Control of Varroa destructor Mite Infestations at Experimental Apiaries Situated in Croatia

: Experimental varroacidal treatments of honey bee colonies were conducted on ﬁve apiaries (EA1–EA5) situated at ﬁve di ﬀ erent geographical and climatic locations across Croatia. The aim of this study was to assess the comparative e ﬃ cacy of CheckMite + (Bayer, Germany), Apiguard (Vita Europe Ltd.; England), Bayvarol, C, (Bayer, Germany), Thymovar, (Andrma BioVet GmbH, Germany), and ApiLife Var, (Chemicals Laif SPA; Vigonza, Italy) for controlling the honey bee obligatory parasitic mite Varroa destructor in di ﬀ erent conditions in the ﬁeld during summer treatment. The relative varroa mite mortality after treatments with applied veterinary medicinal products were EA1 (59.24%), EA2 (47.31%), EA3 (36.75%), EA4 (48.33%), and EA5 (16.78%). Comparing the relative e ﬃ cacy of applied varroacides, the best e ﬀ ect was achieved with CheckMite + , and the lowest for honey bee colonies treated with Apiguard (statistically signiﬁcant di ﬀ erence was conﬁrmed; p < 0.05). Considering the lower e ﬃ cacy of thymol-based veterinary medicinal products observed on all EA in these study conditions, it may be concluded that their use is limited under di ﬀ erent treatment regimes. Despite unfavourable weather and environmental conditions, with exceptions of EA5 / EA5 (cid:48) and EA1, the relative varroacidal e ﬃ cacy of authorized veterinary medicinal product treatments in moderately infested honey bee colonies ensured normal overwintering and colony development during next spring.


