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Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, how- ever, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial trans- mission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV dissemi- nated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replica- tion. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and hori- zontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Small mammals are potential bio-indicators of various ecosystems and their populations are often studied. However, many small mammal species are difficult to detect due to their small size and elusive behaviour. Camera trapping and live trapping are commonly employed survey techniques, but they both have their limitations. Recently developed techniques such as adjusted short-focal camera trapping and environmental DNA (eDNA) are promising new approaches, but their relative performance remains poorly quantified. We compared the effectiveness of three survey protocols for detecting a semi-aquatic and elusive small mammal, the Eurasian water shrew (Neomys fodiens), by (1) short-focal camera trapping, (2) live trapping, and (3) soil eDNA. During September and October 2022, we surveyed 20 transects of each 100 m in length alongside the Kleine Dommel, a lowland brook in the Netherlands. The effectiveness of the three survey protocols was compared based on detection probabilities. Short-focal camera trapping yielded a significantly higher detection probability than the eDNA protocol. Detection probabilities between short-focal camera trapping and live trapping and, between the eDNA protocol and live trapping, were not significantly different. Short-focal camera trapping is an effective technique to survey Eurasian water shrews. Furthermore, this method detected additional species compared to live trapping and is non-invasive and less labour-intensive. Short-focal camera trapping showed a promising method for small mammal surveys in general and we recommend further evaluation of its applicability for other small mammal species.

Understanding which factors determine tick-borne disease hazard can contribute to effective disease control. In Europe, the hazard of the pathogens Borrelia burgdorferi s.l. and Anaplasma phagocytophilum is determined by local tick densities (mainly Ixodes ricinus) and the reservoir competence of the host species community. Sand lizards (Lacerta agilis) are common hosts for larvae and nymphs of I. ricinus and non-competent reservoirs for both pathogens. Consequently, high relative abundance of L. agilis is hypothesized to be associated with lower infection prevalence in nymphs. Here, we aimed to test whether this effectively occurs in natural settings. We sampled different habitat types within a heterogenous dune landscape at the Dutch coast and estimated (1) L. agilis densities, (2) host community competence, (3) the density and infection prevalence of questing I. ricinus ticks and (4) the number and infection prevalence of ticks feeding on L. agilis. Captured L. agilis had high tick burdens and contributed substantially to feeding I. ricinus larvae in their natural habitat. B. burgdorferi s.l. and A. phagocytophilum were virtually absent from feeding larvae and nymphs. The nymphal infection prevalence of both pathogens in questing ticks was lower in habitat types where L. agilis was more abundant. Hence, L. agilis strongly reduced community competence. The density of questing nymphs was higher in habitat types with denser vegetation and also varied more between habitat types than infection prevalence. As a result, nymphal density had a stronger effect on the density of infected ticks than did nymphal infection prevalence. Synthesis and applications. Coastal dune habitats favourable for L. agilis have lower densities of questing nymphs and a lower human infection hazard. These results might be applicable to similar ecosystems where L. agilis is present. From a public health perspective, this underlines the importance of preserving early successional habitat, as enc

Ca. Neoehrlichia mikurensis is widely prevalent in I. ricinus across Europe and has been associated with human disease. However, diagnostic modalities are limited, and much is still unknown about its biology. Here, we present the first complete Ca. Neoehrlichia mikurensis genomes directly derived from wildlife reservoir host tissues, using both long-and short-read sequencing technologies. This pragmatic approach provides an alternative to obtaining sufficient material from clinical cases, a difficult task for emerging infectious diseases, and to expensive and challenging bacterial isolation and culture methods. Both genomes exhibit a larger chromosome than the currently available Ca. Neoehrlichia mikurensis genomes and expand the ability to find new targets for the development of supportive laboratory diagnostics in the future. Moreover, this method could be utilized for other tick-borne pathogens that are difficult to culture.

