University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations and Student Research in Entomology Entomology, Department of Summer 7-28-2011 Genetic variability of the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) assessed on a global scale using Amplified Fragment Length Polymorphism Kathleen M. Kneeland University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/entomologydiss Part of the Entomology Commons Kneeland, Kathleen M., "Genetic variability of the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) assessed on a global scale using Amplified rF agment Length Polymorphism" (2011). Dissertations and Student Research in Entomology. 11. https://digitalcommons.unl.edu/entomologydiss/11 This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations and Student Research in Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Genetic variability of the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) assessed on a global scale using Amplified Fragment Length Polymorphism By Kathleen M. Kneeland A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Entomology Under the Supervision of Professors Steven R. Skoda and John E. Foster Lincoln, Nebraska August, 2011 ii Genetic variability of the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) assessed on a global scale using Amplified Fragment Length Polymorphism Kathleen M. Kneeland, Ph.D. University of Nebraska, 2011 Advisors: Steven R. Skoda and John E. Foster The stable fly, Stomoxys calcitrans, is a cosmopolitan pest of livestock and humans. It is a major pest in livestock facilities, where exist excellent breeding sites such as spilled feed mixed with manure. The pestiferous nature and painful bite cause stress to cattle and other animals. Cattle perform avoidance behaviors such as bunching together, standing in water, tail swishing, ear flicking and leg stamping. The stress and avoidance behaviors result in reductions in weight gain or milk production, with an estimated annual economic loss of > $1billion. Therefore, the development of more efficient control methods would benefit the global economy, as well as the animals. Studying the population genetics of stable flies could provide information on their population dynamics, origins of outbreaks, and geographical patterns of insecticide resistance. Many studies have been conducted on a local scale, most reporting a high level of gene flow between locations. To date, few studies have been conducted on a global scale. Here I report a study of samples acquired from 4 biogeographical regions: Nearctic, Neotropical, Palearctic and Australian. No samples were acquired from the Oriental region. The results indicate a high level of gene flow on a global scale. FST and GST values are low, and Nm values very high. The tests of neutrality suggest population expansion, and tests for genetic differentiation simply reported “no differentiation”. AMOVA results show the majority of genetic diversity is within groups, and very little among groups. These results suggest that stable flies have a panmictic population, with no isolation by distance or across geographical barriers. iv DEDICATION This dissertation is dedicated to loved ones lost along this journey. To my dad, Earl Richard Kneeland, who taught me that common sense was as important as „book learning‟, and truth, honesty and loyalty more treasured than gold. To my mom, Gloria Ann Kneeland, a little lady with a huge heart, who taught me to go for what I want. I couldn‟t have chosen better parents, and I will love you always. To Clayton Blaine Bomber, my free-spirited cousin. May you play your guitar for a choir of angels. And to all my family and friends who are gone but remain in my heart. I miss you all very much, and I am a better person for having known and loved you. v ACKNOWLEDGMENTS A simple “thank you” cannot begin to express the deep gratitude, admiration and respect I have for my co-advisors, Dr. Steven R. Skoda and Dr. John E. Foster. Dr. Skoda has supported me every step of the way, offering advice but making me solve my own problems. He has been a shoulder to lean on, an ear to bend, and he was always available to listen, even from Panama. He is dedicated to his family and his career, and cares deeply about the success of his students. Dr. Foster takes mentoring far above and beyond the call of duty, being available for counseling, advising or simply chatting over coffee on a daily basis, giving his time to help me through the best and worst times of the last 4 years. Dr. Skoda and Dr. Foster are a team, and I am most fortunate to have them as mentors and friends. I would also like to thank my committee members, Dr. Dennis Berkebile, Dr. David Carter, and Dr. Rodger Johnson from Animal Sciences. Dr. Berkebile, for