Bio-efficacy of permethrin/tetramethrin and lambda-cyhalothrin treatments in habitats of hard ticks (Acari, Ixodidae) populations with confirmed Borrelia spp. infection

The aim of the study was to evaluate the bio-efficacy of two different acaricides against mobile stages of hard ticks Ixodes ricinus, Dermacentor marginatus, and Haemaphysalis punctata in their natural habitats. The study was conducted during 2020 and 2021 at localities populated by I. ricinus as the predominant species, at which the presence of Borrelia afzelii, Borrelia garinii, and Borrelia lusitaniae was confirmed. During the first investigation year, a combination of two pyrethroids, permethrin, and tetramethrin, with an insecticide synergist piperonyl butoxide (trade name: Perme Plus®) was tested. At the first evaluation, 24 h after the treatment with Perme Plus®, the efficacy expressed as a reduction rate of the population density was within the interval of satisfying performance (70–90%) at all localities, while the highest efficacy (97.8%) was recorded on the 14th post-treatment day. In the second investigation year, the formulation based on lambda-cyhalothrin (trade name: Icon® 10CS) was used. On the first post-treatment evaluation day, satisfying effects were also demonstrated. The highest recorded efficacy rate of lambda-cyhalothrin (94.7%) was recorded on the 14th post-treatment day. Both tested acaricides manifested satisfying initial acaricidal effects against mobile stages of ticks and provided long-term effects. Comparison of the regression trend lines of population reduction revealed that satisfying effects of treatment with Perme Plus® lasted until the 17th post-treatment day, while in the case of Icon® 10CS, the residual effects were significantly prolonged (30 days).


Introduction
Frequent occurrence and increasing population density of ticks in urban green areas frequented by humans have been emerging as a significant problem over the past years in Serbia. The presence of ticks of the Ixodidae family poses a potential risk of human and animal infections with various pathogenic agents (Pavlović et al. 1999). The species Ixodes ricinus Linnaeus 1758, associated with deciduous and mixed forests, is the principal vector of several viruses, bacteria, and protozoa (Rizzoli et al. 2014). The contact with the host is crucial for the survival of ticks and also for the pathogens' transmission. The finding of a suitable host is determined by diverse factors such as temperature, light, relative air humidity, physiological status, and the presence of vertebrates (Sonenshine 1993). According to the guidelines of the World Health Organization (WHO 1997) and the European Centre for Disease Prevention and Control (ECDC 2021), vector surveillance and control are required in the prevention of vector-borne disease transmission. Within the integrated control approach, long-term monitoring and surveillance, as well as an understanding of the seasonal dynamics of tick species are prerequisites for the optimization of tick control practices. The final objective of the integrated approach to tick control is the reduction of human and animal tick-borne disease cases, environmental protection, and the reduction of associated economic costs. In Serbia, ticks have been intensively investigated as indicators of the natural-focal viral infections such as Crimean-Congo Hemorrhagic Fever (CCHF), Lyme disease (Obradović 1985;Vujošević 1995), and Tick-Borne Encephalitis Virus-TBEV (Potkonjak et al. 2017). Tick-borne diseases of humans and animals were reported worldwide, many of them being described as zoonoses.
Tick-borne encephalitis and Lyme disease occur most commonly in the central and eastern regions of Europe (Estrada-Peña and Jongejan 1999). In the past few years, a range of investigations has been conducted worldwide with an aim of determining the most effective measures for monitoring and control of ticks as vectors of diverse pathogens (Gray et al. 2009).
Common practices of hard and soft tick control in animals include the application of acaricides. However, such methods have serious shortcomings manifested by potential milk and meat contamination, as well as the development of acaricide resistance in some tick species (Jongejan and Uilenberg 2004;Walker 2011;Abbas et al. 2014). Generally, it is more effective to control ticks on the body of animals, or in the case of soft ticks (Argasidae), it is better to apply acaricides with long residual effects indoors, because that way, overuse of acaricides on pastures is avoided, and harmful effects on the environment are reduced.
The protection of humans against ticks predominantly relies on personal protection by applying repellents and chemical agents (biocides) onto potential contact sites and areas -most commonly walking tracks close to the rivers, picnic areas, and parks. An effective and timely protection of human and animal health is based on relevant scientific knowledge and adequate surveillance strategies. According to the Centers for Disease Control and Prevention (CDC), the incidence of diseases associated with tick bites shows an increasing tendency (CDC 2013;Hinckley et al. 2014;Nelson et al. 2015). Lyme disease is the most common tickborne disease with a yearly incidence of over 85,000 new cases registered only in the region of Europe, though the number is largely underestimated as the reporting was highly inconsistent with many Lyme disease cases undiagnosed (Lindgren and Jaenson 2006;CDC 2013). Vandekerckhove et al. (2019) also found that the Lime disease incidence in Western Europe is increasing.
