A University of Sussex PhD thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details 1 Chemicals on the cuticle of ants: their role in hygiene, navigation and kairomone signalling to termites Thesis submitted by Alan Gallagher For the degree of Master of Philosophy School of Life Sciences University of Sussex August 2017 2 Declaration I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Signature:……………………………………… 3 University of Sussex Alan John Gallagher, Master of Philosophy Chemicals on the cuticle of ants: their role in hygiene, navigation and kairomone signalling to termites Summary This thesis describes investigations of how chemicals present on the cuticle of ants impact three important features of social living in insects: hygiene and disease resistance; navigation; and interspecies chemical signalling. Eusociality brings many benefits, but also has the potential to make insect colonies vulnerable to disease. In Chapter 2 of this thesis I investigate the role of the antimicrobial agent micromolide, in the Yellow meadow ant, Lasius flavus. Micromolide is found to be present on the cuticle of L. flavus workers, and is also found to be deposited onto a substrate by walking ants, revealing a possible mechanism for maintenance of sanitary nest conditions. Chapter 3 of this thesis focuses on navigation in L. flavus, specifically route-memory formation and the possibility of home-range markings providing a chemical cue via which ants can navigate from a food source to the nest. It was found that allowing ants to follow a pheromone trail to food increased the number of navigational errors made by returning ants, and that home-range markings did not provide effective guidance to ants returning to the nest. In Chapter 4, I report on a project undertaken during field work in Brazil into how cuticular hydrocarbons (CHCs) of the ant Camponotus arborious can act as kairomones when detected by Nasutitermes corniger, a common termite species. Experiments showed that N. corniger is less likely to repair experimentally opened tunnels in the presence of C. arborious CHCs, with 4 of 7 colonies tested blocking up tunnels, rather than rebuilding over CHC marked areas. Finally, Chapter 5 of this thesis discusses potential future projects, following on from the work presented in Chapters 2, 3 and 4. 4 Acknowledgements Firstly, I would like to say a huge thank you to my main supervisor Jonathan Bacon. It is safe to say that without his guidance, and considerable patience, this thesis would never have been completed. I know that he has put considerable time and effort into helping me through this MPhil, and for that I will be eternally grateful. My second supervisor, Liz Hill, was also hugely important, allowing me lab space and access to equipment that made this degree possible. I was very fortunate to work alongside a number of fellow social insect enthusiasts, whose advice was invaluable, foremost amongst whom is Tom Butterfield, another of Jonathan’s students. Working with Tom was a pleasure, and his advice on experimental design, and especially on using GC-MS, helped me get to grips with the techniques needed to complete this degree. Kyle Shackleton helped immensely with his advice on statistics, and all the members of LASI contributed by making my working environment such a fun and inspiring place to be. Last but not least, thank you to my parents, Paul and Julie Gallagher, who have supported me throughout my time studying. They are the ones who first impressed upon me the importance of education, and without them I would never have been able to undertake this MPhil, let alone complete it. 5 Chemicals on the cuticle of ants: their role in hygiene, navigation and kairomone signalling to termites Contents 1. Introduction .......................................................................................................................... 7 2. Micromolide on the cuticle of Lasius flavus and passive deposition of micromolide onto a substrate ..................................................................................................................................... 11 Abstract ................................................................................................................................... 11 2.1 Background ................................................................................................................. 12 2.1.1 Behavioural Defences ......................................................................................... 13 2.1.2 Chemical Defences .............................................................................................. 14 2.2 Materials & Methods .................................................................................................. 16 2.2.1 Housing and maintenance of ants ...................................................................... 16 2.2.2 Method for extraction of micromolide from paper substrate ............................ 16 2.2.3 Method for extraction of micromolide from the cuticle .................................... 17 2.2.4 Statistical analysis .................................................................................................... 18 2.3 Results ......................................................................................................................... 19 2.3.1 Identification of compounds of interest ............................................................. 19 2.3.2 Passive deposition of micromolide by L. flavus workers on paper substrate ..... 20 2.3.3 Substrate and cuticle extraction: ants with blocked or unblocked acidopores .. 22 2.4 Discussion .................................................................................................................... 25 3. Getting Home: How food is found has effects on Lasius flavus nest bound navigation .... 29 Abstract ................................................................................................................................... 29 3.1 Background ................................................................................................................. 30 3.2 Materials & Methods .................................................................................................. 36 3.2.1 Housing and maintenance of the ants ................................................................ 36 3.2.2 Conditioning paper with CHCs for T-maze experiment and Treatment 4 .......... 36 3.2.3 Experimental setup for T-maze: detection of CHC home range markings ......... 37 3.2.4 Experimental setup for H-maze: Treatments 1-4 ............................................... 38 6 3.2.5 Method for rectal gland extraction and preparation of trail-pheromone extract 39 3.2.6 Encouraging ants to enter the maze ................................................................... 39 3.2.7 Method Treatment 1: ants led to feeder with pheromone ................................ 40 3.2.8 Method Treatment 2: ants allowed to explore the maze to find the feeder ..... 40 3.2.9 Method Treatment 3: ants provided with pheromone trail to and from the food 41 3.2.10 Method Treatment 4: ants led to feeder with pheromone, return journey marked with home-range markings .................................................................................... 41 3.2.11 Statistical analysis method .................................................................................. 41 3.3 Results ......................................................................................................................... 42 3.3.1 L. flavus foragers can detect paper marked with CHCs ...................................... 42 3.3.2 Total number of attempts taken to complete the maze .................................... 42 3.3.3 Time taken to complete maze ............................................................................ 44 3.3.4 Turns encountered earlier on the outward journey are remembered more accurately on the return journey ........................................................................................ 46 3.4 Discussion .................................................................................................................... 47 4. Kairomone communication between Brazilian predator ants and their predated termites 53 Abstract ................................................................................................................................... 53 4.1 Background ................................................................................................................. 54 4.2 Materials & Methods .................................................................................................. 59 4.2.1 Extraction of ant cuticular hydrocarbons ........................................................... 59 4.2.2 Termites detecting ant cuticular hydrocarbons .................................................