Ticks of the H. marginatum complex are known vectors of diseases of veterinary and public health importance, and able to survive in a wide range of climatic conditions and a variety of habitats . In this study, we report the first citizens' notifications of Hyalomma adults in the Netherlands. Results of this study raise questions about the frequency of introductions and the ability to establish endemic Hyalomma populations in The Netherlands and warrants for vigilance because of the risks for human and animal health.
Even though Hyalomma ticks have been found and reported before in The Netherlands [15, 24, 25, 26], the numbers of adult Hylalomma ticks reported in the last few years seem unprecedented. In the weeks preceding the findings in 2019, the media reported about adult Hyalomma ticks in horse farms in Germany  in a sensationalistic way. The finding of the first Hyalomma ticks in The Netherlands that year boosted the media attention. There, the Hyalomma ticks were classified as ‘giant’, ‘monster’ or ‘horror’ ticks and were connected to ‘a deadly Ebola-like virus’ (meaning CCHFv), which probably led to an increased awareness among the public. Fifteen out of seventeen adult Hyalomma ticks were discovered on horses. This is most likely due to the fact that horses, in contrast to other hosts for Hyalomma adults, are on a regular base closely inspected and handled by their carers during grooming and saddling.
Of the thirteen available adult Hyalomma ticks (Table 2), eight could be morphologically identified as H. marginatum and one as H. rufipes. Four could not be morphologically identified to species level with certainty, due to the poor state (dried out and/or moldy) of the received specimens. Also, the engorged nymph that was found on a migratory bird in 2012  could not morphologically be identified to species level, because of the difficulties arising morphological identification of Hyalomma nymphs to species level [27, 35].
Molecularly, twelve adults, including the eight ticks that were morphologically identified as H. marginatum, clustered mainly with H. marginatum (24). Also in this cluster were two other members of the H. marginatum species complex, H. turanicum (5) and H. rufipes (1) (Fig. 2). The one adult that was morphologically identified as H. rufipes, and the nymph clustered mainly with H. rufipes (9). One H. dromedarii and one H. truncatum were also in this cluster, which also clustered together in their own cluster (Fig. 2). Ticks from the H. marginatum species complex are known to be taxonomically challenging to identify . Also, cryptic hybridization in Hyalomma ticks might, at least partly, account for the apparent incongruence between morphology and molecular clustering .
H. rufipes has also been found in western and northern European countries [13, 18]. To our knowledge, H. dromedarii or H. truncatum specimens have not been found in western and northern European countries. In our consideration, we also took into account the fact that H. rufipes is a two host tick of which the immatures are most likely to seek (migratory) birds as hosts, in contrast to H. dromedarii and H. truncatum [35, 36]. Combining morphological and molecular identification, we interpret that the twelve adults are H. marginatum, and both the one adult and nymph are H. rufipes.
The ticks were most likely introduced via migratory birds. In general, migrating birds that are breeding in The Netherlands and wintering in warmer climates can be divided into two groups. Birds belonging to the first group migrate over long distances, and winter in Africa (Sahel to more southern regions). The mass arrival in The Netherlands of these birds, such as Garden warbler (Sylvia borin), Common whitethroat (S. communis), Willow warbler (Phylloscopus trochilus) and Common redstart (Phoenicurus phoenicurus) is from half of April to May. The second group consists of birds that winter much closer by, in southern Europe and northern Africa. These birds, such as European robin (Erithacus rubecula), Song thrush (Turdus philomelos), Common chiffchaff (Phylloscopus collybita) and Eurasian blackcap (Sylvia atricapilla) arrive from March onwards, leaving the African and Mediterranean European countries already in February. In the wintering and stop-over areas of both groups, Hyalomma ticks are endemic and can be dispersed via returning migrating birds . Indeed, Hyalomma immatures have been found on bird species of both groups returning to or present in their breeding area [5, 6, 7, 8, 9, 10, 11, 12, 13, 50, 51, 52], also in The Netherlands . The Hyalomma ticks reported in this study were most likely introduced at the locations in early spring by migratory birds as engorged nymphs, and moulted to adult stage. The horses and persons on which the adult ticks were found had not (recently) been abroad. Also, in case of introduction of adult ticks with animal hosts (e.g. introduced via imported horses), more specimens found at one location would be expected [31, 33, 46].