Causes of the increased incidence of tick-borne diseases should be considered in relation to climatic changes, increased abundance of primary tick-host population, changes in human behavior, and the lack of adequate control and protection strategies.
Many countries still have not developed long-term strategies and tick monitoring programs which are required to be supported by substantial and consistent funding by governmental authorities and are expected to greatly contribute to the awareness of public health hazards associated with ticks and tick-borne pathogens (Eisen and Paddock 2020). Public health authorities in many European countries have clearly identified the need for improved tick surveillance that would enable better assessment of public and veterinary risks. Considering a highly complex and still unelucidated tick-pathogen-environment relationship, some authors emphasized that the One Health concept should be raised to a higher level towards EcoHealth, which would allow the development of effective and sustainable tick control (Dantas-Tores et al. 2012;Destoumieux-Garzón et al. 2018). In Serbia, such a concept is still missing at the national level. However, it is applied in some regions and areas. Chemical control of ticks mainly includes acaricide application in a targeted area (i.e., tick habitat) or acaricide treatment of the hosts.
The development of tick resistance to some groups of acaricides required the need for new acaricides with improved efficacy and selectivity, and with less adverse effects on the environment (Jeschke 2021). The development of vaccines to control hard and soft ticks is an alternate prevention method (Bogovic and Strle 2015;van Oosterwijk and Wikel 2021). Bio-control of ticks emerges from a need for sustainable and safe control measures, which can be applied in an integrated management of vectors (Klingen and van Duijvendijk 2016). Recent studies address the perspectives of biological control of ticks; however, investigations are still limited to laboratory conditions (Lu et al. 2015). Despite the shortcomings of numerous pyrethroids due to residual contamination of milk and meat and environmental pollution, they are still the most commonly used acaricides in tick control. According to EU directives, synthetic pyrethroids: permethrin, cypermethrin, deltamethrin, and flumethrin are the mainly used compounds to control harmful arthropods (Durel et al. 2015).
The main objective of this study was the evaluation of the bio-efficacy of two synthetic pyrethroid acaricides, Perme Plus® and Icon® 10CS, against mobile stages of ixodid ticks in natural tick habitats with a high risk of transmission of tick-borne diseases, as well as the evaluation of long-term effects in treated habitats.

Biocide products used in the study
The two synthetic pyrethroid products applied in the study were as follows: Perme Plus® (producer: ORMA SRL, Trofarello (TO), Italy; provider Bio Spin d.o.o. Novi Sad, Serbia) based on two active ingredients, permethrin (15.2%) and tetramethrin (0.95%), with the synergist piperonyl butoxide (5.2%), in a formulation of a concentrated aqueous microemulsion; and Icon® 10CS (producer: Syngenta Crop Protection Basel, Switzerland; provider: Syngenta Agro d.o.o. Belgrade, Serbia) based on Lambda-cyhalothrin, in a formulation of an aqueous liquid microencapsulated suspension (10%).

Localities
The efficacy of acaricides was investigated in two different groups of localities, which were sufficiently isolated, distantly located from each other, and characterized by different ecological features.
According to ecological category and habitat type, the first group of localities belongs to a category of forests. Within this group, the chosen localities for this study were situated in Kaćka Forest, north-east of the city of Novi Sad (Vojvodina Province, Serbia), near the village of Kać, extending across an area of 220 ha. Kaćka Forest represents an experimental estate of the Institute of Lowland Forestry and Environment, Novi Sad, Serbia, and serves for studies of biological and ecological characteristics of lowland tree species such as poplar, willow, black locust, narrow-leafed ash, common oak, and wild cherry. In addition, this forest is used for the commercial production of poplar and willow wood, as well as for the production of a high-quality propagation material. This ecosystem is characterized by a rich fauna of amphibians, reptiles, and mammals, which under favorable climatic conditions facilitate the development and persistence of tick populations. The personnel engaged in wood and propagation material production are at the highest risk of exposure to tick bites throughout the year. The geographical position (longitude and latitude) of the chosen localities in this study were the following: N 45° 17.667′ E 19° 54.019′ (locality 1); N 45° 17.586′ E 19° 53.754′ (locality 2); N 45° 17.582′ E 19° 53.750′ (locality 3); and N 45° 17.582′ E 19° 54.049′ (locality 4), N 45° 17.588′ E 19° 53.758′ (locality 5). Chemical treatment of ticks in Kaćka Forest was conducted in localities 1-4, and the fifth served as negative (untreated) control.
