. The influence of external (a)biotic factors on the maintenance of symbionts in tick populations depends much on symbionts’ mode of transmission. (this thesis) . Ticks choose their symbionts rather than symbionts choose their ticks. (this thesis) . An infection that is a burden for an individual can be beneficial for a population. The !AS rRNA amplicon sequencing technique is a powerful tool for forming hypotheses. We are selective about who or what should be a member of the (bio)diversity we want to preserve. Nowadays, it is difficult to distinguish what we really think from what we have read online. Propositions belonging to the thesis, entitled: Questioning microbiotics: Interactions of microbes, ticks, vertebrates, and the environment Aleksandra I. Krawczyk Wageningen, =Q March Q<! Questing microbioticks: Interactions of microbes, ticks, vertebrates, and the environment Aleksandra I. Krawczyk Thesis committee Promotor Prof. Dr W. Takken Personal chair at the Laboratory of Entomology Wageningen University & Research Co-promotor Dr H. Sprong Research coordinator wildlife and vector-borne diseases National Institute for Public Health and the Environment, Bilthoven Other members Prof. Dr G. Smant, Wageningen University & Research Prof. Dr S.B.J. Menken, University of Amsterdam Dr T. Pollet, French National Institute for Agriculture, Food and the Environment (INRA), Montpellier, France Dr H. Esser, Wageningen University & Research This research was conducted under the auspices of the C.T. de Wit Graduate School for Production Ecology and Resource Conservation Questing microbioticks: Interactions of microbes, ticks, vertebrates, and the environment Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Tuesday 30 March 2021 at 4 p.m. in the Aula. Aleksandra I. Krawczyk Aleksandra I. Krawczyk Questing microbioticks: Interactions of microbes, ticks, vertebrates, and the environment A6, 224 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2021) With references, with summary in English ISBN: 978-94-6395-720-5 DOI: https://doi.org/10.18174/542129 Table of contents Chapter 1 General Introduction 7 Chapter 2 Large-scale quantitative microbial population study reveals regional 15 differences in bacterial symbionts ofIxodes ricinus Chapter 3 Co-infection analysis identifies new associations between horizontally- 47 and vertically-transmittedIxodes ricinus microorganisms Chapter 4 Effect of rodent density on tick and tick-borne pathogen populations: 81 consequences for infectious disease risk Chapter 5 Spatiotemporal variation of heritable Ixodes ricinus symbionts is 113 associated with climatic factors Chapter 6 Tripartite interactions among Ixodiphagus hookeri, Ixodes ricinus, and 157 deer: differential interference with transmission cycles of tick-borne pathogens Chapter 7 General discussion 175 References 189 Summary 211 Acknowledgements 215 Curriculum vitae 217 Publication 219 PE&RC Education statement 221 Chapter 1 Please click here (for the online version) or scan this QR code to Please click here (for the online version) or scan this QR code tolisten listen toto aa music music track track entitled entitled ‘Call and ‘Call Answer’. and TheAnswer’. music The music has hasbeen been inspired inspired by by this this thesis thesis and composedand composed by Michał by Michał Szablowski. Szablowski.The full album The full is album available is available on: ticksandmicrobes.bandcamp.comon: ticksandmicrobes. bandcamp.com Chapter 2 Please click here (for the online version) or scan this QR code to listen to an immersive soundscape of a forest in Amsterdamse Waterleidingduinen, the Netherlands recorded by Vitalij Kuzkin. The Amsterdamse Waterleidingduinen forest is one of the study sites where I have collected samples for this research. Listen with headphones for the best experience. The full album is available on: ticksandmicrobes.bandcamp.com Chapter 3 Please click here (for the online version) or scan this QR code to listen to an immersive soundscape of a forest in Duin en Kruidberg, the Netherlands recorded by Vitalij Kuzkin. The Duin en Kruidberg forest is one of the study sites where I have collected samples for this research. Listen with headphones for the best experience. The full album is available on: ticksandmicrobes.bandcamp.com Chapter 4 Please click here (for the online version) or scan this QR code to listen to an immersive soundscape of a forest in Buunderkamp, the Netherlands recorded by Vitalij Kuzkin. The Herperduin forest is one of the study sites where I have collected samples for this research. Listen with headphones for the best experience. The full album is available