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\begin{document}

\title{Emergence of Hyalomma marginatum and Hyalomma rufipes adults revealed by
citizen science tick monitoring in Hungary}



\author[1]{Gabor Foldvari}%
\author[1]{Éva Szabó}%
\author[2]{Gábor Endre Tóth}%
\author[2]{Zsófia Lanszki}%
\author[2]{Brigitta Zana}%
\author[2]{Zsaklin Varga}%
\author[2]{Gabor Kemenesi}%
\affil[1]{Okologiai Kutatokozpont}%
\affil[2]{University of Pecs Biologia Intezet}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
\emph{Hyalomma} ticks are important vectors of Crimean-Congo
Haemorrhagic Fever Virus (CCHFV) and other pathogens. They are
frequently carried as immatures from Africa, the Middle East and
Mediterranean areas to temperate Europe via migratory birds and
emergence of its adults has been reported in many countries where it has
so far been non-endemic. Our aim was to implement the first steps of the
DAMA (Document, Assess, Monitor, Act) protocol by monitoring the
potential arrival of adult \emph{Hyalomma} ticks in Hungary applying
citizen-science methods. Ticks were collected from April-December 2021
by asking volunteer participants through a self-made website to look for
unusual hard ticks on themselves, their pets and livestock. Owing to the
intensive media campaign, the project website had over 31 thousand
visitors within seven months and 137 specimens and several hundreds of
photos of hard ticks were submitted by citizen scientists from all over
the country. Beside \emph{Ixodes ricinus}, \emph{Dermacentor
reticulatus,} \emph{Dermacentor marginatus} and \emph{Haemaphysalis
inermis,} a specimen from a dog was morphologically identified as a male
\emph{Hyalomma marginatum} and another removed from a cattle as a male
\emph{Hyalomma rufipes}. The dog and the cattle had never been abroad,
they were approximately 280 km apart, thus the two \emph{Hyalomma}
observations can be considered as separate introductions. Amplification
of the partial mitochondrial cytochrome C oxidase subunit I gene was
successful for both specimens. Sequencing verified the previous
morphological identification for both ticks. Based on the phylogenetic
analyses the \emph{Hy. marginatum} individual most likely belongs to the
Eurasian population and the \emph{Hy. rufipes} to a clade of mixed
sequences from Europe and Africa. We summarize the scattered historical
reports about the occurrence of \emph{Hyalomma} ticks and CCHFV in
Hungary. Our data highlight the effectiveness of citizens science
programmes in the monitoring and risk assessment of CCHFV emergence and
preparedness in our region.%
\end{abstract}%



\sloppy


\textbf{Emergence of \emph{Hyalomma marginatum} and \emph{Hyalomma
rufipes} adults revealed by citizen science tick monitoring in Hungary}

\selectlanguage{ngerman}\textbf{Gábor Földvári\textsuperscript{1}} *\textbf{, Éva
Szabó\textsuperscript{1}, Gábor Endre Tóth\textsuperscript{2,3}, Zsófia
Lanszki\textsuperscript{2,3}, Brigitta Zana\textsuperscript{2,3},
Zsaklin Varga\textsuperscript{2,3}, Gábor Kemenesi\textsuperscript{2,3}}

\textsuperscript{1}Institute of Evolution, Centre for Ecological
Research, 1121 Budapest, Konkoly-Thege Miklós út 29-33.

\textsuperscript{2}National Laboratory of Virology, Szentágothai
Research Centre, University of Pécs, 7624 Pécs, Hungary

\textsuperscript{3}Institute of Biology, Faculty of Sciences, University
of Pécs, 7624 Pécs, Hungary