The second group of localities was located in the Fruška Gora National Park (Vojvodina Province, Serbia), and according to ecological category and habitat type, it included the shrub and meadow type of flora. Based on a previous long-term tick-monitoring result and eco-epidemiological indicators of tick born diseases risks, four localities were selected for chemical treatment of ticks: Letenka (N45° 08′ 07.94″ E19° 40′ 46.39″), Andrevlje (N45° 10′ 25.83″ E19° 38′ 49.76″), Testera (N45° 11′ 01.69″ E19° 38′ 59.29″), Stražilovo (N45° 10′ 9.06″ E19° 55′ 01.94″) and one served as untreated control locality (N45° 08′ 14.43″ E19° 41′ 05.2″). The selected localities are characterized by green areas that represent frequently visited picnic sites, walking and hiking trails, and recreation spots. With more than 60 registered mammalian species and due to favorable microclimatic conditions, Fruška Gora provides abundant ideal habitats for the establishment and growth of tick populations, thus posing a high potential risk for the contact of humans and animals with ticks, especially with I. ricinus as the main vector of transmission of Lyme disease. At these localities, chemical treatment of ticks with Icon® 10 CS was conducted.

Product application
The application was performed using the equipment Igeba, model U-15 HD-M (Mobilstar E fogging equipment for spraying products in LV formulation), specially designed for vector control in accordance with the WHO specifications on equipment for vector control WHO/VBC/89.972 (WHO 1989). The equipment is mounted on a vehicle that driving with a velocity of 10-15 km/h may provide the flow rate from 5-100 L/h of spray liquid. The apparatus is adjustable to precisely apply a given amount of spray liquid per hectare. The equipment includes 2-4 aerosol nozzles, individually operable, adjustable to 360° rotation, and working in a low volume (LV) regime. The adjustable treatment width (15-60 m) is provided through variable angle-positioning of nozzles. Spray liquid of Perme Plus® was applied at a 4:1 ratio (800 ml Perme Plus® and 200 ml water per hectare). For Icon® 10 CS, the spray liquid was applied at a 1:7 ratio (100 ml of Icon® 10CS and 700 ml water). For both products, the output dosage of spray liquid was calibrated to 1 L/ha. Treatment of ticks on green areas was performed by working under a LV regime using an adequate droplet size that ranged from 75 to 100 µm.

Collection and identification of ticks
The density of the tick population in each locality was determined by the number of ticks sampled by the flag/hour method (Maupin 1991), where 1 m 2 large white flannel cloth was dragged for 1 h over a soil surface with low vegetation. After every 20 m of dragging, the cloth was inspected on both sides. At each locality during each sampling date, the collection was performed in 6 repetitions. Field route marking was completed using the GPS device GARMIN Nuvi. Sampling was carried out in the period April-May 2020 and 2021. In the pre-treatment period, ticks were sampled 2 days before treatment. After conducting the treatments (on 26 April 2020 and 29 April 2021), the effects were evaluated by sampling at each of the four post-treatment intervals, comprising of day 1 (24 h after the treatment), day 7, day 14, and day 21. The tick abundance was expressed as the average number of ticks sampled by flag/hour for each locality and each day of evaluation.
Tick identification to species level was performed according to the identification keys of Estrada-Peña et al. (2004) and Walker et al. (2003). After the identification of species and developmental stage, nymphs and adult specimens of I. ricinus were examined for the presence of Borrelia spp.

Examination of ticks for the presence of Borrelia
The presence of Borrelia species in I. ricinus ticks collected in the natural environment of Fruška Gora (localities Letenka, Testera, Andrevlje, Stražilovo) and Kaćka Forest included DNA extraction followed by amplification of the RecA gene using a commercial quantitative real-time PCR assay (Lyme Disease genesig® Easy Kit, Primerdesign Ltd), and identification of Borrelia spp. by applying a quantitative real-time PCR Light Mix Modular Borrelia spp. kit (TIB MolBiol).