Introduction
Honey bee colonies are valuable ecological and economical insects. They are fundamental for the food production and ecosystem biodiversity through their important role in pollination of cultivated crops and wild plants [1,2]. Different adverse environmental conditions, including pathogens and pests, have been implicated in the dissipated health of honey bee colonies or even their losses [3]. Although causes of extensive honey bee colony losses appear to be multifactorial [4], the obligate ectoparasitic mite Varroa destructor of honey bees is a major contributing factor [5]. As the V. destructor mite feeds on the haemolymph and fat body of adults [6] and developing stages of honey bees, and additionally facilitates the transmission of certain viruses [7][8][9], this seems to have a significant negative effect on the host immune response [10]. Because the aforementioned possible consequences of mite infestation consist in damages on the individual and colony level, the V. destructor mite population in honey bee colonies requires regular control during the whole year.
Moreover, honey bee colonies are highly influenced by beekeepers' management practices, socioeconomic conditions, and the level of implementation of policies supporting beekeeping activities [11] in the particular country. In view of the spread of varroosis across Europe and the problems which this disease has brought in the beekeeping sector, the European Union (EU) has been encouraged to set up national programs aimed at improving the general conditions for the sustainable beekeeping production and management in its ecological, economic, and social dimensions [12].
In Croatia, as a new member of the EU, such apicultural programs have existed for several years. These national beekeeping programs (2011-2013, before accessing EU; 2014-2016; 2017-2019) employed a high participation rate of beekeepers [13] but failed to a certain extent in terms of the efficacy of the varroosis control by using authorized veterinary medicinal products (VMP). This is followed by the beekeepers' explanation that there are very few acaricide active substances on the market [14]. This is evident from the fact that in the past, beekeepers have not made any kind of notes or records of evidence regarding the use of varroacides as active ingredients and its efficacy on V. destructor mites. Over a few consecutive years, there has been a visible decreasing trend in procuration of authorized VMPs for usage in beekeeping [14], despite their total number, as well as the number of active substances, increasing by more than double [15].
According to the Varroa Control Program, which is a part of the Croatian National Regulation on animal protection measures against infectious and parasitic diseases and related financing from 2011 till today (with annual minor modifications) [16], beekeepers nationwide are required to implement one obligatory treatment of honey bee colonies against V. destructor mites using an authorized VMP in combination with other measures of integrated pest management implemented in an appropriate time of year [17]. Application of VMPs is recommended in the period from 1 July to 31 August, after main honey harvesting, and with the schedule dependent on the geographical, climatic and honey bee forage conditions in different regions. The main model is also to conduct treatments on all colonies and apiaries in the same area and during the same period. In this way, reinfestation and horizontal mite transfer between apiaries during robbing behavior [18,19] can be avoided.
Experimental apiaries (EA) in Croatia were established in 2013 with the purpose of conducting different research studies with a possible comparison of results linked to different environmental impacts.
There are numerous active acaricidal substances available incorporated into different formulations of medicines, different methods of application, and techniques for V. destructor mite population control. A variety of synthetic varroacides have been widely used in the last few decades with variable effects, due to geographical regions or often in response to bad beekeeping practices, such as multiple consecutive and repeated use of the same acaricide, sub-or overdosage, too short or prolonged treatment duration, improper time and way of acaricidal product application, too few active substances in the same time treatment, etc., which has led to increased tolerance to most of them. V. destructor mites have developed resistance to the most widely used synthetic varroacides [20][21][22]. In order to avoid the accumulation of chemical residues in honey and beeswax, beekeepers are very interested in treatments of natural, ecofriendly, so-called soft acaricides, which are often inconsistent, more variable, or effectiveness or therapeutically limited under different climatic conditions [23][24][25][26].
The aim of this study was to evaluate and compare the varroacidal efficacy and mite mortality dynamic during summer treatment of honey bee colonies situated at EA influenced by different environmental conditions, treated with five different available authorized VMPs (CheckMite+, Apiquard, Bayvarol, Thymovar, and ApiLife Var) used simultaneously and compared with negative control (untreated group of honey bee colonies). The percentages of V. destructor mite mortality by the experimental treatments were estimated according to recommendations of the European Medicines Agency (EMA) [27]. Efficacy of treatments was also compared between EA. Additionally, the commonly used oxalic acid was used for follow-up winter treatment in broodless honey bee colonies to establish the final parasitic mite drop.
Each EA consisted of 30 honey bee colonies (Apis mellifera carnica Pollmann, 1879) placed in standard Langsthrot Root (LR) hives. Experimental colonies were queen right, had combs occupied with adult honey bees, and were fully developed and productive. Prior to the experimental period, all experimental honey bee colonies were uniformed in respect to brood size, the comb area covered with adult bees, and amount of stored food [28]. Honey bee queens were one year old. Experimental colonies were also visually inspected for the presence of pathology signs on adult bees and brood. Adult bees showed normal behavior, and there were no visible signs of infectious diseases on brood. No acaricidial treatment of the honey bee colonies was done prior to the start of experimental treatments. After inspection, the honeybee colonies situated on EA1-EA5 were divided into six experimental groups (A, B, C, D, E, and O), and each group consisted of five beehives (Table 1). All beehives were equipped with varroa mite screen boards for monitoring mite fall counts. In early spring, metal sheets were placed on the bottom board of each bee hive in order to record the natural mite mortality prior to treatments, and later in the season, the mite drop after the experimental treatments. Above the sheets, wire screens were installed to prevent contact of the adult bees with debris and to prevent ants from removing dropped V. destructor mites. Experimental and control colonies were monitored for mites mortality in prior, during, and after treatments performed in brood and broodless (winter) periods. Mite counts were carried out every day during summer treatment (A, C, D, and O groups-42 reads; B and E groups-28 reads were recorded for each Each EA consisted of 30 honey bee colonies (Apis mellifera carnica Pollmann, 1879) placed in standard Langsthrot Root (LR) hives. Experimental colonies were queen right, had combs occupied with adult honey bees, and were fully developed and productive. Prior to the experimental period, all experimental honey bee colonies were uniformed in respect to brood size, the comb area covered with adult bees, and amount of stored food [28]. Honey bee queens were one year old. Experimental colonies were also visually inspected for the presence of pathology signs on adult bees and brood. Adult bees showed normal behavior, and there were no visible signs of infectious diseases on brood. No acaricidial treatment of the honey bee colonies was done prior to the start of experimental treatments. After inspection, the honeybee colonies situated on EA1-EA5 were divided into six experimental groups (A, B, C, D, E, and O), and each group consisted of five beehives (Table 1).
All beehives were equipped with varroa mite screen boards for monitoring mite fall counts. In early spring, metal sheets were placed on the bottom board of each bee hive in order to record the natural mite mortality prior to treatments, and later in the season, the mite drop after the experimental treatments. Above the sheets, wire screens were installed to prevent contact of the adult bees with debris and to prevent ants from removing dropped V. destructor mites. Experimental and control colonies were monitored for mites mortality in prior, during, and after treatments performed in brood and broodless (winter) periods. Mite counts were carried out every day during summer treatment (A, C, D, and O groups-42 reads; B and E groups-28 reads were recorded for each colony), and seven days after winter treatment. At EA5 , the emergency autumn treatment was performed on 16 September by the commonly used amitraz on a one-time basis.