* \textbf{Corresponding author} \textbf{FoldvariGabor@gmx.de}

\textbf{Summary}

\emph{Hyalomma} ticks are important vectors of Crimean-Congo
Haemorrhagic Fever Virus (CCHFV) and other pathogens. They are
frequently carried as immatures from Africa, the Middle East and
Mediterranean areas to temperate Europe via migratory birds and
emergence of its adults has been reported in many countries where it has
so far been non-endemic. Our aim was to implement the first steps of the
DAMA (Document, Assess, Monitor, Act) protocol by monitoring the
potential arrival of adult \emph{Hyalomma} ticks in Hungary applying
citizen-science methods. Ticks were collected from April-December 2021
by asking volunteer participants through a self-made website to look for
unusual hard ticks on themselves, their pets and livestock. Owing to the
intensive media campaign, the project website had over 31 thousand
visitors within seven months and 137 specimens and several hundreds of
photos of hard ticks were submitted by citizen scientists from all over
the country. Beside \emph{Ixodes ricinus} , \emph{Dermacentor
reticulatus,} \emph{Dermacentor marginatus} and \emph{Haemaphysalis
inermis,} a specimen from a dog was morphologically identified as a
male\emph{Hyalomma marginatum} and another removed from a cattle as a
male\emph{Hyalomma rufipes} . The dog and the cattle had never been
abroad, they were approximately 280 km apart, thus the two
\emph{Hyalomma}observations can be considered as separate introductions.
Amplification of the partial mitochondrial cytochrome C oxidase subunit
I gene was successful for both specimens. Sequencing verified the
previous morphological identification for both ticks. Based on the
phylogenetic analyses the \emph{Hy. marginatum} individual most likely
belongs to the Eurasian population and the \emph{Hy. rufipes} to a clade
of mixed sequences from Europe and Africa. We summarize the scattered
historical reports about the occurrence of \emph{Hyalomma} ticks and
CCHFV in Hungary. Our data highlight the effectiveness of citizens
science programmes in the monitoring and risk assessment of CCHFV
emergence and preparedness in our region.

\textbf{Acknowledgements}

We are grateful to all citizen scientists who took part in the project
and helped with submitting photos, ticks or data. We would like to
acknowledge Kornél Baráth and Attila Ádám for providing
the\emph{Hyalomma} specimens. We thank Zoltán Soltész for his advice on
the citizen science project and the excellent work of Eszter Draskóczy
who played a crucial role in reaching society through the appropriate
media appearances. The help of Sonia A. Olmeda and Félix Valcárcel in
confirming our morphological identification of the two
\emph{Hyalomma}specimens is much appreciated.

\textbf{Keywords:} Crimean-Congo Haemorrhagic Fever, DAMA protocol,
emerging infectious diseases, imported ticks, Hungary, Hyalomma adult

\textbf{Background}

Emergence of pathogens and vectors in new hosts and new geographical
areas poses great threats for public and veterinary health (Jones et
al., 2008). Ticks are responsible for the spread of numerous pathogens
worldwide and species belonging to the genus \emph{Hyalomma} have been
shown to have a particular epidemiologic role (Estrada-Peña, D\selectlanguage{english}Amico, \&
Fern\selectlanguage{ngerman}ández-Ruiz, 2020; Hubálek, Sedlá\selectlanguage{polish}č\selectlanguage{english}ek, Estrada-Pe\selectlanguage{ngerman}ña, Vojtí\selectlanguage{polish}š\selectlanguage{english}ek, \&
Rudolf, 2020). The tick species \emph{Hyalomma marginatum}
and\emph{Hyalomma rufipes} are the main vectors of Crimean-Congo
haemorrhagic fever virus (CCHFV). This is a negative-sense
single-stranded RNA virus belonging to the \emph{Nairoviridae} family
(order \emph{Bunyavirales)} . It can cause debilitating human disease
with no available casual treatment or vaccine and has a case fatality
rate up to 30\% (Bente et al., 2013). The public health significance of
Crimean-Congo haemorrhagic fever is demonstrated by the fact that this
infection is geographically the most widespread tick-transmitted viral
disease in humans. It is found in the southern parts of Asia, the Middle
East, Europe and Africa. The emergence of the virus is mainly determined
by the distribution of \emph{Hyalomma} ticks being responsible for the
spread of the disease agent (Bente et al., 2013).