After washing ticks in 70% ethanol solution and then in distilled water, the lysis of whole ticks in phosphate-saline buffer applying TissueLyser LT (Qiagen) was done, and subsequently, the genomic DNA was extracted using the Gen-esig® Easy extraction kit (Primedesign Ltd), as described by Žekić Stošić et al. (2021). The detection and quantification of Borrelia-DNA were performed using quantitative real-time PCR for the RecA gene (Lyme Disease Genesig® Easy Kit, Primerdesign Ltd). A 50-cycle qPCR protocol was used. Each cycle comprised a 2-min enzyme activation at 95 °C, 10-s denaturation at 95 °C, and 1 min data collection at 60 °C. DNA samples positive for the presence of Borrelia species were again amplified and identified by applying the quantitative real-time PCR Light Mix Modular Borrelia spp. kit (TIB MolBiol). The ospA gene fragment of Borrelia was amplified using specific primers and detected by hybridization probes according to the manufacturer's instruction. Identification of Borrelia spp. was performed by analysis of melting temperature (Tm). The PCR reaction was monitored by an additional PCR product which allows for the detection of amplification inhibition (Cerar et al. 2015).

Statistical analysis
Verification of the bio-efficacy of the applied pyrethroids was performed by applying the standard WHO method (WHO 2006). The treatment efficacy rate was calculated by evaluation of the reduction of the tick population density in each treated locality and on each post-treatment day, according to the modified formula of Abbott (1925) given by Fleming and Retnakaran (1985) as follows: where R (%) is the reduction of tick population, T1 and T2 are the pre-and post-treatment number of live ticks in a treated plot, and C1 and C2 are the pre-and post-treatment number of live ticks in the control (untreated) plot.
Classification of the treatment efficacy of tested pyrethroids was defined according to the following criteria: -Weak effects (efficacy < 70%) -Satisfying effects (efficacy 70-90%) -Good effects (efficacy > 90%) Statistical analysis of the obtained data was conducted in the statistical software package Statistica version 14.0.0.15 © 2020 TIBCO Software Inc., applying descriptive statistics and ANOVA analysis of variance and post-hoc Duncan's multiple range test. The data on tick population abundance (number of ticks per sample) before and after the treatment was used for calculation of the achieved rate of reduction of tick population density at the prospected localities. For statistical analysis of population reduction, the percentage data was transformed into arcsin √%, and afterwards, for easier interpretation of the results, the mean values and standard deviation were converted into percentages. The analysis of variance was used to establish the statistical significance of differences between the mean numbers of ticks as a dependent variable, influenced by the day of testing and by the locality as independent variables. The significance of differences between the groups was determined using Duncan's multiple range test with a significance level α = 0.05. A comparison of the effects of the two applied products to tick population reduction, in terms of duration of treatment effects, was conducted by regression analysis of the mean values of population reduction for each treated locality in each evaluation term.

Results
The examination performed before the chemical treatment revealed the presence of ixodid ticks at all prospected localities. At the localities of Kaćka Forest in 2020, two tick species were identified: I. ricinus (nymph and adult stages) and Dermacentor marginatus (Sulzer 1776) in the adult stage. At localities of Fruška Gora National Park in 2021, three tick species were identified: nymph and adult stages of I. ricinus and adult stage of D. marginatus and Haemaphysalis punctata (Canestrini and Fanzago 1878). Ixodes ricinus was the dominant species at all investigated localities. Before treatment, all prospected localities included in the study were characterized by a high density of tick population expressed as the number of ticks sampled by flag/hour (Tables 1 and 2).
The presence of only one Borrelia species, Borrelia afzelii, was identified in I. ricinus ticks collected at the localities of Letenka, Testera, and Andrevlje, whereas two Borrelia species, Borrelia garinii and Borrelia lusitaniae, were identified in ticks collected from Kaćka Forest. In I. ricinus collected at the locality of Stražilovo, the presence  of Borrelia DNA was detected, yet without successful identification at the species level. In all localities intended for the treatment with Perme Plus®, both adults and nymphs were recorded with adults representing the dominant mobile stage. The mean number of ticks recorded in localities of Kaćka Forest on the pretreatment day was ranging between 25.0 ± 2.1 and 49.0 ± 1.9 ticks per flag hour (Table 1).
One day after the treatment with Perme Plus®, the mean number of sampled ticks was significantly lower at all treated localities (from 6.0 ± 1.4 to 9.0 ± 0.9) while in the untreated control locality, no significant change could be observed (48.0 ± 2.6). Duncan's multiple range test demonstrated that the number of ticks in the control locality was significantly higher than in all treated localities. With respect to the composition of developmental stages, both adult and nymphs were present at all treated localities except for Kaćka Forest 4, where only adults were recorded.
