The density and peaks of seasonal activity of Dermacentor reticulatus ticks in eastern Poland

Background: Next to Ixodes ricinus , Dermacentor reticulatus ticks are the most important reservoir and vector of infectious diseases in Europe. In recent years, progressing expansion of D. reticulatus ticks into new territories and an increase in their population size in areas of their previous occurrence have been observed. Methods: The studies on tick population density were conducted in Lublin Province, eastern Poland. The investigated area was divided into 101 equal squares (plots). Additionally, studies on seasonal activity in the preferred habitat of the ornate cow ticks were conducted from 2014 to 2019 in the Polesie National Park. Results: Throughout the study on the tick population density, 19559 adult D. reticulatus specimens were collected. An average density of the ornate cow ticks in Lublin Province was 96.8 specimens/100 m 2 . A significant impact on the tick population density is exerted by meadow areas (Rs=0.281, p=0.0044), while weather conditions have no effect (H=6.08, p=0.1931). The local population of D. reticulatus ticks in the eastern and middle regions of Lublin Province exhibits predominance of spring activity peaks, while autumnal seasonal activity peaks prevail in the rest of the region. Multiannual monitoring of the seasonal activity of D. reticulatus in the Polesie National Park shows the prevalence of autumnal activity peaks in 2014-2019 and spring activity peaks in 2019. Conclusions: The area of Lublin Province is characterized by high density of the ornate cow ticks. Local populations of D. reticulatus show two peaks of seasonal activity. Dynamics of seasonal activity of D. reticulatus is influenced by availability of their hosts. The highest risk of ornate cow tick attacks on animals and humans occurs during spring in the eastern part and in autumn in the western part of Lublin Province. The mosaic character of the landscape offers beneficial conditions for D. reticulatus populations. Forests provide availability of host species for adult specimens, while meadows and wastelands are preferred habitats for rodents – hosts of juvenile forms of ticks.


Background
Dermacentor reticulatus belongs to the most widely spread tick species throughout Europe. According to many authors, two geographically separated populations of ornate cow ticks are distinguished -the "western European" and "eastern European" population. This phenomenon is not found in other widely distributed ticks species in Europe i.e. Ixodes ricinus or D. marginatus [1,2].
In Western Europe, the compact area of ornate cow tick distribution includes the southern part of the British Islands, France, Switzerland, Benelux, and Germany [2][3][4][5]. Specimens of this species are most commonly collected from plants as well as wild, farm, and domestic animals in Central Europe. The lowlands of the Czech Republic, western Slovakia, Hungary, Slovenia, and Austria should be considered as endemic areas for this species. D. reticulatus from the regions mentioned above are included into the western European population of this species [6][7][8][9][10].
The compact range of the eastern European D. reticulatus population covers eastern Poland, Ukraine, Belarus, Lithuania, Latvia, Estonia, and the north-west of the European part of Russia. Numerous sites of ornate cow tick occurrence have been reported from the Caucasus and Asia to Omsk and Novosibirsk [4,[11][12][13][14].
Until the beginning of the 21 st century, it was thought that the compact area of D. reticulatus distribution in Poland is limited to the territory located to the east of the Vistula River and in northeastern part of the country, while western regions were considered free from this species.
Simultaneously, it was accepted that the border between the eastern and western population of D.
reticulatus ran through Poland [12,15,16]. Studies on tick ecology and distribution, which have been intensified in recent years by Polish scientists, confirmed the occurrence of the species also in the northern, western and south-western territory of Poland [17][18][19][20][21][22][23][24]. As suggested by some authors, this phenomenon should be referred to as expansion of ornate cow ticks to new areas [21,25].
Similar changes in the dynamics and distribution of local D. reticulatus populations were observed in other European countries, e.g. in Germany where before the 70s of the 20 th century its distribution was limited to western Lands, and the described population was not numerous [26]. In the next years, expansion to eastern Lands bordering Poland was observed [7]. Many new D. reticulatus occurrence sites were reported in the Czech Republic. In Slovakia, new sites of distribution of ornate cow ticks representing both eastern European and western European populations were reported [27,28]. In recent years, expansion of D. reticulatus ticks was observed in Hungary, where only two sites of occurrence were documented before the 70s of the past century [8]. A similar process of expansion was also observed in Romania [10,29].
Clustered populations of D. reticulatus were confirmed in the south of the continent, i.e. in the Balkan [30][31][32][33] and Iberian peninsulas [34]. The spread of ornate cow ticks to Scandinavia [35] and northern Italy, which were regarded as tick-free previously has been observed during the last years [36]. The studies on the density of D. reticulatus ticks in eastern Poland were conducted in Lublin Province ( Fig. 1). A grid of 101 squares with 15.8-km long sides (plots) was applied to a physical map of the investigated area (Fig. 2). An average area of each square was 250 km 2 , which represents 1% of Lublin Province. Plots 1,2,19,27,35,44,53,63,81,89,96,97,100, and 101 were the exceptions, as they had smaller areas due to the course of administrative boundaries of the Province. At the same time, they constituted more than 60% of the area marked by the described grid.
The next stage included the use of satellite pictures available in Google Maps [38] to determine potential sites of species collection. Meadows undergoing ecological succession located on the edge of forests or in its vicinity and with access to watercourses were regarded as preferred tick habitats (Fig. 3).

