experiment, samples of the control polyp were taken at every sampling step of the
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experiment. There are several factors that characterize a healthy polyp for this
experiment, as can be demonstrated by Figures 4 and 5. Healthy polyps contain no faded
tissue; in other words, polyp tissue is tinted darker shades than bleached counterparts.
Healthy Symbiodinium are in general maintained in one of two places: either along the
tentacles near the top of the polyp (Figure 4, 400x, Figure 5, Day 12, 400x) or maintained
in gastrodermis in the mesenteries of the polyp (Figure 5, Day 14 400x Polyps 1 and 3).
Small numbers of Symbiodinium could also be found maintained in vesicles within the
polyp epidermis (Figure 5, Day 14 Polyp 3). Finally, there is no sign of Symbiodinium
lysis; in other words, all cells of Symbiodinium are whole and contain plastids (Figure 4,
400x, Figure 5, all 400x images show relatively healthy Symbiodinium). Not every
control polyp displayed these characteristics; bleaching occurred in the control treatment
of the second experiment on the 14th day.
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Effects of Elevated Nitrogen
Figure 6. Images of polyps taken from Treatments 2 and 3 (elevated Nitrogen treatments) at 40x throughout the experiment. Polyps in treatment 3 days 4, 8 and 14 exhibit moderate bleaching.
Figure 7. Images of polyps taken from Treatments 2 and 3 (elevated Nitrogen treatments) at 400x throughout the experiment. Within elevated nitrogen treatments, there was a major difference between these
treatments and the control treatments. Full bleaching of coral tissue did not occur;
however, there was a trend of gastrointestinal pockets of Symbiodinium among the
15
tentacles and mesenteries being expelled into the coelenteron of the polyp (Figure 7).
Near the end of the experiments, some of the Symbiodinium had lysed within Treatment 3
(Figure 7; Treatment 3, Day 14).
Figure 8. A comparison of Symbiodinium on the last day of the experiment for the control (treatment 1) and elevated nitrogen treatments (2 and 3) at 400x. There appears to be a trend with time between different levels of elevated nitrate.
Small levels of nitrate may stimulate growth of Symbiodinium (Figure 8) to an unhealthy
degree. Higher levels of nitrate cause coral tissue to bleach partially, but retain those
Symbiodinium within their system.
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Figure 9. Box plots comparing Symbiodinium Feret diameter among Control, Treatment 2 and Treatment 3. Matching colors represent insignificant differences (ANOVA, P>0.05). In the case of Day 12, Treatment 2, the mean was significantly different from Treatment 1, but not from Treatment 3.
Elevated nitrate affected Symbiodinium Feret diameter, however, how nitrate
affected the size of the algae cells varies between time and treatment (Figure 9). For the
first half of the experiment, there was a clear trend of increasing size with increased
nitrate levels within the water. As the experiment continued however, the average Feret
diameter of treatment 3 decreased from almost 10 µm in diameter on Day 4 at the highest
to as low as 9 µm on Day 12. Within nitrate treatments, Symbiodinium Feret diameter
remains between 8 and 10 µm. For both treatment 2 and 3, diameter increased in the first
half of the experiment and appears to decrease in the second half.
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Effects of Elevated Temperature
Figure 10. Images of polyps taken from Treatments 4 and 5 (elevated temperature treatments) at 40x throughout the experiment. Treatment 4 on day 14, and treatment 5 on day 4 exhibit symptoms associated with bleaching.
Figure 11. Images of polyps taken from Treatments 4 and 5 (elevated temperature treatments) at 400x throughout the experiment.
Elevated temperature in general caused minor bleaching (Figure 10, Treatment 4
Day 14, Treatment 5 Day 4 and Day 14). In general, similar to nitrogen treatments, there
was a trend of Symbiodinium being expelled from the polyp tissues to coagulate in spaces
18
among tentacles and within the coelenteron of the coral (Figure 11, Treatment 4 Day 14,
Treatment 5 Day 4). Some breakdown of gastrointestinal tissue occurred in particularly
unhealthy polyps (Figure 10, Treatment 4 Day 14, Figure 9, Treatment 4), however the
major trend remained change in Symbiodinium location, moving out of gastrointestinal
lining into spaces inside the polyp (Figure 10). Bleaching remained minimal, with only
one polyp in treatment 4 and one polyp in treatment 5 displaying signs of moderate
bleaching.
