Olfactory Responses of Two Coleopteran Species: The Stored Product Pest Tribolium castaneum and The Forest Pest Predator Dastarcus helophoroides DISSERTATION In Partial Fulfilment of the Requirements for the Degree Doctor of Philosophy (PhD) of the Faculty of Forest Sciences and Forest Ecology Georg-August-University Göttingen submitted by Karthi Balakrishnan born on 13th May 1984, Nambarai, Tamil Nadu, India Göttingen, 2019 Thesis committee --------------------------------------------------------------------------------------------------------------------- 1st Reviewer Prof. Dr. Andreas Schuldt Department of Forest Nature Conservation Georg-August-University, Göttingen 2nd Reviewer Prof. Dr. Ernst A. Wimmer Department of Developmental Biology Georg-August-University, Göttingen Members of the Examination Board 1st Examiner Prof. Dr. Andreas Schuldt Department of Forest Nature Conservation Georg-August-University, Göttingen 2nd Examiner Prof. Dr. Ernst A. Wimmer Department of Developmental Biology Georg-August-University, Göttingen 3rd Examiner Prof. Dr. Michael Rostás Department for Crop Sciences, Agricultural Entomology Georg-August University Goettingen Date of Oral Examination: 10th May 2019 Abstract --------------------------------------------------------------------------------------------------------------------- Olfaction is unavoidable in guiding insect behaviors. Detection of odorant molecules by the principle anatomical structure, the antenna, is thought to result from direct activation of odorant receptors (ORs) by odor molecules. Besides ORs, several other olfactory components are involved in insect olfaction, e.g. odorant binding proteins (OBPs) are highly expressed in insect olfactory tissues, mainly antenna and involved in the first step of odorant reception. Improved knowledge of the olfaction of pest insects or beneficial insects may contribute to identifying new attractants/repellents and support in the development of pest insect control strategies. The overall objective of this study was to identify volatile organic compounds (VOCs) that could modulate the olfactory responses of the stored product pest Tribolium castaneum and the forest insect predator Dastarcus helophoroides. The first part of this research work, began with the investigation of the electroantennographic (EAG) responses of the most destructive stored product pest insect T. castaneum. Despite the importance of this beetle as “model organism” in population ecology, evolution, and mathematical studies, there is no systemic olfactory information available on this beetle. EAG responses from both sexes of T. castaneum to 94 VOCs were recorded and identified more than 90% of tested compounds to elicit antennal responses at high concentration (10-1). Specifically, the volatile compounds undecane, 1-hexen-3-ol, octanal, 2-heptanone, 2-pentanone, hexanoic acid, and ethyl hexanoate were eliciting the strongest EAG responses within the compound group of chemical similarity. Additionally, comparison of vapor pressure with the strength of the EAG amplitudes revealed these compounds were outstanding within a homologous series of compounds. This effort is the first to identify several perceived compounds by both sexes of T. castaneum beetles. RNAi experiments and EAG recordings were performed to investigate the roles of the highly expressed OBPs TcasOBP9A and TcasOBP9B in the olfactory reception of both sexes of T. castaneum. RNAi-treated dsTcasOBP9A and dsTcasOBP9B beetles were injected into the conjunctivum between fourth and fifth abdominal segments of each T. castaneum pupae. It was found that the dsTcasOBP9A injected beetles showed highly significant EAG response reduction to 4,8-dimethyldecanal, (E)-2-heptenal, 2-hexanone, 6-methyl-5-hepten-2-one, cis-3-hexen-1-ol, and β-ionone at most tested dilutions (10-1-10-8). In male beetles, knock-down of TcasOBP9B shows less effective and both gene knock-down revealed a similar effect as the TcasOBP9A single knock-down. But in female beetles, the TcasOBP9B knock-down caused a significant EAG response reduction to all tested VOCs. Similar to the double knock-down situation, which reveal significantly high pronounced response reduction to all tested VOCs at all dilutions. These results provide the first evidence that both TcasOBP9A and TcasOBP9B play important crucial roles in the perception of diverse VOCs. i A practical application of using OBPs is to develop a portable biosensor device with sensing element for detecting disease marker compounds. The above EAG results and the RNAi knockdown studies enabled me to select two OBPs, TcasOBP9A and TcasOBP9B, from T. castaneum and their respective binding ligand 6-methyl-5-hepten-2-one and 3-octanol. Consequently, for a proof of principle study. These