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Master's Thesis – E. Millar; Mcmaster University – Biology the EFFECTS of WASTEWATER TREATMENT PLANT EFFLUENT on THE

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Master's Thesis – E. Millar; Mcmaster University – Biology the EFFECTS of WASTEWATER TREATMENT PLANT EFFLUENT on THE Master’s Thesis – E. Millar; McMaster University – Biology THE EFFECTS OF WASTEWATER TREATMENT PLANT EFFLUENT ON THE GUT MICROBIOME OF AQUATIC AND RIPARIAN INVERTEBRATES IN THE GRAND RIVER, ON Master’s Thesis – E. Millar; McMaster University – Biology THE EFFECTS OF WASTEWATER TREATMENT PLANT EFFLUENT ON THE GUT MICROBIOME OF AQUATIC AND RIPARIAN INVERTEBRATES IN THE GRAND RIVER, ON By ELISE MILLAR Bachelor of Science in Biochemistry and Molecular Biology, Trent University, 2018 A Thesis Submitted to the School of Graduate Studies in Partial Fulfilment of the Requirements for the Degree of Master of Science in the Graduate Academic Unit of Biology Supervisor: Karen Kidd, Ph.D., Dept of Biology and School of Earth, Environment, and Society at McMaster University, and Canadian Rivers Institute Examining Patricia Gillis, Ph.D., Aquatic Contaminants Research Division, Committee: Environment and Climate Change Canada Michael Surette, Ph.D., Dept of Biochemistry and Biomedical Sciences, McMaster University McMaster University © Copyright by Elise Millar, August 2020 Master’s Thesis – E. Millar; McMaster University – Biology McMaster University MASTER OF SCIENCE (2020) Hamilton, Ontario (Biology) TITLE: The Effects of Wastewater Treatment Plant Effluent on the Gut Microbiome of Aquatic and Riparian Invertebrates in the Grand River, ON AUTHOR: Elise Millar, B.Sc. (Trent University) SUPERVISOR: Professor K.A. Kidd NUMBER OF PAGES: xix, 144 ii Master’s Thesis – E. Millar; McMaster University – Biology ABSTRACT The composition of gut microbes affects host weight, immune function, and disease status, and is sensitive to diet, environment, and pharmaceutical exposure. The gut microbiome modulates the toxicity and bioavailability of chemical stressors, however the effects of chemicals on the gut microbiome of aquatic biota are largely unknown. The Waterloo and Kitchener wastewater treatment plants (WWTPs) release effluents containing antibiotics, pharmaceuticals, and other contaminants into the Grand River (ON) that may negatively affect the gut microbiome of downstream organisms. In this study done in Fall 2018, I collected freshwater mussels (Lasmigona costata), several species of insect larvae, and riparian spiders (Tetragnathidae) from sites upstream and downstream of these WWTPs. The gut microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16S rRNA genetic barcode. Changes in the relative abundance of major gut microbiome phyla were observed in all targeted aquatic organisms downstream of WWTPs except Hydropsychidae. Shannon alpha diversity, a measure of bacterial abundance and evenness, differed significantly among sites for mussels (one-way ANOVA: F=7.894, p=0.001), spiders (F=4.788, p=0.01), Perlidae (F=3.1, p=0.0056), Hydropsychidae (F=3.674, p=0.0014), and Heptageniidae (F=2.715, p=0.0143), but not for Baetidae and Ephemerellidae. In sites downstream of the Waterloo WWTP, alpha diversity decreased in spiders, while in sites downstream of the Kitchener WWTP diversity decreased in mussels and Perlidae, while increasing for spiders. Bray- Curtis beta diversity, a measure of dissimilarity between bacterial communities, was significantly dissimilar among sites in all invertebrate taxa (Permanova: p<0.02). Upstream sites differed from downstream Waterloo sites in spiders, Perlidae, and Hydropsychidae (Adonis pairwise: p<0.05), while upstream mussels, spiders, Perlidae, and Hydropsychidae differed from downstream Kitchener sites (p<0.05). Additionally, effluent-derived bacteria were found in the microbiomes of aquatic invertebrates downstream of the WWTPs and not upstream. Taxa was also a significant driver of iii Master’s Thesis – E. Millar; McMaster University – Biology bacterial composition and diversity in invertebrates. These results indicate that the gut microbiome of downstream organisms differed from the bacterial composition observed in the same invertebrate taxa upstream of the WWTPs, potentially leading to altered host health. This adds to our understanding of how chemical stressors impact the gut microbiome of aquatic and riparian biota; however, future studies are needed to investigate linkages between the gut microbiome and health of these species. iv Master’s Thesis – E. Millar; McMaster University – Biology ACKNOWLEDGEMENTS Firstly, I would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC), the Jarislowski Foundation, McMaster University, and the McMaster Graduate Student Association (GSA) for funding myself and this research project. I would like to thank my supervisor, Dr. Karen Kidd, for her patience and support