Figure 12. A comparison of Symbiodinium on the last day of the experiment for the control and elevated temperature treatments at 40x.
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Figure 13. Box plots comparing Symbiodinium Feret diameter between Control, Treatment 4 and Treatment 5. Matching colors represent insignificantly different samples (ANOVA, P>0.05). The pattern of Symbiodinium diameter size change in elevated temperature
treatments was more random than elevated nitrate treatments but displayed a similar
pattern. In general, treatments 4 and 5 had significantly different means than the control
(Figure 13). In the case of the beginning of the experiment, both treatment 4 and 5 had
larger Feret diameters on average, with treatment 4 being the largest of the three.
However, by the end of the experiment, the average Feret diameter of treatments 4 and 5
had decreased. By Day 12, there was no significant difference between treatment 4 and
the control. By Day 14, there was no significant difference between treatment 5 and the
control, and treatment 4 had decreased significantly to below the average Feret diameter
of the control.
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Variance Among Treatments
Figure 14. Box plots of average Feret diameter within each individual treatment throughout the experiment. Matching colors represent insignificantly different samples (ANOVA, P>0.05). In the case of multiply colored samples, a significant difference occurred for at least one treatment comparison. For treatment 2, Day 12, Feret diameter was significantly different from Day 14 but equivalent to Days 4 and 8. In Treatment 4, Day 14 was not significantly different from either Day 4 or Day 12, but both Day 4 and Day 12 varied from each other. Finally in Treatment 5, Day 8 and 14 were significantly different than Day 12, but not Day 4. There was significant variance among individual tank Feret diameters as time
passed during the experiment. Among treatments 1, 2 and 3, treatment Feret diameter
remained consistent for several days at a time but still varied at different points of the
experiment. Treatments 4 and 5 varied to a higher extent, with Treatment 4 containing the
most variation among Feret diameters within the experiment.
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Experiment Two
Figure 15. Images of the control polyps throughout experiment 2 and 40x and 400x.
Control polyps of experiment 2 matched the healthy characteristics from
experiment 1 with little change. Healthy Symbiodinium were maintained between
tentacles, among mesentarial tissues and within vesicles along the epidermis of the polyp.
Bleaching occurred within the control tank on Day 14.
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Effects of Combined Elevated Nitrogen and Temperature
Figure 16. Images of polyps from treatments 3 (low temperature, high nitrate) and 4 (high temperature, low nitrate) at 40x.
Figure 17. Images of polyps from treatments 3 (low temperature, high nitrate) and 4 (high temperature, low nitrate) at 400x. Within varying combined levels of stress, there was a clear trend that higher
temperatures in combination with nitrate stress of any level resulted in increased
bleaching (Figure 16, Figure 17). High elevated nitrate levels and lower elevated
23
temperature levels did not cause bleaching to occur in treatment 3 (whereas moderate and
some severe bleaching occurred in highest nitrate levels in experiment 1). The most
severe cases of bleaching occurred on Day 12 within treatments 4 and 5 as samples were
too damaged to be histologically fixed. Within treatment 3, however, high temperature in
combination with stress resulted not only in more frequent bleaching, but more severe
bleaching when it did occur, though there was no bleaching on the final day of
experiment 2 in either treatment 3 or 4. Bleaching generally resulted in Symbiodinium
still being maintained within the coral coelenteron. Fading of coral tissues was more
obvious within experiment 2 (Figure 17 Treatment 4 Days 4 and 8).
Figure 18. Images of polyps from treatments 2 (low temperature, low nitrate) and 5 (high temperature, high nitrate) at 40x.
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Figure 19. Images of polyps from treatments 2 (low temperature, low nitrate) and 5 (high temperature, high nitrate) at 400x. Treatment 2 contained no bleached samples, generally maintaining relatively
healthy tissues and Symbiodinium compared to control treatments. Treatment 5 contained
the most occurrences of bleaching, with bleaching occurring severely in every sample
taken (Figure 19, Figure 20, Treatment 5), and the samples from Day 12 were too
damaged to even histologically fix. Movement of Symbiodinium bleaching is most
obvious within these treatments. Tissues are completely pale, Symbiodinium are found
exclusively in either the coelenteron or between tentacles, and contain several instances
of what may be lysed cell material among surviving Symbiodinium (Figure 19, Treatment
5, Day 14, Figure 19).
