The effects of metal nanoparticles on the microbiome and immune responses of earthworms Elmer Swart 2020 A thesis presented for the degree of Doctor of Philosophy Supervisory group: Prof. P. Kille1, D.J. Spurgeon2 and Dr. C. Svendsen2 1 Cardiff University, School of Biosciences, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK 2 UK Centre for Ecology and Hydrology, Maclean Building, Benson lane, Wallingford, OX10 8BB, UK This page intentionally left blank ii Summary Metal nanomaterials are increasingly applied as an antimicrobial agent in consumer products, coatings and pesticides. Through environmental release, non-target microbes, including symbiotic microbiota associated with animals, may be at risk due to exposure to these nanomaterials. Here, the effects of biocidal nanomaterial exposures (i.e. copper oxide and silver) on the gut microbiome of three earthworm species (i.e. Eisenia fetida, Lumbricus terrestris and Aporrectodea calignosa) and associated soils are studied using a metabarcoding approach. Further, the consequences of a microbiome disruption by nanomaterials on the E. fetida immune responses and the resilience of E. fetida to an infection by the bacterium Bacillus subtilis are investigated. This thesis provides a unique and in-depth view of the gut bacterial microbiome of three environmentally relevant earthworm species and shows how metal pollutants can affect these host-associated bacterial communities. It is shown that the resident component of the earthworm gut microbiome is largely independent from the associated soil bacterial communities. Further, through high replication (within and across concentrations), this thesis shows that the earthworm resident gut microbiome is largely resilient to exposure to antimicrobial nanomaterials. However, a key earthworm symbiont (i.e. the Mollicutes ‘Candidatus Lumbricincola’) that likely plays a role in earthworm digestion is negatively affected by exposure to copper oxide nanomaterial. These adverse effects on this symbiont were recorded in both short-term lab exposures and long-term outdoor soil mesocosm studies. Despite the impact of copper oxide nanomaterial on the earthworm gut microbiome, no evidence for an effect of nanomaterial exposure on host immunity or host susceptibility to a bacterial infection was found. This thesis demonstrates that pollutants can adversely affect crucial earthworm microbes, highlighting the need for further testing for effects of pollutants on microbiomes. The methodological approach adopted in this thesis should guide such future studies. iii Acknowledgments First and foremost, my gratitude goes to my supervisory team, Pete, Dave and Claus. Thank you for your continuous support and friendship over the last four years. I feel privileged to have you as my mentors. I would also like to thank Diana Boraschi and the whole of the PANDORA consortium for an amazing experience. It has been a real pleasure to work with such excellent researchers. Further, my thanks goes out to Carolin Schultz, Stephen Short, Elma Lahive, Marianne Matzke and rest of the ‘Ecotox’ team at the UK CEH. I cannot image a nicer and more supportive group of people to work with. Also thanks to Tim Goodall, Lindsay Newbold and Anna Oliver for your patience, kindness and help. In Prague, Petra Procházková and Jiří Dvořák, thank you for a great collaboration, I had a truly wonderful time working with you. Also thanks to Szabolcs Hernádi for helping me out on numerous occasions. My office mates, Holly, Denny and Ilze, thank you so much for your friendship which has meant a lot to me. Many thanks to my parents, Sjaak and Joke, whose everlasting love and support allowed me to pursue a career in science. Lastly, thank you Jack for being with me from the beginning and pulling me through the most difficult parts along the way. The last four years has been an amazing period in my life thanks to you. This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 671881. iv Contents Summary................................................................................................................................. iii Acknowledgments ................................................................................................................... iv Declarations ............................................................................................................................ ix Statement on publications......................................................................................................... x Statement on research collaborations ....................................................................................... xi 1. Introduction ......................................................................................................................... 2 1.1 General introduction .................................................................................................. 2 1.2 The role of microbial symbionts in the health and functioning of invertebrate animals... 3 1.2.1 Common beneficial roles of microbes in invertebrate health ................................. 4 1.2.2 Regulation of the microbiome ............................................................................. 7 1.2.3 Consequences of the microbiome disruption (dysbiosis) ....................................... 9 1.3 Biocidal metal nanoparticles in the soil environment.................................................. 10 1.3.1 The antimicrobial activity of Ag-NPs and Cu-based-NPs ....................................... 10 1.3.2 Route of exposure and transformations of antimicrobial NPs in soils ................... 12 1.3.3 Effects of antimicrobial NPs on soil bacterial communities .................................. 13 1.4 The earthworm microbiome ..................................................................................... 15 1.4.1 The nephridial bacterial community ................................................................... 15 1.4.2 The transient and resident gut bacterial community ........................................... 16 1.4.3 The earthworm microbiome under chemical stress............................................. 19 1.5 Innate immunity in earthworms and the interaction with NPs..................................... 19 1.5.1 NPs and the innate immune system ................................................................... 20 1.5.2 Innate immunity in earthworms ........................................................................ 22 1.5.3 The interactions of NMs with the earthworm immune system............................. 23 1.6 Research question and hypotheses ........................................................................... 23 2. Methods ............................................................................................................................ 25 2.1 Earthworm tissue sampling ....................................................................................... 26 2.1.1 Dissection and sampling of earthworm gut tissue ............................................... 26 2.1.2 Sampling of soil samples for microbiome analysis ............................................... 26 2.2 Metabarcoding ........................................................................................................ 26 2.2.1 DNA extraction from earthworm tissue and soil samples..................................... 26 2.2.2 Amplification and sequencing of the bacterial 16S-rRNA gene ............................. 27 2.2.3 Bioinformatics .................................................................................................. 27 3. The midgut of the earthworm Eisenia fetida harbours a resident bacterial community independent from soil ....................................................................................................... 29 3.1 Introduction............................................................................................................. 30 3.2 Methods .................................................................................................................. 31 3.2.1 Culturing of Eisenia fetida ................................................................................. 31 3.2.2 Sampling procedure of the midgut, cast and soil ................................................. 32 3.2.3 Soil transfer experiment 1 ................................................................................. 32 3.2.4 Soil transfer experiment 2 ................................................................................. 33 3.2.5 DNA extraction, 16S-rRNA amplification and sequencing..................................... 34 3.2.6 Bioinformatics and data analysis ........................................................................ 34 3.3 Results..................................................................................................................... 35 3.3.1 The effect of rinsing of the midgut on the bacterial community signal.................. 35 3.2.2 Soil transfer experiment 1 ................................................................................. 37 3.3.3 Characterization of soils and mediums used in soil transfer experiment 2 ............ 37 3.3.4 The impact of soil transfer on the resident earthworm midgut bacterial community ...................................................................................................................... 39 3.4 Discussion ..............................................................................................................