Behavioural Aspects of the Neonicotinoid Resistance of Myzus persicae Lucy Fray Imperial College London Ecology and Evolution Department PhD 1 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Declaration of originality All of the work contained in this PhD thesis is my own, as described in my own words. All references to the works of others have been appropriately distinguished from my own work and referenced. 2 Acknowledgements During the course of this PhD I have worked in three groups under four supervisors, and so I have a lot of people to thank. First, I wish to thank my supervisors, Dr Glen Powell, Dr Rob Lind, Dr Simon Leather and Dr Dennis Wright, all of whom gave me invaluable support in what turned out to be a fairly tumultuous PhD... There is no way I could have finished this without them. Secondly, I wish to thank the BBSRC and Syngenta Crop Protection for jointly funding me to work at Imperial College London. The Royal Entomological Society and The Society for Experimental Biologists also provided me with grants to attend several conferences and present my work, for which I am very grateful. Next I would like to thank my PhD advisors, Dr Colin Turnbull and Dr Alex Grabov, who gave me much useful help and advice. I wish to thank all of the people at Jealott’s Hill who have helped me to complete this challenge. I can’t name you all, but I would like to give special thanks to Miriam Daniels, Dave Bartlett and the Biokinetics team. Jenny Pennack gave a lot of her time and was invaluable in helping me with the work in chapter five. I’d like to thank Penny Turner and her team for all of the help in maintaining the aphid cultures, and also Rosie Woodruff and her team for sowing an awful lot of Chinese cabbage. Chris Beckingham needs a special mention for all of his help with the FRC culture and generally keeping me sane. Also Emily Marchant, Steph Osbourne, Alison Fraser, Emily Hawkes, Steve Johnson, James Allanson and everyone else who kept me going over lunchtime cups of tea and listened to all of my complaining in good humour. Oh, and the climbers as well. Next I must thank all of the people at Imperial College who were with me when this study began, especially my group members Simon Hodge, Martin Selby, Sadia Kanvil and Sophie Stewart. Thank you all for showing me where to begin, and keeping me going. There are also many other people in the Plant Science department who gave me invaluable help and advice, and again I can’t name them all but Joe McKenna, David Charles, Rosa Lopez-Cobollo, Leanne Williams, Lars Kjaer and Gianna Anton all deserve a special mention. Also Fiona May and Ian Morris, both of whom always had a cheery word to give. And also all of the attendees of the Friday beer sessions –you know who you are. 3 Thanks go to Anthony Fitzgerald and Christine Short, who patiently went through the ordering system multiple times with me, and Diana Anderson who sorted out all of the admin associated with my move to Silwood Park. I think I should also thank my housemate Helen Cockerton, and Sarah and Simon of The Barge. I want to thank my Mum and Dad, who were always ready to help and listen despite not being entirely sure what I was doing, and of course my dogs. The dogs were particularly generous in making me go outside at least once a day during the writing up stage. Finally, I want to thank my partner, James, who went through every single step of this journey alongside me, and always had time to give despite making the same journey for himself. James, I love you for a reason. Oh, and I also need to thank the aphids. 4 “Aphids, known as greenfly, are very friendly bugs, They live in great big colonies and give each other hugs, They all get on most famously until one needs more space, And then like other sisters, kick each other in the face. Aphids like to feed on plants, they drink up all the sap, Hanging underneath the leaves into the phloem they tap, Their mouth is like a needle, the cuticle it pierces, It travels to the vessels via intercellular spaces. As they try to find the phloem, the other cells they puncture, Sometimes they transmit viruses but sap feeding's their juncture. When they finally reach the phloem they first will salivate, This unblocks all the forisomes and seals the poor plant's fate. Aphids will feed for hours on the phloem and just think, And when they tire of thinking from the xylem they will drink. As a pest of plants they do one other thing of point, They fire out lots of honeydew and fungus up the joint. They reproduce asexually, they don't go in for dates, 80 daughters born