25
Figure 20. A comparison of Symbiodinium between all four treatments of experiment 2 at 400x on the final day of the experiment.
Figure 21. Box plots comparing average Feret diameter within each individual treatment throughout the experiment. Matching colors represent insignificantly different samples (ANOVA, P>0.05). In the case of Treatment 4, Day 4, Feret was significantly different from treatment 5, but not treatment 1. Within Day 8, treatments 2, 3 and 5 were significantly different than treatment 1, but not treatment 4. Within Day 14, treatments 2, 3 and 4 were significantly different than treatment 5, but not from treatment 1.
26
Within experiment 2 Symbiodinium Feret diameter varied generally differently
than the control treatment (with the exception of Day 14) with no other discernible
pattern. It seems with multiple stressors on the polyp, the Feret diameter generally
increases in size, with the major exception of samples from Day 14.
27
Variance Among Treatments
Figure 22. Box plots comparing average Feret diameter within each individual treatment throughout the experiment. Matching colors represent insignificantly different samples (ANOVA, P>0.05). In general, Feret diameter varied strongly within each treatment more so than in
experiment 1. In the case of all experimental treatment, there is strong variation
throughout the experiment with nearly every average value varying significantly from the
previous sample with the most variation occurring in treatments 2 and 3.
28
DISCUSSION
Nitrogen Effects on P. toxica
There is a general trend of size increase among elevated nitrate treatments in the
Feret diameters of Symbiodinium; however, this trend is inconsistent throughout the
experiment. Furthermore, there is a trend of variation within each system. There is
significant variation within the control treatment with a general trend of Feret size
increase; however these conditions were kept constant. This is potentially a sign that
when P. toxica is maintained at constant condition, healthy Symbiodinium will grow,
causing the increase in size (Figure 14, Figure 22). Both treatments displayed a similar
trend: near the beginning of the experiment, size was elevated directly with size, though
by the end of the experiment both treatments dropped to either an insignificantly different
size to the control, or significantly lower. This could mean that with elevated nitrogen
there is a promotion of growth due to these excess nutrients, then a collapse of the
system, resulting in the cell lysis displayed in both nitrate treatments and an overall
decrease in size.
Size trends within each treatment varied significantly as well. The control
treatment varied significantly, increasing with time during the experiment. Originally it
was thought that if conditions remained constant and healthy, Symbiodinium size would
not be affected. However, this could support the idea that a healthy coral displays
Symbiodinium growth. Meanwhile, Treatment 2 remained consistent, with a slight
insignificant decrease. Treatment 3 varied significantly, decreasing and then increasing.
29
Nitrate uptake in Symbiodinium has been studied in the past, and there is a
consensus of Symbiodinium nitrogen uptake in forms of ammonium and nitrate, with a
preference for ammonium (D’Elia et. al. 1983). Furthermore, sudden increases in
nitrogen cause an increase in uptake of nitrogen in Symbiodinium and storage as uric
crystals (Kopp et al 2013). This could be an explanation of the sudden increase and
subsequent decrease in size of the Symbiodinium, however, within this experiment the
timeline of Symbiodinium increase and decrease within treatments 2 and 3 occurs more
slowly than formation of uric crystals, and these crystals were not examined within the
cell. Corals generally display a nutrient limitation on their symbionts, disrupted by
organic carbon (Wang and Douglas 1998), and the effects of nutrient stimulation on
corals is potentially helpful in the short term, promoting growth in the symbionts, but
eventually leading to a collapse in growth, especially with compounded stressors. There
is also evidence that the coral microbiome plays a large role in the nitrogen cycle within
corals (Pogourez et al 2017), so there is good reason to believe that there is more
happening within this pattern of Symbiodinium growth and collapse than what this study
examined. Recent studies have begun to emphasize the importance of nitrogen as a
bleaching trigger (Wiedenmann et al 2012).
Bleaching occurred for the samples enriched in nitrate with a total of eleven cases
of bleaching out of the total of twenty one samples, about 50%. However, the bleaching
that did occur was moderate in general, with most Symbiodinium colonies remaining in
the coelentron of the coral. Only by the very end of the experiment in the highest nitrate
treatment did severe bleaching occur in Day 12 and Day 14. Therefore, according to the
30
results of this experiment, elevated nitrate triggers the bleaching mechanism of corals to
some extent.
