Mechanistic bases of metal tolerance: linking phenotype to genotype. Craig Anderson 2012 PhD Thesis Cardiff University School of Biological Sciences Abstract Ecotoxicology is currently undergoing a revolution as the result of new technological advances in molecular biology, capable of finely resolving metabolic mechanisms associated with exposure. These high-throughput analyses can detail the evolutionary and ecological implications of exposure in non-model organisms, such as the earthworm, Lumbricus rubellus. This terrestrial sentinel has been observed across former mine sites that are highly contaminated with arsenic and have been found to mitigate toxicity at soil concentrations that cause mortality in unadapted populations. This is indicative of the adaptive capacity of natural populations recently exposed to persistent and strong selection pressure. However, mechanisms surrounding adaptation to arsenic in L. rubellus have yet to be characterised, and so the effects of exposure are broadly reported with the aim of distinguishing resistance from phenotypic plasticity in natural populations. Unadapted earthworms were initially used to derive basal phenotypic variation associated with arsenic exposure. Variation in life-history parameters was observed among adult and juvenile L. rubellus, establishing relative sensitivity and population- level inferences. A systems biology approach was employed to describe molecular mechanisms associated with arsenic metabolism, encompassing transcriptomic and metabolomic analyses, underpinned by arsenic speciation. Insight into the genetic bases of arsenic resistance, which enable persistence of L. rubellus at highly contaminated sites, was sought. Recombinant inbred lineages derived from adapted populations, were cultivated and their phenotypes relative to arsenic exposure determined. Phylogeographic analyses were used to interrogate genetic variation among populations inhabiting former mine sites as well as proximal control sites. A mitochondrial marker defined cryptic species across the UK, but did not establish soil chemical profiles relative to clade occurrence. RADseq better resolved genetic variation at these sites, determining that soil geochemistry is strongly associated with genetic variation. Furthermore, genomic markers inferred genetic erosion, found to selectively reduce variation at sites relative to a single clade. DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. 28/09/2012 Signed ……………………………………… (candidate) Date ………………………… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of a PhD. 28/09/2012 Signed ………………………………………(candidate) Date ………………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. 28/09/2012 Signed ……………………………………… (candidate) Date ………………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. 28/09/2012 Signed ……………………………………… (candidate) Date ………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. 28/09/2012 Signed …………………………………… (candidate) Date ………………………… Acknowledgements Primarily, thanks tremendously to my supervisors Dave, Claus and Pete for donating to me generous portions of their time, effort and talent. I wouldn’t have gotten here without their guidance and enthusiasm. Thanks so much to my collaborators for all of their help and infinite patience- Mark Hodson, Ben Elsworth, Jake Bundy, Manuel Liebeke, Karim Gharbi, John Davey and Pablo Orozco ter Wengel. A massive thanks to Dan Pass and Pier Francesco Sechi, who have been both great friends and colleagues. Thanks to all the great students at CEH, including Rory O’Connor, Fingers Phil, Leah Cuthbertson, Christine Tansey and especially Fraser Sinclair. Thanks to the Danish contingent at CEH and Copenhagen- Nils, Hanna, Lisbeth and Laura. Cheers to Chris Goulebourne for all of the seriously good times and posh meals. Thanks to the guys from Plymouth- Matt, Alex, Raul. Maybe Lenny. Big thanks to Harry, Mel, Stu, Yaser, Luis and Ceri for time well spent in Cardiff. I’m also extremely grateful to all the fantastic support I received at Cardiff, including Steve Turner, Esh, Julian, Mike O’Reily, Joan, Carsten and Mike Bruford. Thanks to the Oxford skaters for all of the amazing times and trips- Sam, Tom, Hugo Boss, Louie, Doddy, Leddy and JK. Thanks to Aaron and James for everything. Cheers to John Morgan, for showing everyone how it’s done. Thanks to my parents and sister for taking an interest, supporting me and putting up with me as I wrote up. Thanks to my wonderful girlfriend Sarah, though this is probably worse because of her... Finally, I’d like to thank Tom and Jenny. I absolutely wouldn’t be where I am without their friendship, generosity and company. I am forever indebted. Contents Chapter 1 .................................................................................................................................... 1 Introduction ............................................................................................................................... 1 1.1 General Introduction ......................................................................................................... 1 1.2 Phenotypic Plasticity ........................................................................................................ 2 1.3 Maternal effects ................................................................................................................. 4 1.4 Epigenetics ......................................................................................................................... 5 1.5 Resistance ........................................................................................................................... 8 1.6 Investigating adaptation in natural populations ..................................................... 11 1.7 Research Aims ................................................................................................................. 16 Chapter 2 Life-History Effects of Arsenic Toxicity in Clades of the Earthworm, Lumbricus rubellus. .................................................................................................................................... 18 2.1 Introduction....................................................................................................................... 18 2.2 Materials and methods ................................................................................................... 19 2.2.1 Test Medium ................................................................................................................... 19 2.2.2 Adult Arsenic Exposure ................................................................................................. 20 2.2.3 Juvenile Arsenic Exposure............................................................................................ 20 2.2.4 Juvenile Genotyping ...................................................................................................... 21 2.2.5 Soil Arsenic Analysis ..................................................................................................... 21 2.2.6 Tissue Metal Analysis .................................................................................................... 22 2.2.7 Statistics........................................................................................................................... 22 2.2.8 Population Model ............................................................................................................ 23 2.3.1 Soil Physico-Chemistry ................................................................................................. 24 2.3.2 Adult and Juvenile Mortality .......................................................................................... 24 2.3.3 Cocoon Production ......................................................................................................... 25 2.3.4 Adult Earthworm Tissue Arsenic Concentrations ...................................................... 26 2.3.5 Growth and Maturation .................................................................................................. 26 2.3.6 Effects of Arsenic on Intrinsic Rate of Population Increase ..................................... 29 2.3.7 Genetic Variation, Juvenile Development and Arsenic Sensitivity ......................... 29 2.4 Discussion......................................................................................................................... 31 2.5 Conclusion ........................................................................................................................ 35 Chapter 3 Arsenic Toxicity and Metabolism in the Earthworm, Lumbricus rubellus. ............ 36 3.1 Introduction....................................................................................................................... 36 3.2 Methods ............................................................................................................................