Human babesiosis in Europe has been attributed to infection with Babesia divergens and, to a lesser extent, with Babesia venatorum and Babesia microti, which are all transmitted to humans through a bite of Ixodes ricinus. These Babesia species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these Babesia species, their presence in reservoir hosts and in I. ricinus was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of Babesia species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of Babesia species were used to test 25,849 questing I. ricinus. Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. Babesia microti was found in two widespread rodent species: Myodes glareolus and Apodemus sylvaticus, whereas B. divergens was detected in the geographically restricted Cervus elaphus and Bison bonasus, and occasionally in free-ranging Ovis aries. B. venatorum was detected in the ubiquitous Capreolus capreolus, and occasionally in free-ranging O. aries. Species-specific PCR revealed co-infections in C. capreolus and C. elaphus, resulting in higher prevalence of B. venatorum and B. divergens than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic Babesia species found were Babesia capreoli, Babesia vulpes, Babesia sp. deer clade, and badger-associated Babesia species. The infection rate of zoonotic Babesia species in questing I. ricinus ticks was higher for Babesia clade I (2.6%) than Babesia clade X (1.9%). Co-infection of B. microti with Borr

Habitat fragmentation is a primary driver of wildlife loss, and establishment of biological corridors is a common strategy to mitigate this problem. A flagship example is the Mesoamerican Biological Corridor (MBC), which aims to connect protected forest areas between Mexico and Panama to allow dispersal and gene flow of forest organisms. Because forests across Central America have continued to degrade, the functioning of the MBC has been questioned, but reliable estimates of species occurrence were unavailable. Large mammals are suitable indicators of forest functioning, so we assessed their conservation status across the Isthmus of Panama, the narrowest section of the MBC. We used large-scale camera-trap surveys and hierarchical multispecies occupancy models in a Bayesian framework to estimate the occupancy of 9 medium to large mammals and developed an occupancy-weighted connectivity metric to evaluate species-specific functional connectivity. White-lipped peccary (Tayassu pecari), jaguar (Panthera onca), giant anteater (Myrmecophaga tridactyla), white-tailed deer (Odocoileus virginianus), and tapir (Tapirus bairdii) had low expected occupancy along the MBC in Panama. Puma (Puma concolor), red brocket deer (Mazama temama), ocelot (Leopardus pardalis), and collared peccary (Pecari tajacu), which are more adaptable, had higher occupancy, even in areas with low forest cover near infrastructure. However, the majority of species were subject to ≥1 gap that was larger than their known dispersal distances, suggesting poor connectivity along the MBC in Panama. Based on our results, forests in Darien, Donoso–Santa Fe, and La Amistad International Park are critical for survival of large terrestrial mammals in Panama and 2 areas need restoration.

The dataset is part of the TBEV-COMEIN project (ZonMW project number 522003004). One of the goals of the project was to characterize how common co-feeding is between larval and nymphal ticks on rodents hosts in the Netherlands. The dataset summarizes multiple years of data collection regarding ticks on rodents (2007 - 2019). These data were collected during several PhD projects at Wageningen University and during fieldwork within the TBEV-COMEIN project.

This dataset is part of the TBEV-COMEIN project (ZonMW project number 522003004). One of the goals of this project was to determine the prevalence of tick-borne encephalitis virus (TBEV) in rodents in the Netherlands. This dataset includes data on 320 collected rodents: species, sex, weight, number of ticks, and whether the rodent tested positive for TBEV via serology and PCR.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used for proteomic fingerprinting and taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method to complement other well-established techniques for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens.

The ongoing loss of global biodiversity is unprecedented in both magnitude and pace, raising urgent questions as to how this loss will affect ecosystem functioning and human well-being. Control of infectious diseases has been proposed as an important ecosystem service that is likely to be affected by biodiversity loss. A negative relationship between biodiversity and disease risk could offer a win-win situation for nature conservation and human health. However, the generality of this relationship remains the subject of contentious debate. The aim of this thesis was to contribute to a better understanding of the interactions between ticks and their vertebrate hosts in a biodiversity hotspot, and how loss of biodiversity affects these interactions and ultimately, tick-borne disease risk. My study was unique in that I simultaneously considered and directly assessed broader communities of Neotropical wildlife, ticks, and tick-borne pathogens across an anthropogenic disturbance gradient. Determining whether and how biodiversity loss affects tick-borne disease risk in tropical forests requires a thorough understanding of tick-host associations, which are a function of tick-host specificity as well as host biological and ecological traits. In chapter 2, I therefore quantified the degree to which adult ticks are host-specific in my study region: Panama. Using quantitative network analyses and phylogenetic tools with null model comparisons, I found that the adult life stages of most tick species were specific to a limited number of host species that were phylogenetically closely related. In Chapter 4 I showed that species assemblages of adult ticks became increasingly diverse on larger-bodied host species, indicating that adult ticks in Panama tend to select for large reproduction hosts. In contrast to adult ticks, understanding the ecological interactions between immature ticks and their hosts in the tropics has long been hampered by a lack of morphological identification k