Drugs and Treatments
Treatments were conducted during the summer season immediately after the main honey harvesting. The authorized VMPs were used in the recommended doses. CheckMite+ (Bayer, Leverkusen, Germany), based on coumaphos as the active ingredient, was applied in the form of two beehive pest control strips inserted between frames, with waxcombs sealed with honeybee brood in the brood chamber for a 42 day period. Apiguard (Vita Europe Ltd.; Basingstoke, England) is an authorized thymol-based acaricide packed in an aluminum tray, so its coated sheets were placed on the top bars of the bee hive frames of each brood chamber, one tray during two weeks and the second one during the consecutive two weeks. The treatment with flumethrin in an authorized VMP-Bayvarol (Bayer)-was applied as four pest control strips were inserted between frames with sealed honey bee brood in the brood hive chamber for a 42 day period. Thymovar (Andermatt BioVet GmbH, Lörrach, Germany), formulated on cellulose wafer, contains 15 g of thymol and was used as one piece cut in two parts, which were separately placed on the top bars of frames on a two-time-basis for 42 days in total. ApiLife Var (Chemicals Laif SPA; Vigonza, Italy) is based on a few active ingredients (thymol, eucalyptus oil, levomenthol, and camphor) imbibed in vermiculite tablets. One tablet per bee hive was applied every eight days with four applications in total. Every portion of the tablet was placed in a corner of the brood hive chamber and remained in the honey bee colonies to be chewed and removed by the adult bees.
The oxalic acid solution was prepared using 1 L of sugar syrup to dissolve 35 g of oxalic acid dehydrate (Kemika, Zagreb, Croatia). The sugar syrup was prepared by mixing hot freshwater (70-80 • C) with commercial sugar (Viro, Virovitica, Croatia) (1:1). Prepared oxalic acid solution was administered to the honey bee colonies cold with a syringe trickling 5 mL for each intercomb space occupied by adult bees, from the top. The number of phoretic mites fallen after the winter treatment was counted by using metal label sheets on the bottom boards, checked every day for one week.
For autumn treatment, Varidol (TolnAgro Kft., Szekszárd, Hungary) was used once, in honeybee colonies at location EA5 , by fumigation according to instructions for use.

Estimating the Strength of Honeybee Colonies
To estimate the strength of honey bee colonies, a Liebefeld method was performed, with visual determination of number of adult honey bees and brood sealed in beeswax combs [28]. The estimation Diversity 2020, 12, 12 5 of 14 of honey bee colonies was conducted three times (prior-I, during-II, and after experimental summer treatments-III), during morning hours, between 9:00 and 10:00 a.m. before the first massive forage flights of honey bees. Strength of honey bee colonies was estimated as follows: EA1-19 May, 25 July, and 16 September; EA2-29 April, 17 July, and 8 September; EA3-14 May, 16 July, and 7 September; EA4-20 May, 10 July, and 2 September; and EA5-28 April, 15 July, and 10 September. Owing to easier assessment with adult honey bees or brood-covered comb areas, the frame for the LR hive was used and prior divided with a plastic grid into 1 dm 2 quadrants.

Colony Examinations, Mite Counts and Treatments Efficacy
Clinical examination of honeybee colonies included visual inspection of honeybee brood and adult bees, the behaviour of adult bees, as well as activity of bees at the entrances of hives. Fallen V. destructor mites were counted during the pretreatment, treatment, and a particular number of days after each treatment, and the sum of those results calculated after the final treatment was considered the total mite drop. The proportion of mites falling after each treatment to the total number of fallen mites was estimated in percentages (%). The efficiency of each experimental treatment (A, B, C, D, E) was estimated according to the recommendations of the EMA [27].

Data Analysis
The data analyses were performed by one-way analysis of variance (ANOVA) using statistical software package Statistica-StatSoft v.7. (StatSoft, Inc, Tulsa, OK, U.S.A.). The results were presented as the mean values, standard deviations, and standard errors. To assess the statistical differences in honeybee colonies' strength (estimated number of honeybees per colony), they were compared between groups and three estimation dates (April/May, July, and September), and the number of fallen V. destructor mites between experimental groups (A, B, C, D, E) and control group (O) at different locations and estimation dates, the Kruskal-Wallis test was performed (α = 0.05). Multisample comparison using Kruskal-Wallis H test was carried out to compare the mean values of the number of fallen V. destructor mites between experimental and control groups in the pretreatment, treatment, and after-treatment period.