\emph{Hyalomma marginatum} and \emph{Hy. rufipes} are two-host ticks
(Estrada-Pe\selectlanguage{ngerman}ña, Mihalca, \& Petney, 2018). The larva moults into a nymph
on the same first host, feeding on that animal at both stages of
development. For these two \emph{Hyalomma} species, the first host is
very often a bird. The fact that they do not change host before becoming
a nymph greatly contributes to the successful geographical spread of
these ticks, as it allows them to travel longer distances by attaching
to the same migratory bird host. The adults moult from the engorged
nymphs dropping off the first host and actively look for the second
host, which is usually a large wild or domesticated mammal or human
(Valcárcel et al., 2020; Walker et al., 2003).

Although the yearly arrival of immature \emph{Hyalomma} ticks with
migratory birds in temperate Europe has been a frequent phenomenon
(Capek et al., 2014), the emergence of its adults has only been detected
in higher numbers and wider distribution recently. Adults of \emph{Hy.
marginatum} were found in Slovakia (Nosek, Ko\selectlanguage{polish}ž\selectlanguage{english}uch, \& Lys\selectlanguage{ngerman}ý, 1982) and
Austria (Duscher et al., 2018), \emph{Hyalomma rufipes} adults were
found in Hungary (S Hornok \& Horváth, 2012). The adults of both tick
species have recently been detected the Czech Republic (Hubálek et al.,
2020; Lesiczka et al., 2022; Rudolf et al., 2021) Germany
(Chitimia-Dobler et al., 2019), Sweden (Grandi et al., 2020), United
Kingdom (Hansford et al., 2019; McGinley et al., 2021) and the
Netherlands (Uiterwijk et al., 2021).

Citizen science, or community research, is a method that is gaining in
popularity and involvement in many research fields today. The basic idea
is that the data are collected or generated with the help of
non-researchers but are analysed by researchers specializing in the
given topic (Tran et al., 2021). Citizen science has many benefits, both
for the researchers who evaluate the data and for the citizens who
collect them. With the help of citizen science, for example,
professionals have access to the data they need without having to pay
for the work of hundreds of civilian researchers involved in the
project, and the travel, accommodation and education that may be
associated with it. It is also a great advantage that a project can be
carried out over a large geographical area for many years (MacPhail \&
Colla, 2020). This approach has been successfully used in searching for
the emergence of \emph{Hyalomma} adults in several European countries
(Grandi et al., 2020; Lesiczka et al., 2022; Uiterwijk et al., 2021).

In Hungary, we have limited published information about the occurrence
of \emph{Hyalomma} ticks. An engorged nymph of \emph{Hy. marginatum} has
been reported from a hedgehog (\emph{Erinaceus roumanicus} ) living in a
Budapest city park (G Földvári et al., 2011) and two larvae and a nymph
were identified from robins (\emph{Erithacus rubecula} ) (S Hornok et
al., 2013) sampled to the south of the capital. Three \emph{Hy.
rufipes}nymphs were reported from a common whitethroat (\emph{Sylvia
communis} ) in another study (S. Hornok et al., 2016). The presence of
antibodies against CCHFV has also been detected in Hungarian hares
(\emph{Lepus europaeus} ) (Németh et al., 2013), rodents (Földes et al.,
2019) and recently also in humans (Magyar et al., 2021).

Based on these previous findings, the aim of our research was to obtain
a comprehensive, nationwide picture of the occurrence of
adult\emph{Hyalomma} ticks in Hungary through a citizen science
approach. This kind of documentation is of great epidemiological
importance because this is the first step for the prevention of emerging
pathogens according to the DAMA (Document, Assess, Monitor, Act)
protocol (Brooks et al., 2014). Based on this, we plan to continuously
document the appearance of pathogens, their vectors and reservoirs
hosts, then, after the appropriate scientific analyses (Assess), we will
be able to identify the organisms that pose a threat to us and manage
their targeted surveillance (Monitor). Thus, it will be possible to make
adequate proposals to decision makers to take appropriate preventive
measures (Act) (Brooks, Hoberg, \& Boeger, 2019).