on: ticksandmicrobes.bandcamp.com Chapter 1 General introduction Introduction In the northern hemisphere, the sheep tick, Ixodes ricinus, transmits a plethora of pathogens to humans and animals, posing severe health concerns and economic losses (Heyman et al., 2010; Randolph & Šumilo, 2007; Sprong et al., 2018). For decades, in many European countries, the incidences of tick-borne diseases, most notably Lyme borreliosis and tick-borne encephalitis, have been increasing (Jaenson et al., 2012; Mysterud et al., 2016; Sykes & Makiello, 2017; Walter et al., 2020). The distribution of agents that cause these and many other diseases is changing on a spatiotemporal scale along with their tick vectors, and therefore it is of great importance to understand tick ecology (Beugnet & Marie, 2009; Parola, 2004). The ecology of ticks involves their interactions with biotic and abiotic parts of their natural environment, which impose physiological and evolutionary pressures (Eisen et al., 2017; Hartemink et al., 2019). The abiotic environment includes climatic factors, while the biotic environment consists of vertebrate hosts, vegetation as well as microorganisms inhabiting ticks. Ixodes ricinus is a hematophagous arthropod species; thus, its survival depends largely on its ability to find and successfully feed on a vertebrate host. It utilizes a multitude of vertebrate species for a blood meal; however, a minimum requirement is animals’ presence (mainly deer), on which adult female ticks can feed, meet a male and propagate (Figure 1; Gray et al., 1998b; Hofmeester et al., 2017b). Like their main propagation hosts, I. ricinus ticks are predominantly found in forested areas, however, they can also survive in other habitats such as parks and gardens, as long as the leaf litter layer is of suitable thickness, pH, and humidity (Gray, 1998a). Thus, local relative humidity and temperature are substantial determinants for tick survival and behaviour (Gassner et al., 2011; Macleod, 1936; Perret et al., 2000; Randolph, 2004). Further, the climate can also indirectly affect tick survival and activity by affecting tick hosts, vegetation, and soil traits. Many of the above-mentioned interactions have been extensively studied, and the general mechanisms behind them are well understood. More recently, there is increasing interest in the role of microorganisms in the life cycle of I. ricinus, and its vectorial capacity and competence (de la Fuente et al., 2017; Gall et al., 2016; Narasimhan & Fikrig, 2015; Weiss & Aksoy, 2011). Besides known human pathogens such as tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Neoehrlichia mikurensis, and Babesia spp. (Heyman et al., 2010; Randolph et al., 2007; Rizzoli et al., 2011), I. ricinus harbours many other microorganisms. Some of these microorganisms have developed close and long-term biological interactions with their tick hosts. These symbioses lie on a continuum of interactions that ranges from obligate mutualistic, through commensal to exclusively parasitic (Noda et al., 1997; Sacchi et al., 2004; Scoles, 2004). The functional significance of symbiotic microorganisms for ticks and how they circulate in tick populations remains largely unknown. Lastly, it is poorly understood how tick microorganisms interact with each other, with tick vertebrate hosts and the environment (Figure 2). In the long- term, understanding these interactions can lead to the development of novel and sustainable ways to combat ticks and tick-borne diseases. Recent research on host-symbiont interactions in mosquitoes has been an inspiration for strategies that aim at reducing the mosquito vectorial capacity. For instance, these strategies include paratransgenesis that relies on genetically-modified mosquito symbionts interfering with malaria parasite development and transmission (Mancini et al., 2016; Ricci et al., 2011). 1 Figure 1. Schematic representation of the life cycle of I. ricinus. Ixodes ricinus needs from 2 to 4 years to complete its life cycle and consists of four life stages: egg, larva, nymph, and adult. A proportion of female adult ticks mate in the vegetation; however, they also mate on a host (Gray, 1987). After mating and successful feeding, females of I. ricinus lay on average 2,000-2,500 eggs in the leaf litter or on the forest floor (Balashov, 1972). During unfavourable environmental conditions, ticks are in so-called diapause, which can delay the hatching of eggs, development of one stage to the next, or delay the onset of host-seeking following the moult (Belozerov, 1982). When the temperature is ≥ 7 ºC, larvae, which hatched from the eggs, nymphs, and adult females