Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 1-15-2018 Determining Bioindicators for Coastal Tidal Marsh Health Using the Food Web of Larvae of the Greenhead Horse Fly (Tabanus nigrovittatus) Devika Rajeev Bhalerao 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 Bioinformatics Commons, Ecology and Evolutionary Biology Commons, Entomology Commons, and the Genomics Commons Recommended Citation Bhalerao, Devika Rajeev, "Determining Bioindicators for Coastal Tidal Marsh Health Using the Food Web of Larvae of the Greenhead Horse Fly (Tabanus nigrovittatus)" (2018). LSU Master's Theses. 4377. https://digitalcommons.lsu.edu/gradschool_theses/4377 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]. DETERMINING BIOINDICATORS FOR COASTAL TIDAL MARSH HEALTH USING THE FOOD WEB OF LARVAE OF THE GREENHEAD HORSE FLY (Tabanus nigrovittatus) 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 Entomology by Devika Rajeev Bhalerao B.Sc., Pune University, 2008 M.Sc., Pune University, 2010 May 2018 Acknowledgements My heartfelt gratitude to Claudia Husseneder for providing an environment conducive to research and her unwavering patience especially during the thesis writing phase. I will always be grateful to her for the stress free environment in the lab and the all the motivation for conducting the mind boggling bioinformatics analyses. It was a nice feeling to know that I could show up at her office door and always be greeted with a smile. Lane Foil has been a constant source of inspiration and expertise. I would like to thank him for his perspective which made me envision my study as a part of a larger project with an overarching goal which would direct future research. Daniel Swale’s expert dissection skills paved the way for a dependable gut content analyses creating the foundation of this thesis. I will always be grateful for his friendly demeanor and honest advice. A deep appreciation to Kelly Thomas and his team especially Devin Thomas, Holly Bik and Taruna Aggarwal for providing bioinformatics training and continual help. None of my research would have been possible without the unconditional love and prayers of my parents, in-laws and husband Chinmay Tikhe. A special note of thanks to family and friends back home in India. My heartwarming gratitude to Jong-Seok Park, Chinmay Tikhe, Micheal Becker, Raymond Frasier, Ashley Reddicks and all the members of the Husseneder and Foil labs for their support, encouragement and jokes. Last but not the least, I would like to thank all my friends at LSU for being a family and for providing a sense of belonging. ii Table of Contents Acknowledgements ......................................................................................................................... ii Abstract .......................................................................................................................................... vi Chapter 1. Literature review ........................................................................................................... 1 1.1 Wetlands, Coastal Wetlands and Salt Marshes ..................................................................... 1 1.2 Importance of Wetlands, Coastal Wetlands and the Salt Marshes of Louisiana ................... 1 1.3 Vulnerability of Coastal Wetlands globally and within the United States with special emphasis on the Salt Marshes of Louisiana ................................................................................ 2 1.4 Oil spills – a major anthropogenic stressor of Coastal Wetlands .......................................... 2 1.5 Impacts and restoration efforts of damaged Coastal Wetlands within the United States with special emphasis on the Salt Marshes of Louisiana .................................................................... 3 1.6 Impacts and restoration effects after oil spills on marine and coastal ecosystems ............... 4 1.7 Deepwater Horizon Oil Spill – A wake up call for urgent assessment methods .................. 5 1.7.1. Distribution of oil .......................................................................................................... 5 1.7.2. PAH assessment ............................................................................................................ 5 1.7.3 Impact of the spill ........................................................................................................... 6 1.8 Need for assessment tools for marsh health and role of bioindicators .................................. 9 1.9 Environmental monitoring using invertebrate bioindicators with special emphasis on insects and food webs ................................................................................................................ 10 1.10 Food webs for understanding ecosystem dynamics .......................................................... 12 1.11 Deciphering food webs...................................................................................................... 13 1.12 Current methods for environmental monitoring................................................................ 14 1.13 DNA metabarcoding and Next generation sequencing ..................................................... 15 Chapter 2. Introduction ................................................................................................................. 18 2.1 Objectives of the study ........................................................................................................ 24 iii Chapter 3. Material and Methods.................................................................................................. 25 3.1 Site selection ....................................................................................................................... 25 3.2 Collection of sediments ....................................................................................................... 25 3.3 Larval collection .................................................................................................................. 27 3.4 Tabanid larval identification ............................................................................................... 27 3.5 Generating a molecular identity for Tabanus nigrovittatus Macquart ................................ 29 3.6 Tabanid larva dissection, DNA extraction and amplification ............................................. 31 3.7 Molecular identification of tabanid larvae .......................................................................... 32 3.8 Cloning to detect extent of tabanid DNA contamination in the gut contents ..................... 33 3.9 DNA extraction from voucher sediments............................................................................ 34 3.10 DNA amplification and sequencing .................................................................................. 35 3.11 Bioinformatic and statistical analyses ............................................................................... 36 3.12 Measuring Sediment PAH levels ...................................................................................... 41 3.13 Measuring Sediment chemistry Parameters ...................................................................... 42 3.14 Sediment Toxicity assay using the model organism Hyallela azteca ............................... 43 Chapter 4. Results ......................................................................................................................... 46 4.1 Molecular identification of tabanid larvae .......................................................................... 46 4.2 Tabanid DNA and preliminary data on food web components in larva gut contents ......... 46 4.3 General assessment of next generation sequence data ........................................................ 49 4.4 Assessment of sample sizes and OTU capture .................................................................... 50 4.5 OTU richness....................................................................................................................... 52 4.6 OTU diversity analysis in sediments and guts .................................................................... 54 4.7 Diet of the top invertebrate predator (tabanid larvae) in Louisiana marsh soil ................... 57 4.8 Identification of insect species in the tabanid larval gut ..................................................... 65 4.9 Variation in tabanid larval diets across locations ................................................................ 66 4.10 Comparison of OTU composition in sediments and larvae .............................................. 70 4.11 Variation in sediment OTU composition across locations ................................................ 75 4.12 Alpha and Beta diversity among sediments based on locations with statistical tests ....... 77 iv 4.13 Differences in sediment OTU composition between the East and West........................... 79 4.14 Difference in OTU composition of sediments