\textbf{Materials and methods}

\emph{Tick and data collection}

Ticks were collected from April-December 2021 within the tick monitoring
project https://www.kullancsfigyelo.hu, a citizen-science project aimed
at monitoring the emergence and possible establishment of\emph{Hyalomma}
ticks in Hungary. We were asking participants through the self-made
website to look for unusual hard ticks on themselves, their pets and
their livestock. We called attention to specimens larger than usual with
visible stripes on their legs and with apparently quick movement. To
enhance visibility, the project was intensively communicated in local
and national on-line media outlets, local and national television and
radio channels and social media platforms.

Ticks were reported either by high-quality images sent by e-mail or
physically delivered to the Institute of Evolution, Centre for
Ecological Research, personally or by mail. Information about safe
removal, storage and postage of tick specimens was provided on the
project website.

\emph{Morphological identification of ticks}

Ticks received by mail were kept at 4 ° C and examined alive. Specimens
were identified with a Nikon SMZ800N stereomicroscope using appropriate
morphological characters and identification keys (Estrada-Peña et al.,
2018; Walker et al., 2003). The species, sex, developmental stage,
engorgement state, host and place of origin of the ticks were recorded.

\emph{Molecular identification of Hyalomma ticks}

\emph{Hyalomma} specimens were homogenised with Bertin Minilys (Bertin
Instruments, Montigny-le-Bretonneux, France) after 3 times
freeze-thawing in liquid nitrogen. We used multiple beads in various
sizes with the addition of quartz sand after the freeze-thaw cycles and
before proceeding to . .Samples were sequenced at a commercial centre
(Eurofins Genomics, Ebersberg, Germany).

Sequence data were subjected for phylogenetic analysis, using the MEGA11
(Tamura, Stecher, \& Kumar, 2021). The visualisation of final data was
performed with iTOL v6 online tool.

\textbf{Results}

This tick monitoring project is still ongoing but the data for the
present study are from the period between 28 April and 7 December 2021.
During this time interval, the
website\emph{https://www.kullancsfigyelo.hu/}had 31325 individual
visitors. A total of 137 hard tick specimens and several hundreds of
photos were received. Some other arthropods, e.g. soft ticks, spiders,
bugs and a beetle were also sent by participants as a by-catch. Most of
the ticks were identified as \emph{Ixodes ricinus} , but
\emph{Dermacentor reticulatus,} \emph{Dermacentor marginatus}
and\emph{Haemaphysalis inermis} also occurred (Table 1.). Some
participants sent only photos from specimens, in these cases the
identification was usually possible only to the genus level.

Two specimens were morphologically identified as \emph{Hyalomma} ticks.
The first \emph{Hyalomma} was discovered in a garden of the small town
Bük (47°23'09.2''N 16°44'29.8''E) in Western Hungary (Vas County) on
August 10. The tick was crawling on a dog's leg when the submitter
noticed it. This tick was morphologically identified as a
male\emph{Hyalomma marginatum} . The second specimen was found feeding
on a cattle at a farm near Kiskunmajsa (46°26'09.6''N 19°41'13.4''E) in
South-Eastern Hungary (Bács-Kiskun County) on September 10. This
individual was morphologically identified as a \emph{Hyalomma
rufipes}male. The dog and the cattle had never been abroad. Following
morphological identification, \emph{Hyalomma} ticks were stored in
liquid nitrogen until molecular analyses. Amplification of the COX1 gene
was successful for both specimens. Sequencing verified the previous
morphological identification as \emph{Hy. marginatum} for the first
specimen. Based on the phylogenetic analysis with cognate sequences this
individual most likely belongs to the Eurasian population (Figure 1.).
Sequencing of the COX1 gene fragment of the second specimen
confirmed\emph{Hy. rufipes} with the highest sequence similarity to a
clade of mixed sequences from Europe and Africa. (Figure 2.)

