University of Texas at Tyler Scholar Works at UT Tyler Biology Theses Biology Fall 11-1-2015 The oD wnregulation of Short Neuropeptide Receptor (SNPFR) in the Red Imported Fire Ant Solenopsis Invicta (Hymenoptera: Formicidae) and the Tawny Crazy Ant Nylanderia Fulva (Hymenoptera: Formicidae) using RNA Interference, and the Use of an Anthranilic Diamide as a Novel Management Technique Megan Rudolph Follow this and additional works at: https://scholarworks.uttyler.edu/biology_grad Part of the Biology Commons Recommended Citation Rudolph, Megan, "The oD wnregulation of Short Neuropeptide Receptor (SNPFR) in the Red Imported Fire Ant Solenopsis Invicta (Hymenoptera: Formicidae) and the Tawny Crazy Ant Nylanderia Fulva (Hymenoptera: Formicidae) using RNA Interference, and the Use of an Anthranilic Diamide as a Novel Management Technique" (2015). Biology Theses. Paper 31. http://hdl.handle.net/10950/307 This Thesis is brought to you for free and open access by the Biology at Scholar Works at UT Tyler. It has been accepted for inclusion in Biology Theses by an authorized administrator of Scholar Works at UT Tyler. For more information, please contact [email protected]. THE DOWNREGULATION OF SHORT NEUROPEPTIDE F RECEPTOR (SNPFR) IN THE RED IMPORTED FIRE ANT SOLENOPSIS INVICTA (HYMENOPTERA: FORMICIDAE) AND THE TAWNY CRAZY ANT NYLANDERIA FULVA (HYMENOPTERA: FORMICIDAE) USING RNA INTERFERENCE, AND THE USE OF AN ANTHRANILIC DIAMIDE AS A NOVEL MANAGEMENT TECHNIQUE by MEGAN RUDOLPH A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science of Biology Department of Biology Blake Bextine, Ph.D., Committee Chair College of Arts and Sciences The University of Texas at Tyler November 2015 Acknowledgements I would like to begin my thanking my amazing family, whose unconditional love and support has helped me through this challenging and rewarding endeavor. I cannot sufficiently describe the depth of gratitude I have for the opportunities you have provided me and the intellectually stimulating environment I was lucky enough to have. To my mother and father, I attribute my success and motivation to the work ethic you instilled in me and the warmth and love I have always been surrounded with. Thank you for believing in me and lifting my spirits when I felt hopeless and didn’t believe in myself. Without your unwavering support, I would not be where I am today. To Terra, thank you for supporting me and caring for me on my most challenging days. Without your positivity and love, I don’t know how I would have made it through many of my sleepless nights. Thank you for tolerating my countless breakdowns and listening to my rambling late-night thoughts and thank you for standing by my side through this tumultuous journey. Blake Bextine, thank you for accepting me into your amazing lab and giving me this incredible opportunity. The past two years have been the most challenging and rewarding years of my life, and for that, I am eternally grateful. I have learned so much and have undergone immense personal growth that I don’t think I would have experienced otherwise. You have continued to be nothing but supportive and positive, and have helped me develop academically and professionally. Thank you for pushing me to think independently and for being a great mentor and advisor. I greatly appreciate everything you have done for me and will always cherish the opportunities you have provided. I would like to thank the University of Texas at Tyler and all of the other members of my thesis committee, Brent Bill, Jon Seal, Danny McDonald, and Srini Kambhampati. Your continued support, collaboration, and guidance has been truly appreciated and it has been a pleasure to work and collaborate with each one of you. To Maequeta Humber and Brad Lowe, without your hard work and dedication, the biology department would undoubtedly not be as exceptional as it is today. Thank you for always being there for me when I needed help. Members of the biology faculty, you have all taught me so much and I feel honored to have received your instruction. To the Bextine laboratory members, thank you for your collaboration and friendship. I wish I could thank each one of you individually, but I would not be able to sufficiently illustrate my gratitude for you. Thank you for sharing you immense knowledge and being so supportive throughout my time here. Each and every one of you was instrumental in my growth as a graduate student, and I have thoroughly enjoyed surrounding myself with driven, brilliant individuals such as yourselves. Good luck in all of your aspirations. I would like to give a special thanks to Chris Powell; it would have been impossible to finish this work without the guidance and support you have provided. To my fellow graduate students, I have thoroughly enjoyed the conversations and meals shared together. You are among the most intelligent men and women I have had the pleasure of meeting, and I wish you luck with your accomplished academic careers. Thank you all. -Megan Rudolph Table of Contents List of Tables………………………………………………………………………………………ii List of Figures……………………………………………………………………...