Meteorological Conditions
The average values of air temperatures, relative air moisture, and number of days with rain for each location of EA during the observed period of beekeeping season 2014 is shown in Table 2. Obviously, these harms encountered in beekeeping may indicate that this acaricidal therapy of honey bee colonies with authorised VMPs had the obtained effect instead of especially detrimental environmental circumstances.

Estimating the Strength of Honeybee Colonies
Despite the equalization of honeybee colonies before pretreatment period with respect to colony strength, some statistical differences were determined between experimantal and control groups of honey bee colonies at different estimation days (I, II, or III) at EA1, EA2, and EA5, as follows: EA1: I (p < 0.001; F = 9.87), II (p < 0.05; F = 3.32), III (p < 0.01; F = 3.94); E2: III (p < 0.001; F = 9.87); and E5: III (p < 0.05; F = 3.55). Variations in the average number of honey bees per group during three estimation terms are shown in Figure 2.

Treatment Period
During the summer treatment period, the estimated total V. destructor mite mortality that resulted was significantly higher than natural mite drop. Results of summer and winter treatment (to determine the residual amount of mites) of honey bee colonies with VMPs are shown in Figure 4. Because of very low efficacy of summer treatments in all groups of experimental colonies situated on EA5 (Figure 5), the necessary follow-up autumn treatment was done at EA5′ (Figure 5), and calculations of treatments efficacy was based on those mite drops (A-47.52%; B-3.11%; C-25%; D -5.18%; E-3.11%). Results of this EA were analyzed separately from those of other EAs.
The mean values of V. destructor mortality on EA1 did not differ significantly between the experimental groups (A, B, C, D, E), but it was different in comparison with the control group (p < 0.05; h = 14.2). Summer treatment results were significantly different from those of winter treatment (p < 0.05; F = 2.74). Efficacy of varoacidal treatment is presented in Table 3, and it was decreased as follows: A > E > D > C > B. Prior to winter treatment, nine of the treated honey bee colonies were dead: 3 colonies from B group, 3 colonies from E group, 2 colonies from D group and 1 colony from C group.
At the location of EA2, each treatment (A, B, C, D, E) induced a significantly higher (p < 0.05; h = 12.9) V. destructor mite mortality in the parallel untreated control colonies (O). The summer treatments and winter treatments (p < 0.005; F = 2.83) were also statistically significantly different. Efficacy of different VMP treatments (Table 3) decreased as follows: A > D > E > B > C. All honey bee colonies survived.
The mortality rates in the treated colonies of EA3 apiary were significantly different from those in the control honey bee colonies (p < 0.05; h = 12.77), and between the summer and winter treatments

Treatment Period
During the summer treatment period, the estimated total V. destructor mite mortality that resulted was significantly higher than natural mite drop. Results of summer and winter treatment (to determine the residual amount of mites) of honey bee colonies with VMPs are shown in Figure 4. Because of very low efficacy of summer treatments in all groups of experimental colonies situated on EA5 (Figure 5), the necessary follow-up autumn treatment was done at EA5 (Figure 5), and calculations of treatments efficacy was based on those mite drops (A-47.52%; B-3.11%; C-25%; D -5.18%; E-3.11%). Results of this EA were analyzed separately from those of other EAs.
Total V. destructor mite drop after the treatments were significantly higher (p < 0.005; h = 16.6) than number of dead mites counted on the bottom boards hive inserts of untreated control honey bee colonies situated at EA4. Also, the differences between experimental groups were determinated (p < 0.01; F = 5.95), during the summer treatments. Order of VMPs treatment efficacy decreased as follows: A, C, E, D, and B.    The mean values of V. destructor mortality on EA1 did not differ significantly between the experimental groups (A, B, C, D, E), but it was different in comparison with the control group (p < 0.05; h = 14.2). Summer treatment results were significantly different from those of winter treatment (p < 0.05; F = 2.74). Efficacy of varoacidal treatment is presented in Table 3, and it was decreased as follows: A > E > D > C > B. Prior to winter treatment, nine of the treated honey bee colonies were dead: 3 colonies from B group, 3 colonies from E group, 2 colonies from D group and 1 colony from C group. At the location of EA2, each treatment (A, B, C, D, E) induced a significantly higher (p < 0.05; h = 12.9) V. destructor mite mortality in the parallel untreated control colonies (O). The summer treatments and winter treatments (p < 0.005; F = 2.83) were also statistically significantly different. Efficacy of different VMP treatments (Table 3) decreased as follows: A > D > E > B > C. All honey bee colonies survived.
The mortality rates in the treated colonies of EA3 apiary were significantly different from those in the control honey bee colonies (p < 0.05; h = 12.77), and between the summer and winter treatments (p < 0.001; F = 5.79). Efficacy of acaricidal treatments (Table 3) decreased in the same order as on the EA1.
Total V. destructor mite drop after the treatments were significantly higher (p < 0.005; h = 16.6) than number of dead mites counted on the bottom boards hive inserts of untreated control honey bee colonies situated at EA4. Also, the differences between experimental groups were determinated (p < 0.01; F = 5.95), during the summer treatments. Order of VMPs treatment efficacy decreased as follows: A, C, E, D, and B.
Varroacidal efficacy of applied VMPs was significantly different for treatments of experimental honey bee colonies from groups A and B (Figure 6a), at each EA (p < 0.001, F = 6.933). As a consequence of nontreated honey bee colonies during the summer period, all control groups were also significatly lower regarding percentages of varroacidal efficacy. Overall varroacidal efficacy at individual EA did not differ significantly between EA1, EA2, EA3, and EA4. These results are presented in Figure 6b.

