OXYGEN OPTODE ANALYSIS OF CORAL-ALGAL INTERACTIONS

71 Pages·2013·3.67 MB·English

OXYGEN OPTODE ANALYSIS OF CORAL‐ALGAL INTERACTIONS
____________________
A Thesis
Presented to the
Faculty of
San Diego State University
____________________
In Partial Fulfillment
of the Requirements for the Degree
Master of Science in Biology
with a Concentration in
Molecular Biology
____________________
by
Allison Kathleen Gregg
Fall 2013

iii
Copyright © 2013
by
Allison Kathleen Gregg
All Rights Reserved
iv
DEDICATION
This thesis is dedicated to my parents, Karin and Roy, whose unwavering
support is why I was able to accomplish this. And to my Dad, Chris, who passed down to
me a love of the ocean I never realized I had.
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We shall not cease from exploration, and the end of all our exploring
will be to arrive where we started and know the place for the first time.
‐‐ T.S. Eliot
Little Gidding
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ABSTRACT OF THE THESIS
Oxygen Optode Analysis of Coral‐Algal Interactions
by
Allison Kathleen Gregg
Master of Science in Biology with a Concentration in Molecular
Biology
San Diego State University, 2013

Coral reefs contain a diverse consortium of benthic macro‐organisms including
scleractinian corals, fleshy algae, calcifying algae, and their associated microbiotas.
These organisms are in a constant battle, competing for space and resources. Over the
last 50 years, fleshy algae have increasingly been shown to outcompete corals;
however, the mechanisms are not completely known. Algal‐derived dissolved organic
matter (DOM) can induce mortality of reef building corals. One proposed killing
mechanism is a zone of hypoxia created by rapidly growing microbes. To investigate
this hypothesis, oxygen optodes were used to investigate dissolved oxygen
concentrations at coral‐algal interfaces, as well as how the biological oxygen demand
(BOD) of coral‐associated microbial communities are affected by algal DOM. Oxygen
optodes were visualized with a novel, low‐cost Submersible Oxygen Optode Recorder
(SOOpR) system, which is capable of accurately measuring oxygen concentrations in the
lab or in situ. The BOD studies examined the effects of two types of algal organic matter;
turf algae, shown to be detrimental to corals, and crustose coralline algae (CCA), which
have been shown to be beneficial. This investigation of oxygen dynamics of coral‐algal
interactions shows that algae can create complex oxygen dynamics that change with
flow, and when in close contact with coral, the interface becomes hypoxic. When
microbial communities from coral are exposed to turf algal exudate and CCA exudate,
exudates from turf algae elicit the greatest BOD. Together, the results of these studies
show that coral‐algal interaction zones can be hypoxic, and this is due in some part to
microbial activity.
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TABLE OF CONTENTS
PAGE
ABSTRACT ........................................................................................................................................................... vi
LIST OF FIGURES ................................................................................................................................................ x
CHAPTER
1 INTRODUCTION .................................................................................................................................. 1
1.1 Microbes and DOC— Quantity or Quality? ............................................................... 1
1.2 Microbially‐Mediated Hypoxia— The DDAM Model ............................................ 2
1.3 Using Planar Optodes to Study Coral Reefs .............................................................. 3
2 VISUALIZATION OF OXYGEN AT CORAL‐ALGAL INTERFACE ........................................ 5
2.1 Introduction ........................................................................................................................... 5
2.2 Materials and Methods ...................................................................................................... 6
2.2.1 Experimental Design ............................................................................................... 6
2.2.2 Planar Optode Images ............................................................................................. 7
2.2.3 Preparation of Optodes .......................................................................................... 9
2.2.4 Image Analysis ........................................................................................................... 9
2.3 Results and Discussion .................................................................................................... 10
2.4 Conclusions .......................................................................................................................... 15
2.5 Acknowledgements ........................................................................................................... 16
3 BIOLOGICAL OXYGEN DEMAND OF CORAL‐ASSOCIATED MICROBES ..................... 17
3.1 Introduction ......................................................................................................................... 17
3.2 Materials and Methods .................................................................................................... 18
3.2.1 Lighting and Camera System ............................................................................. 18
3.2.2 Construction of BOD Optode Plates ................................................................ 19
3.2.3 Calibration of Optodes .......................................................................................... 19
3.3 Experiment 1‐ Algal Exudate Studies ........................................................................ 19
3.4 Experiment 2‐ Single Strain Studies .......................................................................... 22
3.5 Experiment 3‐ Different Organism‐Associated Bacteria
Assemblage Study .............................................................................................................. 23
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3.5.1 Sequencing of M. Annularis Isolates ............................................................... 23
3.5.2 Image Analysis ......................................................................................................... 24
3.5.3 Statistical Analyses ................................................................................................ 24
3.6 Results and Discussion .................................................................................................... 25
3.6.1 Validation of Camera System ............................................................................. 25
3.6.2 Bacterial Isolates Respond Differently to Turf Algal Exudate ............. 25
3.6.3 Bacterial Communities Cultured from Coral, Turf Algae, and
CCA Respond Similarly to Turf Algal Exudate ............................................ 26
