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Understanding Mechanisms Behind the Toxicity of Ethanedinitrile, an Alternative Fumigant to Methyl Bromide

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Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the author. UNDERSTANDING MECHANISMS BEHIND THE TOXICITY OF ETHANEDINITRILE, AN ALTERNATIVE FUMIGANT TO METHYL BROMIDE A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Entomology (Plant Sciences) at Massey University, Manawatū, New Zealand Godwin Nutifafa Gidiglo 2021 Thesis Supervisors Dr. Adriana J. Najar-Rodriguez (Plant and Food Research, Palmerston North, New Zealand) Dr. Matthew M.K.D Hall (Department of Industry, Tourism and Trade, Darwin, Australia) Dr. Maria Minor (Massey University, Palmerston North, New Zealand) Professor Wang Qiao (Massey University, Palmerston North, New Zealand) Thesis examiners Dr. Andrea Clavijo-McCormick (Massey University, Palmerston North, New Zealand) Professor Phil Lester (Victoria University of Wellington, Wellington, New Zealand) Professor Vaclav Stejskal (Czech Agricultural University, Czech Republic) Abstract Ethanedinitrile (EDN, syn. cyanogen, EDN Fumigas®) has the potential to replace methyl bromide for the phytosanitary treatment of export timber and logs. Previous studies have shown that the toxicity of EDN differs between insect species and their life stages. For example, adults of the golden-haired bark beetle, Hylurgus ligniperda (Fabricius) (Coleoptera: Curculionidae) are highly tolerant to EDN, while those of the burnt pine longhorn beetle, Arhopalus ferus (Mulsant) (Coleoptera: Cerambycidae) are highly susceptible, implying that the mode of action might be species-specific. To understand the mechanisms behind the toxicity of the fumigant to insects, I examined the anatomy of the cuticle and spiracles of adults of both beetles. I found that H. ligniperda possessed smaller total spiracle area, suggesting the possibility of less EDN uptake; and more cuticle layers, suggesting the possibility of delayed penetration of the fumigant. These morphological features might limit the uptake of EDN and the related toxic effect. Further studies have shown that the main route of entry for EDN into the insect body was through the spiracles for A. ferus, and through the cuticle for H. ligniperda. The fumigant caused a reduction in walking activity, narcosis, and paralysis in both species. Reductions in walking activities and narcosis occurred earlier in H. ligniperda, suggesting that this species may be able to quickly decrease metabolism and respiration rates in response to EDN, which could minimize uptake and toxic effect of the fumigant. Using attenuated total reflection - fourier transformed infrared (ATR - FTIR) spectroscopy, I identified higher cyanide (CN-) peaks in the insect ganglia in adult A. ferus, confirming the higher susceptibility of this species to EDN. This study has contributed to the knowledge of mechanisms behind the variable toxicity of EDN to adult A. ferus and H. ligniperda. The findings will provide a platform for future studies of the mode of action of EDN against insect pests. i Acknowledgements I will give thanks to you with all my heart, oh Lord. I will remind you about all your wonderful deeds upon my life (Psalm 9:1). There is none to be compared unto thee (Jeremiah 10:6). Indeed, I have felt your intervention all these while. The unflinching support and blessings you offered me were unmatched. I say, “akpe na wo”. I would like to register my special thanks and appreciation to Prof. Qiao Wang (my primary supervisor), for his amazing mentorship and guidance throughout this work. Prof., I appreciate your encouragement and the opportunity to develop as a researcher, under your distinguished supervision. Your directives regarding the conduct of this research and life after studies have prepared me to face the future with a broad smile. I also owe my co-supervisors: Dr. Adriana J. Najar-Rodriguez, Dr. Matthew K.D. Hall and Dr. Maria Minor lots of gratitude for their support throughout my PhD studies. I appreciate your words of encouragement, guidance, patience, motivation and dedication that have seen me through this journey successfully. Your unprecedented supervision had kept me on track throughout the entire period of study and writing of this thesis. I am utterly indebted to Plant and Food Research (PFR), Stakeholders in Methyl Bromide Reduction (STIMBR), the Massey University Doctoral Research Scholarship, Helen E. Akers Scholarship and Draslovka (manufacturers of EDN) for funding my entire PhD studies. I also acknowledge Dr. Duncan Hedderley of Plant and Food Research for helping with statistical advice. The beauty of this work is a clear manifestation of your various degrees of support. Once again, many thanks to Plant and Food Research, for providing me everything I needed for my laboratory work and the environment to write this thesis. To members of the Disinfestation team: Emma Noakes, Steven Burgess, Dr. Saeedeh Afsar, Dr. Kambiz Esfandi, and the entire research team, I appreciate your assistance and encouragement throughout the entire study period. I would also like to thank Massey University for the opportunity to study and acquire an extensive range of opportunities to develop intellectually, professionally and personally. Special thanks go to Prof. Mark Waterland for access to ATR-FTIR spectroscope. ii To my lovely friends and relatives in New Zealand: Robert Kofi Fugah, Emmanuel Kyere, Moses Kakrada, Tily Kordieh, Effah Evans, Dr. Nicholas Amponsah, Carol Dolman and others, I appreciate the fantastic moments we shared together. I ask God’s blessings upon your lives and I look forward to the continuity of our fun moments. Also to my friends in the diaspora, Setutsi Tetteh, Dumegah Moses Tademe, Godwin Attitsogbui, Rev. Sesi, Rev. D.M. Banini, Rev. Daniel Magnus-George, Abigail Ahiable, Dr. Hormenu Thomas, Dr. Songwe Fanuel, Dr. Emmanuel Edutwum, and Prof. Wisdom Akpalu for your efforts in checking on me from time to time. God bless you all. Many thanks to Dr. Vern Collette, his family and the entire membership of Elijah Centre church for their prayers and support. I also thank my family, particularly my lovely mom, Felicia Gidiglo, my brother (Pastor Wilson Selasi Gidiglo) for the prayers and sacrifices they offered on my behalf. iii Table of Contents Thesis Supervisors ...................................................................................................................... i Abstract .......................................................................................................................................i Acknowledgements .................................................................................................................... ii Table of Contents ....................................................................................................................... iv List of Tables ........................................................................................................................... vii List of Figures ......................................................................................................................... viii List of Abbreviations ............................................................................................................... xiii CHAPTER 1 General Introduction .............................................................................................. 1 1.1 Background ................................................................................................................... 1 1.2 Importance and relevance of this research ...................................................................... 3 1.3 Aims and objectives of this study .................................................................................. 5 CHAPTER 2 Literature Review .................................................................................................. 6 2.1 Introduction ................................................................................................................... 6 2.2 The burnt pine longhorn beetle, Arhopalus ferus (Mulsant) ........................................... 7 2.2.1 Classification and distribution .............................................................................. 7 2.2.2 Host plants and importance................................................................................... 7 2.2.3 Morphology and life cycle .................................................................................... 8 2.3 The golden-haired pine bark beetle, Hylurgus ligniperda (Fabricius) ........................... 12 2.3.1 Classification and distribution ............................................................................ 12 2.3.2 Host plants and importance................................................................................. 12 2.3.3 Morphology and life cycle .................................................................................. 13 2.4 Ethanedinitrile (EDN).................................................................................................. 17 2.4.1 Physical and chemical properties of EDN ........................................................... 17 2.4.2 The efficacy of EDN against insect pests ............................................................ 19 2.5 Insecticide mode of entry into insects .......................................................................... 22 2.5.1 The insect spiracle
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