Discussion
Varroa mites, acting simultaneously with other stressors, are known to be a main cause of honey bee colony weakening and collapsing [5,29]. The V. destructor mite population requires regular control, correctly and in a timely manner, of honey bee colony management. Five different VMPs, only authorized in Croatia in 2014, were tested at five apiaries to test their performance in different field conditions because control of varroosis by using VMPs is widely acknowledged to be an essential part of beehive management [30].
The 2014 active beekeeping season was generally very weak in different aspects. There were more days with rain, very low average year air temperatures, along with a mild winter, and consequently, a very early honey bee brood development was noticed, even in January. As one replication cycle of V. destructor mites is about 13 days, multiple replications of mites can be expected to have occurred [17]. Weather circumstances were also very favorable for a fast-increasing growth trend of V. destructor mite populations in honey bee colonies with early reared brood [31]. Additionally, lack of natural food in the environment effected a decreasing of immunological status of the honey bee colonies and induced stress caused by hunger. All these detrimental impacts of environmental factors, high V. destructor mite infestations, and weak main pastures ended very early, so in most regions, the first summer varroacidal treatment was essential.
A different number of V. destructor mite drops was detected during the pretreatment period, which was probably a consequence of a different level of parasitic colony invasions at different EAs. Meeting the demand of no varroacidal treatments that year, before the start of the described study means that they were treated the last time in the winter preceding the experimental period. This may have implicated a situation where establishing new summer generations in all honey bee colonies coincided with a high level of V. destructor mite infestation, which may have damaged colonies in each experimental group. Similar findings were published before [32]. Because of extremely low amounts of stored honey in hives, at EA2 and EA5, the honey extraction was not done for the whole year.
Treatments against varroa mites started at different dates for each EA because the blooming of plants that make up the main bee pastures finished at different times. The best relative varroacidal efficacy was seemingly achieved at EA1 (59.24%), but if an analysis of the survival of treated colonies is included, then it is clear that 30% of them collapsed before the next active beekeeping season. Most of the lost honey bee colonies were treated with VMPs based on thymol as active ingredients. Here it must be stressed that first summer treatments on EA1 started only towards the end of July, which is relatively late but still in accordance with advised varroosis control schedules in Croatia.