Tick collection
The potential areas of tick collection were surveyed previously. Field studies were conducted during seasonal peaks (spring and autumn) of tick activity in Lublin Province. pictures, the percentage structure of the area in each square was calculated. The surface of the cultivated land, forests, and meadows was analyzed. The term "other" means buildings, water resource types, and areas that could not be classified clearly.

Analysis of the weather conditions
The historical data on the weather conditions from 2015-2019 in different regions of Lublin Province were retrieved from the Internet service tutiempo.net [43]. Data of the Terespol weather station were accepted as representative for the north-eastern region of Lublin Province, while the stations in Włodawa, Zamość, and Kozienice provided relevant data on the eastern, southern, and western regions, respectively (Fig. 4). The methods of collection and identification of specimens to the species level and developmental forms were the same as described above. Ticks were collected in equal periods from March to November. Throughout the study period, ticks were collected in the same habitat. In all statistical tests, the significance level was p<0.05. The statistical analysis was conducted with Statistica 10PL software.

Study area
Lublin Province has an area of 25,122 km 2 , which accounts for 8.02% of the territory of Poland. The border between three megaregions differing in the geomorphological structure, water features, and climate, i.e East European Plain, Extra-Alpine Central Europe, and Carpathian Region, runs across the province area [44].
The eastern and north-eastern parts of Lublin Province are located in western Polesie and Volhynian Polesie (Fig. 4). These regions are located in the basin of the Bug -one of the longest rivers in Poland.
The subsoil consists mainly of marls and chalk, hence the presence of many lakes and bogs. The regions are characterized by high woodiness and very low average air temperature values. At the same time, this area is exposed to the lowest anthropopressure in Poland [44].
The western part of the study area belongs to the South Podlasie Plain. This region is typically agricultural with high ratio of wastelands. The climate is more temperate, especially in the western regions. The central area of Lublin Province is part of Lublin Upland. Fawn and podzol soils form the subsoil, while brown soils are the substrate in the river valleys. Landform is pleated. This part of the province is intensively used for agriculture and has been almost deforested [44].
The southern outskirts of the study area are part of Roztocze. This is the highest area of the region, with climate close to continental and marls, opoka rocks, and limestone in its subsoil. Rivers in this area have a mountain character [44].