As shown by the results of the cumulative daily temperature analyses, the accounted temperatures between September and December of 2018 and 2019 do not correspond with the areas where H. marginatum has permanent populations. As suggested by Gray et al. (2009), these parameters (average of 800°C in places where the tick has permanent populations, and below 400°C in sites not colonized) are related to the local factors that affect the moulting of nymphs to adult stage before the onset of winter. These parameters are not connected to (extreme) cold winter temperatures that prevent wintering adults surviving into the next year, because unfed adults of Hyalomma are very capable of surviving even harsh winter circumstances in continental climate, probably hidden deeply in the litter layer [32, 46]. So, temperatures in late summer and autumn are more critical for potential Hyalomma survival and establishment of permanent populations than temperatures in winter.
Results according to the criteria of Emylyanova et al. (2005)  show that the early findings (before August) of adult Hyalomma ticks in The Netherlands do not match with the suggested temperature accumulation. According to these criteria, introduced engorged nymphs in 2019 and 2020 could not moult to adults within this time period, suggesting that adult Hyalomma ticks that were notified before August wintered at these locations. A period of at least four months would have been necessary in 2019 for engorged nymphs to moult into adults. Taking into consideration the probability of survival of the moulting ticks during such a four months period exposed to (environmental) factors such as predation, parasitism, fungal infections, drowning or desiccation [31, 53, 54], this period can be considered too long and the proposed parameters less appropriate. Applying the criterion proposed by Gale et al. (2012) , engorged nymphs that arrived at the end of March or in April, could moult to adults in the first days of May in 2019 and 2020, matching with the findings of the first adults. The criterion used by Gale et al. (2012)  and accepted for the H. marginatum populations maintained in Spain (Estrada-Peña personal communication) is based on temperature as the main factor affecting the seasonal pattern of the H. marginatum tick (Estrada-Pena et al. 2011) and is therefore used as the sole parameter for moulting. Other critical parts of the life cycle necessary for establishment of permanent populations besides nymphal moulting and wintering of adults [46, 49, 53], such as oviposition by the adult females and questing and moulting activity of all stages, are depending heavily on (micro)climatic circumstances as well . This explains why in areas where climatic circumstances are in favour of moulting from nymph to adult, this does not automatically lead to permanent populations of Hyalomma ticks.
Besides climatic factors, tick densities and dispersal are also important factors for possible establishment of Hyalomma [28, 53]. All findings of Hyalomma ticks so far were singular, and no other Hyalomma ticks were reported by the citizens or detected by us in the vicinity of the reported adult Hyalomma tick. Also, in the horse screening part of this study no Hyalomma ticks were found. Although it is likely that more Hyalomma ticks are present than noticed, we hypothesize that these ticks are still too widespread to find a mating partner (Allee effect [55, 56]). With the current seemingly occasional introduction on migration birds, the chance for adult females to encounter adult males for mating on the same mammalian host can be considered very low. However, we cannot conclude that this event is impossible or could not already have happened. To detect attached larvae or nymphs, active (more site-directed) monitoring on small mammals and resident birds would need to be implemented in early spring, at least in areas where Hyalomma has been notified. Besides the previous mentioned factors, the availability and densities of suitable hosts are also of importance. Since in The Netherlands birds, rodents, lagomorphs and wild and domestic ungulates are abundant, this will probably not be the most limiting factor.
