Diversity and Dynamics of Algal Viruses in the Bay of Quinte - T-Space

82 Pages·2013·2.14 MB·English

Diversity and Dynamics of Algal Viruses
in the Bay of Quinte

By

Robin Marie Rozon

A thesis submitted in conformity with the requirements
for the degree of Master of Science
Ecology and Evolutionary Biology
University of Toronto

© Copyright by Robin Marie Rozon 2013
Diversity and Dynamics of Algal Viruses in the Bay of Quinte

Robin Marie Rozon
Master of Science
Ecology and Evolutionary Biology
University of Toronto
2013

Abstract
To initiate algal virus research in the Bay of Quinte, three stations were sampled biweekly
throughout 2011. By targeting algal virus DNA polymerase, major capsid protein genes (MCP),
and a Microcystis aeruginosa cyanophage (Ma-LMM01) tail sheath protein gene, PCR
amplification revealed diverse and unique Phycodnaviruses (viruses of eukaryotic algae) and
cyanophage. When analysed statistically, patterns of virus abundance suggested that the
seasonality of any one virus cannot be generalised to predict that of other viruses, even among
closely related viruses. This study also demonstrated a strong relationship between algal virus
abundance and host biomass. It was found that despite the apparent heterogeneity of virus
abundance across the Bay, virus abundance patterns clustered by sampling date and geographic
location. By providing evidence for diverse algal viruses with complex seasonality, this work
highlights significant gaps in the current understanding of Bay of Quinte phytoplankton
ecology.

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Acknowledgements
First and foremost I would like to thank my supervisor Dr. Steven Short for making this
‘drive-by Masters’ the most rewarding and intellectually challenging experience of my life. It is
because of your enthusiasm that I came to UTM, it was with your encouragement that I learned
to learn from my mistakes, and without your guidance I would never had been able to finish this
thesis. Because of your passion for science I understand what it means to work hard and to truly
love what you do; it’s viral! I am forever grateful for your patience and commitment to my
education. Thank you for welcoming me into your lab family and will never forget what we’ve
accomplished here.
Thank you to the members of my supervisory committee, Dr. Peter Kotanen and Dr.
Nick Collins. Your insights, recommendations and comments throughout my graduate degree
have improved my thesis and my skills as a scientist. I hope I can be as beneficial to others in
the future as you have both been to me.
To Cindy Short, Michael Staniewski, and all the past and present members of the Short
lab. While my stay with all of you was ‘short’-er than other programs, you deserve more than a
‘short’ expression of gratitude. In all seriousness, I thank you from the bottom of my heart for
putting up with my endless questions and helping me at every step of my journey. You are the
heart and soul of our work; the lab would have been lonely without you. I will miss our lab
lunches and geeking out with other like-minded souls. I wish you all the best for your future
endeavours and expect many great things from all of you. Finally, thank you for teaching me
that “adversity builds character; if everything went right all the time you’d be as interesting as a
carrot”. People like you are rare in this world and I look forward to many lasting friendships.
A very big thank you goes to Dr. Mohi Munawar and the people at DFO-GLLFAS who
made this collaboration possible. Without the encouragement received from Mark Fitzpatrick
and Heather Niblock I would never have come this far. To the GLLFAS field and lab crew,
Robert Bonnell, Ashley Bedford and Michele Burley, the DFO samples were collected,
processed and analysed on your backs and I am forever grateful. To the smiling faces of Lisa
Elder, Jennifer Lorimer, Ron Dermott, Kelly Bowen, Marten Koops, and the staff at CCIW
Burlington, thank you for your encouragement throughout my undergraduate and graduate
degrees, and for making my time with you so unforgettable.
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Last but not least thank you to my family and friends who supported me through the
good times and the bad. Thank you for being a sympathetic ear when I needed it and a kick in
the pants when I needed that too. Special thanks to Nathaniel Costa for your understanding and
patience; I owe my current sanity to you. Also, thank you for use of your ‘workspace’ and
assorted computer programs. To my parents, thank you for tolerating my rants against physics,
statistics, traffic jams and incidences of personal electrical interference. Most of all, I truly
appreciate your support over the last six years; it’s the best feeling in the world when your
parents can tell other people, “my daughter is working on her Masters degree. Something about
the little dudes in the water… I think.”

