Using DNA to explore predator diet in temperate marine ecosystems Kevin Scott Redd BSc Honours, University of Tasmania Bachelors in Biology, University of California Santa Cruz Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy. University of Tasmania 7 April 2017 1 Declaration of originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of the my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Kevin Scott Redd Statement of authority of access This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. The publishers of the papers comprising Chapters 2, 3, 5 and 7 hold the copyright for that content, and access to the material should be sought from the respective journals. The remaining non published content of the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968 Kevin Scott Redd 2 Thesis Abstract This thesis describes the utility of molecular techniques to detect and identify predator/prey interactions in temperate marine ecosystems with an emphasis on the southern rock lobster, (Jasus edwardsii) in Tasmania. A range of DNA-based methods are developed and implemented to better understand the role of this important benthic predator in shaping reef communities. The design and testing of numerous general and species-specific PCR primers is detailed and the utility of these PCR-based assays for monitoring trophic interactions is explored. Captive feeding experiments examine the efficacy of single- species-specific prey detection assays for detection of predation by southern rock lobsters (Jasus edwardsii), and also determine the longevity of the DNA signal and the possibility of quantitative PCR to infer the relative amount of prey consumed by these decapod crustaceans. Based upon these results, the molecular methodologies were tested in a large scale manipulative field experiment to investigate the predation capacity of southern rock lobsters on sea urchins (Centrostephanus rodgersii and Heliocidaris erythrogramma) to understand the shaping of benthic habitats via rock lobster predation in the wild. The results of the dietary component of this experiment are presented and the implications for management are explored briefly. To understand the role of Jasus edwardsii in the marine environment, a broad scale molecular prey inventory approach was used to determine the overall diet of southern rock lobsters both spatially at fished and unfished locations and temporally at sites over several years in Tasmania by using molecular cloning and the use of the 454 Next Generation / pyrosequencing platform. The results of these southern rock lobster prey inventories are presented as well as several other examples of situations with other marine animals using related applications of molecular dietary methods where prey information is difficult to obtain (cephalopods) or is problematic due to the challenges of capturing predators (elasmobranchs). Also provided is a critical review of the utility of molecular prey detection in examining the diet of marine crustaceans, cephalopods and chondrychthians and the thesis concludes by summarising the benefits and pitfalls in using DNA-based dietary methods to address both specific predator-prey issues and more general broad scale trophic webs. Key Words: Molecular prey detection, molecular trophic interactions, prey inventory, rock lobster diet, Jasus edwardsii, rock lobsters, sea urchins, Centrostephanus rodgersii, Heliocidaris erythrogramma, polymerase chain reaction (PCR), quantitative PCR, Real Time PCR, seven gilled shark, Notorynchus cepedianus, Octopus vulgaris, paralarvae, prey detection 3 Acknowledgements These projects came about primarily due to the extensive experience of my supervisors and their ongoing scientific endeavours to further our understanding of the marine environment. The initial work on rock lobsters in particular was the result of discussions between Simon Jarman and Stewart Frusher about the possibility of using the newly emerging molecular techniques to better understand the diet of wild rock lobsters in Tasmania. The sea urchin component began with the fundamental Tasmanian reef ecology work of Craig Johnson and Scott Ling, two of the most enthusiastic and inspiring marine ecologists I have ever met. They both motivated me to think about the complex issues of marine ecosystems in new ways and were the primary drivers for the completion of this thesis. For their support throughout the process I am eternally grateful. Once the PhD was underway, I was fortunate to meet several other postgraduates who also had interests in these molecular techniques and offered their predators as side projects to tackle. It has been a pleasure to work with Álvaro Roura Labiaga and Adam Barnett on these additional endeavours and to share their enthusiasm when we achieved the results contained in those manuscripts. All the technical molecular work in this thesis was done at the University of Tasmania’s Molecular Genetics Laboratory. This facility was an excellent platform for the development of new techniques as a wide range of researchers from other disciplines also shared the space and their expertise quite feely. In particular, I am indebted to the sage advice of both Dr Bob Elliott and Dr Valérie Hecht whose collective knowledge of molecular biology is incredible. The Laboratory manager, Adam Smolenski also provided ongoing support and assistance as I went along. The field work has been accomplished in conjunction with the UTAS Marine Ecology (KZA 356) Unit, the Centrostephanus project as well as the ongoing sampling which takes place at IMAS/TAFI/MRL. It has been great fun to work alongside the adept and experienced people who also share a passion for the marine environment. In particular David ‘Irish’ Faloon, Ruari Colquhoun, Kylie Cahill, Sarah Pyke, Dan Haley and the multitude of keen undergraduates who helped us pull lobster pots in the Maria Island Marine Protected Area, as well as at the St Helens and North Bay field sites. The funding was provided by the Fisheries Research and Development Corporation through two projects; Towards integrated multi-species management of Australia’s SE reef fisheries: A Tasmanian example (FRDC 2004/013) and Rebuilding Ecosystem Resilience-Assessment of management options to minimize formation of “barrens“ habitat by the long-spined sea urchin (Centrostephanus rodgersii) in Tasmania (FRDC 2007/045). Additional funding came from the ANZ Charitable Trust-Holsworth Wildlife Research Endowment. Of course, none of this would have been possible without the marine organisms which really should be the stars of the show. The southern rock lobster is a very resilient and tenacious creature which I have grown to love over the past few years. I have been accused of keeping these crustaceans as pets, of committing unspeakable acts of torture on them, of cooking them better than anyone else on the planet and of handling them like a commercial fisherman. These highly prized and handsome animals have landed me on the television, in the MoBio ‘Where in the World’ website and reagent catalogues as well as in the local newspaper on many notable occasions. I honestly hope that the management of these fascinating animals improves in Tasmania and that future generations will know what it is like to hold a fully grown adult Jasus edwardsii and to marvel at its beauty. 4 Table of Contents Thesis Abstract ............................................................................................................................... 3 Acknowledgements ........................................................................................................................ 4 Table of Contents ........................................................................................................................... 5 Chapter 1: General introduction ...................................................................................................... 10 1.1 Abstract .................................................................................................................................. 10 1.2 Introduction ............................................................................................................................ 10 1.3 Traditional approaches ........................................................................................................... 11 1.4 Rise of technology: new approaches ...................................................................................... 12 1.5 Molecular techniques and applications ................................................................................. 12 1.6 Overall structure of this thesis ............................................................................................... 14 Chapter 2 overview .................................................................................................................. 15 Chapter 3 overview .................................................................................................................. 15 Chapter 4 overview .................................................................................................................