Tick specimens were sent by citizen scientists from all over the
country, the largest number from Budapest and its surroundings. The
least data were received from the south-east of the country (Figure 3).
Ticks were either collected from the environment or from hosts. Most
ticks were removed by citizen scientists from humans, with a total of 45
such cases. The second most common hosts were dogs with 18 submissions
(including the \emph{Hy. marginatum} specimen) and then cats with seven.
A single specimen was received from a cattle, this was the animal in
which the \emph{Hy. rufipes} was discovered.

\textbf{Discussion}

Within seven months from its start, our tick monitoring citizen science
research succeeded to reveal the emergence of two adult
\emph{Hyalomma}tick species in two different locations of Hungary. Our
project highlights the importance of public relations in the success of
a citizen science project. We could reach relatively high visibility
(over 31000 individual website visitors) within seven months (data from
Google Analytics not shown). The highest number of daily visitors, 5453
could be reached after an extensive media release of our project.
Participating civilians also benefit from initiatives that use citizen
science. First, in the optimal case, all such research is intended to
serve the interests of society directly or indirectly, so the interest
of civilian researchers is the success of the program. In addition, the
increased social awareness caused by various citizen science projects is
a positive side effect. Due to their involvement in the survey,
participants pay more attention to nature and to monitoring non-endemic
tick species in their environment and the relationship between
participating citizens, scientists and public health agencies may also
become closer. Our participants received reply from us regarding their
questions and samples within 24 hours. This was important because some
secondary media appearances, emphasizing the seriousness of
Crimean-Congo haemorrhagic fever, were frightening some of our
responders, and in most of the cases they were comforted by the fact
that the specimen they found was not a \emph{Hyalomma} tick.

In addition to the advantages, we must not forget about the limitations
and disadvantages of the method. For example, at the beginning of any
program, it is important to pay close attention to accurately describing
the information requested from the public, failing which it can be very
time-consuming to select relevant data from the set of information
received. It may also be the case that, due to the incorrect or
unverifiable data received, the decision-making bodies may not take into
account the results of such a project. It is particularly difficult to
filter out or prevent the creation of such erroneous data, perhaps
because citizen science is still often characterized by a lack of
universal, reproducible methods. The introduction and strict adherence
to these methods can easily dampen the enthusiasm of the participating
civilians, which is why the high level of attention in project design is
important (MacPhail \& Colla, 2020).

The form of the requested data may also be crucial for the preparation
of the research. In monitoring the distribution of tick species, where,
for example, citizen scientists may try to identify an animal found, the
participants have three ways to submit the requested information. They
can only send the name of the species identified, the photographic
documentation of the individual, or the specimen itself. All three
options have advantages and disadvantages, the first case favours large
amounts of incoming data, but there is no way to verify them. In the
second case, a lot depends on whether the person making the shot is
aware of what morphological characters make the identification possible,
e.g., from what angle and at what resolution it is worth taking
pictures. The exact identification is most secure when citizen
scientists send the specimen to be identified by professionals. We
advised the participants to do so, however, in addition to being the
most time consuming, this case raises other issues. Submission might
involve the destruction of the specimen, and on the other hand, it
increases the risk of human-tick contact (Eisen \& Eisen, n.d.).
Therefore, we particularly made our participants aware of the risks and
suggested the safe removal, storage and postage of tick specimens.

The most commonly submitted tick species was \emph{I. ricinus} . This is
in line with the widespread distribution of this species, however, it
also shows that participants do frequently not recognise the most common
tick species. \emph{Dermacentor} ticks were also submitted in large
numbers. This is again partly due to the common occurrence of this tick
species throughout the country (G. Földvári et al., 2007), however the
on-line media also biased the study in this aspect. Unfortunately,
several news portals used images that featured a
\emph{Dermacentor}species instead of a \emph{Hyalomma} tick. As a
consequence, many citizen scientists, especially dog owners, sent
\emph{Dermacentor}specimens that are larger and quicker compared to
\emph{I. ricinus} and, in addition, matched the mistakenly included
photograph of some online portals. We archived all tick specimens and
data for further analyses, thus this by-catch might provide important
epidemiological data in the future.