throughout my time as a graduate student. She provided me with invaluable guidance throughout our mentor-student relationship, which was integral for the success of my research. She has also provided me with numerous opportunities to showcase my research and I feel that she has prepared me for success in my field as a young scientist. I would also like to thank my advisory committee members, Dr. Patty Gillis and Dr. Michael Surette. Their breadth of knowledge and expertise have helped guide my project and expand my own understanding in these fields. Their input and comments on my project have been invaluable in the development of my research. In particular, I would like to thank Dr. Gillis for collaborating with me on method development experiments regarding the effect of dissection time on the digestive gland microbiome of mussels as well as feeding experiments to better understand their digestive passage. I would like to thank Jim Bennett and Joe Salerno from the Gillis Lab at Environment and Climate Change Canada for their help in collecting mussels (no matter the weather), as well as the support and knowledge they provided me throughout my project. I would also like to thank Michelle Shah and Laura Rossi from the Surette Lab at McMaster University for their immense help and guidance in the collection, processing, and navigation of my microbiome data. I would like to thank Dr. Mark Servos, Hadi Dhiyebi, Leslie Bragg, and the rest of the Servos Lab for their help with site selection, sampling, and field work logistics. I would also like to thank Dr. Paul Sibley for his help in the taxonomic identification of some of my more difficult invertebrates, as well as Dr. Jonathon Pruitt for his guidance in the capture of riparian spiders in the field. v Master’s Thesis – E. Millar; McMaster University – Biology I would like to thank everyone who helped me along the way with field work, logistics, data analysis, and presentation practice including Kathy Aleksa, Eliza Wu, Shawn Kroetsch, Craig Irwin, Kelli Charbonneau, Celine Lajoie, Ellie Weir, Alex Chan, Graydon McKee, Stephanie Varty, and Lauren Negrazis. I would also like to thank my family, especially my parents Nancy and Glenn, and friends for their constant encouragement and support, as well as my partner Cole, who found himself helping me catch more creepy crawlers than I’m sure he’d planned on during my university career. Finally, I would like to give a special thank you to Victoria Restivo who helped me every step of the way, my partner in diving into the world of the gut microbiome. I could not have completed this project without your knowledge, support, and friendship these last two years. vi Master’s Thesis – E. Millar; McMaster University – Biology Table of Contents ABSTRACT ........................................................................................................................ iii ACKNOWLEDGEMENTS ................................................................................................. v Table of Contents ............................................................................................................... vii List of Tables ...................................................................................................................... xi List of Figures .................................................................................................................... xv The effects of wastewater treatment plant effluent on the gut microbiome of aquatic and riparian macroinvertebrates in the Grand River, ON ........................................................... 1 1. Introduction .................................................................................................................. 1 1.1. General Introduction to the Gut Microbiome ....................................................... 1 1.2. Gut Microbiome of Aquatic Invertebrates and Riparian Spiders ......................... 2 1.2.1. Aquatic Macroinvertebrates ........................................................................... 2 1.2.2. Mussels .......................................................................................................... 4 1.2.3. Riparian Spiders ............................................................................................. 5 1.3. Disruptions of the Gut Microbiome ...................................................................... 7 1.4. Wastewater and The Grand River Waterway ....................................................... 9 1.5. Study Rationale ................................................................................................... 13 1.6. Study Objectives ................................................................................................. 14 2. Methods .....................................................................................................................
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