On the 7th post-treatment day with Perme Plus®, a significant reduction of the mean number of ticks compared to the previous evaluation term was recorded in the control and at the localities Kaćka Forest 1 and 2. At localities Kaćka Forest 3 and 4, the mean number of ticks also decreased, but not significantly. Regarding the developmental stage composition of samples, both adults and nymphs were recorded at all treated localities. The number of ticks at the control locality remained significantly higher (42.0 ± 1.4) compared to treated localities (from 4.0 ± 1.6 to 5.0 ± 1.4).
On the 14th post-treatment day, all sampled ticks were in the nymphal stage. Within the treated localities, the lowest number of ticks was recorded at Kaćka Forest 3 (1.0 ± 0.9), and the highest at Kaćka Forest 1 (12.0 ± 2.1). A significant increase in population density was demonstrated in the control locality but also in Kaćka Forest 1 and 4. At the same time, the number of ticks in the remaining two treated localities declined significantly further.
Regardless of the increasing trend of the tick number recorded at all treated localities on the 21st post-treatment day, the obtained mean values (4.0 ± 1.3 to 18.0 ± 3.4) remained significantly lower than in the untreated control locality (33.0 ± 2.0). The qualitative and quantitative stage composition of collected ticks again revealed that the population consisted exclusively of ticks in the nymphal stage.
In the second group of localities (Fruška Gora) where the efficacy of Icon® 10CS was tested, the mean number of ticks on the pre-treatmant day was ranging between 21.0 ± 1.3 and 31.0 ± 0.9 ticks per flag hour (Table 2). Collected samples of ticks consisted of adult and nymphal stages at all localities.
One day after the treatment with Icon® 10CS, the mean number of sampled ticks at all treated localities was significantly reduced (from 4.3 ± 1.0 to 6.0 ± 0.6). Although the mean number of ticks at the control locality (27.0 ± 2.4) was lower in comparison with the pre-treatment day, it was still significantly higher than in all localities treated with Icon® 10CS. The adult stage was present at all treated localities, while only at the locality Letenka the nymphal stage could be recorded.
Seven days after treatment with Icon® CS, the mean number of ticks continued to decrease at all treated localities. The recorded mean number of ticks at the treated localities was between 2.0 ± 0.6 and 4.0 ± 1.4, and in all cases, it was significantly lower than in the control locality (25.0 ± 2.0). The adult stage was present in samples, while the nymphal stage was only present in the samples from the one treated locality (Andrevlje).
On the 14th evaluation day, the mean number of ticks at all treated localities with Icon® CS10 was still significantly lower (from 1.0 ± 0.6 to 3.0 ± 0.9) than in the control locality (22.5 ± 1.9). Compared to the 7th post-treatment day, the population density at all treated localities did not significantly change. At this evaluation term, the stage composition changed. The nymphal stage was present in all treated localities, while the adult stage was recorded in only one treated locality (Andrevlje) and in the control site.
On the 21st post-treatment day, the mean number of ticks at all localities did not significantly change when compared to the previous evaluation term. Additionally, between the treated localities, no significant differences could be observed and all showed a significantly lower number of ticks than the control. The nymphal stage was present in all of the treated localities, while only at the Stražilovo locality adults and nymphs could be recorded.
Treatment effects of applied biocide Perme Plus®, expressed by the reduction of the number of ticks during the post-treatment evaluation period, revealed highly significant differences at each treated locality of Kaćka Forest (Table 3).
Post-treatment results obtained 24 h after the treatment indicated that the tick population reduction at all four treated localities of Kaćka Forest was within the interval of satisfying effects (70 to 90%). The highest reduction of tick population was recorded at the locality Kaćka forest 3 (80.9%), and according to Duncan's test, here, the reduction was significantly different from the values obtained at all other treated localities of Kaćka Forest.
On the 7th post-treatment day, the reduction of the tick population was still satisfying at all localities (from 76.6 to 85.8%). The highest reduction of tick population was recorded again at the locality Kaćka Forest 3. Compared to the first post-treatment day, the reduction rate increased in all localities, but the increase was significant only at Kaćka Forest 1.
On the 14th post-treatment day, the reduction rate in localities Kaćka Forest 2 and 3 significantly increased, while at Kaćka Forest 1 and 4, the reduction showed a significant decreasing tendency. The highest reduction rate, classified as good (> 90%), was recorded at Kaćka Forest 3 (97.8%), followed by Kaćka Forest 2 with satisfying reduction (89.2%), while at Kaćka Forest 1 and 4, the reduction was weak (< 70%).
