POSSIBILITY OF TREATING VARROA ACARIASIS ( Varroa jacobsoni Oud ) WITH NATURAL SUBSTANCES

Bee acariasis, caused by the mite Varroa jacobsoni Oudemans Š261, represents a serious problem for apiculture. It is caused primarily due to the application of synthetic chemical miticides, which under certain conditions may result in the parasitism of bees. They become vulnerable to chemical treatment, along with probable deterioration of the quality of honey produced. This assay examines the possibility of treating bee Varroasis with a mixture of thymol, menthol and ethereal oils of eucalyptus and citronella. This mixture, in different doses, was applied to experimental hives and to regular Langstroh hives by means of specially-prepared pieces of moss. The greatest efficacy (89.71-90.20%) was found in both hives when the mixture was used in a dosage of 15 grams of thymol to one gram of menthol, 3 ml eucalyptus oil and 1 ml citronella.

In Greece, Varroa acariasis was firstly described in 1979 Š28, 11, 18¹.That year, the damage to Greek apiculture was extensive.Even now, the disease remains unsolved causing serious problems.
All methods used so far have been based on the extensive and frequent application of various synthetic chemical substances.This resulted in the appearance of resistance of these parasite strains, such as to fluvalinate Š34¹ and the deterioration of the quality of the honey goods produced, due to the presence of residues in them.By the development of protective science of the bee, other natural substances were discovered, more friendly to the ecosystem of bees.Among them, ethereal plant oils and the natural substances derived from them have been employed by many researchers with interesting results Š36, 12, 29, 17, 8, 30, 23, 35, 20¹.This study examines the possibility of treating this bee parasite through the application of a mixture of various natural substances.The experiment included preliminary attempts in order to determine the right proportions of the natural substances in the mixture and was carried out in specially prepared experimental model hives.In the second phase, the mixture of natural substances was employed in regular beehives.

Preliminary trials
In this phase the trials were carried out with model cube-shaped hives up to 18 cm in length.Each hive had the capacity to accommodate four frames and contained honeycombs 15 cm in length, 11 cm in height and 12 cm in thickness.These frames contained unworked masses of honeycomb.The population of each cell was homogeneous of 500 bees.The first infection affected between 12 and 14 percent of the bee swarm.The natural substances, employed in six different mixtures, were kindly provided by APIVITA S.A. and BIORYL, and were the following: Thymol, extracted from the ethereal oils of lavender (Lavadula officinalis), thyme (Corydothymus capitatus) and oregano (Origanum vulgare).The mixture is characterised by its distinctive perfume and antiseptic qualities.
Menthol, derived from the oils of olives and various types of mint (Mentha spp) Ethereal oil of eucalyptus (Eucalyptus globulus), which is obtained by distillation of partially dried-out leaves and twigs.
Citronella, the ethereal oil of the plant Cymbpogon nardus.

Materials and methods / Materijal i metode rada
The doses used are shown in Table 1.For the achievement of the greatest possible effectiveness, the mixtures were injected into speciallyprepared pieces of moss of dimensions 5 x 1 x 3 cm for the small doses and 5 x 1 x 5 for the large ones.The moss in the control cells was soaked in water.These moss pieces were then enclosed in an airtight plastic wrapping.To avoid disturbance to the bees, the wrapping around the moss was not removed the first day, but a slit about two centimetres long was cut.After 24 hours, the above-mentioned wrapping was removed entirely.
In the course of the experiment, the bee swarms in all the cells were fed on a syrup composed 50/50 of sugar and water.The sugar was placed in purpose-designed small flasks designed to fit the special holes no. 1 and 2 (Figure 1).
The experimental plan provided for six hives per operation with one control, repeated three times.To calculate the amount of mite infection remaining in the bees after application of the special preparation, the miticide Bromopropilate was used in the form of the commercial preparation Folbex VA.This preparation was not used previously for dealing with mite infections in the hives' mother swarm.One third of the Folbex VA strip was placed in each cell, morning and evening.
In order to evaluate the effectiveness of the mixtures used, five days after their application, readings were taken of the mites found on the bottom of the hives.The linear alternation method was used for statistical analysis of the data Š21¹.