……………..iii Abstract…………………………………………………………………………………………....v Chapter One: Literature Review Introduction………………………………………………………………………………..1 References………………………………………………………………………..16 Chapter Two: Management of S. Invicta using cyclaniliprole Introduction………………………………………………………………………………19 Materials and Methods…………………………………………………………...23 Results……………………………………………………………………26 Discussion………………………………………………………..31 References………………………………………………..34 Chapter Three: The downregulation of short Neuropeptide F Receptors (sNPFRs) in Solenopsis invicta using RNA interference Introduction………………………………………………………………………………36 Materials and Methods…………………………………………………………...39 Results……………………………………………………………………44 Discussion………………………………………………………..51 References………………………………………………..54 Chapter Four: The downregulation of short Neuropeptide F Receptors (sNPFRs) in Nylanderia fulva using RNA interference Introduction………………………………………………………………………………56 Materials and Methods…………………………………………………………...61 Results……………………………………………………………………63 Discussion………………………………………………………..66 References………………………………………………..67 Chapter Five: Conclusions and Future Research Conclusion……………………………………………………………………………….69 i List of Tables Table 2.1 Treatments and concentrations of cyclaniliprole……………………………………..24 Table 3.1. A list of Ct values, replicate Ct values, and calculated standard deviation obtained by running qPCR on S. invicta castes………………………………………………………………..47 Table 4.1 qPCR primers designed for sNPFR in N. fulva……………………………………….62 ii List of Figures Figure 1.1 Potential global range expansion for S. invicta in the western hemisphere………..…2 Figure 1.2 S. invicta sexuals and polymorphic workers……………………………………….…3 Figure 1.3 The chemical structure of ryanodine and the ryanodine receptor…………………….5 Figure 1.4 S. invicta eggs, larvae, and pupae……………………………………………………..6 Figure 1.5 Profile view of N. fulva worker from Texas…………………………………………..8 Figure 1.6 Diagram of an insect’s central nervous system……………………………………...10 Figure 1.7 Dicer and RNA-induced Silencing Complex………………………………………..13 Figure 1.8 Potential transformation of S. invicta management techniques……………………..14 Figure 2.1 Profile view of S. invicta worker………………………………………………….…20 Figure 2.2 2D structure of cyclaniliprole……………………………………….…………….....22 Figure 2.3 Schematic of the ryanodine receptor……………………………….……………...…22 Figure 2.4 Experimental setup of the preliminary rate-determining bioassay…..…………….....25 Figure 2.5 Red imported fire ant percent mortality, observed over a period of 7 days……….....27 Figure 2.6 S. invicta percent mortality, observed over a period of 7 days…………………….…29 Figure 2.7 Dose response curves produced using mortality data from the bioassay…………….30 Figure 2.8 Probit transformation modeled using SPSS………………………………………….30 Figure 3.1 Profile view of S. invicta minor worker……………………………………………...37 Figure 3.2 Experimental setup of the S. invicta adult mortality study…………………………...43 Figure 3.3 Confirmation of sNPFR sequence in S. invicta workers by 1% agarose gel…………45 Figure 3.4 18S gene expression normalization indexes of sNPFR gene expression…………….46 iii List of Figures (continued) Figure 3.5 Use of fluorescent dsRNA in S. invicta brood……………………………………….48 Figure 3.6 Fire ant mortality study………………………………………………………………49 Figure 3.7 18S gene expression normalization indexes of sNPFR gene expression…………….50 Figure 4.1 Profile view of N. fulva worker………………………………………………………57 Figure 4.2 Native (blue triangles) and invasive (red circles) occurrences of N. fulva…………...58 Figure 4.3 Confirmation of sNPFR gene sequence in N. fulva workers by 1% agarose gel…….64 Figure 4.4 PCR optimization for S. invicta sNPFR primer sets…………………………………65 Figure 4.5 Ct values from qPCRs run with N. fulva cDNA……………………………………..65 iv Abstract THE DOWNREGULATION OF SHORT NEUROPEPTIDE F RECEPTOR (SNPFR) IN THE RED IMPORTED FIRE ANT SOLENOPSIS INVICTA (HYMENOPTERA:FORMICIDAE) AND THE TAWNY CRAZY ANT NYLANDERIA FULVA (HYMENOPTERA:FORMICIDAE) USING RNA INTERFERENCE, AND THE USE OF AN ANTHRANILIC DIAMIDE AS A NOVEL MANAGEMENT TECHNIQUE Megan Rudolph Thesis Chair: Blake Bextine, Ph.D. The University of Texas at Tyler November 2015 The red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), is an invasive species endemic to South America that was inadvertently introduced into the United States. This invasive species costs over six billion dollars (USD) annually and causes