On the 21st post-treatment day, the reduction rate of tick population density significantly decreased in all treated localities of Kaćka Forest. However, satisfying effects were still recorded at localities Kaćka Forest 2 and 3 (79.5% and 72.2%, respectively), while at localities Kaćka Forest 1 and 4, the efficacy substantially decreased (25.9% and 13.6%, respectively).
Based on the presented results in Table 3, it can be observed that at two treated localities (Kaćka Forest 2 and 3), the treatment efficacy expressed as reduction rate had the increasing trend until the 14th post-treatment day and afterwards, the efficacy significantly decreased, although the reduction at the 21st day was still satisfying. In the other two localities, the satisfying treatment effects of Perme Plus® were observed until the 7th post-treatment day, and afterwards, the reduction significantly decreased to values classified as weak.
Post-treatment results for the applied biocide Icon® 10CS (Table 4) recorded at the first assessment (24 h after the treatment) demonstrated that the efficacy among the four treated localities was not significantly different. The effects at all localities were classified as satisfying, ranging between 74.3 and 76.4%.
On the 7th post-treatment day, the increasing trend of reduction of tick population was recorded at all localities. Despite significant differences between the localities, the reduction rate at all localities was classified as satisfying (from 78.2 to 88.6%). Fourteen days after the treatment, the highest efficacy, classified as good, was recorded at the locality Testera (94.7%), while in other localities, the treatment effects were lower but still satisfying (from 81.5 to 84.7%).
On the final, 21st post-treatment evaluation day, the highest reduction rate, classified as good, was recorded at Andrevlje (91.3%), while the reduction rate at other treated localities was lower but still satisfying (79.8 to 86.2%). Compared to the results obtained on the 14th day, the increase in the population reduction was only recorded at Andrevlje, but Duncan's test revealed that the increase was not significant.
In contrast to the efficacy results for Perme Plus®, Icon® 10CS provided a satisfying efficacy rate during the entire evaluation period of 21 days.
Comparison of the efficacy trends of Perme Plus® and Icon® 10CS (Fig. 1) demonstrated similar initial effects until the 7th post-treatment day, when the reduction rate of treated tick populations for both applied products achieved the values which, transformed to percentages was about 84%. In the following post-treatment period, the reduction of population density treated with Icon® 10CS continued to increase until day 14.5 when the maximal reduction of 87.1% was achieved. Later on, the population reduction rate gradually decreased. According to the resulting trend formula for Icon® 10CS, the expected efficacy 1 month after the treatment (on the 30th day) was still satisfying (70.7%). The trend line for Perme Plus® demonstrated that the maximal reduction rate of the population density was achieved on day 8.5 with the corresponding value of 84.4%. In the following period, the decrease of the treatment efficacy demonstrated by the trend line for Perme Plus® was intensive and more expressed than in the case of Icon® 10CS. According to the resulting trend formula for Perme Plus®, the population reduction rate on the 17th post-treatment day declined to the corresponding value of 68.6%; thus, the treatment effects starting from this post-treatment day were weak. Finally, on the 30th day, any post-treatment effects of Perme Plus® could be expected (reduction equal to 0).

Discussion
The increased number of reported cases of tick-borne diseases is closely related to altered behavior patterns and the zoogeographical prevalence of some tick species during the past few years (Paddock et al. 2018). In this study, three tick species were identified, with I. ricinus being the predominant species at all localities.
The present study confirmed the infection of I. ricinus ticks with three Borellia species: B. afzelii, B. garinii, and B. lusitaniae. In ticks collected at the study area of Kaćka Forest, two species B. garinii and B. lusitaniae were identified, while B. afzelii was present in the study area of Fruška Gora. According to previous studies (Savić et al. 2010;Potkonjak et al. 2014Potkonjak et al. , 2016, the same species of Borrelia were identified in the geographical region of Vojvodina Province, which is considered an endemic area. Savić et al. (2010) used a species-specific PCR to confirm the presence of B. afzelii and B. burgdorferi s.s. in I. ricinus ticks. Subsequently, Potkonjak et al. (2014) identified B. afzelii as the predominant Borrelia species by applying the MluI-LRFP and realtime PCR assays that target the hbb gene. Moreover, using molecular diagnostics, Potkonjak et al. (2016) confirmed the presence of five Borrelia species (B. luisitaniae, B. afzelii, B. valaisiana, B. garinii, and B. miyamotoi) in I. ricinus ticks collected from natural habitats of Vojvodina.