Application in regular hives
For the purpose of this phase, regular hives of a contemporary design were used (Langstroh).These hives provided a special moveable base at the bot-361 Vet.glasnik 58 (3 -4) 359 -368 (2004)  tom, with net-like latticework at the top.Under the lattice there was a sliding plate spread with vaseline.Ventilation of the hive was effected through only half of the opening.During the experiment, the bees were fed with honey and had very few offspring.The temperature was recorded by a special thermograph.The population of the swarms perhive was about 30,000 bees.The infected population was 5.9 -6.7%.
The mixture of natural substances used was the optimum one from those tested during the preliminary trials.This particular mixture contained 15 g.thymol, 1 g menthol, 3 ml eucalyptus oil and 1 ml citronella.The application of the mixture was carried out in this phase with the assistance of moss pieces 5 x 1 x 5 cm, which were enclosed in special airtight plastic wrapping and placed inside the upper part of the honeycombs.In order to facilitate the diffusion of the vapours from the mixture to the hive, each moss piece was divided into three subfragments, placed in different positions.This procedure was carried out twice with an interval of a fortnight.For the control hives, the moss was saturated in tap water.
For the mites remaining in the hives after the application of the mixture, the commercial preparation Folbex VA was re-employed.On the 33 rd day of the experiment, two strips were set in place, one in the morning and one in the evening.Measurement of the mite infestation was carried out on the 16 th , 32 nd and 38 th days.The washing of 200 bees from each hive with benzine and 98% ethanol was aimed at the precise assessment of the efficacy of the miticide.
The experimental plan was used for two repetitions of the procedure.For each experiment, a group of 10 hives were used, while a similar group was used as control.Assessment of the effectiveness and processing of the data was carried out as in the preceding phase.Ulje eukaliptusa 1 ml 1 ml 3 ml 3 ml 3 ml 4.5 ml Citronella / Citronela 1 ml 1 ml 1 ml 1 ml 1 ml 1.5 ml

Preliminary trials
The highest rate of efficacy, of the natural substances mixtures employed, in terms of the proportion of mites exterminated, was found in Mixture 5.This mixture, with a total mite fall-off rate of 46.66 per cell (Table 2) controlled the bee parasite with a level of efficacy reaching 76.60%, and showed statistically significant differences in companison with mixtures 1, 2, 3 and 4 (Table 3).Mixture 6, with an efficacy of 96.60%, precipitated an unacceptable 15% casualty rate among bees at a temperature of 27 o C. By the application of linear alternation of the type y=bx (where y= the number of mites falling off and x= the dose of thymol applied) in mixtures 1 and 2 in which the dose of the other active substances was unchanged, the equation was arrived at which best conveyed the experimental data (R2 = 96.64%),which is as follows: Y = 3.69 (+0.24)x This equation determines the dosage of thymol, in relation to the number of dead mites falling off the bees.

Tests in regular hives
Mixture 5 applied in regular hives is able to control the mite with an efficacy ranging between 93.3 and 95.6% , which is very satisfactory (Table 4), when the low death rate from poisoning (0%) is also taken into consideration.Due to the damage caused by the mite to Apis melifera, the treatment of Varroa acariasis occupied and shall occupy many researchers throughout the world.The chemical method, involving synthetic substances, raises problems concerning deterioration of the quality of honey products, as well as the development of parasite strains which showed greater resistance to these substances.
The utilisation of natural substances in an integrated approach to the disease has become of particular interest nowadays.These are substances for which mechanisms of decomposition exist in the natural environment in wellknown transfigurations.
The combined use of thymol, menthol, camphor and eucalyptus oil can induce a 93.7%-97.7%extermination rate in the mite Š20, 7¹.In other experimental trials, thymol or camphor controlled the mite with an efficacy of 99.14% and 71.96% respectively, while menthol did not give satisfactory results Š16, 15¹.Depending on the manner of use, thymol may reduce mite populations by 13.9% up to 97.6% Š16, 15¹.Moreover, if it is applied five times every 3-4 days, the reduction should rise up to a high 98.6% Š15¹.The mixture of thymol, camphor, eucalyptus oil and menthol, known under the commercial name of Apilife-Var, compared to the mixture of formic, 15% lactic and 3.5% oxalic acids, yielded an extermination rate of 51.1%-68.7%,when the levels for the organic acids were 49.2, 98.8, 83.2 and 98.3 respectively Š25¹.
Other tests with thymol, menthol and ethereal oils of eucalyptus, lavender and thyme, oregano and citronella in which various different dosages and techniques were employed, thymol, menthol, eucalyptus oil and citronella were the most efficacious Š1, 2, 3¹.
Following the preliminary tests in special cells, this study indicated, in outdoor experiments with regular Langstroh hives, that the mixture of 15 g.thymol, 1 g.menthol, 3 ml.eucalyptus oil and 1 ml.citronella administered twice at a two-week interval can control the bee parasite V. jacobsoni at acceptable levels of efficacy.These results are of particular interest because they provide apiculturers with a simple, serviceable and economical method of treatment which replaces synthetic chemical miticides, well-known for their side-effects on product quality and for the ecosystems of bees.Thus, our study opens new paths for investigation, raising the possibility of treating other enemies and diseases of bees, by substances and methods friendlier to their ecosystem.Varroa acariasis predstavlja danas ozbiljan p~elarski patolo{ki problem za svetsko p~elarstvo.Izaziva ga parazit Varroa jacobsoni Oudemans (1904).Le~enje se zasniva prvenstveno na kori{}enju hemijskih sinteti~kih sredstava protiv parazita koja pod odre|enim uslovima umanjuju osetljivost parazita p~ele, a mogu} je i pad kvaliteta proizvedenog meda.

Table 3 .
H. Bacandritsos et al: Possibility of treating Varroa acariasis (Varroa jacobsoni Oud) with natural substances Efficacy of mixtures of natural substances in Varroa jacobsoni mite control /