two OBPs were used as sensing element functionalized in a reduced graphene oxide field effect transistor (rGO-FET). Real-time binding affinity measurements by the rGO-FET sensor with selected OBPs to their ligands (in-vitro) and antennal responses of T. castaneum (in-vivo) to the aforementioned ligands revealed to follow the Langmuir model for ligand-OBP interactions. The results demonstrate that TcasOBP9A and TcasOBP9B are able to bind selected odorants when immobilized on rGO-FET and discriminate between ligands binding within a range of concentrations. These results led to a promising possibility to develop a portable sensor system based on rGO-FET and OBP as a sensing element to detect the compound that acts as an indicator for the toxin, early insect infestation on stored products, food contamination, and food degradation by microbial organisms. The second part of this research work, the bothriderid predatory beetle D. helophoroides was selected for examining its olfactory perception, since this beetle’s life cycle is strongly related to several of the most important cerambycid forest tree insect pests. For instance, Monochamus spp., Anoplophora glabripennis, Massicus raddei, Apriona germari, A. swainsoni and Batocera horsfieldi were considered major prey species for D. helophoroides. Antennae of both sexes of D. helophoroides were tested for EAG and behavioral responses to VOCs emitted from their prey infested trees, prey perceiving host volatiles, and larval frass of many preys. Both sexes of the beetle elicited EAG responses to nonanal, octanal, cis-3-hexenol, 3-carene, (R)-(+)-α-pinene, (S)- (-)-α-pinene, (R)-(+)-limonene, and (S)-(-)-limonene. In addition, behavioral tests of D. helophoroides showed that both sexes were significantly attracted to nonanal, cis-3-hexenol, 3- carene, or (R)-(+)-limonene even at low concentrations but were repelled to high concentrations of cis-3-hexenol or (S)-(-)-limonene. These highly attracted compounds might be useful either individually or in mixtures for developing efficient attractants to lure this predatory beetle into forest stands damaged by different cerambycids. By using all these results together with available molecular information of both coleopterans, it should be possible to develop semiochemicals, RNAi approach, or rGO-FET biosensor based integrated pest management (IPM) strategies for controlling stored product pest like T. castaneum and forest insect pests. Furthermore, understanding the regulation of olfactory responses in the model organism T. castaneum will enhance our understanding of the olfaction in beetles in general, and in the long term may lead to developing new pest control strategies. ii Acknowledgements --------------------------------------------------------------------------------------------------------------------- I would like to express my sincere gratitude to Prof. Dr. Ernst A. Wimmer. His endless support in difficult situation, encouragement and resourcefulness make him the ultimate co-supervisor. I wish to thank my supervisor, Prof. Dr. Andreas Schuldt, for his support in hard time. I wish to also thank Prof. Dr. Michael Rostás for accepting as an examiner of my PhD defense. I would like to also thank my former supervisor, Prof. Dr. Stefan Schütz, for providing me with guidance, ideas, support, encouragement and assistance during my research studies. I wish to express my deepest gratitude to Dr. Gerrit Holighaus and Dr. Bernhard Weissbecker for their timely help, critical comments, support and discussions during my whole research studies. During my PhD studies the following individuals have contributed resources, ideas and support: Prof. Dr. Niko Balkenhol, Prof. Dr. Wolfgang Rohe, Dr. Martin Gabriel, Dr. Heinrich Lehmann- Danzinger, Lena Marie Simon, Christine Rachow, Jan Seelig, Dr. Maximilian von Fragstein, Thi Phuong Hoang, Andrea Hopf-Biziks, Daniel Jestrzemski, Yadanar Oo, Karly Rayne, Sara Nicke Mühlfeit, Ulrike Eisenwiener, Evelin Kistner, and Andreas Teichmann. I further like to thank all the external persons who supported my research work Dr. Stefan Dippel, Dr. Alice Christine Montino née Metzger, Dr. Lili Ren, Prof. Wolfgang Knoll, Prof. Dr. Petr Karlovsky, Dr. Maria Vinas and Elke Küster. I would also like to thank my beloved parents and sisters for all their love and support Finally, I thank my dear wife Revathi Selvam and my dear child Kathrina Rizpah for giving me forever moral support and love. iii CONTENTS Abstract i Acknowledgements iii Contents iv Abbreviations ix Lists of figures xi Lists of tables xiv Chapter 1 General Introduction 1 1.1. The nature and mechanism of the insect olfaction 1 1.2. Components of the insect olfactory system 3 1.2.1. Odorant binding proteins (OBPs) 3 1.2.2. Chemosensory proteins (CSPs)