THE IMPORTANCE OF MICROBIAL AND PRIMARY COLONIZER INTERACTIONS ON AN EPHEMERAL RESOURCE A Dissertation by JENNIFER LYNNE PECHAL Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY May 2012 Major Subject: Entomology THE IMPORTANCE OF MICROBIAL AND PRIMARY COLONIZER INTERACTIONS ON AN EPHEMERAL RESOURCE A Dissertation by JENNIFER LYNNE PECHAL Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Jeffery K. Tomberlin Committee Members, M. Eric Benbow Tawni L. Crippen Micky D. Eubanks Aaron M. Tarone Head of Department, David W. Ragsdale May 2012 Major Subject: Entomology iii ABSTRACT The Importance of Microbial and Primary Colonizer Interactions on an Ephemeral Resource. (May 2012) Jennifer Lynne Pechal, B.S., Sam Houston State University; M.S., Texas A&M University Chair of Advisory Committee: Dr. Jeffery K. Tomberlin Carrion decomposition is an essential ecosystem function as it is an important component of nutrient cycling. Carrion decomposition has primarily been attributed to insect consumption, with little attention given to microbial communities or their potential interactions with insects. The first objective was to use passive insect-trapping methods to assess primary colonizer communities on swine carcasses between two treatments: 1) carrion with access to insects and 2) carrion excluded from insect access for five days using exclusion cages. Despite similarities between succession patterns within each treatment, carcasses initially exposed to insects had significantly fewer insect taxa. Therefore, collections of adult insect communities associated with carrion are promising as an indication of whether or not there has been a delay in insect colonization of a resource. There has yet to be a study documenting bacterial communities during carrion decomposition. The second objective was to describe bacterial community succession and composition during decomposition in the presence and absence of naturally occurring insects. Total genomic DNA was used to identify bacterial community composition via a modified bacterial tagged encoded FLX amplicon pyrosequencing. I obtained 378,904 sequences and documented distinct bacterial community successional trajectories associated with insect access and exclusion carcasses. By the fifth day of decomposition, Proteus was the dominant (72%) bacterial genus on exclusion carcasses iv while Psychrobacillus (58%) and Ignatzschineria (18%) were dominant bacterial genera on insect carcasses. These data are the first to document bacterial community composition and succession on carrion. My final objective was to assess microbial community function in response to carrion insect colonization using metabolic profiling. I characterized microbial community metabolic function in the presence and absence of the primary necrophagous insects. I documented significant microbial community metabolic profile changes during active decomposition of carcasses. Mean carcass microbial community metabolic function with insect access continuously decreased over decomposition during both field seasons. Thus demonstrating microbial metabolic activity may have discriminatory power to differentiate early and late stages of decomposition. Overall, my data contributes to an understudied area of microbial research important to organic matter decomposition, forensic entomology, and microbial-insect ecological interactions. v ACKNOWLEDGEMENTS I would like to thank my chair Jeff Tomberlin and committee members M. Eric Benbow, Tawni “TC” Crippen, Aaron Tarone, and Micky Eubanks for their guidance, support and advice throughout my dissertation. This was an incredible (and at times terrifying) committee to have for my Ph.D. work. My committee has instilled a passion, work ethic, and continued interesting in striving becoming a better researcher and person. A very special thanks to Dr. Jimmy Olson whom quickly became an advisor of life, as he was there to ask the what, why, why not and offer honest advice about any situation. The Whole Systems Genomics Initiative for Improved Human, Animal, and Environmental Well-Being Graduate Traineeship provided funding for portions of my research. Of course none of this work could be done with out the support of many, many people. First, I want to thank present and past lab mates of the forensic laboratory for investigative entomological sciences (F.L.I.E.S.) facility: Micah Flores, Michelle Sanford, Christine Picard, Adrienne Brundage, Wenqui Liu, Rachel Mohr, Charity Owings, Meaghan Pimsler, and Baneshwar Singh for all of their assistance both in the field and in the lab. I feel privileged to share the past 3.5 years experiences with you and am able to call you colleagues. My field research could not be conducted without the assistance of the Benbow Lab at the University of Dayton: Andy Lewis, Tiffany Blair, Jon White, Allissa Blystone, Jen Lang, Maureen Berg, Allison Gansel, Carolyn Teter, Jamie Alferi, and Michael Diaz. I am grateful for each of you “volunteering” your time and efforts, which made this research successful. A special thanks to the Blair family for graciously allowing me repeatedly use their land to study how dead carcasses rot. Finally, words cannot express the love, gratitude, and thanks to my dad, mom, and sisters for their support throughout this entire process. I could not have done this with out y’all. vi NOMENCLATURE EXC Insect exclusion carcasses ACC Insect access carcasses ADH Accumulated degree hours RM-ANOVA Repeated measures analysis of variance NMDS Nonmetric multidimensional scaling MRPP Multiple-response permutation procedure ISA Indicator species analysis MMCPs Microbial metabolic community profiles vii TABLE OF CONTENTS Page ABSTRACT ............................................................................................................. iii ACKNOWLEDGEMENTS ...................................................................................... v NOMENCLATURE ................................................................................................. vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES .................................................................................................. x LIST OF TABLES .................................................................................................... xv CHAPTER I INTRODUCTION AND LITERATURE REVIEW ........................... 1 Introduction ............................................................................ 1 Decomposition ecology .............................................. 1 Carrion decomposition rates........................................ 2 Consumer interactions on carrion ................................ 4 Role of microbial communities in decomposition ................................................. 5 Insects associated with carrion ........................ 6 Role of insects in decomposition ..................... 8 Microbe-insect interactions ............................. 8 Novel methodologies in decomposition ecology ......... 10 Objectives ................................................................... 12 II INSECT COMMUNITY ARRIVAL PATTERNS DURING DELAYED COLONIZATION OF PATCHY, EPHEMERAL RESOURCES .................................................................................... 13 Introduction ............................................................................ 13 Methods ................................................................................. 15 Site description and experimental design .................... 15 Statistical analyses ...................................................... 19 viii CHAPTER Page Results ................................................................................... 21 Abiotic conditions....................................................... 21 Decomposition stages ................................................. 22 Insect arrival patterns .................................................. 24 2010 field season ............................................. 24 2011 field season ............................................. 27 Insect community composition ................................... 30 2010 and 2011 field seasons combined ............ 30 2010 field season ............................................. 30 2011 field season ............................................. 37 Discussion .............................................................................. 47 III CHANGES IN CARRION BACTERIAL COMMUNITIES THROUGHOUT DECOMPOSITION CHARACTERIZED BY 16S rRNA AMPLICON PYROSEQUENCING ................................. 51 Introduction ............................................................................ 51 Methods ................................................................................. 53 Site description and experimental design .................... 53 Bacteria sampling protocol ......................................... 55 DNA extraction .......................................................... 55 Massive parallel bTEFAP ........................................... 56 Pyrosequencing data analysis ...................................... 57 Bacterial community analysis ..................................... 58 Results ..................................................................................