Discussion
Varroa mites, acting simultaneously with other stressors, are known to be a main cause of honey bee colony weakening and collapsing [5,29]. The V. destructor mite population requires regular control, correctly and in a timely manner, of honey bee colony management. Five different VMPs, only authorized in Croatia in 2014, were tested at five apiaries to test their performance in different field conditions because control of varroosis by using VMPs is widely acknowledged to be an essential part of beehive management [30].
The 2014 active beekeeping season was generally very weak in different aspects. There were more days with rain, very low average year air temperatures, along with a mild winter, and consequently, a very early honey bee brood development was noticed, even in January. As one replication cycle of V. destructor mites is about 13 days, multiple replications of mites can be expected to have occurred [17]. Weather circumstances were also very favorable for a fast-increasing growth trend of V. destructor mite populations in honey bee colonies with early reared brood [31]. Additionally, lack of natural food in the environment effected a decreasing of immunological status of the honey bee colonies and induced stress caused by hunger. All these detrimental impacts of environmental factors, high V. destructor mite infestations, and weak main pastures ended very early, so in most regions, the first summer varroacidal treatment was essential.
A different number of V. destructor mite drops was detected during the pretreatment period, which was probably a consequence of a different level of parasitic colony invasions at different EAs. Meeting the demand of no varroacidal treatments that year, before the start of the described study means that they were treated the last time in the winter preceding the experimental period. This may have implicated a situation where establishing new summer generations in all honey bee colonies coincided with a high level of V. destructor mite infestation, which may have damaged colonies in each experimental group. Similar findings were published before [32]. Because of extremely low amounts of stored honey in hives, at EA2 and EA5, the honey extraction was not done for the whole year.
Treatments against varroa mites started at different dates for each EA because the blooming of plants that make up the main bee pastures finished at different times. The best relative varroacidal efficacy was seemingly achieved at EA1 (59.24%), but if an analysis of the survival of treated colonies is included, then it is clear that 30% of them collapsed before the next active beekeeping season. Most of the lost honey bee colonies were treated with VMPs based on thymol as active ingredients. Here it must be stressed that first summer treatments on EA1 started only towards the end of July, which is relatively late but still in accordance with advised varroosis control schedules in Croatia.
Comparing the relative efficacy of the used varroacides with different active ingredients (Figure 6a), the best effect was achieved for honey bee colonies from an A experimental group, and lowest for colonies from a B experimental group, where statistically significant difference was affirmed (p < 0.05; F = 6.93). Although the organic acaricides have certain advantages after repeated use, their efficacy may be inconsistent and more variable compared with synthetic acaricide formulations [24,[33][34][35][36], which is also confirmed with our results.
Changes in honey bee colonies' strength trends were different between apiary locations, but within the expected ranges under the study and environmental conditions, as well as beekeeping practices (EA1, EA2, EA3, and EA4).
At location EA5 at the beginning of active beekeeping season, honey bee colonies were very weak with low numbers of adult bees ( Figure 2). Then, in the middle of the active season, there was an opposite situation: a high number of adult bees without brood or very few comb cells were sealed with development stages of bees. At the same time there was non food in nature, and honey bees ate away almost all food storages in their hives. After emergency varroacidal treatment, which produced higher efficacy compared to earlier treatments because larger proportion of phoretic mites being on adult bees [37], and transport at island Vis (EA5 ), they came in with much better environmental circumstances with plenty of natural food. In the next few weeks, the strength of colonies increased quickly, but because of serious damages, bees got high varroa infestation and insufficient efficacy of VMPs treatment during the summer treatment (except A group-47.52%), most of the colonies from other treated groups died before the next spring (63.4%). The efficacy of the VMP treatments was under expectations, probably due to fast reproduction of the surviving mites, but also because of possible reinfestations during experiments [35].

Conclusions
Varroa mite infestations control using CheckMite+ (Bayer, Germany), Apiguard (Vita Europe Ltd.; England), Bayvarol (Bayer, Germany), Thymovar (Andrma BioVet GmbH, Germany), and ApiLife Var (Chemicals Laif SPA; Vigonza, Italy) at five different EAs in Croatia during the beekeeping season of 2014 induced a higher mite mortality compared to control, as well as in comparison with mite drop in the pretreatment period. Despite unfavourable weather and environmental conditions, with the exceptions of EA5/EA5 and EA1, the relative varroacidal efficacy of authorized VMP treatments in moderately infested colonies ensured normal overwintering and colony development during the next spring. Due to a lower efficacy of thymol-based VMPs observed at all EAs in this study conditions, it may be concluded that their use is limited under different treatment regimes. The results of this study imply that efficacy of used varroacidals strongly depends on geography, but also on timely manner, and can vary from season to season.
It can be concluded that an adequate V. destructor mite control must include a few measures, primarily good beekeeping maintenance techniques in combination with appropriate use of authorized VMPs. Different treatment regimes should also be applied with continuous parasitic mite mortality monitoring. Application of varroacides should be performed after the main honey flow, on all apiaries of the same epizootiology area, and in all honey bee colonies with mite infestations levels above the economic threshold. The same and appropriate treatment timing will ensure honey bee colonies surviving and prevent reinfestations. In specific situations, it is possible to use emergency treatments and alternate synthetic acaricides with food additives with acaricidal effect in rotation programs in order to decelerate the resistance of varroa mites to multiply used acaricides and to reduce the impact of increasing comb wax contamination. All varroacidal treatments must be performed in accordance and in combination with other specific regulations ordered by the national authorities.