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Investigating the lignocellulolytic gut microbiome of huhu grubs A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences at The University of Waikato by JAYADEV PAYYAKKAL VISWAM _________ The University of Waikato 2016 Abstract New Zealand’s endemic longhorned beetle (Prionoplus reticularis) larvae, huhu grub, are xylophagous and feed on a broad range of dead trees. Since insects are not believed to have the ability to fully degrade lignocellulose, it is likely that microbes present in the gut assist in degrading lignocellulose to release energy rich sugars. This process is considered the rate limiting step in the utilisation of woody material in biofuel production. To better understand the gut consortium present while feeding on the lignocellulose and cellulose components, huhu grubs were initially collected and reared on non-degraded pine blocks (lignocellulose) or cotton (cellulose). The gut fungal and bacterial community of huhu grubs was investigated using high-throughput sequencing and analysis techniques. Furthermore, the effects of a dietary switch on both fungal and bacterial communities was explored by switching huhu grubs initially reared on pine to cotton, and vice versa. Cellulose- and lignocellulose-reared huhu grubs were expected to host a wide range of bacteria and fungi in their gut, some of which produce lignocellulose degrading enzymes. Assessment of the gut fungal community composition confirmed that cellulose-reared huhu grubs were significantly distinct from lignocellulose-reared grubs. The fungal community of lignocellulose-fed huhu grubs was more functionally diverse, with several yeasts, soft-rot fungi and potential hemicellulose and cellulose degraders present. Whereas, the fungal community of cellulose-reared grubs was mostly abundant in yeasts and potential cellulose degraders. When cotton-reared grubs where switched to pine (lignocellulose), lignocellulose was observed to have a ‘bottleneck effect’ or selective pressure on the fungal gut community. The gut bacterial community of huhu grubs reared on cellulose or lignocellulose diets seemed to be unaffected by the selective pressure of the diet, and no significant change in the bacterial community was observed following dietary switch. Therefore it was concluded that the bacterial community while still important is playing a secondary role to the fungi. Finally, bioprospecting studies conducted using the frass (excretia) of these huhu grubs resulted in the isolation of a bacterium, Acinetobacter H23, capable of producing an extracellular laccase. Overall, the increased understanding of structure and composition of the gut microbiome of huhu grubs reared on cellulose or lignocellulose diets, along with ii isolation of novel lignocellulolytic isolates could prove to be significant for the biofuel industry. iii Acknowledgements First and foremost I would like to gratefully acknowledge my supervisors Assoc Prof. Ian McDonald and Dr. Charles lee for their support, advice and encouragement. In particular, I would like to express my appreciation for their support during the hectic “final phase” and for always opening their door whenever I needed help. I would like to extend my thanks to Prof. Craig Cary for his insights and for providing me with opportunities to work. I also would like to thank Prof. Hugh Morgan for introducing me to this project, for his valuable guidance in the early stages of my PhD and his inputs into this thesis. I extend my sincere gratitude to Prof. Roberta Farrell for providing me with valuable advice. I wish to gratefully acknowledge several individuals that helped me to endure this long but fruitful journey. Especially, I wish to thank Lynne Parker for her incredible support throughout my time in Thermophile Research Unit. I will cherish all the random conversations, word wheels, life advices and coffees. Above all I am thankful for gently guiding me back to right paths whenever I strayed and for always looking out for me. Further, I wish to thank Sarah Campbell for her support and cheerful coffee sessions. I extend my thanks to everyone in TRU and Proteins and Microbes laboratory for all your help and support. I wish to express my appreciation to Erica for helping me learn about the magic of enzymes. Additionally, I would like to thank Gloria Edwards, Vicki Smith, Tanya Wakefield, John Longmore, Ivan Bell, Roanna Richards and other staff members for their continued support. I appreciate the encouragement, support and help provided by Kev and Stephen. Thank you for being there with the readiness to help at all times. Thank you Josh (BH) and family, Courts, Cantona, Chels, Steve, Claire, Cherbo, Stace, Knoxy, Magda, Mateusz, Mafalda, Bekkah AP, Chris, Qi, Mitchy, Brad, Ethan, Toma, Tama, Tauks, Centry, Tens, James, Charlie, Ez, Aaron, Hugo, Simone, my touch rugby group and netcafix group for being part of my story in this beautiful country. I wish to thank Maria for her support, joyful craziness, squash sessions and specially for the delicious triple- egg omelettes. Flo (DQ), I will treasure the memories of the time we have shared together. I would like to thank you for the crazy laughing sessions, random conversations, for getting concerned and caring about me, for pushing me to finish my thesis and for iv your extended support. I could not have asked for anyone better to be around in these few challenging months. This acknowledgement would be incomplete without expressing my thanks to Tif. Thank you for always being there through my struggles and happiness. Without you, this journey would not have been the same. Your unconditional care, crazy giggles, random hugs and extended support throughout these years have definitely been among the highlights of this journey. Thank you for being such an amazing person. Finally I would like to thank my parents, sister and other family members for your constant support and unconditional love. Without you this journey would have been impossible. I would love to dedicate this thesis to my grandparents whom I am certain would have been extremely proud of my achievements throughout my life. v Table of Contents Abstract .................................................................................................................... ii Acknowledgements ................................................................................................. iv Table of Contents .................................................................................................... vi List of Figures .......................................................................................................... x List of Tables.......................................................................................................... xii List of Abbreviations............................................................................................. xiv Chapter One: Introduction........................................................................................1 1.1 General background ......................................................................................1 1.2 Lignocellulose ...............................................................................................3 1.2.1 Cellulose ................................................................................................4 1.2.2 Hemicellulose ........................................................................................4 1.2.3 Lignin ....................................................................................................5 1.3 Lignocellulose degradation ...........................................................................5 1.4 Lignocellulolytic fungi and bacteria .............................................................7 1.5 Lignocellulolytic enzymes ............................................................................8 1.5.1 Cellulose degrading enzymes ................................................................8 1.5.2 Hemicellulose degrading enzymes ........................................................8 1.5.3 Lignin degrading enzymes ....................................................................9 1.6 Wood-feeding insects..................................................................................12 1.7 Huhu............................................................................................................14 1.8 Diet-induced gut microbiome alterations....................................................15 1.9 Significance of high-throughput sequencing ..............................................16 1.10 Objectives....................................................................................................17 1.11 Summary .....................................................................................................18