Synergistic Effects of Temperature and Salinity on the Gene Expression
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Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 10-23-2018 Synergistic Effects of Temperature and Salinity on the Gene Expression and Physiology of Crassostrea virginica Hollis Jones Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Marine Biology Commons, Molecular Genetics Commons, and the Other Physiology Commons Recommended Citation Jones, Hollis, "Synergistic Effects of Temperature and Salinity on the Gene Expression and Physiology of Crassostrea virginica" (2018). LSU Master's Theses. 4819. https://digitalcommons.lsu.edu/gradschool_theses/4819 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. SYNERGISTIC EFFECTS OF TEMPERATURE AND SALINITY ON THE GENE EXPRESSION AND PHYSIOLOGY OF CRASSOSTREA VIRGINICA A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College In partial fulfillment of the requirements for the degree of Master of Science in The Department of Biological Sciences by Hollis Renee Jones B.S., Northeastern University, 2015 December 2018 Acknowledgements I would first like to thank my major advisor, Dr. Morgan Kelly, for her expert guidance throughout the past two and half years and all of my lab mates within the Kelly Lab – Dr. Kevin Johnson, Joanna Griffiths, Kyle Sirovy, and Scott Riley for their support through the project design, execution, and analysis. My thanks also go out to the undergraduate students in the Kelly Lab that helped with the lab work associated with my project – Michelle Gautreaux, Bennett Thomas, Devin Comba, and Yasmeen Kawji. I would also like to thank my co-advisor Dr. Megan La Peyre for her help designing the project and for her connections with the Louisiana Department of Wildlife and Fisheries who were instrumental to my oyster collection efforts. I would also like to thank my committee member Dr. Maheshi Dassanayake for her input on my transcriptome assembly. My thanks also go out to Dr. Jerome La Peyre and Dr. Sandra Casas Liste who helped enormously with oyster collection, experimental design, execution, and analysis. This project would not have been possible without them. Finally, I would like to thank the Louisiana State University Biological Sciences and Renewable Natural Resources Graduate Faculty and Student network that has been instrumental to the completion of this work. ii Table of Contents Acknowledgments………………………………………………………………………………...ii List of Tables……………………...……………………………………………………………...iv List of Figures…………………………………………………………………………..………....v Abstract…………………………………………………………………………………………...vi Introduction………………………………………………………………………………..……....1 Materials and Methods……………………………………………………………….…………....5 Comparative Transcriptomics………………………….…………………….…………....5 Physiology…………………………………………………...……………….…………...8 Genes of Interest ………...…………………………………………………..…………....9 Results ……………………………………………………………………………………….…..13 Comparative Transcriptomics………………..…………………………………………..13 Physiology………………………………………………………..…………..………….16 Genes of Interest………………..………………………………………………………..17 Discussion………………………………………………………………………………………..21 Synergistic Effects of Temperature and Salinity Stress…………….………..…………..22 Contrasting Laboratory and Wild Expression……………………………......…………..23 Future Directions………………..……………………………………………………….24 Limitations and Considerations………………...……………………..……..…………..25 Conclusions………………..……………………………………………………………..25 Appendix. Oyster Measurements……………………………………………………..……….27 Literature Cited…………………………………………………………………………………..29 Vita…………………………………………………………………………………...…………..35 iii List of Tables 1. Genes of Interest Sampling Locations Temperature and Salinity Information……...……......10 2. Enriched Gene Ontology Terms……...…………………………………………………...…..16 3. Genes of Interest……...…………………………………………………………………….....18 A.1. Oyster Shell Height, Dry Meat Weight, and Individual Physiological Measurements....….27 iv List of Figures 1. Study Area Map ……...……………..…………………………………………………….…....5 2. Experimental Design……...……………….…………………………………………………....6 3. Genes of Interest Sampling Locations Daily Mean Salinity and Temperature for 2017……...10 4. Differentially Expressed Gene Counts……...………………………………………..………..14 5. Principle Coordinate Analysis of Transcriptomes by Treatment……………......…...………..14 6. Log Fold Change of Genes Responding to Low Salinity at Warm Temperatures………..…..15 7. Box Plot of Respiration and Clearance Rate……..................................................…….……...17 8. Heat Map of 2-ΔΔ Values for Genes of Interest……...………………………………….……..19 9. Principle Coordinate Analysis of 2-ΔΔ Values by Location and Season…………….