Mitochondrial gene-based genetic analysis is highly important to reveal
dispersal patterns and introduction routes of \emph{Hyalomma} ticks
(Capek et al., 2014; Wallménius et al., 2014). Novel mitochondrial
sequence data for \emph{Hyalomma} spp. is increasingly reported (Ciloglu
et al., 2021; Lang, Shan, Zhang, Liu, \& Wang, 2022). The importance of
providing sequence data of these vector organisms is getting more
relevant in the light of disease emergence, especially the geographic
expansion of Crimean-Congo Haemorrhagic Fever virus during the last
decades (Akyildiz, Bente, Keles, Vatansever, \& Kar, 2021; Arteaga et
al., 2021; Moraga-Fernández et al., 2021; Sánchez-Seco et al., n.d.). We
further highlight the importance of sequence data by integrating this
activity into the citizens science programme and also into the risk
assessment of CCHFV emergence and preparedness in our region. The two
sites were \emph{Hyalomma} ticks were found in our study are
approximately 280 kilometres apart, with the first specimen discovered
on August 10 and the second on September 10, so exactly one month apart.
Due to the large geographical distance and the time difference, the two
specimens must have arrived in the country separately as a result of two
separate introductions. Although much more published sequence data would
be necessary, we suspect that the probable source of introduction was a
Eurasian population for the detected \emph{Hy. marginatum} and a
population along the Africa-Europe bird migration route for \emph{Hy.
rufipes} .

Another important issue is whether the specimens found entered the
country in the same year, e.g. as engorged nymphs dropping off from
migratory birds, or overwintered before finding them in the next year,
or even already hatched here as larvae in the previous year. The latter
possibility is of greatest concern because it would indicate the
establishment of a local population, making this species able to
successfully complete its entire life cycle in Hungary. This seems
unlikely currently, because a critical number of
conspecific\emph{Hyalomma} adults would be needed to yield egg-laying
fertilized females. The moulting into adult in the year of drop-off,
among many other factors, is affected mainly by temperature. On the one
hand, hot springs are preferred by \emph{Hyalomma} ticks, where the
average daily temperature above 8 ° C for \emph{Hy. marginatum} ticks
can be determined as a criterion for moulting from nymph to adult (Gale
et al., 2012). Due to the colder-than-average April weather in Hungary
in 2021, this was only achieved in mid-May (National Meteorological
Service). Only adults found before this could have unambiguously
overwintered in the country, giving the opportunity to develop a local
population. However, during our program, both specimens of
\emph{Hyalomma} were discovered well after May, so these adults were
presumably introduced the same year. Our tick monitoring program was not
running during the spring, and there is a possibility for overwintering
adult\emph{Hyalomma} in Hungary, as it has recently been shown in the
Czech Republic (Rudolf et al., 2021). Unfed adult \emph{Hyalomma} ticks
tolerate the cold months well, with the most critical period being late
summer and autumn due to the heat sensitivity of engorged nymphs
(Uiterwijk et al., 2021; Valcárcel et al., 2020). Thus, it is not the
cold winter but the cold autumn that is critical in shaping the
geographical spread of \emph{Hyalomma} species. Climate change might
easily enhance establishment of new local \emph{Hyalomma} populations
with elevating autumn temperatures in temperate Europe as exemplified by
the rising presence of the tick \emph{Hy. marginatum} in Southern France
(Vial et al., 2016). Once imported, establishment of \emph{Hy.
marginatum} on common local hosts, wild and domesticated mammals and
even urban ones like hedgehog has been shown in Hungary (G Földvári et
al., 2011) and also recently in Bulgaria (Arnaudov, Mikov, \& Georgiev,
2022).