Temperature Effects on P. toxica
The effect of temperature on the Feret diameter of Symbiodinium has a similar
trend to the nitrogen enriched treatments. At the beginning of the experiment, there is a
trend of increasing Symbiodinium size in the elevated temperature treatments. By Day 8,
treatment 4 (held at 30˚C) had the highest Feret diameter, before crashing in size and
falling to a significantly smaller diameter than the control treatment. Treatment 5 had a
similar trend of increasing in size and decreasing in size, though these changes were less
extreme. By the end of the experiment Treatment 5 had fallen back to an insignificantly
different size than the control.
These could be following a similar pattern of the nitrogen enrichment experiment.
It has been shown in Pocillopora coral that slight temperature elevation can increase the
size of Symbiodinium for short periods of time (Edmunds et al 2005), however this effect
ceased at 27˚C. There was significant variation in the temperature of the two treatment
tanks during the experiment, so this may have contributed to the slight increase in size
and eventual decrease. Furthermore, this effect was only noted for short-term exposure
(24 hours), whereas this experiment incubated coral polyps for 14 days.
Within treatments, similar to the nitrogen enriched treatments, there was a trend
of variance through time. Symbiodinium size in both treatments increased significantly
compared to the control (for treatment 4 this occurs on Day 8; for treatment 5 this
occurred on Day 12), before a collapse, resulting in sizes significantly lower than the
control.
31
There is very little literature on the dynamics of how Symbiodinium change size
during temperature elevation, as this method for measuring Symbiodinium size change
was a novel method developed from coral reproduction methods (Waller, Tyler 2005).
However, it has been shown that Symbiodinium photosynthesis decreases in response to
elevated temperature (Goulet et al 2005), though this response varies depending on the
coral acclimation to temperature (Kemp 2006). This experiment on these zoanthids are
the first case of a more extended length study on elevated temperature on Symbiodinium
size, and it is clear that there is a linkage between length of time in treatment and
Symbiodinium size.
There is an indication that temperature is a bleaching trigger in P. toxica, with the
highest temperature treatment resulting in six cases of bleaching, the most bleaching
occurring at the end of the experiment, linking time to bleaching as well. Prolonged
exposure to elevated temperature is the most consistent trigger of coral bleaching and has
been studied numerous times, mostly in scleractinian corals (Glynn and D’Elia 1990,
Brown 1997, Hughes et al 2017) but in zoanthids as well (Kemp et al, 2006). This
experiment serves further confirmation of the bleaching response in P. toxica.
Combined Stress Effects
Nitrate and temperature enrichment in combination mostly continue the pattern in
Symbiodinium size displayed in the first experiment. In the most stressed conditions in
treatment 5, there is no significant difference from the control. Treatment 4 displayed the
pattern of Symbiodinium increase in size and decrease in size, which again supports the
idea that low amounts of nitrogen stimulate Symbiodinium growth. However, for the most
32
part there is not much significant difference in Symbiodinium sizes between treatments
within experiment 2.
It is clear that combined stressors within an environment cause further damage to
systems than singular stressors alone (Paine et al 1998, Hughes et al 1999). There has
been a movement in coral research to understand multiple stressors on coral in
combination, especially in combination with temperature (Coles and Jokiel, 1978, Porter
et al 1999, Wiedenmann et al 2012). It has been shown that elevated nitrogen increases
coral’s susceptibility to bleaching (Widenmann et al 2012). There has been some debate
in the past as to whether or not elevated nitrogen is unhealthy for coral reefs, with studies
that support very different ideas about nitrogen and coral. One study supports the idea
that low amounts of nitrogen stimulate coral growth (Atkinson et al 1995). Other studies
show that elevated nitrogen does not have a negative effect on coral growth (Szmant
2002, Bongiorni et al 2003). More recently it was shown that elevated nitrogen increases
bleaching severity and disease susceptibility (Vega-Thurber et al 2014).