Robin

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Dedication
To my parents
Je t’aime toujours
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Table of Contents
Abstract ii
Acknowledgements iii
Table of Contents vi
List of Tables viii
List of Figures ix
List of Abbreviations x
Chapter 1: Introduction
The Bay of Quinte
A Historical Perspective 1
Environmental Degradation 1
Project Quinte 3
Phytoplankton in the Bay of Quinte
Research History 3
Historical Trends 5
Aquatic Viruses
Historical Perspective on Aquatic Virus Research 6
Virus Diversity and Dynamics in Aquatic Systems 9
Objectives and Research Questions 11
Chapter 2: Materials and Methods
Study Sites and Sample Collection 12
PCR, Cloning and Sequencing
AVS 13
MCP 14
Sheath 14
Sequence Analysis and Primer Design 16
Quantitative PCR 17
Analysis 18
vi
Chapter 3: Results
Algal Virus Diversity Study
Algal virus DNA polymerase genes 20
Algal virus major capsid protein genes 20
Microcystis phage sheath protein genes 21
Quantitative Analysis of Viral Dynamics
Virus Dynamics across Time and Space 21
Statistical Analysis of Viral Dynamics 23
Virus Abundance and Host Biomass 24
Clustering by Taxonomic Classification 25
Clustering by Spatial Distribution 26
Chapter 4: Discussion
Algal Virus Diversity Study 27
Quantitative Analysis of Viral Dynamics
Virus Dynamics across Time and Space 31
Statistical Analysis of Viral Dynamics 35
Virus Abundance and Host Biomass 37
Clustering by Taxonomic Classification 39
Clustering by Spatial Distribution 41
Summary 42
Future Directions 44
Literature Cited 45
vii
List of Tables
Table 1. Limnological Divisions of the Bay of Quinte 52
Table 2. Sample Locations 53
Table 3. Reagents used in PCR reactions 54
Table 4. Thermocycling parameters for PCR reactions 55
Table 5. Quantitative Primers and Probes 56
Table 6. Reagents used in quantitative PCR 58
Table 7. Test of Primer and Probe Specificity 59
Table 8. Friedman analysis of virus abundances between stations 60
viii
List of Figures
Figure 1. Map of the Bay of Quinte 61
Figure 2. Neighbor joining phylogeny polB 62
Figure 3. Neighbor joining phylogeny MCP 63
Figure 4. Abundances of individual virus genes plotted against time 64
Figure 5. Virus gene abundances at each station plotted against time 65
Figure 6. Virus gene abundance 66
Figure 7. Regression of the sum of virus gene abundances on chlorophyll a. 67
Figure 8. Cluster analysis of virus abundance 68
Figure 9. A comparison of different proximity measures 69
Figure 10. Cluster analysis of biweekly stations 70
Figure 11. Cluster analysis of spatial stations 71

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List of Abbreviations
ºC – Degree(s) Celsius GLLFAS – Great Lakes Laboratory for
Fisheries and Aquatic Sciences
µ - micro (10-6)
GLWQA – Great Lakes Water Quality
µL – microliter(s) Agreement
µm – micrometers GPS – Global Positioning System
µM – micromolar HAB(s) – Harmful Algal Blooms
AOC – Area of Concern HB – Hay Bay sampling station
B – Belleville sampling station HCl – Hydrogen chloride
BLAST – Basic Local Alignment Search Tool IBM SPSS – International Business
Machines’s Statistical Package
bp – Base pairs for the Social Sciences
BQ – Bay of Quinte IJC – International Joint Commission
cm – centimeters IPTG - Isopropyl β-D-1-
thiogalactopyranoside
Ct – Cycle threshold
JTT – Jones-Taylor-Thornton
DFO – Fisheries and Oceans Canada
L – liter(s)
DH5α – E. coli strain of competent cells
LB agar - Lysogeny broth agar
DNA – deoxyribonucleic acid
LE agarose – Low electroendosmosis agarose
dNTP – deoxyribonucleotide triphosphate
LO – Lake Ontario
dsDNA – Double stranded DNA
M – Molarity
EDTA – ethylenediaminetetraacetic acid
m – meter(s)
FAM – 6-carboxyfluorescein
Ma-LMM01 – Strain of Microcystis
aeruginosa cyanophage
FQ – Fluorescein quencher
MCP – Major capsid protein
g – gram(s)
x