The ultimate reason for monitoring the emergence of adult\emph{Hyalomma}
ticks is the possible introduction of tick-borne pathogens, most
importantly Crimean-Congo Haemorrhagic Fever Virus. There are scattered
historical data about the occurrence of\emph{Hyalomma} ticks and CCHFV
in Hungary as summarised in Figure 4. Immature \emph{Hy. marginatum}
from a hedgehog in a city park (G Földvári et al., 2011), immature
\emph{Hy. rufipes} and \emph{Hy. marginatum} from songbirds (S Hornok et
al., 2013; S. Hornok et al., 2016) and two adult \emph{Hy. rufipes} from
cattle were previously detected in the country. Reported occurrence of
CCHFV spans from isolation from \emph{Ixodes ricinus} ticks (Molnár,
1982), to seropositive cattle, sheep (S. Horváth, 1974), brown hares
(Németh et al., 2013) and wild rodents (Földes et al., 2019). Most
interestingly, humans seropositive for CCHFV were reported well before
the first observation of adult \emph{Hyalomma} ticks (L. B. Horváth,
1976). There was also a reported human case of CCHF with unknown origin
in 2004 (Országos Epidemiológiai Központ, 2008) and CCHFV seropositives
were detected in 12 healthy blood donors collected between 2008-2017
(Magyar et al., 2021). All these data indicate that CCHFV might be
transmitted by tick species other than \emph{Hyalomma} spp. and that the
earlier emergence of adult \emph{Hyalomma} ticks might have been
overlooked in Hungary. In any case, we will continue with the citizen
science based systematic monitoring of \emph{Hyalomma} ticks in the
future. We also plan to analyse these and future \emph{Hyalomma}
specimens for the occurrence of CCHFV and other pathogens.

\textbf{Conclusions}

In the course of our research, we examined the occurrence of
non-endemic\emph{Hyalomma} ticks in Hungary using the citizen science
method. The use of community research was successful in terms of both
website traffic and number of submitters. Participants reacted
positively, people took the program noticeably seriously. Due to the
importance of the topic and the good reception, our goal is to continue
the research next spring, so the first years' experience is also useful
for fine-tuning the project. One striking result of our research was
that one of the two \emph{Hyalomma} found was discovered in the only
cattle involved in our research. \emph{Hyalomma} often parasitize large
domesticated mammals in their adult stages, as was previously known (S
Hornok \& Horváth, 2012; Uiterwijk et al., 2021). It would therefore be
worthwhile next year for our program to target inspections of
establishments affected in this respect. This could be done, for
example, by finding journals, online platforms and social media groups
for animal husbandry, horse riding, or by looking for farms specializing
in these. Data were received from across the country, but most
submissions are clearly from the capital or a narrower neighbourhood. In
the future, greater emphasis should be placed on rural areas, especially
livestock farms, villages and towns. As media presence has proven to be
key to our research, in the future we will try to focus on the print and
electronic media presence also in rural areas.

In addition, it would be worthwhile to launch a media campaign for the
project as early as the end of the winter, ensuring that people take
pictures of the ticks already discovered in the spring and send them via
mail. This would be important because in such cases, there would
probably only be individuals of the potentially
overwintering\emph{Hyalomma} ticks in the country, so such an
observation would indicate that the group may be able to form local
populations in the future. It is of utmost importance to continue
systematic monitoring of\emph{Hyalomma} ticks in the future. We will
analyse these and future\emph{Hyalomma} specimens for the occurrence of
CCHFV and other pathogens to be able to monitor and prepare for the
introduction of transboundary and emerging diseases.

\textbf{Conflict of interest}

The authors declare that they have no conflict of interest.

\textbf{Author contribution}

GF conceptualized, designed and coordinated the study and drafted the
original manuscript. GF and ÉSz coordinated the citizen science project,
morphologically identified ticks. ÉSz designed and managed the project
website\emph{https://www.kullancsfigyelo.hu/}. GET, ZSL, ZSV performed
laboratory work and sequencing. BZ, GK performed phylogenetic analysis.
ZSL did data visualization. GK supervised the molecular and sequencing
works. GET and GK edited and reviewed the manuscript and carried out
data analysis. All authors read and approved the final manuscript.