According to the results of this study, however, there is a clear detrimental effect
of combined nitrate and temperature stress on coral. The instances of bleaching within the
second experiment outnumbered the instances of bleaching in experiment one, with
fourteen instances of bleaching across all treatments, as opposed to the eleven cases of
bleaching in experiment one. Furthermore, the vast majority of bleaching in experiment
one was moderate, with only a slight fading in zoanthid tissue color. Symbiodinium cells
were maintained within the coelenteron of the polyp rather than being expelled from the
polyp completely. Within the second experiment however, nearly every case of bleaching
was severe, with high degradation of coral tissues (to the point where samples were
33
unable to be histologically fixed). Finally, Symbiodinium were for the most part lysed,
and zoanthid tissue was nearly clear (Figure 16, 18), especially in high temperature
treatments. According to the results of this experiment, then, there is evidence that
elevated nitrate in combination with temperature is a bleaching trigger within Palythoa
toxica.
Conclusions
Overall, the results of this experiment confirm the effect of elevated temperature
as a bleaching trigger for P. toxica and provide evidence that elevated nitrate is a
potential bleaching trigger in prolonged exposure. Furthermore, there is evidence that
combined elevated nitrate and temperature compound bleaching mechanisms, with the
highest amount of bleaching occurring in the most stressed treatments.
A pattern of Symbiodinium size changes emerges within this experiment as well.
When exposed to low levels of nitrate and moderate temperature elevation, there is a
trend of Symbiodinium Feret diameter increase. With time, however, there is an eventual
collapse of growth, resulting in Symbiodinium diameter decrease and an increase of algal
mass potentially from cell lysis within bleached Symbiodinium remaining between
tentacles and within the coelenteron.
However, these conclusions come with major caveats. There is no replication
within this experiment between coral polyps with only two genetically identical polyps
sampled from the same water treatment. In future experiments, it will be necessary to
replicate treatments, polyps and genetic individuals to provide for good replication.
Furthermore, there was a significant amount of variation within treatments, so further
water quality maintenance methods must be developed, as coral prove very delicate in
34
care (as demonstrated by the bleaching that occurred in the control treatment near the end
of experiment 2). That being said, this experiment does provide preliminary evidence of
Symbiodinium growth dynamics when exposed to prolonged periods of nitrate and
temperature stress. It is likely that prolonged stress results in different dynamics of coral
adaptation to stressed conditions, and this project is part of that effort to understand
sublethal prolonged chronic stress conditions. Future experiments building on the
knowledge gained from this project are essential to understanding the true dynamic
between nitrogen, temperature and Symbiodinium.
35
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APPENDICES
41
APPENDIX 1: IMAGEJ FERET DIAMETER CALCULATIONS
Feret diameter, Feret’s diameter, or the caliper diameter of an object is a
measurement used for a non-circular object. It is the measurement between two furthest
points on the surface of the object. ImageJ was used to calculate this measurement, and in
this case, it was used to measure Symbiodinium.
Figure 1. A Hypothetical Symbiodinium
The perimeter of the Symbiodinium was used by the program to calculate Feret
diameter. The maximum and minimum Feret diameter is calculated by ImageJ by rotating
the object and drawing diameters every 2˚ of rotation. The coordinates of the two furthest
points are recorded, and the smallest diameter, the Feret minimum, is located. The angle
on which these two lines intersect is the Feret angle (Figure 2). These two Feret
measurements were then averaged for each Symbiodiniu, which were used for all size
analysis.
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Figure 2. The Feret measurements imageJ calculates.
However, ImageJ’s calculation of Feret diameter is not the true Feret
measurement, it is a very close approximation. Because the program measures by
rotation, there is a slight chance it can miss the true furthest points.
In order to find the true Feret maximum of a small object, in this case a
Symbiodinium, the perimeter of the object must be drawn. Because of the noncircular
nature of Symbiodinium, there are places where the cell has concave imperfections,
however the only points in the Symbiodinium that matter are convex imperfections, as the
calculation is for Feret maximum, and the maximum point will not occur at a concave
point. A perimeter can be traced where only the convex points of the cell shape touch the
perimeter, and all of these measurements can be compared until the Feret maximum is
calculated (Figure 4). This object is called the convex hull.
43
Figure 4. The convex hull of the hypothetical Symbiodinium.
However, this method is tedious for near-circular objects. In just this simple
example, the number of pairs a program would have to remember would be 7 choose 2,
or 21. In an object with several hundred convex points, for example, 150, a program
would have to measure 150 choose 2, or 11175.
An applicable theorem states that “the diameter of a convex figure is the greatest
distance between parallel lines of support” [Theorem 4.18, Preparata and Shamos,
Computational Geometry, 1985] reduces the number of pairs a program would have to
measure. A pair of points in an object that follows this theorem is called antipodal
vertices (Figure 3).