…….…..20 v Abstract Crassostrea virginica, the eastern oyster, forms reefs that provide critical services and benefits to the resiliency of the surrounding ecosystem. Changes in environmental conditions, including salinity and temperature, can dramatically alter the services oysters provide by affecting their population dynamics. Climate warming may further exacerbate the effects of salinity changes as precipitation events increase in frequency, intensity, and duration. Temperature and salinity independently and synergistically influence gene expression and physiology in marine organisms. We used comparative transcriptomics, physiology, and a field assessment experiment to investigate whether Louisianan oyster are changing their phenotypes to cope with increased temperature and salinity stress in Gulf of Mexico. Oysters from Sister Lake, Louisiana were exposed to fully crossed temperature (20°C and 30°C) and salinity (25ppt, 15ppt, and 7ppt) treatments. We found a higher number of genes were differentially expressed (downregulated) in response to low salinity at warmer temperatures – suggesting metabolic suppression. Gene ontology terms for ion transport and microtubule based processes were significantly enriched among upregulated genes in response to low salinity. Ion transport plays a role in osmolyte regulation which is important to maintain cell volume during low salinity. Microtubule based processes play a role in ciliary action which can improve fluid transport, prolonging aerobic metabolism and survival at low salinities. Oyster respiration rate significantly increased between 20°C and 30°C but, despite the higher energetic demands the clearance rate did not comparably increase. To investigate transcriptional differences in wild populations, we collected tissue from three locations across the Louisiana Gulf. We determined the expression levels of seven target genes and found an upregulation of genes that function in osmolyte transport, oxidative stress mediation, apoptosis, and protein synthesis at our low salinity site and sampling time point. Overall, oysters altered their phenotype more in response to low salinity at higher temperatures as evidenced by a higher number of differentially expressed genes during laboratory exposure, increased respiration (higher energetic demands), differentially expression by season and location. Warm temperatures lower the eastern oyster’s ability to cope with low salinities; the timing and length of low salinity exposure is important for understanding oyster recruitment, mortality, and growth. vi Introduction Average temperatures are rising in response to a changing global climate; in the northern Gulf of Mexico (GOM) bottom waters experienced an average warming of 0.046˚C/year since 1985 (Turner, Rabalais, & Justić, 2017). Warmer temperatures result in more evaporation and regional changes in precipitation patterns (Biasutti, Sobel, Camargo, & Creyts, 2012). The Continental United States has experienced recent droughts in the West, while Southeastern states have been experiencing record wet years (Blunden, Arndt, Blunden, & Arndt, 2016). This rise of precipitation in the Southeastern states combined with increased snow and ice melt has led to greater freshwater outflow from two large rivers that flow into the GOM through the state of Louisiana. The Mississippi and Atchafalaya Rivers account for over 80% of the total freshwater input to the Gulf of Mexico (NOAA, 2016), resulting in Louisiana’s coast having the lowest salinities in the GOM (NOAA, 2016). Warming water temperatures and increased freshwater input are of special concern for aquatic invertebrates in the northern GOM because simultaneous stressors may have greater effects than either stressor experienced individually. Environmental conditions can change rapidly and simultaneously along multiple axes, resulting in shifts in species distribution limits and lethal tolerances as well as constraining both plastic and evolutionary responses to global climate change (DeBiasse & Kelly, 2016; Kelly, Pankey, Debiasse, & Plachetzki, 2016; Somero, 2010). Most marine invertebrates are osmoconformers with internal osmolarities close to that of the surrounding seawater, which allows them to tolerate a wide salinity gradient (La Peyre, Eberline, Soniat, & La Peyre, 2013) while simultaneously making them sensitive to environmental changes in salinity (Zhao, Yu, Kong, & Li, 2012). For instance, exposure to acute or long term low salinity causes mass mortality in osmoconforming marine invertebrates (Gardner & Thompson, 2001; Laing, 2002; Y. Li, Qin, Abbott,