The majority of recent studies worldwide analyze the change in regional and seasonal patterns of abundance of individual tick species and the prevalence of specific Borrelia species in I. ricinus ticks (Oechslin et al. 2017). In conditions of temperate climate in Vojvodina, the highest tick population density and prevalence of Borellia infection are observed in early summer (Jurišić et al. 2010). The occurrence of ticks throughout the year is determined by highly complex diapause mechanisms (Milutinović et al. 2002). At any stage of the tick's life cycle, the diapause is influenced either by the development of engorged ticks (developmental diapause) or by the host-seeking activity of unfed ticks (behavioral diapause). Belozerov et al. (2002) reported that developmental diapause in nymphs, manifested as an arrest of the metamorphosis of engorged nymphs, is common and regulated by photoperiodity. Understanding seasonal adaptations is of great importance in predicting the dynamics of ticks and the implementation of effective control measures. The assessment of the population density of ticks depends on the sampling methodology applied and does not provide information about the presence of all tick species in a studied locality (Barandika et al. 2006). The presence and activity of ticks at particular localities are closely associated with soil humidity, temperature, precipitation, altitude, and vegetation structure. Ixodes ricinus is able to modify its seasonal activity depending on the microclimatic conditions of the habitat (Daniel et al. 2015). Liebisch et al. (1996) reported that habitats of the tick I. ricinus can be found up to 1500 m altitude. Gray (1991) stated that I. ricinus ticks prefer forest habitats since they provide a wider range of hosts and more suitable microclimatic conditions compared to field and urban environments. Our research confirmed a higher abundance of tick populations, including I. ricinus, in forest habitats (Kaćka Forest) compared to shrub/meadow vegetation (Fruška Gora). The lesser number of ticks collected at recreation sites of Fruška Gora may be attributed to anthropogenic impact.
In Serbia, chemical treatment of ticks is commonly conducted before May 1st (International Working Day, traditionally celebrated at a picnic and recreational sites) in order to reduce the risk of contacts between ticks and humans. Criteria on which the decision of acaricide treatment is based include the abundance of I. ricinus ticks and the infection rate of the pathogens that cause Lyme disease.
The abundance of ticks, as basic information, can be considered from two perspectives: (1) The number of active ticks is important for risk assessment for the human population because only active ticks represent a potential risk to humans; (2) the total abundance of tick population is important information for the evaluation of the applied acaricides efficacy. Juvenile tick stages, especially nymphs, may interrupt the period of inactivity (diapause or resting) during the period of testing the efficacy of pyrethroids, particularly during the period of residual activity. Indeed, our study demonstrated the increasing number of ticks in nymphal stages in some treated localities on the 14th and 21st post-treatment days.
Our research on acaricide efficacy was conducted in the period from the end of April to the second half of May 2020 and 2021. This part of the season in Serbia is mainly characterized by the variation of precipitation amounts and temperature oscillations. Variable environmental conditions influence the formation of specific patterns of daily and periodical behavioral dynamics of ticks in their habitat, which is manifested by the frequent switch between dormancy and activity periods. Therefore, especially in the period April-May characterized by unfavorable weather conditions for ticks, a high percentage of nymphs are usually found in developmental or behavioral diapause. This may result in the false assumption of lower tick activity or lower population density in the period of sampling, as well as a wrong assessment of acaricide efficacy. We assume that this phenomenon played a role at the localities of Kaćka Forest 1 and 4, where the lowest efficacy of the combined pyrethroids permethrin, tetramethrin, and piperonyl butoxide was recorded.
The efficacy of the majority of pyrethroids such as flumethrin, permethrin, lambda-cyhalothrin, and deltamethrin was frequently tested against a range of insect species, while the assessment of their performance against ticks has been poorly studied (Liebisch and Liebisch 2008;Mehlhorn et al. 2010). Commonly, acaricides applied for the control of mobile tick stages in their natural environment and on the host animals include organophosphate insecticides and pyrethroids. According to EU regulations, tick control in their natural environment is limited to ground treatment techniques, exclusively.
Research on the efficacy of different acaricidal active ingredients in the prevention and control of tick infestation of hosts has been mainly focused on the treatment of the animals (El-Bahy et al. 2015;Hagimori et al. 2005;Spencer et al. 2003;Kužner et al. 2013;Krämer et al. 2020;Stanneck and Fourie 2013;Dantas-Torres et al. 2013;Mehlhorn et al. 2011;Young et al. 2003).