Figure 4. All of the antipodal vertex pairs of the hypothetical Symbiodinium.
44
The number of antipodal pairs the program would now have to measure is
reduced to four pairs, a much easier number of distances to measure and store. A similar
method is used to find the Feret minimum with concave points. Hypothetically, if there
was a small number of Symbiodinium, this method could be used to measure the actual
Feret diameter of each Symbiodinium cell.
That being said, there are a lot of data in this project, and that data was measured
under ImageJ’s assumptions of Feret diameter approximation, and is assumed to be
“close enough”.
References: Eddins, S. (2017). Feret Diameter: Introduction. Retrieved from https://blogs.mathworks.com/steve/2017/09/29/feret-diameter-introduction/ Ferriera, T., & Rasband, W. (2012). ImageJ. Retrieved from https://imagej.nih.gov/ij/docs/guide/146.html
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APPEDIX 2: R SCRIPT FOR STATISTICAL ANALYSIS
##ANOVA SET I #Trial 1 #fetching data #transfer all cvs from folder to desktop #note: Feret diameter was misspelled as ‘ferret’ in data column organization #thus ‘ferret’ was used during this program script in place of ‘feret’ setwd("~/Desktop") T1D4 <- read.csv("T1D4.csv", header = TRUE) T1D8 <- read.csv("T1D8.csv", header = TRUE) T1D12 <- read.csv("T1D12.csv", header = TRUE) T1D14 <- read.csv("T1D14.csv", header = TRUE) #make some boxplots library(ggplot2) ggplot(T1D4, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() ggplot(T1D8, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() ggplot(T1D12, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() ggplot(T1D14, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() #Trial 2 #fetching setwd("~/Desktop") T2D4 <- read.csv("T2D4.csv", header = TRUE) T2D8 <- read.csv("T2D8.csv", header = TRUE) T2D12 <- read.csv("T2D12.csv", header = TRUE) T2D14 <- read.csv("T2D14.csv", header = TRUE) #more box plots library(ggplot2) ggplot(T2D4, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() ggplot(T2D8, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() ggplot(T2D12, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() ggplot(T2D14, aes(x = factor(Tank), y = Ferret)) + geom_boxplot() #removed a bunch of outliers in excel to normalize #test for symmetry library(lawstat) levene.test(T1D4$Ferret, T1D4$Tank) levene.test(T1D8$Ferret, T1D8$Tank)
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levene.test(T1D12$Ferret, T1D12$Tank) levene.test(T1D14$Ferret, T1D14$Tank) levene.test(T2D4$Ferret, T2D4$Tank) levene.test(T2D8$Ferret, T2D8$Tank) levene.test(T2D12$Ferret, T2D12$Tank) levene.test(T2D14$Ferret, T2D14$Tank) #test for normality shapiro.test(T1D4$Ferret [T1D4$Tank=="A"]) shapiro.test(T1D4$Ferret [T1D4$Tank=="B"]) shapiro.test(T1D4$Ferret [T1D4$Tank=="C"]) shapiro.test(T1D4$Ferret [T1D4$Tank=="D"]) shapiro.test(T1D4$Ferret [T1D4$Tank=="E"]) shapiro.test(T1D8$Ferret [T1D8$Tank=="A"]) shapiro.test(T1D8$Ferret [T1D8$Tank=="B"]) shapiro.test(T1D8$Ferret [T1D8$Tank=="C"]) shapiro.test(T1D8$Ferret [T1D8$Tank=="D"]) shapiro.test(T1D8$Ferret [T1D8$Tank=="E"]) shapiro.test(T1D12$Ferret [T1D12$Tank=="A"]) shapiro.test(T1D12$Ferret [T1D12$Tank=="B"]) shapiro.test(T1D12$Ferret [T1D12$Tank=="C"]) shapiro.test(T1D12$Ferret [T1D12$Tank=="D"]) shapiro.test(T1D12$Ferret [T1D12$Tank=="E"]) shapiro.test(T1D14$Ferret [T1D14$Tank=="A"]) shapiro.test(T1D14$Ferret [T1D14$Tank=="B"]) shapiro.test(T1D14$Ferret [T1D14$Tank=="C"]) shapiro.test(T1D14$Ferret [T1D14$Tank=="D"]) shapiro.test(T1D14$Ferret [T1D14$Tank=="E"]) shapiro.test(T2D4$Ferret [T2D4$Tank=="A"]) shapiro.test(T2D4$Ferret [T2D4$Tank=="B"]) shapiro.test(T2D4$Ferret [T2D4$Tank=="C"]) shapiro.test(T2D4$Ferret [T2D4$Tank=="D"]) shapiro.test(T2D4$Ferret [T2D4$Tank=="E"]) shapiro.test(T2D8$Ferret [T2D8$Tank=="A"]) shapiro.test(T2D8$Ferret [T2D8$Tank=="B"]) shapiro.test(T2D8$Ferret [T2D8$Tank=="C"]) shapiro.test(T2D8$Ferret [T2D8$Tank=="D"]) shapiro.test(T2D8$Ferret [T2D8$Tank=="E"]) shapiro.test(T2D12$Ferret [T2D12$Tank=="A"]) shapiro.test(T2D12$Ferret [T2D12$Tank=="B"]) shapiro.test(T2D12$Ferret [T2D12$Tank=="C"]) shapiro.test(T2D14$Ferret [T2D14$Tank=="A"]) shapiro.test(T2D14$Ferret [T2D14$Tank=="B"]) shapiro.test(T2D14$Ferret [T2D14$Tank=="C"]) shapiro.test(T2D14$Ferret [T2D14$Tank=="D"]) shapiro.test(T2D14$Ferret [T2D14$Tank=="E"]) #boom #anova time anova.results <- aov(Ferret ~ Tank, data=T1D4) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Tank, data=T1D8)
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summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Tank, data=T1D12) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Tank, data=T1D14) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Tank, data=T2D4) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Tank, data=T2D8) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Tank, data=T2D12) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Tank, data=T2D14) summary(anova.results) TukeyHSD(anova.results) ##ANOVA SET II #bring in the data setwd("~/Desktop") T1 <- read.csv("T1.csv", header = TRUE) T2 <- read.csv("T2.csv", header = TRUE) T3 <- read.csv("T3.csv", header = TRUE) T4 <- read.csv("T4.csv", header = TRUE) T5 <- read.csv("T5.csv", header = TRUE) #all the data is already normally distributed and symmetrical so anova time anova.results <- aov(Ferret ~ Day, data=T1) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Day, data=T2) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Day, data=T3) summary(anova.results) TukeyHSD(anova.results) anova.results <- aov(Ferret ~ Day, data=T4) summary(anova.results) TukeyHSD(anova.results)
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anova.results <- aov(Ferret ~ Day, data=T5) summary(anova.results) TukeyHSD(anova.results) ##now for the final figures #Experiment 1: Nitrogen Figures setwd("~/Desktop") ND4 <- read.csv("ND4.csv", header = TRUE) ND8 <- read.csv("ND8.csv", header = TRUE) ND12 <- read.csv("ND12.csv", header = TRUE) ND14 <- read.csv("ND14.csv", header = TRUE) library(ggplot2) ggplot(ND4, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() ggplot(ND8, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() ggplot(ND12, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() ggplot(ND14, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() #Experiment 1: Temperature setwd("~/Desktop") TD4 <- read.csv("TD4.csv", header = TRUE) TD8 <- read.csv("TD8.csv", header = TRUE) TD12 <- read.csv("TD12.csv", header = TRUE) TD14 <- read.csv("TD14.csv", header = TRUE) library(ggplot2) ggplot(TD4, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() ggplot(TD8, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() ggplot(TD12, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() ggplot(TD14, aes(x=factor(Treatment), y=Feret)) + theme_classic() + geom_boxplot() #end script
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AUTHOR’S BIOGRAPHY
Molly Westbrook grew up in Ithaca, NY with a huge passion for the natural
sciences. They went on to attend the University of Maine, majoring in Marine Science
with concentrations in Aquaculture and Marine Biology, and minoring in Mathematics.
Molly’s done several things while at UMaine, including co-founding the UMaine Coral
Club and acting as Treasurer for the first year, DJing at WMEB 91.9 fm, performing as a
singer-songwriter in the greater Orono area, studying abroad at James Cook University in
Australia, working in many labs, analyzing many datasets, and playing many board
games. They had to write a lot of bios when they were a stage manager throughout
middle school and high school, and ten years later they’re still not great at it.