3.6.4 Turf Algae Elicit the Greatest Oxygen Drawdown by Coral‐
Associated Bacteria ................................................................................................ 26
3.6.5 Caveats ........................................................................................................................ 31
3.7 Conclusions .......................................................................................................................... 32
3.8 Acknowledgements ........................................................................................................... 33
4 OXYGEN OPTODES FOR IN SITU APPLICATION .................................................................. 34
4.1 Introduction ......................................................................................................................... 34
4.2 Materials and Methods .................................................................................................... 34
4.2.1 Preparation of Optode Strips and Contour Gauge .................................... 35
4.2.2 Lighting and Camera Settings ............................................................................ 36
4.2.3 Image Analysis ......................................................................................................... 37
4.2.4 Calibration of Optodes .......................................................................................... 37
4.3 Results and Discussion .................................................................................................... 38
4.4 Conclusions .......................................................................................................................... 38
5 CONCLUSIONS AND FUTURE DIRECTIONS .......................................................................... 42
5.1 Recommendations for Future Directions ................................................................ 43
5.2 Conclusion............................................................................................................................. 44
ACKNOWLEDGEMENTS ............................................................................................................................... 46
REFERENCES ..................................................................................................................................................... 49
APPENDICES
A SCRIPTS ................................................................................................................................................ 56
B SUPPLEMENTARY DATA ............................................................................................................... 60
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LIST OF FIGURES
PAGE
Figure 2.1. Experimental setup. .................................................................................................................. 8
Figure 2.2. Oxygen patterns over coral and algae during light and dark. ............................... 11
Figure 2.3. Algae generated oxygen patterns under flow conditions. ...................................... 13
Figure 2.4. Oxygen patterns generated by coral algae interaction. ........................................... 14
Figure 3.1. Schematic of the SOOpR system. ....................................................................................... 20
Figure 3.2. Dissolved oxygen drawdown by four single bacterial strains isolated
from M. annularis and exposed to turf exudate. .................................................................. 21
Figure 3.3. Dissolved oxygen drawdown by bacterial communities cultured from
turf, coral and CCA. .......................................................................................................................... 27
Figure 3.4. Experimental biological oxygen demand (BOD) plates. .......................................... 28
Figure 3.5. Dissolved oxygen drawdown by coral‐associated bacteria when
exposed to different algal exudate treatments. ................................................................... 29
Figure 4.1. In situ oxygen optode strips. ............................................................................................... 35
Figure 4.2. In situ oxygen patterns generated above algal bed. .................................................. 39
Figure 4.3. In situ oxygen patterns generated around coral‐algal interface. ......................... 40
Figure A.1. Matlab script. ............................................................................................................................. 57
Figure A.2. Ultra intervalometer CHDK script. ................................................................................... 58
Figure B.1. Example of optode calibration curve. ............................................................................. 61
Figure B.2. Spectrum of oxygen optode excited by lighting system. ......................................... 62
1
CHAPTER 1
INTRODUCTION
Coral reefs are home to a rich diversity of life forms, from microscopic bacteria
to Goliath groupers and sharks. The reef structure itself is made up of calcifying
organisms such as Scleractinian corals and crustose coralline algae (CCA), as well as
sponges, fleshy macroalgae, and turf algal mats. These organisms are in a constant
battle competing for resources and space, with the outcome of these battles shaping the
benthos (Lang and Chornesky 1990, Tanner 1995, McCook et al. 2001, Chadwick and
Morrow 2011, Barott and Rohwer 2012). When anthropogenic impacts such as
overfishing and eutrophication increase, fleshy algae gain a competitive advantage over
calcifying organisms (Lirman 2001, Littler et al. 2006, Barott et al. 2012). These
stressors are associated with coral‐ algal phase shifts worldwide (McClanahan et al.
2002, Dulvy et al. 2004, Sandin et al. 2008, Vermeij et al. 2010), whereby abundances of
fleshy macro‐ and turf algae increase and subsequently lead to coral mortality (Done
1992, Harvell et al. 1999, Pandolfi et al. 2003, Harvell et al. 2007, Hughes et al. 2007).
1.1 MICROBES AND DOC— QUANTITY OR QUALITY?
Algal‐dominated reefs are associated with elevated microbial abundances
(Dinsdale et al. 2008, Bruce et al. 2012) and greater microbial energy usage (McDole et
al. 2012). Increased microbial growth on algal‐dominated reefs have been attributed to
higher release rates of bioavailable dissolved organic carbon (DOC) by fleshy algae
compared to calcifying reef organisms such as coral and CCA, as this algal‐ derived DOC
promotes microbial growth and respiration (Wild et al. 2010, Haas et al. 2011, Nelson et
al. 2013). Further, experimental additions of bioavailable DOC (e.g., glucose, algal
exudates) resulted in increased microbial growth followed by coral mortality (Kline et
al. 2006). These effects were prevented by the addition of antibiotics (Smith et al.
2006), suggesting microbially mediated mechanisms are involved.