Results
The density and peaks of D. reticulatus population activity In total, 19559 adult D. reticulatus specimens were collected throughout the study period, including 11598 females and 7961 males (Table 1). An average density of the ornate cow tick population in Lublin Province was 96.8 specimens/100m 2 with statistically significant dominance of females (Z=8.19, p<0.001). The ticks population density was higher in autumn than in spring ( Table 2). The greatest population density was observed in the northern part of the study area (plots 1-18). It varied from 86 specimens to 311 specimens/100 m 2 throughout the study period. The lowest density of the D. reticulatus population was noted in the central part of the province. However, the lowest number of ticks were collected in square 67, where the population density was 22 specimens/100 m 2 ( Table 1, The predominance of active D. reticulatus ticks in autumn was observed in the northern, southern, and western part of Lublin Province, while in the middle and eastern parts of region, the highest number of ticks were collected in spring (Table 1, Fig. 2).
In the regression model of the density of the ornate cow ticks, none of the land structure variables (arable lands, forests, meadows) has statistical significance (F (3.97) =1.05, p=0.3728). However, Spearman's Rank correlation coefficient indicates that the density of active D. reticulatus females and males rises with the increase in the meadow area (Rs=0.281, p=0.0044).
The weather conditions observed in the particular parts of the region in 2019 were not significantly different (average air temperature H=0.18, p=0.9962; relative air humidity H=2.00, p=0.7354; total precipitation H=0.82, p=0.9361; number of days with snow cover H=0.06, p=0.9995; number of days with fog H=6.08, p=0.1931). These parameters had no statistically significant impact on differences in the tick density between the particular study areas (average air temperature p=0.2848; relative air humidity p=0.1881; total precipitation p=0.3910; number of days with snow cover p=0. 3910).
The statistical analysis of the weather parameters in 2015-2019 did not show statistically significant differences between the parts of Lublin Province (H=0.91, p=0.9901).

Multiannual monitoring of the seasonal activity of D. reticulatus ticks
In the Polesie National Park (51º22'N, 30º30'E), a tendency towards an increase in the number of active D. reticulatus ticks was observed in 2014-2018. The substantial dominance of the average number of active ticks in autumn over spring was revealed. Simultaneously, more females were collected. In 2019, more active ticks were collected in spring than in autumn (331 to 93 specimens, respectively) ( Table 3).
In each year from 2014 to 2018, a statistically significant difference between the number of active D. reticulatus ticks in spring and autumn was confirmed (H=6.98, p=0.047). However, there was no statistically significant difference in the number of active ticks between the study years (H=3.94, p=2678). Moreover, a statistically significant difference in the number of active ticks in 2014-2018 was revealed (H=4.87, p<0.014). The seasonal activity of D. reticulatus ticks is significantly influenced by the air temperature prevailing during tick collection (H=12.887, p=0.002).

Discussion
The dynamics and size of the D. reticulatus population, as in the case of other hard ticks, depend on many biotic and abiotic factors. The most important include the presence of potential hosts, ecological type of occupied habitats, climatic factors, photoperiod, excreted semiochemical substances and hormones controlling the rhythms of seasonal activity, diapause periods, and biology of tick reproduction and development [13,39,45,46].  Table 1). These areas have the largest meadows areas in the entire province. An increase in the meadow area positively correlates and has a statistically significant impact on the number of collected ticks (Rs = 0.281, p = 0.0044). It should be noticed that a significant percentage of this area is occupied by wastelands and forest islands ( Table 1). The mosaic character of the landscape offers beneficial conditions for D. reticulatus populations. Forests provide availability of host species for adult specimens, while meadows and wastelands are preferred habitats for rodents -hosts of juvenile forms of ticks.
The greatest density of D. reticulatus ticks throughout the whole study period was observed in the experimental field located in plot 69 placed in Lublin Upland (Fig. 4). During single collection in the spring activity peak, 516 specimens of ornate cow ticks were harvested (297 females and 219 males) per 100 m 2 area (Table 1, Fig. 5, Fig. 6). Attention should be drawn to the lowest density of tick populations in the neighboring sites (located in the same macroregion) ranging from 10 to 100 specimens/100 m 2 (Fig. 5). Therefore, the high density of ornate cow ticks is a result of the ecological character of the habitat. The site where the greatest number of ticks was collected is surrounded by an uncut meadow, a watercourse, and a forest. The forest cover is < 8% in the scale of the whole macroregion and 22% for the province (Table 1). With its fertile soils, the area is intensively used for Considering the survivability of adult specimens in a habitat over 2 years [13], the predominance of tick activity peaks in spring in areas where the autumn peak was presented previously [41] (Table 3) can be caused by an insufficient amount of hosts for adult ticks.

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Photos used in Fig. 3 and Fig. 6