Taking the abovementioned into account, we hypothesize that engorged nymphs arriving on migratory birds in spring are able to moult to adults under the current Dutch climatic circumstances of spring and summer. H. marginatum is currently not able to establish permanent populations due to the climate. For H. rufipes, which is distributed in drier areas of Africa and prefers arid conditions , it seems even more unlikely that permanent populations will be established in The Netherlands, which classifies as Cfb (temperate oceanic), according to the Köppen–Geiger climate classification .
In none of the tested ticks (13 adults and 1 nymph) CCHFv was detected. This is in accordance with other studies performed in western and northern countries which tested for CCHFv in Hyalomma ticks [11, 16, 17, 18, 20, 22]. CCHFv positive Hyalomma ticks can be found in ticks collected in regions in and around Europe that lay closer to CCHFv endemic regions [8, 9, 58]. CCHFv seems to have a focal geographic distribution range, also in the regions where competent vector tick species are established . Complex interactions between tick vector species, reservoir hosts, climate (change) and social changes leading to alterations in vegetation and use of landscape determines the enzootic cycle of CCHFv and human outbreaks . Widespread occurrence of vectors and reservoirs do not necessarily lead to outbreaks of CCHFv in humans and even though climate may favour competent tick populations, it is not directly linked to presence of CCHFv . This makes it difficult to predict the dynamics of CCHFv in areas where potential reservoirs are present, the competent vectors are occasionally introduced and climate is changing, as is the current situation in The Netherlands. Recently, CCHFv was found in Hyalomma ticks in Spain  and at a later stage evidence of circulation of the virus in wildlife in parts of Spain was found, including human cases [62, 63]. The virus strain found in Spain belongs to a cluster of strains isolated in western Africa and it was calculated that it was introduced in Spain around 50 years prior to the first human cases . This finding shows that enzootic cycles can be present unnoticed for quite some time. Although other tick species are also involved in epidemiology of CCHFv, H. marginatum is considered the principal vector in Europe [33, 65]. Because the virus can be transmitted in ticks both transstadial and transovarial, Hyalomma ticks can also form a reservoir of CCHFv . To estimate the risks of introduction of CCHFv via Hyalomma ticks imported by migratory birds, the dynamics between the virus, the tick and feeding on a non-viremic host need to be clarified. In summary, taking into account that i) only a small percentage of larvae in endemic areas are infected via transovarial transmission [45, 60, 66], ii) viremic transmission of ticks via migratory birds [60, 67] and transmission of CCHFv via co-feeding on migratory birds seems unlikely , iii) transstadial transmission is only successful in over a third of the ticks and perhaps lower in ticks feeding on birds [66, 68], iv) most probably only occasionally H. marginatum immatures are dropped off in northern and western European regions, and v) no CCHFv has been found so far in Hyalomma ticks in northern and western European countries, the chance of human exposure to CCHFv via Hyalomma ticks in The Netherlands is currently considered very low.
Presence of the strictly intracellular bacterium Rickettsia, of which the species aeschlimannii was found in our study in one tick, is much more often reported in Hyalomma ticks [14, 17, 18, 19, 23, 51, 58, 69, 70]. The reason for this might be that Rickettsia spp. are considered heritable symbionts from invertebrates [71, 72].
The ticks tested negative for the parasites Babesia and Theileria. This is in accordance with other studies that tested Hyalomma ticks for Babesia and Theileria species in northern and western European countries [17, 18, 20]. Bovine tropical theileriosis (T. annulata) is only present in southern parts of Europe and has to our knowledge not been detected in north western European countries, but equine piroplasmosis (B. caballi/T. equi) is occasionally diagnosed outside its endemic range . In fact, a study revealed piroplasmosis in horses in the south western part of The Netherlands in 2010. Not all horses had been abroad, suggesting autochthonous infections, most likely caused by Dermacentor reticulatus ticks [74, 75]. Because Theileria parasites are not transmitted transovarial, the risk of introduction of Theileria with Hyalomma ticks seem negligibly low . We estimate the risk of introduction of Babesia through Hyalomma ticks also very low, although it must be taken into account that this protozoan can be transmitted transovarially .