\textbf{Ethics}

The authors confirm that the ethical policies of the journal, as noted
on the journal's author guidelines page, have been adhered to. All
relevant guidelines for the use of animals in scientific studies were
followed.

\textbf{Data availability statement}

Sequence data is available in NCBI GenBank database under the accession
numbers: OM527159-OM527160. The data that support the findings of this
study are available from the corresponding author upon reasonable
request.

\textbf{ORCID}

GF: 0000-0001-5297-9036

ÉSZ: 0000-0002-2848-1577

GET: 0000-0002-7201-9646

ZSL: 0000-0003-3116-4633

BZ: 0000-0002-3503-8901

ZSV: 0000-0002-0432-3654

GK: 0000-0001-9775-3065

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\textbf{Tables}

Table 1. Species, stage, sex and host of tick specimens received within
the tick monitoring citizen science project between 15 April and 7
December 2021

\textbf{Figure legends}

\textbf{Figure 1.} Phylogenetic tree of 22 partial gene sequences of the
mitochondrial cytochrome oxidase c I gene of \emph{Hyalomma marginatum}
. The tree was constructed with the Neighbour-Joining method using the
Tamura 3-parameter method to compute evolutionary distances. We applied
the bootstrap test with 1000 replicates. Novel sequence data is
highlighted with bold letters and an arrow.

\textbf{Figure 2.} Phylogenetic tree of 24 partial gene sequences of the
mitochondrial cytochrome oxidase c I gene of \emph{Hyalomma rufipes} .
The tree was constructed with the Neighbour-Joining method using the
Tamura 3-parameter method to compute evolutionary distances. We applied
the bootstrap test with 1000 replicates. Novel sequence data is
highlighted with bold letters and an arrow.

\textbf{Figure 3.} Geographical distribution of the ticks received by
the tick monitoring citizen science
project\emph{https://www.kullancsfigyelo.hu}. Each dot represents a tick
sent in by the civil participants of the research.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 4.} Historical published data about the occurrence
of\emph{Hyalomma} ticks and Crimean-Congo Haemorrhagic Fever Virus
(CCHFV) in Hungary in chronological order.

1 CCHFV isolation in 1972 from two \emph{I. ricinus} ticks in Veszpr\selectlanguage{ngerman}ém
county (Molnár, 1982)

2 CCHFV seropositive cattle and sheep in 1973 in Hajdú-Bihar county ,

3 CCHFV antibody in 17 human sera. One from a slaughterhouse worker in
Budapest, all others from Hajdú-Bihar county (L. B. Horváth, 1976).

4 Human CCHF infection from unknown source in Baranya county in 2004
(Országos Epidemiológiai Központ, 2008)

5 CCHFV seropositive brown hares collected between 2008 and 2009 from
near Dévaványa village ,

6 An engorged \emph{Hyalomma marginatum} nymph found on a Northern
white-breasted hedgehog in 2009 in an urban park of Budapest (G Földvári
et al., 2011)

7 Two \emph{Hy. rufipes} males found on two cows in September 2011 in
south-western Hungary ,

8 CCHFV seropositive wild rodents collected between 2011 and 2013 in the
Mecsek Mountain region ,

9 Three \emph{Hy. marginatum} (two larvae and a nymph) collected from an
European robin at the Ócsa Ringing Station in 2011 and three \emph{Hy.
rufipes} nymphs from a common whitethroat (\emph{Sylvia communis} ) in
2014 (S Hornok et al., 2013; S. Hornok et al., 2016)

Red crosses indicate origin of CCHFV seropositive blood donors collected
between 2008-2017 (Magyar et al., 2021)

Red dots indicate the origin of the two \emph{Hyalomma} specimens of the
present study.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig1/Fig1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig2/Fig2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig3/Fig3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig4/Fig4}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`Table1.docx`  available at \url{https://authorea.com/users/462203/articles/557693-emergence-of-hyalomma-marginatum-and-hyalomma-rufipes-adults-revealed-by-citizen-science-tick-monitoring-in-hungary}

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