The control of ticks in natural and urban environments with a high risk of human infestation and tick-borne disease transmission is required to improve public health (Talbert et al. 1998). For this purpose, mainly the pyrethroids are used. In our study, the efficacy of lambda-cyhalothrin (Icon® 10CS), evaluated during a period of 3 weeks, demonstrated that a population reduction could be achieved that ranged from 74.3 to 94.7%, depending on the locality and the evaluation time. In the same evaluation period, the results for Perme Plus® showed a weak population reduction after 2 weeks in two localities (< 70%), while in the remaining localities, the efficacy was satisfying or good (from 71.8 to 97.8%) during the entire evaluation period.
Synthetic pyrethroids, such as lambda-cyhalothrin, demonstrated an efficacy of up to 100% (Curran et al. 1993;Jurišić et al. 2010). Cetin et al. (2009) tested spinosad, deltamethrin, permethrin + esbiothrin, chlorpyrifos-methyl, and a mixture of alpha-cypermethrin/tetramethrin/piperonyl butoxide in laboratory conditions, treating larvae of Rhipicephalus turanicus Pomerantsev 1936 and Argas persicus Oken 1818. They reported that a mixture of alpha-cypermethrin/tetramethrin with piperonyl butoxide demonstrated the fastest action against larvae of A. persicus resulting in a mortality rate of 100% after 15 min post-exposure. A field trial conducted by Brites-Neto et al. (2017) in a riparian forest habitat demonstrated that the application of a product based on alpha-cypermethrin (3%) and flufenoxuron (3%) was effective in reducing the environmental infestation of adult and immature stages of Amblyomma ticks (43 days after treatment a population reduction of 87% was observed), and the residual effect was presented for 2 months. The study of Curran et al. (1993) who evaluated the acaricidal efficacy of cyfluthrin applied on the lawn, ornamental plants, and surrounding vegetation recorded a high average efficacy of 92.2% reduction. The same authors recorded a population reduction of 95.4% after 15 days, 87.5% after 31 days, and 93.1% after 41 days. Solberg et al. (1992) carried out the evaluation of different formulations of cyfluthrin in an oak-dominated forest and demonstrated a high reduction of treated tick population with 96% after 10 days, and later increase to 100%, lasting for 2 months.
In our study, the evaluation of acaricidal effects was conducted in a shorter period of 3 weeks after treatment. However, analyses of the results demonstrated higher and more consistent efficacy of lambda-cyhalothrin compared to permethrin + tetramethrin. During the entire period, Icon® 10CS provided satisfying/good results of population reduction in all treated localities. Perme Plus® treatment also provided satisfying/good results over the entire evaluation period in two localities, while in the remaining two localities, the satisfying efficacy was only observed till the 7th post-treatment day but not later. The low population reduction in these two localities on the 14th and 21st post-treatment days may be explained by the occurrence of a high number of activated nymphs. Anyhow, the estimated population reduction trend demonstrated that satisfying treatment efficacy could be projected for the period until the 17th post-treatment day for Perme Plus® and until the 30th post-treatment day for Icon® 10CS.

Conclusion
The application of acaricides based on a combination of pyrethroids permethrin/tetramethrin with synergist piperonyl butoxide (Perme Plus®) and the formulation based on lambda-cyhalothrin (Icon® 10CS) provided satisfying acaricidal efficacy in natural habitats of three tick species (I. ricinus, D. marginatus, and H. punctata). The dominant tick species was I. ricinus, and Borellia infection of this tick species was detected in all localities where the study was conducted. Icon® 10CS and Perme Plus® manifested residual activity and satisfying acaricidal performance.
Although both Perme Plus and Icon® 10CS could be considered a good choice for the treatment of hard ticks in natural habitats, Icon® 10CS seems to be preferable since it provided more consistent residual effects and a higher efficacy rate.
According to the One Health approach to protect humans, animals, and the environment, it is necessary to implement comprehensive and effective strategies to reduce tick population density, especially of those tick species which are confirmed vectors of pathogens. Principally, adequate long-term continual surveillance strategies are required in order to identify tick species composition, abundance of vector species, and prevalence of tick-borne diseases in a targeted area. Therefore, the control of ticks should be focused on areas with a high risk of tick-borne disease transmission. The selection of the acaricide should be based on appropriate active ingredients and formulations ensuring good residual efficacy and a low hazardous risk to untargeted organisms and the environment.