**1. 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 [7–9], 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' managemen<sup>t</sup> 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 managemen<sup>t</sup> 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 managemen<sup>t</sup> 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 [20–22]. 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 [23–26].

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<sup>+</sup>, 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]. <sup>E</sup>fficacy 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.

#### **2. Materials and Methods**

#### *2.1. Locations of Experimental Apiaries and Field Trail Design*

The study was conducted during active beekeeping season of 2014 at five di fferent apiaries: EA1–EA5 (Figure 1; http://geoportal.dgu.hr/) in Croatia. EAs were located in di fferent geographical regions: EA1 (46◦ 5 37" N, 15◦ 53 34" E); EA2 (45◦ 48 16" N, 18◦ 39 54" E); EA3 (45◦ 13 45" N, 13◦ 56 29" E); EA4 (44◦ 36 54" N, 43◦ 55 46" E), and EA5 (43◦ 55 47" N, 16◦ 26 18" E). EA1 was situated in grassland surrounded by fields where intensive agriculture practice is in use; EA2 was on the lea surrounded with vegatable gardens; EA3 was located in a rural area surrounded with orchards and vineyards; EA4 was in the deciduous wood, and EA5 was situated on the grassland prairie. Due to the lack of natural food during July and August on the EA5 location, the honeybee colonies were moved after summer treatment against *V. destructor* mites to a new, more favourable location on the Adriatic sea island Vis. The new position of this apiary was annotated as EA5 (43◦ 2 45" N, 16◦ 9 14" E).

**Figure 1.** Locations of experimental apiaries in Croatia.

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.


**Table 1.** Field trail design.

Note: On every experimental apiary (EA), the negative control was included (untreated group of honeybee colonies, O; *n* = 5/apiary); treatment of broodless honeybee colonies with oxalic acid was carried out on all survivals during the winter period, except in EA5.

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#### *2.2. 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.
