ABSTRACT CARR, ANN LOUISE. Profiling of Acarine Attractants And
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ABSTRACT CARR, ANN LOUISE. Profiling of Acarine Attractants and Chemosensation. (Under the direction of Dr. R. Michael Roe.) Acarine Attractants. The Acari are of significant economic and health importance, and consist of two major groups, the mites that demonstrate a wide variety of life strategies, i.e., herbivory, predation and ectoparasitism, and ticks which have evolved obligatory hematophagy. The major sites of chemoreception in the acarines are the palps and tarsi on the forelegs. A unifying name, the “foretarsal sensory organ” (FSO), is proposed for the first time for the sensory site on the forelegs of all acarines. Over the past three to four decades’ significant progress has been made in the chemistry and analysis of function for acarine attractants in mites and ticks. This has been made possible by the variety of laboratory and field bioassay methods that have been developed for the identification and characterization of attractants including a novel ultrasonic micro-dissection method that was developed. In mites, attractants include aggregation, immature female, female sex and alarm pheromones; in ticks, the attraction-aggregation-attachment, assembly and sex pheromones; in mites and ticks host kairomones and plant allomones; and in mites, fungal allomones. There are still large gaps in our knowledge of chemical communication in the acarines compared to insects. However, the use of lure-and-kill and lure-enhanced biocontrol strategies have been investigated for tick and mite control, respectively, with significant environmental advantages which warrant further study. Haller’s Organ Olfactory Mechanism. Next generation sequencing and comparative analyses suggest that the chemosensory mechanism in the Haller’s organ of ticks is a G- protein coupled receptor (GPCRs) signal cascade. Only olfactory GPCRs were identified suggesting, despite conflicting morphological data, that the Haller’s organ functions only in olfaction and not gustation. Each component of the olfactory GPCR signal cascade was identified, except for the odorant binding proteins. This suggests that ticks rely on either a novel, unknown class of binding proteins or other methods of sensilla lymph solubilization to deliver odorant molecules from the environment to chemoreceptors. qPCR experiments documenting the expression profile of the olfactory transcripts GPCR, Gαo, and β-arrestin in unfed and blood-fed adult D. variabilis determined that there is hormonal regulation of the olfactory system in ticks. Behavioral assays confirmed the role of the Haller’s organ in chemical avoidance, in addition to its known role is chemical attraction. Remarkably, it was determined that the Haller’s organ is not required for host attachment or blood-feeding. Sex Pheromones of the Black-legged Tick. Copulation in I. scapularis involves physical contact between the male and female (on or off the host), male mounting of the female, insertion/maintenance of the male chelicerae in the female genital pore (initiates spermatophore production), and the transfer of the spermatophore by the male into the female genital pore. Bioassays determined that male mounting behavior/chelicerae insertion required direct contact with the female likely requiring non-volatile chemical cues with no evidence of a female volatile sex pheromone to attract males. Unfed virgin adult females and replete mated adult females elicited the highest rates of male chelicerae insertion with part fed virgin adult females exhibiting a much lower response. Whole body surface hexane extracts of unfed virgin adult females and males, separately analyzed by GC-MS, identified a number of novel tick surface associated compounds: fatty alcohols (1-hexadecanol and 1-heptanol), a fatty amide (erucylamid), aromatic hydrocarbons, a short chain alkene (1-heptene), and a carboxylic acid ester (5β-androstane). These compounds are discussed in terms of their potential role in female-male communication. The two most abundant fatty acid esters found were butyl palmitate and butyl stearate present in ratios that were sex specific. Only 6 n- saturated hydrocarbons were identified in I. scapularis ranging from 10-18 carbons. © Copyright 2015 Ann Louise Carr All Rights Reserved Profiling of Acarine Attractants and Chemosensation by Ann Louise Carr A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Entomology Raleigh, North Carolina 2015 APPROVED BY: _______________________________ _______________________________ Dr. R. Michael Roe Dr. Coby Schal Committee Chair _______________________________ _______________________________ Dr. Wes Watson Dr. Daniel E. Sonenshine DEDICATION This dissertation is dedicated to my grandmother, Martha Carr. Her generosity and support have allowed me to pursue my education to the fullest, and for that I will always be grateful. ii BIOGRAPHY Ann Carr was born and raised in Washington, DC making her a 6th generation Washingtonian. She graduated from the Holton-Arms School for Girls and made her way down south to attend Texas A & M University. She has always had an interest in science and research, interning both her freshman and sophomore years are the Walter Reed Army Institute of Research analyzing the genetic variances between SHIV positive and negative rhesus monkeys. She was fortunate enough to obtain a lab technician position her junior and senior years with Dr. Albert Mulenga in the Texas A & M University medical and veterinary entomology research lab. It was during her time spent helping Dr. Mulenga with his research on tick salivary proteins that she first became fascinated with ticks, and developed a desire to pursue a career researching them. Ann graduated from Texas A & M University in 2009 with a BS double major in biomedical science and entomology. She arrived at North Carolina State University and obtained a master’s in entomology in 2011 under the direction of Dr. Charles Apperson, Dr. R. Michael Roe and Dr. Coby Schal studying host kairomones and chemical lures for ticks. She continued on for her pHD in Dr. Roe’s lab sequencing the first transcriptome to the Haller’s organ and researching tick chemical communication and mating behaviors. In addition to her research, Ann is actively involved in EGSA student outreach and the Triangle Beagle Rescue. She also enjoys skiing and kiteboarding in her free time. iii ACKNOWLEDGMENTS I would first and foremost like to thank my advisor Dr. R. Michael Roe for all his support and guidance, in addition to providing me an opportunity to stay on after my master’s and pursue a pHD. Dr. Roe’s knowledge and insight has been an invaluable resource, and it has been a privilege to work in his lab. I would also like to thank all the members of my graduate committee Dr. Daniel E. Sonenshine, Dr. Charles Apperson, Dr. Coby Schal, and Dr. Wes Watson, as well as my graduate representatives Dr. Asimina Mila and Dr. Wesley Everman. I very much appreciate their expertise, advice, and participation on my committee. I would like to thank all the members of the Roe lab for creating such a positive and welcoming work environment. I truly consider all my work colleagues to be friends and have enjoyed all of our collaborations immensely. Lastly I would like to thank my family for their unwavering love and support throughout my doctoral studies. iv TABLE OF CONTENTS List of Tables .................................................................................................................. vi List of Figures ................................................................................................................. vii Acarine Attractants: Chemoreception, Bioassay, Chemistry, and Control ............. 1 Abstract ............................................................................................................... 2 Introduction ........................................................................................................ 3 Chemosensory Structure and Function ............................................................ 5 Bioassay Methods for Acarine Attractants...................................................... 10 Chemistry of Aggregation and Assembly Pheromones .................................. 18 Chemistry of Reproductive (Sex) Pheromones ............................................... 23 Chemistry of Alarm Pheromones ..................................................................... 26 Chemistry of Host Kairomones ........................................................................ 27 Chemistry of Plant Allomones .......................................................................... 32 Chemistry of Fungal Allomones ....................................................................... 35 Acarine Trapping: Attraction and Kill ............................................................ 36 Summary and Conclusions ............................................................................... 39 Acknowledgments .............................................................................................. 41 References ........................................................................................................... 41 Tables .................................................................................................................. 55 Figures ................................................................................................................. 66 Putative GPCR Chemosensory Pathway Identified