In the tropics, ticks parasitize many classes of vertebrate hosts. However, because many tropical tick species are only identifiable in the adult stage, and these adults usually parasitize mammals, most attention on the ecology of tick-host interactions has focused on mammalian hosts. In contrast, immature Neotropical ticks are often found on wild birds, yet difficulties in identifying immatures hinder studies of birds' role in tropical tick ecology and tick-borne disease transmission. In Panama, we found immature ticks on 227 out of 3,498 individually-sampled birds representing 93 host species (24% of the bird species sampled, and 13% of the Panamanian land bird fauna). Tick parasitism rates did not vary with rainfall or temperature, but did vary significantly with several host ecological traits. Likewise, Neotropical-Nearctic migratory birds were significantly less likely to be infested than resident species. Using a molecular library developed from morphologically-identified adult ticks specifically for this study, we identified eleven tick species parasitizing birds, indicating that a substantial portion of the Panamanian avian species pool is parasitized by a diversity of tick species. Tick species that most commonly parasitized birds had the widest diversity of avian hosts, suggesting that immature tick species are opportunistic bird parasites. Although certain avian ecological traits are positively associated with parasitism, we found no evidence that individual tick species show specificity to particular avian host ecological traits. Finally, our data suggest that the four principal vectors of Rocky Mountain Spotted Fever in the Neotropics rarely, if ever, parasitize Panamanian birds. However, other tick species that harbor newly-discovered rickettsial parasites of unknown pathogenicity are frequently found on these birds. Given our discovery of broad interaction between Panamanian tick and avian biodiversity, future work on tick ecology and the dynamics of

Identifying the factors that influence the species diversity and distribution of ticks (Acari: Ixodida) across vertebrate host taxa is of fundamental ecological and medical importance. Host body size is considered one of the most important determinants of tick abundance, with larger hosts having higher tick burdens. The species diversity of tick assemblages should also be greater on larger-bodied host species, but empirical studies testing this hypothesis are lacking. Here, we evaluate this relationship using a comparative dataset of feeding associations from Panama between 45 tick species and 171 host species that range in body size by three orders of magnitude. We found that tick species diversity increased with host body size for adult ticks but not for immature ticks. We also found that closely related host species tended to have similar tick species diversity, but correcting for host phylogeny did not alter the relationships between host body size and tick species diversity. The distribution of tick species was highly aggregated, with approximately 20% of the host species harboring 80% of all tick species, following the Pareto principle or 20/80 Rule. Thus, the aggregated pattern commonly observed for tick burdens and disease transmission also holds for patterns of tick species richness. Our finding that the adult ticks in this system preferentially parasitize large-bodied host species suggests that the ongoing anthropogenic loss of large-bodied vertebrates is likely to result in host-tick coextinction events, even when immature stages feed opportunistically. As parasites play critical roles in ecological and evolutionary processes, such losses may profoundly affect ecosystem functioning and services.

Background: Host specificity is a fundamental determinant of tick population and pathogen transmission dynamics, and therefore has important implications for human health. Tick host specificity is expected to be particularly high in the tropics, where communities of ticks, hosts and pathogens are most diverse. Yet the degree to which tropical tick species are host-specific remains poorly understood. Combining new field data with published records, we assessed the specificity of tick-host associations in Panama, a diverse Neotropical region.Methods: The resulting dataset includes 5,298 adult ticks belonging to 41 species of eight genera that were directly collected from 68 vertebrate host species of 17 orders. We considered three important aspects of tick hostspecificity: (i) the relative ecological importance of each host species (structural specificity); (ii) relatedness amonghost species (phylogenetic specificity); and (iii) spatial scale-dependence of tick-host relationships (geographicalspecificity). Applying quantitative network analyses and phylogenetic tools with null model comparisons, weassessed the structural and phylogenetic specificity across three spatial scales, ranging from central Panama tocountrywide. Further, we tested whether species-rich tick genera parasitized a wider variety of hosts thanspecies-poor genera, as expected when ticks specialize on different host species.Results: Most tick species showed high structural and/or phylogenetic specificity in the adult stage. However, aftercorrecting for sampling effort, we found little support for geographical specificity. Across the three scales, adult tickstended to be specific to a limited number of host species that were phylogenetically closely related. These hostspecies in turn, were parasitized by tick species from distinct genera, suggesting switching among distantly relatedhosts is common at evolutionary timescales. Further, there was a strong positive relationship between thetaxonomic richnes