from each no wonder they need space! The babies are born naturally, for aphids give birth live, And inside the newborn young there's more daughters inside! When it gets to winter, the aphids they grow wings, They fly off to their winter home, where they now mate and things. They like to overwinter as tiny small black eggs, And when it's spring they rise again, as horrible plant-based cleggs!” -Lucy Fray 5 Abstract The peach potato aphid Myzus persicae (Sulzer) is an agricultural pest of high economic importance distributed across much of the globe. The main method of control of this pest is the application of neonicotinoid insecticides such as imidacloprid and thiamethoxam (TMX). Until recently no significant resistance to neonicotinoids was known in M. persicae. In 2009 however, a clone exhibiting severe resistance to neonicotinoids was collected from peach orchards in France. This resistant clone was designated ‘FRC’. Published literature has shown the high levels of neonicotinoid resistance exhibited by the FRC clone to be due to a combination of adaptations including enhanced overexpression of the detoxifying cytochrome P450 CYP6CY3, a target site mutation in the nicotinic acetylcholine receptor and reduced cuticular penetration. No studies however have investigated the possibility of a behavioural neonicotinoid resistance mechanism in M. persicae. This study describes an investigation into the presence or absence of a behavioural component to the neonicotinoid resistance of the FRC clone. Two different aspects of the behaviour of the FRC clone, feeding and dispersal, were compared to that of a clone of low neonicotinoid resistance (5191A) and a clone of complete neonicotinoid susceptibility (US1L). A significant difference was found in the dispersal behaviour of the FRC clone which could potentially contribute to its high levels of neonicotinoid resistance. Further investigation was then undertaken to determine whether this altered behaviour influenced the efficiency of the FRC clone as a vector of the persistently transmitted Turnip Yellows virus. No difference in vector ability was detected between the US1L and FRC clones. This study concludes that there is significant evidence of a potential behavioural aspect to the neonicotinoid resistance of the M. persicae FRC clone. 6 Table of Contents Copyright Declaration and Declaration of originality.……………………..……….…………2 Acknowledgements……………………………………………………………….….………..3 Abstract……………………………………………………………………….….……………6 Chapter 1: Introduction………………………………………………………………………12 1.1 Lifecycle and formation of clones..………………………………………....…....12 1.2 Control and resistance of Myzus persicae…………………………….………….13 1.3 Neonicotinoids…………………………………………………………………...14 1.4 Neonicotinoid resistance…………………………………………………………15 1.5 Molecular understanding of neonicotinoid resistance……………………...……16 1.6 Neonicotinoid metabolism and resistance.……………………………………….21 1.7 Behavioural effects of insecticides…………………………………………...….23 1.8 Hemipteran feeding physiology………………………………………………….24 1.9 Hemipteran feeding terminology……………………………………………...…26 1.10 Hemipteran feeding and plant virus transmission………………………………28 1.11 Aims and objectives…………………………………………………………….33 Chapter 2: Discerning appropriate sublethal doses.………………………………………….34 2.1 Introduction..………………………………………………………….............….34 2.2 Methods.……………………………………………………………………....….36 2.2.1 Insects and plant material………………………………………………36 2.2.2 Plant drench dose-response…………………………………………….37 2.2.3 Preparation of artificial diet……………………………………………37 2.2.4 Formation of diet rings…………………………………………………38 7 2.2.5 Artificial diet dose-response set-up…………………………………….39 2.2.6 Wide range artificial diet dose-response.……………………………….40 2.2.7 Narrow range artificial diet dose-response.…………………………….40 2.2.8 Statistical analysis.……………………………………………………...40 2.3 Results.....…………………………………………………………………………41 2.4 Discussion.………………………………………………………………………..46 Chapter 3: Dispersal behaviour of neonicotinoid resistant and susceptible Myzus persicae…51 3.1 Introduction………………………………………………………………………51 3.2 Methods…………………………………………………………………………..53 3.2.1 Insect and Chinese cabbage cultures…………………………………...53 3.2.2 Experimental set-up…………………………………………………….53 3.2.3 Statistical analysis.……………………………………………………...55 3.3 Results…………………………………………………………………………....56 3.3.1 Rate of movement……………………………………………………...56 3.3.2 Aphid location: leaf vs. disc……………………………………………63 3.3.3 Aphid location: leaf tissue vs. agar.…………………………………….72 3.3.4 Aphid mortality and reproduction……………………………………...72 3.4 Discussion.………………………………………………………………………..78