keeping me supplied with stable flies. Dr. Carter, you got me off to a running start during your forensic science class. I have based my subsequent work on the quality you expected from me. Dr. Johnson, I want to thank you for recognizing my persistence, if not my mastery of statistics. I owe a debt of gratitude to my colleagues around the world who supplied the stable flies for my research. Henrik Skovgaard: Denmark; Gerard Duvallet: France, Morocco, Gabon and La Reunion Island; Geoff Brown: Queensland, Australia; Steve Skoda: Panama and Texas; Steve Mihok: Ontario, Canada; Kevin Floate: Alberta, Canada; Dennis Berkebile and Dave Boxler: Nebraska; Holly vi Ferguson: Washington; Tanja McKay: Arkansas; Greg Johnson and Kristina Hale: Montana; Stephen Young: Idaho; Ralph Williams: Indiana; Wes Watson: North Carolina; and Ludek Zurek: Kansas. Without their help this project would not have been possible. The faculty, staff and students of the Entomology department have been a joy to work with and I have made some special friends. I especially want to thank my lab partners, Drs. Difabachew Belay, Bamphitli “Banks” Tiroesele and Erica Lindroth, for helping me get past the same problems they encountered in their research, and for their friendship. I would never have made it this far if not for my family. My parents were so very proud of me, and I know that if it‟s possible, they are watching my progress. Others have done their best to be there for me in my parents‟ place, especially Aunt Loy. You have been my rock. Audrey, Ray and Sherry, Wayne and Sherry, Mary and Phil, Dot, Lee, Dennis and Richard, I would not have made it through the frustration and loneliness without having you at the other end of my phone. Also my brother, Larry, who was left to take care of my animals when I took off for Nebraska, and my sons, Aaron and Cliff Bortoletto, who have backed me all the way. And I give special thanks for my granddaughter Abby, who has inherited my love of insects. Some special friends also helped me reach my goals and I am forever grateful. Mary Jane, you are the best sister anyone could have. John and Kay, thanks for listening when I needed a shoulder to cry on. And I especially want to vii thank Mary Lou Peltier, my undergrad mentor, who knew I could do it even when I didn‟t. Lastly, thank you, Gil, for your support. You were there in the beginning, through most of the journey, and although you didn‟t stay until the end, your confidence in me sometimes kept me going. Most of all, I thank God for giving me the stubbornness and determination to keep going through good times and bad, and for giving me the circle of support I needed to make it. viii TABLE OF CONTENTS ABSTRACT……………………………………………………………….ii DEDICATION…………………………………………………………….iv ACKNOWLEDGMENTS………………………………………………...v TABLE OF CONTENTS………………………………………………..viii LIST OF TABLES………………………………………………………..xii LIST OF FIGURES……………………………………………………...xiii CHAPTER 1: Literature review Introduction 2 Life history and biology 4 Distribution and climatic variables 8 Stable fly parasitism of livestock 10 Control methods 11 Stable flies and disease 14 Stable fly genetics 15 Genetic analysis techniques 20 Protein electrophoresis 21 RFLP and PCR-RFLP 21 RAPDs 22 AFLP 23 DNA sequencing 24 Other techniques 24 Conclusions 24 ix Significance of research 27 Research objectives 27 Biological questions 28 Dissertation hypotheses 28 CHAPTER 2: An Ethanol Preservation Technique Suitable for Shipping Stable Flies Long Distance While Maintaining the Quantity of DNA 30 Introduction 31 Materials and Methods 33 Experimental design 33 DNA Extraction 34 Data Collection 35 Statistical Analysis 36 Results 36 Discussion 38 CHAPTER 3: Genetic variability of the stable fly Stomoxys calcitrans (L) (Diptera: Muscidae) within North America and on a global scale, using Amplified Fragment Length Polymorphism 40 Introduction 41 Materials and methods 44 Samples 44 DNA Extraction 49 x AFLP-PCR 50 Gel Electrophoresis 55 Data Analysis 56 Results 57 Arlequin Analyses 58 AMOVA 59 Tests of Neutrality 60 Mismatch Distribution 61 Mantel Test 63 Popgene 65 Nei‟s Genetic Diversity 65 Dendrograms 66 Discussion 67 LITERATURE CITED 77 APPENDICES 104 I. DNA extraction protocol 104 II. Reagent and buffer preparation 107 III. AFLP PCR protocol: Lab version 110 IV. Optimized AFLP protocol 119 V. Gel Preparation, Loading and Scanning 124 VI. Scoring Gels 128 VII. Stable fly gels 131 VIII. Dendrograms 153 xi IX. Data Analysis 157 X. Lab supply list 176 XI Troubleshooting 180 xii LIST OF TABLES Table 1. Experimental design 34 Table 2. Type 3 test of fixed effects 36 Table 3. Differences of LSMeans 38 Table 4. Collection sites of stable fly samples 47 Table 5. Coordinates and elevation of collection sites 48 Table 6.