Characterizing Potential Bacterial Pathogens of Pisaster Ochraceus Sea Stars with Wasting Disease
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Western Washington University Western CEDAR WWU Graduate School Collection WWU Graduate and Undergraduate Scholarship Fall 2018 Characterizing potential bacterial pathogens of Pisaster ochraceus sea stars with wasting disease Chelsea Hutchinson Western Washington University, [email protected] Follow this and additional works at: https://cedar.wwu.edu/wwuet Part of the Biology Commons Recommended Citation Hutchinson, Chelsea, "Characterizing potential bacterial pathogens of Pisaster ochraceus sea stars with wasting disease" (2018). WWU Graduate School Collection. 803. https://cedar.wwu.edu/wwuet/803 This Masters Thesis is brought to you for free and open access by the WWU Graduate and Undergraduate Scholarship at Western CEDAR. It has been accepted for inclusion in WWU Graduate School Collection by an authorized administrator of Western CEDAR. For more information, please contact [email protected]. Characterizing potential bacterial pathogens of Pisaster ochraceus sea stars with wasting disease By Chelsea Hutchinson Accepted in Partial Completion of the Requirements for the Degree Master of Biology ADVISORY COMMITTEE Dr. Marion Brodhagen, Chair Dr. Benjamin Miner Dr. Deborah Donovan GRADUATE SCHOOL Dr. Gautam Pillay, Dean Master’s Thesis In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Western Washington University, I grant to Western Washington University the non-exclusive royalty-free right to archive, reproduce, distribute, and display the thesis in any and all forms, including electronic format, via any digital library mechanisms maintained by WWU. I represent and warrant this is my original work and does not infringe or violate any rights of others. I warrant that I have obtained written permissions from the owner of any third party copyrighted material included in these files. 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Chelsea Hutchinson September 13, 2018 Characterizing potential bacterial pathogens of Pisaster ochraceus sea stars with wasting disease A Thesis Presented to The Faculty of Western Washington University In Partial Fulfillment Of the Requirements for the Degree Master of Science by Chelsea Hutchinson September 13, 2018 Abstract In 2013 sea star wasting disease (SSWD) caused an epizootic in over 20 species of asteroids along the west coast of North America. To see if SSWD was still affecting wild populations, we surveyed populations of the sea star, Pisaster ochraceus, in Birch Bay, WA. Our surveys indicated that advanced symptoms (lesion formation) increased 73% from July to September in 2017. To understand the role of bacteria in SSWD, we isolated bacteria with tissue-degrading potential from epidermal tissues of P. ochraceus animals in Birch Bay, WA. Next, we identified these isolates via 16S rRNA gene sequencing. Our results indicated that the community structure differed between SSWD-affected and healthy stars. In addition, we found that many of the Vibrio species isolated primarily from diseased sea stars lyse red blood cells, avidly degrade gelatin, and are related to species that cause disease in other marine animals. Other researchers have suggested that SSWD is caused by a microbial agent smaller than most bacterial cells (0.2 microns). Although none of the bacteria we isolated were smaller than 0.2 microns, we theorized that their signaling molecules could transmit disease. Quorum sensing signals (QSSs) are bacterially-produced, autoinducing molecules that regulate gene expression in a population- dependent manner within bacterial populations. We hypothesized that quorum sensing signals from diseased animals could facilitate development of SSWD on healthy individuals by triggering expression of virulence genes in opportunistic bacterial pathogens creating a state of dysbiosis. We tested bacterial isolates and tissue extracts from diseased and healthy sea stars for the presence of a subset of QSSs, acyl homoserine lactones (AHLs). We found that organic extracts from healthy and diseased stars differed in how strongly they activated a QSS bioreporter, and that multiple of the bacterial isolates associating with diseased stars produced AHLs. Thus, quorum sensing might contribute to transmission of SSWD. iv Acknowledgements Many undergraduate research assistants donated their time to facilitate this thesis work and we wholeheartedly thank them for their contributions. Fran Carpenter provided valuable assistance in the sea star surveys as well as caring for animals housed in aquaria. Trevor Grimm assisted to PCR amplify the 16S rRNA gene from bacteria isolates. Kayla Russell assisted with aligning sequences and optimizing techniques to sterilize the surfaces of sea star epidermis tissues for use in bioassays. Emma Kooyman contributed her time monitoring sea stars and maintaining aquaria for experiments. Serena Riley, Alastair Laing, Nathan Hannah, and Logan Wallace assisted in multiple experiments involving testing the abilities of isolated bacteria to degrade gelatin and other media. Cassandra McHugh assisted with many aspects of this study including testing the isolates for production of hemolysins and SSWD surveys and sample collection. Beth Loper played a key role in determining morphology of our bacterial isolates and provided valuable assistance in many experiments. A special thanks goes to stockroom employees Peter Thut, Sam Danforth, Erin Macri, and Joe Somera for their assistance with getting materials and equipment to complete this thesis work. In addition, I would like to thank Western Washington University and specifically the Biology Department of WWU for the use of facilities as well as generously providing me with funding for Sanger sequencing of the 16S rRNA gene and quorum sensing bioassay reagents. We would also like to thank the North Cascades Audubon Society for funding the 16S rRNA gene sequencing. Zoë Zilz contributed to this thesis work by teaching molecular biologists how to survey sea stars and she generously helped us collect animals for use in this study. Finally, we would like to thank the entire Pete Greenberg lab at the University of Washington and especially Amy Schaefer for her invaluable insights, teaching us the techniques for testing our samples for quorum sensing activity, and providing us with the quorum sensing bioreporter A. tumefaciens strain KYC55 (pJZ410)(pJZ384)(pJZ372) used in this study. v Table of Contents Abstract .......................................................................................................................................... iv Acknowledgements ..........................................................................................................................v List of Tables and Figures............................................................................................................. vii Introduction .................................................................................................................................. 1-6 Methods...................................................................................................................................... 7-16 Results ...................................................................................................................................... 17-21 Discussion ................................................................................................................................ 21-32 Works Cited ............................................................................................................................. 33-39 Tables and Figures ................................................................................................................... 40-51 Appendix A ....................................................................................................................................52 vi List of Tables and Figures Title Page # Table 1. Primers used for PCR and Sanger sequencing of the 16S rRNA gene 40 Table 2. Results of Sanger sequencing of the 16S rRNA genes of bacterial isolates 41-43 Table 3. Phenotypes of representative bacterial isolates 44 Figure 1. Geographic region sampled in surveys 45 Figure 2. SSWD scoring system for P. ochraceus stars 46 Figure 3. Sequence alignment strategy of the 16S rRNA gene 47 Figure 4. SSWD progression at Point Whitehorn, WA 48 Figure 5. Genera of gelatin-degrading bacteria by tissue zone 49 Figure 6. Prevalence, gelatin degradation, and AHL production of isolates from the 50 epidermal tissues of P. ochraceus. Figure 7. Activation of the bioreporter Agrobacterium tumefaciens KYC55 51 (pJZ410)(pJZ384)(pJZ372) by organic tissue extracts taken from P. ochraceus arms or seawater Supplementary Table 1. Results of Sanger sequencing of the 16S rRNA genes of 52 bacterial isolates, sequences that were short or lacked two-fold coverage vii Introduction Sea star wasting disease (SSWD). Sea star wasting disease describes a particular set of signs that researchers have observed in asteroid populations since the 1970’s