Questing microbioticks : Interactions of microbes, ticks, vertebrates, and the environment

The sheep tick, Ixodes ricinus, transmits many pathogens to humans and animals, posing serious health concerns and economic losses. There is an increasing interest in if and how microorganisms affect the fitness and physiology, and possibly, the vectorial capacity of ticks.Little is known about the members, other than the pathogens, of the microbial community of I. ricinus: Specifically, who they are, what they do, and how they are acquired and propagated in ticks. The research presented in this thesis focused on describing the microbiome of I. ricinus and aimed to elucidate how it is generated. Therefore, I studied the bacterial communities of the different life stages of ticks from several geographical locations, which display different vertebrate communities and climatic conditions.I first showed that most ticks have a limited microbiome, which was surprising given that ticks are blood-sucking arthropods. The bacterial communities of ticks were strongly associated with geographical origin rather than the tick life stage, and differences in their Rickettsia abundance mostly determined the clusters.The most prominent tick symbionts were Rickettsia, Rickettsiella spp., Midichloria and Spiroplasma, all of which turned out to be vertically-transmitted. The distribution of symbionts was investigated in ~17,000 questing nymphs from 19 locations around the Netherlands. Their prevalences in ticks differed between the regions in which the symbionts occurred in different proportions. Therefore, I speculate that selective pressures on a regional scale determine infection rates of some heritable symbionts in I. ricinus.In a follow-up study, I studied whether the underlying microbiome of a tick could affect the acquisition of zoonotic pathogens and how a blood meal and pathogens might contribute to the overall change in the bacterial community. In the study, tick bacterial communities clustered poorly by pathogen infection status but better by geographic regions. As the presence of a pathogen in nymphs indicates a specific vertebrate host on which it fed as a larva, our findings imply that the vertebrate host does not, or hardly, contributes to the bacterial community of ticks.Nevertheless, by studying individual questing nymphs, I detected positive associations of vertically-transmitted symbionts with horizontally-transmitted pathogens. Among the most striking associations were M. mitochondrii and Rickettsiella spp. with Borrelia burgdorferi s.l., suggesting that ticks harbouring bacterial symbionts are more likely to acquire and propagate pathogens.Although I did not observe any effects of the vertebrate community on the overall tick microbiome, vertebrates strongly determine the distribution and abundance of ticks and the prevalence of tick-borne pathogens. Particularly, rodents contribute strongly to the risk of tick-borne diseases by feeding I. ricinus larvae and by acting as amplifying hosts for pathogens. Therefore, in an experimental study, I tested to what extent these two processes depend on rodent density.The strongest associations were found between rodent density and rodent-associated pathogens B. afzelii and N. mikurensis that rely on horizontal transmission. Overall, the associations of rodent density with other studied tick microorganisms varied, likely due to contrasts in microorganisms’ biology, including differences in the transmission mode and efficiency, and host specificity. Nevertheless, even for rodent-associated pathogens, it seems impossible to predict disease risk solely based on rodent density since other factors, independent from our experiment, strongly affected tick density.Although the environmental requirements for tick survival are well established, it is not known if and how fluctuating climatic conditions influence transmission dynamics of tick symbionts and pathogens. In the longitudinal study, I investigated spatio-temporal dynamics of the infection prevalence of the vertically-transmitted symbionts, M. mitochondrii and R. helvetica, and the pathogens B. burgdorferi s.l., B. miyamotoi, and N. mikurensis concerning climatic conditions such as precipitation, relative humidity, temperature, and evaporation rate.All but B. miyamotoi presented spatio-temporal variation. Rickettsia helvetica was the most variable and positively associated with all climatic factors, except for temperature. Among the 12 studied locations, the five forest sites with the highest R. helvetica prevalence in questing nymphs expressed the highest relative humidity in spring and summer (and often the lowest in autumn and winter). Although a short-term positive effect of relative humidity was detected, I propose that the spatial distribution of R. helvetica prevalence in Dutch tick populations derives from long-term, subtle differences in climatic conditions between locations, most notably the relative humidity.Another possible biotic factor shaping the tick microbiota is the presence and abundance of a natural tick enemy, the parasitoid wasps Ixodiphagus hookeri. This wasp infests both questing and feeding larvae and nymphs, but the egg development only occurs in fully-engorged nymphs. Since the emergence of wasps kills its host, a tick cycle is disrupted. Therefore, I examined whether this phenomenon affects the transmission dynamics of zoonotic pathogens. The ecological interactions among I. hookeri, I. ricinus, and two vertebrate species groups (ungulates and rodents) were explored.I found higher than expected co-occurrence rates of I. hookeri with deer-associated A. phagocytophilum, and lower than expected rates with rodent-associated B. afzelii and N. mikurensis. The prevalence of I. hookeri was positively correlated with the encounter probability of ungulates and the densities of all life stages of I. ricinus. Our observation support that female wasps are attracted to deer odour, and subsequently infest ticks that feed on these animals, which increases chances for a wasp and A. phagocytophilum to co-occur. Therefore, the presence of I. hookeri may directly interfere with the transmission cycle of A. phagocytophilum, and probably other deer-associated pathogens.Based on the results from my research and the literature published previously, I interpret that individual I. ricinus ticks have a limited microbiome and often lack symbionts. Nevertheless, given the relatively high prevalence of symbionts in tick populations, symbiotic bacteria appear to be essential for this tick species as a community to thrive under different (a)biotic conditions. Tick-associated microorganisms, including human pathogens, may utilize different transmission routes and play various roles in tick fitness and physiology, which defines to what extent (a)biotic factors influence microorganisms’ dynamics. I propose that to improve our ability to assess and control the risk of tick-borne diseases, we should describe the transmission route of disease agents and their symbiosis with ticks.